Editor's evaluation: The effects of caloric restriction on adipose tissue and metabolic health are sex- and age-dependent

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Caloric restriction (CR) reduces the risk of age-related diseases in numerous species, including humans. CR’s metabolic effects, including decreased adiposity and improved insulin sensitivity, are important for its broader health benefits; however, the extent and basis of sex differences in CR’s health benefits are unknown. We found that 30% CR in young (3-month-old) male mice decreased fat mass and improved glucose tolerance and insulin sensitivity, whereas these effects were blunted or absent in young females. Females’ resistance to fat loss was associated with decreased lipolysis, energy expenditure and fatty acid oxidation, and increased postprandial lipogenesis, compared to males. The sex differences in glucose homeostasis were not associated with differential glucose uptake but with altered hepatic ceramide content and substrate metabolism: compared to CR males, CR females had lower TCA cycle activity and higher blood ketone concentrations, a marker of hepatic acetyl-CoA content. This suggests that males use hepatic acetyl-CoA for the TCA cycle whereas in females it accumulates, stimulating gluconeogenesis and limiting hypoglycaemia during CR. In aged mice (18-months old), when females are anoestrus, CR decreased fat mass and improved glucose homeostasis similarly in both sexes. Finally, in a cohort of overweight and obese humans, CR-induced fat loss was also sex- and age-dependent: younger females (<45 years) resisted fat loss compared to younger males while in older subjects (>45 years) this sex difference was absent. Collectively, these studies identify age-dependent sex differences in the metabolic effects of CR and highlight adipose tissue, the liver and oestrogen as key determinants of CR’s metabolic benefits. These findings have important implications for understanding the interplay between diet and health, and for maximising the benefits of CR in humans. Editor's evaluation This manuscript breaks new ground in old soil; i.e. sex differences in mouse and human studies and one of the greatest challenges facing translational investigators is the remarkable difference in phenotypes by sex. Defining those differences has been relatively straightforward, but understanding the underlying basis for the phenotypic effects continues to be difficult. This paper demonstrates the sex-and age-dependent effects of calorie restriction on adipose tissue and body composition. It then goes further in describing the metabolic distinctions that arise between males and females. As such, this study will open up new opportunities to explore these sex differences and to test the hypothesis that estrogen in younger animals and humans may be at the center of these effects. Furthermore, it provides a basis for determining why weight loss may be difficult for some individuals with calorie-restricted diets. https://doi.org/10.7554/eLife.88080.sa0 Decision letter eLife's review process Introduction Caloric restriction (CR) is a therapeutic nutritional intervention involving a sustained decrease in calorie intake whilst maintaining adequate nutrition. CR extends lifespan and reduces the risk of age-related diseases in numerous species, ranging from yeast to primates (Lin et al., 2002; Weindruch et al., 1986; Mattison et al., 2017). CR can have detrimental effects, including bone loss (Villareal et al., 2016) and increased susceptibility to infections (Speakman and Mitchell, 2011), and therefore it may not be suitable for all individuals; however, trials of CR in humans show numerous health benefits, including the prevention of cardiovascular disease, hypertension, obesity, type 2 diabetes, chronic inflammation, and risk of certain cancers (Most et al., 2017). Thus, the ability of CR to promote healthy ageing is now also recognised in humans. In addition to these health benefits, many effects of CR reflect evolutionary adaptations that confer a survival advantage during periods of food scarcity (Speakman and Mitchell, 2011). Establishing the extent and basis of CR’s effects may thereby reveal new knowledge of healthy ageing and the interplay between food, nutrition, and health. A key contributor to the health benefits of CR is its impact on metabolic function. Ageing is characterised by hepatic insulin resistance, hyperinsulinaemia, and excessive accumulation of white adipose tissue (WAT), particularly visceral WAT (Barzilai et al., 1998; López-Otín et al., 2016; Mancuso and Bouchard, 2019). The latter is coupled with adipose dysfunction, whereby WAT becomes unable to meet the demands for safe lipid storage. This results in ectopic lipid accumulation in the liver and other tissues, contributing to insulin resistance and metabolic dysregulation (Barzilai et al., 1998; Mancuso and Bouchard, 2019). CR counteracts these effects, decreasing WAT mass, increasing fatty acid (FA) oxidation, preventing ectopic lipid deposition and enhancing insulin sensitivity and glucose tolerance (Speakman and Mitchell, 2011). Notably, studies in rodents show that removal of visceral WAT, independent of CR, is sufficient to prevent insulin resistance, improve glucose tolerance and increase lifespan (Gabriely et al., 2002; Muzumdar et al., 2008; Tran et al., 2008). Visceral WAT loss in humans is also strongly associated with CR’s metabolic benefits (Larson-Meyer et al., 2006). Thus, the ability of CR to decrease visceral WAT, and the resultant improvements in hepatic insulin sensitivity, are likely central to CR’s effects on metabolic function and healthy ageing. This importance of adiposity and metabolic function raises the possibility of sex differences in the CR response. Indeed, it is now well established that metabolic homeostasis and adipose biology differ substantially between males and females (Mauvais-Jarvis, 2018; Oliva et al., 2020; Valencak et al., 2017; Maggi and Della Torre, 2018); however, most preclinical rodent studies have used males only (Prendergast et al., 2014), suggesting that much of the CR literature may have overlooked sex as a potential determinant of CR’s effects. Nevertheless, some clinical and preclinical CR studies from our lab and others have identified sexually dimorphic responses (Kane et al., 2018; Redman et al., 2007; Shi et al., 2007; Cawthorn et al., 2016), whereby males lose fat mass to a greater extent than females. Oestrogens underlie many metabolic sex differences (Mauvais-Jarvis, 2018; Della Torre et al., 2018), suggesting that oestrogen may contribute to females’ resistance to fat loss during CR. However, the extent and basis of sexual dimorphism in CR’s metabolic effects remains to be firmly established. Herein, we first systematically review the CR literature to establish the degree to which sex has been overlooked a determinant of the CR response. To further elucidate the extent and basis of sex differences, we studied CR in male and female mice and humans at ages where oestrogen is physiologically active or absent. Together, our findings show that the CR field has largely overlooked sex differences and reveal that both mice and humans display age-dependent sexual dimorphism in the metabolic effects of CR. Results Sex is routinely overlooked as a biological variable in the CR literature One review of the recent CR literature found that most rodent studies use males only, with females typically used only to address female-specific experimental questions (Kane et al., 2018); however, the extent of this bias in earlier CR studies, and whether it applies to human CR research, has not been assessed. To address these issues, we first systematically quantified the use of males and females, and the consideration of sex as a biological variable, in mouse and human CR studies (Figure 1, Figure 1—figure supplement 1). We focused on research published since 2003, when the European Commission first highlighted the importance of addressing sex as a biological variable (Lee, 2018). We also excluded studies that necessarily focused on a single sex, such as those addressing effects of CR on female reproductive function, leaving only studies in which there is no scientific rationale for ignoring potential sexual dimorphism. We found that male-only studies predominated for mice, accounting for ~64% of all mouse CR studies since 2003 (Figure 1A, Figure 1—figure supplement 1B). This is consistent with previous analyses of the more-recent CR literature (Kane et al., 2018). Fewer than 20% of studies used females only, while around 7.3% combined males and females, suggesting an assumption that sex would not influence experimental outcomes. In contrast,~60% of human studies combined males and females, while ~23% used only females and ~12% only males (Figure 1C, Figure 1—figure supplement 1B). Strikingly, by the end of 2021, studies that included both sexes and analysed data with sex as a variable were in a minority for both mice (~3.4%) and humans (~4.5%) (Figure 1A and C). Moreover, the proportion of studies using each sex or combination of sexes has remained relatively constant since 2003 (Figure 1A and C). Thus, efforts to increase the consideration of sex in experimental design, as promoted by the European Commission (Lee, 2018), Canadian Institutes of Health Research (CIHR) and National Institutes of Health (NIH) (Johnson et al., 2014), appear to have had little impact on the field of CR research. Figure 1 with 1 supplement see all Download asset Open asset Summary of sex differences in mouse and human CR research. PubMed was searched using MeSH terms to identify research articles that studied caloric restriction in vivo in mice (A,B) or humans (C,D), published between 2003 and 2021. Search results were then classified into the following groups depending on the sexes included in each study: ‘Male only’=male subjects used exclusively; ‘Female only’=female subjects used exclusively; ‘Male & Female separate’=male and female subjects used with data reported respective to each sex, allowing sex differences to be addressed; ‘Male & female combined’=male and female subjects used with data from both sexes combined either in part or in full; ‘N/A’=no sex data available. (A,C) Cumulative publications for studies within each group. The boxes between (A) and (C) highlight the dates of funders’ policies highlighting the importance of sex as a biological variable. (B,D) Pie charts of those studies in the ‘Male & Female separate’ group that considered sex a biological variable, and the proportion of these that did or did not detect sex differences. Source data are provided as a Source Data file. See also Figure 1—figure supplement 1. Figure 1—source data 1 Literature search to identify sex differences in mouse and human CR research. Values for the data presented in Figure 1A–D are shown in the file named ‘Figure 1_Source_Data.xlsx’. Raw data for the literature searches for mice and humans are contained in the files named ‘Literature_Search_Mice.xlsx’ and ‘Literature_Search_Humans.xlsx’. https://cdn.elifesciences.org/articles/88080/elife-88080-fig1-data1-v2.zip Download elife-88080-fig1-data1-v2.zip We next focused on the minority of studies that did consider sex in their experiments (‘M&F separate’) and that included analysis of metabolic parameters. We found that results in ~74% of mouse studies (Figure 1B) and ~62% of human studies (Figure 1D) indicated a sex difference in the CR response. Thus, sex differences have been described in the CR literature, but the continuing dearth of studies that include both sexes suggests that this issue continues to be overlooked in the CR field. Females resist CR-induced weight loss and fat loss To explore sex differences in the CR response, we assessed the effects of 30% CR, implemented from 9 to 15 weeks of age, in C57BL/6 J and C57BL/6 N mice. As expected, CR decreased body mass in both males and females (Figure 2A) but this effect was greater in males, with ANOVA confirming a significant sex-diet interaction. This sex difference in response to CR is particularly clear when body mass is presented relative to baseline, pre-CR levels (Figure 2B). Figure 2 with 4 supplements see all Download asset Open asset Female mice resist weight loss, fat loss, and lipolysis during CR. Male and female mice on a C57BL/6NCrl or C57BL/6 J background were fed ad libitum (AL) or a 30% CR diet from 9 to 15 weeks of age (0–6 weeks of CR). (A–F) Each week mice were weighed (A,B) and body composition was determined by TD-NMR (C–F). Body mass, fat mass, and lean mass are shown as absolute masses (A,C,E) or fold-change relative to baseline (B,D,F). (G) The mass of gWAT (gonadal WAT) was recorded at necropsy and is shown as % body mass. (H,I) Micrographs of H&E-stained sections of gWAT (H) were used for histomorphometric analysis of adipocyte area (I); in (H), scale bar = 100 µm. (J) Plasma was sampled at 15 weeks of age and the concentration of non-esterified fatty acids (NEFA) was assayed. (K,L) At 10 weeks of age, during the period of maximal weight and fat loss, a separate cohort of mice were culled and iWAT was collected for analysis of the indicated proteins by Western blotting. Fluorescent Western blots (K) were quantified using LICOR software (L); L=protein ladder. Data in (H) and (K) show representative micrographs (H) and immunoblots (K). Data in (A–F) are shown as mean ± SEM of the following numbers of mice per group: male AL, n=42; female AL, n=43; male CR, n=44; female CR, n=52. Data in (G), (J) and (L) are shown as violin plots of the following numbers of mice per group: male AL, n=29(G), 13 (J) or 11 (L); female AL, n=28(G), 14 (J) or 10 (L); male CR, n=33 (G), 14 (J) or 11 (L); female CR, n=34 (G) or 13 (J,L). Data in (H–I) are shown as representative micrographs or violin plots from 5 (male AL) or 6 (female AL, male CR, female CR) mice per group. For (A–F), significant effects of diet, sex and/or time, and interactions thereof, were determined by three-way ANOVA or a mixed-effects model. For (G), (J) and (L), significant effects of diet and/or sex were determined by two-way ANOVA with Tukey’s or Šídák’s multiple comparisons tests. For (I), significant effects of diet and/or sex on adipocyte area (Ad.Ar) were determined using a mixed-effects model, while significant differences in median Ad.Ar between AL and CR mice were determined two-way ANOVA with Šídák’s multiple comparisons test. p Values from ANOVA or mixed models are shown beneath the graphs, as indicated. For (G), (I), (J) and (L), significant differences between comparable groups are indicated by * (p<0.05), ** (p<0.01), or *** (p<0.001). Source data are provided as a Source Data file. See also Figure 2—figure supplements 1–4. Figure 2—source data 1 Female mice resist weight loss, fat loss, and lipolysis during CR. Values for the data presented in Figure 2A–G1–J and L are shown in the file named ‘Figure 2_Source_Data.xlsx’. Raw uncropped images of immunoblots in Figure 2K are shown in the files named ‘Figure 2 blots lower beta-actin.tif’, ‘Figure 2 blots lower HSL.png’, ‘Figure 2 blots lower P-HSL.png’, ‘Figure 2 blots upper beta-actin.tif’, ‘Figure 2 blots upper HSL.png’, and ‘Figure 2 blots upper P-HSL.png’. Annotated images of uncropped immunoblots from Figure 2K are shown in the file named ‘Figure_2 K_Uncropped_blots.eps’. https://cdn.elifesciences.org/articles/88080/elife-88080-fig2-data1-v2.zip Download elife-88080-fig2-data1-v2.zip To determine how fat and lean mass contribute to these diet and sex effects, body composition was assessed weekly using time-domain nuclear magnetic resonance (TD-NMR; Figure 2C–F, Figure 2—figure supplement 1A–B). CR decreased fat and lean mass in males, whereas females maintained fat mass and lost only lean mass (Figure 2C–F). A significant sex-diet interaction was apparent for absolute fat mass (Figure 2C) and for fat mass relative to baseline (Figure 2D); the latter showed that CR decreased fat mass in males, whereas AL males and AL or CR females increased fat mass to a similar extent over the 6-week duration (Figure 2D). For lean mass, a significant sex-diet interaction occurred for absolute mass, with losses being greater in males than in females (Figure 2E). However, when compared to baseline lean mass, the CR vs AL effect was similar between the sexes, in part because AL females continued to increase lean mass over time (Figure 2F). Given that these body composition changes coincide with changes in overall body mass, we also assessed fat and lean mass as % body mass. Males preferentially lost fat mass and preserved lean mass in response to CR, with % lean mass being greater in CR vs AL-fed males (Figure 2—figure supplement 1A–B). In contrast, diet did not alter % fat or % lean mass in females, indicating that changes in body composition were proportionate to the overall changes in body mass (Figure 2—figure supplement 1A–B). To determine how these changes relate to regional adiposity, we measured adipose depot masses after 6 weeks of AL or CR diet (Figure 2G, Figure 2—figure supplement 1C, Figure 2—figure supplement 2A). We found that in males but not females CR decreased the absolute mass of gonadal (gWAT), inguinal (iWAT), mesenteric (mWAT), and perirenal (pWAT) WAT depots, as well as brown adipose tissue (BAT; Figure 2—figure supplement 1C). CR also tended to decrease the absolute mass of pericardial WAT (pcWAT) in males only (Figure 2—figure supplement 1C). To determine if these changes were proportionate to changes in overall body mass, we further analysed the mass of each adipose depot as % body mass (Figure 2G, Figure 2—figure supplement 2A). This showed that the significant sex-diet interaction persisted for gWAT, iWAT, mWAT and pWAT. CR did not affect % BAT mass in either sex, although % BAT mass was significantly greater in males than in females (Figure 2—figure supplement 2A). Thus, both in absolute terms and as % body mass, CR decreases WAT mass in males but not in females. We next analysed the masses of other tissues to determine if this sex-dependent effect of CR is unique to WAT. CR significantly decreased the absolute mass of the liver, pancreas, kidneys, gastrocnemius muscle (gastroc), heart, spleen, and thymus (Figure 2—figure supplement 2B). Significant sex-diet effects were detected for the liver and kidney, with CR causing greater decreases in males than in females; however, for each of the other tissues the CR effect was similar between the sexes (Figure 2—figure supplement 2B). In contrast to these effects on absolute mass, CR did not significantly affect the relative masses of each of these tissues, nor did sex influence the CR effect (Figure 2—figure supplement 2C). This indicates that the absolute masses of these tissues decreased in proportion to the changes in overall body mass. One notable exception is the adrenal glands, the mass of which was increased with CR to a greater extent in males than in females (Figure 2—figure supplement 2B–C). Together, these data show that the sex differences in CR-induced loss of body mass and fat mass are driven primarily by decreased WAT mass in males, which females robustly resist. Females resist adipocyte hypotrophy and lipolysis during CR Differences in WAT mass can be driven by changes in adipocyte size and/or adipocyte number. Thus, given the marked sex differences in the effect of CR on WAT mass, we next investigated this effect at the level of adipocyte size (Figure 2H–I, Figure 2—figure supplement 3A–B). CR significantly decreased average adipocyte area in males but not in females, both for gWAT (Figure 2H–I) and for mWAT (Figure 2—figure supplement 3A–B). This suggests that females resist lipolysis during CR. Consistent with this, CR increased plasma NEFA concentrations in males but not in females (Figure 2J). To further assess adipocyte lipolysis, we analysed phosphorylation of hormone-sensitive lipase (HSL) in iWAT. CR stimulated HSL phosphorylation in males but not in females (Figure 2K–L, Figure 2—figure supplement 3C). Moreover, across both diets HSL phosphorylation was lower in females whereas total HSL was increased by CR in females only (Figure 2K–L, Figure 2—figure supplement 3C). Together, these observations show that females resist lipolysis during CR. Females suppress energy expenditure and increase postprandial lipogenesis more than males during CR We next investigated if altered energy expenditure also contributes to the sex differences in the CR response. To do so, we used indirect calorimetry to analyse mice during week 1 and week 3 of CR, corresponding to periods of weight loss and weight maintenance, respectively (Figure 2A). This revealed that CR decreased total energy expenditure in both sexes, with greater decreases occurring during week 3 compared to week 1, and during nighttime compared to daytime (Figure 3A–B). Notably, during week 1, CR females had lower daytime, nighttime, and total energy expenditure than CR males (Figure 3A–B), likely explaining females’ resistance to weight loss and fat loss during the first week of CR (Figure 2A–D). The daytime diet differences disappeared when normalised to lean body mass (Figure 3—figure supplement 1A), suggesting that they are driven primarily by the loss of lean mass and a consequent reduction in basal metabolic rate. In contrast, CR still decreased in nighttime and total energy expenditure even when normalised to lean body mass (Figure 3—figure supplement 1A). The relationship between lean mass and total energy expenditure (P, Slope) did not differ among the groups; however, the intercepts of the best-fit lines (P, Intercept) did differ significantly, both during week 1 (Figure 3C) and week 3 (Figure 3—figure supplement 1B). Thus, for a given lean body mass, CR-fed males and females had significantly lower energy expenditure than their AL-fed counterparts (Figure 3C, Figure 3—figure supplement 1B). In contrast to these decreases in energy expenditure, CR increased total and daytime physical activity in both sexes, with CR females having higher activity than CR males during week 1 (Figure 3—figure supplement 1C). Together, these data show that CR decreases energy expenditure more in females than in males, despite increasing physical activity, and that factors beyond decreased lean mass contribute to this CR effect. Figure 3 with 2 supplements see all Download asset Open asset CR decreases energy expenditure and stimulates postprandial lipogenesis more in female than in male mice. Male and female mice were fed AL or CR diets, as described for Figure 2. In week 1 and week 3 after beginning AL or CR diets, mice were housed for 4 days in Promethion CORE System cages for indirect calorimetry. Energy expenditure (A–C) and respiratory exchange ratio (RER; D–F) was recorded every minute throughout the 4 days. (A,D) Average energy expenditure (A) (kcal) or RER (D) per hour over the 24 hr light (Day) and dark (Night) periods, based on the average for days 2–4 of Promethion housing, for Week 1 (left) and Week 3 (right). (B) Overall energy expenditure (kcal) during the day, night, or day +night (Total) for Week 1 and Week 3. (C) Linear regression of lean mass vs total energy expenditure (kcal/24 h) during Week 1. (E) Average total RER (day +night) in Weeks 1 and 3. (F) Average RER during the postprandial period, from 12.00 to 17.00, for CR mice. (G) Absolute FA oxidation was determined based on energy expenditure and RER as described (Bruss et al., 2010). Data are from 10 (female AL), 9 (female CR, male AL) or 8 (male CR) mice per group. In (A) and (D), data are shown as mean ± SEM. In (B) and (E–G), data are shown as violin plots overlaid with individual data points; within each time period (day, night, or total), significant effects of sex, diet, week, and interactions thereof, were determined by three-way (B,E,G) or two-way ANOVA (F), with p values shown beneath each graph. Statistically significant differences between comparable groups were further assessed by Šídák’s (B,E,G) or Tukey’s (F) multiple comparisons tests and are indicated by * (p<0.05), ** (p<0.01), or *** (p<0.001). For linear regression in (C), ANCOVA was used to test if the relationship between lean mass and total energy expenditure differs significantly across all of the individual diet-sex groups (i. All mice); between AL and CR mice, irrespective of sex (ii. AL vs CR (M+F)); and between males and females fed AL diet (iii) or CR diet (iv) only. ANCOVA p values for differences in slope and intercept are reported beneath the graph. See also Figure 3—figure supplements 1–2. Figure 3—source data 1 CR decreases energy expenditure and stimulates postprandial lipogenesis more in female than in male mice. Values for the data presented in Figure 3A–G are shown in the file named ‘Figure 3_Source_Data.xlsx’. https://cdn.elifesciences.org/articles/88080/elife-88080-fig3-data1-v2.zip Download elife-88080-fig3-data1-v2.zip We next analysed the respiratory exchange ratio (RER) to assess how diet and sex influence carbohydrate and lipid oxidation, and if these effects differ as CR progresses. In AL mice, RER peaked during the night, when most food consumption and physical activity occurs; in contrast, RER for CR mice peaked from 12.00 to 17.00 in the daytime, following provision of the daily ration of CR diet (Figure 3D–E; Figure 3—figure supplement 2A). The effects of CR on average daytime, night-time or total RER did not differ between the sexes (Figure 3E, Figure 3—figure supplement 2A); however, the dynamic changes in RER during these periods, and the strong influence of feeding and fasting on RER in the CR mice, make such average RER measurements difficult to interpret. Thus, we further investigated if RER in the postprandial and/or fasted states differs between CR males and females. Postprandial RER, calculated as the average RER from 12.00 to 17.00, exceeded 1 in both sexes (Figure 3D and F), indicating that CR mice use dietary carbohydrates for FA synthesis (Bruss et al., 2010). Notably, this postprandial RER was greater in females than in males, particularly during week 1 (Figure 3F). Fasting RER, calculated as the average from 04.00 to 09.00, was below 0.8 for both sexes, indicating preferential oxidation of lipids rather than carbohydrates during this fasted state (Figure 3—figure supplement 2B). Fasting RER was lower during week 1 than during week 3 of CR but, unlike for postprandial RER, no sex differences were observed (Figure 3—figure supplement 2B). This suggests that relative lipid oxidation is greater in the first week of CR and is similar in males and females. However, given that females have lower absolute energy expenditure, we hypothesised that they would also have lower absolute FA oxidation (Bruss et al., 2010). As shown in Figure 3G and Figure 3—figure supplement 2C, CR increased absolute FA oxidation in both sexes during week 1, but not during week 3. Across both timepoints there was a significant sex-diet interaction, with CR increasing FA oxidation more in males than females (Figure 3G). Together, these data show that females have greater postprandial FA synthesis and lower absolute FA oxidation than males, particularly during the first week of CR. This highlights further mechanisms through which females maintain fat mass during CR. Females resist CR-induced improvements in glucose homeostasis We next investigated if other metabolic effects of CR also differ between the sexes. We found that CR decreased blood glucose to a greater extent in males than in females (Figure 4A). Consistent with this, oral glucose tolerance tests (OGTT) revealed that CR improved glucose tolerance in both sexes, but this effect was greater in males (Figure 4B). These diet and sex effects were reflected by the OGTT total area under the curve (tAUC), calculated against 0 mM blood glucose; however, they were not apparent for the incremental AUC (iAUC), calculated against fasting blood glucose for each mouse (Figure 4C). This suggests that differences in fasting glucose are the main driver of CR-induced improvements in glucose tolerance and the sex differences therein. Figure 4 with 1 supplement see all Download asset Open asset The effects of CR on glucose homeostasis differ between young male and female mice. Male and female C57BL6/NCrl mice were fed AL or CR diet from 9 to 15 weeks of age, as described for Figure 2. (A) Random-fed blood glucose was recorded each week. (B–D) At 13 weeks of age, mice underwent an oral glucose tolerance test (OGTT). (B) Blood glucose readings during the OGTT. (C) Area under the curve (AUC) during the OGTT was determined relative to 0 mmol/L (total AUC: tAUC) and relative to baseline (incremental AUC: iAUC). (D) Glucose-stimulated insulin secretion in mice during OGTT was assessed using an insulin ELISA. (E) HOMA-IR and Matsuda indices of mice calculated from glucose and insulin concentrations during the OGTT. Data in (A), (B), and (D) are presented as mean ± SEM. Data in (C) and (E) are presented as violin plots overlaid with individual data points. For each group and timepoint, the following numbers of mice were used: (A): male AL, n=42 (Wk 0), 36 (Wk2, Wk 2), 34 (Wk 3), 29 (Wk 4), 26 (Wk 5), or 22 (Wk 6); female AL, n=43 (Wk 0), 28 (Wk 1), 35 (Wk 2), 31 (Wk 3), 27 (Wk 4), 26 (Wk 5), or 21 (Wk 6); male CR, n=44 (Wk 0), 40 (Wk 1), 38 (Wk 2), 35 (Wk 3), 31 (Wk 4), 29 (Wk 5), or 26 (Wk 6); female CR, n=51 (Wk 0), 44 (Wk 1, Wk 2), 41 (Wk 4), 35 (Wk 4), 33 (Wk 5), or 27 (Wk 6). (B): male AL, n=27 (T0, T15, T30, T60, T120); female AL, n=26 (T15, T30, T120), or 25