Synergistic effects of leucine and resveratrol on insulin sensitivity and fat metabolism in adipocytes and mice

Abstract

Background: Sirtuins are important regulators of glucose and fat metabolism, and sirtuin activation has been proposed as a therapeutic target for insulin resistance and diabetes. We have shown leucine to increase mitochondrial biogenesis and fat oxidation via Sirt1 dependent pathways. Resveratrol is a widely recognized activator of Sirt; however, the biologically-effective high concentrations used in cell and animal studies are generally impractical or difficult to achieve in humans. Accordingly, we sought to determine whether leucine would exhibit synergy with low levels of resveratrol on sirtuin-dependent outcomes in adipocytes and in diet-induced obese (DIO) mice.

Methods: 3T3-L1 mouse adipocytes were treated with Leucine (0.5 mM), β-hydroxy-β-methyl butyrate (HMB) (5 μM) or Resveratrol (200 nM) alone or in combination. In addition, diet-induced obese mice were treated for 6-weeks with low (2 g/kg diet) or high (10 g/kg diet) dose HMB, Leucine (24 g/kg diet; 200% of normal level) or low (12.5 mg/kg diet) or high (225 mg/kg diet) dose resveratrol, alone or as combination with leucine-resveratrol or HMB-resveratrol.

Results: Fatty acid oxidation, AMPK, Sirt1 and Sirt3 activity in 3T3-L1 adipocytes and in muscle cells, were significantly increased by the combinations compared to the individual treatments. Similarly, 6-week feeding of low-dose resveratrol combined with either leucine or its metabolite HMB to DIO mice increased adipose Sirt1 activity, muscle glucose and palmitate uptake (measured via PET/CT), insulin sensitivity (HOMAIR), improved inflammatory stress biomarkers (CRP, IL-6, MCP-1, adiponectin) and reduced adiposity comparable to the effects of high dose resveratrol, while low-dose resveratrol exerted no independent effect.

Conclusion: These data demonstrate that either leucine or its metabolite HMB may be combined with a low concentration of resveratrol to exert synergistic effects on Sirt1-dependent outcomes; this may result in more practical dosing of resveratrol in the management of obesity, insulin-resistance and diabetes.

Figure 1

Leucine and HMB Synergize with Resvetratrol in activation of Sirt1 and Sirt 3 activity. a) Sirt1 in Muscle Cells. C2C12 muscle cells were incubated with indicated treatments under low glucose (5 mM) conditions and Sirt1 activity was measured. Data are expressed as mean ± SE (n = 7 to 9) and are expressed as % change from control; control = 1085 ± 41 AFU/mg protein. * indicates significant difference compared to control (p < 0.04), ** indicates significant difference compared to control, leucine and HMB (p < 0.01). b) Sirt1 in Adipocytes. 3T3-L1 mouse adipocytes were incubated with indicated treatments under low glucose (5 mM) conditions and Sirt1 activity was measured. Data are expressed as mean ± SE (n = 7 to 9) and are expressed as % change from control; control = 759 ± 63 AFU/mg protein. * indicates significant difference compared to control (p < 0.05), ** indicates significant difference compared to control, leucine and HMB (p < 0.01).c) Sirt3 in Muscle Cells. C2C12 muscle cells were incubated with indicated treatments under low glucose (5 mM) conditions and Sirt1 activity was measured. Data are expressed as mean ± SE (n = 7 to 9) and are expressed as % change from control; control = 410 ± 57 AFU/mg protein. * indicates significant difference compared to control (p < 0.03), ** indicates significant difference compared to control, leucine and HMB (p < 0.02). d) Sirt3 in Adipocytes. 3T3-L1 mouse adipocytes were incubated with indicated treatments for 4 hours under low glucose (5 mM) conditions . Mitochondrial protein was isolated and Sirt3 activity was measured. Data are presented as mean ± SE (n = 6) and are expressed as % change from control; control = 507.8 ± 20.5 AFU/mg protein. * indicates significant difference compared to control (p = 0.03).

Figure 2

Leucine and HMB Synergize with Resveratrol to stimulate AMPK activity in 3T3L1 adipocytes. 3T3-L1 mouse adipocytes were incubated with indicated treatments for 24 hours and AMPK activity was measured. Data are presented as mean ± SE (n = 4). ^a indicates significant difference to control (p < 0.04), ^b indicates significant difference to control, HMB and leucine (p < 0.03), ^c indicates significant difference to control and leucine (p < 0.03).

Figure 3

Leucine and HMB Synergize with Resveratrol to Stimulate Fatty Acid Oxidation under a) Low Glucose Conditions and b) High Glucose Conditions. 3T3-L1 mouse adipocytes were incubated with indicated treatments for 4 hours under a) low glucose (5 mM) or b) high glucose (25 mM) conditions. Data are presented as mean ± SE (n = 6) and are expressed as % stimulation over control, where low glucose control = 193 ± 39 cpm/ng DNA and high glucose control = 302 ± 24 cpm/ng DNA. Stars above the bars indicate significant difference * compared to control (p < 0.05), ** compared to control, Leucine, and HMB (p < 0.005).

Figure 4

Resveratrol-HMB synergy in glucose uptake using FDG-PET. Mice were fed a high fat-diet with indicated treatments for 6 weeks. At the end of the treatment period, Fluorine-18-deoxy-glucose (FDG) or palmitate PET/CT scans were performed to measure whole body glucose (a) or fat uptake (b). Representative images of control diet group and resveratrol/low HMB diet group are shown

Figure 5

Effects of resveratrol, leucine and HMB on adipose tissue Sirt1 activity in diet-induced obese mice. Mice were fed a high fat-diet with indicated treatments for 6 weeks. At the end of the treatment period, Sirt1 activity in adipose tissue was measured. Data are presented as means ± SE (n = 9 to 10). Stars above the bars indicate significant difference compared to control (p < 0.02).