Tyrosine Phosphorylation Of Insulin Receptor Research Articles

Arsenic-induced IGF-1 signaling impairment and neurite shortening: The protective roles of IGF-1 through the PI3K/Akt axis.

We recently reported that arsenic caused insulin resistance in differentiated human neuroblastoma SH-SY5Y cells. Herein, we further investigated the effects of sodium arsenite on IGF-1 signaling, which shares downstream signaling with insulin. A time-course experiment revealed that sodium arsenite began to decrease IGF-1-stimulated Akt phosphorylation on Day 3 after treatment, indicating that prolonged sodium arsenite treatment disrupted the neuronal IGF-1 response. Additionally, sodium arsenite decreased IGF-1-stimulated tyrosine phosphorylation of the IGF-1 receptor β (IGF-1Rβ) and its downstream target, insulin receptor substrate 1 (IRS1). These results suggested that sodium arsenite impaired the intrinsic tyrosine kinase activity of IGF-1Rβ, ultimately resulting in a reduction in tyrosine-phosphorylated IRS1. Sodium arsenite also reduced IGF-1 stimulated tyrosine phosphorylation of insulin receptor β (IRβ), indicating the potential inhibition of IGF-1R/IR crosstalk by sodium arsenite. Interestingly, sodium arsenite also induced neurite shortening at the same concentrations that caused IGF-1 signaling impairment. A 24-h IGF-1 treatment partially rescued neurite shortening caused by sodium arsenite. Moreover, the reduction in Akt phosphorylation by sodium arsenite was attenuated by IGF-1. Inhibition of PI3K/Akt by LY294002 diminished the protective effects of IGF-1 against sodium arsenite-induced neurite retraction. Together, our findings suggested that sodium arsenite-impaired IGF-1 signaling, leading to neurite shortening through IGF-1/PI3K/Akt.

289-OR: Defining Pathway-Selective Insulin Resistance in Type 2 Diabetes Using iPS Cell-Derived Hepatocytes

Hepatic insulin resistance is central to type 2 diabetes (T2D), fatty liver disease, and metabolic syndrome. To identify the fundamental, cell-autonomous defects underlying hepatic insulin resistance in T2D, we have used inducible human pluripotent stem (iPS) cells derived from 16 individuals with and without T2D differentiated to hepatocytes (iHeps) and studied in vitro. Consistent with pathway selective insulin resistance, we find that insulin failed to suppress gene expression of critical gluconeogenic enzymes (PCK1, G6PC) but continued to stimulate increased expression of the lipogenic enzymes, fatty acid synthase and acetyl Co-A carboxylase 1 in T2D iHeps. iHeps from T2D also displayed reduced insulin receptor tyrosine phosphorylation and reduced phosphorylation through the IRS/Akt pathway. Global phosphoproteomics in control and T2D iHeps identified 378 unique phosphosites regulated by insulin, and many dysregulated phosphosites within the classical insulin signaling pathway (IRS, AKT1/2, GSK3, FOXO1, RPS6KB1, TSC2) and components of gene transcription regulation, membrane trafficking, Rho-GTPases, vesicle transport. Using a kinome-wide data set created with synthetic peptide libraries to profile almost all functional known human serine/threonine kinases, we found the alterations in insulin signaling mapped to AKT, P70S6K, P90S6K, RSK, PKCτ, and SGK kinases. Thus, iHeps from T2D patients show pathway-selective insulin resistance with a loss of suppression of gluconeogenesis by insulin yet persistent activation of lipogenesis, even in vitro, in the absence of any circulating factor exposure. T2D iHeps also show a dysregulated phosphorylation network that provides new clues to the kinases whose action is altered in T2D and hepatic insulin resistance. Disclosure A.Gattu: None. A.Krook: None. J.R.Zierath: None. M.Mann: None. C.Kahn: None. Funding National Institutes of Health (T32DK007260-46)

Phosphatase protector alpha4 (α4) is involved in adipocyte maintenance and mitochondrial homeostasis through regulation of insulin signaling

Insulin signaling is mediated via a network of protein phosphorylation. Dysregulation of this network is central to obesity, type 2 diabetes and metabolic syndrome. Here we investigate the role of phosphatase binding protein Alpha4 (α4) that is essential for the serine/threonine protein phosphatase 2A (PP2A) in insulin action/resistance in adipocytes. Unexpectedly, adipocyte-specific inactivation of α4 impairs insulin-induced Akt-mediated serine/threonine phosphorylation despite a decrease in the protein phosphatase 2A (PP2A) levels. Interestingly, loss of α4 also reduces insulin-induced insulin receptor tyrosine phosphorylation. This occurs through decreased association of α4 with Y-box protein 1, resulting in the enhancement of the tyrosine phosphatase protein tyrosine phosphatase 1B (PTP1B) expression. Moreover, adipocyte-specific knockout of α4 in male mice results in impaired adipogenesis and altered mitochondrial oxidation leading to increased inflammation, systemic insulin resistance, hepatosteatosis, islet hyperplasia, and impaired thermogenesis. Thus, the α4 /Y-box protein 1(YBX1)-mediated pathway of insulin receptor signaling is involved in maintaining insulin sensitivity, normal adipose tissue homeostasis and systemic metabolism.

Extracellular vesicle biomarkers for cognitive impairment in Parkinson's disease.

Besides motor symptoms, many individuals with Parkinson's disease develop cognitive impairment perhaps due to coexisting α-synuclein and Alzheimer's disease pathologies and impaired brain insulin signalling. Discovering biomarkers for cognitive impairment in Parkinson's disease could help clarify the underlying pathogenic processes and improve Parkinson's disease diagnosis and prognosis. This study used plasma samples from 273 participants: 103 Parkinson's disease individuals with normal cognition, 121 Parkinson's disease individuals with cognitive impairment (81 with mild cognitive impairment, 40 with dementia) and 49 age- and sex-matched controls. Plasma extracellular vesicles enriched for neuronal origin were immunocaptured by targeting the L1 cell adhesion molecule, then biomarkers were quantified using immunoassays. α-Synuclein was lower in Parkinson's disease compared to control individuals (P = 0.004) and in cognitively impaired Parkinson's disease individuals compared to Parkinson's disease with normal cognition (P < 0.001) and control (P < 0.001) individuals. Amyloid-β42 did not differ between groups. Phosphorylated tau (T181) was higher in Parkinson's disease than control individuals (P = 0.003) and in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P < 0.001) and controls (P < 0.001). Total tau was not different between groups. Tyrosine-phosphorylated insulin receptor substrate-1 was lower in Parkinson's disease compared to control individuals (P = 0.03) and in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.02) and controls (P = 0.01), and also decreased with increasing motor symptom severity (P = 0.005); serine312-phosphorylated insulin receptor substrate-1 was not different between groups. Mechanistic target of rapamycin was not different between groups, whereas phosphorylated mechanistic target of rapamycin trended lower in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.05). The ratio of α-synuclein to phosphorylated tau181 was lower in Parkinson's disease compared to controls (P = 0.001), in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P < 0.001) and decreased with increasing motor symptom severity (P < 0.001). The ratio of insulin receptor substrate-1 phosphorylated serine312 to insulin receptor substrate-1 phosphorylated tyrosine was higher in Parkinson's disease compared to control individuals (P = 0.01), in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.02) and increased with increasing motor symptom severity (P = 0.003). α-Synuclein, phosphorylated tau181 and insulin receptor substrate-1 phosphorylated tyrosine contributed in diagnostic classification between groups. These findings suggest that both α-synuclein and tau pathologies and impaired insulin signalling underlie Parkinson's disease with cognitive impairment. Plasma neuronal extracellular vesicles biomarkers may inform cognitive prognosis in Parkinson's disease.

CD36 gene deletion reduces muscle insulin sensitivity in mice by up-regulating PTP1B expression

To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. Wild-type (WT) mice and systemic CD36 knockout (CD36-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. CD36-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36-/- mice (P>0.05). In the muscle tissue of CD36-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36-/- mice. CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.

Development of Botanical Pesticides: Exploration on the Phenotype of Vestigial Wings of Insect Pests Induced by Plant Natural Products or Their Derivatives by Blocking Tyrosine Phosphorylation of Insulin Receptor 1

Unlike faster-acting conventional insecticides, some botanical insecticides exhibit growth inhibitory activity against some insect pests. One of the distinguishing features of growth inhibitory activity appears to be in malformed moths with vestigial wings. However, the molecular mechanism underlying vestigial wings of insect pests induced by plant natural products or their derivatives is still elusive. In this work, based upon the phenotype of the vestigial wings of Mythimna separata Walker (as a model pest) induced by a podophyllotoxin derivative 2a (as a model compound), we found that compound 2a not only resulted in 22.1% of malformed moths with vestigial wings but also significantly decreased the fecundity of vestigial-winged female moths in the P generation; the trait of vestigial wings caused by 2a in the P generation can be inherited by the F1 generation; compound 2a may target insulin receptor 1 (InR1), suppress the InR1 mRNA level, and block InR1-pY1229 and InR1-pY1233/1234 phosphorylation levels in a tissue-specific manner "head/thorax/wing tissues". Notably, compound 2a can also induce the vestigial wings of Spodoptera frugiperda (another seriously harmful migratory lepidoptera pest). It is noteworthy that this insect insulin receptor can be used as a new kind of target receptors for the design of novel green insecticides.

Bardet-Biedl syndrome proteins regulate intracellular signaling and neuronal function in patient-specific iPSC-derived neurons

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1M390R and BBS10C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of BBS1M390R hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that BBS1M390R and BBS10C91fsX95 mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS10C91fsX95 neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in BBS1M390R and BBS10C91fsX95 iPSC-derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis.

PTP1B Inhibitors as Potential Target for Type II Diabetes

Diabetes mellitus is a metabolic disorder characterized by elevated blood sugar levels resulting primarily from either a lack of insulin production or insulin resistance. Insulin elicits its effects by activating the Insulin Receptor (IR) which is responsible for glucose uptake and promotes insulin signaling. Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of IR signaling and is responsible for dephosphorylating the tyrosine phosphorylated IR. Inhibition of PTP1B could thus promote glucose uptake [1]. Early PTP1B inhibitors, such as charged active site-directed analogs, showed poor cell membrane permeability. Subsequent uncharged mimetics overcome this initial hurdle however specificity over T-cell protein tyrosine phosphatase (TCPTP) was another challenge that needed to be overcome. Herein is an overview of the challenges faced for targeting PTP1B. These include the evaluation of compounds that target the active site, allosteric site, and finally covalent inhibition of this potentially viable drug target.

Astaxanthin Inhibits p70 S6 Kinase 1 Activity to Sensitize Insulin Signaling.

Astaxanthin (AST) is a carotenoid with therapeutic values on hyperglycemia and diabetic complications. The mechanisms of action of AST remain incompletely understood. p70 S6 kinase 1 (S6K1) is a serine/threonine kinase that phosphorylates insulin receptor substrate 1 (IRS-1)S1101 and desensitizes the insulin receptor (IR). Our present study aims to determine if AST improves glucose metabolisms by targeting S6K1. Western blot analysis revealed that AST inhibited the phosphorylation of two S6K1 substrates, S6S235/236 and IRS-1S1101, but enhanced the phosphorylation of AKTT308, AKTS473, and S6K1T389 by feedback activation of the phosphatidylinositol-3 (PI-3) kinase in 3T3-L1 adipocytes and L6 myotubes. In vitro kinase assays revealed that AST inhibited S6K1 activity with an IC50 value of approximately 13.8 μM. AST increased insulin-induced IR tyrosine phosphorylation and IRS-1 binding to the p85 subunit of PI-3 kinase. Confocal microscopy revealed that AST increased the translocation of the glucose transporter 4 (GLUT4) to the plasma membrane in L6 cells. Glucose uptake assays using a fluorescent dye, 2-NBDG (2-N-(Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), revealed that AST increased glucose uptake in 3T3-L1 adipocytes and L6 myotubes under insulin resistance conditions. Our study identifies S6K1 as a previously unrecognized molecular target of AST and provides novel insights into the mechanisms of action of AST on IR sensitization.

Loss of CD36 impairs hepatic insulin signaling by enhancing the interaction of PTP1B with IR.

A contradictory role of CD36 in insulin resistance was found to be related to the nutrient state. Here, we examined that the physiological functions of CD36 in insulin signal transduction in mice fed a low-fat diet. CD36 deficiency led to hepatic insulin resistance and decreased insulin-stimulated tyrosine phosphorylation of insulin receptor β (IRβ) in mice fed a low-fat diet. The ability of insulin to bind with IR did not differ between WT and CD36-deficient hepatocytes. CD36 formed a complex with IRβ and dissociation of CD36/Fyn complex or inhibition of Fyn only partially reversed the effects of CD36 on hepatic insulin signaling. Furthermore, we found that CD36 deficiency led to abnormally increased hepatic protein-tyrosine phosphatase 1B (PTP1B) expression and enhanced PTP1B and IR interactions, which contributed to the decreased insulin signaling and disordered glucose metabolism. In addition, increased endoplasmic reticulum (ER) stress was found in the livers of the CD36-deficient mice, while inhibited ER stress normalized the PTP1B expression and restored insulin signaling in the CD36-deficient mice. Our findings suggest that the loss of CD36 impairs hepatic insulin signaling by enhancing the PTP1B/IR interaction that is induced by ER stress, indicating a possible critical step in the progression of hepatic insulin resistance.

Abstract P2-06-03: Insulin receptor isoform signaling in breast cancer

Abstract Recent data shows that insulin receptor (IR) and the type I insulin-like growth factor receptor (IGF1R) play important roles in breast cancer cell biology. Targeting only IGF1R has not been successful perhaps due to compensation by IR. IR exists in two isoforms, fetal isoform IR-A is a splice variant of IR which excludes exon 11. The adult/metabolic isoform IR-B is the predominant species expressed in normal tissues, while the fetal form IR-A is more highly expressed in breast cancer. IR-A mRNA in endocrine resistant cells is expressed at levels 24-fold higher than IGF1R expression (Gradishar, et al. Clin Cancer Res 22:301 2016 PMID: 26324738). In addition to its homodimer, IR-A can also dimerize with IGF1R to form a hybrid. Homodimer IR-A responds to IGF-II and insulin, while IGF1R/IR-A hybrid can also respond to IGF-I. Previous studies in our lab showed down regulation of IGFIR increases the sensitivity of breast cancer cells to insulin. To further investigate the roles of IR-A and IR-B in breast cancer biology, we cloned IR-A and IR-B isoforms into pLV-mCherry and pLJM1EGFP lentiviral expression vectors. Then MCF-7L breast cancer cells were infected with IR-A, IR-A vector control, IR-B and IR-B vector control. Both pooled and single clones were studied. Our results showed that both IR-A and IR-B were highly expressed in MCF-7L cells with the introduced IR tagged species present at a higher molecular weight confirmed by immunoprecipitation and immunoblot. The lower migrating species was endogenous IR. IR-A-pool/Clones had strong basal IR tyrosine phosphorylation, while IR-B-pool/clones did not. This basal phosphorylation did not activate downstream signaling as measured by pAKT and pErk1,2. IR-A basal phosphorylation was completely inhibited by BMS754807 (0.3μM), a dual IGFIR/IR tyrosine kinase inhibitor. Total levels of IRS1, IRS2, IGF1R, ERa, AKT and Erk1,2 levels were not changed in the over-expressing cells. Ligands-IGF-I (5nM), IGF-II (10nM) and Insulin (10nM) stimulated IGF1R/IR, IRS, AKT and Erk1,2 phosphorylation in IR-A-pool and high expression clone IR-A-G5. Cells expressing IR-A activated downstream signaling (IRS, AKT and Erk1,2) at 0.1nM insulin, while IR-A vector control cells required 1nM insulin. IR-B-pool overexpressing cells were not more sensitive to insulin compared to parental cells. Insulin significantly increased Erk1,2 phosphorylation in IR-A-G5 cell while IGF-I stimulation was minimal. In contrast, Erk1,2 phosphorylation in parental cells and vector control cells was primarily mediated by IGF-I, not insulin. IR-A overexpressing cells were stimulated in monolayer growth by insulin. These data show that IR-A expression, as seen in endocrine resistant breast cancer cells, sensitizes breast cancers to low concentrations of insulin. Thus, IR-A expression could serve as a target in breast cancer. Citation Format: Zhang X, Chan JY, Pan Y, Dong C, Yee D. Insulin receptor isoform signaling in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-03.

Utility of Neuronal-Derived Exosomes to Examine Molecular Mechanisms That Affect Motor Function in Patients With Parkinson Disease

Exenatide, a glucagon-like peptide 1 agonist used in type 2 diabetes, was recently found to have beneficial effects on motor function in a randomized, placebo-controlled trial in Parkinson disease (PD). Accumulating evidence suggests that impaired brain insulin and protein kinase B (Akt) signaling play a role in PD pathogenesis; however, exploring the extent to which drugs engage with putative mechnisms in vivo remains a challenge. To assess whether participants in the Exenatide-PD trial have augmented activity in brain insulin and Akt signaling pathways. Serum samples were collected from 60 participants in the single-center Exenatide-PD trial (June 18, 2014, to June 16, 2016), which compared patients with moderate PD randomized to 2 mg of exenatide once weekly or placebo for 48 weeks followed by a 12-week washout period. Serum extracellular vesicles, including exosomes, were extracted, precipitated, and enriched for neuronal source by anti-L1 cell adhesion molecule antibody absorption, and proteins of interest were evaluated using electrochemiluminescence assays. Statistical analysis was performed from May 1, 2017, to August 31, 2017. The main outcome was augmented brain insulin signaling that manifested as a change in tyrosine phosphorylated insulin receptor substrate 1 within neuronal extracellular vesicles at the end of 48 weeks of exenatide treatment. Additional outcome measures were changes in other insulin receptor substrate proteins and effects on protein expression in the Akt and mitogen-activated protein kinase pathways. Sixty patients (mean [SD] age, 59.9 [8.4] years; 43 [72%] male) participated in the study: 31 in the exenatide group and 29 in the placebo group (data from 1 patient in the exenatide group were excluded). Patients treated with exenatide had augmented tyrosine phosphorylation of insulin receptor substrate 1 at 48 weeks (0.27 absorbance units [AU]; 95% CI, 0.09-0.44 AU; P = .003) and 60 weeks (0.23 AU; 95% CI, 0.05-0.41 AU; P = .01) compared with patients receiving placebo. Exenatide-treated patients had elevated expression of downstream substrates, including total Akt (0.35 U/mL; 95% CI, 0.16-0.53 U/mL; P < .001) and phosphorylated mechanistic target of rapamycin (mTOR) (0.22 AU; 95% CI, 0.04-0.40 AU; P = .02). Improvements in Movement Disorders Society Unified Parkinson's Disease Rating Scale part 3 off-medication scores were associated with levels of total mTOR (F4,50 = 5.343, P = .001) and phosphorylated mTOR (F4,50 = 4.384, P = .04). The results of this study are consistent with target engagement of brain insulin, Akt, and mTOR signaling pathways by exenatide and provide a mechanistic context for the clinical findings of the Exenatide-PD trial. This study suggests the potential of using exosome-based biomarkers as objective measures of target engagement in clinical trials using drugs that target neuronal pathways.

Gingerenone A Sensitizes the Insulin Receptor and Increases Glucose Uptake by Inhibiting the Activity of p70 S6 Kinase.

The bioactive constituents in ginger extract are responsible for anti-hyperglycemic effects and the underlying mechanisms are incompletely understood. Gingerenone A (Gin A) has been identified as an inhibitor of p70 S6 (S6K1), a kinase that plays a critical role in the pathogenesis of insulin resistance. This study aims to evaluate if Gin A can sensitize the insulin receptor by inhibiting S6K1 activity. Western blot analysis reveals that Gin A induces phosphatidylinositide-3 kinase (PI3K) feedback activation in murine 3T3-L1 adipocytes and rat L6 myotubes, as evidenced by increased AKTS473 and S6K1T389 but decreases S6S235/236 and insulin receptor substrate 1 (IRS-1)S1101 phosphorylation. Western blot and immunoprecipitation analysis reveal that Gin A increases insulin receptor tyrosine phosphorylation in L6 myotubes and IRS-1 binding to the PI3K in 3T3-L1 adipocytes. Confocal microscopy reveals that Gin A enhances insulin-induced translocation of glucose transporter 4 (GLUT4) into the cell membrane in L6 cells. 2-NBDG (2-N-(Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) Fluorescent assay reveals that Gin A enhances insulin-stimulated glucose uptake in 3T3-L1 adipocytes and L6 myotubes. Gin A overcomes insulin resistance and increases glucose uptake by inhibiting S6K1 activity. Gin A or other plant-derived S6K1 inhibitors could be developed as novel antidiabetic agents.

Sub-toxic exposure to lindane activates redox sensitive kinases and impairs insulin signaling in muscle cell culture: The possible mechanism of lindane-induced insulin resistance

Lindane exposure is claimed to be involved in pathogenesis of type 2 diabetes mellitus (T2DM) and insulin resistance state by an as yet unknown mechanism. The redox sensitive kinases (RSKs) and heat shock proteins (HSPs) interfere with insulin signaling and induce insulin resistance. The present study was designed to explore the mechanism of insulin resistance induced by sub-toxic lindane exposure. In an in vitro study, exposure to 60 mg/L and 120 mg/L of lindane for 18 h on rat L6 myoblasts derived myotubes significantly increased malondialdehyde level & superoxide dismutase activity, decreased total antioxidant level and insulin-induced glucose uptake in a dose dependent manner. The extent of activation of RSKs and HSP25 as measured by western blot from the extent of phosphorylation of IκBα, p38 MAPK, JNK & HSP25 in lindane-exposed myotubes was higher. HSP70 was induced and insulin signaling as measured from tyrosine phosphorylation of insulin receptor (IR) & insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Akt was attenuated in comparison to those in untreated myotubes. We conclude that sub-toxic lindane exposure induces oxidative stress, activates RSKs & HSP25 and induces HSP25. These in turn, impair insulin signaling to impart insulin resistance in myotubes induced by sub-toxic lindane exposure.

Treatment of type 2 diabetes mellitus via reversing insulin resistance and regulating lipid homeostasis invitro and invivo using cajanonic acid A.

The present study investigated the effects of cajanonic acid A (CAA), extracted from the leaves of Cajanus cajan (L.) Millsp with a purity of 98.22%, on the regulatory mechanisms of glucose and lipid metabolism. HepG2 cells transfected with a protein-tyrosine phosphatase 1B (PTP1B) overexpression plasmid were established. The cells, induced with insulin resistance by dexamethasone (Dex) treatment, together with type 2 diabetes mellitus (T2DM) model rats and ob/ob mice, were used in the present study. The effects of CAA treatment on the differentiation of 3T3-L1 adipocytes were determined using Oil Red O. The expression levels of insulin signaling factors were detected via reverse transcription-quantitative polymerase chain reaction and western blot analyses. The results revealed that the overexpression of PTP1B contributed to insulin resistance, which was reversed by CAA treatment via inhibiting the activity of PTP1B and by regulating the expression of associated insulin signaling factors. The treatment of cell lines with Dex led to increased expression of PTP1B but decreased glucose consumption, and decreased tyrosine phosphorylation of insulin receptor, insulin receptor substrate 1, and phosphoinositide 3-kinase. Treatment with CAA not only reduced the fasting blood glucose levels and protected organs from damage, but also reduced the serum fasting levels of total cholesterol, triglycerides and low-density lipoprotein cholesterol in the T2DM rats. CAA treatment also inhibited adipocyte differentiation and decreased the mRNA levels of various adipogenic genes. Furthermore, CAA treatment restored the transduction of insulin signaling by regulating the expression of PTP1B and associated insulin signaling factors. Treatment with CAA also reduced the problems associated with hyperglycemia and hyperlipidemia. In conclusion, CAA may be used to cure T2DM via restoring insulin resistance and preventing obesity.

High-fat diet-induced obesity and insulin resistance in CYP4a14-/- mice is mediated by 20-HETE.

20-Hydroxyeicosatetraenoic acid (20-HETE) has been shown to positively correlate with body mass index, hyperglycemia, and plasma insulin levels. This study seeks to identify a causal relationship between 20-HETE and obesity-driven insulin resistance. Cyp4a14-/- male mice, a model of 20-HETE overproduction, were fed a regular or high-fat diet (HFD) for 15 wk. 20-SOLA [2,5,8,11,14,17-hexaoxanonadecan-19-yl 20-hydroxyeicosa-6( Z),15( Z)-dienoate], a 20-HETE antagonist, was administered from week 0 or week 7 of HFD. HFD-fed mice gained significant weight (16.7 ± 3.2 vs. 3.8 ± 0.35 g, P < 0.05) and developed hyperglycemia (157 ± 3 vs. 121 ± 7 mg/dl, P < 0.05) and hyperinsulinemia (2.3 ± 0.4 vs. 0.5 ± 0.1 ng/ml, P < 0.05) compared with regular diet-fed mice. 20-SOLA attenuated HFD-induced weight gain (9.4 ± 1 vs. 16.7 ± 3 g, P < 0.05) and normalized the hyperglycemia (157 ± 7 vs. 102 ± 5 mg/dl, P < 0.05) and hyperinsulinemia (1.1 ± 0.1 vs. 2.3 ± 0.4 ng/ml, P < 0.05). The impaired glucose homeostasis and insulin resistance in HFD-fed mice evidenced by reduced insulin and glucose tolerance were also ameliorated by 20-SOLA. Circulatory and adipose tissue 20-HETE levels significantly increased in HFD-fed mice correlating with impaired insulin signaling, including reduction in insulin receptor tyrosine (Y972) phosphorylation and increased serine (S307) phosphorylation of the insulin receptor substrate-1 (IRS-1). 20-SOLA treatments prevented changes in insulin signaling. These findings indicate that 20-HETE contributes to HFD-induced obesity, insulin resistance, and impaired insulin signaling.

Hepatic fatty acid biosynthesis in KK-Ay mice is modulated by administration of persimmon peel extract: A DNA microarray study.

ScopePreviously, we showed that the intake of a persimmon peel (PP) extract altered hepatic gene expression associated with the insulin signaling pathway and enhanced tyrosine phosphorylation of insulin receptors in nonobese type 2 diabetic Goto‐Kakizaki rats. Our objective was to evaluate the effect of fat‐soluble PP extract on obese type 2 diabetic KK‐Ay mice with insulin resistance.Methods and results KK‐Ay mice were fed a diet mixed with 0.1% of the extract for 8 weeks. The total ketone body levels in the plasma of PP extract‐fed mice were significantly lower than those in the normal diet‐fed mice. Hepatic nonesterified palmitic acid content was higher in the PP extract‐fed mice than in normal diet‐fed mice. The hepatic gene expression profiles of the treated mice indicated upregulation of fatty acid synthesis and downregulation of inflammation‐associated genes, predicting SREBP‐1c and PPARγ activation.ConclusionThese results suggest that the PP extract enhances hepatic fatty acid synthesis via SREBP‐1c and PPARγ, as well as anti‐inflammatory activity in KK‐Ay mice.

Regulation of Insulin Receptor Pathway and Glucose Metabolism by CD36 Signaling.

During reduced energy intake, skeletal muscle maintains homeostasis by rapidly suppressing insulin-stimulated glucose utilization. Loss of this adaptation is observed with deficiency of the fatty acid transporter CD36. A similar loss is also characteristic of the insulin-resistant state where CD36 is dysfunctional. To elucidate what links CD36 to muscle glucose utilization, we examined whether CD36 signaling might influence insulin action. First, we show that CD36 deletion specific to skeletal muscle reduces expression of insulin signaling and glucose metabolism genes. It decreases muscle ceramides but impairs glucose disposal during a meal. Second, depletion of CD36 suppresses insulin signaling in primary-derived human myotubes, and the mechanism is shown to involve functional CD36 interaction with the insulin receptor (IR). CD36 promotes tyrosine phosphorylation of IR by the Fyn kinase and enhances IR recruitment of P85 and downstream signaling. Third, pretreatment for 15 min with saturated fatty acids suppresses CD36-Fyn enhancement of IR phosphorylation, whereas unsaturated fatty acids are neutral or stimulatory. These findings define mechanisms important for muscle glucose metabolism and optimal insulin responsiveness. Potential human relevance is suggested by genome-wide analysis and RNA sequencing data that associate genetically determined low muscle CD36 expression to incidence of type 2 diabetes.

Control of hyperglycemia in male mice by leflunomide: mechanisms of action.

p70 S6 kinase (S6K1) is a serine/threonine kinase that phosphorylates the insulin receptor substrate-1 (IRS-1) at serine 1101 and desensitizes insulin receptor signaling. S6K1 hyperactivation due to overnutrition leads to hyperglycemia and type 2 diabetes. Our recent study showed that A77 1726, the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide, is an inhibitor of S6K1. Whether leflunomide can control hyperglycemia and sensitize the insulin receptor has not been tested. Here we report that A77 1726 increased AKTS473/T308 and S6K1T389 phosphorylation but decreased S6S235/236 and IRS-1S1101 phosphorylation in 3T3-L1 adipocytes, C2C12 and L6 myotubes. A77 1726 increased insulin receptor tyrosine phosphorylation and binding of the p85 subunit of the PI-3 kinase to IRS-1. A77 1726 enhanced insulin-stimulated glucose uptake in L6 myotubes and 3T3-L1 adipocytes, and enhanced insulin-stimulated glucose transporter type 4 (GLUT4) translocation to the plasma membrane of L6 cells. Finally, we investigated the anti-hyperglycemic effect of leflunomide on ob/ob and high-fat diet (HFD)-induced diabetes mouse models. Leflunomide treatment normalized blood glucose levels and overcame insulin resistance in glucose and insulin tolerance tests in ob/ob and HFD-fed mice but had no effect on mice fed a normal chow diet (NCD). Leflunomide treatment increased AKTS473/T308 phosphorylation in the fat and muscle of ob/ob mice but not in normal mice. Our results suggest that leflunomide sensitizes the insulin receptor by inhibiting S6K1 activity in vitro, and that leflunomide could be potentially useful for treating patients with both RA and diabetes.

Arsenic impairs insulin signaling in differentiated neuroblastoma SH-SY5Y cells

A strong correlation between chronic arsenic exposure and neuropsychological disorders leads to a growing concern about a potential risk of arsenic related neurodegeneration. Evidently, brain insulin signaling contributes to physiological effects, including energy homeostasis, and learning and memory. Arsenic has been shown to impair insulin signaling in adipocytes and myocytes, however, this impairment has not yet been explored in neurons. Here we showed that NaAsO2 caused significant reduction in basal levels of glucose, plasma membrane glucose transporter, GLUT 3 and Akt phosphorylation in differentiated human neuroblastoma SH-SY5Y cells. NaAsO2 significantly decreased insulin-mediated glucose uptake, as well as GLUT1 and 3 membrane translocation. Furthermore, the ability of insulin to increase Akt phosphorylation, a well-recognized insulin signaling response, was significantly lessened by NaAsO2 treatment. In addition, the classical tyrosine phosphorylation response of insulin was reduced by NaAsO2, as evidenced by reduction of insulin-induced tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1(IRS-1). Moreover, NaAsO2 lowered the ratio of p110, a catalytic subunit to p85, a regulatory subunit of PI3K causing an imbalance between p110 and p85, the conditions reported to contribute to insulin sensitivity. Additionally, increment of IRS-1 interaction with GSK3β, and p85-PI3K were observed in NaAsO2 treated cells. These molecular modulations may be mechanistically attributed to neuronal insulin signaling impairment by arsenic.