Establishment and verification of insulin resistance model in mouse skeletal muscle cells

AIM:To establish a stable and repeatable insulin resistance model of skeletal muscle cells in vitro, so as to promote the exploration of the pathological mechanism of insulin resistance and the development and screening of related drugs. METHODS:C2C12 mouse myoblasts were used to induce differentiation in normal differentiation medium and differentiation medium containing glucose at 40 and 60 mmol/L, respectively. The effects of glucose at different concentrations on cell convergence, fusion and formation of multinucleated myotubes were observed under phase contrast microscope every day. After 1, 3, 5 and 7 d of differentiation, 2-NBDG assay was used to detect the effects of different interventions on C2C12 basal glucose uptake and insulin-stimulated glucose uptake. The effects of different interventions on the protein expression of glucose transporter 4 (GLUT4) after 5 d and 7 d of differentiation were determined by Western blot. The effects of different interventions on the distribution of GLUT4 protein after 5 d of differentiation were detected by immunofluorescence staining. RESULTS:After treated with glucose at 60 mmol/L, the morphological observation showed that high glucose treatment significantly inhibited the growth and differentiation of C2C12 cells after 3 d. High glucose treatment significantly inhibited basal glucose uptake and insulin-stimulated glucose uptake of the C2C12 cells after 5 d and 7 d (P<0.01). No difference between insulin-stimulated GLUT4 expression and basal GLUT4 expression after 5 d and 7 d of high glucose treatment was observed (P>0.05), but there was significant difference between control group and 60 mmol/L group (P<0.05) determined by Western blot. Immunofluorescence staining observation showed that the distribution of GLUT4 protein in the C2C12 cell membrane was significantly decreased after 5 d of high glucose treatment (P<0.01). Glucose treatment (40 mmol/L) also played a role to some extent, but the effect was not as obvious and stable as 60 mmol/L glucose. CONCLUSION:A stable insulin resistance model of mouse skeletal muscle cells in vitro was successfully established by high glucose stimulation. The treatment of glucose at 60 mmol/L for 5 d was the best. Morphological observation and detection of basic and insulin-stimulated glucose uptake and GLUT4 protein expression and distribution evaluates the insulin resistance level of skeletal muscle cells in vitro.     

Effect of rosiglitazone on expressions of insulin receptor substrate-1 and glucose transporter 4 in skeletal muscle of type 2 diabetic rats with hyperlipemia

AIM: To investigate the effect of rosiglitazone on the expressions of insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) in skeletal muscles of type 2 diabetic rats with hyperlipemia, and to explore the different pharmacological mechanism. METHODS: The model of type 2 diabetic rats with hyperlipemia was established by injecting low dosage of streptozotocin (STZ) and feeding with high fat diet. Then the diabetic rats were divided into two groups: untreated diabetic group and rosiglitazone-intervened diabetic group. The course of treatment lasted for 4 weeks. The expressions of IRS-1 and the GLUT4 proteins in the cell membrane of isolated rats skeletal muscles were detected by Western blotting. RESULTS: The fasting blood glucose, insulin and triglyceride contents in rosiglitazone-intervened diabetic group were lower than those in untreated diabetic group, but they were still higher than those in control group. The result of Western blotting showed that the expression of GLUT4 protein in rosiglitazone-intervened diabetic group was increased compared with untreated diabetic group, but its level was still lower than that in control group. The protein expression and tyrosine phosphorylation of IRS-1 in rosiglitazone-intervened diabetic group were significantly higher than those in untreated diabetic group and their levels were lower than those in control group. CONCLUSION: The effect of rosiglitazone on GLUT4 protein may link to its ability to induce the protein expression and tyrosine phosphorylation of IRS-1 in skeletal muscles in type 2 diabetic rats.     

Shenmai injection improves insulin resistance in 3T3-L1 cells

AIM: To discuss the effect of Shenmai injection on insulin resistance (IR) in 3T3-L1 cells and its mechanisms. METHODS: 3T3-L1 preadipocytes were induced by chemical reagents to differentiate into fully differentiated adipocytes. Oil red O staining was used to detect the differentiation level of the adipocytes. The insulin-resistant 3T3-L1 cell model was demonstrated using insulin, which was confirmed by glucose concentration in cell supernatant. The IR cell model was given 10 μmol/L rosiglitazone, 25 and 50 g/L Shenmai injection and normal saline for comparison. MTT assay was used to assess the cell activity of 3T3-L1 cells which was treated with drugs for 8, 16, 24 and 36 h. Glucose oxidase method was used to detect the glucose concentration in the cell supernatant at 8, 16 and 24 h. The protein levels of glucose transporter-4 (GLUT4), phosphatidylinositol 3-kinase (PI3K), AKT and p-AKT were determined by Western blot. RESULTS: 3T3-L1 adipocytes were successfully induced as shown by the positive oil red O staining. The IR cell model was demonstrated, and glucose concentration in the cell supernatant after treatment with Shenmai injection showed that Shenmai injection reduced the IR in 3T3-L1 cell model. The protein levels of GLUT4, PI3K and p-AKT increased compared to control group. CONCLUSION: Shenmai injection reduces the IR in 3T3-L1 cell model, which functions by increasing the protein levels of GLUT4, PI3K and p-AKT.     

Icariin attenuates high glucose-induced insulin resistance in C2C12 myotubes

AIM: To observe the potential effects of icariin on high glucose-induced insulin resistance in C2C12 myotubes and to investigate its underlying mechanisms. METHODS: The insulin resistance model was induced by high glucose (25 mmol/L) in the C2C12 myotubes. The effects of icariin on Akt phosphorylation at T308, glucose transporter 4 (GLUT4) membrane translocation, and glucose uptake were investigated in high glucose-treated C2C12 myotubes. The protein levels of phosphorylated proteins were determined by Western blot. The glucose uptake was measured by colorimetric method. The small interfering RNA (siRNA) was used to knockdown the expression of p38 MAPK. RESULTS: Icariin significantly increased insulin-stimulated Akt T308 phosphorylation in C2C12 myotubes treated with high glucose. Treatment with icariin at 25, 50 and 75 μmol/L for 24 h increased Akt T308 phosphorylation in a dose-dependent manner (P<0.05 or P<0.01). Treatment with icariin at 50 μmol/L for 12, 24 and 36 h increased Akt T308 phosphorylation in a time-dependent manner (P<0.05 or P<0.01). In addition, treatment with icariin at 50 μmol/L for 24 h significantly enhanced the expression of GLUT4 on plasma membrane (P<0.01) and 2-deoxyglucose (2-DG) uptake (P<0.01). Treatment with icariin recovered high glucose-reduced p38 MAPK phosphorylation (P<0.01). Pharmacological or genetic inhibition of p38 MAPK abolished the protective impacts of icariin on insulin-stimulated Akt T308 phosphorylation (P<0.01), GLUT4 plasma membrane translocation (P<0.01), and 2-DG uptake under high glucose condition (P<0.05). CONCLUSION: Icariin attenuates high glucose-induced insulin resistance in C2C12 myotubes by activating p38 MAPK.     

Correlation of serum omentin-1 level and insulin resistance in rats

AIM：To establish the insulin resistance rat model for evaluating the correlation of omentin-1 level and insulin resistance. METHODS：SPF male Wistar rats (n=30) were randomly divided into normal control group (NC, n=15) and high-fat diet group (HF, n=15). The rats in NC group were fed with basic diet. The insulin resistant model was established by feeding the rats with high-fat diet in HF group. After 10 weeks, 5 rats in each group were assessed by the technique of hyperinsulinaemic-euglycaemic clamp. After the insulin resistant model was successfully established, the body weight and fasting blood glucose were detected. The concentration of fasting serum omentin-1 was analyzed by ELISA. Fasting serum insulin was measured by radioimmunoassay. RESULTS：No difference of fasting blood glucose between the 2 groups was observed. The level of fasting serum insulin in HF group was significantly higher than that in NC group (P<0.05). The level of serum omentin-1 in HF group were significantly decreased compared with NC group (P<0.01). Pearson’s correlation analysis showed that negative correlations between serum omentin-1 and fasting serum insulin (r=-0.654,P<0.01), serum omentin-1 and free fatty acid (r=-0.446, P<0.05) was found. CONCLUSION：In rats, serum omentin-1 level began to decrease at insulin resistance stage. As serum omentin-1 level decreased, the basal insulin level increased, indicating that decreased serum omentin-1 level may be an early factor of IR, diabetes and cardiovascular diseases.     

Association of serum soluble E-selectin concentrations with insulin resistance in essential hypertension patients

AIM: To investigate the relationship between serum soluble E-selectin (sE-selectin) and insulin resistance, serum uric acid, serum lipid in essential hypertension patients. METHODS: Fasting serum sE-selectin concentration, plasma glucose, serum insulin, serum uric acid, total cholesterol, triglycerides, high density lipoprotein-cholesterol, low density lipoprotein-cholesterol were determined in 186 patients with essential hypertension (75 males, 111 females). Homeostasis model assessment was applied to assess the status of insulin resistance (HOMA-IR). RESULTS: Based on the HOMA-IR, the essential hypertension patients were divided into insulin-sensitive individuals (IS) and insulin resistant subjects (IR). The serum sE-selectin concentration was significantly higher in male group [(50.1±17.8)g/L]than in female group [(40.6±16.6)g/L](P<0.01). No difference between IR group [(51.6±16.8)g/L]and IS group [(48.5±18.8)g/L] in male, and significantly higher in IR group [(45.1±18.0)g/L]than in IS group [(36.0±13.7)g/L](P<0.01) in female were observed. A stepwise multiple linear regression analysis showed that HOMA-IR was an independent predictor of serum sE-selectin concentrations in female group and not in male group, and both serum uric acid and serum lipid were not independent predictors of serum sE-selectin concentrations. CONCLUSION: Serum sE-selectin concentrations were directly related to insulin resistance in females with essential hypertension and not in males with essential hypertension. Both serum uric acid and serum lipid were not directly related to serum sE-selectin concentrations.     

Effect of calories restriction on ER stress in the liver of high fat diet rats

AIM: To study the effect of calories restriction on endoplasmic reticulum(ER) chaperone protein 78-kD glucose regulated protein (GRP78) mRNA expression in the liver of high fat diet rats, in order to explore the mechanism of how calories restriction improves insulin resistance. METHODS: Wistar rats (n=24) were randomly divided into 3 groups: normal chow (NC) group, was fed free normal chow (18.94% of calories as fat) for 12 weeks; high fat group (HF) was fed high fat diet (50.55% of calories as fat) for 12 weeks; calories restriction group (CR) was fed high fat diet for 8 weeks at first, then given 50% of diet consumed by the same age NC group. Changes of body weight, height, and food intake were recorded. At the end of experiment, HOMAIR, the rate of visceral fat (including perirenal fat and epididymal fat) vs weight, plasma protein, blood lipid (including total cholesterol and triglyceride), hepatic GRP78 mRNA and hepatic histological changes (including light microscopic studies and electron microscopic studies) were detected. RESULTS: (1) Animals in HF group had an obviously elevation of fasting insulin (27.51±3.51) mU/L vs (15.46±2.25) mU/L, triglyceride (1.35±0.25) mmol/L vs (0.67±0.10) mmol/L, total cholesterol (2.59±0.34) mmol/L vs (1.41±0.28) mmol/L and insulin resistance index HOMAIR (5.85±0.23 vs 2.85±0.60) compared with NC group, and also had obviously lipid accumulations in the liver. (2) After calories restriction, all the abnormal elevated biochemical indicators were decreased to normal levels, the hepatic lipid accumulations were also improved. (3) The changes of liver ultrastructure in HF group showed rough endoplasmic reticulum enlargement, fragmentation, taking off grain, and with glycogen solution. The changes in CR group were nearly the same as those in NC group. (4) High fat diet induced the expression of GRP78 mRNA, calories restriction might reverse it. CONCLUSION: Reasonable food calories restriction is a good method to improve insulin resistance, partly due to improvement of endoplasmic reticulum stress in liver.     

Oxymatrine ameliorates high fat-induced insulin resistance in ApoE-/- mice

ATM: To investigate the effect of oxymatrine (OXY) on high fat-induced insulin resistance in mice, and to investigate the mechanism. METHODS: ApoE^-/-mice with high-fat diet for 16 weeks were divided into insulin resistance group, and OXY groups at concentrations of 25, 50 and 100 mg/kg. C57BL/6J mice served as normal control group. The mice in OXY groups were gavaged with OXY for 8 weeks. Glucose tolerance test in the mice was performed. Fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), fatty acid (FFA) and fasting insulin (FINS) in the plasma were measured. The mRNA expression of insulin receptor (INSR), insulin receptor substrate-2 (IRS-2), glucose transporter 2 (GLUT2) in the liver tissues was examined by RT-qPCR. The protein levels of GLUT2, INSR, IRS-2, p-INSR, p-IRS-2, PI3K, p-PI3K, serine/threonine protein kinase (AKT) and p-AKT were examined by Western blot.RESULTS: OXY reduced the levels of FBG, TC, TG, FFA and FINS, and attenuated insulin resistance. Compared with insulin resistance group, the mRNA expression of INSR, IRS-2 and GLUT2 significantly increased in OXY groups (P<0.05). The protein levels of p-INSR/INSR, p-IRS-2/IRS-2, p-PI3K/PI3K, p-AKT/AKT and GLUT2 also increased in OXY groups (P<0.05).CONCLUSION: OXY ameliorates high fat-induced insulin resistance in mice via PI3K/AKT pathway.     

Hypothalamic expression of orexin A and lipid metabolism disorder in rats with alimentary obesity

AIM: To evaluate the effect of orexin A in rat hypothalamus on lipid metabolism disorder in rats with alimentary obesity induced by high-fat diet.METHODS: The rat model of alimentary obesity was induced by high-fat diet. The levels of insulin, triglyceride (TG) and total cholesterol (TC) in the serum were detected by luminescent immunoassay and enzymic method. The mRNA expression of orexin A in rat hypothalamus was determined by real-time PCR.RESULTS: There were statistically significant differences of weight, body fat content, and Lee's index between high-fat diet group and control group after 8-week feeding of high-fat diet. Compared to control animals, the levels of insulin, TG and TC in the rats with alimentary obesity significantly increased by 50％, 94％ and 43％, respectively (P<0.05). The expression of orexin A in rat hypothalamus significantly decreased by 57％, and had significant negative correlation with Lee's index, insulin, TG and TC. Their correlation coefficients were r=-0.798 (P<0.05), r=-0.868 (P<0.05), r=-0.981(P<0.05) and r=-0.815 (P<0.05), respectively. CONCLUSION: Alimentary obesity and lipid metabolism disorder induced by high-fat diet are correlated with down-regulation of orexin A expression in rat hypothalamus.     

Hyperlipidemia induces endothelial dysfunction in rats by increasing oxidative and nitrative stresses

AIM: To observe the effect of high-fat diet on the endothelial functions in rats. METHODS: Male SD rats (8 week old) were randomly divided into 2 groups to receive a regular or a high-fat diet, respectively. After 14 weeks, the animals were anesthetized and caval blood was collected to determine the lipid profile, fasting blood glucose and insulin levels. The aorta of the animals was isolated to observe the response of vasorelaxation to endothelium-dependent vasodilator acetylcholine (ACh) and endothelium-independent vasodilator SNAP(S-nitroso-N-acetyl penicillamine). In addition, the production of nitric oxide(NO) and superoxide, the expression of gp91^phox, and the activity of NO synthase(NOS) in the aortic tissues were measured. RESULTS: The lipid profile, the levels of fasting blood glucose and insulin were significantly increased in the plasma of rats fed with high-fat diet. A dose-dependent vasorelaxation to ACh was reduced, and the expression of gp91^phox, the production of superoxide and the activity of iNOS were enhanced in the aortic tissues of the rats fed with high-fat diet. CONCLUSION: High-fat diet induces endothelial dysfunction by increasing the oxidative and nitrative stresses.     

Tetramethylpyrazine combined with aminoguanidine improves renal functions in n0-STZ Wistar rats

AIM: To investigate the effects of tetramethylpyrazine combined with aminoguanidine on the renal functions of neonatal-0 streptozotocin-induced (n0-STZ) rats. METHODS: Neonatal Wistar rats were intraperitoneally injected with a single dose of streptozotocin (STZ) to establish the n0-STZ rat model. The n0-STZ rats were divided into 4 groups: normal control group, insulin resistance group, metformin treatment group and tetramethylpyrazine+aminoguanidine treatment group. Fasting plasm glucose, fasting insulin, insulin resistance index, blood urea nitrogen, serum creatinine, urine albumin and glomerular filtration rate were measured at the 32nd week. The mRNA content of inducible nitric oxide synthase (iNOS) in peripheral blood leukocytes was detected by the technique of in situ hybridization. Nitric oxide (NO) concentration, iNOS activity, the protein expression of iNOS and 3-nitrotyrosine(3-NT) were also assessed in the renal tissues. RESULTS: At the 8th week after the administration of STZ, 82.5% of Wistar rats showed that the fasting plasm glucose level was ≥7.0 mmol/L and the renal functions were seriously damaged. Although both metformin and the combined treatment reduced fasting plasm glucose, fasting insulin and insulin resistance index, the combined treatment was superior in improving the insulin resistance. The damaged renal functions were improved by the combined treatment as reducing blood urea nitrogen and creatinine, increasing glomerular filtration rate were observed. Furthermore, the combined treatment reduced NO concentration, decreased iNOS activity and diminished mRNA content of iNOS, resulting in depressing the generation of 3-NT and iNOS, which surpassed the treatment of metformin. CONCLUSION: Tetramethylpyrazine combined with aminoguanidine improves the renal functions of n0-STZ rats by depressing nitrative stress and enhancing the effect of metformin.     

Protective effect on hepatic insulin resistance of Astragalus polysaccharide in the high fat-fed mice model

AIM: To investigate the effect and mechanism of Astragalus polysaccharide (APS) on the amelioration of hepatic insulin resistance in high fat-fed mouse model.METHODS: C57BL/6J mice (n=26) were divided into three groups randomly: C group (an animal model for control,n=10);IR group ( an animal model of insulin resistance,n=8) and IA group (an animal model in high-fat diet with APS treatment for12 weeks,700mg·kg^-1·d^-1,ig).High-fat diet was used to induce the formation of insulin resistant.The parameters and insulin sensitivity of the animals were observed.The pathological features of the liver were presented through microscope and TEM.The expression changes of hepatic GSK3β were measured by Western blotting.RESULTS: In this study,the fat-fed mouse model of insulin resistance was established successfully.The mice in IA group responded to the 12-week APS therapy with a significant decrease in the level of blood glucose,plasma insulin,body weight,hepatic TG/FFA and improved glucose tolerance compared with those in IR group.In addition,the expression and the activity of GSK3β were lower in IA group (vs IR group，P<0.05).We also found the hepatic steatosis could be significantly alleviated with APS therapy.CONCLUSION: These results indicate that APS prevents the occurrence of insulin resistance and the hepatic steatosis induced by high-fat diet,at least in part by inhibiting the expression and activity of the hepatic GSK3β.     

Expression of GLUT4 in endometrium of rats with polycystic ovary syndrome

AIM:To explore the expression of glucose transporter 4 (GLUT4) in the endometrium of rats with polycystic ovarian syndrom (PCOS) and evaluate the relationship between GLUT4 expression and insulin resistance (IR). METHODS:54 female SD rats of 85 days were randomized to control group (n=20), PCOS model group (n=17) and metformin treatment group (n=17). The rats in the latter two groups were induced by Poretsky’s method for PCOS model, followed by placebo or metformin, respectively. After 14 days of treatment, the rats were sacrificed and the expression of GLUT4 in endometrium was detected by Elivision^TM Plus two steps immunohistochemical staining. RESULTS:The expression of GLUT4 and insulin receptor(INS-R) proteins of endometrial glandulan epitheliu in PCOS rats were significantly lower (P<0.01,P<0.05) than those in control group, however, the expression of insulin(INS) protein in PCOS rats was higher than that in control group (P<0.01). The expression of GLUT4 in the treatment group increased (P<0.01), but was still lower than that in control group (P<0.01). However, compared with PCOS group, the expression of INS protein was decreased (P<0.05), but was still higher than that in control group (P<0.05). There was no GLUT4 expression in interstitial cells in endometrium, and the changes of the expressions of INS and INS-R proteins in those cells were similar with those in glandulan epitheliu. CONCLUSION:The decrease in GLUT4 expression of endometrium in PCOS rats is related with endometrial insulin resistance.     

Influence of metformin on LPIN1 expression and AMPK signaling in high-fat diet-induced insulin resistant rats

AIM: To explore the regulatory mechanism of LPIN1 in hepatic insulin resistance by investigating the influence of metformin on the expression of LPIN1 and AMP-activated protein kinase(AMPK) signaling in the rats with high-fat diet-induced insulin resistance. METHODS: Thirty-six 4-week-old male Wistar rats were randomly divided into 2 groups: control group and high-fat diet (HF) group. The rats in HF group were fed with high-fat diet for 8 weeks and then were randomly divided into 2 subgroups: HF group and metformin intervention group, and the animals were continuously raised for 8 months. The mRNA levels of α1 and α2 subunit of AMPK as well as LPIN1 were measured by real-time RT-PCR. Phospho-AMPKα (Thr-172) was detected by Western blotting to evaluate the activity of AMPK. RESULTS: After 4 months, the rats in HF group showed significant increase in the levels of body weight, fast plasma glucose and insulin, and the levels of triglyceride and total cholesterol significantly elevated.Significant decrease in LPIN1 and phospho-AMPKα (Thr-172) expression in the rat livers were also observed. After treated with metformin, the metabolic indexes of the HF rats were improved. The mRNA and protein expression of AMPKα1 and AMPKα2 had no significant difference among the 3 groups. Metformin treatment also increased the expression of LPIN1 in the liver tissues of HF rats. CONCLUSION: The decrease in LPIN1 expression and AMPK activity may contribute to hepatic insulin resistance in diet-induced obese rats. Metformin improves the LPIN1 expression and AMPK activity through the interaction between LPIN1 and AMPK signal pathways.     

Mechanisms of hyperglycemia induced by immunosuppressant FK506

AIM：To investigate the effect of immunosuppressant FK506 on serum glucose in rats and to explore its mechanism. METHODS：Sprague-Dawley rats (n=12) were randomly divided into drug group and normal group. The rats in drug group were intraperitoneally injected with FK506 at dose of 1 mg·kg-1·d-1 and the rats in normal group received saline (1 mL·kg-1·d-1, ip) for 14 d. The fasting weight and fasting glucose were regularly measured every 2 d. Visceral fat was isolated from the rats at the end of experiment. The mRNA expression of adiponectin, leptin, visfatin, resistin, retinol-binding protein 4 (RBP4) and peroxisome proliferator-activated receptors γ (PPAR-γ) was determined by real-time fluorescence quantitative PCR. The protein expression of PPAR-γ and adiponectin was measured by Western blotting. RESULTS：Compared with normal group, the concentration of fasting blood glucose in model group was significantly increased from the 10th day (P<0.05). At day 14, the fasting blood glucose of the model group increased from (5.10±062) mmol/L to (7.73 ± 0.73) mmol/L. No significant change of blood glucose in normal group between the 10th day and the 14th day ［from (4.66 ± 0.32) mmol/L to (5.80±0.10) mmol/L］ was observed. Compared with normal group, the mRNA expression of PPAR-γ, adiponectin and leptin in the adipose tissue of model group was significantly decreased (P<001), whereas the expression of visfatin, resistin and RBP4 was significantly increased (P<005). Compared with normal group, the expression of PPAR-γ and adiponectin in model group was decreased (P<001). CONCLUSION：FK506 may decrease the expression of PPAR-γ to change the expression of adipocytokines and induce hyperglycemia in rats.     

Correlation between post-operation insulin resistance and intestinal barrier dysfunction in obstructive jaundice rats

AIM：To investigate the correlation between post-operation insulin resistance (IR) and intestinal barrier dysfunction in obstructive jaundice (OJ) rats. METHODS：The rat model of OJ was set up. The rats were randoml divided into control group (sham operation), OJ group, glucagon-like peptide 2 (GLP-2) group (challenged with GLP-2 by intraperitoneal injection) and insulin group (insulin subcutaneous injection). Peripheral blood was collected 1 d before operation and 2 h, 24 h, 48 h, 3 d and 7 d after operation. The IR index and the ratio of lactulose/mannitol (L/M) were determined. The concentration of serum resistin-like molecule (RELM) β was detected by ELISA. The relative mRNA level of RELMβ in terminal ileum enterocytes was measured by semi-quantitative RT-PCR. RESULTS：The IR index and the ratio of L/M in OJ group 3 d after operation were 10.1±1.8 and 0.66±0.08, respectively, which were higher than those at other time points (all P<0.05). The correlation coefficient between the changes of IR index and ratio of L/M was 0.86 (P<0.05). The IR index in GLP-2 group descended by 37.0% 7 d after operation (7.33±1.07 vs 4.62±0.53, P<0.05). The highest concentration of serum RELMβ was observed in OJ group 3 d after operation (0.69 μg/L±0.05 μg/L). The relative mRNA level of RELMβ in terminal ileum enterocytes was also increased obviously. The expression of RELMβ was obviously decreased in GLP-2 group and insulin group. CONCLUSION：There is closed relationship between post-operation insulin resistance and intestinal barrier dysfunction. The RELMβ is the point of intersection. Enterocytes are not only the source of post-operation insulin resistance but also the target organ.     

Early high-protein diet improves glucose metabolism in SGA rats via adiponectin-AMPK signaling

AIM: To investigate the effect of early high-protein diet on glucose metabolism in small-for-gestational-age (SGA) rats and the role of adiponectin-AMP-activated protein (AMPK) signaling in this process. METHODS: Forty-eight neonatal male SGA rats were established by maternal food restriction throughout the period of pregnancy. The animals were randomly divided into SGA control group (CS group, 24 rats) and high-protein intervention SGA group (HPS group, 24 rats) when born. Twenty-four normal neonatal male rats were used as normal control group (CN group). The rats in CN group and CS group were breastfed for 3 weeks and their mothers were provided free access to basic diet. After weaning, they were provided free access to basic diet until 12 weeks of age. The rats in HPS group were breastfed for 3 weeks and their mothers were provided free access to high-protein diet. After weaning, they were provided free access to high-protein diet until 4 weeks of age. At the 4th week of age, they were provided free access to basic diet until 12 weeks of age. From 4 to 12 weeks of age, fasting blood glucose and insulin were measured and homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated. The serum adiponectin level and the visceral fat mass (VFM) were detected. The percentage of VFM to body weight (VFM%) was calculated. The visceral fat was paraffin-embedded and the adipocyte area was determined. The expression of AMPKα,phosphorylated AMPKα (p-AMPKα) and glucose transporter 4 (GLUT4) in skeletal muscle was analyzed. At the 12th week of age, the oral glucose tolerance test (OGTT) was performed. RESULTS: At the 4th week of age, no significant difference of HOMA-IR among groups was observed. At the 12th week of age, HOMA-IR in CS group was significantly higher than that in CN group and HPS group. No significant difference of HOMA-IR between CN group and HPS group was found. From 4 to 12 weeks of age, VFM% and adipocyte area of visceral fat in CS group were significantly higher than those in CN group and HPS group. No significant difference of VFM% and adipocyte area of visceral fat between CN group and HPS group was observed. The level of serum adiponectin, and the expression of p-AMPKα and GLUT4 in skeletal muscle were significantly lower in CS group than those in CN group and HPS group. The protein levels of p-AMPKα and GLUT4 were not significantly different between CN group and HPS group. No significant difference in the expression of AMPKα in skeletal muscle among groups was observed. At the 12th week of age, the area under the curve (AUC) of glucose level in OGTT was higher in CS group than that in CN group and HPS group. No significant difference of AUC of glucose level between CN group and HPS group was found. CONCLUSION: Early high protein diet may improve glucose metabolism in SGA rats, partly by avoiding excessive accumulation of visceral fat and possibly by activating adiponectin-AMPK signal pathway to increase GLUT4 expression.     

Effect of benazepril on expression of insulin receptor and its substrate-1 protein in renal tissue cell membrane in diabetic rats

AIM: To study effect of benazepril (an ACE inhibitor) on expression of insulin receptor (IR) and its substrate-1 (IRS-1) protein in renal tissue cell membrane in diabetic rats. METHODS: The rats were randomly divided into following groups: control (n=6),streptozotocin induced diabetic (n=7) and diabetic treated with benazepril (n=7). Body weight, kidney weight and kidney weight/body weight were observed after 4 weeks of treatment. ACE activities in plasma, renal tissue were measured by the fluorimetric assay. The expressions of IR and IRS-1 protein were determined by Western blot analysis in renal tissue cell membrane. RESULTS: After 4 weeks of treatment,benazepril significantly ameliorated kidney hypertrophy in diabetic rats. ACE activities in plasma,renal tissue were reduced by approximately 92.00% and 88.77%,respectively. Western blot analysis showed that the expressions of IR and IRS-1 protein were increased by 2.1 and 1.5 folds in renal tissue cell membrane in diabetic rats. However, benazepril reduced expression of IR and IRS-1 protein by 45.74% and 47.66%, respectively. CONCLUSIONS: Increased the expression of IR and IRS-1 protein might be related to abnormally active glucose metabolism in diabetic rat kidney. Down-regulation of expression of IR and IRS-1 protein might be one of important machnisms of Benazepril nephroprotection on diabetic rats.     

Intranasal insulin treatment ameliorates Alzheimer disease-like changes in hippocampus in a rat model of type 2 diabetes

AIM： To investigate Alzheimer disease (AD)-like changes and 2 key components of the insulin signaling pathway in the brain of a rat model of type 2 diabetes (T2D) after insulin treatment. METHODS：The rat model of T2D was established by feeding a high-protein, high-glucose and high-fat diet followed by intrasubcutaneous injection of streptozocin. Intranasal insulin treatment (T2D+I-I) and subcutaneous insulin injection (T2D+S-I) were applied to elevate the insulin level in the brain. The insulin levels in plasma and cerebrospinal fluid as well as the concentration of plasma glucose were measured. Total tau level, the phosphorylation level of tau at some phosphorylation sites, and the activation of GSK-3β and Akt in subcutaneous of the rats were also analyzed by Western blotting.RESULTS：AD-like changes, decreased Akt activation and over-activation of GSK-3β in the hippocampus of the T2D rats were observed. Intranasal insulin treatment for 4 weeks normalized the levels of Akt and GSK-3β, as well as reduced the AD-like changes in the hippocampus of the T2D rats, whereas the treatment with insulin by subcutaneous injection for 4 weeks had minimal effects on the levels of GSK-3β and tau phosphorylation in the hippocampus. CONCLUSION： Intranasal insulin treatment, but not subcutaneous insulin treatment, might decrease the risk of AD in T2D rats by reducing AD-like changes and up-regulating the impaired insulin signaling pathway in the hippocampus,indicating the potential use of intranasal insulin delivery for treatment of AD.     

Rat urotensin-II-induced main pulmonary artery contractions are mediated by MAPK

AIM: To investigate rat Urotensin-II(rat U-II)-induced vasoconstriction of rat main pulmonary arteries and the role of mitogen-activated protein kinase(MAPK). METHODS: The main pulmonary artery was dissected from the male Sprague-Dawley rats and artery ring width was 3-4 mm. Concentration-response curves were generated to rat U-II(0.03 nmol/L-30 nmol/L).Inhibitor of MAPK, PD 98059(0.1 μmol/L-10 μmol/L) were added into the medium after rat U-II(30 nmol/L)induced vasoconstriction had reached plateau to construct the relaxant concentration-response curves and their EC₅₀ and E_max. RESULTS：Rat U-II was a potent vasoconstrictor of isolated rat main pulmonary arteries [EC₅₀=7.95±0.40, E_max=(14.28±6.34)% of the response to 60 mmol/L KCl]; PD 98059 caused concentration-dependent relaxations of rat U-II precontracted arteries [EC₅₀=5.91±0.45, E_max=(81.39±13.65)%]. CONCLUSION: Rat U-II was a potent vasoconstrictor of rat main pulmonary arteries and this response was mediated through MAPK.