Members of the peroxisome proliferator-activated receptor (PPAR) family might be involved in pathologies with altered lipid metabolism. They participate in the control of the expression of genes involved in lipid metabolism and adipocyte differentiation. In addition, thiazolidinediones improve insulin resistance in vivo by activating PPAR gamma. However, little is known regarding their tissue distribution and relative expression in humans. Using a quantitative and sensitive reverse transcription (RT)-competitive polymerase chain reaction (PCR) assay, we determined the distribution and relative mRNA expression of the four PPARs (alpha,beta, gamma1, and gamma2) and liver X receptor-alpha (LXR alpha) in the main tissues implicated in lipid metabolism. PPAR alpha and LXR alpha were mainly expressed in liver, while PPAR gamma1 predominated in adipose tissue and large intestine. We found that PPAR gamma2 mRNA was a minor isoform, even in adipose tissue, thus causing question of its role in humans. PPAR beta mRNA was present in all the tissues tested at low levels. In addition, PPAR gamma mRNA was barely detectable in skeletal muscle, suggesting that improvement of insulin resistance with thiazolidinediones may not result from a direct effect of these agents on PPAR gamma in muscle. Obesity and NIDDM were not associated with change in PPARs and LXR alpha expression in adipose tissue. The mRNA levels of PPAR gamma1, the predominant form in adipocytes, did not correlate with BMI, leptin mRNA levels, or fasting insulinemia in 29 subjects with various degrees of obesity. These results indicated that obesity is not associated with alteration in PPAR gene expression in abdominal subcutaneous adipose tissue in humans.

Defective regulation of gene expression may be involved in the pathogenesis of type 2 diabetes. We have characterized the concerted regulation by insulin (3-h hyperinsulinemic clamp) of the expression of 10 genes related to insulin action in skeletal muscle and in subcutaneous adipose tissue, and we have verified whether a defective regulation of some of them could be specifically encountered in tissues of type 2 diabetic patients. Basal mRNA levels (determined by reverse transcriptase-competitive polymerase chain reaction) of insulin receptor, insulin receptor substrate-1, p85␣ phosphatidylinositol 3-kinase (PI3K), p110␣PI3K, p110␤PI3K, GLUT4, glycogen synthase, and sterol regulatory-element-binding protein-1c (SREBP-1c) were similar in muscle of control (n ‫؍‬ 17), type 2 diabetic (n ‫؍‬ 9), type 1 diabetic (n ‫؍‬ 9), and nondiabetic obese (n ‫؍‬ 9) subjects. In muscle, the expression of hexokinase II was decreased in type 2 diabetic patients (P < 0.01). In adipose tissue, SREBP-1c (P < 0.01) mRNA expression was reduced in obese (nondiabetic and type 2 diabetic) subjects and was negatively correlated with the BMI of the subjects (r ‫؍‬ ؊0.63, P ‫؍‬ 0.02). Insulin (؎1,000 pmol/l) induced a two-to threefold increase (P < 0.05) in hexokinase II, p85␣PI3K, and SREBP-1c mRNA levels in muscle and in adipose tissue in control subjects, in insulin-resistant nondiabetic obese patients, and in hyperglycemic type 1 diabetic subjects. Upregulation of these genes was completely blunted in type 2 diabetic patients. This study thus provides evidence for a specific defect in the regulation of a group of important genes in response to insulin in peripheral tissues of type 2 diabetic patients. Diabetes 50: 1134 -1142, 2001 I nsulin resistance is the main metabolic feature of type 2 diabetes (1,2), and several studies indicate that it generally precedes the onset of the disease (2,3). In vivo, skeletal muscle is the major site for insulin-dependent glucose disposal, and type 2 diabetic patients are characterized by a marked decrease in insulinstimulated glucose utilization in muscle mainly due to reduced glucose uptake and storage (1,2). Insulin stimulates glucose uptake by increasing the translocation of GLUT4-containing vesicles to the plasma membrane and by modifying the activity of enzymes involved in glucose metabolism (4). Insulin action is initiated by binding of the hormone to cell membranes and activation of the insulin receptor tyrosine kinase that results in the stimulation of intracellular signaling cascades (4). Among these cascades, the phosphatidylinositol 3-kinase (PI3K) pathway is thought to play a crucial role in the effects of insulin on glucose metabolism (5). Several defects in the insulin signaling pathways have been identified in skeletal muscle of type 2 diabetic patients. Impaired phosphorylation of insulin receptor and insulin receptor substrate (IRS)-1 in response to insulin has been reported (6 -8), and the induction of PI3K and Akt kinase activities have been found to be reduced (8 -10). The stimulat...

To understand better the defects in the proximal steps of insulin signaling during type 2 diabetes, we used differentiated human skeletal muscle cells in primary culture. When compared with cells from control subjects, myotubes established from patients with type 2 diabetes presented the same defects as those previously evidenced in vivo in muscle biopsies, including defective stimulation of phosphatidylinositol (PI) 3-kinase activity, decreased association of PI 3-kinase with insulin receptor substrate (IRS)-1 and reduced IRS-1 tyrosine phosphorylation during insulin stimulation. In contrast to IRS-1, the signaling through IRS-2 was not altered. Investigating the causes of the reduced tyrosine phosphorylation of IRS-1, we found a more than twofold increase in the basal phosphorylation of IRS-1 on serine 636 in myotubes from patients with diabetes. Concomitantly, there was a higher basal mitogen-activated protein kinase (MAPK) activity in these cells, and inhibition of the MAPKs with PD98059 strongly reduced the level of serine 636 phosphorylation. These results suggest that IRS-1 phosphorylation on serine 636 might be involved in the reduced phosphorylation of IRS-1 on tyrosine and in the subsequent alteration of insulininduced PI 3-kinase activation. Moreover, increased MAPK activity seems to play a role in the phosphorylation of IRS-1 on serine residue in human muscle cells.