Insulin Receptor Substrate Research Articles

Insulin receptor substrate 1(IRS1) is related with lymph node metastases and prognosis in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer-related mortality globally with a high risk of lymph node metastasis (LNM). In this study, weighted gene co-expression network analysis (WGCNA) showed the identification of 10 modules among which the significant module (turquoise), including 1352 genes, was correlated with LNM. A group 52 overlapping differentially expressed genes (DEGs) was identified based on the comparison of turquoise module with GSE23400 and GSE20347 datasets. Using Ctyohubba plugin, we identified 7 hub genes (ACTG2, SORBS1, MYH11, CXCL12, CNN1, IRS1 and CXCL8). IRS1 displayed significant correlation with metastasis. The decreased expression of IRS1 was also a predictor of poor OS of ESCC patients whereas the hub genes namely ACTG2, MYH11, CXCL8, CXCL12, IRS1 and CNN1 were associated with RFS of ESCC patients. These findings suggest that the altered expression of these hub genes are associated with prognosis and thus can be used as potential biomarkers for ESCC. Moreover, immunohistochemical staining and cell functional experiments displayed that the overexpression of IRS1 was negatively associated with metastasis in ESCC. In general, our research revealed several novel genes in ESCC especially the association of IRS1 with LNM in ESCC, which could provide novel insights into the initiation and progression of ESCC.

Molecular Mechanisms of Hawthorn Extracts in Multiple Organs Disorders in Underlying of Diabetes: A Review.

Diabetes mellitus (DM) is one of the most important metabolic disorders associated with chronic hyperglycemia and occurs when the body cannot manage insulin secretion, insulin action, or both. Autoimmune destruction of pancreatic beta cells and insulin resistance are the major pathophysiological factors of types 1 and 2 of DM, respectively. Prolonged hyperglycemia leads to multiple organs dysfunctions, including nephropathy, neuropathy, cardiomyopathy, gastropathy, and micro- and macrovascular disorders. The basis of the metabolic abnormalities in carbohydrate, fat, and protein in diabetes is insufficient action of insulin on various target tissues. Medicinal plants are rich sources of bioactive chemical compounds with therapeutic effects. The beneficial effects of leaves, fruits, and flowers extracts of Crataegus oxyacantha, commonly called hawthorn, belonging to the Rosaceae family, are widely used as hawthorn-derived medicines. Data in this review have been collected from the scientific articles published in databases such as Science Direct, Scopus, PubMed, Web of Science, and Scientific Information Database from 2000 to 2021. Based on this review, hawthorn extracts appear both therapeutic and protective effects against diabetic-related complications in various organs through molecular mechanisms, such as decreasing triglyceride, cholesterol, very low density lipoprotein and increasing the antioxidant activity of superoxide dismutase, catalase, glutathione peroxidase, total antioxidant capacity, decreasing malondialdehyde level, and attenuating tumor necrosis factor alpha, interleukin 6 and sirtuin 1/AMP-activated protein kinase (AMPK)/nuclear factor kappa B (NF-κB) pathway and increasing the phosphorylation of glucose transporter 4, insulin receptor substrate 1, AKT and phosphoinositide 3-kinases, and attenuating blood sugar and regulation of insulin secretion, insulin resistance, and improvement of histopathological changes in pancreatic beta cells. Collectively, hawthorn can be considered as one new target for the research and development of innovative drugs for the prevention or treatment of DM and related problems.

Normalization of maternal adiponectin in obese pregnant mice prevents programming of impaired glucose metabolism in adult offspring.

Infants born to obese mothers have a greater risk for childhood obesity and insulin resistance. However, the underlying biological mechanism remains elusive, which constitutes a significant roadblock for developing specific prevention strategies. Maternal adiponectin levels are lower in obese pregnant women, which is linked with increased placental nutrient transport and fetal overgrowth. We have previously reported that adiponectin supplementation to obese dams during the last four days of pregnancy prevented the development of obesity, glucose intolerance, muscle insulin resistance, and fatty liver in three months old offspring. In the present study, we tested the hypothesis that 6-9-month-old offspring of obese dams show glucose intolerance associated with muscle insulin resistance and mitochondrial dysfunction and that normalization of maternal adiponectin in obese pregnant mice prevents the development of this phenotype in the offspring. Male and female offspring of obese mice exhibited in vivo glucose intolerance and insulin resistance at 6 and 9months of age. In gastrocnemius muscles ex vivo, male and female offspring of obese dams showed reduced phosphorylation of insulin receptor substrate 1Tyr-608 , AktThr-308 , and decreased Glut4 plasma membrane translocation upon insulin stimulation. These metabolic abnormalities in offspring born to obese mice were largely prevented by normalization of maternal adiponectin levels in late pregnancy. We provide evidence that low circulating maternal adiponectin is a critical mechanistic link between maternal obesity and the development of metabolic disease in offspring. Strategies aimed at improving maternal adiponectin levels may prevent long-term metabolic dysfunction in offspring of obese mothers.

Participation of Magnesium in the Secretion and Signaling Pathways of Insulin: an Updated Review.

Several studies have demonstrated the participation of various minerals in mechanisms involving insulin. Magnesium, in particular, plays an important role in the secretion and action of this hormone. Therefore, this review aimed to examine the latest insights into the biochemical and molecular aspects of the participation of magnesium in insulin sensitivity. Magnesium plays a vital role in the activity of intracellular proteins involved in insulin secretion in β-pancreatic cells, such as glucokinase, ATPase, and protein kinase C. In addition, evidence suggests that this mineral participates directly in insulin sensitivity and signaling in peripheral tissues, acting in the phosphorylation of the receptor tyrosine kinase and the insulin receptor substrates 1, insulin receptor substrates 2, phosphatidylinositol 3-kinase, and protein kinase B, and indirectly by reducing oxidative stress and chronic low-grade inflammation, which also lead to insulin resistance. Thus, magnesium deficiency is associated with glucose intolerance, while magnesium supplementation stimulates insulin secretion in pancreatic cells and improves insulin sensitivity in peripheral tissues. However, studies must consider assess short- and long-term nutritional status of mineral before performing intervention, the relevance of the balance of other nutrients that influence hormone secretion and sensibility, and health status of the assessed population.

Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice.

Icariin, a major prenylated flavonoid found in Epimedium spp., is a bioactive constituent of Herba Epimedii and has been shown to exert neuroprotective effects in experimental models of Alzheimer's disease. In this study, we investigated the neuroprotective mechanism of icariin in an APP/PS1/Tau triple-transgenic mouse model of Alzheimer's disease. We performed behavioral tests, pathological examination, and western blot assay, and found that memory deficits of the model mice were obviously improved, neuronal and synaptic damage in the cerebral cortex was substantially mitigated, and amyloid-β accumulation and tau hyperphosphorylation were considerably reduced after 5 months of intragastric administration of icariin at a dose of 60 mg/kg body weight per day. Furthermore, deficits of proteins in the insulin signaling pathway and their phosphorylation levels were significantly reversed, including the insulin receptor, insulin receptor substrate 1, phosphatidylinositol-3-kinase, protein kinase B, and glycogen synthase kinase 3β, and the levels of glucose transporter 1 and 3 were markedly increased. These findings suggest that icariin can improve learning and memory impairments in the mouse model of Alzheimer's disease by regulating brain insulin signaling and glucose transporters, which lays the foundation for potential clinical application of icariin in the prevention and treatment of Alzheimer's disease.

The Release of Exosomes from Astrocytes in Response to Fatty Acid Alters the Metabolism of Proopiomelanocortin (POMC) Neurons

Exosomes are extracellular micro-vesicles with a complex content that include a vast heterogeneity of molecules such as growth factors, cytokines and RNAs, including micro-RNAs (mi-RNAs) that affect target cells. Neurons are influenced by neighboring astrocytes through the release of exosomes amongst other signals. We postulated that the metabolic status is communicated by hypothalamic astrocytes via exosomes to neighboring POMC neurons to modify their functions in the promotion of energy expenditure and satiety. With this aim, cultures of primary hypothalamic astrocytes were treated with both palmitic or oleic acid (PA or OA; 0.5 mM) for 24 hours. Exosomes were isolated and purified from the media culture and used to treat (1.25 or 2.50 µg/mL) a POMC neuronal cell line for 24 hours. Exosomes released in response to PA (E-PA) or (E-OA) did not change the expression of the markers of inflammation [interleukin (IL)-6] and Endoplasmic Reticle stress (CHOP) comparing to exosomes released in response to vehicle (E-V) or with no exosomes (control). Next-Generation miRNA sequencing analysis was performed to reveal modifications in miRNAs contained in exosomes. The content of miR-199a-3p and miR-145-5p was higher in E-PA compared to E-V. POMC neurons transfected with a mimetic of miR-199a-3p (1.5 pmol) increased insulin-like growth factor 1 receptor (IGF1r) protein levels (p < 0.05), as well as POMC expression (ns). Moreover, levels of mTOR and p70S6k, reported targets of miR-199a-3p, were decreased (p < 0.05, both). Mimetic overexpression of miR- 145-5p reduced POMC expression (p < 0.001) and protein levels of insulin receptor substrate 1 (IRS1; p < 0.001), which is a known target of this miRNA. To determine modifications in cellular metabolism in the POMC neurons in response to exosomal astrocytes, Seahorse Cell Mito Stress test was performed. The mitochondrial spare respiratory capacity of neurons was increased (p < 0.0001) in response to both doses (1.25 or 2.50 µg/mL) of E-PA and E-OA, with the maximal respiration (p < 0.0001) also increasing with E-PA or E-OA (both doses) compared to E-V or control. Our results suggest that astrocytes may communicate directly with neurons via exosomes, and that the exosomes content is modulated by the source cell in response to the nutritional environment. The messages contained in exosomes from astrocytes can directly modulate the levels of cellular receptors and factors involved in cell proliferation, protection, metabolism, and nutrient sensing. Specific miRNAs participate in these processes as well as on the neuropeptide expression in targeted neurons. In addition, cellular respiration is affected in POMC neurons treated with exosomes from astrocytes in response to fatty acid in a way that suggests that cells are preparing for a possible respiratory stress by increasing their spare respiratory capacity and maximal respiration.

Effect of Danlou Tablets on alleviating hepatic adipogenesis, inflammatory response, and insulin resistance in db/db mice

This study explored the effect and potential mechanism of Danlou Tablets(DLT) on insulin resistance in db/db mice with type 2 diabetic mellitus(T2 DM). The db/db male mice were randomly assigned into model control(MC) group, metformin(MET, tablet, 100 mg·kg~(-1)) group, and DLT(1 g·kg~(-1)) group, and C57 BL/6 J mice were taken as normal control(NC) group. The mice in the MET group and DLT group were given corresponding drugs by gavage once a day for 16 weeks. The fasting blood glucose, glucose tolerance, and insulin tolerance were measured to evaluate the effect of DLT on blood glucose and insulin resistance in diabetic mice. The serum free fatty acid, triacylglycerol, and total cholesterol levels were determined to evaluate the effect of DLT on blood lipids in diabetic mice. The liver index and perirenal fat index were calculated to measure the effect of DLT on lipid accumulation in non-adipose tissue and adipose tissue. Western blot was performed to determine the protein levels of insulin receptor-β(IRβ), phospho-IRβ(p-IRβ), phosphatidylinositol 3-kinase(PI3 K), and insulin receptor substrate-1(IRS-1) involved in IRS-1/PI3 K/Akt signaling pathway in the livers of mice to reveal the mechanism of DLT in alleviating insulin resistance in diabetic mice. The protein levels of sterol regulatory element binding protein-1(SREBP-1) and the mRNA levels of sterol regulatory element binding protein-1 c(SREBP-1 c), fatty acid synthase(FAS), acetyl-CoA carboxylase(ACC), diacylglycerol acyltransferase-1(Dgat1), and diacylglycerol acyltransferase-2(Dgat2) involved in the SREBP-1/FAS signaling pathway were detected to evaluate the effect of DLT on lipid metabolism in diabetic mice. Real-time quantitative PCR was employed to determine the mRNA levels of galectin-3(Gal-3), interleukin-6(IL-6), and monocyte chemoattractant protein-1(MCP-1) in mouse liver to evaluate the effect of DLT on inflammatory response in diabetic mice. The results showed that DLT significantly reduced the blood glucose and lipid levels and alleviated the insulin resistance in diabetic mice. Compared with the MC group, DLT significantly up-regulated the protein levels of p-IRβ, PI3 K, and IRS-1(P<0.05 or P<0.01), and down-regulated the protein level of SREBP-1 in liver tissues of diabetic mice(P<0.05). DLT lowered the mRNA levels of SREBP-1 c, FAS, ACC, Dgat1, and Dgat2 related to lipid metabolism as well as those of Gal-3, IL-6, and MCP-1 associated with inflammation in the livers of diabetic mice(P<0.05 or P<0.01). The findings of this study suggest that DLT may alleviate insulin resistance in diabetic mice by regulating IRS-1/PI3 K/Akt signaling pathway and SREBP-1/FAS signaling pathway to reduce lipid production and inhibit inflammatory response.

Changes in the Expression of Insulin Pathway, Neutrophil Elastase and Alpha 1 Antitrypsin Genes from Leukocytes of Young Individuals with Insulin Resistance.

BackgroundChronic hyperinsulinemia is a hallmark of insulin resistance that affects a diversity of cells, including leukocytes modifying the expression of some genes involved in insulin signaling.PurposeThe aim of this study was to evaluate how hyperinsulinemia affects the expression of genes involved in the proximal insulin signaling pathway in leukocytes from 45 young individuals grouped: normal weight with not insulin resistance (NIR), with insulin resistance (IR) and with obesity (OB-IR).MethodsqPCR was performed to analyze the expression of insulin receptor (INSR), insulin receptor substrate 1 and 2 (IRS-1 and IRS-2), neutrophil elastase (NE), alpha 1 antitrypsin (A1AT), glucose transporters 1, 3 and 4 (GLUT-1, GLUT-3 and GLUT-4) by the 2−ΔCt method, and the correlation between the genes was determined by Spearman’s test.ResultsThe mRNA expression analysis of all genes between NIR and IR individuals revealed no differences. However, when comparing NIR and IR individuals with OB-IR, an increase in NE and A1AT expression and a clear trend towards a decrease in IRS-2 expression was observed, whereas the comparison of IR and OB-IR showed a decrease in GLUT-3 expression. Overall, the correlation analysis showed that in the IR group there was a positive correlation only between NE with IRS-1 (r = 0.72, p = 0.003), while in the OB-IR group, there was a positive correlation between the NE and A1AT with INSR (r = 0.62, p = 0.01 and r = 0.74, p = 0.002, respectively) and with IRS-2 (r = 0.74, p = 0.002 and r = 0.76, p = 0.001, respectively).ConclusionThese results suggest that hyperinsulinemia and obesity are associated with changes in the expression of genes in leukocytes involved in the insulin pathway that are related to NE and A1AT.

Estrogen Protects Cardiac Function and Energy Metabolism in Dilated Cardiomyopathy Induced by Loss of Cardiac IRS1 and IRS2.

Type 2 diabetes (T2D) is a high-risk factor for incident of cardiovascular diseases. Women at young ages show a reduced incidence of both T2D and cardiovascular diseases compared with men, but these disparities disappear in postmenopausal women versus age-matched men. Thus, ovaries and ovarian hormones, such as estrogen, are expected to protect from T2D and cardiovascular diseases. In this study, we aimed to investigate the role of ovaries and ovarian hormone estrogen in cardiac function and energy metabolism using the cardiac IRS (insulin receptor substrate) 1 and IRS2 double genes knockout mice that mimic cardiac insulin resistance. Control and heart-specific IRS1/2 double genes knockout mice were treated with placebo or 17β-estradiol (E2) pellets, respectively, through subcutaneous implantation. Female mice were subjected to a bilateral ovariectomy surgery to remove endogenous E2. The cardiac function and energy metabolism were determined using echocardiography and indirect calorimeter, respectively. All male heart-specific IRS1/2 double genes knockout mice died of heart failure at 6 to 8 weeks as we previously described (Qi et al), but all female heart-specific IRS1/2 double genes knockout mice survived >1 year. Removal of ovaries in heart-specific IRS1/2 double genes knockout female mice resulted in cardiac dysfunction, and ultimately animal death. However, E2 supplementation prevented the dilated cardiomyopathy, improved cardiac function and energy metabolism, and enhanced lifespan in both male and ovariectomy female mice deficient for cardiac IRS1 and IRS2 genes, largely owing to the activation of Akt (protein kinase B)-Foxo1 (O1 class of forkhead/winged helix transcription factor) signaling cascades. These results show that estrogen protects mice from cardiac insulin resistance-induced diabetic cardiomyopathy. This may provide a fundamental mechanism for the gender difference for the incidence of both T2D and cardiovascular diseases. This study highlights that estrogen signaling could be a potential target for improving cardiac function and energy metabolism in humans with T2D.

Bioactives From Coffee By-Products Stimulate Liver Metabolism and Mitochondrial Bioenergetics via AMPK/PGC-1α/Nrf2 and IRS/AKT/GLUT2 Pathways In Vitro

ObjectivesThis study aimed to investigate the effect of the main bioactive compounds from the coffee silverskin and coffee husk on hepatic lipid and glucose metabolism and mitochondrial bioenergetics using an in vitro model mimicking non-alcoholic fatty liver disease. MethodsHepG2 cells were treated with 50 μmol L–1 caffeine, chlorogenic, caffeic, protocatechuic, or gallic acids, or kaempferol, or the aqueous extracts from the coffee silverskin (CSE, 100 μg mL–1) or the coffee husk (CHE, 100 μg mL–1) in the presence of palmitic acid (500 μmol L–1). Cell metabolism biomarkers were assessed 24 h after the co-treatment in cell lysates and supernatants using chemical and immunochemical techniques. Differences among treatments were considered significant at p < 0.05. ResultsBioactive compounds in coffee by-products, CSE, and CHE decreased lipid accumulation (53–115%) by reducing fatty acid synthase expression (44–76%) and stimulated β-oxidation by triggering carnitine palmitoyltransferase I activity (1.6 to 2.6-fold) and up-phosphorylating AMP-activated protein kinase (AMPK, 1.5–2.4-fold). Glucose uptake was promoted via the increase in the insulin receptor substrate (IRS)-1 (1.5 to 2.1-fold) and protein kinase B (Akt1, 1.7–2.4-fold) phosphorylation, and glucose transporter (GLUT)-2 expression (2.1 to 3.7-fold). Mitochondrial function was enhanced (ATP production and O2 consumption rate, 1.7–2.5-fold and 1.8 to 2.5-fold, respectively), and oxidative phosphorylation complexes and peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α expression restored (30–105%). Oxidative stress was diminished (56 – 85%) through augmented superoxide dismutase (1.3 to 1.7-fold) and catalase (1.5 to 2.0-fold) activities, and nuclear factor-erythroid 2-related factor 2 (Nrf2) expression (2.3 to 3.8-fold). ConclusionsThe major bioactive compounds in coffee by-products, primarily chlorogenic and protocatechuic acids, could regulate hepatic lipid and glucose metabolism and prevent oxidative stress and mitochondrial dysfunction by activating AMPK/PGC-1α/Nrf2 and IRS/AKT/GLUT2 signaling pathways. Altogether, our results suggest the use of coffee by-products as a sustainable source of natural bioactives to counteract non-alcoholic fatty liver disease. Funding SourcesUSDA-NIFA-HATCH and the Spanish Ministry of Science and Innovation.

158-OR: Perinatal Hypercaloric Diet Leads to Cardiometabolic Imbalance via Hypothalamic Methylation Events in Male Mice

Cardiometabolic diseases are among the highest prevalent diseases worldwide. We have reported that perinatal maternal programming through hypercaloric diet (HCD) leads to an elevation in systolic BP and blood glucose levels exclusively in male mice offspring. These findings led us to investigate epigenetic modulations in the brain of the offspring that support the cardiometabolic phenotype. To elucidate the underlying mechanisms, we challenged the programmed offspring with postnatal HCD, speculating that it would exaggerate the cardiometabolic phenotype. C57BL6/J dams were fed on HCD or regular diet (RD) for 1 month then mated with RD-fed males. From weaning, all pups were maintained on HCD for 3 months resulting in RD-HCD and HCD-HCD groups of male mice. Results showed that HCD-HCD mice have higher systolic BP when compared to RD-HCD (125 ±1.9 vs. 115 ±2.8 mmHg) . Surprisingly, a glucose tolerance test showed that HCD-HCD mice were more glucose tolerant when compared with RD-HCD mice (AUC 29632 ±3516 vs. 604±4724, p=0.003, n=6-10/group) . Whole-genome methylation sequencing of the hypothalamus in offspring revealed differentially methylated regions (DMR) in HCD-HCD mice. Pathway analysis showed that DMR are annotated to genes involved in insulin signaling, neurotransmitter binding, and inflammatory pathways. Insulin receptor substrate (Irs1) gene exon was hypomethylated in HCD-HCD mice when compared to RD-HCD (DMR= -0.12, p=0.03) . Protein expression showed an increase in Irs1 protein fold change (FC) levels in both HCD-HCD and RD-HCD (FC=1.45 and 1.33 p=0.005, 0.03 respectively) compared to RD-fed mice. However, serine phosphorylated Irs1 (pSer307-Irs1) , an indicator for disturbed insulin signaling, showed decreased levels only in HCD-HCD mice compared to RD-HCD males (p=0.003) . We conclude that perinatal HCD programming leads to methylation events in the hypothalamus that modulate the expression of key genes regulating cardiometabolic functions. Disclosure M.Elgazzaz: None. C.Berdasco: None. E.D.Lazartigues: None. Funding American Diabetes Association (1-19-IBS-291) ;

557-P: Effect of Monocarbonyl Curcumin Analogues C66 and B2BrBC on Pancreatic Expression of Genes Related to Insulin Signaling Pathway and Oxidative Stress in Streptozotocin-Induced Diabetes

Curcumin has well documented beneficial effects against diabetes but its poor bioavailability limits its use. Here, we investigated the effects of two monocarbonyl curcumin analogues - (2E,6E) -2,6-bis[ (2-trifluoromethyl) benzylidene]cyclohexanone (C66) and (2E,6E) -2,6-bis (2-bromobenzylidene) cyclohexanone) (B2BrBC) - on the expression of genes involved in insulin signaling and oxidative stress in pancreas samples from streptozotocin (STZ) -induced diabetic rats. Animals were divided into two experiments: (Experiment 1) STZ injection (60 mg/kg) followed by 30 days treatment with C66 or B2BrBC (both 50 µmol/kg, daily) or (Experiment 2) single-dose treated with C66 or B2BrBC (125 µmol/kg) , 6 hours before STZ injection, and sacrifice after 72 hours. Quantitative RT-PCR analysis was used to assess the expression of: insulin (Ins1) , insulin receptor substrate 2 (Irs2) , Slc2a2 solute carrier family 2 member 2 (Slc2a2) (s. Glut2) , mitogen-activated protein kinase 14 (Mapk14) , protein kinase AMP-activated catalytic subunit alpha 1 (Prkaa1) , peroxisome proliferator-activator receptor gamma (Pparg) , BCL2 apoptosis regulator (Bcl2) , BCL2-associated X apoptosis regulator (Bax) , and NF-κB inhibitor alpha (Nfkbia) . Results demonstrated that when C66 and B2BrBC were administered at lower, chronic doses after STZ application (Experiment 1) , they both reversed the effect of STZ on Irs2 mRNA levels in the pancreas, with the effect of C66 being significantly higher. At higher, acute doses (Experiment 2) , C66 ameliorated Ins1 mRNA levels after STZ treatment. Also, although not statistically significant, at higher doses, both C66 and B2BrBC showed a trend to normalize the suppressing effect of STZ on Glut2 mRNA levels. Overall, our data demonstrate that these two monocarbonyl curcumin analogues have direct effects on the expression of insulin signaling pathway-related genes in the pancreas. Disclosure N. Hadzi-Petrushev: None. M. Mladenov: None. J.B. Bogdanov: None. L. Poretsky: None. D. Avtanski: None. Funding Gerald J. and Dorothy R. Friedman New York Foundation for Medical Research

Interplay of retinol binding protein 4 with obesity and associated chronic alterations (Review).

Obesity is a multifactorial disease, defined as excessive fat deposition in adipose tissue. Adipose tissue is responsible for the production and secretion of numerous adipokines that induce metabolic disorders. Retinol-binding protein 4 (RBP4) is an adipokine that transports vitamin A or retinol in the blood. High levels of RBP4 are associated with development of metabolic disease, including obesity, insulin resistance (IR), metabolic syndrome, and type 2 diabetes (T2D). The present review summarizes the role of RBP4 in obesity and associated chronic alterations. Excessive synthesis of RBP4 contributes to inflammatory characteristic of obesity by activation of immune cells and release of proinflammatory cytokines, such as TNFα and ILs, via the Toll-like receptor/JNK pathway. The retinol-RBP4 complex inhibits insulin signaling directly in adipocytes by activating Janus kinase 2 (JAK2)/STAT5/suppressor of cytokine signaling 3 signaling. This mechanism is retinol-dependent and requires vitamin A receptor stimulation by retinoic acid 6 (STRA6). In muscle, RBP4 is associated with increased serine 307 phosphorylation of insulin receptor substrate-1, which decreases its affinity to PI3K and promotes IR. In the liver, RBP4 increases hepatic expression of phosphoenolpyruvate carboxykinase, which increases production of glucose. Elevated serum RBP4 levels are associated with β-cell dysfunction in T2D via the STRA6/JAK2/STAT1/insulin gene enhancer protein 1 pathway. By contrast, RBP4 induces endothelial inflammation via the NF-κB/nicotinamide adenine dinucleotide phosphate oxidase pathway independently of retinol and STRA6, which stimulates expression of proinflammatory molecules, such as vascular cell adhesion molecule 1, E-selectin, intercellular adhesion molecule 1, monocyte chemoattractant protein 1 and TNFα. RBP4 promotes oxidative stress by decreasing endothelial mitochondrial function; overall, it may serve as a useful biomarker in the diagnosis of obesity and prognosis of associated disease, as well as a potential therapeutic target for treatment of these diseases.

222-LB: Switching Palmitoyl and Oleoyl Position in Sn-2 and Sn-3 of a Triacylglycerol Led to Differential Body Weight Gain Rates and Hepatic Gene Expressions in Mice Fed a High-Fat Diet

Alterations of fatty acid metabolism have been associated with diabetes. Triacylglycerol (TAG) can be obtained from dietary sources and synthesized de novo. The composition and location of the fatty acyl groups in a TAG can affect its physicochemical features such as melting temperature, and physiological characteristics such as digestion, absorption, and lipid metabolism. We hypothesize that the TAG fatty acyl position affects the body weight gain and lipid metabolism of mice in a high-fat diet (HFD) setting. Twenty-seven 6-week-old male C57/BL6J mice were randomly assigned to 3 dietary treatment groups, 1,2-dipalmitoyl-3-oleoylglycerol (PPO, 36 % w/w diet) , 1,3-dipalmitoyl-2-oleoylglycerol (POP, 36% w/w diet) , and the chow diet control groups. Mice in each group were fed their respective diet for 6 weeks, and food intake and body weight were measured daily. Mice fed the PPO diet had significantly higher food intake, calories intake and body wight gain rate, and lower liver to body weight ratio than those fed the POP diet. Mice fed the chow diet had significantly higher total grams of food intake, but lower calories intake, liver and epididymal fat weight, and body weight gain rate than those fed the PPO, but not the POP, diet. Mice in the PPO group had significantly higher serum glucose than that in other two groups, whereas mice fed the chow diet had significantly lower plasma cholesterol level than those HFD groups. The plasma TAG levels were not different among them. The hepatic levels of insulin receptor substrate 1, and fatty acid synthase, and phosphorylated acetyl CoA carboxylase in mice fed the chow diet are significantly higher than those fed the PPO or POP diet, whereas the hepatic expression level of stearoyl CoA desaturase 1 in mice fed the POP diet was lower than those fed the PPO or chow diet. We conclude that feeding a HFD affects the expression levels of hepatic proteins for lipid metabolism in mice. In addition, the position of fatty acyl group in a TAG affects the food intake, the body weight gain rate and lipid homeostasis in mice. Disclosure X. Hu: None. T. Wang: None. G. Chen: None.

N-Acetylcysteine Enhances the Recovery of Ischemic Limb in Type-2 Diabetic Mice

Critical limb ischemia (CLI) is a severe complication of diabetes mellitus that occurs without effective therapy. Excessive reactive oxygen species (ROS) production and oxidative stress play critical roles in the development of diabetic cardiovascular complications. N-acetylcysteine (NAC) reduces ischemia-induced ROS production. The present study aimed to investigate the effect of NAC on the recovery of ischemic limb in an experimental model of type-2 diabetes. TALLYHO/JngJ diabetic and SWR/J non-diabetic mice were used for developing a CLI model. For NAC treatment, mice received NAC (1 mg/mL) in their drinking water for 24 h before initiating CLI, and continuously for the duration of the experiment. Blood flow, mechanical function, histology, expression of antioxidant enzymes including superoxide dismutase (SOD)-1, SOD-3, glutathione peroxidase (Gpx)-1, catalase, and phosphorylated insulin receptor substrate (IRS)-1, Akt, and eNOS in ischemic limb were evaluated in vivo or ex vivo. Body weight, blood glucose, plasma advanced glycation end-products (AGEs), plasma insulin, insulin resistance index, and plasma TNF-a were also evaluated during the experiment. NAC treatment effectively attenuated ROS production with preserved expressions of SOD-1, Gpx-1, catalase, phosphorylated Akt, and eNOS, and enhanced the recovery of blood flow and function of the diabetic ischemic limb. NAC treatment also significantly decreased the levels of phosphorylated IRS-1 (Ser307) expression and plasma TNF-α in diabetic mice without significant changes in blood glucose and AGEs levels. In conclusion, NAC treatment enhanced the recovery of blood flow and mechanical function in ischemic limbs in T2D mice in association with improved tissue redox/inflammatory status and insulin resistance.

Retracted] MicroRNA‑766 inhibits papillary thyroid cancer progression by directly targeting insulin receptor substrate2 and regulating the PI3K/Akt pathway.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that various panels showing the data from flow cytometry experiments in Figs.2D,4D and5D, and certain of the tumor images featured in Fig.7A, were strikingly similar to data appearing in different form in other articles by different authors. Furthermore, overlapping data panels were identified within this paper comparing the cell migration assay images between Figs.2C and4F. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to International Journal of Oncology, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in International Journal of Oncology 54: 315‑325, 2019; DOI: 10.3892/ijo.2018.4615].

Reduced insulin signaling and high glucagon in early insulin resistance impaired fast-fed regulation of renal gluconeogenesis via insulin receptor substrate.

Gluconeogenesis is one of the key processes through which the kidney contributes to glucose homeostasis. Urinary exosomes (uE) have been used to study renal gene regulation noninvasively in humans and rodents. Recently, we demonstrated fast-fed regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme for gluconeogenesis, in human uE. The regulation was impaired in subjects with early insulin resistance. Here, we studied primary human proximal tubule cells (hPT) and human uE to elucidate a potential link between insulin resistance and fast-fed regulation of renal PEPCK. We demonstrate that fasted hPTs had higher PEPCK and insulin receptor substrate-2 (IRS2) mRNA and protein levels, relative to fed cells. The fast-fed regulation was, however, attenuated in insulin receptor knockdown (IRKO) hPTs. The IRKO was confirmed by the blunted insulin-induced response on PEPCK, PGC1α, p-IR, and p-AKT expression in IRKO cells. Exosomes secreted by the wild-type or IRKO hPT showed similar regulation to the respective hPT. Similarly, in human uE, the relative abundance of IRS-2 mRNA (to IRS1) was higher in the fasted state relative to the fed condition. However, the fast-fed difference was absent in subjects with early insulin resistance. These subjects had higher circulating glucagon levels relative to subjects with optimal insulin sensitivity. Furthermore, in hPT cells, glucagon significantly induced PEPCK and IRS2 gene, and gluconeogenesis. IR knockdown in hPT cells further increased the gene expression levels. Together the data suggest that reduced insulin sensitivity and high glucagon in early insulin resistance may impair renal gluconeogenesis via IRS2 regulation.

Angiotensin II Inhibits Insulin Receptor Signaling in Adipose Cells

Angiotensin II (Ang II) is a critical regulator of insulin signaling in the cardiovascular system and metabolic tissues. However, in adipose cells, the regulatory role of Ang II on insulin actions remains to be elucidated. The effect of Ang II on insulin-induced insulin receptor (IR) phosphorylation, Akt activation, and glucose uptake was examined in 3T3-L1 adipocytes. In these cells, Ang II specifically inhibited insulin-stimulated IR and insulin receptor substrate-1 (IRS-1) tyrosine-phosphorylation, Akt activation, and glucose uptake in a time-dependent manner. These inhibitory actions were associated with increased phosphorylation of the IR at serine residues. Interestingly, Ang II-induced serine-phosphorylation of IRS was not detected, suggesting that Ang II-induced desensitization begins from IR regulation itself. PKC inhibition by BIM I restored the inhibitory effect of Ang II on insulin actions. We also found that Ang II promoted activation of several PKC isoforms, including PKCα/βI/βII/δ, and its association with the IR, particularly PKCβII, showed the highest interaction. Finally, we also found a similar regulatory effect of Ang II in isolated adipocytes, where insulin-induced Akt phosphorylation was inhibited by Ang II, an effect that was prevented by PKC inhibitors. These results suggest that Ang II may lead to insulin resistance through PKC activation in adipocytes.

Preparation, Physicochemical and Hypoglycemic Properties of Natural Selenium-Enriched Coarse Tea Glycoproteins.

Various functional components in tea have been well developed, but less research has been explored on glycoproteins in tea. In this paper, three types of glycoprotein fractions, namely tea selenium-binding glycoprotein1-1 (TSBGP1-1), TSBGP2-1, and TSBGP3-1, respectively, were extracted and purified from selenium-enriched coarse green tea. Chemical analysis revealed that three fractions were glycoproteins, but their selenium content, molecular weight, and monosaccharide composition were significantly different. Fourier transforms infrared (FT-IR) analysis indicated that three fractions contained characteristic absorption peaks of glycoproteins but differed in secondary structural composition. Thermogravimetric (TG) analysis showed that the thermal stability of the three fractions was dramatically distinct. The in vitro hypoglycemic activity showed that TSBGPs significantly activated the insulin receptor substrate 2 (IRS2)/protein kinase B (Akt) pathway in LO2 cells, then enhanced glucose metabolism and inhibited gluconeogenesis, and finally ameliorated insulin resistance (IR) and glucose metabolism disorders. Furthermore, Pearson correlation analysis reveals that the hypoglycemic activity was significantly correlated with Se, protein, monosaccharide composition (especially glucose), molecular weight, and secondary structure. Our results show that Se-enriched tea glycoprotein is a desirable candidate for developing anti-diabetic food, and TSBGP-2 and TSBGP-3 had a better regulation effect. Our results can provide a research reference for the extraction, physicochemical property, and function of selenium-enriched plant glycoproteins.

Kutkin, iridoid glycosides enriched fraction of Picrorrhiza kurroa promotes insulin sensitivity and enhances glucose uptake by activating PI3K/Akt signaling in 3T3-L1 adipocytes

BackgroundTherapeutic failure and drug resistance are common sequelae to insulin resistance associated with type 2 diabetes mellitus (T2DM). Consequently, there is an unmet need of alternative strategies to overcome insulin resistance associated complications. PurposeTo demonstrate whether Kutkin (KT), iridoid glycoside enriched fraction of Picrorhiza kurroa extract (PKE) has potential to increase the insulin sensitivity vis à vis glucose uptake in differentiated adipocytes. MethodsMolecular interaction of KT phytoconstituents, picroside-I (P-I) & picroside- II (P-II) with peroxisome proliferator-activated receptor gamma (PPARγ), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) were analyzed in silico. Cellular viability and adipogenesis were determined by following 3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-Diphenyltetrazolium bromide (MTT) assay and Oil Red-O staining. Further, ELISA kit based triglycerides and diacylglycerol-O-Acyltransferase-1 (DGAT1) were assessed in differentiated adipocytes. ELISA based determination were performed to check the levels of adiponectin and tumor necrosis factor alpha (TNF-α). However, Flow cytometry and immunofluorescence based assays were employed to measure the glucose uptake and glucose transporter 4 (glut4) expression in differentiated adipocytes, respectively. Further to explore the targeted signaling axis, mRNA expression levels of PPARγ, CCAAT/enhancer binding protein α (CEBPα), and glut4 were determined using qRT-PCR and insulin receptor substrate-1 (IRS-1), Insulin receptor substrate-2 (IRS-2), PI3K/Akt, AS160, glut4 followed by protein validation using immunoblotting in differentiated adipocytes. ResultsIn silico analysis revealed the binding affinities of major constituents of KT (P-I& P-II) with PPARγ/PI3K/Akt. The enhanced intracellular accumulation of triglycerides with concomitant activation of PPARγ and C/EBPα in KT treated differentiated adipocytes indicates augmentation of adipogenesis in a concentration-dependent manner. Additionally, at cellular level, KT upregulated the expression of DAGT1, and decreases fatty acid synthase (FAS), and lipoprotein lipase (LPL), further affirmed improvement in lipid milieu. It was also observed that KT upregulated the levels of adiponectin and reduced TNFα expression, thus improving the secretory functions of adipocytes along with enhanced insulin sensitivity. Furthermore, KT significantly promoted insulin mediated glucose uptake by increasing glut4 translocation to the membrane via PI3/Akt signaling cascade. The results were further validated using PI3K specific inhibitor, wortmannin and findings revealed that KT treatment significantly enhanced the expression and activation of p-PI3K/PI3K and p-Akt/Akt even in case of treatment with PI3K inhibitor wortmannin alone and co-treatment with KT in differentiated adipocytes and affirmed that KT as activator of PI3K/Akt axis in the presence of inhibitor as well. ConclusionCollectively, KT fraction of PKE showed anti-diabetic effects by enhancing glucose uptake in differentiated adipocytes via activation of PI3K/Akt signaling cascade. Therefore, KT may be used as a promising novel natural therapeutic agent for managing T2DMand to the best of our knowledge, this is the first report, showing the efficacy and potential molecular mechanism of KT in enhancing insulin sensitivity and glucose uptake in differentiated adipocytes.