BackgroundSirtuins 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.Methods3T3-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.ResultsFatty 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.ConclusionThese 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.

Leucine stimulates tissue protein synthesis and may also attenuate adiposity by increasing fatty acid oxidation and mitochondrial biogenesis in muscle and adipocytes. Accordingly, the effects of a nutraceutical containing 2.25 g leucine and 30 mg pyridoxine (Vitamin B6) (NuFit active blend) were tested in cell culture and in a clinical trial. 3T3L1 adipocytes were treated with leucine (0.25 mM or 0.5 mM) and/or Pyridoxal Phosphate (PLP) (50 nM or 100 nM) for 48 h. For the clinical trial, twenty overweight or obese subjects received the NuFit active blend or placebo three times/day for 4 weeks without energy restriction. Leucine decreased fatty acid synthase (FAS) expression and triglyceride content in adipocytes, and PLP addition significantly augmented this effect. Administration of NuFit active blend in the clinical trial increased fat oxidation by 33.6 g/day (p < 0.04), decreased respiratory quotient, improved HOMAIR, reduced oxidative and inflammatory biomarkers (plasma MDA, 8-isoprostane-F2α, TNF-α, C-reactive protein), and increased the anti-inflammatory marker adiponectin. These data indicate that the NuFit active blend significantly increased fat oxidation and insulin sensitivity, and reduced oxidative and inflammatory stress. Therefore, the NuFit active blend appears to be a useful nutraceutical in the management of obesity and associated co-morbidities.

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The AMPK-Sirt1 pathway is an important regulator of energy metabolism and therefore a potential target for prevention and therapy of metabolic diseases. We recently demonstrated leucine and its metabolite β-hydroxy-β-methylbutyrate (HMB) to synergize with low-dose resveratrol (200 nM) to activate sirtuin signaling and stimulate energy metabolism. Here we show that leucine exerts a direct effect on Sirt1 kinetics, reducing its Km for NAD+ by >50% and enabling low doses of resveratrol to further activate the enzyme (p = 0.012). To test which structure elements of resveratrol are necessary for synergy, we assessed potential synergy of structurally similar and dissimilar polyphenols as well as other compounds converging on the same pathways with leucine using fatty acid oxidation (FAO) as screening tool. Dose-response curves for FAO were constructed and the highest non-effective dose (typically 1–10 nM) was used with either leucine (0.5 mM) or HMB (5 µM) to treat adipocytes and myotubes for 24 h. Significant synergy was detected for stilbenes with FAO increase in adipocytes by 60–70% (p<0.05) and in myotubes >2000% (p<0.01). Sirt1 and AMPK activities were stimulated by ∼65% (p<0.001) and ∼50% (p<0.03), respectively. Similarly, hydroxycinnamic acids and derivatives (chlorogenic, cinnamic, and ferulic acids) combined with leucine/HMB increased FAO (300–1300%, p<0.01), AMPK activity (50–150%, p<0.01), and Sirt1 activity (∼70%, p<0.001). In contrast, more complex polyphenol structures, such as ellagic acid and epigallocatechin gallate required higher concentrations (>1 µM) and exhibited little or no synergy. Thus, the six-carbon ring structure bound to a carboxylic group seems to be a necessary element for leucine/HMB synergy with other stilbenes and hydroxycinnamic acids to stimulate AMPK/Sirt1 dependent FAO; these effects occur at concentrations that produce no independent effects and are readily achievable via oral administration.

BackgroundRecent data from this laboratory suggest that components of dairy foods may serve as activators of SIRT1 (Silent Information Regulator Transcript 1), and thereby participate in regulation of glucose and lipid metabolism. In this study, an ex-vivo/in-vitro approach was used to examine the integrated effects of dairy diets on SIRT1 activation in two key target tissues (adipose and muscle tissue).MethodsSerum from overweight and obese subjects fed low or high dairy diets for 28 days was added to culture medium (similar to conditioned media) to treat cultured adipocytes and muscle cells for 48 hours.ResultsTreatment with high dairy group conditioned media resulted in 40% increased SIRT1 gene expression in both tissues (p < 0.01) and 13% increased enzyme activity in adipose tissue compared to baseline. This was associated with increased gene expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF1), cytochrome oxidase c subunit 7 (Cox 7), NADH dehydrogenase and uncoupling protein 2 (UCP2) in adipocytes as well as uncoupling protein 3 (UCP3), NRF1 and Cox 7 in muscle cells (p < 0.05). Further, direct incubation of physiological concentrations of leucine and its metabolites α-Ketoisocaproic acid (KIC) and β-hydroxy-methylbuteric acid (HMB) with recombinant human SIRT1 enzyme resulted in 30 to 50% increase of SIRT1 activity (p < 0.05).ConclusionsThese data indicate that dairy consumption leads to systemic effects, which may promote mitochondrial biogenesis in key target tissues such as muscle and adipose tissue both by direct activation of SIRT1 as well as by SIRT1-independent pathways.