Resistance to the orexigenic effect of ghrelin in dietary-induced obesity in mice: reversal upon weight loss.

BACKGROUND: Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), is known to increase food intake in lean humans and rodents. In addition, ghrelin levels are increased by fasting in lean rodents and are elevated before meals in humans, suggesting an important role for ghrelin in meal initiation. However, in obese human, circulating ghrelin levels were found to be significantly reduced as compared to lean individuals. OBJECTIVES: To evaluate whether circulating ghrelin levels, as well as ghrelin sensitivity, are decreased in obese individuals in order to limit its effect on food intake. DESIGN: : Lean C57BL/6J mice fed a chow, a low- (LFD) or a high-fat diet (HFD) were used to determine ghrelin regulation and secretion as well as ghrelin sensitivity. MEASUREMENTS: Plasma ghrelin levels were measured in low- and high-fat fed mice. Ghrelin-induced food intake was measured in chow, low- and high-fat fed mice. RESULTS: We measured ghrelin levels in lean and diet-induced obese mice, fed on an LFD or an HFD, respectively. We observed that not only ghrelin secretion was reduced in obese mice but its diurnal regulation was also lost. In addition, we failed to observe any change in ghrelin secretion upon fasting and refeeding. Moreover, we observed that the sensitivity to the orexigenic effects of exogenous ghrelin was reduced in obese mice when compared to lean mice fed a chow or a LFD. The insensitivity of obese mice to ghrelin was improved upon weigh loss. CONCLUSION: : Altogether, these results indicate that ghrelin secretion and regulation is impaired in dietary-induced obesity in mice and suggest that ghrelin inhibition could prevent weight regain after weight loss.

Biochemical mechanisms responsible for the attenuation of diabetic and obese conditions in ob/ob mice treated with dopaminergic agonists.

OBJECTIVE: We previously reported that a two week treatment with SKF 38393 (SKF, a dopamine D1 receptor agonist), plus bromocriptine (BC, a dopamine D2 receptor agonist) acted synergistically to normalize hyperphagia, body fat, hyperglycaemia and hyperlipidaemia in ob/ob mice. The present study further investigates the biochemical mechanisms triggered by this drug treatment. DESIGN: Six week old female C57BL/6J ob/ob mice were divided into three groups and treated for two weeks with either BC and SKF, vehicle (control), or vehicle and pair fed to match the drug-treated group's daily food intake. RESULTS: BC/SKF treatment reduced food consumption by 55%, and treated mice weighed less than either pair fed or ad libitum fed controls after two weeks of treatment. Moreover, oxygen consumption was increased by 2.4-fold and the respiratory quotient (RQ) decreased from 1.23 to 0.96 (indicating a reduction in de novo lipogenesis) by drug treatment relative to ad libitum fed controls, but these parameters were unaffected by pair feeding control mice. The treatment also reduced blood glucose and free fatty acids (FFA) relative to pair fed and ad libitum fed controls. BC/SKF treatment (but not pair feeding) concurrently reduced lipolysis, lipogenic enzyme activities and hepatic gluconeogenic enzyme activities. Treatment also increased hepatic concentrations of glycogen and xylulose-5-phosphate (X-5-P), a key stimulator of glycolysis. Finally, BC/SKF, but not pair feeding, reduced the circulating concentrations of thyroxine and corticosterone, two hormones known to increase lipolysis, lipogenesis and hyperglycaemia. Drug treatment also increased serum dehydroepiandrosterone (DHEA) sulfate concentrations, an inhibitor of body fat store accumulation. CONCLUSION: These findings demonstrate that BC/SKF treatment not only normalizes hyperphagia of ob/ob mice, but also redirects several metabolic and endocrine activities, independent of its effects on feeding to improve the obese-diabetic syndrome in ob/ob mice.

Amelioration of insulin resistance in streptozotocin diabetic mice by transgenic overexpression of GLUT4 driven by an adipose-specific promoter.

In diabetic rodents and humans, glucose transporter 4 (GLUT4) expression is suppressed in adipocytes in association with insulin resistance. Transgenic mice overexpressing GLUT4 selectively in fat have enhanced glucose disposal in vivo and massively increased glucose transport in adipocytes. To determine whether overexpression can be maintained in diabetes and whether it can prevent insulin resistance, we rendered wild-type and transgenic mice diabetic with streptozotocin. After 12-14 days, blood glucose was more than 21.4 mM and plasma insulin was 1.06 ng/ml or less in both diabetic groups in the fed state. Body weight was reduced and gonadal fat pad weight and adipocyte size were 52-75% smaller in both nontransgenic and transgenic diabetic mice, compared with nondiabetic. Basal and maximally-stimulated rates of lipolysis were similar in adipocytes from nontransgenic and transgenic mice, but the ED50 for isoproterenol stimulation was 50% lower in transgenic mice. There was no difference in the sensitivity to insulin to inhibit lipolysis. In adipocytes of nontransgenic diabetic mice, GLUT4 protein was reduced 34%, with a 46% reduction in insulin stimulated glucose transport. In contrast, in adipocytes of transgenic diabetic mice, GLUT4 remained 21-fold overexpressed, resulting in 21-fold increased basal and 10-fold increased insulin stimulated glucose transport. Injection of insulin (0.7 mU/g BW) resulted in a 35% decrease in blood glucose in transgenic diabetic mice (P < 0.05), with no effect in nontransgenic diabetic mice. Thus, high-level overexpression of GLUT4 driven by a fat specific promoter can be maintained with insulinopenic diabetes, even when fat cell metabolism is markedly altered. Overexpression of GLUT4 in adipocytes prevents insulin resistant glucose transport at the cellular level and improves insulin action in vivo, even with overt diabetes.

Bromocriptine/SKF38393 treatment ameliorates obesity and associated metabolic dysfunctions in obese (ob/ob) mice.

It has been postulated that dopaminergic activities comprise a major functional component of a central regulatory system for metabolism which can be manipulated by dopamine modulating drugs. The present study is aimed at delineating the role and importance of pharmacological dopaminergic activation in the regulation of metabolism during obesity and diabetes. We treated C57BL/6J ob/ob mice for 2 weeks with bromocriptine (dopamine D2 agonist), SKF38393 (dopamine D1 agonist), both drugs combined or vehicle and monitored the effects of such treatment on body composition, food consumption, and serum metabolites. Bromocriptine and SKF38393 individually produced moderate improvements in obesity, hyperglycemia, and hyperinsulinemia. However, a combination of bromocriptine plus SKF38393 resulted in major reductions in body weight (7.5 g), body fat (40%), food consumption (42%), and serum concentrations of glucose (59%), triglyceride (37%), free fatty acid (45%) and insulin (49%) while increasing protein mass (8%). These results indicate that regulatory components of metabolism in the ob/ob mouse are modulated by and/or are comprised of dopaminergic activities. Importantly, dopaminergic D1/D2 receptor coactivation maximizes this dopaminergic response (i.e., improvement of metabolic abnormalities) in these mice.

Overexpression of Ha-ras selectively in adipose tissue of transgenic mice. Evidence for enhanced sensitivity to insulin.

To determine the role of Ras-dependent signaling pathways in adipocyte function, we created transgenic mice that overexpress Ha-ras in adipocytes using the aP2 fatty acid-binding protein promoter/enhancer ligated to the human genomic ras sequence. ras mRNA was increased 8-17-fold and Ras protein 4-5-fold in white and brown fat, with no overexpression in other tissues. The subcellular distribution of overexpressed Ras paralleled that of endogenous Ras. [U-14C]Glucose uptake into isolated adipocytes was increased approximately 2-fold in the absence of insulin, and the ED50 for insulin was reduced 70%, with minimal effect on maximally stimulated glucose transport. Expression of Glut4 protein was unaltered in transgenic adipocytes, but photoaffinity labeling of transporters in intact cells with [3H]2-N-[4-(1-azi-Z,Z,Z-trifluoroethyl)benzoyl]-1,3-bis-(D-mann os-4- yloxy)-2-propylamine revealed 1.7-2.6-fold more cell-surface Glut 4 in the absence of insulin and at half-maximal insulin concentration (0.3 nM) compared with nontransgenic adipocytes. With maximal insulin concentration (80 nM), cell-surface Glut4 in nontransgenic and transgenic adipocytes was similar. Glut1 expression and basal cell-surface Glut1 were increased 2-2.9-fold in adipocytes of transgenic mice. However, Glut1 was much less abundant than Glut4, making its contribution to transport negligible. These in vitro changes were accompanied by in vivo alterations including increased glucose tolerance, decreased plasma insulin levels, and decreased adipose mass. We conclude that ras overexpression in adipocytes leads to a partial translocation of Glut4 in the absence of insulin and enhanced Glut4 translocation at physiological insulin concentration, but no effect with maximally stimulating insulin concentrations.

Glut4 is targeted to specific vesicles in adipocytes of transgenic mice overexpressing Glut4 selectively in adipose tissue.

Adipocytes of transgenic mice overexpressing Glut4 selectively in adipose tissue (Shepherd, P.R., Gnudi, L., Tozzo, E., Yang, H., Leach, F., and Kahn, B.B. (1993) J. Biol. Chem. 268, 22243-22246) have 15-20-fold more Glut4 than normal adipocytes. To study compartmentalization of intracellular Glut4 in these cells, we fractionated light microsomes prepared from transgenic and normal adipocytes in velocity and density sucrose gradients. Glut4-containing intracellular membranes from both cell types have a specific and narrow distribution in these gradients, i.e. behave as homogeneous vesicles with identical sedimentation coefficients and different buoyant densities. Immunoadsorption of Glut4-containing vesicles with covalently immobilized monoclonal anti-transporter antibody demonstrated that the total polypeptide composition of these vesicles from transgenic and normal cells was identical, with the exception of Glut4 itself, which was much more abundant in the transgenic cells. Both preparations also had comparable levels of secretory carrier membrane proteins and of aminopeptidase activity (gp160). Glut4-containing vesicles from both normal and transgenic adipocytes excluded Glut1, which in both cell types formed a different vesicle population. Thus, even under conditions of high level overexpression, Glut4 is still specifically targeted to the same unique type of structurally defined insulin-sensitive vesicles as in normal cells.

Adipose-specific overexpression of GLUT-4 in transgenic mice alters lipoprotein lipase activity.

Transgenic mice overexpressing GLUT-4 selectively in adipose tissue using the aP2 promoter/enhancer develop obesity, enhanced glucose tolerance, and increased insulin sensitivity. The current study was designed to determine whether altering glucose transport affects lipoprotein lipase (LPL) activity. Female transgenic mice (10-12 mo old) have increased parametrial fat pad weight, adipocyte size, total body lipid and fasting plasma triglycerides, fatty acids, and glycerol compared with nontransgenics. Stimulation of LPL activity by feeding is blunted in parametrial and perirenal fat from 15- and 22-fold in nontransgenic mice to three- to sevenfold in transgenics. LPL activity in the fed state in transgenic mice is reduced 60-75% in fat. In heart and skeletal muscle of transgenic mice, LPL activity in the fasted state is 55-65% lower than in nontransgenics and feeding induces an unexpected rise in LPL activity. Muscle LPL activity is strongly and inversely correlated with glucose transport in adipocytes (r = -0.942, P < 0.005), which is increased 15- to 27-fold in the basal state and 4.5- to 6.9-fold in the insulin-stimulated state in transgenics. Whereas stimulation of adipose LPL may be blunted by lower plasma insulin levels in transgenics, fasting muscle LPL may be suppressed by elevated plasma lipids. Thus altering the partitioning of glucose between adipose tissue and muscle alters a critical step for the partitioning of lipoprotein fatty acids between these tissues.

Targeted disruption of the beta 3-adrenergic receptor gene.

beta 3-Adrenergic receptors (beta 3-ARs) are expressed predominantly in white and brown adipose tissue, and beta 3-selective agonists are potential anti-obesity drugs. However, the role of beta 3-ARs in normal physiology is unknown. To address this issue, homologous recombination was used to generate mice that lack beta 3-ARs. This was accomplished by direct injection of a DNA-targeting construct into mouse zygotes. Twenty-three transgenic mice were generated, of which two had targeted disruption of the beta 3-AR gene. Mice that were homozygous for the disrupted allele had undetectable levels of intact beta 3-AR mRNA, as assessed by RNase protection assay and Northern blotting, and lacked functional beta 3-ARs, as demonstrated by complete loss of beta 3-agonist (CL 316,243)-induced stimulation of adenylate cyclase activity and lipolysis. beta 3-AR-deficient mice had modestly increased fat stores (females more than males), indicating that beta 3-ARs play a role in regulating energy balance. Importantly, beta 1 but not beta 2-AR mRNA levels up-regulated in white and brown adipose tissue of beta 3-AR-deficient mice (brown more than white), strongly implying that beta 3-ARs mediate physiologically relevant signaling under normal conditions and that "cross-talk" exists between beta 3-ARs and beta 1-AR gene expression. Finally, acute treatment of normal mice with CL 316,243 increased serum levels of free fatty acids (FFAs) (3.2-fold) and insulin (140-fold), increased energy expenditure (2-fold), and reduced food intake (by 45%). These effects were completely absent in beta 3-AR-deficient mice, proving that the actions of CL are mediated exclusively by beta 3-ARs. beta 3-AR-deficient mice should be useful as a means to a better understanding of the physiology and pharmacology of beta 3-ARs.

Transgenic GLUT-4 overexpression in fat enhances glucose metabolism: preferential effect on fatty acid synthesis.

GLUT-4 expression varies widely among normal humans and those with obesity and diabetes. Using the alpha P2 promoter/enhancer ligated to the human GLUT-4 gene, we created transgenic mice to study the impact of alterations in GLUT-4 expression selectively in adipocytes on glucose homeostasis and body composition. Here we investigated molecular mechanisms for enhanced glucose tolerance and obesity in these mice. [U-14C]glucose incorporation into triglycerides, glyceride-glycerol, glyceride-fatty acids, CO2, and lactate was measured in adipocytes incubated at 3, 0.5, and 3 microM glucose with or without maximally stimulating insulin. In nontransgenic and transgenic mice, the major pathway for glucose metabolism shifts from lipogenesis at tracer glucose concentration to glycolysis at physiological glucose concentration. In transgenic adipocytes incubated at 3 microM glucose, metabolism via all major pathways is enhanced by 8.6- to 38-fold in the absence of insulin and 3- to 13-fold in the presence of insulin. At physiological glucose concentration, constitutive metabolism to triglycerides, CO2, and lactate is two- to threefold greater in transgenic than in nontransgenic adipocytes. De novo fatty acid synthesis is preferentially increased: 31-fold for basal and 21-fold for insulin-stimulated compared with nontransgenic adipocytes. Thus overexpression of GLUT-4 in adipocytes of transgenic mice results in increased glucose metabolism in all major pathways, with differential regulation of the pathways involved in lipogenesis.

Expression of the hepatic insulin receptor gene in the rat during postnatal development.

Changes in the expression of the liver insulin receptor are known to occur in the rat during postnatal development. To assess whether such changes occur at the level of gene expression, steady-state levels of insulin receptor mRNA and transcription rates of the receptor gene have been measured in livers of rats from birth (1 day) to adulthood (60 days). Northern blot analysis of liver RNA revealed two major insulin receptor mRNA species of 9.5 and 7.5 kb. When normalized to beta actin mRNA, insulin receptor mRNA levels increased 4-fold between 1 and 15 days, remained stable between 15 and 30 days, and decreased 2-fold between 30 and 60 days. These changes were fully suppressed by in vivo treatment with actinomycin D, an inhibitor of gene transcription. In vitro nuclear transcription assays showed that the rate of transcription of the insulin receptor gene increased 2-fold between 1 and 30 days. Insulin receptor concentration in liver membrane fractions did not exactly parallel insulin receptor mRNA levels since it increased by 20-30% from 1 to 10 days and decreased 2-fold from 10 to 60 days. During the suckling-weaning transition, insulin receptor mRNA level decreased 2-fold in rats weaned onto a high carbohydrate diet but remained unchanged in rats weaned onto a high fat diet. Throughout postnatal life, an inverse relationship was observed between liver insulin receptor mRNA and plasma insulin levels. These results show that transcriptional changes in insulin receptor gene expression occur postnatally and suggest that such changes may be insulin-related.

High level overexpression of glucose transporter-4 driven by an adipose-specific promoter is maintained in transgenic mice on a high fat diet, but does not prevent impaired glucose tolerance.

High fat feeding is associated with impaired insulin action, an obese body composition, and down-regulation of glucose transporter-4 (GLUT4) expression in adipocytes. We recently showed that overexpression of GLUT4 selectively in adipocytes of transgenic mice using the aP2 (fatty acid-binding protein) promoter/enhancer results in enhanced glucose tolerance and adipocyte hyperplasia. Here, we fed these GLUT4-overexpressing transgenic mice a high fat (55%) or a low fat (10%) diet for 13-15 weeks to determine the role of alterations in GLUT4 expression in adipocytes in the development of insulin resistance and obesity, which are characteristic of high fat consumption. In nontransgenic mice, high fat feeding results in 45-50% reduction of GLUT4 levels in white and brown adipose tissue, with a parallel decrease in insulin-stimulated glucose transport. In transgenic mice receiving the low fat diet, GLUT4 is overexpressed 20-fold in white and 4-fold in brown adipose tissue. Glucose transport in epididymal adipocytes is increased 20-fold in the basal state and 6-fold in the insulin-stimulated state. Even after transgenic mice are fed a high fat diet, GLUT4 expression and glucose transport in their adipocytes remains 14- to 30-fold greater than that in nontransgenic mice receiving the same diet. Despite these marked effects at the adipose cell level, glucose tolerance is not improved, probably due to insulin resistance in skeletal muscle and liver, where the transgene is not expressed. During the low fat diet, transgenic mice have 80% more body lipid than nontransgenics. High fat feeding increases body lipid 76% and adipocyte size 65% in nontransgenic mice, but has no effect in transgenic mice. Thus, overexpression of GLUT4 selectively in adipocytes protects against a further increase in adiposity. Furthermore, by using a heterologous promoter, high level overexpression of GLUT4 can be maintained even under metabolic conditions where it is normally down-regulated in adipocytes. This overexpression results in markedly increased glucose transport at the cellular level, but adipose-specific GLUT4 overexpression does not prevent the decrease in glucose tolerance associated with high fat feeding.

Adipose cell hyperplasia and enhanced glucose disposal in transgenic mice overexpressing GLUT4 selectively in adipose tissue.

To gain insight into the molecular pathogenesis of obesity and specifically the role of nutrient partitioning in the development of obesity, we overexpressed the insulin-responsive glucose transporter (GLUT4) in transgenic mice under the control of the fat-specific aP2 fatty acid-binding protein promoter/enhancer. Two lines of transgenic mice were generated, which overexpressed GLUT4 6-9-fold in white fat and 3-5-fold in brown fat with no overexpression in other tissues. In vivo glucose tolerance was enhanced in transgenic mice. In isolated epididymal, parametrial, and subcutaneous adipose cells from transgenic mice, basal glucose transport was 20-34-fold greater than in nontransgenic littermates. Insulin-stimulated glucose transport was 2-4-fold greater in cells from transgenic mice. Total body lipid was increased 2-3-fold in transgenic mice overexpressing GLUT4 in fat. Surprisingly, fat cell size was unaltered and fat cell number was increased > 2-fold. This is the first animal model in which increased fat mass results solely from adipocyte hyperplasia and it will be a valuable model for understanding the mechanisms responsible for fat cell replication and/or differentiation in vivo.

[Regulation of insulin receptor expression and its gene]. / Régulation de l'expression du récepteur de l'insuline et de son gène.

The insulin receptor is a membrane macromolecule whose expression on the cell surface is essential for cell sensitivity to insulin. Current knowledge on the regulation of expression of the insulin receptor and its gene in human and animal cells is presented. Although ubiquitously distributed, the insulin receptor and its messenger RNA (mRNA) are mainly expressed in metabolically active cells such as hepatocytes and adipocytes. Two receptor isoforms, generated by alternative splicing of exon 11, have been identified. Isoform B (exon 11+) predominates in liver and adipocytes, and isoform A (exon 11-) in brain, spleen and leukocytes. In vivo and in several cell models, the expression of the insulin receptor and/or its mRNA is under positive regulation by glucocorticoid hormones and negative regulation by insulin. Glucocorticoid hormones stimulate receptor gene transcription and receptor protein synthesis. Insulin stimulates receptor protein degradation and, in certain cell types, decreases receptor mRNA level. Vanadate (an insulinomimetic agent) corrects, in vivo, the hyperexpression of the liver receptor observed in experimental insulinopenic diabetes, but its effects on receptor expression in vitro are complex and vary with the cell type. In vivo the insulin receptor and/or its mRNA are expressed early in fetal development with a high level, in liver, of isoform A. Maximal expression is reached at the end of gestation and then decreases after birth. In several cell models, receptor protein and/or mRNA expression is affected by cell growth and/or differentiation. Several cis- and trans-acting factors regulating the expression of the human insulin receptor gene and its response to glucocorticoid hormones have been identified.

Effects of STZ-induced diabetes and fasting on insulin receptor mRNA expression and insulin receptor gene transcription in rat liver.

Insulinopenic states in rodents are known to cause an increase in the number of hepatic insulin receptors. To determine if this change is related to an abnormality in insulin receptor gene expression, insulin receptor binding, insulin receptor mRNA levels, and insulin receptor gene transcription rates have been measured in livers from rats rendered hypoinsulinemic by STZ administration (65 mg/kg) or fasting. In the two groups of experimental rats, insulin binding to liver plasma membranes was increased (approximately 40 and 25%, respectively) relative to control, normoinsulinemic animals. Northern blot analysis of either total or poly (A)+ RNA from livers of hypo- and normoinsulinemic rats revealed two major insulin receptor mRNA species of 9.5 and 7.5 kbs. In hypoinsulinemic rats, insulin receptor mRNA levels were increased > or = 10-fold, with similar effects on the two mRNA species. The effects of STZ administration and fasting on insulin receptor binding and insulin receptor mRNA levels were fully reversed by insulin treatment or refeeding, respectively. Injection of ACT D, an inhibitor of gene transcription, decreased insulin receptor mRNA levels by > or = 80% in control and diabetic rats and suppressed the overexpression of mRNA seen in diabetic rats. In vitro nuclear transcription assays showed that the rate of transcription of the insulin receptor gene was increased 2-fold in STZ-induced diabetic rats and fasted rats relative to control animals. Taken together, these results suggest that the upregulation of the insulin receptor induced by chronic insulinopenia results, at least in part, from an increase in insulin receptor gene transcription.

DNA polymorphism analysis of HLA class II genes in unrelated children and in first-degree relatives with type I diabetes.

Eighty unrelated diabetic children, seventy healthy controls and hundred and ten affected and unaffected first-degree relatives of twenty multiplex families were investigated by restriction fragment length polymorphism analysis of HLA class II genes using five probe/enzyme systems: DRB and DQB/Taq I, DRB and DQB/EcoRI and DQB/BamHI according to standard procedures described in the 10th Histocompatibility Workshop protocol. Comparison between the unrelated diabetic patients and the controls confirmed the positive association of type 1 diabetes with DR3(w17)DQw2 Dw24 or Dw25 and DR4DQw8 and the negative association with DR2(w15)DQw6, DR4DQw7 and DR7DQw2 haplotypes. In multiplex families, similar allele associations were found and the distinction between haplotypes present in diabetic patients and those that segregated to healthy family members allowed to observe striking differences between the "affected" and "unaffected" haplotypes, particularly for the subtypes of DR3(w17) DQw2, DR4DQw3 and DR2DQw1 haplotypes. Heterozygous siblings who carried both DR3DQw2 and DR4DQw8 subtypes disclosed a highly increased risk and more than 80% of DR3/DR4 affected siblings received a paternal DR4DQw8 together with a maternal DR3DQw2. These observations indicate that several genetic aspects influence susceptibility to type 1 diabetes: 1) some particular HLA class II subsets; 2) the parental origin of the predisposing genes; 3) the synergistic effect of both haplotypes, in particular DR3DQw2 and DR4DQw8. These results may help to better specify susceptibility markers for risk prediction in siblings.