Insulin Receptor Substrate-1 mRNA Research Articles

ASPSCR1::TFE3-mediated upregulation of insulin receptor substrate 2 (IRS-2) activates PI3K/AKT signaling and promotes malignant phenotype

The ASPSCR1::TFE3 fusion gene, resulting from chromosomal translocation, is detected in alveolar soft part sarcoma (ASPS) and a subset of renal cell carcinomas (RCC). The ASPSCR1::TFE3 oncoprotein, functioning as an aberrant transcription factor, contributes to tumor development and progression by inappropriately upregulating target genes. Here, we identified insulin receptor substrate 2 (IRS-2), a cytoplasmic adaptor protein, as a novel transcriptional target of ASPSCR1::TFE3. Ectopic expression of ASPSCR1::TFE3 led to increased IRS-2 mRNA and protein levels. Chromatin immunoprecipitation and luciferase assays demonstrated that ASPSCR1::TFE3 bound to the IRS-2 promoter region and enhanced its transcription. Moreover, IRS-2 was highly expressed in the ASPSCR1::TFE3-positive RCC cell line FU-UR1, while small interfering RNA-mediated depletion of ASPSCR1::TFE3 markedly decreased IRS-2 mRNA and protein levels. Functionally, IRS-2 knockdown attenuated activation of the PI3K/AKT pathway and reduced proliferation, migration, invasion, adhesion, and clonogenicity in FU-UR1 cells. Pharmacological inhibition of IRS-2 also reduced AKT activation as well as cell viability, clonogenicity, migration, invasion, and adhesion. These findings suggest that IRS-2, regulated by ASPSCR1::TFE3, promotes tumor progression by activating PI3K/AKT signaling and enhancing the malignant phenotype.

Renal gluconeogenesis induction by glucagon and insulin receptor deletion in humans

Gluconeogenesis is one of the key processes through which the kidney contributes to glucose homeostasis. Recently, we provided evidence to suggest that early insulin resistance may impair fast‐fed regulation of Phosphoenolpyruvate Carboxykinase (PEPCK), rate‐limiting enzyme for gluconeogenesis in humans. To confirm this and to elucidate a link between insulin resistance and fast‐fed regulation of PEPCK in the kidneys, we studied primary proximal tubule cells (hPT) and urinary extracellular vesicles (uEVs) from humans. We demonstrate that fasted hPTs had higher PEPCK and insulin receptor substrate‐2 (IRS2) mRNA levels than fed cells. The fast‐fed regulation was, however, attenuated in insulin receptor knockdown hPTs (IRKO). The insulin receptor knockdown was confirmed by a blunted insulin‐induced suppression in PEPCK and PGC1α mRNA and p‐IR and p‐AKT protein expression in IRKO cells. Exosomes secreted by the wild‐type or IRKO hPT showed similar regulation as observed in the respective hPT. Similarly, in human uE, the relative abundance of IRS‐2 mRNA (to IRS1) was higher in the fasted state relative to the fed condition. However, the fast‐fed difference was absent in subjects with early insulin resistance. These subjects had higher circulating glucagon levels relative to subjects with optimal insulin sensitivity. Furthermore, in hPT cells, glucagon significantly induced PEPCK mRNA, IRS2 mRNA, and gluconeogenesis. Insulin receptor deletion further increased their levels. Together the data suggest that reduced insulin sensitivity and high glucagon in early insulin resistance may impair renal gluconeogenesis via IRS2 regulation. Funding from ICMR‐CARE and SGPGI intramural to ST and CSIR to RS.

Low-frequency electroacupuncture improves disordered hepatic energy metabolism in insulin-resistant Zucker diabetic fatty rats via the AMPK/mTORC1/p70S6K signaling pathway.

Disordered hepatic energy metabolism is found in obese rats with insulin resistance (IR). There are insufficient experimental studies of electroacupuncture (EA) for IR and type 2 diabetes mellitus (T2DM). The aim of this study was to probe the effect of EA on disordered hepatic energy metabolism and the adenosine monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase, 70-kDa (p70S6K) signaling pathway. Zucker Diabetic Fatty (ZDF) rats were randomly divided into three groups: EA group receiving EA treatment; Pi group receiving pioglitazone gavage; and ZF group remaining untreated (n = 8 per group). Inbred non-insulin-resistant Zucker lean rats formed an (untreated) healthy control group (ZL, n = 8). Fasting plasma glucose (FPG), fasting insulin (FINS), C-peptide, C-reactive protein (CRP) and homeostatic model assessment of insulin resistance (HOMA-IR) indices were measured. Hematoxylin-eosin (H&E) staining was used to investigate the liver morphologically. The mitochondrial structure of hepatocytes was observed by transmission electron microscopy (TEM). Western blotting was adopted to determine protein expression of insulin receptor substrate 1 (IRS-1), mTOR, mTORC1, AMPK, tuberous sclerosis 2 (TSC2) and p70S6K, and their phosphorylation. RT-PCR was used to quantify IRS-1, mTOR, mTORC1, AMPK and p70S6K mRNA levels. Compared with the ZF group, FPG, FINS, C-peptide, CRP and HOMA-IR levels were significantly reduced in the EA group (p < 0.05, p < 0.01). Evaluation of histopathology showed improvement in liver appearances following EA. Phosphorylation levels of AMPK, mTOR and TSC2 decreased, and IRS-1 and p70S6K increased, in hepatocytes of the ZF group, while these negative effects appeared to be alleviated by EA. EA can effectively ameliorate IR and regulate energy metabolism in the ZDF rat model. AMPK/mTORC1/p70S6K and related molecules may represent a potential mechanism of action underlying these effects.

Insulin receptor substrate-1 and dishevelled 2 are negatively regulated by microRNA-144 and inhibit nasopharyngeal carcinoma cell malignancy.

Insulin receptor substrate-1 (IRS-1) is reported to play a critical role in the development, progression, invasion and metastasis of several types of tumors and is abnormally expressed in nasopharyngeal carcinoma (NPC). Although IRS-1 is predicted to be targeted by microRNA (miR)-144, the biological roles and potential mechanisms of miR-144 in NPC remain unclear. In the present study, the expression levels of miR-144 and IRS-1 in several NPC cell lines were first examined, and found that they were negatively correlated. Following the introduction of the miR-144 mimic, IRS-1 was downregulated at the protein level without affecting the mRNA level. The Cell Counting Kit-8 assay showed that the miR-144 mimic and siRNA targeting IRS-1 mRNA significantly decreased cell proliferation by arresting the cell cycle at the G1/G0 phase. The malignant behaviours of NPC cell lines, including migration, invasion and tumour formation in soft agar, were then analyzed after regulating miR-144 levels; as expected, the results showed that both the miR-144 mimic and siIRS-1 decreased these malignant behaviours. Furthermore, the downregulation of IRS-1 by miR-144 decreased the expression level of dishevelled 2 (Dvl2) protein without affecting its mRNA level, and Dvl2 overexpression abolished the inhibitory effect of the miR-144 mimic in NPC, indicating that miR-144 potentially regulates NPC by indirectly regulating Dvl2. Taken together, the present study results suggest that miR-144 acts as a tumour suppressor in NPC cell lines by regulating IRS-1 and Dvl2, which indicates that it is a potential therapeutic target for NPC treatment.

Agriophyllum Oligosaccharides Ameliorate Diabetic Insulin Resistance Through INS-R/IRS/Glut4-Mediated Insulin Pathway in db/db Mice and MIN6 Cells.

We have previously reported that Agriophyllum oligosaccharides (AOS) significantly enhance glycemic control by increasing the activation of insulin receptor (INS-R), insulin receptor substrate-2 (IRS-2), phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), peroxisome proliferator-activated receptor (PPAR)-γ, and glucose transporter 4 (Glut4) proteins in hepatic tissues. However, the effect of glucose control by AOS on the regulation of pancreatic tissues in db/db mice and MIN6 cells remains to be determined. An oral dose of AOS (380 or 750 mg/kg) was administered to type-2 diabetic db/db mice for 8 weeks to determine whether AOS regulates glucose by the INS-R/IRS/Glut4-mediated insulin pathway. Meanwhile, the effects of AOS on glucose uptake and its related signaling pathway in MIN6 cells were also investigated. The results showed that the random blood glucose (RBG) level in the AOS-treated group was lower than that in the control group. AOS reduced the levels of glycated hemoglobin (HbA1c) and free fatty acid (FFA) and significantly improved the pathological changes in the pancreatic tissues in db/db mice. Moreover, immunohistochemical analysis revealed that the expression of INS-R, IRS-1, IRS-2, and Glut4 was increased in the AOS-treated group than in the model group. Further, in vitro experiments using MIN6 cells showed that AOS regulated INS-R, IRS-1, IRS-2, and Glut4 protein and mRNA levels and attenuated insulin resistance and cell apoptosis. The results of both in vitro and in vivo experiments were comparable. Ultra-performance liquid chromatography coupled with time-of-flight mass spectrometric analysis of AOS with precolumn derivatization with 3-amino-9-ethylcarbazole (AEC) tentatively identified five types of sugars: glucose, lactose, rutinose, glucuronic acid, and maltotriose. Our present study clearly showed that AOS is efficacious in preventing hyperglycemia, possibly by increasing insulin sensitivity and improving IR by regulating the INS-R/IRS/Glut4 insulin signal pathway. Therefore, AOS may be considered as a potential drug for diabetes treatment.

Palmitic Acid Regulates miRNA-3148 via Insulin Receptor Substrate-1 and is Involved in Insulin Resistance

Previous studies reported that saturated fatty acid palmitic acid (PA) is closely related to insulin resistance. miR-3148 regulates insulin receptor substrate-1 (IRS1) predicted by MiRDB analysis. However, whether PA regulates IRS1 via miR-3148 remains to be elucidated. Therefore, in this work, we assessed whether PA regulates miRNA-3148 via IRS1 in insulin resistance. We cultured HepG2 cells in vitro and classified them into control group (NC group), miR-3148 Mimics group, and miR-3148 Mimics+ pFBD-IRS1 group. We used qRT-PCR to detect miR-3148 and IRS1 mRNA; used Dual-Luciferase Reporter Assays to detect miR-3148 with 3′-UTR region of IRS1 mRNA; and utilized Western blot (WB) to detect IRS1, p-AKT, AKT and Tubulin. Our results showed that PA could increase miR-3148 and decrease IRS1 which is a target protein of miR-3148, as shown by Dual-Luciferase Reporter assays. miR-3148 significantly inhibited the impact of insulin on p-AKT level (P &lt; 0.01) and over-expression of IRS1 by pFBD-IRS1 can partially alleviate the inhibitory effect of miR-3148 mimics on p-AKT. In HepG2 cells, PA regulates miR-3148. Via targeting IRS1 mRNA, miR-3148 impairs insulin signaling pathway, leading to insulin resistance. Over-expression of IRS1 by pFBD-IRS1 alleviates miR-3148-induced insulin resistance.

The role of serum inflammatory cytokines and berberine in the insulin signaling pathway among women with polycystic ovary syndrome.

ObjectiveTo study the role of selected serum inflammatory cytokines and berberine in the insulin signaling pathway among women with polycystic ovary syndrome (PCOS).MethodsSelected serum inflammatory cytokines were analyzed in the particle cells, which were interfered by berberine, from 78 infertile women who were to be treated with In Vitro Fertilization (IVF) /Intracytoplasmic Sperm Injection-Embryo Transfer (icsi-et). Among them, 49 patients had PCOS infertility, and 29 were non-PCOS patients whose infertility resulted from fallopian tube and male factors. The elisa method was used to detect the changes in the expression levels of inflammatory factors in the cells. The correlations between the serum inflammatory cytokine expression levels and the corresponding clinical hormones were analyzed. The changes in the expression (mRNA and protein) levels of the serum inflammatory cytokines were studied by real-time quantitative PCR and protein printing. Fluorescence microscope and flow cytometry were used to detect the glucose uptake capacity of ovarian granulosa cells in PCOS patients under the action of insulin after berberine.ResultsIn the PCOS group, IL-17a (P = 0.001), IL-1Ra (P<0.0001), and IL-6 (P = 0.035) were significantly higher than those in the non-PCOS group. In the non-PCOS group, AMH level was negatively correlated with inflammatory cytokines IL-17a (r = -0.819;P = 0.004), IL-1a (r = -0.716;P = 0.0.02), IL-1b (r = -0.678;P = 0.031), IL-2 (r = -0.765;P = 0.01), and IL-8 (r = -0.705;P = 0.023). However, in the PCOS group, AMH levels were not significantly correlated with the levels of the examined inflammatory cytokines. Berberine significantly reduced the expression level of mTOR mRNA (P = 0.001), and increased the expression level of IRS-1 mRNA (P = 0.009) in the PCOS granule cells.ConclusionIn this study, we find that the elevated levels of serum inflammatory factors IL-17a, IL-1Ra, and IL-6 cause women to be in a subclinical inflammatory state for a long time. Abnormal changes in inflammatory factors alter their original negative correlations with AMH levels, thereby weakening the metabolism of glycolipids, promoting insulin resistance, destroying the normal ovulation and fertilization system of women, leading to polycystic ovary syndrome characterized by menstrual thinning and abnormal ovulation. Berberine can improve the sensitivity of insulin by regulating the signal pathway of insulin receptor substrate-1 (IRS-1) and mammalian target of rapamycin (mTOR) in PCOS patients and achieve a therapeutic effect of treating PCOS.

Downregulation of microRNA‑126 is inversely correlated with insulin receptor substrate‑1 protein expression in colorectal cancer and is associated with advanced stages of disease

Colorectal cancer (CRC) is a common human malignant tumor, and the fourth most common cause of cancer-associated mortality in China. However, the pathogenesis of CRC is not yet fully understood. The present study aimed to investigate the expression and clinical significance of microRNA (miR)-126 and insulin receptor substrate-1 (IRS-1), as well as the role of miR-126 in the prognosis of patients with CRC. A total of 86 colorectal tissue specimens, including 40 CRC and adjacent normal tissue, 26 colorectal adenoma tissue and 20 normal colorectal tissue samples, were collected for the present study. Reverse transcription-quantitative PCR analysis was performed to determine miR-126 and IRS-1 mRNA expression levels, while western blotting and immunohistochemistry (IHC) analyses were performed to determine IRS-1 protein expression levels. The correlation between miR-126 and IRS-1 expression, as well as the association between altered miR-126 and IRS-1 expression levels and clinicopathological characteristics, and the overall survival time of patients with CRC were assessed. The results demonstrated that miR-126 expression was significantly downregulated, while IRS-1 protein expression was upregulated in CRC tissues compared with that in adjacent normal tissues, colorectal adenoma tissues and normal colorectal tissues, respectively. IHC analysis exhibited strong positive staining of IRS-1 protein in CRC tissues, while absent or weak staining of IRS-1 protein was detected in adjacent normal tissues, colorectal adenoma tissues and normal colorectal tissues. miR-126 expression was inversely correlated with IRS-1 protein expression in CRC tissues (r=−0.420; P<0.05). Furthermore, downregulated miR-126 expression was associated with advanced clinicopathological characteristics of the disease and a shorter overall survival time in patients with CRC. Taken together, the results of the present study suggest that miR-126 downregulation may be a candidate molecular marker predictive of poor prognosis of patients with CRC.

MiR-145 is involved in the proliferation of bovine mammary epithelial cells and regulates bovine insulin receptor substrate 1

MicroRNAs (miRNAs) are short noncoding RNAs that can regulate target gene expression at the posttranscriptional level. It has been reported that bta-miR-145 shows differential expression between the different lactation stages of bovine mammary glands, but the function of bta-miR-145 in normal mammary glands remains unknown. The overexpression of bta-miR-145 significantly decreased the proliferation of mammary epithelial cells (p < .05), and this effect was reversed upon bta-miR-145 inhibition. Using several miRNA prediction programmes, insulin receptor substrate 1 (IRS1) was predicted to be a potential target of miR-145. Using the DAVID database, many predicted targets of bta-miR-145 were found to be involved in the MAPK signalling pathway associated with cell proliferation, including IRS1. Bovine IRS1 3’RACE sequencing and reference comparison revealed that possible binding sites for bta-miR-145 still exist in IRS1. The IRS1 mRNA level was not affected by the transfection of primary bovine mammary epithelial cells with 150 pmol of a bta-miR-145 mimic or 300 pmol of a bta-miR-145 inhibitor for 24 h (pBMEC, p > .05), whereas the IRS1 protein level was changed significantly after bta-miR-145 mimic or inhibitor transfection for 72 h (p < .05). Taken together, these results suggest that bta-miR-145 is involved in the proliferation of bovine mammary epithelial cells by targeting IRS1, which is related to the MAPK signalling pathway. Highlights Bta-miR-145 affects bovine mammary epithelial cell vitality in primary bovine mammary epithelial cells. IRS1 was verified as target gene of bta-miR-145. IRS1 belongs to MAPK signaling pathway, which regulate cell proliferation.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A ameliorates insulin resistance in neurons by up-regulating IRS-1 expression

Insulin resistance in the brain is a pathological mechanism that is shared between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Although aberrant expression and phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to insulin resistance, the underlying mechanism remains elusive. In this study, we used several approaches, including adeno-associated virus-based protein overexpression, immunoblotting, immunoprecipitation, immunohistochemistry, and in situ proximal ligation assays, to investigate the function of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) in IRS-1 regulation and the downstream insulin signaling in neurons. We found that DYRK1A overexpression up-regulated IRS-1 expression by slowing turnover of the IRS-1 protein. We further observed that DYRK1A directly interacted with IRS-1 and phosphorylated IRS-1's multiple serine residues. Of note, DYRK1A and IRS-1 were coordinately up-regulated in the prefrontal cortex of db/db mice brain. Furthermore, DYRK1A overexpression ameliorated chronic high insulin-induced insulin resistance in SH-SY5Y cells as well as in primary rat neurons. These findings suggest that DYRK1A protects against insulin resistance in the brain by elevating IRS-1 expression.

Centella asiatica (L.) extract attenuates inflammation and improve insulin sensitivity in a coculture of lipopolysaccharide (LPS)-induced 3T3-L1 adipocytes and RAW 264.7 macrophages.

Insulin resistance in obese condition is related to chronic low-grade inflammation which leads to insulin signaling impairment. Centella asiatica (L.) is an herb that exhibits anti-inflammatory and blood sugar-lowering activity (hypoglycemia). The study aims to investigate the molecular mechanism of C. asiatica extract in insulin sensitivity improvement in a coculture of lipopolysaccharide (LPS)-induced 3T3-L1 adipocytes and RAW 264.7 macrophages. A coculture of 3T3-L1 adipocytes and RAW 264.7 macrophages were incubated with LPS to induce insulin resistance in the adipocytes. An extract of C. asiatica was added to coculture cells and after 24 hours, insulin sensitivity and inflammatory response were determined, including glucose consumption, glucose transporter-4 (GLUT-4), insulin receptor substrate-1 (IRS-1), and interleukin-6 (IL-6) mRNA expression. C. asiatica extract at a concentration of 500 µg/mL increased glucose consumption and induced GLUT-4 and IRS-1 mRNA expression significantly in a coculture of LPS-induced 3T3-L1 adipocytes and RAW 264.7 macrophages. The pro-inflammatory cytokines IL-6 mRNA expression was decreased in the coculture cells after treatment with C. asiatica extract at a concentration of 500 µg/mL. This result indicates that C. asiatica has an effect to stimulate glucose consumption in the coculture cells that might be mediated via GLUT-4/IRS-1 pathway as a result of IL-6 inhibition. These findings suggest that the C. asiatica extract inhibits inflammation and improves insulin sensitivity in a coculture of LPS-induced 3T3-L1 adipocytes and RAW 264.7 macrophages.

Effects of Aryl Hydrocarbon Receptor Deficiency on PCB-77-Induced Impairment of Glucose Homeostasis during Weight Loss in Male and Female Obese Mice.

Background:Lipophilic polychlorinated biphenyls (PCBs) accumulate with obesity, but during weight loss, liberated PCBs act as ligands of the aryl hydrocarbon receptor (AhR) to negatively influence health. Previous studies demonstrated that PCB-77 administration to obese male mice impaired glucose tolerance during weight loss. Recent studies indicate higher toxic equivalencies of dioxin-like PCBs in exposed females than males.Objectives:We compared effects of PCB-77 on weight gain or loss and glucose homeostasis in male vs. female mice. We defined effects of AhR deficiency during weight gain or loss in male and female mice exposed to PCB-77.Methods:Study design was vehicle (VEH) or PCB-77 administration while fed a high-fat (HF) diet for 12 wk, followed by weight loss for 4 wk. The following groups were examined: male and female C57BL/6 mice administered VEH or PCB-77, female and mice administered VEH or PCB-77, and male and mice administered PCB-77. Glucose tolerance was quantified during weight gain (week 11) and loss (week 15); liver and adipose AhR and IRS2 (insulin receptor substrate 2) mRNA abundance, and PCB-77 concentrations were quantified at week 16.Results:PCB-77 attenuated development of obesity in females but not males. During weight loss, PCB-77 impaired glucose tolerance of males. AhR-deficient females (VEH) were resistant to diet-induced obesity. Compared with VEH-treated mice, HF-fed females treated with PCB-77 has less weight gain, and females had greater weight gain. During weight loss, females but not males treated with PCB-77 exhibited impaired glucose tolerance. In females administered PCB-77, IRS2 mRNA abundance was lower in adipose tissue compared with VEH-treated mice.Conclusion:Male and female mice responded differently to PCB-77 and AhR deficiency in body weight (BW) regulation and glucose homeostasis. AhR deficiency reversed PCB-77-induced glucose impairment of obese males losing weight but augmented glucose intolerance of females. These results demonstrate sex differences in PCB-77-induced regulation of glucose homeostasis of mice. https://doi.org/10.1289/EHP4133

Effects of Agriophyllum squarrosum extracts on glucose metabolism in KKAy mice and the associated underlying mechanisms

Ethnopharmacological relevanceAgriophyllum squarrosum (L.) Moq. is a traditional Mongol medicine commonly used in the treatment of diabetes. Aim of the studyTo examine the effects of Agriophyllum squarrosum extract (ASE) on glucose metabolism in type 2 diabetic KKAy mice, and to investigate the mechanisms underlying these effects. Material and methodsKKAy mice were divided into a model control group (MCG), a low-dose Agriophyllum squarrosum extract group (LASEG), a medium-dose Agriophyllum squarrosum extract group (MASEG), a high-dose Agriophyllum squarrosum extract group (HASEG), and a metformin group (MEG). Syngeneic C57BL/6 mice were used as a normal control group (NCG). Drugs were administered to all mice by gavage for 8 weeks. Random blood glucose levels were measured in the mice at baseline and after 2, 4, and 8 weeks of treatment. Glucose tolerance was measured after 6 weeks of drug administration. After 8 weeks, glycated serum proteins (GSP) and advanced glycation end-products (AGEs) in the serum of all mice were measured. Sections of mouse liver tissues were used for periodic acid-Schiff staining (PAS) and the content of hepatic glycogen was determined. Immunohistochemistry was used to determine the effects of ASE on liver phospho-insulin receptor substrate 2 (P-IRS2) protein expression. Western blotting was used to quantify the protein expression levels of phosphatidylinositol 3-kinase (PI3K), AKT, phospho-AKT (S473) (P-AKT), glycogen synthase kinase 3β (GSK3β), and glucose transporters 4 (GLUT4), while PCR was used to quantify the mRNA expression levels of insulin receptor substrate 2 (IRS2), PI3K, AKT, GSK3β, and GLUT4. ResultsASE treatment decreased random blood glucose levels in type 2 diabetic KKAy mice; increased glucose tolerance; decreased serum GSP and AGEs content; increased glycogen synthesis in liver tissues; upregulated the protein expression levels of PI3K, AKT, GLUT4, and P-IRS2; downregulated the protein expression level of GSK3β in liver tissues; upregulated the mRNA expression levels of IRS2, PI3K, AKT, and GLUT4; and downregulated the mRNA expression level of GSK3β in liver tissues. ConclusionASE treatment may increase glucose metabolism in KKAy mice and improve glucose tolerance. The underlying mechanisms of the beneficial effects of ASE may be associated with the increase of glycogen synthesis, the inhibition of AGEs production, the upregulation of IRS2, PI3K, AKT, and GLUT4 protein and mRNA expression, and the downregulation of GSK3β protein and mRNA expression.

Yunpi Heluo decoction attenuates insulin resistance by regulating liver miR-29a-3p in Zucker diabetic fatty rats

Background and objectiveYunpiheluo (YPHL) decoction is a Chinese herbal formula with unique advantages for the treatment of type 2 diabetes mellitus (T2DM). The aim of the present study was to investigate changes in miRNA expression and downstream gene expression in Zucker diabetic fatty (ZDF) rats treated with YPHL to determine whether YPHL could be used as an adjuvant treatment of T2DM. MethodsSerum and liver total cholesterol (TC) and triglycerides (TG) levels, insulin resistance index (IR) and differentially expressed miRNAs were detected in a T2DM ZDF rat model. miRNA target prediction was based on bioinformatic algorithms and dual luciferase reporter assay. Protein expression of genes in the insulin receptor signaling pathway was detected by Western blot. The IR cell model was established and the effects of lyophilized YPHL powder on the protein expressions were observed by transfecting specific miRNA mimics and inhibitors. ResultsThe miR-29a-3p expression level was significantly increased in the liver of ZDF rats. Insulin receptor substrate 1 (IRS1) was the target gene of miR-29a-3p. IRS1 mRNA and protein expressions of IRS1, IRS1 (phospho S307), protein kinase B (Akt), Akt (phosphor ser473) and pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1) were decreased significantly. miR-29a-3p over-expression decrease IRS1 and the others protein expressions in the HepG2 IR cell model while anti-miR-29a-3p showed the opposite result. The miR-29a-3p level was decreased, and the expressions of IRS1 mRNA and the above proteins were all increased after YPHL treatment. ConclusionmiR-29a-3p played a functional role in insulin receptor signaling in the liver of ZDF rats. YPHL decoction attenuated IR in T2DM probably by down-regulating or maintaining the miR-29a-3p level, increasing the expression of IRS1 mRNA and its phosphorylated proteins, and regulating the expression of insulin receptor signaling-related proteins. YPHL may prove to be an alternative treatment for T2DM.

Treatment of type 2 diabetes mellitus with the stem and leaf of blacktartary buckwheat and its effects on the pancreas and spleen

To investigate the therapeutic effects of the black buckwheat leaf (BBL) in type 2 diabetes mellitus mice and its effects on pancreas and spleen. Forty male C57 / B16 mice (SPF) were randomly divided into normal control (NC) group (n=10) and the experimental group (n=30), the experimental group were fed with high sugar and high fat, combined with intraperitoneal injection of streptozotocin in small dose to establish the model of type 2 diabetes mellitus (T2DM). Those thirty model mice were randomly divided into 3 groups (n=10), diabetes mellitus group (DM), low dose of BBL (DM+L) treated group, high dose of BBL (DM+H) treated group. The mice in the NC group and the DM group were given normal saline per day, and the DM+L group and DM+H group were treated with black tartary buckwheat at the doses of 0.21g/kg·d-1 and 0.42g/kg·d-1 respectively. After 14 days. All mice were executed by cervical dislocation, then blood samples were collected, pancreas and spleen were removed for subsequent experiments. The serum levels of fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TCH) and insulin were detected. TNF-α protein in spleen tissue was detected by ELISA kit. The morphology of pancreas tissue was observed by HE staining, and the spleen coefficient was calculated. The expression levels of insulin receptor substrate-1(IRS-1) mRNA and IRS-1 protein in pancreatic tissue were detected. Compared with the control group, the serum levels of FBG, TC and TCH in the model group were increased significantly, while the serum level of insulin was decreased significantly (P＜0.05), the expression of TNF-α protein in spleen tissues was obviously raised, the expressions of IRS-1 mRNA and IRS-1 protein in pancreatic tissue in model group were decreased significantly (P＜0.05). Compared with the model group, the serum levels of FBG, TC and TCH were decreased significantly in the BBL treated groups. The serum insulin level, spleen coefficient, TNF-α protein expression level in spleen tissue, IRS-1 mRNA expression and IRS-1 protein expression levels in pancreatic tissue in BBL treated group were increased significantly (P＜ 0.05). High-dose black tartary buckwheat leaves (0.42g/kg·d-1) exerted a more significant effect. Stem and leaf of black bitter buckwheat has significant therapeutic effects on reducing blood sugar and blood fat in type 2 diabetic mice, and has certain protective effects on pancreas and spleen of diabetic mice.

Protective effects of rutin on liver injury in type 2 diabetic db/db mice

The aim of this study was to evaluate the protective effect of rutin on the liver of type 2 diabetic mice and explore the correlation mechanism. The db/db mice, selected as the type 2 diabetes mellitus (T2DM) models, have random blood glucose (RBG) and glucose level after 2 h of oral glucose loading of more than 16.7 mmol/L. After administration of 120 mg/kg or 60 mg/kg rutin, to T2DM mice, RBG, oral glucose tolerance, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, and advanced glycation end products (AGEs) in vivo and vitro of different groups were detected. The liver pathological changes were observed under light and electron microscopy. Western blotting was used to detect the protein expression of insulin receptor substrate 2 (IRS-2) and phosphorylation of phosphatidylinositol 3 kinase (PI3K) on p85, Akt on Ser473, glycogen synthase kinase 3β (GSK-3β) on Ser9, real-time quantitative PCR was used to detect IRS-2 mRNA expression. Moreover, dynamically observing the effect of rutin on the generation of AGEs in non-enzymatic protein glycosylated system, Cell Counting Kit-8 (CCK-8) method was used to detect the effect of rutin on proliferation activity of HepG2 liver cells. The results showed that RBG and glucose levels of oral glucose tolerance test (OGTT) of mice in model group were significantly higher than that of normal group, which were significantly reduced after the rutin treatment. Rutin could reduce the ALT, AST activities and AGEs level in serum and potentiate the expression of IRS-2, P-PI3K (p85), P-Akt (Ser473), P-GSK-3β (Ser9) protein and IRS-2 mRNA in the liver tissue of db/db mice. Moreover, rutin could significantly alleviate the structure disorder of liver, reduce the degeneration and necrosis of liver cells and formation of collagen fibers of db/db mice. The results in vitro also showed that rutin could obviously inhibit the generation of AGEs, and promoted the proliferation activity of high glucose-stimulating HepG2 cells. In general, the protective effect of rutin on the liver of T2DM may be mediated by facilitating signal transduction and activated state of insulin IRS-2/PI3K/Akt/GSK-3β signal pathway, promoting hepatocyte proliferation, reducing blood glucose level and generation of AGEs.

TAZ enhances mammary cell proliferation in 3D culture through transcriptional regulation of IRS1

WW domain-containing transcriptional regulator 1 (TAZ) is a transcriptional co-activator and effector of the Hippo signaling pathway. In certain breast cancer subtypes, Hippo signaling is dysregulated leading to activation of TAZ and altered expression of TAZ transcriptional targets. Over the past decade, we and others have found that TAZ transcriptionally regulates genes that affect multiple aspects of breast cancer cell behaviour. However, while cancer cell-intrinsic oncogenic functions of TAZ have emerged, less is known about whether TAZ might also contribute to tumourigenesis by sensitizing tumour cells to factors present in the tumour microenvironment or in systemic circulation. Here, we show that TAZ directly regulates the expression of insulin receptor substrate 1 (IRS1) in breast cancer cells. TAZ or IRS1 overexpression induces a similar proliferative transformation phenotype in MCF10A mammary epithelial cells. TAZ enhances IRS1 mRNA, protein levels and downstream signaling in MCF10A. Mechanistically, TAZ interacts with the IRS1 promoter through the TEAD family of transcription factors and enhances its activity. Critically, TAZ-induced IRS1 upregulation contributes to the proliferation of TAZ-overexpressing MCF10A in 3-dimensional (3D) Matrigel culture. Therefore, we offer compelling evidence that TAZ regulates signaling through the insulin pathway in breast cancer cells. These findings highlight an additional mechanism by which TAZ may promote breast cancer tumourigenesis and progression by modulating cancer cell responses to exogenously produced factors.

The intervention effects of AdipoRon on renal injury in type 2 diabetic mice

To study the effects of adiponin receptor agonist (AdipoRon) on renal injury in type 2 diabetic mice. The experiment was carried out on 40 SPF C57/BL6 male mice and they were randomly divided into normal control group (n=10) and experimental group (n=30). Mice in experimental group were given with high sugar and high fat feed in combination with only an intraperitoneal injection of small dose of streptozotocin to build the model of type 2 diabetes (T2DM), which were randomly divided into three groups, model control group (DM), low dose AdipoRon group (DM + L) and high dose AdipoRon group (DM+H)(n=10). Then the change of blood glucose was detected. The serum levels of insulin receptor (INSR), insulin receptor substrate-1 (IRS-1) and tumor necrosis factor-α (TNF-α) in mice were measured by ELASA. Pathological changes of renal tissues were observed with a light microscope after HE staining. The expressions of pancreatic duodenal homebox-1 (PDX-1) and insulin mRNA in renal tissues were detected by RT-PCR. The content of phosphated insulin receptor substrate-1 (p-IRS-1) protein in the kidney was determined by Western blot. Compared with DM mice, blood glucose and TNF-α levels in DM + H mice and DM + L mice were significantly reduced (P<0.05), while the expressions of INSR,IRS-1 and the content of p-IRS-1 were increased markedly(P<0.05), and the expressions of PDX-1 and insulin mRNA in renal tissue were increased significantly(P<0.05, P<0.01). Mice treated with AdipoRon have lower blood glucose and TNF-α levels, and higher protein expression levels of INSR, IRS-1, and higher mRNA expression levels of PDX-1 and insulin, and the content of p-IRS-1. All of these indicate that AdipoRon has a certain effects on renal injury in type 2 diabetic mice.

Effect of ursolic acid on obesity-induced insulin resistance in rat liver

Purpose: To determine the expression of protein tyrosine phosphatase-1B (PTP-1B) and insulin receptor substrate-2 (IRS-2) in the liver tissue of obesity-induced insulin-resistant rats.Methods: Insulin resistance (IR) was induced in Wistar rats by placing them on a high fat diet for 6weeks, and ursolic acid (UA) was administered. Metformin served as positive control drug. The rats were divided into 5 groups based on the treatments given: normal group, positive control group, metformin group, high-dose UA group, and low-dose UA group. The general conditions of the rats were assessed 4 and 8 weeks after the various treatments. Liver glycogen levels were measured, and liver histological examination carried out after tissue processing and staining with hematoxylin and eosin (H &amp; E). Real-time polymerase chain reaction (RT-PCR) was employed for the determination of hepatic expressions of PTP-1B and IRS-2 mRNAs, while expressions of PTP-1B protein and IRS-2 protein, and phosphorylation of IRS-2 tyrosine were assayed by Western blotting.Results: Liver glycogen levels were significantly increased in the UA-treated groups (p &lt; 0.05). Moreover, UA provoked reductions in the expression of PTP-1B protein (p &lt; 0.05), but up-regulated the expression of IRS-2 protein (p &lt; 0.05), and enhanced IRS-2 tyrosine phosphorylation (p &lt; 0.05).Conclusion: These results suggest that UA mitigates IR through blockage of PTP-1B expression and up-regulation of the expression of IRS-2 mRNA. Therefore, PTP-1B is a potential target for the treatment of type 2 diabetes.Keywords: Ursolic acid, Insulin resistance, Liver, Protein tyrosine phosphatase-1B, Insulin receptor substrate-

Effect of Metformin-Induced Stimulation on the Expression of Insulin Receptor Substrate 1 through Negative Regulation of P70S6k

Objective: The aim is to study the effects of metformin on the expression of 70 kDa ribosomal protein S6 kinase (P70S6k), insulin receptor substrate 1 (IRS-1), and IRS-1Ser307 phosphorylation in human luteinized granulosa cells. Methods: Granulosa cells in the experimental group were cultured in M199 medium containing 0.1 mmol/L metformin for 24 h and those in control group were cultured in M199 medium. The expression levels of P70S6k and IRS-1 mRNA were detected by reverse-transcriptiom polymerase chain reaction (RT-PCR) and real-time PCR. P70S6k, IRS-1, p-ser307-IRS-1, and p-thr389-P70S6k protein expression levels were detected by immunofluorescence and western blotting. Results: P70S6k mRNA level was higher and IRS-1 was significantly lower in the experimental group than those in the control group. IRS-1 and p-ser307-IRS-1 were expressed in cell plasma, and P70S6k and p-thr389-P70S6k were expressed in cell nucleus. The results of Western blot analysis indicated that the expression levels of P70S6k, p-thr389-P70S6k, IRS-1, and p-ser307-IRS-1 proteins had significant difference between the experimental group and the control group. Compared to the control group, the relative intensity illustrated that the expression levels of P70S6K and p-thr389-P70S6k significantly increased in the experimental group; however, those of IRS-1 and p-ser307-IRS-1 proteins significantly decreased. Conclusion: Metformin can inhibit the P70S6k mRNA and protein expression levels in the granulosa cells and improve insulin sensitivity by regulating IRS-1 expression through Akt/P70S6k/IRS-1-dependent pathway.