| Research BackgroundInsulin is a protein hormone secreted by the pancreatic beta cells when stimulated by endogenous or exogenous substances such as glucose,glucagon,etc.Insulin can reduce blood glucose levels and also promote synthesis of glycogen,fat and protein.Insulin signaling as an important signal of metabolism regulation is strictly regulated under normal conditions.Since the insulin signaling plays an important role in the processes of cell metabolism,cycle regulation,proliferation and apoptosis,therefore,insulin signal abnormalities are associated with various diseases.Insulin promotes cell proliferation and mutation,particularly when there is abnormal activation of insulin signal,defective glands and cells proliferation are especially stimulated and lead to occurrence of tumor.On the other hand,insulin signaling when inhibited is also associated with the insulin resistance(IR)related diseases.Insulin resistance refers to decreased insulin sensitivity of the insulin target cells such as liver,muscle and fat cells and reduced or disappeared response to the insulin physiological function,causing a series of pathological changes and clinical symptoms.Studies have shown that insulin resistance is closely related to the occurrence and development of various metabolic diseases,such as diabetes,obesity,nonalcoholic fatty liver disease,hypertension etc.There are also many reports on the relationship between insulin resistance and a variety of tumors,such as liver cancer,colorectal cancer,and pancreatic cancer.However,the occurrence mechanism of these diseases associated with insulin resistance is still unclear.There are many insulin signal pathways,main including PI3K/Akt and MAPK/ERK signal pathways.The former one is related to a variety of metabolic regulation,while the latter mainly relates to cell growth and proliferation and the regulation of gene transcription.Phosphatidylinositol 3-kinase(PI3K)composed of a regulatory subunit p85 and catalytic subunit p110,which is a transit molecular of cellular metabolism,growth and survival related cell signal pathway.Under normal circumstances,insulin and insulin receptor(IR)are combined to cause phosphorylation of itself,then activation of insulin receptor substrate-1(IRS-1)which is phosphorylated on tyrosine residues and binding to p85 subunits,afterwards activation of the P110 subunit.The activation of PI3K turns phosphatidyl inositol 4,5 diphosphate(PIP2)into phosphatidyl inositol 3,4,5 triphosphate(PIP3),then PIP3 make protein kinase B(PKB/Akt)allosteric to be activated.PTEN is the main factor of negative regulation for insulin signaling pathway.The tumor suppressor gene PTEN(Phosphatase and tensin homologue,also known as MMAC1,mutated in multiple advanced cancers 1)protein expression product has dual activities inclusive of protein phosphatases and lipid phosphatase,which regulates signaling and cell life process through the dephosphorylation of lipid and protein.As mentioned above abnormal enhancement and inhibition of insulin signal are associated with tumor occurrence and insulin resistance respectively,and the phosphatase PTEN different regulating abnormal corresponds respectively to either tumorigenesis or insulin resistance.Studies showed that PTEN mutants appeared in a variety of primary tumor tissues,while wild ecological excessive expression of PTEN appeared in the chronic insulin resistance related diseases.Regulatory subunit p85 of PI3K is another molecular playing an important role in regulation of insulin signaling pathway.It can control downstream molecules by regulating PI3K,and abnormal regulation of p85 is also associated with tumor occurrence and insulin resistance respectively.For the first time,our team reported the phosphatase PTEN bound with PI3K regulatory subunit p85,followed by regulation of insulin/PI3K/Akt signal pathway,which provided a new field for pathological study of insulin signaling pathway.While PTEN and p85 are simultaneously associated with tumorigenesis and insulin resistance,the inner mechanism is not clear.Our team discovered the direct interaction between the two molecules may reveal that their direct effect jointly decides the fate of the cells towards cancerous or resistance of insulin signal.The subject of this research is to study the mechanism of relationship between phosphatase PTEN interaction with the PI3Kp85 subunit and insulin resistance related diseases on the basis of their interaction.This study was based on relationship between phosphatase PTEN and PI3Kp85 subunit with the insulin resistance related diseases.We chose and modeled two different hepatic pathological conditions with insulin resistance caused by clinical long-time growth hormone(GH)therapy and chronic HCV infection.Part 1.Impact of interaction between PTEN and p85 on insulin signaling pathwayBackground and purposeInsulin signaling is an important signal of metabolism regulation,and insulin resistance is related to the development of various diseases.PI3K/Akt is an important downstream pathway of insulin regulation of cell metabolism,while phosphatase PTEN is its important regulatory molecule.Therefore,we verified the role of PTEN and p85 in Inuslin/PI3K/Akt signaling pathway and explore the regulation mechanism of their interaction in vitro.Methods1.HepG2 cells were conventionally cultured,and the corresponding plasmids were transfected,while equivalent liposomes were added into the control hole according to the experiment scheme.After 12 hours’serum starvation,the cells were stimulated by rhINS before the lysis;the lysates of cellular protein were collected for western blot to detect Akt phosphorylation and gene expression and CoIP test to detect the binding between PTEN and p85.Results1.Insulin signaling was inhibited with the overexpression of wild-type PTEN plasmid(P=0.011),while this inhibition disappeared after transfection with PTENC124S plasmid and silence RNA.2.Insulin signal was also suppressed after p85 plasmid transfeced(t=2.877,P=0.045),and the inhibition disappeared after transfection with p50 plasmid.3.The binding between PTEN and p85 was reduced with overexpression of C124S compared to wild-type PTEN(t=5.265,P=0.006).ConclusionWe verified that PTEN and p85 both played negative regulation roles in Insulin/PI3K/Akt signaling pathway,which was referred to their combination that enabled PTEN and p85 to have an impact on insulin/PI3K/Akt signaling pathway..Part 2.Impact of interaction between PTEN and p85 on growth hormone-induced hepatic insulin resistanceBackground and purposeGrowth hormone(GH)as one of the most important hormones regulating cell growth and metabolism has been widely used in clinical replacement therapy of hormone deficiency and reverse of infection,trauma,etc.While the long-time treatment of GH can lead to insulin resistance,the mechanism is not clear.On the basis of our previous research,we studied the effect of molecular behavior changes of PTEN and PI3Kp85 in development of insulin resistance by long-time stimulation of GH in vitro and in vivo,using human hepatocellular carcinoma cell line HepG2 cells,human embryo kidney cell line HEK293T cells and Balb/c mice.Methods1.HepG2 and HEK293T cells were conventionally cultured,then the corresponding plasmid were transfected,while equivalent liposomes were added into the control hole according to the experiment scheme.After 12 hours of serum starvation,the cells were stimulated by rhGH and insulin before the lysis;The lysates of cellular protein were collected for western blot to detect Akt phosphorylation and gene expression.2.Balb/c mice were randomly divided into control group and rhGH group.Animals in rhGH group received the intraperitoneal injection of rhGH at 1μg/g body weight for 3 weeks,while mice in control group received normal saline at the same volume.Weight and fasting glucose were measured.All groups received either intraperitoneal injection of recombinant human insulin or sterile saline of the same volume 10 minutes before sacrificed randomly.Mice were sacrificed after anesthetized with Barbital sodium.Heart blood was collected for testing plasma insulin concentrations using ELISA kits.Hepatic tissues were ground on ice,and were lysed to have the protein collected for western blot to detect Akt phosphorylation and gene expression and CoIP test to detect the binding between PTEN and p85..Results1.It was found that there was no inhibitory effect on insulin signal when stimulated HepG2 with short-time(30m)GH,But the signal was inhibited when given long-time(4h)GH stimulation(P<0.001),which well reproduced the conditions showed in literature.2.Transfected with wild-type PTEN plasmid,the insulin pathway in cells still remained inhibition similar with above phenomenon(t=4.469,P=0.011).However,the inhibition disappeared when transfected with mutant PTEN C124S plasmid and PTEN siRNA.3.We further tested that long-time GH stimulation still inhibited insulin signaling after transfection with p85(P=0.024).When cells were transfected with p50 which has N terminal deletion of p85 to cut off the interaction between PTEN and p85,insulin inhibition disappeared after long-time GH stimulation(P=0.415).Same phenomenon was observed when cells were transfected with p85 siRNA.4.We chose 293T cells transfected with different mutants for further studies,the same results with HepG2 were obtained.5.In order to verify the experimental results in cells,we further used Balb/c mice.After 3 weeks injection of rhGH,as expected,body weight of mice in long time GH stimulation group were significantly increased.Hepatic insulin signal of the mice in the control group did not change,while the signal in GH stimulation group was significantly inhibited in mice6.Then we did experiments to further detect PTEN function.The results showed that the expression level of PTEN in liver tissue was significantly increased in GH stimulation group(t=-3.209,P=0.033).We also found the adhesion between PTEN and p85 was enhanced significantly in liver tissue in GH stimulation group.ConclusionOur study showed that hepatic insulin signaling was inhibited by long-time GH stimulation,which presented that exogenous GH treatment resulted in hepatic insulin resistance.In our study of HEK293T cells,long-time GH stimulation also resulted in the inhibition of insulin signaling.It can be inferred that inhibition of insulin signal by long time GH stimulation may be a relatively common phenomenon,which was not just in the liver tissueWe well verified that inhibition of insulin signal by long time GH stimulation might be closely related to the abnormal regulation of PTEN and p85 in vivo and in vitro experiments.Therefore,this study provided a new idea and reference for further researches on GH in clinical dose control and following pathological mechanism of insulin resistance.In addition,the long-time stimulation of GH may also lead to the occurrence of tumor,meanwhile PTEN expression increased after long-time GH stimulation,which may be referred to self-body adjustment ability to resist tumor.Part 3.Impact of interaction between PTEN and p85 on hepatitis C induced hepatic insulin resistanceBackground and purposeThe chronic infection of Hepatitis C virus(HCV)can lead to chronic hepatitis C,which is an important infectious disease and can lead to liver fibrosis,cirrhosis or hepatocellular carcinoma that are often associated with insulin resistance.HCV non structural protein 5A(NS5A)is involved in cell signal transduction,resistance to apoptosis in HCV infection and viral replication,playing an important role in the pathogenic happen.NS5A interactions with N terminal of p85 same as the PTEN binding site of p85.But the mechanism of insulin resistance caused by NS5A infection liver cells is not clear.We explored the mechanism of insulin resistance in hepatitis c virus(HCV)infection with the interaction among PTEN,NS5A and PI3Kp85 subunits in vitro.MethodsBecause there was a lack of effective mice model of human HCV chronic infection,we chose HepG2 cells transfected with hepatitis C virus NS5A for research.HepG2 cells were conventional cultured,and then the corresponding plasmids were transfected,while equivalent liposomes were added into control hole according to the experiment scheme.After 12 hours of serum starvation or not,the cells were stimulated by rhINS or rhEGF before the lysis,The lysates of cellular protein were collected for western blot to detect Akt phosphorylation and gene expression,IP test to detect phosphorylation of p85 and CoIP test to detect the binding between PTEN and p85.Results1.It appeared into the two cases after transfected with NS5A that NS5A lead to increased sensitivity of cells to insulin when after serum starvation and NS5 A lead to inhibition of cell insulin signal without serum starvation(t=-4.235,P=0.013).Thus,we found that there was no need for cell serum starvation in the in vitro model of insulin resistance induced by NS5A(t=4.870,P=0.008).2.The binding between PTEN and p85 was weakened after transfected with NS5A.This suggested that NS5A and PTEN may be of a competitive relationship of binding the same part.3.The inhibition of the interaction between PTEN and p85 suppressed the dephosphorylation of PTEN on p85.Correspondingly,our results showed that p85 phosphorylation is increased.4.To study the role of PTEN,cells were transfected with wild-type PTEN and mutant PTENC124S.The results showed the inhibitory effect of PTEN on PI3K was weakened(t=-5.953,P=0.004),which mean PTEN and p85 interactions was down-regulated in cells expressing NS5A.In conclusion,NS5A had a stronger binding ability with p85.ConclusionIt was firstly discovered from our study that the in vitro research model of hepatitis C induced insulin resistance that experiments were conducted without serum starvation corresponding to the in vivo condition in presence of serum.In this section,we found that PTEN and NS5A were competitively binding with p85,while NS5A has an advantageous binding ability.Therefor,the role of PTEN as a negative regulateor of the PI3K with signaling normality,thus PI3K signaling pathway appeared abnormality.Because of the advantageous binding between NS5A and p85,p85 showed a high state of phosphorylation in cells transfected with NS5A.But the specific phosphorylation sites were not clear in present studies.But the activation of phosphorylation on p85 caused by NS5A was obviously associated with inhibited Akt signal.We proposed a good explanation of the mechanism of insulin resistance lead by HCV infection that NS5A bound with p85 which weakened the interaction between PTEN and p85 thus caused the cell insulin signal abnormalitySummaryThe interaction between PTEN and p85 in insulin signal was highlighted recently,which provided a new idea to study insulin signaling related diseases.This subject was based on the latest research progress,and explored the molecular mechanism of insulin resistance.This was the first study to show that insulin resistance induced by GH stimulation was relevant to the increase of interaction between PTEN and p85.The long-time inhibitory effect of GH on insulin signal disappeared when cells were transfected with the gene mutation or silencing of PTEN and p85.Similarly,the insulin resistance caused by HCV infection was related to maladjustment of the interaction between PTEN and p85.The advantageous binding ability of NS5A on p85 resulted in abnormally binding between PTEN and p85,which may be the main cause of insulin resistance.We firstly established the insulin resistance cell model infected with HCV nonstructural protein NS5A.And for the first time,we found the competition effect between NS5A and PTEN on p85,while NS5A had the advantages of combining ability.We built up a new field for researches on the resistance of insulin signal induced by hepatitis C virus. |
PDF Full Text Request