APS improves free fatty acid metabolism by activating AMPK and promoting translocation of FAT/CD36 in C2C12 myoblasts

AIM：To investigate the effect of Astragalus polysaccharides (APS) on the metabolism of free fatty acids (FFAs) in C2C12 myoblasts. METHODS：Cultured C2C12 myoblasts were used in the study. The viability of C2C12 myoblasts treated with FFAs at different concentrations for different time was observed by MTT assay. The concentrations of FFAs in the medium were detected by acetyl-CoA synthase (ACS)-acetyl-CoA oxidase (ACOD) method. The expression of fatty acid translocase (FAT/CD36), AMPK and p-AMPK protein was examined by Western blotting. RESULTS：FFAs decreased the viability of C2C12 myoblasts in a time- and concentration-dependent manner. Compared with FFAs group, the expression of cellular membrane FAT/CD36 and p-AMPK proteins increased in FFAs+APS group, but total AMPK and FAT/CD36 protein expression was not significantly changed. Meanwhile, the concentration of FFAs in the medium decreased and the cell viability increased in FFAs+APS group as compared with the group. CONCLUSION：APS improves the metabolism of FFAs by activating AMPK and promoting translocation of FAT/CD36 in C2C12 myoblasts.     

3,4-Dihydroxyacetophenone reduces TG content in L02 cells and hepatic tissue via AMPK pathway

AIM: To investigate the mechanism of 3,4-dihydroxyacetophenone (3,4-DHAP) regulating lipid metabolism in human hepatic cells (L02 cells) and rabbit hepatic tissue. METHODS: L02 cells were divided into normal control group, model group, 3,4-DHAP group and simvastatin group. The cells were collected after treated with drugs for 8 h. Triglyceride (TG) content in the cells was detected by TG kit. RT-qPCR was applied to detect the mRNA expression level of AMP-activated protein kinase (AMPK). The protein levels of AMPK, phosphorylated AMPK (p-AMPK), phosphorylated sterol regulatory element binding protein-1c (p-SREBP-1c) and phosphorylated acetyl-CoA carboxylase (p-ACC) were detected by Western blot. Male New Zealand white rabbits (n=32) were randomized into normal control group, model group, 3,4-DHAP group and simvastatin group. The rabbits were treated with the drugs from week 2 to week 12. At the end of week 12, all rabbits were sacrificed. The liver lipids were measured by oil red O staining, and TG content was analyzed by TG kit. The protein levels of AMPK, p-AMPK, p-SREBP-1c and p-ACC in hepatic tissue were detected by Western blot. RESULTS: In L02 cells, compared with model group, TG content in 3,4-DHAP group was significantly decreased, and the expression of AMPK at mRNA and protein levels and the protein levels of p-AMPK, p-SREBP-1c and p-ACC were significantly increased. In rabbits of 3,4-DHAP group, the TG content was significantly decreased compared with model group, and the protein levels of AMPK, p-SREBP-1c and p-ACC were significantly increased. CONCLUSION: The AMPK signaling pathway may be involved in the mechanism of 3,4-DHAP to reduce TG content in L02 cells and rabbit hepatic tissue.     

Astragalus polysaccharides protects against free fatty acid-induced human vascular endothelial cell dysfunction via AMPK-eNOS pathway

AIM: To study the protective effect of Astragalus polysaccharides (APS) on free fatty acid-induced injury in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs were divided into control group, APS group [APS (200 mg/L) treated for 24 h], free fatty acid group [free fatty acid (0.25 mmol/L) treated for 24 h], free fatty acid plus APS group [free fatty acid (0.25 mmol/L) and APS (200 mg/L) treated for 24 h], and compound C group [free fatty acid (0.25 mmol/L) and APS (200 mg/L) and AMPK inhibitor compound C (10 μmol/L) treated for 24 h]. The cell viability was detected by MTT assay. Nitric oxide (NO) content in the medium was determined by nitrate reductase assay. The protein levels of total adenosine monophosphate-activated protein kinase (AMPK), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), endothelial nitric oxide synthase (eNOS) and phosphorylated endothelial nitric oxide synthase (p-eNOS) were measured by Western blot. RESULTS: No significant difference of all indexes between APS group and control group was observed. The cell viability in free fatty acid group decreased significantly compared with control group. The cell viability in free fatty acid plus APS group was significantly improved as compared with free fatty acid group. The cell viability in compound C group was almost the same as that in free fatty acid group. The No content and protein levels of p-AMPK and p-eNOS in free fatty acid group decreased obviously as compared with control group, while the NO content and protein levels of p-AMPK and p-eNOS in free fatty acid plus APS group increased obviously compared with free fatty acid group. No significant difference of the p-AMPK and p-eNOS protein levels between free fatty acid plus APS group and free fatty acid group was observed. No significant difference of the AMPK and eNOS protein levels in all groups was found. CONCLUSION: APS attenuates the free fatty acid-induced injury, and its mechanism is related to the AMPK-eNOS signal pathway.     

Insulin and gliclazide therapies improve liver lipid deposition in type 2 diabetic rats

AIM：To investigate the effect of insulin and gliclazide therapies on the liver fat accumulation in type 2 diabetic rats. METHODS：A high-fat diet plus low-dose streptozotocin was implemented to establish a type 2 diabetic rat model, and the rats were randomly divided into diabetes mellitus (DM) group, diabetic rats treated with insulin (INS) group, diabetic rats treated with gliclazide per os （PO） group, and normal control (NC) group. The diabetic rats in INS group and PO group were given insulin and gliclazide for 3 weeks, respectively. The changes of the liver fatty were evaluated with oil red O staining. Fasting plasma adiponectin concentration was measured by ELISA. The expression of adiponectin receptor 1 (AdipoR1) was detected by real-time PCR. The protein levels of AMP-activated protein kinase (AMPK), phosphorylated AMPK on threonine 172 (Thr172p-AMPK), sterol regulatory element-binding protein 1c (SREBP-1c), phosphorylated SREBP-1c on serine 372 (Ser372p-SREBP-1c), acetyl-CoA carboxylase (ACC), phosphorylated ACC on serine79 (Ser79p-ACC) and immunoglobulin-binding protein (BiP) in the liver homogenate were determined by Western blotting. RESULTS：Compared with the normal rats, in DM group, the presence of cytoplasmic lipid deposits was confirmed by oil red O staining. In INS group, these changes were significantly lower than those in DM group. Similar results were obtained in PO group. Insulin therapy significantly increased the plasma concentration of diponectin and liver tissue levels of AdipoR1 compared with DM group. At the same time, these 2 indicators returned to normal levels after gliclazide therapy. Thr172p-AMPK/AMPK, Ser372p-SREBP-1c/SREBP-1c and Ser79p-ACC/ACC expression ratios were significantly reduced in DM group compared with control values. The expression of BiP was increased on the contrary. After insulin therapy, Thr172p-AMPK/AMPK and Ser372p-SREBP-1c/SREBP-1c were significantly increased, and Ser79p-ACC/ACC and BiP returned to the normal levels. After gliclazide treatment, Thr172p-AMPK/AMPK and Ser372p-SREBP-1c/SREBP-1c returned to the normal levels, the expression ratio of Ser79p-ACC/ACC had no significant improvement compared with DM group, and the expression of BiP significantly declined. CONCLUSION：Both the insulin and gliclazide therapies reduce the lipid deposition in the liver of rats with type 2 diabetes by activating AMPK, but the extent and mechanism are not the same. In insulin therapy, AMPK restrains the expression of SREBP-1c directly, increases the phosphorylation of SREBP-1c, and affects SREBP-1c by inhibiting the endoplasmic reticulum stress. Gliclazide treatment, which has no effect on the lipid oxidation, reduces lipid deposition in the liver only through the phosphorylation of SREBP-1c and the suppression of the endoplasmic reticulum stress.     

Astragalus polysaccharides attenuate endothelial cell injury caused by homocysteine via AMPK pathway

AIM:To investigate the protective effect of Astragalus polysaccharides (APS) on human umbilical vein endothelial cells (HUVECs) injured by homocysteine (Hcy) and its mechanism. METHODS:HUVECs cultured in vitro were divided into 4 groups:control group, APS group[APS (200 mg/L) treatment for 24 h], Hcy group[Hcy (1 mmol/L) treatment for 24 h], and Hcy+APS group[Hcy (1 mmol/L) and APS (200 mg/L) co-treatment for 24 h]. The cell viability were measured by MTT assay. The activity of lactate dehydrogenase (LDH) and superoxidase dismutase (SOD), and the content of malondialdehyde (MDA) in HUVECs were detected by the commercial kits. The mRNA expression of SOD1, catalase (CAT) and NADPH oxidase 2 (NOX2) was detected by RT-qPCR. The protein levels of AMP-activated protein kinase α (AMPKα) and phosphorylated AMPKα (p-AMPKα) were determined by Western blot. RESULTS:Compared with control group, the cell viability, the activity of SOD, and the mRNA expression of SOD1 and CAT in the HUVECs were decreased, but the activity of LDH, the content of MDA, and the mRNA expression of NOX2 were increased significantly in Hcy group(P<0.05). APS inhibited the decrease in cell viability, and the increases in LDH acti-vity and MDA content induced by Hcy. APS increased SOD activity and the mRNA expression of SOD1 and CAT, but reduced the mRNA expression of NOX2. Compound C, an AMPK inhibitor, reduced the protective effect of APS on HUVECs injured by Hcy. CONCLUSION:APS protects HUVECs from Hcy-induced injury via AMPK signaling pathway to regulate intracellular oxidative stress.     

Effect of sitagliptin on autopaghy in mesangial cells induced by AGEs

AIM: To investigate the effect of sitagliptin on the autopaghy and the expression of extracellular matrix in mesangial cells induced by advanced glycation end products (AGEs). METHODS: The cells were divided into 5 groups:control group, AGE group, and sitagliptin (5, 10 and 20 μmol/L) groups. After 48 h, the cell viability was measured by MTT assay, and the content of collagen (Col) Ⅳ in the supernatant of the cell culture was detected by ELISA. The protein levels of beclin-1, adenosine monophosphate-activated protein kinase (AMPK), p-AMPK, p70S6K and p-p70S6K were determined by Western blot. RESULTS: Compared with control group, the viability and the expression of Col IV induced by AGEs in the cultured mesangial cells were significantly increased (P<0.01). Sitagliptin decreased the viability and the expression of Col IV induced by AGEs in the mesangial cells in a dose-dependent manner. AGEs significantly inhibited the protein levels of beclin-1 and p-AMPK, but significantly increased the protein level of p-p70S6K. Compared with AGE group, sitagliptin significantly reversed the above results in a dose-dependent manner. CONCLUSION: Autophagy may mediate the protective effect of sitagliptin on mesangial cells induced by AGEs.     

Astragalus polysaccharides improve chronic heart failure by promoting myocardial FFA metabolism via AMPK pathway

AIM: To investigate the effect of Astragalus polysaccharides (APS) on chronic heart failure and its mechanism. METHODS: Male SD rats (n=32) were randomly divided into control group, sham group, model group and APS group (8 rats in each group). The left coronary artery ligation in the rats was conducted to establish myocardial infarction heart failure model. After modeling, the rats in APS group were given APS (3 g·kg^-1·d^-1) by intragastric administration for 6 weeks. Left ventricular diastolic diameter (LVD), left ventricular systolic diameter (LVS), left ventricular ejection fraction (LVEF) and fractional shortening (FS) were detected by echocardiography. HE staining was used to observe the pathological changes. The concentrations of free fatty acid (FFA) in the serum and myocardium were observed by the method of acetyl coenzyme A synthetase and acetyl coenzyme A oxidase (ACS-ACOD). The protein levels of total AMP-activated protein kinase (AMPK), phosphorylated AMP-activated protein kinase (p-AMPK), fatty acid translocase (FAT/CD36) and carnitine palmitoyltransferase I (CPT-1) were measured by Western blotting. RESULTS: No significant difference in each index between sham group and control group was observed. Compared with control group, LVEF and FS in model group was significantly decreased, while LVD and LVS was significantly increased (P<0.05). The LVEF and FS in APS group were significantly improved compared with model group (P<0.05), and there was no significant difference between APS group and control group. LVD and LVS in APS group were obviously improved compared with mo-del group (P<0.05), and the difference was significant compared with control group (P<0.05). Compared with control group, focal myocardial necrosis increased, and residual myocardial cells reduced in model group, while those was much better in APS group as compared with model group (P<0.05). The FFA concentrations in the serum and myocardium in model group increased significantly compared with control group (P<0.05), while those decreased significantly in APS group as compared with model group (P<0.05). The protein levels of p-AMPK, CPT-1, and cell membrane FAT/CD36 in model group decreased significantly compared with control group (P<0.05), and those in APS group increased obviously compared with control group (P<0.05). CONCLUSION: APS improves chronic heart failure by activating the AMPK pathway and promoting myocardial ingestion and utiliation of FFA.     

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.     

Adiponectin inhibits decrease in autophagy of rat H9c2 cardiomyocytes induced by β1-adrenergic receptor autoantibodies

AIMTo investigate whether adiponectin inhibits the decrease in autophagy of rat H9c2 cardiomy?ocytes induced by β₁-adrenergic receptor (β₁-AR) autoantibodies (β₁-AA), and to explore its mechanism. METH?ODS: SD rats were actively immunized with β₁-AR extracellular second loop (β₁-AR-ECII) antigen peptide. Affinity chromatography was used to purify β₁-AA in serum of the SD rats. The viability of H9c2 cells was measured by CCK-8 assay. The mRNA levels of LC3B and beclin-1 in the H9c2 cells were detected by real-time PCR. The protein levels of LC3-II, P62, AMP-activated protein kinase (AMPK) and phosphorylated AMPK (p-AMPK) were determined by Western blot. RESULTSPretreatment with adiponectin at 10 μg/L for 1 h reversed the decreased viability of H9c2 cells induced by β₁-AA. Compared with control group, β₁-AA decreased the mRNA expression of LC3B and beclin-1, decreased the protein level of LC3-II, and increased the expression of P62 protein in the H9c2 cells, suggesting that β₁-AA decreased the autophagic flux in cardiomyocytes. Adiponectin obviously reversed β₁-AA-induced decline in autophagic flux, and up-regulated the phosphorylation level of AMPK decreased by β₁-AA. Treatment with AMPK inhibitor Compound C for 30 min, we observed that the mRNA expression of LC3B and beclin-1 and the protein level of LC3-II in the H9c2 cells decreased by β₁-AA were not effectively reversed by adiponectin, but the increase in P62 protein expression was still effectively reversed, indicating that adiponectin increased autophagosome production dependent on the AMPK pathway, but increased autophagosome clearance independent on the AMPK pathway. CONCLUSION Adiponectin inhibits the decreased autophagy of H9c2 cardiomyocytes induced by β₁-AA.     

Effect of metformin combined with paclitaxel on apoptosis and AMPK signaling in human breast cancer cells

AIM:To investigate the effect of metformin combined with paclitaxel on the viability and apoptosis of breast cancer cell line MCF-7 and its possible mechanism. METHODS:MCF-7 cells were treated with metformin at different concentrations (2, 5, 10, 20, 40 and 80 mmol/L) and in vitro cultured. The viability of MCF-7 cells was measured by MTT assay. Metformin at 2 mmol/L or paclitaxel at 2.4 mg/L alone or in combination was used to treat the cells, and compound C, an inhibitor of adenosine monophosphate-activated protein kinase (AMPK) signaling transduction pathway, was also used. The cells were divided into control group, metformin group, paclitaxel group, combination group, and combination +compound C group. The apoptosis of the cells was analyzed by flow cytometry. The expression of Bax, Bcl-2 and caspase-3 at mRNA and protein levels was determined by RT-qPCR and Western blot. The protein levels of AMPK and P21 were examined by Western blot. RESULTS:Metformin at different concentrations (2, 5, 10, 20, 40 and 80 mmol/L) significantly inhibited the cell viability in a concentration-dependent manner (P<0.05). Compared with control group, treatment with metformin at 2 mmol/L or paclitaxel at 2.4 mg/L alone or in combination significantly inhibited the cell viability, induced apoptosis (P<0.05), decreased the level of Bcl-2 (P<0.05), increased the levels of Bax and caspase-3 (P<0.05), and promoted the protein expression of AMPK and P21 (P<0.05). The effects of metformin and paclitaxel in combination were better than those of single drug treatment, while AMPK inhibitor weaken these effects. CONCLUSION:Metformin combined with paclitaxel inhibits the viability and induces the apoptosis of breast cancer MCF-7 cells by activating AMPK signaling pathway and regulating apoptosis signaling pathway.     

Losartan inhibits LPS-induced GFAP expression via AMPK activation in mouse hippocampus

AIM: To investigate the effects of losartan on lipopolysaccharide (LPS)-induced glial fibrillary acidic protein (GFAP) expression, and to determine whether adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation is involved in the mechanism.METHODS: Adult male KM mice were divided into control group, LPS model group, losartan treatment group, and losartan and Compound C co-treatment group. To establish a model of central nervous system inflammation, the mice received daily intracerebroventricular injection of LPS (24 μg/d) for 2 d. Daily losartan administration (0.5, 1 or 5 mg·kg^-1·d^-1, ip) initiated at 14 d prior to LPS injection. Compound C (10 mg/kg, ip), a selective AMPK inhibitor, started to be injected daily at 2 d prior to LPS injection. The hippocampal tissues in each group were isolated at 3 d after the last LPS injection, and then the protein levels of GFAP, AMPK, p-AMPK, mammalian target of rapamycin (mTOR) and p-mTOR were determined by Western blot.RESULTS: Twice LPS injections significantly increased the expression of GFAP in the hippocampus (P<0.01). Losartan inhibited LPS-induced GFAP expression in a concentration-dependent way, and losartan at 5 mg·kg^-1·d^-1 significantly inhibited GFAP expression and AMPK activation (P<0.05), but it had no obvious effect on mTOR activation. Furthermore, Compound C significantly reversed the effect of losartan treatment on LPS-induced GFAP expression and AMPK phosphorylation (P<0.05).CONCLUSION: Losartan inhibits LPS-induced GFAP expression in the mouse hippocampus, and AMPK activation but not mTOR, is involved in the mechanism.     

Protective effect of hydrogen sulfide on PC12 cells injured by ATP

AIM: To prove the purinergic signaling mechanism of the neuroprotective action of hydrogen sulfide by observing the effects of sodium hydrosulfide (NaHS), a donor of hydrogen sulfide, on the cell viability, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the change of membrane permeability in the PC12 cells injured by adenosine triphosphate (ATP). METHODS: PC12 cells in logarithmic growth phase were randomly divided into 4 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treated with ATP after cultured for 24 h. In NaHS+ATP group, the cells were incubated with NaHS for 30 min before treated with ATP, and NaHS always existed in the reaction system. In KN-62+ATP group, the cells were pretreated with KN-62 for 30 min, and the other treatments were as the same as those in NaHS+ATP group. The cell viability was assessed by MTT assay. The [Ca²⁺]_i was detected by Fura-2/AM staining. The membrane permeability was observed by staining with fluorescent dye YO-PRO-1.RESULTS: ATP at concentration of 0.3 mmol/L showed no injury effect on the cells. However, the cell viability was dropped gradually in a dose-dependent manner as the ATP at doses of 1, 3, 5 and 10 mmol/L. The decline of cell viability by ATP was obviously reversed by 200 μmol/L of NaHS in the PC12 cells (P<0.05), but exasperated by 800 μmol/L of NaHS (P<0.05). At the same time, ATP evoked the increase in [Ca²⁺]_i in a dose-dependent manner, which was inhibited by NaHS (P<0.05). Furthermore, the YO-PRO-1 uptake induced by ATP in a dose-dependent and time-dependent manner was also reduced by NaHS (P<0.05). CONCLUSION: Hydrogen sulfide has protective effect on the PC12 cells injured by ATP. The mechanism may be related to the reverse of the increased [Ca²⁺]_i and YO-PRO-1 uptake.     

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 chronic intermittent hypoxia on AMPK pathway in young rats

AIM: To investigate the effect of chronic intermittent hypoxia on AMP-activated protein kinase(AMPK) pathway in the brain of young rats. METHODS: Part one: SD mice(3~4 weeks old) were randomly divided into 4 groups(n=8): simulated air control group for 2 weeks(2AC), chronic intermittent hypoxia group for 2 weeks(2IH), simulated air control group for 4 weeks(4AC) and chronic intermittent hypoxia group for 4 weeks(4IH). Part two: SD mice(3~4 weeks old) were randomly divided into 2 groups(n=8): chronic intermittent hypoxia group for 4 weeks(4IH) and chronic intermittent hypoxia group treated with AMPK inhibitor for 4 weeks(4IHI). After modeling, the eight-arm maze test was performed. TUNEL method was used to detect the neuronal apoptosis in the hippocampal and prefrontal cortical tissues. The mRNA expression of adenosine A2a receptor was examined by RT-qPCR, and the protein levels of phosphorylated AMPK(p-AMPK) and mammalian target of rapamycin(p-mTOR) were determined by Western blot. RESULTS: Compared with control group, the numbers of reference memory error(RME), working memory error(WME) and total error(TE) in 2IH group and 4IH group significantly increased(P<0.01). Compared with 2IH group, the numbers of errors in 4IH group also increased significantly(P<0.01). Compared with 4IH group, the values in 4IHI group significantly decreased. Compared with control group, the neuronal apoptosis of hippocampus and prefrontal cortex in 2IH group and 4IH group increased, and that in 4IH group was more evident(P<0.05). In 4IHI group, the neuronal apoptosis decreased. The mRNA expression of adenosine A2a receptor in the hippocampal and cortical tissues in 2IH group and 4IH group was higher than that in control group. The protein level of p-AMPK was higher, and p-mTOR was lower in 2IH group and 4IH group, and those in 4IH group were more evident(P<0.05). Compared with 4IH group, the protein level of p-AMPK was lower, and p-mTOR was higher in 4IHI group. CONCLUSION: Chronic intermittent hypoxia induces neuronal apoptosis, resulting in impairment of learning and memory in a time-dependent manner by upregulating adenosine A2a receptor, activating AMPK activity, and inhibiting mTOR phosphorylation in rats.     

Role of retinoic acid X receptor in regulation of autophagy in rat alveolar type Ⅱ epithelial cells induced by hypoxia/reoxygenation

AIM To investigate the regulatory effect of retinoic acid X receptor （RXR） on autophagy induced by hypoxia/reoxygenation （H/R） in rat alveolar type Ⅱ epithelial cells （AEC Ⅱ） and its molecular mechanism. METHODS AEC Ⅱ were cultured in normoxia. The cells growing to logarithmic growth phase were randomly divided into 5 groups： （1） control （Con） group： cells were cultured for 30 h under normal operation; （2） H/R group： cells were cultured in hypoxia condition for 6 h and then in reoxygenation condition for 24 h; （3） DMSO group： cells were pretreated 1.5 h with medium containing less than 0.1% DMSO before modeling, and the rest were treated the same as the H/R group; （4） 9-cis-retinoic acid （9-RA） group： cells were pretreated for 1 h with 9-RA （100 nmol/L） before hypoxia; （5） HX531 group： cells were treated with 9-RA （100 nmol/L） for 0.5 h, then treatment with HX531 （2.5 μmol/L） for 1 h. CCK-8 assay was used to detect the cell viability. Immunofluorescence staining was used to observe the expression of RXRα. Transmission electron microscope was used to observe the changes of intracellular ultrastructure, and the mRNA expression of adenosine AMP-activated protein kinase （AMPK）, beclin 1, LC3, mammalian target of rapamycin （mTOR） and P62 was detected by RT-PCR. Western blot was used to detected the protein levels of p-AMPK, beclin 1, LC3-Ⅱ, p-mTOR and P62. RESULTS Compared with Con group, the cell viability in H/R, DMSO, 9-RA and HX531 groups were significantly decreased. The mRNA expression of AMPK, beclin 1 and LC3 was significantly increased, and the protein levels of p-AMPK, beclin 1 and LC3-Ⅱ were also increased. The mRNA expression of mTOR and P62 was decreased, and the protein levels of p-mTOR and P62 were also decreased （P<0.05）. The cell injury in 9-RA group was alleviated and autophagy level was significantly lower than that in H/R, DMSO and HX531 groups （P<0.05）, and no significant difference among H/R, DMSO and HX531 groups was observed （P>0.05）. CONCLUSION H/R induces autophagy of AEC Ⅱ. Activating RXR reduce the damage of AEC Ⅱ cells induced by H/R, and its mechanism may be related to the inhibition of autophagy.     

Proliferative effect of PDGF and anti-proliferative activity of AMPK on vascular smooth muscle cells

AIM: To investigate the proliferative effect of platelet-derived growth factor (PDGF) and anti-proliferative activity of AMP-activated protein kinase (AMPK) on vascular smooth muscle cells (VSMCs). METHODS: The proliferation of VSMCs cultured with PDGF and activation of AMPK were observed. VSMCs were divided in 4 groups: control group; PDGF group; 5-aminoimidazole-4 -carboxamide-1-β-D-riboside (AICAR) group and AICAR+PDGF group. The time course of AMPK activation was determined. The protein level of mTOR was also measured. RESULTS: Compared with control group, the proliferative rate in PDGF group was significantly increased. The growth of VSMCs was inhibited in a time-dependent manner and the activity of p-mTOR was significantly decreased in AICAR group. Compared with control group, the expression of p-AMPK in PDGF group was significantly decreased, and that in AICAR group and AICAR+PDGF group was significantly increased. The expression of p-AMPK in AICAR+PDGF group was higher than that in PDGF group. The activity of p-mTOR in PDGF group was significantly higher than that in control group, while that of AICAR group and AICAR+PDGF group was significantly decreased. The expression of p-mTOR in AICAR+PDGF group was lower than that in PDGF group. CONCLUSION: Stimulation of VSMCs with PDGF promotes the cell proliferation, which can be inhibited by AICAR. The proliferation of VSMCs activated by AMPK is probably correlated with the down-regulation of mTOR expression.     

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.     

miR-153 promotes LPS-induced myocardial H9C2 cell injury by targeting SORBS2

AIM: To study the effects of microRNA-153 (miR-153) on inflammatory factors, cell viability and apoptosis of embryonic rat H9C2 cardiomyocytes induced by lipopolysaccharide (LPS), and to explore its mechanism. METHODS: The injury model of H9C2 cells was established by LPS stimulation. The H9C2 cells were divided into anti-miR-Con group (transfected with anti-miR-Con), anti-miR-153 group (transfected with anti-miR-153), pcDNA group (transfected with pcDNA), pcDNA-SORBS2 group (transfected with pcDNA-SORBS2), anti-miR-153+si-Con group (co-transfected with anti-miR-153 and si-Con) and anti-miR-153+si-SORBS2 group (co-transfected with anti-miR-153 and si-SORBS2), and treated with LPS after transfection. The expression of miR-153 and SORBS2 mRNA in the cells was detected by RT-qPCR. The viability of H9C2 cells was measured by MTT assay. The protein expression of SORBS2 in the H9C2 cells was determined by Western blot. The contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected by ELISA. The apoptosis of the H9C2 cells was analyzed by flow cytometry. The targeting relationship between miR-153 and SORBS2 was verified by dual-luciferase reporter assay. RESULTS: The LPS-induced H9C2 cell injury model was successfully constructed. Compared with PBS group, the expression of miR-153 was significantly increased and the expression of SORBS2 was significantly decreased in the H9C2 cells treated with LPS. The inhibition of miR-153 and over-expression of SORBS2 decreased the contents of TNF-α and IL-6 and the level of apoptosis, but increased the cell viability. miR-153 inhibited the luciferase activity of the H9C2 cells containing wild-type SORBS2. Inhibition of SORBS2 reversibly inhi-bited the anti-inflammatory effects of miR-153 on LPS-induced H9C2 cells and increased the viability of the cells. CONCLUSION: miR-153 promotes the secretion of inflammatory factors, induces apoptosis, and inhibits the viability of H9C2 cells induced by LPS, thus enhancing the damage. Its mechanism may be related to targeting SORBS2, which will provide new targets for the treatment of myocardial injury.     

Liraglutide ameliorates liver injury of type 2 diabetic mice via micro-RNA-33-AMPK pathway

AIM: To observe the effects of liraglutide on the level of microRNA-33 (miR-33) and the expression of AMP-activated protein kinase (AMPK) and apoptosis-related proteins in mice with type 2 diabetes mellitus (T2DM), and to explore its possible mechanism. METHODS: High-fat diet and intraperitoneal injection of streptozocin were used to establish the type 2 diabetic model in C57BL/6 mice. The mice were randomly divided into 4 groups (n=15):in control group, the normal mice were subcutaneously injected with equivalent volume of saline; in model group, the T2DM mice were subcutaneously injected with equivalent volume of saline; in low-and high-dose liraglutide treatment groups, the T2DM mice were subcutaneously injected with 100 and 200 μg·kg^-1·d^-1, respectively. After 4 weeks of administration, the levels of FBG, TG, TC, HDL-C, LDL-C, ALT and AST were determined. HE staining was used to observe the pathological changes of the liver tissues. The protein level of cleaved caspase-3 in the liver tissue was detected by the technique of immunofluorescence. The protein levels of p-AMPK/AMPK and apoptosis-related proteins were detected by Western blot. The expression of miR-33 in the liver tissues was detected by real-time PCR. RESULTS: Compared with model group, the contents of FBG, TG, TC, LDL-C, ALT and AST were decreased significantly, while the content of HDL-C was increased significantly in low-dose liraglutide group and high-dose liraglutide group (P<0.05). The protein levels of phosphorylated AMPK and Bcl-2 were up-regulated significantly, and the expression of cleaved caspase-3 was down-regulated significantly (P<0.05). The level of miR-33 was decreased significantly (P<0.01). CONCLUSION: Liraglutide alleviates liver injury in type 2 diabetic mice, and the mechanism may be associated with reducing the level of miR-33 and increasing the phosphorylation of AMPK in the liver tissues, thereby inhibiting hepatocyte apoptosis.