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.

Reactivation of the insulin-like growth factor-Ⅱsignaling pathway in human hepatocellular carcinoma

Constitutive activation of the insulin-like growth factor (IGF)-signaling axis is frequently observed in human hepatocellular carcinoma(HCC).Especially the over- expression of the fetal growth factor IGF-Ⅱ,IGF-Ⅰ receptor(IGF-IR),and cytoplasmic downstream effectors such as insulin-receptor substrates(IRS)contribute to proliferation,anti-apoptosis,and invasive behavior. This review focuses on the relevant alterations in this signaling pathway and independent in vivo models that support the central role IGF-Ⅱsignaling during HCC development and progression.Since this pathway has become the center of interest as a target for potential anti-cancer therapy in many types of malignancies,various experimental strategies have been developed,including neutralizing antibodies and selective receptor kinase inhibitors,with respect to the specific and efficient reduction of oncogenic IGF-Ⅱ/IGF-IR-signaling.

Research progress in mulberry leaf polysaccharide in treating diabetic nephropathy

Diabetic nephropathy(DN)is one of the most common complications of diabetes.It is an important cause of diabetes disability and death.DN is a systemic metabolic syndrome.In its pathogenesis,the interaction of various cell activities and a large number of cytokine biological activities,the activation of signal pathways and so on are involved in the development of DN.At present,the clinical treatment of DN is mainly Western medicine,but it has limitations such as strong toxicity,high side effects and poor compliance.Therefore,the discovery of natural anti-DN substances has also become an important means to treat DN.Mulberry leaves are the dry leaves of Morus alba L.It is not only a traditional Chinese medicine,but also a dual-purpose medicinal material for medicine and food.It has the effects of dispelling wind and clearing heat,cooling blood and brightening eyes,tonifying and so on.Mulberry leaf polysaccharide(MLP)is a kind of high molecular compound in mulberry leaves.It has many pharmacological effects,such as hypoglycemic,antioxidant,anti-stress,anti-virus and so on.Therefore,the pharmacological effects of mulberry leaf polysaccharides on diabetic nephropathy are reviewed in this paper,so as to provide references for further research and application.The pathogenesis of DN is complex,and the mechanism of renal injury has not been completely clarified.The current studies believe that DN is closely related to heredity,abnormal glucose metabolism,abnormal lipid metabolism,microcirculation disorder,cytokine action,oxidative stress and so on.Relevant studies show that the pharmacological effects of mulberry leaf polysaccharide in the prevention and treatment of DN mainly include:①Effect on transforming factor-β1(TGF-β1):TGF-β1 has become an important cytokine involved in the formation of renal fibrosis by regulating cell proliferation and differentiation and the production of extracellular matrix(ECM).MLP can significantly inhibit TGF-β1 protein,and then inhibit the synthesis of extracellular matrix by renal interstitial fibroblasts and inhibit the realization of fibrosis.②Effect on insulin receptor substrate(IRS-1):IRS-1 is an important signal molecule at the beginning of IR signal transduction.The decrease of IRS-1 gene expression or the decrease of expression can affect the effective transmission of IR signal and lead to the development and deterioration of diabetes. MPL can significantly increase the expression of IRS-1 mRNA in liver tissue of DN rats, so as to prevent and treat DN. ③ Effect on the expression of resistin protein in adipose tis sue. Resistin is a secretory polypeptide derived from adipose tissue and is specifically expressed in white adipose tissue and is closely related to type 2 diabetes mellitus (T2DM). Experimental studies show that MLP can effectively reduce the expression of resistin protein in white adipose tissue of T2DM rats, indicating that MLP may reduce the level of IR by inhibiting the expression of resistin in adipose tissue, thereby reducing the insulin resistance state of T2DM rats, so as to achieve the goal of treating diabetes. ④ Effect on adiponectin receptor 1 (AdipoR1): adiponectin can improve insulin resistance, reduce blood glucose and lipid. AdipoR1 is mainly expressed in skeletal muscle and kidney. Studies have shown that AdipoR1 is closely related to the occurrence and development of DN. The results showed that MLP could reduce the blood glucose and blood lipid level and up regulate the expression of AdipoR1 mRNA in DN rats, suggesting that MLP may delay the occurrence and development of DN. This article reviewed the pharmacological effects of mulberry leaf polysaccharides on diabetic nephropathy, and provided a useful basis for further development and utilization of mul berry leaf polysaccharides in the treatment of DN.

Clean-DM1, a Korean Polyherbal Formula, Improves High Fat Diet-Induced Diabetic Symptoms in Mice by Regulating IRS/PI3K/AKT and AMPK Expressionsin Pancreas and Liver Tissues

Objective:To investigate the effects of Clean-DM1(C-DM1),a polyherbal formulation of Radix Scrophulariae,Radix Astragali,Rhizoma Atractylodis,and Radix Salviae Miltiorrhizae,on high-fat diet(HFD)-induced diabetes mice.Methods:The information about active components of C-DM1 extract and molecular mechanism was obtained from network pharmacology analysis.Main compounds of C-DM1 extract by high performance liquid chromatography-mass spectrometry(HPLC-MS)analysis were conducted for quality control.For in vivo study,mice were induced diabetes by HFD for 12 weeks.The mice in the normal group(Nor)were maintained with a regular diet and treated with saline by gavage.The HFD model mice were randomly divided into 3 groups,including a HFD diabetic model group,a C-DM1 extract-administered group(C-DM1,500 mg/kg),and metformin-administered groups(Met,500 mg/kg),8 mice in each group.Food intake,body weight(BW),and fasting blood glucose(FBG)levels were recorded weekly for 4 weeks.After 4 weeks of treatment,alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood glucose,low-density lipoprotein cholesterol(LDL-C)were determined using an automated clinical chemistry analyzer,and homeostatic model for assessing insulin resistance(HOMA-IR)levels and oral glucose tolerance test(OGTT)were detected.The histopathological changes of liver and pancreatic tissues were observed by hematoxylin-eosin staining.Insulin receptor substrate(IRS)/phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)and adenosine 5'-monophosphate-activated protein kinase(AMPK)expressions in liver and pancreas tissues were detected by Western blot analysis.Results:HPLC-MS identified dihydroisotanshinone,dihydroisotanshinone I,cryptotanshinone,harpagoside,and atractyloside A in C-DM1 extract.The administration of C-DM1 extract significantly decreased body weight,calorie intake,and the levels of blood glucose and insulin in the diabetic mice(P<0.05 or P<0.01).The C-DM1 extract administration improved the impaired glucose tolerance and insulin resistance in the diabetic mice and significantly decreased the levels of LDL-C,ALT and AST(P<0.01).The C-DM1 extract inhibited the histopathological changes of fatty liver and hyperplasia of pancreatic islets in the diabetic mice.The C-DM1 extract significantly increased the phosphorylation of IRS,AKT,and AMPK and the expression of PI3K in pancreas and liver tissues(P<0.05 or P<0.01),which was consistent with the analysis results of network pharmacology.Conclusion:C-DM1 extract improved diabetes symptoms in longterm HFD-induced mice by regulation of IRS/PI3K/AKT and AMPK expressions in pancreas and liver tissues,suggesting that C-DM1 formulation may help prevent the progression of T2DM.

Effect of Shenzhu Tiaopi granule(参术调脾颗粒) on hepatic insulin resistance in diabetic Goto-Kakizakirats via liver kinase B1/adenosine 5’-monophosphate/mammalian target of rapamycin signaling pathway

OBJECTIVE: To observe the therapeutic effect of Shenzhu Tiaopi granule(参术调脾颗粒, STG) on insulin resistance(IR) in the liver of diabetic Goto-Kakizaki(GK) rat and investigate underlying mechanisms.METHODS: Ten 12-week-old male Wistar rats were assigned as normal control(NC) group, while 4012-week-old male specific-pathogen-free GK rats were randomly divided into four experimental groups, 10 diabetic rats each. Animals were fed with a normal diet. Fasting blood glucose(FBG), water intake, and body weight were recorded during6 weeks of daily single-dose treatment: STG low-dose group, 4.5 g/kg(STG-L);STG high-dose group,9 g/kg(STG-H);metformin group, 0.1 g/kg(MET);model control(MC) and NC groups, equal volume of 0.9% Na Cl solution. The serum fasting insulin(FINS), C-Peptide and IR index(HOMA-IR) were detected every 2 weeks during treatment and glucose tolerance was measured in the 3 rd day before the material was taken. After the 6-week STG treatment, Liver tissues were processed for hematoxylin-eosin staining to perform light microscopy analysis and for assessing expression and distribution of insulin receptor substrates(IRS-1) and glucose transporter(GLUT-4) by immunohistochemistry analysis. Expression levels of liver kinase B1(LKB1)/adenosine 5’-monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) pathway proteins, including LKB1, phospho-AMPK(p-AMPK)/AMPK, phospho-mTOR(p-mTOR)/mTOR, and ribosomal protein S6 kinase polypeptide 1(S6 K1),were detected by Western blotting.RESULTS: STG significantly reduced the FBG level and liver fat deposition in diabetic GK rats. After STG treatment completion, FINS, HOMA-IR, C-Peptide and area under blood glucose curve(AUC)were lower in STG groups than in the MC group, indicating that IR was reduced and liver fat lesions were resolved. In liver tissues, STG groups displayed significantly higher IRS-1 and GLUT-4 expression than the MC group, along with increased LKB1 and p-AMPK/AMPK expression and decreased p-mTOR/mTOR and phospho-S6 K1 expression, suggesting that STG stimulated LKB1 activation of AMPK and suppressed themTOR/S6 K1 downstream pathway.CONCLUSION: Growing GK rats developed hepatic IR, but STG treatment significantly improved hyperglycemia and IR and resolved hepatic fatty lesions.Interestingly, STG treatment stimulated the expression of IRS-1 and GLUT-4 in the liver of diabetic GK rats, indicating a potential involvement in the regulation of the LKB1/AMPK/mTOR signaling pathway.

Curcumin Alleviates Hyperandrogenism and Promotes Follicular Proliferation in Polycystic Ovary Syndrome Rats:Insightson IRS1/PI3K/GLUT4 and PTEN Modulations

Objective:To explore the effect of curcuminon the insulin receptor substrate1(IRS1)/phosphatidylinositol-3-kinase(PI3K)/endometrial expression of glucose 4(GLUT4)signalling pathway and its regulator,phosphatase and tensin homolog(PTEN),in a rat model of polycystic ovarian syndrome(PCOS).Methods:PcoS model was induced by letrozole intragastric administration.Sprague-Dawleyrats were randomized into 4groups according to a random number table:(1)control group;(2)PcoS group,which was subjected to PCOS and received vehicle;(3)curcumin group,which was subjected to PCoS and treated with curcumin(200 mg/kg for 2 weeks);and(4)curcumin+LY294002 group,which was subjected to PCOS,and treated with curcumin and LY294002(a specific PI3K inhibitor).Serum hormone levels(17β-estradiol,follicle stimulating hormone,luteinizinghormone,progesterone,and testosterone)were measured by enzyme linked immunosorbentassay,and insulin resistance(IR)was assessed using the homeostasismodel assessment of IR.Ovarian tissues were stained with haematoxylin and eosin for pathological and apoptosis examination.Expression levels of key transcriptional regulators and downstream targets,including IRS1,Pl3K,protein kinase B(AKT),GLUT4,and PTEN,were measured via reverse transcription polymerase chain reaction and Western blot,respectively.Results:The Pcos group showed impaired ovarian morphology and function.Compared with the PCoS group,curcumin treatment exerted ovarioprotective effects,down-regulated serum testosterone,restored IR,inhibited inflammatory cell infiltration in ovarian tissues,decreased IRS1,PI3K,and AKT expressions,and up-regulated GLUT4 and PTEN expressions in PCOS rats(P<0.05orP<0.01).In contrast,IRS1,PI3K,AKT,and PTEN expression levelswerenot significantly different between PCOS and curcumin+LY294002 groups(P>0.05).Conclusion:The beneficial effects of curcumin on PCOS rats included the alteration of serum hormone levels and recovery of morphological ovarian lesions,in which,PTEN,a new target,may play a role in regulating the IRS1/PI3K/GLUT4 pathway.