Study On Mechanisms Of Insulin Resistance And Impacts Of Knockdown SOCS3 Expression On Glucose Metabolism In Hepatocytes Of IUGR Rats With Catch-up Growth

PART I Isolation, culture and identification of primary rat hepatocytesObjective This study was to explore a relative feasible and simple method of primary rat hepatocytes isolation and culture, and to identify its purity and biological activity.Methods We isolated the primary hepatocytes of SD rats using a modified two-step in situ non-circulating perfusion method followed by multiple filtration and low-speed centrifugation. Thereafter, the hepatocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) with high level of glucose. Before hepatocytes were seeded the viabilities of the cells were evaluated by Trypan blue exclusion. Cellular morphological changes were observed by using an inverted microscope. The status of hepatic glycogen storage was evaluated via Periodic acid Schiff (PAS) staining. The purity of hepatocytes were identified by the immunocytochemical staining targeting cytokeratinl8 (CK18) and PAS staining.Results 1.38*10～1.74*108 hepatocytes were harvested per rat. The viabilities of primary rat hepatocytes were more than 90% when seeded.4 hours after seeding, the hepatocytes with flattened and enlarged round shape attached to the surface of dishes that coated with collagen, and then combined with surrounding cells to form islands or cords. Glycogen preserved in hepatocytes was dyed purple after periodic-acid-Schiff staining. The purity of hepatocytes was estimated more than 95% through the PAS staining and anti-CK18 immunocytochemical staining of hepatocytes.Conclusions Primary rat hepatocytes were successfully isolated and cultured by using a modified two-step in situ non-circulating perfusion method, which is relative simple and feasible. The quantity, viability and purity of isolated and cultured hepatocytes were high. This method is available to be employed in labs with basic cell culture conditions. PART II Expression changes of SOCS3 and molecules involved in post-insulin receptor pathway in hepatocytes of IUGR rats with catch-up growthObjective To explore expression changes of suppressor of cytokine signaling3 (SOCS3), insulin receptor substrates 1 (IRS1), insulin receptor substrate2 (IRS2), phosphatidylinositol 3-kinase (PI3K), Akt2, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in hepatocytes of intrauterine growth retardation rats with catch-up growth (CG-IUGR) for the purpose of elucidating potential mechanism of insulin resistance in CG-IUGR offspring.Methods We employed a CG-IUGR rat model with maternal nutritional restriction during the whole pregnant period and reducing the litter size of IUGR offspring after birth. After we assessed the metabolic profile of glucose and lipid, we isolated and cultured primary rat hepatocytes using a modified two-step in situ non-circulating perfusion method. The mRNA levels of SOCS3, IRS1, IRS2 and PI3K in hepatocytes of CG-IUGR rats were detected at both the basal and insulin-treated states via real-time quantitive PCR and protein levels of above mentioned molecules were also evaluated under the basal and insulin-treated conditions via western blot. Besides, we also assessed the expression changes of phosphorylated Akt2 and transcriptional levels of critical enzymes in gluconeogenesis pathway between CG-IUGR and control rats.Results The mRNA and protein expressions of SOCS3 were both higher in hepatocytes of CG-IUGR offspring than in controls at the basal and insulin-treated states (P＜0.05). IRS1 and PI3K expression at the transcriptional and protein levels were lower in CG-IUGR hepatocytes compared with the control cells at both the basal and insulin-treated states (P＜0.05), whereas the transcriptional and protein levels of IRS2 had no statistical difference between CU-IUGR and control groups irrespective of the conditions (P> 0.05). The phosphorylated expressions of Akt2 were lower in CG-IUGR hepatocytes than that of control cells. After insulin stimulated, the decrease in transcriptional expression of gluconeogenesis genes were more obvious in control hepatocytes compared with CG-IUGR cells.Conclusions The increased levels of SOCS3 expression in hepatocytes of CG-IUGR rats might subsequently decrease the expression of IRS1 and the activities of downstream molecules. Increases in transcriptional levels of gluconeogenesis genes implied that the insulin sensitivity was decreased and hepatic glucose production was elevated in CG-IUGR hepatocytes. This may be an underlying mechanism for the development of insulin-resistance related diseases in IUGR individuals with catch-up growth. PARTIII Effects of knockdown expression of SOCS3 on post-insulin receptor signaling and glucose metabolism in hepatocytes of IUGR rats with catch-up growthObjective To explore the impacts of down-regulating the expression of suppressor of cytokine signaling3 (SOCS3) in transcriptional level on post-insulin receptor signaling and glucose metabolism of IUGR rats with catch-up growth in vitro.Methods We employed a CG-IUGR rat model with maternal nutritional restriction during the whole pregnant period and reducing the litter size of IUGR offspring after birth. We isolated and cultured primary rat hepatocytes using a modified two-step in situ non-circulating perfusion method. Specific small interference RNA (siRNA) targeting rat SOCS3 gene (siSOCS3) were designed and delivered to primary rat hepatocytes by liposome. Most effective siSOCS3 was picked up via real-time quantitive PCR (RT-PCR) and western blotting.48h after transfection, the protein levels of IRS1, PI3K and phosphorylated Akt2 in hepatocytes of CG-IUGR rats were detected at both the basal and insulin-treated states via western blot. Transcriptional expression changes of critical enzymes in gluconeogenesis pathway were also assessed after transfection under the basal and insulin-treated conditions via RT-PCR.Results 48h after transfection, the protein expressions of IRS1 and PI3K were both higher in hepatocytes of CG-IUGR offspring than that of cells without transfection at both the basal and insulin-treated states (P＜0.05). The phosphorylated expressions of Akt2 were higher in CG-IUGR hepatocytes treated with siSOCS3 compared with siSOCS3-untreated cells regardless of the conditions (P＜0.05). At the basal states, after transfection the transcriptional levels of PEPCK and G6Pase genes were lower in hepatocytes of CG-IUGR rats, but the differences were not statistically significant. However, after insulin stimulation the decreases in transcriptional expression of gluconeogenesis genes were statistically more obvious in hepatocytes treated with siSOCS3.Conclusions Down-regulating the expression of SOCS3 via siSOCS3 could ameliorate insulin sensitivity via increasing expression of downstream molecules involved in post-insulin receptor pathway and improve the glucose metabolism via decreasing the expression of gluconeogenesis genes in hepatocytes of CG-IUGR rats. This might be a potential genetic therapy for the insulin-resistance related diseases.