Insulin receptor substrate-1 inhibits high-fat diet-induced obesity by browning of white adipose tissue through miR-503.

Genetic variation of insulin receptor substrate 1 (IRS-1) was found to modulate the insulin resistance of adipose tissues, but the underlying mechanism was not clear. To investigate how the IRS-1 was involved in the browning of white adipose tissue through miRNA, we identified a mutated Irs-1 (Irs-1-/- ) mice model and found that this mice had a reduced subcutaneous WAT (sWAT) and increased brown adipose tissue (BAT) in the interscapular region. So we isolated the bone marrow stromal cells and analyzed differentially expressed miRNAs and adipogenesis-related genes with miRNA arrays and PCR arrays. Irs-1-/- mice showed decreased miR-503 expression, but increased expression of its target, bone morphogenetic protein receptor type 1a (BMPR1a). Overexpression of miR-503 in preadipocytes downregulated BMPR1a and impaired adipogenic activity through the phosphotidylinositol 3-kinase (PI3K/Akt) pathway, while the inhibitor had the opposite effect. In both Irs-1-/- and cold-induced models, sWAT exhibited BAT features, and showed tissue-specific increased BMPR1a expression, PI3K expression, and Akt phosphorylation. Thus, our results showed that IRS-1 regulated brown preadipocyte differentiation and induced browning in sWAT through the miR-503-BMPR1a pathway, which played important roles in high-fat diet-induced obesity.

MiRNA-7b-5p attenuates the progression of osteoporosis by inhibiting adipose differentiation of hMSCs via regulating IRS2.

OBJECTIVE: To elucidate whether microRNA-7b-5p (miRNA-7b-5p) could inhibit adipose differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) through regulating IRS2, thereby alleviating the progression of osteoporosis. MATERIALS AND METHODS: Expression levels of miRNA-7b-5p and IRS2 in hMSCs at different stages of adipogenic differentiation and osteogenic differentiation were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. After transfection of miRNA-7b-5p mimic or pcDNA-IRS2 in hMSCs, lipid droplet formation in cells was observed by oil red O staining. Expressions of C/EBPα and PPARγ were detected by qRT-PCR and Western blot. The potential target gene of miRNA-7b-5p was predicted by bioinformatics and verified by dual-luciferase reporter gene assay. Finally, expressions of IRS2 in hMSCs transfected with miRNA-7b-5p-NC, miRNA-7b-5p mimic or co-transfected with miRNA-7b-5p mimic and pcDNA-IRS2 were examined. RESULTS: Expressions of miRNA-7b-5p and IRS2 gradually decreased with the prolongation of adipogenic differentiation, but increased during osteogenic differentiation of hMSCs. Transfection of miRNA-7b-5p mimic reduced oil red O staining after adipogenic differentiation and downregulated mRNA and protein levels of C/EBPα and PPARγ. Transfection of pcDNA-IRS2 increased oil red O staining after osteogenic differentiation and upregulated mRNA and protein levels of C/EBPα and PPARγ. Dual-luciferase reporter gene results showed that miRNA-7b-5p could bind to IRS2. Overexpression of IRS2 reversed the downregulated mRNA and protein levels of adipogenic-related genes C/EBPα and PPARγ due to the overexpression of miRNA-7b-5p. CONCLUSIONS: MiRNA-7b-5p inhibits the adipogenic differentiation of hMSCs through IRS2, thus alleviating the development of osteoporosis.

AMPK-mediated activation of Akt protects renal tubular cells from stress-induced apoptosis in vitro and ameliorates ischemic AKI in vivo.

We have reported that preconditioning renal tubular cells (RTCs) with A-769662 [a pharmacological activator of AMP-activated protein kinase (AMPK)] reduces apoptosis of RTCs induced by subsequent stress and ameliorates the severity of ischemic acute kidney injury (AKI) in mice. In the present study, we examined the role of the phosphoinositide 3-kinase (PI3K)/Akt pathway in mediating these effects. Using shRNA, we developed knockdown (KD) RTCs to confirm that any novel effects of A-769662 are mediated specifically by AMPK. We reduced expression of the total ß-domain of AMPK in KD RTCs by >80%. Control RTCs were transfected with "scrambled" shRNA. Preconditioning control RTCs with A-769662 increased both the phosphorylation (activity) of AMPK and survival of these cells when exposed to subsequent stress, but neither effect was observed in KD cells. These data demonstrate that activation of AMPK by A-769662 is profoundly impaired in KD cells. A-769662 activated PI3K and Akt in control but not KD RTCs. These data provide novel evidence that activation of the PI3K/Akt pathway by A-769662 is mediated specifically through activation of AMPK and not by a nonspecific mechanism. We also demonstrate that, in control RTCs, Akt plays a role in mediating the antiapoptotic effects of A-769662. In addition, we provide evidence that AMPK ameliorates the severity of ischemic AKI in mice and that this effect is also partially mediated by Akt. Finally, we provide evidence that AMPK activates PI3K by inhibiting mechanistic target of rapamycin complex 1 and preventing mechanistic target of rapamycin complex 1-mediated inhibition of insulin receptor substrate-1-associated activation of PI3K.

Insulin resistance disrupts epithelial repair and niche-progenitor Fgf signaling during chronic liver injury.

Insulin provides important information to tissues about feeding behavior and energy status. Defective insulin signaling is associated with ageing, tissue dysfunction, and impaired wound healing. In the liver, insulin resistance leads to chronic damage and fibrosis, but it is unclear how tissue-repair mechanisms integrate insulin signals to coordinate an appropriate injury response or how they are affected by insulin resistance. In this study, we demonstrate that insulin resistance impairs local cellular crosstalk between the fibrotic stroma and bipotent adult liver progenitor cells (LPCs), whose paracrine interactions promote epithelial repair and tissue remodeling. Using insulin-resistant mice deficient for insulin receptor substrate 2 (Irs2), we highlight dramatic impairment of proregenerative fibroblast growth factor 7 (Fgf7) signaling between stromal niche cells and LPCs during chronic injury. We provide a detailed account of the role played by IRS2 in promoting Fgf7 ligand and receptor (Fgfr2-IIIb) expression by the two cell compartments, and we describe an insulin/IRS2-dependent feed-forward loop capable of sustaining hepatic re-epithelialization by driving FGFR2-IIIb expression. Finally, we shed light on the regulation of IRS2 and FGF7 within the fibrotic stroma and show-using a human coculture system-that IRS2 silencing shifts the equilibrium away from paracrine epithelial repair in favor of fibrogenesis. Hence, we offer a compelling insight into the contribution of insulin resistance to the pathogenesis of chronic liver disease and propose IRS2 as a positive regulator of communication between cell types and the transition between phases of stromal to epithelial repair.

Sequential testicular atrophy involves changes in cellular proliferation and apoptosis associated with variations in aromatase P450 expression levels in Irs-2-deficient mice.

Insulin receptor substrate 2 (Irs-2) is an intracellular protein susceptible to phosphorylation after activation of the insulin receptor. Its suppression affects testis development and its absence induces peripheral resistance to insulin. The aim of this study was to identify changes induced by the deletion of Irs-2 in the testicular structure and by the altered expression of cytochrome P450 aromatase, a protein necessary for the development and maturation of germ cells. Adult knockout (KO) mice (Irs-2-/- , 6 and 12 weeks old) and age-matched wild-type (WT) mice were used in this study. Immunohistochemistry and Western blot analyses were performed to study proliferation (PCNA), apoptosis (active caspase-3) and P450 aromatase expression in testicular histological sections. Deletion of Irs-2 decreased the number of epithelial cells in the seminiferous tubule and rete testis. Aberrant cells were frequently detected in the epithelia of Irs-2-/- mice, accompanied by variations in spermatogonia, which were shown to exhibit small hyperchromatic nuclei as well as polynuclear and anuclear structures. The amount of cell proliferation was significantly lower in Irs-2-/- mice than in WT mice, whereas apoptotic processes were more common in Irs-2-/- mice. Aromatase P450 reactivity was higher in 6-week-old KO mice than in WT mice of the same age and was even higher at 12 weeks. Our results suggest that Irs-2 is a key element in spermatogenesis because silencing Irs-2 induces the sequential development of testicular atrophy. The effects are observed mainly in germ cells present in the seminiferous tubule, which may be due to changes in cytochrome P450 aromatase expression.

Insulin Substrate Receptor (IRS) proteins in normal and malignant hematopoiesis.

The insulin receptor substrate (IRS) proteins are a family of cytoplasmic proteins that integrate and coordinate the transmission of signals from the extracellular to the intracellular environment via transmembrane receptors, thus regulating cell growth, metabolism, survival and proliferation. The PI3K/AKT/mTOR and MAPK signaling pathways are the best-characterized downstream signaling pathways activated by IRS signaling (canonical pathways). However, novel signaling axes involving IRS proteins (noncanonical pathways) have recently been identified in solid tumor and hematologic neoplasm models. Insulin receptor substrate-1 (IRS1) and insulin receptor substrate-2 (IRS2) are the best-characterized IRS proteins in hematologic-related processes. IRS2 binds to important cellular receptors involved in normal hematopoiesis (EPOR, MPL and IGF1R). Moreover, the identification of IRS1/ABL1 and IRS2/JAK2V617F interactions and their functional consequences has opened a new frontier for investigating the roles of the IRS protein family in malignant hematopoiesis. Insulin receptor substrate-4 (IRS4) is absent in normal hematopoietic tissues but may be expressed under abnormal conditions. Moreover, insulin receptor substrate-5 (DOK4) and insulin receptor substrate-6 (DOK5) are linked to lymphocyte regulation. An improved understanding of the signaling pathways mediated by IRS proteins in hematopoiesis-related processes, along with the increased development of agonists and antagonists of these signaling axes, may generate new therapeutic approaches for hematological diseases. The scope of this review is to recapitulate and review the evidence for the functions of IRS proteins in normal and malignant hematopoiesis.

Ablation of insulin receptor substrates 1 and 2 suppresses Kras-driven lung tumorigenesis.

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer death worldwide, with 25% of cases harboring oncogenic Kirsten rat sarcoma (KRAS). Although KRAS direct binding to and activation of PI3K is required for KRAS-driven lung tumorigenesis, the contribution of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) in the context of mutant KRAS remains controversial. Here, we provide genetic evidence that lung-specific dual ablation of insulin receptor substrates 1/2 (Irs1/Irs2), which mediate insulin and IGF1 signaling, strongly suppresses tumor initiation and dramatically extends the survival of a mouse model of lung cancer with Kras activation and p53 loss. Mice with Irs1/Irs2 loss eventually succumb to tumor burden, with tumor cells displaying suppressed Akt activation and strikingly diminished intracellular levels of essential amino acids. Acute loss of IRS1/IRS2 or inhibition of IR/IGF1R in KRAS-mutant human NSCLC cells decreases the uptake and lowers the intracellular levels of amino acids, while enhancing basal autophagy and sensitivity to autophagy and proteasome inhibitors. These findings demonstrate that insulin/IGF1 signaling is required for KRAS-mutant lung cancer initiation, and identify decreased amino acid levels as a metabolic vulnerability in tumor cells with IR/IGF1R inhibition. Consequently, combinatorial targeting of IR/IGF1R with autophagy or proteasome inhibitors may represent an effective therapeutic strategy in KRAS-mutant NSCLC.

Inhibition of p53 Relieves Insulin Resistance in Fetal Growth Restriction Mice with Catch-Up Growth via Activating IGFBP3/IGF-1/IRS-1/Akt Signaling Pathway.

To investigate insulin resistance of the fetal growth restriction (FGR) mice with catch-up growth (CUG) and the underlying mechanism, in this study, low protein diet was used during pregnancy to establish the FGR mice model, and high fat diet was applied to establish the CUG model of FGR mice. The insulin and Pifithrin-α stimulation was performed via intraperitoneal injection. The physical characters, biochemical parameters, expression of related molecules in each group were detected via ELISA, RT-PCR, WB, etc. The results showed FBG, FINS and HOAM-IR in CUG-FGR group were higher than those in high fat feeding control group (NC+HF), but the content of IGF-1 in blood was lower than that in NC + HF group. Meanwhile, RT-PCR and WB showed that the expression of IGF was negatively correlated with the expression of P53/IGFBP3. Moreover, the expression of P-IRS/p-PI3K/p-Akt decreased with the increasing of HOAM-IR in IGF signaling pathway. When the mice were injected with Pifithrin-α, the phosphorylation level of IGF signaling pathway and insulin resistance index in the CUG-FGR group were increased and decreased, respectively. In conclusion, insulin resistance in CUG-FGR mice is correlated with the IGFBP3/IGF-1/IRS-1/Akt signaling pathway and inhibited p53 could activate this signaling pathway and relieve insulin resistance.

Molecular and Structural Traits of Insulin Receptor Substrate 1/LC3 Nuclear Structures and Their Role in Autophagy Control and Tumor Cell Survival.

Insulin receptor substrate 1 (IRS-1) is a common cytosolic adaptor molecule involved in signal transduction from insulin and insulin-like growth factor I (IGF-I) receptors. IRS-1 can also be found in the nucleus. We report here a new finding of unique IRS-1 nuclear structures, which we observed initially in glioblastoma biopsy specimens and glioblastoma xenografts. These nuclear structures can be reproduced in vitro by the ectopic expression of IRS-1 cDNA cloned in frame with the nuclear localization signal (NLS-IRS-1). In these structures, IRS-1 localizes at the periphery, while the center harbors a key autophagy protein, LC3. These new nuclear structures are highly dynamic, rapidly exchange IRS-1 molecules with the surrounding nucleoplasm, disassemble during mitosis, and require a growth stimulus for their reassembly and maintenance. In tumor cells engineered to express NLS-IRS-1, the IRS-1/LC3 nuclear structures repress autophagy induced by either amino acid starvation or rapamycin treatment. In this process, IRS-1 nuclear structures sequester LC3 inside the nucleus, possibly preventing its cytosolic translocation and the formation of new autophagosomes. This novel mechanism provides a quick and reversible way of inhibiting autophagy, which could counteract autophagy-induced cancer cell death under severe stress, including anticancer therapies.

Insulin Substrate Receptor (IRS) proteins in normal and malignant hematopoiesis

The insulin receptor substrate (IRS) proteins are a family of cytoplasmic proteins that integrate and coordinate the transmission of signals from the extracellular to the intracellular environment via transmembrane receptors, thus regulating cell growth, metabolism, survival and proliferation. The PI3K/AKT/mTOR and MAPK signaling pathways are the best-characterized downstream signaling pathways activated by IRS signaling (canonical pathways). However, novel signaling axes involving IRS proteins (noncanonical pathways) have recently been identified in solid tumor and hematologic neoplasm models. Insulin receptor substrate-1 (IRS1) and insulin receptor substrate-2 (IRS2) are the best-characterized IRS proteins in hematologic-related processes. IRS2 binds to important cellular receptors involved in normal hematopoiesis (EPOR, MPL and IGF1R). Moreover, the identification of IRS1/ABL1 and IRS2/JAK2V617F interactions and their functional consequences has opened a new frontier for investigating the roles of the IRS protein family in malignant hematopoiesis. Insulin receptor substrate-4 (IRS4) is absent in normal hematopoietic tissues but may be expressed under abnormal conditions. Moreover, insulin receptor substrate-5 (DOK4) and insulin receptor substrate-6 (DOK5) are linked to lymphocyte regulation. An improved understanding of the signaling pathways mediated by IRS proteins in hematopoiesis-related processes, along with the increased development of agonists and antagonists of these signaling axes, may generate new therapeutic approaches for hematological diseases. The scope of this review is to recapitulate and review the evidence for the functions of IRS proteins in normal and malignant hematopoiesis.

Genome-Wide Association Study of the Modified Stumvoll Insulin Sensitivity Index Identifies BCL2 and FAM19A2 as Novel Insulin Sensitivity Loci.

Genome-wide association studies (GWAS) have found few common variants that influence fasting measures of insulin sensitivity. We hypothesized that a GWAS of an integrated assessment of fasting and dynamic measures of insulin sensitivity would detect novel common variants. We performed a GWAS of the modified Stumvoll Insulin Sensitivity Index (ISI) within the Meta-Analyses of Glucose and Insulin-Related Traits Consortium. Discovery for genetic association was performed in 16,753 individuals, and replication was attempted for the 23 most significant novel loci in 13,354 independent individuals. Association with ISI was tested in models adjusted for age, sex, and BMI and in a model analyzing the combined influence of the genotype effect adjusted for BMI and the interaction effect between the genotype and BMI on ISI (model 3). In model 3, three variants reached genome-wide significance: rs13422522 (NYAP2; P = 8.87 × 10(-11)), rs12454712 (BCL2; P = 2.7 × 10(-8)), and rs10506418 (FAM19A2; P = 1.9 × 10(-8)). The association at NYAP2 was eliminated by conditioning on the known IRS1 insulin sensitivity locus; the BCL2 and FAM19A2 associations were independent of known cardiometabolic loci. In conclusion, we identified two novel loci and replicated known variants associated with insulin sensitivity. Further studies are needed to clarify the causal variant and function at the BCL2 and FAM19A2 loci.

Effect of mulberry leaf (Folium Mori) on insulin resistance via IRS-1/PI3K/Glut-4 signalling pathway in type 2 diabetes mellitus rats.

CONTEXT: Folium Mori, the leaf of Morus alba L. (Moraceae), has been used in traditional Chinese medicine (TCM) for treating diabetes. However, it is unclear which components in the mulberry leaf are effective for the treatment of type 2 diabetes mellitus (T2DM). OBJECTIVE: To investigate the flavonoids and polyphenols in mulberry leaves and their antihyperglycemic and antihyperlipidemic effects in T2DM rats. MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into five groups: normal control (NC), diabetic control (DBC), diabetic group with 0.3 mg/kg b.w./day rosiglitazone (RSG), diabetic group with 7 g/kg b.w./day TCM formula and diabetic group with 2 g/kg b.w./day Folium Mori extract (FME). After 4 weeks, the rats were sacrificed; biochemical parameters, gene and protein expression were measured. RESULTS: The FBG level was significantly lower in the FME group than in the DBC group (p < 0.05). In oral glucose tolerance test, the AUC was significantly lower in the FME group (p < 0.05). The HOMA-IR level was significantly decreased in the FME group (p < 0.05). FME decreased the total cholesterol (TC), triglyceride (TG) and low density lipoprotein (LDL) levels (p < 0.05). FME increased the mRNA and protein expression of IRS-1, PI3K p85α and Glut-4 increased significantly (p < 0.05). Histological analysis revealed amelioration of lipid accumulation following FME treatment. Additionally, immunohistochemical analysis displayed stronger staining of Glut-4 in the FME group compared to the DBC group. DISCUSSION AND CONCLUSION: FME could decrease the body weight, blood glucose, TG, TC and LDL levels, and improve insulin resistance. FME possessed significant antihyperglycemic and antihyperlipidemic activities via the IRS-1/PI3K/Glut-4 signalling pathway.

Activation of signal transduction pathways during hepatic oncogenesis.

BACKGROUND AND AIMS: Understanding the molecular pathogenesis of hepatocellular carcinoma (HCC) is essential to identify therapeutic targets. A hepatitis B virus (HBV) related double transgenic murine model was developed. METHODS: Liver specific expression of HBV X protein (HBx) and insulin receptor substrate 1 (IRS1) was achieved and transgenic mice were followed from birth to age 21 months. Liver and tumor tissue were assessed for histologic changes as well as activation of signal transduction pathways by qRT-PCR and multiplex ELISA protein assays. RESULTS: Overexpression of HBx and IRS1 stimulates liver cell proliferation in the double transgenic mice. Only the male mice developed HCC starting at age 15-18 months. The IN/IGF1/IRS1/MAPK/ERK and IN/IGF1/IRS1/PI3K/AKT/GSK3ß cascades were activated early (6-9 months) in the liver followed by WNT/ß-catenin and Notch signaling. Aspartate ß-hydroxylase (ASPH) was found to link these upstream growth factor signaling pathways to downstream Notch activation in tumor tissues. CONCLUSIONS: Sustained overexpression of HBx and IRS1 led to constitutive activation of a tripartite growth factor signal transduction cascade in the liver and was necessary and sufficient to promote HCC development and progression.

IRS2 and PTEN are key molecules in controlling insulin sensitivity in podocytes.

Insulin signaling to the glomerular podocyte is important for normal kidney function and is implicated in the pathogenesis of diabetic nephropathy (DN). This study determined the role of the insulin receptor substrate 2 (IRS2) in this system. Conditionally immortalized murine podocytes were generated from wild-type (WT) and insulin receptor substrate 2-deficient mice (Irs2(-/-)). Insulin signaling, glucose transport, cellular motility and cytoskeleton rearrangement were then analyzed. Within the glomerulus IRS2 is enriched in the podocyte and is preferentially phosphorylated by insulin in comparison to IRS1. Irs2(-/-) podocytes are significantly insulin resistant in respect to AKT signaling, insulin-stimulated GLUT4-mediated glucose uptake, filamentous actin (F-actin) cytoskeleton remodeling and cell motility. Mechanistically, we discovered that Irs2 deficiency causes insulin resistance through up-regulation of the phosphatase and tensin homolog (PTEN). Importantly, suppressing PTEN in Irs2(-/-) podocytes rescued insulin sensitivity. In conclusion, this study has identified for the first time IRS2 as a critical molecule for sensitizing the podocyte to insulin actions through its ability to modulate PTEN expression. This finding reveals two potential molecular targets in the podocyte for modulating insulin sensitivity and treating DN.

A liver Hif-2α-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition.

Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis. The liver possesses a rich sinusoidal capillary network with a higher degree of hypoxia and lower gluconeogenesis in the perivenous zone as compared to the rest of the organ. Here, we show that diverse vascular endothelial growth factor (VEGF) inhibitors improved glucose tolerance in nondiabetic C57BL/6 and diabetic db/db mice, potentiating hepatic insulin signaling with lower gluconeogenic gene expression, higher glycogen storage and suppressed hepatic glucose production. VEGF inhibition induced hepatic hypoxia through sinusoidal vascular regression and sensitized liver insulin signaling through hypoxia-inducible factor-2α (Hif-2α, encoded by Epas1) stabilization. Notably, liver-specific constitutive activation of HIF-2α, but not HIF-1α, was sufficient to augment hepatic insulin signaling through direct and indirect induction of insulin receptor substrate-2 (Irs2), an essential insulin receptor adaptor protein. Further, liver Irs2 was both necessary and sufficient to mediate Hif-2α and Vegf inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between Hif-2α-mediated hypoxic signaling and hepatic insulin action through Irs2 induction, which can be co-opted by Vegf inhibitors to modulate glucose metabolism. These studies also indicate distinct roles in hepatic metabolism for Hif-1α, which promotes glycolysis, and Hif-2α, which suppresses gluconeogenesis, and suggest new treatment approaches for type 2 diabetes mellitus.

[Cross-talk between insulin signaling and cell proliferation pathways]. / Dialogue entre les voies de signalisation de l'insuline et les voies de prolifération cellulaire.

Epidemiological studies provide evidence for a close relationship between diabetes and cancer. Insulin is in fact a growth factor, and its binding to its membrane receptor activates intracellular signaling pathways involved in the regulation of both metabolism and cell proliferation. The balance between mitogenic and metabolic actions of insulin can be modulated by various mechanisms, including the way the ligand binds to its receptor or to the closely related insulin-like growth factor-1 (IGF-1) receptor. Cross-talks with other signaling pathways implicated in cell proliferation have also been described, like the Wnt/ß catenin pathway, and involve the activation of common downstream effectors such as insulin receptor substrate-1 (IRS-1). Finally, the identification of new proteins activated by insulin and involved in intracellular signaling would allow a better understanding of the complex connections linking metabolic and proliferative regulatory pathways. As an example, the molecular adaptor Grb14, which is a specific inhibitor of insulin receptor catalytic activity, also controls insulin-induced metabolic and mitogenic signaling pathways through post-receptor mechanisms that remain to be fully elucidated.

Chronic activation of a designer G(q)-coupled receptor improves ß cell function.

Type 2 diabetes (T2D) has emerged as a major threat to human health in most parts of the world. Therapeutic strategies aimed at improving pancreatic ß cell function are predicted to prove beneficial for the treatment of T2D. In the present study, we demonstrate that drug-mediated, chronic, and selective activation of ß cell G(q) signaling greatly improve ß cell function and glucose homeostasis in mice. These beneficial metabolic effects were accompanied by the enhanced expression of many genes critical for ß cell function, maintenance, and differentiation. By employing a combination of in vivo and in vitro approaches, we identified a novel ß cell pathway through which receptor-activated G(q) leads to the sequential activation of ERK1/2 and IRS2 signaling, thus triggering a series of events that greatly improve ß cell function. Importantly, we found that chronic stimulation of a designer G(q)-coupled receptor selectively expressed in ß cells prevented both streptozotocin-induced diabetes and the metabolic deficits associated with the consumption of a high-fat diet in mice. Since ß cells are endowed with numerous receptors that mediate their cellular effects via activation of G(q)-type G proteins, our findings provide a rational basis for the development of novel antidiabetic drugs targeting this class of receptors.

Diet-induced obesity impairs AKT signalling in the retina and causes retinal degeneration.

Retinopathy, a common complication of diabetes, is characterized by an unbalanced production of nitric oxide (NO), a process regulated by nitric oxide synthase (NOS). We hypothesized that retinopathy might stem from changes in the insulin receptor substrate (IRS)/PI3K/AKT pathway and/or expression of NOS isoforms. Thus, we analysed the morphology and apoptosis index in retinas of obese rats in whom insulin resistance had been induced by a high-fat diet (HFD). Immunoblotting analysis revealed that the retinal tissue of HFD rats had lower levels of AKT(1) , eNOS and nNOS protein than those of samples taken from control animals. Furthermore, immunohistochemical analyses indicated higher levels of iNOS and 4-hydroxynonenal and a larger number of apoptotic nuclei in HFD rats. Finally, both the inner and outer retinal layers of HFD rats were thinner than those in their control counterparts. When considered alongside previous results, these patterns suggest two major ways in which HFD might impact animals: direct activity of ingested fatty acids and/or via insulin-resistance-induced changes in intracellular pathways. We discuss these possibilities in further detail and advocate the use of this animal model for further understanding relationships between retinopathy, metabolic syndrome and type 2 diabetes.

Acute effects of different diet compositions on skeletal muscle insulin signalling in obese individuals during caloric restriction.

OBJECTIVE: The cellular effects of restricting fat versus carbohydrate during a low-calorie diet are unclear. The aim of this study was to examine acute effects of energy and macronutrient restriction on skeletal muscle insulin signalling in obesity. MATERIALS/METHODS: Eighteen obese individuals without diabetes underwent euglycemic-hyperinsulinemic clamp and skeletal muscle biopsy after: (a) 5days of eucaloric diet (30% fat, 50% carbohydrate), and (b) 5days of a 30% calorie-restricted diet, either low fat/high carbohydrate (LF/HC: 20% fat, 60% carbohydrate) or high-fat/low carbohydrate (HF/LC: 50% fat, 30% carbohydrate). RESULTS: Weight, body composition, and insulin sensitivity were similar between groups after eucaloric diet. Weight loss was similar between groups after hypocaloric diet, 1.3±1.3kg (p<0.0001 compared with eucaloric). Whole-body insulin sensitivity was unchanged after calorie restriction and similar between groups. However, ex vivo skeletal muscle insulin signalling differed depending on macronutrient composition of calorie-restricted diet. Skeletal muscle of the LF/HC group had increased insulin-stimulated tyrosine phosphorylation of IRS-1, decreased insulin-stimulated Ser307 phosphorylation of IRS-1, and increased IRS-1-associated phosphatidylinositol (PI)3-kinase activity. Conversely, insulin stimulation of tyrosine phosphorylated IRS-1 was absent and serine 307 phosphorylation of IRS-1 was increased on HF/LC, with blunting of IRS-1-associated PI3-kinase activity. CONCLUSION: Acute caloric restriction with an LF/HC diet alters skeletal muscle insulin signalling in a way that improves insulin sensitivity, while acute caloric restriction with an HF/LC diet induces changes compatible with insulin resistance. In both cases, ex vivo changes in skeletal muscle insulin signalling appear prior to changes in whole body insulin sensitivity.

miR-370 modulates insulin receptor substrate-1 expression and inhibits the tumor phenotypes of oral carcinoma.

BACKGROUND: MicroRNAs play important roles in carcinogenesis. A preliminary screening study suggested that down-regulation of miR-370 occurs in oral squamous cell carcinoma (OSCC) tissue. Insulin receptor substratre-1 (IRS-1) is the substrate of insulin-like growth factor receptor (IGFR), which modulates AKT/mTOR activation in malignancies. The relationship between miR-370 and IRS-1, and their functional roles in OSCC pathogenesis are unclear. MATERIALS AND METHODS: Primary OSCC specimens were examined for miR-370 expression. Exogenous expression of miR-370 was established using both stable subclones and transient expression, and these were used to gain insights into miR-370's functions in OSCC cells. Knockdown of miR-370 and IRS-1 was also carried out in OSCC cells using a small interference oligonucleotide approach. RESULTS: Squamous cell carcinoma tissues with perineural invasion had lowered miR-370 expression compared with contrasting OSCC. OSCC cells also exhibited lower miR-370 expression than normal oral keratinocytes, and this can be reversed by treatment with 5-aza-2'-deoxycytidine. Exogenous miR-370 expression decreases the migration and anchorage-independent growth of OSCC cells, which implies a suppressor role for miR-370. The enhancement of anchorage-independent growth of OSCC cells through miR-370 inhibiting can be reduced by knockdown of IRS-1 expression. CONCLUSION: This study concludes that miR-370 is able to target IRS-1 for oral tumorigenesis.