Reduced Plasma Insulin Research Articles

Overexpression of c-Myc in β-Cells of Transgenic Mice Causes Proliferation and Apoptosis, Downregulation of Insulin Gene Expression, and Diabetes

To test the hypothesis that c-Myc plays an important role in beta-cell growth and differentiation, we generated transgenic mice overexpressing c-Myc in beta-cells under control of the rat insulin II promoter. F(1) transgenic mice from two founders developed neonatal diabetes (associated with reduced plasma insulin levels) and died of hyperglycemia 3 days after birth. In pancreata of transgenic mice, marked hyperplasia of cells with an altered phenotype and amorphous islet organization was displayed: islet volume was increased threefold versus wild-type littermates. Apoptotic nuclei were increased fourfold in transgenic versus wild-type mice, suggesting an increased turnover of beta-cells. Very few cells immunostained for insulin; pancreatic insulin mRNA and content were markedly reduced. GLUT2 mRNA was decreased, but other beta-cell-associated genes (IAPP [islet amyloid pancreatic polypeptide], PDX-1 [pancreatic and duodenal homeobox-1], and BETA2/NeuroD) were expressed at near-normal levels. Immunostaining for both GLUT2 and Nkx6.1 was mainly cytoplasmic. The defect in beta-cell phenotype in transgenic embryos (embryonic days 17-18) and neonates (days 1-2) was similar and, therefore, was not secondary to overt hyperglycemia. When pancreata were transplanted under the kidney capsules of athymic mice to analyze the long-term effects of c-Myc activation, beta-cell depletion was found, suggesting that, ultimately, apoptosis predominates over proliferation. In conclusion, these studies demonstrate that activation of c-Myc in beta-cells leads to 1) increased proliferation and apoptosis, 2) initial hyperplasia with amorphous islet organization, and 3) selective downregulation of insulin gene expression and the development of overt diabetes.

Anorectic, thermogenic and anti-obesity activity of a selective orexin-1 receptor antagonist in ob/ ob mice

A single dose of the orexin-1 (OX 1) receptor antagonist 1-(2-methylbenzoxazol-6-yl)-3-[1,5] naphthyridin-4-yl urea hydrochloride (SB-334867-A) reduces orexin-A-induced feeding and natural feeding in Sprague Dawley rats. In this study, the anti-obesity effects of SB-334867-A were determined in genetically obese ( ob/ ob) mice dosed with SB-334867-A (30 mg/kg, i.p.) once daily for 7 days, and then twice daily for a further 7 days. SB-334867-A reduced cumulative food intake and body weight gain over 14 days. Total fat mass gain, determined by Dual Emission X-ray Absorptiometry, was reduced, while gain in fat-free mass was unchanged. Fasting (5 h) blood glucose was also reduced at the end of the study, with a trend to reduced plasma insulin. Interscapular brown adipose tissue (BAT) weight was reduced, the tissue was noticeably darker in colour and quantitative PCR (TaqMan) analysis of this tissue showed a trend to an increase in uncoupling protein-1 mRNA expression, suggesting that SB-334867-A might stimulate thermogenesis. This was confirmed in a separate study in which a single dose of SB-334867-A (30 mg/kg, i.p.) increased metabolic rate over 4 h in ob/ ob mice. OX 1 receptor mRNA was detected in BAT, and its expression was increased by 58% by treatment with SB-334867-A. This is the first demonstration that OX 1 receptor antagonists have potential as both anti-obesity and anti-diabetic agents.

Changes in blood glucose and plasma insulin levels induced by bradykinin in anaesthetized rats.

1. The influence of bradykinin (BK) on blood glucose and plasma insulin levels was investigated in anaesthetized rats. 2. Blood glucose level was dose-dependently increased by intravenous infusion of BK. This effect of BK was enhanced by captopril, an inhibitor of angiotensin-converting enzyme (ACE). Des-Arg9-bradykinin (DABK), a kinin B1 receptor agonist, did not modify blood glucose levels while the effect of BK was inhibited by Hoe-140, a kinin B2 receptor antagonist. 3. The effect of BK was reduced by the NO-synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), and by the cyclo-oxygenase inhibitor, indomethacin. The effect of BK was suppressed by the association of propranolol with phentolamine or phenoxybenzamine. It was also reduced by hexamethonium, a ganglion-blocking drug. In adrenalectomized rats, the infusion of BK slightly decreased blood glucose levels. 4. The hyperglycaemic effect of adrenaline was suppressed by propranolol associated with phentolamine or phenoxybenzamine, but it was not modified by L-NAME. 5. Infusion of BK did not modify plasma insulin levels. However, after phentolamine and propranolol, BK induced a transient 2 fold rise in plasma insulin levels. The release of insulin was dose-dependent and inhibited by Hoe-140. 6. We conclude that infusion of BK induces, via a stimulation of B2 receptors, the release of NO and of prostanoids. The latter agents activate through a reflex pathway the release of catecholamines from the adrenal medulla. This release increases blood glucose levels and reduces plasma insulin levels. After adrenoceptor inhibition, BK induces a secretion of insulin, via the stimulation of B2 receptors.

Effects of palm oil and transesterified palm oil on chylomicron and VLDL triacylglycerol structures and postprandial lipid response

The effects of positional distribution of triacylglycerol (TAG) fatty acids to TAG structures in chylomicrons and VLDL, and to postprandial lipemia, were studied in 10 healthy premenopausal women using a 6-h oral fat load test and a randomized, double-blind cross-over design. Molecular level information of TAG regioisomerism was obtained with a tandem mass spectrometric method. The positional distribution of fatty acids in chylomicron TAGs was similar to the respective dietary fat; 79% of the analyzed regioisomers in palm oil and 84% of the analyzed regioisomers in transesterified oil were found in chylomicron TAGs 3 h after the oral fat loads. VLDL TAGs were equal after the two fat loads in all but one regioisomer. Similarities in the fatty acid compositions of chylomicron TAGs suggest that palmitic acid was absorbed equally from both test fats. The proportion of palmitoleic acid in the chylomicrons was increased. Fat with palmitic acid predominantly in the sn-1 and sn-3 positions caused a larger incremental area of total TAGs in plasma and reduced plasma insulin values at the beginning of the postprandial response (0–90 min) compared with fat with palmitic acid randomly distributed. The relationship between TAG molecular structures in dietary fats and in lipoproteins provides new means for understanding the effects of fatty acid positonal distribution on human lipid metabolism.—Yli-Jokipii, K., H. Kallio, U. Schwab, H. Mykkänen, J-P. Kurvinen, M. J. Savolainen, and R. Tahvonen. Effects of palm oil and transesterified palm oil on chylomicron and VLDL triacylglycerol structures and postprandial lipid response. J. Lipid Res. 2001. 42: 1618–1625.

Central but not peripheral glucocorticoid infusion in adrenalectomized male rats increases basal and substrate-induced insulinemia through a parasympathetic pathway.

Glucocorticoids acting through the central nervous system are postulated to play a role in the hyperinsulinemia and increased adiposity of obesity. We investigated the role of parasympathetic activation in glucocorticoid-induced hyperinsulinemia. Plasma pancreatic polypeptide (PP) levels were used as an index of parasympathetic output. Insulinemia and plasma PP levels were measured basally and after intravenous glucose injection (300 mg/kg) in adrenalectomized male rats infused with dexamethasone (7.5 microg/kg per day) intracerebroventricularly (ICV) or subcutaneously (SC) for 3 to 6 days in the presence or absence of acute atropine blockade (1.0 mg/kg). Food intake was controlled between groups. Compared with normal rats, adrenalectomy decreased white adipose tissue depot weights and leptinemia, and these were restored to normal values by ICV but not SC dexamethasone infusion. Adrenalectomy significantly reduced insulinemia below normal levels, which was restored by SC dexamethasone replacement. However, ICV dexamethasone replacement increased insulinemia of adrenalectomized rats to levels higher than normal control values (basal, 500 +/- 40 pM vs. 280 +/- 40 pM; 1-minute postglucose, 2500 +/- 180 pM vs. 1240 +/- 260 pM; p < 0.0001) and increased plasma PP levels, which were correlated with insulinemia. Atropine significantly reduced plasma insulin and PP to levels similar to normal controls but had no effect in any other group. These data show that glucocorticoids act within the brain to increase insulinemia, most likely through activation of parasympathetic efferent fibers. Such an affect would contribute to the adipogenic effects of central glucocorticoids.

Effect of orlistat-assisted weight loss in decreasing coronary heart disease risk in patients with syndrome X

This study describes the changes in risk factors for coronary heart disease in obese persons with syndrome X after orlistat-assisted weight loss. Data were available for 1,700 patients who completed 52 weeks of weight loss; 128 were defined as having syndrome X by being in the quintile with the highest plasma triglyceride levels (>2.2 mM/L) and the lowest high-density lipoprotein cholesterol (HDL, <1.0 mM/L) concentrations. Initial characteristics of those with syndrome X were similar to the 119 subjects (non-syndrome X) in the lowest quintile of plasma triglyceride (<0.975 mM/L) and highest quintile of HDL cholesterol (>1.5 mM/L). Subjects were placed on a calorie-restricted diet, and randomized to receive orlistat or placebo. Initial values were higher in those with syndrome X for diastolic blood pressure (p = 0.03), plasma insulin (p = 0.0001), triglyceride (p = 0.0001) concentrations, and ratio of low-density lipoprotein cholesterol to HDL cholesterol (p = 0.0001), and were lower for HDL cholesterol (p = 0.001) concentrations. Weight loss was greater in both groups of orlistat-treated patients (p = 0.026); in those with syndrome X, it was associated with a significant reduction in plasma insulin (p = 0.019) and triglyceride (p = 0.0001) concentrations, an increase in HDL cholesterol concentration, and a decrease in low-density lipoprotein/HDL cholesterol ratio (p = 0.0001). There were no significant changes in plasma insulin, triglycerides, or HDL cholesterol concentration in the non-syndrome X group. In conclusion, weight loss attenuates coronary heart disease risk factors in obese persons with syndrome X, and the risk factor reduction is enhanced with administration of orlistat.

Characterisation of Ventricular Myocyte Shortening after Administration of Streptozotocin (STZ) to Neonatal Rats

Human and animal studies have shown that diabetes mellitus can be associated with altered cardiac function that is independent of vascular complications. Streptozotocin (STZ) is a diabetogenic agent and when administered to rats causes selective β-cell necrosis which is accompanied by a drastic reduction in plasma insulin and hyperglycaemia. We have investigated the characteristics of shortening in ventricular myocytes isolated from rat heart at 10 months after administration of STZ to neonatal rats at 2 days of age. The characteristics of shortening in myocytes from the STZ-treated neonatal rat compared to shortening in myocytes from the STZ-treated young adult rat are discussed. STZ-treated rats gained significantly less weight compared to age-matched controls. Although non-fasting blood glucose was not significantly different in STZ-treated rats they were found to be markedly glucose intolerant when given an intraperitoneal challenge of glucose (2 g/kg) after an overnight fast. During electrical stimulation (1 or 2 Hz) ventricular myocyte resting length, time to peak shortening, time to half relaxation and amplitude of shortening were not altered after STZ treatment. The imposition of rest periods (2–60 s) after trains of electrically stimulated (1 Hz) steady-state contractions resulted in a potentiation of the contraction, which immediately followed the rest period (post rest potentiation). Post rest potentiation was larger, following rest periods between 20 and 60 s, in myocytes from STZ-treated rats compared to controls. The absence of major alterations to the amplitude and kinetics of contraction in myocytes from STZ-treated neonatal rats at 10 months after treatment might be explained by a partial recovery of the β-cells in the growing animal.

Diazoxide restores beta3-adrenergic receptor function in diet-induced obesity and diabetes.

We previously demonstrated that the expression and function of the adipocyte-specific beta3-adrenergic receptor (beta3AR) are significantly depressed in single gene and diet-induced rodent models of obesity. Furthermore, these models are relatively unresponsive to the anti-obesity effects of beta3AR agonists. Because all of these models are hyperinsulinemic, we hypothesized that hyperinsulinemia could be responsible for this abnormality in beta3AR function. The goal of this study was to determine whether lowering insulin with the K-ATP channel agonist, diazoxide (Dz) would reverse the depressed expression and function of the beta3AR found in a model of diet-induced diabetes and obesity in C57BL/6J (B6) mice. B6 male mice were placed on either high fat (HF) or low fat experimental diets. After 4 weeks, HF-fed mice were assigned to a group: HF or HF containing disodium (R,R)-5- [2-( [2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl-1,3-benzodioxole-2,2-di carboxylate (CL; 0.001%, wt/wt), Dz (0.32%, wt/wt), or their combination (CLDz). Dz animals exhibited significantly reduced plasma insulin levels as well as increased 3pAR expression and agonist-stimulated adenylyl cyclase activity in adipocytes. CLDz was more effective in reducing percent body fat, lowering nonesterified fatty acids, improving glucose tolerance, and reducing feed efficiency than either treatment alone.

Diet in late pregnancy and glucose-insulin metabolism of the offspring 40 years later.

To determine how diets of women in pregnancy influence the glucose-insulin metabolism of their offspring in adult life. A follow up study of men and women born during 1948-1954 whose mothers had taken part in a survey of diet in late pregnancy. Aberdeen, Scotland. One hundred and sixty-eight men and women born in the Aberdeen Maternity Hospital. Plasma glucose and insulin concentrations, fasting and after a standard oral glucose challenge. The offspring of women who had high intakes of fat and protein in late pregnancy had a reduced plasma insulin increment between fasting and 30 min with a 7.0% decrease in increment (P = 0.007) per 10 g increase in protein intake and a 4.9% decrease (P = 0.002) per 10 g increase in fat intake. This was independent of the mother's body mass index or weight gain in pregnancy. A low maternal body mass index in early or late pregnancy was associated with a raised fasting plasma insulin concentration with a decrease of 2.4% (P = 0.05) per 1 kg/m2 increase of maternal body mass. High intakes of protein and fat during pregnancy may impair development of the fetal pancreatic beta cells and lead to insulin deficiency in the offspring. The offspring of thin mothers tend to be insulin resistant.

Metformin Effects on Clinical Features, Endocrine and Metabolic Profiles, and Insulin Sensitivity in Polycystic Ovary Syndrome: A Randomized, Double-Blind, Placebo-Controlled 6-Month Trial, Followed by Open, Long-Term Clinical Evaluation

In the last few years some studies assessed the effects of attenuation of hyperinsulinemia and insulin resistance, obtained by insulin sensitizing agents, in women with polycystic ovary syndrome (PCOS), suggesting potential scope for these drugs in treating the whole spectrum of reproductive, endocrine, and metabolic abnormalities found in such subjects. However, the results of these studies, mostly uncontrolled and short-term, are still inconclusive, and there is no long-term follow-up. In the present study, 23 PCOS subjects [mean (+/- SE) body mass index 30.0+/-1.1 kg/m2] were randomly assigned to double-blind treatment with metformin (500 mg tid) or placebo for 6 months, while maintaining their usual eating habits. Before and after treatment, menstrual history, endocrine and metabolic profiles, serum 17-hydroxyprogesterone response to GnRH-agonist testing, and insulin sensitivity measured by the glucose clamp technique were assessed. Eighteen of these women, as well as 14 additional PCOS patients, were subsequently given metformin in an open trial for 11.0+/-1.3 months (range 4-26), to assess long-term effects of treatment and baseline predictors of metformin efficacy on reproductive abnormalities. After metformin treatment, mean frequency of menstruation improved (P = 0.002), due to striking amelioration of menstrual abnormalities in about 50% of subjects. Women given metformin showed reduced plasma insulin (at fasting: P = 0.057; during the clamp studies: P<0.01) and increased insulin sensitivity (P<0.05). Concurrently, ovarian hyperandrogenism was attenuated, as indicated by significant reductions in serum free testosterone (P<0.05) and in the 17-hydroxyprogesterone response to GnRH-agonist testing (P<0.05). No changes were found in the placebo group. Only comparable minor changes in body mass index were found both in the metformin group and in the placebo group. In the open, long-term trial 17 women (54.8%) showed striking improvements of their menstrual abnormalities and were considered as responders. Logistic regression analysis of baseline characteristics in responders and nonresponders showed that plasma insulin, serum androstenedione, and menstrual history were independent predictors of the treatment's clinical efficacy. In 10 subjects whose menses proved regular after treatment, the great majority of cycles became ovulatory (32 out of 39 assessed, 79%). In conclusion, in women with PCOS metformin treatment reduced hyperinsulinemia and hyperandrogenemia, independently of changes in body weight. In a large number of subjects these changes were associated with striking, sustained improvements in menstrual abnormalities and resumption of ovulation. Higher plasma insulin, lower serum androstenedione, and less severe menstrual abnormalities are baseline predictors of clinical response to metformin.

Endocrine host responses during early and late phases of tumor development.

It is well established that hormones affect tumor growth. Conversely, inoculation of cells obtained from tumors that had been transplanted for many generations causes changes in the concentration of different hormones before and after tumor detection. We aimed at answering the question of whether hormonal alterations also occur during the development of primary tumors and following transplantation of tumors from early generations. Primary tumors were induced in mice by either the carcinogenic agent 3-methylcholanthrene, which produces fibrosarcomas, or the milk-transmitted mammary tumor virus, which induces adenocarcinomas. The results showed that (i) in both models, an early reduction in plasma insulin and prolactin levels occurred, and in the case of insulin, this reduction was sustained for a prolong period prior to tumor detection, indicating that recognition by the host of emergent tumor cells triggers an endocrine response; (ii) in contrast with multiply transplanted tumors, cells from early transplant generations produced no significant endocrine changes during latency; (iii) irrespective of whether they were primary or transplanted, large tumor burdens caused similar hormonal alterations, consisting of increased corticosterone and growth hormone and decreased insulin, thyroxin, prolactin and sex steroid levels in blood. Our comprehensive longitudinal study demonstrates host endocrine responses during different stages of neoplastic development.

Down-regulation of orexin gene expression by severe obesity in the rats: studies in Zucker fatty and Zucker diabetic fatty rats and effects of rosiglitazone

Orexins (hypocretins) are lateral hypothalamic neuropeptides implicated in regulating feeding and the sleep–wake cycle. To study their possible relevance to obesity and diabetes, we measured hypothalamic prepro-orexin mRNA levels in obese, normoglycemic Zucker fatty ( fa/fa) and in hyperglycemic, non-obese Zucker diabetic fatty (ZDF) rats. Hypothalamic prepro-orexin mRNA concentrations in Zucker fatty rats were 31% lower than those in lean controls (0.69±0.06 vs. 1.00±0.10 arbitrary units, P<0.05), but did not differ between ZDF diabetic rats and non-diabetic controls. Treatment of ZDF diabetic rats with rosiglitazone (1 or 3 mg/kg body weight daily for 13 weeks) normalized plasma glucose and significantly reduced plasma insulin, while leptin levels were 67% higher than in untreated ZDF rats (20.2±0.5 vs. 12.1±2.5, P<0.001). Rosiglitazone treatment markedly enhanced weight gain compared with untreated ZDF rats (final weight 732±13 g vs. 409±13 g, P<0.001) even though they were restricted to the same food intake. Rosiglitazone-treated ZDF rats had significantly lower hypothalamic prepro-orexin mRNA levels (0.68±0.07 arbitrary units) than both non-diabetic lean controls (1.00±0.10, P=0.02) and untreated diabetics (1.03±0.14, P=0.03). Our data suggest that prepro-orexin gene expression may be suppressed by substantial weight gain. Obesity-related signals that might mediate this effect have not been identified, but plasma leptin, insulin and glucose are not obviously involved.

Neuropeptide Y receptor alterations in the hypothalamus of lactating rats

Hypothalamic neuropeptide Y (NPY) neurons are influenced by circulating levels of insulin and leptin and are thought to be involved in mediating hunger following underfeeding. We have investigated hypothalamic NPY receptor subtypes in lactating rats, which are markedly hyperphagic throughout the day and night. NPY receptors were measured by using [ 125I] peptide YY, a high-affinity ligand, and Y1 receptors were masked by using the highly specific antagonist BIBP 3226. Freely fed lactating rats showed no changes in the densities of Y1, or non-Y1, NPY binding sites in whole hypothalamic homogenates or in individual hypothalamic regions (measured by quantitative autoradiography) examined during the day or night ( P > 0.05; n = 10/group, and n = 6/group, respectively). However, reducing food intake by 35% had a more profound effect on NPY receptor density in lactating than in control rats, producing down-regulation of non-Y1 receptors in the ventromedial, dorsomedial, and perifornical lateral areas (all P < 0.05; n = 7/group) and reduction of plasma insulin and leptin levels (both P < 0.01). Thus, although the NPY system may not have a major role in the hyperphagia of freely fed lactating rats, it appears to have an important function in the response to undernutrition in such animals.

Acute Effects of Exercise on Circulating Leptin in Lean and Genetically Obesefa/faRats

Mechanisms of regulation of plasma leptin in lean and genetically obese animals are not completely understood. In particular a relation has been proposed between energy metabolism and leptin. However, it is not clear how energy expenditure and leptin are related under exercise in lean and obese animals. To clarify these aspects we investigated lean and genetically obese (fa/fa) Zucker rats undergoing a single bout (30 min) of swimming and measured several biochemical and hormonal parameters of energy metabolism and leptin changes throughout the study. Moreover ob-gene expression in adipose tissue was also measured. Our results showed that plasma leptin is decreased by 30% at the end of exercise in lean animals while resulting unaffected in obese animals. Leptin changes in lean rats are concomitant with the peak of NEFA and glycerol release from adipose tissue rather than with the reduction of plasma insulin. Ob-gene expression in adipose tissue was markedly increased in fa/fa compared to lean rats, but was not modified by exercise both in lean and obese animals. In conclusion our data show that leptin changes during exercise are related to lipolytic events in adipose tissue and support a link between leptin and energy expenditure.

Effects of high-fat diet intake on glucose uptake in central and peripheral tissues of non-obese rats.

We previously demonstrated that plasma glucose concentration was higher while plasma insulin concentration was lower in rats fed a high-fat diet. In the present study, we examined the effects of high-fat diet on glucose uptake in central and peripheral tissues in non-obese rats. Forty male Sprague-Dawley rats were fed high- or low-fat diets for 4 wk. Body weight and body fat accumulation were not different between the two diet groups after 4 wk. Glucose uptake in the skeletal muscles and adipose tissues, estimated by the 2-deoxy-D-glucose method, was lower in the rats fed the high-fat diet than that in the rats fed the low-fat diet, whereas uptake in the liver and pancreas did not differ between the two groups. Glucose uptake in the hypothalamus and cortex was higher in the high-fat diet group as compared with that in the low-fat diet group. These results suggest that increased plasma glucose levels in rats fed the high-fat diet were caused by a decrease in glucose uptake in the skeletal muscles and adipose tissues. Reduced plasma insulin level in the high fat diet group with no difference in glucose uptake in the pancreas may be due to increased sympathetic activity in the pancreas resulting from the increased glucose uptake in the brain regions involved in autonomic functions.

Acute and chronic oral administration of bis(maltolato)oxovanadium(IV) in Zucker diabetic fatty (ZDF) rats

This is a preliminary study in which both acute and chronic oral administration of bis(maltolato)oxovanadium (IV) (BMOV) was examined in the Zucker diabetic fatty (ZDF) rat, an animal model that develops overt hyperglycemia in the presence of hyperinsulinemia followed by β-cell depletion. At 9–10 weeks of age, in the presence of hyperglycemia, hyperinsulinemia and hyperlipidemia, an acute oral gavage dose response was conducted to determine glucose-lowering properties of BMOV, time of response and effect of BMOV on plasma insulin levels. Doses of BMOV greater than 0.2 mmol/kg resulted in plasma glucose levels of less than 9 mmol/l. The highest dose administered (0.8 mmol/kg) significantly reduced plasma insulin (initial: 2.83±0.2, final: 1.23±0.09 nmol/l, P<0.05) and plasma triglyceride (initial: 4.94±0.33, final: 1.55±0.07 mmol/l, P<0.05) levels. At 15 weeks of age, in the presence of hyperglycemia, hyperlipidemia and normal insulin levels, BMOV was administered orally in the drinking water for a 10-week period to determine the effect of treatment on glucose, insulin and lipid levels. BMOV treatment significantly reduced plasma glucose levels (final BMOV-treated: 13.25±1.43, untreated: 28.71±0.6 mmol/l, P<0.05) and effectively preserved pancreatic β-cell function. These data suggest a role for BMOV as a therapeutic agent in non-insulin-dependent diabetes mellitus through improvement in glucose homeostasis and preservation of insulin reserves.

An in vivo model for elucidation of the mechanism of tumor necrosis factor-alpha (TNF-alpha)-induced insulin resistance: evidence for differential regulation of insulin signaling by TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce insulin resistance in cultured cells as well as in animal models. The aim of this study was to map the in vivo mechanism whereby TNF-alpha contributes to the pathogenesis of impaired insulin signaling, using obese and lean Zucker rats in which TNF-alpha activity was inhibited through adenovirus-mediated gene transfer. We employed a replication-incompetent adenovirus-5 (Ad5) vector to endogenously express a TNF inhibitor (TNFi) gene, which encodes a chimeric protein consisting of the extracellular domain of the human 55-kDa TNF receptor joined to a mouse IgG heavy chain. Control animals consisted of rats infected with the same titer of adenovirus carrying the lac-z complementary DNA, encoding for beta-galactosidase. There was a significant reduction in plasma insulin and free fatty acid levels in TNFi obese rats 2 days following Ad5 administration. The peripheral insulin sensitivity index was 50% greater, whereas hepatic glucose output was completely suppressed during hyperinsulinemic glucose clamps in TNFi obese animals, with no differences observed between the two lean groups. The improvement in peripheral and hepatic sensitivity to insulin seen in the obese animals was independent of insulin receptor (IR) number and insulin binding affinity for IR. However, TNF-alpha neutralization led to a 2.5-fold increase in tyrosine phosphorylation of IR in skeletal muscle, whereas this was unchanged in liver. There was also a 4-fold increase in particulate protein tyrosine phosphatase activity of skeletal muscle in TNFi obese animals vs. beta-galactosidase controls, whereas protein tyrosine phosphatase activity in liver was unchanged. These results suggest that TNF-alpha is a mediator of insulin resistance in obesity and may modulate IR signaling in skeletal muscle and liver through different pathways. TNF-alpha may affect insulin action in the liver either at sites distal to the IR or indirectly, possibly because of increased provision of gluconeogenic substrates or altered counterregulation. In addition, the Ad5-mediated gene delivery system employed here provides an in vivo model that is efficient and economical for exploring mechanisms involved in TNF-alpha-induced insulin resistance in various genetic models of obesity-linked diabetes.

Nutritional Status Modulates Rat Liver Cytochrome P450 Arachidonic Acid Metabolism

Alterations in nutritional status affect hepatic cytochrome P450 levels. Since cytochromes P450 participate in the metabolism of arachidonic acid, we hypothesized that changes in liver P450 arachidonic acid metabolism occur during fasting and refeeding. Male Fisher 344 rats were either fed, fasted 48 hr (F48), fasted 48 hr and then refed 6 hr (F48/R6), or fasted 48 hr and then refed 24 hr (F48/R24). F48 rats had reduced body weight, increased plasma beta-hydroxybutyrate, and reduced plasma insulin compared with the other groups. Although there was no significant change in total liver P450 content, there was a significant 20%, 48%, and 24% reduction in total hepatic microsomal arachidonic acid metabolism in F48, F48/R6, and F48/R24 rats, respectively, compared with fed rats. Epoxygenase activity decreased by 28%, 51%, and 26% in F48, F48/R6, and F48/R24 rats, respectively. In contrast, omega-1 hydroxylase activity increased by 126% in F48 rats compared with fed rats. Immunoblotting revealed that levels of CYP2C11 protein were markedly reduced, whereas levels of CYP2E1 protein were markedly increased in the F48 and F48/R6 groups. In contrast, levels of CYP1A1, CYP1A2, CYP2B1, CYP2J3, CYP4A1, and CYP4A3 were unchanged with fasting/refeeding. Northern blots revealed that levels of CYP2C11 mRNAs were decreased, whereas CYP2E1 mRNAs were increased in F48 and F48/R6 rats. Recombinant CYP2C11 metabolized arachidonic acid primarily to epoxides with preference for the 14(S),15(R)-, 11(R), 12(S)-, and 8(S),9(R)- epoxyeicosatrienoic acid enantiomers. We conclude that (1) nutritional status affects hepatic microsomal arachidonic acid metabolism, (2) reduced epoxygenase activity in F48 and F48/R6 rats is accompanied by decreased levels of CYP2C11, (3) increased omega-1 hydroxylase activity is accompanied by augmented levels of CYP2E1, and (4) the effects of fasting on CYP2C11 and CYP2E1 expression occur at the pretranslational level.

NIDDM genes in mice: deleterious synergism by both parental genomes contributes to diabetogenic thresholds.

We used mouse genetics to model how polygenic thresholds for the transition from impaired glucose tolerance (IGT) to NIDDM are reached. NON/Lt and NZO/Hl are inbred mouse strains selected for IGT and polygenic obesity, respectively. Their F1 male progeny consistently developed NIDDM. Genetic analysis of F2 males from both cross directions identified an NON-derived diabetogenic locus, Nidd 1, on chromosome (Chr) 4 near the leptin receptor. This locus was associated with reduced plasma insulin, increased non-fasted blood glucose, and lower body weight. Another NON-derived diabetogenic locus on Chr 18 (Nidd2) that controls blood glucose was identified. An NZO-derived diabetogenic region on Chr 11 (Nidd3), possibly comprising two separate loci, reduced ability to sustain elevated plasma insulin and significantly reduced weight gain over time. Thus, the diabetogenic synergism between genetic loci from strains separately exhibiting subthreshold defects perturbing glucose homeostasis underscores the likely complexity of the inheritance of obesity-associated forms of NIDDM in humans.

NIDDM genes in mice: deleterious synergism by both parental genomes contributes to diabetogenic thresholds

We used mouse genetics to model how polygenic thresholds for the transition from impaired glucose tolerance (IGT) to NIDDM are reached. NON/Lt and NZO/Hl are inbred mouse strains selected for IGT and polygenic obesity, respectively. Their Fl male progeny consistently developed NIDDM. Genetic analysis of F2 males from both cross directions identified an NON-derived diabetogenic locus, Nidd 1, on chromosome (Chr) 4 near the leptin receptor. This locus was associated with reduced plasma insulin, increased non-fasted blood glucose, and lower body weight. Another NON-derived diabetogenic locus on Chr 18 (Nidd2) that controls blood glucose was identified. An NZO-derived diabetogenic region on Chr 11 (Nidd3), possibly comprising two separate loci, reduced ability to sustain elevated plasma insulin and significantly reduced weight gain over time. Thus, the diabetogenic synergism between genetic loci from strains separately exhibiting subthreshold defects perturbing glucose homeostasis underscores the likely complexity of the inheritance of obesity-associated forms of NIDDM in humans.