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

Leucine modulation of mitochondrial mass and oxygen consumption in skeletal muscle cells and adipocytes

<p>Abstract</p> <p>Background</p> <p>The effects of dairy on energy metabolism appear to be mediated, in part, by leucine and calcium which regulate both adipocyte and skeletal muscle energy metabolism. We recently demonstrated that leucine and calcitriol regulate fatty acid oxidation in skeletal muscle cells <it>in vitro</it>, with leucine promoting and calcitriol suppressing fatty acid oxidation. Moreover, leucine coordinately regulated adipocyte lipid metabolism to promote flux of lipid to skeletal muscle and regulate metabolic flexibility. We have now investigated the role of mitochondrial biogenesis in mediating these effects.</p> <p>Methods</p> <p>We tested the effect of leucine, calcitriol and calcium in regulation of mitochondrial mass using a fluorescence method and tested mitochondrial biogenesis regulatory genes as well mitochondrial component genes using real-time PCR. We also evaluated the effect of leucine on oxygen consumption with a modified perfusion system.</p> <p>Results</p> <p>Leucine (0.5 mM) increased mitochondrial mass by 30% and 53% in C2C12 myocytes and 3T3-L1 adipocytes, respectively, while calcitriol (10 nM) decreased mitochondrial abundance by 37% and 27% (p < 0.02). Leucine also stimulated mitochondrial biogenesis genes SIRT-1, PGC-1α and NRF-1 as well as mitochondrial component genes UCP3, COX, and NADH expression by 3–5 fold in C2C12 cells (p < 0.003). Adipocyte-conditioned medium reduced mitochondrial abundance (p < 0.001) and decreased UCP3 but increased PGC-1α expression in myocytes, suggesting a feedback stimulation of mitochondrial biogenesis. Similar data were observed in C2C12 myocytes co-cultured with adipocytes, with co-culture markedly suppressing mitochondrial abundance (p < 0.02). Leucine stimulated oxygen consumption in both C2C12 cells and adipocytes compared with either control or valine-treated cells. Transfection of C2C12 myocytes with SIRT-1 siRNA resulted in parallel suppression of SIRT-1 expression and leucine-induced stimulation of PGC-1α and NRF-1, indicating that SIRT-1 mediates leucine induced mitochondrial biogenesis in muscle cells.</p> <p>Conclusion</p> <p>These data suggest that leucine and calcitriol modulation of muscle and adipocyte energy metabolism is mediated, in part, by mitochondrial biogenesis.</p

Dietary fat and not calcium supplementation or dairy product consumption is associated with changes in anthropometrics during a randomized, placebo-controlled energy-restriction trial

Insufficient calcium intake has been proposed to cause unbalanced energy partitioning leading to obesity. However, weight loss interventions including dietary calcium or dairy product consumption have not reported changes in lipid metabolism measured by the plasma lipidome

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

Dairy attentuates oxidative and inflammatory stress in metabolic syndrome123

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

Effects of dairy intake on weight maintenance

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Effects of dairy intake on weight maintenance

Background: To compare the effects of low versus recommended levels of dairy intake on weight maintenance and body composition subsequent to weight loss. Design and Methods: Two site (University of Kansas-KU; University of Tennessee-UT), 9 month, randomized trial. Weight loss was baseline to 3 months, weight maintenance was 4 to 9 months. Participants were maintained randomly assigned to low dairy ( 3 servings/d) diets for the maintenance phase. Three hundred thirty eight men and women, age: 40.3 ± 7.0 years and BMI: 34.5 ± 3.1, were randomized; Change in weight and body composition (total fat, trunk fat) from 4 to 9 months were the primary outcomes. Blood chemistry, blood pressure, resting metabolism, and respiratory quotient were secondary outcomes. Energy intake, calcium intake, dairy intake, and physical activity were measured as process evaluation. Results: During weight maintenance, there were no overall significant differences for weight or body composition between the low and recommended dairy groups. A significant site interaction occurred with the low dairy group at KU maintaining weight and body composition and the low dairy group at UT increasing weight and body fat. The recommended dairy group exhibited reductions in plasma 1,25-(OH)2-D while no change was observed in the low dairy group. No other differences were found for blood chemistry, blood pressure or physical activity between low and recommended dairy groups. The recommended dairy group showed significantly greater energy intake and lower respiratory quotient compared to the low dairy group. Conclusion: Weight maintenance was similar for low and recommended dairy groups. The recommended dairy group exhibited evidence of greater fat oxidation and was able to consume greater energy without greater weight gain compared to the low dairy group. Recommended levels of dairy products may be used during weight maintenance without contributing to weight gain compared to diets low in dairy products. Trial Registration: ClinicalTrials.gov NCT0068642

From Molecular Signal Activation to Locomotion: An Integrated, Multiscale Analysis of Cell Motility on Defined Matrices

The adhesion, mechanics, and motility of eukaryotic cells are highly sensitive to the ligand density and stiffness of the extracellular matrix (ECM). This relationship bears profound implications for stem cell engineering, tumor invasion and metastasis. Yet, our quantitative understanding of how ECM biophysical properties, mechanotransductive signals, and assembly of contractile and adhesive structures collude to control these cell behaviors remains extremely limited. Here we present a novel multiscale model of cell migration on ECMs of defined biophysical properties that integrates local activation of biochemical signals with adhesion and force generation at the cell-ECM interface. We capture the mechanosensitivity of individual cellular components by dynamically coupling ECM properties to the activation of Rho and Rac GTPases in specific portions of the cell with actomyosin contractility, cell-ECM adhesion bond formation and rupture, and process extension and retraction. We show that our framework is capable of recreating key experimentally-observed features of the relationship between cell migration and ECM biophysical properties. In particular, our model predicts for the first time recently reported transitions from filopodial to “stick-slip” to gliding motility on ECMs of increasing stiffness, previously observed dependences of migration speed on ECM stiffness and ligand density, and high-resolution measurements of mechanosensitive protrusion dynamics during cell motility we newly obtained for this study. It also relates the biphasic dependence of cell migration speed on ECM stiffness to the tendency of the cell to polarize. By enabling the investigation of experimentally-inaccessible microscale relationships between mechanotransductive signaling, adhesion, and motility, our model offers new insight into how these factors interact with one another to produce complex migration patterns across a variety of ECM conditions

De Novo Mutations in SLC1A2 and CACNA1A Are Important Causes of Epileptic Encephalopathies

Epileptic encephalopathies (EEs) are the most clinically important group of severe early-onset epilepsies. Next-generation sequencing has highlighted the crucial contribution of de novo mutations to the genetic architecture of EEs as well as to their underlying genetic heterogeneity. Our previous whole-exome sequencing study of 264 parent-child trios revealed more than 290 candidate genes in which only a single individual had a de novo variant. We sought to identify additional pathogenic variants in a subset (n = 27) of these genes via targeted sequencing in an unsolved cohort of 531 individuals with a diverse range of EEs. We report 17 individuals with pathogenic variants in seven of the 27 genes, defining a genetic etiology in 3.2% of this unsolved cohort. Our results provide definitive evidence that de novo mutations in SLC1A2 and CACNA1A cause specific EEs and expand the compendium of clinically relevant genotypes for GABRB3. We also identified EEs caused by genetic variants in ALG13, DNM1, and GNAO1 and report a mutation in IQSEC2. Notably, recurrent mutations accounted for 7/17 of the pathogenic variants identified. As a result of high-depth coverage, parental mosaicism was identified in two out of 14 cases tested with mutant allelic fractions of 5%–6% in the unaffected parents, carrying significant reproductive counseling implications. These results confirm that dysregulation in diverse cellular neuronal pathways causes EEs, and they will inform the diagnosis and management of individuals with these devastating disorders