Target Of Insulin Action Research Articles

Insulin inhibits the ubiquitin-dependent degrading activity of the 26S proteasome.

A major metabolic effect of insulin is inhibition of cellular proteolysis, but the proteolytic systems involved are unclear. Tissues have multiple proteolytic systems, including the ATP- and ubiquitin-dependent proteasome pathway. The effect of insulin on this pathway was examined in vitro and in cultured cells. Insulin inhibited ATP- and ubiquitin-dependent lysozyme degradation more than 90% by reticulocyte extract, in a dose-dependent manner (IC50 approximately 50 nM). Insulin did not reduce the conjugation of ubiquitin to lysozyme and was not itself ubiquitin-conjugated. In HepG2 cells, insulin increased ubiquitin-conjugate accumulation 80%. The association between the 26S proteasome and an intracellular protease, the insulin-degrading enzyme (IDE), was examined by a purification scheme designed to enrich for the 26S proteasome. Copurification of IDE activity and immunoreactivity with the proteasome were detected through several chromatographic steps. Glycerol gradient analysis revealed cosedimentation of IDE with the 20S proteasome and possibly with the 26S proteasome. The proteasome-associated IDE was displaced when the samples were treated with insulin. These results suggest that insulin regulates protein catabolism, at least in part, by decreasing ubiquitin-mediated proteasomal activity, and provides a new target for insulin action. The displacement of IDE from the proteasome provides a mechanism for this insulin action.

Food Deprivation Decreases mRNA and Activity of the Rat Dopamine Transporter

We have hypothesized that the midbrain dopamine (DA) neurons are a target for insulin action in the central nervous system (CNS). In support of this hypothesis, we have previously demonstrated that direct intracerebroventricular infusion of insulin results in an increase in mRNA levels for the DA reuptake transporter (DAT). In this study, 24- to 36-hour food deprivation was used as a model of decreased CNS insulin levels, to test whether DAT mRNA levels, DAT protein concentration or DAT functional activity would be decreased. DAT mRNA levels, assessed by in situ hybridization, were significantly decreased in the ventral tegmental area/substantia nigra pars compacta (VTA/SNc) (77 ± 7% of controls, p < 0.05) of food-deprived (hypoinsulinemic) rats. Binding of a specific high-affinity DAT ligand (¹²⁵I-RTI-121) to membranes from brain regions of fasted or free-feeding rats provided an estimate of DAT protein, which was unchanged in both of the major terminal projection fields, the striatum and nucleus accumbens (NAc). In addition, we utilized the rotating disk electrode voltametry technique to assess possible changes in the function of the DAT in fasting (hypoinsulinemic) rats. The V_max of DA uptake was significantly decreased (87 ± 7% of control, p < 0.05), without a change in the K_m of uptake, in striatum from fasted rats. In vitro incubation with a physiological concentration (1 nM) of insulin resulted in an increase of striatal DA uptake to control levels. We conclude that striatal DAT function can be modulated by fasting and nutritional status, with a contribution by insulin.

Insulin Stimulates Guanine Nucleotide Exchange on Rab4 via a Wortmannin-sensitive Signaling Pathway in Rat Adipocytes

Rab4, a member of the Rab family of Ras-related small GTP-binding proteins, has been shown to be associated with GLUT4-containing vesicles and implicated in the insulin action on glucose transport in rat adipocytes. In the present study, we investigated the insulin effects on the guanine nucleotide exchange on Rab4. In electrically permeabilized rat adipocytes, the amount of [35S]guanosine 5'-O-(3-thiotrisphosphate) (GTPgammaS) bound to Rab4 increased in a time-dependent manner during 45 min of the incubation period. Addition of insulin resulted in about a 2-fold stimulation of the binding of [35S]GTPgammaS to Rab4, indicating that insulin stimulated the guanine nucleotide exchange on the GTPase. Pretreatment of the cells with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, completely abolished the stimulatory effect of insulin on [35S]GTPgammaS binding to Rab4. Wortmannin also attenuated the nucleotide binding to Rab4 in the basal cells, suggesting that phosphatidylinositol 3-kinase activity may be essential for regulation of guanine nucleotide exchange on the GTPase and insulin may up-regulate the exchange activity by stimulating the lipid kinase. Insulin-induced subcellular redistribution of Rab4 from the microsomal fraction to the soluble fraction was also inhibited by wortmannin. These results suggest that insulin stimulates the guanine nucleotide exchange on Rab4 via a phosphatidylinositol 3-kinase-dependent signaling pathway and that Rab4 is one of possible targets of insulin action on intracellular vesicle traffic in rat adipocytes.

Insulin Enhances Glucocorticoid Receptor-Mediated Induction of Gene Expression Independent of a Specific Insulin Response Element

We have established a system in which we observe a synergistic interaction between insulin and glucocorticoids, This includes chimeric genes constructed to contain synthetic glucocorticoid-responsive elements, 5′ of the HSV thymidine kinase promoter and the chloramphenicol acetyltransferase reporter gene, The magnitude of induction of gene expression by glucocorticoid was dependent on the number of GREs, Insulin alone had virtually no effect on the expression of any of these genes but together with dexamethasone acted in a synergistic manner, This synergy diminished as the number of GREs in the promoter increased, The synergy is independent of promoter sequences other than the GREs and a functional TATAA box. Three different approaches demonstrate that the effect of insulin is not directly on the glucocorticoid signal transduction pathway. Insulin does not change the dose-response relationship for dexamethasone. The effect of insulin is independent of the intracellular concentration of glucocorticoid receptor, The effect is independent of any specific domain of the glucocorticoid receptor. The target of insulin action is likely to be part of the normal host cell transcriptional initiation complex or a putative adaptor molecule.

Insulin Therapy in Maturity-Onset Diabetes

A prime target of insulin action in patients with maturity onset diabetes is suppression of hepatic glucose production in both the fasting and postprandial states. In patients who fail to respond to oral treatment insulin improves glycaemic control, has an antiatherogenic effect on the serum lipoprotein pattern, and increases well being. The beneficial long-term effects of insulin therapy on morbidity and mortality are, however, still unproved. The regimens of insulin therapy are not established. In patients with normal or increased basal serum C-peptide concentration treatment may start with intermediate or long-acting insulin at bedtime together with oral medication, or in the morning with or without oral medication. If this fails two injections of intermediate-acting insulin or multiple injections of short-acting insulin should be started. Hypoglycaemia is uncommon in maturity onset diabetes treated with insulin, but moderate weight gain usually occurs. The possible role of hyperinsulinemia as a predisposing factor for atherogenesis remains unresolved. Despite controversies over treatment, any patient with maturity onset diabetes not responding to oral agents should be regarded as a candidate for insulin therapy.

Insulin Rapidly Induces Rat Liver S14 Gene Transcription

We evaluated the role insulin plays in the regulation of hepatic S14 gene transcription using the streptozotocin-induced diabetic rat model. Nuclear run-on activity and mRNAS14 levels were reduced by more than 85% in diabetic rats compared to those in intact animals. After the administration of insulin, both S14 run-on activity and mRNAS14 levels were rapidly induced. Within 1 h of insulin administration, S14 run-on activity and mRNAS14 were induced 5- and 8-fold, respectively. S14 gene expression was restored to intact levels within 4 h, with overall increases in run-on activity and mRNAS14 of 7- and 20-fold, respectively. The full induction of mRNAS14 cannot be accounted for solely by activation of S14 gene transcription, implicating insulin effects at the posttranscriptional level. However, our results show that the principal target for insulin action on the S14 gene is transcriptional. Administration of dibutryl cAMP and theophylline fully blocked the insulin-mediated increase in S14 gene transcription, indicating that hepatic cAMP levels play a dominant negative role in regulating S14 gene transcription in vivo. Fructose administration to starved diabetic rats induced only a marginal 60% increase in mRNAS14 and S14 run-on activity within 4 h, while insulin plus fructose or insulin plus glucose fully restored S14 gene expression to intact levels within the same time period. Thus, dietary fructose or a metabolite generated from fructose alone cannot induce S14 gene transcription or mRNAS14 to intact levels in the starved diabetic rat. Acute effects of dietary carbohydrate on hepatic S14 gene transcription are insulin dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Contrasting interactions between phorbol ester and insulin on the regulation of glycogen synthase activity and p33 mRNA accumulation in rat hepatoma cells

We have compared the effect of phorbol 12-myristate 13-acetate (PMA) with that of insulin on three targets of insulin action in H4IIEC3 (H4) rat hepatoma cells. These parameters are the phosphorylation state and tyrosine kinase activity of the insulin receptor, the activation state of glycogen synthase, and the accumulation of p33 mRNA. Under conditions where insulin treatment of H4 cells clearly activated receptor serine and tyrosine phosphorylation on the insulin receptor β-subunit in situ, activated receptor tyrosine kinase activity in vitro, and activated glycogen synthase and p33 mRNA accumulation in situ, PMA alone did not influence the insulin receptor phosphorylation state or tyrosine kinase activity and did not affect glycogen synthase activity, but markedly increased p33 mRNA accumulation. When PMA was added in the presence of insulin, particularly if PMA was preincubated, the receptor phosphorylation state and the tyrosine kinase activity again were not affected, but insulin-activated glycogen synthase was significantly diminished or abolished. In contrast, increased p33 mRNA accumulation by PMA was additive with that of insulin. Thus, under conditions where no effect was observed on the insulin receptor phosphorylation state or the tyrosine kinase activity, PMA acted in an insulin-antagonistic manner on glycogen synthase and in an insulin-like manner on p33 mRNA accumulation, indicating that these actions of PMA are unrelated to early events in the pathway of the insulin action. Effects on glycogen synthase are most readily explained by an effect of protein kinase C-activated phosphorylation of glycogen synthase.

The role of insulin and glucocorticoids in the regulation of hepatic glycogen metabolism: effect of fasting, refeeding, and adrenalectomy.

The roles of insulin, adrenal corticol hormones, and nutritional factors in the regulation of hepatic glycogen metabolism were investigated by means of fasting and refeeding normal and adrenalectomized (ADX) rats. More specifically, the hypothesis in question in this study is that certain hepatic phosphoprotein phosphatases are targets of insulin action in liver. In anesthetized rats, the hepatic glycogen concentration and the activities of hepatic glycogen synthase, glycogen synthase phosphatase, glycogen phosphorylase, and phosphorylase phosphatase were correlated with peripheral plasma glucose and immunoreactive insulin levels. Hepatic phosphatase activities were measured in (soluble) the high speed supernatant and smooth endoplasmic reticulum (SER). Fasting resulted in expected diminutions in circulating glucose and insulin levels and loss of hepatic glycogen. These changes were greater in ADX rats. The percentage of hepatic glycogen synthase in the active or I form increased with fasting in normal rats, but did not change in ADX rats. Hepatic synthase phosphatase activities were decreased in SER by fasting in both normal and ADX rats, but to a much greater extent in the latter; soluble synthase phosphatase was much less affected by fasting. The percentage of phosphorylase in the active or a form was significantly decreased in normal, but not ADX, rats. Phosphorylase phosphatase activities were not significantly changed by fasting in any of the subcellular fractions in normal liver, but were increased in the hepatic SER of ADX rats. Refeeding fasted rats for 2 and 6 h resulted in increased hepatic glycogen, activation of glycogen synthase, and increased circulating levels of both insulin and glucose. Refeeding also caused increases in SER-associated synthase phosphatase activity in ADX animals. SER phosphorylase phosphatase activities were significantly increased by refeeding in normal rats, but were decreased in ADX rats. Regression analysis of the data suggested statistically significant positive correlations between insulin levels and SER synthase phosphatase activity in ADX animals, on the one hand, and SER synthase phosphatase and the percentage of synthase in the I form, on the other. No statistically significant correlation between insulin levels and phosphorylase phosphatase activities could be demonstrated. These results are compatible with the hypothesis that glycogen synthase phosphatase activity in liver, especially that associated with SER, is subject to physiological regulation by circulating levels of insulin. In contrast, phosphorylase phosphatase activity seems to be much less influenced by changes in the circulating insulin level. The results are compatible with the proposition that SER-associated phosphoprotein phosphatases are physiologically relevant in the regulation of hepatic glycogen metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

Photoaffinity labeling of insulin receptors in viable cultured human lymphocytes. Demonstration of receptor shedding and degradation.

A photosensitive derivative of radiolabeled insulin, SANAH-125I-insulin, was prepared by reacting N-succinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate (SANAH) with 125I-insulin. Cultured IM-9 cells were incubated with SANAH-125I-insulin at 16 degrees C in the dark. They were then washed, photolyzed, solubilized, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Under disulfide reducing conditions, a single specific band of Mr 125,000 was obtained. The characteristics of the labeling of this band with SANAH-125I-insulin (specificity, time course, concentration effect) were the same as that of 125I-insulin interaction with the IM-9 cells and the labeling process did not affect cell viability. The solubilized photolabeled insulin receptor fraction was enriched by first adsorbing to agarose-bound wheat germ agglutinin and the material eluted with N-acetyl-D-glucosamine was then analyzed by SDS-PAGE and autoradiography. Under nonreducing conditions, a major receptor band of Mr 320 K and a minor band of 280 K were obtained. Upon disulfide bond reduction with increasing concentrations of dithiothreitol, a major band of Mr 125 K and two minor bands of Mr 210 K and 94 K were seen. When cells photolabeled at 16 degrees C were further incubated at 37 degrees C, there was a time-dependent loss of intact receptors into the incubation buffer. In contrast, no similar shedding of labeled receptors was observed from isolated rat adipocytes. Following shedding, the labeled IM-9 insulin receptors rapidly disappeared from the incubation buffer (half-time approximately 1.5 h). These results demonstrate the feasibility of photoaffinity labeling, characterizing, and following the fate of insulin receptor in viable cells. Thus receptor photoaffinity labeling should provide a suitable approach for studies of the biologic fate of insulin receptors in cells that are targets for insulin action.