OBJECTIVES: High fat-induced podocyte injury is one of the important factors leading to obesity related nephropathy (ORG), but the mechanism is not clear. This study aims to explore the mechanism of period circadian clock 3 (PER3) in the oxidative stress and inflammation induced by palmitic acid (PA) in podocytes. METHODS: The C57BL/6J mice were fed with chow and high-fat diet for 16 weeks. The PER3 expression in kidney tissues were detected in the normal body weight group and the obesity group. The PER3 mRNA and protein expression were detected after the podocytes were induced with different concentrations (0, 50, 150 and 300 μmol/L) of PA for 48 h. The PER3 mRNA and protein expression were detected after the podocytes were induced with 150 μmol/L PA for 0, 24, 36, and 48 h. Triglyceride (TG) levels were examined in the PA group, the adenovirus (ad)-PER3+PA group, and the siRNA-PER+PA group after the podocytes were transfected by Ad-PER3 or small interfering RNA (siRNA)-PER3 for 48 h and subsequently were induced with 150 μmol/L PA for 48 h. The differential gene expression was detected using RNA sequencing (RNA-seq) after podocytes were transfected by siRNA-PER3 (siRNA-PER3 group) and siRNA-control (siRNA-control group), respectively. The mRNA levels of nephrin, podocin, podocalyxin, podoplanin, superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), catalase (CAT), and the levels of malondialdehyde (MDA), glutathione (GSH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and interleukin-2 (IL-2) were detected after podocytes were transfected with Ad-PER3 or Ad-control for 48 h and then they were induced by 150 μmol/L PA for 48 h. RESULTS: The PER3 was down-regulated in the obesity group compared with the normal body weight group ( CONCLUSIONS PER3 can decrease the PA-induced oxidative stress and inflammatory factor secretion via inhibiting the lipogenesis in podocytes.
Animals ; Circadian Clocks ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; Palmitic Acid/toxicity* ; Podocytes/metabolism*

The objective of this work is to investigate how dilution rate and carbon-to-nitrogen (C/N) ratio affects lipid accumulation by Rhodosporidium toruloides AS 2.138 9 in continuous culture. Under steady-state conditions, the increase in dilution rate led to the decrease in lipid content and lipid yield. The highest lipid yield and lipid content at D = 0.02 h(-1) were 0.18 g lipid/g sugar and 57.1%, respectively, while the highest lipid productivity and biomass productivity were obtained at D = 0.14 h(-1). The increase in C/N ratio led to the increase in lipid content. The highest lipid content of 38% was obtained at C/N = 237. The highest lipid yield of 0.12 g lipid/g sugar was obtained at C/N = 92. However, the highest lipid productivity of 0.12 g/(L x h) was obtained at C/N = 32. No significant changes were observed in terms of fatty acid composition of the lipid produced under different C/N ratios, and these three fatty acids, palmitic acid, stearic acid and oleic acid, took over 85% in all samples.
Basidiomycota ; growth & development ; metabolism ; Batch Cell Culture Techniques ; Carbon ; metabolism ; Culture Media ; Fatty Acids ; metabolism ; Glucose ; metabolism ; Lipids ; biosynthesis ; Nitrogen ; metabolism ; Oleic Acid ; biosynthesis ; Palmitic Acid ; metabolism

Protein palmitoylation, one of post-translation modifications, refers to the addition of saturated 16-carbon palmitic acid to cysteine residues via the thioester bond. It plays key roles in various functional activities, such as the interaction, stability and location of proteins. Heat shock protein 90 (Hsp90), an important molecular chaperone, has been reported to be involved in sperm capacitation. However, it remains unclear whether protein palmitoylation exists in sperm and whether Hsp90 in sperm is palmitoylated under different physiological conditions. In this study, we examined whether the protein palmitoylation is present in mouse cauda epididymis sperm using acyl-biotin exchange method, predicted the potential palmitoylated sites of Hsp90 by the software CSS-Palm 4.0 and detected the palmitoylated Hsp90 in the mouse sperm from caput epididymis and cauda epididymis by immunoprecipitation. We found that some proteins, approximately 50, 65, 72, 85 and 130 kDa, were palmitoylated in mouse cauda epididymis sperm. Five sites in two Hsp90 isoforms were predicted to be palmitoylated. The results also showed that Hsp90 in mouse sperm was palmitoylated and its palmitoylation level was involved in different physiological conditions: the palmitoylation level of cauda epididymis sperm was higher than that of caput epididymis sperm; and the palmitoylation level after capacitation was much higher than that before capacitation. In conclusion, this study reveals that protein palmitoylation is present in mouse sperm and the palmitoylated Hsp90 is associated with different physiological conditions in sperm.
Animals ; Epididymis ; HSP90 Heat-Shock Proteins ; metabolism ; Lipoylation ; Male ; Mice ; Palmitic Acid ; chemistry ; Sperm Capacitation ; Spermatozoa ; metabolism

OBJECTIVETo investigate the protective mechanism of stearoyl-CoA desaturase 1 (SCD1) over-expression against the pro-apoptotic affects of palmitic acid on hepatocytes using the rat BRL cell line.

METHODSConcentration effect curves were generated using the trypan blue exclusion test to assess the death rate of BRL cells upon exposure to a dilution series of palmitic acid. The multiplicity of infection (MOI) of a lentiviral expression vector, pGC-FU-GFP, was determined for the BRL cells. Unmanipulated BRL cells were divided into two groups: the non-palmitate groups were composed of ordinary cultured cells (CON) alone, infected with lentivirus empty expression vector (negative control, NC), and infected with lentivirus overexpressing SCD1 (SCD1-LV); the palmitate groups were composed of ordinary cultured cells plus palmitate (CON+) alone, infected with lentivirus empty expression vector plus palmitate (NC+), and infected with lentivirus overexpressing SCD1 plus palmitate (SCD1-LV+). SCD1 mRNA expression was detected by real-time PCR. Propidium iodide (PI) single-staining was used to detect apoptosis and assess the cell cycle. Inter-group differences were analyzed statistically.

RESULTSThe death rate of BRL cells increased significantly after 72 h of exposure to 400 mumol/L palmitate (P less than 0.01). The MOI of pGC-FU-GFP in BRL cells was 20. The expression of SCD1 was significantly higher in the SCD1-LV and SCD1-LV+ groups than in the respective controls (vs. CON: F = 289, P less than 0.01; vs. CON+: F = 1522, P less than 0.01). Palmitate exposure led to decreased expression of SCD1 (CON+ vs. CON, F = 22, P less than 0.05 and NC+ vs. NC: F = 34, P less than 0.05). The ratio of S stage cells was similar in all non-palmitate groups (CON, NC and SCD1-LV, P = 0.137). However, there was a significant apoptotic peak and lower ratio of S stage cells in the control palmitate groups (CON+ and NC+) and the activity of cell proliferation was decreased as well. The ratio of apoptotic cells was decreased significantly in the SCD1-LV+ group compared to the CON+ group (P less than 0.01).

CONCLUSIONThe expression of SCD1 and its desaturation activity increased in BRL cells upon infection with the pGC-FU-SCD1-GFP lentiviral vector, suggesting that SCD1 over-expression can decrease palmitic acid-induced toxicity and apoptosis in hepatocytes.

Animals ; Apoptosis ; Cell Line ; Genetic Vectors ; Hepatocytes ; metabolism ; Lentivirus ; genetics ; Palmitic Acid ; toxicity ; Rats ; Stearoyl-CoA Desaturase ; metabolism

Fatty acid binding and oxidation kinetics for wild type P450(BM3) (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than k (cat). The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450(BM3) could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.
Bacterial Proteins ; metabolism ; Cytochrome P-450 Enzyme System ; metabolism ; Fatty Acids ; chemistry ; metabolism ; Lauric Acids ; chemistry ; metabolism ; Myristic Acid ; chemistry ; metabolism ; NADPH-Ferrihemoprotein Reductase ; metabolism ; Osmolar Concentration ; Oxidation-Reduction ; Palmitic Acid ; chemistry ; metabolism ; Structure-Activity Relationship

Non-alcoholic steatohepatitis (NASH), a metabolic disorder consisting of steatosis and inflammation, is considered the hepatic equivalent of metabolic syndrome and can result in irreversible liver damage. Macrophage-stimulating protein (MSP) is a hepatokine that potentially has a beneficial role in hepatic lipid and glucose metabolism via the activation of AMP-activated protein kinase (AMPK). In the current study, we investigated the regulatory role of MSP in the development of inflammation and lipid metabolism in various NASH models, both in vitro and ex vivo. We observed that MSP treatment activated the AMPK signaling pathway and inhibited lipopolysaccharide (LPS)- and palmitic acid (PA)-induced gene expression of pro-inflammatory cytokines in primary mouse hepatocytes. In addition, MSP treatment resulted in a significant reduction in PA-induced lipid accumulation and inhibited the gene expression of key lipogenic enzymes in HepG2 cells. Upon short hairpin RNA-induced knockdown of RON (the membrane-bound receptor for MSP), the anti-inflammatory and anti-lipogenic effects of MSP were markedly ablated. Finally, to mimic NASH ex vivo, we challenged bone marrow-derived macrophages with oxidized low-density lipoprotein (oxLDL) in combination with LPS. OxLDL+LPS exposure led to a marked inhibition of AMPK activity and a robust increase in inflammation. MSP treatment significantly reversed these effects by restoring AMPK activity and by suppressing pro-inflammatory cytokine gene expression and secretion under this condition. Taken together, these data suggest that MSP is an effective inhibitor of inflammation and lipid accumulation in the stressed liver, thereby indicating that MSP has a key regulatory role in NASH.
AMP-Activated Protein Kinases ; Animals ; Cytokines ; Fatty Liver* ; Gene Expression ; Glucose ; Hep G2 Cells ; Hepatocytes ; In Vitro Techniques ; Inflammation* ; Lipid Metabolism ; Lipogenesis* ; Lipoproteins ; Liver ; Macrophages ; Metabolism ; Mice ; Palmitic Acid

The present study observed the effect of Guanxin Zhitong Capsules(GXZT) on the lipotoxicity of vascular endothelial cells and investigated the mechanism of GXZT in atherosclerosis treatment. The lipotoxicity model in human umbilical vein endothelial cells(HUVECs) was induced by palmitic acid(PA) stimulation. These cells were divided into a normal control group(NC, 15% normal serum), a model group(PA, 0.6 mmol·L~(-1) PA+15% normal serum), a high-dose GXZT group(GXZT-H, 0.6 mmol·L~(-1) PA+15% GXZT-medicated serum), a medium-dose GXZT group(GXZT-M, 0.6 mmol·L~(-1) PA+10% GXZT-medicated serum+5% normal serum) and a low-dose GXZT group(GXZT-L, 0.6 mmol·L~(-1) PA+5% GXZT-medicated serum+10% normal serum). HUVECs were detected for cell viability by cell counting kit-8(CCK-8) assay, apoptosis by flow cytometry, mitochondrial membrane potential(MMP) by JC-1 labeled laser scanning confocal microscopy, and total and phosphorylated proteins of p38, ERK1/2, and JNK1/2 in the mitogen-activated protein kinases(MAPK) signaling pathway by Western blot. The phosphorylated level was calcula-ted. Compared with the NC group, the PA group showed decreased cell viability and MMP(P<0.01, P<0.01), elevated apoptosis(P<0.01), and up-regulated phosphorylated levels of p38, ERK1/2, and JNK1/2(P<0.01, P<0.01, P<0.01). Compared with the PA group, the GXZT-H, GXZT-M, and GXZT-L groups showed increased cell viability and MMP(P<0.01, P<0.01, P<0.01), reduced apoptosis(P<0.01), and down-regulated protein expression and phosphorylated levels of p38, ERK1/2 and JNK1/2 in the MAPK signaling pathway(P<0.01, P<0.01, P<0.01). In conclusion, the results suggest that GXZT functions via blocking MAPK signaling pathway to relieve the damage of HUVECs induced by PA.
Apoptosis ; Capsules ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; MAP Kinase Signaling System ; Palmitic Acid/toxicity* ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases/metabolism*

This study was carried out to investigate the efficacy of sn-2 palmitic acid-fortified vegetable oil (Sn2PA) on calcium absorption and to confirm the synergistic effects of fructooligosaccharide on calcium absorption. Male SD rats were fed 6 kinds of casein based diets containing vegetable oil (control), sn-2 palmitic acid-fortified vegetable oil (Sn2PA) and Sn2PA with fructooligosaccharide(Sn2PAFO) in two levels of calcium (normal 0.5% and high 1.0%) for 3 weeks. Total lipids, cholesterol, triglyceride and calcium in blood were measured. Feces were collected using cages for 4 days. Serum concentrations of total lipids and calcium were not significantly different among groups. However, serum triglyceride was significantly decreased by fructooligosaccharide supplementation regardless of dietary calcium level. The lipid absorption was not significantly different among experimental groups. Calcium absorption was significantly higher in Sn2PAFO group than other groups. Calcium solubility of intestine was increased by sn-2 palmitic acid supplementation. These results suggest that sn-2 palmitic acid and fructooligosaccharide supplementation could be beneficial for baby foods including infant formula, with regard to increasing absorption of calcium by more soluble calcium in the small intestinal content.
Absorption ; Animals ; Calcium ; Calcium, Dietary ; Caseins ; Cholesterol ; Diet ; Feces ; Gastrointestinal Contents ; Humans ; Infant Formula ; Intestines ; Lipid Metabolism ; Male ; Oligosaccharides ; Palmitic Acid ; Rats ; Solubility ; Vegetables

Lipids play many structural and metabolic roles, and dietary fat has great impact on metabolism and health. Fatty acid oxidation rate is dependent on tissue types. However there has been no report on the relationship between the rate of fatty acid oxidation and carnitine transport system in outer mitochondrial membrane of many tissues. In this study, the rate of fatty acid oxidation and carnitine palmitoyltransferase (CPT) I activity in the carnitine transport system were measured to understand the metabolic characteristics of fatty acid in various tissues. Palmitic acid oxidation rate and CPT I activity in various tissues were measured. Tissues were obtained from the white and red skeletal muscles, heart, liver, kidney and brain of rats. The highest lipid oxidation rate was demonstrated in the cardiac muscle, and the lowest oxidation rate was in brain. Red gastrocnemius muscle followed to the cardiac muscle. Lipid oxidation rates of kidney, white gastrocnemius muscle and liver were similar, ranging from 101 to 126 DPM/mg/hr. CPT I activity in the cardiac muscle was the highest, red gastrocnemius muscle followed by liver. Brain tissue showed the lowest CPT I activity as well as lipid oxidation rate, although the values were not significantly different from those of kidney and white gastrocnemius muscle. Therefore, lipid oxidation rate was highly (p< 0.001) related to CPT I activity. Lipid oxidation rate is variable, depending on tissue types, and is highly (p< 0.001) related to CPT I activity. CPT I activity may be a good marker to indicate lipid oxidation capacity in various tissues.
Animals ; Brain ; Carnitine O-Palmitoyltransferase ; Carnitine* ; Dietary Fats ; Heart ; Kidney ; Liver ; Metabolism ; Mitochondrial Membranes ; Muscle, Skeletal ; Myocardium ; Palmitic Acid ; Rats* ; Transferases*

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