Insulin Insulin Treatment Improves Growth Hormone Resistance In Septic Adult Male Sprague-Dawley Rats

Hypercatabolism appearing in septic state is one of challenges in the field of nutritional support. Currently, which affects the prognosis of sepsis patients seriously. Since the1980s, regulating metabolic hormone secretion, with the aim of inhibiting catabolic metabolism and promoting anabolic metabolism, has been intensely investigated.Great attention has once been paid to growth hormone (Growth Hormone), one of the representatives of the synthetic hormone which has significantly promote bone and soft tissue growing, body nitrogen retention, protein synthesis, and immunomodulatory role. To date, growth hormone (GH) has been an unsatisfactory therapeutic in critically ill patients with severe sepsis or hypercatabolic diseases. Recent evidence suggests that acquired growth hormone resistance (AGHR), which inhibits the anabolic effects of exogenous growth hormone, may be responsible for the ineffectiveness of GH therapy in these patients. AGHR performance mainly as follows:elevated growth hormone, insulin-like growth factor-1(IGF-1) reduction and exogenous growth hormone anabolic weakened. The reasons for AGHR in sepsis is:ubiquitin-proteasome pathway overactivation of the growth hormone receptor levels; excessive inflammatory cytokine release signaling pathway inside the cell after the receptor blocked suppression.Insulin,another important synthetic hormone,has anti-inflammatory action, inhibiting the ubiquitin-proteasome pathway and enhancing the role of the growth hormone receptor level. Evidence about that continues to grow in our study as well as other domestic and foreign studies. Accordingly, we infer:combined insulin and growth hormone can improve growth hormone resistance in septic rats. We designed this experiment to verify this inference that has not been reported at home and abroad.There is no study about combined insulin with GH to improve AGHR in sepsis.But the studies which provide some theoretical support about this is increasing in recent years. First, GHR synthesized reduction as well as post-receptor signaling blocked in sepsis leaded to AGHR. Insteretingly,Kin-Chuen found that insulin regulates hepatic GHR biosynthesis and surface translocation in a reciprocal manner. The divergent actions of insulin appear to be mediated by the mitogenactivatedprotein kinase and phosphatidylinositol3-kinase pathways, respectively. Of course, there is no similar effects for insulin to skeletal muscle in sepsis requiring further studies,though there are studies about liver cells under normal culture conditions.Second, important reasons for AGHR contain the ubiquitin-proteasome excessive activation, release of inflammatory mediators and so on. In addition, studies at home and abroad supportting insulin has anti-inflammatory and inhibition of the ubiquitin-proteasome pathway activity results in increased. Studies have shown intensive insulin therapy in critically ill patients can reduce skeletal muscle protein breakdown> promote anabolic,but the study failed to make it clear the effect is to control blood sugar or insulin directly. Orellana etc. found that insulin can directly promote skeletal muscle protein synthesis in neonatal endotoxemia recently. These results support our previous findings and may be complement with those, collaborative GH to improve sepsis patients metabolic provide theoretical support for insulin.In view of this, in order to consure whether insulin could improve growth hormone resistance in septic rats,we designed this study by intraperitoneal injection of endotoxin (LPS) to establish a rat model of sepsis. In the first part of this study, we detected the consentration of insulin growth hormone、insulin-like growth factor-1in serum, the mRNA levels of liver growth hormone receptor expression and IGF-1 mRNA expression (RT-PCR method) in liver, growth hormone receptor、pJAK2、 pSTAT5b、tJAK2and tSTAT5b protein content (Western-blotting method) to verify our assume. On the basis of the first part of the experiment, in the second part of the study, we investigated the possible signal pathways that the treatment of combined insulin with growth hormone could improve growth hormone resistance in sepstic rats by blocking the ubiquitin-proteasome pathway and PI3K-Akt pathway.PartⅠInsulin improves growth hormone resistence in septic ratsObjective:To observe whether insulin can improves growth hormone resistence in septic rats.Methods:This study used30adult male Sprague-Dawley rats, weighing200±10g, from the animal center of Jinling Hospital. The Institutional Animal Care Committee approved the study protocol. The Association accredits the animal care facility for Assessment and Accreditation of Laboratory Animal Care. Rats were housed in mesh cages at25℃under alternating12h light-dark cycles. Animals were acclimated in the facility for7d before the study. They were provided with standard rodent chow and water ad libitum.Rats were anesthetized by intraperitoneal (i.p.) injection of sodium phenobarbital (60mg/kg) and catheters (PE-50or PE-10; Becton-Dickinson, Sparks, MD) were implanted into the right jugular vein and the left carotid artery as previously described. A solution of insulin and dextrose was infused through the right jugular vein using a micro-pump (provided by the Research Center for Analytical Instrument, Zhejiang University) and blood glucose measurements were performed by an Elite glucometer (Bayer, Elkhart, IN) on blood from the left carotid artery. The catheters were filled with saline containing sodium heparin.Rats were withheld food for12h and divided randomly into the following seven treatment groups (n=6per group)Rats in the insulin, IG groups received continuous insulin infusion (Humulin R, EliLilly&Co., Indianapolis, IN) at a rate of about4.8mU/min/kg for1h (5u/kg/24h)after LPS stimulation. Rats that did not receive insulin received a sham infusion of sterile saline instead. Rats in the GH, IG groups were subcutaneously injected with GH (1IU/kg, Novo Nordisk, A/S)20min before harvesting while in other groups received a sham injection of sterile saline. Blood glucose was maintained between4.4-6.1mmol/l.The control group received only sham treatments of sterile saline in place of LPS injection, insulin infusion, and GH injection. In the LPS group, rats were injected with LPS.Rats were then infused with insulin1h after injection of LPS and received a sham injection of sterile saline in place of GH20min prior to euthanasia. In the insulin group, rats were injected with LPS. In the GH group, rats were injected with LPS, and then received an injection of GH20min before euthanasia. In the IG group, rats were given LPS. They were then infused with a combination of insulin and GH.The consentration of insulin、growth hormone、insulin-like growth factor-1in serum, the mRNA levels of liver growth hormone receptor expression and IGF-1mRNA expression (RT-PCR method) in liver, growth hormone receptor、pJAK2、pSTAT5b、tJAK2and tSTAT5b protein content (Western-blotting method).Data are expressed as means±standard error (SE). All data were analyzed with SPSS software (Version19.0, SPSS, Chicago, IL). Statistical analyses entailed ANOVA using the Tamhane’s T2M test for post-hoc analysis. P<0.05was considered statically significant.Results:Insulin and GH concentrations in serum were shown by RIA to significantly increase after LPS injection. Additionally, serum insulin levels in septic rats increased gradually (Fig.1)15.25±0.40mU/L vs.16.84±0.64mU/L, P=0.00after insulin administration and GH levels in septic rats increased gradually (Fig.2)1.71±0.85vs.1.90±0.03, P=0.04)after GH administration. ELISA showed that serum IGF-1levels were significantly reduced after LPS injection (Fig.3)(18.13±0.26vs.12.14±0.85, P=0.00). However, serum IGF-1levels increased following the combination treatment with insulin and GH (Fig.3) compared to GH alone (16.63±0.47VS.12.89±0.83, P=0.00). GHR and IGF-1mRNA expression in liver. RT-PCR showed that GHR and IGF-1mRNA decreased significantly in the liver after LPS injection(99.49±0.69vs.64.38±0.72, P<0.01;99.50±0.84vs.52.91±0.88, P<0.01) After administration of both insulin and GH in septic rats, GHR and IGF-1mRNA levels significantly increased compared to GH alone (Figs.4and5)(85.81±2.66vs.68.65±0.83P<0.01;73.53±0.19vs.52.67±0.31, P<0.01. Western blot analysis showed that LPS injection reduced levels of GHR, pJAK2, and pSTAT5b. After insulin treatment however, GHR levels increased (64.38±0.72vs.76.18±0.89, P<0.01). When both insulin and GH were administered, levels of GHR, pJAK2, and pSTAT5b were increased compared to GH alone (Figs.6,7and8)(86.09±3.09vs.64.61±0.52, P=1.00;83.00±1.79vs.49.83±2.23, P<0.01;85.77±2.22VS65.95±1.92, P<0.01).Conclusion:insulin can improves growth hormone resistence in septic rats. Part IIThe mechanism of insulin improves growth hormone resistence in septic ratsObjective:by blocking the PI3K-Akt pathway or the ubiquitin-proteasome system, to observe whether insulin can improves growth hormone resistence in septic rats septic rat.Methods:This study used42adult male Sprague-Dawley rats, weighing200±10g, from the animal center of Jinling Hospital. The Institutional Animal Care Committee approved the study protocol. The Association accredits the animal care facility for Assessment and Accreditation of Laboratory Animal Care. Rats were housed in mesh cages at25℃under alternating12h light-dark cycles. Animals were acclimated in the facility for7d before the study. They were provided with standard rodent chow and water ad libitum.Rats were anesthetized by intraperitoneal (i.p.) injection of sodium phenobarbital (60mg/kg) and catheters (PE-50or PE-10; Becton-Dickinson, Sparks, MD) were implanted into the right jugular vein and the left carotid artery as previously described. A solution of insulin and dextrose was infused through the right jugular vein using a micro-pump (provided by the Research Center for Analytical Instrument, Zhejiang University) and blood glucose measurements were performed by an Elite glucometer (Bayer, Elkhart, IN) on blood from the left carotid artery. The catheters were filled with saline containing sodium heparin.Rats were withheld food for12h and divided randomly into the following seven treatment groups (n=6per group)Rats in the insulin, IG groups received continuous insulin infusion (Humulin R, EliLilly&Co., Indianapolis, IN) at a rate of4.8mU/min/kg for1h after LPS stimulation. Rats that did not receive insulin received a sham infusion of sterile saline instead. Rats in the GH, IG groups were subcutaneously injected with GH (1IU/kg, Novo Nordisk, A/S)20min before harvesting while in other groups received a sham injection of sterile saline. Blood glucose was maintained between4.4-6.1mmol/l.The control group received only sham treatments of sterile saline in place of LPS injection, insulin infusion, and GH injection. In the LPS group, rats were injected with LPS and then injected with LY294002or MG-132through the tail vein at the beginning Rats were then infused with insulin1h after injection of LPS and received a sham injection of sterile saline in place of GH20min prior to euthanasia. In the insulin group, rats were injected with LPS followed by an injection of LY294002or MG-132through the tail vein prior to insulin infusion. In the GH group, rats were injected with LPS followed by LY294002or MG-132through the tail vein, and then received an injection of GH20min before euthanasia. In the IG group, rats were given LPS followed by LY294002or MG-132through the tail vein. They were then infused with a combination of insulin and GH.The LY294002group received an injection of LY294002(1.4mg/Kg) through the tail vein followed by infusion of insulin and GH. The MG-132group received an injection of MG-132(30mg/Kg) into the tail vein followed by infusion of insulin and GH. the consentration of insulin、growth hormone、insulin-like growth factor-1in serum, the mRNA levels of liver growth hormone receptor expression and IGF-1mRNA expression (RT-PCR method) in liver, growth hormone receptor、pJAK、pSTAT5b、 UAK2and tSTAT5b protein content (Western-blotting method).Data are expressed as means±standard error (SE). All data were analyzed with SPSS software (Version19.0, SPSS, Chicago, IL). Statistical analyses entailed ANOVA using the Tamhane’s T2M test for post-hoc analysis. P<0.05was considered statically significant.Results:Serum levels of insulin, GH, and IGF-1 Insulin and GH concentrations in serum were shown by RIA to significantly increase after LPS injection. Additionally, serum insulin levels in septic rats increased gradually (Fig.1)15.25±0.40mU/L vs.16.84±0.64mU/L, P=0.00after insulin administration and GH levels in septic rats increased gradually (Fig.2)1.71±0.85vs.1.90±0.03, P=0.04)after GH administration. ELISA showed that serum IGF-1levels were significantly reduced after LPS injection (Fig.3)(18.13±0.26vs.12.14±0.85, P=0.00). However, serum IGF-1levels increased following the combination treatment with insulin and GH (Fig.3) compared to GH alone (16.63±0.47VS.12.89±0.83, P=0.00). In the PI3K group,serum IGF-1levels were significantly lower than the IG group (16.63±0.47vs.12.53±0.03, P<0.01). GHR and IGF-1mRNA expression in liverRT-PCR showed that GHR and IGF-1mRNA decreased significantly in the liver after LPS injection(99.49±0.69vs.64.38±0.72, P<0.01;99.50±0.84vs.52.91±0.88, P<0.01) After administration of both insulin and GH in septic rats, GHR and IGF-1mRNA levels significantly increased compared to GH alone (Figs.4and5)(85.81±2.66vs.68.65±0.83P<0.01;73.53±0.19vs.52.67±0.31, P<0.01. In the PI3K roup, levels of GHR and IGF-1mRNA were significantly lower than the IG group (85.81±2.66vs.68.64±0.00, P<0.01;73.53±0.19vs.52.69±0.41, P<0.01) Western blot analysis showed that LPS injection reduced levels of GHR, pJAK2, and pSTAT5b. After insulin treatment however, GHR levels increased (64.38±0.72vs.76.18±0.89, P<0.01). When both insulin and GH were administered, levels of GHR, pJAK2, and pSTAT5b were increased compared to GH alone (Figs.6,7, and8)(86.09±3.09vs.64.61±0.52, P=1.00;83.00±1.79vs.49.83±2.23, P<0.01;85.77±2.22VS65.95±1.92, P<0.01).In the PI3K roup, the levels of GHR, pJAK2, and pSTAT5b were significantly lower than the IG group (86.09±3.09vs.64.19±0.66, P<0.01;83.00±1.79vs.50.00±1.41, P<0.01;85.77±2.22vs.64.69±2.06, P<0.01). In the MG-132group, levels of GHR, pJAK2, and pSTAT5b were also significantly lower than in the IG group (86.09±3.09vs.64.19±0.66, P<0.01;83.00±1.79vs.50.00±1.41, P<0.01;85.77±2.22vs.64.69±2.06, P<0.01). Conclusion:Insulin can improve growth hormone resistence in septic rats septic rat.The ubiquitin-proteasome system and PI3K-Akt pathway may involve this process.