Effects of dairy consumption on SIRT1 and mitochondrial biogenesis in adipocytes and muscle cells

<p>Abstract</p> <p>Background</p> <p>Recent 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 <it>ex-vivo/in-vitro </it>approach was used to examine the integrated effects of dairy diets on SIRT1 activation in two key target tissues (adipose and muscle tissue).</p> <p>Methods</p> <p>Serum 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.</p> <p>Results</p> <p>Treatment 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).</p> <p>Conclusions</p> <p>These 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.</p

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

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), 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

Role of Dietary Calcium and Dairy in Modulating Oxidative Stress, Inflammatory Stress and Lifespan

Oxidative stress and inflammatory stress have been implicated as a cause of tissue damage in multiple organ systems, leading to the development of chronic diseases such as obesity, diabetes, hypertony and atherosclerosis. They are also recognized as major factors contributing to the physiological process of aging. Previous studies have demonstrated that dietary calcium regulates reactive oxygen species production (ROS) production in adipocytes in vitro and in vivo, and inhibits adipocyte-derived inflammatory cytokine expression by suppression of calcitriol. In addition, high calcium diets modulate energy metabolism and partitioning between adipose tissue and muscle resulting in a decrease in fat storage and an increase in fat oxidation in muscle. Providing calcium in form of dairy appears to cause greater effects than supplemental calcium in both mice and humans, most likely mediated by additional components in dairy products such as branched-chain amino acids (BCAA) and angiotensin converting enzyme inhibitor (ACEi) peptides. In consideration of the multiple effects of dietary calcium and other components of dairy on adipocyte and muscle metabolism, a high density oligonucleotide microarray approach was used to identify common and differential pathways related to energy metabolism, inflammation and oxidative stress in adipose and muscle tissue in response to milk and milk components. In addition, considering the protective role of dietary calcium against oxidative and inflammatory stress, which otherwise accelerate the process of aging, a lifespan study in a mouse model of diet- induced obesity was conducted to evaluate the effects of dietary calcium from both non-dairy and dairy sources on mouse lifespan and on lifespan-related biomarkers. The results of this research confirm that calcium and BCAA contribute to the alteration of energy partitioning between adipose tissue and muscle and provide new evidence for calcium independent effects of BCAA and ACEi in energy metabolism and inflammation. Further, present data demonstrate that milk diet attenuates adiposity, protects against muscle loss and reduces oxidative and inflammatory stress. Although these did not alter maximum lifespan, they significantly suppressed early mortality

Synergistic Effects of Polyphenols and Methylxanthines with Leucine on AMPK/Sirtuin-Mediated Metabolism in Muscle Cells and Adipocytes

<div><p>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.</p></div

Effects of unspecific PDE inhibitors combined with Resv, Leu or HMB on fatty acid oxidation.

<p>Differentiated cells were treated with indicated treatments for 24 µM palmitate injection and the area under the curve (AUC) at a two-hour measurement point was calculated as % change from baseline. (<b>a</b>) Caffeine (Caff, 10 nM), theophylline (Theo, 1 uM) and chocamine (Choc, 0.1 ug/ml) combinations in 3T3L1 adipocytes (<b>b</b>) IBMX (50 nM), pentoxyphylline (PTX, 5 nM) and chocamine (Choc, 0.1 ug/ml) combinations in C2C12 muscle cells. Data are represented as mean ± SEM (n = 4). **indicates significant difference to control and individual compound, ***indicates significant difference to all other treatment groups (p<0.05).</p

Effects of specific PDE inhibitors combined with Resv, Leu or HMB on fatty acid oxidation.

<p>Differentiated cells were treated with indicated treatments for 24 µM palmitate injection and the percent OCR from baseline (3rd measurement point) was calculated as the area under the curve (AUC) at a two-hour measurement point. (<b>a</b>) Effects of Leu/HMB/Resv combinations with the PDE5-inhibitor sildenafil (Sild, 1 nM) in 3T3L1 adipocytes and C2C12 muscle cells. (<b>b</b>) Effects of Leu/HMB/Resv combinations with the PDE4-inhibitor rolipram (Roli, 1 nM) in C2C12 muscle cells. Data are represented as mean ± SEM (n = 4 to 8). *indicates significant difference to control (p<0.05), **indicates significant difference to control and Sildenafil (p<0.05), ***indicates significant difference to all other treatment groups (p<0.05).</p

Effects of chlorogenic acid-Leu or -HMB combinations on AMPK and Sirt1 activity in adipocytes.

<p>Differentiated cells were treated with CA (0.5 µM), Leu (0.5 µM) or HMB (5 µM) (<b>a</b>) for 24 h for AMPK activity and (<b>b</b>) for 48 h for Sirt1 activity (AFU = arbitrary fluorescence units). Data are represented as mean ± SEM (n = 4). *indicates significant difference to control (p<0.03), **indicates significant difference to control and CA (p<0.05).</p