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. 2012 Nov 15;303(10):E1234-44.
doi: 10.1152/ajpendo.00198.2012. Epub 2012 Sep 11.

Leucine supplementation increases SIRT1 expression and prevents mitochondrial dysfunction and metabolic disorders in high-fat diet-induced obese mice

Hongliang Li  1 , Mingjiang Xu, Jiyeon Lee, Chaoyong He, Zhonglin Xie
Affiliations

Leucine supplementation increases SIRT1 expression and prevents mitochondrial dysfunction and metabolic disorders in high-fat diet-induced obese mice

Hongliang Li et al. Am J Physiol Endocrinol Metab. .

Abstract

Leucine supplementation has been shown to prevent high-fat diet (HFD)-induced obesity, hyperglycemia, and dyslipidemia in animal models, but the underlying mechanisms are not fully understood. Recent studies suggest that activation of Sirtuin 1 (SIRT1) is an important mechanism to maintain energy and metabolic homeostasis. We therefore examined the involvement of SIRT1 in leucine supplementation-prevented obesity and insulin resistance. To accomplish this goal, male C57BL/6J mice were fed normal diet or HFD, supplemented with or without leucine. After 2 mo of treatment, alterations in SIRT1 expression, insulin signaling, and energy metabolism were analyzed. Eight weeks of HFD induced obesity, fatty liver, mitochondrial dysfunction, hyperglycemia, and insulin resistance in mice. Addition of leucine to HFD correlated with increased expression of SIRT1 and NAMPT (nicotinamide phosphoribosyltransferase) as well as higher intracellular NAD(+) levels, which decreased acetylation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) and forkhead box O1 (FoxO1). The deacetylation of PGC1α may contribute to upregulation of genes controlling mitochondrial biogenesis and fatty acid oxidation, thereby improving mitochondrial function and preventing HFD-induced obesity in mice. Moreover, decreased acetylation of FoxO1 was accompanied by decreased expression of pseudokinase tribble 3 (TRB3) and reduced the association between TRB3 and Akt, which enhanced insulin sensitivity and improved glucose metabolism. Finally, transfection of dominant negative AMPK prevented activation of SIRT1 signaling in HFD-Leu mice. These data suggest that increased expression of SIRT1 after leucine supplementation may lead to reduced acetylation of PGC1α and FoxO1, which is associated with attenuation of HFD-induced mitochondrial dysfunction, insulin resistance, and obesity.

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Figures

Fig. 1.
Fig. 1.
Leucine supplementation prevents high-fat diet (HFD)-induced obesity. Mice were fed normal diet (ND) or HFD, supplemented with/without leucine (Leu, 1.5 g/100 ml in drinking water) for 8 wk. A: leucine intake was calculated from the amount of drinking water in individually caged mice; n = 30 in each group. *P < 0.05 vs. ND or HFD. B: serum leucine concentrations were analyzed as described in materials and methods; n = 10 in each group. *P < 0.05 vs. ND or HFD. C: daily caloric intake was calculated from the amount of ingested food in individually caged mice; n = 30 in each group. *P < 0.05 vs. ND or leucine. D: body weights were monitored at weeks indicated; n = 30 in each group. *P < 0.05. E: subcutaneous and visceral fat was measured after 8 wk of treatment; n = 10 in each group. *P < 0.05 vs. ND or Leu; †P < 0.05 HFD-Leu vs. HFD.
Fig. 2.
Fig. 2.
Dietary leucine regulates sirtuin 1 (SIRT1) signaling and NAD+ levels in liver. A: liver homogenates were subjected to Western blot (WB) analysis using an anti-SIRT1 antibody (n = 5). *P < 0.05 vs. ND or HFD. B: real-time PCR analysis of SIRT1 mRNA in liver. C: liver homogenates were subjected to WB analysis using an anti-NAMPT (nicotinamide phosphoribosyltransferase) antibody (n = 5). *P < 0.05 vs. ND; †P < 0.05 vs. HFD. D: NAD+/NADH ratio in hepatic tissues was determined as described in materials and methods. E and F: liver homogenates were immunoprecipitated (IP) by anti-PGC1α (PPARγ coactivator 1α; E) or anti-FoxO1 (forkhead box O1; F) antibody, and acetylated lysine was detected by WB analysis (n = 5). *P < 0.05 vs. HFD.
Fig. 3.
Fig. 3.
Effects of HFD and leucine on hepatic mitochondrial biogenesis and lipid accumulation. A: RNA was extracted from hepatic tissue, and mRNA levels of PGC1α, nuclear respiratory factor 1 (NRF1), NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 (Ndufs8), and mitochondrial (mt)DNA transcription factor A (mTFA) were analyzed by quantitative real-time PCR. B: mtDNA/nuclear DNA ratio in liver was determined by quantitative real-time PCR. C: liver citrate synthase (CS) activity was assayed as described in materials and methods. D: liver ATP levels. E: expression of mRNA controlling fatty acid oxidation (FAO), including carnitine palmitoyltransferase-1b (CPT-1b), medium-chain acyl-CoA dehydrogenase (MCAD) and PPARα in liver was measured by real-time PCR. F and G: plasma cholesterol (F) and triglyceride (G) levels were measured after 16 h of fasting. H: liver lipids were extracted as described in materials and methods, and hepatic triglyceride levels were measured using a commercial kit; n = 5. *P < 0.05 vs. HFD. I: liver sample was embedded in Tissue-Tek O.C.T. compound, and frozen sections were prepared for Oil red O staining. Three mouse livers per group were analyzed. J: immunohistochemical analysis of 3-nitrotyrosine formation in liver from HFD and HFD-Leu mice. Three mouse livers per group were analyzed.
Fig. 4.
Fig. 4.
Effects of dietary leucine on SIRT1 signaling and mitochondrial biogenesis in brown adipose tissue (BAT). A: BAT homogenates were subjected to WB analysis using an anti-SIRT1 antibody, total RNA was extracted from BAT, and mRNA levels of SIRT1 were analyzed by quantitative real-time PCR (n = 5). *P < 0.05 vs. ND or HFD. B: BAT homogenates were subjected to WB analysis using an anti-NAMPT antibody (n = 5). *P < 0.05 vs. ND; †P < 0.05 vs. HFD. C and D: PGC1α or FoxO1 was immunoprecipitated (IP), and WB was performed using indicated antibodies. E: expression of genes related to mitochondrial biogenesis in BAT. F: mtDNA/nuclear DNA ratio in BAT was determined by quantitative real-time PCR. G: BAT CS activity. H: ATP levels in BAT. I: expression of mRNA controlling FAO (n = 5). *P < 0.05 vs. HFD. J: BAT was formalin fixed and embedded in paraffin; sections were stained with hematoxylin and eosin. Three samples in each group were analyzed and showed similar results.
Fig. 5.
Fig. 5.
Effects of HFD and leucine on SIRT1 signaling and expression of FAO genes in skeletal muscle. A: homogenates of gastrocnemius muscle were subjected to WB analysis using an anti-SIRT1 antibody (n = 5). *P < 0.05 vs. ND or HFD. B: RNA was extracted from gastrocnemius muscle, and quantitative real-time PCR was performed to determine SIRT1 mRNA levels. C: homogenates of gastrocnemius muscle were subjected to WB analysis to detect NAMPT expression (n = 5). *P < 0.05 vs. ND; †P < 0.05 vs. HFD. D: PGC1α was immunoprecipitated and Western blotted with an acetylated lysine antibody. E: muscle homogenates were immunoprecipitated by anti-FoxO1 antibody, and acetylated lysine was detected by WB analysis. F: relative mRNA expression levels of genes related to FAO (n = 5 in each group). *P < 0.05 vs. HFD.
Fig. 6.
Fig. 6.
Inhibition of AMPK prevents activation of SIRT1 signaling in HFD-Leu mice. Mice were fed HFD supplemented with or without leucine for 8 wk. After 4 wk of treatment, mice were randomly assigned to be transfected with adenovirus encoding GFP or dominant negative (DN)-AMPK. A: expressions of phospho-AMPK (Thr172), phospho-ACC (Ser79), and SIRT1 in liver were analyzed by WB. B: liver homogenates were subjected to WB to detect expression of NAMPT. C: NAD+/NADH ratio in hepatic tissues was determined as described in materials and methods (n = 5 in each group). *P < 0.05 vs. HFD.
Fig. 7.
Fig. 7.
Leucine suppresses pseudokinase tribble 3 (TRB3) expression and improves insulin signaling in the liver. A: Expression of TRB3 in liver was analyzed by WB using an anti-TRB3 antibody (n = 5 per group). *P < 0.05 vs. HFD. B: liver homogenates were immunoprecipitated with anti-TRB3 antibody and then probed with an anti-Akt antibody by WB (n = 5 in each group). *P < 0.05 vs. HFD. C: liver homogenates were subjected to WB analysis to determine expression of total and phospho-Akt (Ser437; n = 5 per group). *P < 0.05 vs. ND; #P < 0.05 vs. ND/Ins; †P < 0.05 vs. HFD/Ins.
Fig. 8.
Fig. 8.
Leucine supplementation improves insulin sensitivity in mice subjected to HFD. A: fasting blood glucose levels were measured in tail vein blood sample using a glucometer (n = 8 in each group). *P < 0.05 vs. ND; †P < 0.05 vs. HFD. B: an intraperitoneal glucose tolerance test was performed in mice after an overnight fast (n = 5 per group). *P < 0.05. C: area under the curve (AUC) was calculated for each dietary condition (n = 5 per group). *P < 0.05 vs. ND; †P < 0.05 vs. HFD. D and E: hyperinsulinemic-euglycemic clamps were performed over a 120-min period as described in materials and methods. Insulin sensitivity was evaluated through the average glucose infusion rate at equilibrium in a hyperinsulinemic-euglycemic clamp (18 mU insulin·min−1·kg−1; n = 5 in each group). *P < 0.05 vs. ND; †P < 0.05 vs. HFD.
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