Insulin Receptor Kinase Activity Research Articles

Profiling of secondary metabolite and evaluation of anti-diabetic potency of Crotalaria quinquefolia (L): In-vitro, in-vivo, and in-silico approaches

Traditional medicinal plants have played a promising role in the human health system. In folklore medicine, Crotalaria quinquefolia L. is used to treat fever, pain, eczema, impetigo, lung infections, scabies. The present investigation was executed to identify secondary metabolites responsible for anti-diabetic potential of C. quinquefolia L. leaf extract along with their possible mechanistic pathways. The anti-hyperglycemic activity was assessed by in vitro α-amylase and α-glucosidase inhibitory assays and an in vivo oral glucose tolerance test and diabetogenic effect of streptozotocin in mice, followed by an integrative computational analysis. A total of 23 compounds were identified through GCMS and HPLC. The extract showed potent in-vitro α-amylase and α-glucosidase suppressive activity with IC50 values of 12.8 ± 0.1 µg/mL and 36.3 ± 0.07 µg/mL, respectively. In an in vivo oral glucose tolerance test, the extract (400 mg/kg body weight) prompted blood glucose levels to plummet by 18.9 % after 30 min, compared to the normal control and streptozotocin induced diabetes test, maximum glucose reduction was observed 11.67 % by dose of 200 mg/kg compared to the control; glibenclamide and extract (400 mg/kg) reduced blood glucose levels by 1.3 % and 16.7 %, respectively, compared to diabetic control at the end of the trial. Additionally, among the identified compounds, myricetin, quercetin, rutin, and kaempferol revealed good binding affinity as well as stability with the studied anti-diabetic proteins in docking and molecular dynamics simulation studies. Furthermore, QSAR analysis and network pharmacology studies of the identified compounds divulged enhanced insulin secretion stimulation, insulin receptor kinase activity, PPARγ expression; enzyme inhibition (α-glucosidase, α-amylase) and protection of the pancreas -mediated antidiabetic effects. Besides, they proved strong inhibitory potential against the studied antidiabetic proteins in other computational analysis. Based on the present findings, it can be affirmed that C. quinquefolia extract possesses anti-diabetic activity.

Pancreastatin inhibitor PSTi8 ameliorates streptozotocin-induced diabetes by suppressing hepatic glucose production

Elevated plasma glucose concentration, as a consequence of excessive hepatic glucose production, plays a pivotal role in the development of diabetes. A chromogranin A-derived diabetogenic peptide Pancreastatin (PST) enhances hepatic glucose output leading to diabetes. Therefore, here we probed the role of PSTi8, a PST inhibitor in ameliorating diabetes by investigating the effect of high glucose (HG) or PST on glucose metabolism. Further, we also explored the action mechanism of the underlying anti-hyperglycemic effect of PSTi8. PSTi8 treatment rescue cultured L6 and HepG2 cells from HG and PST-induced insulin resistance, respectively. It also enhances insulin receptor kinase activity by interacting with the insulin receptor and enhancing GLUT4 translocation and glucose uptake. Thus, our in-silico and in-vitro data support the PST-dependent and independent activity of PSTi8. Additionally, PSTi8 treatment in streptozotocin-induced diabetic rats improved glucose tolerance by lowering blood glucose and plasma PST levels. Concomitantly, the treated animals exhibited reduced hepatic glucose production accompanied by downregulation of hepatic gluconeogenic genes PEPCK and G6Pase. PSTi8-treated rats also exhibited enhanced hepatic glycogen in line with reduced plasma glucagon concentrations. Consistently, improved plasma insulin levels in PSTi8-treated rats enhanced skeletal muscle glucose disposal via enhanced P-Akt expression. In summary, these findings suggest PSTi8 has anti-hyperglycemic properties with enhanced skeletal muscle glucose disposal and reduced hepatic gluconeogenesis both PST dependent as well as independent.

Upregulation of GLUT4 and PI3K, and downregulation of GSK3 mediate the anti-hyperglycemic effects of proanthocyanidins.

Diabetes mellitus is the most common chronic metabolic disorder worldwide. The present study was designed to investigate the potential role of cinnamon bark extract oligomeric proanthocyanidins (OPCs) in controlling streptozotocin (STZ)-induced hyperglycemia and to clarify the underlying molecular mechanisms underlying its effects. For this purpose, 60 male rats were equally divided into six groups as follows: The normal control group; OPC control group (non-diabetic rats treated with OPC at 300 mg/kg orally for 21 days); the untreated diabetic control group; the wortmannin control group [diabetic rats treated with wortmannin at 1 mg/kg, intraperitoneal (i.p.) on the final day of the experiment]; the OPC diabetic group (diabetic rats treated with OPC at 300 mg/kg orally for 21 days); and the OPC diabetic + wortmannin co-treated group (diabetic rats treated with OPC at 300 mg/kg/day for 21 consecutive days and then 24 h after the final OPC dose treated with a single wortmannin injection at 1 mg/kg, i.p.). The results indicated that OPC ameliorated the diabetic state, as evidenced by a significant decrease in serum glucose levels, and a significant increase in the levels of insulin, amylin, insulin receptor phosphorylation, glycogen and glucose transporter-4 translocation; it also improved the lipid profile in STZ-diabetic rats. On the whole, the findings of the present study provide biochemical evidence that OPC treatment is effective as an anti-diabetic and anti-hyperlipidemic agent by enhancing glucose uptake through the activation of insulin receptor kinase activity and the PI3K/Akt pathway.

The effects of different carbon sources on growth, tyrosine kinase activity and gene expression of insulin receptor 1 in GIFT tilapia

A feeding trial was conducted to evaluate the effects of different sugar sources on growth performance, serum biochemistry, immunity, insulin receptor 1 and tyrosine kinase activity of GIFT tilapia. Four hundred and eighty fish with specifications of 31.70±0.68 g were selected and placed in 16 aquariums. The experiment was divided into four treatment with four replicates of thirty fish per tank. After 50 days of feeding, the results were as follows: From the indexes of final average weight, weight gain rate and specific growth rate, the growth performance was sucrose, glucose, starch and dextrin group in turn, and the sucrose group performed best (P<0.05). The activcities of acid phosphotase, alkaline phosphotase, superoxide dismutase and lysozyme in sucrose group were significantly higher than that of the other three groups (P<0.05). The serum glucose level of sucrose group and glucose group is significantly higher than that of dextrin group and starch group (P < 0.05). In terms of liver glycogen level, the content of sucrose group was significantly higher than that of dextrin group and starch group (P < 0.05). In terms of muscle glycogen level, the content of sucrose group was the highest and that of starch group was the lowest. The tyrosine kinase activity of glucose group was the highest and significantly different from dextrin group (P< 0.05). The tyrosine kinase activity in muscle was always higher than that in liver (P < 0.05). The IR-1 mRNA in starch group and dextrin group was significantly higher than that in sucrose group. Different sugar source diets had a significant effect on the expression of IR-1 mRNA in the muscle of tilapia. In conclusion, compared with dextrin and starch, sucrose and glucose can be better utilized by GIFT tilapia and show good growth performance. Key words: GIFT tilapia; Carbohydrates; Growth; Immunity; Insulin Receptor 1; Tyrosine Kinase

Arsenic disrupts neuronal insulin signaling through increasing free PI3K-p85 and decreasing PI3K activity

Previously, we reported that prolonged arsenic exposure impaired neuronal insulin signaling. Here we have further identified novel molecular mechanisms underlying neuronal insulin signaling impairment by arsenic. Arsenic treatment altered insulin dose-response curve and reduced maximum insulin response in differentiated human neuroblastoma SH-SY5Y cells, suggesting that arsenic hindered neuronal insulin signaling in a non-competitive like manner. Mechanistically, arsenic suppressed insulin receptor (IR) kinase activity, as witnessed by a decreased insulin-activated autophosphorylation of IR at Y1150/1151. Arsenic decreased the level of insulin receptor substrate 1 (IRS1) but increased the protein ratio between PI3K regulatory subunit, p85, and PI3K catalytic subunit, p110. Interestingly, co-immunoprecipitation demonstrated that arsenic did not alter a level of PI3K-p110/PI3K-p85 complex while increased PI3K-p85 levels in a PI3K-p110 depletion supernatant resulted from PI3K-p110 immunoprecipitation. These results indicated that arsenic increased PI3K-p85 which was free from PI3K-p110 binding. In addition, arsenic significantly increased interaction between IRS1 and PI3K-p85 but not PI3K-p110, suggesting that there may be a fraction of free PI3K-p85 interacting with IRS1. In vitro PI3K activity demonstrated that arsenic lowered PI3K activity in both basal and insulin-stimulated conditions. These results suggested that the increase in free PI3K-p85 by arsenic might compete with PI3K heterodimer for the same IRS1 binding site, in turn blocking the activation of its catalytic subunit, PI3K-p110. Taken together, our results provide additional insights into mechanisms underlying the impairment of neuronal insulin signaling by arsenic through the reduction of IR autophosphorylation, the increase in free PI3K-p85, and the impeding of PI3K activity.

Insulin-like growth factor-2 regulates basal retinal insulin receptor activity

The retinal insulin receptor (IR) exhibits basal kinase activity equivalent to that of the liver of fed animals, but unlike the liver, does not fluctuate with feeding and fasting; it also declines rapidly after the onset of insulin-deficient diabetes. The ligand(s) that determine basal IR activity in the retina has not been identified. Using a highly sensitive insulin assay, we found that retinal insulin concentrations remain constant in fed versus fasted rats and in diabetic versus control rats; vitreous fluid insulin levels were undetectable. Neutralizing antibodies against insulin-like growth factor 2 (IGF-2), but not insulin-like growth factor 1 (IGF-1) or insulin, decreased IR kinase activity in normal rat retinas, and depletion of IGF-2 from serum specifically reduced IR phosphorylation in retinal cells. Immunoprecipitation studies demonstrated that IGF-2 induced greater phosphorylation of the retinal IR than the IGF-1 receptor. Retinal IGF-2 mRNA content was 10-fold higher in adults than pups and orders of magnitude higher than in liver. Diabetes reduced retinal IGF-2, but not IGF-1 or IR, mRNA levels, and reduced IGF-2 and IGF-1 content in vitreous fluid. Finally, intravitreal administration of IGF-2 (mature and pro-forms) increased retinal IR and Akt kinase activity in diabetic rats. Collectively, these data reveal that IGF-2 is the primary ligand that defines basal retinal IR activity and suggest that reduced ocular IGF-2 may contribute to reduced IR activity in response to diabetes. These findings may have importance for understanding the regulation of metabolic and prosurvival signaling in the retina.

Analyzing structural differences between insulin receptor (IR) and IGF1R for designing small molecule allosteric inhibitors of IGF1R as novel anti-cancer agents

IR and insulin-like growth factor-1 receptor (IGF-1R) share high degree of sequence and structural similarity that hinders the development of anticancer drugs targeting IGF1R, which is dysregulated in many cancers. Although IR and IGF1R mediate their activities through similar signalling pathways, yet they show different physiological effects. The exact molecular mechanism(s) how IR and IGF1R exert their distinct functions remain largely unknown. Here, we performed in silico analysis and generated GFP-fusion proteins of wild type IR and its K1079R mutant to analyze their subcellular localization, cytoplasmic and nuclear activities in comparison to IGF1R and its K1055R mutant. We showed that, like K1055R mutation in IGF1R, K1079R mutation does not impede the subcellular localization and nuclear activities of IR. Although K1079R mutation significantly decreases the kinase activity of IR but not as much as K1055R mutation, which was seen to drastically reduce the kinase activity of IGF1R. Moreover, K1079 residue in IR is seen to be sitting in a pocket which is different than the allosteric inhibitor binding pocket present in its homologue (IGF1R). This is for the first time such a study has been conducted to identify structural differences between these receptors that could be exploited for designing small molecule allosteric inhibitor(s) of IGF1R as novel anti-cancer drugs

Modulation of Insulin Receptor Kinase Activity by Lipid Environment

Modulation of Insulin Receptor Kinase Activity by Lipid Environment

266-OR: Plasma Membrane sn-1,2 Diacylglycerol Mediates Lipid-Induced Hepatic Insulin Resistance

The identity and subcellular localization of the bioactive lipid species that causes hepatic insulin resistance remain controversial. Though chronic diacylglycerol acyltransferase 2 (DGAT2) knockdown improves hepatic steatosis and insulin action, we hypothesized that acute DGAT2 knockdown would transiently increase hepatic diacylglycerol (DAG) content to specifically assess the role of hepatic DAG on hepatic insulin action. Regular chow-fed rats were treated with a single dose of DGAT2 antisense oligonucleotide and studied 2 days later. The content and subcellular localization of sn-1,2, sn-2,3 and sn-1,3 DAGs were assessed by LC-MS/MS in the endoplasmic reticulum (ER), mitochondrial (MIT), plasma membrane (PM), lipid droplet (LD) and cytosol (CYT) fractions. Acute knockdown of hepatic DGAT2 induced hepatic insulin resistance with impaired suppression of hepatic glucose production during a hyperinsulinemic-euglycemic clamp. Insulin receptor kinase (IRK) activation was impaired, as reflected by reduced insulin-stimulated IRK Y1162 phosphorylation. This was associated with increased PKCε activation, as reflected by increased translocation from CYT to membrane, and increased sn-1,2 DAG content specifically in the PM. In contrast, there were no changes in hepatic ceramide content or sn-2,3 and sn-1,3 DAGs in the five subcellular fractions tested (ER, MIT, PM, LD, CYT). We next infused [13C16] palmitate in 24-hr-fasted rats to track hepatic DAG synthesis in vivo and observed that sn-1,2 DAGs were the major (73%) product of re-esterification from exogenous fatty acids, compared to sn-2,3 (25%) and sn-1,3 (2%) DAGs. We also found that sn-1,2 and sn-2,3 DAGs were primarily synthesized in the ER before translocating to PM, whereas sn-1,3 DAGs were exclusively produced in the LD by lipolysis. Conclusion: These data demonstrate that sn-1,2 DAGs in the PM, derived from re-esterification of exogenous fatty acids, are sufficient to induce hepatic insulin resistance at the level of IRK. Disclosure K. Lyu: None. Y. Zhang: None. D. Zhang: None. M. Kahn: None. Y. Nozaki: None. S. Bhanot: None. J. Bogan: None. G. Cline: None. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health (R01DK116774)

283-LB: Dissociating Insulin Signaling and SREBP1c Action from the Lipogenic Drive Seen in Human and Murine Hepatic Insulin Resistance

The ongoing debate regarding pathway selective hepatic insulin resistance (IR) focuses on potential insulin signaling branchpoints beyond which regulation of glucose metabolism is attenuated and regulation of de novo lipogenesis (DNL) remains intact. These models suggest that impaired insulin receptor kinase activity, proximal to any potential branchpoint, only plays a minor role in clinical hepatic IR. We compared insulin signaling in liver biopsies from human subjects with nonalcoholic fatty liver disease (NAFLD) and hepatic IR vs. weight matched controls, using glucose ingestion to drive insulin secretion and fructose ingestion as a low insulin comparator. Insulin signaling through phosphorylation of insulin receptor β, Akt, and GSK3β was suppressed in NAFLD (all P < 0.05). Insulin regulation of mTORC1 via PRAS40 phosphorylation was absent in NAFLD subjects; no differences were seen between controls and NAFLD subjects in AMPK dependent RAPTOR phosphorylation. Pathway selective IR implies SREBP1c is primarily responsible for DNL in the insulin resistant liver; we probed the transcriptional regulation of DNL by assessing the in vivo occupancy of lipogenic target gene promoters by SREBP1c and ChREBP using ChIP experiments in mice with diet-induced IR. Fasted-refed mice were refed with high fat diet (HFD) and 1% dextrose drinking water. Mice with hepatic IR (9 day HFD fed) or hepatic and peripheral IR (4 week HFD fed) were compared with more insulin sensitive 2 day HFD fed mice. SREBP1c was not bound to the Fasn promoter in insulin resistant livers, while ChREBP was bound to the Fasn promoter equally in all three groups of mice. Conclusions: There is a proximal block in insulin signaling in humans with hepatic IR and NAFLD, dissociating previously measured changes in DNL rates from insulin signaling. SREBP does not mediate DNL in insulin resistant mice while ChREBP action can explain the maintenance of the transcriptional program of lipogenesis. Disclosure D.F. Vatner: None. K.W. ter Horst: None. L. Goedeke: None. D. Zhang: None. V. Samuel: None. M.J. Serlie: Advisory Panel; Self; Fresenius Medical Care. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health

1782-P: Mechanism of Lipid-Induced White Adipose Tissue Insulin Resistance

White adipose tissue (WAT), as an energy storage organ, plays a major role in regulating peripheral insulin resistance and hepatic gluconeogenesis, but the mechanism by which lipid induces insulin resistance in WAT is poorly understood. To examine the potential role of the diacylglycerol (DAG) - protein kinase Cε (PKCε) - insulin receptor (INSR) pathway in mediating lipid-induced WAT insulin resistance, we assessed insulin action in liver, muscle and WAT in 7-day high fat diet (HFD) and regular chow (RC) fed rats by hyperinsulinemic-euglycemic clamp (HEC) combined with [3-3H] glucose, [14C] deoxyglucose and [2H5]glycerol infusions. Using this approach, we found that HFD induced WAT insulin resistance as reflected by impaired suppression (∼20%, P<0.05 vs. RC) of WAT lipolysis during the HEC. WAT insulin resistance was associated with increased PKCε translocation (P<0.05 vs. RC) and increased phosphorylation of perilipin, HSL and ATGL (all P<0.05 vs. RC). To investigate if PKCε induced Thr1150phosphorylation mediates lipid-induced WAT insulin resistance by inhibiting INSR kinase activity, we studied mice with a threonine-to-alanine knockin mutation at INSR Thr1150(InsrT1150Amice). InsrT1150Amice were protected from HFD-induced WAT insulin resistance as reflected by normalization of insulin’s ability to suppress WAT lipolysis and normalization of insulin’s ability to stimulate INSR and Akt2 phosphorylation in WAT, as well as a restoration of insulin’s ability to suppress phosphorylation of perilipin, HSL and ATGL (all P<0.05 vs. WT). Conclusion: The DAG-PKCε-INSR Thr1150pathway plays a major role in mediating lipid-induced WAT insulin resistance and represents a potential therapeutic target to improve insulin sensitivity in WAT. Disclosure D. Zhang: None. K. Lyu: None. J.D. Song: None. X. Li: None. R.J. Perry: Research Support; Self; AstraZeneca. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health (R01DK116774)

A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells

Wolbachia-infected mosquitoes are refractory to super-infection with arthropod-borne pathogens, but the role of host cell signaling proteins in pathogen-blocking mechanisms remains to be elucidated. Here, we use an antibody microarray approach to provide a comprehensive picture of the signaling response of Aedes aegypti-derived cells to Wolbachia. This approach identifies the host cell insulin receptor as being downregulated by the bacterium. Furthermore, siRNA-mediated knockdown and treatment with a small-molecule inhibitor of the insulin receptor kinase concur to assign a crucial role for this enzyme in the replication of dengue and Zika viruses in cultured mosquito cells. Finally, we show that the production of Zika virus in Wolbachia-free live mosquitoes is impaired by treatment with the selective inhibitor mimicking Wolbachia infection. This study identifies Wolbachia-mediated downregulation of insulin receptor kinase activity as a mechanism contributing to the blocking of super-infection by arboviruses.

Membrane sn-1,2 Diacylglycerol Mediates Lipid-Induced Hepatic Insulin Resistance In Vivo

Diacylglycerol (DAG) has been proposed to mediate lipid-induced hepatic insulin resistance. Accumulated hepatic DAG activates PKCε which phosphorylates insulin receptor kinase (IRK) at T1160 to inhibit IRK activation and cause insulin resistance. However, the importance of DAG in lipid-induced hepatic insulin resistance remains controversial. To test the physiological effect of acute DAG accumulation on hepatic insulin sensitivity, we used antisense oligonucleotide (ASO) to specifically inhibit hepatic diglyceride acyltransferase 2 (DGAT2) which catalyzes the conversion of DAG to triglyceride. Previously, chronic DGAT2 inhibition was shown to paradoxically decrease hepatic DAG content due to decreased SREBP-1c-mediated lipogenesis and improve hepatic insulin sensitivity. However, we hypothesized that acute DGAT2 inhibition might provide a narrow window to transiently increase hepatic DAG content and allow us to identify the association between DAG, PKCε activation/translocation and hepatic insulin resistance. As hypothesized, an acute (2-day) DGAT2 inhibition increased hepatic DAG content and PKCε activation (cytosol to membrane translocation). DGAT2 ASO-treated rats on regular chow diet displayed impaired insulin-mediated suppression of hepatic glucose production. This defect of hepatic insulin action was at the level of IRK as indicated by impaired insulin-stimulated IRK Y1158/1162 phosphorylation. Furthermore, measured by a novel LC-MS/MS method, hepatic sn-1,2 DAG, which is the DAG stereoisomer capable of activating PKCε, was increased in the subcellular membrane compartment with DGAT2 ASO treatment. Conclusion: These data support the importance of membrane sn-1,2 DAG in mediating lipid-induced hepatic insulin resistance through “increased membrane sn-1,2 DAG content -> PKCε activation/translocation -> increased IRK T1160 phosphorylation -> decreased IRK Y1158/1162 phosphorylation -> decreased IRK activity” in vivo. Disclosure K. Lyu: None. D. Zhang: None. Y. Nozaki: None. Y. Zhang: None. S. Bhanot: Employee; Self; Ionis Pharmaceuticals, Inc.. G. Cline: None. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc., Novo Nordisk Inc.. Research Support; Self; Gilead Sciences, Inc..

Gastro-Resistant Insulin Receptor-Binding Peptide from Momordica charantia Improved the Glucose Tolerance in Streptozotocin-Induced Diabetic Mice via Insulin Receptor Signaling Pathway.

Momordica charantia is a commonly used food and has been used for the management of diabetes. Our previous study has identified an insulin receptor (IR)-binding protein (mcIRBP) from Momordica charantia. Here we identified the gastro-resistant hypoglycemic bioactive peptides from protease-digested mcIRBP. By in vitro digestion and IR kinase activity assay, we found that a 9-amino-acid-residue peptide, mcIRBP-9, was a gastro-resistant peptide that enhanced IR kinase activities. mcIRBP-9 activated IR signaling transduction pathway, which resulted in the phosphorylation of IR, the translocation of glucose transporter 4, and the uptake of glucose in cells. Intraperitoneal and oral administration of mcIRBP-9 stimulated the glucose clearance by 30.91 ± 0.39% and 32.09 ± 0.38%, respectively, in streptozotocin-induced diabetic mice. Moreover, a pilot study showed that daily ingestion of mcIRBP-9 for 30 days decreased the fasting blood glucose levels and glycated hemoglobin (HbA1c) levels by 23.62 ± 6.14% and 24.06 ± 1.53%, respectively. In conclusion, mcIRBP-9 is a unique gastro-resistant bioactive peptide generated after the digestion of mcIRBP. Furthermore, oral administration of mcIRBP-9 improves both the glucose tolerance and the HbA1c levels in diabetic mice via targeting IR signaling transduction pathway.

Hypoglycemic effects of Trichosanthes kirilowii and its protein constituent in diabetic mice: the involvement of insulin receptor pathway

BackgroundDiabetes is a serious chronic metabolic disorder. Trichosanthes kirilowii Maxim. (TK) is traditionally used for the treatment of diabetes in traditional Chinese medicine (TCM). However, the clinical application of TK on diabetic patients and the hypoglycemic efficacies of TK are still unclear.MethodsA retrospective cohort study was conducted to analyze the usage of Chinese herbs in patients with type 2 diabetes in Taiwan. Glucose tolerance test was performed to analyze the hypoglycemic effect of TK. Proteomic approach was performed to identify the protein constituents of TK. Insulin receptor (IR) kinase activity assay and glucose tolerance tests in diabetic mice were further used to elucidate the hypoglycemic mechanisms and efficacies of TK.ResultsBy a retrospective cohort study, we found that TK was the most frequently used Chinese medicinal herb in type 2 diabetic patients in Taiwan. Oral administration of aqueous extract of TK displayed hypoglycemic effects in a dose-dependent manner in mice. An abundant novel TK protein (TKP) was further identified by proteomic approach. TKP interacted with IR by docking analysis and activated the kinase activity of IR. In addition, TKP enhanced the clearance of glucose in diabetic mice in a dose-dependent manner.ConclusionsIn conclusion, this study applied a bed-to-bench approach to elucidate the hypoglycemic efficacies and mechanisms of TK on clinical usage. In addition, we newly identified a hypoglycemic protein TKP from TK. Our findings might provide a reasonable explanation of TK on the treatment of diabetes in TCM.

In silico Docking Studies on ATP-Sensitive K+Channel, Insulin Receptor and Phosphorylase kinase Activity by Isolated Active Principles of Stereospermum tetragonum DC

: Background: Stereospermum tetragonum is a medicinal plant that grows throughout tropical parts of Indian sub continent, particularly in sandy soils of river beds in Northern India. S. tetragonum is a potential source of metabolites such as coumarines, glycosides, tannins and terpenoids traditionally used in the treatment of Diabetes mellitus. The main objective of the study is to evaluate the interaction of active principles isolated from S. tetragonum with ATP-sensitive K+ channel, Insulin receptor and phosphorylase kinase by in silico docking studies. Methods: Activation by in silico docking studies of ATP-sensitive K+ channel, Insulin receptor, Phosphorylase kinase with1,4a,5,7a-tetrahydro-5-hydroxy-7-(hydroxymethyl)- 1-(tetrahydro-6-(hydroxymethyl)-3,4,5-trimethoxy-2H-pyran-2-yloxy) cyclopentapyran-4-carboxylicacid and 5,8-dihydro-7-isopentyl-2,3,5,8- tetramethoxynaphthalene-1,4,6-triol isolated from the active fraction by solvent fractionation and chromatographic techniques and characterized with spectral data of water extract of S.tetragonum. Results: The isolated compounds showed better interaction than metformin glide score -2.563 through an extensive in silico docking approach with ATP-sensitive K+channel. The glide score is -6.981 Kcal/mol for compound 1 and -9.425 Kcal/mol for compound 2. In Insulin receptor the glide score for standard metformin is -3.359 and for compound 1 is -7.882 and compound 2 is -4.62. in Phosphorylase kinase the glide score of standard metformin is -4.864 and the glide score for compound 1 is -6.981 and compound 2 is -5.253 with the receptors. Conclusion: The work establishes the isolated compounds from the fraction of S. tetragonum as a potential source for Diabetic treatment thus enabling a possibility of this plant as new alternative to existing diabetic approaches.Key words: ATP- sensitive K+channel, Insulin receptor, Phosphorylase kinase, Stereospermum tetragonum.

371 - Improvement of BVR-A Activity Ameliorates Brain Insulin Resistance in Alzheimer Disease Following Intranasal Insulin Administration

Brain insulin resistance (b.i.r.) was proposed as a mechanistic mediator of the cognitive deficits observed in Alzheimer disease (AD). Interestingly, the efficacy of intranasal insulin (I-Ins) administration – which allows to directly transport insulin to the CNS – was proposed as an innovative therapeutic strategy to alleviate cognitive dysfunction in AD. However, significant gaps in our knowledge about the mechanisms underlying these beneficial outcomes still exist. We previously reported about the oxidative-stress (OS) induced impairment of biliverdin reductase A (BVR-A) [a novel direct target of the insulin receptor (IR) kinase activity] as an early event contributing to b.i.r. in AD. Indeed, under normal conditions, once IR-activated BVR-A controls the activation of the insulin receptor substrate (IRS)-1, thus representing an upstream regulator of the insulin signaling cascade. Conversely, along the progression of AD pathology, the OS-induced inactivation of BVR-A promotes the hyper-activation of the IR/IRS-1 axis, which finally lead to b.i.r.. Here we hypothesized that the I-Ins-associated beneficial effects on cognition would be mediated by the restoration of BVR-A activity. To this aim, we evaluated in vivo (i) changes of the insulin signaling cascade (ii) variations of total OS markers (PC, HNE, 3-NT), (iii) changes of AD neuropathology markers e.g., Aβ and tau levels, in the hippocampus of 3xTg-AD and WT mice undergoing an early (4 months) or late (10 moths) intranasal insulin treatment (1 U/day, 3 times per week, for 2 months). Furthermore, changes of cognitive functions assessed through the morris water maze (MWM) and the novel object recognition (NOR) tasks, were evaluated. We found that I-Ins administration rescue the activation of BVR-A both in young and old 3xTg-AD mice. Improved BVR-A activity is associated with (i) reduced OS markers, (ii) a restoration of the insulin signaling cascade and (iii) a reduction of Tau pathology in the hippocampus. All these changes parallel an improved cognition. Interestingly, cell-based experiments confirmed the role of BVR-A by showing no beneficial effects of insulin when BVR-A is knocked-down.

Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance.

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet-induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance.

Insulin Signalling: The Inside Story.

Insulin signalling begins with binding to its cell surface insulin receptor (IR), which is a tyrosine kinase. The insulin receptor kinase (IRK) is subsequently autophosphorylated and activated to tyrosine phosphorylate key cellular substrates that are essential for entraining the insulin response. Although IRK activation begins at the cell surface, it is maintained and augmented following internalization into the endosomal system (ENS). The peroxovanadium compounds (pVs) were discovered to activate the IRK in the absence of insulin and lead to a full insulin response. Thus, IRK activation is both necessary and sufficient for insulin signalling. Furthermore, this could be shown to occur with activation of only the endosomal IRK. The mechanism of pV action was shown to be the inhibition of IRK-associated phosphotyrosine phosphatases (PTPs). Our studies showed that the duration and intensity of insulin signalling are modulated within ENS by the recruitment of cellular substrates to ENS; intra-endosomal acidification, which promotes dissociation of insulin from the IRK; an endosomal acidic insulinase, which degrades intra-endosomal insulin; and IRK-associated PTPs, which dephosphorylate and, hence, deactivate the IRK. Therefore, the internalization of IRKs is central to insulin signalling and its regulation.

CMX-2043 Mechanisms of Action In Vitro.

α-Lipoic acid has been shown to provide cytoprotection in some tissues through antioxidant and antiapoptotic mechanisms. We have enhanced these properties by synthetic modification, resulting in a new chemical entity, CMX-2043, with proven efficacy in an animal model of cardiac ischemia-reperfusion injury. The present studies compare cytoprotective cellular pathways of R-α-lipoic acid and CMX-2043. Biochemical and cellular assays were used to compare antioxidant potency, tyrosine kinase activation, and protein kinase B (Akt) phosphorylation. CMX-2043 was more effective than lipoic acid in antioxidant effect, activation of insulin receptor kinase, soluble tyrosine kinase, and Akt phosphorylation. Activation of insulin-like growth factor 1 receptor was similar for both. CMX-2043 stimulation of Akt phosphorylation was abolished by the phosphatidylinositide 3-kinase inhibitor LY294002. Consistent with Akt activation, CMX-2043 reduced carbachol-induced calcium overload. The S-stereoisomer of CMX-2043 was less active in the biochemical assays than the R-isomer. These results are consistent with cytoprotection through activation of Akt and antioxidant action. CMX-2043 may thus provide a pharmacological approach to cytoprotection consistent with established anti-apoptotic mechanisms.