Resveratrol potentiates rapamycin to prevent hyperinsulinemia and obesity in male mice on high fat diet

Abstract

High doses of rapamycin, an antiaging agent, can prevent obesity in mice on high fat diet (HFD). Obesity is usually associated with hyperinsulinemia. Here, we showed that rapamycin given orally, at doses that did not affect weight gain in male mice on HFD, tended to decrease fasting insulin levels. Addition of resveratrol, which alone did not affect insulin levels, potentiated the effect of rapamycin, so that the combination decreased obesity and prevented hyperinsulinemia. Neither rapamycin nor resveratrol, and their combination affected fasting levels of glucose (despite lowering insulin levels), implying that the combination might prevent insulin resistance. We and others previously reported that resveratrol at high doses inhibited the mTOR (Target of Rapamycin) pathway in cell culture. Yet, as we confirmed here, this effect was observed only at super-pharmacological concentrations. At pharmacological concentrations, resveratrol did not exert 'rapamycin-like effects' on cellular senescence and did not inhibit the mTOR pathway in vitro, indicating nonoverlapping therapeutic mechanisms of actions of rapamycin and resveratrol in vivo. Although, like rapamycin, resveratrol decreased insulin-induced HIF-1-dependent transcription in cell culture, resveratrol did not inhibit mTOR at the same concentrations. Given distinct mechanisms of action of rapamycin and resveratrol at clinically relevant doses, their combination warrants further investigation as a potential antiaging, antiobesity and antidiabetic modality.

Figure 1

Body weight of mice on HFD with or without treatment. Initial body weight of each mouse is taken for 100 percent. Change of the weight was determined once a week. 1: RD; 2: HFD; 3: HFD+resv (resveratrol); 4: HFD+Rapa (rapamycin); 5: HFD+Re+Ra (+resveratrol+rapamycin in combination). Data present mean±S.E.M. Statistically significant differences were assessed using Student's t-test

Figure 2

Effects of HFD and rapamycin on body weight: comparison of male and female mice. Body weight of each mouse was determined at the beginning of the experiment (0 weeks) and 13 weeks after the initiation of HFD, and treatment with rapamycin in females and males of similar age (42 and 38.3 weeks, respectively). Data present mean±S.E.M. Statistically significant differences were assessed using Student's t-test

Figure 3

Blood levels of insulin (a), IGF-1 (b), glucose (c) and triglycerides (d) at the end of treatment. Mice onRD or HFD were treated with resveratrol, rapamycin or a combination of resveratrol and rapamycin for 13 weeks, as described in the legend of Figure 1. 1: RD; 2: HFD; 3: HFD+resv (resveratrol); 4: HFD+Rapa (rapamycin); 5: HFD+Re+Ra (+resveratrol+rapamycin in combination). Fasting levels of insulin, glucose, triglycerides and IGF-1 in blood serum were determined at the end of the treatment. Data present mean±S.E.M for each group of mice. Statistically significant differences were assessed using Student's t-test

Figure 4

Correlation between insulin and weight ((a) untreated groups and (b) all groups) and insulin and glucose (c) in individual mice at the end of treatment. 1: RD; 2: HFD; 3: HFD+resv (resveratrol); 4: HFD+Rapa (rapamycin); 5: HFD+Re+Ra (+resveratrol+rapamycin in combination). Correlation analyses Pearson r coefficient and P value (two tailed) were performed using GraphPad Prism version 5.00 for Windows

Figure 5

Effects of resveratrol on MTOR-dependent induction of HIF-1-responsive transcription in RPE cells. (a), (c): immunoblot analysis. RPE cells were plated in 10% serum-MEM, and the next day, the medium was changed for serum-free MEM for one day before treatment. In serum-free MEM cells were treated with indicated concentrations of resveratrol followed by either insulin (a) or serum (c). Then the cells were lysed and immunoblot for pS6 and S6 was performed. (b), (d): HIF-1-responsive luciferase induction. Cells were transfected with HRE -Luc (HIF-responsive-luciferase construct) as described previously. The next day, cells were treated with were treated with indicated concentrations of resveratrol followed by either insulin (b) or serum (d). After 16 h, cells were lysed and luciferase activity was measured

Figure 6

Effects of resveratrol on p21-induced senescence and ‘chronological senescence' in HT-p21 cells. a, b: Effect of resveratrol on senescence induced by p21 induction in HT-p21 cells. (a). Replicative/regenerative potential. HT-p21 cells were treated with IPTG in the presence or absence of resveratrol and/or rapamycin at 5 and 500 nℳ for 3 days, and then cells were washed and allow to regrow for 7 days. Colonies were stained with Crystal violet. Data present mean±STD from triplicate wells. b. HT-p21 cells were treated with resveratrol, rapamycin or resveratrol+IPTG for 24 h. Immunoblotting was performed with the indicated antibodies c. Effect of resveratrol or its combination with rapamycin on ‘chronological senescence' of HT-p21 cells. Cells were plated in high density in the presence or absence of indicated concentrations of resveratrol (Resv) and/or rapamycin (Rap). Rap in nM and Resv in μM. After 4 days, cells were trypsinized and an aliquots of cells were replated and allowed to regrow. After 6 days cells were counted. Data present mean±S.E.M. from triplicate wells d. HT-p21 cells were treated as in C. Lactate concentration was determined in conditioned medium on Day 3. Data present mean±S.E.M. from triplicate wells