Effects of liraglutide on adiponectin and insulin resistance in rats with non-alcoholic fatty liver disease

AIM：To investigate the effects of glucagon-like peptide 1 analog, liraglutide, on adiponectin and insulin resistance in the rats with diet-induced non-alcoholic fatty liver disease (NAFLD). METHODS：Male rats were randomly divided into 3 groups:normal diet (ND) group (n=10), high-fat diet (HFD) group (n=10), and HFD with intraperitoneal injection of liraglutide group (n=10, first 12 weeks with HFD, later 4 weeks with liraglutide). All treatments continued for 16 weeks, and then the rats were killed ethically and the blood samples and liver tissues were collected. The levels of alanine aminotransferase (ALT), fasting blood glucose (FBG), total cholesterol (TC) and triglyceride (TG) were detected by a biochemical automatic analyzer. The levels of free fatty acids (FFAs), fasting insulin (FINS) and adiponectin were measured by RIA and ELISA. RESULTS：Compared with HFD group, the body weight, liver index, homeostasis assessment-insulin resistance (HOMA-IR), the serum levels of TG, TC, ALT and FBG, and the liver levels of TG, TC and FFAs in the rats in liraglutide group were apparently lower, the degree of hepatic steatosis and inflammatory activity significantly decreased (P<0.05), and the level of adiponectin in the serum and liver homogenate increased ob-viously (P<0.05). The level of adiponectin in the liver homogenate was negatively correlated with the levels of FFAs in the liver homogenate. CONCLUSION:Liraglutide is beneficial for NAFLD rats to improve insulin resistance and reduce hepatic steatosis by increasing the level of adiponectin in the serum and liver tissues.     

GLP-1 down-regulates mRNA expression of SOCS-3 and SREBP-1c in rats with nonalcoholic fatty liver disease

AIM: To investigate the effects of glucagon-like peptide-1(GLP-1) on mRNA expression of SOCS-3 and SREBP-1c in the rats with nonalcoholic fatty liver disease. METHODS: Male SD rats were randomly divided into normal control(NC) group, high fat(HF) group and HF+liraglutide(Lira) group. The rats in HF group and HF+Lira group were given high-fat diet for 16 weeks. After 12 weeks of high-fat diet feeding in HF+Lira group, Lira(600 μg·kg^-1·d^-1) was intraperitoneally injected for 4 weeks. At the end of the 16th week, the rats were killed. The pathological changes of the liver were observed under optical microscope. The serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), triglyceride(TG) and total cholesterol(TC) were detected by automatic biochemical analyzer. TG contents of liver were measured by GPO-PAP method. The fasting insulin(FINS) was determined by ELISA, and insulin resistance index was assessed by homeostasis mode assessment(HOMA-IR). The mRNA expression of SOCS-3 and SREBP-1c in the liver tissues was detected by RT-qPCR. RESULTS: Compared with NC group, HOMA-IR, TG of liver, and the serum levels of ALT, AST, TG, TC and FINS in HF group were obviously increased(P<0.01). Compared with HF group, HOMA-IR, TG of liver, and the serum levels of ALT, AST, TG, TC and FINS in HF+Lira group were all obviously decreased(P<0.05 or P<0.01). The mRNA expression of SOCS-3 and SREBP-1c in HF group was significantly higher than that in NC group(P<0.01). The mRNA expression of SOCSV3 and SREBP-1c in HF+Lira group was significantly decreased as compared with HF group(P<0.05). CONCLUSION: Liraglutide may improve the IR and reduce TG of liver through decreasing the mRNA expression of SOCS-3 and SREBP-1c, so as to play a therapeutic role in nonalcoholic fatty liver disease.     

Role of SIRT1/AMPK pathway in early intervention of Lira alleviating non-alcoholic fatty liver disease caused by high-fat diet in rats

AIM To investigate the effect of early intervention of glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide (Lira) on oxidative stress, glucose tolerance, hepatic steatosis and insulin resistance of the rats with high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD), and to explore the role of silent information regulator 1 (SIRT1)/AMP-activated protein kinase (AMPK) signaling pathway in this process. METHODS Twenty-four male SD rats were randomly divided into normal diet (ND) group, HFD group and HFD+Lira group, with 8 rats in each group. After 1 week of adaptive feeding, the rats in HFD+Lira group were subcutaneously injected with Lira (200 μg/kg) per day at a fixed time point, while the rats in the remaining 2 groups were injected with normal saline at the same volume. During the intervention, the body weight, hair, appetite, defecation and activity of the rats were observed to adjust the dosage timely. The body weight, food intake and blood glucose were recorded weekly. Glucose tolerance test was performed at the end of the 16th week. At the end of the 18th week, hyperinsulinemic euglycemic clamp test was conducted after anesthesia. Blood was taken from the carotid artery. The liver and adipose tissues from different parts were taken after death. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other indicators were detected. HE staining was used to observe the pathological changes of the liver tissue. Lipid accumulation in the liver tissues was observed by oil red O staining. Liver fibrosis was observed by Masson staining and Sirius red staining. Fluorescence staining for reactive oxygen species (ROS) was used to observe the oxidative stress in the liver. The expression of GLP-1 receptor in the liver was observed by immunofluorescence staining. The expression and localization of SIRT1 and phosphorylated AMPK at Thr172 [p-AMPK (Thr172)] were observed by immunohistochemical staining. The protein levels of AMPK, p-AMPK (Thr172), SIRT1, phosphorylated sterol regulatory element binding protein-1c at Ser372 [p-SREBP-1c (Ser372)], phosphorylated acetyl coenzyme A carboxylase at Ser79 [p-ACC (Ser79)], carnitine palmitoyltransferase 1A (CPT1A) and fatty acid synthase (FAS) in liver tissues were determined by Western blot. RESULTS The results of HE and oil red O staining of rat liver tissues in HFD group confirmed the structural disorder and serious lipid accumulation, while Masson and Sirius red staining showed severe fibrosis, suggesting the successful establishment of NAFLD rat model. Compared with ND group, the levels of total cholesterol (TC), triglyceride (TG), AST and ALT in serum, and the levels of malondialdehyde (MDA), TC, TG and ROS in liver tissues in HFD group were significantly increased (P<0.01), while the activity of superoxide dismutase (SOD) was decreased (P<0.01). The protein levels of p-AMPK (Thr172), SIRT1, p-SREBP-1c (Ser372), p-ACC (Ser79) and CPT1A in the liver tissues were significantly reduced (P<0.05 or P<0.01), while the expression of FAS was increased （P<0.01）. Compared with HFD group, lipid accumulation and fibrosis in the liver tissues of the rats in HFD+Lira group were significantly attenuated, the serum levels of TC, TG, AST and ALT, and MDA, TC, TG and ROS in liver tissues were markedly reduced (P<0.05 or P<0.01), while SOD activity was increased (P<0.05). The protein levels of p-AMPK (Thr172), SIRT1, p-SREBP-1c (Ser372), p-ACC (Ser79) and CPT1A in the liver tissues were significantly increased (P<0.05 or P<0.01), while the expression of FAS was decreased （P<0.01）. CONCLUSION Lira attenuates insulin resistance, oxidative stress and fibrosis, and improves liver lipid metabolism in the rats with NAFLD induced by HFD, which may be mediated by SIRT1/AMPK signaling pathway.     

Ginsenoside Rg1 improves liver function by regulating fat metabolism in rats with non-alcoholic fatty liver disease

AIM: To investigate whether ginsenoside Rg1 attenuates high-fat diet (HFD)-induced non-alcoho-lic fatty liver disease (NAFLD) by improving β-oxidation. METHODS: SD rats (n=60) were randomly divided into control group (CON), HFD group, low-dose, medium-dose and high-dose ginsenoside Rg1 groups (LDG, MDG and HDG) and positive drug (sodium ursodeoxycholate) treatment group (PDT). High-fat diet was given for 8 weeks to successfully establish an NAFLD model. The animals were treated with the appropriate medications for 4 weeks and 8 weeks after modeling, and sacrificed to collect the liver tissues for observing the pathologic changes with HE staining and for detecting liver functions and lipid levels. The expression of hepatic acyl-CoA synthetase 1 (CoASH1), carnitine acyltransferase I (CATI) and acyl-CoA oxidase 1 (ACOX1) at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS: After 4-week treatment, the fatty infiltration of the liver tissues in PDT group, LDG group and MDG group was not attenuated except HDG group. After 8 weeks of treatment, a small number of fat particles was observed in PDT group and LDG group, while no infiltration of lipid droplet was found in MDG group and HDG group. Compared with HFD group, the levels of AST, ALT, AKP, TC, TG and LDL-C were significantly decreased after 4-week treatment in PDT group, LDG group, MDG group and HDG group (P<0.05), these indexes were further reduced after 8-week treatment. After 4-week treatment, HDL-C was significantly increased in the 4 treatment groups and almost restored to the level of CON group after 8-week treatment. The levels of CoASH1, CACTI and ACOX1 in the liver tissue of the 4 treatment groups were significantly increased after 4-week treatment (P<0.05) and much improved after 8-week treatment, and those in MDG group and HDG group were better than those in PDT group (P<0.05).CONCLUSION: Ginsenoside Rg1 regulates β-oxidation-related enzymes to improve the fat metabolism, thus playing a therapeutic role in liver injury in the rats with NAFLD.     

Effect of non-alcoholic fatty liver disease on metabolic syndrome-rela-ted type 2 diabetes

AIM: To investigate the effect of non-alcoholic fatty liver disease (NAFLD) on metabolic syndrome (MS)- related type 2 diabetes. METHODS: Sixty-four rats were randomly divided into 8 groups: normal control groups of 3 months (3NC), 6 months (6NC), 9 months (9NC) and 12 months (12NC), and high-sucrose and high-fat groups of 3 months (3H), 6 months (6H), 9 months (9H) and 12 months (12H). The serum levels of alanine transaminase (ALT), free fatty acid (FFA), endotoxin (LPS), tumor necrosis factor alpha (TNFα), C-reactive protein (CRP), monocyte chemotactic protein-1 (MCP-1), fasting plasma glucose (FPG) and fasting plasma insulin (FINS) were measured. The insulin resistance index (HOMA-IR) and beta cell function index(HOMA-β) were also calculated. The specimens of liver and pancreas were prepared for microscopic observation with HE and Sudan Ⅳ staining. The visceral adipose specimens were also prepared with HE staining. The apoptosis of islet cells was detected by TUNEL.RESULTS: Compared with normal control groups, the levels of ALT, FFA, LPS, TNFα, CRP, MCP-1, FPG, FINS and HOMA-IR in high-sucrose and high-fat groups of every phase were higher, but the level of HOMA-β in 6H group showed a compensatory increase first and then progressively decreased. Compared with normal control groups, TUNEL results showed that the number of apoptotic islet cells in high-sucrose and high-fat groups gradually increased from the 3rd to the 12th month. CONCLUSION: The NAFLD plays a trigger role in the start of MS-related type 2 diabetes.     

Effect of insulin resistance on fatty liver in high-fat diet-fed mice

AIM: To study the influence of insulin resistance on fatty liver in the mice fed with high-fat diet (HFD).METHODS: Male 8-week-old C57BL/6J mice were randomly divided into HFD group (with 60% calories by high saturated fatty acid) and control group (with chow diet).The mice in both groups were fed for 12 weeks. The body weight, liver weight, serum triglyceride (TG) and total cholesterol (TC), and blood glucose and insulin levels were measured. Hyperinsulinemic euglycemic clamp experiment was applied to reflect insulin sensitivity. The lipid deposition in the liver was analyzed by HE staining, Sudan IV staining and measurement of liver fat content. The phosphorylation levels of IRS1 and Akt, and the protein levels of SREBP-1 and FAS were determined by Western blot to reflect the activities of insulin signaling and lipid synthesis.RESULTS: Compared with control group, the body weight and liver weight were significantly increased in HFD group. TG and TC contents in serum and liver tissues were remarkably increased in HFD group. High-fat diet induced insulin resistance, as evidenced by increased serum insulin levels, reduced glucose infusion rate and decreases in IRS1 and Akt phosphorylation levels. In livers of HFD group, HE staining showed that the cytoplasm of hepatocytes was filled with vacuoles. Sudan IV staining also displayed that many different sizes of red lipid drops existed in the hepatocytes, and the protein levels of SREBP-1 and FAS were significantly increased. In primary normal hepatocytes with exogenous oleic acid intervention for 48 h, the phosphorylation levels of IRS1 and Akt were reduced, and the protein expression of SREBP-1 and FAS was significantly increased in a dose-dependent manner.CONCLUSION: Feeding with HFD leads to insulin resistance, resulting in activation of lipid synthesis and accumulation of lipid deposition in the liver, thus inducing fatty liver.     

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.     

Radix Pseudostellariae polysaccharide attenuates high fat diet induced hepatic insulin resistance in mice

AIM: To study the role of Radix Pseudostellariae polysaccharide (RPP) in hepatic insulin resistance.METHODS: Six-week-old C57BL/6J mice were randomly divided into low-fat diet (LFD) control group and high-fat diet (HFD) model group. After 16 weeks, intraperitoneal pyruvate tolerance test (IPPTT) was performed to determine the establishment of the HFD-induced hepatic insulin resistance model. HFD containing RPP (500 mg/kg) was given for 4 consecutive weeks. IPPTT, liver malondialdehyde (MDA) level and liver mitochondrial MDA level were measured. The protein levels of p-AKT (Ser473/Thr308), p-AMPK, nuclear factor E2-related factor 2 (Nrf2), NQO1 and IκBα in the liver tissues were measured by Western blot.RESULTS: After administration of RPP, a significant reduction in the levels of blood glucose and hepatic mitochondrial MDA was observed. The levels of p-AKT (Ser473/Thr308) and p-AMPK were significantly elevated in the liver tissues. The hepatic IκBα levels were up-regulated. RPP also enhanced the expression of Nrf2 system-regulated proteins NQO1 and HO-1 in the liver tissues.CONCLUSION: Radix Pseudostellariae polysaccharides effectively reduce HFD-induced hepatic insulin resistance in C57BL/6J mice and improves liver glucose metabolism by ameliorating HFD-impaired hepatic transduction of insulin signaling, activating Nrf2-associated signaling and inhibiting the expression of inflammatory signaling proteins.     

Anthocyanins attenuates hepatic fibrosis and inflammation in non-alcoho-lic fatty liver disease mice

AIM:To explore the therapeutic effect of anthocyanins from Fructus Acanthophorae on high-fat diet-induced non-alcoholic fatty liver disease (NAFLD) in mice and the potential mechanism. METHODS:NAFLD mouse model was established by high-fat diet, and interferred with anthocyanins. The liver weight, and serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC) and low-density li-poprotein cholesterol (LDL-C) were measured. The liver tissues were staining with HE, Oil Red O and Masson's trichrome. The protein levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-10 in the liver tissues were determined by Western blot. The liver macrophage, white blood cell and mononuclear cell infiltration was detected by immunohistochemical method. The chemokines CCL7 and MCP-1 were also measured by immunohistochemical method. RESULTS:Anthocyanins significantly inhibited the increases in the liver weight, ALT, AST, TG, TC and LDL-C induced by high-fat diet. Anthocyanins attenuated the liver fibrosis and inflammatory cell infiltration caused by high-fat diet, and reduced the levels of inflammatory factors TNF-α, IL-1β, IL-6, IL-10 and inflammatory chemokines CCL7 and MCP-1 in the liver tissues. CONCLUSION:Anthocyanins significantly alleviate non-alcoholic fatty liver disease caused by high-fat diet though reducing inflammatory factors, inflammatory cell infiltration and inflammatory chemokines.     

Effect of glucagon-like peptide 1 on Sesn2/AMPK/mTOR signaling pathway in liver of obese rats induced by high-fat diet

AIM: To explored the effect of glucagon-like peptide 1 receptor agonist liraglutide on Sesn2/AMPK/mTOR signaling pathway in the liver of obese rats.METHODS: Male SD rats were divided into normal chow (NC) group (n=12) and high-fat diet (HF) group (n=33). After 12 weeks, 5 rats of each group were used to assess establishment of obese rat model. The rats in HF group were divided into 4 subgroups, HF group, low dose of liraglutide (LG) group, middle dose of liraglutide (MG) group, and high dose of liraglutide (HG) group, and treated with various doses of liraglutide (0, 50, 100 and 200 μg/kg) via hypodermic injection twice a day for 4 weeks. The body weight and epididymal fat index of the rats at the 16th week were measured. The liver tissue fatty degeneration was observed. The protein levels of Sesn2, AMPK, p-AMPK, mTOR and p-mTOR were determined by Western blot.RESULTS: The body weight of rats in HF group was obviously higher than that in NC group (P<0.01). Compared with NC group, the levels of Sesn2 and p-AMPK/AMPK were significantly decreased in HF group (P<0.01), while the level of p-mTOR/mTOR was not changed. After treatment with liraglutide for 4-week, the body weight of the rats in LG, MG and HG groups was obviously lower than that in HF group (P<0.01), and epididymal fat index of the rats in MG and HG groups was obviously lower than that in HF group (P<0.01). The protein level of Sesn2 in HG group was obviously higher than that in HF group (P<0.01). The level of p-AMPK/AMPK was significantly increased in MG and HG groups (P<0.01). The level of p-mTOR/mTOR was significantly increased decreased in LG, MG and HG groups (P<0.01).CONCLUSION: Glucagon-like peptide 1 receptor agonist liraglutide affects energy metabolism and improves the state of obesity through Sesn2/AMPK/mTOR signaling pathway.     

Mechanism of Fuzilizhong decoction alleviating inflammatory damage of rats with non-alcoholic fatty liver disease

AIM To observe the effect of Fuzilizhong decoction on the inflammatory damage of non-alcoholic fatty liver disease （NAFLD） rats and to explore its mechanism. METHODS SPF male SD rats were randomly divided into 6 groups： control group, model group, high dose （20 mg·kg^-1·d^-1）, middle dose （10 mg·kg^-1·d^-1）, low dose （5 mg·kg^-1·d^-1） Fuzilizhong decoction group and Yishanfu （30 mg·kg^-1·d^-1）group, 8 rats in each group. A NAFLD rat modelwas established by intragastric administration of fat emulsion for 4 weeks. Then the drug was given for 4 weeks in each treatment group. HE staining was performed to observe the histopathological changes of the rat liver.The serum levels of interleukin-2（IL-2）, IL-6 and tumor necrosis factor-α（TNF-α） were measured by ELISA. The expression of toll like receptor 4（TLR4） and NF-κB p65 in liver tissues at mRNA and protein levels was determined by RT-qPCR and Western bolt,respectively. RESULTS Compared with control group, the inflammatory damage of liver tissue was more serious, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression TLR4 and NF-κB p65 in liver tissues were significantly increased in model group（P<0.05）. However, compared with model group, the liver pathological changes in each treatment group were significantly relieved, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression of TLR4 and NF-κB p65 in liver tissues were significantly reduced（P<0.05）.In addition, the changes of TLR4 and p-NF-κB p65 protein levels in liver tissue were consistent with the changes of TLR4 and NF-κB p65 mRNA. CONCLUSION Fuzilizhong decoction attenuates the inflammatory damages of NAFLD in rats by inhibiting TLR4/NF-κB p65 signaling pathway.     

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.     

Mechanism of interleukin-6 induced insulin resistance in 3T3-L1 adipocytes

AIM: To investigate the molecular mechanism of interleukin-6 induced insulin resistance in 3T3-L1 adipocytes.METHODS: 3T3-L1 adipocytes were treated with IL-6 at concentration of 20 μg/L within 48 hours. Insulin stimulated glucose uptake was measured by 2-deoxy ［3H］ glucose. Western blotting was used to measure insulin receptor substrate-1(IRS-1), protein kinase B(PKB) expression, tyrosine phosphorylation on IRS-1, and PKB phosphorylation. RESULTS: On basal status, glucose uptake in 3T3-L1 cells, PKB phosphorylation and tyrosine phosphorylation of IRS-1 were all at low level. Insulin stimulation induced a rapid increase in glucose uptake, PKB phosphorylation and IRS-1 tyrosine phosphorylation. IL-6 inhibited insulin-induced glucose uptake and PKB phosphorylation level about 50%. After IL-6 treatment, IRS-1 protein expression and tyrosine phosphorylation of IRS-1 were decreased 35% and 40%, respectively. The inhibitor of mammalian target of rapamycin(mTOR), rapamycin, reversed above effects of IL-6. CONCLUSION: IL-6 induced insulin resistance in 3T3-L1 adipocytes is related to decrease IRS-1 expression and impairs IRS-1 tyrosine phosphorylation. IL-6 induced insulin resistance in adipocytes may be related to the activity of mTOR.     

Total triterpenoids from Psidium Guajava leaf improves insulin resistance in 3T3-L1 adipocytes

AIM: To investigate the effects of total triterpenoids from Psidium guajava leaf (TTPGL) on 3T3-L1 adipocyte insulin resistance (IR) and to explore the possible mechanism. METHODS: 3T3-L1 pre-adipocytes were cultured and induced to differentiate into 3T3-L1 adipocytes, then treated with TTPGL (0.3, 1, 3, 10 μg/L) for 48 h. The cells were divided into 0.1% DMSO group, positive drug sodium orthovanadate (Van, 10 μmol/L) group, model group and control group. The effect of TTPGL on the cell activity of pre-adipocytes was detected by MTT assay and its influence on the cellular differentiation was observed by oil red O staining. The IR model of the 3T3-L1 adipocytes was established successfully and then treated with different drugs for 48 h. The glucose consumption in the supernatant of IR adipocyte's culture medium was assayed by glucose oxidase-peroxidase (GOD-POD), free fatty acid (FFA) levels were measured by colorimetric method, and adipocytokines levels were assayed by ELISA. The mRNA expression of protein tyrosine phosphatase-1B (PTP1B) of IR adipocyte was detected by real-time PCR. The protein levels of phosphorylated insulin receptor substrate 1/insulin receptor substrate 1 (p-IRS-1/IRS-1) and phosphorylated protein kinase B/protein kinase B (p-Akt/Akt) were determined by Western blot. RESULTS: Compared with DMSO group, TTPGL treatment significantly promoted the cell activity of 3T3-L1 pre-adipocytes and inhibited its differentiation (P < 0.01). TTPGL (1~10 μg/L) improved glucose consumption of IR adipocytes significantly (P < 0.01), with or without insulin stimulation, and TTPGL (0.3~3 μg/L) restrained FFA production remarkably(P < 0.01). Compared with model group, TTPGL (0.3 and 3 μg/L) significantly increased the secretion of adiponectin in IR adipocytes (P < 0.05), and inhibited the secretion of tumor necrosis factor-α (TNF-α) (P < 0.01). TTPGL (3 μg/L) restrained the secretion of resistin significantly (P < 0.05), and showed no significant effect on secretion of leptin. It also down-regulated the mRNA expression of protein tyrosine phosphates 1B (PTP1B) in IR adipocytes significantly (P < 0.01), and increased the protein levels of p-IRS-1/IRS-1. TTPGL (0.3 and 3 μg/L) up-regulated the protein level of p-Akt/Akt in IR adipocytes significantly (P < 0.05).CONCLUSION: TTPGL reduces IR in 3T3-L1 adipocytes. The mechanism may be that TTPGL significantly down-regulated mRNA expression of PTP1B and increased the protein levels of p-IRS-1/IRS-1 and p-Akt/Akt in IR adipocytes.     

Effects of glycine on insulin resistance and cognitive impairment induced by high-fructose diet

AIM: To investigate the role of intestinal endotoxemia (IETM) in insulin resistance (IR) and cognitive impairment, and to explore the protective mechanisms of glycine in rats with high-fructose diet. METHODS: The rats in model group were fed with 8% fructose water, and the rats in intervention group were fed with water containing 8% fructose and 1% glycine. The body weight and systolic pressure were measured monthly. After 8 months, plasma glucose, plasma lipids, glucose tolerance and plasma endotoxin (LPS) were detected. Plasma insulin, pro-inflammatory cytokines in plasma and cerebral cortex were determined by ELISA, and HOMA-IR was also calculated. The molecules of insulin signaling pathway in cerebral cortex were determined by Western blotting. The cognitive functions of the rats were tested by Morris water maze. RESULTS: The weight gain in model group was increased from the 3rd month to the 6th month, and systolic pressure was increased after the 3rd month as compared with control group. Glycine significantly reduced the weight gain in the 4th month and the 6th month, and significantly reduced the systolic pressure from the 4th month to the 6th month. Meanwhile, glycine partly attenuated dyslipidemia and glucose intolerance, and lowered the levels of plasma LPS, plasma insulin, HOMA-IR and pro-inflammatory cytokines in plasma and cortex. Furthermore, glycine attenuated the abnormal expression of insulin signaling proteins and cognitive impairment. CONCLUSION: Long-term fructose diet induces the rats to peripheral and neuronal IR, which accompanies IETM and low-grade inflammation. Glycine attenuates IR and cognitive impairment by lowering IETM.     

Effects of resveratrol on levels of ceramide via regulating miRNA-122 in treating non-alcoholic fatty liver disease

AIM: To observe the therapeutical effects of resveratrol on non-alcoholic fatty liver disease and its potential mechanism. METHODS: Male C57BL/6J mice were fed with high-fat and high-cholesterol diet to established non alcoholic fatty liver disease model, and were administrated with resveratrol at doses of 80 mg/kg and 160 mg/kg. After 4-week treatment, the blood sample was collected for determination of total cholesterol (TC) and triglyceride (TG). The liver tissues were harvested for measuring the liver lipid content. The histopathological examination were conducted with hematoxylin and eosin staining. The ceramide levels in the liver tissues were detected by HPLC-MS. The microRNA (mi-RNA)-122 levels in the liver tissues were detected by real-time PCR. The protein levels of serine palmitoyltransferase (SPT) were determined by Western blot. The HepG2 cells were cultured and divided into 5 groups:control group, model group (induced by 0.25 mmol/L oleic acid), model+resveratrol group (treated with 5 μmol/L resveratrol), miRNA-122 siRNA group and resveratrol+miRNA-122 siRNA group. Except control group, the cells in other groups were stimulated with oleic acid and incubated with respective drugs simultaneously for 24 h. The levels of TC, TG and ceramide in the cells of each group were measured. The protein levels of SPT in each group were determined by Western blot. RESULTS: In non-alcoholic fatty liver disease mice, resveratrol dose-dependently reduced the serum TC and TG levels, decreased the lipid deposition, the ceramide level and the SPT protein level, and increased the level of miRNA-122 in the liver tissues. In the in vitro study, compared with model group, resveratrol reduced the serum TC and TG levels, decreased the ceramide level, reduced the SPT protein level. Compared with control group, the levels of TC, TG and ceramide, and the protein expression of SPT were increased in miRNA-122 siRNA group. Compared with miRNA-122 siRNA group, no statistical difference of TC, TG, ceramide and protein expression of SPT in resveratrol combined miRNA-122 siRNA group was observed. CONCLUSION: Resveratrol significantly reduces lipid accumulation by reduction of miRNA-122 and ceramide levels, and decrease in SPT protein levels in the liver.     

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.     

Effect of short-term high-fructose feeding on liver triglyceride content and hepatic insulin sensitivity in mice

AIM: To investigate the effect of short-term high-fructose feeding on liver triglyceride content and hepatic insulin sensitivity in mice. METHODS: Male C57BL/J6 mice were divided into control group and high (HFru) fructose group. After 3-day feeding, intraperitoneal glucose tolerance test (ipGTT) was performed to evaluate whole-body insulin sensitivity. The mice were sacrificed,and the liver samples were collected for measuring the liver triglyceride content and observing the pathological changes of the liver under light microscope with HE staining. The protein levels of lipogenic enzymes in the liver tissues were measured. To evaluate the hepatic insulin sensitivity, the protein levels (expressed as the ratio) of phosphorylated Akt/total Akt (p-Akt/t- Akt) and phosphorylated GSK-3α/β/total GSK-3α/β(p- GSK-3α/β/t- GSK-3α/β) were compared between 2 groups of the mice with or without insulin injection. RESULTS: After 3-day feeding of high-fructose diet, compared with control group, the area under the curve of ipGTT and triglyceride contents in the liver tissues were significantly increased in HFru group. HE staining of the liver in the mice in HFru group showed obvious lipid droplet formation. Compared with control group, the protein expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD-1) was significantly increased in HFru group. After insulin injection, the ratio of p-Akt/t-Akt and p-GSK-3α/β/t-GSK-3α/β was significantly decreased in HFru group as compared with control group. CONCLUSION: A 3-day short-term high-fructose feeding induces liver steatosis, which is related to the increased protein expression of FAS, ACC and SCD-1. Liver steatosis occurs simultaneously with the development of hepatic insulin resistance.     

Advances in research on mechanism of CCN proteins in prevention and treatment of obesity and non-alcoholic fatty liver

CCN protein family plays a role in regulating the formation and remodeling of extracellular matrix, inflammation regulation, injury repair and so on, which is closely related to the occurrence and development of metabolic diseases. This review focuses on the mechanism of CCN proteins in obesity and non-alcoholic fatty liver. The roles of CCN proteins in the adipocyte activation, the fibrosis and inflammatory response in non-alcoholic fatty liver, and the injury repair against non-alcoholic fatty liver and its complications are introduced, providing new ideas for the study of CCN proteins in metabolic diseases such as obesity and non-alcoholic fatty liver.