Intracellular signalling by the insulin receptor

The insulin receptor transduces insulin's biological signal through the tyrosine kinase present in the receptor's B subunit. The activated insulin receptor kinase then phosphorylates a series of intracellular substrate including insulin receptor substrate 1 (IRS-1), which has been shown to be the pivotal substrate for insulin receptor signal transduction. The phosphorylated tyrosine residues in IRS-1 can bind and activate the downstream effectors, many of which are SH2 domain containing proteins such as phosphotidylinositol 3-kinase, growth factor binding protein 2, and SH2 phosphotyrosine phosphatase 2. Phosphorylated synthetic IRS-1 peptides with the corresponding sequences of the IRS-1 have been shown to associate and activate their respective SH2 domain containing proteins. Another important event happening during insulin binding with the insulin receptor is that the insulin receptor rapidly undergoes internalization. However, the insulin receptor signalling and the receptor endocytosis have been studied as two independent processes. The hypothesis of the present thesis is that the insulin receptor endocytosis is involved in insulin receptor signalling and signal termination. The results of the present investigation demonstrate that insulin receptors in the earliest stage of endocytosis contain significantly greater kinase activity towards IRS-1 peptides than the receptors localized at the plasma membrane, indicating that they are potentially more capable of transducing signals. On the other hand, insulin receptors in the middle and late stage of endocytosis lose their kinase activity, suggesting that insulin receptor kinase activity inactivation and signal termination might take place in the late phase of the insulin receptor internalization. In addition, this study also found that the increased insulin receptor kinase activity in the endosomes is related to the tyrosyl phosphorylation of the specific domains of the receptor's $\beta$ subunit. ^

Correlation of α-Fetoprotein Expression in Normal Hepatocytes during Development with Tyrosine Phosphorylation and Insulin Receptor Expression

The molecular mechanism of hepatic cell growth and differentiation is ill defined. In the present study, we examined the putative role of tyrosine phosphorylation in normal rat liver development and in an in vitro model, the α-fetoprotein-producing (AFP+) and AFP-nonproducing (AFP−) clones of the McA-RH 7777 rat hepatoma. We demonstrated in vivo and in vitro that the AFP+ phenotype is clearly associated with enhanced tyrosine phosphorylation, as assessed by immunoblotting and flow cytometry. Moreover, immunoprecipitation of proteins with anti-phosphotyrosine antibody showed that normal fetal hepatocytes expressed the same phosphorylation pattern as stable AFP+ clones and likewise for adult hepatocytes and AFP− clones. The tyrosine phosphorylation of several proteins, including the β-subunit of the insulin receptor, insulin receptor substrate-1, p85 regulatory subunit of phosphatidylinositol-3-kinase, and ras-guanosine triphosphatase-activating protein, was observed in AFP+ clones, whereas the same proteins were not phosphorylated in AFP− clones. We also observed that fetal hepatocytes and the AFP+ clones express 4 times more of the insulin receptor β-subunit compared with adult hepatocytes and AFP− clones and, accordingly, that these AFP+ clones were more responsive to exogenous insulin in terms of protein tyrosine phosphorylation. Finally, growth rate in cells of AFP+ clones was higher than that measured in cells of AFP− clones, and inhibition of phosphatidylinositol-3-kinase by LY294002 and Wortmannin blocked insulin- and serum-stimulated DNA synthesis only in cells of AFP+ clones. These studies provide evidences in support of the hypothesis that signaling via insulin prevents hepatocyte differentiation by promoting fetal hepatocyte growth.

Glucose- and insulin-induced phosphorylation of the insulin receptor and its primary substrates IRS-1 and IRS-2 in rat pancreatic islets

The presence of tyrosine-phosphorylated proteins was studied in cultured rat pancreatic islets, Immunoblotting performed with total extracts of islets cultured in the presence of 1.8 or 5.6 mM glucose revealed at least three distinct tyrosine-phosphorylated bands (25 kDa, 95 kDa and 165-185 kDa). After 12 h incubation in medium containing 1.8 mM glucose, a pulse exposition to 11 or 22 mM glucose or to 10(-7) M insulin led to a substantial increase in the phosphorylation of all three bands, with no appearance of novel bands. Immunoprecipitation with specific antibodies demonstrated that the signal detected at 95 kDa corresponds to the beta subunit of the insulin receptor (IR) while the band at 165-185 kDa corresponds to the early substrates of the insulin receptor, IRS-1 and IRS-2. Immunoprecipitation with IRS-I or IRS-2 antisera detected their association with the lipid metabolizing enzyme phosphatidylinositol 3-kinase (PI 3-kinase), Thus, this is the first demonstration that elements involved in the insulin-signalling pathway of traditional target tissues are also present in pancreatic islets and are potentially involved in auto- and paracrine-signalling in this organ.

Serine residues 1177/78/82 of the insulin receptor are required for substrate phosphorylation but not autophosphorylation

Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

In this study, we evaluated the effects of obesity and insulin resistance induced by a high-fat diet on prostate morphophysiology, focusing on cell proliferation, expression of androgen (AR) and estrogen receptors (ER) and proteins of the insulin signaling pathway. Adult male Wistar rats were fed a high-fat diet (20% fat) for 15 weeks, whereas control animals received a balanced diet (4% fat). Both groups were then divided and treated for 2 weeks with 1 mg/kg body weight/day of the aromatase inhibitor letrozole or vehicle only. The ventral prostate was analyzed with immunohistochemical, histopathological, stereological, and Western blotting methods. Obese rats showed insulin resistance, hyperinsulinemia, and reduced plasma testosterone levels. The incidence of prostatic intraepithelial neoplasia (PIN) was 2.7 times higher in obese rats and affected 0.4% of the gland compared with 0.1% PIN areas found in control rats. Obesity doubled cell proliferation in both prostate epithelium and stroma. AR content decreased in the prostate of obese rats and estrogen receptor beta (ER beta) increased in this group. Protein levels of insulin receptor substrate 1 and protein kinase B diminished in the obese group, whereas phosphatidylinositol 3-kinase (PI3K) increased significantly. Most structural changes observed in the prostate of obese rats normalized after letrozole treatment, except for increased stromal cell proliferation and ER beta expression, which might be associated with insulin resistance. This experimental model of obesity and insulin resistance induced by a high-fat diet increases cell proliferation in rat prostate. Such alterations are associated with decreased levels of AR and increased ER beta and PI3K proteins. This change can facilitate the establishment of proliferative lesions in rat prostate.

Relationship between abnormal maternal inflammation and insulin resistance during pregnancy

Abnormal maternal inflammation during pregnancy is linked to complications such as preeclampsia and fetal growth restriction. There is growing evidence that insulin resistance is also associated with a heightened inflammatory state, and is linked to pregnancy complications such as gestational diabetes. This study tested the hypothesis that abnormal inflammation during pregnancy is causally linked to elevations in blood glucose and insulin resistance. To induce a state of abnormal systemic inflammation, bacterial lipopolysaccharide (LPS) was administered to pregnant rats on gestational days (GD) 13.5-16.5. Dams treated with LPS exhibited an abnormal immune response characterized by an elevation in white blood cells, which was linked to reduced fetal weight and increased glucose levels over pregnancy. Abnormal inflammation is characterized by increased levels of circulating pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF) and interleukin-6, which contribute to insulin resistance by inhibiting the insulin signalling pathway. TNF in particular induces a serine phosphorylation (pSer307) of insulin receptor substrate 1 (IRS-1). In our model, insulin resistance was assessed by measuring the extent of pSer307 of IRS-1 and total IRS-1 expression in skeletal muscle, as well as changes in metabolic parameters and pancreas tissue morphology associated with insulin resistance. LPS-treated dams exhibited a significant reduction in IRS-1 expression, elevation in fasting glucose levels, and reduction in insulin sensitivity indices. There were also biologically relevant increases in fasting plasma insulin levels and insulin resistance indices, but not pSer307 of IRS-1 and pancreatic islet size. To determine whether inflammation plays a role in reducing insulin signalling and the other changes associated with LPS administration, etanercept, a TNF antagonist, was administered on GDs 13.5 and 15.5 prior to LPS injections. With the exception of IRS-1 expression, in rats treated with etanercept all of the measured parameters remained at the levels observed in saline controls, indicating a link between abnormal inflammation and insulin resistance. The results of this study support the practice of monitoring the inflammatory conditions of the mother prior to and during pregnancy, and support further investigation into the potential use of anti-inflammatory agents during pregnancy in women at risk of insulin resistance and gestational diabetes.

Biological effects of insulin on murine melanoma cells and fish erythrophoroma cells: A comparative study

Insulin is the hormone that plays an essential role in metabolism and mitosis of normal and tumor cells, exerting its pleiotropic effects through binding to specific membrane receptors and promoting the phosphorylation of tyrosine residues of the receptor itself and of other components of the signaling pathway. The aim of this study was to investigate the effects of insulin on melanogenesis and cell growth in three different cell lines: the goldfish GEM-81 erythrophoroma cells (undifferentiated and differentiated with 1.5% dimethylsulfoxide-DMSO), and the murine B16F10 and Cloudman S91 melanoma cells. Undifferentiated GEM-81 and B16F10 cells responded to insulin with a small increase of cell proliferation, whereas S91 cells responded with a decrease of growth. In the two mammalian cell lines, and in DMSO-differentiated GEM-81 cells, the hormone strongly inhibited melanogenesis, by decreasing tyrosinase activity. In undifferentiated GEM-81 cells, insulin had no effect on tyrosinase activity. An increase in the tyrosine phosphorylation status of pp 185 (insulin receptor substrate 1 and 2-IRS-1/2) phosphorylation degree was observed in S91 mouse melanoma and in differentiated GEM-81 erythrophoroma cells, suggesting that this specific protein was maintained during transformation process and participates in insulin signaling. Our results imply an ancient and diverse history of the insulin signaling system in vertebrate pigment cells. (C) 2008 Elsevier Inc. All rights reserved.

Protein deficiency and nutritional recovery modulate insulin secretion and the early steps of insulin action in rats

Maternal malnutrition was shown to affect early growth and leads to permanent alterations in insulin secretion and sensitivity of offspring. In addition, epidemiological studies showed an association between low birth weight and glucose intolerance in adult life. To understand these interactions better, we investigated the insulin secretion by isolated islets and the early events related to insulin action in the hind-limb muscle of adult rats fed a diet of 17% protein (control) or 6% protein [low (LP) protein] during fetal life, suckling and after weaning, and in rats receiving 6% protein during fetal life and suckling followed by a 17% protein diet after weaning (recovered). The basal and maximal insulin secretion by islets from rats fed LP diet and the basal release by islets from recovered rats were significantly lower than that of control rats. The dose-response curves to glucose of islets from LP and recovered groups were shifted to the right compared to control islets, with the half-maximal response (EC 50) occurring at 16.9 ± 1.3, 12.4 ± 0.5 and 8.4 ± 0.1 mmol/L, respectively. The levels of insulin receptor, as well as insulin receptor substrate-1 and phosphorylation and the association between insulin receptor substrate-1 and phosphatidylinositol 3-kinase were greater in rats fed a LP diet than in control rats. In recovered rats, these variables were not significantly different from those of the other two groups. These results suggest that glucose homeostasis is maintained in LP and recovered rats by an increased sensitivity to insulin as a result of alterations in the early steps of the insulin signal transduction pathway.

Low ethanol consumption induces enhancement of insulin sensitivity in liver of normal rats

Moderate amounts of alcohol intake have been reported to have a protective effect on the cardiovascular system and this may involve enhanced insulin sensitivity. We established an animal model of increased insulin sensitivity by low ethanol consumption and here we investigated metabolic parameters and molecular mechanisms potentially involved in this phenomenon. For that, Wistar rats have received drinking water either without (control) or with 3% ethanol for four weeks. The effect of ethanol intake on insulin sensitivity was analyzed by insulin resistance index (HOMA-IR), intravenous insulin tolerance test (IVITT) and lipid profile. The role of liver was investigated by the analysis of insulin signaling pathway, GLUT2 gene expression and tissue glycogen content. Rats consuming 3% ethanol showed lower values of HOMA-IR and plasma free fatty acids (FFA) levels and higher hepatic glycogen content and glucose disappearance constant during the IVITT. Neither the phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1), nor its association with phosphatidylinositol-3-kinase (PI3-kinase), was affected by ethanol. However, ethanol consumption enhanced liver IRS-2 and protein kinase B (Akt) phosphorylation (3 times, P < 0.05), which can be involved in the 2-fold increased (P < 0.05) hepatic glycogen content. The GLUT2 protein content was unchanged. Our findings point out that liver plays a role in enhanced insulin sensitivity induced by low ethanol consumption. © 2005 Elsevier Inc. All rights reserved.

Acute exercise decreases PTP-1B protein level and improves insulin signaling in the liver of old rats

It is now commonly accepted that chronic inflammation associated with obesity during aging induces insulin resistance in the liver. In the present study, we investigated whether the improvement in insulin sensitivity and insulin signaling, mediated by acute exercise, could be associated with modulation of protein-tyrosine phosphatase 1B (PTP-1B) in the liver of old rats. Aging rats were subjected to swimming for two 1.5-h long bouts, separated by a 45 min rest period. Sixteen hours after the exercise, the rats were sacrificed and proteins from the insulin signaling pathway were analyzed by immunoblotting. Our results show that the fat mass was increased in old rats. The reduction in glucose disappearance rate (Kitt) observed in aged rats was restored 16 h after exercise. Aging increased the content of PTP-1B and attenuated insulin signaling in the liver of rats, a phenomenon that was reversed by exercise. Aging rats also increased the IRβ/PTP-1B and IRS-1/PTP-1B association in the liver when compared with young rats. Conversely, in the liver of exercised old rats, IRβ/PTP-1B and IRS-1/PTP-1B association was markedly decreased. Moreover, in the hepatic tissue of old rats, the insulin signalling was decreased and PEPCK and G6Pase levels were increased when compared with young rats. Interestingly, 16 h after acute exercise, the PEPCK and G6Pase protein level were decreased in the old exercised group. These results provide new insights into the mechanisms by which exercise restores insulin signalling in liver during aging. © 2013 Moura et al; licensee BioMed Central Ltd.

Resveratrol reduces chronic inflammation and improves insulin action in the myocardium of high-fat diet-induced obeserats

OBJETIVO: Avaliar o efeito do resveratrol sobre a via de sinalização da insulina e melhora do quadro inflamatório no miocárdio de ratos Wistar obesos induzidos por dieta.MÉTODOS: Ratos Wistar foram divididos em grupos: controle (dieta padrão para roedores), obeso (dieta hiperlipídica) e obeso suplementado com resveratrol (20 mg/kg/dia), por 8 semanas (n=10). Ao final do período experimental, realizou-se o teste de tolerância à insulina, nos tempos 0 (sem insulina), 5, 10, 15, 20, 25 e 30 minutos após injeção intraperitoneal de insulina (2 U/kg). O peso corporal e o tecido adiposo epididimal foram mensurados. Fragmentos do miocárdio foram extraídos para análises da via da insulina e moléculas pró-inflamatórias através de Western blot.RESULTADOS: Os resultados indicam que a intervenção com resveratrol aumenta a constante de decaimento da glicose, fosforilação do receptor de insulina, substrato do receptor de insulina e da proteína quinase B. A suplementação de resveratrol também reduziu os níveis proteicos do fator de necrose tumoral alfa e de moléculas envolvidas com a transdução do sinal pró-inflamatório (quinase indutora do kappa B e fator nuclear kappa B). Os resultados ainda sugerem que a melhora na sensibilidade à insulina e a redução das moléculas pró-inflamatórias ocorreram independentemente da perda de peso corporal e da redução do tecido adiposo epididimal.CONCLUSÃO: A suplementação de resveratrol aumenta a sensibilidade à insulina, o que está relacionado à redução de fatores inflamatórios no miocárdio.

Periapical lesions decrease insulin signaling in rat skeletal muscle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metabolic Disorders and Adipose Tissue Insulin Responsiveness in Neonatally STZ-Induced Diabetic Rats Are Improved by Long-Term Melatonin Treatment

Diabetes mellitus is a product of low insulin sensibility and pancreatic beta-cell insufficiency. Rats with streptozotocin-induced diabetes during the neonatal period by the fifth day of age develop the classic diabetic picture of hyperglycemia, hypoinsulinemia, polyuria, and polydipsia aggravated by insulin resistance in adulthood. In this study, we investigated whether the effect of long-term treatment with melatonin can improve insulin resistance and other metabolic disorders in these animals. At the fourth week of age, diabetic animals started an 8-wk treatment with melatonin (1 mg/kg body weight) in the drinking water at night. Animals were then killing, and the sc, epididymal (EP), and retroperitoneal (RP) fat pads were excised, weighed, and processed for adipocyte isolation for morphometric analysis as well as for measuring glucose uptake, oxidation, and incorporation of glucose into lipids. Blood samples were collected for biochemical assays. Melatonin treatment reduced hyperglycemia, polydipsia, and polyphagia as well as improved insulin resistance as demonstrated by constant glucose disappearance rate and homeostasis model of assessment-insulin resistance. However, melatonin treatment was unable to recover body weight deficiency, fat mass, and adipocyte size of diabetic animals. Adiponectin and fructosamine levels were completely recovered by melatonin, whereas neither plasma insulin level nor insulin secretion capacity was improved in diabetic animals. Furthermore, melatonin caused a marked delay in the sexual development, leaving genital structures smaller than those of nontreated diabetic animals. Melatonin treatment improved the responsiveness of adipocytes to insulin in diabetic animals measured by tests of glucose uptake (sc, EP, and RP), glucose oxidation, and incorporation of glucose into lipids (EP and RP), an effect that seems partially related to an increased expression of insulin receptor substrate 1, acetyl-coenzyme A carboxylase and fatty acid synthase. In conclusion, melatonin treatment was capable of ameliorating the metabolic abnormalities in this particular diabetes model, including insulin resistance and promoting a better long-term glycemic control. (Endocrinology 153: 2178-2188, 2012)

Gene expression of tumour necrosis factor and insulin signalling-related factors in subcutaneous adipose tissue during the dry period and in early lactation in dairy cows

Gene expression of adipose factors, which may be part of the mechanisms that underlie insulin sensitivity, were studied in dairy cows around parturition. Subcutaneous fat biopsies and blood samples were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. In the adipose tissue samples, mRNA was quantified by real-time reverse transcription polymerase chain reaction for tumour necrosis factor alpha (TNFalpha), insulin-independent glucose transporter (GLUT1), insulin-responsive glucose transporter (GLUT4), insulin receptor, insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), regulatory subunit of phosphatidylinositol-3 kinase (p85) and catalytic subunit of phosphatidylinositol-3 kinase. Blood plasma was assayed for concentrations of glucose, beta-hydroxybutyric acid, non-esterified fatty acids (NEFA) and insulin. Plasma parameters followed a pattern typically observed in dairy cows. Gene expression changes were observed, but there were no changes in TNFalpha concentrations, which may indicate its local involvement in catabolic adaptation of adipose tissue. Changes in GLUT4 and GLUT1 mRNA abundance may reflect their involvement in reduced insulin sensitivity and in sparing glucose for milk synthesis in early lactation. Unchanged gene expression of IRS1, IRS2 and p85 over time may imply a lack of their involvement in terms of insulin sensitivity dynamics. Alternatively, it may indicate that post-transcriptional modifications of these factors came into play and may have concealed an involvement.