Energy Expenditure and Adaptive Responses to an Acute

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The Journal of Clinical Endocrinology & Metabolism 90(3):1446 –1452 Copyright © 2005 by The Endocrine Society doi: 10.1210/jc.2004-1494

Energy Expenditure and Adaptive Responses to an Acute Hypercaloric Fat Load in Humans with Lipodystrophy David B. Savage, Peter R. Murgatroyd, V. Krishna Chatterjee, and Stephen O’Rahilly Departments of Clinical Biochemistry and Medicine, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom Humans respond to an acute excess of ingested energy by storing the surplus energy as triglyceride in white adipose tissue. To study the energetic response to acute overfeeding in human subjects with limited adipose tissue capacity, we recruited seven subjects with lipodystrophy and seven lean healthy controls. Total fat mass was approximately 70% lower in lipodystrophic subjects (mean, 6.1 kg) than in body mass index-matched lean controls (mean, 22.0 kg). Energy expenditure and macronutrient oxidation rates were assessed in chamber calorimeters on two separate occasions for 40 h, during which time subjects consumed either an energy-balanced diet or a diet incorporating 30% excess energy as fat. On the energy-balanced diet, total daily energy expenditure and basal metabolic rate were linearly associated with lean mass in both groups (r2 ⴝ 0.83) and were not significantly different

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EALTHY HUMANS RESPOND to a state of positive energy balance primarily by storing excess energy as triglycerides in white adipose tissue. Although this ultimately predisposes overnourished humans to weight gain, in the short to medium term, it represents an efficient metabolic response to fluctuating energy balance. Lipodystrophy is a state of reduced adipocyte storage capacity due to a lack of functional adipocytes. This is the result of either a failure of development or premature destruction of adipocytes usually due to genetic or immunological mechanisms (1). It is distinct from leanness, because in that case adipose storage can readily be normalized or even increased above normal by the imposition of a positive energy balance. In lipodystrophic subjects with a significantly reduced adipose tissue storage capacity, a positive energy balance is thought to lead to ectopic fat deposition, insulin resistance, and ultimately diabetes (2). The situation is complicated by hypoleptinemia in many lipodystrophic subjects, a state that drives increased appetite and excess energy intake (3). Ectopic lipid accumulation has been well documented in liver and skeletal muscle in lipodystrophic humans, but is likely to affect other tissues as well, including pancreatic islets (2). However, the combined capacity of all of these sites to store excess energy is very limited compared with adipose tissue, First Published Online December 21, 2004 Abbreviations: BMI, Body mass index; BMR, basal metabolic rate; CHO, carbohydrate; DXA, dual energy x-ray absorptiometry; EB, energy-balanced diet; HF, fat-supplemented diet; LBM, lean body mass; TEE, total energy expenditure. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

between groups when corrected for lean mass. In response to the fat challenge, total energy expenditure did not increase significantly in healthy controls (9,472 ⴞ 1,069 to 9,724 ⴞ 1,114 kJ/d; P ⴝ 0.189). Substrate oxidation results confirm that excess fat was predominantly stored. In contrast, lipodystrophic subjects significantly increased total daily energy expenditure (11,081 ⴞ 1,226 to 11,730 ⴞ 1,374 kJ/d; P < 0.005). This was largely attributable to a 29% increase in fat oxidation. Thus, subjects with lipodystrophy uniquely respond to an acute hypercaloric load with a higher energy expenditure increment and by increasing fat oxidation. Insight into the molecular mechanisms responsible for this phenomenon may yield novel therapeutic approaches for obesity. (J Clin Endocrinol Metab 90: 1446 –1452, 2005)

suggesting that lipodystrophic subjects must have additional strategies for responding to positive energy balance. Energy balance in lipodystrophy has been studied over many years, but information remains limited. What has been reported in subjects with generalized lipodystrophy is a state of hypermetabolism associated with hyperhydrosis (4 – 6). However, none of these studies controlled for body composition, energy intake, or macronutrient balance. In this study we addressed two principal questions. Firstly, is human lipodystrophy really a state in which energy expenditure is increased? Secondly, given the paucity of adipose tissue in lipodystrophic subjects, how do they respond to ingestion of excess fat? We measured energy expenditure and substrate oxidation in the basal state, in response to exercise, and throughout a test day in a chamber calorimeter in seven lipodystrophic subjects and seven lean healthy controls. We also examined the response of these parameters to an acute hypercaloric load, comprising 30% excess energy delivered as fat. Measures of blood lipids and urinary glucose excretion completed our macronutrient account. We found that the apparent increase in basal metabolic rate (BMR) reported in lipodystrophic subjects is, in fact, the result of a relative increase in lean body mass (LBM). We also found that in contrast to normal human subjects, lipodystrophic subjects acutely up-regulate energy expenditure and fat oxidation in response to an acute fat load. Subjects and Methods The study was approved by the Cambridge local research ethics committee, and all subjects provided written, informed consent. The study was conducted in the Metabolic Research Area of the Wellcome Trust Clinical Research Facility (WTCRF; Cambridge, UK).

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Savage et al. • Energy Expenditure in Humans with Lipodystrophy

Subjects Genetically characterized lipodystrophic subjects (referred to as LD1–7) were identified as part of an ongoing program of research into monogenic forms of severe insulin resistance (7). They were selected on the basis of the presence of clinically discernible lipodystrophy as well as the presence of mutations known to be associated with either generalized or partial lipodystrophy. One subject had an acquired form of generalized lipodystrophy. We recruited subjects with a number of different genetic reasons for lipodystrophy because our primary aim was to examine energy regulation in humans with limited adipose tissue capacity. Control volunteers were recruited by advertisement and were age-, gender-, and body mass index (BMI)-matched with the lipodystrophic subjects. All controls were nonsmokers, without any medical conditions likely to influence energy balance, and without a family history of diabetes. All subjects were euthyroid at the time of the study. Descriptive details of both groups are provided in Tables 1 and 2. Each subject was studied on two occasions with randomly presented energybalanced (EB) or fat-supplemented (HF) treatments. Visits for premenopausal female subjects coincided with the follicular phase of the menstrual cycle.

Body composition measurement Body composition was measured by both Lunar Prodigy dual energy x-ray absorptiometry (DXA; GE Lunar Corp., Madison, WI) and by BOD POD (Life Measurement Instruments, Concord, CA). The two measurements reinforced confidence in the very low fat contents observed in some lipodystrophic patients. Mean data from the two measurements are presented.

J Clin Endocrinol Metab, March 2005, 90(3):1446 –1452

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visit were as the same as those for the control diet with additional fat to raise fat intake to 1.30 ⫻ EB energy. The resulting composition was 52% fat, 39.5% CHO, and 8.5% protein by energy. Diets were prepared from ingredients of well established composition (13). Breakfast consisted of porridge, toast, and marmalade; lunch was cheese salad, followed by fruit salad with cream; and dinner was cottage pie, followed by apple pie with cream. Manipulation was predominantly through adjustment of the amount of cream, the type of cheese, and the proportions of whole to skimmed milk and butter to low fat spread. Subjects were required to eat all of the food provided.

Experimental protocol Each visit spanned 3 d, D0 –D2. Subjects arrived at the WTCRF for lunch on D0. Meals followed the treatment formulation throughout. On the first visit, after providing informed written consent and undergoing a medical examination, body composition was measured. Subjects received an evening meal at 1900 h on D0 before entering the whole body calorimeter at 2000 h, where they remained until 1330 h on D2. Each night subjects prepared for bed at 2230 h. Lights out was at 2300 h. Subjects were woken at 0800 h, but remained in bed during the BMR measurement until 0900 h. Subjects washed and dressed between 0900 and 0930 h. Breakfast was served on D1 at 0930 h, lunch at 1330 h, and supper at 1900 h. At 1100, 1500, and 2030 h, subjects exercised on a cycle ergometer at an external work rate of 50 watts for 30 min. During the remainder of each day, subjects were occupied in sedentary activities. The morning of D2 proceeded like that for D1 until the subjects left the calorimeter. Subjects were supervised by nursing staff around the clock.

Biochemical measurements Calorimetry Macronutrient oxidations were measured by whole body indirect calorimetry. The calorimeter rooms have 10.4-m2 floor area and 24-m3 volume. They are constructed as comfortable bed-sitting rooms within the WTCRF. They have outside windows and washbasins, comfortable beds and chairs, TV and video, hifi, and internet computers. Instrumentation and calibration procedures have been previously described (8). O2 consumption and CO2 production were calculated by the method described by Brown et al. (9). Macronutrient oxidation rates were calculated to better than ⫾20 g/d for carbohydrate (CHO) and ⫾9.5 g/d for fat, using the expressions of Murgatroyd et al. (10), with macronutrient respiratory quotients and energy equivalents of oxygen from Elia and Livesey (11).

Fasting blood samples were drawn at 0900 h on D2 of each visit. Biochemical assays, including leptin and insulin measurements, were performed as described previously (14). Plasma adiponectin measurements were undertaken using a RIA kit (HADP-61HK, Linco Research, Inc., St. Charles, MO).

Statistical analysis Analyses were undertaken using Data Desk V 6.02 (Data Description, Inc., Ithaca, NY). Descriptive statistics are presented as the mean ⫾ se in figures and tables. Treatment effects were analyzed by ANOVA with treatment (EB vs. HF) and group (control vs. LD) as factors. Subjects were nested in groups. Treatment-group interactions were tested for significance with Scheffe´ post hoc tests. Significance was accepted for P ⬍ 0.05.

Diets The energy requirement for each subject was predicted from estimates of BMR (12) scaled by a factor of 1.48, derived from energy expenditure levels in previous studies. Energy requirements for the lipodystrophics were scaled by an additional factor of 1.15 to account for their proportionally higher LBM. Diets for the control visit were formulated to maintain EB. The composition of each meal was 35% fat, 53% CHO, and 12% protein by energy. Diets for the fat-supplemented (HF) TABLE 1. Subject characteristics

Age (yr) Gender (males; females) Weight (kg) BMI (kg/m2) LBM (kg) Total body fat (%) Arm fat (%) Leg fat (%) Trunk fat (%)

Lipodystrophic subjects (n ⫽ 7)

Healthy controls (n ⫽ 7)

42 (17– 63) 2; 5 68.7 (57.1– 87.3) 24.2 (21.8 –27.7) 61.5 (50.5–79.3)a 11.5 (2.6 –24.9)a 6.3 (4.0 –25.9)a 7.2 (4.0 –16.9)a 10.3 (4.4 –33.2)a

40 (22– 49) 2; 5 72.3 (58.0 – 82.3) 23.9 (21.4 –29.5) 48.9 (40.4 –59.8) 30.1 (17.5– 43.7) 33.2 (14.2–51.9) 35.0 (24.3– 42.4) 33.2 (23.2– 43.9)

Values are medians (range). LBM and total body fat results represent the average of DXA and BOD POD determinations. Regional fat distribution was determined by DXA alone. a P ⬍ 0.01, lipodystrophic subjects vs. healthy controls.

Results Subject characteristics (Tables 1 and 2)

Lipodystrophic subjects had significantly less total body fat than age-, gender-, weight-, and BMI-matched controls, with corresponding increases in LBM (Table 1). Leg fat was strikingly reduced in all lipodystrophic subjects, whereas arm and trunk fat were variably preserved. Subject LD1 was heterozygous for a novel DNA-binding domain mutation in PPARG (Chatterjee, V. K., unpublished observations). Her phenotype was very similar to that of subjects harboring dominant negative mutations in the ligand-binding domain of PPARG (14), and in vitro characterization of this mutation suggested that it too behaves in a dominant negative manner (Chatterjee, V. K., unpublished observations). Subject LD2 had frameshift premature stop mutations in two unlinked genes (15). Although heterozygous dominant negative mutations in PPARG are associated with partial lipodystrophy (as manifested in LD1), the PPARG frameshift (FS) mutant does not appear to induce a lipodystrophic state in most cases (15). LD2 was the only member of her family with clinically apparent lipodystrophy. This clinical impression was sup-

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TABLE 2. Genotype, phenotype, and biochemical characterization of lipodystrophic subjects Identifier age (yr)/sex

LD1 42F LD2a 49F LD3 30F LD4 63F LD5a 17M LD6 57F LD7a 50M

Genotype

PPARG C131Y PPARG FSb/PPP1R3A FSc LMNA R482W LMNA R482L AGPAT2 LMNA R482W Acquired

HbA1c (%)

TG HDL (mmol/liter) (mmol/liter)

Weight (kg)

Total body fat (%)

Leptin (ng/liter)

Adiponectin (␮g/ml)

FPLD

65.8

18.1

11.5

9.6

5.9

4.5

0.9

Partial LD

74.0

24.9

5.3

6.3

10.5

3.3

0.7

FPLD

71.2

11.2

1.1

3.5

5.9

1.7

1.0

Nil

FPLD

63.1

2.6

0.1

1.4

4.4

0.9

1.0

Nil

CGL

68.7

3.0

0.1

3.5

10.9

3.4

0.8

Insulin

FPLD

57.1

11.5

0.5

6.0

5.6

3.6

1.0

Nil

AGL

87.0

9.2

1.5

1.3

9.2

2.9

0.9

Insulin

5.5–37.8

9.3–26.3

5.0 –5.7

1.0 –2.3

0.6 –1.8

Phenotype

Control range

58.0 – 82.3 17.5– 43.7

Therapy

ACE inhibitor; T4 Insulin; fibrate; ACE inhibitor

LD, Lipodystrophy; FPLD, familial partial lipodystrophy; CGL, congenital generalized lipodystrophy; AGL, acquired generalized lipodystrophy; HbA1c, glycated hemoglobin; TG, triglyceride; HDL, high-density lipoprotein cholesterol; ACE inhibitor, angiotensin-converting enzyme inhibitor; F, female; M, male. To convert triglyceride concentration into mg/dl, multiply by 88.574; to convert HDL concentration into mg/dl, multiply by 38.670. a Diabetic. b PPARG 553delAAAinsT (15). c PPP1R3A 1984delAG (15).

ported by both magnetic resonance imaging (data not shown) and DXA measurement of her leg fat (16.9%), which was well below the range seen in healthy control females (35– 42.4%). Interestingly one of the women with an LMNA mutation (LD4) typically associated with Dunnigan-Kobberling-type partial lipodystrophy had a total body fat measurement as low as 2.6%. This was consistent with our clinical impression of generalized lipodystrophy. She was 63 yr old and reported progressive loss of almost all visible body fat with age. Although this observation has not been formally reported previously in Dunnigan-Kobberling syndrome, it is not particularly surprising given the presence of lamins A and C in all mature adipocytes (16). Total body fat was also very low in both of the other women with LMNA mutations (LD3 and LD6), although in their cases, facial and neck fat was still clinically apparent. As a group, the lipodystrophic subjects had significantly higher blood glucose and insulin concentrations and significantly lower leptin and adiponectin concentrations than the control subjects (Table 3).

Relationships among total daily energy expenditure (TEE), weight, and LBM

TEE was significantly increased in lipodystrophic subjects compared with weight- and BMI-matched healthy controls (P ⬍ 0.05; Fig. 1A). However, because fat contributes very little to human energy expenditure, the principal determinant of TEE is LBM, which was significantly higher in lipodystrophic subjects than in the controls (Table 1). When TEE was expressed per kilogram of LBM (Fig. 1B), we found no significant difference between lipodystrophic and control subjects (P ⫽ 0.11). Response to fat overfeeding

Energy intake was not significantly different from energy expenditure in either group during the EB diet. The fat supplement induced a significant increase in blood glucose concentration in the lipodystrophic group, but not in the controls. There were no significant changes in any other

TABLE 3. Biochemical responses to dietary manipulation Lipodystrophy

Glucose Insulin Triglycerides FFAs Leptin Adiponectin

Control

Reference range

EB

SE

HF

SE

EB

SE

HF

SE

7.6a,b 174a 2.9 338 4.1c 4.5c

0.6 21 0.2 27 0.6 0.4

9.0a 185a 6.8 325 4.2c 3.8d

0.6 17 1.5 19 0.7 0.3

4.6 36 1.4 406 12.9 15.1

0.04 1 0.1 48 0.9 0.9

4.9 45 1.0 322 14.9 11.6

0.1 2 0.1 19 1.4 0.7

3.5– 6.3 mmol/liter ⬍80 pmol/liter ⬍2 mmol/liter, desirable 290 –720 ␮mol/liter ␮g/liter ␮g/ml

Values are the mean ⫾ SE. FFAs, Free fatty acids. To convert glucose concentration into mg/dl, multiply by 18.01477; to convert insulin concentration into mU/liter, multiply by 0.139373; to convert triglyceride concentration into mg/dl, multiply by 88.574. a P ⬍ 0.0001, lipodystrophy vs. control. b P ⬍ 0.0001, EB vs. HF. c P ⬍ 0.002, lipodystrophy vs. control. d P ⬍ 0.005, lipodystrophy vs. control.



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nutrient oxidation rates were also unchanged in the control group, whereas fat oxidation was substantially increased in the lipodystrophic subjects (Fig. 2 and Table 4). This dramatic increase in energy expenditure as fat oxidation was partly offset by a trend toward a reduction in carbohydrate and protein oxidation. BMR and overnight energy expenditure were both significantly increased in lipodystrophic subjects in response to overfeeding, whereas exercise-associated energy expenditure was unchanged (Table 4). Fat oxidation, however, was significantly elevated by HF diet in the lipodystrophic subjects during all the analysis periods. Discussion

FIG. 1. A, TEE vs. weight plot for each individual [lipodystrophic (LD; F and f) and control (C; E and 䡺)] on the EB (f and 䡺) or HF (F and E) diet. B, TEE vs. LBM for all subjects on the EB diet only (r2 ⫽ 0.83; lipodystrophic, F and f; control, E and 䡺). To convert joules to calories, multiply by 0.2388.

biochemical or hormonal parameter measured in either group in response to the fat supplement (Table 3). TEE increased significantly in the lipodystrophic group during the HF diet (Fig. 2 and Table 4). The excess energy expended by LD subjects receiving HF treatment was 22% of the fat supplement. For controls, the corresponding increase was 8.7% of the energy supplement and was not significant. Macro-

FIG. 2. Total (24-h) energy expenditure and macronutrient oxidation rates in lipodystrophic and control subjects on the EB or HF diet (mean ⫾ SE). ns, Not significant. To convert joules to calories, multiply by 0.2388.

These studies were designed to address two questions. Firstly, is human lipodystrophy characterized by abnormally elevated energy expenditure? Secondly, how, in terms of changes in energy expenditure and substrate oxidation, do subjects with lipodystrophy respond to a hypercaloric fat load. By studying lipodystrophic subjects and BMI-matched controls under highly controlled circumstances in a chamber calorimeter, we have been able to obtain more definitive information than has previously been available. Generalized lipodystrophy has been reported by several investigators to be a hypermetabolic state (4 – 6). However, none of these early studies controlled for body composition, energy intake, and macronutrient balance, all of which can significantly change energy expenditure. We studied seven human immunodeficiency virus-negative, euthyroid subjects with lipodystrophy with a view to definitively establishing whether energy expenditure is increased in lipodystrophic humans. DXA and Bodpod measurements of body composition revealed significantly higher LBM in lipodystrophic subjects compared with age-, gender-, weight-, and BMI-matched controls. The relationships among TEE, BMR, and LBM were linear in both healthy controls and lipodystrophic subjects in energy balance, suggesting that the reported elevation in energy expenditure in lipodystrophic subjects is largely attributable to their relatively higher LBM. Thus, it appears that the apparent increase in energy expenditure in lipodystrophic subjects is primarily a consequence of their altered body composition rather than a true increase in the metabolic activity of any particular cell type. One caveat is that we studied subjects with a range of severity of lipodystrophy and a range of etiological mechanisms. It will be of interest to examine larger numbers of each specific subgroup, including those with the different forms of total lipodystrophy, to determine whether any of these subsets of patients deviate from the observed relationship between LBM and energy expenditure. We suspect that the increased energy expenditure previously reported in patients with total lipodystrophy may have been related to the hyperphagia induced in such patients by their severe hypoleptinemia. This idea is supported by the reduction in energy intake and energy expenditure seen in patients with total lipodystrophy in response to leptin replacement (17). The normal response to an acute hypercaloric mixed macronutrient load in humans, given their limited CHO stores, is to adjust CHO oxidation to maintain CHO balance. Thus, the entire energy excess transfers to fat, which can be stored

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TABLE 4. Energy expenditure and macronutrient oxidation data Period

Group

Energy expenditure (kJ)

Fat oxidation (kJ)

CHO oxidation (kJ)

EB

SE

HF

SE

EB

SE

HF

SE

EB

SE

HF

SE

24 h

LD C

11,081a 9,472

463 404

11,730a 9,724

519 421

4,132b 3,490

433 182

5,338b 3,764

368 269

5,400 4,702

264 197

5,047 4,618

240 113

Overnight

LD C

2,120b 1,753

135 70

2,243b 1,764

142 62

785c 796

60 71

998c 817

82 65

950 638

98 53

860 617

77 50

BMR

LD C

349d 301

17 17

364d 287

23 17

143d 150

20 21

176d 133

25 14

149 108

16 25

134 108

15 16

Exercise

LD C

984 851

55 34

47 57

270e 190

48 38

335e 213

45 41

647 602

46 40

624 585

36 55

1,016 856

Values are mean ⫾ SE. Overnight values are the average of two nighttime measurement periods, from 0000 – 0700 h. BMR values are the average of the two 1-h measurement periods between 0800 and 0900 h. Exercise values are the average of four 1-h exercise periods, each with 0.5 h of exercise at 50 watts, followed by periods of standing (10 min) and sedentary recovery. LD, Lipodystrophy; C, control. To convert joules to calories, multiply by 0.2388. a P ⬍ 0.005. b P ⬍ 0.0005. c P ⬍ 0.0001. d P ⬍ 0.05. e P ⬍ 0.01.

as adipose tissue triglyceride (18, 19). When the excess energy is presented purely as fat, more than 90% is stored in adipocytes. Because lipodystrophic subjects have limited fat storage capability, this option is denied them. Alternative strategies for managing an energy excess must be invoked. Hypothetically, these might include thermogenic dissipation of the excess, storage as CHO, excretion as glucose, or ectopic lipid storage. There is a documented propensity for ectopic lipid deposition, lipotoxicity, and diabetes observed in lipodystrophic humans and mice (2, 20, 21), but the other options have not been comprehensively researched. Against this background, we have investigated how lipodystrophic subjects respond to acute overfeeding with fat. In contrast to controls, we found that lipodystrophic subjects significantly increase BMR and total energy expenditure. This increase is largely attributable to an increase in fat oxidation. How lipodystrophic humans increase fat oxidation and energy expenditure in response to overfeeding with fat will require additional study, but the fact that the increase in energy expenditure was apparent during both BMR measurement and sleep suggests that nonexercise-mediated thermogenesis is involved. It is important to appreciate that this response should not be interpreted as an increase in what is frequently referred to as diet-induced or postprandial thermogenesis, nor should it be considered equivalent to long-term adaptation to changes in the macronutrient composition of the diet. Postprandial thermogenesis refers to the thermogenic effect of ingesting a meal and is thought to be a result of heat generated by the digestive process. It was not specifically measured in this study. Because neither plasma leptin nor adiponectin concentrations increased in response to the fat supplement, they are unlikely to have contributed to the observed increase in fat oxidation. Although both hormones are thought to promote fat oxidation in rodents, this response has yet to be demonstrated in humans. In fact, leptin administration consistently reduces energy intake and energy expenditure in leptin-deficient humans (17, 22, 23). Although every lipodystrophic subject in this study in-

creased both TEE and fat oxidation in response to the fat challenge, the proportion of the energy surplus dissipated averaged only 22%, and the magnitude of this response was quite variable (Fig. 3). This diversity probably reflects the heterogeneity within the cohort in terms of the specific etiology responsible for lipodystrophy in each subject. Other than human immunodeficiency virus-associated lipodystrophy, all other forms of lipodystrophy are very rare, necessitating recruitment of a genetically heterogenous cohort of lipodystrophic subjects. In subject LD1, who has a dominant negative mutation in PPARG and partial lipodystrophy, most of the excess fat appeared to remain in plasma for a prolonged period of time. D2 fasting triglycerides increased from 398.6 mg/dl (4.5 mmol/liter) to 2728.1 mg/dl (30.8 mmol/liter) between the two studies (equivalent to 2.2 MJ or 68% of the energy excess). Fat oxidation and TEE were slightly up-regulated, with a corresponding decrease in carbohydrate oxidation. Intriguingly, the patient’s fasting glucose also rose from 95.5 mg/dl (5.3 mmol/liter) to 133.3 mg/dl (7.4 mmol/liter) in response to fat overfeeding. This tendency to substantially increase plasma triglycerides and glucose in response to the fat challenge was not apparent in the rest of the group and is of particular interest for two reasons. Firstly, it is consistent with the idea that in addition to influencing adipose tissue mass, PPAR␥ is involved in modulating the metabolic response to fat ingestion (14, 20), and secondly, it provides some in vivo evidence, albeit anecdotal, in support of the proposal that impaired lipid handling might precede hyperglycaemia in some diabetics. The only other subject to substantially increase her serum glucose in response to the fat challenge was subject LD4, whose serum glucose rose from 100.9 mg/dl (5.6 mmol/liter) to 122.5 mg/dl (6.8 mmol/liter). Remarkably, she disposed of approximately half of the excess energy she ingested by increasing fat oxidation without an increase in serum triglycerides [79.7 mg/dl (0.9 mmol/liter) to 88.6 mg/dl (1.0 mmol/liter)]. Subject LD2, a diabetic, also responded to ingestion of excess fat by substantially increasing fat oxidation,



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FIG. 3. Total (24-h) energy intake (I), macronutrient disposal (D) rates in subjects LD1, LD2, and LD4. Disposal is by oxidation for protein, CHO, and fat; glucose excretion for urine; and energy as stored circulating triglyceride (TG). To convert joules to calories, multiply by 0.2388.

but, in addition, she disposed of a significant proportion (1.2 MJ, 40%) of the energy excess in the form of glucosuria. This was typical of all of the diabetic subjects. Although diabetes might be expected to confound energy balance studies, indirect calorimetry measurements are based upon changes in chamber oxygen and carbon dioxide concentrations as well as urinary nitrogen disposal. They do not, therefore, take into account energy disposal in the form of glucosuria. Overfeeding diabetics tends to increase blood glucose, leading to an increase in glucosuria, which can account for a significant amount of energy disposal. If anything, this would tend to diminish concurrent changes in oxidative energy expenditure and reduce the significant increases we observed in TEE and fat oxidation. Urinary glucose losses were insignificant in all nondiabetic subjects. Thus, we found that the lipodystrophic subjects universally dissipated some of the fat challenge through increased energy expenditure, but invoked a range of strategies, including increased glucose excretion and retention of fat as circulating triglyceride, to manage the remainder. In conclusion, the data presented here suggest that the apparent increase in energy expenditure seen in lipodystrophic subjects in energy balance reflects their relatively high LBM to fat mass. However, they demonstrate a remarkable capacity to rapidly increase energy expenditure and fat oxidation in response to overfeeding. Other than the administration of thyroid hormone (24) or adrenergic agents (25), there are few acute manipulations (apart from physical activity, including shivering) that can induce comparable increases in human energy expenditure. Understanding this response at the molecular level could provide novel ideas for interventions to limit weight gain. Acknowledgments We are very grateful to the subjects who kindly participated in this study, to Elaine Marriott and colleagues of the Wellcome Trust Clinical Research Facility for providing 24-h care throughout all calorimetry visits and for assistance with sample collection, and to Ian Halsall and colleagues of Medical Research Council Human Nutrition Research for urinary nitrogen analysis.

Received July 28, 2004. Accepted December 9, 2004. Address all correspondence and requests for reprints to: Dr. Stephen O’Rahilly, Department of Clinical Biochemistry and Medicine, Level 4, Box 232, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ, United Kingdom. E-mail: [email protected]. This work was supported by the Wellcome Trust (to D.B.S., V.K.C., and S.O.) and the Medical Research Council, United Kingdom (to P.R.M.). D.B.S. and P.R.M. contributed equally to this work.

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Erratum In the article “Cortistatin Rather Than Somatostatin as a Potential Endogenous Ligand for Somatostatin Receptors in the Human Immune System” by Virgil A. Dalm, P. Martin van Hagen, Peter M. van Koetsveld, Anton W. Langerak, Aart-Jan van der Lely, Steven W. Lamberts, and Leo J. Hofland (The Journal of Clinical Endocrinology & Metabolism 88:270 –276, 2003), the primer sequences for cortistatin A (173 bp) and cortistatin B (701 bp) were mistakenly reversed in Table 1. The correct sequences appear in the table below. Primer

Cortistatin Cortistatin Cortistatin Cortistatin

A A B B

(forward) (reverse) (forward) (reverse)

Sequence (5⬘–3⬘)

-CTCCAGTCAGCCCACAAGAT-CAAGCGAGGAAAGTCAGGAG-GCAAATTCGCTCTAAACACAGGA-TTGGGAAGGAGGAGAGGAAAGAT-

Expected size (bp)

173 701

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