IRS-1 Levels Research Articles

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.

Astragalus hamosus Acts as an Insulin Sensitizer in the Treatment of Polycystic Ovary Syndrome Rat Models by Affecting IRS1 Expression.

Polycystic ovary syndrome (PCOS) is the most common endocrine abnormality among women of reproductive age. Insulin resistance is known as the hallmark of PCOS that leads to hyperinsulinemia and type 2 diabetes in PCOS patients. This study aimed to evaluate the expression pattern of IRS1 as a candidate gene in insulin resistance development in the PCOS rat models. In this study, estradiol valerate was used for PCOS induction. Then, all of the rats were divided into five experimental groups and treated with Astragalus hamosus extract. Ethanol was used for extraction by Soxhlet, and extracts were analyzed by GC-MS. Ovarian morphology was analyzed using histological experiments. Finally, the expression of IRS1 and hormonal titration of testosterone and insulin were evaluated using qRT-PCR and ELISA assays, respectively. Induction of PCOS led to an increase in body weight, which decreased after treatment with the extract. Histological assessment declared an increased number of corpora lutea in treated groups and reduced cystic follicles compared to the PCOS group. Astragalus hamosus extract-treated groups exhibited decreased levels of insulin and testosterone compared to the PCOS group. qRT-PCR results showed an increase in the expression levels of IRS1 in the treated groups compared to the PCOS group. This study indicated the impact of Astragalus hamosus extract on PCOS by clarifying the increased levels of IRS1 expression in the treated groups compared to the PCOS group.

Mechanism of Mongolian Medicine Eerdun Wurile in Improving Postoperative Cognitive Dysfunction Through Activation of the PI3K Signaling Pathway.

ObjectiveTo study the effect of Eerdun Wurile (EW), a traditional Mongolian medicine, on the cognitive function of rats by activating the IRS-PI3K-AKT-GLUT4 pathway in an animal model of postoperative cognitive dysfunction (POCD).MethodsFifty clean-grade adults Sprague Dawley (SD) male rats were assigned to one of five groups: (1) a control group with no anesthesia (Group C), (2) a POCD model group with anesthesia only (Group P), (3) POCD group with low-dose EW treated (Group L), (4) a POCD group with high-dose EW treated (Group H), and (5) a POCD model group with dexmedetomidine treated (Group D) for positive control. The study started 7 days after all rats had acclimated to housing. Rats were trained in the Morris Water Maze navigation 5 days before surgery. All rats underwent the same maze for navigation and spatial exploration experiments on the preoperative day 1 and postoperative days 1, 3, 5, and their learning and memory abilities were assessed. At the end of the water maze experiment, rats were sacrificed to obtain hippocampal tissue. The mRNA levels of IRS-2, PI3K, AKT, and GLUT4 were measured in the hippocampus by real-time PCR, and the expression of IRS-2, PI3K, AKT, and GLUT4 protein in the hippocampus was determined by Western blotting to investigate the potential mechanisms at the molecular level.ResultsCompared to control Group C, Group P, L, H, and D showed prolonged escape latency (P < 0.05) and decreased number of times to cross the platform (P < 0.05) at 1, 3 and 5 days after surgery. Compared to Group P, Group L, H, and D showed a decrease in escape latency with an increased number of crossing the platform at all-time points after surgery (P < 0.05). Within individual P, L, H, and D groups, escape latencies decreased (P < 0.05) and the number of times that the platform was crossed increased (P < 0.05) between postoperative days 3 and 5 compared to postoperative 1 day. Compared to Group C, the mRNA expression of IRS-2, PI3K, AKT and GLUT4 in the hippocampus of P, L, H, and D groups were decreased (P < 0.05). Compared to Group P, IRS-2, PI3K, AKT, and GLUT4 in the hippocampus of L, H, and D groups were increased (P < 0.05). Compared with Group D, the expression levels of IRS-2 and AKT in both L and H groups were higher. The expression level of PI3K in Group L was also higher (P < 0.05) vs Group D. The expression of AKT mRNA in Group H was higher than in Group L (P < 0.05). Compared to Group C, the p-IRS-2/IRS-2 ratio in the hippocampus of Group P was higher than that of Group C (P < 0.05). Compared to Group P, the ratios of p-IRS-2/IRS-2 in Group L, Group H, and Group D were lower, and the ratios of the p-PI3K/PI3K, p-AKT/AKT, and p-GLUT4/GLUT4 were higher (P < 0.05).ConclusionAdministration of EW showed the effect on the signaling pathway in rats with POCD. The therapeutic effect was better in the low-dose group. This could be related to the insulin downstream signal molecule PI3K and the IRS-PI3K-AKT-GLUT4 signaling pathway.

Additional effects of simultaneous treatment with C14-Cblin and celastrol on the clinorotation-induced rat L6 myotube atrophy.

Two novel reagents, N-myristoylated Cbl-b inhibitory peptide (C14-Cblin) and celastrol, a quinone methide triterpene, are reported to be effective in preventing myotube atrophy. The combined effects of C14-Cblin and celastrol on rat L6 myotubes atrophy induced by 3D-clinorotation, a simulated microgravity model, was investigated in the present study. We first examined their effects on expression in atrogenes. Increase in MAFbx1/atrogin-1 and MuRF-1 by 3D-clinorotation was significantly suppressed by treatment with C14-Cblin or celastrol, but there was no additive effect of simultaneous treatment. However, celastrol significantly suppressed the upregulation of Cbl-b and HSP70 by 3D-clinorotation. Whereas 3D-clinorotation decreased the protein level of IRS-1 in L6 myotubes, C14-Cblin and celastrol inhibited the degradation of IRS-1. C14-Cblin and celastrol promoted the phosphorylation of FOXO3a even in microgravity condition. Simultaneous administration of C14-Cblin and celastrol had shown little additive effect in reversing the impairment of IGF-1 signaling by 3D-clinorotation. While 3D-clinorotation-induced marked oxidative stress in L6 myotubes, celastrol suppressed 3D-clinorotation-induced ROS production. Finally, the C14-Cblin and celastrol-treated groups were inhibited decrease in L6 myotube diameter and increased the protein content of slow-twitch MyHC cultured under 3D-clinorotation. The simultaneous treatment of C14-Cblin and celastrol additively prevented 3D-clinorotation-induced myotube atrophy than single treatment. J. Med. Invest. 69 : 127-134, February, 2022.

Effects of Different Intensity Exercise on Glucose Metabolism and Hepatic IRS/PI3K/AKT Pathway in SD Rats Exposed with TCDD.

The objective of the study was to investigate the effects of different intensity exercise and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure on glucose metabolism in Sprague Dawley (SD) rats, as well as the action of insulin receptor substrate (IRS)/phosphatidylinositol-3-kinases (PI3K)/protein kinase (AKT) signaling pathway in it. Besides that, we explored whether exercise can alleviate the toxicity induced by TCDD. Sixty male SD rats (8 weeks old) were randomly divided into non-exercise group, none-exercise toxic group, moderate-intensity exercise group, moderate-intensity exercise toxic group, high-intensity exercise group, high-intensity exercise toxic group. The toxic groups were intraperitoneally injected with TCDD, which the dose was 6.4 µg/kg· BW for the first week, then 21% of the above week dose for continuous 8 weeks. The 8-week treadmill running of moderate intensity (15 m/min, 60 min/day) and high intensity (26 m/min, 35 min/day) were implemented separately in exercise groups five times a week. After detecting the concentration of fasting serum glucose, insulin and C-peptide, the index of the homeostasis model assessment of insulin resistance (HOMA-IR) and islet β-cell secretion (HOMA-β) were calculated. We measured the hepatic mRNA expression levels of IRS2, phosphatidylinositol-3-kinases catalytic subunit alpha (PIK3CA), AKT by real-time PCR. The protein expression of total IRS2 (tIRS2), phosphorylated IRS2 at Ser731 (pSer731), total PIK3CA (tPIK3CA), total Akt (tAkt), phosphorylated Akt at Thr308 (pThr308) in liver were analyzed by western blot. We observed that compared to the non-exercise group, insulin and HOMA-IR index were significantly higher in the none-exercise toxic group (p < 0.05), while glucose, insulin, C-peptide and HOMA-IR index were significantly lower in the moderate-intensity exercise group (p < 0.05). In the high-intensity exercise group, the HOMA-IR index was significantly lower and the gene expression of IRS2 was significantly higher than in the non-exercise group (p < 0.05). Besides that, the HOMA-β index in the moderate-intensity exercise toxic group was significantly higher compared to the none-exercise toxic group and moderate-intensity exercise group (p < 0.05). The level of IRS2mRNA was significantly lower in the high-intensity exercise toxic group than in the high-intensity exercise group (p < 0.05). Our results demonstrated that 8-week TCDD exposure could induce insulin resistance in rats, while exercise could improve insulin sensitivity in which moderate intensity was more obvious than high intensity exercise. Meanwhile, both intensity exercise could not effectively alleviate the insulin resistance induced by TCDD, but high intensity exercise could promote compensatory insulin secretion to maintain glucose homeostasis. Although the gene expression of IRS2 was changed in high-intensity exercise groups, the mediation role of the hepatic IRS2/PI3K/AKT pathway in the effects of exercise and TCDD exposure on glucose metabolism remains very limited.

Effects of COVID-19, diabetes mellitus, and cardiovascular diseases on insulin receptor substrate-1 amount in the blood plasma of patients

Insulin receptor substrate (IRS) is a key adapter protein mediating effects of insulin and insulin-like growth factors (IGF) in cells. IRS-1 is a member of the insulin receptor substrate family, which is associated with tumor initiation and progression. The aim of the study is to determine the level of IRS-1 in the blood of patients (n = 81) with diabetes mellitus and COVID-19. IRS-1 was determined with enzyme-linked immunosorbent assay (ELISA) (Elabscience, USA). The measurements were performed at an optical wavelength of 450 nm. The level of IRS-1 in the blood plasma of patients with COVID-19 was much (from 3.5 to more than 6 times) higher than that in the blood of healthy people. The IRS-1 amounts in COVID-19 patients with diabetes and diabetes + CVD were significantly higher than in patients with COVID-19 without concomitant diseases. The level of IRS-1 in blood plasma may be one of the promising markers of COVID-19.

Ameliorative Effect of Annona muricata (Graviola) Extract on Hyperglycemia Induced Hepatic Damage in Type 2 Diabetic Mice.

Annona muricata (AM) is evergreen plant of the Annonaceae family and known to have anticancer and antidiabetic effects. However, anti-diabetic mechanisms of AM extracts (AME) associated with hepatic glucose regulation and lipid metabolism remain unclear. In this study, we investigated the protective effect of AME extracted on hepatic damage in diabetic mice. Diabetes was induced by a high-fat diet with two-times streptozotocin (STZ) injection (60 mg/kg BW) in C57BL/6 male mice. The diabetic mice were daily administered with AME (50 or 100 mg/kg BW) by gavage for 9 weeks. Biomarkers related to energy metabolism and insulin signaling were examined to identify the effect of AME on hyperglycemia induced hepatic damage. AME supplementation reduced levels of FBG, HbA1c, HOMA-IR and hepatic lipid profiles as well as enhanced insulin signaling by increased the protein levels of IRS-1 accompanied GLUT2 in diabetic mice. Especially low dose of AME showed the beneficial effect of reducing oxidative stress (4-HNE, protein carbonyls, Nrf2, NQO1) and improved hepatic morphology demonstrated by lipid droplets along with upregulation of lipophagy (pAMPK, p-mTOR/mTOR, LC3-2/LC3-1) in diabetic mice. Moreover, AME supplementation ameliorated hepatic lipid metabolism (FAS, SREBP1c, C/EBPα, PPARγ, CPT1A, PPARα) and energy metabolism (pAMPK, PGC1α) in diabetic mice. Taken together, this study suggested that AME could be helpful to prevent hepatic abnormality by regulation of insulin signaling associated with energy metabolism and autophagy in diabetes.

Exploration of the lactation function of protein phosphorylation sites in goat mammary tissues by phosphoproteome analysis

BackgroundProtein phosphorylation plays an important role in lactation. Differentially modified phosphorylation sites and phosphorylated proteins between peak lactation (PL, 90 days postpartum) and late lactation (LL, 280 days postpartum) were investigated using an integrated approach, namely, liquid chromatography with tandem mass spectrometry (LC-MS/MS) and tandem mass tag (TMT) labeling, to determine the molecular changes in the mammary tissues during the different stages of goat lactation.ResultsA total of 1,938 (1,111 upregulated, 827 downregulated) differentially modified phosphorylation sites of 1,172 proteins were identified (P values < 0.05 and fold change of phosphorylation ratios > 1.5). Multiple phosphorylation sites of FASN, ACACA, mTOR, PRKAA, IRS1, RPS6KB, EIF4EBP1, JUN, and TSC2 were different in PL compared with LL. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the calcium signaling pathway, oxytocin signaling pathway and MAPK signaling pathway were enriched. The western blot results showed that the phosphorylation levels of ACACA (Ser80), EIF4EBP1 (Thr46) and IRS1 (Ser312) increased and JUN (Ser63) decreased in PL compared with LL. These results were consistent with the phosphoproteome results.ConclusionsIn this study, we identified for the first time the differentially modified phosphorylation sites in goat mammary tissues between PL and LL. These results indicate that the multiple differentially modified phosphorylation sites of FASN, ACACA, mTOR, PRKAA, IRS1, RPS6KB, EIF4EBP1, TSC2, and JUN and proteins involved in the calcium signaling pathway, oxytocin signaling pathway, and MAPK signaling pathway are worthy of further exploration.

FGF15/FGF19 alleviates insulin resistance and upregulates placental IRS1/GLUT expression in pregnant mice fed a high-fat diet

IntroductionThis study aimed to evaluate whether FGF19 can alleviate insulin resistance and change the expression of placental IRS1/GLUTs. MethodsMice transgenic for Fgf15 (the murine homologue of human FGF19) were constructed, and human recombinant FGF19 was administered to pregnant high-fat diet mice. Then, glycolipid metabolism parameters and the weight of foetus and placenta were observed. The expression levels of key molecules of the insulin signalling pathway and glucose transporters in placentae were detected by qRT-PCR and western blotting. Primary trophoblasts and JAR cells were cultured in high-glucose medium, and FGF19 was added to observe its regulatory effects on IRS1/GLUTs. ResultsOverexpressing FGF15 or exogenously administering FGF19 reduced the levels of fasting blood glucose, HOMA-IR, triglycerides, and free fatty acids in pregnant high-fat diet mice compared to control mice (P < 0.05). FGF15/FGF19 did not significantly affect placental weight, foetal weight or litter size (P > 0.05). In addition, FGF15/FGF19 upregulated the expression of p-IRS1 and GLUT4 in the placentae of high-fat diet mice and upregulated GLUT1 levels in the placentae of normal diet-fed mice (P < 0.05), while it did not significantly alter total IRS1 and GLUT3 levels (P > 0.05). Consistent with the results of the animal experiments, FGF19 increased the expression of p-IRS1 and GLUT4 in trophoblast cells cultured in high-glucose medium (P < 0.05). DiscussionOverexpressing FGF15 or administering FGF19 to pregnant high-fat diet mice can improve glycolipid metabolism and alleviate systemic and local insulin resistance. The possible underlying mechanism may involve upregulation of placental expression of p-IRS1 and GLUT4.

Chemical composition, antioxidant, and anti-diabetic activities of ethyl acetate fraction of Stachys riederi var. japonica (Miq.) in streptozotocin-induced type 2 diabetic mice

This work analysed the chemical composition, antioxidant, and enzyme inhibitory activities of solvent extract (SJ-ME) and fractions (SJ-HF, SJ-EAF, and SJ-MF) of the Stachys riederi var. japonica (Miq.) (SJ). Furthermore, the effect of SJ-EAF in STZ induced type 2 diabetic mice was examined. Among the samples, SJ-EAF exhibited a lower IC50 concentration of 64.2 ± 0.48 μg/mL for DPPH and 82.6 ± 0.09 μg/mL for ABTS+. The SJ-EAF concentration of 2.89 ± 0.03 μg and 2.27 ± 0.98 μg was equivalent to 1 μg of acarbose mediated enzyme inhibitory effect against α-amylase and α -glucosidase, respectively. The SJ-EAF did not show cytotoxicity (<80%) to NIH3T3 nor HepG2 cells but enhanced the glucose uptake in the IR-HepG2. LC-MS/MS of SJ-EAF showed the presence of a total of 16 compounds. Among the identified compounds, rosmarinic acid, caffeic acid, oleanolic acid, and ursolic acid showed high catalytic activity of α-amylase and α-glucosidase. The treatments of SJ-EAF restored the level of blood glucose, body weight, insulin, HDL and mRNA level of IRS1, GLUT2, GLUT4 and Akt whereas it reduced the excess elevation of total cholesterol, total triglycerides, LDL, AST, ALT, ALP, BUN, and creatinine in STZ induced diabetic mice. Overall, the present study concluded that the SJ-EAF exhibited promising antidiabetic activity.

Anti-Diabetic Effects of Metformin Nanoemulsion and Cell-Based Therapy on the Insulin Signaling Pathway (IRS1/AKT) and Apoptotic Related Genes in Type 2 Diabetic Rat Model

Restoration of the normoglycemic state is the basic tenet of diabetes research. Nowadays, using nanoemulsion drug delivery systems represent an alternative effective tool in Type 2 diabetes mellitus management. We aimed to investigate the anti-diabetic potential of metformin nanoemulsion and mesenchymal stem cells (MSCs) therapy on streptozotocin (STZ)-induced diabetic rat model. Nanoemulsion formulation was prepared with oil phase (5%v/v), 1gm metformin with a hydrophilic surfactant, then characterized for particle size and zeta potential.Adult male albino rats were induced for diabetes with 60 mg/kg STZ and were classified into 4 groups: STZ control group; STZ+MET group (orally administered daily dose of metformin 18 mg/200 g b.wt.); STZ+MSCs group (mono-therapeutic dose of BM-MSCs 1×106 cells/rat) and STZ+Nano-MET group (daily intraperitoneal dose of metformin nanoemulsion 18 mg/kg b.wt.). After 8 weeks, relative expression levels of hepatic IRS-1, AKT, Bcl-2, and Bax were assessed by qPCR. Also, histopathological investigations were performed. We reported cytoplasmic vacuolation, tissue necrosis, dilated blood sinusoid and congested hepatic vein in STZ group. Experimental animals either treated with metformin nanoemulsion or the mono-therapeutic dose of MSCs exhibited significant up-regulation in IRS-1/AKT pathway and Bcl-2 gene expression levels, as well as significant repression in Bax mRNA levels, besides a remarkable amelioration in a hepatic histological organization.Conclusion: Nano-MET holds an anti-diabetic potential that clearly surpasses metformin via affecting the insulin signaling pathway and apoptotic gene expression.

Molecular characterization, expression and functional analysis of yak IFITM3 gene

IFITM3 is interferon-induced transmembrane 3, which plays an extremely key role in anti-proliferation, anti-virus and anti-tumor diseases.In this study, the yak (Bos grunniens) IFITM3 (BgIFITM3) gene contained a 5′-untranslated region (UTR) (25 bp), a coding region (441 bp), and a 3′-UTR (115 bp). The expression of BgIFITM3 gene in liver was significantly higher than that in heart, spleen, lung and kidney (P < 0.01). BgIFITM3 protein was localized on the yak hepatocyte plasma membrane, and its expression was significantly different between 1 day and 15 months of age (P < 0.05). Moreover, the prokaryotic expression vector of BgIFITM3 protein was constructed and expressed successfully, with a molecular weight of 19.5 kDa. The activities of yak hepatocyte were significantly inhibited after treating with BgIFITM3 protein (10 and 20 μg/mL) (P < 0.01). The expression levels of ERBB-2, IRS-1, PI3KR-1, AKT-1 and MAPK-3 were significantly lower after treating with 20 μg/mL BgIFITM3 protein (P < 0.05). Besides, the activities of HepG2 cells were significantly inhibited after treating with BgIFITM3 protein (1, 10 and 20 μg/mL) (P < 0.05). While, the cloning ability and migration ability of HepG2 cells were significantly inhibited after treating with 10 μg/mL BgIFITM3 protein (P < 0.05). Finally, the mitochondria of HepG2 cells was concentrated, cristae widened, and the double film density of mitochondria was increased after treating with 10 μg/mL BgIFITM3 protein. After 10 μg/mL BgIFITM3 protein treating, the expression levels of VDAC-2, VDAC-3 and p53 genes were significantly increased, but the expression level of GPX-4 gene was significantly decreased (P < 0.01).Taken together, the BgIFITM3 protein could inhibit the proliferations of yak hepatocyte and HepG2 cells by regulating the PI3K/Akt pathway or ferroptosis-related genes, respectively. These results benefit for further study of the function of BgIFITM3 protein.

CMF and MET treatment induce cognitive impairment through upregulation of IL-1α in rat brain.

Cyclophosphamide (CYP), methotrexate (MTX) and 5-fluorouracil (5-FU) (CMF) are chemotherapeutic agents known to cause acute and long-term cognitive impairment in cancer patients. Cognitive function is regulated mainly by neuronal circuitry in the brain, especially the cortex and hippocampus as well as other components of the limbic area. Neuroinflammation mediated by proinflammatory cytokines is a well-known cause of cognitive impairment. Our previous study showed that metformin induced cognitive impairment and neuroinflammation in CMF-treated rats. Understanding the effects and mechanisms of CMF and MET treatment on chemotherapy-related cognitive impairment and the relationship with neuroinflammation may help prevent some of the adverse effects of this type of chemotherapy in cancer patients. Rats were divided into four groups: control (normal saline), CMF (50 mg/kg CYP, 2 mg/kg MTX, 50 mg/kg 5-FU; two doses administered by intraperitoneal injection over two weeks), MET (2.5 mg/ml - oral administration daily), and CMF+MET group. IL-1α, IRS-1, Akt-a and TNF-α levels in brain tissues were measured by ELISA and data were analyzed by one-way ANOVA test followed by Tukey's test. Compared with the control group, IL-1α levels were significantly increased in the CMF+MET group, whereas there were no significant differences in the MET and CMF groups. On the other hand, IRS-1, TNF-α and Akt-a expression and mitochondrial complex 1 activity indicated that systemic CMF and MET treatment did not change the expression of these proteins in the brain compared to the control group. Our results indicate that cognitive function is impaired by the administration of two doses of CMF and MET over a period of two weeks as a result of IL-1α overexpression in the brain.

Anemarrhena saponins attenuate insulin resistance in rats with high-fat diet-induced obesity via the IRS-1/PI3K/AKT pathway

Ethnopharmacological relevanceAnemarrhena asphodeloides is the dry rhizome of Anemarrhena asphodeloides Bge. Anemarrhena Saponins isolated from Anemarrhena asphodeloides are one of the pharmacologically active components of this plant and have blood lipid reduction and blood glucose reduction properties. These facts suggest that these saponins might be helpful in the treatment of insulin resistance. Aim of the studyTo determine the therapeutic effect of anemarrhena saponins on insulin resistance and the probable underlying mechanism. Materials and methodsInsulin-resistant rats were used as the experimental subject, to observe the therapeutic effect of anemarrhena saponins. The blood glucose and blood lipid parameters were determined using the relevant kits. We used hematoxylin and eosin (H&E) staining to observe the protective effect of anemarrhena saponins on the livers of insulin-resistant rats and reverser transcripition polymerase chain reaction (RT-PCR) to analyze the mRNA expressions patterns of genes related to glucose metabolism and inflammatory factors. The toxicity of anemarrhena saponins to HepG2 cells was calculated using the MTT assay. Further, we conducted in vivo and in vitro experiments, and Western-blot analysis to study the effects of anemarrhena saponins on the IRS-1/PI3K/AKT pathway. ResultsAnemarrhena saponins were found to improve dyslipidemia, reduce obesity and inflammation, and alleviate liver injury in insulin-resistant rats. Anemarrhena saponins also reduced the mRNA expression of gluconeogenesis-related genes sunch as G6pase, PEPCK, and GSK3β in the liver. Moreover, anemarrhena saponins up-regulated the phosphorylation levels of IRS-1, PI3K and AKT, promoted insulin signal transduction, and reduced liver injury induced by insulin resistance. ConclusionsThese findings suggest that anemarrhena saponins could promote insulin signal transduction through the IRS-1/PI3K/AKT pathway, thereby reducing the damage caused by insulin resistance.

Saponins of Momordica charantia increase insulin secretion in INS-1 pancreatic β-cells via the PI3K/Akt/FoxO1 signaling pathway

Saponins are the main bioactive substances with anti-hyperglycemic activities of Momordica charantia. This study aimed to verify the effects of M. charantia saponins on insulin secretion and explore the potential underlying mechanisms in INS-1 pancreatic β-cells. We injured INS-1 cells with 33.3mM glucose and then treated them with saponins. Saponins improved cell morphology and viability as demonstrated by inverted microscopy and CCK8 detection and significantly increased insulin secretion in a concentration-dependent manner as shown by ELISA. Thus, we obtained the optimal concentration for the subsequent experiments. Potential mechanisms were explored by immunofluorescence, western blotting, and RT-qPCR techniques. First, saponins increased the mRNA and protein levels of IRS-2 but decreased the serine 731 phosphorylation level of IRS-2. Moreover, saponins increased the phosphorylation of Akt protein and decreased the protein level of FoxO1, which were both reversed by the PI3K inhibitor ly294002. Furthermore, saponins increased the protein level of the downstream molecule and insulin initiating factor PDX-1, which was also reversed by ly294002. Saponins also increased Akt and PDX-1 mRNA and decreased FoxO1 mRNA, which were both reversed by ly294002. Saponins increased glucose-stimulated insulin secretion (GSIS) and intracellular insulin content, which were reversed by ly294002, as determined by ELISA. The immunofluorescence results also confirmed this tendency. In conclusion, our findings improve our understanding of the function of saponins in INS-1 pancreatic β-cells and suggest that saponins may increase insulin secretion via the PI3K/Akt/FoxO1 signaling pathway.

Rosiglitazone promotes glucose metabolism of GIFT tilapia based on the PI3K/Akt signaling pathway.

The study aimed to explore the effects of rosiglitazone on glucose metabolism of GIFT tilapia based on the PI3K/Akt signaling pathway. The experiment was divided into five groups: normal starch group (32%, LC), high starch group (53%, HC), high starch +rosiglitazone group 1 (10 mg/kg, R1), high starch + rosiglitazone group 2 (20 mg/kg, R2), and high starch + rosiglitazone group 3 (30 mg/kg, R3). The results showed that a high starch diet supplemented with 10–20 mg/kg rosiglitazone had a better specific growth rate and protein efficiency that was beneficial for the growth of the tilapia. Rosiglitazone had no significant effect on the contents of crude lipid, crude protein, crude ash, and moisture of the whole fish body (p > 0.05). The contents of triglycerides and total cholesterol in the R1, R2, and R3 groups were lower than those in the HC group. The levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in R1 and R2 groups were significantly lower than those in the HC groups (p < 0.05). However, the GOT and GPT levels in the R3 groups were significantly higher than those in the R1 and R2 groups (p < 0.05). With an increase in the rosiglitazone concentration, the contents of serum glucose, insulin, and hepatic glycogen in the R1, R2, and R3 groups decreased gradually. Meanwhile, the muscle glycogen content in the R1, R2, and R3 groups increased gradually. The mRNA expression of the IRS‐1, PI3K, GLUT‐4, and Akt proteins in the R1, R2, and R3 groups was significantly higher than that in the HC group (p < 0.05). Compared with the HC group, the expression of the GSK‐3 mRNA in the R1, R2, and R3 groups was significantly reduced (p < 0.05). The protein expression of p‐Akt in the R1 and R2 groups was higher than that in the HC group (p > 0.05). The protein expression of p‐GSK‐3β in the R1 and R2 groups was significantly higher than that in the HC group (p < 0.05). In conclusion, a high starch diet supplemented with rosiglitazone can improve growth, enhance the serum biochemical indices, and increase the muscle glycogen content in the GIFT tilapia. It benefits in upregulating the IRS‐1, PI3K, and GLUT‐4 mRNA levels in the skeletal muscle and promotes glucose uptake. Meanwhile, the phosphorylation of Akt and GSK‐3β increased significantly and resulted in the inactivation of GSK‐3β and alleviation of insulin resistance.

Abstract PS4-08: Biomarker analysis of paclitaxel, ganitumab, and metformin (PGM) therapy in the I-SPY2 neoadjuvant clinical trial

Abstract I-SPY2 is a neoadjuvant trial evaluating experimental therapies in combination with cytotoxic chemotherapy compared to chemotherapy alone with the primary endpoint of pathologic complete response (pCR). Abundant preclinical evidence suggested the type I insulin-like growth factor receptor (IGF-1R) regulated breast cancer growth, although multiple clinical trials did not show benefit. We were the first to report the results of a monoclonal IGF-1R antibody ganitumab (G) in combination with chemotherapy. PGM followed by doxorubicin/cyclophosphamide (AC) did not result in substantial increases in pCR when compared to P followed by AC. In this report, we examined several potential predictive biomarkers. IGF-1R inhibitors induce hyperglycemia and we examined hemoglobin A1C (HgbA1c) as a measure of glucose control in patients before and after PGM therapy. 106 patients received PGM and 104 patients had baseline HgbA1c with a median of 5.4%. However, 27% (28/104) had levels greater than 5.7% the upper limit of normal as defined by the NIDDK. 4 of 104 had HgbA1c greater than 6.5%, a level associated with type 2 diabetes. pCR rates are similar between patients with baseline HgbA1c ≤5.7% (21%) vs. &amp;gt;5.7% (25%) (Fisher test p=0.79). 72 of these patients had an additional HgbA1c during the course of PGM therapy. For patients with HgbA1c ≤5.7%, 27% (14/52) had subsequent elevation above 5.7% after PGM. For patients with a baseline HgbA1C &amp;gt;5.7%, all 20 patients continued to have elevated levels through PGM. We also examined pre-treatment tumor gene expression profiles derived from custom Agilent 44K full-genome microarrays. We studied 11 genes associated with the IGF-1R signaling (IGF1, IGF2, IGF1R, INSR, IGFBP2, IRS1, IRS2, IGFBP3, IGFBP4, IGFBP5, CDH1), the IGFBP5/IGFBP4 ratio, and two IGFR expression signatures (Creighton, et al. J Clin Oncol 26:4078 2008 PMID: 18757322; Mu, et al. Breast Cancer Res Treat 133:321 2012 PMID: 22297468). The 2 signatures evaluated: the IGF1 ligand score and the IGF1-R signature are anti-correlated (Rp= - 0.79). In the population as a whole, lower levels of IRS1 and IGFBP5 significantly associated with response to PGM (likelihood ratio test (LR) p&amp;lt; 0.05), as do lower levels of the IGF1 ligand score and higher levels of the IGF-1R signature. However, levels of IRS1 and the two expression signatures also trend toward or are significantly associated with response in the control arm; and treatment interactions for all four biomarkers are non-significant (LR p&amp;gt;0.05). Therefore, none of these biomarkers qualify as specific predictors of response to PGM. Similarly, high MammaPrint scores (MP2) were associated with higher pCR scores in both PGM and Control arms. Previous gene expression profiles were divided into tertiles (low, intermediate, high). Similar to the continuous case, IGF1Rsig-class associates with pCR in both the PGM and control arms (Fisher test p=0.033 and 0.044, respectively), and thus also fails as a specific predictor of response to PGM. We conclude that PGM therapy results in worsening of glucose control and likely increases serum insulin levels. While IGF gene expression profiling associated with treatment response, they were not specific for PGM. Further, biomarker analysis and strategies to control glucose will be needed to optimize anti-IGF-1R therapies. Citation Format: Douglas Yee, Paul Haluska, Denise M Wolf, Christina Yau, Amy Wilson, I-SPY2 TRIAL Consortium, Angela DeMichele, Claudine Isaacs, Jane Perlmutter, Joan Venticinque, Hope S Rugo, Richard Schwab, Nola M Hylton, W Fraser Symmans, Michelle E Melisko, Teresa L Helsten, Laura J van't Veer, Donald A Berry, Laura J Esserman. Biomarker analysis of paclitaxel, ganitumab, and metformin (PGM) therapy in the I-SPY2 neoadjuvant clinical trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-08.

MiR-7a Targets Insulin Receptor Substrate-2 Gene and Suppresses Viability and Invasion of Cells in Diabetic Retinopathy Mice via PI3K-Akt-VEGF Pathway.

IntroductionDiabetic retinopathy (DR) is one of the major leading causes for vision loss globally. Current study illustrates the role of miR-7a in DR.Material and MethodsRetinal pericytes (RPs) and Endothelial cells (ECs) were isolated from mouse model of DR. qRT-PCR was done for expression of miR-7a and target gene mRNA, Western blot for protein expression. Identification of miR-7a target gene was done by TargetScan and Luciferase assay. Cell viability and invasion was done by MTT and Transwell chamber assay.ResultsThe expression of miR-7a was down-regulated whereas level of IRS-2 was unregulated in isolated RPs and ECs. Luciferase assay suggested correlation between miR-7a and IRS-2, over-expression of miR-7a using a mimic resulted in suppression in viability and invasion capacity of RPs and ECs and inhibited the protein levels of PI3K/Akt cascade and IRS-2, and however the inhibitor reversed them respectively. Transfection of siRNA targeting IRS-2 caused alteration in miR-7a mediated changes in ECs suggesting that miR-7a may decrease angiogenesis in DR by inhibiting the levels of IRS-2.ConclusionmiR-7a suppresses PI3K/Akt cascade via targeting IRS-2, thus decreasing the viability and invasion capacity of RPs and ECs, suggesting an interesting treatment target for DR.

Altered Insulin Receptor Substrate 1 Phosphorylation in Blood Neuron-Derived Extracellular Vesicles From Patients With Parkinson's Disease.

IntroductionDiabetes increases the risk of Parkinson’s disease (PD). The phosphorylation of type 1 insulin receptor substrate (IRS-1) determines the function of insulin signaling pathway. Extracellular vesicles (EVs) are emerging as biomarkers of human diseases. The present study investigated whether PD patients exert altered phosphorylation IRS-1 (p-IRS-1) inside the blood neuron-derived extracellular vesicles (NDEVs).Research Design and MethodsIn total, there were 94 patients with PD and 63 healthy controls recruited and their clinical manifestations were evaluated. Blood NDEVs were isolated using the immunoprecipitation method, and Western blot analysis was conducted to assess total IRS-1, p-IRS-1, and downstream substrates level in blood NDEVs. Statistical analysis was performed using SPSS 19.0, and p < 0.05 was considered significant.ResultsThe isolated blood EVs were validated according to the presence of CD63 and HSP70, nanoparticle tracking analysis and transmission electron microscopy. NDEVs were positive with neuronal markers. PD patients exerted significantly higher level of p-IRS-1S312 in blood NDEVs than controls. In addition, the p-IRS-1S312 levels in blood NDEVs was positively associated with the severity of tremor in PD patients after adjusting of age, sex, hemoglobin A1c, and body mass index (BMI).ConclusionPD patients exerted altered p-IRS-1S312 in the blood NDEVs, and also correlated with the severity of tremor. These findings suggested the association between dysfunctional insulin signaling pathway with PD. The role of altered p-IRS-1S312 in blood NDEVs as a segregating biomarker of PD required further cohort study to assess the association with the progression of PD.

Quantitative proteomics and phosphoproteomic analyses of mouse livers after tick-borne Babesia microti infection

Babesia microti is a tick-borne protozoan parasite that infects the red blood cells of mice, humans, and other mammals. The liver tissues of BALB/c mice infected with B. microti exhibit severe injury. To further investigate the molecular mechanisms underlying liver injury and liver self-repair after B. microti infection, data-independent acquisition (DIA) quantitative proteomics was used to analyse changes in the expression and phosphorylation of proteins in liver tissues of BALB/c mice during a B. microti infection period and a recovery period. The expression of FABP1 and ACBP, which are related to fatty acid transport in the liver, was downregulated after infection with B. microti, as was the expression of Acox1, Ehhadh and Acaa1a, which are crucial rate-limiting enzymes in the process of fatty acid β oxidation. The phosphorylation levels of AMP-activated protein kinase (AMPK) and Hormone-sensitive lipase (HSL) were also downregulated. In addition, the expression of PSMB9, CTSC, and other immune-related proteins was increased, reflecting an active immune regulation mechanism in the mice. The weights of mice infected with B. microti were significantly reduced, and the phosphorylation levels of IRS-1, c-Raf, mTOR, and other proteins related to growth and development were downregulated.