Previous works performed in collaboration with the group of D. Langin (INSERM U317), have led us to hypothesize that free fatty acids could play a major role in the regulation of uncoupling protein 2 and 3 (UCP2 et UCP3) in vivo in humans (Millet et al, J Clin Invest, 1997; Vidal et al., Amer J Physiol, 1999). This hypothesis was strongly supported by the findings that an infusion of a triglyceride emulsion for 6 hours induced a significant increase in UCP-3 mRNA levels in skeletal muscle of healthy subjects. The changes in UCP-3 expression correlated with changes in plasma free fatty acid concentrations (Khalfallah et al. Diabetes, 2000). The mechanism of action of fatty acids has then been studied in vitro in primary cultures of human muscle cells (myotubes). We found that omega-6, but not omega-3, polyunsaturated fatty acids are potent activators of UCP2 gene expression. Their mechanisms of action involve the production of prostaglandins E2 et I2 that act, at least in part, through activation of adenylate cyclase and the protein kinase A pathway. In addition to this mechanism, we found that PGI2 could also increase UCP2 expression through a direct activation of the nuclear receptor PPARbeta (Chevillotte et al, J Biol Chem, submitted).

We have also participated in collaborative works with FATLINK partners to demonstrate the regulation of UCP gene expression by thyroid hormone in vivo in human tissues (Barbe et al, FASEB J, 2000) and to characterize the effect of weight reduction on UCP expression in skeletal muscle of type 2 diabetic patients (Schrauwen et al, Diabetologia, 2000).

We have previously demonstrated that the basal expression of the different nuclear receptors of the PPARs’ family is not altered in adipose tissue and in skeletal muscle of obese subjects with or without type 2 diabetes (Auboeuf D et al, Diabetes, 1997; Lefebvre et al. Diabetes, 1998; Rieusset J et al, Diabetes, 1999). The PPARgamma isoform can be activated by thiazolidinediones (TZD), a new class of antidiabetic agents. In vivo treatment with TZD leads to improved insulin sensitivity both in animal models and in type 2 diabetic patients. We have found that activation of PPARgamma by TZD induces a significant increase in the expression (mRNA and protein) of the p85alpha? regulatory subunit of PI-3 kinase in isolated human adipocytes, without affecting the expression of other important genes of insulin action (Rieusset et al, BBRC, 1999). Increased expression of p85alpha PI-3 kinase results in enhanced insulin-stimulated PI3-kinase and PKB activities associated with an improved antilipolytic action of insulin in adipocytes (Rieusset et al, 2000, submitted to Diabetologia). The PPARgamma-dependent regulation of the expression of p85alpha PI-3 kinase may thus be involved in the beneficial action of the TZD on insulin sensitivity in vivo.

In a collaborative study with the group of C. Bocos and E. Herrera (Madrid), the nutritional and hormonal regulation of PPARalpha expression has been characterized during the perinatal period of the rats (Panadero et al, Biochem J, submitted).

To identify genes with altered regulation in peripheral tissues of type 2 diabetic patients, we have submitted volunteers to nutritional and hormonal interventions and taken biopsies (skeletal muscle and abdominal subcutaneous adipose tissue) before and at the end of the interventions. Gene expression was estimated by measuring specific mRNA levels by RT-competitive PCR. Using two different protocols (hyperinsulinemic clamp and severe calorie restriction), we have found that the regulation of p85alpha PI-3 kinase is altered in muscle and adipose tissue of type 2 diabetic patients (Andreelli et al, Diabetologia 1999; Andreelli et al, Diabetologia, 2000). We have now strongly strengthened these data. Firstly we have found that the regulation of hexokinase II gene expression is impaired, in addition to p85alpha PI-3 kinase, in the tissue of diabetic patients. Secondly we have demonstrated that the defective regulation of these two genes is specific of type 2 diabetes and not related to obesity-associated insulin resistance (investigation of non diabetic obese subjects) or to chronic hyperglycemia (investigation of type 1 diabetic patients) (Ducluzeau et al, Diabetes, submitted). The study of the promoters of these two genes is in progress in cultured myotubes obtained from diabetic patients.

1 Laville M, Auboeuf D, Khalfallah Y, Vega N, Riou JP, Vidal H. Acute regulation by insulin of phosphatidylinositol-3-kinase, Rad, Glut 4 and lipoprotein lipase mRNA levels in human muscle. J Clin Invest, 1996, 98, 43-49.