OBJECTIVE: To investigate the effect of palmitic acid (PA) on autophagy in neonatal rat cardiomyocytes (NRCMs) and explore the underlying mechanism. METHODS: NRCMs were isolated and cultured for 24 h before exposure to 10% BSA and 0.1, 0.3, 0.5, or 0.7 mmol/L PA for 24 h. After the treatments, the expressions of Parkin, PINK1, p62, LC3Ⅱ/ LC3Ⅰ, cGAS, STING and p-IRF3/IRF3 were detected using Western blotting and the cell viability was assessed with CCK8 assay, based on which 0.7 mmol/L was selected as the optimal concentration in subsequent experiments. The effects of cGAS knockdown mediated by cGAS siRNA in the presence of PA on autophagy-related proteins in the NRCMs were determined using Western blotting, and the expressions of P62 and LC3 in the treated cells were examined using immunofluorescence assay. RESULTS: PA at different concentrations significantly lowered the expressions of Parkin, PINK1, LC3 Ⅱ/LC3 Ⅰ and LC3 Ⅱ/LC3 Ⅰ+Ⅱ (P < 0.05), increased the expression of p62 (P < 0.05), and inhibited the viability of NRCMs (P < 0.05). Knockdown of cGAS obviously blocked the autophagy-suppressing effect of PA and improved the viability of NRCMs (P < 0.05). CONCLUSION PA inhibits autophagy by activating the cGAS-STING-IRF3 pathway to reduce the viability of NRCMs.
Animals ; Animals, Newborn ; Autophagy ; Myocytes, Cardiac ; Nucleotidyltransferases/pharmacology* ; Palmitic Acid/pharmacology* ; Rats

OBJECTIVETo study the effects of palmitic acid (PA) on the proliferation of peripheral blood-derived endothelial progenitor cells (EPCs) in vitro.

METHODSThe mononuclear cells (MNCs) were isolated from the peripheral blood by Ficoll density-gradient centrifugation. The isolated EPCs were characterized by Di-LDI uptake and FITC-lectin binding assay using laser confocal microscope, and further identified by detection of CD34, CD133 and VEGFR2 expression using flow cytometry. The cultured EPCs were incubated in the presence of PA at the concentrations of 0, 50, 100, 200, 400 and 800 micromol/L for different durations (0, 12, 24, 36, 48 and 60 h). The cell morphology was observed and cell proliferation determined with CCK-8 assay.

RESULTSIncubation with 400 and 800 micromol/L of PA significantly inhibited the proliferative ability of EPCs as compared with the control group (P < 0.05). PA at 400 micromol/L had the strongest effect on the cell proliferation, and this effect was intensified with the passage of time, reaching the peak at 48 h with the growth inhibition rate of 58.59% (P < 0.05).

CONCLUSIONHigh-concentration PA can significantly inhibit the proliferation of EPCs in vitro.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endothelial Cells ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Palmitic Acid ; pharmacology ; Stem Cells ; cytology

BACKGROUNDPodocyte apoptosis is recently indicated as an early phenomenon of diabetic nephropathy. Pancreatic β-cells exposed to saturated free fatty acid palmitate undergo irreversible endoplasmic reticulum (ER) stress and consequent apoptosis, contributing to the onset of diabetes. We hypothesized that palmitate could induce podocyte apoptosis via ER stress, which initiates or aggravates proteinuria in diabetic nephropathy.

METHODSPodocyte apoptosis was detected by 4',6-diamidio-2-phenylindole (DAPI) stained apoptotic cell count and Annexin V-PI stain. The expressions of ER molecule chaperone glucose-regulated protein 78 (GRP78), indicators of ER-associated apoptosis C/EBP homologous protein (CHOP), and Bcl-2 were assayed by Western blotting and real-time PCR. GRP78 and synaptopodin were co-localized by immunofluorescence stain.

RESULTSPalmitate significantly increased the percentage of cultured apoptotic murine podocytes time-dependently when loading 0.75 mmol/L (10 hours, 13 hours, and 15 hours compared with 0 hour, P < 0.001) and dose-dependently when loading palmitate ranging from 0.25 to 1.00 mmol/L for 15 hours (compared to control, P < 0.001). Palmitate time-dependently and dose-dependently increased the protein expression of GRP78 and CHOP, and decreased that of Bcl-2. Palmitate loading ranging from 0.5 to 1.0 mmol/L for 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to control (P < 0.001), with the maximum concentration being 0.75 mmol/L. Palmitate 0.5 mmol/L loading for 3 hours, 8 hours, and 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to 0 hour (P < 0.001), with the maximum effect at 3 hours. Confocal microscopy demonstrated that GRP78 expression was significantly increased when exposed to 0.5 mmol/L of palmitate for 8 hours compared to control.

CONCLUSIONPalmitate could induce podocyte apoptosis via ER stress, suggesting podocyte apoptosis and consequent proteinuria caused by lipotoxic free fatty acid could be ameliorated by relief of ER stress.

Apoptosis ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; physiology ; Heat-Shock Proteins ; analysis ; physiology ; Humans ; Insulin Resistance ; Palmitic Acid ; pharmacology ; Podocytes ; drug effects ; pathology

The purpose of the study is to investigate the effect of 8-hydroxy-dihydroberberine on insulin resistance induced by high free fatty acid (FFA) and high glucose in 3T3-L1 adipocytes and its possible molecular mechanism. Palmic acid or glucose in combination with insulin was used to induce insulin resistance in 3T3-L1 adipocytes. 8-Hydroxy-dihydroberberine and berberine were added to the cultured medium separately, which were considered as treated group and positive control group. The rate of glucose uptake was determined by 2-deoxy-[3H]-D-glucose method. The amount of glucose consumption in the medium was measured by glucose oxidase method. Cell growth and proliferation of 3T3-L1 adipocytes were detected with Cell Counting Kit-8 (CCK-8) assay. After incubated with palmic acid for 24 hours or glucose with insulin for 18 hours, the rate of glucose transport in 3T3-L1 adipocytes was inhibited by 67% and 58%, respectively. The amount of glucose consumption in 3T3-L1 adipose cells was decreased by 41% after cells were incubated with palmic acid for 24 h. However, the above changes were reversed by pretreatment with 8-hydroxy-dihydroberberine for 24 and 48 h. Significant difference existed between groups. Insulin resistance in 3T3-L1 adipocytes, which is induced by high FFA and high glucose, could be ameliorated by 8-hydroxy-dihydroberberine.
3T3-L1 Cells ; Adipocytes ; cytology ; drug effects ; metabolism ; Animals ; Berberine ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; Cell Differentiation ; drug effects ; Glucose ; metabolism ; pharmacology ; Hypoglycemic Agents ; chemical synthesis ; chemistry ; pharmacology ; Insulin ; pharmacology ; Insulin Resistance ; Mice ; Molecular Structure ; Palmitic Acid ; pharmacology

OBJECTIVE: To investigate the effect of biflavonoid 4'-O-methylochnaflavone (MF) on palmitic acid-induced endothelial dysfunction in rat cavernous endothelial cells (RCECs). METHODS: The isolated RCECs were commercially available and randomly divided into four groups: normal+BSA group (NC group), palmitic acid (PA) group, MF group, and icariside Ⅱ (ICA Ⅱ) group. The protein expression levels of protein kinase B (PKB/AKT) and endothelial nitric oxide synthase (eNOS) in each group were evaluated via Western blotting. The differences in the intracellular nitric oxide of RCECs treated by MF or ICA Ⅱ were detected by DAF-FM DA that served as a nitric oxide fluorescent probe. Effects of MF and ICA Ⅱ on cell proliferation of PA-stimulated RCECs were determined via CCK-8 assay. RESULTS: The content of nitric oxide in RCECs was significantly increased after the treatment of MF and ICA Ⅱ in comparison with the NC group (P < 0.05). Moreover, compared with ICA Ⅱ group, MF demonstrated a more obvious effect in promoting nitric oxide production (P < 0.05). Compared with the NC group, the expression levels of eNOS and AKT in the PA group were significantly decreased, indicating that a model for simulating the high-fat environment in vitro was successfully constructed (P < 0.05). Meanwhile, the intervention of MF and ICA Ⅱ could effectively increase the expression of eNOS and AKT, suggesting that MF and ICA Ⅱ could promote the recovery of endothelial dysfunction caused by high levels of free fatty acids (P < 0.05). The results of CCK-8 assays showed that PA could significantly reduce the proli-feration ability of RCECs (P < 0.05). Furthermore, the decreased cell viability induced by PA was significantly elevated by treatment with ICA Ⅱ and MF (P < 0.05). CONCLUSION In RCECs, MF and ICA Ⅱ could effectively increase the content of nitric oxide. The down-regulation of the expression of proteins associated with the AKT/eNOS pathway after PA treatment revealed that this pathway was involved in the development of endothelial dysfunction, which could be effectively reversed by MF and ICA Ⅱ. In addition, the cell proliferation ability was significantly decreased following PA treatment, but MF and ICA Ⅱ could restore the above changes. Overall, biflavonoid MF has an obvious repairing effect on PA-stimulated endothelial dysfunction.
Animals ; Biflavonoids/pharmacology* ; Cells, Cultured ; Endothelial Cells/metabolism* ; Nitric Oxide/pharmacology* ; Nitric Oxide Synthase Type III/pharmacology* ; Palmitic Acid/pharmacology* ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats ; Signal Transduction ; Sincalide/pharmacology*

OBJECTIVE: To investigate effects of Jiangtang Sanhuang tablet (JTSHT) for regulating blood glucose and alleviating islet cell damage in db/db mice and its protective effects against endoplasmic reticulum stress (ERS) and autophagy induced by glycolipid toxicity. METHODS: Forty db/db mice were randomized into 4 groups for daily intragastric administration of saline, JTSHT of 2.64 and 1.32 g/kg, and metformin at 0.225g/kg for 8 weeks, using 10 C57BL/6J mice as the normal control. After the treatments, the metabolic indexes of the mice were measured, and morphological changes of the islet cells were observed. A mouse islet cell line (MIN6) was exposed to high glucose (22 mmol/L glucose) and 0.1 mmol/L palmitic acid, followed by treatment with the sera from JTSHT- or saline- treated SD rats, alone or in combination with SP600125, and the changes in cell apoptosis, ERS and autophagy were evaluated using flow cytometry, RT-qPCR and Western blotting. RESULTS: In db/db mice, treatment with JTSHT significantly improved glucose and lipid metabolism (P < 0.05) and suppressed progressive weight gain (P < 0.05) without significant effect on drinking water volume (P > 0.05). JTSHT was also found to promote repair of islet cell injuries. In the cell experiments, high glucose exposure significantly increased apoptosis rate of MIN6 cells (P < 0.05), which was obviously lowered by treatment with JTSHT-treated rat serum (P < 0.05). Western blotting showed that JTSHT significantly reduced the level of ERS and autophagy caused by glycolipid toxicity in MIN6 cells (P < 0.05). Interference with ERS using SP600125 significantly attenuated the protective effect of JTSHT against MIN6 cell injury, apoptosis and autophagy induced by glycolipid toxicity (P < 0.05). CONCLUSION JTSHT has protective effects against glycolipid toxicity in MIN6 cells possibly by inhibiting ERS and autophagy.
Animals ; Anthracenes ; Apoptosis ; Autophagy ; Blood Glucose ; Diabetes Mellitus ; Drinking Water ; Drugs, Chinese Herbal ; Endoplasmic Reticulum Stress ; Glucose/pharmacology* ; Glycolipids/pharmacology* ; Islets of Langerhans ; Metformin ; Mice ; Mice, Inbred C57BL ; Palmitic Acid/pharmacology* ; Tablets/pharmacology*

BACKGROUNDThe levels of long-term elevated serum or intracellular free fatty acid (FFA) induce insulin resistance associated with central obesity. The insulin-mimetic protein visfatin is preferentially produced by visceral adipose tissues and has been implicated in obesity and insulin resistance. To identify that FFA is capable of inducing insulin resistance and to clarify the role of FFA on visfatin, we examined the effect of monounsaturated FFA oleate (C18:1) and saturated FFA palmitate (C16:0) on glucose transport and visfatin gene expression in cultured 3T3-L1 adipocytes or preadipocytes.

METHODSFFA-free DMEM/F12, 0.125 mmol/L, 0.5 mmol/l and 1.0 mmol/L oleate or palmitate was added to cultured 3T3-L1 adipocytes or preadipocytes and incubated overnight. Glucose transport was assessed as (3)H-2-deoxy-glucose uptake. Total RNA was extracted and subjected to RT-PCR for the measurement of visfatin mRNA levels. Statistical comparisons between control group and other groups were performed with the two-tailed paired t test, and one-way ANOVA was used to compare the mean values among the groups.

RESULTSInsulin increased specific membrane glucose transport in 3T3-L1 preadipocytes. Upregulation was evident from 15 minutes to 1 hour exposure to insulin. However, after 6-hour exposure to insulin, there was a downregulation in the response to insulin. Dose response studies demonstrated that 2-deoxy glucose transport was increased by 336% at 50 nmol/L insulin (P < 0.01), and reached a maximal effect at 100 nmol/L insulin (P < 0.01). Oleate and palmitate treatment did not influence basal glucose transport (without insulin stimulation), whereas insulin-stimulated glucose transport was inhibited after overnight oleate and palmitate treatment in preadipocytes and adipocytes. In 3T3-L1 preadipocytes, insulin resistance could be achieved at 0.125 mmol/L oleate or palmitate (P < 0.05, respectively), and the inhibition was dose dependent. In adipocytes, the inhibition was noted at 0.5 mmol/L oleate or 1.0 mmol/L palmitate. Visfatin mRNA expression increased during differentiation more than 1.5-fold. Bovine serum albumin (BSA) did not influence visfatin mRNA expression compared with the control group. Dose-response studies demonstrated that addition of 0.125 mmol/L oleate and palmitate to 3T3-L1 adipocytes decreased visfatin mRNA expression significantly (78%, 77%, respectively, relative to untreated control, P < 0.05), and further to 65% (relative to untreated control, P < 0.05) and 55% (relative to untreated control, P < 0.01) at 1.0 mmol/L FFA. Furthermore, the suppression on preadipocytes was similar to that of adipocytes, which reached a maximal reduction of 44% (oleate, P < 0.05) and 47% (palmitate, P < 0.05) at 1.0 mmol/L FFA.

CONCLUSIONSOleic acid and palmitic acid may induce insulin resistance in 3T3-L1 adipocytes and preadipocytes. Downregulation of visfatin mRNA may contribute to impair insulin sensitivity caused by oleate and palmitate.

3T3-L1 Cells ; Adipocytes ; cytology ; metabolism ; Animals ; Cell Differentiation ; Cytokines ; genetics ; Dose-Response Relationship, Drug ; Gene Expression Regulation ; drug effects ; Insulin Resistance ; Mice ; Nicotinamide Phosphoribosyltransferase ; Oleic Acid ; pharmacology ; Palmitic Acid ; pharmacology ; RNA, Messenger ; analysis ; Stem Cells ; metabolism

BACKGROUNDGlucagon-like peptide-1 (GLP-1) reduces fatty acid-induced beta-cell lipotoxicity in diabetes; however, the explicit mechanisms underlying this process are not fully understood. This study was designed to investigate the involvement of microRNA, which regulates gene expression by the sequence-specific inhibition of mRNA transcription in the GLP-1 mediation of beta-cell function.

METHODSThe cell viability and apoptosis were determined using an methyl thiazoleterazolium (MTT) assay and flow cytometry. The expression of genes involved in beta-cell function, including microRNA-34a and sirtuin 1, were investigated using real-time PCR. The underlying mechanisms of microRNA-34a were further explored using cell-transfection assays.

RESULTSA 24-hours incubation of INS-1 cells with palmitate significantly decreased cell viability, increased cell apoptosis and led to the activation of microRNA-34a and the suppression of sirtuin 1. A co-incubation with GLP-1 protected the cells against palmitate-induced toxicity in association with a reduction in palmitate-induced activation of microRNA-34a. Furthermore, palmitate-induced apoptosis was significantly increased in cells that were infected with microRNA-34a mimics and decreased in cells that were infected with microRNA-34a inhibitors.

CONCLUSIONMicroRNA-34a is involved in the mechanism of GLP-1 on the modulation of beta-cell growth and survival.

Animals ; Apoptosis ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Fatty Acids, Nonesterified ; toxicity ; Glucagon-Like Peptide 1 ; pharmacology ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; MicroRNAs ; genetics ; metabolism ; Palmitic Acid ; pharmacology ; Rats ; Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate the effects of JAZF1 overexpression on the pro-inflammatory cytokines in hepatic steatosis.

METHODSThe model of hepatic steatosis was established by incubating hepatocytes with palmitic acid (PA) at 0, 0.125, 0.25, 0.5 and 1 mM dose and for 0, 6, 12, 24 and 48 hours, after which recombinant adenovirus expressing JAZF1 (Ad-JAZF1) was introduced to up-regulate expression. Triglyceride level was measured by GOD. Cell viability was detected by CCK-8. The mRNA and protein expression of TNF-alpha, MCP-1, IL-8 and JAZF1 was examined by RT-PCR, ELISA, and western blotting.

RESULTSThe PA-treated hepatocytes showed dose-dependent significant increases in TNF-alpha, MCP-1 and IL-8 mRNA expression for doses up to 0.25 mM; there were no significant increases for the highest doses of 0.5 and 1 mM. The 0.25 mM PA-treated hepatocytes showed time-dependent significant increases in TNF-alpha, MCP-1 and IL-8 mRNA expressions (FTNF-alpha = 26.51, FMCP-1 = 57.20, FIL-8 = 353.85, P less than 0.01), with the maximum level reached at 12 h and followed by a gradual decrease with longer treatment times. JAZF1 mRNA and protein expression was markedly increased in hepatocytes infected with Ad-JAZF1 (P less than 0.01). However, the AP-treated hepatocytes with JAZF1 overexpression showed down-regulation of TNF-alpha, MCP-1 and IL-8 mRNA expression (decreased by 89.69%, 77.68%, and 83.21%, respectively) and secretion (37%, 37% and 41%, respectively, P less than 0.01).

CONCLUSIONStimulation of hepatocytes by the PA fatty acid in vitro promotes mRNA expression of TNF-alpha, MCP-1 and IL-8, but overexpression of JAZF1 inhibits the PA-induced expression and secretion of these factors.

Cell Survival ; Chemokine CCL2 ; metabolism ; Cytokines ; metabolism ; Fatty Liver ; Hepatocytes ; drug effects ; metabolism ; Humans ; Interleukin-8 ; metabolism ; Neoplasm Proteins ; metabolism ; Palmitic Acid ; pharmacology ; RNA, Messenger ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Up-Regulation

To reveal the roles of uncoupling proteins (UCPs) in disorder of mitochondrial oxidative phosphorylation induced by free fatty acid during hypoxic exposure, the effects of palmitic acid on activity of UCPs, proton leak and mitochondrial membrane potential in hypoxia-exposed rat brain mitochondria were observed in vitro. Adult Sprague-Dawley (SD) rats were set randomly into control, acute hypoxia and chronic hypoxia groups (n=8 in each group). The acute and chronic hypoxic rats were exposed to simulated 5000 m high altitude in a hypobaric chamber 23 h/d for 3 d and 30 d, respectively. The brain mitochondria were isolated by centrifugation. UCP content and activity were detected by [(3)H]-GTP binding method. The proton leak was measured by TPMP(+) electrode and oxygen electrode. The membrane potential of mitochondria was calculated by detecting the fluorescence from Rodamine 123. Hypoxic exposure resulted in an increase in UCP activity and content as well as proton leak, but a decrease in the membrane potential of rat brain mitochondria. Palmitic acid resulted in further increases in UCP activity and content as well as proton leak, and further decrease in membrane potential of brain mitochondria in vitro from hypoxia-exposed rats, but hypoxic exposure decreased the reactivity of cerebral mitochondria to palmitic acid, especially in the acute hypoxia group. There was a negative correlation between mitochondrial proton leak and K(d) value (representing derivative of UCP activity, P<0.01, r = -0.906), and a positive correlation between proton leak and B(max) (representing the maximal content of UCPs in mitochondrial inner membrane, P<0.01, r = 0.856). Cerebral mitochondrial membrane potential was negatively correlated with proton leak (P<0.01, r = -0.880). It is suggested that hypoxia-induced proton leak enhancement and membrane potential decrease are correlated with the increased activity of UCPs. Hypoxia can also decrease the sensitivity of cerebral mitochondria to palmitic acid, which may be a self-protective mechanism in high altitude environment.
Altitude ; Animals ; Brain ; metabolism ; Cell Hypoxia ; Ion Channels ; metabolism ; Membrane Potential, Mitochondrial ; Mitochondria ; metabolism ; Mitochondrial Proteins ; metabolism ; Oxidative Phosphorylation ; Oxygen ; metabolism ; Palmitic Acid ; pharmacology ; Protons ; Rats ; Rats, Sprague-Dawley ; Uncoupling Protein 1