Long-lived crowded-litter mice exhibit lasting effects on insulin ...

Am J Physiol Endocrinol Metab 306: E1305–E1314, 2014. First published April 15, 2014; doi:10.1152/ajpendo.00031.2014.

Long-lived crowded-litter mice exhibit lasting effects on insulin sensitivity and energy homeostasis Marianna Sadagurski,1 Taylor Landeryou,2 Manuel Blandino-Rosano,3 Gillian Cady,2 Lynda Elghazi,3 Daniel Meister,3 Lauren See,3 Andrzej Bartke,4 Ernesto Bernal-Mizrachi,3,5 and Richard A. Miller2 1

Department of Internal Medicine, Division of Geriatric and Palliative Medicine, University of Michigan, Ann Arbor, Michigan; 2Department of Pathology and Geriatrics Center, University of Michigan, Ann Arbor, Michigan; 3Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, Michigan; 4 Department of Internal Medicine-Geriatrics Research, Southern Illinois University School of Medicine, Springfield, Illinois; and 5Endocrinology Section, Medical Service, Veterans Affairs Medical Center, Ann Arbor, Michigan Submitted 23 January 2014; accepted in final form 14 April 2014

Sadagurski M, Landeryou T, Blandino-Rosano M, Cady G, Elghazi L, Meister D, See L, Bartke A, Bernal-Mizrachi E, Miller RA. Long-lived crowded-litter mice exhibit lasting effects on insulin sensitivity and energy homeostasis. Am J Physiol Endocrinol Metab 306: E1305–E1314, 2014. First published April 15, 2014; doi:10.1152/ajpendo.00031.2014.—The action of nutrients on early postnatal growth can influence mammalian aging and longevity. Recent work has demonstrated that limiting nutrient availability in the first 3 wk of life [by increasing the number of pups in the crowdedlitter (CL) model] leads to extension of mean and maximal lifespan in genetically normal mice. In this study, we aimed to characterize the impact of early-life nutrient intervention on glucose metabolism and energy homeostasis in CL mice. In our study, we used mice from litters supplemented to 12 or 15 pups and compared those to control litters limited to eight pups. At weaning and then throughout adult life, CL mice are significantly leaner and consume more oxygen relative to control mice. At 6 mo of age, CL mice had low fasting leptin concentrations, and low-dose leptin injections reduced body weight and food intake more in CL female mice than in controls. At 22 mo, CL female mice also have smaller adipocytes compared with controls. Glucose and insulin tolerance tests show an increase in insulin sensitivity in 6 mo old CL male mice, and females become more insulin sensitive later in life. Furthermore, ␤-cell mass was significantly reduced in the CL male mice and was associated with reduction in ␤-cell proliferation rate in these mice. Together, these data show that early-life nutrient intervention has a significant lifelong effect on metabolic characteristics that may contribute to the increased lifespan of CL mice. longevity; caloric restriction; insulin; leptin; crowded litter CALORIE RESTRICTION (CR) during adult life slows aging, prevents or delays age-related disease, and increases lifespan in mice and rats (23, 40, 44) and perhaps in primates (15). On the other hand, most studies of undernutrition imposed during gestation and/or lactation document detrimental effects in the offspring (63, 68). In mice, protein restriction during lactation was reported to extend the lifespan of the offspring (52), although CR during the same period was without effect (13, 64, 74). Recent work from our laboratory showed that transient reduction in food availability limited to the period of suckling [producing “crowded-litter” (CL) mice] was sufficient to in-

Address for reprint requests and other correspondence: M. Sadagurski, Dept. of Internal Medicine, Div. of Geriatric and Palliative Medicine, Univ. of Michigan, Rm. 3003 BSRB, 109 Zina Pitcher Pl., Ann Arbor, MI 48109-2200 (e-mail: [email protected]). http://www.ajpendo.org

crease mean and maximal longevity of genetically normal mice (67). The mechanism responsible for extended longevity in the CL system is unknown. Several long-lived mouse mutants exhibit a combination of reduced insulin levels and enhanced insulin sensitivity, and these physiological changes are postulated to contribute to the extended longevity in these mice (5). For example, long-lived mice with growth hormone (GH) deficiency, mice with targeted disruption of the GH receptor gene [GH receptor knockout (GHRKO)], and Ames (Prop1df) and Snell (Pit1dw) dwarfs all show improved glucose tolerance and insulin sensitivity (1, 3, 7, 9, 18, 25). Similarly, persistent decreases in blood glucose, insulin, and IGF-I, along with increased insulin sensitivity, may contribute to the beneficial effects of CR in mice (5). Monkeys show similar effects of CR on glucose and insulin as well as indications of reduced mortality (14, 42). Human studies show glucose tolerance declining as a function of age, typically beginning in the third decade of life and continuing throughout the entire adult lifespan (17). In contrast, extreme human longevity seen in centenarians is associated with a low degree of insulin resistance, suggesting that long-lived people are protected, perhaps genetically, from an age-related decline of insulin action (10, 58, 66). In addition, people with GHR deficiency (Laron syndrome) are especially sensitive to insulin and protected from diabetes and cancer despite the prevalence of obesity in this group (20, 33, 34). Because our previous studies (67) have shown extended lifespan in mice subjected to transient milk restriction in the 3 wk between birth and weaning, we have now evaluated indices of energy balance, glucose homeostasis, and leptin sensitivity in CL mice to test the idea that this early-life intervention leads to long-lasting changes in metabolic status. METHODS

Animals. Mice involved in this study were approved by the Institutional Animal Care and Use Committee of the University of Michigan. Mice from the UM-HET3 stock were produced as described previously (47). Litters were culled to eight, after which either 0, 4, or 7 additional pups were added from another litter, thus increasing the total litter size. At 20 days of age, pups were weaned onto a normal chow diet (Purina 5001, 23% protein). Metabolic analysis. Intraperitoneal glucose tolerance tests were performed on mice fasted for 16 h overnight. Blood glucose levels were measured on random-fed or overnight-fasted animals in mouse tail blood using Glucometer Elite (Bayer), and serum samples were collected for the insulin measurements. Animals E1305

CL MICE EXHIBIT LONG-LASTING CHANGES IN METABOLIC STATUS

RESULTS

The effect of CL on growth and nutrient homeostasis. To investigate the effect of litter size on metabolic status, we adjusted litters in normal, genetically heterogeneous (UMHET3) mice (47) within 24 h of birth to produce litters of eight pups (CL8; the control group) or of 12 or 15 pups (CL12 and CL15; the 2 experimental groups). Mice were weaned at 20 days of age and fed ad libitum for the remainder of their lives. As shown previously for CL12 mice (67), both males and females raised in crowded litters in both the CL12 and CL15 groups were significantly lighter in weight than those from control CL8 litters throughout their lifespan (Fig. 1A). DEXA showed reduced percent of adipose mass in 8-wk-old CL12 and

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were then injected intraperitoneally (ip) with D-glucose (2 g/kg), and blood glucose levels were measured (60). For insulin tolerance tests, mice were fasted for a 4-h period in the light cycle before ip injections of insulin (0.8 U/kg; Humulin R) diluted in sterile saline. Blood glucose concentrations were measured at the indicated time points. Blood insulin and leptin levels were determined on serum from tail vein bleeds using a Mouse Insulin ELISA kit and Mouse Leptin ELISA kit (Crystal Chem). For food intake measurements, mice were singly housed, and food intake was measured for 6 consecutive days. For peripheral leptin treatment, mice were treated with either recombinant mouse leptin (10 mg/kg; provided by Dr. A. Parlow, National Hormone and Pituitary Program, Torrance, CA) or vehicle and injected ip twice/day for 12 consecutive days. Food and body weights were recorded before injection and during the treatment period. Lean and fat body mass were assessed by dual-energy X-ray absorptiometry (DEXA; GE Lunar, Madison, WI). Energy expenditure. As described previously (59), energy expenditure was measured over 72-h period with a Comprehensive Laboratory Animal Monitoring System (CLAMS; Oxymax Windows 3.0.3, Columbus Instruments). Mice were housed individually at room temperature (22°C) under an alternating 12:12-h light-dark cycle. This portion of the study was conducted by personnel at the University of Michigan Animal Phenotyping Core. Histology and morphometric analysis. Histological analysis was performed on white adipose tissues isolated from the animals as described previously (60). Morphometric analysis of white adipose tissue from 400 cells from six to seven different animals per genotype was performed with National Institutes of Health Image J software (http://rsb.info.nih.gov/ij/). The ␤-cell mass was calculated by pointcounting morphometry from five insulin-stained sections (5 ␮m) separated by 200 ␮m using the BQ Classic98 MR software package (Bioquant) as described (19). Proliferation was assessed in sections stained with insulin and Ki-67. The proliferation of ␤-cells was expressed as a proliferation index that was determined as the number of Ki-67-positive ␤-cells over the total number of ␤-cells. At least 3,000 insulin-stained cells were counted for each animal in a blinded fashion. RNA extraction and quantitative PCR. Total RNA was extracted from brown adipose tissue with Trizol (Gibco-BRL), and 1-mg samples were converted to complementary DNA (cDNA) with the iScript cDNA kit (Bio-Rad Laboratories). Sample cDNAs were analyzed in triplicate via quantitative RT-PCR for Ucp1 and Ppargc1a in brown adipose tissue with customized primers, as described previously (59). Statistical analysis. Unless otherwise stated, means ⫾ SE are presented in the figures, and significance was determined by a Student t-test. A P value of ⬍0.05 was considered statistically significant. Generalized linear regression (SPSS version 19) was used to identify significant differences in mouse body weight and energy expenditure parameters.

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Fig. 1. A: average body weights of male and female mice; groups of 12 (CL12) 15 mice (CL15) and controls (CL8) on regular chow diet were determined in each age group by ANOVA with Sidak post hoc test (P ⬍ 0.001, CL8 vs. CL12 or CL15). Mice were measured at several ages: 23 days, 3 wk, 6 wk, 9 wk, 12 wk, and 18 mo; n ⫽ 32– 40 for each sex at each age. B–E: %body fat (B and D) and %lean body mass (C and E) were determined by dual-energy X-ray absorptiometry in 8-wk-old male (n ⫽ 6) and female (n ⫽ 6) mice (means ⫾ SE; *P ⬍ 0.05 by Student’s t-test). CL, crowded litter.

CL15 males and females (Fig. 1, B and D) and an increase in percentage of lean body mass (Fig. 1, C and E). Hematoxylin and eosin staining revealed that in 22-moold females, perigonadal adipocytes were 20% smaller in CL12 mice (P ⬍ 0.05) and almost 40% smaller in CL15 mice (P ⬍ 0.001) compared with CL8 mice (Fig. 2, A and B), suggesting that changes in adipocyte size might contribute to the lower mass and metabolic phenotypes of CL mice. When tested at 6 mo of age, CL12 or CL15 males consumed the same amount of food as control CL8 males but had dramatically lower levels of circulating leptin (Fig. 3, A and B). Circulating leptin concentrations were also reduced in 6-mo-old CL12 and CL15 females (Fig. 3C). Food intake was slightly reduced in CL12 females and significantly reduced in CL15 females, although whether this is a cause or a consequence of lower body mass is impossible to decide on the basis of current evidence. Leptin

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00031.2014 • www.ajpendo.org

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Fig. 2. A: representative hematoxylin and eosin staining of perigonadal adipose tissue of CL8, CL12, and CL15 female mice aged 22 mo. Scale bar, 500 ␮m. B: morphometric analysis of adipose tissue (n ⫽ 5 different animals/genotype). Means ⫾ SE, CL8 vs. CL12 or CL15. *P ⬍ 0.05 by Student’s t-test. WAT-F, white adipose tissue-females.

levels were significantly low in CL males and females (P ⬍ 0.01) as early as 6 wk of age (data not shown). To evaluate the response of CL mice to leptin more directly, we administrated leptin by ip injections into 12wk-old female mice for 12 days. All leptin-treated mice lost weight compared with saline-treated mice. Compared with control mice, leptin administration produced a greater decrease in body weight (P ⬍ 0.01) and in the amount of food consumed (P ⬍ 0.05) by 12-wk-old CL females (Fig. 3, E and F). These observations suggest that leptin action is enhanced in CL mice. Energy balance in CL mice. To determine the effect of CL on the control of energy utilization, we analyzed components of energy expenditure in 6-mo-old female mice. At 6 mo of age, individually housed CL8, CL12, and CL15 mice were monitored for 72 h in the CLAMS. CL12 and CL15 mice consumed significantly more O2 and produced more CO2 per kilogram of body mass than CL8 control mice during the dark phase of the diurnal cycle (Fig. 4, A and B). We saw no differences among groups in the light phase and no differences when mice were evaluated at 8 wk of age (not shown). In addition, we detected no differences among groups in food consumption (Fig. 4C), voluntary movement, or respiratory quotient (not shown). Heat production was slightly but significantly elevated in CL12 and CL15 female mice compared with controls (Fig. 4D), suggesting potential changes in brown adipose tissue function in these animals. However, the expression of mitochondrial uncoupling protein 1 (UCP1) and Pgc-1a [peroxisome proliferator-activated receptor-␥ coactivator-1␣ (Ppargc1a)] in 6-mo-old CL12 and CL15 female mice was comparable with controls

(Fig. 4E). We conclude that CL mice as adults have higher mass-adjusted energy expenditure in addition to lower leptin levels and reduced adiposity. Glucose tolerance and insulin sensitivity in CL mice. At 6 mo of age there were no differences in fasting blood glucose among CL8 (control), CL12, and CL15 male mice (143 ⫾ 15.7, 139 ⫾ 24.7, and 140 ⫾ 17.4, respectively), but the CL12 and CL15 mice had substantially lower fasting insulin levels than control mice (Fig. 5A). Male CL12 and CL15 mice at this age also showed better glucose tolerance (Fig. 5B) and stronger responses to injected insulin (Fig. 5D) compared with controls. The insulin sensitivity index measured by HOMA2 was significantly elevated in both CL12 and CL15 male mice (Fig. 5C). To see if this improved insulin sensitivity was accompanied by diminished insulin production, we assessed plasma insulin levels before and after ip glucose administration. Insulin levels after overnight fasting were significantly lower in CL12 and CL15 males than in control mice (Fig. 5E). Serum insulin remained relatively low in CL12 and CL15 males in the first 15 min after glucose challenge and returned almost to basal levels by 30 min after glucose injection (Fig. 5E). Female CL mice showed a somewhat different pattern. At 6 mo of age, fasting insulin levels were lower in both CL12 and CL15 females but reached statistical significance only in the CL15 mice (Fig. 6A). In parallel, the HOMA2%S index was significantly elevated only in the CL15 female mice (Fig. 6C). Glucose and insulin tolerance in 6-mo-old CL12 female mice were similar to the CL8 controls (Fig. 6, B and D), although CL15 females showed some improvement in glucose tolerance (Fig. 6B). However, by 22 mo, both CL12 and CL15 females


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showed clear and dramatic improvements in glucose tolerance (Fig. 6E). Pancreatic islet morphology and assessment of ␤-cell proliferation in CL mice. Immunofluorescence staining for insulin showed that pancreatic islets from 6-mo-old CL mice exhibited normal architecture consisting of insulin-producing cells in the core and non-␤-cells in the periphery (Fig. 7). ␤-Cell mass was significantly reduced in CL12 and CL15 male mice compared with controls (P ⬍ 0.03; Fig. 7, A and B). Proliferation of ␤-cells measured by Ki-67 immunostaining showed a twofold decrease in frequency of proliferating ␤-cells in 6-mo-old CL12 and CL15 male mice compared with control mice (P ⬍ 0.01; Fig. 7, C and D). Thus, the reduction in pancreatic islet mass may be related to decreases in ␤-cell proliferation in adult CL males. In contrast, ␤-cell mass and ␤-cell proliferation in females did not differ among groups at 6 mo of age (Fig. 7, E and F, and data not shown). DISCUSSION

It is well established that prenatal and immediate postnatal variations in nutrient availability can modulate metabolic status and disease susceptibility in rodents and in humans, but most previous studies have focused on factors that impair adult health, often by increased obesity and impairments in glucose and insulin homeostasis (11, 41). A largely separate research

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Fig. 3. Serum leptin levels of 6-mo-old male (M; n ⫽ 6) (A) and female (F; n ⫽ 6) (C) mice. Food intake over 6 days in 6-mo-old male (n ⫽ 6; B) and female (n ⫽ 6; D) mice fed regular chow. E and F: cumulative food intake and reduction in body weight were measured over a 12-day period of treatment with leptin or vehicle in 12-wk-old female mice of the indicated genotypes. Data are presented as means ⫾ SE. *P ⬍ 0.05.

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tradition has shown that restriction of caloric intake (21, 22, 28, 39, 43), or in some cases intake of the amino acid methionine (49), throughout adult life can prevent or delay a wide range of late-life illnesses and thereby increase mean and maximal longevity. A recent publication from our group (67) has shown that a mere 3 wk of food restriction imposed by litter crowding between birth and weaning can extend mean and maximal lifespan in genetically heterogeneous mice despite ad libitum availability of food from 3 wk of age onward. In this report, we have taken initial steps to evaluate the idea that the improved longevity of CL mice is accompanied by and may reflect lifelong alterations in metabolic status and in particular in glucose and insulin homeostasis. Our data show that 3 wk of postnatal nutrient restriction has long-lasting metabolic effects in adult mice. We had shown previously that CL mice had altered patterns of expression of genes for xenobiotic metabolizing enzymes at 12 and 22 mo of age, suggesting long-term effects reminiscent of those seen in CR and long-lived dwarf models (65). In this study, we have evaluated long-term effects of the CL intervention on a cluster of connected metabolic end points in the long-lived CL mice. Our data show that CL mice have less fat, lower leptin levels, better response to leptin, and lower insulin levels than control mice. They are more tolerant to glucose challenge and are more sensitive to insulin, with lower insulin secretion needed to



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Fig. 4. Energy expenditure in CL mice. Sixmonth-old female mice were monitored for 72 h in the Comprehensive Laboratory Animal Monitoring System (n ⫽ 8/genotype) to assess ox˙ O2; ml·kg⫺1·h⫺1) (A), carygen consumption (V ˙ CO2; ml·kg⫺1·h⫺1) bon dioxide production (V (B), food intake (g/h; C), and heat production (kcal·kg⫺1·h⫺1; D). E: levels of mRNA by semiquantitative RT-PCR of ucnoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor-␥ coactivator-1␣ (Ppargc1a) from brown adipose tissue of 6-mo-old CL and control female mice (n ⫽ 6). Data are presented as means ⫾ SE.

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restore glucose to baseline levels; these differences become apparent by 6 mo of age in males and by 22 mo of age in females. Furthermore, in CL males, ␤-cell mass and proliferation are lower, consistent with lower demand for insulin in these mice. Improved insulin sensitivity combined with low insulin and glucose levels is characteristic of a series of long-lived mutant mice with mutations in the GH/IGF axis and of mice exposed to long-term CR (3). Long-lived Ames dwarf and Snell dwarf mice, for example, show increased insulin sensitivity along with decreased levels of serum glucose and insulin (3, 6). Mice homozygous for the targeted disruption of the GH receptor gene (GHRKO) are hypoinsulinemic, highly insulin sensitive, normoglycemic, and long-lived (1, 8). Similarly, long-lived mice on a methionine-deficient (Meth-R) diet have significantly lower levels of serum IGF-I, insulin, and glucose (67). Acarbose, a drug that blunts postprandial glucose spikes and extends lifespan in both male and female mice, and which is used clinically to improve glucose control in diabetic patients (57), significantly reduces blood glucose levels in mice (27). Similarly, long-term treatment with another diabetes drug, metformin, extends mice lifespan and mimics some of the benefits of CR, such as increased insulin sensitivity, and reduces cholesterol levels without a decrease in caloric intake (29, 42).

Thus our data on CL mice are consistent with models in which improved glucose homeostasis leads to slower aging and extended lifespan in mice. However, most of the mice in such experiments die of some form of neoplastic illness, rather than of diabetes or other metabolic disease, and the connections between the metabolic effects and the postponement of fatal illnesses are likely to be indirect. In contrast to the strong association of improved insulin signaling with extended longevity in the majority of long-lived mice models, several varieties of mutant or drug-treated mice are long-lived but insulin resistant (70). Brain insulin receptor substrate 2-deficient mice and Klotho transgenic mice are insulin resistant but live longer than their littermates (38, 69). Similarly, long-lived rapamycin-treated mice are insulin resistant (32, 51), suggesting a complex interaction between insulin signaling and aging. Furthermore, wild-derived mice of the Majuro stock live longer than laboratory-derived mice despite profound impairment in glucose control (48). Majuro mice also display dramatically high levels of leptin, in contrast to the low leptin levels and increased leptin sensitivity in CL mice, although the significance of this difference to the long lifespan remains to be clarified. Thus, it is difficult to conclude that enhanced insulin sensitivity and reduced glucose levels are required for increased lifespan in mice, although it seems plausible that improved glucose control contributes to post-


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Fig. 5. Glucose homeostasis in 6-mo-old CL male mice assessed for fasting serum insulin levels (A), intraperitoneal glucose tolerance test (B), homeostasis model assessment (HOMA) index of insulin resistance as presented by HOMA2 %sensitivity (HOMA2%S; C), insulin tolerance test (D), and in vivo insulin secretion after intraperitoneal glucose injection (E). We used the same mice for the studies shown in each part of this figure. Data are expressed as means ⫾ SE (n ⫽ 4/genotype); CL8 vs. CL12 or CL15 mice. *P ⬍ 0.01.

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ponement of lethal illnesses, including neoplasia (4, 46, 67), in CL, CR, and methionine-deficient mice as well as in mice with disruption of GH and IGF-I signals. It is also possible that insulin resistance mimics some of the effects of hypoinsulinemia by protecting the cells from excessive insulin stimulation (62, 69). The weeks just before and just after weaning may represent a brief window of opportunity during which interventions can have a critical long-lasting impact on longevity and insulin sensitivity (5). For example, GH administered to Ames dwarf mice between 2 and 8 wk of age significantly shortened their lifespan, impaired cellular stress sensitivity, and significantly reduced insulin sensitivity (54). In contrast, GH injections initiated at 4 wk of age did not diminish the extended lifespan of Snell dwarf mice (72), consistent with a model in which sensitivity to GH injections is lost by 4 wk. Studies in which caloric restriction was initiated at 6 wk and then terminated at 6 mo of age led to a statistically significant increase in lifespan in rats; however, this was much smaller than that seen in rats in which CR diet was continued beyond 6 mo of age (76).

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Further work to define the effects of transient vs. continuous nutritional deficits on late-life diseases and lifespan is likely to be rewarding. There is growing evidence that improved lifespan is associated with changes in the relative proportions of specific fat depots or with inflammatory pathways within fat tissue (31). In particular, reduction of intra-abdominal (“visceral”) adipose tissue by surgical removal (24) or as a result of CR (12) is associated with a significant improvement in glucose tolerance in mice. Differences in distribution of adipose tissue could, in principle, influence circulating levels of adipocyte-derived cytokines (adipokines) such as adiponectin and resistin (45), which modulate insulin sensitivity. Thus, it has been suggested that enhanced insulin sensitivity of long-lived GHR-KO mice may be due to the altered secretory profile of visceral fat and, in particular, to enhanced adiponectin secretion by these fat depots (45). At this point, we have only fragmentary evidence on the status of adipocytes in CL mice, with indications of diminished adipocyte size in at least one depot of female mice by the age of 22 mo. Data on other depots, on markers of



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Fig. 6. Glucose homeostasis in 6-mo-old CL female mice assessed for fasting serum insulin levels (A), glucose tolerance test (B), HOMA index of insulin resistance as presented by HOMA2%S (C), and insulin tolerance test (D). We used the same mice for the studies shown in each part of the figure. E: glucose tolerance test for 22-mo-old female mice. Data are expressed as means ⫾ SE (n ⫽ 4/genotype); CL8 vs. CL12 or CL15 mice. *P ⬍ 0.01.

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senescence (71) and inflammation (2), on adipokine production, and on responses to diets high in fat will help to clarify the potential role of altered adipose tissue in the CL longevity model. Since hypoinsulinemia promotes insulin sensitivity and vice versa, it could be debated which of these characteristics is primary and which is secondary in CL mice. Our evidence suggests that early-life CL intervention affects both the secretory function of insulin-producing ␤-cells and peripheral actions of insulin. Pancreatic islet mass is reduced and ␤-cell proliferation diminished in CL mice. Similarly, multiple animal models, including maternal protein restriction, caloric restriction, and intrauterine placental ligation models, have identified common metabolic phenotypes in offspring and have identified alterations in insulin sensitivity and the pancreatic ␤-cell (26, 30, 36, 53, 56, 61). ␤-Cell mass and proliferation were also reduced in GHR-deficient mice (37), and the number of large islets was reduced in Ames dwarf mice (55). Our data indicate that, even at 22 mo of age, CL mice have increased glucose tolerance, suggesting that early-life nutrient intervention continues to modulate metabolism well into old age. It

remains to be determined whether this “protective” effect of nutritional constraint before the weaning period persists in the presence of a high-caloric diet. There are major sex differences in insulin sensitivity in 6-mo-old CL males and females. Whereas CL males are significantly insulin sensitive compared with controls at this age, CL female mice demonstrate increased insulin sensitivity only much later in life. Previous studies in different rodent models of glucose intolerance and insulin resistance have demonstrated that female mice are less prone to diet-induced insulin resistance (16, 77), and many genetically induced forms of insulin resistance have a milder phenotype in females compared with males (35, 50). Indeed, we find that the increased insulin sensitivity of female vs. male mice can be detected using intraperitoneal glucose and insulin tolerance tests. These sex-related differences in insulin sensitivity could be attributable in part to actions of estrogen and testosterone, or they may reflect sex-specific differences in growth hormone pulse patterns (73). It has been suggested that differences in visceral and subcutaneous adipose tissue depots between females and males may also contribute to differences in insulin


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Fig. 7. Islet morphology in CL mice. A and E: immunofluorescence staining for insulin (green) and 4,6-diamidino-2-phenylindole (blue) in representative pancreatic sections from 6-mo-old CL8, CL12, and CL15 male (A) and female (E) mice. B and F: quantitation of ␤-cell mass in males (B) and females (F). C and D: proliferation index in sections stained for Ki-67 and insulin. Frequency of ␤-cell proliferation was assessed by Ki-67 staining in insulin-stained sections from 6-mo-old CL8, CL12, and CL15 male mice. Data are presented as means ⫾ SE (n ⫽ 4/genotype); CL8 vs. CL12 or CL15 mice. *P ⬍ 0.05.

sensitivity, allowing female mice to remain insulin sensitive longer in life. Our study also demonstrates an effect of the CL intervention on energy metabolism. Twenty-four-hour recordings of oxygen consumption and carbon dioxide output revealed that oxygen consumption and carbon dioxide production are significantly increased in CL mice. Heat production was also increased in CL mice. Intriguingly, both GHRKO and Ames dwarf mice have increased oxygen consumption per gram of body weight and decreased respiratory quotient (RQ), which provides an indication of the relative levels of fat and carbohydrate oxidation (75). Our data show that RQ did not change in CL mice. ˙ O2 in CL mice was not due merely to The increase in V expressing the data per unit of total body mass, because differences between CL and control mice were still significant when data were recalculated per unit of lean body mass (as determined by DEXA). The current work reveals long-lasting endocrine and metabolic effects of transient early-life nutrient restriction, alterations that could contribute to the long lifespan of these CL mice. Further exploration of the CL system and the factors that lead to altered physiological status at both the metabolic and cellular levels should shed light on the mechanisms by which

aging and lifespan are regulated by early nutritional interventions. ACKNOWLEDGMENTS We thank Dr. A. Parlow, National Hormone and Pituitary Program, for recombinant mouse leptin. GRANTS This project was supported by National Institute on Aging grant R01AG-019899 (to A. Bartke), by a Senior Scholar Award from the Ellison Medical Foundation (to R. A. Miller), and by the UM Nathan Shock Center, P30-AG-013283 (to R. A. Miller). M. Sadagurski was supported by Shock Center Grant AG-13283 (pilot grant) and Pepper Center Grant AG-024824, and E. Bernal-Mizrachi was supported by Shock Center Grant DK-084236 and by a Feasibility Grant from the Michigan Diabetes Research Center (P30-DK-020572). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS M.S., A.B., E.B.-M., and R.A.M. conception and design of research; M.S., T.L., M.B.-R., G.C., L.E., D.M., and L.S. performed experiments; M.S., M.B.-R., L.S., E.B.-M., and R.A.M. analyzed data; M.S., M.B.-R., G.C., A.B., E.B.-M., and R.A.M. interpreted results of experiments; M.S. and R.A.M.


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