Mechanisms Study Of Insulin Resistance In IUGR Rats

Intrauterine growth retardation (IUGR), also called fetal growth restriction (FGR), is a significant complication of pregnancy. In addition to perinatal morbidity and mortality, epidemiological studies suggest that IUGR may contribute to adverse health effects in adulthood including obesity, cardiovascular disease, impaired glucose tolerance, type 2diabetes, hypertension and other complications. Although the biochemical bases of these deleterious consequences are not well defined, it has been indicated that a poor nutrition during gestation results in metabolic changes in the offspring which can lead to permanent alterations in the structure and/or function of certain organs and tissues. The "thrifty phenotype hypothesis" proposed by Hales and Barker postulates that these changes occur in order to limit the use of nutrients by certain tissues, to ensure sufficient supply of them to the brain and other vital organs . Insulin resistance means the biological effect decreased after insulin combined with its specific receptor. It associated with abnormity and polymorphism of signal transduction and effecter molecule. Dysfunction of genes which regulate glucose and lipid metabolism can also cause insulin resistance. PI-3K pathway is the most principal way for insulin metabolism effect. Insulin combined and activated insulin receptor, subsequently phosphorylated insulin receptor substrates (IRS). Phosphorylated IRS activated phosphatidyliphosphatidylinositol 3-kinase (PI3-kinase). Through insulin-signaling cascade the phosphoinositide-dependent kinase-1 (PDK1) activated protein kinase B (PKB) . Phosphorylated PKB can made glycogen synthase kinase (GSK) 3 serine residue phosphorylation and deactivation subsequently accelerate glycogen synthesis. An important component of the peripheral insulin resistance associated with type 2 diabetes is impaired suppression of endogenous hepatic glucose production, which is predominately the result of gluconeogenesis. The enzymes glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and fructose-1, 6-bisphosphatase (FBPase) determine the rate of gluconeogenesis. In gluconeogenesis, the regulation of the rate-limiting (PEPCK) and the last committed (G6Pase) enzymes occurs mainly at the transcription level. It has been recently demonstrated that hepatic gene expression of these enzymes is controlled by peroxisome proliferator-activated receptor-γcoactivator-1 (PGC-1).Protein and amino acids deficiency are critical factors in chronic malnutrition. We established IUGR rat model by protein malnutrition during pregnancy. The incidence of IUGR and average birth weight were calculated. We observed the body weight, organ weight and perinephric fat pads at different weeks. Fasted serum glucose was determined by glucose oxidase method. Fasted serum insulin concentration were measured by enzyme-linked immunoabsordent assay (ELISA).in addition, glycogen and free fatty acid(FFA) of serum and hepatic were measured to study glucose lipid metabolism in IUGR rats.At 8 weeks of age, male rats were subjected to an insulin tolerance test (ITT), to evaluate insulin sensitivity in IUGR animals. Western blot analysis were undertaken to assess hepatic expression of IRS-2,PI-3K,PKB, phosphorylated Ser473-PKB protein and GSK-3 .the hepatic mRNA level of PEPCK and G6Pase and that of transcription factor PGC-1 which promote glucogenesis were determined . The present studies were performed to reveal the underlying mechanism responsible for insulin resistance in IUGR rats.Materials and Methods一,Animal modelHealthy Wistar rats (weight: 230-280g) .Female and male rats were raised in same cages by the proportion of four ratio one. Female rats were randomly divided into 2 groups: control group, IUGR group. Control maternal rats were fed with standard rat chow(caloriel583KJ/100g, protein 23%);animals of IUGR group were fed with low protein rat chow(calorie1558KJ/100g, protein 8%) since 1day of gestation. All the maternal rats were allowed to deliver spontaneously. Neonate rats were weighed on a scale with an accuracy of 0.01g.二,Experiment objectsJuvenal rats were weaned at 3 weeks of age, and then all fed with standard rat chow.1,Sample collection and treatmentOn 3 week and 8 week, after fasted for 12 hour, 0.2～0.5ml blood samples were collected from rats of each group. Blood samples were placed for 15 min and centrifuged at 3000 rpm for 10 minutes. The serum was stored at -70℃for later detection. The brain, heart, liver, kidney, pancreas and perinephric fat pads were harvested and weighted on a scale with an accuracy of 0.001g, quickly snap-frozen in lipid nitrogen and stored at -70℃.2,ITTOn 8 week, after fasted for 12 hour, in certain adult male animals, jugular vein catheters were placed surgically after intraperitoneal injection of ethyl carbamate. The animals received 0.5U/kg human insulin via intraperitoneal injection, and blood was obtained at 0, 15, 30, and 60 min subsequently to measure glucose concentration.三,Experiment Methods1,The body weights of rats since birth to 8 weeks of age were weighted. The weight of brain, heart, liver, kidney, pancreas and perinephric fat pads at W3 and W8 were examined. The diagnostic standard of IUGR is generally assigned to the Neonate rats measured with two standard deviation lower than the average birth weight of control group.2,The measurement of glycogen and FFA in the serum and hepatic: biochemical methods. 3,Serum glucose was determined by glucose oxidase method.4,Fasted serum insulin concentration was measured by ELISA.5,The measurement of PGC-1α,PEPCK and G6Pase mRNA levels in the hepatic: RT-PCR. The measurement of PGC-1αcprotein levels in the hepatic: Western blot.6,The measurement of IRS-2,PI-3K,PKB ,phosphorylated Ser473-PKB and GSK-3βprotein levels in the hepatic: Western blotResults一,The effects of protein deficiency during of pregnancy ongrowth and developmentThe incidence of IUGR in model group were significantly higher than control groups (P<0.01) . The average birth weight of IUGR group were significantly lower than that of control groups (P<0.05), until 4 weeks of age, when IUGR rats caught up to controls. Between 4～7 weeks of age, the growth of IUGR rats accelerated and surpassed that of controls at 8 weeks of age (P<0.05). IUGR rats were obese at 8 weeks of age with perinephric fat pads significantly increased compared with control rats (P<0.01) .二,Abnormal metabolism and insulin sensitivity in IUGR ratsNo significant differences were observed in blood glucose and insulin concentration at 3 week. Nor was the insulin resistance index (IRL). Hepatic concentration of FFA and glycogen did not differ at 3 week and 8 week between IUGR and control. But serum FFA concentration significantly increased compared with control. By 8 week IUGR rats developed hyperinsulinemia and high IRI (P<0.05) . Insulin tolerance test showed a significantly blunted glycemic response to exogenous insulin in IUGR rats (P<0.05) .三,Changes of PI-3K pathways signaling molecules in hepatic of adult IUGR rats 1,Hepatic protein levels of IRS-2,PI-3K and GSK-3βBasal levels of IRS-2 protein did not differ between IUGR and control rats (P=0.371) . The expression of PI-3K protein are decreased in adult IUGR rats compared with control (P<0.01) .whereas GSK-3βprotein level was significantly increased in IUGR rats (P<0.01) .2,Hepatic protein levels of PKB and phosphorylated PKBBoth PKB and phosphorylated Ser473-PKB protein levels are significantly decreased in liver of IUGR animals compared with controls (P<0.01) . After administration of insulin, phosphorylation at Ser473 of PKB significantly increased to 182% of basal level in control liver (P<0.01) ; However, phosphorylation of PKB in responded to insulin was markedly blunted in IUGR rats compared with controls, only increased to 123% of basal level (P=0.024) .四,Variation of PGC-1α,PEPCK and G6Pase in hepatic of IUGRrats1,Hepatic protein and mRNA levels of PGC-1αUsing the traditional RT-PCR method, we found that W 3 and W 8 hepatic mRNA levels of PGC-1 were significantly increased in IUGR livers compared with control values (P<0.05) . Hepatic protein levels of PGC-1 at W 3 and W 8 were significantly increased to 162 % and 230%of control values, respectively (P < 0.05)2,Hepatic mRNA levels of PEPCK and G6PaseHepatic mRNA levels of G6Pase at W 3 and W 8 were significantly increased in the IUGR animals (P<0.01) .Slimily, hepatic mRNA levels of PEPCK at W 3 and W 8 were increased in the IUGR animals compared with control rats. Moreover, when the extent of these increases was analyzed in comparison with the rise in PGC-1 mRNA levels on a sample-to-sample basis, we found that the G6Pase (r =0.726, P<0.01) , PEPCK (r =0.907, P<0.01) mRNA levels correlated directly to PGC-1 mRNA levels. Conclusions1,Exposed to protein malnutrition during pregnancy can induce IUGR. The IUGR rats catch up growth after birth, and to be fatter than normal rats when grown up. They appear to have more fatty deposit in internal organs and abnormal glucose and lipid metabolism2,No significant differences were observed in blood glucose and plasma insulin levels at 3 week of age. However, at age 8weeks, IUGR rats developed hyperinsulinemia and high IRI. Insulin tolerance test confirmed that IUGR rats developed insulin resistance.3,The expression of PI-3K protein are decreased in livers of adult IUGR rats. Both PKB and phosphorylated Ser473-PKB protein levels are significantly decreased in hepatic of IUGR animals compared with controls. After administration of insulin, phosphorylation of PKB in responded to insulin was markedly blunted in IUGR rats compared with controls. These data suggest there are postreceptor defect in the intracellular insulin -signaling cascade in IUGR animals.4,The expression levels of GSK-3P protein are increased in livers of adult IUGR rats. It may inhibit the activity of glycogen synthase and subsequent promote the glucose production.5,Hepatic protein and mRNA levels of PGC-1αare significantly increased in IUGR rats. It contributed to the increased mRNA levels of gluconeogenic enzymes PEPCK and G6Pase, and subsequently promotes hepatic gluconeogenesis. These changes may hasten the development of hyperglycaemia.