Uncoupling protein-3 gene expression in skeletal muscle during development is regulated by nutritional factors that alter circulating non-esterified fatty acids.

Uncoupling protein-3 gene expression in skeletal muscle is up-regulated during postnatal development of mice. A high-carbohydrate diet at weaning induces a decrease in uncoupling protein-3 mRNA levels that does not occur when mice were weaned onto a high-fat diet. Uncoupling protein-3 mRNA levels do not increase in response to fasting in young pups. Only after day 15 of life, when fasting increases serum non-esterified fatty acids, uncoupling protein-3 mRNA is up-regulated by starvation. Over-nutrition or under-nutrition during lactation increases or decreases, respectively, uncoupling protein-3 mRNA expression in skeletal muscle. Regulation of uncoupling protein-3 gene expression in skeletal muscle during development is mediated by ontogenic and nutritional factors determining changes in circulating non-esterified fatty acids.     

Reduced expression of PGC-1 and insulin-signaling molecules in adipose tissue is associated with insulin resistance

Peroxisome proliferator-activated receptor gamma (PPAR gamma) co-activator 1 (PGC-1) regulates glucose metabolism and energy expenditure and, thus, potentially insulin sensitivity. We examined the expression of PGC-1, PPAR gamma, insulin receptor substrate-1 (IRS-1), glucose transporter isoform-4 (GLUT-4), and mitochondrial uncoupling protein-1 (UCP-1) in adipose tissue and skeletal muscle from non-obese, non-diabetic insulin-resistant, and insulin-sensitive individuals. PGC-1, both mRNA and protein, was expressed in human adipose tissue and the expression was significantly reduced in insulin-resistant subjects. The expression of PGC-1 correlated with the mRNA levels of IRS-1, GLUT-4, and UCP-1 in adipose tissue. Furthermore, the adipose tissue expression of PGC-1 and IRS-1 correlated with insulin action in vivo. In contrast, no differential expression of PGC-1, GLUT-4, or IRS-1 was found in the skeletal muscle of insulin-resistant vs insulin-sensitive subjects. The findings suggest that PGC-1 may be involved in the differential gene expression and regulation between adipose tissue and skeletal muscle. The combined reduction of PGC-1 and insulin signaling molecules in adipose tissue implicates adipose tissue dysfunction which, in turn, can impair the systemic insulin response in the insulin-resistant subjects.     

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyl-transferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARbeta/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.     

Intramyocellular lipid and insulin resistance: differential relationships in European and African Americans

Insulin resistance has been associated with the accumulation of fat within skeletal muscle fibers as intramyocellular lipid (IMCL). Here, we have examined in a cross-sectional study the interrelationships among IMCL, insulin sensitivity, and adiposity in European Americans (EAs) and African Americans (AAs). In 43 EA and 43 AA subjects, we measured soleus IMCL content with proton-magnetic resonance spectroscopy, insulin sensitivity with hyperinsulinemic-euglycemic clamp, and body composition with dual-energy X-ray absorptiometry. The AA and EA subgroups had similar IMCL content, insulin sensitivity, and percent fat, but only in EA was IMCL correlated with insulin sensitivity (r = -0.47, P < 0.01), BMI (r = 0.56, P < 0.01), percent fat (r = 0.35, P < 0.05), trunk fat (r = 0.47, P < 0.01), leg fat (r = 0.40, P < 0.05), and waist and hip circumferences (r = 0.54 and 0.55, respectively, P < 0.01). In a multiple regression model including IMCL, race, and a race by IMCL interaction, the interaction was found to be a significant predictor (t = 1.69, DF = 1, P = 0.0422). IMCL is related to insulin sensitivity and adiposity in EA but not in AA, suggesting that IMCL may not function as a pathophysiological factor in individuals of African descent. These results highlight ethnic differences in the determinants of insulin sensitivity and in the pathogenesis of the metabolic syndrome trait cluster.     

Metabolic and anthropometric factors related to skeletal muscle UCP3 gene expression in healthy human adults

This cross-sectional investigation sought to determine the relationship between selected metabolic, endocrine, and anthropometric factors and skeletal muscle UCP3 mRNA in healthy adult humans. Twenty-four healthy adults (13 male and 11 female) across a range of aerobic capacity, age, and body composition were studied. Muscle biopsies were obtained from the vastus lateralis, from which UCP3 mRNA was quantified by Northern blot, and fiber type was determined by use of the myosin ATPase staining procedure. In addition, resting energy expenditure and maximum rate of oxygen consumption were determined by indirect calorimetry, body composition was determined by dual-energy X-ray absorptiometry, and fasting plasma leptin and insulin were determined by ELISA. UCP3 mRNA was correlated positively with the percent type I fibers (r = 0.842, P < 0.001), plasma leptin (r = 0.454, P = 0.026), and plasma insulin (r = 0.615, P < 0.001) and inversely to age (r = -0.411, P = 0.046). Stepwise multiple regression analysis determined that percent type I muscle fibers was the best predictor of vastus lateralis UCP3 mRNA, and no other variable entered the equation (model r(2) = 0.66). This study suggests that of the variables measured, UCP3 mRNA is primarily related to skeletal muscle fiber type in healthy adults. The factors that contribute to fiber-specific differences in UCP3 mRNA expression will need to be examined in future studies.     

Overexpression of UCP-3 in skeletal muscle of mice results in increased expression of mitochondrial thioesterase mRNA

Mice overexpressing human UCP-3 in skeletal muscle (UCP-3tg) are lean despite overeating, have increased metabolic rate, and their skeletal muscle mitochondria show increased proton conductance. The true function of UCP-3 however, has yet to be determined. It is assumed that UCP-3tg mice have increased fatty acid beta-oxidation to fuel their increased metabolic rate. In this study we have quantified skeletal muscle mRNA levels of a number of genes involved in fatty acid metabolism. mRNA levels of uncoupling protein-2, carnitine palmitoyl transferase-1beta and fatty acid binding proteins, and transporters were unchanged when compared to wild-type mice. Lipoprotein lipase mRNA was slightly, but significantly, increased by 50%. The most notable change in gene expression was a threefold increase in mitochondrial thioesterase (MTE-1) expression. In the face of a chronic increase in mitochondrial uncoupling these changes suggest that increased flux of fatty acids through the beta-oxidation pathway does not necessarily require marked changes in expression of genes involved in fatty acid metabolism. The large increase in MTE-1 both confirms the importance of this gene in situations where mitochondrial beta-oxidation is increased and supports the hypothesis that UCP-3 exports fatty acids generated by MTE-1 in the mitochondrion.     

Effect of pioglitazone on skeletal muscle lipid deposition in the insulin resistance rat model induced by high fructose diet under AMPK signaling pathway

To study the changes of lipid deposition in skeletal muscle of insulin resistance rat and the effect of pioglitazone intervention on the expression of AMPK pathway related genes in rat, a rat model of insulin resistance was induced and constructed by high fructose diet as an test group, and normal rats were used as a control group. First, the effect of pioglitazone intervention on serum lipids-related indicators and mRNA expression levels of fat-related genes in skeletal muscle in rats was investigated. Then skeletal muscle sections were made and stained with oil red O to investigate the effect of pioglitazone intervention on lipid deposition in skeletal muscle of rats. Finally, the effects of pioglitazone intervention therapy on the mRNA and protein expression of related genes in the AMPK signaling pathway in skeletal muscle tissue of rat were explored by real-time quantitative PCR (qRT-PCR) and Western-blotting technology. The results showed that the blood glucose (BG), insulin (INS), adiponectin (ADPN), free fatty acid (FFA), triglyceride (TG), and cholesterol (TC) levels in serum of the test group were higher than the control group (P < 0.05); the visceral fat weight and abdominal fat index of the test group were significantly higher than the control group (P < 0.01); after the pioglitazone intervention, all blood lipid-related indexes in the rat model were significantly lower than before the intervention (P < 0.05); skeletal muscle section staining results showed that the number of lipid droplets in skeletal muscle of rat model was significantly reduced after pioglitazone intervention; and pioglitazone intervention can significantly increase the mRNA and protein expression levels of p-ACC, GLUT7, PGC-1α, and CPT1 genes in the skeletal muscles of experimental rats (P < 0.05). Accordingly, it can be concluded that pioglitazone can play a role in treating insulin resistance by regulating the expression of related genes of AMPK, ACC, etc. in the AMPK signaling pathway.     

Nobiletin improves obesity and insulin resistance in high-fat diet-induced obese mice

Nobiletin (NOB) is a polymethoxylated flavone present in citrus fruits and has been reported to have antitumor and anti-inflammatory effects. However, little is known about the effects of NOB on obesity and insulin resistance. In this study, we examined the effects of NOB on obesity and insulin resistance, and the underlying mechanisms, in high-fat diet (HFD)-induced obese mice. Obese mice were fed a HFD for 8 weeks and then treated without (HFD control group) or with NOB at 10 or 100 mg/kg. NOB decreased body weight gain, white adipose tissue (WAT) weight and plasma triglyceride. Plasma glucose levels tended to decrease compared with the HFD group and improved plasma adiponectin levels and glucose tolerance. Furthermore, NOB altered the expression levels of several lipid metabolism-related and adipokine genes. NOB increased the mRNA expression of peroxisome proliferator-activated receptor (PPAR)-γ, sterol regulatory element-binding protein-1c, fatty acid synthase, stearoyl-CoA desaturase-1, PPAR-α, carnitine palmitoyltransferase-1, uncoupling protein-2 and adiponectin, and decreased the mRNA expression of tumor necrosis factor-α and monocyte chemoattractant protein-1 in WAT. NOB also up-regulated glucose transporter-4 protein expression and Akt phosphorylation and suppressed IκBα degradation in WAT. Taken together, these results suggest that NOB improves adiposity, dyslipidemia, hyperglycemia and insulin resistance. These effects may be elicited by regulating the expression of lipid metabolism-related and adipokine genes, and by regulating the expression of inflammatory makers and activity of the insulin signaling pathway.     

Correlation of apolipoprotein M with leptin and cholesterol in normal and obese subjects

Apolipoprotein M (apoM) is a recently characterized apolipoprotein that is exclusively expressed in the liver and kidney. In plasma it is present predominantly in high-density lipoprotein (HDL). The physiological function of apoM is not yet known. In the present study we investigated relationships between plasma apoM levels and leptin levels, body mass index (BMI), as well as fasting glucose and other lipid parameters in women with a wide range of BMI (18.9-57.1 kg/m(2), n = 51). In univariate analysis, apoM correlated significantly with leptin (r = 0.54, P < 0.001), BMI (r = 0,70, P < 0.001), fasting insulin (r = 0.33, P = 0.025), total cholesterol (r = -0.41, P = 0.016), and LDL-cholesterol (r = -0.39, P = 0.018). The correlations between apoM and cholesterol and between apoM and leptin remained significant after adjustment for the influence of BMI. Forward stepwise multiple regressions when leptin, BMI, insulin, and cholesterol were entered into a model as independent variables and apoM as the dependent variable, showed that cholesterol and leptin were independent predictors of circulating apoM. These two parameters yielded a value of r(2) = 0.28, thereby explaining approximately 30% of the variance in apoM. Hence, we show that apoM is positively related to leptin and negatively related to cholesterol in humans.     

Amyloid precursor protein expression is upregulated in adipocytes in obesity

The aim of this study was to determine whether amyloid precursor protein (APP) is expressed in human adipose tissue, dysregulated in obesity, and related to insulin resistance and inflammation. APP expression was examined by microarray expression profiling of subcutaneous abdominal adipocytes (SAC) and cultured preadipocytes from obese and nonobese subjects. Quantitative real-time PCR (QPCR) was performed to confirm differences in APP expression in SAC and to compare APP expression levels in adipose tissue, adipocytes, and stromal vascular cells (SVCs) from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) specimens. Adipose tissue samples were also examined by western blot and immunofluorescence confocal microscopy. Microarray studies demonstrated that APP mRNA expression levels were higher in SAC (approximately 2.5-fold) and preadipocytes (approximately 1.4) from obese subjects. Real-time PCR confirmed increased APP expression in SAC in a separate group of obese compared with nonobese subjects (P=0.02). APP expression correlated to in vivo indices of insulin resistance independently of BMI and with the expression of proinflammatory genes, such as monocyte chemoattractant protein-1 (MCP-1) (R=0.62, P=0.004), macrophage inflammatory protein-1alpha (MIP-1alpha) (R=0.60, P=0.005), and interleukin-6 (IL-6) (R=0.71, P=0.0005). Full-length APP protein was detected in adipocytes by western blotting and APP and its cleavage peptides, Abeta40 and Abeta42, were observed in SAT and VAT by immunofluorescence confocal microscopy. In summary, APP is highly expressed in adipose tissue, upregulated in obesity, and expression levels correlate with insulin resistance and adipocyte cytokine expression levels. These data suggest a possible role for APP and/or Abeta in the development of obesity-related insulin resistance and adipose tissue inflammation.     

Tissue-specific expression and regulation of GSK-3 in human skeletal muscle and adipose tissue

Glycogen synthase kinase-3 (GSK-3) is a ubiquitous kinase implicated in both insulin action and adipogenesis. To determine how these multiple roles may relate to insulin resistance, we studied the regulation of GSK-3 protein expression and phosphorylation in skeletal muscle and isolated adipocytes from nonobese healthy control (HC), obese control (OC), and obese type 2 diabetic (OT2D) subjects. At baseline there were no differences in the GSK-3 protein expression in adipocytes. OC subjects underwent a 6-mo caloric restriction resulting in a 7% decrease in body mass index (BMI) and a 21% improvement in insulin-stimulated whole body glucose disposal rate (GDR). GSK-3alpha and GSK-3beta expression decreased in adipocytes (P < 0.05), whereas GSK-3alpha protein expression increased in skeletal muscle (P < 0.05). OT2D subjects were treated with troglitazone or metformin for 3-4 mo. After troglitazone treatment GDR improved (P < 0.05) despite an increase in BMI (P < 0.05), whereas metformin had no significant effect on GDR. There was no significant change in GSK-3 expression in adipocytes following troglitazone, whereas both GSK-3alpha and -beta were decreased in skeletal muscle (P < 0.05). Metformin treatment had no significant impact on GSK-3 protein expression in either adipocytes or skeletal muscle. Neither treatment influenced GSK-3 serine phosphorylation in skeletal muscle or adipocytes. These results suggest that there is tissue specificity for the regulation of GSK-3 in humans. In skeletal muscle GSK-3 plays a role in control of metabolism and insulin action, whereas the function in adipose tissue is less clear.