USP19 Stabilizes TAK1 to Regulate High Glucose/Free Fatty Acid-induced Dysfunction in HK-2 Cells

Objective Obesity-induced kidney injury contributes to the development of diabetic nephropathy(DN).Here,we identified the functions of ubiquitin-specific peptidase 19(USP19)in HK-2 cells exposed to a combination of high glucose(HG)and free fatty acid(FFA)and determined its association with TGF-beta-activated kinase 1(TAK1).Methods HK-2 cells were exposed to a combination of HG and FFA.USP19 mRNA expression was detected by quantitative RT-PCR(qRT-PCR),and protein analysis was performed by immunoblotting(IB).Cell growth was assessed by Cell Counting Kit-8(CCK-8)viability and 5-ethynyl-2′-deoxyuridine(EdU)proliferation assays.Cell cycle distribution and apoptosis were detected by flow cytometry.The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation(Co-IP)assays and IB.Results In HG+FFA-challenged HK-2 cells,USP19 was highly expressed.USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells.Moreover,USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1(PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species(ROS)generation in HK-2 cells.Mechanistically,USP19 stabilized the TAK1 protein through deubiquitination.Importantly,increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells.Conclusion The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1,providing a potential therapeutic strategy for combating DN.

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.

Correlation between serum free fatty acids levels and Gensini score in elderly patients with coronary heart disease

Objectives To investigate the relationship between serum fxee fatty acids （FFAs） levels and the severity of coronary artery lesions in elderly patients with coronary heart disease （CAD）. Methods A total of 172 elderly patients who underwent coronary angiography were divided into CAD group （n = 128） and non-CAD group （n = 44） according to the results of coronary angiography. Serum FFAs and lipid levels were measured and the Gensini score were calculated. Results No matter the differences between age, gender and the usage of stat- ins or not, there was no statistical significance in FFAs levels （P 〉 0.05）. In terms of the Gensini score, it was higher in patients aged 70-79 years than in patients 60-69 years old [15.00 （5.00, 34.00） vs. 10.00 （2.00, 24.00）, P 〈 0.05], higher in men than women [14.00 （4.00, 34.00） vs. 7.00 （2.50, 19.75）, P 〈 0.05], and higher in patients on statins [13.50 （4.25, 33.50）vs. 6.50 （2.00, 18.00）, P 〈 0.05]. The serum FFAs lev- els [449.50 （299.00, 624.75） mEq/L vs. 388.00 （258.50, 495.25） mEq/L, P 〈 0.05J and Gensini score [17.50 （8.00, 41.75） vs. 1.00 （0, 5.00）, P 〈 0.05] were higher in the CAD group than in the non-CAD group. In the CAD group, there was no statistical significance in FFAs levels among patients with different numbers of diseased coronary vessels （P 〉 0.05）. Furthermore, the FFAs levels were positively correlated with the Gensini score （r = 0.394, P = 0.005）. Regression analysis showed that the FFAs levels were related to the Gensini score independently after adjusting for the other risk factors. Conclusions The serum FFAs levels were associated with the Gensini score in elderly patients with CAD. It might indicate FFAs as a biomarker predicting the severity of coronary artery lesions.

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.

The Kinetics of the Esterification of Free Fatty Acids in Waste Cook- ing Oil Using Fe2（SO4）3/C Catalyst

The esterification of free fatty acids(FFA) in waste cooking oil with methanol in the presence of Fe2(SO4)3/C(ferric sulfate/active carbon) catalyst was studied.The effects of different temperature,methanol/FFA mole ratio and amount of catalyst on the conversion of FFA were investigated.The results demonstrated that under optimal esterification conditions the final acid value of the resultant system can be reduced to ～1(mg KOH)·g-1,which met fully the requirements in post-treatment for efficient separation of glycerin and biodiesel.The kinetics of the esterification were also investigated under different temperatures.The results indicated that the rate-control step could be attributed to the surface reaction and the esterification processes can be well-depicted by the as-calculated kinetic formula in the range of the experimental conditions.

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.

Inhibitory effect of schisandrin B on free fatty acid-induced steatosis in L-02 cells

AIM:To investigate the effects of schisandrin B (Sch B) on free fatty acid (FFA)-induced steatosis in L-02 cells.METHODS:Cellular steatosis was induced by incubating L-02 cells with a FFA mixture (oleate and palmitate at the ratio of 2:1) for 24 h.Cytotoxicity and apoptosis were evaluated by 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and Annexin V/propidium iodide staining,respectively.Cellular total lipid was determined using a photocolorimetric method after Nile red staining,and triglyceride content was measured using an enzymatic kit.To study the effects of Sch B on steatosis,L-02 cells were treated with Sch B (1-100 μmol/L) in the absence or presence of 1 mmol/L FFA for 24 h,and cellular total lipid and triglyceride levels were measured.To explore the mechanisms of action of Sch B in the steatotic L-02 cells,mRNA levels of several regulators of hepatic lipid metabolism including adipose differentiation related protein (ADRP),sterol regulatory element binding protein 1 (SREBP-1),peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ were measured by quantitative real-time polymerase chain reaction (PCR),and protein levels of ADRP and SREBP-1 were measured by immunoblotting.RESULTS:Treatment with 1 mmol/L FFA for 24 h induced intracellular lipid accumulation in L-02 cells comparable to that in human steatotic livers without causing apparent apoptosis and cytotoxicity.Sch B mitigated cellular total lipid and triglyceride accumulations in the steatotic L-02 cells in a dose-dependent manner.Quantitative real-time PCR and Western blot analyses revealed that treatment of L-02 cells with 100 μmol/L Sch B reverted the FFA-stimulated up-regulation of ADRP and SREBP-1.CONCLUSION:Sch B inhibits FFA-induced steatosis in L-02 cells by,at least in part,reversing the up-regulation of ADRP and SREBP-1.

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.

Kinetic Modeling of Esterification Reaction of Free Fatty Acids Present in Macauba Oil Using a Cationic Resin as Catalyst

The cost of raw materials has the largest contribution to the final price of biodiesel produced by traditional routes, currently adopted in most industrial scale processes. That contribution comes from the need to use edible and noble oils, with low acidity, such as soybean oil. This work proposes＇the use of Macauba oil, a vegetable oil in focus in the State of Minas Gerais, Brazil, in which the current extractive yield generates a raw material with high acidity, therefore, not suitable to be used in biodiesel production. To make it technically feasible, a cationic exchange resin, the Purolite CT275DR, was used as a catalyst for esterification reaction with samples of Macauba oil, aiming to reduce its acidity. The resin can be reused, regenerated and easily removed from the reaction product, reducing costs with catalyst and purification stages. As a result of this work, in a sample of oil with an initial acidity of about 10% m/m were achieved acidity reductions up to 97% by using cationic resins as catalyst, demonstrating its potential use in the oil pretreatrnent step. Additionally, the data collected during all the analysis made it possible to define the chemical kinetic of the esterification reaction.

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.

Identification of crucial roles of transcription factor IhfA on high production of free fatty acids in Escherichia coli

Transcription factor engineering has unique advantages in improving the performance of microbial cell factories due to the global regulation of gene transcription.Omics analyses and reverse engineering enable learning and subsequent incorporation of novel design strategies for further engineering.Here,we identify the role of the global regulator IhfA for overproduction of free fatty acids(FFAs)using CRISPRi-facilitated reverse engineering and cellular physiological characterization.From the differentially expressed genes in the ihfALstrain,a total of 14 beneficial targets that enhance FFAs production by above 20% are identified,which involve membrane function,oxidative stress,and others.For membrane-related genes,the engineered strains obtain lower cell surface hydrophobicity and increased average length of membrane lipid tails.For oxidative stress-related genes,the engineered strains present decreased reactive oxygen species(ROS)levels.These gene modulations enhance cellular robustness and save cellular resources,contributing to FFAs production.This study provides novel targets and strategies for engineering microbial cell factories with improved FFAs bioproduction.

Inhibitive effects of glucose and free fatty acids on proliferation of human vascular endothelial cells in vitro

s To investigate the effects of glucose and free fatty acids (FFAs) on the proliferation and cell cycle of human vascular endothelial cells in vitro , and to examine whether the combined presence of elevated FFAs and glucose may cross amplify their individual injurious effects Methods Cultured human vascular endothelial cells (ECV304) were incubated with various concentrations of glucose and/or FFAs (palmitate and/or oleate) for 24-96 h Morphologic alterations were observed using a phase contrast microscope and an electron microscope Inhibition of proliferation was measured by a colorimetric 3 [4, 5 dimethyl thiazol 2 yl] 2, 5 diphenyltetrazolium bromide (MTT) assay Cell viability was determined using trypan blue exclusion Distribution of cells along phases of the cell cycle was analyzed by flow cytometry Results Glucose 15 or 30 mmol/L, palmitate (PA) 0 25 or 0 5 mmol/L, and oleate (OA) 0 5 mmol/L inhibited proliferation and accelerated death of endothelial cells in a dose and time dependent manner After treatment with elevated glucose and/or FFAs, the G 0/G 1 phase cells increased, whereas S phase cells decreased, suggesting that high glucose and/or FFAs mainly arrested endothelial cells at G 0/G 1 phase The inhibitive rates of proliferation and population of dead cells in endothelial cells incubated with glucose plus FFAs (glucose 30 mmol/L+PA 0 25 mmol/L, glucose 30 mmol/L+OA 0 5 mmol/L, glucose 30 mmol/L+PA 0 25 mmol/L+OA 0 5 mmol/L) increased more markedly than those treated with high glucose or FFAs (PA and/or OA) alone Conclusion Both high ambient glucose and FFAs can inhibit proliferation and accelerate death of endothelial cells in vitro These changes were cross amplified in the combined presence of high levels of glucose and FFAs

Berberine reverses free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKβ

AIM: To investigate the effects and molecular mechanisms of berberine on improving insulin resistance induced by free fatty acids (FFAs) in 3T3-L1 adipocytes. METHODS: The model of insulin resistance in 3T3-L1 adipocytes was established by adding palmic acid (0.5 mmol/L) to the culture medium. Berberine treatment was performed at the same time. Glucose uptake rate was determined by the 2-deoxy-[3H]-D-glucose method. The levels of IkB kinase beta (IKKβ) Ser181 phosphorylation, insulin receptor substrate-1(IRS-1) Ser307 phosphorylation, expression of IKKβ, IRS-1, nuclear transcription factor kappaB p65 (NF-κB p65), phosphatidylinositol-3-kinase p85 (PI-3K p85) and glucose transporter 4 (GLUT4) proteins were detected by Western blotting. The distribution of NF-κB p65 proteins inside the adipocytes was observed through confocal laser scanning microscopy (CLSM). RESULTS: After the intervention of palmic acid for 24 h, the insulin-stimulated glucose transport in 3T3-L1 adipocytes was inhibited by 67%. Meanwhile, the expression of IRS-1 and PI-3K p85 protein was reduced, while the levels of IKKβ Ser181 and IRS-1 Ser307 phosphorylation, and nuclear translocation of NF-κB p65 protein were increased. However, the above indexes, which indicated the existence of insulin resistance, were reversed by berberine although the expression of GLUT4, IKKβ and total NF-κB p65 protein were not changed during this study. CONCLUSION: Insulin resistance induced by FFAs in 3T3-L1 adipocytes can be improved by berberine. Berberine reversed free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKβ.

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.

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.