Dietary conjugated linoleic acid increases PPAR gamma gene expression in adipose tissue of obese rat, and improves insulin resistance.

Conjugated linoleic acid (CLA) is a class of positional, geometric, conjugated dienoic isomers of linoleic acid. Dietary CLA supplementation has resulted in a dramatic decrease in body fat mass in mice. However, some but not all studies in mice and humans have found that CLA promoted insulin resistance, and there were conflicting reports on the effects of CLA on peroxisomal proliferator-activated receptor-gamma (PPAR gamma) activation and expression. The objective of present study was to investigate the effect of CLA on insulin resistance and its molecular mechanisms. Fifty male Wistar rats were randomly designed to the control, high-fat and high-fat with CLA (0.75, 1.50, and 3.00 g in per 100 g diet) groups. The effect of CLA on insulin sensitivity and the mechanism of resisting diabetes by CLA were investigated by RT-PCR assay. The results showed that supplementation with CLA significantly reduced body weight gain and white fat pad weight in the rats, the levels of plasma free fatty acids (FFA), triglycerides (TGs), cholesterin (TC), leptin, insulin and blood glucose concentration in the obese rats of CLA group were also decreased compared to the rats in the high-fat group. Dietary CLA increased the mRNA expression of PPAR gamma, fatty acid binding proteins (aP2), fatty acid transporter protein (FATP), acyl-CoA synthetase (ACS) and adiponectin in the adipose tissues of obese rats. The results suggest that CLA may ameliorate insulin resistance by activating PPAR gamma, and increasing the expression of PPAR gamma target genes such as ap2, FATP, FAT, and adiponectin in the white adipose tissue.     

Thiazolidinediones: metabolic actions in vitro

To unravel the molecular mechanisms and the causal chain of how thiazolidinediones (TZDs) affect glucose homeostasis, it is helpful to analyse their direct influence on isolated specimens of fat, muscle, and liver in vitro. Studies on isolated adipocytes have shown that the nuclear peroxisome proliferator-activated receptor-gamma (PPAR gamma) is an important molecular target for TZDs, through which they trigger adipocyte differentiation and adipose tissue remodelling. It is not clear, however, if the activation of PPAR gamma in adipose tissue is the cause of all the metabolic actions of TZDs. Based on in vitro studies, two hypotheses have been developed. The first emphasizes PPAR gamma-mediated actions on adipose tissue, suggesting that insulin sensitization of skeletal muscle and liver is triggered indirectly by changes in circulating concentrations of adipocyte-derived non-esterified fatty acids and peptide hormones. The second states that TZDs improve glucose homeostasis independently from adipose tissue actions by the direct interaction with muscle and liver. This hypothesis is supported by direct TZD actions on fuel metabolism of skeletal muscle and liver in vitro, which seem to be independent from PPAR gamma signalling. Major progress has been made in understanding the mechanisms involved in the effects of TZDs on adipose tissue but the causal chain responsible for their antihyperglycaemic action is still not clear. The involvement of other molecular targets in addition to PPAR gamma, of adipocyte-derived messengers, and of direct interaction with skeletal muscle and liver have yet to be clarified.     

Compensation by the muscle limits the metabolic consequences of lipodystrophy in PPAR gamma hypomorphic mice

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor, which controls adipocyte differentiation. We targeted with homologous recombination the PPAR gamma 2-specific exon B, resulting in a white adipose tissue knockdown of PPAR gamma. Although homozygous (PPAR gamma hyp/hyp) mice are born with similar weight as the WT mice, the PPAR gamma hyp/hyp animals become growth retarded and develop severe lipodystrophy and hyperlipidemia. Almost half of these PPAR gamma hyp/hyp mice die before adulthood, whereas the surviving PPAR gamma hyp/hyp animals overcome the growth retardation, yet remain lipodystrophic. In contrast to most lipodystrophic models, the adult PPAR gamma hyp/hyp mice only have mild glucose intolerance and do not have a fatty liver. These metabolic consequences of the lipodystrophy are relatively benign because of the induction of a compensatory gene expression program in the muscle that enables efficient oxidation of excess lipids. The PPAR gamma hyp/hyp mice unequivocally demonstrate that PPAR gamma is the master regulator of adipogenesis in vivo and establish that lipid and glucose homeostasis can be relatively well maintained in the absence of white adipose tissue.     

Lowering plasma free fatty acids with Acipimox mimics the antidiabetic effects of the beta 3-adrenergic agonist CL-316243 in obese Zucker diabetic fatty rats

We previously reported that long-term treatment of Zucker diabetic fatty (ZDF) rats with the selective beta(3) agonist CL-316243 normalizes glycemia, decreases plasma free fatty acids (FFA) concentration, improves insulin responsiveness, and increases glucose uptake, not only in brown and white adipose tissues, but also in skeletal muscles. Because muscles do not express typical beta(3) adrenoceptors, we postulated that the muscle effect was indirect and that it was possibly mediated by an activation of the glucose-fatty acid cycle. To test this hypothesis, we investigated the effects of Acipimox, a potent inhibitor of lipolysis in adipose tissue. Similar to CL-316243, Acipimox (150 mg/kg orally) markedly decreased plasma FFA, glucose, and insulin concentrations and improved glucose tolerance while reducing the insulin response in obese (350 to 400 g) ZDF rats. Plasma FFA concentrations were significantly correlated with plasma glucose and insulin concentrations (r =.72 and.83, respectively; P <.01), indicating strong metabolic relationships between these parameters. Euglycemic-hyperinsulinemic clamps combined with the 2-[(3)H]deoxyglucose method revealed that Acipimox markedly improved insulin responsiveness and significantly increased glucose uptake (Rg') in the diaphragm, the heart, and various skeletal muscles. Unlike CL-316243, Acipimox did not increase glucose use in brown or white adipose tissues. This selectivity shows that it is possible to improve diabetes in obese ZDF rats without necessarily stimulating thermogenesis in adipose tissues. Thus, decreasing plasma FFA with 2 drugs (Acipimox or CL-316243) that act via different mechanisms (acute inhibition of lipolysis or chronic stimulation of FFA oxidation) is associated with increased glucose uptake in muscles and enhanced insulin responsiveness. These observations support the hypothesis that CL-316243 may indirectly stimulate glucose uptake in muscles of type II diabetic rats by first stimulating brown adipose tissue (increasing uncoupling protein content and fatty acid oxidation) and progressively decreasing the levels of circulating FFA, resulting in activation of the glucose-fatty acid cycle or other mechanisms regulating insulin responsiveness in skeletal muscles.     

Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding

PPARs are members of the nuclear hormone receptor superfamily and are primarily involved in lipid metabolism. The expression patterns of all 3 PPAR isotypes in 22 adult rat organs were analyzed by a quantitative ribonuclease protection assay. The data obtained allowed comparison of the expression of each isotype to the others and provided new insight into the less studied PPAR beta (NR1C2) expression and function. This isotype shows a ubiquitous expression pattern and is the most abundant of the three PPARs in all analyzed tissues except adipose tissue. Its expression is especially high in the digestive tract, in addition to kidney, heart, diaphragm, and esophagus. After an overnight fast, PPAR beta mRNA levels are dramatically down-regulated in liver and kidney by up to 80% and are rapidly restored to control levels upon refeeding. This tight nutritional regulation is independent of the circulating glucocorticoid levels and the presence of PPAR alpha, whose activity is markedly up-regulated in the liver and small intestine during fasting. Finally, PPAR gamma 2 mRNA levels are decreased by 50% during fasting in both white and brown adipose tissue. In conclusion, fasting can strongly influence PPAR expression, but in only a few selected tissues.     

Physiological difference between obese (fa/fa) Zucker rats and lean Zucker rats concerning adiponectin

Obese (fa/fa) Zucker rat is a spontaneous genetic obesity model and, by comparison with lean Zucker rat, exhibits hyperphagia, hyperinsulinemia, and hyperlipidemia. The aim of this study was to examine the physiological difference concerning adiponectin between obese (fa/fa) Zucker rats and control lean Zucker rats. We therefore measured plasma adiponectin level and analyzed adiponectin and adiponectin receptor 1 mRNA expression in retroperitoneal white adipose tissue (RT WAT), brown adipose tissue (BAT), liver, and soleus muscle. We also examined the tissue mRNA expression of peroxisome proliferator-activated receptor alpha (PPAR alpha), PPAR delta, and PPAR gamma, which regulate adiponectin expression sensitivity to a PPAR gamma agonist shown by brown adipocytes from obese (fa/fa) Zucker rats and lean Zucker rats, by measuring adiponectin release from these cells. Plasma adiponectin levels of obese (fa/fa) Zucker rats were significantly higher than those of lean Zucker rats. Adiponectin mRNA expression levels in RT WAT were lower in obese (fa/fa) Zucker rats than in lean Zucker rats, but those in BAT were higher. Adiponectin receptor 1 expression levels in RT WAT, BAT, and liver of obese (fa/fa) Zucker rats were lower than in lean Zucker rats. The expression level of PPAR alpha, PPAR delta, and PPAR gamma in BAT was lower in obese (fa/fa) Zucker rats than in lean Zucker rats. Moreover, the PPAR gamma agonist increased adiponectin release only from the brown adipocytes isolated from lean Zucker rats. It is the conclusive difference between obese (fa/fa) Zucker rats and lean Zucker rats that plasma adiponectin levels of obese (fa/fa) Zucker rats are significantly higher than those of lean Zucker rats. Moreover, we clarified that mRNA expression level of adiponectin receptor 1 in RT WAT, BAT, and liver of obese (fa/fa) Zucker rats is low despite high plasma adiponectin level, and low expression of PPARs in BAT leads to less sensibility of adiponectin release from brown adipocytes to a PPAR gamma agonist in obese (fa/fa) Zucker rats.     

A mutation in the peroxisome proliferator-activated receptor γ-binding site in the gene for the cytosolic form of phosphoenolpyruvate carboxykinase reduces adipose tissue size and fat content in mice

Regulation of the turnover of triglycerides in adipose tissue requires the continuous provision of 3-glycerophosphate, which may be supplied by the metabolism of glucose or by glyceroneogenesis, the de novo synthesis of 3-glycerophosphate from sources other than hexoses or glycerol. The importance of glyceroneogenesis in adipose tissue was assessed in mice by specifically eliminating the expression of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C), an enzyme that plays a pivotal role in the pathway. To accomplish this, we mutated the binding site for the peroxisome proliferator-activated receptor gamma (PPAR gamma) called the peroxisome proliferator-activated receptor element (PPARE), in the 5' flanking region of the PEPCK-C gene in the mouse by homologous recombination. The mutation abolished expression of the gene in white adipose tissue and considerably reduced its expression in brown adipose tissue, whereas the level of PEPCK-C mRNA in liver and kidney remained normal. Epididymal white adipose tissue from these mice had a reduced triglyceride deposition, with 25% of the animals displaying lipodystrophy. There was also a greatly reduced level of lipid accumulation in brown adipose tissue. A strong correlation between the hepatic content of triglycerides and the size of the epididymal fat pad in PPARE(-/-) mice suggests that hepatic triglyceride synthesis predominantly utilizes free fatty acids derived from the adipose tissue. Unlike other models, PPARE(-/-) mice with lipodystrophy did not exhibit the lipodystrophy-associated features of diabetes and displayed only moderate hyperglycemia. These studies establish the importance of the PPARE site for PEPCK-C gene expression in adipose tissue and the role of PEPCK-C in the regulation of glyceroneogenesis, a pathway critical for maintaining the deposition of triglycerides in adipose tissue.     

Thiazolidinediones improve insulin sensitivity in adipose tissue and reduce the hyperlipidaemia without affecting the hyperglycaemia in a transgenic model of type 2 diabetes

Aim/hypothesis The aim of this study was to examine the effects of thiazolidinediones on the MKR mouse model of type 2 diabetes.Methods Six-week-old wild-type (WT) and MKR mice were fed with or without rosiglitazone or pioglitazone for 3 weeks. Blood was collected from the tail vein for serum biochemistry analysis. Hyperinsulinaemic–euglycaemic clamp analysis was performed to study effects of thiazolidinediones on insulin sensitivity of tissues in MKR mice. Northern blot analysis was performed to measure levels of target genes of PPAR agonists in white adipose tissue and hepatic gluconeogenic genes.Results Thiazolidinedione treatment of MKR mice significantly lowered serum lipid levels and increased serum adiponectin levels but did not affect levels of blood glucose and serum insulin. Hyperinsulinaemic–euglycaemic clamp showed that whole-body insulin sensitivity and glucose homeostasis failed to improve in MKR mice after rosiglitazone treatment. Insulin suppression of hepatic endogenous glucose production failed to improve in MKR mice following rosiglitazone treatment. This lack of change in hepatic insulin insensitivity was associated with no change in the ratio of HMW : total adiponectin, hepatic triglyceride content, and sustained hepatic expression of PPAR and stearoyl-CoA desaturase 1 mRNA. Interestingly, rosiglitazone markedly enhanced glucose uptake by white adipose tissue with a parallel increase in CD36, aP2 and GLUT4 gene expression.Conclusions/interpretation These data suggest that potentiation of insulin action on tissues other than adipose tissue is required to mediate the antidiabetic effects of thiazolidinediones in our MKR diabetic mice.     

Effect of cold acclimation on the expression of glucose transporter Glut 4.

Glucose uptake by brown adipose tissue, measured following deoxyglucose injection in vivo, was increased by 6- and 11-fold following 2 and 14 days of cold exposure, respectively. To look for the possible mechanism of these modifications, the glucose transporter Glut 4 has been characterized at the protein and mRNA levels in brown adipose tissue, skeletal muscle and white adipose tissue following cold acclimation. Crude membranes were prepared from those tissues, and Glut 4 was studied by Western blot analysis. In brown adipose tissue, the total Glut 4 amount was increased by 52 +/- 7% and by 104 +/- 12% following 2 and 14 days of cold exposure, respectively. By contrast, in white adipose tissue of 14-day-cold-exposed mice the total Glut 4 content was decreased by 42 +/- 5%. However, Glut 4 concentration, expressed per mg of membrane protein, was unchanged in both brown and white adipose tissues following cold exposure, since the membrane protein content increased in brown but decreased in white adipose tissue. No modification in Glut 4 content was observed in skeletal muscle from cold-exposed mice. Total RNA were prepared and analyzed for Glut 4, glyceraldehyde phosphate dehydrogenase (GAPDH) and actin. Glut 4 and GAPDH mRNA were increased 2-fold in brown adipose tissue from cold-exposed mice, while actin mRNA content was unmodified. Glut 4 mRNA content was not changed in white adipose tissue and skeletal muscle from cold-exposed mice. Our results suggest that Glut 4 expression is differently modulated in the three insulin-responsive tissues during cold acclimation.     

Up-regulation of uncoupling protein 3 gene expression by fatty acids and agonists for PPARs in L6 myotubes

Uncoupling protein 3 (UCP3), which uncouples electron transport from ATP synthesis, is expressed at high levels in the skeletal muscle, an important organ in glucose and lipid metabolism. Because several reports proposed that fatty acids induced UCP3 gene expression in skeletal muscle in vivo, in the present study we examined the regulation of UCP3 gene expression by various fatty acids using L6 myotubes. UCP3 gene expression was increased in L6 myotubes by various fatty acids or by alpha-bromopalmitate, a nonmetabolized derivative of palmitic acid. Because fatty acids are also known as agonists for PPARs, we examined the involvement of PPARs in the regulation of the UCP3 gene expression. L-165041, a PPAR delta agonist, increased UCP3 gene expression in L6 myotubes, whereas neither Wy 14,643, a PPAR alpha agonist, nor Pioglitazone, a PPAR gamma agonist, increased it. Therefore, we conclude that UCP3 gene expression is increased by the activation of PPAR delta in L6 myotubes and postulate that PPAR delta mediates at least some part of the increased UCP3 gene expression by fatty acids in skeletal muscle in vivo.