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K Grzelkowska, D Dardevet, M Balage and J Grizard

Insulin resistance in 3-day streptozotocin (STZ)-treated rats was manifested by the lack of antiproteolytic action of insulin as well as by a reduction of its stimulatory effect on protein synthesis (-60% compared with the control group) in epitrochlearis muscle incubated in vitro. In the present study, we have investigated the diabetes-associated alterations in the insulin signalling cascade, especially the phosphatidylinositol-3 kinase (PI-3 kinase)/p70 S6 kinase (p70(S6K)) pathway, in rat skeletal muscle. LY 294002, a specific inhibitor of PI-3 kinase, markedly decreased the basal rate of protein synthesis and completely prevented insulin-mediated stimulation of this process both in control and diabetic rats. Thus, PI-3 kinase is required for insulin-stimulated muscle protein synthesis in diabetic rats as in the controls. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), had no effect on the basal rate of protein synthesis in either of the experimental groups. In control rats, the stimulatory action of insulin on muscle protein synthesis was diminished by 36% in the presence of rapamycin, whereas in diabetic muscles this reduction amounted to 68%. The rapamycin-sensitive pathway makes a relatively greater contribution to the stimulatory effect of insulin on muscle protein synthesis in diabetic rats compared with the controls, due presumably to the preferential decrease in the rapamycin-insensitive component of protein synthesis. Neither basal nor insulin-stimulated p70(S6K) activity, a signalling element lying downstream of mTOR, were modified by STZ-diabetes.

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EP Araujo, ME Amaral, E Filiputti, CT De Souza, TL Laurito, VD Augusto, MJ Saad, AC Boschero, LA Velloso and EM Carneiro

Autocrine and paracrine insulin signaling may participate in the fine control of insulin secretion. In the present study, tissue distribution and protein amounts of the insulin receptor and its major substrates, insulin receptor substrate (IRS)-1 and IRS-2, were evaluated in a model of impaired glucose-induced insulin secretion, the protein-deficient rat. Immunoblot and RT-PCR studies showed that the insulin receptor and IRS-2 expression are increased, whilst IRS-1 protein and mRNA contents are decreased in pancreatic islets of protein-deficient rats. Immunohistochemical studies revealed that the insulin receptor and IRS-1 and -2 are present in the great majority of islet cells; however, the greatest staining was localized at the periphery, suggesting a co-localization with non-insulin-secreting cells. Exogenous insulin stimulation of isolated islets promoted higher insulin receptor and IRS-1 and -2 tyrosine phosphorylation in islets from protein-deficient rats, as compared with controls. Moreover, insulin-induced IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activity are increased in islets of protein-deficient rats. The reduction of IRS-1 and IRS-2 protein expression in islets isolated from protein-deficient rats by the use of antisense IRS-1 or IRS-2 phosphorthioate-modified oligonucleotides partially restored glucose-induced insulin secretion. Thus, the impairment of insulin cell signaling through members of the IRS family of proteins in isolated rat pancreatic islets improves glucose-induced insulin secretion. The present data reinforced the role of insulin paracrine and autocrine signaling in the control of its own secretion.

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Y Ito, M Ariga, S-I Takahashi, A Takenaka, T Hidaka and T Noguchi


The binding of insulin to its receptor rapidly induces intrinsic insulin receptor tyrosine kinase activity, resulting in tyrosine phosphorylation of various cytosolic substrates, such as insulin receptor substrate-1 (IRS-1) which, in turn, associates with a p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) followed by activation of this enzyme.

In the present study, we have examined these early steps of insulin signalling in rat liver in vivo after food ingestion. After fasting for 22 h, a 12% casein diet was available ad libitum throughout the 8-h experimental period. Plasma insulin concentrations increased within 45 min after feeding, reached a maximum at 1·5 h and gradually decreased until 8 h. Autophosphorylation of the insulin receptor β-subunit in liver was detected even during fasting and increased about 1·5-fold at 1·5 h after feeding. Basal tyrosine phosphorylation of IRS-1 was detectable during starvation, increased about twofold at 3 h after feeding and levels were maintained until 8 h. The content of the p85 subunit of PI 3-kinase associated with IRS-1 also increased after feeding in parallel with the changes in tyrosine phosphorylation of IRS-1.

Because tyrosine phosphorylation of the insulin receptor β-subunit and IRS-1 and the association of the p85 subunit of PI 3-kinase with IRS-1 in liver were closely correlated with the changes in the plasma concentration of insulin, we concluded that endogenous insulin secreted in response to eating caused these insulin-dependent intracellular changes in the liver.

Journal of Endocrinology (1997) 154, 267–273

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F Shi and PS LaPolt

FoxO1 is a transcription factor implicated in a growing number of physiological processes, including apoptosis, cell cycle progression, and insulin signaling. Recent findings indicate that FSH and growth factors influence ovarian functions in part through regulation of FoxO1. The present study utilized immunohistochemical analysis to determine the ovarian localization and regulation of FoxO1 protein levels in neonatal rats, immature rats during gonadotropin-induced follicular development, ovulation, and luteinization, and in spontaneously developing ovarian cysts of aging rats. In postnatal rats, FoxO1 immunoreactivity was very faint in ovaries of 5- and 10-day-old females. In contrast, strong immunoreactivity was observed in granulosa cells of larger developing follicles at 25 days of age. To stimulate follicle development, immature female rats received equine chorionic gonadotropin (eCG) followed 52 h later by an ovulatory dose of human chorionic gonadotropin (hCG). Prior to gonadotropin treatment, moderate FoxO1 immunoreactivity was observed in granulosa cells of small follicles. Subsequently, treatment with eCG markedly decreased FoxO1 protein levels in granulosa cells of healthy antral and preovulatory follicles. Interestingly, FoxO1 staining was observed in cumulus and antral, but not mural granulosa cells of preovulatory follicles. Induction of ovulation and luteinization with hCG further decreased ovarian FoxO1 levels, with no staining evident in corpora lutea. At all time points, the most intensive FoxO1 staining was observed in granulosa cells of atretic follicles, with predominantly nuclear localization. Similarly, while FoxO1 levels were low in granulosa cells of preovulatory follicles in proestrous rats, FoxO1 staining was intense in granulosa cells of spontaneously developing cystic follicles in aged, acyclic females. Together, these findings indicate that FoxO1 is expressed in a regulated, cell-specific manner during ovarian follicular development, atresia and luteinization, suggesting roles in these physiological processes.

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M Fasshauer, S Kralisch, M Klier, U Lossner, M Bluher, J Klein and R Paschke

Various cytokines, including tumor necrosis factor (TNF) alpha, growth hormone (GH) and interleukin (IL)-6, induce insulin resistance. Recently, it was demonstrated that induction of suppressor of cytokine signaling (SOCS)-3 by TNFalpha and GH is an important mechanism by which these cytokines impair insulin sensitivity. The current study investigated in 3T3-L1 adipocytes whether TNFalpha and GH also upregulate SOCS-1 and SOCS-6, which have both been shown to inhibit insulin signaling potently, and whether IL-6 might alter synthesis of SOCS-1, -3 and -6. Interestingly, 10 ng/ml TNFalpha, 500 ng/ml GH and 30 ng/ml IL-6 induced SOCS-1 mRNA time-dependently with maximal stimulation detectable after 8 h of TNFalpha and 1 h of GH and IL-6 addition respectively. Furthermore, TNFalpha and GH caused sustained upregulation of SOCS-1 for up to 24 h, whereas stimulation by IL-6 was only transient, with SOCS-1 mRNA returning to basal levels 2 h after effector addition. Induction of SOCS-1 was dose-dependent, and significant stimulation was detectable at concentrations as low as 3 ng/ml TNFalpha, 50 ng/ml GH and 10 ng/ml IL-6. Furthermore, stimulation experiments and studies using pharmacologic inhibitors suggested that the positive effect of TNFalpha, GH and IL-6 on SOCS-1 mRNA is, at least in part, mediated by Janus kinase (Jak) 2. Finally, SOCS-3 expression was dose- and time-dependently induced by IL-6, at least in part via Jak2, but none of the cytokines affected SOCS-6 expression. Taken together, our results show a differential regulation of SOCS mRNA by insulin resistance-inducing hormones, and suggest that SOCS-1, as well as SOCS-3, may be an important intracellular mediator of insulin resistance in fat cells and a potential pharmacologic target for the treatment of impaired insulin sensitivity.

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A Acitores, N Gonzalez, V Sancho, I Valverde and ML Villanueva-Penacarrillo

Glucagon-like peptide-1 (GLP-1), an incretin with glucose-dependent insulinotropic and insulin-independent antidiabetic properties, has insulin-like effects on glucose metabolism in extrapancreatic tissues participating in overall glucose homeostasis. These effects are exerted through specific receptors not associated with cAMP, an inositol phosphoglycan being a possible second messenger. In rat hepatocytes, activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB), protein kinase C (PKC) and protein phosphatase 1 (PP-1) has been shown to be involved in the GLP-1-induced stimulation of glycogen synthase. We have investigated the role of enzymes known or suggested to mediate the actions of insulin in the GLP-1-induced increase in glycogen synthase a activity in rat skeletal muscle strips. We first explored the effect of GLP-1, compared with that of insulin, on the activation of PI3K, PKB, p70s6 kinase (p70s6k) and p44/42 mitogen-activated protein kinases (MAPKs) and the action of specific inhibitors of these kinases on the insulin- and GLP-1-induced increment in glycogen synthase a activity. The study showed that GLP-1, like insulin, activated PI3K/PKB, p70s6k and p44/42. Wortmannin (a PI3K inhibitor) reduced the stimulatory action of insulin on glycogen synthase a activity and blocked that of GLP-1, rapamycin (a 70s6k inhibitor) did not affect the action of GLP-1 but abolished that of insulin, PD98059 (MAPK inhibitor) was ineffective on insulin but blocked the action of GLP-1, okadaic acid (a PP-2A inhibitor) and tumour necrosis factor-alpha (a PP-1 inhibitor) were both ineffective on GLP-1 but abolished the action of insulin, and Ro 31-8220 (an inhibitor of some PKC isoforms) reduced the effect of GLP-1 while completely preventing that of insulin. It was concluded that activation of PI3K/PKB and MAPKs is required for the GLP-1-induced increment in glycogen synthase a activity, while PKC, although apparently participating, does not seem to play an essential role; unlike in insulin signaling, p70s6k, PP-1 and PP-2A do not seem to be needed in the action of GLP-1 upon glycogen synthase a activity in rat muscle.

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FP Dominici, A Balbis, A Bartke and D Turyn

Overexpression of bovine growth hormone (bGH) in transgenic (PEPCK-bGH) mice induces resistance to insulin, which is compensated by a major increase in insulin levels. In these animals, hepatic insulin receptors (InsRs) are downregulated while tyrosine kinase activity of wheat germ agglutinin (WGA)-purified InsRs towards exogenous substrates is unexpectedly increased. By normalizing insulinemia, we attempted to determine whether the alterations detected in the early steps of insulin signal transduction are due to exposure to chronically high GH levels or are secondary to hyperinsulinemia. Transgenic PEPCK-bGH animals were treated with a single intraperitoneal administration of streptozotocin (STZ) or were deprived of food for 48 h, to normalize insulin levels. Both fasting and STZ treatment were effective in reducing insulin blood levels to control values or below, while GH levels remained unchanged (STZ treatment) or increased (fasted animals). In the liver of untreated transgenic mice, the number of InsRs as determined by 125I-insulin binding was significantly diminished (65+/-5% and 60+/-6% of normal values in microsomes and solubilized membranes respectively;P<0.01 vs control mice). In treated transgenic mice, the number of InsRs increased to values similar to or slightly higher than those found in normal control mice (STZ-treated: 139+/-26% and 126+/-8%; fasted: 128+/-5% (P<0.05) and 102+/-1.5%, for microsomes and solubilized membranes respectively). Neither treatment altered InsR affinity. InsR concentration in liver as determined by immunoblotting using an antibody against the beta-subunit of the insulin receptor was found to be reduced in transgenic mice (69+/-3% of normal values,P<0.001) and was normalized after both STZ treatment (105+/-4%) and fasting (109+/-4%). Insulin-stimulated autophosphorylation activity of InsRs in transgenic mice was increased (154+/-13%,P<0.01 compared with the control group), essentially normalized by STZ treatment (96+/-14%), and reduced by fasting, to below the values measured in normal control mice (56+/-15%,P<0.05). The potential influence of basal serine/threonine (Ser/Thr) phosphorylation of the InsR beta-subunit on the regulation of the InsRs from transgenic mice was also investigated. The autophosphorylation activity of WGA-purified InsRs from all groups of mice studied was essentially unchanged after dephosphorylation with alkaline phosphatase or mild trypsinization. Consequently, our results suggest that the observed changes in InsR number and autophosphorylation activity in the liver of bGH transgenic mice are directly related to changes in insulin blood levels, and that Ser/Thr phosphorylation is apparently not involved in the regulation of the InsR autophosphorylation activity in this model of insulin resistance.

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D Patiag, X Qu, S Gray, I Idris, M Wilkes, JP Seale and R Donnelly

Angiotensin II (ANGII) increases insulin sensitivity in diabetic and non-diabetic subjects, even at subpressor doses, and because there is 'crosstalk' between ANGII and insulin-signaling pathways the underlying mechanism may not be due solely to changes in regional blood flow. A series of experimental studies was undertaken to evaluate the effects of ANGII on glucose and lipid metabolism in vivo and in vitro. Groups of fructose-fed, insulin-resistant Sprague-Dawley (SD) rats were pre-treated with 0.3 mg/kg per day of the AT(1)-receptor antagonist L-158 809 (n=16), or vehicle (n=16), by oral gavage. This was prior to an oral glucose tolerance test (day 5) and measurement of the effects of ANGII infusion (20 ng/kg per min i.v. for 3 h) on whole-body insulin sensitivity using the insulin suppression test (day 7). The effect of ANGII infusion on total triglyceride secretion rate (TGSR) was evaluated in normal SD rats pretreated for 7 days with L-158 809 (n=12) or vehicle (n=12). AT(1)- and AT(2)- receptor mRNA expression and [(3)H]2-deoxyglucose uptake were assessed in cultured L6 myoblasts. Short-term treatment with L-158 809 had no effect on glucose tolerance or fasting triglyceride levels in fructose-fed rats. ANGII infusion had no effect on insulin sensitivity in fructose-fed rats pretreated with vehicle (steady-state plasma glucose (SSPG) values 8.1+/-1.6 vs 8. 4+/-0.4 mmol/l), but pretreatment with L-158 809 resulted in ANGII having a modest insulin antagonist effect in this insulin-resistant model (SSPG values 9.6+/-0.3 vs 7.1+/-0.6, P<0.03). ANGII infusion had no significant effect on TGSR (e.g. 24.6+/-1.4 vs 28.4+/-0.9 mg/100 g per h in vehicle-treated animals). RT-PCR analysis showed that L6 cells express both AT(1)- and AT(2)-receptor mRNA. Incubation with ANGII (10(-9) and 10(-8) M) had no significant effect on the dose-response curve for insulin-stimulated [(3)H]2-deoxyglucose uptake. For example, C(I200) values (dose of insulin required to increase glucose uptake by 200%) were 4.5 x 10(-9) M (control) vs 3.9 x 10(-9) M and 6.2 x 10(-9) M, whereas the positive control (glucagon-like peptide-1) increased insulin sensitivity. Thus, ANGII infusion may have a modest insulin antagonist effect on glucose disposal in insulin-resistant fructose-fed rats pretreated with an AT(1)-blocker, but ANGII has no effect on TGSR or in vitro glucose uptake in L6 myoblasts. These findings are relevant to recent clinical discussions about the metabolic effects of ANGII and renin-angiotensin system blockade.

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Shaodong Guo

focus on studies using genetically engineered mouse models, which have provided detailed information with respect to the inactivation of the insulin signaling cascade in the brain, adipose tissue, pancreas, muscle, and liver, as well as other tissues

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Márcio Pereira-da-Silva, Cláudio T De Souza, Alessandra L Gasparetti, Mário J A Saad and Lício A Velloso

-term i.c.v. treatment with exogenous MCH upon whole-body glucose homeostasis, and upon insulin signal transduction in liver, skeletal muscle, and white and brown adipose tissues of non-genetically manipulated, non-diabetes prone Wistar rats