We have investigated the acute regulation by insulin of the mRNA levels of nine genes involved in insulin action, in muscle biopsies obtained before and at the end of a 3-h euglycemic hyperinsulinemic clamp. Using reverse transcriptioncompetitive PCR, we have measured the mRNAs encoding the two insulin receptor variants, the insulin receptor substrate-1, the p85 ␣ subunit of phosphatidylinositol-3-kinase, Ras associated to diabetes (Rad), the glucose transporter Glut 4, glycogen synthase, 6-phosphofructo-1-kinase, lipoprotein lipase, and the hormone-sensitive lipase. Insulin infusion induced a significant increase in the mRNA level of Glut 4 ( ϩ 56 Ϯ 13%), Rad ( ϩ 96 Ϯ 25%), the p85 ␣ subunit of phosphatidylinositol-3-kinase ( ϩ 92 Ϯ 18%) and a decrease in the lipoprotein lipase mRNA level ( Ϫ 49 Ϯ 5%), while the abundance of the other mRNAs was unaffected. The relative expression of the two insulin receptor variants was not modified. These results demonstrate an acute coordinated regulation by insulin of the expression of genes coding key proteins involved in its action in human skeletal muscle and suggest that Rad and the p85 ␣ regulatory subunit of phosphatidylinositol-3-kinase can be added to the list of the genes controlled by insulin. ( J. Clin. Invest. 1996. 98:43-49.)

Background:Contrasting with obesity, constitutional thinness (CT) is a rare condition of natural low bodyweight. CT exhibits preserved menstruation in females, no biological marker of undernutrition, no eating disorders but a bodyweight gain desire. Anorexigenic hormonal profile with high peptide tyrosine tyrosine (PYY) was shown in circadian profile. CT could be considered as the opposite of obesity, where some patients appear to resist diet-induced bodyweight loss.Objective:The objective of this study was to evaluate appetite regulatory hormones in CTs in an inverse paradigm of diet-induced weight loss.Methods:A 4-week fat overfeeding (2640 kJ excess) was performed to compare eight CT women (body mass index (BMI)<17.5 kg m−2) to eight female controls (BMI 18.5–25 kg m−2). Appetite regulatory hormones profile after test meal, food intake, bodyweight, body composition, energy expenditure and urine metabolomics profiles were monitored before and after overfeeding.Results:After overfeeding, fasting total and acylated ghrelin were significantly lower in CTs than in controls (P=0.01 and 0.03, respectively). After overfeeding, peptide tyrosine tyrosine (PYY) and glucagon-like-peptide 1 both presented earlier (T15 min vs T30 min) and higher post-meal responses (incremental area under the curve) in CTs compared with controls. CTs failed to increase bodyweight (+0.22±0.18 kg, P=0.26 vs baseline), contrasting with controls (+0.72±0.26 kg, P=0.03 vs baseline, P=0.01 vs CTs). Resting energy expenditure increased in CTs only (P=0.031 vs baseline). After overfeeding, a significant negative difference between total energy expenditure and food intake was noticed in CTs only (−2754±720 kJ, P=0.01).Conclusion:CTs showed specific adaptation to fat overfeeding: overall increase in anorexigenic hormonal profile, enhanced post prandial GLP-1 and PYY and inverse to controls changes in urine metabolomics. Overfeeding revealed a paradoxical positive energy balance contemporary to a lack of bodyweight gain, suggesting yet unknown specific energy expenditure pathways in CTs.

To study the effect of nonesterified fatty acids (NEFA s ) on uncoupling protein-2 (UCP-2) and uncoupling protein-3 (UCP-3) gene expression, a triglyceride emulsion was infused for 5 h in 14 healthy volunteers. A euglycemic-hyperinsulinemic clamp was administered concomitantly in 7 of the 14 subjects. The mRNA levels of UCP-2 and of the short (UCP-3S) and long (UCP-3L) isoforms of UCP-3 were quantified by reverse transcription-competitive polymerase chain reaction in tissue biopsies taken before and at the end of the infusion periods. Plasma NEFA concentrations increased from 362 ± 52 to 989 ± 157 µmol/l (P = 0.018) during triglyceride infusion. UCP-3L (8 ± 1 vs. 19 ± 2 amol/µg total RNA, P = 0.018) and UCP-3S (11 ± 2 vs. 17 ± 3 amol/µg total RNA, P = 0.027) mRNA levels increased in skeletal muscle during triglyceride infusion. UCP-3L mRNA levels were positively correlated with plasma NEFA concentrations (r = 0.53, P = 0.005) and with lipid oxidation rates (r = 0.56, P = 0.004) determined by indirect c a l o r i m e t r y. In contrast, the expression of UCP-2 was not affected by lipid infusion in skeletal muscle or in subcutaneous adipose tissue. During the hyperinsulinemic clamp (plasma insulin concentrations 202 ± 1 2 pmol/l), NEFA levels (405 ± 49 vs. 648 ± 77 µmol/l, P = 0.063) and lipid oxidation rates (0.67 ± 0.09 vs. 0.84 ± 0 . 1 0 m g · k g -1 · min -1 , P = 0.091) were not significantly increased during triglyceride infusion. Under such conditions, the induction of UCP-3L and UCP-3S mRNA expression was totally prevented (8 ± 2 vs. 8 ± 1 and 8 ± 2 vs. 9 ± 2 amol/µg total RNA, respectively). We conclude that increased plasma NEFA levels by lipid infusion for 5 h induces the expression of UCP-3 but not UCP-2 in humans. During triglyceride infusion, physiological hyperinsulinemia appears to prevent the induction of UCP-3 mRNA levels. Diabetes 4 9 :2 5-31, 2000

Despite intensive efforts, the nature of the molecular defects leading to the development of Type II (noninsulin-dependent) diabetes mellitus is poorly understood. Whereas both alterations in insulin secretion and action are involved in established diabetes [1,2], it has been suggested that defects in insulin action precede the onset of the disease [3]. There is good evidence that insulin resistance in Type II diabetes is dependent, at least in part, on genetic factors [4,5]. The mutations identified so far in the sequences of candidate genes can, however, explain only a small proportion of the different forms of Type II diabetes [5]. Changes in biological functions in response to environmental factors are also involved in the development of the disease [4]. The modulation of the expression of specific key genes is one of the main mechanisms for changing biological function in response to the environment. Several lines of evidence indicate that the expression of some important genes in insulin action is abnormal in peripheral tissues in the common form of Type II diabetes. For example, the basal concentration of the insulin sensitive glu- Diabetologia (1999) Summary We investigated the regulation of the mRNA expression of the insulin receptor, insulin receptor substrate-1 (IRS-1) and p85a-phosphatidylinositol-3-kinase (PI-3K), three major actors of insulin action, in skeletal muscle from 10 healthy lean volunteers, 13 obese patients with Type II (non-insulindependent) diabetes mellitus and 7 non-diabetic obese subjects. The in vivo regulation by insulin was studied using a 3-h euglycaemic, hyperinsulinaemic clamp. There were no differences in the basal concentrations of the three mRNAs in skeletal muscle between groups. Insulin infusion produced a twofold reduction in insulin receptor substrate-1 mRNA expression in the three groups (p < 0.02). In contrast, insulin increased p85a-phosphatidylinositol-3-kinase mRNA expression in muscle from non-diabetic subjects ( + 98 ± 22 % in lean and + 127 ± 16 % in obese, p < 0.02) but this effect was totally impaired in Type II diabetic patients ( + 5 ± 12 %, NS). A similar defect in insulin action on p85a-phosphatidylinositol-3-kinase mRNA expression was observed in abdominal subcutaneous adipose tissue ( + 138 ± 25 %, p < 0.01 in lean and + 46 ± 14 %, p < 0.02 in obese and + 29 ± 11 %, NS in Type II diabetic patients). The lack of action of insulin on p85a-phosphatidylinositol-3-kinase mRNA in diabetic subjects was probably not due to a deleterious effect of hyperglycaemia since improvement of the glycaemic control for 10 days did not restore the response in muscle or in adipose tissue. This study provides evidence for a defect in the regulation by insulin of PI-3K gene expression in Type II diabetic patients, thus reinforcing the concept that alterations at the gene expression might be involved in the pathogeny of Type II diabetes. [Diabetologia (1999)