11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) catalyzes the interconversion of inactive cortisone to active cortisol. Overexpression of 11beta-HSD-1 in murine adipose tissue results in glucocorticoid receptor (GR)alpha overexpression, central obesity, and insulin resistance. It is controversial whether 11beta-HSD-1 or GRalpha expression are increased in human adipose tissue in obesity. We studied effects of acquired obesity on 11beta-HSD-1 gene (real-time PCR) and protein (Western blotting) expression in sc adipose tissue in 17 monozygotic twin pairs aged 24-27 yr with a mean intrapair difference in body mass index (BMI) of 3.8 kg/m(2) (range 0.4-10.1 kg/m(2)). Intrapair correlations were calculated to study effects of acquired obesity on 11beta-HSD-1 expression. Western blot analysis of adipose tissue homogenates identified approximately 50- and approximately 68-kDa proteins specific for 11beta-HSD-1. Both structural forms correlated positively with 11beta-HSD-1 mRNA concentrations. Intrapair differences in 11beta-HSD-1 mRNA, and the 50- and 68-kDa proteins in sc adipose tissue correlated positively with those in BMI (kilograms per square meter) (r = 0.78 for 11beta-HSD-1 mRNA, P = 0.0002; r = 0.87 for the 11beta-HSD-1 50-kDa protein, P = 0.0003; and r = 0.62 for the 11beta-HSD-1 68-kDa protein, P = 0.033), total body fat (percent) (r = 0.65, P = 0.005; r = 0.83, P = 0.001; and r = 0.69, P = 0.013, respectively) and sc fat (cubed centimeters) (r = 0.66, P = 0.004; r = 0.94, P = 0.0001; and r = 0.71, P = 0.009, respectively). Furthermore, 11beta-HSD-1 mRNA and 50-kDa protein expression, but not 68-kDa protein expression, correlated positively with intrapair differences in intraabdominal fat mass (cubed centimeters) (r = 0.62, P = 0.008; r = 0.69, P = 0.013; r = 0.48, P = 0.112) and serum fasting insulin concentration (milliunits per liter) (r = 0.76, P = 0.0004; r = 0.60, P = 0.037; and r = 0.43, P = 0.160, respectively). Intrapair differences in GRalpha expression were significantly inversely correlated with those in BMI and total and sc fat mass. In conclusion, expression of 11beta-HSD-1 in sc adipose tissue is increased in human acquired obesity and is closely related to accumulation of sc and intraabdominal fat and features of insulin resistance.

Objective-The objective of this work was to explore the role of peroxisome proliferator-activated receptor delta (PPARD) in lipid metabolism in humans. Methods and Results-PPARD is a nuclear receptor involved in lipid metabolism in primates and mice. We screened the 5Ј-region of the human gene for polymorphisms to be used as tools in association studies. Four polymorphisms were detected: Ϫ409C/T in the promoter region, ϩ73C/T in exon 1, ϩ255A/G in exon 3, and ϩ294T/C in exon 4. The frequencies of the rare alleles were 4.2%, 4.2%, 1.2% and 15.6%, respectively, in a population-based group of 543 healthy men. Only the ϩ294T/C polymorphism showed significant association with a metabolic trait. Homozygotes for the rare C allele had a higher plasma LDL-cholesterol concentration than homozygotes for the common T allele, which was verified in an independent cohort consisting of 282 healthy men. Transfection studies showed that the rare C allele had higher transcriptional activity than the common T allele. Electrophoretic mobility shift assays demonstrated that the ϩ294T/C polymorphism influenced binding of Sp-1. An interaction with the PPAR alpha L162V polymorphism was also detected for several lipid parameters. Key Words: genetics Ⅲ cholesterol Ⅲ peroxisome proliferator-activated receptor Ⅲ polymorphism P eroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors involved in the regulation of lipid and glucose metabolism. Three distinct PPARs, termed alpha (PPARA), gamma (PPARG), and delta (PPARD), have been isolated. They are encoded by separate genes and characterized by distinct tissue and developmental distribution patterns. PPARD is, in contrast with the other two PPARs, ubiquitously expressed. 1 PPARA is involved in lipid metabolism and fatty acid oxidation whereas PPARG influences adipocyte differentiation and insulin action. 2 The function of PPARD is not yet fully understood, but it has been shown to play a role in cholesterol metabolism in animal models. Treatment with a PPARD agonist increased plasma cholesterol concentrations in mice with a dysfunctional leptin receptor (db/db mice). 3 A recent study showed that treatment with a potent selective PPARD agonist increased plasma cholesterol concentrations, decreased plasma triglyceride concentrations, increased HDL cholesterol, and decreased the fraction of small and dense LDLs in obese rhesus monkeys. 4 Furthermore, PPARD plays a central role in fatty acidcontrolled differentiation of preadipocytes, where it promotes induction of PPARG and other genes involved in adipocyte differentiation. 5,6 Taken together, these data suggest that PPARD may play a role in the development of metabolic perturbations associated with dyslipidemia and predisposing to atherosclerosis. Conclusions-TheseBecause the promoter and 5Ј-untranslated region (5Ј-UTR) of the gene contain important regulatory regions for gene expression 7 one might speculate that nucleotide changes in these regions of PPARD may result in altered mRNA and protein levels wh...

In mature human skeletal muscle, insulin-stimulated glucose transport is mediated primarily via the GLUT4 glucose transporter. However, in contrast to mature skeletal muscle, cultured muscle expresses significant levels of the GLUT1 glucose transporter. To assess the relative contribution of these two glucose transporters, we used a novel photolabelling techniques to assess the cell surface abundance of GLUT1 and GLUT4 specifically in primary cultures of human skeletal muscle. We demonstrate that insulin-stimulated glucose transport in cultured human skeletal muscle is mediated by GLUT4, as no effect on GLUT1 appearance at the plasma membrane was noted. Furthermore, GLUT4 mRNA and protein increased twofold (p < 0.05), after differentiation, whereas GLUT1 mRNA and protein decreased 55% (p < 0.005). Incubation of differentiated human skeletal muscle cells with a non-peptide insulin mimetic significantly (p < 0.05) increased glucose uptake and glycogen synthesis. Thus, cultured myotubes are a useful tool to facilitate biological and molecular validation of novel pharmacological agents aimed to improve glucose metabolism in skeletal muscle.

Multiple alterations characterize gene expression in the subcutaneous adipose tissue of patients with HAART-associated lipodystrophy compared with HIV-positive, HAART-treated patients without lipodystrophy. The low expression of transcription factors inhibits adipocyte differentiation. The low expression of PGC-1 may contribute to mitochondrial defects. In addition, IL-6 and CD45 expressions are increased, the latter implying an excessive number of cells of leukocyte origin in lipodystrophic adipose tissue. Mitochondrial injury and an excess of proinflammatory cytokines may lead to increased apoptosis. All these changes may contribute to the loss of subcutaneous fat in HAART-associated lipodystrophy.

Activators of peroxisome proliferator-activated receptor (PPAR)␥ have been studied intensively for their insulin-sensitizing properties and antidiabetic effects. Recently, a specific PPAR␦ activator (GW501516) was reported to attenuate plasma glucose and insulin levels when administered to genetically obese ob/ob mice. This study was performed to determine whether specific activation of PPAR␦ has direct effects on insulin action in skeletal muscle. Specific activation of PPAR␦ using two pharmacological agonists (GW501516 and GW0742) increased glucose uptake independently of insulin in differentiated C2C12 myotubes. In cultured primary human skeletal myotubes, GW501516 increased glucose uptake independently of insulin and enhanced subsequent insulin stimulation. PPAR␦ agonists increased the respective phosphorylation and expression of AMP-activated protein kinase 1.9-fold (P < 0.05) and 1.8-fold (P < 0.05), of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (MAPK) 2.2-fold (P < 0.05) and 1.7-fold (P < 0.05), and of p38 MAPK 1.2-fold (P < 0.05) and 1.4-fold (P < 0.05). Basal and insulinstimulated protein kinase B/Akt was unaltered in cells preexposed to PPAR␦ agonists. Preincubation of myotubes with the p38 MAPK inhibitor SB203580 reduced insulin-and PPAR␦-mediated increase in glucose uptake, whereas the mitogen-activated protein kinase kinase inhibitor PD98059 was without effect. PPAR␦ agonists reduced mRNA expression of PPAR␦, sterol regulatory element binding protein (SREBP)-1a, and SREBP-1c (P < 0.05). In contrast, mRNA expression of PPAR␥, PPAR␥ coactivator 1, GLUT1, and GLUT4 was unaltered. Our results provide evidence to suggest that PPAR␦ agonists increase glucose metabolism and promote gene regulatory responses in cultured human skeletal muscle. Moreover, we provide biological validation of PPAR␦ as a potential target for antidiabetic therapy.

No abstract

Acquired obesity independent of genetic influences is able to increase expression of macrophage and inflammatory markers and decrease adiponectin expression in adipose tissue.

Aims/hypothesis: The aim of this study was to determine the effect of several antidiabetic agents on insulin-stimulated glycogen synthesis, as well as on mRNA expression. Methods: Cultured primary human skeletal myotubes obtained from six healthy subjects were treated for 4 or 8 days without or with glucose (25 mmol/l), insulin (400 pmol/l), rosiglitazone (10 μmol/l), metformin (20 μmol/l) or the AMP-activated kinase activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (200 μmol/l). After this, insulin-stimulated glycogen synthesis was determined. mRNA levels of the glucose transporters GLUT1 and GL UT4, the peroxisomal proliferator activator receptor gamma (PPAR gamma) co-activator 1 (PGC1) and the myocytespecific enhancer factors (MEF2), MEF2A, MEF2C and MEF2D were determined using real-time PCR analysis after 8 days exposure to the various antidiabetic agents. Results: Insulin-stimulated glycogen synthesis was significantly increased in cultured human myotubes treated with insulin, rosiglitazone or metformin for 8 days, compared with non-treated cells. Furthermore, an 8-day exposure of myotubes to 25 mmol/l glucose impaired insulin-stimulated glycogen synthesis. In contrast, treatment with AICAR was without effect on insulin-mediated glycogen synthesis. Exposure to insulin, rosiglitazone or metformin increased mRNA expression of PGC1 and GLUT4, while AICAR or 25 mmol/l glucose treatment increased GLUT1 mRNA expression. Metformin also increased mRNA expression of the MEF2 isoforms. Conclusions/ interpretation: Enhanced insulin-stimulated glycogen synthesis in human skeletal muscle cell culture coincides with increased GLUT4 and PGC1 mRNA expression following treatment with various antidiabetic agents. These data show that chronic treatment of human myotubes with insulin, metformin or rosiglitazone has a direct positive effect on insulin action and mRNA expression.