Xiaocun Sun

Calcium and 1,25-Dihydroxyvitamin d3 regulation of adipokine expression

Objective: Obesity is associated with elevated oxidative stress and low‐grade systemic inflammation. We have demonstrated recently that 1α,25‐(OH)2‐D3 promotes reactive oxygen species production in cultured adipocytes, whereas suppression of 1α,25‐(OH)2‐D3 by increasing dietary calcium down‐regulates diet‐induced oxidative stress in aP2‐agouti transgenic mice. However, whether the anti‐obesity effect of dietary calcium plays a role in regulation of obesity‐associated inflammation is not clear.

Effects of dairy compared with soy on oxidative and inflammatory stress in overweight and obese subjects

BACKGROUND We recently showed that calcitriol increases oxidative and inflammatory stress; moreover, inhibition of calcitriol with high-calcium diets decreased both adipose tissue and systemic oxidative and inflammatory stress in obese mice, whereas dairy exerted a greater effect. However, these findings may be confounded by concomitant changes in adiposity. OBJECTIVE The objective of this study was to evaluate the acute effects of a dairy-rich diet on oxidative and inflammatory stress in overweight and obese subjects in the absence of adiposity changes. DESIGN Twenty subjects (10 obese, 10 overweight) participated in a blinded, randomized, crossover study of dairy- compared with soy-supplemented eucaloric diets. Two 28-d dietary periods were separated by a 28-d washout period. Inflammatory and oxidative stress biomarkers were measured on days 0, 7, and 28 of each dietary period. RESULTS The dairy-supplemented diet resulted in significant suppression of oxidative stress (plasma malondialdehyde, 22%; 8-isoprostane-F(2alpha), 12%; P < 0.0005) and lower inflammatory markers (tumor necrosis factor-alpha, 15%, P < 0.002; interleukin-6, 13%, P < 0.01; monocyte chemoattractant protein-1, 10%, P < 0.0006) and increased adiponectin (20%, P < 0.002), whereas the soy exerted no significant effect. These effects were evident by day 7 of treatment and increased in magnitude at the end of the 28-d treatment periods. There were no significant differences in response to treatment between overweight and obese subjects for any variable studied. CONCLUSION An increase in dairy food intake produces significant and substantial suppression of the oxidative and inflammatory stress associated with overweight and obesity. This trial was registered at clinicaltrials.gov as NCT00686426.

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

BackgroundThe 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 in vitro, 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.MethodsWe 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.ResultsLeucine (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.ConclusionThese data suggest that leucine and calcitriol modulation of muscle and adipocyte energy metabolism is mediated, in part, by mitochondrial biogenesis.

Role of uncoupling protein 2 (UCP2) expression and 1α, 25-dihydroxyvitamin D3 in modulating adipocyte apoptosis

We previously found that 1α, 25‐dihydroxyvitamin D3 [1α, 25‐(OH)2‐D3] modulates adipocyte lipid metabolism via a Ca2+‐dependent mechanism and inhibits adipocyte UCP2 expression, indicating that the anti‐obesity effects of dietary calcium are mediated by suppression of 1α, 25‐(OH)2‐D3 levels. However, because UCP2 reduces mitochondrial potential, we have evaluated the roles of UCP2, mitochondrial uncoupling, and 1α, 25‐(OH)2‐D3 in adipocyte apoptosis. Overexpressing UCP2 in 3T3‐L1 cells induced marked reductions in mitochondrial potential (∆ψ) and ATP production (P<0.01), increases in the expression of caspases (P<0.05), and a decrease in Bcl‐2/Bax expression ratio (P<0.01). Physiological doses of 1α, 25‐(OH)2‐D3 (0.1–10 nM) restored mitochondrial ∆ψ in LI‐UCP2 cells and protected against UCP2 overexpression‐induced apoptosis (P<0.01), whereas a high dose (100 nM) stimulated apoptosis in 3T3‐L1 and L1‐UCP2 cells (P<0.05). 1α, 25‐(OH)2‐D3 stimulated cytosolic Ca2+ dose‐dependently in both 3T3‐L1 and L1‐UCP2 cells. However, physiological doses suppressed mitochondrial Ca2+ levels by ~50% whereas the high dose increased mitochondrial Ca2+ by 25% (P<0.05); this explains stimulation of apoptosis by the high dose of 1α, 25‐(OH)2‐D3. Using high‐calcium diets to suppress 1α, 25‐(OH)2‐D3 stimulated adipose tissue apoptosis in aP2 transgenic mice (P<0.01), suggesting that increasing dietary calcium stimulates adipose apoptosis and thereby further contributes to an anti‐obesity effect of dietary calcium.

Effects of dairy intake on weight maintenance

BackgroundTo compare the effects of low versus recommended levels of dairy intake on weight maintenance and body composition subsequent to weight loss.Design and MethodsTwo 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 (< 1 dairy serving/d) or recommended 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.ResultsDuring 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.ConclusionWeight 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 RegistrationClinicalTrials.gov NCT00686426

Calcitriol and energy metabolism

Calcitriol, a calcitrophic hormone that can be suppressed by high dietary calcium, favors fatty acid synthesis and inhibits lipolysis via non-genomic modulation of Ca(2+) influx. Calcitriol also suppresses UCP2 expression via the nVDR and thereby increases energy efficiency. Calcitriol exerts a dose-dependent impact on adipocyte apoptosis and regulates adipose tissue fat depot location and expansion by promoting glucocorticoid production and release. Recent data also demonstrate a pivotal role of calcitriol in the modulation of cytokines, with potential roles in energy metabolism in adipocytes, macrophages, and skeletal muscle.

1α,25‐Dihydroxyvitamin D3 Modulation of Adipocyte Reactive Oxygen Species Production

Objective: We have previously shown 1α,25‐dihydroxyvitamin D3 [1α,25‐(OH)2D3] to inhibit mitochondrial uncoupling protein 2 (UCP2) expression in adipocytes and that in vivo suppression of calcitriol levels with calcium‐rich diets increases UCP2 expression. Because UCP2 plays a significant role in the clearance of reactive oxygen species (ROS), we studied the effect of calcitriol on ROS production and ROS‐induced adipocyte proliferation.

Role of Calcitriol and Cortisol on Human Adipocyte Proliferation and Oxidative and Inflammatory Stress: A Microarray Study

Dietary calcium inhibits adiposity, and a key underlying mechanism is suppression of calcitriol, which modulates Ca2+ signaling and mitochondrial uncoupling in adipocytes. We demonstrated that calcitriol directly regulates adipocyte 11β-HSD-1 expression and cortisol production in human adipocytes in vitro and dietary calcium inhibits visceral adipose tissue 11β-HSD-1 expression in mice, indicating an interaction of calcitriol and cortisol in obesity. Consequently, we have evaluated the gene expression profile of human subcutaneous adipocytes treated with calcitriol and/or cortisone. Data analysis demonstrated significant calcitriol modulation of gene expression toward inhibition of the adipocyte apoptosis (e.g., VEGF and STC-2) and promotion of adipocyte proliferation (e.g., IGF-1 and IGF-1R). Calcitriol also up-regulated oxidative stress and inflammatory genes such as NOX-4 and TLR-3. The calcitriol/cortisone combination resulted in significant additional up-regulation of 11β-HSD-1 and down-regulation of adiponectin expression, while cortisone exerted little independent effect in the absence of calcitriol. Overall, calcitriol stimulated a pattern of adipocyte gene expression which favored adipocyte proliferation, oxidative and inflammatory stress and visceral adiposity, and these effects were amplified in the presence of cortisone; however, this conclusion must be tempered by the adipocyte source (subcutaneous) and requires confirmation in visceral adipocytes.

Effects of mitochondrial uncoupling on adipocyte intracellular Ca2+ and lipid metabolism

Previous data from this laboratory demonstrate that increased intracellular Ca(2+) ([Ca(2+)]i) coordinately regulates human and murine adipocyte lipid metabolism by stimulating lipogenesis and inhibiting lipolysis. However, recent data demonstrate metabolic uncoupling increases [Ca(2+)]i but inhibits lipogenesis by suppressing fatty acid synthase (FAS) activity. Accordingly, we have evaluated the interaction between mitochondrial uncoupling, adipocyte [Ca(2+)]i, and adipocyte lipid metabolism. Pretreatment of 3T3-L1 cells with mitochondrial uncouplers (DNP or FCCP) amplified the [Ca(2+)]i response to depolarization with KCl by 2-4 fold (p <0.001), while this increase was prevented by [Ca(2+)]i channel antagonism with lanthanum. Mitochondrial uncouplers caused rapid (within 4hr) dose-dependent inhibition of FAS activity (p <0.001), while lanthanum caused a further additive inhibition. The suppression of FAS activity induced by uncoupling was reversed by addition of ATP. Mitochondrial uncouplers increased FAS expression significantly while [Ca(2+)]i antagonism with lanthanum decreased FAS expression (P <0.001). In contrast, mitochondrial uncouplers independently inhibited basal and isoproterenol-stimulated lipolysis (20-40%, p <0.001), while this inhibition was fully reversed by lanthanum. Thus, mitochondrial uncoupling exerted short-term regulatory effects on adipocyte [Ca(2+)]i and lipogenic and lipolytic systems, serving to suppress lipolysis via a Ca(2+) -dependent mechanism and FAS activity via a Ca(2+)-independent mechanism.

Vitamin D Modulation of Adipocyte Function

Calcitriol has recently been demonstrated to play an important role in modulating adipocyte function by regulating adipocyte lipid metabolism and energy homeostasis via both genomic and non-genomic actions. Physiological concentrations of calcitriol dose-dependently inhibit adipocyte apoptosis, although supra-physiological concentrations stimulate adipocyte apoptosis; the former is mediated by inhibition of mitochondrial uncoupling and the latter by mitochondrial calcium overload. Calcitriol also regulates adipose tissue fat depot location and expansion by promoting glucocorticoid production and release. Finally, calcitriol also modulates the cross talk between adipose tissue and both skeletal muscle and macrophages. Calcitriol modulation of adipocyte–macrophage cross talk results in a synergistic increase in expression and release of reactive oxygen species and inflammatory cytokines from both cell types, while calcitriol regulation of adipocyte–skeletal muscle cross talk results in inhibition of skeletal muscle fatty acid oxidation and preferential energy storage in adipocytes. Accordingly, conditions which chronically increase calcitriol levels, such as low-calcium diets, increase obesity risk, decrease metabolic flexibility, and increase oxidative and inflammatory stress.