Leucine and Sildenafil Combination Therapy Reduces Body Weight and Metformin Enhances the Effect at Low Dose: A Randomized Controlled Trial.

BACKGROUND: This study evaluated the potential of activating the fuel-sensing enzymes Adenine monophosphate (AMP)-activated protein kinase and the deacetylase sirtuin1, to promote weight loss. We tested the efficacy of a fixed dose combination of the amino acid leucine and 2 well-characterized agents with established safety profiles to modulate energy metabolism and facilitate weight loss. STUDY QUESTION: Will a combination of l-leucine with low-dose metformin and sildenafil produce a novel synergistic interaction that reduces body weight? STUDY DESIGN: We conducted a 24-week randomized controlled trial evaluating the effect on weight loss of leucine 1.1 g and sildenafil 1.0 mg or 4.0 mg, with and without metformin 500 mg (Leu/Sil 1.0, Leu/Sil 4.0, Leu/Met/Sil 1.0, and Leu/Met/Sil 4.0 twice/day). We enrolled 267 participants who were 18-65 years of age without diabetes and with the body mass index (BMI) of 30-45 kg/m2. MEASURES AND OUTCOMES: The primary endpoint was percentage weight change after 24 weeks. Adverse events were evaluated. The primary analysis was performed using the perprotocol population analysis of covariance estimation. Subgroup analyses of patients residing above certain threshold limits at baseline and in populations at increased risk of obesity were assessed post-hoc as exploratory end points. RESULTS: Placebo-adjusted mean bodyweight reductions in the Leu/Met/Sil 1.0, Leu/Met/Sil 4.0, and Leu/Sil 4.0 groups were -1.99%, -1.69%, and -1.67% (P = 0.015, 0.035, and 0.036, respectively). The most common adverse events were gastrointestinal-related and occurred in the metformin-treated groups consistent with metformin treatment. In African Americans, Leu/Met/Sil 1.0 produced 5.4% mean weight loss. In participants with BMI <40 kg/m2 treated with Leu/Met/Sil 1.0, the weight loss increased to 2.84%, particularly in participants with baseline insulin ≥12mU/L (3.5%). CONCLUSIONS: Leu/Met/Sil 1.0 and 4.0 and Leu/Sil 4.0 reduced body weight, but Leu/Met/Sil 1.0 was associated with robust weight loss in African Americans, and individuals with BMI 30-39.9 kg/m2, especially participants with hyperinsulinemia.

Effect of a Leucine/Pyridoxine Nutraceutical on Caloric Intake and Body Composition of Obese Dogs Losing Weight.

The aim of this 29-week randomized, positively and negatively controlled study was to investigate whether a nutraceutical containing 1 g leucine and 13 mg pyridoxine can enhance weight loss while maintaining lean muscle mass in obese dogs. Twenty-four healthy, 2-year-old beagles were initially divided into obesification (n = 18) or ideal body weight groups (n = 6). After obesification, the 18 dogs were divided into three weight loss groups and fed one of the following over 12 weeks: nutraceutical with canned adult diet (CAD; ObN), placebo with CAD (ObP), or a canned therapeutic weight loss diet (WLD). Dogs in the ideal body weight (IBW) group were fed maintenance calorie requirements with CAD over 12 weeks. Based on MANOVA, ObN and WLD lost similar amounts of total weight (3.6 ± 0.9 vs. 4.4 ± 1.1 kg, respectively) and fat mass (3.1 ± 0.6 vs. 3.9 ± 0.8 kg, respectively) after 12 weeks of treatment, and more than ObP (1.1 ± 1.2 kg weight; 0.9 ± 1.0 kg fat; p < 0.0001). These data show the nutraceutical is a promising option for successful weight loss in dogs. Maintenance levels of CAD were able to induce weight loss without risk of hypo- or anorexia, or the need to switch diets or restrict energy intake.

Modulation of Energy Sensing by Leucine Synergy with Natural Sirtuin Activators: Effects on Health Span.

Sirt1 and 5' adenosine monophosphate-activated protein kinase (AMPK) are energy-sensing systems that work cooperatively and regulate mitochondrial biogenesis and fuel metabolism, and mediate, in part, the salutary effects of caloric restriction on lifespan and healthspan. We have shown that leucine activates Sirt1 and enables synergy with sirtuin co-activators. Resveratrol is a widely recognized activator of Sirt1; however, poor bioavailability and rapid metabolism limit effective clinical translation of promising animal data. However, we found that combining low resveratrol doses with leucine increased skeletal muscle and adipocyte Sirt1 activity, mitochondrial biogenesis and fatty acid oxidation; these effects result in increased lifespan and marked reductions in insulin resistance, inflammatory markers, body weight, and visceral adiposity in preclinical models. To translate these data to humans, we assessed the effects of resveratrol (50 mg)/leucine (1.11 g) on glucose dynamics in a 4-week placebo-controlled trial of 36 prediabetic subjects. Leucine-resveratrol reduced insulin resistance (homeostatic model assessment for insulin resistance) 33% with corresponding reductions in glucose and insulin area under the curve in oral glucose tolerance tests. We extended these concepts in preclinical studies using both direct Sirt1 activators and Sirt1 pathway activators. Low-dose (10 nM) NAD+ precursors (nicotinic acid, nicotinamide mononucleotide, and nicotinamide riboside) synergized with leucine to increase Sirt1 activity in adipocytes, hepatocytes, and muscle cells (30-100%, P < .01) and lifespan in Caenorhabditis elegans (25%, P = .025) and to significantly regress atherosclerotic lesion size and macrophage infiltration in a mouse model of atherosclerosis. Thus, synergistic activation of Sirt1 using leucine and a co-activator exerts pleiotropic effects impacting cardiometabolic endpoints.

Biphasic Effect of Sildenafil on Energy Sensing is Mediated by Phosphodiesterases 2 and 3 in Adipocytes and Hepatocytes.

Sirt1 (Sirtuin 1), AMPK (AMP-activated protein kinase), and eNOS (endothelial nitric oxide synthase) modulate hepatic energy metabolism and inflammation and play a major role in the development of NASH. Cyclic nucleotide phosphodiesterases (PDEs) play an important role in signal transduction by modulating intracellular levels of cyclic nucleotides. We previously found the PDE5 inhibitor sildenafil to synergize with leucine and leucine-metformin combinations in preclinical studies of NASH and obesity. However, efficacy is diminished at higher sildenafil concentrations. Herein, we have successfully modeled the U-shaped sildenafil dose-response in vitro and utilized this model to assess potential mechanisms of this dose-response relationship. Adipocytes and liver cells were treated with leucine (0.5 mM) and different concentrations of sildenafil (1 nM to 100 µM). cAMP, cGMP, and P-AMPK protein expression were used to demonstrate the biphasic response for increasing concentrations of sildenafil. The reversal with higher sildenafil levels was blunted by PDE2 inhibition. These data indicate that sildenafil-mediated increases in cGMP inhibits PDE3 at lower concentrations, which increases cAMP. However, further increases in cGMP from higher sildenafil concentrations activate PDE2 and consequently decrease cAMP, which demonstrates crosstalk between cAMP and cGMP via PDE2, PDE3, and PDE5. These changes in cAMP concentration are further reflected in downstream effects, including AMPK activation.

Randomized Controlled Trial of a Leucine-Metformin-Sildenafil Combination (NS-0200) on Weight and Metabolic Parameters.

OBJECTIVE: Leucine was previously demonstrated to allosterically activate mammalian sirtuin 1 and synergize with other sirtuin 1/AMP-activated protein kinase/nitric oxide pathway activators to modulate energy metabolism. The objective of this study was to evaluate the effects of a triple combination of leucine, metformin, and sildenafil (NS-0200) on body weight and obesity comorbidities in a phase 2 randomized trial. METHODS: A total of 91 subjects with obesity were randomized to placebo, low dose (1.1 g leucine/0.5 g metformin/0.5 mg sildenafil), or high dose (1.1 g leucine/0.5 g metformin/1.0 mg sildenafil) twice daily for 16 weeks. Seventy subjects completed the trial and met all a priori compliance criteria. Hypertensive (n = 35) and hypertriglyceridemic (n = 22) subcohorts were also analyzed. RESULTS: NS-0200 dose-responsively reduced weight; high dose reduced weight by 2.4 and 5.0 kg in the full and high-triglyceride cohorts, respectively (P < 0.0001). High-dose NS-0200 treatment also decreased blood pressure (-5.5 mm Hg diastolic pressure; P = 0.011), with greater effects among hypertensive subjects. NS-0200 also significantly reduced triglycerides and hemoglobin A1c. Significant improvement in ≥ 2 comorbidities was exhibited by 54% of subjects in the high-dose arm versus 5% of placebo subjects (P = 0.0009). Treatment-emergent adverse events did not significantly differ among groups. CONCLUSIONS: These data support further study of NS-0200 as a therapy for obesity and associated comorbidities.

Erratum: Leucine-nicotinic acid synergy stimulates AMPK/Sirt1 signaling and regulates lipid metabolism and lifespan in Caenorhabditis elegans, and hyperlipidemia and atherosclerosis in mice.

[This corrects the article on p. 33 in vol. 7, PMID: 28533928.].

Leucine-nicotinic acid synergy stimulates AMPK/Sirt1 signaling and regulates lipid metabolism and lifespan in Caenorhabditis elegans, and hyperlipidemia and atherosclerosis in mice.

BACKGROUND/AIMS: Nicotinic acid (NA), a lipid-lowering drug, serves as a source of NAD+, the cofactor for Sirt1. Leucine (Leu) stimulates the AMPK/Sirt1 axis and amplifies the effects of other AMPK/Sirt1 activating compounds. Therefore, we tested the interactive effects of leucine and low dose NA on AMPK/Sirt1 signaling and downstream effects of lipid metabolism in cell culture, C. elegans and mice. METHODS: LDL-receptor knockout mice were fed an atherogenic Western diet supplemented with leucine (24 g/kg diet) and sub-therapeutic NA combinations (50 mg/kg diet and 250 mg/kg diet) or low therapeutic NA (1000 mg/kg diet) for 8 weeks to evaluate markers of hyperlipidemia and atherosclerosis. RESULTS: NA-Leu increased P-AMPK and Sirt1 in adipocytes and myotubes. In C. elegans, NA-Leu increased P-AMPK and DAF-16 (FOXO), reduced lipid accumulation and increased median survival under mild oxidative stress conditions. In the mice, NA-Leu reduced total cholesterol, cholesterol esters, plasma triglycerides, atherosclerotic lesion size, lipid area, and aortic macrophage infiltration, similar to the therapeutic NA dose. CONCLUSION: Leu amplifies the effects of NA on lipid metabolism, hyperlipidemia and atherosclerosis in mice, at least in part by activation of the AMPK/Sirt1 axis. This combination may be a potential therapeutic alternative for hyperlipidemia and atherosclerosis.

Activation of the AMPK/Sirt1 pathway by a leucine-metformin combination increases insulin sensitivity in skeletal muscle, and stimulates glucose and lipid metabolism and increases life span in Caenorhabditis elegans.

BACKGROUND: We have previously shown leucine (Leu) to activate Sirt1 by lowering its KM for NAD+, thereby amplifying the effects of other sirtuin activators and improving insulin sensitivity. Metformin (Met) converges on this pathway both indirectly (via AMPK) and by direct activation of Sirt1, and we recently found Leu to synergize with Met to improve insulin sensitivity and glycemic control while achieving ~80% dose-reduction in diet-induced obese mice. Accordingly, we sought here to define the mechanism of this interaction. METHODS: Muscle cells C2C12 and liver cells HepG2 were used to test the effect of Met-Leu on Sirt1 activation. Caenorhabditis elegans was used for glucose utilization and life span studies. RESULTS: Leu (0.5mmol/L)+Met (50-100µmol/L) synergistically activated Sirt1 (p<0.001) at low (≤100µmol/L) NAD+ levels while Met exerted no independent effect. This was associated with an increase in AMPK and ACC, phosphorylation, and increased fatty acid oxidation, which was prevented by AMPK or Sirt inhibition or silencing. Met-Leu also increased P-IRS1/IRS1 and P-AKT/AKT and in insulin-independent glucose disposal in myotubes (~50%, p<0.002) evident within 30 min as well as a 60% reduction in insulin EC50. In addition, in HepG2 liver cells nuclear CREB regulated transcription coactivator 2 (CRTC2) protein expression and phosphorylation of glycogen synthase was decreased, while glycogen synthase kinase phosphorylation was increased indicating decreased gluconeogenesis and glycogen synthesis. We utilized C. elegans to assess the metabolic consequences of this interaction. Exposure to high glucose impaired glucose utilization and shortened life span by ~25%, while addition of Leu+Met to high glucose worms increased median and maximal life span by 29 and 15%, respectively (p=0.023), restored normal glucose utilization and increased fat oxidation ~two-fold (p<0.005), while metformin exerted no independent effect at any concentration (0.1-0.5mmol/L). CONCLUSION: Thus, Leu and Met synergize to enable Sirt1 activation at low NAD+ concentrations (typical of energy replete states). Sirt1 and AMPK activations are required for Met-Leu's full action, which result in improvements in energy metabolism and insulin sensitivity.

A Combination of Leucine, Metformin, and Sildenafil Treats Nonalcoholic Fatty Liver Disease and Steatohepatitis in Mice.

Sirt1, AMPK, and eNOS modulate hepatic energy metabolism and inflammation and are key players in the development of NASH. L-leucine, an allosteric Sirt1 activator, synergizes with low doses of metformin or sildenafil on the AMPK-eNOS-Sirt1 pathway to reverse mild NAFLD in preclinical mouse models. Here we tested a possible multicomponent synergy to yield greater therapeutic efficacy in NAFLD/NASH. Liver cells and macrophages or an atherogenic diet induced NASH mouse model was treated with two-way and three-way combinations. The three-way combination Sild-Met-Leu increased hepatic fatty acid oxidation and reduced lipogenic gene expression and inflammatory marker in vitro. In mice, Sild-Met-Leu reduced the diet induced increases of ALT, TGFß, PAI-1, IL1ß, and TNFα, hepatic collagen expression, and nearly completely reversed hepatocyte ballooning and triglyceride accumulation, while all two-way combinations had only modest effects. Therefore, these data provide preclinical evidence for therapeutic efficacy of Sild-Met-Leu in the treatment of NAFLD and NASH.

Interaction between metformin and leucine in reducing hyperlipidemia and hepatic lipid accumulation in diet-induced obese mice.

BACKGROUND: Leucine stimulates Sirt1 and AMPK signaling in vitro and in vivo. Since metformin converges on the same pathway, we have tested the ability of leucine to amplify the effects of metformin on AMPK-mediated hepatic lipid metabolism in diet-induced-obese insulin-resistant mice. METHODS: Mice were fed high leucine (24 g/kg diet) with or without sub-therapeutic levels of metformin (0.05-0.50 g/kg diet) or therapeutic levels of metformin (1.5 g/kg diet; ~300 mg/kg body weight). RESULTS: High-fat diet produced a 10-fold increase in inguinal fat pad weight and 25% increase in liver weight, histologically confirmed as steatosis. The leucine-metformin combinations reduced fat pad mass, normalized liver weight, liver and plasma lipids and inflammatory markers (interleukin 6, interleukin 1 beta, tumor necrosis factor alpha, monocyte chemotactic protein-1, C-reactive protein) comparable to the effects of therapeutic metformin. Moreover, the highest sub-therapeutic levels of metformin with leucine exerted significantly greater effects than therapeutic levels of metformin and fully reversed hepatic steatosis. These effects were mediated by upregulation of hepatic AMPK and associated changes in lipogenic gene expression (fatty acid synthase, stearoyl CoA desaturase, acetyl CoA carboxylase) in the liver. CONCLUSION: A low-dose leucine-metformin combination exerts comparable effects on adiposity to therapeutic doses of metformin and fully reverses hepatic steatosis in diet-induced-obese mice.

Interaction between leucine and phosphodiesterase 5 inhibition in modulating insulin sensitivity and lipid metabolism.

PURPOSE: Leucine activates SIRT1/AMP-activated protein kinase (AMPK) signaling and markedly potentiates the effects of other sirtuin and AMPK activators on insulin signaling and lipid metabolism. Phosphodiesterase 5 inhibition increases nitric oxide-cGMP signaling, which in turn exhibits a positive feedback loop with both SIRT1 and AMPK, thus amplifying peroxisome proliferator-activated receptor γ co-activator α (PGC1α)-mediated effects. METHODS: We evaluated potential synergy between leucine and PDE5i on insulin sensitivity and lipid metabolism in vitro and in diet-induced obese (DIO) mice. RESULTS: Leucine (0.5 mM) exhibited significant synergy with subtherapeutic doses (0.1-10 nM) of PDE5-inhibitors (sildenafil and icariin) on fat oxidation, nitric oxide production, and mitochondrial biogenesis in hepatocytes, adipocytes, and myotubes. Effects on insulin sensitivity, glycemic control, and lipid metabolism were then assessed in DIO-mice. DIO-mice exhibited fasting and postprandial hyperglycemia, insulin resistance, and hepatic steatosis, which were not affected by the addition of leucine (24 g/kg diet). However, the combination of leucine and a subtherapeutic dose of icariin (25 mg/kg diet) for 6 weeks reduced fasting glucose (38%, P<0.002), insulin (37%, P<0.05), area under the glucose tolerance curve (20%, P<0.01), and fully restored glucose response to exogenous insulin challenge. The combination also inhibited hepatic lipogenesis, stimulated hepatic and muscle fatty acid oxidation, suppressed hepatic inflammation, and reversed high-fat diet-induced steatosis. CONCLUSION: These robust improvements in insulin sensitivity, glycemic control, and lipid metabolism indicate therapeutic potential for leucine-PDE5 inhibitor combinations.

A combination of probiotics and whey proteins enhances anti-obesity effects of calcium and dairy products during nutritional energy restriction in aP2-agouti transgenic mice.

Lactobacillus rhamnosus GG, Lactobacillus paracasei TMC0409, Streptococcus thermophilus TMC1543 and whey proteins were used to prepare fermented milk. For the experiment aP2- agouti transgenic mice were pre-treated with a high-sucrose/high-fat diet for 6 weeks to induce obesity. The obese mice were fed a diet containing 1·2% Ca and either non-fat dried milk (NFDM) or probiotic-fermented milk (PFM) with nutritional energy restriction for 6 weeks. The animals were examined after the treatment for changes in body weight, fat pad weight, fatty acid synthase (FAS) activity, lypolysis, the expression levels of genes related to lipid metabolism, insulin sensitivity in adipocytes and skeletal muscle and the presence of biomarkers for oxidative and inflammatory stress in plasma. It was found that the PFM diet significantly reduced body weight, fat accumulation, and adipocyte FAS activity, and increased adipocyte lipolysis as compared with the effects of the NFDM diet (P<0·05). The adipose tissue gene expression of 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) was significantly suppressed in mice that were fed PFM as compared with those that were fed NFDM (P<0·05). PFM caused a greater up-regulation of skeletal muscle PPARα, PPARδ, uncoupling protein 3 (UCP3) and GLUT4 expression and a significant decrease in the plasma concentration of insulin, malondialdehyde, TNF-α, monocyte chemotactic protein-1 and C-reactive protein as compared with the effects of NFDM (P<0·05). Fermentation of milk with selected probiotics and supplementation of milk with whey proteins may thus enhance anti-obesity effects of Ca and dairy products by the suppression of adipose tissue lipogenesis, activation of fat oxidation in skeletal muscle and reduction of oxidative and inflammatory stress.

Leucine amplifies the effects of metformin on insulin sensitivity and glycemic control in diet-induced obese mice.

BACKGROUND AND OBJECTIVE: The Sirt1/AMPK signaling pathway is a key sensor of energy status and regulates glucose and lipid metabolism. Leucine (Leu) activates Sirt1 by lowering its Km for NAD(+) and potentiates other sirtuin/AMPK-activators, resulting in improvement of insulin sensitivity. Since metformin (Met) converges on this pathway, we hypothesized that leucine would amplify its gluco-regulatory effects. MATERIALS AND METHODS: The effects of Leu (24 g/kg diet)+Met (0.05-0.5 g/kg diet) combinations were compared to standard therapeutic Met (1.5 g/kg diet; ~300 mg/kg BW) on glycemic control in high fat diet induced insulin resistant mice for 6 weeks. The effects of Leu on Met stimulation of Sirt1 and AMPK activities were further evaluated in adipocytes. RESULTS: Sub-therapeutic levels of Met combined with Leu resulted in increases in Sirt1 activity and in tissue P-AMPK/AMPK ratio and corresponding dose-responsive improvements in fasting and post-prandial glucose, in glucose response to an insulin tolerance test and in the area under the curve in glucose tolerance tests. Changes were evident within 7 days of treatment and sustained throughout the 6-week study duration. The Leu+Met (0.25 g/kg)-combinations produced a comparable effect to a standard therapeutic Met dose, while the Leu+Met (0.5 g/kg diet) resulted in greater improvements. Since resveratrol also synergizes with leucine to augment sirtuin signaling and insulin sensitivity, we tested the addition of resveratrol to Leu-Met and found no additional benefit. CONCLUSION: These data demonstrate that adding Leu to Met enables a dose reduction of 66% with improved efficacy and of 83% with comparable efficacy to standard metformin in diet-induced obese mice, and addition of resveratrol does not provide further benefit.

Leucine Modulates Mitochondrial Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes.

Previous studies from this laboratory demonstrate that dietary leucine protects against high fat diet-induced mitochondrial impairments and stimulates mitochondrial biogenesis and energy partitioning from adipocytes to muscle cells through SIRT1-mediated mechanisms. Moreover, ß-hydroxy-ß-methyl butyrate (HMB), a metabolite of leucine, has been reported to activate AMPK synergistically with resveratrol in C2C12 myotubes. Therefore, we hypothesize that leucine-induced activation of SIRT1 and AMPK is the central event that links the upregulated mitochondrial biogenesis and fatty acid oxidation in skeletal muscle. Thus, C2C12 myotubes were treated with leucine (0.5 mM), alanine (0.5 mM), valine (0.5 mM), EX527 (SIRT1 inhibitor, 25 µM), and Compound C (AMPK inhibitor, 25 µM) alone or in combination to determine the roles of AMPK and SIRT1 in leucine-modulation of energy metabolism. Leucine significantly increased mitochondrial content, mitochondrial biogenesis-related genes expression, fatty acid oxidation, SIRT1 activity and gene expression, and AMPK phosphorylation in C2C12 myotubes compared to the controls, while EX527 and Compound C markedly attenuated these effects. Furthermore, leucine treatment for 24 hours resulted in time-dependent increases in cellular NAD(+), SIRT1 activity, and p-AMPK level, with SIRT1 activation preceding that of AMPK, indicating that leucine activation of SIRT1, rather than AMPK, is the primary event.

Synergistic effects of polyphenols and methylxanthines with Leucine on AMPK/Sirtuin-mediated metabolism in muscle cells and adipocytes.

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 ß-hydroxy-ß-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.

Effects of a leucine and pyridoxine-containing nutraceutical on body weight and composition in obese subjects.

BACKGROUND: We recently demonstrated leucine to modulate energy partitioning between adipose tissue and muscle. Further, leucine exhibits a synergy with B6, resulting in reduced adipocyte lipid storage coupled with increased muscle fat oxidation. Accordingly, a nutraceutical (NuShape™) containing 2.25 g leucine and 30 mg B6 increased fat oxidation by >30 g/day in a 28-day randomized controlled trial. The present study evaluated the long-term efficacy of this combination in modulating body weight and composition. METHODS: Two 24-week, placebo-controlled, randomized trials, one with weight maintenance (n = 20) and one hypocaloric (-500 kcal/day; n = 24), were conducted using the nutraceutical Nushape in obese subjects. RESULTS: The supplement resulted in fat loss in the maintenance study (-1.12 ± 0.36 and -1.82 ± 0.70 kg at 12 and 24 weeks, P < 0.01 versus placebo) while no change was found in the placebo group. In the hypocaloric study, the supplement group lost up to twice as much weight (6.18 ± 1.02 versus 3.40 ± 0.81 kg at 12 weeks and 8.15 ± 1.33 versus 5.25 ± 1.13 kg at 24 weeks, P < 0.01) and fat (4.96 ± 0.61 versus 2.31 ± 0.53 kg at 12 weeks and 7.00 ± 0.95 versus 4.22 ± 0.74 kg at 24 weeks, P < 0.01) than the placebo group. CONCLUSION: This nutraceutical combination results in significant fat loss in the absence of caloric restriction and markedly enhances weight and fat loss by 50%-80% over a 24-week period.

Synergistic effects of metformin, resveratrol, and hydroxymethylbutyrate on insulin sensitivity.

BACKGROUND: The purpose of this study was to determine whether a mixture of the polyphenol, resveratrol, and the leucine metabolite, hydroxymethylbutyrate (HMB), acts synergistically with low doses of metformin to impact insulin sensitivity and AMP-activated protein kinase-dependent outcomes in cell culture and in diabetic mice. METHODS: C2C12 skeletal myotubes and 3T3-L1 adipocytes were treated with resveratrol 0.2 µM, HMB 5 µM, and metformin 0.1 mM alone or in combination. db/db mice were treated for 2 weeks with high (1.5 g/kg diet), low (0.75 g/kg diet), or very low (0.25 g/kg diet) doses of metformin alone or in combination with a diet containing resveratrol 12.5 mg and CaHMB 2 g/kg. RESULTS: The combination of metformin-resveratrol-HMB significantly increased fat oxidation, AMP-activated protein kinase, and Sirt1 activity in muscle cells compared with metformin or resveratrol-HMB alone. A similar trend was found in 3T3L1 adipocytes. In mice, the two lower doses of metformin exerted no independent effect but, when combined with resveratrol-HMB, both low-dose and very low-dose metformin improved insulin sensitivity (HOMA(IR)), plasma insulin levels, and insulin tolerance test response to a level comparable with that found for high-dose metformin. In addition, the metformin-resveratrol-HMB combination decreased visceral fat and liver weight in mice. CONCLUSION: Resveratrol-HMB combined with metformin may act synergistically on AMP-activated protein kinase-dependent pathways, leading to increased insulin sensitivity, which may reduce the therapeutic doses of metformin necessary in the treatment of diabetes.