Abstract

We previously demonstrated that male, but not female, Swiss Webster mice are susceptible to diabetes, with incidence increased by early overnutrition and high-fat diet (HFD). In this study, we investigated how HFD in Swiss Webster males and females during preweaning, peripubertal, and postpubertal periods alters glucose homeostasis and diabetes susceptibility. In males, HFD throughout life resulted in the highest diabetes incidence. Notably, switching to chow postpuberty was protective against diabetes relative to switching to chow at weaning, despite the longer period of HFD exposure. Similarly, HFD throughout life in males resulted in less liver steatosis relative to mice with shorter duration of postpubertal HFD. Thus, HFD timing relative to weaning and puberty, not simply exposure length, contributes to metabolic outcomes. Females were protected from hyperglycemia regardless of length or timing of HFD. However, postpubertal HFD resulted in a high degree of hepatic steatosis and adipose fibrosis, but glucose regulation and insulin sensitivity remained unchanged. Interestingly, peri-insulitis was observed in the majority of females but was not correlated with impaired glucose regulation. Our findings reveal critical periods of HFD-induced glucose dysregulation with striking sex differences in Swiss Webster mice, highlighting the importance of careful consideration of HFD timing relative to critical developmental periods.

Introduction

Consumption of an obesogenic diet is associated with metabolic dysfunction, including obesity and impairments in glucose homeostasis that can lead to diabetes (1). Intuitively, longer exposure to an obesogenic diet is expected to be more detrimental. However, the developmental time period of diet exposure is an additional key factor to consider since tissues and organ systems may be more susceptible to an adverse nutritional environment during periods of rapid growth. Long-term detrimental effects of adverse prenatal and perinatal environments, the so-called Developmental Origins of Health and Disease hypothesis, have long been recognized particularly in relation to undernutrition, which increases incidence of obesity and type 2 diabetes in offspring (2,3). Similarly, prenatal exposure to maternal obesity, high-fat diet (HFD), and diabetes increase offspring risk of developing obesity and diabetes (4,5). With human studies, it is difficult to isolate the role of early postnatal nutrition from potential confounding effects of prenatal environment and genetics. However, studies in rodents have shown that exposure to overnutrition and HFD that is limited to the lactation/preweaning period (first 3 weeks of life) can lead to lifelong impairments in glucose homeostasis and increased adiposity (6–13). Although most studies have been conducted in the inbred C57BL/6 strain and in males only, Masuyama and Hiramatsu (8) found that both male and female adults of the outbred ICR mouse strain had impaired glucose and insulin tolerance after HFD exposure during the preweaning period, while Hafner et al. (14) found that only male C57BL/6 but not female mice had impaired glucose and insulin tolerance, suggesting strain differences in sex effects. Notably, 8-week-old male offspring from dams exposed to HFD only during lactation exhibited reduced hypothalamic densities of α–melanocyte-stimulating hormone and agouti-related peptide fibers, indicating long-term effects on hypothalamic neurocircuitry critical for energy homeostasis, as well as decreased parasympathetic outputs to islets (11); however, females were not examined. In line with the importance of offspring nutrient exposure during preweaning, we previously showed that overnutrition during this period, induced by reducing the litter size, increased susceptibility to diabetes in male, but not female, Swiss Webster mice and that this was exacerbated by HFD exposure (15,16). Together, these studies indicate that the preweaning period is a critical time for programming body composition and glucose homeostasis.

Numerous studies have examined long-term effects of HFD exposure in the prenatal and early postnatal periods; however, there is a paucity of studies specifically examining the effects of dietary fat content in diets initiated at weaning. In rodents, weaning onto standard chow typically results in a significant decrease in dietary fat intake, from ∼55% of calories from fat in maternal milk to 10–20% with chow. The World Health Organization recommends dietary fat intake constitute <30% of daily calories (17), while an obesogenic western diet in humans may constitute 35–60% fat. Thus, infants transitioning to solid food may experience varying degrees of shift in dietary macronutrient composition, including fat content, depending on the diet. However, whether a dramatic shift in diet composition is beneficial or detrimental to developing metabolic systems is unclear.

The final major developmental period is puberty. Given the extensive cross talk among endocrine tissues, the dramatic changes in sex hormones during puberty subsequently give rise to alterations throughout the endocrine system. Of relevance to glucose regulation, transient insulin resistance is observed during puberty in humans with lower insulin sensitivity in girls than boys, although the underlying mechanisms are unclear (18,19). Obesity during puberty further exacerbates insulin resistance, and overweight during puberty is associated with a higher incidence of type 2 diabetes in adulthood even when heavier body weight resolves (19,20). However, there is a scarcity of data on the role that dietary fat may play on glucose regulation during puberty. With diet-induced mouse models of obesity and glucose intolerance, HFD exposure is often initiated at 6–10 weeks of age since some studies have shown that initiating HFD immediately after weaning or during the peripubertal period may attenuate detrimental metabolic effects (21,22). These observations suggest that puberty may be a critical developmental period for HFD-mediated metabolic impairments.

Given the many unanswered questions regarding dietary fat exposure in relation to weaning and puberty, we took an unbiased approach to explore the impact of HFD during three broad postnatal periods designated as the preweaning period from postnatal day (P) 2 to 21, the peripubertal period from P21 to 35, and the postpubertal period from P35 onwards, with HFD exposure limited to either zero, one, two, or all three of these periods, resulting in eight different groups. We chose to conduct these studies in the outbred Swiss Webster mouse strain, as we have previously observed that Swiss Webster males are susceptible to HFD and early overnutrition-induced β-cell dysfunction and diabetes (15,16). Moreover, the superior nurturing traits of Swiss Webster dams ensures consistent nutrition for suckling pups and thus minimizes variation due to maternal behavior. Examination of resulting body weight gain, glucose regulation, and diabetes susceptibility in offspring revealed critical periods of HFD-induced glucose dysregulation, diabetes susceptibility, and liver steatosis, as well as striking sex differences in Swiss Webster mice.

Research Design and MethodsAnimals and Diets

Diets were modified (either chow or HFD) during three broad periods in offspring: preweaning, P2–21; peripubertal, P21–35; and postpubertal, P35 onward as illustrated in Supplementary Fig. 1. First, pregnant Swiss Webster females (Taconic Biosciences, Rensselaer, NY) were maintained on chow diet (13 kcal% fat; NIH-31M; Ziegler Bros., Gardners, PA). Two days postdelivery (P2 for pups), litter size was adjusted to 10 pups to ensure consistent nutrition among litters, and half of the dams were switched to HFD (45 kcal% fat; #D12451; Research Diets, Inc., New Brunswick, NJ). Offspring were weaned at P21 and either remained on the same diet as their mother or had their diet switched (chow to HFD or vice versa). At P35, offspring either remained on the same diet or underwent a diet switch (from chow to HFD or vice versa). This resulted in eight diet groups in which group names indicate the period of HFD exposure (otherwise chow diet was consumed): CHOW, HFDP21–35, HFDP2–21, HFDP2–35, HFDP35+, HFDP21+, HFDP2–21,P35+, and HFDP2+. Although we did not systematically evaluate puberty onset (e.g., by day of vaginal opening in females and balanopreputial separation in males), several studies have shown earlier onset in response to HFD and wide differences among strains (23–26). Thus, we chose a larger window of P21–35 for our peripubertal period to cover a wide range of onset. Mice also show a second growth spurt around this time (∼P20–33), after an earlier perinatal growth spurt that ends at ∼P16 (27); thus, we hypothesized that during this time period mice may be more sensitive to dietary changes. Two independent cohorts were generated. In cohort 1, only male mice were monitored to 6 months. In cohort 2, both males and females were studied, with a subset euthanized at P35 (with no diet change at P35). Remaining males were euthanized at P72 to obtain adult tissues without the potential confounding effects of high diabetes incidence that would be more likely at a later age. Females were monitored up to P325. Although the initial plan was to monitor female mice to 6 months, as we did for males, we observed some cases of hyperglycemia in females and therefore followed females to 1 year in case diabetes in females occurred at a later age. Mice were housed in Optimice cages (Animal Care Systems, Centennial, CO) in a barrier facility (Centre for Disease Modeling, University of British Columbia Life Sciences Institute) with a 12-h light/12-h dark cycle and controlled temperature and humidity. All mice had ad libitum access to food and water as well as shelter huts, cotton nestlets, and crinkle paper. All animal procedures were approved by the University of British Columbia Animal Care Committee and carried out in accordance with the Canadian Council of Animal Care guidelines.

Glucose Monitoring

Beginning at P30, 4-h fasted blood glucose measures were taken weekly from the saphenous vein using a OneTouch Ultra 2 glucometer (LifeScan, Burnaby, British Columbia, Canada). Note that the coefficient of variation of glucose measures with this glucometer is estimated at 9.3% (28). Day of diabetes onset was defined as the first 4-h fasted glucose value ≥20 mmol/L and confirmed by a second fasting glucose value ≥20 mmol/L the following week. For oral glucose tolerance tests, mice were fasted for 6 h and glucose (2 g/kg) administered by oral gavage with glucose measured at 0, 10, 20, 30, 60, 90, and 120 min. For insulin tolerance tests, mice were fasted for 4 h and 0.75 units/kg insulin (Novolin ge Toronto; Novo Nordisk Canada, Mississauga, Ontario, Canada) was injected intraperitoneally with glucose measured at 0, 10, 20, 30, 60, 90, and 120 min.

Tissue Harvest and Immunohistochemistry

For tissue harvests, mice were fasted for 4 h and blood collected by cardiac puncture under isoflurane anesthesia, followed by cervical dislocation. Insulin (ALPCO Mouse Ultrasensitive Insulin ELISA; ALPCO, Salem, NH) and leptin (Mouse Leptin ELISA Kit; Crystal Chem, Downers Grove, IL) were measured in plasma. Tissues were immediately dissected, weighed, stored in 4% paraformaldehyde (pH 7.4) overnight, and then transferred to 70% ethanol. Tissues were paraffin-embedded and sectioned at 5 µm by Wax-it Histology Services Inc. (Vancouver, British Columbia, Canada), with hematoxylin-eosin (H-E) and Masson’s trichrome staining on a subset of slides. Immunohistochemistry was performed as previously described (16) using rabbit insulin (1:200; #3014; Cell Signaling Technology, Danvers, MA) (RRID:AB_2126503) and mouse glucagon (1:1,000; #G2654; Sigma-Aldrich, St. Louis, MO) (RRID:AB_259852) antibodies. For β-cell mass analysis, three sections per sample, 200 µm apart, were stained and scanned on an ImageXpress Micro imager and analyzed with MetaXpress software (Molecular Devices, San Jose, CA) by measuring thresholded insulin-positive area over pancreas area multiplied by pancreas mass. Islet mass was measured by multiplying pancreas mass by islet over pancreas area, determined from one scanned slide per mouse, and analyzed using ImageJ 1.52v.

Statistical Analyses

Measures at P35 were collected in parallel from males and females, and data were analyzed as two-way ANOVAs for factors of sex and treatment. For single time point measures in older mice, experiments in males and females were conducted separately at different ages and/or from different cohorts; thus, one-way ANOVAs were conducted within each sex. Mixed-effects models within each sex were used for all time course data. Tukey correction for multiple comparisons was used for all post hoc comparisons. For Figs. 2 and 3 and Supplementary Fig. 2, in which only four of eight groups are shown for clarity, statistical analyses were performed on all eight groups with Tukey correction for multiple comparisons. Correlations were assessed by Pearson correlation coefficients. Statistical analyses were performed using GraphPad Prism 8, with significance defined as P < 0.05.

Data and Resource Availability

All data generated or analyzed during this study are included in the published article (and its Supplementary Material online). No applicable resources were generated or analyzed during the current study.

ResultsPreweaning and Peripubertal HFD in Adolescent Males and Females

In mice examined until adolescence at P35 (Fig. 1A) (i.e., no P35 diet switch), both male and female HFDP2–21 and HFDP2+ offspring had higher body weights than CHOW and HFDP21+ counterparts (Fig. 1B and C) from P5 to P25. Insulin tolerance tests at P30 (Fig. 1D and E) revealed that HFDP2+ males had a faster return toward basal glucose compared with CHOW after insulin administration, but females overall had a higher area under the curve (AUC) compared with males (Fig. 1F), indicative of lower insulin sensitivity at this age. By P35 (Table 1), male body weights were overall higher than females (main effect of sex, P < 0.0001) but were no longer significantly different among groups. While there was no sex difference in fat pad weights, HFDP2+ females had higher adiposity than CHOW females, reflected in gonadal (gWAT) (Fig. 1G), mesenteric (mWAT) (Table 1), and retroperitoneal white adipose tissue (rpWAT) (Table 1) weights. Peripubertal HFD in the HFDP21+ group also increased gWAT and rpWAT weight in females (Fig. 1G and Table 1), but leptin levels were not significantly different among females (Fig. 1H) and were correlated with adipose tissue weights (r = 0.7864, P < 0.0001 for gWAT; r = 08312, P < 0.0001 for mWAT; and r = 0.6229, P = 0.0011 for rpWAT). Despite a lack of difference in adipose tissue weight among male groups, HFDP21+ males had higher leptin levels than chow males. Interestingly, HFDP2+ males had lower leptin levels despite longer exposure to HFD. Leptin levels are typically proportional to fat mass, and indeed, we did see a positive correlation between leptin and mass of gWAT (r = 0.6353; P = 0.0009), mWAT (r = 0.7022; P = 0.0001), and rpWAT (r = 0.6204; P = 0.0012). Liver weight was overall higher in males versus females and, when normalized to body weight, was highest in CHOW males and females (Table 1). Islet mass and islet number per section did not differ among groups nor between males and females, and islet morphology was similar in all mice (Fig. 1I–L). In addition, no sex or group differences were observed in fasting insulin levels (Table 1).

Figure 1

Preweaning and peripubertal HFD effects in adolescent males and females. A: Schematic of diet treatment groups for mice terminated at P35. Dams were maintained on chow diet (14 kcal% fat, designated by white bars) through pregnancy and until P2, at which time some dams were placed on HFD (45 kcal% fat, designated by black bars). Pups were weaned at P21 and either kept on the same diet as their mothers (CHOW and HFDP2+ groups) or diet was switched (HFDP2–21 and HFDP21+groups). B: Growth curves of male offspring from P2 to 30: *HFDP2+ and HFDP2–21 > HFDP21+ and CHOW, P values <0.001; †HFDP21+ > CHOW, P < 0.05; ‡HFDP2+ and HFDP2–21 > CHOW, P values <0.001; §HFDP2+and HFDP2–21 > HFDP21+, P values <0.05. n = 17–20/group. C: Growth curves of female offspring from P2 to 30: *HFDP2+ and HFDP2–21 > HFDP21+ and CHOW, P values <0.005; †HFDP2+ > HFDP21+, P < 0.005; ‡HFDP2+ and HFDP2–21 > CHOW, P values <0.005. n = 18–20/group. Glucose responses (normalized to 0 min value) during insulin tolerance tests in male (D) and female (E) offspring at P30–31 (*HFDP2–21 > CHOW, P < 0.05; †HFDP2+ > HFDP2–21, P < 0.05; ‡HFDP2+ > CHOW, P < 0.01) and corresponding AUC measures (F). G: gWAT weight in P35 males and females, left fat pad only. *HFDP21+ > CHOW and HFDP2–21, P values <0.05; †HFDP2+ > CHOW, HFDP21+, and HFDP2–21, P values <0.05. H: Plasma leptin levels in P35 males and females. *HFDP21+ > CHOW, P < 0.05. Islet mass (I) and number of islets per section (J) in P35 males and females (lines represent mean). Representative H-E pancreas sections from HFDP2+ male (K) and HFDP2+ female (L) at P35. Scale bars = 100 µm. Body weights in B and C are in nonfasted mice except for P30, which followed a 4-h fast. Arrow in B and C indicates age (P21) of diet switch in HFDP2–21 and HFDP21+ mice. Body weight and glucose responses are mean ± SEM. Boxes in AUC and tissue weight panels represent interquartile range with line at the median, and whiskers represent minimum and maximum values.

Timing of Dietary Fat During Development Impacts Body Weight and Diabetes Incidence

Body weights at P66 (Supplementary Fig. 1B and C) were significantly higher in males versus females (main effect of sex, P < 0.0001), with higher levels in HFDP21+ and HFDP2–35 males compared with CHOW and higher body weight in HFDP2–21,P35+ females compared with CHOW and HFDP21–35. In males, all groups had at least one diabetic mouse with highest incidence in HFDP2–21,P35+ and HFDP2+ males (Supplementary Fig. 1D). In contrast, after tracking fasting glucose to P316, only one female, from the HFDP35+ group, developed diabetes (Supplementary Fig. 1E). Interestingly, three other females (one HFDP21+, one HFDP35+, and one HFDP2+) had transient elevations in fasting glucose in the 16 to 19 mmol/L range but returned to normal levels. Otherwise, females maintained normal fasting glucose levels throughout life.

Effects of Preweaning and Peripubertal HFD on Glucose Homeostasis

Monitoring of diabetes incidence, glucose tolerance, and insulin tolerance was performed in males and females of all eight treatment groups, and statistical analyses were performed with all eight groups within each sex to correct for multiple comparisons. However, for clarity, we present the data as groups of four in order to address specific questions, with the same CHOW and HFDP2+ groups included in all as controls. Figure 2 addresses the role of HFD exposure that is limited to early development, either just the preweaning period (HFDP2–21) or extending through the peripubertal period (HFDP2–35) (Fig. 2A). Diabetes incidence (Fig. 2B) was highest in the HFDP2+ group and lowest in the CHOW and HFDP2–35 groups, with intermediate incidence in the HFDP2–21 group. Glucose (Fig. 2C and D) and insulin (Fig. 2E and F) tolerance were not impaired by HFD limited to preweaning (P2–21) or extending through puberty (P2–35), with only HFDP2+ females showing glucose impairment. Notably, unlike at P30 when HFDP2+ females had poor insulin sensitivity compared with males, at P66, all females had a robust insulin response with AUCs overall lower in females versus males (main effect of sex, P = 0.0033). When mice on chow diet were transiently placed on HFD only during the 2-week peripubertal period (HFDP21–35 group), diabetes incidence, glucose tolerance, and insulin sensitivity were similar to CHOW mice that had remained on normal chow throughout life (Supplementary Fig. 2). Similarly, mice on HFD that were placed on normal chow only during the peripubertal period (HFDP2–21,P35+ group) had similar diabetes incidence, glucose tolerance, and insulin sensitivity to HFDP2+ mice that were on HFD throughout life. Thus, a brief peripubertal diet switch did not impact these measures.

Figure 2

Effects of preweaning and peripubertal HFD on glucose homeostasis in adults. A: Schematic of diet treatment groups. Dams were maintained on chow diet (14 kcal% fat, designated by white bars) through pregnancy and until pup P2, at which time some dams were placed on HFD (45 kcal% fat, designated by black bars). Pups were weaned at P21. CHOW and HFDP2+ pups were kept on the same diet as their mothers from P2 onwards. HFDP2–21 offspring were switched from HFD to chow diet at P21, and HFDP2–35 offspring were switched at P35. B: Diabetes incidence to 23 weeks of age in males. n = 10–11/group. HFDP2+ > HFDP2–35, P = 0.003; HFDP2+ > CHOW, P = 0.003 by log-rank tests. Glucose response and AUC during oral glucose tolerance test in males at P50 (C) and females at P88 (D). *HFDP2+ > HFDP2–35, HFDP2–21, and CHOW, P values <0.05. Glucose response and AUC during insulin tolerance test in males (E) and females (F) at P66. Note that data from CHOW and HFDP2+ control groups are the same in Fig. 3 and Supplementary Fig. 2, with all statistical analyses controlling for multiple comparisons of all eight diet groups.

Timing of HFD Onset Affects Glucose Homeostasis and Diabetes Incidence

Compared with continuous HFD exposure throughout life, postponing HFD onset reduced and delayed diabetes incidence (Fig. 3A and B) with a greater delay seen when HFD was initiated at P35 (HFDP35+ group) compared with P21 (HFDP21+). Despite the improvements in diabetes susceptibility, however, HFDP35+ males (Fig. 3C) had a mild impairment in glucose tolerance, such that glucose levels remained higher at the 120-min time point relative to CHOW males. However, this group difference was not reflected in corresponding AUCs likely due to the difference occurring at only the one time point. In females, mild impairments in glucose tolerance were observed in the HFDP21+, HFDP35+, and HFDP2+ groups relative to CHOW (Fig. 3D); however, only HFDP2+ females had a corresponding significant increase in AUC. Similar to glucose tolerance, HFDP35+ males had elevated glucose levels at a single time point (60 min) during an insulin tolerance test (Fig. 3E), with a faster return toward baseline relative to CHOW suggesting reduced insulin sensitivity. However, this difference again was not reflected in AUC. Females all showed similar glucose responses during the insulin tolerance test (Fig. 3F).

Figure 3

Timing of HFD onset affects glucose homeostasis and diabetes incidence in adults. A: Schematic of diet treatment groups. Dams were maintained on chow diet (14 kcal% fat, designated by white bars) through pregnancy and until pup P2, at which time some dams were placed on HFD (45 kcal% fat, designated by black bars). Pups were weaned at P21. CHOW and HFDP2+ pups were kept on the same diet as their mothers from P2 onwards. HFDP35+ offspring were switched to HFD at P35, and HFDP21+ offspring were switched to HFD at P21. B: Diabetes incidence to 23 weeks of age in males. n = 10–11/group. HFDP2+ > HFDP35+, P = 0.009; HFDP2+ > HFDP21+, P = 0.052; HFDP2+ > CHOW, P = 0.003 by log-rank tests. Glucose response and AUC during oral glucose tolerance tests in males at P50 (C) and females at P88 (D) and glucose response and AUC during insulin tolerance test in males (E) and females (F) at P66. *HFDP35+ > CHOW, P values <0.05; †HFDP21+ > CHOW, P values <0.05; ‡HFDP2+ > CHOW, P < 0.05. Note that data from CHOW and HFDP2+ control groups are the same in Fig. 2 and Supplementary Fig. 2, with all statistical analyses controlling for multiple comparisons of all eight diet groups.

Effects of Diet Exposure on Plasma Hormones and Metabolic Tissues in Adult Males

There were no differences in insulin levels among the eight treatment groups at P47 (Fig. 4A). Insulin increased overall at P80 relative to P47, with higher levels mainly seen in mice that were on HFD postpuberty, including increases in HFDP35+, HFDP2–21,P35+, and HFDP2+ compared with other groups. However, β-cell mass did not differ among groups and islet architecture as well as insulin and glucagon immunoreactivity were similar among all groups (Fig. 4B–D). β-Cell mass was, however, positively correlated to gWAT weight (r = 0.530; P = 0.002) and body weight (r = 0.364; P = 0.040), suggesting some ability of β-cells in males to compensate for HFD-induced obesity. Since HFD typically increases adiposity and leptin levels, it is not surprising that mice consuming HFD postpuberty had elevated leptin levels relative to groups consuming normal chow postpuberty (Fig. 4E). Unexpectedly, HFDP2+ males that consumed HFD throughout life did not show such an elevation. Plasma leptin levels are typically proportional to fat mass, but we did not observe differences in gWAT (Fig. 4F) or mWAT (Fig. 4G) weights among treatment groups and leptin levels remained elevated after normalizing to body weight, in HFDP21+, HFDP35+, and HFDP2–21,P35+ mice relative to HFDP2–21 and HFDP21–35 (P < 0.05), with levels in HFDP21+ also greater than in CHOW mice (P < 0.05). A similar pattern was observed with liver weight and liver steatosis (Fig. 4H–K) in which HFDP35+, HFDP21+, and HFDP2–21,P35+ groups had higher liver weight and/or steatosis relative to mice on normal chow postpuberty and HFDP2+ mice. Leptin levels were also positively correlated to percentage of liver steatosis (r = 0.617; P = 0.0002).

Figure 4

Effects of diet exposure on plasma hormones and metabolic tissues in adult males. A: Plasma insulin levels in males at P47 and P80. At P80, **HFDP35+ > HFDP21–35, P < 0.005; †HFDP2–21,P35+ > HFDP21–35 and CHOW, P values <0.05; *HFDP2+ > CHOW, HFDP2–21, HFDP21–35, and HFDP2–35, P values <0.05, with no differences among treatment groups at P47 by mixed-effects analysis. Dotted lines connect values from the same mouse across age. B: β-Cell mass in males at P72 and representative pancreas sections of P72 males from CHOW group (C) and HFDP2+ group (D) with insulin in magenta, glucagon in green, and DAPI in white. E: Plasma leptin in P72 males: #HFDP35+>HFDP21–35 and HFDP2–21, P values <0.05; §HFDP21+ > CHOW, HFDP21–35, HFDP2–21, and HFDP2+, P values <0.05; ‡HFDP2–21,P35+ > HFDP21–35 and HFDP2–21, P values <0.05. gWAT weight (F), mWAT weight (G), and liver weight (H) in P72 males. I: Percentage of liver section area, based on examination of H-E–stained slide, showing steatosis. Representative liver H-E sections of P72 males from CHOW group showing normal histology (J) and HFDP21+ group showing widespread steatosis (K). Scale bars = 100 µm. Insulin values are from cohort 1; remaining data are from cohort 2.

Glucose Homeostasis Remains Stable as Females Age

Despite continued weight gain as female mice aged, plasma insulin levels (Fig. 5A) did not change significantly and did not differ among treatment groups at P149 or P325. Insulin and glucose tolerance also remained relatively stable. Insulin tolerance tests (Fig. 5B–E) generated similar AUC measurements at P129 and P275, although HFDP21+ females had the lowest insulin sensitivity. CHOW females had the best insulin sensitivity with a robust response persisting at P275. Glucose tolerance (Fig. 5F and G) did not differ among groups at P290 with similar overall AUC levels to P88 (Figs. 2 and 3 and Supplementary Fig. 2), suggesting that glucose tolerance remained similar as females aged. Body weight (Fig. 5H) was not statistically different among groups at P316.

Figure 5

Glucose homeostasis remains stable as females age. A: Plasma insulin levels in females at P149 and P325. Insulin tolerance test and AUC in females at P129 (B and C) and P275 (D and E). F and G: Oral glucose tolerance test and AUC in females at P290. H: Body weight in females at P316; note that only three HFDP35+ females and four HFDP2–21,P35+ females remained at this age. *HFDP21+ > CHOW, P < 0.05; †HFDP2–21,P35+ > CHOW, P < 0.05; ‡HFDP2–35 > CHOW, P < 0.05; §HFDP2–21,P35+ > HFDP2–35, P < 0.05.

Liver Steatosis, Dysfunctional Adipose Tissue, and Islet Hyperplasia in Aged Females

At P325, higher liver weight and extensive liver steatosis were seen in females maintained on HFD postpuberty (i.e., post-P35) and highest in HFDP2–21,P35+ females that were exposed to chow diet for 2 weeks during puberty (Fig. 6A–D). Immune cell clusters were observed adjacent to veins within the liver in some mice (Fig. 6D and Supplementary Table 1), including those with mild or absent steatosis and all HFDP21–35 mice (i.e., HFD exposure limited to the peripubertal period). Leptin levels were highest in HFDP2–21,P35+ females (Fig. 6E). In P325 females, mWAT weight did not vary among groups (Fig. 6F). However, gWAT weight (Fig. 6G) was surprisingly lower in HFDP21+, HFDP2–21,P35+, and HFDP2+ females compared with CHOW females and generally lower in females consuming HFD postpuberty (i.e., post-P35). This lower gWAT weight was associated with gross abnormalities of fat pads (Supplementary Table 1) with a yellowish appearance as opposed to the typical white appearance. This was mainly observed in gWAT, but some mice also exhibited similar abnormalities in mWAT and perirenal WAT. Histological examination of abnormal gWAT by Masson’s trichrome staining (Fig. 6H) revealed extensive fibrosis together with some enlarged adipocytes. H-E examination of P325 female pancreas revealed a surprisingly high area covered by islets relative to acinar tissue in some mice (Fig. 6I). Islet architecture appeared normal with similarly robust insulin and glucagon staining among groups in P325 females (Fig. 6J and K). Analysis of islet mass revealed no significant differences among groups (Fig. 6L); however, some mice had remarkably high islet mass (up to 76 mg in an HFDP2–21 mouse), although this was not consistent within treatment group. There was, however, a strong positive correlation between islet mass and AUC during insulin tolerance tests, both at P129 (r = 0.578; P < 0.0001) and P275 (r = 0.585; P < 0.0001). Peri-insulitis was observed in some islets and was observed in all but the HFDP35+ group (Fig. 6M and Supplementary Fig. 3A), although only three mice remained in this group. Degree of insulitis was assigned a grade of 0 to 4, with no difference among treatment groups by grade (Supplementary Fig. 3B). However, grouping together all islets with any degree of insulitis together revealed highest levels in the HFDP21–35 group (Fig. 6N), which were exposed to HFD only during 2 weeks peripuberty. Percent insulitis was positively correlated to spleen weight even when normalized to body weight (r = 0.471; P = 0.002), and parallels were seen between treatment groups exhibiting immune cell accumulation in the liver and pancreas (Supplementary Table 1), suggesting a systemic inflammation. Although insulitis did not manifest as diabetes in females, we questioned whether Swiss Webster males may be prone to autoimmune destruction of β-cells despite not previously observing immune infiltration in male Swiss Webster islets. However, we did not observe cell proliferation of male Swiss Webster splenocytes in response to insulin. Furthermore, administration of cyclophosphamide, at a dose that induces diabetes in NOD mice (29), did not induce diabetes in Swiss Webster males (Supplementary Fig. 4).