Expression of IRS2 in the female reproductive system during the estrous cycle in mice.

Insulin receptor substrate 2 (IRS2) participates in reproduction; however, the location and expression of IRS2 in the reproductive system of female mice is not clear. We used real-time quantitative polymerase chain reaction (RT-PCR), western blot and immunohistochemical staining to investigate the expression of IRS2 in the ovary, oviduct and uterus of female mice during the estrous cycle. We found that IRS2 was expressed in all reproductive organs of mouse and that the expression level changed with the estrous phases. The expression of IRS2 in reproductive organs was greatest during estrus.

El tratamiento con sofosbuvir mejora la resistencia a la insulina Inducida por el virus de la hepatitis C / Sofosbuvir treatment improves hepatitis C virus-induced insulin resistance

La infección crónica por el virus de la hepatitis C (VHC) está asociada con la resistencia a la insulina y la diabetes tipo 2. El objetivo general de este estudio fue evaluar los efectos del sofosbuvir sobre la resistencia a la insulina inducida por el VHC. Para ello, se realizó un estudio clínico que incluyó 42 pacientes con VHC resistentes a la insulina, que fueron tratados con diferentes regímenes antivirales basados en sofosbuvir. Asimismo, se utilizó una línea de hepatocitos humanos que expresan un replicón del VHC de manera estable para determinar los mecanismos moleculares implicados en la acción de la insulina regulada por sofosbuvir. Todos los pacientes alcanzaron una respuesta virológica sostenida después del tratamiento con sofosbuvir y se observó una reducción significativa en los marcadores de daño hepático, así como en el estadio de fibrosis. El índice de resistencia a la insulina (HOMA) mejoró significativamente a lo largo del estudio. A nivel molecular, el tratamiento con sofosbuvir mejoró la activación de la cascada de señalización de la insulina tras la estimulación con dicha hormona en los hepatocitos con VHC, y, en consecuencia, revirtió la expresión elevada de genes gluconeogénicos, el aumento de la producción de glucosa y la deficiencia de la síntesis de glucógeno en estas células. En conclusión, estos resultados sugieren que el sofosbuvir mejora la respuesta deficiente a la insulina originada por la infección del VHC, proporcionando novedosas evidencias en cuanto a los mecanismos moleculares implicados en la sensibilización a la insulina inducida por este tratamiento

Chronic hepatitis C virus (HCV) infection is associated with insulin resistance and type 2 diabetes. The overall aim of this study was to evaluate the effects of sofosbuvir (SOF) on HCV-induced insulin resistance. Clinical parameters were recorded and insulin resistance index (HOMA) calculated from 42 insulin-resistant HCV-patients who underwent SOF-based regimens, at baseline, at the end of treatment (EoT), and at one year after the EoT. Likewise, Huh7 cells expressing full-length HCV replicons were used to elucidate the molecular mechanisms involved in insulin action regulated by SOF. All patients reached a sustained virological response after SOF treatment and, as expected, a significant reduction in liver damage markers and fibrosis stage was observed at the EoT that remained one year later. HOMA significantly improved throughout the study time period. Besides, an increase of total cholesterol, low-density lipoprotein cholesterol and apolipoprotein B levels were maintained over time after the EoT. At the molecular level, SOF treatment improved the activation of the insulin signalling cascade after stimulation with the hormone in HCV-hepatocytes and, accordingly, reversed the elevated expression of gluconeogenic genes, the increased glucose production and the impairment of glycogen synthesis induced by HCV. Furthermore, SOF challenge induced an increase of insulin receptor substrate 1 (IRS1) content parallel to a reduction in its serine phosphorylation in HCV-hepatocytes. These results provide novel evidence about the molecular mechanisms involved in the hepatic insulin sensitization induced by SOF treatment involving the recovery of IRS1 protein levels as a hallmark of SOF effects

Night-Shift Work Duration and Risk of Colorectal Cancer According to IRS1 and IRS2 Expression.

BACKGROUND: We hypothesized that the risk of colorectal cancer in night-shift workers might be different according to insulin receptor substrate status. METHODS: Among 77,470 eligible women having night work assessed in the Nurses' Health Study, we documented a total of 1,397 colorectal cancer cases, of which 304 or 308 had available data on IRS1 and IRS2, respectively. We used duplication-method Cox proportional hazards regression analysis for competing risks to calculate HRs and 95% confidence intervals (CI) for each colorectal cancer subtype. We measured tumor IRS1 or IRS2 expression by immunohistochemistry (IHC). RESULTS: Compared with women who never worked night shifts, those working ≥15 years night shifts had a marginal trend of increased overall risk of colorectal cancer (P trend = 0.06; multivariable HR = 1.20; 95% CI, 0.99-1.45). Longer duration of night-shift work was associated with a higher risk of IRS2-positive tumors (multivariable HR = 2.69; 95% CI, 1.48-4.89; P trend = 0.001, ≥15 years night shifts vs. never) but not with IRS2-negative tumors (multivariable HR = 0.90; 95% CI, 0.54-1.51; P trend = 0.72; P heterogeneity for IRS2 = 0.008). Similarly, the corresponding multivariable HRs were 1.81 for IRS1-positive tumors (95% CI, 0.94-3.48; P trend = 0.06) and 1.13 for IRS1-negative tumors (95% CI, 0.71-1.80; P trend = 0.56; P heterogeneity for IRS1 = 0.02). CONCLUSIONS: Our molecular pathologic epidemiology data suggest a potential role of IRS in mediating carcinogenesis induced by night-shift work. IMPACT: Although these findings need validation, rotating night shift might increase colorectal cancer risk in women with abnormal insulin receptor pathways.

Roles of Autophagy and Protein Kinase C-epsilon in Lipid Metabolism of Nonalcoholic Fatty Liver Cell Models.

OBJECTIVE: To investigate interaction between autophagy and PKC-ε in lipid metabolism of NAFLD cell models. METHODS: HL-7702 cells and SK-HEP-1 cells were cultured in vitro as NAFLD cell models and treated with RAPA to induce autophagy. 3-MA was used to inhibit cell autophagy. And HL-7702 and SK-HEP-1 cell were ordinary cultured as control groups. Cell viability was determined by MTT colorimetric assay. The levels of TG, TC and PKC-ε were detected by ELISA. PKC-ε was detected by IF. LC3-II/LC3-I, P62, IRS-1, IRS-2, PI3Kp85, mTOR were detected by Western-blot. SPSS 20 software was used for statistical analysis. RESULTS: The values of PKC-ε were the highest in the steatosis groups (HL-7702 cells were 91.10%, SK-HEP-1 cells were 98.20%). Compared with the steatosis groups, the LC3-II/LC3-I ratio in the induced autophagy groups increased obviously (p <0.05). P62/ß-actin grayscale ratio of the induced autophagy groups decreased significantly compared with the steatosis group (p <0.05). MTOR/ß-actin grayscale ratio in the induced autophagy groups were significantly lower than those in the steatosis groups (p <0.05). PI3Kp85, IRS-1 and IRS-2/ß-actin grayscale ratio of the induced autophagy groups increased significantly compared with the steatosis group (p <0.05). CONCLUSION: Up-regulation of autophagy can promote the elimination of liver fat; while down-regulation can promote lipid accumulation. The expression of PKC-ε is positively related to the degree of hepatic steatosis. PI3K was involved in both autophagy and IR induced by PKC-ε. PKC-ε might participate in hepatocyte autophagy by regulating PI3K.

Selective insulin resistance with differential expressions of IRS-1 and IRS-2 in human NAFLD livers.

BACKGROUND/OBJECTIVE: Insulin signals, via the regulation of key enzyme expression, both suppress gluconeogenesis and enhance lipid synthesis in the liver. Animal studies have revealed insulin signaling favoring gluconeogenesis suppression to be selectively impaired in steatotic livers. However, whether, and if so how, such selective insulin resistance occurs in human steatotic livers remains unknown. Our aim was to investigate selective insulin resistance in human livers with non-alcoholic fatty liver disease (NAFLD). SUBJECTS/METHODS: We examined mRNA expressions of key molecules for insulin signaling, gluconeogenesis and lipogenesis in human liver biopsy samples obtained from 51 non-diabetic subjects: 9 healthy controls and 42 NAFLD patients, and analyzed associations of these molecules with each other and with detailed pathological and clinical biochemistry data. RESULTS: In NAFLD patients, insulin receptor substrate (IRS)-2 expression was decreased, while those of key enzymes for gluconeogenesis were increased. These alterations of IRS-2 and gluconeogenesis enzymes were induced both in simple steatosis (SS) and non-alcoholic steatohepatitis (NASH), while these expression levels did not differ between SS and NASH. Furthermore, alterations in the expressions of IRS-2 and gluconeogenesis enzymes showed strong negative correlations and were concurrently induced in the early histological stage of NAFLD. In contrast, fatty acid synthase (FAS) expression was not decreased in NAFLD, despite IRS-2 downregulation, but correlated strongly with IRS-1 expression. Furthermore, no histological scores were associated with these molecules. Thus, IRS-1 signaling, which is not impaired in NAFLD, appears to modulate FAS expression. CONCLUSION: These analyses revealed that selective insulin resistance is present in human NAFLD livers and occurs in its early phases. The effect of insulin, during the IRS step, on gene expressions for lipogenesis and gluconeogenesis are apparently distinct and preferential downregulation of IRS-2 may contribute to selective resistance to the suppressive effects of insulin on gluconeogenesis.

Expressions of IGF-1, ERK, GLUT4, IRS-1 in metabolic syndrome complicated with colorectal cancer and their associations with the clinical characteristics of CRC.

BACKGROUND: Epidemiological data have revealed that colorectal cancer (CRC) risk is increased in patients with Metabolic syndrome. OBJECTIVE: To explore the expressions of IGF-1, ERK, GLUT4, IRS-1 in MS patients with CRC and their associations with the clinical characteristics of CRC. METHODS: We investigated the expressions of IGF-1, ERK, GLUT4 and IRS-1 in greater omental adipose tissues of 168 MS patients with/without CRC, 85 CRC patients without MS and 98 healthy controls by RT-PCR, and analyzed the relationships between their expressions and clinical characteristics of CRC. RESULTS: The expression levels of IGF-1 and ERK in MS patients with/without CRC were higher while the expression levels of GLUT4 were lower compared with CRC patients without MS and healthy controls (P< 0.01). The expression levels of IGF-1 and ERK in MS patients with CRC were higher while expression levels of GLUT4 were lower compared to MS patients without CRC (P< 0.01). Expression levels of ERK, IGF-1, GLUT4 were associated with clinical characteristics of CRC, including tumor size, distant metastasis and advanced stages (III/IV) (P< 0.05). CONCLUSIONS: Expressions of IGF-1, ERK and GLUT4 in greater omental adipose tissues might be useful biomarkers and predictive targets in the diagnosis of CRC.

Elevated expression of IRS-1 associates with phosphorylated Akt expression and predicts poor prognosis of breast invasive ductal carcinoma.

Overexpression of insulin receptor substrate 1 (IRS-1) has been reported to promote cell growth, atypical hyperplasia, and carcinogenesis, and phosphorylated Akt (p-Akt) is certified to be involved in many types of cancers such as breast invasive ductal carcinoma (BIDC). However, the relationship between IRS-1 and Akt, as well as the role of expression of IRS-1 in BIDC, has never been reported. The purpose of this research is to investigate the association between expression of IRS-1 and p-Akt proteins and clinicopathological features of BIDC by immunohistochemistry, as well as the survival status. The results showed that the percentage of either elevated expression of IRS-1 or positive p-Akt expression in BIDC was significantly higher than that in control breast tissue from noncancer patients (P < .001 and P = .001, respectively). Overexpression of IRS-1 was evidently associated with positive expression of p-Akt (r = 0.337, P < .001). Also, positive percentage of p-Akt expression was statistically different among different molecular subtypes of BIDC (highest in luminal B BIDC, P = .009). Furthermore, significantly worse overall survival was found in BIDC patients with high expression of IRS-1 and p-Akt than in patients with low expression (P = .006 and P = .004, respectively). The multivariate Cox proportional hazard regression analysis showed that high expression of IRS-1 and positive expression of p-Akt protein were independent poor prognostic factors for patients with BIDC (P = .022 and P = .046, respectively). In conclusion, we report for the first time that overexpression of IRS-1 protein is associated with expression of p-Akt, and overexpression of IRS-1 and positive expression of p-Akt might be independent biomarkers for poor prognosis in BIDC.

Overexpression of insulin receptor substrate-4 is correlated with clinical staging in colorectal cancer patients.

Insulin receptor substrate-4 (IRS-4), a poorly studied member of the IRS family, may play an important role in colorectal cancer (CRC) tumourigenesis. The aim of this pilot study was to elucidate the potential role of IRS-4 in colorectal carcinogenesis by evaluating IRS-4 expression in different types of colorectal tumours (n = 20) and comparing its expression to normal mucosa (n = 20). Tissue samples were collected from 18 patients with CRC and 2 with precancerous lesions (tubulovillous adenomas), all of whom were undergoing potentially curative surgery. IRS-4 expression was evaluated using immunohistochemical staining and compared to clinicopathological features. In normal colonic crypts, the subcellular localization of IRS-4 varied from the crypt base compartment to the surface epithelium. Nuclear IRS-4 staining decreased while non-nuclear IRS-4 increased as cells approached the top of the crypt. In the patients studied, colorectal tumours showed a significant (p < 0.001) increase of IRS-4 expression compared with adjacent normal tissue. Furthermore, nuclear IRS-4 intensity of CRC patients was significantly (p < 0.0001) higher in colonic tumoural tissue than in paired normal specimens. Tumour expression of IRS-4 in CRC patients was positively associated with T (p < 0.0001) and N (p < 0.05), of TNM (tumour and nodes and metastasis) staging system. Taken together, these results suggest that increase of IRS-4 expression may be involved to some extent in colorectal cancer.

High-fructose corn syrup-induced hepatic dysfunction in rats: improving effect of resveratrol.

PURPOSE: The increased consumption of high-fructose corn syrup (HFCS) may contribute to the worldwide epidemic of fatty liver. In this study, we have investigated whether HFCS intake (20% beverages) influences lipid synthesis and accumulation in conjunction with insulin receptor substrate-1/2 (IRS-1; IRS-2), endothelial nitric oxide synthase (eNOS), sirtuin 1 (SIRT1) and inducible NOS (iNOS) expressions in liver of rats. Resveratrol was tested for its potential efficacy on changes induced by HFCS. METHODS: Animals were randomly divided into four groups as control, resveratrol, HFCS and resveratrol plus HFCS (resveratrol + HFCS). HFCS was given as 20% solutions in drinking water. Feeding of all rats was maintained by a standard diet that enriched with or without resveratrol for 12 weeks. RESULTS: Dietary HFCS increased triglyceride content and caused mild microvesicular steatosis in association with up-regulation of fatty acid synthase and sterol regulatory element binding protein (SREBP)-1c in liver of rats. Moreover, HFCS feeding impaired hepatic expression levels of IRS-1, eNOS and SIRT1 mRNA/proteins, but did not change iNOS level. Resveratrol promoted IRS, eNOS and SIRT1, whereas suppressed SREBP-1c expression in rats fed with HFCS. CONCLUSIONS: Resveratrol supplementation considerably restored hepatic changes induced by HFCS. The improvement of hepatic insulin signaling and activation of SIRT1 by resveratrol may be associated with decreased triglyceride content and expression levels of the lipogenic genes of the liver.

Increased expression of IRS-1 is associated with lymph node metastasis in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a head and neck malignant tumor rare throughout most of the world but common in Southeast Asia, especially in Southern China, which is with characteristics of early cervical lymph node metastasis and high incidence rate of distant metastasis. Insulin receptor substrate 1 (IRS-1) is a signaling adapter protein that is encoded by the IRS-1 gene in humans, plays an important role in the development, progression, invasion and metastasis of tumors. The aim of the present study was to investigate the association between the expression of IRS-1 protein and clinicopathological characteristics in NPC by immunohistochemistry. The results showed that the expression level of IRS-1 was significant higher in NPC than that in the control nasopharyngeal epithelia (P = 0.042). The positive percentage of IRS-1 expression in NPC with lymph node metastasis was also significantly higher than those without lymph node metastasis (P = 0.008). Positive expression of IRS-1 was proved to be the independent predicted factor for lymph node metastasis of NPC (P = 0.025) regardless of age, gender, histological type and clinical stages by multivariate logistic regression analysis. In addition, results showed higher sensitivity and agreement rate of IRS-1 for predicting lymph node metastasis of NPC patients. Taken together, high expression of IRS-1 might be closely correlated with lymph node metastasis in NPC and positive expression of IRS-1 could be used as an independent biomarker for predicting lymph node metastasis of NPC.

Altered gene expression patterns during the initiation and promotion stages of neonatally diethylstilbestrol-induced hyperplasia/dysplasia/neoplasia in the hamster uterus.

Neonatal treatment of hamsters with diethylstilbestrol (DES) induces uterine hyperplasia/dysplasia/neoplasia (endometrial adenocarcinoma) in adult animals. We subsequently determined that the neonatal DES exposure event directly and permanently disrupts the developing hamster uterus (initiation stage) so that it responds abnormally when it is stimulated with estrogen in adulthood (promotion stage). To identify candidate molecular elements involved in progression of the disruption/neoplastic process, we performed: (1) immunoblot analyses and (2) microarray profiling (Affymetrix Gene Chip System) on sets of uterine protein and RNA extracts, respectively, and (3) immunohistochemical analysis on uterine sections; all from both initiation stage and promotion stage groups of animals. Here we report that: (1) progression of the neonatal DES-induced hyperplasia/dysplasia/neoplasia phenomenon in the hamster uterus involves a wide spectrum of specific gene expression alterations and (2) the gene products involved and their manner of altered expression differ dramatically during the initiation vs. promotion stages of the phenomenon.

Beneficial effects of Ginkgo biloba extract on insulin signaling cascade, dyslipidemia, and body adiposity of diet-induced obese rats

Ginkgo biloba extract (GbE) has been indicated as an efficient medicine for the treatment of diabetes mellitus type 2. It remains unclear if its effects are due to an improvement of the insulin signaling cascade, especially in obese subjects. The aim of the present study was to evaluate the effect of GbE on insulin tolerance, food intake, body adiposity, lipid profile, fasting insulin, and muscle levels of insulin receptor substrate 1 (IRS-1), protein tyrosine phosphatase 1B (PTP-1B), and protein kinase B (Akt), as well as Akt phosphorylation, in diet-induced obese rats. Rats were fed with a high-fat diet (HFD) or a normal fat diet (NFD) for 8 weeks. After that, the HFD group was divided into two groups: rats gavaged with a saline vehicle (HFD+V), and rats gavaged with 500 mg/kg of GbE diluted in the saline vehicle (HFD+Gb). NFD rats were gavaged with the saline vehicle only. At the end of the treatment, the rats were anesthetized, insulin was injected into the portal vein, and after 90s, the gastrocnemius muscle was removed. The quantification of IRS-1, Akt, and Akt phosphorylation was performed using Western blotting. Serum levels of fasting insulin and glucose, triacylglycerols and total cholesterol, and LDL and HDL fractions were measured. An insulin tolerance test was also performed. Ingestion of a hyperlipidic diet promoted loss of insulin sensitivity and also resulted in a significant increase in body adiposity, plasma triacylglycerol, and glucose levels. In addition, GbE treatment significantly reduced food intake and body adiposity while it protected against hyperglycemia and dyslipidemia in diet-induced obesity rats. It also enhanced insulin sensitivity in comparison to HFD+V rats, while it restored insulin-induced Akt phosphorylation, increased IRS-1, and reduced PTP-1B levels in gastrocnemius muscle. The present findings suggest that G. biloba might be efficient in preventing and treating obesity-induced insulin signaling impairment.

Beneficial effects of Ginkgo biloba extract on insulin signaling cascade, dyslipidemia, and body adiposity of diet-induced obese rats.

Ginkgo biloba extract (GbE) has been indicated as an efficient medicine for the treatment of diabetes mellitus type 2. It remains unclear if its effects are due to an improvement of the insulin signaling cascade, especially in obese subjects. The aim of the present study was to evaluate the effect of GbE on insulin tolerance, food intake, body adiposity, lipid profile, fasting insulin, and muscle levels of insulin receptor substrate 1 (IRS-1), protein tyrosine phosphatase 1B (PTP-1B), and protein kinase B (Akt), as well as Akt phosphorylation, in diet-induced obese rats. Rats were fed with a high-fat diet (HFD) or a normal fat diet (NFD) for 8 weeks. After that, the HFD group was divided into two groups: rats gavaged with a saline vehicle (HFD+V), and rats gavaged with 500 mg/kg of GbE diluted in the saline vehicle (HFD+Gb). NFD rats were gavaged with the saline vehicle only. At the end of the treatment, the rats were anesthetized, insulin was injected into the portal vein, and after 90s, the gastrocnemius muscle was removed. The quantification of IRS-1, Akt, and Akt phosphorylation was performed using Western blotting. Serum levels of fasting insulin and glucose, triacylglycerols and total cholesterol, and LDL and HDL fractions were measured. An insulin tolerance test was also performed. Ingestion of a hyperlipidic diet promoted loss of insulin sensitivity and also resulted in a significant increase in body adiposity, plasma triacylglycerol, and glucose levels. In addition, GbE treatment significantly reduced food intake and body adiposity while it protected against hyperglycemia and dyslipidemia in diet-induced obesity rats. It also enhanced insulin sensitivity in comparison to HFD+V rats, while it restored insulin-induced Akt phosphorylation, increased IRS-1, and reduced PTP-1B levels in gastrocnemius muscle. The present findings suggest that G. biloba might be efficient in preventing and treating obesity-induced insulin signaling impairment.

Obesity-associated Gingival Vascular Inflammation and Insulin Resistance.

Obesity is a risk factor for periodontitis, but the pathogenic mechanism involved is unclear. We studied the effects of insulin in periodontal tissues during the state of obesity-induced insulin resistance. Gingival samples were collected from fatty (ZF) and lean (ZL, control) Zucker rats. Endothelial nitric oxide synthase (eNOS) expression was decreased, and activities of protein kinase C (PKC) α, ß2, δ, and ϵ isoforms were significantly increased in the gingiva from ZF rats compared with those from ZL rats. Expression of oxidative stress markers (mRNA) and the p65 subunit of NF-κB was significantly increased in ZF rats. Immunohistochemistry revealed that NF-κB activation was also increased in the gingival endothelial cells from transgenic mice overexpressing NF-κB-dependent enhanced green fluorescent protein (GFP) and on a high-fat vs. normal chow diet. Analysis of the gingiva showed that insulin-induced phosphorylation of IRS-1, Akt, and eNOS was significantly decreased in ZF rats, but Erk1/2 activation was not affected. General PKC inhibitor and an anti-oxidant normalized the action of insulin on Akt and eNOS activation in the gingiva from ZF rats. This provided the first documentation of obesity-induced insulin resistance in the gingiva. Analysis of our data suggested that PKC activation and oxidative stress may selectively inhibit insulin-induced Akt and eNOS activation, causing endothelial dysfunction and inflammation.

Effect of IRS4 levels on PI 3-kinase signalling.

Insulin receptor substrate 1 (IRS1) and IRS2 are well-characterized adapter proteins that relay signals from receptor tyrosine kinases to downstream components of signalling pathways. In contrast, the function of IRS4 is not well understood. IRS4 overexpression has been associated with acute lymphoblastic leukaemia and subungual exostosis, while point mutations of IRS4 have been found in melanomas. Here, we show that while IRS4 expression is low in most cancer cell lines, IRS4 mRNA and protein levels are markedly elevated in certain cells including the NCI-H720, DMS114, HEK293T and HEK293AAV lines. Surprisingly, IRS4 expression was also strongly induced when HEK293 cells were infected with retroviral particles and selected under puromycin, making IRS4 expression a potential off-target effect of retroviral expression vectors. Cells with high expression of IRS4 displayed high phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, as well as elevated Akt and p70 S6 kinase activities, even in the absence of growth factors. PI 3-kinase (PI3K) signalling in these cells depends on IRS4, even though these cells also express IRS1/2. Knockdown of IRS4 also inhibited cell proliferation in cells with high levels of IRS4. Together, these findings suggest IRS4 as a potential therapeutic target for cancers with high expression of this protein.

Periapical lesions decrease insulin signal and cause insulin resistance.

INTRODUCTION: Inflammatory cytokines are associated with decreased insulin signal transduction. Moreover, local oral inflammation, such as that accompanying periodontal disease, is associated with insulin resistance and type 2 diabetes mellitus. The aim of this study was to evaluate the effect of periapical lesions (PLs) on insulin signaling and insulin sensitivity in rats. We hypothesized that PLs alter systemic insulin signaling and insulin sensitivity via elevated plasmatic tumor necrosis factor α (TNF-α). METHODS: Wistar rats were divided into control (CN) and PL groups. PLs were induced by exposing pulpal tissue to the oral environment. After 30 days, insulin sensitivity was measured using the insulin tolerance test. After euthanization, maxillae were processed for histopathology. Plasmatic concentrations of tumor necrosis factor α (TNF-α) were determined via the enzyme-linked immunosorbent assay. Insulin signal transduction was evaluated using insulin receptor substrate tyrosine phosphorylation status and serine phosphorylation status in periepididymal white adipose tissue via Western blotting. For insulin signaling and insulin tolerance tests, the analyses performed were analysis of variance followed by the Tukey post hoc test. For TNF-α analysis, the Student's t test was used. In all tests, P < .05 was considered significant. RESULTS: The rats with PLs showed higher plasmatic TNF-α, lower constant rate for glucose disappearance values, and reduced pp185 tyrosine phosphorylation status but no change in serine phosphorylation status in white adipose tissue after insulin stimulation. CONCLUSIONS: PLs can cause alterations to both insulin signaling and insulin sensitivity, probably because of elevation of plasmatic TNF-α. The results from this study emphasize the importance of the prevention of local inflammatory diseases, such as PLs, with regard to the prevention of insulin resistance.

Development of a protocol for selection of genes fit for the in vivo knockdown method and its application to insulin receptor substrate genes in mice.

Prediabetes model mice in which more than one gene associated with diabetes is knocked down simultaneously are potentially useful for pharmaceutical and medical studies of diabetes. However, the effective conditions for sufficient knockdown in vivo are dependent on the intrinsic properties of the target genes. It is necessary to investigate which genes are applicable or not to the in vivo knockdown method. In this study, insulin receptor substrate 1 and 2 (Irs-1, Irs-2) were selected as target genes. Effective siRNAs against the respective genes were designed, and their efficacy was confirmed by cell-based experiments. Based on the results of siRNAs, shRNA expression vectors against Irs-1 and Irs-2 were constructed, respectively. Their efficacy was also confirmed by cell-based experiments. A hydrodynamic method was applied to the delivery of the vectors to mice. This method was found to be effective for predominant delivery to the liver by demonstrative delivery of an EGFP expression vector and successive histochemical analysis. Fifty micrograms of the shRNA expression vector was injected into the tail vein. After 24 h, the liver, pancreas, and muscle were isolated, and the expression levels of Irs-1 and Irs-2 were analyzed by quantitative RT-PCR. In the liver, Irs-2 was effectively knocked down to 60% of the control level, but Irs-1 was not influenced even under the same conditions. The protocol developed here is feasible for the selection of genes fit for in vivo knockdown method.

Haloperidol and olanzapine mediate metabolic abnormalities through different molecular pathways.

The pathogenesis of antipsychotic-induced disturbances of glucose homeostasis is still unclear. Increased visceral adiposity has been suggested to be a possible mediating mechanism. The aim of this study was to investigate, in an animal model, the differential effects of olanzapine and haloperidol on visceral fat deposition (using magnetic resonance imaging(MRI)) and on critical nodes of the insulin signaling pathway (liver-protein levels of IRS2 (insulin receptor substrate 2), GSK3α (glycogen synthase kinase-3α), GSK3ß, GSK3α-Ser21, GSK3ß-Ser9). To this end, we studied male Sprague-Dawley rats treated with vehicle (n=8), haloperidol (2 mg kg(-1) per day, n=8), or olanzapine (10 mg kg(-1)per day, n=8), using osmotic minipumps, for 8 weeks. The haloperidol group showed a higher percentage of visceral fat than both the olanzapine group and the vehicle group, whereas there was no difference between the olanzapine and the vehicle group. In terms of insulin signaling pathway, the olanzapine group showed significantly reduced IRS2 levels, reduced phosphorylation of GSK3α and increased phosphorylation of GSK3ß, whereas there was no difference between the haloperidol and the vehicle group. Our data suggest that different molecular pathways mediate the disturbances of glucose homeostasis induced by haloperidol and olanzapine with a direct effect of olanzapine on the insulin molecular pathway, possibly partly explaining the stronger propensity of olanzapine for adverse effects on glucose regulation when compared with haloperidol in clinical settings.

Insulin receptor and IRS-1 co-immunoprecipitation with SOCS-3, and IKKα/ß phosphorylation are increased in obese Zucker rat skeletal muscle.

AIMS: We evaluated if selected pro-inflammatory cytokines and/or the protein suppressor of cytokine signaling 3 (SOCS-3) could account for decreased insulin-stimulated phosphatidylinositol 3-kinase (PI3-K) activity in the skeletal muscle of the obese Zucker rat. MAIN METHODS: Eight lean and eight obese Zucker rats ~4weeks of age were obtained and allowed to feed ad libitum for 4weeks before undergoing hind limb perfusion in the presence of 500µU/ml insulin. KEY FINDINGS: Insulin-stimulated skeletal muscle PI3-K activity and 3-O-methylglucose transport rates were reduced (P<0.05) in obese compared to lean animals. IRS-1 concentration remained unchanged although IRS-1 tyrosine phosphorylation was decreased (P<0.05), and IRS-1 serine phosphorylation (pS) was increased (P<0.05) in obese animals compared to lean animals. IKKα/ß pS and JNK theronine/tyrosine phosphorylation was increased (P<0.05) in the obese animals. IκBα concentration was decreased (P<0.05) and IκBα pS was increased (P<0.05) in the obese compared to lean Zucker animals. SOCS-3 concentration and SOCS-3 co-immunoprecipitation with both insulin receptor ß-subunit (IR-ß) and IRS-1 were elevated (P<0.05) in obese compared to lean animals. IRS-1 co-immunoprecipitation with IR-ß was reduced 56% in the obese animals. SIGNIFICANCE: Increased IKKα/ß and JNK serine phosphorylation may contribute to increasing IRS-1 serine phosphorylation, while concurrent co-localization of SOCS-3 with both IR-ß and IRS-1 may prevent IRS-1 from interacting with IR-ß. These two mechanisms thusly may independently contribute to impairing insulin-stimulated PI3-K activation in the skeletal muscle of the obese Zucker rat.

Long-term intermittent feeding, but not caloric restriction, leads to redox imbalance, insulin receptor nitration, and glucose intolerance.

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance.