Evidence for an interaction between the insulin receptor and Grb7. A role for two of its binding domains, PIR and SH2.

The molecular adapter Grb7 is likely to be implicated in the development of certain cancer types. In this study we show that Grb7 binds the insulin receptors, when they are activated and tyrosine phosphorylated. This interaction is documented by two-hybrid experiments, GST pull-down assays and in vivo coimmunoprecipitations. In addition, our results argue in favor of a preferential association between Grb7 and the insulin receptors when compared to other tyrosine kinase receptors like the EGF receptor, the FGF receptor and Ret. Interestingly, Grb7 is not a substrate of the insulin receptor tyrosine kinase activity. Grb7 binds the activated tyrosine kinase loop of the insulin receptors. Two domains of Grb7 are implicated in the insulin receptor binding: the SH2 domain and the PIR (phosphotyrosine interacting region). The role of these two domains in the interaction with the insulin receptor was already reported for Grb10 and Grb14, the other members of the Grb7 family of proteins. However, the relative importance of these domains varies, considering the receptor and the Grb protein. These differences should be a determinant of the specificity of the receptor tyrosine kinase-Grbs binding, and thus of the implication of Grb7/10/14 in signal transduction.

Identification of the rat adapter Grb14 as an inhibitor of insulin actions.

We cloned by interaction with the beta-subunit of the insulin receptor the rat variant of the human adapter Grb14 (rGrb14). rGrb14 is specifically expressed in rat insulin-sensitive tissues and in the brain. The binding of rGrb14 to insulin receptors is insulin-dependent in vivo in Chinese hamster ovary (CHO) cells overexpressing both proteins and importantly, in rat liver expressing physiological levels of proteins. However, rGrb14 is not a substrate of the tyrosine kinase of the receptor. In the two-hybrid system, two domains of rGrb14 can mediate the interaction with insulin receptors: the Src homology 2 (SH2) domain and a region between the PH and SH2 domains that we named PIR (for phosphorylated insulin receptor-interacting region). In vitro interaction assays using deletion mutants of rGrb14 show that the PIR, but not the SH2 domain, is able to coprecipitate insulin receptors, suggesting that the PIR is the major binding domain of rGrb14. The interaction between rGrb14 and the insulin receptors is almost abolished by mutating tyrosine residue Tyr1150 or Tyr1151 of the receptor. The overexpression of rGrb14 in CHO-IR cells decreases insulin stimulation of both DNA and glycogen synthesis. These effects are accompanied by a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1, but insulin receptor autophosphorylation is unaltered. These findings suggest that rGrb14 could be a new downstream signaling component of the insulin-mediated pathways.

Evidence for a direct interaction between insulin receptor substrate-1 and Shc.

Insulin receptor substrate-1 (IRS-1) and Shc are two proteins implicated in intracellular signal transduction. They are activated by an increasing number of extracellular signals, mediated by receptor tyrosine kinases, cytokine receptors, and G protein-coupled receptors. In this study we demonstrate that Shc interacts directly with IRS-1, using the yeast two-hybrid system and an in vitro interaction assay. Deletion analysis of the proteins to map the domains implicated in this interaction shows that the phosphotyrosine binding domain of Shc binds to the region of IRS-1 comprising amino acids 583-661. An in vitro association assay, performed with or without activation of tyrosine kinases, gives evidence that tyrosine phosphorylation of IRS-1 and Shc drastically improves the interaction. Site-directed mutagenesis on IRS-1 583-693 shows that the asparagine, but not the tyrosine residue of the N625GDY628motif domain, is implicated in the IRS-1-Shc-phosphotyrosine binding interaction. Mutation of another tyrosine residue, Tyr608, also induced a 40% decrease in the interaction. This study, describing a phosphotyrosine-dependent interaction between IRS-1 and Shc, suggests that this association might be important in signal transduction.

Induction of fatty-acid-synthase gene expression by glucose in primary culture of rat hepatocytes. Dependency upon glucokinase activity.

Fatty acid synthase (FAS) expression is low in liver and adipose tissue of suckling rats and increases markedly after weaning on to a high-carbohydrate low-fat diet. It has been shown previously that glucose alone, via an increase in intracellular glucose-6-phosphate level, stimulated the accumulation of FAS mRNA in cultured white adipose tissue of suckling rats. The regulation of FAS expression by glucose and hormones (insulin, dexamethasone and triiodothyronine) was studied in cultured hepatocytes from suckling rats. In hepatocytes cultured for 48 h in the absence of hormones and glucose, FAS mRNA, as well as glucokinase mRNA, levels remained undetectable. Glucose alone was unable to stimulate FAS expression. The combination of hormones, in the absence of glucose, has a marginal effect on FAS mRNA levels. However, FAS mRNA levels were increased in the presence of both glucose and the combination of hormones. This demonstrated that the hormonal induction of FAS mRNA was dependent on the presence of glucose in the culture medium. We have then investigated if glucokinase expression could be a prerequisite for the stimulation of FAS expression in response to glucose. Hepatocytes were cultured for 48 h in the absence of glucose but in the presence of insulin, dexamethasone and triiodothyronine. In these conditions, glucokinase mRNA and activity were markedly increased but there was no accumulation of FAS mRNA. When these hepatocytes were then exposed to various levels of glucose, FAS mRNA rapidly accumulated. Glucose stimulation of FAS expression was observed only in hepatocytes which expressed glucokinase activity. The importance of glucokinase expression for the induction of FAS mRNA by glucose is supported by the striking correlation between glucose-6-phosphate concentrations and the levels of FAS mRNA. This study clearly demonstrates that: (a) glucose metabolism is directly involved in the regulation of FAS gene expression; (b) the effect of hormones is partly due to their capacity to induce in the hepatocytes the capacity for glucose phosphorylation.

[Regulation of expression of genes coding for lipogenesis enzymes during development in the rat]. / Régulation de l'expression des gènes codant les enzymes de la lipogénèse au cours du développement chez le rat.

In vivo studies in the rat during the weaning period and in vitro studies on cultured cells have shown that nutriments and pancreatic hormones play a major role in lipogenic enzyme gene expression in liver and adipose tissue. Activation of lipogenic enzyme gene expression in liver and adipose tissue is dependent upon the increased supply of glucose and the concomitant hyperinsulinemia that occur after weaning. In contrast, an elevated supply of polyunsaturated or an increased plasma glucagon level prevent lipogenic enzyme gene expression in liver and adipose tissue. Glucose-6-phosphate seems to be the intracellular metabolite that mediates the transcriptional activation of lipogenic enzyme genes in response to glucose.

Regulation of lipogenic enzyme and phosphoenolpyruvate carboxykinase gene expression in cultured white adipose tissue. Glucose and insulin effects are antagonized by cAMP.

In cultured adipose tissue of suckling rats, glucose alone is able to induce the appearance of fatty-acid synthase and acetyl-CoA carboxylase mRNA by a mechanism involving glucose-6-phosphate accumulation; insulin alone has no effect but potentiates the effect of glucose. In the present study, we have analysed in cultured adipose tissue the effects of other hormones on the expression of these enzymes as well as on phosphoenolpyruvate carboxykinase. Triiodothyronine has only a marginal effect on fatty-acid synthase expression, in the absence or presence of glucose and insulin. A synthetic glucocorticoid, dexamethasone, opposes the inductive effect of glucose and insulin on fatty-acid synthase expression but increases the expression of phosphoenolpyruvate carboxykinase. A beta-agonist, isoproterenol totally inhibits the inductive effect of glucose and insulin on acetyl-CoA carboxylase and fatty-acid synthase expression whereas it increases the expression of phosphoenolpyruvate carboxykinase. Similarly, glucagon and cAMP have antagonistic effects on glucose and insulin-induced fatty-acid synthase expression. These inhibitory effects cannot be explained only by a reduction in glucose-6-phosphate concentration. We conclude that, in adipose tissue, dexamethasone and cAMP-generating hormones are negative regulators of lipogenic enzyme expression. Finally, the regulation of phosphoenolpyruvate carboxykinase expression in adipose tissue is similar to that found in the liver, i.e. inhibition by insulin and glucose and activation by glucocorticoids and cAMP.

Regulation of lipogenic enzyme gene expression by nutrients and hormones.

In vivo and in vitro experiments strongly support the view that marked increases in the levels of mRNA and in the activities of lipogenic enzymes that occur in liver and white adipose tissue of the rat after weaning to a high-carbohydrate diet are dependent on an increase in plasma glucose and insulin concentrations. An increased glucose metabolism is necessary for the expression of insulin effects on fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) mRNA accumulation in white adipose tissue, as insulin is ineffective in vitro in the absence of glucose. It is suggested that intracellular glucose-6-phosphate could play an important role in the effect of insulin on lipogenic enzyme gene expression in white adipose tissue. Other hormones and substrates could also play a role in the surge of lipogenesis after weaning. The fall in plasma glucagon after weaning to a high-carbohydrate diet could reinforce the insulin-induced accumulation of FAS and ACC mRNA, as this hormone inhibits the accumulation of lipogenic enzyme mRNA in liver and white adipose tissue. The decrease in the dietary supply of fat after weaning to a high-carbohydrate diet could also potentiate the accumulation of FAS and ACC mRNA in liver because long-chain poly-unsaturated fatty acids are potent inhibitors of the expression of the genes encoding liver lipogenic enzymes. A direct effect of fatty acids on a cis-acting element of the lipogenic enzyme genes could be involved, as the regulatory region of FAS gene contains a polyunsaturated fatty acid response element that shares some similarity with the peroxisome proliferator-activated receptor recently described.

Effect of acarbose on glucose homeostasis, lipogenesis and lipogenic enzyme gene expression in adipose tissue of weaned rats.

Acarbose is a potent intestinal glucosidase inhibitor which could have an anti-obesity property by reducing postprandial plasma glucose and insulin levels, potentially responsible for high rates of lipid synthesis in adipose tissue. We have tested this hypothesis by studying rats during the weaning period, when the lipogenic capacity of the adipose tissue develops. Rats were treated from age 19 days onwards with acarbose (10 mg/100 g diet) and studied at age 30 days. Acarbose was efficient in reducing postprandial excursions of both blood glucose and plasma insulin. Acarbose-treated rats behave like rats continuously infused with glucose with no metabolic signs of carbohydrate deprivation since gluconeogenesis was not activated. There was no massive caecal fermentation of carbohydrate since volatile fatty acids did not significantly increase in the portal blood. One of the most striking features of the acarbose-treated rats was the reduction of adipose tissue weight due to a reduced adipocyte size. This was concomitant with a reduced lipogenic capacity from glucose in isolated adipocytes under insulin stimulation. The activity of fatty acid synthase and acetyl-CoA carboxylase was decreased concomitantly with a reduced expression of their specific mRNA. This study allows the conclusion that postprandial hyperinsulinaemia and hyperglycaemia have a major role in the control of expression of lipogenic enzymes and thus on adipose tissue lipogenic capacity.

Glucose stimulation of lipogenic enzyme gene expression in cultured white adipose tissue. A role for glucose 6-phosphate.

The expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) is low in the adipose tissue of suckling rats and increases markedly at weaning to a high carbohydrate diet. We have studied in vitro the factors regulating this phenomenon. Inguinal adipose tissue pieces from 19-day-old suckling rats were cultured for 6 or 24 h in minimal essential medium. Insulin (100 nM) added in the presence of lactate and pyruvate did not stimulate the expression of FAS and ACC. Glucose (20 mM) alone resulted in a 5-7-fold increase of FAS and ACC mRNA. Insulin potentiated the effect of glucose. 3-O-Methylglucose, a glucose analog that is transported into the cell but not metabolized, had no effect on FAS and ACC mRNA accumulation. However, 2-deoxyglucose (1 mM), a glucose analog which is phosphorylated to 2-deoxyglucose 6-phosphate, stimulated the expression of FAS and ACC to the same extent as 20 mM glucose. Glucose 6-phosphate concentrations in adipose tissue pieces cultured in various conditions changed in parallel with the FAS and ACC mRNA levels. We conclude that glucose 6-phosphate could be the metabolite involved in the stimulation of lipogenic enzyme gene expression in response to glucose.

Effect of diets rich in medium-chain and long-chain triglycerides on lipogenic-enzyme gene expression in liver and adipose tissue of the weaned rat.

The activity and mRNA concentrations of two lipogenic enzymes, fatty-acid synthase and acetyl-CoA carboxylase were measured in the liver and white adipose tissue of rats weaned to a carbohydrate-rich diet containing either long-chain or medium-chain fatty acids, and compared to those of rats weaned on a diet containing less than 1% (total energy) fat (high-carbohydrate diet). In the liver, the diet containing long-chain fatty acids inhibited the increase of both lipogenic-enzyme mRNA concentrations and activities seen at weaning on the high-carbohydrate diet but did not prevent the decrease in phosphoenolpyruvate carboxykinase mRNA and activity. In contrast, the diet containing medium-chain fatty acids induced a slower but finally similar increase in lipogenic-enzyme mRNA concentrations and activities. In adipose tissue, a similar trend was observed, although the inhibitory effect of the diet containing long-chain fatty acids was considerably less marked than in liver. It is concluded that medium-chain and long-chain fatty acids have not the same inhibitory potency of the gene expression of lipogenic enzymes, and that long-chain fatty acids have a more marked effect in the liver.

Increased gene expression of lipogenic enzymes and glucose transporter in white adipose tissue of suckling and weaned obese Zucker rats.

Previous experiments have shown that insulin-induced glucose utilization is increased in white adipose tissue of young obese Zucker rats. We have investigated the possible role of over-expression of the muscle/fat glucose transporter (Glut 4) and key lipogenic enzymes in this increased insulin-responsiveness. The amount or activity and the mRNA concentrations of Glut 4, fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were measured before and after weaning in white adipose tissue of obese and lean Zucker rats. Comparison of the levels of Glut 4 and lipogenic-enzyme expression in 15-day-old suckling and 30-day-old weaned rats on a high-carbohydrate diet shows a marked increase in the latter group. The increase was, in lean and obese rats respectively, 6- and 7-fold for the amount of Glut 4 and 2- and 3-fold for its mRNA concentrations, 40- and 100-fold for the activity of lipogenic enzymes (FAS and ACC) and 30- and 10-fold for their mRNA concentrations. Furthermore, all these parameters, except the amount of Glut 4, were 2-5-fold higher in obese rats, both before and after weaning. Changes at weaning were largely blunted when rats were weaned on to a high-fat diet, although the differences between lean and obese rats persisted, and even became significant for the amount of Glut 4. Whatever the experimental conditions, plasma insulin levels were significantly higher in obese than in lean rats. These results indicate the existence of an enhanced expression of Glut 4, FAS and ACC in white adipose tissue of young obese fa/fa rats which could be related to the increased plasma insulin levels.

Hormonal regulation of liver phosphoenolpyruvate carboxykinase and glucokinase gene expression at weaning in the rat.

During the suckling period, the rats are fed continuously with milk, which is a high-fat low-carbohydrate diet (HF). At weaning, milk is progressively replaced by the rat's laboratory chow which is a high-carbohydrate low-fat diet (HCHO), and this is accompanied by large hormonal modifications: an increase in plasma insulin and a decrease in plasma glucagon concentrations, and by marked changes in metabolic pathways in liver: decrease in hepatic gluconeogenesis and increase in glycolysis and lipogenesis. Most of the data concerning these changes are related to maximal activity of enzymes. The recent availability of specific cDNA probes for phosphoenolpyruvate carboxykinase (PEPCK), and glucokinase (GK) has allowed the study of the role of pancreatic hormones and nutrition in the changes of the expression of these genes at weaning in the rat. Regarding phosphoenolpyruvate carboxykinase gene transcription, the concentration of mRNA as well as the activity of PEPCK are elevated in the liver of suckling rat until the onset of weaning, 21 d after delivery. After weaning to a HCHO diet, both mRNA and activity of PEPCK rapidly decrease to a very low level. In contrast, weaning on an HF diet, which maintains high plasma glucagon and low plasma insulin levels, does not decrease in plasma glucagon concentration and a 90% decrease in PEPCK gene transcription and PEPCK mRNA concentration in 1 h. Regarding glucokinase gene transcription, the concentration of mRNA as well as the activity of GK are not detectable before 15 d after birth in the liver of the rat. They markedly increase when the newborn are weaned on an HCHO diet but not when they are weaned on an HF diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal control of specific gene expression in the rat liver during the suckling-weaning transition.

In the rat, the suckling-weaning transition is accompanied by marked changes in nutrition. During the suckling period, the pups are fed with milk which is a high-fat low-carbohydrate diet. At weaning, milk is progressively replaced by the rat chow which is a high-carbohydrate low-fat diet. This is accompanied by considerable hormonal modifications: an increase in plasma insulin and a decrease in plasma glucagon concentrations, as well as by marked changes in metabolic pathways in liver: decrease in hepatic gluconeogenesis, increase in lipogenesis, and appearance of liver glucokinase. Most of the data concerning these changes are related to maximal activity of enzymes. The recent availability of specific cDNA probes for phosphoenolpyruvate carboxykinase, acetyl-CoA carboxylase, fatty acid synthase and glucokinase has allowed study of the role of pancreatic hormones and of nutrition in the changes of the expression of these genes at weaning in the rat.

Lipogenic enzyme activities and mRNA in rat adipose tissue at weaning.

The activities and mRNA concentrations of two lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase, and one enzyme involved in glyceroneogenesis, phosphoenolpyruvate carboxykinase (PEPCK), were measured in rat white adipose tissue during the suckling-weaning transition. Activities and mRNA concentrations of lipogenic enzymes were low in suckling rats, whereas activity and mRNA concentration of PEPCK were high. At weaning to a high-carbohydrate diet, the rapid increase in lipogenic enzymes mRNA (10- to 20-fold) and decrease in PEPCK mRNA (10-fold) were followed by parallel changes in enzyme activities. In contrast, weaning to a high-fat diet prevented these modifications. Force feeding suckling rats with carbohydrates induced a rise in blood glucose and plasma insulin concentrations. During these experiments, mRNA concentrations increased 10- to 20-fold for lipogenic enzymes and decreased 5-fold for PEPCK in less than 6 h, whereas all enzyme activities did not vary. This suggests a pretranslational regulation of gene expression. Force feeding suckling rats with a mixture of fat devoid of carbohydrate induced a slight increase in plasma insulin concentration and a fall in PEPCK mRNA but was not accompanied by a rise in lipogenic enzyme mRNAs. This suggested that insulin is a prime regulator of PEPCK gene expression, whereas glucose and insulin act synergistically in the regulation of lipogenic enzyme gene expression.

Genetic variations in serum lipid levels of inbred mice and response to hypercholesterolemic diet.

The serum lipid contents of a number of inbred and congenic strains of mice were measured. There were inter-strain variations in each of the lipid fractions in mice fed a normal diet. Male and female C3H mice had the highest total cholesterol level; AKR mice showed the lowest values. Serum phospholipids were correlated well with cholesterolemia. The greatest variations between strains were in the triglyceride levels. There also was significant variation in the high density lipoprotein cholesterol serum levels (from 73-88% of the total cholesterol). The response to a hypercholesterolemic diet (1% cholesterol) was tested in seven inbred strains. All strains showed changes in serum cholesterol and in the proportions of the lipoproteins fractions. There was a large increase in the low density lipoprotein + very low density lipoprotein fractions. Feeding the diet revealed marked interstrain differences in the responses of the serum cholesterol and electrophoretic lipoprotein profiles. The C57BL/6 and B10.D2 strains were hyperresponders to the hypercholesterolemic diet with 71% and 63% of their serum cholesterol in the low density lipoprotein plus very low density lipoprotein fractions, respectively.