Comparison of the Catabolic Rates of Linoleic and Oleic Acid Hydroperoxides Using 13CO2 Expired from Mice.

Unsaturated fatty acids, such as oleic and linoleic acids, are easily oxidized by exposure to temperature and light in the presence of air to form unsaturated fatty acid hydroperoxides as primary oxidation products. However, the catabolic rates of unsaturated fatty acid hydroperoxides in the human body remain unknown. In this study, ethyl esters of 13C-labeled linoleic acid (*C18:2-EE) and oleic acid (*C18:1-EE) and their hydroperoxides (*C18:2-EE-OOH and *C18:1-EE-OOH, respectively) prepared by the photo-oxidation of *C18:2-EE and *C18:1-EE, respectively, were administered to mice and their catabolic rates were determined by measuring the expired 13CO2 levels. *C18:2-EE-OOH and *C18:1-EE-OOH were ß-oxidized faster than *C18:2-EE and *C18:1-EE, respectively. Notably, rapid ß-oxidation of *C18:2-EE-OOH and *C18:1-EE-OOH was similar to that of medium-chain fatty acids, such as octanoic acid. Then, degradation products of C18:2-EE-OOH and C18:1-EE-OOH were analyzed under gastric conditions by gas chromatography/mass spectrometry. Major decomposition products of C18:2-EE-OOH and C18:1-EE-OOH were medium-chain compounds, such as octanoic acid ethyl ester, 9-oxo-nonanoic acid ethyl ester, and 10-oxo-8-decenoic acid ethyl esters, indicating that C18:2-EE-OOH and C18:1-EE-OOH isomers formed during photo-oxidation were decomposed under acidic conditions. These findings support previous reports that dietary lipid hydroperoxides are not absorbed into the intestine as lipid hydroperoxides but as degradation products. This is the first study to suggest that dietary lipid hydroperoxides decompose during gastric digestion to form medium-chain compounds that are directly absorbed into the liver via the portal vein and rapidly catabolized via ß-oxidation.

Upregulation of Hepatic Glutathione S-Transferase Alpha 1 Ameliorates Metabolic Dysfunction-Associated Steatosis by Degrading Fatty Acid Binding Protein 1.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common metabolic disease of the liver, characterized by hepatic steatosis in more than 5% of hepatocytes. However, despite the recent approval of the first drug, resmetirom, for the management of metabolic dysfunction-associated steatohepatitis, decades of target exploration and hundreds of clinical trials have failed, highlighting the urgent need to find new druggable targets for the discovery of innovative drug candidates against MASLD. Here, we found that glutathione S-transferase alpha 1 (GSTA1) expression was negatively associated with lipid droplet accumulation in vitro and in vivo. Overexpression of GSTA1 significantly attenuated oleic acid-induced steatosis in hepatocytes or high-fat diet-induced steatosis in the mouse liver. The hepatoprotective and anti-inflammatory drug bicyclol also attenuated steatosis by upregulating GSTA1 expression. A detailed mechanism showed that GSTA1 directly interacts with fatty acid binding protein 1 (FABP1) and facilitates the degradation of FABP1, thereby inhibiting intracellular triglyceride synthesis by impeding the uptake and transportation of free fatty acids. Conclusion: GSTA1 may be a good target for the discovery of innovative drug candidates as GSTA1 stabilizers or enhancers against MASLD.

Decoding the role of oxidative stress resistance and alternative carbon substrate assimilation in the mature biofilm growth mode of Candida glabrata.

BACKGROUND: Biofilm formation is viewed as a vital mechanism in C. glabrata pathogenesis. Although, it plays a significant role in virulence but transcriptomic architecture and metabolic pathways governing the biofilm growth mode of C. glabrata remain elusive. The present study intended to investigate the genes implicated in biofilm growth phase of C. glabrata through global transcriptomic approach. RESULTS: Functional analysis of Differentially expressed genes (DEGs) using gene ontology and pathways analysis revealed that upregulated genes are involved in the glyoxylate cycle, carbon-carbon lyase activity, pre-autophagosomal structure membrane and vacuolar parts whereas, down- regulated genes appear to be associated with glycolysis, ribonucleoside biosynthetic process, ribosomal and translation process in the biofilm growth condition. The RNA-Seq expression of eight selected DEGs (CgICL1, CgMLS1, CgPEP1, and CgNTH1, CgERG9, CgERG11, CgTEF3, and CgCOF1) was performed with quantitative real-time PCR (RT-qPCR). The gene expression profile of selected DEGs with RT-qPCR displayed a similar pattern of expression as observed in RNA-Seq. Phenotype screening of mutant strains generated for genes CgPCK1 and CgPEP1, showed that Cgpck1∆ failed to grow on alternative carbon substrate (Glycerol, Ethanol, Oleic acid) and similarly, Cgpep1∆ unable to grow on YPD medium supplemented with hydrogen peroxide. Our results suggest that in the absence of glucose, C. glabrata assimilate glycerol, oleic acid and generate acetyl coenzyme-A (acetyl-CoA) which is a central and connecting metabolite between catabolic and anabolic pathways (glyoxylate and gluconeogenesis) to produce glucose and fulfil energy requirements. CONCLUSIONS: The study was executed using various approaches (transcriptomics, functional genomics and gene deletion) and it revealed that metabolic plasticity of C. glabrata (NCCPF-100,037) in biofilm stage modulates its virulence and survival ability to counter the stress and may promote its transition from commensal to opportunistic pathogen. The observations deduced from the present study along with future work on characterization of the proteins involved in this intricate process may prove to be beneficial for designing novel antifungal strategies.

Fatty acyl-coenzyme A activates mitochondrial division through oligomerization of MiD49 and MiD51.

Mitochondrial fission occurs in many cellular processes, but the regulation of fission is poorly understood. We show that long-chain acyl-coenzyme A (LCACA) activates two related mitochondrial fission proteins, MiD49 and MiD51, by inducing their oligomerization, which activates their ability to stimulate the DRP1 GTPase. The 1:1 stoichiometry of LCACA:MiD in the oligomer suggests interaction in the previously identified nucleotide-binding pocket, and a point mutation in this pocket reduces LCACA binding and LCACA-induced oligomerization for MiD51. In cells, this LCACA binding mutant does not assemble into puncta on mitochondria or rescue MiD49/51 knockdown effects on mitochondrial length and DRP1 recruitment. Furthermore, cellular treatment with BSA-bound oleic acid, which causes increased LCACA, promotes mitochondrial fission in an MiD49/51-dependent manner. These results suggest that LCACA is an endogenous ligand for MiDs, inducing mitochondrial fission and providing a potential mechanism for fatty-acid-induced mitochondrial division. Finally, MiD49 or MiD51 oligomers synergize with Mff, but not with actin filaments, in DRP1 activation, suggesting distinct pathways for DRP1 activation.

Nuclear lipid droplets in Caco2 cells originate from nascent precursors and in situ at the nuclear envelope.

Intestinal epithelial cells convert excess fatty acids into triglyceride (TAG) for storage in cytoplasmic lipid droplets and secretion in chylomicrons. Nuclear lipid droplets (nLDs) are present in intestinal cells but their origin and relationship to cytoplasmic TAG synthesis and secretion is unknown. nLDs and related lipid-associated promyelocytic leukemia structures (LAPS) were abundant in oleate-treated Caco2 but less frequent in other human colorectal cancer cell lines and mouse intestinal organoids. nLDs and LAPS in undifferentiated oleate-treated Caco2 cells harbored the phosphatidate phosphatase Lipin1, its product diacylglycerol, and CTP:phosphocholine cytidylyltransferase (CCT)α. CCTα knockout Caco2 cells had fewer but larger nLDs, indicating a reliance on de novo PC synthesis for assembly. Differentiation of Caco2 cells caused large nLDs and LAPS to form regardless of oleate treatment or CCTα expression. nLDs and LAPS in Caco2 cells did not associate with apoCIII and apoAI and formed dependently of microsomal triglyceride transfer protein expression and activity, indicating they are not derived from endoplasmic reticulum luminal LDs precursors. Instead, undifferentiated Caco2 cells harbored a constitutive pool of nLDs and LAPS in proximity to the nuclear envelope that expanded in size and number with oleate treatment. Inhibition of TAG synthesis did affect the number of nascent nLDs and LAPS but prevented their association with promyelocytic leukemia protein, Lipin1α, and diacylglycerol, which instead accumulated on the nuclear membranes. Thus, nLD and LAPS biogenesis in Caco2 cells is not linked to lipoprotein secretion but involves biogenesis and/or expansion of nascent nLDs by de novo lipid synthesis.

Comparative evaluation of physical and chemical enhancement techniques for transdermal delivery of linagliptin.

Linagliptin is a dipeptidyl peptidase-4 inhibitor used for the management of type-2 diabetes. US FDA-approved products are available exclusively as oral tablets. The inherent drawbacks of the oral administration route necessitate exploring delivery strategies via other routes. In this study, we investigated the feasibility of transdermal administration of linagliptin through various approaches. We compared chemical penetration enhancers (oleic acid, oleyl alcohol, and isopropyl myristate) and physical enhancement techniques (iontophoresis, sonophoresis, microneedles, laser, and microdermabrasion) to understand their potential to improve transdermal delivery of linagliptin. To our knowledge, this is the first reported comparison of chemical and physical enhancement techniques for the transdermal delivery of a moderately lipophilic molecule. All physical enhancement techniques caused a significant reduction in the transepithelial electrical resistance of the skin samples. Disruption of the skin's structure post-treatment with physical enhancement techniques was further confirmed using characterization techniques such as dye binding, histology, and confocal microscopy. In vitro permeation testing (IVPT) demonstrated that the passive delivery of linagliptin across the skin was < 5 µg/sq.cm. Two penetration enhancers - oleic acid (93.39 ± 8.34 µg/sq.cm.) and oleyl alcohol (424.73 ± 42.86 µg/sq.cm.), and three physical techniques - iontophoresis (53.05 ± 0.79 µg/sq.cm.), sonophoresis (141.13 ± 34.22 µg/sq.cm.), and laser (555.11 ± 78.97 µg/sq.cm.) exceeded the desired target delivery for therapeutic effect. This study established that linagliptin is an excellent candidate for transdermal delivery and thoroughly compared chemical penetration and physical transdermal delivery strategies.

Oleic acid induces lipogenesis and NLRP3 inflammasome activation in organotypic mouse meibomian gland and human meibomian gland epithelial cells.

The accumulation of oleic acid (OA) in the meibum from patients with meibomian gland dysfunction (MGD) suggests that it may contribute to meibomian gland (MG) functional disorder, as it is a potent stimulator of acne-related lipogenesis and inflammation in sebaceous gland. Therefore, we investigate whether OA induces lipogenesis and inflammasome activation in organotypic cultured mouse MG and human meibomian gland epithelial cells (HMGECs). Organotypic cultured mouse MG and HMGECs were exposed to OA or combinations with specific AMPK agonists 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Lipogenic status, ductal keratinization, squamous metaplasia, NLRP3/ASC/Caspase-1 inflammasome activation, proinflammatory cytokine IL-1ß production, and AMPK pathway phosphorylation in MG were subsequently examined by lipid staining, immunofluorescence staining, immunohistochemical staining, ELISA assay, and Western blot analyses. We found that OA significantly induced lipid accumulation, ductal keratinization, and squamous metaplasia in organotypic cultured MG, as evidenced by increased lipids deposition within acini and duct, upregulated expression of lipogenic proteins (SREBP-1 and HMGCR), and elevation of K10/Sprr1b. Additionally, OA induced NLRP3/ASC/Caspase-1 inflammasome activation, cleavage of Caspase-1, and production of downstream proinflammatory cytokine IL-1ß. The findings of lipogenesis and NLRP3-related proinflammatory response in OA-stimulated HMGECs were consistent with those in organotypic cultured MG. OA exposure downregulated phospho-AMPK in two models, while AICAR treatment alleviated lipogenesis by improving AMPK/ACC phosphorylation and SREBP-1/HMGCR expression. Furthermore, AMPK amelioration inhibited activation of the NLRP3/ASC/Caspase-1 axis and secretion of IL-1ß, thereby relieving the OA-induced proinflammatory response. These results demonstrated that OA induced lipogenic disorder and NLRP3 inflammasome activation in organotypic cultured mouse MG and HMGECs by suppressing the AMPK signaling pathway, indicating OA may play an etiological role in MGD.

Creation of an Atherosclerosis Model Using Palmitic Acid and Oleic Acid in the Vascular Smooth Muscle Cells of Rats.

BACKGROUND: Atherosclerosis (AS) is a chronic vascular inflammatory disease resulting from vascular endothelial injury and lipid deposition, closely linked to abnormal lipid metabolism within the body. The critical processes involved in atherosclerosis encompass lipid deposition, oxidation, metabolic disruptions, and inflammatory stimulation within the inner vessel wall. Lipid deposition emerges as a pivotal factor triggering these pathological changes, with vascular smooth muscle cells (VSMCs) playing a significant role in the development of AS. Therefore, the goal was to employ lipids, specifically palmitic acid (PA) and oleic acid (OA) solutions, to stimulate VSMCs and create an in vitro atherosclerosis model. This approach allows for the establishment of a rapid and efficient cell model for simulating atherosclerosis in vitro. METHODS: Primary vascular smooth muscle cells (VSMCs) were isolated and cultured from the thoracic aorta of healthy rats using the tissue-block method. VSMCs were identified through cell climbing slices and immunofluorescence. The growth of VSMCs was observed using light microscopy. The logarithmic growth phase of VSMCs was induced and stimulated by various concentrations of palmitic acid (PA) and oleic acid (OA) ranging from 0 to 650 µmol/L, with a gradient dilution of 50 µmol/L. VSMC activity was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Intracellular lipid deposition was visualized through Oil Red O staining. The levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) within VSMCs were quantified using commercially available kits. RESULTS: The optimal conditions for VSMC proliferation were determined to be an OA concentration of 500 µmol/L, a PA concentration of 300 µmol/L, and a culture duration of 48 hours. In comparison to the control group, the presence of lipid droplets within VSMCs became significantly evident following treatment with OA or PA. Furthermore, the levels of TC, TG, and LDL-C increased, while the HDL-C content decreased after treatment with OA or PA. CONCLUSIONS: A research model for atherosclerosis (AS) and the early stages of cardiovascular events, specifically lipid deposition, was successfully established through the use of OA and PA solutions. This model has the potential to open up new research avenues for gaining a deeper understanding of the pathogenesis and progression of AS.

The Role of Neutral Sphingomyelinase-2 (NSM2) in the Control of Neutral Lipid Storage in T Cells.

The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied. Here, we identified the presence of the LD marker protein perilipin 3 (PLIN3) in the intracellular nano-environment of NSM2 using the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. In line with this, super-resolution structured illumination microscopy (SIM) shows NSM2 and PLIN3 co-localization in LD organelles in the presence of increased extracellular concentrations of oleic acid (OA). Furthermore, the association of enzymatically active NSM2 with isolated LDs correlates with increased Cer levels in these lipid storage organelles. NSM2 enzymatic activity is not required for NSM2 association with LDs, but negatively affects the LD numbers and cellular accumulation of long-chain unsaturated triacylglycerol (TAG) species. Concurrently, NSM2 expression promotes mitochondrial respiration and fatty acid oxidation (FAO) in response to increased OA levels, thereby shifting cells to a high energetic state. Importantly, endogenous NSM2 activity is crucial for primary human CD4+ T cell survival and proliferation in a FA rich environment. To conclude, our study shows a novel NSM2 intracellular localization to LDs and the role of enzymatically active NSM2 in metabolic response to enhanced FA concentrations in T cells.

Sesamin alleviates lipid accumulation induced by oleic acid via PINK1/Parkin-mediated mitophagy in HepG2 cells.

Sesamin, a special compound present in sesame and sesame oil, has been reported a role in regulating lipid metabolism, while the underlying mechanisms remain unclear. Autophagy has been reported associated with lipid metabolism and regarded as a key modulator in liver steatosis. The present work aimed to investigate whether sesamin could exert its protective effects against lipid accumulation via modulating autophagy in HepG2 cells stimulated with oleic acid (OA). Cell viability was evaluated using the CCK-8 method, and triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein, cholesterol (LDL-C), alanine aminotransferase (ALT), along with aspartate aminotransferase (AST) were assessed by oil red O staining, transmission electron microscopy (TEM), and biochemical kits to investigate the lipid-lowering effects of sesamin. Differentially expressed genes were screened by RNA sequencing and validated using real-time quantitative PCR and Western blot. Autophagy and mitophagy related molecules were analyzed employing TEM, Western blot, and immunofluorescence. The data shows that in HepG2 cells stimulated by OA, sesamin reduces levels of TG, TC, LDL-C, ALT, and AST while elevating HDL-C, alleviates the lipid accumulation and improves fatty acid metabolism through modulating the levels of fat metabolism related genes including PCSK9, FABP1, CD36, and SOX4. Sesamin restores the suppressed autophagy in HepG2 cells caused by OA, which could be blocked by autophagy inhibitors. This indicates that sesamin improves fatty acid metabolism by enhancing autophagy levels, thereby mitigating the intracellular lipid accumulation. Furthermore, sesamin significantly enhances the mitophagy and improves mitochondrial homeostasis via activating the PINK/Parkin pathway. These data suggest that sesamin alleviates the excessive lipid accumulation in HepG2 caused by OA by restoring the impaired mitophagy via the PINK1/Parkin pathway, probably playing a preventive or therapeutic role in hepatic steatosis.

Analysis of the therapeutic potential of miR-124 and miR-16 in non-alcoholic fatty liver disease.

BACKGROUNDS: Non-alcoholic fatty liver disease (NAFLD) is a common condition affecting >25 % of the population worldwide. This disorder ranges in severity from simple steatosis (fat accumulation) to severe steatohepatitis (inflammation), fibrosis and, at its end-stage, liver cancer. A number of studies have identified overexpression of several key genes that are critical in the initiation and progression of NAFLD. MiRNAs are potential therapeutic agents that can regulate several genes simultaneously. Therefore, we transfected cell lines with two key miRNAs involved in targeting NAFLD-related genes. METHODS: The suppression effects of the investigated miRNAs (miR-124 and miR-16) and genes (TNF, TLR4, SCD, FASN, SREBF2, and TGFß-1) from our previous study were investigated by real-time PCR in Huh7 and HepG2 cells treated with oleic acid. Oil red O staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay were utilized to assess cell lipid accumulation and cytotoxic effects of the miRNAs, respectively. The pro-oxidant-antioxidant balance (PAB) assay was undertaken for miR-16 and miR-124 after cell transfection. RESULTS: Following transfection of miRNAs into HepG2, oil red O staining showed miR-124 and miR-16 reduced oleic acid-induced lipid accumulation by 35.2 % and 28.6 % respectively (p < 0.05). In Huh7, miR-124 and miR-16 reduced accumulation by 23.5 % and 31.3 % respectively (p < 0.05) but without impacting anti-oxidant activity. Real-time PCR in HepG2 revealed miR-124 decreased expression of TNF by 0.13-fold, TLR4 by 0.12-fold and SREBF2 by 0.127-fold (p < 0.05). miR-16 decreased TLR4 by 0.66-fold and FASN by 0.3-fold (p < 0.05). In Huh7, miR-124 decreased TNF by 0.12-fold and FASN by 0.09-fold (p < 0.05). miR-16 decreased SCD by 0.28-fold and FASN by 0.64-fold (p < 0.05). MTT assays showed, in HepG2, viability was decreased 24.7 % by miR-124 and decreased 33 % by miR-16 at 72 h (p < 0.05). In Huh7, miR-124 decreased viability 42 % at 48 h and 29.33 % at 72 h (p < 0.05), while miR-16 decreased viability by 32.3 % (p < 0.05). CONCLUSION: These results demonstrate the ability of miR-124 and miR-16 to significantly reduce lipid accumulation and expression of key pathogenic genes associated with NAFLD through direct targeting. Though this requires further in vivo investigation.

Scutellarin inhibits oleic acid induced vascular smooth muscle foam cell formation via activating autophagy and inhibiting NLRP3 inflammasome activation.

Abnormalities in vascular smooth muscle cells (VSMCs) are pivotal in the pathogenesis of cardiovascular pathologies such as atherosclerosis and hypertension. Scutellarin (Scu), a flavonoid derived from marigold flowers, exhibits a spectrum of biological activities including anti-inflammatory, antioxidant, antitumor, immunomodulatory and antimicrobial effects. Notably, Scu has demonstrated the capacity to mitigate vascular endothelial damage and prevent atherosclerosis via its antioxidative properties. Nevertheless, the influence of Scu on the formation of VSMC-derived foam cells remains underexplored. In this study, Scu was evidenced to efficaciously attenuate oleic acid (OA)-induced lipid accumulation and the upregulation of adipose differentiation-associated protein Plin2 in a dose- and time-responsive manner. We elucidated that Scu effectively diminishes OA-provoked VSMC foam cell formation. Further, it was established that Scu pretreatment augments the protein expression of LC3B-II and the mRNA levels of Map1lc3b and Becn1, concurrently diminishing the protein levels of the NLRP3 inflammasome compared to the OA group. Activation of autophagy through rapamycin attenuated NLRP3 inflammasome protein expression, intracellular lipid droplet content and Plin2 mRNA levels. Scu also counteracted the OA-induced decrement of LC3B-II levels in the presence of bafilomycin-a1, facilitating the genesis of autophagosomes and autolysosomes. Complementarily, in vivo experiments revealed that Scu administration substantially reduced arterial wall thickness, vessel wall cross-sectional area, wall-to-lumen ratio and serum total cholesterol levels in comparison to the high-fat diet model group. Collectively, our findings suggest that Scu attenuates OA-induced VSMC foam cell formation through the induction of autophagy and the suppression of NLRP3 inflammasome activation.

Scd-1 deficiency promotes the differentiation of CD8+ T effector.

The impact of various fatty acid types on adaptive immunity remains uncertain, and their roles remain unelucidated. Stearoyl-CoA desaturase (Scd) is a Δ-9 desaturase, which is a key rate-limiting enzyme for the conversion of saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA) in the fatty acid de novo synthesis. Scd-1 converts stearic acid (SA) and palmitic acid (PA) to oleic acid (OA) and palmitoleic acid (PO), respectively. In this study, through a series of experiments, we showed that Scd-1 and its resulting compound, OA, have a substantial impact on the transformation of CD8+ naïve T cells into effector T cells. Inactivation of Scd-1 triggers the specialization of CD8+ T cells into the Teff subset, enhancing the effector function and mitochondrial metabolism of Teff cells, and OA can partially counteract this. A deeper understanding of lipid metabolism in immune cells and its impact on cell function can lead to new therapeutic approaches for controlling the immune response and improving prognosis.

Identification of oleic acid as an endogenous ligand of GPR3.

Although GPR3 plays pivotal roles in both the nervous system and metabolic processes, such as cold-induced thermogenesis, its endogenous ligand remains elusive. Here, by combining structural approach (including cryo-electron microscopy), mass spectrometry analysis, and functional studies, we identify oleic acid (OA) as an endogenous ligand of GPR3. Our study reveals a hydrophobic tunnel within GPR3 that connects the extracellular side of the receptor to the middle of plasma membrane, enabling fatty acids to readily engage the receptor. Functional studies demonstrate that OA triggers downstream Gs signaling, whereas lysophospholipids fail to activate the receptor. Moreover, our research reveals that cold stimulation induces the secretion of OA in mice, subsequently activating Gs/cAMP/PKA signaling in brown adipose tissue. Notably, brown adipose tissues from Gpr3 knockout mice do not respond to OA during cold stimulation, reinforcing the significance of GPR3 in this process. Finally, we propose a "born to be activated and cold to enhance" model for GPR3 activation. Our study provides a starting framework for the understanding of GPR3 signaling in cold-stimulated thermogenesis.

(+)-Lipoic acid reduces mitochondrial unfolded protein response and attenuates oxidative stress and aging in an in vitro model of non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a liver disorder characterized by the ac-cumulation of fat in hepatocytes without alcohol consumption. Mitochondrial dysfunction and endoplasmic reticulum (ER) stress play significant roles in NAFLD pathogenesis. The unfolded protein response in mitochondria (UPRmt) is an adaptive mechanism that aims to restore mitochondrial protein homeostasis and mitigate cellular stress. This study aimed to investigate the effects of ( +)-Lipoic acid (ALA) on UPRmt, inflammation, and oxidative stress in an in vitro model of NAFLD using HepG2 cells treated with palmitic acid and oleic acid to induce steatosis. RESULTS: Treatment with palmitic and oleic acids increased UPRmt-related proteins HSP90 and HSP60 (heat shock protein), and decreased CLPP (caseinolytic protease P), indicating ER stress activation. ALA treatment at 1 µM and 5 µM restored UPRmt-related protein levels. PA:OA (palmitic acid:oleic acid)-induced ER stress markers IRE1α (Inositol requiring enzyme-1), CHOP (C/EBP Homologous Protein), BIP (Binding Immunoglobulin Protein), and BAX (Bcl-2-associated X protein) were significantly reduced by ALA treatment. ALA also enhanced ER-mediated protein glycosylation and reduced oxidative stress, as evidenced by decreased GPX1 (Glutathione peroxidase 1), GSTP1 (glutathione S-transferase pi 1), and GSR (glutathione-disulfide reductase) expression and increased GSH (Glutathione) levels, and improved cellular senescence as shown by the markers ß-galactosidase, γH2Ax and Klotho-beta. CONCLUSIONS: In conclusion, ALA ameliorated ER stress, oxidative stress, and inflammation in HepG2 cells treated with palmitic and oleic acids, potentially offering therapeutic benefits for NAFLD providing a possible biochemical mechanism underlying ALA beneficial effects.

Chronic exposure to parabens promotes non-alcoholic fatty liver disease in association with the changes of the gut microbiota and lipid metabolism.

Non-alcoholic fatty liver disease (NAFLD) has become a serious public health issue due to changing dietary patterns and composition. However, the relationship between NAFLD occurrence and food additives, such as preservatives, remains unknown. This study aimed to evaluate the toxicity of parabens, namely methylparaben (MeP) and ethylparaben (EtP), in relation to NAFLD occurrence in mice under different dietary conditions. Exposure to MeP and EtP exacerbated high-fat diet (HFD)-induced obesity, glucose intolerance, higher serum lipid concentrations, and fat accumulation by upregulating genes involved in lipid metabolism. Untargeted metabolomics revealed that arachidonic acid (AA) metabolism was the top enriched pathway upon MeP and EtP exposure in the presence of HFD. 11,12-Epoxyeicosatrienoic acid (11,12-EET) was the most abundant AA metabolite and was significantly reduced upon exposure to MeP or EtP. Moreover, an integrative analysis of differential fecal taxa at the genus level and serum AA metabolites revealed significant associations. In addition, MeP and EtP enhanced lipid accumulation in AML12 cells and HepG2 cells cultured with oleic acid. 11,12-EET supplementation could significantly alleviate lipid accumulation by suppressing the expression of lipid metabolism-related genes and proteins. The present study suggests that chronic exposure to MeP and EtP promoted NAFLD via gut microbiota-dependent AA metabolism. These results highlight the need for reducing oral exposure to synthetic preservatives to improve metabolic disturbance under HFD conditions.

Protective effect of oleic acid against very long-chain fatty acid-induced apoptosis in peroxisome-deficient CHO cells.

Very long-chain fatty acids (VLCFAs) are degraded exclusively in peroxisomes, as evidenced by the accumulation of VLCFAs in patients with certain peroxisomal disorders. Although accumulation of VLCFAs is considered to be associated with health issues, including neuronal degeneration, the mechanisms underlying VLCFAs-induced tissue degeneration remain unclear. Here, we report the toxic effect of VLCFA and protective effect of C18: 1 FA in peroxisome-deficient CHO cells. We examined the cytotoxicity of saturated and monounsaturated VLCFAs with chain-length at C20-C26, and found that longer and saturated VLCFA showed potent cytotoxicity at lower accumulation levels. Furthermore, the extent of VLCFA-induced toxicity was found to be associated with a decrease in cellular C18:1 FA levels. Notably, supplementation with C18:1 FA effectively rescued the cells from VLCFA-induced apoptosis without reducing the cellular VLCFAs levels, implying that peroxisome-deficient cells can survive in the presence of accumulated VLCFA, as long as the cells keep sufficient levels of cellular C18:1 FA. These results suggest a therapeutic potential of C18:1 FA in peroxisome disease and may provide new insights into the pharmacological effect of Lorenzo's oil, a 4:1 mixture of C18:1 and C22:1 FA.

Engineering Yarrowia lipolytica for sustainable ricinoleic acid production: A pathway to free fatty acid synthesis.

Ricinoleic acid (C18:1-OH, RA) is a valuable hydroxy fatty acid with versatile applications. The current industrial source of RA relies on the hydrolysis of castor bean oil. However, the coexistence of the toxic compound ricin and the unstable supply of this plant have led to an exploration of promising alternatives: generating RA in heterologous plants or microorganisms. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce RA in the form of free fatty acids (FFA). First, we overexpressed fungal Δ12 oleate hydroxylase gene (CpFAH12) from Claviceps purpurea while deleting genes related to fatty acid degradation (MEF1 and PEX10) and oleic acid desaturation (FAD2). Since Δ12 oleate hydroxylase converts oleic acid (C18:1) located at the sn-2 position of phosphatidylcholine (PC), we next focused on increasing the PC pool containing oleic acid. This objective was achieved thorough implementing metabolic engineering strategies designed to enhance the biosynthesis of PC and C18 fatty acids. To increase the PC pool, we redirected the flux towards phospholipid biosynthesis by deleting phosphatidic acid phosphatase genes (PAH1 and APP1) and diacylglycerol acyltransferase gene (DGA1), involved in the production of diacylglycerol and triacylglycerol, respectively. Furthermore, the PC biosynthesis via the CDP-DAG pathway was enhanced through the overexpression of CDS1, PSD1, CHO2, and OPI3 genes. Subsequently, to increase the oleic acid content within PC, we overexpressed the heterologous fatty acid elongase gene (MaC16E) involved in the conversion of C16 to C18 fatty acids. As RA production titer escalated, the produced RA was mainly found in the FFA form, leading to cell growth inhibition. The growth inhibition was mitigated by inducing RA secretion via Triton X-100 treatment, a process that simultaneously amplified RA production by redirecting flux towards RA synthesis. The final engineered strain JHYL-R146 produced 2.061 g/L of free RA in a medium treated with 5% Triton X-100, constituting 74% of the total FFAs produced. Generating free RA offers the added benefit of bypassing the hydrolysis stage required when employing castor bean oil as an RA source. This achievement represents the highest level of RA synthesis from glucose reported thus far, underscoring the potential of Y. lipolytica as a host for sustainable RA production.

Assessment of endocrine disruptor impacts on lipid metabolism in a fatty acid-supplemented HepaRG human hepatic cell line.

The incidence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing worldwide. This disease encompasses several stages, from steatosis to steatohepatitis and, eventually, to fibrosis and cirrhosis. Exposure to environmental contaminants is one of the risk factors and an increasing amount of evidence points to a role for endocrine disrupting compounds (EDCs). This study assesses the impact of selected EDCs on the formation of lipid droplets, the marker for steatosis in a hepatic model. The mechanisms underlying this effect are then explored. Ten compounds were selected according to their obesogenic properties: bisphenol A, F and S, butyl-paraben, cadmium chloride, p,p'-DDE, DBP, DEHP, PFOA and PFOS. Using a 2D or 3D model, HepaRG cells were exposed to the compounds with or without fatty acid supplementation. Then, the formation of lipid droplets was quantified by an automated fluorescence-based method. The expression of genes and proteins involved in lipid metabolism and the impact on cellular respiration was analyzed. The formation of lipid droplets, which is revealed or enhanced by oleic acid supplementation, was most effectively induced by p,p'-DDE and DEHP. Experiments employing either 2D or 3D culture conditions gave similar results. Both compounds induced the expression of PLIN2. p,p'-DDE also appears to act by decreasing in fatty acid oxidation. Some EDCs were able to induce the formation of lipid droplets, in HepaRG cells, an effect which was increased after supplementation of the cells with oleic acid. A full understanding of the mechanisms of these effects will require further investigation. The novel automated detection method described here may also be useful in the future as a regulatory test for EDC risk assessment.

Oleic acid as modulator of oxidative stress in European sea bass (Dicentrarchus labrax) juveniles fed high dietary lipid levels.

Although the benefits of oleic acid (OA) have been established in mammals, its effects on fish remain understudied. The aim of this study was to evaluate the antioxidant potential of OA in the liver, intestine, and muscle of European sea bass juveniles fed diets containing different lipid levels. For that purpose, six diets with 16 or 22% lipids and 0, 1, and 2% OA were formulated and triplicate groups of European sea bass juveniles (21.4 g) were fed with these experimental diets for 9 weeks. Increasing dietary lipid levels or OA supplementation did not affect antioxidant enzyme activity in the liver and muscle. Superoxide dismutase (SOD) activity in the intestine increased with both the dietary lipid and OA levels, while glucose 6 phosphate dehydrogenase (G6PDH) activity increased only with dietary OA supplementation. Reduced glutathione (GSH) and total glutathione (tGSH) content were higher in the liver and intestine of fish fed the low-lipid diets, while in the high-lipid diets it was lower in the muscle than in the liver and intestine. Present findings suggest that OA plays a role in the antioxidant defense mechanisms of European sea bass, particularly at the intestine level, but additional research is required to further assess the potential benefits of incorporating OA into the diets.