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.

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

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), ß-hydroxy-ß-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.

Dairy attentuates oxidative and inflammatory stress in metabolic syndrome.

BACKGROUND: Oxidative and inflammatory stress are elevated in obesity and are further augmented in metabolic syndrome. We showed previously that dairy components suppress the adipocyte- and macrophage-mediated generation of reactive oxygen species and inflammatory cytokines and systemic oxidative and inflammatory biomarkers in obesity. OBJECTIVE: The objective of this study was to determine the early (7 d) and sustained (4 and 12 wk) effects of adequate-dairy (AD) compared with low-dairy (LD) diets in subjects with metabolic syndrome. DESIGN: Forty overweight and obese adults with metabolic syndrome were randomly assigned to receive AD (3.5 daily servings) or LD (<0.5 daily servings) weight-maintenance diets for 12 wk. Oxidative and inflammatory biomarkers were assessed at 0, 1, 4, and 12 wk as primary outcomes; body weight and composition were measured at 0, 4, and 12 wk as secondary outcomes. RESULTS: AD decreased malondialdehyde and oxidized LDL at 7 d (35% and 11%, respectively; P < 0.01), with further decreases by 12 wk. Inflammatory markers were suppressed with intake of AD, with decreases in tumor necrosis factor-α at 7 d and further reductions through 12 wk (35%; P < 0.05); decreases in interleukin-6 (21%; P < 0.02) and monocyte chemoattractant protein 1 (14% decrease at 4 wk, 24% decrease at 12 wk; P < 0.05); and a corresponding 55% increase in adiponectin at 12 wk (P < 0.01). LD exerted no effect on oxidative or inflammatory markers. Diet had no effect on body weight; however, AD significantly reduced waist circumference and trunk fat (P < 0.01 for both), and LD exerted no effect. CONCLUSION: An increase in dairy intake attenuates oxidative and inflammatory stress in metabolic syndrome. This trial was registered at clinicaltrials.gov as NCT01266330.