Accumulation Of Free Fatty Acids Research Articles

In Vitro Influence of Specific Bacteroidales Strains on Gut and Liver Health Related to Metabolic Dysfunction-Associated Fatty Liver Disease.

Metabolic dysfunction-associated fatty liver disease (MAFLD) has become a major health risk and a serious worldwide issue. MAFLD typically arises from aberrant lipid metabolism, insulin resistance, oxidative stress, and inflammation. However, subjacent causes are multifactorial. The gut has been proposed as a major factor in health and disease, and over the last decade, bacterial strains with potentially beneficial effects on the host have been identified. In vitro cell models have been commonly used as an early step before in vivo drug assessment and can confer complementary advantages in gut and liver health research. In this study, several selected strains of the order Bacteroidales were used in a three-cell line in vitro analysis (HT-29, Caco-2, and HepG2 cell lines) to investigate their potential as new-generation probiotics and microbiota therapeutics. Antimicrobial activity, a potentially useful trait, was studied, and the results showed that Bacteroidales can be a source of either wide- or narrow-spectrum antimicrobials targeting other closely related strains. Moreover, Bacteroides sp. 4_1_36 induced a significant decrease in gut permeability, as evidenced by the high TEER values in the Caco-2 monolayer assay, as well as a reduction in free fatty acid accumulation and improved fatty acid clearance in a steatosis HepG2 model. These results suggest that Bacteroidales may spearhead the next generation of probiotics to prevent or diminish MAFLD.

Lipid droplets, autophagy, and ER stress as key (survival) pathways during ischemia-reperfusion of transplanted grafts.

Ischemia-reperfusion injuryis an event concerning any organ under a procedure of transplantation. The early result of ischemia is hypoxia, which causes malfunction of mitochondria and decrease in cellular ATP. This leads to disruption of cellular metabolism. Reperfusion also results in cell damage due to reoxygenation and increased production of reactive oxygen species, and later by induced inflammation. In damaged and hypoxic cells, the endoplasmic reticulum(ER) stress pathway is activated by increased amount of damaged or misfolded proteins, accumulation of free fatty acids and other lipids due to inability of their oxidation (lipotoxicity). ER stress is an adaptive response and a survival pathway, however, its prolonged activity eventually lead to induction of apoptosis. Sustaining cell functionality in stress conditions is a great challenge for transplant surgeons as it is crucial for maintaining a desired level of graft vitality. Pathways counteracting negative consequences of ischemia-reperfusion are autophagy and lipid droplets (LD) metabolism. Autophagy remove damaged organelles and molecules driving them to lysosomes, digested simpler compounds are energy source for the cell. Mitophagy and ER-phagy results in improvement of cell energetic balance and alleviation of ER stress. This is important in sustaining metabolic homeostasis and thus cell survival. LD metabolism is connected with autophagy as LD are degraded by lipophagy, a source of free fatty acids and glycerol-thus autophagy and LD can readily remove lipotoxic compounds in the cell. In conclusion, monitoring and pharmaceutic regulation of those pathways during transplantation procedure might result in increased/improved vitality of transplanted organ.

Use of thistle seeds of modified composition in chocolate mass technology

The object of the study is the analytical values of lipids of milk thistle seeds of a modified composition, namely acid and peroxide values, as well as the induction period of lipids of chocolate mass with the addition of milk thistle seeds. The paper substantiates rational parameters of thistle seed processing for the inactivation of lipases and lipoxygenases. Approximate dependences of the acid and peroxide values of thistle seed lipids on the pH of the wetting solution and the wetting degree of seeds were obtained. This makes it possible to substantiate such rational processing parameters of thistle seeds that inhibit the accumulation of free fatty acids and primary oxidation products in seeds during storage. An increase in the induction period of the oxidation of the lipid component of the chocolate mass with the use of thistle seeds of a modified composition was proven, which is 2.5 times higher than the induction period of the chocolate mass sample with thistle seeds with a native enzyme complex. The research results make it possible to develop a technology of healthy chocolate mass using thistle seeds of a modified composition, the lipid component of such mass is stable to oxidation and hydrolysis. The data obtained in the work are explained by an increase in the ability to inactivate lipolytic and lipoxygenase enzymes of milk thistle at high humidity in an acidic environment under the influence of microwave radiation. The advantage of the obtained results is the possibility of not violating the integrity of thistle seeds during the inactivation of the enzyme complex, which allows extending the shelf life of this raw material. An applied aspect of using the scientific result is the possibility of expanding the range of healthy chocolate masses with the use of thistle seeds

The Interconnection between Hepatic Insulin Resistance and Metabolic Dysfunction-Associated Steatotic Liver Disease-The Transition from an Adipocentric to Liver-Centric Approach.

The central mechanism involved in the pathogenesis of MAFLD is insulin resistance with hyperinsulinemia, which stimulates triglyceride synthesis and accumulation in the liver. On the other side, triglyceride and free fatty acid accumulation in hepatocytes promotes insulin resistance via oxidative stress, endoplasmic reticulum stress, lipotoxicity, and the increased secretion of hepatokines. Cytokines and adipokines cause insulin resistance, thus promoting lipolysis in adipose tissue and ectopic fat deposition in the muscles and liver. Free fatty acids along with cytokines and adipokines contribute to insulin resistance in the liver via the activation of numerous signaling pathways. The secretion of hepatokines, hormone-like proteins, primarily by hepatocytes is disturbed and impairs signaling pathways, causing metabolic dysregulation in the liver. ER stress and unfolded protein response play significant roles in insulin resistance aggravation through the activation of apoptosis, inflammatory response, and insulin signaling impairment mediated via IRE1/PERK/ATF6 signaling pathways and the upregulation of SREBP 1c. Circadian rhythm derangement and biological clock desynchronization are related to metabolic disorders, insulin resistance, and NAFLD, suggesting clock genes as a potential target for new therapeutic strategies. This review aims to summarize the mechanisms of hepatic insulin resistance involved in NAFLD development and progression.

Exposure to Chlorpyrifos Alters Proliferation, Differentiation and Fatty Acid Uptake in 3T3-L1 Cells.

Organophosphorus pesticides (OPs) are important factors in the etiology of many diseases, including obesity and type 2 diabetes mellitus. The aim of this study was to investigate the effect of a representative of OPs, chlorpyrifos (CPF), on viability, proliferation, differentiation, and fatty acid uptake in 3T3-L1 cells. The effect of CPF exposure on preadipocyte proliferation was examined by the MTT, NR, and BrdU assays. The impact of CPF exposure on the differentiation of preadipocytes into mature adipocytes was evaluated by Oil Red O staining and RT-qPCR. The effect of CPF on free fatty acid uptake in adipocytes was assessed with the fluorescent dye BODIPY. Our experiments demonstrated that exposure to CPF decreased the viability of 3T3-L1 cells; however, it was increased when the cells were exposed to low concentrations of the pesticide. Exposure to CPF inhibited the proliferation and differentiation of 3T3-L1 preadipocytes. CPF exposure resulted in decreased lipid accumulation, accompanied by down-regulation of the two key transcription factors in adipogenesis: C/EBPα and PPARγ. Exposure to CPF increased basal free fatty acid uptake in fully differentiated adipocytes but decreased this uptake when CPF was added during the differentiation process. Increased free fatty acid accumulation in fully differentiated adipocytes may suggest that CPF leads to adipocyte hypertrophy, one of the mechanisms leading to obesity, particularly in adults. It can therefore be concluded that CPF may disturb the activity of preadipocytes and adipocytes, although the role of this pesticide in the development of obesity requires further research.

Multi-lipase gene knockdown in Chinese hamster ovary cells using artificial microRNAs to reduce host cell protein mediated polysorbate degradation.

A large number of companies observe polysorbate (PS) degradation and associated (sub-)visible particle formation in biological drug formulations, which compromise the stability of the drug product, ultimately posing a risk toward delivering innovative medicines to patients. The main culprits of PS degradation are hydrolytic host cell proteins (HCPs) originating from the production cell lines, which are mostly Chinese hamster ovary (CHO) cell derived. Here, a small portion of particularly difficult-to-remove HCPs-mainly lipases-cause hydrolytic cleavage of PS resulting in the accumulation of free fatty acid aggregates/particles. One possible mitigation strategy is the removal of such critical HCPs in the production cell line. Multigene regulation can be achieved via microRNAs (miRNAs) thereby serving as a smart tool to reduce the expression of different target genes using a single miRNA. To enable a tailored gene regulation of multiple specific target lipases self-designed and non-naturally occurring artificial miRNAs (amiRNA) can be designed. Based on micro-conserved regions in the mRNA sequence of two sets of target HCPs, we provide a proof-of-concept for a simultaneous multi-lipase knockdown in CHO cells using single amiRNAs. By this, we were not only able to reduce PS degradation but laid the foundation to expand this tool to other areas of cell line phenotype engineering.

Impeding DNA Polymerase β Activity by Oleic Acid to Inhibit Base Excision Repair and Induce Mitochondrial Dysfunction in Hepatic Cells.

Free fatty acids (FFAs) hepatic accumulation and the resulting oxidative stress contribute to several chronic liver diseases including nonalcoholic steatohepatitis. However, the underlying pathological mechanisms remain unclear. In this study, we propose a novel mechanism whereby the toxicity of FFAs detrimentally affects DNA repair activity. Specifically, we have discovered that oleic acid (OA), a prominent dietary free fatty acid, inhibits the activity of DNA polymerase β (Pol β), a crucial enzyme involved in base excision repair (BER), by actively competing with 2'-deoxycytidine-5'-triphosphate. Consequently, OA hinders the efficiency of BER, leading to the accumulation of DNA damage in hepatocytes overloaded with FFAs. Additionally, the excessive presence of both OA and palmitic acid (PA) lead to mitochondrial dysfunction in hepatocytes. These findings suggest that the accumulation of FFAs hampers Pol β activity and contributes to mitochondrial dysfunction, shedding light on potential pathogenic mechanisms underlying FFAs-related diseases.

Oxidative stress mediates the inhibitory effects of Manzamine A on uterine leiomyoma cell proliferation and extracellular matrix deposition via SOAT inhibition

Uterine fibroids, the most common benign tumors of the myometrium in women, are characterized by abnormal extracellular matrix deposition and uterine smooth muscle cell neoplasia, with high recurrence rates. Here, we investigated the potential of the marine natural product manzamine A (Manz A), which has potent anti-cancer effects, as a treatment for uterine fibroids. Manz A inhibited leiomyoma cell proliferation in vitro and in vivo by arresting cell cycle progression and inducing caspase-mediated apoptosis. We performed target prediction analysis and identified sterol o-acyltransferases (SOATs) as potential targets of Manz A. Cholesterol esterification and lipid droplet formation were reduced by Manz A, in line with reduced SOAT expression. As a downstream target of SOAT, Manz A also prevented extracellular matrix deposition by inhibiting the β-catenin/fibronectin/metalloproteinases axis and enhanced autophagy turnover. Excessive free fatty acid accumulation by SOAT inhibition led to reactive oxygen species to impair mitochondrial oxidative phosphorylation and trigger endoplasmic reticulum stress via PERK/eIF2α/CHOP signaling. The inhibitory effect of ManzA on cell proliferation was partially restored by PERK knockdown and eliminated by tauroursodeoxycholic acid, suggesting oxidative stress plays a critical role in the mechanism of action of Manz A. These findings suggest that targeting SOATs by Manz A may be a promising therapeutic approach for uterine fibroids.

Comparison of the Effects of Monounsaturated Fatty Acids and Polyunsaturated Fatty Acids on Liver Lipid Disorders in Obese Mice.

Obesity is a recognized epidemic worldwide, and the accumulation of excess free saturated fatty acids (SFAs) in cells induces cellular lipotoxic damage and increases the risk of a wide spectrum of metabolic diseases including type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD). Monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) have been reported to combat SFA-induced cellular damage. However, the comparative studies of the two types of unsaturated fatty acids (UFAs) are still limited. We investigated the effects of different MUFAs and PUFAs in the human hepatocyte line L-02 cells in vitro, and in high-fat-diet (HFD)-induced obese C57BL/6 mice in vivo. The results of the in vitro study showed that SFAs induced significant cellular lipotoxic damage, but the combination of MUFAs/PUFAs with SFAs significantly improved the impaired cell viability. Particularly, oleic acid (OA) was superior to eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA), and arachidonic acid (AA) in terms of its anti-apoptotic effect and inhibition of endoplasmic reticulum (ER) stress. In vivo, both olive-oil-enriched (HFD + OO) and fish-oil-enriched high-fat diets (HFD + FO) reduced hepatic steatosis and improved insulin sensitivity in obese mice. However, FO induced an abnormal increase in serum aspartate aminotransferase (AST) and an increase in the oxidative stress indicator Malondialdehyde (MDA). Liver-targeted lipidomic analysis showed that liver lipid metabolites under the two types of UFA dietary interventions differed from the HFD group, modulating the abundance of some lipid metabolites such as triglycerides (TGs) and glycerophospholipids. Furthermore, the FO diet significantly increased the abundance of the associated FA 20:5 long-chain lipid metabolites, whereas the OO diet regulated the unsaturation of all fatty acids in general and increased the abundance of FA 18:1 in the overall lipid metabolites, especially TGs, which may primarily contribute to the FO, and OO drove protection in NAFLD.

Poster 124: Elevated Lipid Metabolites in Stored Clinical OCA Media are Associated with Chondrocyte Death

Objectives: A major limitation of osteochondral allografts (OCA) is the deterioration of cartilage health associated with cell death during prolonged storage. It is well established that chondrocyte viability in OCA is a critical factor for repair and clinical outcomes, but little is known about the mechanisms that contribute to chondrocyte death during storage. Elevated bioactive lipid metabolites and free fatty acids (FFAs) have been associated with cell death and tissue damage for various pathologies through lipotoxicity. However, the levels and effect of FFA accumulation in OCA media during prolonged storage are unknown. The purpose of this study was to determine if bioactive lipid metabolites accumulate in the storage media of OCA and are associated with a loss of chondrocyte viability during prolonged storage. We hypothesized that FFAs will accumulate over time in the storage media of OCA and adversely impact cartilage health during storage. Methods: To elucidate potential mechanisms of cartilage health decline during storage, 21 fresh human hemi-condyle osteochondral allograft tissues and media provided by a US tissue bank were analyzed after 7, 28, and 68 days of prolonged cold storage. Targeted metabolomics analysis measured bioactive FFAs, and primary (lipid hydroperoxide, ROOH) and secondary (malondialdehyde, MDA) lipid oxidation products. Chondrocyte viability was measured using standard LIVE/DEAD assay and confocal microscopy. Results: All fatty acid metabolites in storage media significantly increased with increased storage time (Figure 1; p<0.05). ROOH was significantly higher at Day 28 of storage. No difference in secondary lipid hydroperoxide products in storage media were observed. Chondrocyte viability significantly declined in both the en face and vertical cross-section analysis with increased storage time (Figure 2) and inversely correlated with fatty acid metabolites (Figure 3; p<0.05). Conclusions: It is well established that elevated levels of certain free fatty acids and lipid oxidation products can alter cell function and cause cell death through lipotoxicity and other mechanisms. This work is the first to identify elevated levels of FFA metabolites and primary oxidation lipid product in the storage media from clinical OCA. The concentrations of FFA metabolites were measured at levels (>100µM) known to induce cell death and were directly correlated with chondrocyte viability. These findings provide important targets for understanding why cartilage health declines during storage to better optimize media formulations and improve graft health.

Intrahepatic macrophage reprogramming associated with lipid metabolism in hepatitis B virus-related acute-on-chronic liver failure

BackgroundAcute-on-chronic liver failure (ACLF) is a severe syndrome with high short-term mortality, but the pathophysiology still remains largely unknown. Immune dysregulation and metabolic disorders contribute to the progression of ACLF, but the crosstalk between immunity and metabolism during ACLF is less understood. This study aims to depict the immune microenvironment in the liver during ACLF, and explore the role of lipid metabolic disorder on immunity.MethodsSingle-cell RNA-sequencing (scRNA-seq) was performed using the liver non-parenchymal cells (NPCs) and peripheral blood mononuclear cells (PBMCs) from healthy controls, cirrhosis patients and ACLF patients. A series of inflammation-related cytokines and chemokines were detected using liver and plasma samples. The lipid metabolomics targeted free fatty acids (FFAs) in the liver was also detected.ResultsThe scRNA-seq analysis of liver NPCs showed a significant increase of monocytes/macrophages (Mono/Mac) infiltration in ACLF livers, whereas the resident Kupffer cells (KCs) were exhausted. A characterized TREM2+ Mono/Mac subpopulation was identified in ACLF, and showed immunosuppressive function. Combined with the scRNA-seq data from PBMCs, the pseudotime analysis revealed that the TREM2+ Mono/Mac were differentiated from the peripheral monocytes and correlated with lipid metabolism-related genes including APOE, APOC1, FABP5 and TREM2. The targeted lipid metabolomics proved the accumulation of unsaturated FFAs associated with α-linolenic acid (α-LA) and α-LA metabolism and beta oxidation of very long chain fatty acids in the ACLF livers, indicating that unsaturated FFAs might promote the differentiation of TREM2+ Mono/Mac during ACLF.ConclusionsThe reprogramming of macrophages was found in the liver during ACLF. The immunosuppressive TREM2+ macrophages were enriched in the ACLF liver and contributed to the immunosuppressive hepatic microenvironment. The accumulation of unsaturated FFAs in the ACLF liver promoted the reprogramming of the macrophages. It might be a potential target to improve the immune deficiency of ACLF patients through regulating lipid metabolism.

Effect of Spoilage by Lipolytic Fungi Strains on Free Fatty Acids (FFA) Formation in Fermented and Dry Cocoa Beans

Aim: High free fatty acids (FFA) content of cocoa beans is high lead to alter quality of butter fraction and chocolate manufactured. During primary post-harvest processing, cocoa beans harbor lipase producing mold speciess could trigger the accumulation of FFA. This study investigated the improvement of the quality of raw cocoa material sourced from Côte d’Ivoire regarding its high FFA concentration.
 Methods: Fermented and dry cocoa were sampled on-farm level in some main cocoa producing regions of Côte d'Ivoire. Contaminating mold strains were isolated using Sabouraud chloramphenicol agar incubated at 25°C for 7 days. Morphological identification of mold isolates was preformed from younger fungal culture. Biochemical study about lipolytic properties of mold isolates was carried out using Cazpeck-Dox Agar medium enriched with olive oil as carbon source and rhodamine B as indicator of lipase activity. Extraction, PCR amplification using a specific transcribed spacer primer (ITS4/ITS5) and sequencing of DNA were performed for fungal species molecular identification.
 Results: Approximately 154 mold isolates belonging to 8 genera were found in tested cocoa beans. Only Rhizopus oryzae, Absidia corymbifera, Mucor heimalis, Aspergillus niger, A. tubingensis, A. aculeatus, A. flavus, A. tamari, A. fumigatus, Nigrospora sphaerica, Curvularia geniculate and Penicillium chrysogenum species produced greater lipolytic properties. A. corymbifera, A. niger, A. fumigatus and R. orysae were the most frequent and predominant species. Cocoa beans inoculated with R. oryzae, A. corymbifera or A. niger recorded higher FFA content above 1.75%.
 Conclusion: Reducing of the mold species growth during cocoa primary post-harvest processing could sustainably produce raw cocoa material with less FFA.

Homologous Overexpression of Acyl-CoA Thioesterase 8 Enhanced Free Fatty Acid Accumulation in Oleaginous Fungus Mucor circinelloides WJ11

Thioesterases play an essential role in the metabolism of fatty acids since they are considered one of the key enzymes to change the total amount and composition of fatty acid in an organism’s cells. Acyl-coenzyme A thioesterase 8 (ACOT8) exhibits substrate specificity mainly for short- to long-chain acyl-CoA. To identify and characterize the ACOT8 enzyme’s superfamily in Mucor circinelloides, three genes were characterized and homologously expressed in M. circinelloides WJ11 which has been used as a model organism to investigate the mechanism of lipid accumulation. Multiple sequence alignment showed that McACOT8s had significant conserved motifs in the ACOT8 family. Experimental data indicated that the biomass of the three recombinant strains (McACOT8a, McACOT8b and McACOT8c) was slightly lower than that of the control strain Mc2075, but the total fatty acid (TFA) content was significantly increased by 30.3, 21.5 and 23.9%, respectively, and the free fatty acid production increased from 12.9% (control strain) to 19.2, 25.2 and 26.2% (recombinant types), respectively, with a maximum increase of 103.1% in McACOT8c. The three strains did not show any significant improvement in fatty acid composition compared to the control strain. However, in terms of fatty acid composition of free fatty acid, McACOT8b and McACOT8c contained γ-linolenic acid (C18:3), which was not detected in the control strain or in McACOT8a, indicating that ACOT8b and ACOT8c had substrate specificity for C18:3. These results displayed that ACOT8 can increase TFA accumulation and that it may be an important target of genetic manipulation for microbial oil production.

Free fatty acid exacerbates diabetic kidney injury by suppressing autophagy-lysosomal function via ER stress

Diabetic kidney disease (DKD), the leading cause of chronic kidney disease, is characterized by accumulation of renal tubule free fatty acids (FFAs) and triglyceride (TG)-enriched lipid droplets (LDs) and cell death. The present study aimed to investigate how FFA or TG induces renal tubular cell apoptosis, thereby contributing to the progression of DKD. Cell studies were conducted to define the causal role and underlying mechanism of FFA-activated ER stress and organelle dysfunction in tubular cell injury. Six-week-old wild-type (WT) and eNOS-deficient C57BL/6J mice were placed on normal diet (ND, 5% fat calories) or high-fat diet (HFD, 42% fat calories) for two weeks, and then rendered diabetic with alloxan. Alloxan injection resulted in equivalent hyperglycemia in ND and HFD-fed WT and eNOS-deficient mice. However, a massive increase in urinary albumin was observed in HFD plus alloxan-treated eNOS-deficient mice, which was accompanied by excessive accumulation of neutral lipids in damaged proximal tubules. Moreover, in the diabetic eNOS-deficient kidneys, HFD feeding resulted in suppression of autophagy-lysosomal function and increase in tubular cell apoptosis. Cell studies revealed that saturated FFA palmitate but not unsaturated oleate induced eukaryotic initiation factor (eIF)2α phosphorylation, and then stimulated the activating transcription factor (ATF)4 and C/EBP homologous protein (CHOP), which, in turn, impaired autophagy-lysosomal function, leading to cell apoptosis. In addition, we observed a dramatic upregulation of LD-associated protein ADRP expression and TG-enriched LD formation in oleate-treated renal tubular cells, which was not apparent in palmitate-treated ones. Together, this study demonstrates that accumulation of renal FFA, rather than TG, plays a crucial role in the pathogenesis of diabetic kidney injury by suppressing autophagy-lysosomal function through ER stress signaling. This work was supported by the National Institutes of Health Grants SC1DK112151 and S21MD000101. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Free Fatty Acid Formation Points in Palm Oil Processing and the Impact on Oil Quality

Background: The level of free fatty acids (FFAs) is an important oil quality index that is consistently measured at mills and refineries to ensure that palm oil is within specification limits. FFAs can accumulate at any point throughout the process, for example, during fresh fruit bunch (FFB) harvesting or during the mill process before sterilisation. Another key contributor to FFA build-up is loose fruit (LF), which is collected following FFB harvesting and is commonly processed together with FFB into crude palm oil (CPO) at the mill. The aim of this study was to identify pivotal points of FFA formation during the process of crude palm oil production. Results: The present study shows that the highest FFA accumulation occurred during the conveying process at the mill before sterilisation due to significant fruit damage. The rapid formation of FFA occurred during the first 15 min of oil palm fruit bruising. A minimum temperature of 60 °C for one hour was needed to deactivate the lipase activity, which is responsible for FFA formation. Blending high-FFA CPO with standard CPO affected indices of palm oil quality, such as the deteriorated peroxide value (PV) and anisidine value (AV), and particularly worsened the bleachability index (DOBI). Conclusions: This study suggests that the conveyor system in the mill could be the prime area to focus on in terms of FFA reduction, along with minimising bruising events. In addition, loose fruits (LF) with high FFA content should be processed separately from FFB, and high-FFA CPO derived from LF should not be mixed with standard CPO.

Immune regulation in obesity: a narrative review

Obesity is a condition triggered by many risk factors, but the main one is due to high calorie intake and low physical activity. Various studies have explored the relationship between obesity and metabolic complications, namely chronic degenerative disorders. In the condition of obesity, there are biochemical changes including chronic inflammation. This condition mainly occurs due to the load on the metabolic tissue due to weight gain and fat tissue dysfunction. These changes result in changes in the distribution of leukocytes, lymphocyte activity, and in general the immune defense system. The changes that occur include the occurrence of a chronic inflammatory process. Among the various inflammatory mediators, there are three mediators that play an important role in the regulation of the immune system due to obesity, namely TNF-α, IL-6, and adiponectin. In addition, the accumulation of free fatty acids in obesity can activate a cascade of a series of pro-inflammatory kinases, such as IkB kinase and c-Jun N-terminal kinase, which triggers fatty tissue to release IL-6. Other effects include activation of the inflammasome, and hyperleptinemia. Leptin is then associated with cell survival, cytokine release, and chemotaxis. In addition, leptin can also be related to the proliferation of T cells.

ETNPPL impairs autophagy through regulation of the ARG2-ROS signaling axis, contributing to palmitic acid-induced hepatic insulin resistance.

Excessive free fatty acids (FFAs) accumulation is a leading risk factor for the pathogenesis of insulin resistance (IR) in metabolic tissues, including the liver. Ethanolamine-phosphate phospho-lyase (ETNPPL), a newly identified metabolic enzyme, catalyzes phosphoethanolamine (PEA) to ammonia, inorganic phosphate, and acetaldehyde and is highly expressed in hepatic tissue. Whether it plays a role in regulating FFA-induced IR in hepatocytes has yet to be understood. In this study, we established an in vitro palmitic acid (PA)-induced IR model in human HepG2 cells and mouse AML12cells with chronic treatment of PA. Next, we overexpressed ETNPPL by using lentivirus-mediated ectopic to investigate the effects of ETNPPL per se on IR without PA stimulation. We show that ETNPPL expression is significantly elevated in PA-induced IR and that silencing ETNPPL ameliorates this IR in hepatocytes. Inversely, overexpressing ETNPPL under normal conditions without PA promotes IR, reactive oxygen species generation, and ARG2 activation in both HepG2 and AML12cells. Moreover, ETNPPL depletion markedly down-regulates ARG2 expression in hepatocytes. Besides, silencing ARG2 prevents ETNPPL-induced ROS accumulation and inhibition of autophagic flux and IR in hepatocytes. Finally, we found that phytopharmaceutical disruption of ETNPPL by quercetin ameliorates PA-induced IR in hepatocytes. Our study discloses that ETNPPL inhibiting autophagic flux mediates insulin resistance triggered by PA in hepatocytes via ARG2/ROS signaling cascade. Our findings provide novel insights into elucidating the pathogenesis of obesity-associated hepatic IR, suggesting that targeting ETNPPL might represent a potential approach for T2DM therapy.

Role of serum free fatty acids in determining severity of diabetic nephropathy: A case control study

Introduction and Aim: The Diabetic Nephropathy (DN) is a result of impaired renal function in Type 2 diabetes. At the onset of diabetes, microvascular complication increases due to accumulation of free fatty acids (FFA) causing renal damage. A study was conducted to estimate the concentration of serum FFA causing severity of diabetic nephropathy.
 
 Materials and Methods: 90 Type 2 diabetic subjects and 30 study controls (age group 35 to 65 years) were selected from the medicine OPD of S N Medical College and HSK Hospital, Bagalkot. Based on the presence of microalbuminuria, the 90 Type 2 diabetic patients were equally divided in to 3 groups, named as stage I to stage III. The serum FFA was estimated by ELISA method in these three groups and control subjects. The statistical analysis was done using SPSS software version 19 utilizing unpaired “t” test for quantitative data and Pearson’s correlation tests.
 
 Results: The estimated serum FFA levels in stage I to III was found to be higher and highly significant as compared to control (p=0.001). We find the best cut off value of serum FFA was 4.75 mmol/L, causing severity of diabetic nephropathy. The area under the curve (AUC) is 0.92 with the specificity of 86%, sensitivity 89% and the diagnostic accuracy was found to be of 87%.
 
 Conclusion: Serum free fatty acid levels were higher in diabetic nephropathy subjects, which could be used as diagnostic marker for the severity of renal damage with cut off value of 4.75 mmol/L.

Canagliflozin ameliorates the development of NAFLD by preventing NLRP3-mediated pyroptosis through FGF21-ERK1/2 pathway

Recent studies have suggested that sodium-glucose co-transporter2 inhibitors go beyond their glycemic advantages to ameliorate the development of NAFLD. However, little research has been done on the underlying mechanisms. Here, we took deep insight into the effect of canagliflozin (CANA), one of the sodium-glucose co-transporter2 inhibitor, on the progression of NAFLD, and explored the molecular mechanisms. Our findings showed that CANA-treated ob/ob and diabetic mice developed improved glucose and insulin tolerance, although their body weights were comparable or even increased compared with the controls. The CANA treatment ameliorated hepatic steatosis and lipid accumulation of free fatty acid-treated AML12 cells, accompanied by decreased lipogenic gene expression and increased fatty acid β oxidation-related gene expression. Furthermore, inflammation and fibrosis genes decreased in the livers of CANA-treated ob/ob and diabetic mice mice. FGF21 and its downstream ERK1/2/AMPK signaling decreased, whereas NLRP3-mediated pyroptosis increased in the livers of the ob/ob and diabetic mice mice, which was reversed by the CANA treatment. In addition, blocking FGF21 or ERK1/2 activity antagonized the effects of CANA on NLRP3-mediated pyroptosis in lipopolysaccharide plus nigericin-treated J774A.1 cells. We conclude that CANA treatment alleviated insulin resistance and the progression of NAFLD in ob/ob and diabetic mice mice independent of the body weight change. CANA protected against the progression of NAFLD by inhibiting NLRP3-mediated pyroptosis and enhancing FGF21-ERK1/2 pathway activity in the liver. These findings suggest the therapeutic potential of sodium-glucose co-transporter2 inhibitors in the treatment of NAFLD.

How do engineered Yarrowia lipolytica strains secrete free fatty acids: hints from comparative transcriptomics.

Yarrowia lipolytica has been considered one of the most promising platforms for the microbial production of fatty acids and derived products. The deletion of the faa1 gene coding for an acyl-CoA synthetase leads to the accumulation and secretion of free fatty acids (FFAs) into the extracellular space. The secretion of products is beneficial for the development of microbial cell factories to avoid intracellular inhibitory effects and reduce downstream processing costs. However, the mechanism behind the secretion of fatty acids is not well known. As a starting point, we compared the transcriptome of this mutant showing FFA secretion to a wildtype-like strain not showing this phenotype. The 12 most upregulated genes were evaluated for involvement in FFA secretion by the creation of deletion and overexpression mutants, among them MCH2, YMOH, three cell wall proteins CWP3, CWP4, and CWP11, M12B, and three proteins with unknown functions YUP1, YUP2, and YUP3. None of these proteins take a clear or isolated role in FFA export. As the transcriptomic data revealed an overrepresentation of cell wall-related proteins, some of them were further examined on a theoretical and experimental way. Surprisingly, overexpression of Ygpi led to the production of FFAs in the wildtype-like genetic background. Finally, some of the evaluated genes showed involvement in resistance to FFA toxicity.