Free fatty acids receptors 2 and 3 control cell proliferation by regulating cellular glucose uptake

BACKGROUND Colorectal cancer(CRC)is a worldwide problem,which has been associated with changes in diet and lifestyle pattern.As a result of colonic fermentation of dietary fibres,short chain free fatty acids are generated which activate free fatty acid receptors(FFAR)2 and 3.FFAR2 and FFAR3 genes are abundantly expressed in colonic epithelium and play an important role in the metabolic homeostasis of colonic epithelial cells.Earlier studies point to the involvement of FFAR2 in colorectal carcinogenesis.AIM To understand the role of short chain FFARs in CRC.METHODS Transcriptome analysis console software was used to analyse microarray data from CRC patients and cell lines.We employed short-hairpin RNA mediated down regulation of FFAR2 and FFAR3 genes,which was validated using quantitative real time polymerase chain reaction.Assays for glucose uptake and cyclic adenosine monophosphate(cAMP)generation was done along with immunofluorescence studies to study the effects of FFAR2/FFAR3 knockdown.For measuring cell proliferation,we employed real time electrical impedancebased assay available from xCELLigence.RESULTS Microarray data analysis of CRC patient samples showed a significant down regulation of FFAR2 gene expression.This prompted us to study the FFAR2 in CRC.Since,FFAR3 shares significant structural and functional homology with FFAR2,we knocked down both these receptors in CRC cell line HCT 116.These modified cell lines exhibited higher proliferation rate and were found to have increased glucose uptake as well as increased level of glucose transporter 1.Since,FFAR2 and FFAR3 signal through G protein subunit(Gαi),knockdown of these receptors was associated with increased cAMP.Inhibition of protein kinase A(PKA)did not alter the growth and proliferation of these cells indicating a mechanism independent of cAMP/PKA pathway.CONCLUSION Our results suggest role of FFAR2/FFAR3 genes in increased proliferation of colon cancer cells via enhanced glucose uptake and exclude the role of PKA mediated cAMP signalling.Alternate pathways could be involved that would ultimately result in increased cell proliferation as a result of down regulated FFAR2/FFAR3 genes.This study paves the way to understand the mechanism of action of short chain FFARs in CRC.

Effect of sodium salicylate on oxidative stress and insulin resistance induced by free fatty acids

BACKGROUND: It has been reported that high-dose salicylates improve free fatty acids (FFAs)-induced insulin resistance and beta-cell dysfunction in vitro, but the mechanism remains uncertain. In insulin-resistant rats, we found that the supplementation of sodium salicylate is associated with a reduction of plasma malondialdehyde (MDA), a marker of oxidative stress. Few studies have investigated the effects of salicylates on oxidative stress levels in insulin-resistant animal models. This study aimed to assess the effect of sodium salicylate on insulin sensitivity and to explore the potential mechanism by which it improves hepatic and peripheral insulin resistance. METHODS: Intralipid+heparin (IH), saline (SAL), or intralipid+heparin+sodium salicylate (IHS) were separately infused for 7 hours in normal Wistar rats. During the last 2 hours of the infusion, hyperinsulinemic-euglycemic clamping was 3 performed with [6-(3)H] glucose tracer. Plasma glucose was measured using the glucose oxygenase method. Plasma insulin and C-peptide were determined by radioimmunoassay. MDA levels and glutathione peroxidase (GSH-PX) activity in the liver and skeletal muscle were measured with colorimetric kits. RESULTS: Compared with infusion of SAL, IH infusion increased hepatic glucose production (HGP), and decreased glucose utilization (GU) (P<0.05). The elevation of plasma free fatty acids increased the MDA levels and decreased the GSH-PX activity in the liver and muscle (P<0.01). Sodium salicylate treatment decreased HGP, elevated GU (P<0.05), reduced MDA content by 60% (P<0.01), and increased the GSH-PX activity by 35% (P<0.05). CONCLUSIONS: Short-term elevation of fatty acids induces insulin resistance by enhancing oxidative stress levels in the liver and muscle. The administration of the anti-inflammatory drug sodium salicylate reduces the degree of oxidative stress, therefore improving hepatic and peripheral insulin resistance. IKK-beta and NF-kappa B provide potential pathogenic links to oxidative stress.

Effects of different free fatty acids on insulin resistance in rats

BACKGROUND: Much evidence demonstrates that elevated free fatty acids (FFAs) are associated with insulin resistance. However, it is not clear whether different FFAs can cause different degrees of peripheral insulin resistance. This study aimed to investigate the effects of short-term elevation of FFAs on hepatic and peripheral insulin action, and determine whether FFAs with different degrees of saturation have differential effects on hepatic insulin resistance. METHODS: Intralipid+heparin (IH, polyunsaturated fatty acids), oleate (OLE), lard oil+heparin (LOH), and saline (SAL) were separately infused intravenously for 7 hours in normal Wistar rats. During the last 2 hours of the fat/saline infusion, a hyperinsulinemic-euglycemic clamping was performed with [6-H-3] glucose tracer. Plasma glucose was measured using the glucose oxygenase method. Plasma insulin and C-peptide were determined by radioimmunoassays. Plasma FFAs were measured using a colorimetric method. RESULTS: Compared with infusion of SAL, plasma FFA levels were significantly elevated by infusions of IH, OLE, and LOH (P<0.001). All three fat infusions caused remarkably higher hepatic glucose production (HGP) than SAL (P<0.001). OLE and LOH infusions induced much higher HGP than IH (P<0.01). Glucose utilization (GU) was decreased with all three fat infusions relative to SAL (P<0.001), but GU did not differ among the three types of fat infusions. CONCLUSIONS: Short-term elevation of FFAs can induce hepatic and peripheral insulin resistance. Polyunsaturated fatty acids induced less hepatic insulin resistance than monounsaturated or saturated fatty acids. However, IH, OLE, and LOH infusions induced similar peripheral insulin resistance.

Genistein Reverses Free Fatty Acid-induced Insulin Resistance in HepG2 Hepatocytes through Targeting JNK

This study investigated the effects and molecular mechanisms of genistein in improving insulin resistance induced by free fatty acids （FFAs） in HepG2 hepatocytes. A model of insulin resistance in HepG2 cells was established by adding palmitic acid （0.5 mmol/L） to the culture medium and the cells were treated by genistein. Glucose consumption of HepG2 cells was determined by glucose oxidase method. The levels of c-jun N-terminal kinase （JNK） phosphorylation, insulin receptor substrate-1 （IRS-1） Ser307 phosphorylation, JNK, IRS-1, phosphatidylinositol-3-kinase p85 （PI-3K p85） and glucose transporter 1 （GLUT1） proteins were detected by Western blotting. The results showed that after the treatment with palmitic acid for 24 h, the insulin-stimulated glucose transport in HepG2 cells was inhibited, and the glucose consumption was substantially reduced. Meanwhile, the expressions of IRS-1, PI-3K p85 protein and GLUT1 were obviously reduced, while the levels of JNK phosphorylation and IRS-1 Ser307 phosphorylation and the expression of JNK protein were significantly increased, as compared with cells of normal control. However, the aforementioned indices, which indicated the existence of insulin resistance, were reversed by genistein at 1-4 μmol/L in a dose-dependent manner. It was concluded that insulin resistance induced by FFAs in HepG2 hepatocytes could be improved by genistein. Genistein might reverse FFAs-induced insulin resistance in HepG2 cells by targeting JNK.

Development of a Derivatization-free GC-FID Method for Evaluation of Free Fatty Acid Levels in Plasma of Diabetic Nephropathy Patients

Metabolism of free fatty acids（FFAs） is related to several important physiological events and therefore their quantitaion in biological samples arouses extensive interest and efforts.Existing gas chromatography with flame ionization detector（GC-FID） methods for the analysis of FFAs normally require derivatization of them in order to lower boiling points.But this extra procedure tends to induce additional error and it is laborious and time-consuming.A derivatization-free method was therefore established in the present investigation to determine FFAs in human plasma by capillary（GC-FID）.After extraction of FFAs from plasma,a highly polar FFAP（free fatty acid in plasma） column was employed to directly quantitate FFAs concentration,free from derivatization reaction.All sample pretreatments were carried out at room temperature,improving recovery of short-chain FFAs.Heptadecanoic acid（C17：0） was employed as internal standard,and the proposed method was validated for recovery,precision,sensitivity,stabi-lity,and linearity.Validation data show that it is suitable for clinical study that has been applied to the evaluation of FFAs levels in plasma of diabetic nephropathy（DN） patients during the course of treatment.Forty-seven patients diagnosed with DN were admitted to the double-blind experiment.Control group（n=17） underwent solely basic treatment and the patients did not show significant change in FFAs concentration during six months of treatment.Experiment group（n=30） was supplied with traditional Chinese medicine besides basic treatment.After six months of medication,their plasma concentration of palmitic acid（C16：0）,stearic acid（C18：0） and oleic acid（C18：1n-9） decreased while linolenic acid（C18：3n-3） increased significantly（P〈0.05）.These four compounds could be served as biomar-kers in the evaluation of drug efficacy,and their quantitation in plasma may provide additional information for disease progression in DN patients.

Free fatty acids, glucose, and insulin in type 2 diabetes mellitus

Xu et al used the HOMA2 model to estimate theβ-cell function and insulin resistance levels in an individual from simultaneously measured fasting plasma glucose and fasting plasma insulin levels.This method is based on the assumption that the glucose-insulin axis is central for the metabolic activities,which led to type 2 diabetes.However,significant downregulation of both the NKX2-1 gene and the TPD52L3 gene force an increase in the release of free fatty acids(FFAs)into the blood circulation,which leads to a marked reduction in membrane flexibility.These data favor a FFA-glucose-insulin axis.The authors are invited to extend their study with the introduction of the saturation index(number of carbon-carbon double bonds per 100 fatty-acyl chains),as observed in erythrocytes.

Synthesis of spinel ferrite and its role in the removal of free fatty acids from deteriorated vegetable oil

Deterioration and loss of quality of vegetable oil is a big challenge in the food industry.This study investigated the synthesis of nickel ferrite(Ni Fe_(2)_(O4))via co-precipitation method and its use for the removal of free fatty acids(FFAs)in deteriorated vegetable oil.Ni Fe2 O4 was characterized using Fourier transformed infrared spectroscopy(FTIR),X-ray diffraction(XRD),thermogravimetric(TG)analysis,Brunauer–Emm ett–Teller(BET)surface area,transmission electron microscopy(TEM),scanning electron microscopy(SEM)and energy-dispersive X-ray spectroscopy(EDX).Synthesis of Ni Fe_(2)_(O4)was confirmed by characterization,which revealed a BET surface area of 16.30 m^(2)·g^(-1)and crystallite size of 29 nm.Ni Fe_(2)_(O4)exhibited an adsorption capacity of 145.20 L·kg^(-1)towards FFAs with an 80.69%removal in a process,which obeys Langmuir isotherm and can be described by the pseudo-second-order kinetic model.The process has enthalpy(DH)of 11.251 k Jámolà1 and entropy(DS)of 0.038 k J·mol^(-1)K^(-1)with negative free energy change(DG),which suggests the process to be spontaneous and endothermic.The quantum chemical computation analysis via density functional theory further revealed the sorption mechanism of FFAs by Ni Fe_(2)_(O4)occurred via donor–acceptor interaction,which may be described by the lowest unoccupied molecular orbital(LUMO)and the highest occupied molecular orbital(HOMO).The study showed Ni Fe_(2)_(O4)to be a potential means that can remove FFAs from deteriorated vegetable oil.

Certain sulfonylurea drugs increase serum free fatty acid in diabetic patients:A systematic review and meta-analysis

BACKGROUND Sulfonylurea(SU)is a commonly used antidiabetic drugs effective for type 2diabetes mellitus.Previous studies have reported that the SU treatment could alter the serum free fatty acid(FFA)concentration in diabetic patients;however,their exact effects remain unknown.AIM To assess the impact of SU on the FFA level in diabetic patients.METHODS A systematic literature search was conducted by consulting the PubMed,EMBASE,Cochrane Library,Reference Citation Analysis(https://www.referencecitationanalysis.com/),and Web of Science databases from January 1,1991 to July 30,2021.Either a fixed-effects model or random-effects model was applied to study the association between SU treatment and FFA concentration according to the heterogeneity test.Two investigators independently performed data extraction.The mean difference(MD)and corresponding 95%confidence interval(CI)were used to measure effect size.R3.5.1 software was utilized for conducting statistical analyses.RESULTS A total of 13 studies with 2273 individuals were selected.Results indicated that FFA concentration increased slightly after treatment with SU(MD=0.08,95%CI:0.03-0.12,P<0.01).In addition,we found that SU treatment combined with other antidiabetics could also increase the concentration of serum FFA(MD=0.14,95%CI:0.01-0.28,P<0.01).Regarding the type of SU,there was no significant difference in FFA concentration with glimepiride or glibenclamide.FFA concentration was higher at≥12 wk(MD=0.09,95%CI:0.04-0.13)but not at<12 wk(MD=0.01,95%CI:-0.07-0.09).CONCLUSION SU treatment could increase the serum FFA concentration in diabetic patients.The fundamental underlying mechanism still needs further investigation.

Rapid semi-quantification of triacylglycerols,phosphatidylcholines,and free fatty acids in the rice bran of one grain

We developed a microplate assay method for determining the contents of triacylglycerols（TAGs）, phosphatidylcholines（PCs）, and free fatty acids（FFAs） in the rice bran of one grain using enzymatic reactions. In this method, enzymes from commercially available kits were used. Optimum reaction conditions were established. It was found that Nonidet P-40 was the optimal among the three surfactants used（Triton X-100, Tween 40, and Nonidet P-40） when lipid was dissolved in a reaction solution. Using this method, it was possible to quantify TAGs, PCs, and FFAs in concentration ranges of 7–150, 5–70, and 8–200 mg L-（–1）, respectively. Furthermore, when the TAG contents in the rice bran were measured, the values closely corresponded to those obtained by extracting from large amounts of rice bran. However, sufficient data on the PC and FFA contents in rice bran are not available for valid comparisons. Although this method can accurately quantify the TAG contents in the rice bran of one grain, the accuracy of the PC and FFA contents has not been verified. Hence, future study is necessary.

Yap1 plays a protective role in suppressing free fatty acid-induced apoptosis and promoting beta-cell survival

Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes （T2D）. Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids （FFAs） such as palmitate. In this work, we demonstrated that Yes-associated protein （Yap1） is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor （CTGF）. Our gain-offunction analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.

Beneficial effects of a diabetes specific formula on insulin sensitivity and free fatty acid in patients with type 2 diabetes mellitus

Background This prospective, randomized, controlled study was designed to investigate the effects of a diabetes specific formula （Diason low energy： 313.8 k J/100 ml）, compared with a standard formula, on insulin sensitivity, serum C peptide, serum lipids and free fatty acid （FFA） in type 2 diabetics. Methods In total of 71 type 2 diabetics completed the study. Enteral formulas were given orally as the sole source of nutrition to the subjects for 6 days. Venous blood samples （0.5, 1, 2, 3 hours） were collected at day -7 after a 75 g oral glucose tolerance test （OGTT）, day 1 after a standard test meal （1673.6 k J） and after 6 days of either the test diabetes specific formula or a standard formula. Plasma glucose, serum insulin, C peptide and lipids were.measured. Results After the intervention period, the diabetes specific formula resulted in a significantly lower postprandial rise in blood glucose concentrations at 0.5 hour （P 〈0.05） and 1 hour （P 〈0.01）; significantly lower peak height of plasma glucose （P=0.05）; significantly lower plasma insulin concentrations at 0.5 hour （P〈0.01）, 1 hour （P〈0.01） and 2 hours （P 〈0.01）; and a significantly lower plasma insulin peak compared to controls; both OGTT and a standard test meal （P 〈0.05）. The glucose and insulin area under the curve after the diabetes specific formula compared to the standard formula were significantly lower. The C peptide level was lower after 6 days of both nutrition formulas compare to 75 g OGTT, but not different from the standard mixed meal. Both formulas were well tolerated. Conclusions In summary the diabetes specific formula with a relatively high monounsaturated fatty acid and high multi fiber proportion significantly improved glycemic control. On top of this, the insulin sensitivity （HOMA-IS） was significantly improved and may therefore directly improve the impact on long term complications. The disease specific formula should therefore be the preferred option to be used by diabetic and hyperglycemic patients in need of nutritional support.

Dietary free fatty acids complex with amylose creating another form of resistant starch: Gastrointestinal formation with fowl and swine

Fat added to poultry and swine feeds often contains abundant free fatty acids(FFA)that can impair digestible energy(DE).Placement of the fatty acid(FA)hydrocarbon chain in the helix core reformed from amylose creates a complex of both nutrients.Resulting modifications create a new structure termed the V-helix that becomes resistant toα-amylase.Granules in grain naturally contain minimal amounts of these complexes with more being generated during food manufacturing when moisture and heat release amylose in the presence of FFA.A paucity of FFA usually exists in complete feeds without sources of poorquality fat.Animal fats and by-product meals from rendering are prominent in their saturated FFA content which favorably complex within the helix.V-helix-FA complexes may arise during their concurrent encounter of FFA together with amylose during feed manufacture,particularly pelleting.FFA in the gastrointestinal tract(GIT)are speculated to further form complexes when present together with amylose.Although amylose may be dissolved in the gastric and small intestinal milieu,FFA separately coalesce into hydrophobic fat droplets along with other dietary lipids.Formation of complexes is likely restricted until FFA are released into the aqueous phase during fat digestion.Althoughα-amylase may be prominent,V-helix-FA complexes being resistant to enzymic attack pass into the large intestine.Subsequent microbial catabolism of V-helices may generate volatile fatty acids that are absorbed by the mucosa;however,an inability to use FFA once released leads to their excretion and basis for decreased DE.Immature microbial populations with young animals usually lack the capacity to fully catabolize the V-helix,further extending the loss in DE.

Qushi Huayu Decoction (祛湿化瘀方) Inhibits Protein and Gene Expression of Cathepsin B in HepG2 Cells Induced by Free Fatty Acids

Objective： To study the experimental efficacy of Qushi Huayu Decoction （祛湿化瘀方,QHD） on protein and gene expression of cathepsin B （ctsb） in HepG2 cells induced by free fatty acids （FFAs）.Methods： The model of HepG2 steatosis and tumor necrosis factor-α （TNF-α） secretion was induced by long-chain FFAs.HepG2 cells were divided into 4 groups： control group （group C）,model group （group M）,low-dose QHD group （group L） and high-dose QHD group （group H ）.Long-chain FFAs were added to groups M,L and H.The 10% blank-control serum was added to group C and M,while 5% and 10% QHD-containing sera were added to group L and H,respectively.The levels of serum TNF-α and cellular triglyceride （TG） were detected.Cellular p-IκB and ctsb expression were detected using Western blot and PCR.The expression and distribution of ctsb were observed by immunofluorescence.Results： After incubating with FFA for 24 h,TG deposition in HepG2,TNF-α content in cell supernatant,the protein expression of cellular ctsb and P-IκB,as well as mRNA expression of ctsb increased markedly in group M compared with group C （P〈0.05,P〈0.01）.Compared with group M,TG deposition,the expression of cellular ctsb,P-IκB and ctsb mRNA in groups L and H,as well as TNF-α content in group H,decreased significantly （P〈0.05）.Cell immunochemical fluorescence studies showed that ctsb was released from lysosomes and distributed in the cytoplasm extensively and diffusedly after being stimulated with FFA.In this study,these above-mentioned changes were inhibited markedly in groups L and H.Conclusion： QHD might have a direct inhibitory effect on the ctsb target in the FFA-ctsb-TNFα pathway of hepatic lipotoxicity.

Efficacy of Zaozhu Yinchen recipe for treating non-alcoholic steatohepatitis and its effect on free fatty acid and TNF-α

Objective To observe the efficacy of Zaozhu Yinchen Recipe(ZZYCR)on non-alcoholic steatohepatitis(NASH)patients,and to explore its effect on serum free fatty acid(FFA)and tumor necrosis factorα(TNF-α).Methods Totally 120 patients with NASH were randomly assigned to the treatment group(60 cases,treated with ZZYCR,one dose per day)and the control

Role of short chain fatty acids in gut health and possible therapeutic approaches in inflammatory bowel diseases

Inflammatory bowel diseases(IBDs) are characterized by inflammation in the gastrointestinal tract and include Ulcerative Colitis and Crohn’s Disease.These diseases are costly to health services,substantially reduce patients’ quality of life,and can lead to complications such as cancer and even death.Symptoms include abdominal pain,stool bleeding,diarrhea,and weight loss.The treatment of these diseases is symptomatic,seeking disease remission.The intestine is colonized by several microorganisms,such as fungi,viruses,and bacteria,which constitute the intestinal microbiota(IM).IM bacteria promotes dietary fibers fermentation and produces short-chain fatty acids(SCFAs) that exert several beneficial effects on intestinal health.SCFAs can bind to G protein-coupled receptors,such as GPR41 and GPR43,promoting improvements in the intestinal barrier,anti-inflammatory,and antioxidant effects.Thus,SCFAs could be a therapeutic tool for IBDs.However,the mechanisms involved in these beneficial effects of SCFAs remain poorly understood.Therefore,this paper aims to provide a review addressing the main aspects of IBDs,and a more detailed sight of SCFAs,focusing on the main effects on different aspects of the intestine with an emphasis on IBDs.

Evaluation of Circulating Fatty Acid Synthase as a Biomarker in Non-Alcoholic Fatty Liver Disease

Background: The liver is the corner stone in lipid metabolism, free fatty acid uptake, synthesizing, storing and exporting lipids;non-alcoholic fatty liver disease (NAFLD) develops if there is any interruption or derangements in lipid metabolim. Fatty acid synthase (FAS) is the major enzyme in lipogenesis, and its circulating level is a bi-omarker of metabolically demanding human diseases. Aim of the Work: To evaluate the level of circulating FAS in NAFLD patients and to correlate it to serum lipid pa-rameters. Materials and Methods: The study included forty NAFLD patients and forty age and sex-matched healthy subjects as controls. Results: FAS levels were signifi-cantly higher in NAFLD patients compared to their level in the controls (P < 0.05). Ad-ditionally, a positive correlation was found between the levels of FAS and BMI (r = 0.57), and between FAS levels and triglycerides and low density lipoprotein cholesterol levels in NAFLD patients (r = 0.79 & 0.53, respectively). Conclusion: Elevated levels of circulating FAS can be considered as a biomarker of fatty liver disease.

Fast Fatty Acid Analysis by Core-Shell Reversed-Phase Liquid Chromatography Coupled to Evaporative Light-Scattering Detector

A high-performance liquid chromatography analysis method with an evaporative light-scattering detector has been developed for the separation and quantitative analysis of free fatty acids in biological matrices. Core-shell reversed-phase high-performance liquid chromatography separation of 10 free fatty acids is achieved within 10.5 min using a methanol/water (0.05% trifluoroacetic acid) eluent gradient. After optimization, the drift tube and nebulization temperature of the evaporative light-scattering detector was set at 35°C, nitrogen flow-rate at 1.1 standard liter per minute and column temperature at 25°C. All calibration curves showed good regression (r2 > 0.9975). A validation procedure following the International Conference on Harmonisation guidelines was implemented to certify the method. Relative standard deviations did not exceed 1.5% and 4.25% for repeatability and reproducibility respectively.

Molecular mechanism of non-alcoholic fatty liver disease induced and aggravated by chronic stress through HSL/ATGL-FFA which promotes fat mobilization

Objective:To study the effects of social stress(CS)and social stress combined with high-fat diet on fat mobilization as a candidate mechanism for the induction or aggravation of non-alcoholic fatty liver disease(NAFLD).Methods:Thirty-two male Wistar rats(250±10 g)were randomly allocated to a blank control group(BC),a high-fat diet group(HFD),a CS group,and a combined CS and high-fat diet group(CS t HFD).Rats were sacrificed and tissues were collected after 8 weeks.Liver and body mass were measured and used to calculate the liver index.Serum aspartate aminotransferase(AST),alanine aminotransferase(ALT),and free fatty acids(FFAs)were measured.Liver sections were examined microscopically after oil red O and hematoxylin and eosin staining.The relative mRNA expression of acetyl-CoA carboxylase(ACCase),hydroxymethylglutaryl coenzyme A(HMG-CoA)reductase in liver,and hormone-sensitive lipase(HSL),and adipose triglyceride lipase(ATGL)in subcutaneous adipose tissue,were measured by real-time PCR.The liver concentrations of triglyceride,reactive oxygen species,and ACCase were measured by ELISA and HSL activity was determined using turbidimetry.Results:NAFLD developed in the CS,HFD,and CS t HFD groups,with the most severe NAFLD being in the CS t HFD group.Serum AST,ALT,and FFA,liver index,and hepatic triglyceride,FFA,and tumor necrosis factor-a were significantly higher in both the CS and CS t HFD groups.However,food intake and ACCase mRNA expression were lower.The mRNA expression of HSL and ATGL in adipose tissue was much higher,and HSL activity was higher in the CS group than in the BC group,and in the CS t HFD group than in the HFD group.Conclusion:We have successfully established two models of stress-induced NAFLD,suggesting stress can induce and aggravate NAFLD by promoting fat mobilization through upregulation of HSL and ATGL.

Optimization of pretreatment of Jatropha oil with high free fatty acids for biodiesel production

A central composite rotatable design and response surface methodology were used in order to investigate the individual and combined effects of the ethanol-to-oil ratio, H2SO4 concentration, temperature and time of reaction on the reduction of free fatty acid （FFA） in jatropha oil. A quadratic polynomial model relating the reaction variables with FFA reduction was developed, presenting a good coefficient of determination （R2= 0.893）. For reducing FFA to less than 1%, the optimal combination was found to be 0.62 v.v^-1 ethanol-to-oil ratio （14.9 v.v^-1 ethanol-to-FFA ratio）, 1.7% v.vI H2SO4 concentration, and 79 rain reaction time at a reaction temperature of 54℃. These results are of great relevance to maximize methyl esters formation by transesterification using an alkaline catalyst.

Endoplasmic reticulum stress is involved in podocyte apoptosis induced by saturated fatty acid palmitate

Background Podocyte 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. Methods Podocyte 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. Results Palmitate 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. Conclusion Palmitate 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.