The Interaction Of Growth Hormone And Insulin On Both Signaling Pathways And Its Molecular Mechanisms

BackgroundGrowth hormone (GH) is one of the most important hormones that promote growth, development and metabolism in the body. By means of the extraction of GH from the human body, the limited quantity of production could only met the clinical requirement of the growth hormone deficient (GHD) children. Thanks to the development of bioengineering technology, nowadays the recombinant human growth hormone (RHGH) is available, and being widely used for clinical research as well as medical treatment. Apart from the treatment of GHD, it is also being applied for the following fields:Turner’s syndrome (TS), congestive heart failure, chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), decompensate liver cirrhosis, burn and etc.And insulin is another vital hormone in vivo, which is closely related to the cell proliferation, survival, metabolism, aging, cancerization and so on. It is being widely used for the treatment of diabetes and the like. But after a long-term use, the patients tended to suffer from high blood sugar, hyperinsulinemia and other suppression phenomenons of insulin signaling. And consequently the diabetes patients’s GH signal transduction was also impairedin the liver. Therefore we could definitely conclude that these two vital hormones have an intimate relationship. Studying the interaction between the two and the interrelationship of their signaling pathway was of great importance for the clinical application and the understanding of illness mechanism.First, GH binds to growth hormone receptor (GHR) of the cell membrane and activates cytoplasmic tyrosine kinase-Janus activating kinase2(JAK2), which is consequently phosphorylated, and the phosphorylating of GHR. The combined action of JAK2and GHR activates signal transducer and activator of transcriptions (STATS). Consequently STAT5enters into the cell’s nucleus and combines with the kinase domain receptor promoter in a certain order, and then activates the transcription of downstream genes so as to regulate the cell proliferation, differentiation, apoptosis, immunity and etc.The combination of insulin and insulin receptors (IR) leads to the change of IR conformation, and results in the auto-phosphoylatin of tyrosine, which can boost its combination with insulin receptor substrate (IRS), and activate phosphorylation reaction. The phosphorylated IRS mediates the downstream signals such as phosphoinositide-3-kinase (PI3K). The activated PI3K turns PIP2into PIP3that activates serine-threonine protein kinase B (Akt), and then mediates glycose synthase kinase (GSK), mammalian target of rapamycin (mTOR), bad protein and other downstream signal molecules, so as to regulate metabolism, proliferation, differentiation and apoptosis.There were numerous studies pertaining to the impact of GH on insulin signal, but very few studies showing the impact of insulin on GH signal. The research indicated that GH and insulin shared the same signaling pathway, and the two interact through common mechanism. The liver is the vital organ to regulate metabolism, and regulated by various hormones and cell factors, such as GH and insulin. In order to have a better study on the relationship between these two hormones, under the same technical condition, we studied the interaction between the two. Hereby we took liver Insulin/PI3K/Akt signaling path and GH/JAK2/STAT5signaling pathway as the research objects. And also we studied the interaction and reciprocal effect between liver GH and insulin both in vivo and vitro.The combination of phosphatase and tension homology deleted on chromosome10(PTEN), namely the major negative regulatory factor of insulin signaling pathway, with p85can lead to the dephosphorylation of p85, and then mediate a serial of downstream signals. The abnormal expression of this molecular is related to many diseases, such as insulin resistance, tumor and etc. The mutation of PTEN can result in the abnormal activation of insulin signal. The latest research showed that a long-term stimulation by GH could lead to the function change of PTEN and had a sort of impact on the insulin signal. It clearly indicated that insulin and GH interact, and the key mechanism of the interaction had something to do with PTEN. Therefore, here we adopted siRNA technology, PTEN feral or mutant plasmid technology to study the interaction and possible mechanisms of growth hormone and insulin on both signaling pathways in the liver.ObjectiveUnder the same technical background, this text studied the impact of the preconditioning growth hormone on the insulin signal p-Akt, and the impact of the preconditioning insulin on the growth hormone signal p-STAT5. And then by means of observation of the signal variation along with the functionality change of PTEN, this text demonstrates the functionality of PTEN during the interaction of these two hormones.MethodsPart one:Cellular culture study Taking HepG2as the research targets, cells were cultured in DMEM medium containing10%fetal bovine serum. Hungry for12hours before the cell signal experiment. Cells were lysed with RIPA buffer, and total protein was prepared for the test the level of activation of p-Akt、Akt、p-STAT5、and STAT5with western blot protein analytical method.1. Study the time effect of GH on the insulin signal1).The cells were cultured in DMEM medium containing10%fetal bovine serum, and then the cells were evenly passaged to six wells in twelve well plates, divided into three groups:namely GH Control group,30min GH and4h GH preconditioning group; two samples from each group. Stimulate the insulin (insulin saving concentration was100IU/ml, working concentration was0.15IU/ml, diluted in serum free DMEM medium) or PBS for10minutes before cell lysis.2).In order to study the impact of GH on the insulin signal time effect, here we amplified or eliminated the PTEN, which is the key negative control molecule in insulin signals. The cells of experiment one were transfected with plasmid PTEN, siPTEN or PTEN non-function mutation gene C124S respectively. Divide the cells into three groups just like experiment one, GH Control group,30min GH and4h GH preconditioning group. Stimulate the insulin or PBS for10minutes before cell lysis.2. Study the time effect of insulin on the GH signalCells were cultured in DMEM medium containing10%fetal bovine serum, and then the cells were evenly passaged to eight wells in twelve well plates. The cells were divided into four groups:control group of insulin, ten minutes insulin stimulation group, hirty minutes insulin stimulation group and four hour insulin stimulation group. Stimulate the GH (GH saving concentration was1.33mg/ml, working concentration was2000ng/ml, diluted in serum free DMEM medium) or PBS for30minutes before cell lysis.3. These cellular experiments were repeated for3times. 4. Statistical analysis. All data were expressed as the metic mean＋standard deviation (M±SD) through SPSS13.0statistical software analysis and Excel2010. Akt phosphorylation and STAT5phosphorylation among groups were compared using One-Way ANOVA whether variance was homogeneous or heterogeneous, followed by post hoc test using Bonferronic or Dunnett’s T. P values lower than0.05were considered as significant.Part two:Animal studyA total of32male Balb/c mice aging from6to8weeks were purchased from the Southern Medical University. The experiment was conducted in the animal center of Southern Medical University.1. The mice were randomly divided into4groups:namely GH control group (group A), GH preconditioning treatment group (group B), insulin control group (group C), and insulin preconditioning treatment group (group D). During the experiment, mice in group B were injected RhGH (lug/g weight) in the afternoon, while group B were injected sterile saline solution. After three-week administration of RhGH and PBS, ten minutes before put the mice to death, injected recombinant human insulin (21U/Kg weight) into the mice. For the rest two groups, four hours before put them to death, injected recombinant human insulin into Group D, and did same amount of normal saline into Group C. Thirty minutes before put the mice to death, injected GH into the mice. Collect the liver tissues of all the mice, and froze them with liquid nitrogen, finally preserved them in a freezer at a temperature of-80centigrade refrigerator.a. Comparison study:Conducted between GH preconditioning treatment (group B) and PBS control group (group A), checked the weight gain of the mice, eight mice from each group, as well as the liver Akt and P-Akt related protein expression level.b. Comparison study:conducted between GH preconditioning treatment group (group B) and GH control group (Group A), checked the liver PTEN expression level and protein P-actin protein expression level.c. Comparison study:conducted between insulin preconditioning group (group D) and GH control group (group C), and checked the STAT5and p-STAT5related protein expression level.d. Statistical analysis. All data were expressed as the metic mean±standard deviation (M±SD) through SPSS13.0statistical software analysis and Excel2010. The difference between two groups was analyzed by independent t-test under the condition of homogeneity of variance or Satterthwaite approximate t-test if the varirance was not eaqual. P values lower than0.05were considered as significant.ResultsPart one:Cell culture study1.In HepG2cells, compared to the level of phosphorylation of Akt in control group of growth hormone,30min GH couldn’t change the level of phosphorylated Akt significantly (P>0.05), while4h GH could get the level of Akt phosphorylation down significantly (P<0.05).2.Compared to the control group of growth hormone, after amplification of wtPTEN gene,30min GH could reduce the level of phosphorylated Akt in HepG2cells significantly(P<0.05); while4h GH could also make the level of Akt phosphorylation down more significantly compared with30min GH stimulation (P <0.05).3.Com pared to the control group of growth hormone, after knockdown of PTEN gene,30min GH slightly increased the level of Akt phosphorylation (P>0.05);4h GH did not down-regulate but up-regulate Akt phosphorylation significantly, even to a greater extent compared with30min GH (P<0.05).4.Compared to the control group of growth hormone, after amplifying the loss-of-function PTEN mutant C124S,30min GH also midly increased the level of Akt phosphorylation (P>0.05);4h GH did not down-regulate but also up-regulate Akt phosphorylation (P<0.05).The results of knockdown of PTEN gene were consistent with those results of the loss-of-function PTEN mutant C124S.5.Compared with the level ofSTAT5phosphorylation in the control group of insulin,10min30min and4hinsulin stimulation could not change the level ofSTAT5phosphorylation in HepG2cells significantly(P>0.05).Part two:Animal study1. After three weeks of GH stimulation, contrast to the control group of growth hormone, weight gain rate of mice with three-week GH was significantly increased(P<0.05). Compared with the control group of growth hormone, the stable level of Akt phosphorylation was significantly reduced in the liver tissue of GH pretreatment group (P<0.05). But the expression level of PTEN in GH pretreatment group of mice were also significantly increased (P<0.05).2. In contrast to the control group of insulin, the level of STAT5phosphorylation was lower seen in the insulin pretreatment group (P<0.05).That was not consistent with the results obtained in cell experiments after the stimulation of insulin.Conclusions1. In vivo and in vitro, GH stimulation can lead to the restrain of Insulin/PI3K/Akt signaling path. The amplified cancer inhibition gene wtPTEN can increase the impact of GH on the restrain of insulin signaling pathway. And silent PTEN gene or amplified PTEN no-function mutation gene C124S can reduce the impact of GH on the insulin signaling path. In vivo, the liver P-Akt expression level was reduced dramatically with the GH preconditioning treatment, and the PTEN expression level was raised up dramatically. It clearly shows the impact of GH on the mice liver Insulin/PI3K/Akt signal and cancer suppressor gene PTEN function regulation.2. As for the study of the impact of insulin on GH, with insulin preconditioning treatment, STAT5phosphorylation expression level in HepG2cells did not change much. The STAT5phosphorylation level of the mice with insulin stimulation was lower than that of the mice in the control group. In vivo and vitro, the experiment results were not consistent, possibly because in the body there was a complex signaling pathways network of interaction between the hormones or growth factors; the vivo signals keep steady-state relationship between the signals of the diversity and complexity.