OBJECTIVETo establish an in vitro cell model to investigate hepatic steatosis of non-alcoholic fatty liver disease.

METHODSHepG2 cells cultured in MEM containing 10 % fetal bovine serum were divided into control group and model group. At 7 0%-80 % confluency, HepG2 cells in model group were exposed to a long-chain mixture of free fatty acids (oleate and palmitate) for 24 h, cells in control group were subject to fresh medium. Lipid droplets were observed with oil red O stain and electron microscope, triglyceride and malonaldehyde were detected by respective assay kits.

RESULTA large number of lipid droplet were detected in model HepG2 cells; the level of triglyceride increased. However,malonaldehyde did not increase significantly compared with control group.

CONCLUSIONA large number of lipid droplet were detected in model HepG2 cells; the level of triglyceride increased. However, malonaldehyde did not increase significantly compared with control group.

Culture Media ; pharmacology ; Fatty Acids, Nonesterified ; pharmacology ; Fatty Liver ; Hep G2 Cells ; Humans ; Models, Biological ; Non-alcoholic Fatty Liver Disease ; Oleic Acid ; pharmacology ; Palmitates ; pharmacology

BACKGROUNDGlucolipotoxicity might play an important role in the β cell decompensation stage during the development of obesity-associated type 2 diabetes. Tissue inhibitor of metalloproteinase-1 (TIMP-1) inhibits matrix metalloproteinase (MMP) activity and regulates proliferation and apoptosis of a variety of cell types, including pancreatic β-cells. In the present study, we investigated whether TIMP-1 counteracts glucolipotoxicity in the pancreatic β-cell line INS-1.

METHODSINS-1 cells were incubated in normal or high glucose, with or without palmitate (0.4 mmol/L), in the presence of TIMP-1 or MMP inhibitor GM60001. In some experiments, cells were pretreated with phosphatidylinositol-3 (PI-3) kinase inhibitor, LY294002 or wortmannin. The amount of dead INS-1 cells was determined by HO342 and propidium iodide staining. Akt phosphorylation was evaluated by Western blotting analysis to investigate a possible mechanism of TIMP-1's action.

RESULTSTIMP-1 protected INS-1 cells from glucolipotoxicity independent of MMP inhibition. TIMP-1 stimulated Akt phosphorylation. Inhibition of the PI-3 kinase pathway abolished the survival effect of TIMP-1.

CONCLUSIONTIMP-1 may counteract glucolipotoxicity induced β-cell death via a PI-3 kinase pathway.

Animals ; Cell Line ; Glucose ; pharmacology ; Insulin-Secreting Cells ; drug effects ; metabolism ; Palmitates ; pharmacology ; Phosphatidylinositol 3-Kinases ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Signal Transduction ; Tissue Inhibitor of Metalloproteinase-1 ; pharmacology

Heterotrimeric guanine nucleotide binding regulatory proteins (G proteins) transduce extracellular signals into intracellular signals by coupling receptors and effectors. Because most of the G protein-coupled receptors are integral proteins, the G proteins need to have a membrane binding capacity to receive signals from the receptors. The alpha subunit of G protein binds tightly to the cytoplasmic face of the plasma membrane without any membrane spanning domain. Fatty acylation of G alpha with myristic acid or palmitic acid, in addition to the beta gamma subunits, plays an important role in anchoring the G alpha subunit. The reversible and dynamic palmitoylation of the alpha subunit of stimulatory G protein (Gs alpha) has been suggested as essential for its membrane attachment. However, in our previous experiments, Gs alpha deleted in the amino terminus containing palmitoylation site, retained its binding capacity when expressed in COS cells. Thus, to evaluate the role of palmitoylation in Gs alpha membrane binding, we constructed and expressed non-palmitoylated mutants of Gs alpha and analyzed their subcellular distributions in COS-1 cells. We found that non-palmitoylated mutants of Gs alpha, C3S- and G2A/C3S Gs alpha, retained their membrane binding capacities in COS-1 cells, demonstrating that palmitoylation is not essential for membrane binding of Gs alpha in COS-1 cells. We also found that the palmitoylation did not change significantly the distribution of Gs alpha in Triton X-114 partition. These results suggest that the palmitoylation of Gs alpha may produce different effects on membrane binding depending on cell types.
Animal ; Blotting, Western ; COS Cells ; Cell Membrane/metabolism ; Detergents/pharmacology ; G-Protein, Stimulatory Gs/metabolism* ; G-Protein, Stimulatory Gs/genetics ; Immunoblotting ; Isoproterenol/metabolism ; Mutagenesis ; Palmitates/metabolism* ; Polyethylene Glycols/pharmacology ; Rats ; Transfection

BACKGROUND: Pancreatic β-cells elevate insulin production and secretion through a compensatory mechanism to override insulin resistance under metabolic stress conditions. Deficits in β-cell compensatory capacity result in hyperglycemia and type 2 diabetes (T2D). However, the mechanism in the regulation of β-cell compensative capacity remains elusive. Nuclear factor-Y (NF-Y) is critical for pancreatic islets' homeostasis under physiological conditions, but its role in β-cell compensatory response to insulin resistance in obesity is unclear. METHODS: In this study, using obese ( ob/ob ) mice with an absence of NF-Y subunit A (NF-YA) in β-cells ( ob , Nf-ya βKO) as well as rat insulinoma cell line (INS1)-based models, we determined whether NF-Y-mediated apoptosis makes an essential contribution to β-cell compensation upon metabolic stress. RESULTS: Obese animals had markedly augmented NF-Y expression in pancreatic islets. Deletion of β-cell Nf-ya in obese mice worsened glucose intolerance and resulted in β-cell dysfunction, which was attributable to augmented β-cell apoptosis and reactive oxygen species (ROS). Furthermore, primary pancreatic islets from Nf-ya βKO mice were sensitive to palmitate-induced β-cell apoptosis due to mitochondrial impairment and the attenuated antioxidant response, which resulted in the aggravation of phosphorylated c-Jun N-terminal kinase (JNK) and cleaved caspase-3. These detrimental effects were completely relieved by ROS scavenger. Ultimately, forced overexpression of NF-Y in INS1 β-cell line could rescue palmitate-induced β-cell apoptosis, dysfunction, and mitochondrial impairment. CONCLUSION Pancreatic NF-Y might be an essential regulator of β-cell compensation under metabolic stress.
Rats ; Mice ; Animals ; Reactive Oxygen Species/metabolism* ; Diabetes Mellitus, Type 2/metabolism* ; Insulin Resistance ; Insulin ; Insulin-Secreting Cells/metabolism* ; Apoptosis ; Stress, Physiological ; Transcription Factors/metabolism* ; Palmitates/pharmacology* ; Obesity/metabolism*

OBJECTIVETo investigate the attenuating effect of curcumin, an anti-inflammatory compound derived from dietary spice turmeric (Curcuma longa) on the pro-inflammatory insulin-resistant state in 3T3-L1 adipocytes.

METHODSGlucose uptake rate was determined with the [3H] 2-deoxyglucose uptake method. Expressions of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were measured by quantitative RT-PCR analysis and ELISA. Nuclear transcription factor kappaB p65 (NF-kappa p65) and mitogen-activated protein kinase (MAPKs) were detected by Western blot assay.

RESULTSThe basal glucose uptake was not altered, and curcumin increased the insulin-stimulated glucose uptake in 3T3-L1 cells. Curcumin suppressed the transcription and secretion of TNF-alpha and IL-6 induced by palmitate in a concentration-dependent manner. Palmitate induced nuclear translocation of NF-kappaB. The activities of Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase1/2 (ERK1/2) and p38MAPK decreased in the presence of curcumin. Moreover, pretreatment with SP600125 (inhibitor of JNK) instead of PD98059 or SB203580 (inhibitor of ERK1/2 or p38MAPK, respectively) decreased the up-regulation of TNF-alpha induced by palmitate.

CONCLUSIONCurcumin reverses palmitate-induced insulin resistance state in 3T3-L1 adipocytes through the NF-kappaB and JNK pathway.

3T3-L1 Cells ; Animals ; Anthracenes ; pharmacology ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Curcumin ; pharmacology ; Glucose ; metabolism ; Insulin ; pharmacology ; Insulin Resistance ; Interleukin-6 ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; metabolism ; MAP Kinase Signaling System ; Mice ; NF-kappa B ; metabolism ; Palmitates ; pharmacology ; Protein Kinase Inhibitors ; pharmacology ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; Up-Regulation

OBJECTIVETo study the effect of palmitate on toll-like receptor 4 (TLR4) expression and signaling in vascular endothelial cells.

METHODSPig iliac endothelial cells (PIECs) were incubated with palmitate. TLR4 gene expression levels were measured by quantitative real-time PCR, and TLR4 and IκBα protein expressions by Western blotting. The expression levels of TLR4 protein on the surface of PIECs were quantified using flow cytometry. ELISA was employed to detect tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) concentrations in the cell medium.

RESULTSPalmitate treatment significantly increased TLR4 mRNA and protein expression levels in PIECs compared with those in the control cells (4.73∓0.61 vs 1.25∓0.90, P<0.05; 5.79∓0.05 vs 4.07∓0.31, P<0.05). The expression levels of TLR4 on the cell surface significantly increased (38.070∓3.907 vs 29.390∓1.072, P<0.05), while IκBα protein level was significantly lowered in PIECs after palmitate treatment as compared with those in the control cells (2.04∓0.22 vs 3.98∓0.18, P<0.05). Palmitate treatment significantly elevated TNF-α (2.52∓0.30 vs 1.38∓0.26, P<0.05) and IL-6 (IL-6: 3.28∓0.32 vs 1.44∓0.28, P<0.05) concentrations in the cell culture medium.

CONCLUSIONPalmitate can enhance TLR4 expression and signaling in porcine vascular endothelial cells.

Animals ; Blotting, Western ; Endothelial Cells ; drug effects ; metabolism ; I-kappa B Proteins ; metabolism ; Interleukin-6 ; metabolism ; NF-KappaB Inhibitor alpha ; Palmitates ; pharmacology ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Swine ; Toll-Like Receptor 4 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

The saturated free fatty acid (FFA), palmitate, could induce apoptosis in various cell types, but little is known about its effects on human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). Here, we investigated whether palmitate induced apoptosis and endoplasmic reticulum (ER) stress in hUC-MSCs. hUC-MSCs were stained by labeled antibodies and identified by flow cytometry. After administration with palmitate, apoptotic cell was assessed by flow cytometry using the Annexin V-FITC/7-AAD apoptosis detection kit. Relative spliced XBP1 levels were analyzed using semi-quantitative RT-PCR. The mRNA of BiP, GRP94, ATF4 and CHOP were analyzed by real-time PCR. Relative BiP and CHOP protein were analyzed using Western blot analysis. The results showed that hUC-MSCs were homogeneously positive for MSC markers; palmitate increased apoptosis of hUC-MSCs and activated XBP1 splicing, BiP, GRP94, ATF4 and CHOP transcription. These findings suggest that palmitate induces apoptosis and ER stress in hUC-MSCs.
Activating Transcription Factor 4 ; metabolism ; Apoptosis ; DNA-Binding Proteins ; metabolism ; Endoplasmic Reticulum Stress ; Heat-Shock Proteins ; metabolism ; Humans ; Membrane Glycoproteins ; metabolism ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Palmitates ; pharmacology ; Regulatory Factor X Transcription Factors ; Transcription Factor CHOP ; metabolism ; Transcription Factors ; metabolism ; Umbilical Cord ; cytology ; X-Box Binding Protein 1