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Effect of variation in maternal protein intake during gestation and lactation on fuel metabolism in the offspring – studies in the rat

Matthew Philip Greig Barnett

A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biological Sciences The University of Auckland 2004

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Abstract There is increasing evidence that events during early life can cause permanent changes in an organism, a concept known as programming. In mammals, the period of development from conception to weaning is particularly susceptible, and poor nutrition during this period may programme the metabolism of the offspring to survive on a limited food supply. When this phenomenon is combined with normal or supra-normal nutrition in later life (the so-called Thrifty Phenotype hypothesis), the outcome may be such diseases as hypertension, cardiovascular disease, and type-2 diabetes. In experiments described in this thesis, a rat model system was used to examine the effect of altered maternal nutrition during gestation and lactation on metabolic responses in the offspring. Specific responses investigated were pancreatic function, insulin sensitivity, and glucose intolerance, all of which may be associated with the development of type-2 diabetes. Two studies were performed, one using diets prepared in the laboratory, and one using commercially prepared diets of the same specification. In both cases, changes were evident in the insulin and glucose metabolism of offspring of mothers fed a lowprotein diet during gestation and lactation. Results were not consistent between the two trials, in part because of mortality of offspring in the first trial reducing statistical significance. Overall, poor nutrition in the form of insufficient protein during early life is shown to have a significant effect on insulin and glucose metabolism, and this effect may persist throughout the life of an organism. However, a number of other factors, both genetic and environmental, are also influential in the development of such diseases as type-2 diabetes, and the evidence here is not sufficient in itself to conclusively support the idea that early nutrition is the key or critical factor in the development of this disease.

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Table of Contents Abstract...................................................................................................................................... iii Dedication ............................................................................... Error! Bookmark not defined. Acknowledgements ............................................................... Error! Bookmark not defined. Table of Contents ...................................................................................................................... ix List of Figures ...........................................................................................................................xv List of Tables .......................................................................................................................... xvii Abbreviations .......................................................................................................................... xix Company abbreviations and addresses ............................................................................. xxii Chapter 1 Introduction ........................................................................................................ 1 1.1 Diabetes Mellitus........................................................................................................ 3 1.1.1 History ..................................................................................................................... 3 1.1.2 Definition of diabetes ............................................................................................ 4 1.1.3 Classification of diabetes mellitus ....................................................................... 5 1.1.4 Factors contributing to type-2 diabetes .............................................................. 6 1.2 The fetal/infant origins of adult disease ................................................................ 8 1.2.1 Background ............................................................................................................. 8 1.2.2 The intra-uterine environment ............................................................................. 9 1.2.3 Metabolic programming ....................................................................................... 9 1.2.4 The Thrifty Phenotype hypothesis .................................................................... 11 1.2.5 Rehabilitation........................................................................................................ 11 1.2.6 Animal models ..................................................................................................... 13 1.2.7 An over-stretched hypothesis?........................................................................... 13 1.2.8 Relevance to the current thesis........................................................................... 15 1.3 The pancreas ............................................................................................................. 15 1.3.1 Development of the Endocrine Pancreas with emphasis on the -cell. ....... 15 1.3.1.1 Molecular basis of pancreatic development ............................................. 17 1.3.1.2 Differentiation of endocrine and exocrine cell types during pancreatic development ................................................................................................................... 19 1.3.1.3 Post-natal Homeostasis of the -cell Mass ............................................... 20 1.4 Insulin ........................................................................................................................ 21 1.4.1 Discovery............................................................................................................... 21 1.4.2 General description.............................................................................................. 22 1.4.3 Biosynthesis and secretion .................................................................................. 23 1.4.4 The insulin receptor ............................................................................................. 23 1.4.5 Signal transduction .............................................................................................. 24 1.4.6 Insulin resistance .................................................................................................. 25 1.4.6.1 The insulin resistance syndrome ............................................................... 26 1.4.7 Prediction of Type-2 Diabetes ............................................................................ 27 1.4.7.1 Clinical models ............................................................................................. 27 1.4.7.2 Measures of Insulin Resistance as Clinical Predictors of Type-2 Diabetes ......................................................................................................................... 28 1.5 Amylin ....................................................................................................................... 30 1.5.1 Comparison and contrast of the secretion of insulin and of amylin ............ 31 1.5.2 Other pancreatic hormones ................................................................................ 31 1.6 Insulin and the fetal/infant origins of adult disease .......................................... 32 ix

1.6.1 Insulin production ............................................................................................... 32 1.6.2 Insulin action ........................................................................................................ 33 1.7 Investigative model and hypothesis...................................................................... 33 1.7.1 Background and rationale................................................................................... 33 1.7.2 Hypotheses............................................................................................................ 35 1.7.3 Expected original contribution ............................................................................... 36 Chapter 2 General Methods .............................................................................................. 37 2.1 Animals ...................................................................................................................... 39 2.1.1 General ................................................................................................................... 39 2.1.2 Calculation of daily food requirements ............................................................ 39 2.2 Composition of experimental diets ....................................................................... 40 2.3 Isolated Perfused Pancreas ..................................................................................... 44 2.3.1 Background ........................................................................................................... 44 2.3.2 Solutions ................................................................................................................ 44 2.3.2.1 Pancreas Perfusate ....................................................................................... 44 2.3.2.1.1 Stock solutions: ....................................................................................... 44 2.3.2.1.2 Perfusate preparation ............................................................................ 44 2.3.2.2 Infusion solutions ......................................................................................... 45 2.3.3 Surgical procedure ............................................................................................... 45 2.4 Insulin RIA ................................................................................................................ 48 2.4.1 Background ........................................................................................................... 48 2.4.2 Reagents................................................................................................................. 48 2.4.2.1 Phosphate buffer stock solution ................................................................. 48 2.4.2.2 Insulin Assay Buffer .................................................................................... 48 2.4.2.3 Insulin Primary Antibody ........................................................................... 48 2.4.2.4 Tracer ............................................................................................................. 49 2.4.2.5 PBS-EDTA ..................................................................................................... 49 2.4.2.6 Secondary Antibody .................................................................................... 49 2.4.2.7 Normal Guinea Pig Serum .......................................................................... 49 2.4.2.8 PEG buffer ..................................................................................................... 49 2.4.2.9 Insulin Standard ........................................................................................... 49 2.4.3 Samples .................................................................................................................. 51 2.4.4 The Insulin Assay ................................................................................................. 51 2.4.4.1 Day One ......................................................................................................... 51 2.4.4.2 Day Three ...................................................................................................... 51 2.4.4.3 Insulin Assay Characteristics ..................................................................... 52 2.5 Amylin RIA ............................................................................................................... 53 2.5.1 Background ........................................................................................................... 53 2.5.2 Reagents................................................................................................................. 53 2.5.2.1 Amylin Assay Buffer ................................................................................... 53 2.5.2.2 Primary Antibody ........................................................................................ 53 2.5.2.3 Tracer ............................................................................................................. 53 2.5.2.4 Secondary Antibody .................................................................................... 53 2.5.2.5 Normal Rabbit Serum (NRS) ...................................................................... 53 2.5.2.6 Amylin PEG Buffer ...................................................................................... 53 2.5.2.7 Amylin Standard .......................................................................................... 54 2.5.3 Samples .................................................................................................................. 54 2.5.4 The Amylin Assay ................................................................................................ 54 x

2.5.4.1 Day One ......................................................................................................... 54 2.5.4.2 Day Two ........................................................................................................ 56 2.5.4.3 Day Four ........................................................................................................ 56 2.5.4.4 Amylin Assay Characteristics .................................................................... 56 2.6 Preptin RIA ............................................................................................................... 56 2.6.1 Reagents ................................................................................................................ 56 2.6.1.1 Primary Antibody ........................................................................................ 57 2.6.1.2 Tracer ............................................................................................................. 57 2.6.1.3 Preptin Standard .......................................................................................... 58 2.6.2 The Preptin Assay ................................................................................................ 59 2.6.2.1 Day one .......................................................................................................... 59 2.6.2.2 Day three ....................................................................................................... 59 2.6.2.3 Day five.......................................................................................................... 59 2.6.2.4 Preptin Assay Characteristics..................................................................... 59 2.7 Glucose uptake assay in the isolated soleus muscle ........................................... 59 2.7.1 Background ........................................................................................................... 59 2.7.2 Reagents ................................................................................................................ 60 2.7.2.1 Krebs-Henseleit buffer ................................................................................ 60 2.7.2.2 Normal Dulbecco’s Modified Eagle Medium (nDMEM) ....................... 61 2.7.2.3 Insulin Stock Solution .................................................................................. 61 2.7.2.4 Insulin Working Solutions .......................................................................... 61 2.7.2.5 U-[14C]-D-Glucose ....................................................................................... 61 2.7.3 Experimental Protocol ......................................................................................... 64 2.7.3.1 Preparation of muscle strips ....................................................................... 64 2.7.3.2 Incubation ..................................................................................................... 64 2.7.3.3 Glycogen extraction and liquid scintillation counting ........................... 65 2.8 Statistical Methods ................................................................................................... 66 2.9 Ethical Approval ...................................................................................................... 67 Chapter 3 Trial 1: The effect of maternal protein intake on glucose metabolism and pancreatic function in the offspring ...................................................................................... 69 3.1 Introduction .............................................................................................................. 71 3.2 Aims ........................................................................................................................... 71 3.3 Methods ..................................................................................................................... 72 3.3.1 Diets ....................................................................................................................... 74 3.3.2 Preparation of experimental diets ..................................................................... 74 3.3.2.1 Dietary analysis ............................................................................................ 75 3.3.2.2 Dietary energy content ................................................................................ 75 3.3.2.3 Dietary protein content ............................................................................... 76 3.3.3 Animals.................................................................................................................. 76 3.3.3.1 Food intake and bodyweight ..................................................................... 76 3.3.3.2 Mass-corrected food intake ........................................................................ 77 3.3.3.3 Efficiency calculations ................................................................................. 77 3.3.4 Physical characteristics ........................................................................................ 77 3.3.5 Isolated perfused pancreas ................................................................................. 78 3.3.5.1 Measurement of pancreatic hormone secretion ...................................... 78 3.3.5.2 The amylin/insulin ratio ............................................................................ 79 3.3.5.3 Area under the curve ................................................................................... 79 3.3.5.4 Relative insulin response to stimulation .................................................. 79 xi

3.3.5.5 First phase insulin secretion ....................................................................... 79 3.3.5.6 Weight-matched control group .................................................................. 82 3.3.6 Glucose uptake in the isolated soleus muscle .................................................. 82 3.4 Results ........................................................................................................................ 83 3.4.1 Dietary analyses ................................................................................................... 83 3.4.2 Dams ...................................................................................................................... 83 3.4.2.1 Food intake .................................................................................................... 83 3.4.2.2 Bodyweights ................................................................................................. 87 3.4.2.3 Mass-corrected food intake......................................................................... 88 3.4.2.4 Feeding efficiency ........................................................................................ 90 3.4.3 Litter size ............................................................................................................... 92 3.4.4 Offspring ............................................................................................................... 92 3.4.4.1 Mortality ........................................................................................................ 92 3.4.4.2 Physical characteristics ................................................................................ 92 3.4.4.3 Food Intake ................................................................................................... 92 3.4.4.4 Bodyweight ................................................................................................. 100 3.4.4.5 Mass-corrected food intake....................................................................... 100 3.4.4.6 Feeding efficiency ...................................................................................... 100 3.4.4.6.1 Total post-weaning period.................................................................. 100 3.4.4.6.2 Between 49 g and 75 g body weight .................................................. 100 3.4.4.7 Isolated perfused pancreas ....................................................................... 100 3.4.4.7.1 Glucose and lactate measurements ................................................... 101 3.4.4.7.2 Insulin production ............................................................................... 104 3.4.4.7.3 Amylin production .............................................................................. 106 3.4.4.7.4 Amylin/insulin ratio ........................................................................... 106 3.4.4.7.5 Relative responses to infusions .......................................................... 109 3.4.4.8 Uptake of glucose into glycogen .............................................................. 110 3.5 Discussion ............................................................................................................... 114 3.5.1 Dams .................................................................................................................... 114 3.5.1.1 Food intake .................................................................................................. 114 3.5.1.2 Diets ............................................................................................................. 114 3.5.1.3 Energy intake .............................................................................................. 117 3.5.1.4 Body weight ................................................................................................ 117 3.5.2 Offspring performance ...................................................................................... 118 3.5.2.1 Mortality ...................................................................................................... 118 3.5.2.2 Food intake .................................................................................................. 119 3.5.2.3 Body weight ................................................................................................ 119 3.5.3 Isolated perfused pancreas ............................................................................... 120 3.5.3.1 Insulin .......................................................................................................... 120 3.5.3.2 Amylin ......................................................................................................... 122 3.5.3.3 Amylin/insulin ratio ................................................................................. 123 3.5.4 Glucose uptake into glycogen .......................................................................... 124 3.6 Conclusions ............................................................................................................. 125 Chapter 4 Trial 2: Further studies on the effect of maternal protein intake during gestation and lactation on glucose metabolism in the offspring ..................................... 127 4.1 Introduction ............................................................................................................ 129 4.2 Aims ......................................................................................................................... 129 4.2.1 Additional outcome measures ......................................................................... 130 xii

4.2.1.1 Intra-peritoneal glucose tolerance test (IPGTT)..................................... 130 4.2.1.2 Secretion of preptin from the isolated perfused pancreas ................... 130 4.2.2 Second generation (F2) studies ........................................................................ 130 4.3 Methods ................................................................................................................... 132 4.3.1 Experimental diets ............................................................................................. 132 4.3.2 Animals................................................................................................................ 132 4.3.2.1 F2 studies ..................................................................................................... 134 4.3.3 Glucose Tolerance Test ...................................................................................... 135 4.3.3.1 Background ................................................................................................. 135 4.3.3.2 Materials ...................................................................................................... 135 4.3.3.3 Procedure .................................................................................................... 135 4.3.4 Isolated perfused pancreas ............................................................................... 136 4.3.4.1 Pancreas function ....................................................................................... 136 4.3.4.2 The effect of amylin on pancreatic function ........................................... 136 4.3.4.3 Amylin 8-37 ................................................................................................... 136 4.3.4.4 Solutions ...................................................................................................... 137 4.3.4.4.1 Bovine Serum Albumin (0.1 %) .......................................................... 137 4.3.4.4.2 Amylin 8-37 ............................................................................................. 137 4.3.4.5 Infusion of amylin 8-37 in the perfused pancreas system ...................... 137 4.3.5 Glucose uptake in the isolated soleus muscle ................................................ 138 4.4 Results ...................................................................................................................... 139 4.4.1 Dietary analyses ................................................................................................. 139 4.4.2 Dams .................................................................................................................... 139 4.4.2.1 Food intake.................................................................................................. 139 4.4.2.2 Bodyweight ................................................................................................. 140 4.4.2.3 Mass-corrected food intake ...................................................................... 147 4.4.2.4 Feeding efficiency ...................................................................................... 148 4.4.3 Litter size ............................................................................................................. 149 4.4.4 Offspring ............................................................................................................. 150 4.4.4.1 Mortality ...................................................................................................... 151 4.4.4.2 Food Intake ................................................................................................. 151 4.4.4.3 Bodyweight ................................................................................................. 151 4.4.4.4 Mass-corrected food intake ...................................................................... 151 4.4.4.5 Feeding efficiency ...................................................................................... 151 4.4.4.6 Data between 49 g and 75 g body weight ............................................... 153 4.4.4.7 Intra-peritoneal glucose tolerance test .................................................... 157 4.4.4.8 Isolated perfused pancreas ....................................................................... 162 4.4.4.8.1 Glucose and lactate measurements ................................................... 162 4.4.4.8.2 Hormone secretion: F1 generation studies ....................................... 162 4.4.4.8.3 Hormone secretion: F2 generation studies ....................................... 168 4.4.4.8.4 Infusion of amylin 8-37 .......................................................................... 171 4.4.4.9 Uptake of glucose into glycogen measured in the isolated soleus muscle ....................................................................................................................... 174 4.5 Discussion ............................................................................................................... 178 4.5.1 Maternal effects .................................................................................................. 178 4.5.2 Offspring responses ........................................................................................... 179 4.5.2.1 Mortality ...................................................................................................... 179 4.5.2.2 Food intake and body weight .................................................................. 180 xiii

4.5.2.3 Catch-up growth ........................................................................................ 180 4.5.2.4 Intra-peritoneal glucose tolerance test .................................................... 181 4.5.2.5 Isolated perfused pancreas ....................................................................... 182 4.5.2.5.1 Insulin .................................................................................................... 182 4.5.2.5.2 Amylin ................................................................................................... 183 4.5.2.5.3 Preptin.................................................................................................... 184 4.5.2.5.4 F2 studies ............................................................................................... 185 4.5.2.6 Effect of amylin 8-37 on the secretion of insulin ...................................... 186 4.5.3 Uptake of glucose into glycogen ...................................................................... 188 4.6 Conclusions ............................................................................................................. 190 Chapter 5 General Discussion ........................................................................................ 193 5.1 Maternal effects ...................................................................................................... 195 5.2 Offspring ................................................................................................................. 195 5.2.1.1 Timing of the nutritional insult ................................................................ 198 5.3 Results in relation to the Thrifty Phenotype hypothesis .................................. 199 5.4 Improvements and further research .................................................................... 202 5.4.1 Power analyses ................................................................................................... 202 5.4.2 Diets ..................................................................................................................... 203 5.4.3 Additional outcome measures ......................................................................... 204 5.5 Conclusions ............................................................................................................. 205 Chapter 6

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List of References ........................................................................................... 207

List of Figures Figure 1.1: The Thrifty Phenotype hypothesis ..................................................................... 12 Figure 2.1: Food intake as a function of body weight: data from a previous study in pregnant female Wistar rats fed standard chow (NRM Diet 86)................................ 42 Figure 2.2: Side-arm infusion ................................................................................................. 47 Figure 2.3: Representative standard curve of the insulin RIA .......................................... 52 Figure 2.4: Representative standard curve of the amylin RIA .......................................... 57 Figure 2.5: Representative standard curve of the preptin RIA.......................................... 60 Figure 3.1: Experimental design of the first trial ................................................................. 73 Figure 3.2: Protocol for isolated perfused pancreas experiments performed on the offspring of mothers fed diets varying in protein content and type during gestation and lactation ....................................................................................................................... 80 Figure 3.3: Pattern of calculation of area under the curve (AUC) data for fractions collected from the isolated perfused pancreases of offspring of female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation. ..................................................................................................... 81 Figure 3.4 Phases of insulin secretion measured in the isolated perfused rat pancreas 82 Figure 3.5: Daily food intake of female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation. .............. 86 Figure 3.6: Daily bodyweight of female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation ............... 89 Figure 3.7: Daily mass-corrected food intake of female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation .............................................................................................................................................. 91 Figure 3.8: Survival curve for offspring of female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation .............................................................................................................................................. 93 Figure 3.9: Daily food intake of offspring of female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation .............................................................................................................................................. 95 Figure 3.10: Bodyweights of offspring of female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation .............................................................................................................................................. 98 Figure 3.11: The relationship between food intake and bodyweight in offspring of female rats fed standard rat chow or chemically defined diets varying in protein content during gestation and lactation .......................................................................... 99 Figure 3.12: Levels of hormones measured in fractions obtained from the isolated perfused pancreas of offspring of mothers fed chemically defined diets varying in whey protein content during gestation and lactation ................................................ 102 Figure 3.13: Levels of hormones measured in fractions obtained from the isolated perfused pancreas of offspring of mothers fed normal rat chow through gestation and lactation ..................................................................................................................... 103 Figure 3.14: Total insulin production by the perfused pancreases isolated from offspring of mothers fed standard rat chow or chemically defined diets varying in protein content through gestation and lactation ........................................................ 105

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Figure 3.15: Total amylin production measured in the perfused pancreases isolated from offspring of mothers fed standard rat chow or chemically defined diets varying in protein content through gestation and lactation ..................................... 107 Figure 3.16: Amylin/insulin ratios in perfused pancreases isolated from offspring of mothers fed standard rat chow or chemically defined diets varying in protein content through gestation and lactation ...................................................................... 108 Figure 3.17: Trellis plot of individual litter data for uptake of glucose into glycogen in soleus muscle strips isolated from offspring of mothers fed standard rat chow or chemically defined diets varying in protein content through gestation and lactation ............................................................................................................................................ 111 Figure 3.18: Uptake of labelled glucose into glycogen in soleus muscle strips isolated from offspring of mothers fed standard rat chow or chemically defined diets varying in protein content through gestation and lactation ..................................... 112 Figure 4.1: Experimental design of the second feeding trial............................................ 133 Figure 4.2: Protocol for isolated perfused pancreas with infusion of amylin 8-37. ........ 138 Figure 4.3: Mean daily food intake of female rats fed commercially prepared experimental diets differing in whey protein content during gestation and lactation ............................................................................................................................. 143 Figure 4.4: Daily bodyweight measurements of female rats fed commercially prepared experimental diets varying in whey protein content during gestation and lactation. ............................................................................................................................................ 146 Figure 4.5: Mean daily mass-corrected food intake for female rats fed commercially prepared experimental diets varying in whey protein content during gestation and lactation ............................................................................................................................. 148 Figure 4.6: Survival curves for offspring of female rats fed commercially prepared experimental diets varying in whey protein content during gestation and lactation. ............................................................................................................................................ 152 Figure 4.7: Mean daily consumption measured in the offspring of female rats fed commercially available experimental diets varying in protein content during gestation and lactation .................................................................................................... 154 Figure 4.8: Mean bodyweight of first (F1) and second (F2) generation offspring of female rats fed commercially available experimental diets varying in protein content during gestation and lactation......................................................................... 155 Figure 4.9: Food intake as a function of bodyweight for offspring of female rats fed commercially prepared experimental diets varying in protein content during gestation and lactation .................................................................................................... 156 Figure 4.10: Results of glucose tolerance tests performed on first generation (F1) offspring of mothers fed commercially prepared experimental diets varying in protein content during gestation and lactation. .......................................................... 158 Figure 4.11: Area under the curve data calculated from glucose tolerance tests performed in the first generation (F1) offspring of female rats fed commercially prepared experimental diets varying in protein content during gestation and lactation ............................................................................................................................. 160 Figure 4.12: Results of glucose tolerance tests performed on second generation (F2) offspring of mothers fed commercially prepared experimental diets varying in protein content during gestation and lactation ........................................................... 161 Figure 4.13: Levels of hormones measured in fractions obtained from the isolated perfused pancreas of first generation (F1) offspring of mothers fed commercially xvi

prepared chemically defined diets varying in whey protein content during gestation and lactation .................................................................................................... 166 Figure 4.14: Ratios of hormones secreted from the isolated perfused pancreases of first generation (F1) offspring of mothers fed commercially prepared chemically defined diets varying in protein content through gestation and lactation. ............ 167 Figure 4.15: Levels of hormones measured in fractions obtained from the isolated perfused pancreas of second generation (F2) offspring of mothers fed commercially prepared chemically defined diets varying in whey protein content during gestation and lactation....................................................................................... 169 Figure 4.16: Levels of hormones measured in fractions obtained from ‘control’ (infused with 0.1% BSA) perfused pancreases isolated from first generation (F1) offspring of mothers fed commercially prepared experimental diets varying in whey protein content during gestation and lactation ........................................................................ 170 Figure 4.17: Effect of the infusion of amylin 8-37 on insulin secretion measured in perfused pancreases isolated from first generation (F1) offspring of mothers fed commercially prepared experimental diets varying in whey protein content during gestation and lactation .................................................................................................... 173 Figure 4.18: Trellis plot of individual litter data for uptake of glucose into glycogen in soleus muscle strips isolated from offspring of mothers fed chemically defined diets varying in protein content through gestation and lactation............................ 175 Figure 4.19: Incorporation of labeled glucose into glycogen in soleus muscle strips isolated from offspring of mothers fed commercially prepared chemically defined diets varying in protein content through gestation and lactation............................ 177

List of Tables Table 2.1: General composition of experimental diets fed to pregnant and lactating female rats .......................................................................................................................... 43 Table 2.2: Serial dilutions by which insulin RIA standard solutions were prepared .... 50 Table 2.3: Serial dilutions by which amylin RIA standard solutions were prepared .... 55 Table 2.4: Serial dilutions by which preptin RIA standard solutions were prepared ... 58 Table 2.5: Stock solutions for the preparation of Krebs-Henseleit buffer ........................ 62 Table 2.6: Preparation of insulin working solutions for use in the glucose uptake assay .............................................................................................................................................. 63 Table 3.1: Dietary analyses performed on all diets fed to mothers and offspring during the first experimental trial ................................................................................................ 84 Table 3.2: Summary of numbers of female rats mated for use in the first feeding trial, and the outcome of these matings .................................................................................. 85 Table 3.3: Food intake and feeding efficiency data for female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation............................................................................................................................... 87 Table 3.4: Statistical analysis of maternal bodyweight ....................................................... 89 Table 3.5: Growth characteristics and the day on which they occurred within offspring of female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation............................................................. 94

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Table 3.6: Effect of maternal diet on the bodyweight of offspring of female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation ...................................................................................................... 96 Table 3.7: Food intake and feeding efficiency data for offspring of female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation ...................................................................................................... 97 Table 3.8: Insulin production in the isolated perfused pancreas: response to glucose and glucose + arginine stimulations as a function of arginine response in the offspring of mothers fed standard rat chow or chemically defined diets varying in protein content through gestation and lactation ........................................................ 109 Table 3.9: Nonlinear regression analysis of four parameter logistic model fitted to glucose uptake data derived from offspring of mothers fed standard rat chow or chemically defined diets varying in protein content through gestation and lactation ............................................................................................................................................ 113 Table 4.1: Dietary analyses performed on diets fed to mothers and offspring during the second experimental trial ............................................................................................... 141 Table 4.2: Summary of numbers of female rats mated for use in the second feeding trial, and the outcome of these matings ....................................................................... 142 Table 4.3: Statistical analysis of total food intake and feeding efficiency for female rats fed commercially prepared experimental diets differing in whey protein content during gestation and lactation ....................................................................................... 144 Table 4.4: Statistical analysis of bodyweight data from mothers fed commercially prepared experimental diets varying in whey protein content during gestation and lactation ............................................................................................................................. 147 Table 4.5: Summary of numbers, survival, and outcome measures performed on offspring of mothers fed commercially prepared experimental diets during gestation and lactation .................................................................................................... 150 Table 4.6: Statistical analysis of total food intake and feeding efficiency for offspring of female rats fed commercially prepared experimental diets differing in whey protein content during gestation and lactation ........................................................... 153 Table 4.7: Summary of data from intra-peritoneal glucose tolerance tests performed on offspring of mothers fed chemically defined diets differing in protein content during gestation and lactation. ...................................................................................... 159 Table 4.8: Levels of hormones measured in fractions obtained from the isolated perfused pancreas of offspring of mothers fed chemically defined diets varying in whey protein content during gestation and lactation ................................................ 164 Table 4.9: Ratios of pancreatic hormones measured in fractions obtained from the isolated perfused pancreas of offspring of mothers fed chemically defined diets varying in whey protein content during gestation and lactation............................. 165 Table 4.10: The effect of the infusion of amylin 8-37 on isolated perfused pancreases from donor rats, the mothers of which had been fed isocaloric semi-synthetic diets varying in protein content during gestation and lactation ....................................... 172 Table 4.11: Nonlinear regression analysis of four parameter logistic model fitted to glucose uptake data derived from offspring of mothers fed commercially prepared chemically defined diets varying in protein content through gestation and lactation ............................................................................................................................................ 176 Table 5.1: Power analyses for various outcome measures, based on experimental data obtained from the second experimental trial .............................................................. 203 xviii

Abbreviations 

alpha

ADP

adenosine diphosphate

Amylin8-37

the 8-37 amino acid fragment of amylin

ATP

adenosine triphosphate

AUC

area under the curve



beta

BSA

bovine serum albumin

14C

carbon 14 isotope

cAMP

cyclic adenosine monophosphate

CGRP

calcitonin gene-related peptide

Ci

Curies

cpm

counts per minute

Da

Daltons

DAP

diabetes associated peptide

DMEM

Dulbecco’s modified Eagle medium

EC50

50% of the effective concentration

EDTA

ethylenediaminetetraacetic acid

EEU

efficiency of energy utilization

eIF-2B

eukaryotic initiation factor 2B

F0

parent generation of F1 offspring

F1

first generation offspring

F2

second generation offspring

FCE

food conversion efficiency

GLUT

glucose transporter xix

GSK3

glycogen synthase kinase 3

GTT

glucose tolerance test

GUA

glucose uptake assay

hCGRP

human calcitonin gene-related peptide

125I

iodine 125 isotope

IGF

insulin-like growth factor

IgG

immunoglobulin G

IP

intra-peritoneal

IPP

isolated perfused pancreas

IRS

insulin receptor substrate

LDL

low density lipoprotein



micro (1 x 10-6)

min

minutes

mTOR

mammalian target of rapamycin

nDMEM

normal Dulbecco’s modified Eagle medium

NGPS

normal guinea-pig serum

NME

net metabolisable energy

NRS

normal rabbit serum

NS

not significant

NSB

non specific binding

PBS

phosphate buffered saline

PDK1

3-phosphoinositide-dependent protein kinase-1

PEG

polyethylene glycol

PEPCK

phosphoenolpyruvate carboxykinase

PI-3 kinase

phosphatidylinositol 3 kinase

PI-3,4,5-P3

phosphatidylinositide 3,4,5 triphosphate

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PI-4,5-P2

phosphatidylinositide 4,5 bisphosphate

PKB

protein kinase B

REML

residual maximum likelihood

RIA

radio immunoassay

RNA

ribonucleic acid

SED

standard error of the difference

SEM

standard error of the mean

SUR

sulphonylurea receptor

TC

total counts

TDI

time-dependent inhibition

TDP

time-dependent potentiation

TNF-

tumour necrosis factor alpha

w/v

weight per unit volume

Zdf-drt

Zucker diabetic fatty rat

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Company abbreviations and addresses

AMCO

Goodman Fielder New Zealand Goodman Fielder House Cnr Springs & East Tamaki Roads (7 Springs Road) East Tamaki, Auckland New Zealand www.uncletobys.com.au

ARC:

American Radiolabeled Chemicals, Inc. 11624 Bowling Green Drive St. Louis, MO 63146 USA www.arc-inc.com

Auspep

Auspep Pty Ltd P.O. Box 806 Parkville Victoria 3052 Australia www.auspep.com.au

Avon

Penford New Zealand Ltd. 319 Church St Onehunga Auckland New Zealand E-mail: [email protected]

Baxter

Baxter Healthcare Corporation One Baxter Parkway Deerfield, IL 60015 USA www.baxterdrugdelivery.com

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B-D

Beckton, Dickinson and Company 1 Becton Drive Franklin Lakes, NJ 07417 USA www.bd.com

BDH

VWR International Ltd - Poole Merck House Poole Dorset BH15 1TD England www.bdh.com

Beckman

Beckman Coulter, Inc. 4300 N. Harbor Boulevard P. O. Box 3100 Fullerton, CA 92834-3100 USA www.beckman.com

Biomed

Biomed Limited PO Box 44 069 Pt. Chevalier Auckland New Zealand

BOC

BOC Gases New Zealand Limited 988 Great South Road Penrose Auckland www.boc.co.nz

xxiii

Chelsea

New Zealand Sugar Company Ltd Chelsea Refinery 60 Colonial Rd Birkenhead Auckland New Zealand www.chelsea.co.nz

Crop and Food

Crop & Food Research Private Bag 11600 Palmerston North New Zealand www.cropandfood.co.nz

Eppendorf

Eppendorf AG Barkhausenweg 1 22331 Hamburg Germany www.eppendorf.com

FTS:

FTS Systems Kinetics Group, Inc. 2805 Mission College Blvd. Santa Clara, CA 95054 USA www.kineticsgroup.com

Gelman Sciences

(A subsidiary of Pall Corporation) 600 S. Wagner Rd Ann Arbor, MI 48103 USA www.pall.com

xxiv

Hybaid

Thermo Hybaid Action Court Ashford Road Ashford, Middlesex TW15 1XB U.K. www.thermohybaid.com

IEC

International Equipment Company 300 Second Avenue Needham Heights, MA 02494 USA www.labcentrifuge.com

ICN

ICN Biomedicals Inc 1263 South Chillicothe Rd Aurora, Ohio 44202 USA www.icnbiomed.com

Jouan

Jouan Incorporated 170 Marcel Drive Virginia 22602 USA www.jouaninc.com

Kent Scientific

The Kent Scientific Corporation 1116 Litchfield Street Torrington, CT 06790 USA www.kentscientific.com

Mettler

Mettler-Toledo Inc. 1900 Polaris Parkway Columbus, OH 43240-4035 USA www.mt.com xxv

Novo Nordisk

Novo Nordisk A/S Novo Allé 2880 Bagsværd Denmark www.novonordisk.com

NRM

NRM New Zealand Ltd Private Bag 99-927 Newmarket Auckland New Zealand www.nrm.co.nz

Nunc

Nalge Nunc International 75 Panorama Creek Drive Rochester, NY 14625-2385 USA www.nuncbrand.com

Nutritech

Nutritech International Ltd 12 Fisher Crescent Mt Wellington Auckland New Zealand www.nutritech.co.nz

NZ Dairy

NZMP Head Office (New Zealand) PO Box 417 Pastoral House 25 The Terrace Wellington New Zealand www.nzmp.com

xxvi

PCL

PCL Industries Limited Don Buck Rd Massey PO Box 79048 Royal Heights Auckland New Zealand

Peninsula

Peninsula Laboratories Inc. 601 Taylor Way San Carlos, CA 94070 USA www.penlabs.com

Riedel-de Haën

Sigma-Aldrich Laborchemikalien GmbH P.O. Box 100262 30918 Seelze Germany Email: [email protected] www.sigmaaldrich.com

Roche

F. Hoffmann-La Roche Ltd Grenzacherstrasse 124 CH-4070 Basel Switzerland www.roche.com

Sigma

Sigma-Aldrich Corporation St. Louis, Missouri USA www.sigmaaldrich.com

Sorvall

Kendro Laboratory Products 31 Pecks Lane Newtown, CT 06470-2337 USA www.sorvall.com xxvii

Techno-Plas

Techno-Plas Pty Ltd 8 Benjamin Street St Marys South Australia 5042 www.technoplas.com.au

Wallac

LKB Wallac Wallac Oy PO Box 10 20101 Turku Finland www.perkinelmer.com

YSI

Yellow Springs Instrument Co., Inc. Yellow Springs, Ohio 45387 USA www.ysi.com

xxviii

Chapter 1

Introduction

Voltaire (1694-1778) at his desk, spare pen at the ready in what appears to be a combination inkwell/pen holder with space for up to four pens. Detail from an engraving by Baquoy (ca. 1795). Reproduced in Age of Enlightenment (1966), Peter Gay. From: http://www.jasa.net.au/quillpen.htm

1

2

1.1 Diabetes Mellitus 1.1.1 History As early as 1500 BC an illness was described in the Egyptian Ebers Papyrus (Haas, 1999), of which one symptom was excessive passing of urine. What seems to be the same disease was, in the second century AD, described by the physician Aretaeus of Cappadocia as: “a melting down of the flesh and limbs into urine” (Reed, 1954). He is generally accepted as being the first to name the disease “diabetes”, from the Greek word meaning “a siphon” which is in turn derived from diabainō (Brown, 1993), meaning “to go through” (Reed, 1954). Thomas Willis in 1674 described this excessive urine as “being exceedingly sweet, as if there had been Sugar or Honey in it.” ((Willis, 1679), as quoted by Allan (Allan, 1953)). Thus the disease was given the name diabetes mellitus, mellitus meaning sweet in Latin (Brown, 1993). In 1776, Matthew Dobson showed that this sweetness, which was also present in the blood, was due to the presence of sugar ((Dobson, 1776), as discussed in an Editoral from the Journal of the American Medical Association (No Authors Listed, 1968)) and so demonstrated an abnormality in carbohydrate metabolism associated with the disease. The elucidation of the involvement of the pancreas in diabetes was a critical step in understanding the pathogenesis of the disease, and involved some luck. In 1689, Johann Conrad Brunner surgically removed part of the pancreas from a dog (a subtotal pancreatectomy) and described the resulting symptoms, but failed to associate these symptoms with diabetes (Dittrich and von Dorsche, 1978; Keck and Pfeiffer, 1989). It was not until 200 years later in Strasbourg that Joseph Freiherr Von Mering and Oskar Minkowski also serendipitously performed a pancreatectomy (this time total) on a dog. At the time they were studying the digestive absorption of lipids, but Minkowski correctly associated the symptoms they observed as a result of the pancreatectomy with diabetes, and thus elucidated the central role of the pancreas in the development of the disease ((von Mering and Minkowski, 1889), as reviewed by Luft (Luft, 1989)). This discovery stimulated research in a number of countries to find the “internal secretion from the pancreas which controlled carbohydrate metabolism” that many 3

researchers now believed to exist (Tattersall, 1995). In 1922 this search led to the discovery of insulin, for which Frederick G Banting and John JR MacLeod received the 1923 Nobel Prize for Physiology and Medicine (Raju, 1998; Rosenfeld, 2002). While there is still some controversy relating to the key researchers involved in the discovery of insulin (see section 1.4.1), there is little doubt as to the importance of this discovery, both in terms of understanding diabetes and successful treatment of the disease. Prior to the discovery of insulin, treatment of diabetes was based on rigorous dietary management or ‘starvation treatment’ (Tattersall, 1995) in order to prevent emaciation and death (Leeds, 1979). Without treatment, juvenile diabetics usually died within a year of diagnosis (Broad, 1982). Insulin, in spite of certain associated problems, provided the first truly effective treatment of diabetes (Tattersall, 1995). The final piece to the diabetes puzzle, insulin resistance, was first referred to by Himsworth in the Goulstonian Lectures on ‘Mechanisms of Diabetes Mellitus’, presented to the Royal College of Physicians of London in 1939. In these lectures, Himsworth questioned the commonly held belief that “all cases of human diabetes could be explained by deficiency of insulin”, instead proposing that “the diminished ability of the tissues to utilize glucose is referable either to a deficiency of insulin, or to insensitivity to insulin, although it is possible that both factors may operate simultaneously”. He further suggested a division of diabetes into two categories based on this insulin resistance, pre-empting the National Diabetes Data Group by 40 years (Reaven, 1998).

1.1.2 Definition of diabetes The most recent definition and description of diabetes mellitus comes from the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, and is as follows: Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels.

4

Abnormalities in carbohydrate, fat and protein metabolism occur in diabetes, and all are as a result of deficient insulin action in these target tissues. This deficient action results from either inadequate secretion of insulin from the pancreas, or diminished responses of the target tissues to the secreted insulin, or a combination of both (Expert committee on the diagnosis and classification of diabetes mellitus, 2000).

1.1.3 Classification of diabetes mellitus In order to allow clinical management of diabetes, and for the purposes of epidemiological and clinical research, an appropriate system of classification is required. The first generally accepted systematic categorization of diabetes was that of the National Diabetes Data Group (NDDG) in 1979 (National Diabetes Data Group, 1979). This was based on the knowledge available at that time, and it was anticipated that as this knowledge increased, so the system of classification would require amendment. This amendment is described in the report made by the Expert Committee (Expert committee on the diagnosis and classification of diabetes mellitus, 2000). The current classification is on the basis of etiology, rather than treatment, and is made up of four categories. Type-1 diabetes (immune-mediated diabetes): Previously encompassed by the terms insulin-dependent diabetes, type 1 diabetes, or juvenile onset diabetes. Results from a cellular-mediated auto-immune destruction of the -cells of the pancreas. The rate of destruction is variable, and while most commonly occurring in childhood and adolescence, it can arise at any age. Many individuals with this type of diabetes become dependent on insulin for survival. Type-2 diabetes: Previously referred to as non-insulin-dependent diabetes, type-2 diabetes, or adult-onset diabetes, it is characterized by insulin resistance and usually a relative (rather than absolute) insulin deficiency. Initially, and often throughout their lifetime, such individuals do not require insulin treatment to survive. Most type-2 diabetics are obese. A degree of hyperglycemia sufficient to cause pathologic and functional changes in various target tissues, but without clinical symptoms, may be present for a long period of time before type-2 diabetes is detected. During this asymptomatic period, it is possible to demonstrate an abnormality in carbohydrate metabolism by measurement of plasma glucose in the fasting state or after a challenge with an oral glucose load. 5

Other specific types of diabetes: This includes genetic defects of both -cell function and insulin action, diseases of the exocrine pancreas, drug or chemical-induced diabetes, and infections. Gestational diabetes mellitus: Defined as any degree of glucose intolerance with onset or first recognition during pregnancy, regardless of whether insulin is used for treatment and whether the condition persists after pregnancy. Of these four classes, the vast majority of diabetes cases fall into the first two, with type-2 being by far the most prevalent of these.

1.1.4 Factors contributing to type-2 diabetes Genetic and environmental factors contribute to the pathogenesis of type-2 diabetes, (Katagiri et al., 1992; Kuzuya et al., 2002; Martin et al., 1992). Evidence for a genetic basis comes from both human studies and from animal models. Early studies were conducted examining twins, both mono and dizygotic, both of which show a strong concordance for diabetes (Barnett et al., 1981; Matsuda and Kuzuya, 1994). Diabetes often exhibits a family history, particularly with first-degree relatives. There is also a wide variation in rates of diabetes among different ethnic populations, being very high in the North American Pima Indians (Parks and Waskow, 1961) and Nauruan Islanders in the Pacific (Zimmet et al., 1984; Zimmet et al., 1977). Genetic studies carried out in such populations have resulted in the identification of a number of genetic loci demonstrating associations or linkages to type-2 diabetes, including the apolipoprotein D (Baker et al., 1994) and glucokinase (McCarthy et al., 1993) genes. Some of these genetic loci encode proteins expressed within the -cells of the pancreas. One such gene is that encoding the amylin protein. Amylin, as will be discussed in more detail later (section 1.5), is a pancreatic hormone co-secreted with insulin. Within the Japanese population a missense (S20G) mutation in the amylin gene has been identified in type 2 diabetic patients, but not found in either nondiabetics or sufferers of type-1 diabetes (Sakagashira et al., 1996). Another such example is that of the sulfonylurea receptor (SUR), a subunit of the ATP-sensitive potassium channel (Hansen et al., 1998) which plays an important role in glucosestimulated insulin secretion from the pancreatic -cell (Hani et al., 1997; Inoue et al., 6

1996). Variants in the gene encoding this receptor have shown association with type2 diabetes in a number of populations, including Danish (Hansen et al., 1998), French (Reis et al., 2000) and Northern European (Inoue et al., 1996) Caucasians. Other genes which have shown evidence of an association with type-2 diabetes encode products expressed in insulin target cells, and include the insulin receptor (McClain et al., 1988), glycogen synthase (Kuroyama et al., 1994), and the 3-adrenergic receptor (Walston et al., 1995). Animal genetic models of type-2 diabetes include the Goto-Kakizaki (GK) rat (Tourrel et al., 2002), in which a severely reduced beta cell mass may be the primary deficit leading to type-2 diabetes (Serradas et al., 2002), and the Otsuka Long-Evans Tokushima fatty (OLETF) rat, a model in which the pathological features closely resemble

those

seen

in

humans

(Shima

et

al.,

1999)

characterized

by

hyperinsulinaemia, hyperglycaemia, insulin resistance and obesity (Watanabe et al., 1999). There is also clearly an environmental component, evidence for which also comes from human studies and those using animal models of type-2 diabetes. Studies in human populations have shown an increased risk of the development of type-2 diabetes associated with factors such as age, obesity (Kenny et al., 2002), and lack of physical activity (Hu et al., 2001). The insulin resistance which is an integral part of the disease may be at least partially ameliorated by weight loss, exercise, diet and a number of drug treatments. There is also evidence to suggest that diet and exercise may delay the onset of type-2 diabetes by up to 3 years in humans (Knowler et al., 2002). Both exercise and the nutritional environment are therefore important in the development of type-2 diabetes, and it is nutrition on which the studies described in this thesis focus (see sections 1.2.8 and 1.7). An animal model of nutritionally-induced diabetes is Psammomys obesus (the sand rat, or Israeli desert gerbil) which, when subjected to overnutrition by feeding of a high-energy diet, progresses through hyperinsulinaemia and hyperglycaemia (Cerasi et al., 1997) to an irreversible diabetic state (Jorns et al., 2002). Exercise has been shown to prevent the progression of the disease in Psammomys (Heled et al., 2002).

7

Psammomys obesus is an example of an animal in which the metabolism has adapted to a food-scarce desert environment, becoming what is known as a ‘thrifty metabolism’ (Shafrir et al., 2002). When exposed to a high nutrient intake, this metabolism is compromised, and the result is disease. This phenomenon is the basis of the so-called Thrifty Phenotype hypothesis.

1.2 The fetal/infant origins of adult disease 1.2.1 Background The link between nutrition and disease is well established. Particularly clear examples of this are the various vitamin deficiencies: Vitamin A, in the absence of which disorders of the skin and eye develop; insufficient vitamin C, causing scurvy, a disease characterized by loosened teeth, swollen and bleeding gums and bleeding under the skin and in deep tissues; vitamin D, the lack of which causes rickets, resulting in improper bone formation. In fact, clear-cut definitions of scurvy exist from the time of the medieval Crusades (Manchester, 1998), and in 1747 the Scottish naval surgeon James Lind successfully treated sailors afflicted with scurvy by supplementing their diets with oranges and lemons, and thus established that the disease resulted directly from insufficient nutrition (Manchester, 1998). These diseases can generally be cured and subsequently managed by the inclusion of the missing vitamin in the diet (Manchester, 1998). However, if the deficiency occurs at an early age, or persists for a prolonged period of time, irreversible changes can occur – i.e. poor nutrition can result in permanent changes in an organism which may be detrimental to that organism’s health. Rickets is an example of this, where the improper deposition of calcium during bone growth leads to insufficient hardening of the bones, which can therefore bend and twist, and become permanently altered when they do eventually harden. The dates referred to above demonstrate that the concept of the relationship between a period of poor nutrition and permanent changes in an organism is not a new one. What is relatively new is the idea that the environment prior to birth may be of critical importance.

8

1.2.2 The intra-uterine environment Early evidence of the importance of the intra-uterine environment was obtained in studies of the birth weights of half-siblings, siblings, and twins (both monozygotic and dizygotic) (Morton, 1955). The conclusion after analysis of data from tens of thousands of babies was that “the genotype of the fetus is of less importance in determining the resemblance between sibs and cousins than some characteristic of the maternal constitution or maternal environment”. Variation in birth weight was considered a particular example of this phenomenon (Morton, 1955). An attempt to separate the effects of genetics from maternal environment was made by crossing large Shire horses with the much smaller Shetland ponies. The conclusion from this experiment was that the size of the foal at birth depended largely upon the size of the mare, i.e. her uterus and its blood supply and therefore the environment of the fetus, while later growth depended more upon the size of the sire and therefore the genotype of the fetus (Walton and Hammond, 1938). While the work of Morton focused solely on the effect of the fetal environment on birth weight, McCance and Widdowson reviewed the effect carried through to adulthood, using evidence from a number of their earlier animal studies (McCance and Widdowson, 1974). In guinea pigs, a period of fetal under-nutrition led to a low birth weight, and the animals remained small throughout their lives. This led to two hypotheses: (i) the earlier in the life of an animal that a short period of undernutrition (or over-nutrition) occurs, the more likely it would have permanent effects on stature, (ii) the subsequent rate of growth of an animal is determined by its rate of growth during the period of development when the regulatory centres of the hypothalamus are being coordinated with the rate of growth.

1.2.3 Metabolic programming Further studies in both animals and humans have shown that this permanent effect on size is not just restricted to the organism as a whole, but also to certain organs. An example is the kidney, which has a rapid development phase late in gestation. If there is a period of under-nutrition at this time, the replication of kidney cells is reduced, and there is evidence to suggest that the change in cell number is permanent, as there seems to be no capacity for further renal cell division later in life

9

(Barker, 1998). This is the concept of ‘metabolic programming’, where cell division within the organ must occur in a certain time-frame. If it does not, the organ remains small throughout the animal’s life, and this change can also affect the metabolic function of the organ. Thus early adaptations to nutritional stress or stimulation may permanently alter the physiology and metabolism of an organ such that the changes continue to be expressed in the absence of the original causative events (Patel and Srinivasan, 2002). Further evidence relating early growth to specific health outcomes came from a number of retrospective human studies carried out in Britain, in which accurate measurements of body weight, length, and head circumference at birth had been recorded in certain hospitals in the early part of the 20th century. The individuals in question were traced and studied in middle age for a number of health factors. These studies demonstrated a correlation between low birth weight and/or thinness at birth and an increased susceptibility of the subjects as adults to hypertension (Godfrey et al., 1993; Law et al., 1993), cardiovascular disease (Barker et al., 1993; Fall et al., 1995a), coronary heart disease (Barker, 1995a; Barker et al., 1995a; Fall et al., 1995b), high blood LDL cholesterol levels (Fall et al., 1992), and even depression (Barker et al., 1995b). As low birth weight and thinness at birth were interpreted as indicative of poor fetal nutrition, these results suggested a direct link between poor maternal nutrition and a number of adult onset diseases. Many of these studies were carried out by DJP Barker and his colleagues at the MRC Environmental Epidemiology Unit, University of Southampton, and thus the concept of adult diseases originating in the fetal or infant environment is sometimes referred to as the ‘Barker hypothesis’. It is therefore appropriate to use Barker’s own summary from the Wellcome Foundation Lecture of 1994: Recent findings suggest that many human fetuses have to adapt to a limited supply of nutrients and in doing so they permanently change their physiology and metabolism. These ‘programmed’ changes may be the origins of a number of diseases in later life, including coronary heart disease and the related disorders: stroke, diabetes and hypertension (Barker, 1995b).

10

1.2.4 The Thrifty Phenotype hypothesis While poor fetal nutrition is one piece of the puzzle, the Thrifty Phenotype hypothesis (Hales and Barker, 1992) also considers the nutrition and growth of an organism throughout life (see Figure 1.1). It is thought that an organism subjected to poor fetal nutrition ‘resets’ its growth and metabolism in anticipation of a similar postnatal environment (a ‘thrifty metabolism’, such as that seen in Psammomys obesus). If there is a mismatch between the early and later nutrition, i.e. a poor maternal nutritional environment but adequate or supranormal postnatal nutrition, the ‘programmed’ organs may be unable to deal effectively with an increased burden, and the result is adult disease. Thus, small or thin babies who grow up to be obese as adults are particularly at risk of metabolic disease. This seems to be particularly so when catch-up growth has occurred. This term refers to the situation where a period of poor nutrition (and consequent slowing of growth) is followed by the re-establishment of adequate nutrition (and a consequent increase in the growth trajectory) such that the individual reaches a ‘normal’ body weight (the weight it would have reached had it followed the original growth trajectory without a period of poor nutrition) (Hales and Ozanne, 2003). In human studies, the incidence of type-2 diabetes, coronary heart disease and hypertension was found to increase in association with low in utero and infant growth rates and accelerated growth during childhood (Barker et al., 2002), while an early increase in body mass index in children (‘adiposity rebound’) was also associated with an increased incidence of type-2 diabetes (Eriksson et al., 2003).

1.2.5 Rehabilitation Because of the influence of this mismatch between prenatal and postnatal environments, it is important to consider, albeit briefly, the role of nutritional rehabilitation. This term refers to the recovery of an animal from some kind of nutritional insult. The pattern of this recovery is variable depending on the level and timing of the nutritional insult, as well as the manner of rehabilitation. Catch-up growth, as described in section 1.2.4, is a particular example of nutritional rehabilitation. The conditions under which rehabilitation occurred in the current studies are outlined in section 1.7.

11

Figure 1.1: The Thrifty Phenotype hypothesis

Mother’s development and growth impaired

Maternal malnutrition

Maternal hyperglycaemia

Other maternal/placental influences

Fetal and infant malnutrition

Changes in growth, and vasculature A recent representation of the thriftymetabolism phenotype hypothesis produced by the original authors (Hales and Barker, 2001).

Kidney

Hypertension

 Pancreatic -cells Renal failure

Muscle, liver, adipose tissue

 Adult -cell function

Metabolic syndrome

12

Hypothalamic-pituitaryadrenal axis and sympathetic system

Insulin resistance

1.2.6 Animal models While the use of retrospective studies in humans provided some evidence for the fetal or infant origins of human disease, little information regarding the mechanisms for this phenomenon was available. It is for this reason that much work has been carried out using animal model systems, which have resulted in the elucidation of many aspects of the underlying mechanisms by which programmed changes occur (Ozanne et al., 1998). Animal models fall into three broad categories, defined by the method used to establish growth restriction; dietary manipulation, hormonal insult, or surgical intervention. Using these models, the relationships between maternal nutrition and offspring bodyweight, blood pressure (Langley and Jackson, 1994; Langley-Evans, 1997), organ size, glucose tolerance (Wilson and Hughes, 1997), pancreatic development (Snoeck et al., 1990), insulin sensitivity (Ozanne et al., 1996) and lipid metabolism (Lucas et al., 1996) have been established and the underlying mechanisms elucidated. Each of these studies was carried out using a rat model system. Effects of particular relevance to this thesis, such as those relating to pancreatic function and insulin sensitivity, are discussed in more detail in section 1.6.

1.2.7 An over-stretched hypothesis? While there is a large body of evidence in support of the Thrifty Phenotype hypothesis, there are also several contradictory studies, and a number of critics of the ideas. There seems to be little doubt that the intra-uterine environment has a role in disease, but questions remain as to extent of this role. One of the main assumptions of the hypothesis is that low birth weight and/or ponderal index (a measure of proportionality or ‘thinness’ at birth) reflect poor maternal nutrition, and this is the basis of the majority of human studies. Ponderal index in particular is thought to represent poor fetal growth during the latter stages of gestation. However, there appears to be a low correlation between birth size and various antenatal measures, thus birth shape phenotypes cannot necessarily be used as indicators of adverse events (nutritional or otherwise) at a particular stage of gestation (Hindmarsh et al., 2002).

13

Questions have also been raised as to the validity of some of the human studies. For example, in a review of 55 studies which had reported a correlation between adult blood pressure and birth weight, the association became weaker the larger the number of individuals examined, suggesting a selective emphasis. This report also questioned the validity of certain adjustments made (for example, taking into account current body weight and various confounding factors) and concluded that birth weight was of little relevance to blood pressure levels in later life (Huxley et al., 2002). Other environmental factors may have also been under-emphasized when considering the importance of the fetal environment. Obesity may be the most important permissive factor in the development of type-2 diabetes (i.e. the disease is unlikely to develop in the absence of obesity) and it may also play an active role, as the severity of type-2 diabetes is proportional to the size of the excess fat mass (Prentice, 2001). It is therefore possible that obesity is required for the effect of poor fetal nutrition to manifest as adult disease. Another important environmental factor is exercise, which has been shown to ameliorate or prevent type-2 diabetes in both animals and humans (Heled et al., 2002; Knowler et al., 2002). Finally, there are a number of possible genetic factors involved. While no single mutation has been found that is present in the majority of type-2 diabetes cases, there are a number associated with specific populations (section 1.1.4), and this number continues to grow. There is also recent research highlighting the importance of the interaction of genetic and environmental factors. An example of this is the peroxisome

proliferator-activated

receptor

(PPAR)-gamma2

gene

and

its

relationship with type-2 diabetes. There are two polymorphisms of this gene (Pro12Pro and Pro12Ala), of which the former is referred to as a ‘high-risk’ allele. In a study of elderly individuals, only those with the high-risk Pro12Pro allele showed an association between low birth weight and insulin resistance (Eriksson et al., 2002), thus demonstrating the potential importance of the gene-environment interactions in the development of adult disease. Regardless of the relative contribution of poor fetal nutrition to the development of adult disease, the Thrifty Phenotype hypothesis provides a useful experimental basis by which these associations can be studied (Hales and Ozanne, 2003). 14

1.2.8 Relevance to the current thesis The studies in this thesis make use of an animal model system to study the relationship between fetal/infant nutrition, the results of rehabilitation, and the development of risk factors associated with type-2 diabetes. Before a more detailed description of this model, it is important to consider some of the organs and molecules that are fundamental to the pathogenesis of type-2 diabetes. In particular, the pancreas and hormones produced by it, and tissues such as muscle which are the targets of these hormones.

1.3 The pancreas The pancreas is a large, elongated, irregularly shaped gland in vertebrates, lying behind the stomach, between the spleen and the duodenum. It consists of two distinct subsets of cells; exocrine and endocrine. The exocrine pancreas consists of secretory units called pancreatic acini, and produces and secretes pancreatic juice containing digestive enzymes directly into the duodenum (upper small intestine). The endocrine pancreas, consisting of small clusters of special cells called the islets of Langerhans, produces and secretes insulin, glucagon and somatostatin into the bloodstream (Samols, 1991). For the purposes of the current study, it is the endocrine pancreas which is of importance. During the development of the pancreas, the late fetal and early post-natal periods are critical (Aalinkeel et al., 2001). In rodent models where the mother is fed a lowprotein diet, it has been observed that weight gain of the fetus is affected principally during the last two days of gestation, i.e. a critical period of pancreatic development (Snoeck et al., 1990). This suggests that the pancreas may be an organ particularly susceptible to programming as a result of poor fetal nutrition. In the following section, the development of the endocrine pancreas is discussed in greater detail in order to introduce the factors (i.e. possible mechanisms) affecting pancreatic -cell development.

1.3.1 Development of the Endocrine Pancreas with emphasis on the cell. The following discussion focuses on mechanisms of pancreatic development that have been elucidated mainly through experiments involving genetic manipulations, 15

especially gene knockouts, in mice. Additional important information is becoming available from other vertebrate species, notably the zebrafish, which has emerged as an important model for developmental studies (Grunwald and Eisen, 2002). The following section presents information mainly derived from these sources. Where relevant, the specific origin of that information is stated. The approach taken here has been first to review the factors leading to generation of the pancreas itself, and then to summarise information concerning islet cell differentiation, with particular emphasis on the origins of the islet -cell. This is an active area of work at present, driven by its relevance both to the origins and development of diabetes mellitus, and also of pancreatic cancer. The adult mammalian pancreas is a heterogeneous organ composed of three major cell types (Murtaugh and Melton, 2003). Exocrine cells produce digestive enzymes, such as trypsin, pancreatic lipase and amylase, which are synthesized by exocrine acinar cells and passed into the gut via the pancreatic juice, which is transmitted via the pancreatic duct system. Exocrine tissue, organized into the epithelial acini, comprises at least 95% of the pancreatic cells. Digestive enzymes are secreted into the acinar lumens, which drain into small ducts. These merge and feed into progressively larger structures, finally connecting to the common bile duct. The endocrine cells of the pancreas are organized into islets of Langerhans, which are spheroidal clusters of cells dispersed throughout the exocrine tissue. Each of the endocrine cell types produces a distinct peptide hormone: α-cells make glucagon, cells produce insulin, δ-cells make somatostatin, and PP-cells make pancreatic polypeptide (Murtaugh and Melton, 2003). The islets make up only a small fraction of the total organ mass, about 1–2% in adult mammals. Within the islets, the endocrine cell types are present in varying proportions: -cells make up the majority (60–80%) and in many mammals form a core around which the others are arranged. α -cells typically constitute 15–20% of the islet mass, and the remaining cells are of δ and PP type. Histologically, the islets resemble colonies of endocrine tissue suspended within the acinar matrix, as if derived from single progenitor cells; however, lineage tracing shows that each islet is polyclonal in origin (Deltour et al., 1991). Lineage tracing has provided many key insights into pancreas development,

16

often inconsistent with models developed on the basis of histology or immunohistochemistry.

1.3.1.1 Molecular basis of pancreatic development The basis of pancreatic development has been subjected to molecular dissection by gene-disruption in species including, in particular, mice and zebrafish. The latter model is useful, in that these fish have a isolated endocrine pancreas present in single, paired or multiple organs known as Brockman bodies, which can be directly visualized and studied (Ober et al., 2003). Such mutants can be compared according to the stage at which pancreatic defects appear; combining this information with available lineage tracing data, generates a time-course of gene action in islet development. This methodology has certain limitations imposed by the roles of multiple genes in pancreatic development, as well as sequential action of particular genes at different times during the course of development, but nevertheless has provided a comprehensive picture of gene action in islet development. The pancreas derives from two patches of epithelium that bud dorsally and ventrally from the gut epithelium, between the stomach and duodenum, beginning (in the mouse) at approximately embryonic day 9 (E9) (Murtaugh and Melton, 2003; Samols, 1991). This is also the site of the development of the Brockman bodies in, for example, the zebrafish. During budding, the organ expresses the homeodomain protein PDX1, and all pancreatic cell types derive from PDX1+ progenitors (Gu et al., 2002). Recent work in zebrafish has begun to identify earlier regulators of pancreatic specification. Inhibition of retinoic acid (RA), bone morphogenetic protein (BMP), or hedgehog (Hh) signaling disrupts pancreas development; conversely, whereas excess activity of these pathways results in expanded or ectopic pancreas development (Tiso et al., 2002). BMP and RA regulate global anterior-posterior (A-P) patterning of the gut, whereas Hh is selectively required for pancreatic endocrine development. In amniotes (mouse and chick), excess Hh signaling inhibits pancreas development (Hebrok et al., 2000). In mice, several transcription factors expressed in the epithelium are now known to be required for pancreas specification. Among the earliest specific markers of the

17

definitive endoderm is Sox17, which is expressed throughout the endoderm after gastrulation (Kanai-Azuma et al., 2002). Although its expression subsequently shifts to the hindgut, Sox17 is required for pancreas formation, as assessed by Pdx1 expression. This defect is relatively specific insofar as the nearby liver primordium is spared; presumably this reflects a role for Sox17 very early in specification. The Hlxb9 gene (encoding the Hb9 homeoprotein) is expressed throughout both early pancreatic buds, and in the dorsal bud it precedes Pdx1 (Li et al., 1999). In mice lacking Hlxb9, the dorsal pancreas is entirely absent, Pdx1 is not expressed, and the epithelium does not bud. The ventral pancreas, remarkably, develops normally until -cell maturation. Conversely, mice lacking the Ptf1a/p48 bHLH transcription factor exhibit normal dorsal bud formation, whereas the ventral pancreas not only fails to bud but actually becomes integrated into the surrounding duodenum (Kawaguchi et al., 2002). Together, these data suggest that extrinsic factors regulate A-P patterning of the endoderm to establish the position of the pancreas; this positional information is interpreted by transcription factors that specify pancreatic fate, promoting Pdx1 expression and epithelial budding. Following budding, the pancreatic primordia begin dramatic growth and branching, meanwhile reorienting and fusing into a single, bipolar organ. The growing epithelium is surrounded by mesenchymal cells, and explant experiments showed that mesenchyme was important for growth and exocrine differentiation (Wessells and Cohen, 1967). At least four mesenchymal factors are currently known to mediate this growth-promoting effect, although their interrelationship remains uncertain: these are the transcription factor Isl1, which is expressed throughout the dorsal mesenchyme (Ahlgren et al., 1997); the homeodomain gene Pbx1, whose knockout causes severe hypoplasia of the dorsal pancreas, coupled with lack of acinar development (Kim et al., 2002); fibroblast growth factor-10, Fgf10, whose knockout causes hypoplastic dorsal and ventral pancreata (Bhushan et al., 2001); and Pdx1, whose mutants lead to growth arrest following budding (Holland et al., 2002). Finally, epithelial growth per se is also required for normal pancreatic differentiation: in particular, N-cadherin, which is also expressed and functionally

18

required in the dorsal pancreatic mesenchyme, is required for normal pancreatic growth and development (Esni et al., 2001).

1.3.1.2 Differentiation of endocrine and exocrine cell types during pancreatic development The next section of this review focuses mainly on endocrine differentiation, which is already apparent at the initiation of pancreatic budding (Murtaugh and Melton, 2003). Exocrine development has been mentioned only when directly relevant to endocrine differentiation, since little is yet known about the relationship between these two processes from this time forward in development. During the early stages of murine pancreatic development, E9.5 – E12.5, the majority of cells formed are α -cells (Pictet and Rutter, 1972). Newly formed endocrine cells arise from the epithelium and subsequently aggregate into islets; studies of cell kinetics indicate that new endocrine cells continue to arise throughout embryonic life (Kaung, 1994). The transcription factors involved in pancreatic differentiation have recently been reviewed in depth (Wilson et al., 2003). The following is a brief overview of key points. During bud outgrowth from Pdx1+ progenitors, Pdx1 expression changes to biphasic, with high levels characterizing cells (Jensen et al., 2000); inactivation of Pdx1 after bud formation prevents both islet and acinar development (Holland et al., 2002). The bHLH protein Neurogenin3 (Ngn3) is key regulator of endocrine development, being expressed exclusively in endocrine precursor cells, and subsequently down-regulated during differentiation (Gu et al., 2002), misexpression of Ngn3 was sufficient to induce endocrine differentiation throughout the gut epithelium in chick embryos (Grapin-Botton et al., 2001). If signaling in the Notch pathway becomes disrupted through loss of one of its elements, then pancreatic-endocrine potential becomes spatially unrestricted; such studies have identified Ngn3 as a negative regulator of Notch signaling (Lee et al., 2001), although in vivo Ngn3 misexpression induces mainly excess α-cells. By contrast, -cell competence factors may include the NK-homeodomain genes Nkx2.2 and Nkx6.1 (Sander et al., 2000). Nkx2.2 mutants completely lack insulin expression and cells seem to arrest prior to full -cell development. Adult -cells generally derive from progenitors that have never expressed glucagon (Herrera, 2000).

19

In summary, a model has been developed in which early Pdx1+ cells serve as bipotential stem cells: in the presence of mitogens and Notch activity, they expand and are available for later exocrine and endocrine differentiation; if the cells escape these signals, they upregulate Ngn3 and undergo endocrine differentiation.

1.3.1.3 Post-natal Homeostasis of the -cell Mass It is now thought that the -cell mass is dynamic and that it increases and decreases both in function and mass to maintain the glycaemic level within a narrow physiological range; the changes in mass can be in both number (hyperplasia) and individual volume of -cells (hypertrophy); when the mass cannot increase adequately, diabetes ensues (Bonner-Weir, 2000). In rodents, generation of newlyformed endocrine cells (islet neogenesis) usually ceases shortly after birth; further islet growth presumably occurs via division of existing differentiated cells (Kaung, 1994). In rats, -cell mass increases linearly from weaning until 3–4 months of age (Finegood et al., 1995). Another physiological situation of -cell mass expansion is pregnancy. During pregnancy in the rat, -cell mass increases 50% due to increased -cell proliferation induced by placental lactogen and increased cell volume (cell hypertrophy) as a functional adaptation (Parsons et al., 1992). Two mechanisms of -cell formation from the embryo, neogenesis (or differentiation from ductal precursor cells) and replication of differentiated -cells, are maintained postnatally and even in the adult. After weaning, a low level of replication is maintained, but this can be stimulated: with 2–3%/24 hours (or 0.5–0.7%/6 hours) replication of -cells found in the adult rodent pancreas, the -cell mass can double in about 1 month (Bonner-Weir, 2000). Between 1–2 and 2–3 months of age, the rat cell mass almost doubles but this monthly doubling does not continue, suggesting a loss of cells as there is renewal. Experimentally, increased proliferation of differentiated -cells occurs in a number of models: the 96-hour glucose infused rat; the GLP-1/exendin 4-treated rat; and the sub-totally pancreatectomized rat (BonnerWeir et al., 1989; Brockenbrough et al., 1988). Adult pancreatic duct cells expand and differentiate during pancreatic regeneration in rats; these studies led to the identification of PDX-1/IDX-1 1 duct cells that can transiently regain their pluripotency, which have been hypothesised as the true endocrine precursor cells in the adult pancreas (Sharma et al., 1999). It remains unclear whether the stimulus for 20

-cell expansion is something other than mild transient hyperglycemia, since mildly elevated glucose is a potent stimulus for -cell growth: however, chronic or severe hyperglycemia is detrimental, in that it evokes loss of glucose-induced insulin secretion and even loss of specific -cell differentiation (Jonas et al., 1999). Several genes have been identified in various MODY syndromes that affect aspects of -cell growth and development; however, these have no established role in the development the common form of type-2 diabetes. The recent demonstration that fibrillogenic amylin mediates -cell apoptosis through a JNK1/c-jun-mediated caspase cascade (Zhang et al., 2003) accompanied by its excessive production in early type-2 diabetes (Cooper, 2001) and the finding of a diabetes-associated amylin polymorphism in New Zealand Māori (Poa et al., 2003)}, indicate that amylin-mediated apoptosis could play a significant role in islet -cell failure in some subjects with type-2 diabetes. Hence, conditions that mediate elevated or

deregulated amylin production are

of potential interest

in

understanding the origins of islet -cell failure in type-2 diabetes. These may include nutritional conditions such as total energy or protein deficiency, or more subtly protein quality and quantity at key windows of susceptibility.

1.4 Insulin 1.4.1 Discovery Much has been written on the discovery of insulin, and to whom this achievement should be credited. While the Romanian scientist Nicolas Constantin Paulescu had published an account of an antidiabetic hormone present in the pancreas in 1921 (Ionescu-Tirgoviste, 1996), it is generally accepted that the team based at the University of Toronto under the leadership of JJR MacLeod, and which included Banting, medical student Charles H Best and biochemist James B Collip was responsible for the discovery of insulin. The controversy centres on the role of MacLeod who, when approached by Banting with an idea of obtaining pancreatic islet-cell extracts, was initially reluctant to offer his assistance. Banting persisted, and MacLeod eventually agreed to provide facilities and resources for the experiments, as well as offering significant suggestions at an early stage (Broad, 1982; Raju, 1998). It was, however, during July 1921 while MacLeod was away in 21

Scotland that Banting and Best performed crucial experiments, successfully treating depancreatized dogs with their pancreatic extract, which they had christened ‘isletin’ due to its origin the the islets of Langerhans. The name was changed to ‘insulin’ (from the Latin word ‘insula’ meaning island (Raju, 1998)) on the recommendation of MacLeod, who subsequently wrote that “Banting and Best deserved complete credit for the initial work” (Broad, 1982). The first use of insulin in a human diabetic subject was during January 1922, when daily injections of insulin were administered to a 14 year old diabetic boy, resulting in rapid improvement in the boy’s condition. Credit must in this case go to Collip, who refined the purification procedure, thus allowing the treatment to be administered to human subjects (Broad, 1982). This revolutionary treatment resulted in world-wide publicity, and a talk of a Nobel Prize. It was at this stage that MacLeod appears to have changed his story, implying that the work would have foundered without his guidance, and in the end it was MacLeod and Banting who were, rightly or wrongly, awarded the Nobel Prize. In recognition of the important contribution of their fellow researchers, both shared their prize money; Banting with Best, and MacLeod with Collip (Broad, 1982; Raju, 1998).

1.4.2 General description Insulin is a 51 amino acid (5.8 kDa) peptide hormone made up of two chains, the A and B chain, linked by disulfide bonds. Its function can be broadly said to signal the fed state, thereby acting to promote the uptake of fuel substrates into certain cells, the storage of fuels such as lipids and glycogen, and the biosynthesis of macromolecules. The uptake of glucose into muscle and adipose tissue is increased by stimulation of insulin-sensitive glucose transporters. Glycogen synthesis in muscle and liver is increased by activation of the glycogen synthase enzyme and deactivation of glycogen phosphorylase. Synthesis of fatty acids and triacylglycerols is increased in liver and adipose tissue, while glycolysis is activated and gluconeogenesis deactivated in liver. Uptake of amino acids into muscle is augmented, with consequent activation of muscle protein synthesis and inactivation of protein degradation. These effects occur in response to a food stimulus (Samols, 1991).

22

1.4.3 Biosynthesis and secretion Insulin is synthesized from a single precursor peptide called preproinsulin. Preproinsulin contains an N-terminal leader sequence by which it is targeted to the -cell secretory granule. Once it has entered the endoplasmic reticulum, the leader sequence is cleaved off, yielding proinsulin, which subsequently adopts a threedimensional structure via the formation of disulfide bonds. The intervening sequence of proinsulin between the A and B chains (known as the insulin C-peptide) is cleaved as the secretory granule matures, resulting in the final, active form of insulin within the dense core of the granule, and the C-peptide within the matrix (Samols, 1991). Glucose acts as the major physiological regulator of insulin biosynthesis (Nesher and Cerasi, 2002). Insulin is secreted from the islet -cell of the pancreas in response to a number of nutrient stimuli, glucose being principal among these (Nesher and Cerasi, 2002). When the extracellular glucose concentration is elevated, it is rapidly taken up into the -cell via GLUT2 glucose transporters. Subsequent degradation of glucose increases the cytoplasmic ATP/ADP ratio, which causes ATP-regulated K channels to close. Outflow of the K

+

2+

(KATP)

ions is thus prevented, and the membrane of

the cell depolarizes. This in turn opens voltage-gated Ca concentration of Ca

+

2+

channels, the

increases, vesicles containing insulin move to the plasma

membrane of the -cell, and the insulin is released by exocytosis into the lumen of the Islet of Langerhans, and subsequently enters the bloodstream (Rorsman et al., 2000). Interestingly, the sulfonylureas, which have been used to treat diabetes for over 40 years, cause the closure of the KATP channels independently of the cell’s metabolic state, thus causing membrane depolarization and opening of the Ca

2+

calcium

channels. This effect is mediated by binding to the so-called sulfonylurea receptor (SUR), part of the functional KATP channels (Rorsman et al., 2000).

1.4.4 The insulin receptor The insulin receptor is a transmembrane glycoprotein with an 22 tetrameric structure stabilized by disulfide bonds. Both the -chain, of 735 residues, and the chain, of 620 residues, are translated from a single mRNA (Samols, 1991). The 23

chain, which is thought not to span the membrane, contains the insulin binding site, through which it binds the C-terminal region of the insulin molecule. The -chain has a transmembrane domain, with its C-terminus in the cell interior. Within this region

is

a

protein

tyrosine

kinase

activity,

by

which

the

-subunit

autophosphorylates at multiple tyrosine residues (Lee and Pilch, 1994).

1.4.5 Signal transduction While certain aspects of the signaling pathway are still unclear, much progress has been made in characterizing the molecular events leading from the insulin receptor to the final targets of insulin’s action (Summers et al., 1999). The kinase activity of the insulin receptor’s -subunit is stimulated by binding of insulin to the extracellular part of the receptor. The resultant autophosphorylation causes an interaction between specific tyrosine residues within the -subunit and the so-called insulin receptor substrate molecules, IRS-1 and IRS-2, which are in turn phosphorylated at many tyrosine residues (Cohen et al., 1997). Thus activated, the IRS molecules recruit SH2-domain-containing proteins to the plasma membrane. Key among these is phosphatidylinositol 3-kinase (PI-3 kinase), which binds to IRS-1 via SH2-domains in its p85 regulatory subunit (Rordorf-Nikolic et al., 1995). This binding activates the p110 catalytic subunit of the enzyme, which converts the plasma membrane inositol phospholipid phosphatidlyinositide 4,5 bisphosphate (PI-4,5-P2) to phosphatidylinositide 3,4,5 trisphosphate (PI-3,4,5-P3). It is this molecule, or a metabolite of it, which appears to be the long sought after second messenger for insulin (Cohen et al., 1997). Both PI-3,4,5-P3 and PI-3,4-P2 activate a 67 kDa kinase which has consequently been named 3-phosphoinositide-dependent protein kinase-1 (PDK1) (Dong et al., 1999). PDK1 has been shown to phosphorylate (and thus activate) protein kinase B (PKB) at Thr-308 (Alessi et al., 1997). PKB in turn phosphorylates (and thus deactivates) glycogen synthase kinase 3 (GSK3), which prevents GSK3 from phosphorylating the glycogen synthase enzyme (Shaw et al., 1997). The consequent dephosphorylation of glycogen synthase results in its activation (Lawrence and James, 1984), a fact that has been known since the 1960s (Hizukuri and Larner, 1964), as has the role of insulin in this activation (Bishop and Larner, 1967).

24

Other effects of insulin are also believed to be modulated by some of the enzymes described above. Insulin stimulates the uptake of glucose into skeletal muscle and adipose tissue by promoting translocation of the GLUT4 glucose transporter to the plasma membrane, an effect which some evidence suggests is dependent on PKB (Wang et al., 1999). PKB, possibly via the mammalian target of rapamycin (mTOR) (Proud and Denton, 1997), has also been shown to induce activation of the translation factor p70 S6 kinase, by which insulin activates the ribosomal S6 protein and stimulates the translation of certain types of messenger RNA (Cohen et al., 1997). There is also evidence that GSK3 plays a role in protein synthesis, with its inhibition leading to the dephosphorylation and activation of the translation factor eIF-2B (Welsh and Proud, 1993), which is required for the initiation of protein translation (Proud and Denton, 1997).

1.4.6 Insulin resistance Insulin resistance is, as the term suggests, an insensitivity of target tissues to the effects of insulin. While the pioneering work of Himsworth in this field took place over 60 years ago ((Reaven, 1998), and also see section 1.1.1), considerable research continues in an attempt to increase understanding of the mechanisms underlying insulin resistance, and its consequences. A number of factors, including obesity and lack of physical activity (Godsland and Stevenson, 1995), have been identified as contributing to insulin resistance. The importance of diet is demonstrated by a number of rat models. For example, feeding rats a diet high in either fat (Martin et al., 2000) (particularly saturated fats and ω-6 fatty acids) or sucrose (Lavigne et al., 2001; Mori et al., 1999) leads to major and widespread

impairment

of

insulin

action.

There

is

also

evidence

that

undernourishment in utero may be a factor in the development of insulin resistance, both in human populations (Godsland and Stevenson, 1995) and in a rat model system (Martin et al., 2000). Insulin resistance in type-2 diabetes is, at least in part, due to impaired stimulation of the glucose transport system in muscle and fat (Shepherd and Kahn, 1999). In skeletal muscle, expression of the GLUT4 glucose transporter is normal, but insulinmediated translocation of the transporter to the plasma membrane is impaired. In adipocytes, this translocation is also impaired, but the main cause of insulin 25

resistance in this case is due to the markedly reduced expression of GLUT4 (Maianu et al., 2001). It has been suggested that the amino acid glutamine may promote insulin resistance by routing glucose through the hexosamine pathway (Rumberger et al., 2003), while tumour necrosis factor alpha (TNF-α) has been implicated as a candidate mediator of obesity-associated insulin resistance (de Alvaro et al., 2004). It is also possible that transport of insulin from plasma to the interstitial fluid, binding of insulin to its receptor, signal transduction by the receptor, and the activity of a range of post-receptor signaling pathways may all be important in insulin resistance (Godsland and Stevenson, 1995). Insulin resistance has been linked to a number of other disease states. While one view is that insulin resistance is merely a permissive factor in the development, or even a secondary manifestation, of serious vascular disease (Godsland and Stevenson, 1995), a more central role in a variety of “diseases of Western civilization” has also been proposed (Reaven, 1998).

1.4.6.1 The insulin resistance syndrome This term describes a cluster of diseases (type-2 diabetes, essential hypertension, abnormal lipid metabolism, cardiovascular disease, and obesity) for which insulin resistance is considered to be the central abnormality (Mori et al., 1999). Approximately 50 % of patients with hypertension can be considered to have insulin resistance and hyperinsulinaemia (Mori et al., 1999; Reaven, 1998), and blood pressure is directly related to both insulin resistance and insulin concentration, independent of age, gender, and degree of obesity (Reaven, 1998). Hyperlipidaemia is closely linked with insulin resistance and/or hyperinsulinaemia (Reaven et al., 1996); all three have been shown to be present in both dietary-induced and genetic models of rodent hypertension (Reaven, 1998). In the context of this thesis, type-2 diabetes is of greatest interest with respect to insulin resistance, which is considered to be a major abnormality. Evidence for this includes the presence of both insulin resistance and hyperinsulinaemia in subjects who subsequently develop type-2 diabetes (Reaven, 1998), and the fact that a ‘prediabetic’ state, consisting of glucose intolerance and insulin resistance, seems to be highly prevalent among severely obese children regardless of ethnic group (Ebbeling et al., 2002). There is also greater insulin resistance within non-diabetic 26

Pima Indians (a group with particularly high levels of type-2 diabetes) compared with non-diabetic Caucasians, suggesting a possible genetic basis of insulin resistance. The importance of understanding its causes and mechanisms is underlined by the fact that approximately 25 % of apparently healthy people are estimated to be insulin resistant (Godsland and Stevenson, 1995) to a degree comparable with that seen in type-2 diabetes (Reaven, 1998), and that the insulin resistance syndrome has been identified in children as young as five years of age (Ebbeling et al., 2002). Thus, prevention of insulin resistance is important before development of the associated diseases of the insulin resistance syndrome. Diet is one factor which shows some promise, with feeding of ω-3 polyunsaturated fatty acids (such as those found in fish) preventing the development of insulin resistance induced by high-fat feeding in rats (Mori et al., 1999; Storlien et al., 1987). This effect may be due to these particular fatty acids altering fluidity of the cell membrane (Mori et al., 1999), thus facilitating post-binding effects of the insulin receptor (Storlien et al., 1987). There is also some evidence that the protein component of fish may have beneficial effects on insulin resistance, possibly through an effect of specific amino acids on glucose transport (Lavigne et al., 2001). It is relevant here to briefly consider predictors of type-2 diabetes, as the rat model studied in this thesis has its basis in the observation of factors which predispose an individual to this disease.

1.4.7 Prediction of Type-2 Diabetes 1.4.7.1 Clinical models Several approaches to the prediction of type-2 diabetes have been examined, but none is particularly effective (Tabaei and Herman, 2002). Here, I begin by discussing clinical approaches to this problem. The oral glucose tolerance test identifies highrisk subjects for diabetes, but it is costly, inconvenient and not particularly efficient. To attempt to improve on it, a number of predictive clinical models have been developed, although none has yet gained wide acceptance. These generally employ algorithms based on various risk factors, such as age, sex, ethnicity, BMI, systolic blood pressure, fasting plasma glucose (fpg), HDL cholesterol, and family history of

27

diabetes at baseline. Some recent examples are now presented for purposes of description. For example, in Japanese Americans aged 55 years, a clinical model was better than FPG for predicting diabetes after 5–6 years but not after 10 years; the model was not useful in older Japanese Americans, whereas 2-hour glucose was useful for predicting diabetes risk regardless of age (McNeely et al., 2003). In a similar vein, a predictive model based on multiple logistic regression analysis has been developed using data collected from 1,032 Egyptian subjects with no history of diabetes, which incorporated age, sex, BMI, postprandial time (self-reported number of hours since last food or drink other than water), and random capillary plasma glucose as independent covariates for prediction of undiagnosed diabetes; it was subsequently validated using data collected from an independent sample of 1,065 American subjects (Tabaei and Herman, 2002). Its performance was also compared with that of recommended and proposed static plasma glucose cut points for diabetes-screening. This equation’s sensitivity was 65%, specificity 96%, and positive predictive value, 67%.

1.4.7.2 Measures of Insulin Resistance as Clinical Predictors of Type-2 Diabetes A related approach has employed measures of insulin resistance to predict the development of type-2 diabetes (Hanley et al., 2004; Lorenzo et al., 2003). Insulin resistance is central in the development of type-2 diabetes (Weyer et al., 2001) and essential hypertension (Ferrannini et al., 1987). Results from three large prospective studies have been combined to compare the ability of simple indices of insulin resistance (IR) to predict type-2 diabetes (Lorenzo et al., 2003), and to validate an optimal approach (Hanley et al., 2003). In those studies, Poisson regression was used to assess the ability of each of several candidate indices to predict incident diabetes at the follow-up examination (during which 343 of 3,574 subjects developed diabetes); areas under the receiver operator characteristic (AROC) curves for each index were calculated and statistically compared. In pooled analysis, the insulin sensitivity index at 0 and 120 min (ISI0, 120) (Gutt et al., 2000) displayed the largest AROC (78.5%). This index was significantly more predictive (P < 0.0001) than a number of related indices. The authors concluded that the ISI0, 120 may reflect other

28

aspects of diabetes pathogenesis in addition to insulin resistance, which might explain its strong predictive abilities despite its moderate correlation with direct measures of insulin resistance. Thus, there is strong evidence that measures of insulin resistance can be employed to predict the development of type-2 diabetes. A related approach has employed Cluster Analysis and an augmented definition of the Metabolic Syndrome with inclusion of non-traditional cardiovascular disease risk factors associated with vascular inflammation, including plasminogen activator inhibitor-I, fibrinogen and C-reactive protein, in algorithms designed to predict type-2 diabetes (Hanley et al., 2004). Principal factor analysis of data from nondiabetic subjects at baseline (1992-1994) identified three factors, which explained 28.4, 7.4, and 6% of the total variance in the dataset, respectively. Based on factor loadings of ≥ 0.40, these factors were interpreted as: (1) a ‘metabolic’ factor, with positive loadings of BMI, waist circumference, 2-h glucose, log triglyceride, and log PAI-1, and inverse loadings of log Si + 1 and HDL (Pacini and Bergman, 1986); (2) an ‘inflammation’ factor, with positive loadings of BMI, waist circumference, fibrinogen, and log CRP and an inverse loading of log Si + 1; and 3) a ‘blood pressure’ factor, with positive loadings of systolic and diastolic blood pressure. In a prospective analysis, each of the factors was a significant predictor of diabetes after a median follow-up period of 5.2 years, and each factor remained significant in a multivariate model that included all three factors, although this three-factor model was not significantly more predictive than models using either impaired glucose tolerance or conventional CVD risk factors. Factor analysis identified three underlying factors among a group of inflammation and metabolic syndrome variables, with insulin sensitivity loading on both the metabolic and inflammation variable clusters. Each factor significantly predicted diabetes in multivariate analysis. The findings support the emerging hypothesis that chronic subclinical inflammation is associated with insulin resistance and comprises a component of the metabolic syndrome. The results of these studies support the use of measures of insulin resistance in the prediction of type-2 diabetes, and provide a rationale for the use of measures of insulin resistance in the current thesis studies.

29

1.5 Amylin Amylin is a 37 amino acid peptide hormone first isolated from amyloid-rich pancreatic deposits found in three unrelated individuals with type-2 diabetes (Cooper et al., 1987). These deposits are present in approximately 95 % of type 2 diabetic patients. Initially named diabetes-associated peptide (DAP), it was subsequently discovered in individuals not affected by type-2 diabetes, indicating that the peptide was a naturally occurring hormone. It was therefore renamed ‘amylin’ (Cooper et al., 1988). Human amylin shows 43 % and 49 % sequence homology to the human calcitonin gene-related peptides hCGRP-1 and hCGRP-2 respectively, as well as eliciting similar biological responses (Cooper, 1994). This suggests a possible evolutionary and functional relationship between these peptides. With respect to carbohydrate metabolism, in muscle amylin acts as a noncompetitive, insurmountable antagonist of insulin action (Young et al., 1992). More specifically, in isolated muscle preparations amylin inhibits insulin-stimulated glucose transport (Castle et al., 1998) and incorporation of glucose into glycogen (by inhibiting glycogen synthase and activation of glycogen phosphorylase (Deems et al., 1991; Lawrence and Zhang, 1994)). Amylin does not oppose the action of insulin in fat, increases hepatic glucose production and lactate flux, and impairs insulin secretion from the pancreatic β-cell (Young, 1994). In vivo effects include increasing plasma lactate (Young et al., 1994) and glucose (Wang et al., 1991) levels. Thus the actions of amylin are all consistent with features of insulin resistance and early type2 diabetes (Young, 1994), and indeed amylin has been shown to induce states of insulin resistance in whole animals, as well as inhibiting glucose-stimulated insulin secretion in certain conditions (Cooper, 1994). The occurrence of islet amyloid associated with type-2 diabetes is further evidence that the disease may involve abnormal amylin metabolism (Cooper, 1989), although there is debate as to whether amylin is involved in the disease progression. It has been proposed that amylin levels are elevated early in the pathogenesis of type-2 diabetes, and subsequently fall during the progression of the disease. Amylin metabolism was investigated in the current studies to ascertain if poor maternal nutrition through gestation and lactation resulted in an increase in its secretion from the pancreas, thereby indicating an early stage of type-2 diabetes pathogenesis. 30

1.5.1 Comparison and contrast of the secretion of insulin and of amylin Regulation of amylin secretion has recently been exhaustively reviewed (Cooper, 2001). Amylin is synthesized in pancreatic islet β-cells (Moore and Cooper, 1991), from which it is secreted in response to stimuli, such as glucose and amino acids, which also evoke insulin release (Cooper, 1994). In general, it appears that there are many common factors between the secretory apparatus that regulates the secretion of both hormones. Both hormones are present within secretory granules, from which they have been co-isolated by physical techniques (Buchanan et al., 2001). Under a number of circumstances, regulation and secretion of insulin and amylin are closely linked. In insulin resistant rodents, expression and secretion of both hormones are markedly augmented (reviewed in Cooper, 1994; Huang et al., 1992). There is also evidence that the two hormones can be released through distinct pathways (Gedulin et al., 1991; Verchere et al., 2000). In one study, glucose reportedly stimulated amylin but not insulin secretion from neonatal rat β-cells when regulated secretion was prevented by use of calcium-free media; these findings were interpreted to suggest that amylin secretion can occur via a constitutive secretory pathway (Kahn et al., 1993). In another study employing the isolated perfused rat pancreas, it was shown that amylin secretion was dissociated from that of insulin, and that this dissociation was enhanced in insulin-resistant diabetic rats (Gedulin et al., 1991). Recent evidence indicates that constitutive secretion of amylin is largely confined to immature β -cells, and is minimal in mature β -cells. Thus, a significant proportion of glucose-stimulated amylin secretion from neonatal, but not adult, rat islet cells occurs via a constitutive secretory pathway (Verchere et al., 2000). It is not yet known whether insulin resistance is accompanied by a change in the balance of secretion of amylin and insulin through the constitutive and regulated pathways.

1.5.2 Other pancreatic hormones While insulin and amylin are the hormones of particular interest in this thesis, there are other pancreatic hormones which also play a role in glucose metabolism. Glucagon is a 3.5 kDa peptide hormone secreted from the pancreatic -cells in response to low blood glucose levels, thus acting in the opposite manner to insulin. 31

Its principal effect is to increase levels of cyclic AMP (cAMP) in liver cells. This causes a rise in blood glucose levels by two distinct mechanisms: stimulation of the regulatory cascade that causes glycogen breakdown, and the decrease of fructose2,6-bisphosphate levels, which in turn leads to a reduction of glycolysis and stimulation of gluconeogenesis. Glucagon also causes fat breakdown and release, which leads ultimately to fatty acid accumulation in other cells (Samols, 1991). A recently discovered hormone, preptin, has been purified from the matrix of the cell secretory granule. Early experiments have demonstrated that preptin increases insulin secretion from glucose-stimulated beta TC6-F7 cells, and increases the second phase of glucose-mediated insulin secretion in the isolated perfused pancreas (Buchanan et al., 2001). Preptin thus has a putative role as a physiological amplifier of glucose-mediated insulin secretion.

1.6 Insulin and the fetal/infant origins of adult disease A number of studies have been performed which demonstrate the effect of maternal diet during gestation and/or lactation on the insulin metabolism in the offspring. These studies have been carried out both in human populations, and using animal models, and address both the production and action of insulin.

1.6.1 Insulin production Offspring of mothers fed a low protein (8 % protein) diet during gestation and lactation show lower pancreatic insulin and amylin contents than the offspring of mothers fed a control diet (20 % protein). Offspring were weaned onto the same diet (either control or low-protein) as their mothers, so the change in pancreatic hormone occurred without reverting to adequate nutrition following the period of poor nutrition (Petry et al., 2000). Offspring of mothers fed a low-protein (8 % casein) diet during pregnancy and lactation also show reduced pancreatic -cell proliferation and islet size, and dramatically reduced islet vascularization compared to the offspring of mothers fed a control diet (20 % casein) (Snoeck et al., 1990). Glucose-stimulated insulin release is impaired in islets from previously malnourished animals fed highly palatable fat or carbohydrate diets (Wilson and

32

Hughes, 1997). These diets presumably place additional demands on the pancreatic beta-cells and hence unmask a secretory defect induced by poor nutrition during the fetal and neonatal period. Islet insulin content is not reduced and the response to arginine is not impaired. These results suggest that the defect is not related to a reduced capacity to secrete insulin. The effect of diet during the post-weaning period can also influence insulin production. After 4 weeks of an altered diet post-weaning, food-restricted lowprotein animals show severely blunted insulin secretion in response to glucose, both in vivo and when studied in vitro using the isolated perfused pancreas system (Picarel-Blanchot et al., 1995).

1.6.2 Insulin action Offspring of mothers fed low-protein diets (8 % protein) have better glucose tolerance, as assessed by intra-peritoneal glucose tolerance test (Petry et al., 2000). Offspring of mothers fed a low-protein (8 %) diet show a 50 % decrease in glucokinase activity and a 100 % increase in phosphoenolpyruvate carboxykinase (PEPCK) activity compared to offspring of mothers fed a control (20 % protein) diet. Changes are evident at 21 days of age, and persist until 11 months of age. Parallel changes are evident in the respective mRNA levels of glucokinase and PEPCK, suggesting that the programming effect extends to the regulation of gene expression (Desai et al., 1997). As is the case with insulin production, the diet of the offspring, rather than the mother, can cause significant changes in insulin action. In rats weaned onto either a normal (15% casein) diet ad libitum, a normal diet at 65 % of the ad libitum food intake per day, or a low-protein (5% casein) diet at 65 % of the ad libitum food intake per day, both food-restricted groups show enhanced insulin-mediated total glucose uptake after 3 weeks (Picarel-Blanchot et al., 1995).

1.7 Investigative model and hypothesis 1.7.1 Background and rationale As mentioned previously, a rat model was chosen for the investigations described within this thesis, in which pregnant females were fed diets containing different 33

levels of protein intakes, as models of sufficient or insufficient maternal protein nutrition. In rodents, it has been well established that low protein intake during gestation can result in low birth weight and subsequently leads to various metabolic disturbances in adulthood, including high blood pressure, impaired glucose tolerance and insulin resistance (Metges, 2001). By contrast, high levels of maternal protein intake during gestation can also result in low birth weight and subsequent energy imbalance and adiposity (Daenzer et al., 2002). Additional studies are warranted to explore the relationships linking protein nutrition in early life to the postnatal development of obesity and disease in animals, as a model for the human condition (Metges, 2001). Effects of maternal low protein have recently been reviewed (Bertram and Hanson, 2001). Results from rodent studies indicate that low protein intake during gestation (i.e. 80 – 100 g protein/kg diet vs. 200 g protein/kg diet) can result in low birth weight or thinness at birth and, subsequently, the development of metabolic disturbances in adult life, such as high blood pressure, impaired glucose tolerance and insulin resistance (Lucas, 1998). These effects of maternal protein restriction have been reproduced by several workers (Dahri et al., 1991; Langley-Evans, 2000; Rees et al., 1999), and are thought to provide a reliable stimulus for induction of a variant of the metabolic syndrome in adult offspring. Amino acids have been shown to be important in fetal growth, pancreatic -cell development, and insulin secretion (Hales and Barker, 1992). Protein is also often scarce and expensive in communities with a high prevalence of diabetes and the metabolic syndrome, and for this reason is thought to contribute to the development of the metabolic syndrome in human communities affected by protein restriction (Hales et al., 1997). For these reasons, protein restriction was selected as the nutritional variable to induce variable within the diets. Levels of protein restriction were based on values presented in these references, with 200 g protein/kg representing normal protein intake, and 50 g protein/kg representing severe maternal protein restriction. In this study, all offspring were weaned onto a control diet containing 200 g protein/kg; they were thus receiving a protein intake sufficient to support growth and therefore possibly undergoing nutritional rehabilitation. The period of insult was through both gestation and lactation, and the recovery was in the form of ad 34

libitum feeding of a control diet from weaning throughout the remainder of the trial. The relationship between food intake and bodyweight during the post-weaning nutritional period was examined to determine whether there was a relationship between rehabilitation and other outcome measures. The study was designed to investigate the effect of both protein quantity and quality within the maternal diet on the health of the offspring. Protein quality in this context refers to the balance of the various amino acids present within the protein component of the diet; proteins containing a greater proportion of essential amino acids having a greater biological value and therefore referred to as being of higher quality (Friedman, 1996). Lactalbumin (a major component of whey protein) is considered to be a protein of higher nutritional quality (Friedman, 1996), and thus a whey protein component was used as the “control” protein at the level of 20 % within the diet. In order to examine the protein quantity, whey protein was also used at the level of 5 % within the diet, and to investigate the effect of protein quality, a 5% casein diet group was also included. The relative quality of whey and casein is further discussed in section 2.2.

1.7.2 Hypotheses The hypotheses to be tested were: (1) That a decreased protein-intake in the maternal diet (maternal low protein) during pregnancy and lactation causes impaired glucose tolerance and insulin resistance in the offspring after they have reached maturity. (2) That the impaired glucose tolerance and insulin resistance in the adult offspring of mothers with low protein intakes during pregnancy and lactation leads to reproducible changes in the function of skeletal muscle (insulin resistance) and the endocrine pancreas (impaired hormone secretion in response to standard nutritional stimuli) that account for the observed insulin resistance and impaired glucose tolerance. (3) That the changes in skeletal muscle and endocrine pancreatic function induced in the offspring by maternal low protein can be 35

measured in isolated organs using well characterised in vitro models of skeletal muscle and endocrine pancreatic function. The models to be employed were: (i)

insulin-stimulated glucose uptake in the isolated soleus muscle incubated in vivo;

(ii)

the quantity and quality of hormone (insulin, amylin, preptin) secretion from the isolated, perfused rat pancreas as measured in time-dependent samples by hormone-specific RIAs.

1.7.3 Expected original contribution As discussed above, it has been established in whole animals that maternal low protein causes insulin resistance and impaired glucose tolerance in the adult offspring of affected mothers. However, models for the effect of maternal low protein in the organs of adult offspring had not been well characterised at the time these studies were initiated. Since the interplay between the endocrine pancreas (insulin secretion) and skeletal muscle (insulin-mediated glucose disposal) is central in the regulation of body carbohydrate homeostasis, it was chosen to employ available, well-characterised organ preparations to study the effects of maternal low protein on the adult offspring. These studies would provide an important link between whole animal physiology and studies of molecular mechanisms in these organs.

It was therefore expected that these studies would provide models in

isolated muscle and the isolated pancreas to examine the effects of maternal low protein in the adult offspring, as a prelude to investigating the molecular mechanisms underlying these effects.

36

Chapter 2

General Methods

37

38

2.1 Animals 2.1.1 General Virgin female Sprague-Dawley rats were housed at 21 ± 1 oC, with a 12 hour light cycle. Diet was standard laboratory chow (NRM Diet 86) provided ad libitum prior to mating. A positive pregnancy was determined by the appearance of a vaginal plug, at which stage the rats were transferred to individual cages, and the diet changed to one of the two experimental diets. Appearance of the vaginal plug was defined as day 1 of gestation. Throughout gestation the rats were fed synthetic diets containing a variety of protein contents. These diets are fully described in section 2.2. Animals were not fed ad libitum during gestation. Instead, the amount fed daily to each pregnant rat was calculated from the body weight of the rat, and the particular day of gestation (see section 2.1.2) in order to prevent low-protein mothers consuming more food. All animals had free access to water. Following spontaneous delivery of pups (day 22-23), mothers were maintained on the same diet as had been supplied during gestation, without restriction on intake. Parturition was defined as day 1 and gestation days were defined retrospectively relative to this, to allow for any variation in the length of gestation. Thus, the first day of a 22 day gestation would be day –21 (denoting 21 days prior to day 0), and the final day of gestation day 0. At 21 days of age, pups from all litters were weaned onto the same diet, and this was supplied ad libitum for the remainder of the trial.

2.1.2 Calculation of daily food requirements Because the investigation focused on protein, the experimental diets used in each trial were formulated to be iso-caloric, so any observed differences could be attributed solely to the protein component of the diets. However, pregnant rats fed a protein-deficient diet have been shown to consume up to 50 % more food by weight than those fed a protein-sufficient diet (Rogers, 1979). While this would not fully compensate for the protein deficiency in the low-protein diets, it would result in a significant difference in the caloric intake between the two diet groups, and

39

confound the protein/energy responses. It was therefore decided to control the food intake of both groups. The basis for this controlled feeding was a set of data obtained from a trial in which pregnant female rats were fed normal rat chow (NRM Diet 86) ad libitum throughout gestation1. The information included daily measurements of body weight and food intake for a group of 15 rats for the duration of the gestation period. From these data a graph was constructed with food intake per gram of body weight as a function of day of gestation (Figure 2.1). From the equation of the line of best fit, a factor was derived for each day of gestation such that when multiplied by the body weight of the pregnant rat, the theoretical food requirement for that day could be calculated.

2.2 Composition of experimental diets The level of protein in the diets used in these experiments was either ‘normal’ or ‘low-protein’. There is some variation in the literature with respect to both of these diets. For example, a “normal” diet may contain 20 % (Grigor et al., 1987; Lucas et al., 1996; Ozanne et al., 1996), 18 % (Langley and Jackson, 1994; Langley-Evans, 1997; Rees et al., 1999), or 15 % protein by weight (Bertin et al., 1999). The protein contents (by weight) in “low-protein” diets range from 12 % (Langley and Jackson, 1994), through 9 % (Langley and Jackson, 1994; Langley-Evans, 1997; Rees et al., 1999), 8 % (Lucas et al., 1996), and 6 % (Langley and Jackson, 1994; Langley-Evans et al., 1997) to 5 % (Bertin et al., 1999; Muaku et al., 1997; Ozanne et al., 1996). In the current study, a protein content of 20 % by weight was used as the control, being the standard level commercially available, and also most closely matching the level of protein present in the standard rat chow (NRM Diet 86). The low-protein diet contained 5 % protein by weight, thus providing an extreme comparison most likely to uncover any differences in the outcome measures under investigation. Levels of the remainder of the dietary components were based on diets reported in the literature, and commercially available rodent diets. The main consideration was whether the carbohydrate in the diet was to be starch-based (e.g. cornflour), or based on simple sugars (glucose or sucrose). As the study focuses on insulin 1

Jansen V:, unpublished data entitled “IUGR rat study part 1: gestation weights” from a trial carried out at

the University of Auckland small animal facility.

40

resistance and impaired glucose tolerance, starch was selected as an optimal carbohydrate source in order that the glucose was available from a more slowlyreleasing source. As outlined in the Introduction (see. Section 1.7.1), protein content was chosen as the major nutritional variable in the experimental diets. It has been suggested that the balance of protein with other nutrients, as well as protein level, may be a critical determinant of the long-term health effects of maternal under-nutrition in pregnancy (Langley-Evans, 2000). As the diets were formulated to be iso-caloric, a change in the level of protein would necessarily result in a change in the balance of protein with other dietary components. The use of additional carbohydrate in the low-protein diets to balance the energy deficiency due to protein reduction also altered the carbohydrate: lipid ratio. This approach has been used in a number of published studies, and was considered to be the best approach given the focus on the effect of low dietary protein. The final dietary composition, with manufacturers of individual components, is described in Table 2.1, and the dietary preparation is described in sections 3.3.2 (first experimental trial) and 4.3.1 (second experimental trial). Diets were also analyzed once prepared to ensure that the actual composition was as specified. These analyses are shown in sections 3.4.1 (Table 3.1) and 4.4.1 (Table 4.1). There has been a progressive reassessment in recent decades of the role of amino acids in nutrition. In particular, it has become apparent that relatively few amino acids are nonessential: in humans, these are now thought to comprise only glutamic acid, alanine, serine, aspartic acid, and asparagines. Moreover, there is evidence that one or more of glutamate, alanine, or aspartate may be required as obligatory sources of -amino nitrogen (Food and Nutrition Board, 2002). Diets containing predominantly casein are relatively deficient in tryptophan, one of the absolutely essential amino acids (Dupont, 2003), whereas diets comprising predominantly whey are sufficient in this amino acid. While casein is often used as a reference protein because of its high biological value, studies in Sprague-Dawley rats have shown lactalbumin to be of higher quality than casein as measured by protein efficiency ratio (PER), net protein ratio (NPR), and actual protein utilization (APU) (Mercer and Gustafson, 1984). For these reasons, the sufficient-protein diet and one 41

of the two low-protein diets contained whey as their protein source. All three diets were supplemented with dl-methionine, another absolutely essential amino acid that contains sulphur, at 0.2 % of total diet (w/w).

Figure 2.1: Food intake as a function of body weight: data from a previous study in pregnant female Wistar rats fed standard chow (NRM Diet 86)

Food intake (g) / bodyweight (g)

0.11

0.1

0.09

0.08

y = 0.0006x + 0.0828

0.07

0.06 0

6

12

18

24

Gestation (days)

Data represent the mean ± SD for n = 15. Results were from a previous experimental feeding trial (Jansen V, see footnote on p40). From the equation of the line of best fit, a value of the food intake per unit bodyweight was calculated for each day of gestation, and this value was used to determine the amount of food given to each pregnant female in the current studies. The purpose of this was to prevent the previously observed (Rogers, 1979) increase in food intake of pregnant female rats fed a low-protein diet, and thus energy intake during gestation was standardized across all diet groups.

42

Table 2.1: General composition of experimental diets fed to pregnant and lactating female rats

Ingredient

Content (g/100 g) Control (20 % protein)

Low (5 %) protein

Cellulose (PCL)

5

5

Cornflour (Avon)

47.8

62.8

DL-methionine (ICN)

0.2

0.2

Mineral mix (ICN)

3.9

3.9

Protein (NZ Dairy)

20

5

Soya-bean oil (AMCO)

7

7

Sucrose (Chelsea)

15

15

Vitamin mix (Nutritech)

1.1

1.1

Values represent the % composition by weight of experimental diets used to study the effect of maternal protein insufficiency on health outcomes in the offspring. In the first trial, these were produced in the laboratory using ingredients the listed, with a protocol described in section 3.3.2. In the second trial, diets were commercially prepared to these specifications by Crop and Food Research, as further described in section 4.3.1.

43

2.3 Isolated Perfused Pancreas 2.3.1 Background This protocol is one that, coupled with radio immunoassay (RIA), allows accurate measurement of pancreatic hormone output, and consequently provides a detailed picture of the function of this organ. Due to the technically demanding nature of the surgery, the length of the perfusion process, and the labour-intensive nature of the RIA, the throughput of animals for this assay was a limiting experimental factor.

2.3.2 Solutions 2.3.2.1 Pancreas Perfusate 2.3.2.1.1 Stock solutions: [1] 1.128 M NaCl: Prepared by dissolving 65.92 g solid NaCl (BDH AnalaR) in MilliQ water to a final volume of 1 L. [2] 0.293 M NaHCO3: 24.61 g dissolved in MilliQ water to a final volume of 1 L. [3] 0.44 M KCl (Riedel de Haën): 16.40 g dissolved in MilliQ water to a final volume of 500 mL. [4] 0.15 M KH2PO4 (BDH): 10.21 g dissolved in MilliQ water to a final volume of 500 mL. [5] 0.12 M MgSO4: Prepared by dissolving 14.79 g of MgSO4 (Sigma) in MilliQ water to a final volume of 500 mL. [6] 0.23 M CaCl2: 16.91g CaCl2.2H2O dissolved in MilliQ water to a final volume of 500 mL. 2.3.2.1.2 Perfusate preparation For one litre of the perfusate: [1] The following volumes of stock solutions were mixed together in a large beaker: 100 mL NaCl, 100 mL NaHCO3, 10 mL KCl, 10 mL KH2PO4, 10 mL MgSO4. 650 mL MilliQ water was added. [2] Food-grade CO2 (BOC Gases) was bubbled through the solution for approximately 1 minute.

44

[3] A further 100 mL of MilliQ water, 10 mL of CaCl2 stock, 5 g BSA, 40 g Dextran, and 0.54 g D-glucose (final concentration 3 mM) were added, with stirring. Once all solids had dissolved, the pH was adjusted to 7.4 with either NaOH or HCl. [4] The solution was sterile-filtered (Gelman Vacucap® 60/90, 0.2 m) into a 1 L Schott bottle, and stored at 4 oC until required. Due to the possibility of CaSO4 precipitation and bacterial contamination, the perfusate was not made up more than one week prior to use.

2.3.2.2 Infusion solutions [1] Glucose stock: a 1 M solution of glucose was prepared by dissolving 54.05 g of Dglucose (BDH Analar) in 300 mL of MilliQ water. The solution was sterile filtered (Gelman Acrodisc® syringe filter, 25 mm, 0.2 m) into 50 mL conical-bottomed tubes (Nunc). [2] Arginine stock: a 500 mM solution of L-arginine was prepared by dissolving 26.34 g of L-arginine hydrochloride (Sigma) in 250 mL of MilliQ water. The solution was sterile filtered into 50 mL conical-bottomed tubes. [3] Working solutions: Glucose: stock glucose was diluted 1:1 with MilliQ H2O Arginine: stock arginine was diluted 1:1 with MilliQ H2O Glucose + Arginine: The two stock solutions were mixed 1:1 All three solutions were delivered by side-arm infusion (Figure 2.2) using syringe pumps (Kent Scientific, model YA-12) at the rate of 3 mL.hr-1 (0.05 mL.min-1). With a perfusate flow rate of 1.2 mL.min-1, this gave a final concentration of ~21.7 mM for glucose and ~10.85 mM for arginine.

2.3.3 Surgical procedure Weaned male and female Sprague-Dawley rats from each treatment group described in section 2.2 were used as pancreas donors (age at experimentation 42 ± 2 d). They were anaesthetised initially by halothane induction (5 % + 2 L.min-1 O2) followed by injection of sodium phenobarbitone (Nembutal, 50 mg.kg-1, ip), and pancreases prepared according to the method of Grodsky and Fanska, with minor modifications (Grodsky and Fanska, 1975). Briefly, the pancreas, stomach, and 45

duodenal remnant were isolated en bloc, the aorta cannulated, and the preparation transferred to a perfusion chamber. Perfusion was through the coeliac and superior mesenteric arteries (flow rate 1.2 mL.min-1), and effluent was collected without recycling from the cannulated portal vein. Perfusate was equilibrated against a gas phase of carbogen (95 % CO2 : 5 % O2, BOC gases) for a final pH of 7.4 and temperature of 37 oC. Organs were equilibrated for 20 minutes. For 90 minutes thereafter, effluent fractions were collected at one-minute intervals and stored on ice. Each gland was stimulated during the experiment by means of side-arm infusions (0.1 mL.min-1, Figure 2.2) of either glucose (final concentration, 21.7 mM), arginine (final concentration 10.85 mM), or a simultaneous infusion of both. Both glucose and lactate were measured simultaneously on every second sample during the stimulation phases (every 5th sample during basal periods) (YSI 2300 Stat Plus glucose/lactate analyser) to ensure that the achieved glucose concentration was as expected (glucose) and to confirm pancreas viability (lactate) (Gedulin et al., 1991). Samples were stored at –80 oC until analysis. The two variations in perfusion protocol utilized in these studies are described in full detail in sections 3.2.3 and 4.2.3.5. Fractions obtained as described were analysed for insulin (section 2.4), amylin (section 2.5) and preptin (section 2.6) using the appropriate RIA.

46

Figure 2.2: Side-arm infusion

perfusate

clamp

Infusion solution

to pancreas

The infusion solution was delivered to the perfusion system via the side-arm of a T-junction within the perfusate tubing. The syringe containing the appropriate infusion solution was attached to this side-arm, and the rate of infusion was controlled by a syringe pump. A clamp was applied to the tubing in the area indicated before and after infusion events to prevent mixing of the infusion solution with the perfusate at inappropriate times.

47

2.4 Insulin RIA 2.4.1 Background The insulin radioimmunoassay (Yalow and Berson, 1960), relies on the competitive binding of 125 I labelled insulin and unlabelled insulin to a limited quantity of insulin antibody. The amount of

125

I labelled insulin is constant, thus the amount of label

bound on the addition of secondary antibody is dependent on the amount of ‘cold’ insulin present – if more unlabelled insulin is present, less 125 I-insulin is able to bind to the antibody, and vice versa. At the end of the assay, bound insulin remains in the assay tube and is counted in a gamma counter. Thus there is an inverse relation between the measured counts and the amount of cold insulin present in an assay tube. The quantity present in an unknown sample is determined by comparison to a standard curve prepared using known quantities of unlabelled insulin.

2.4.2 Reagents 2.4.2.1 Phosphate buffer stock solution [1] Solution A: 0.5 M disodium hydrogen phosphate [2] Solution B: 0.5 M sodium dihydrogen phosphate For 1 L of stock buffer, Solution B was added to 800 mL of solution A until the pH was 7.4. This generally required approximately 190 mL of solution B. If the solutions were stored for long periods, 0.24 g of sodium azide was added to the stock solution.

2.4.2.2 Insulin Assay Buffer 3 g Bovine Serum Albumin, Fraction V (BSA) (Sigma A-6793) 100 mL phosphate buffer stock solution (2.4.2.1) MilliQ water was added to a final volume of 1 L.

2.4.2.3 Insulin Primary Antibody Guinea pig anti-[bovine insulin] was prepared in our laboratory. Aliquots (200 L) of a 1/100 dilution in insulin assay buffer were stored at –80 oC until required.

48

Working solution was prepared by a further 1/1 000 dilution in insulin assay buffer, giving a final dilution of 1/100 000.

2.4.2.4 Tracer Bovine insulin was iodinated according to the Chloramine T method (Hunter and Greenwood, 1962). The 125 I- bovine insulin thus produced was diluted in insulin assay buffer to give an activity of between 9 and 10 000 cpm per 100 L.

2.4.2.5 PBS-EDTA 6.93 g sodium chloride 3.75 g disodium EDTA 20 mL phosphate buffer stock solution MilliQ water was added to a final volume of 1 L.

2.4.2.6 Secondary Antibody Goat Anti-Guinea Pig IgG (Sigma G-5393) stock solution was stored at –80 oC until required. Working solution was prepared by diluting the stock 1/20 in PBS-EDTA.

2.4.2.7 Normal Guinea Pig Serum This was stored at –80 oC until required. A working solution was prepared as a 1/100 dilution with PBS-EDTA.

2.4.2.8 PEG buffer 50 g polyethylene glycol 6000 (PEG-6000) (BDH) was added to 100 mL of phosphate buffer stock solution. This was made up to 1 L with MilliQ water.

2.4.2.9 Insulin Standard Actrapid, 100 units.mL-1 (Novo Nordisk) was serially diluted using insulin assay buffer as described in Table 2.2.

49

Table 2.2: Serial dilutions by which insulin RIA standard solutions were prepared

Standard

Serial Dilution

Concentration

-

1/25

4 U.mL-1

-

1/100

40 mU.mL-1

G

1/100

400 U.mL-1 (2840 pM)

F

1/2

200 U.mL-1 (1420 pM)

E

1/2

100 U.mL-1 (710 pM)

D

1/2

50 U.mL-1 (355 pM)

C

1/3..3

15 U.mL-1 (106.5 pM)

B

1/3

5 U.mL-1 (35 pM)

A

Insulin assay buffer

0 U.mL-1 (0 pM)

Insulin stock solution (Actrapid, Novo Nordisk) was diluted according to the scheme described above using insulin assay buffer (section 2.4.2.2). The standards labelled A to G were those used to generate an insulin RIA standard curve, and the insulin concentration of the various experimental samples was determined relative to this.

50

2.4.3 Samples Initial assays of perfused pancreas fractions showed the insulin production during a period of stimulation exceeding the limits of the assay standards. It was therefore necessary to dilute the fractions during which a stimulus had been applied, as well as the five subsequent fractions, collected during the post-phase, when insulin production was returning to basal levels. A variety of dilutions were used, ranging from 1/5 to 1/20. The appropriate dilution was established by trial and error. In general, a 1/5 dilution was performed directly in the assay tube, with 10 L of sample pipetted in and 40 L of insulin assay buffer added to this. If this did not produce a value within the working range of the standard curve, the sample was assayed again using a more appropriate dilution.

2.4.4 The Insulin Assay 2.4.4.1 Day One In addition to the standards described in table 2.2, total counts (TC) and non-specific binding (NSB) standards were included in the assay. Each sample and standard was assayed in duplicate, with 50 µL being pipetted into 5 mL polypropylene tubes (Techno-Plas). The assay was started by the addition of 200 L of primary antibody to all tubes except NSB (into which 250 L of insulin assay buffer was pipetted) and TC. Following addition of primary antibody, tubes were shaken

2

and left at room

temperature for 5 hours. Following incubation, 100 L of tracer was added to all tubes, which were shaken and transferred to 4 oC for 48 hours. Two total counts (TC) tubes were included at this point in the assay.

2.4.4.2 Day Three To all tubes except TC, 100 L of secondary antibody and 100 L of normal guinea pig serum were added. Tubes were shaken, and left at room temperature for one

2

After addition of each reagent, tubes were shaken while still in the rack (up to 90 at a time) using a

standard method

51

hour. One mL of PEG buffer was then added, again to all tubes except TC, and tubes were shaken and transferred to 4 oC for a further hour. Following this final incubation, tubes were centrifuged for 30 minutes at 3000 rpm, 4 oC

(Sorvall RT7) and the supernatant decanted by inversion.

Radioactivity was determined by counting for one minute in a gamma counter (WizardTM, Wallac, Finland).

2.4.4.3 Insulin Assay Characteristics Characteristics of the insulin RIA were derived from 58 independent standard curves. Mean (SEM) characteristics were: Minimum Detectable Concentrations, 124.3 (17.6) pM; EC50 values, 487.6 (43.4); Coefficient of Variation at the EC50, 19.6 (1.5)%. A representative standard curve is included below (Figure 2.3). Figure 2.3: Representative standard curve of the insulin RIA

1

Response (B / Bmax)

0.8

0.6

0.4

0.2

0 10

100

1000

10000

Insulin concentration (pM)

Insulin concentrations used to derive the standard curve were as described in Table 2.2. Curve fitting used a four-parameter logistic model, and was carried out using MultiCalc® software (Wallac, Finland).

52

2.5 Amylin RIA 2.5.1 Background This assay, like the insulin RIA, is a competitive binding assay.

2.5.2 Reagents 2.5.2.1 Amylin Assay Buffer 10 g BSA Fraction V 2.5 g Disodium EDTA 2 g Triton X-100 10 mL Aprotonin 100 mL phosphate buffer stock solution (2.4.2.1) MilliQ water was added to a final volume of 1 L.

2.5.2.2 Primary Antibody Rabbit anti-[rat amylin] antibody was prepared in our laboratory. Working solution was prepared in the same manner as the insulin primary antibody (final dilution 1/100 000), but dilutions were made in amylin assay buffer.

2.5.2.3 Tracer Rat amylin was iodinated according to the Chloramine T method. The

125

I- rat amylin thus produced was diluted in amylin assay buffer to give an

activity of between 10 and 12 000 cpm per 100 L.

2.5.2.4 Secondary Antibody Goat anti-rabbit IgG (Sigma R-1131) was diluted 6.6/100 in amylin assay buffer.

2.5.2.5 Normal Rabbit Serum (NRS) This was stored at -80 oC until required. A working solution was prepared as a 1/100 dilution in amylin assay buffer.

2.5.2.6 Amylin PEG Buffer 80 g polyethylene glycol 6000 (PEG-6000) (BDH).

53

100 mL phosphate buffer stock (2.4.2.1) MilliQ water was added to a final volume of 1 L.

2.5.2.7 Amylin Standard A stock solution was prepared by dissolving 12.8 g rat amylin (Peninsula Laboratories, RIK-7323, Mr = 3920.45) in 1mL of amylin assay buffer. This gave a starting concentration of 3.26 M. Working standards for the calibration curve were prepared by serial dilution of the stock standard as described in Table 2.3.

2.5.3 Samples All samples were used undiluted.

2.5.4 The Amylin Assay 2.5.4.1 Day One As for the insulin RIA (section 2.4), total counts (TC) and non-specific binding (NSB) standards were included in the amylin assay. Each sample and standard was assayed in duplicate, with 100 µL being pipetted into 5 mL polypropylene tubes (Techno-Plas). The assay was started by adding 100 L of amylin primary antibody to all tubes except NSB (200 µL of amylin assay buffer, section 2.5.2.1) and TC. Tubes were shaken, and incubated at 4 oC for 24 hours.

54

Table 2.3: Serial dilutions by which amylin RIA standard solutions were prepared

Standard

Serial Dilution

Concentration

-

1:99

32.6 nM

I

1:99

326 pM

H

1:1

163.2 pM

G

1:1

81.6 pM

F

1:1

40.8 pM

E

1:1

20.4 pM

D

1:1

10.2 pM

C

1:1

5.1 pM

B

1:1

2.55 pM

A

Amylin assay buffer

0 pM

Amylin stock solution (section 2.5.2.7) was diluted in amylin assay buffer (section 2.5.2.1) according to the scheme described above. The standards labelled A to I were those used to generate an amylin RIA standard curve, and the amylin concentration of the various experimental samples was determined relative to this.

55

2.5.4.2 Day Two 100 L of amylin tracer was added to all tubes (including TC), which were then shaken and returned to 4 oC for a further 48 hours.

2.5.4.3 Day Four To all tubes except TC, 100 L of secondary antibody and 100 L of normal guinea pig serum were added. Tubes were shaken, and left at room temperature for one hour. Amylin PEG buffer (500 L) was then added, again to all tubes except TC, and tubes were shaken and transferred to 4 oC for a further hour. Following this final incubation, tubes were centrifuged for 30 minutes at 3000 rpm, 4 oC

(Sorvall RT7) and the supernatant decanted.

Radioactivity was determined by counting for one minute in a gamma counter (WizardTM, Wallac, Finland).

2.5.4.4 Amylin Assay Characteristics Characteristics of the amylin RIA were derived from 31 independent standard curves. Mean (SEM) characteristics were: Minimum Detectable Concentrations, 25.3 (4.4) pM; EC50 values, 71.1 (5.3); Coefficient of Variation at the EC50, 22.4 (3.2) %. A representative standard curve is included below (Figure 2.4).

2.6 Preptin RIA The preptin RIA is also a competitive binding assay, and was developed in our laboratory following the discovery of the preptin molecule, as described in section 1.5.2. The assay is based on the amylin RIA, and therefore uses many of the same reagents.

2.6.1 Reagents Amylin assay buffer, normal rabbit serum, second antibody and PEG buffer were all used for both preptin and amylin RIAs. The following were used only in the preptin assay system.

56

Figure 2.4: Representative standard curve of the amylin RIA 1

Response (B / Bmax)

0.8

0.6

0.4

0.2

0 1

10

100

1000

Amylin concentration (pM)

Amylin concentrations used to derive the standard curve were as described in Table 2.3. Curve fitting used a four-parameter logistic model, and was carried out using MultiCalc® software (Wallac, Finland).

2.6.1.1 Primary Antibody Rabbit anti-[rat preptin] antibody was prepared in our laboratory. Working solution was prepared in the same manner as the insulin primary antibody, but dilutions were made in amylin assay buffer.

2.6.1.2 Tracer Rat preptin was iodinated according to the Chloramine T method. The

125

I- rat preptin thus produced was diluted in amylin assay buffer to give an

activity of approximately 6000 cpm per 100 L.

57

2.6.1.3 Preptin Standard A stock solution was prepared by dissolving 15 g of rat preptin (Auspep, custom synthesis) in 2 mL of amylin assay buffer. This gave a starting concentration of 1.9 M. Working standards for the calibration curve were prepared by serial dilution of the stock standard as described in Table 2.4.

Table 2.4: Serial dilutions by which preptin RIA standard solutions were prepared

Standard

Serial Dilution

Concentration

-

1/19

100 nM

I

1/20

5 nM

H

1/5

1 nM

G

1/2.5

400 pM

F

1/2

200 pM

E

1/2.5

80 pM

D

1/2

40 pM

C

1/2

20 pM

B

1/2.5

8 pM

A

Amylin assay buffer

O pM

Preptin stock solution was diluted in amylin assay buffer (section 2.5.2.1) according to the scheme described above. The standards labelled A to I were those used to generate a preptin RIA standard curve, and the preptin concentration of the various experimental samples was determined relative to this.

58

2.6.2 The Preptin Assay The procedure for the preptin RIA was carried out as described for the amylin RIA, with the following exceptions:

2.6.2.1 Day one The assay was started by adding 100 L of preptin primary antibody to all tubes except TC and NSB. Tubes were shaken, and incubated at 4 oC for 48 hours.

2.6.2.2 Day three 100 L of preptin tracer was added to all tubes, which were then shaken and returned to 4 oC for a further 48 hours.

2.6.2.3 Day five As day four for the amylin RIA (section 2.5.4.3).

2.6.2.4 Preptin Assay Characteristics Characteristics of the amylin RIA were derived from 3 independent standard curves. Mean (SEM) characteristics were: Minimum Detectable Concentrations, 26.4 (6.0) pM; EC50 values, 146.6 (41.1); Coefficient of Variation at the EC50, 18.5 (6.9) %. A representative standard curve is included below (Figure 2.5).

2.7 Glucose uptake assay in the isolated soleus muscle 2.7.1 Background This assay measures the uptake of radioactively labelled glucose into glycogen in the soleus muscle. A variety of insulin concentrations were used in order to determine whether an altered maternal diet had any effect on glucose metabolism, in particular insulin sensitivity. The soleus muscle was used in this study for a number of reasons. It is easily exposed and has a well-defined tendon at each end allowing it to be quickly removed without damage. The tendons also allow the muscle to be split into strips, which is required to ensure a sufficiently small crosssectional area for substrate diffusion, and also oxygen diffusion to prevent the occurrence of regional hypoxia during the incubation (Bonen et al., 1994; Maltin and Harris, 1986; Newsholme et al., 1986; Segal and Faulkner, 1985). Finally, this muscle 59

Figure 2.5: Representative standard curve of the preptin RIA 1

Response (B / Bmax)

0.8

0.6

0.4

0.2

0 1

10

100

1000

10000

Preptin concentration (pM)

Preptin concentrations used to derive the standard curve were as described in Table 2.4. Curve fitting used a four-parameter logistic model, and was carried out using MultiCalc® software (Wallac, Finland).

has been used in numerous studies of carbohydrate metabolism and/or insulin sensitivity (Cooper et al., 1988; Espinal et al., 1983; Kim et al., 1996), and was therefore considered the most appropriate in the context of this study.

2.7.2 Reagents 2.7.2.1 Krebs-Henseleit buffer Ten-fold stock solutions were prepared by dissolving the amounts listed in Table 2.6 in 1 L of MilliQ water. 100 mL of each of the stock solutions except the calcium chloride was mixed in a 1 L Schott bottle, followed by the addition of 300 mL of MilliQ water. The calcium chloride stock (100 mL) was then added (last, in order to avoid CaSO4 precipitation), and the solution mixed by inversion. Thus 1 L was prepared, with 500 mL used in each of two dissection trays – one for removing the muscle from the hindlimb, and one for splitting the muscle into strips (see section 2.7.3.1, step [4]). 60

2.7.2.2 Normal Dulbecco’s Modified Eagle Medium (nDMEM) Stock nDMEM powder (Sigma D-5523) was dissolved according to the manufacturer’s instructions.

2.7.2.3 Insulin Stock Solution This was prepared by diluting 10 L of Actrapid insulin (100 units.mL-1, Novo Nordisk) in 10 mL of nDMEM.

2.7.2.4 Insulin Working Solutions The insulin stock solution was further diluted in nDMEM as described in Table 2.6, to give the required range of final insulin concentrations for calculation of a doseresponse curve.

2.7.2.5 U-[14C]-D-Glucose Stock solution was 1 mCi.mL-1, with a specific activity of 300 mCi.mmol-1 (ARC). The working solution was prepared by diluting the stock solution 1/20 with 70 % ethanol.

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Table 2.5: Stock solutions for the preparation of Krebs-Henseleit buffer

Solute:

Amount (g)

Stock Concentration

Final Buffer Concentration

NaCl

69.25

1.185 M

118.5 mM

KCl

3.54

47.5 mM

4.75 mM

MgSO4∙7H2O

2.90

11.8 mM

1.18 mM

NaHCO3

20.83

248 mM

24.8 mM

KH2PO4

1.6

11.8 mM

1.18 mM

CaCl2

3.73

25.4 mM

2.54 mM

D-glucose∙H2O

19.82

100 mM

10 mM

The listed stock solutions which are the components of Krebs-Henseleit buffer for use in the glucose uptake assay were prepared by dissolving the amounts of solute listed in MilliQ water to a final volume of 1 L.

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Table 2.6: Preparation of insulin working solutions for use in the glucose uptake assay

Insulin Concentration (nM)

Insulin Stock Solution (L)

Normal DMEM (mL)

0 (basal)

0

10

0.71

10

10

2.37

33.3

9.97

7.1

100

9.90

23.7

333

9.67

71.0

1000

9.0

237.0

3333

6.67

The solutions in which soleus muscle strips were incubated in order to determine the uptake of glucose into glycogen were prepared using the volumes described above.

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2.7.3 Experimental Protocol 2.7.3.1 Preparation of muscle strips [1] The animal to be studied was fasted overnight, with free access to water. [2] On the day of the experiment, the animal was anaesthetized by phenobarbitone injection (IP, 27 g needle, 80 mg.kg-1 bodyweight Nembutal). Once a deep anaesthesia had been established, the animal was sacrificed by cervical dislocation. [3] The skin was removed from each hind leg, and the leg removed from the body. The leg was placed in a tray containing Krebs-Henseleit buffer bubbled with carbogen. [4] The soleus muscle was rapidly dissected out from each hind limb under carbogen-saturated Krebs-Henseleit buffer. Once removed, the muscle was placed into a separate tray containing carbogen-saturated Krebs-Henseleit buffer with approximately 1 L of insulin (Actrapid, Novo Nordisk). [5] Once each soleus muscle had been dissected out, it was split into either two or three strips, depending on the muscle size.

2.7.3.2 Incubation [1] Muscle strips were transferred to 50 mL Erlenmeyer conical flasks, each containing insulin ranging in concentration from 0 to 237 nM dissolved in 10 mL of normal DMEM (described in section 2.7.2.4). Not more than 5 strips were placed in any one flask. [2] Flasks were incubated at 30 oC in a shaking waterbath for twenty minutes in order to equilibrate the muscle strips. Following equilibration, 10 L of the [U]14 CD-glucose working solution was added to each flask, with two minute intervals between subsequent additions. [3] Flasks were incubated for exactly two further hours. Muscle strips were then removed from the flask, blotted dry, and snap-frozen in liquid N2. Each strip was placed into a pre-weighed (Mettler MT5) 2 mL eppendorf tube. The lid of each eppendorf tube had two holes punched into it (using an 18 gauge needle) in order to allow water out during the freeze-drying process. The holes were connected by a slit

64

made using a scalpel blade. This allowed addition of reagents during the extraction procedure without needing to open the lid, thus simplifying the procedure. [4] Muscle strips were freeze-dried (FTS EZ-dry or FTS Dura-dry) for a minimum of 24 hours.

2.7.3.3 Glycogen extraction and liquid scintillation counting [1] Tubes were removed from the freeze-drying vial and re-weighed (Mettler MT5), giving an accurate measure of the muscle dry weight. [2] 250 L of 60 % (w/v) KOH was added to each sample, and the tubes incubated for 45 minutes at 70 oC, with gentle shaking after 15 and 30 minutes incubation. This resulted in complete digestion of the muscle material. [3] Tubes were centrifuged briefly to bring all the liquid to the bottom, and allowed to cool to room temperature. [4] Three volumes (750 L) of 96 % ethanol were added to each sample. The tubes were vortex-mixed briefly, then left at –20 oC overnight to allow glycogen to precipitate. [5] Samples were centrifuged (15 minutes, 10 000 rpm, 0 oC) to pellet the glycogen (Jouan MR 18 22 refrigerated centrifuge). [6] Supernatant was aspirated, leaving approximately 100 L (to ensure that no glycogen was lost). The pellet was washed with 750 L of cold (–20 oC) 96 % ethanol, briefly vortex-mixed, and centrifuged (15 minutes, 10 000 rpm, 0 oC). [7] Step [6] was repeated twice. [8] The supernatant was aspirated, again leaving approximately 100 L, and the tubes transferred to a 60 oC oven (Hybaid Maxi 14) to dry for a period of two hours. [9] The dried glycogen pellet was redissolved in 200 L of MilliQ water, then the tube briefly vortex-mixed and centrifuged for 10 seconds (IEC Micromax) to bring all the liquid to the bottom. [10] The redissolved glycogen was transferred to a scintillation vial, and 1.8 mL of scintillation fluid added. Samples were counted for 5 minutes each on a Beckman LS 3801 beta-counter or a Wallac Rackbeta beta-counter.

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2.8 Statistical Methods All statistical analyses were performed using GenStat® for Windows 5th edition (GenStat Release 4.22 (PC/Windows XP), © 2001, Lawes Agricultural Trust), 6th edition (Version – 6.1.0.210 © 2002 Lawes Agricultural Trust), or 7th edition (GenStat for Windows, Release 7. Seventh Edition. VSN International Ltd., Oxford). Unless otherwise stated, the analysis used was Residual Maximum Likelihood (REML). In the case of the glucose uptake assay, a four-parameter logistic model was fitted to the data. The general equation (one of the family of logistic curves, also known as sigmoidal curves) is described as: y = ( a - b ) / ( 1 + exp (x/c) **d ) + b Where:

a is the asymptote as x ---> 0 for all d > 0, b is the asymptote as x diverges, c is the predicted response midway between the asymptotes, d is a function of the rate of change (or slope) of the fitted curve

(Barron, 1999). In this particular case, the equation was expressed as: y = ( a – b ) / ( 1 + (insulin/c) **d ) + b Where:

a is the minimum value b is the maximum value c is the effective insulin concentration at which the half-maximal

response occurs (EC50) d is the slope at the point of inflection insulin refers to the concentration of insulin (nM), which in this case is the x-axis

66

Where possible, the model was fitted to the data from individual litters. However, if the number of animals was insufficient, then the fitting failed to converge (i.e. the curve was unable to be fitted), thus a statistical analysis could not be performed for each of the components listed above. In all cases, a curve was fitted to the overall data from each diet group in order to obtain an overall value for each of the parameters described by the curve, and thus examine any general effects, while statistical analyses (REML) were performed on the data at each insulin concentration. The statistical analyses associated with glucose uptake for each of the two experimental trials performed are further described in sections 3.4.4.8 and 4.4.4.9.

2.9 Ethical Approval All work described in this thesis was approved by the University of Auckland Animal Ethics Committee, under applications N633 and N857.

67

68

Chapter 3 Trial 1: The effect of maternal protein intake on glucose metabolism and pancreatic function in the offspring

69

70

3.1 Introduction Strategies for studying early growth restriction fall into three broad categories – a dietary manipulation of some kind, a hormonal insult such as exposure to glucocorticoids during gestation, or surgical intervention such as uterine artery ligation (Ozanne, 2001). Dietary manipulations include total calorie restriction, or a deficiency in specific dietary components such as iron or protein. The maternal low protein model is one of the most extensively studied (Ozanne, 2001) and induces a phenotype which shows remarkable similarities to the human metabolic syndrome and type-2 diabetes (Hales and Barker, 2001), possibly due to the importance of amino acids in fetal growth, pancreatic -cell development, and insulin secretion (Hales and Barker, 1992). Protein is also often scarce and expensive in communities with a high prevalence of diabetes and the metabolic syndrome (Hales et al., 1997). As type-2 diabetes is the metabolic disorder on which this thesis focuses, protein was selected as the nutritional variable to be studied. Protein quality refers to the balance of the various amino acids present in the protein component of the diet; proteins containing a greater proportion of essential amino acids have an increased biological value and are therefore of higher quality (Friedman, 1996). In this trial, both the level and type of protein used in the maternal diets were varied as is described in section 2.2.

3.2 Aims The global aim of the experiments reported in this chapter was: To develop an in vivo model in rats, based upon previously published work, for the effects of maternal low protein on the regulation of glucose metabolism and insulin sensitivity in adult offspring. The specific aims by which the global aim was to be accomplished were: 1. To measure the effect of maternal low protein on bodyweights of mothers and offspring compared with normal controls; 2. To measure ex vivo the effects of maternal low protein on the nutrient (glucose, arginine, glucose/arginine)-stimulated secretion of islet hormones (insulin, amylin) from the isolated perfused pancreas of adult offspring; and

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3. To measure ex vivo the effects of maternal low protein on basal and insulinstimulated glucose uptake and incorporation into glycogen in the isolated incubated soleus muscle of adult offspring. The global aim was accomplished by the feeding of 200 g protein/kg diets (normal protein) or 50 g protein/kg diet (low protein) to female rats during their pregnancies and lactation periods and serially weighing the mothers and offspring. The diets were analysed to verify protein content. In addition, because questions of protein quality have been raised in the context of such experiments, it was determined to feed either a standard diet (Diet 86, NRM) or a diet rich in whey protein. Dietary composition has been discussed in Chapter 2. The specific aims were accomplished by making organ preparations (isolated soleus muscle; isolated perfused pancreas) from the adult offspring of mothers subjected to maternal low protein or control diets, and measuring organ responses under defined conditions, according to the methods described in Chapter 2.

3.3 Methods The overall design employed in this trial is shown in Figure 3.1. This includes the diets, general timing of mating, parturition and weaning, as well as the times at which principal outcome measures were performed.

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Figure 3.1: Experimental design of the first trial Timed mating

0

Lactation

Parturition

10

Offspring

30

40

Trial end

50

Offspring food intake

20

Weaning

IPP (W)

Offspring body weights

-10

Maternal food intake and body weights

Mothers

Gestation

-20

IPP

GUA

Scale represents the time (in days) relative to parturition. Major time milestones are indicated on the left hand side, while the times at which outcome measures were performed are indicated on the right. Abbreviations for these are IPP (Isolated perfused pancreas, where (W) refers to NRM weightmatched offspring as described in section 3.3.4), and GUA (glucose uptake assay). Colour codes represent diets fed during each phase of the trial (C =, LP =, LPC =, Diet 86 =). While all offspring were weaned onto the C diet, their intake during lactation was determined by maternal diet, which is indicated by the corresponding semi-transparent colour.

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3.3.1 Diets While casein is the protein source most often used for trials of this nature, whey protein contains more lactalbumin, which is considered a protein of higher nutritional quality (Friedman, 1996). The reasons for selecting whey as well as casein as the protein source in experimental diets have been addressed in Chapter 2, section 2.2. Therefore, whey protein was selected as the protein component of the control diet, while both whey protein and casein were used in the two respective ‘low-protein’ diets. Amino acid analyses of the protein components of these diets are presented in Table 3.1. Three diets varying in protein content were prepared, these being: 20 % whey protein by weight

(Control, C)

5 % whey protein by weight

(Low-protein, LP)

5 % casein by weight

(Low-protein casein, LPC)

Both casein and whey were from New Zealand Dairy (NZMP), with the specific products being AlacidTM and AlatalTM respectively. It was intended that total dietary energy would be balanced across diets so that the major effect would be due to variation in the protein component. Diets were prepared based on those commonly used in the literature, as described in Section 2.2 and Table 2.1. Individual ingredients and suppliers are also listed Table 2.1. Prior to the trial, all rats were maintained on standard rat chow (NRM Diet 86 pellets). A fourth experimental group of females, fed Diet 86 throughout gestation and lactation, provided a ‘standard diet’ control against which to compare outcomes obtained from the three experimental diets.

3.3.2 Preparation of experimental diets Each diet was prepared separately in the laboratory from the same individual components (described in Section 2.2 and Table 2.1). Dry ingredients were mixed together in a large plastic bowl, and soya-bean oil added. After manual mixing, the mixture was transferred in lots of approximately 500 g to a blender. Water was added to the mixture and the blender used in short bursts until the consistency of 74

dough was achieved. This was spread onto a large steel tray covered with a sheet of plastic, cut into rectangular blocks of approximately 1.5 x 3 cm and the tray transferred to a commercial freeze-drier (McFarlane Laboratories) for a freezedrying cycle of 16 hours. Total dry weight of food from each tray was approximately 5 kg. Freeze-dried food was stored in sealed plastic bags at –20 oC until required.

3.3.2.1 Dietary analysis In order to confirm the dietary composition of the experimental diet, and to have a comparative composition of the NRM Diet 86 used in this trial, each of the diets were analysed for total nitrogen, carbohydrate, fat, amino acid contents. Nitrogen determination was by the instrumental combustion method (Carlo Erba Nitrogen analyser Series 1500), lipid determination by the Soxhlet method (Christie, 2003) and carbohydrate determination by an enzymatic and spectrophotometric method (Blakeney and Mutton, 1980; Koziol, 1981; Southgate, 1976). Acid stable amino acids were analyzed by acid hydrolysis; sulfur-containing amino acids by performic acid oxidation followed by acid hydrolysis (Cunniff, 1997c). All analyses were performed on an “as-fed” basis, rather than a dry matter basis. This was because subsequent energy calculations were based on the food intake in grams for the various diet groups, and this intake data was not calculated on a dry matter basis.

3.3.2.2 Dietary energy content On the basis of the data from the dietary analyses, a calculation of the energy content of the diets was carried out. Net metabolisable energy (NME) was selected as the best measurement of dietary energy because it accounts for the efficiency of fuel utilization in metabolism and it has been shown to be a robust method which is valid and applicable to each source of food energy, not just carbohydrates (Livesey, 2001). The general equation used was: 36.6 F + 13.3 P + 15.7 AC + 6.2 DF + 26.4 Alc

(Livesey, 2001)

Where F = fat (g), P = protein (g), AC = available carbohydrate (g), DF = dietary fibre (g), and Alc = alcohol (g).

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3.3.2.3 Dietary protein content For Diet 86, a value of protein content was provided by the manufacturer (NRM). For all diets, protein content was calculated from the nitrogen content as follows: Diet 86:

Crude protein = N x 6.25 (Cunniff, 1997a)

Experimental diets:

Crude protein = N x 6.38 (Cunniff, 1997b)

The alternate calculation for the experimental diets was used because the protein within these diets was derived from milk.

3.3.3 Animals General conditions and mating protocols are described in section 2.1.1. Time-mated females were randomly allocated to each diet group – numbers are shown in Table 3.2.

3.3.3.1 Food intake and bodyweight During gestation, dams were weighed daily to calculate required food intake (as described in Section 2.1.2), and excess food weighed daily to determine actual food intake. Total food intake for each mother was calculated, as well as mass-corrected food intake (section 3.3.3.2). During lactation, dams and offspring were weighed daily, as was excess food. Following weaning, offspring and excess food were weighed daily until the end of the trial. Since offspring were not housed in individual cages, average food intake was calculated by dividing total daily food intake for each litter by the number of offspring in that litter on that day. As full data were available for these parameters, a number of analyses were employed. These included plots of food intake and bodyweight as a function of time (including regression analyses) and total food intake and weight. Because the pattern of food intake could be as important as the quantity consumed through gestation, the gestational period was divided into thirds analogous to trimesters in human pregnancy. These periods have been referred to as the first,

76

middle, and last (or final) thirds of gestation. Separate comparison of these provided information on the pattern of food intake through gestation. For offspring separate values for pre- and post-weaning were generated to allow for the change in diet at weaning.

3.3.3.2 Mass-corrected food intake Previous studies suggest that LP offspring are of lower body weight compared to those fed a normal protein diet (Kanarek et al., 1986; Rogers, 1979). In order to investigate whether any observed differences in food intake were due to variation in bodyweight, the relationship between food intake and bodyweight was examined. This was done in two ways: a direct plot of bodyweight as a function of food intake, and mass-corrected food intake (Donnelly et al., 2003; Swallow et al., 2001) plotted against time. During gestation mass-corrected food intake represented total food consumed on a particular day divided by maternal bodyweight on that day. During lactation, this value was calculated as total maternal food intake divided by the combined body weights of the mother and all offspring. For offspring post-weaning, total daily food intake for each litter was divided by the sum of the body weights of all offspring in that litter. It is important to note that the relationship between food intake and bodyweight is not used to investigate “appetite control”, which involves the complex central regulation of hunger and satiety.

3.3.3.3 Efficiency calculations Two calculations of efficiency were employed, based on weight gain during a set period relative to total food intake or total energy intake during the same time period. The two calculations used were food conversion efficiency (FCE) and efficiency of energy utilization (EEU). FCE = total food intake (g) / total weight gain (g) (Lopez-Aliaga et al., 2003) EEU = total weight gain (g) / total energy intake (kJ) (Donnelly et al., 2003)

3.3.4 Physical characteristics In addition to bodyweight, other growth characteristics were also recorded. These were the day at which hair first began to appear on offspring within a litter, the day 77

on which the eyes of offspring within a litter first opened, and the day on which the eyes of all offspring within a litter were open.

3.3.5 Isolated perfused pancreas The basic perfusion protocol outlined in section 2.3 was used in this trial with the following modifications. Following a 20 minute period of equilibration, during which time a flow-rate was measured and the gland examined for leaks, fractions were collected for a 10 minute period to establish a baseline. A side-arm infusion (Figure 2.2) of glucose (21.7 mM) was then introduced for a period of 10 minutes, this being the first stimulatory phase. Following a further 15 minutes without stimulation, there was a 10 minute infusion of arginine (10.85 mM) (the second stimulatory phase). Another fifteen minutes of basal perfusion led to the third and final stimulatory phase, being a combination of 21.7 mM glucose and 10.85 mM arginine. A final basal period of 20 minutes duration completed the perfusion. This protocol is shown in Figure 3.2. Fractions were collected into pre-weighed 1.5 mL eppendorf tubes. Every fifth tube was weighed and the effluent volume determined by weight. Having established the flow into the pancreas prior to perfusion, this allowed each pancreas to be monitored accurately for leaks during the perfusion. Glucose and lactate were measured as described in section 2.3.3 to monitor infusions and pancreas viability respectively. Glucose measurements were performed to independently verify the actual [glucose]perfusate achieved during the perfusions, and lactate in the pancreatic effluent was measured to verify pancreatic viability (Gedulin et al., 1991)

3.3.5.1 Measurement of pancreatic hormone secretion Insulin and amylin were measured in each effluent fraction using radio immunoassay, as described in sections 2.4 and 2.5 respectively. This information, combined with the fraction volumes, was used to calculate the approximate total hormone production of each gland throughout the perfusion and, more importantly, during the various stimulatory phases, enabling comparison between experimental groups (section 3.4.4.7).

78

3.3.5.2 The amylin/insulin ratio The hormone ratio (amylin/insulin) was also calculated to provide a measure of the differential secretion of these hormones. This was derived independently for each pancreas by dividing amylin concentration in each effluent fraction by the corresponding insulin concentration in the same fraction.

3.3.5.3 Area under the curve To provide an overall measure to compare pancreas data between groups, the area under the curve was calculated during the various basal and stimulatory phases, both for hormone production, and the amylin/insulin ratio data. The trapezium rule was used for this calculation, and fraction volumes were incorporated in the case of the hormone data to provide a more accurate assessment of the total output from the gland. Because the response of insulin and amylin occurred at different times relative to the stimulations, a different set of fractions were pooled for the summary data in each case, as is shown in Figure 3.3.

3.3.5.4 Relative insulin response to stimulation Another comparison was that of the total insulin production in response to each of the three stimulatory infusions. In each case, the response to arginine was the smallest, so production of insulin in response to each of the three stimulations was calculated relative to this. For example, if the total pmol of insulin produced in response to glucose, arginine, and glucose + arginine were 45, 10, and 56 respectively, the relative infusion values would be 4.5, 1, and 5.6. This measure was useful in that it standardized the responses across each pancreas, and thus provided information on the pattern of secretion in response to the various stimulations applied to the gland.

3.3.5.5 First phase insulin secretion The normal insulin response to stimulation is biphasic, as shown in Figure 3.4 and described in some detail in section 3.5.3.1. A loss of first phase insulin secretion is a feature of the early stages of type-2 diabetes (Nesher and Cerasi, 2002). In order to determine if any of the offspring had impaired insulin secretion during the first phase, the area under the curve was calculated for each phase as shown in the 79

Figure 3.4. Thus, in addition to total insulin secretion and insulin secretion for the various phases described in section 3.3.5.3 and Figure 3.3, data was obtained on the first and second phase secretion during each of the stimulatory phases.

Figure 3.2: Protocol for isolated perfused pancreas experiments performed on the offspring of mothers fed diets varying in protein content and type during gestation and lactation

-20 equilibration 0

30

60

90

Effluent fractions collected

Perfusion time (minutes)

glucose

arginine

glucose

arginine

infusions

Graphical representation of the protocol described in section 3.2.5, showing the equilibration phase, fraction collection, and various infusions as a function of time-course of the perfusion process. Note that the third infusion of glucose + arginine was a single infusion solution, as described in section 2.3.2.2.

80

Figure 3.3: Pattern of calculation of area under the curve (AUC) data for fractions collected from the isolated perfused pancreases of offspring of female rats fed standard rat chow or chemically defined diets differing in protein content during gestation and lactation.

a

b

c

These figures are schematic representations of the various phases of insulin (a) and amylin (b) secretion, and the ratio of amylin/insulin (c), as determined using the isolated perfused rat pancreas. The vertical lines define the fractions in each case for which an area under the curve was calculated using the trapezium rule.

81

Figure 3.4 Phases of insulin secretion measured in the isolated perfused rat pancreas Changes in first phase insulin secretion were determined by measuring the area under the curve during each stimulatory phase, as shown in the figure (), and expressing this as a % of the total secretion during the stimulation. This measure was performed because a loss in first phase insulin

3.3.5.6 Weight-matched control groupsecretion is a feature of the early stages of type-2 diabetes (Nesher and Cerasi, 2002).

It was clear from an early age that the bodyweights of the offspring in the Diet 86 group were significantly higher than those of the two whey-protein groups. It was therefore possible that any differences observed between the Diet 86 offspring an those from the experimental diet groups would be due to this large difference in body weight at the same age. In order to allow for this, offspring from one of the Diet 86 litters were used as weight-matched controls, i.e. the experiment was performed on these animals when they were the same weight, rather than the same age, as animals from the C and LP groups. This gave two comparisons: first, between control and experimental animals matched for weight, and second, between Diet 86 animals at two different time points, thus providing information on the effects of both maternal diet and age of offspring on the response of the perfused pancreas system to nutrient stimuli.

3.3.6 Glucose uptake in the isolated soleus muscle This was performed as described in section 2.7.

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3.4 Results 3.4.1 Dietary analyses The total nitrogen, fat, carbohydrate and amino acid contents are shown in Table 3.1, along with calculated values of protein content (based on N) and energy (NME) as described in section 3.3.2.2. All dietary analyses were performed on an “a-fed” basis (rather than dry matter), thus energy intake could be calculated directly on the basis of mass of food intake. NRM Diet 86 contained 12% water and 3.5% dietary fibre.

3.4.2 Dams The total number and outcome of timed matings for each group are shown in Table 3.2.

3.4.2.1 Food intake Although restrictions were put in place, food intake during gestation differed between experimental groups. While the overall trend of intake during the second third showed some differences, these did not contribute to an overall difference in food intake through gestation. The period of gestation during which the most significant change in food intake occurred was during the last third, when each of the experimental diet groups exhibited decreased daily intake compared to Diet 86 dams (Figure 3.5). As a result Diet 86 dams had significantly greater total intake throughout gestation compared to all of the experimental diet groups (p