Influence of dietary fat on postprandial glucose metabolism (exogenous and endogenous) using intrinsically (13)C-enriched durum wheat.

The present study evaluates the influence of different amounts of fat added to starch on postprandial glucose metabolism (exogenous and endogenous). Nine women (24 (se 2) years old, BMI 20.4 (se 0.7) kg/m(2)) ingested 1 week apart 75 g glucose equivalent of (13)C-labelled starch in the form of pasta without (low fat; LF) or with 15 (medium fat; MF) or 40 (high fat; HF) g sunflower oil. During the 7 h following meal consumption, plasma glucose, non-esterified fatty acids, triacylglycerols (TG) and insulin concentrations, and endogenous (using [6,6-(2)H(2)]glucose) and exogenous glucose turnover were determined. With MF and HF meals, a lower postprandial glucose peak was observed, but with a secondary recovery. A decrease in exogenous glucose appearance explained lower glycaemia in HF. At 4 h after the HF meal the insulin, insulin:glucose and postprandial blood TG were higher than those measured after the LF and MF meals. Despite higher insulinaemia, total glucose disappearance was similar and endogenous glucose production was suppressed less than after the LF and MF meals, suggesting insulin resistance. Thus, the addition of a large amount of fat appears to be unfavourable to glucose metabolism because it leads to a feature of insulin resistance. On the contrary, the MF meal did not have these adverse effects, but it was able to decrease the initial glycaemic peak.

Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes.

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, p85alpha phosphatidylinositol 3-kinase (PI3K), p110alphaPI3K, p110betaPI3K, 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, p85alphaPI3K, 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.

The expression of the p85alpha subunit of phosphatidylinositol 3-kinase is induced by activation of the peroxisome proliferator-activated receptor gamma in human adipocytes.

AIMS/HYPOTHESIS: Thiazolidinediones are new oral antidiabetic drugs that activate the nuclear receptor PPARgamma. Our aim was to identify potential target genes of PPARgamma in the human adipocyte in order to clarify how thiazolidinediones improve insulin sensitivity. METHODS: The effect of BRL 49653 (Rosiglitazone) on the mRNA expression of insulin receptor, insulin receptor substrate-1, p85alpha, p110alpha and p110beta subunits of phosphatidylinositol 3-kinase, Glut 4 and hormone sensitive lipase was examined in isolated adipocytes. Target mRNA levels were determined by RT-competitive PCR. RESULTS: The BRL 49653 (1 micromol/l) increased the mRNA concentrations of p85alphaPI-3 K (264 +/- 46 vs 161 +/- 31 amol/microg total RNA, p = 0.003) whithout affecting the expression of the other mRNAs of interest. This effect was dose-dependent (K0.5 = 5 nmol/l) and was reproduced by a specific activator of RXR, indicating that it was probably mediated by the PPARgamma/RXR heterodimer. The BRL 49653 also increased the amount of p85alphaPI-3K protein in adipose tissue explants (71 +/- 19%). In addition, BRL 49653 produced a more than twofold increase in insulin stimulation of phosphatidylinositol 3-kinase activity and significantly enhanced the antilipolytic action of insulin. CONCLUSION/INTERPRETATION: This work demonstrates that the gene of p85alphaPI-3K is probably a target of PPARgamma and that thiazolidinediones can improve insulin action in normal human adipocytes. Although the precise mechanism of action of BRL 49653 on PI3-Kinase activity is not completely clear, these findings improve our understanding of the insulin-sensitizing effects of the thiazolidinediones, possible drugs for the treatment of Type II (non-insulin-dependent) diabetes mellitus.

Subcutaneous adipose tissue expression of tumour necrosis factor-alpha is not associated with whole body insulin resistance in obese nondiabetic or in type-2 diabetic subjects.

BACKGROUND: An association with subcutaneous adipose tissue TNFalpha expression and insulin resistance has been suggested in obesity/type-2 diabetes, but this has not been examined directly. In the first part of the study we investigated whether this association is present in 7 lean, 10 obese nondiabetic and 9 type-2 diabetic men. In the second part of the study we examined the relationship between adipose tissue TNFalpha mRNA levels and BMI in 81 nondiabetic subjects spanning a wide range of BMIs. METHODS: Subcutaneous adipose tissue TNFalpha mRNA levels and insulin sensitivity were determined with quantitative RT-competitive PCR and hyperinsulinaemic clamp, respectively. RESULTS: Subcutaneous adipose tissue TNFalpha mRNA levels were similar in 7 lean and 10 obese nondiabetic and 9 type-2 diabetic men (P = 0.68), and did not change in response to 240-min hyperinsulinaemia. TNFalpha mRNA levels and insulin sensitivity were not correlated. Unexpectedly, no correlation between TNFalpha mRNA and BMI was found. The relationship between adipose tissue TNFalpha mRNA and BMI was examined further in 31 male and 50 female nondiabetic subjects. The subcutaneous adipose tissue TNFalpha mRNA level correlated with BMI in all subjects (rS = 0.32, P < 0.01), and in a subgroup analysis in men (rS = 0.55, P < 0.01) but not in women (rS = - 0.08). The correlation in men was dependent on a fourfold higher TNFalpha mRNA level in 5 morbidly obese men while there was no difference in TNFalpha mRNA levels in lean or obese men. CONCLUSIONS: Subcutaneous adipose tissue TNFalpha expression does not correlate with insulin sensitivity in nondiabetic or type-2 diabetic men; is not regulated by acute hyperinsulinaemia; and is increased only in morbidly obese men.

Regulation of gene expression during severe caloric restriction: lack of induction of p85 alpha phosphatidylinositol 3-kinase mRNA in skeletal muscle of patients with type II (non-insulin-dependent) diabetes mellitus.

AIMS/HYPOTHESIS: Alterations in the regulation of gene expression could be involved in the development of Type II (non-insulin-dependent) diabetes mellitus. METHODS: We compared the mRNA concentrations of eight genes encoding proteins involved in insulin action and intermediary metabolism in skeletal muscle of healthy volunteers and Type II diabetic patients. The in vivo regulation of the expression of these genes was investigated after 5 days of hypocaloric diet (1045 kJ/day). RESULTS: In the basal state, diabetic muscle showed reduced insulin receptor (-38%), hexokinase II (-73%), glycogen synthase (-45%) and lipoprotein lipase (-70%) mRNA expression. There was no difference in the mRNA abundances of IRS-1, GLUT 4, p85 alpha phosphatidylinositol-3-kinase (p85 alpha PI3K) or Rad. In both groups, caloric restriction induced weight loss, reduced glycaemia and increased plasma ketone body concentrations. The diet also increased plasma concentrations of fatty acids and decreased whole-body insulin sensitivity in control subjects. In control subjects, the diet increased p85 alpha PI3K (+146%), insulin receptor (+100%) and Rad (+40%) mRNA concentrations in muscle. In Type II diabetic patients, the diet increased insulin receptor (+41%) and Rad (+31%) mRNAs but the expression of p85 alpha PI3K was not modified. CONCLUSION/INTERPRETATION: The regulation of the expression of p85 alpha PI3K is altered during caloric restriction in skeletal muscle of Type II diabetic patients. Because we have shown in an earlier study that there is also a defective regulation of p85 alpha PI3K gene expression in response to insulin, these data support the hypothesis that alterations in the regulation of gene expression could be involved in the pathogenesis of Type II diabetes.

Normal reproductive function in leptin-deficient patients with lipoatropic diabetes.

To further examine the relationships between leptin and female reproductive axis, we conducted hormonal studies in two patients with lipoatropic diabetes that occurred before puberty. Despite complete atrophy of sc and visceral adipose tissue, menarche occurred in these two patients between 11-12 yr of age, followed by regular menstrual cycles. One patient had been pregnant three times, giving birth to children who did not develop the disease. In our two patients, repeated analysis revealed leptin levels below 1 ng/mL (normal range for 20 insulin-treated diabetic women, 2-23 ng/mL for body mass index of 14-39 kg/m2; personal data). We measured peripheral levels of estradiol, progesterone, FSH, LH, free testosterone, and androstenedione within the first 5 days of the menstrual cycle, and we tested the reactivity of pituitary after iv injection of 100 microg GnRH. The variation in body temperature in the morning before arising was also analyzed. We showed that 1) all measured levels of hormones were in the normal range for both patients; and 2) low levels of leptin did not impair the development of reproductive function in one patient and was associated with normal gonadal function in both patients. We conclude that puberty and fertility can occur despite chronic low serum levels of leptin. This suggests that leptin is not fundamental to the maintenance of normal reproductive function in humans.

Postmenopause hormone treatment in women with NIDDM or impaired glucose tolerance: the MEDIA randomized clinical trial.

OBJECTIVE: To assess the biological safety of four hormone replacement treatment (HRT) combinations in women with non insulin dependent diabetes mellitus (NIDDM) or impaired glucose tolerance (IGT). SUBJECTS AND METHODS: Randomized, double-blind, placebo-controlled trial to analyze the variation of fibrinogen, factor VII, PAI1, and TG blood levels in women (n=99), with NIDDM or IGT, receiving a 3-month course of either oral oestradiol (1 or 2 mg) combined with Chlormadinone Acetate 5 mg, or transdermal oestradiol 50 microg/24 h in association with Norethisterone Acetate (11.2 or 22.4 mg), or placebo. Follow-up lasted 3 months. RESULTS: Ninety nine patients, mean age 56 years (SD 5), mean diabetes duration 7 years (S.D. 7), mean glycated hemoglobin (7.3%) were enrolled. There was no significant difference between the groups for any of the primary hemostasis criteria (n=77). Triglycerides (TG) variation significantly differed between groups, P=0.01, from -21% in the large patch group, to +22% in the placebo group (n=82). Treatment administration routes did not significantly differ for any of the criteria. There was a significant difference in the total cholesterol variation between groups, from +8.7% in the placebo group to -10.8% in the oral 1 mg group (P=0.001). CONCLUSION: The treatments had no highly deleterious effect in these patients with NIDDM or with IGT. Long-term trials can be performed with such patients, and an hormone treatment can be prescribed to relieve symptoms. Since these patients had a well-controlled NIDDM, results might be different in less well-controlled diabetes. The data do not support the hypothesis of an impaired oestrogen effect in patients with NIDDM.

Insulin acutely regulates the expression of the peroxisome proliferator-activated receptor-gamma in human adipocytes.

Peroxisome proliferator-activated receptor (PPAR)-gamma is one of the key actors of adipocyte differentiation. This study demonstrates 1) that PPAR-gamma mRNA expression is not altered in subcutaneous adipose tissue (n = 44) or in skeletal muscle (n = 19) of subjects spanning a wide range of BMIs (20-53 kg/m2) and 2) that insulin acutely increases PPAR-gamma mRNA expression in human adipocytes both in vivo and in vitro. The effect of insulin was investigated in abdominal subcutaneous biopsies obtained before and at the end of a 3-h euglycemic-hyperinsulinemic clamp. Insulin significantly increased PPAR-gamma mRNA levels in lean subjects (88 +/- 17%, n = 6), in type 2 diabetic patients (100 +/- 19%, n = 6), and in nondiabetic obese patients (91 +/- 20%, n = 6). Both PPAR-gamma1 and PPAR-gamma2 mRNA variants were increased (P < 0.05) after insulin infusion. In isolated human adipocytes, insulin induced the two PPAR-gamma mRNAs in a dose-dependent manner, with half-maximal stimulation at a concentration of approximately 1-5 nmol/l. However, PPAR-gamma2 mRNA was rapidly (2 h) and transiently increased, whereas a slow and more progressive induction of PPAR-gamma1 was observed during the 6 h of incubation. In explants of human adipose tissue, PPAR-gamma protein levels were significantly increased (42 +/- 3%, P < 0.05) after 12 h of incubation with insulin. These data demonstrate that PPAR-gamma belongs to the list of the insulin-regulated genes and that obesity and type 2 diabetes are not associated with alteration in the expression of this nuclear receptor in adipose tissue.

Defective regulation of phosphatidylinositol-3-kinase gene expression in skeletal muscle and adipose tissue of non-insulin-dependent diabetes mellitus patients.

We investigated the regulation of the mRNA expression of the insulin receptor, insulin receptor substrate-1 (IRS-1) and p85alpha-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-insulin-dependent) 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 p85alpha-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 p85alpha-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 p85alpha-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.

Germ-line mutation analysis in patients with multiple endocrine neoplasia type 1 and related disorders.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome predisposing to tumors of the parathyroid, endocrine pancreas, anterior pituitary, adrenal glands, and diffuse neuroendocrine tissues. The MEN1 gene has been assigned, by linkage analysis and loss of heterozygosity, to chromosome 11q13 and recently has been identified by positional cloning. In this study, a total of 84 families and/or isolated patients with either MEN1 or MEN1-related inherited endocrine tumors were screened for MEN1 germ-line mutations, by heteroduplex and sequence analysis of the MEN1 gene-coding region and untranslated exon 1. Germ-line MEN1 alterations were identified in 47/54 (87%) MEN1 families, in 9/11 (82%) isolated MEN1 patients, and in only 6/19 (31.5%) atypical MEN1-related inherited cases. We characterized 52 distinct mutations in a total of 62 MEN1 germ-line alterations. Thirty-five of the 52 mutations were frameshifts and nonsense mutations predicted to encode for a truncated MEN1 protein. We identified eight missense mutations and five in-frame deletions over the entire coding sequence. Six mutations were observed more than once in familial MEN1. Haplotype analysis in families with identical mutations indicate that these occurrences reflected mainly independent mutational events. No MEN1 germ-line mutations were found in 7/54 (13%) MEN1 families, in 2/11 (18%) isolated MEN1 cases, in 13/19 (68. 5%) MEN1-related cases, and in a kindred with familial isolated hyperparathyroidism. Two hundred twenty gene carriers (167 affected and 53 unaffected) were identified. No evidence of genotype-phenotype correlation was found. Age-related penetrance was estimated to be >95% at age >30 years. Our results add to the diversity of MEN1 germ-line mutations and provide new tools in genetic screening of MEN1 and clinically related cases.

Influence of human obesity on the metabolic fate of dietary long- and medium-chain triacylglycerols.

The metabolic fate of an oral long-chain-triacylglycerol (LCT) load and of a mixed oral LCT and medium-chain-triacylglycerol (MCT) load was followed for 6 h in eight control and eight obese subjects with normal postabsorptive triacylglycerol concentrations. Labeled triacylglycerol and indirect calorimetry were used. Results showed that LCTs were less oxidized in obese than in control subjects (3.2+/-0.5 compared with 6.0+/-0.4 g, P < 0.01). Moreover, the amount of LCT oxidized was negatively correlated with fat mass (r = -0.77, P < 0.01). Appearance in plasma of dietary triacyglycerol-derived long-chain fatty acids was blunted in obese subjects and it was negatively related to fat mass (r = -0.84, P < 0.01) and positively to LCT oxidation (r = 0.70, P < 0.01). On the contrary, MCT oxidation was not altered in obese subjects compared with control subjects. Furthermore, the proportion of MCTs oxidized was higher in both groups compared with LCTs (x+/-SEM: 57.5+/-2.6% compared with 15.2+/-1.6%, P < 0.01, n = 16). Our conclusion is that obesity is associated with a defect in the oxidation of dietary LCTs probably related to an excessive uptake by the adipose tissue of meal-derived long-chain fatty acids. MCTs, the oxidation of which is not altered in obesity, could therefore be of interest in the dietary treatment of obesity.

Depot-specific differences in adipose tissue gene expression in lean and obese subjects.

Intra-abdominal and subcutaneous adipose tissue display important metabolic differences that underlie the association of visceral, but not subcutaneous, fat with obesity-related cardiovascular and metabolic problems. Because the molecular mechanisms contributing to these differences are not yet defined, we compared by reverse transcription-polymerase chain reaction the expression of 15 mRNAs that encode proteins of known importance in adipocyte function in paired omental and subcutaneous abdominal biopsies. No difference in mRNA expression between omental and subcutaneous adipose tissue was observed for hormone sensitive lipase, lipoprotein lipase, 6-phosphofructo-1-kinase, insulin receptor substrate 1, p85alpha regulatory subunit of phosphatidylinositol-3-kinase, and Rad. Total amount of insulin receptor expression was significantly higher in omental adipose tissue. Most of this increase was accounted for by expression of the differentially spliced insulin receptor lacking exon 11, which is considered to transmit the insulin signal less efficiently than the insulin receptor with exon 11. Perhaps consistent with a less efficient insulin signaling, a twofold reduction in GLUT4, glycogen synthase, and leptin mRNA expression was observed in omental adipose tissue. Finally peroxisome proliferator activated receptor-gamma (PPAR-gamma) mRNA levels were significantly lower in visceral adipose tissue in subjects with a BMI <30 kg/m2, but not in obese subjects, indicating that relative PPAR-gamma expression is increased in omental fat in obesity. This suggests that altered expression of PPAR-gamma might play a role in adipose tissue distribution and expansion.

Total body water and body composition in chronic peritoneal dialysis patients.

In this investigation, total body water (TBW) in ten chronic peritoneal dialysis patients was studied by deuterium (TBW-2H), skinfold thickness (TBW-ST), Watson formula (TBW-WA), 58% of body weight (TBW-58%), and bioelectrical impedance (TBW-BIA), and these results were compared with the reference oxygen18 (TBW-18O) method. We also analyzed the fat-free mass (FFM) by skinfold thickness (FFM-ST), bioelectrical impedance (FFM-BIA), oxygen18 (FFM-18O), and creatinine kinetics method (FFM-CK). In addition, resting metabolic rate was measured by indirect calorimetry. Compared with TBW-18O, TBW-58% and TBW-BIA were significantly different (P < 0.01). TBW-2H overestimated TBW-18O by 4.3%. TBW-ST and TBW-WA gave slightly greater values than TBW-18O, although these values were nonstatistically significant. The best prediction of total body water from these methods was obtained with the Watson formula. When Kt/V was calculated from these results, the values obtained were statistically greater (BIA, P < 0.001) and smaller (58% BW, P < 0.01) than those obtained with either 18O or Watson formula. The fat-free mass estimation also led to discrepant findings. Indeed, FFM-CK was significantly lower (P < 0.05) as compared with FFM-ST, FFM-BIA, or FFM-18O. Resting metabolic rate was strongly correlated with FFM estimated by skinfold thickness (r = 0.91, P < 0.001), bioelectrical impedance (r = 0.85, P < 0.005), and 18O (r = 0.77, P < 0.01), but not when fat-free mass was estimated by the creatinine kinetic method. The water content of fat-free mass estimated by skinfold thickness was found to be 69.7 +/- 6.9% in these patients, a value lower than the standard 73.2% found in healthy adults. This study confirms that there is an abnormal water distribution in chronic peritoneal dialysis patients. However, when compared with the oxygen18 reference method, the Watson formula allows a reliable estimation of Kt/V.

Tissue distribution and quantification of the expression of mRNAs of peroxisome proliferator-activated receptors and liver X receptor-alpha in humans: no alteration in adipose tissue of obese and NIDDM patients.

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.

Endogenous glucose production, gluconeogenesis and liver glycogen concentration in obese non-diabetic patients.

Resting, post-absorptive endogenous glucose production (EGP), fractional gluconeogenesis and liver glycogen concentration were assessed in 6 lean and 5 obese non-diabetic subjects undergoing elective abdominal surgery. During the 2 days preceding these measurements, 0.3 g/day U-13C glucose had been added to their usual diet to label their endogenous glycogen stores. On the morning of day 3, EGP was measured with 6,6-2H glucose. Their endogenous 13C glycogen enrichment was calculated from 13CO2 and respiratory gas exchanges. Fractional gluconeogenesis was assessed as 1-(13C glucose/13C glycogen)100. EGP was similar in lean subjects (113 +/- 5 mg/min) and in obese subjects (111 +/- 6). Fractional gluconeogenesis was higher in obese (59 +/- 10%) than in lean subjects (29 +/- 8%). However, overall EGP remained constant due to a decrease in glycogenolysis. Since an increased gluconeogenesis and a decreased glycogenolysis may both contribute to increase liver glycogen concentration in obesity, hepatic glycogen concentrations were assessed in hepatic needle biopsies obtained during surgery. Hepatic glycogen concentrations were increased in obese patients (515 +/- 38 mg/g protein) compared to lean subjects (308 +/- 58, p < 0.05). It is concluded that in obese patients: a) fractional gluconeogenesis is increased; b) overall EGP is unchanged due to a proportional inhibition of glycogenolysis; c) liver glycogen concentration is increased.

Insulin receptor mRNA splicing and altered metabolic control in aged and mildly insulin-deficient rats.

Using reverse transcription-competitive polymerase chain reaction, we measured the abundance of the mRNAs encoding the two spliced isoforms of insulin receptor in aged and mildly insulin-deficient rats. Twelve-month-old rats were characterized by peripheral insulin resistance and decreased glucose tolerance. Mild insulin deficiency, obtained by neonatal streptozotocin treatment, was associated with glucose intolerance due to reduced glucose-stimulated insulin response. Both models were associated with a decrease in the relative abundance of the mRNA with exon 11 in liver, heart, adipose tissue, and tibialis muscle, whereas a slight increase was seen in the extensor digitorum longus and no change in the soleus muscle. In the three muscles, the expression of the form without exon 11 largely predominated (>90%). In heart and adipose tissue, the two isoforms were expressed at a similar level in control rats. In both tissues, the form without exon 11 increased in streptozotocin-treated rats, whereas the absolute level of the form with exon 11 decreased in old rats. Although a decreased level of the variant with exon 11 correlated with insulin resistance of whole body glucose uptake, our results indicated that changes in the expression of the insulin receptor variants were secondary events and thus not the cause of the insulin resistance in old and mildly insulin-deficient rats.

Increased uncoupling protein-2 and -3 mRNA expression during fasting in obese and lean humans.

Uncoupling protein-2 and -3 (UCP2 and UCP3) are mitochondrial proteins that show high sequence homology with the brown adipocyte-specific UCP1. UCP1 induces heat production by uncoupling respiration from ATP synthesis. UCP2 is widely expressed in human tissues, whereas UCP3 expression seems restricted to skeletal muscle, an important site of thermogenesis in humans. We have investigated the regulation of UCP2 and UCP3 gene expression in skeletal muscle and adipose tissue from lean and obese humans. UCP2 and -3 mRNA levels were not correlated with body mass index (BMI) in skeletal muscle, but a positive correlation (r = 0.55, P < 0.01, n = 22) was found between UCP2 mRNA level in adipose tissue and BMI. The effect of fasting was investigated in eight lean and six obese subjects maintained on a hypocaloric diet (1,045 kJ/d) for 5 d. Calorie restriction induced a similar 2-2.5-fold increase in UCP2 and -3 mRNA levels in lean and obese subjects. To study the effect of insulin on UCP gene expression, six lean and five obese subjects underwent a 3-h euglycemic hyperinsulinemic clamp. Insulin infusion did not modify UCP2 and -3 mRNA levels. In conclusion, the similar induction of gene expression observed during fasting in lean and obese subjects shows that there is no major alteration of UCP2 and -3 gene regulation in adipose tissue and skeletal muscle of obese subjects. The increase in UCP2 and -3 mRNA levels suggests a role for these proteins in the metabolic adaptation to fasting.