Caloric restriction chronically impairs metabolic programming in mice

Abstract

Although obesity rates are rapidly rising, caloric restriction remains one of the few safe therapies. Here we tested the hypothesis that obesity-associated disorders are caused by increased adipose tissue as opposed to excess dietary lipids. Fat mass (FM) of lean C57B6 mice fed a high-fat diet (HFD; FMC mice) was "clamped" to match the FM of mice maintained on a low-fat diet (standard diet [SD] mice). FMC mice displayed improved glucose and insulin tolerance as compared with ad libitum HFD mice (P < 0.001) or SD mice (P < 0.05). These improvements were associated with fewer signs of inflammation, consistent with the less-impaired metabolism. In follow-up studies, diet-induced obese mice were food restricted for 5 weeks to achieve FM levels identical with those of age-matched SD mice. Previously, obese mice exhibited improved glucose and insulin tolerance but showed markedly increased fasting-induced hyperphagia (P < 0.001). When mice were given ad libitum access to the HFD, the hyperphagia of these mice led to accelerated body weight gain as compared with otherwise matched controls without a history of obesity. These results suggest that although caloric restriction on a HFD provides metabolic benefits, maintaining those benefits may require lifelong continuation, at least in individuals with a history of obesity.

FIG. 1.

A: Study design and FM clamping. B: FM was measured by nuclear magnetic resonance technology and did not differ between SD and FMC mice. C: BW of SD, HFD fed, and FMC mice was measured daily. Mice from the SD and HFD group were fed with SD or HFD ad libitum. Mice from the FMC group were fed defined amounts of HFD to match FM to the SD group. D: Absolute food intake was lowest in FMC mice (P < 0.001). E: Caloric intake was similar in FMC and SD mice and highest in mice of the HFD group. All mice are male (n = 16 per group). All data are represented as mean ± SEM. ITT, insulin tolerance test; FI, food intake.

FIG. 2.

FM clamping normalizes energy expenditure. Absolute (A) and average energy expenditure (B), RQ (C), and locomotor activity (D) of SD, HFD, and FMC mice was measured hourly. Mice from the SD and HFD group were fed with SD or HFD ad libitum. Mice from the FMC group were fed defined amounts of HFD to match FM to the SD group. All mice are male (n = 16 per group). All data are represented as mean ± SEM. ***P < 0.001.

FIG. 3.

FM clamping improves glucose tolerance and insulin sensitivity. Glucose tolerance was assessed at week 13 via intraperitoneal GTT. After 6 h of fasting mice were injected with 2.0 g/kg glucose. FMC mice had significantly lower blood glucose excursion (A) and area under the curve (AUC; C) compared with lean SD and obese HFD mice. Insulin tolerance was assessed at week 13 via intraperitoneal insulin challenge (1 unit/kg). Insulin tolerance was significantly improved in FMC mice as compared with HFD mice when assessed by either blood glucose excursion (B) or rate of disappearance (K_d) over the initial 30 min (D). All data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

FIG. 4.

FM clamping rescues hyperinsulinemia and hyperleptinemia and normalizes expression of inflammatory markers. Plasma hormone levels measured after 16-h fast at week 14 (A–C) are shown. All mice are male (n = 14 to 15 per group). Expression of inflammatory markers (D), liver (E), epididymal white adipose tissue (eWAT), and (F) hypothalamic tissues was suppressed in FMC mice. All mice are male (n = 8 per group). All data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

FIG. 5.

CR restores leanness independent of the diet. A: BW of mice that were fed with SD or HFD ad libitum in comparison with calorically restricted mice on SD or HFD. The SD and HFD groups were fed ad libitum, whereas the groups CR-SD and CR-HFD were calorically restricted and fed 50% of calories (given as SD or HFD) that were consumed on average in the SD group. FMC mice were fed defined amounts of HFD to match FM to the SD group. FM (B) and lean mass (C) curves of mice fed ad libitum or that were calorically restricted are shown. D: intraperitoneal GTT conducted after 6 h of fasting at day 45 of CR. E: Area-under-the-curve (AUC) analysis of GTT. F: C-peptide secretion at 30 min after intraperitoneal glucose challenge. All mice are male (n = 10–15 per group). All data are represented as mean ± SEM. Lowercase letters above bars (A–F) denote statistically similar (P > 0.05) groups.

FIG. 6.

CR normalizes plasma cholesterol. Plasma leptin (A) and insulin levels (B) measured after 16-h fast at day 44 are shown. Total plasma cholesterol (C), cholesterol profile (D), and total plasma triglycerides (E) of mice that were fed with SD or HFD ad libitum in comparison with FMC or CR-SD or CR-HFD are shown. All mice are male (n = 6–10 per group). All data are represented as mean ± SEM. *P < 0.05; ***P < 0.001.

FIG. 7.

CR induces persistent hyperphagia. A: At day 45 CR was ended and mice of all groups were fasted for 24 h. After the fast mice were given free access to SD or HFD and food intake was measured after 15 h. B: After the fasting-induced hyperphagia experiment mice were allowed to eat ad libitum to study rebound of obesity. Additional control groups (FMC-SD and previously SD-fed ad libitum and switched to HFD ad libitum [SD-HFD]) were introduced. FMC-SD mice were previously fed HFD and were switched to SD, whereas SD-HFD mice were previously fed SD and were switched to HFD. C: BW curves of the ad libitum eating period. D: FM during the period of ad libitum eating. All mice are male (n = 7 to 8 per group). All data are represented as mean ± SEM. Lowercase letters denote statistically similar (P > 0.05) groups.

FIG. 8.

Rebound from CR induces glucose intolerance. Intraperitoneal GTT (A) and area-under-the-curve (AUC) analysis (B) are shown. GTT was conducted after 6 h of fasting following 58 days of rebound. All mice are male (n = 6–10 per group). All data are represented as mean ± SEM. **P < 0.01.