Dietary sn-2 palmitate influences cognitive behavior by increasing the transport of liver-produced lysophosphatidylcholine VLCPUFAs to the brain.

Investigations indicated that sn-2 palmitate have positive effects on brain development, although its mechanism remains largely unexamined. This research delved into how a diet abundant in sn-2 palmitate influenced the cognitive behavior of mice and elucidated the associated mechanisms using metabolomics and lipidomics. The study demonstrated that dietary sn-2 palmitate led to improved working memory and cognition in mice, as well as an increase in brain BDNF concentration when compared to those fed blend vegetable oil (BVO). This was because sn-2 palmitate feeding promoted the synthesis of very long-chain fatty acids (VLCPUFAs) for the lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) in the liver. This led to more efficient delivery of VLCPUFAs to the brain, as indicated by elevated concentration of LPC/LPE-VLCPUFAs in the liver and heightened expression of the major facilitator superfamily domain containing 2a (MFSD2A). In essence, this paper offered a potential mechanism by which sn-2 palmitate enhanced mouse neurodevelopment.

ANT-Mediated Inhibition of the Permeability Transition Pore Alleviates Palmitate-Induced Mitochondrial Dysfunction and Lipotoxicity.

Hyperlipidemia is a major risk factor for vascular lesions in diabetes mellitus and other metabolic disorders, although its basis remains poorly understood. One of the key pathogenetic events in this condition is mitochondrial dysfunction associated with the opening of the mitochondrial permeability transition (MPT) pore, a drop in the membrane potential, and ROS overproduction. Here, we investigated the effects of bongkrekic acid and carboxyatractyloside, a potent blocker and activator of the MPT pore opening, respectively, acting through direct interaction with the adenine nucleotide translocator, on the progression of mitochondrial dysfunction in mouse primary lung endothelial cells exposed to elevated levels of palmitic acid. Palmitate treatment (0.75 mM palmitate/BSA for 6 days) resulted in an 80% decrease in the viability index of endothelial cells, which was accompanied by mitochondrial depolarization, ROS hyperproduction, and increased colocalization of mitochondria with lysosomes. Bongkrekic acid (25 µM) attenuated palmitate-induced lipotoxicity and all the signs of mitochondrial damage, including increased spontaneous formation of the MPT pore. In contrast, carboxyatractyloside (10 µM) stimulated cell death and failed to prevent the progression of mitochondrial dysfunction under hyperlipidemic stress conditions. Silencing of gene expression of the predominate isoform ANT2, similar to the action of carboxyatractyloside, led to increased ROS generation and cell death under conditions of palmitate-induced lipotoxicity in a stably transfected HEK293T cell line. Altogether, these results suggest that targeted manipulation of the permeability transition pore through inhibition of ANT may represent an alternative approach to alleviate mitochondrial dysfunction and cell death in cell culture models of fatty acid overload.

Collagen I Increases Palmitate-Induced Lipotoxicity in HepG2 Cells via Integrin-Mediated Death.

Various strategies have been employed to improve the reliability of 2D, 3D, and co-culture in vitro models of nonalcoholic fatty liver disease, including using extracellular matrix proteins such as collagen I to promote cell adhesion. While studies have demonstrated the significant benefits of culturing cells on collagen I, its effects on the HepG2 cell line after exposure to palmitate (PA) have not been investigated. Therefore, this study aimed to assess the effects of PA-induced lipotoxicity in HepG2 cultured in the absence or presence of collagen I. HepG2 cultured in the absence or presence of collagen I was exposed to PA, followed by analyses that assessed cell proliferation, viability, adhesion, cell death, mitochondrial respiration, reactive oxygen species production, gene and protein expression, and triacylglycerol accumulation. Culturing HepG2 on collagen I was associated with increased cell proliferation, adhesion, and expression of integrin receptors, and improved cellular spreading compared to culturing them in the absence of collagen I. However, PA-induced lipotoxicity was greater in collagen I-cultured HepG2 than in those cultured in the absence of collagen I and was associated with increased α2ß1 receptors. In summary, the present study demonstrated for the first time that collagen I-cultured HepG2 exhibited exacerbated cell death following exposure to PA through integrin-mediated death. The findings from this study may serve as a caution to those using 2D models or 3D scaffold-based models of HepG2 in the presence of collagen I.

Greater oxidation of dietary linoleate compared to palmitate in humans following an acute high-carbohydrate diet.

BACKGROUND: We have previously demonstrated that dietary saturated fatty acids (SFA), when compared to polyunsaturated fatty acids (PUFA), are preferentially partitioned into oxidation pathways. However, it remains unclear if this preferential handling is maintained when hepatocellular metabolism is shifted toward fatty acid (FA) esterification and away from oxidation, such as when hepatic de novo lipogenesis (DNL) is upregulated. AIM: To investigate whether an acute upregulation of hepatic DNL influences dietary FA partitioning into oxidation pathways. METHODS: 20 healthy volunteers (11 females) underwent a fasting baseline visit followed by two study days, 2-weeks apart. Prior to each study day, participants consumed an isocaloric high-carbohydrate diet (to upregulate hepatic DNL) for 3-days. On the two study days, participants consumed an identical standardised test meal that contained either [U13C]palmitate or [U13C]linoleate, in random order, to trace the fate of dietary FA. Blood and breath samples were collected over a 6h postprandial period and 13C enrichment in breath CO2 and plasma lipid fractions were measured using gas-chromatography-combustion-isotope ratio mass spectrometry. RESULTS: Compared to the baseline visit, fasting plasma triglyceride concentrations and markers of hepatic DNL, the lipogenic and stearyl-CoA desaturase indices, were significantly (p < 0.05) increased after consumption of the high-carbohydrate diet. Appearance of 13C in expired CO2 and tracer recovery were significantly (p < 0.05) higher after consumption of the meal containing [U13C]linoleate compared to [U13C]palmitate (5.1 ± 0.5% vs. 3.7 ± 0.4%), respectively. Incorporation of 13C into the plasma triglyceride and non-esterified fatty acid pool was significantly (p < 0.001) greater for [U13C]palmitate compared to [U13C]linoleate. CONCLUSION: Dietary PUFA compared to SFA appear to be preferentially partitioned into oxidation pathways during an acute upregulation of hepatic DNL, thus consumption of a PUFA-enriched diet may help mitigate intrahepatic triglyceride accumulation in individuals at risk of cardiometabolic disease.

Glutathione transferase P1 is modified by palmitate.

Glutathione transferase P1 (GSTP1) is a multi-functional protein that protects cells from electrophiles by catalyzing their conjugation with glutathione, and contributes to the regulation of cell proliferation, apoptosis, and signalling. GSTP1, usually described as a cytosolic enzyme, can localize to other cell compartments and we have reported its strong association with the plasma membrane. In the current study, the hypothesis that GSTP1 is palmitoylated and this modification facilitates its dynamic localization and function was investigated. Palmitoylation is the reversible post-translational addition of a 16-C saturated fatty acid to proteins, most commonly on Cys residues through a thioester bond. GSTP1 in MCF7 cells was modified by palmitate, however, GSTP1 Cys to Ser mutants (individual and Cys-less) retained palmitoylation. Treatment of palmitoylated GSTP1 with 0.1 N NaOH, which cleaves ester bonds, did not remove palmitate. Purified GSTP1 was spontaneously palmitoylated in vitro and peptide sequencing revealed that Cys48 and Cys102 undergo S-palmitoylation, while Lys103 undergoes the rare N-palmitoylation. N-palmitoylation occurs via a stable NaOH-resistant amide bond. Analysis of subcellular fractions of MCF7-GSTP1 cells and a modified proximity ligation assay revealed that palmitoylated GSTP1 was present not only in the membrane fraction but also in the cytosol. GSTP1 isolated from E. coli, and MCF7 cells (grown under fatty acid free or regular conditions), associated with plasma membrane-enriched fractions and this association was not altered by palmitoyl CoA. Overall, GSTP1 is modified by palmitate, at multiple sites, including at least one non-Cys residue. These modifications could contribute to regulating the diverse functions of GSTP1.

Ferulic acid restores mitochondrial dynamics and autophagy via AMPK signaling pathway in a palmitate-induced hepatocyte model of metabolic syndrome.

Mitochondrial dysfunction, characterized by elevated oxidative stress, impaired energy balance, and dysregulated mitochondrial dynamics, is a hallmark of metabolic syndrome (MetS) and its comorbidities. Ferulic acid (FA), a principal phenolic compound found in whole grains, has demonstrated potential in ameliorating oxidative stress and preserving energy homeostasis. However, the influence of FA on mitochondrial health within the context of MetS remains unexplored. Moreover, the impact of FA on autophagy, which is essential for maintaining energy homeostasis and mitochondrial integrity, is not fully understood. Here, we aimed to study the mechanisms of action of FA in regulating mitochondrial health and autophagy using palmitate-treated HepG2 hepatocytes as a MetS cell model. We found that FA improved mitochondrial health by restoring redox balance and optimizing mitochondrial dynamics, including biogenesis and the fusion/fission ratio. Additionally, FA was shown to recover autophagy and activate AMPK-related cell signaling. Our results provide new insights into the therapeutic potential of FA as a mitochondria-targeting agent for the prevention and treatment of MetS.

Effectiveness and Biocompatibility Evaluation of a Novel Absorbable Bone Wax Used in Bone Tissue.

This study aims to determine the hemostatic effectivity and biocompatibility of a novel absorbable bone wax in comparison with a commercially available product. Eighteen small fat-tail sheep were used to simulate clinical surface bleeding of sternal injury. Hemostasis effectiveness, the degree of bone healing, micro-computed tomography, and histopathology were evaluated over a period after the application of the material to the surgically created wound. The absorbable bone wax used in the study stopped bleeding immediately and did not affect bone healing. The histopathological results also showed that there were no complications associated with the new material. The results showed that the new absorbable bone wax used in this study was effective and biocompatible.

2-Bromopalmitate treatment attenuates senescence phenotype in human adult cells - possible role of palmitoylation.

Cells may undergo senescence in response to DNA damage, which is associated with cell cycle arrest, altered gene expression and altered cell morphology. Protein palmitoylation is one of the mechanisms by which the DNA damage response is regulated. Therefore, we hypothesized that protein palmitoylation played a role in regulation of the senescent phenotype. Here, we showed that treatment of senescent human vascular smooth muscle cells (VSMCs) with 2-bromopalmitate (2-BP), an inhibitor of protein acyltransferases, is associated with changes in different aspects of the senescent phenotype, including the resumption of cell proliferation, a decrease in DNA damage markers and the downregulation of senescence-associated ß-galactosidase activity. The effects were dose dependent and associated with significantly decreased total protein palmitoylation level. We also showed that the senescence-modifying properties of 2-BP were at least partially mediated by the downregulation of elements of DNA damage-related molecular pathways, such as phosphorylated p53. Our data suggest that cell senescence may be regulated by palmitoylation, which provides a new perspective on the role of this posttranslational modification in age-related diseases.

Extract of Phyllanthus emblica L. fruit stimulates basal glucose uptake and ameliorates palmitate-induced insulin resistance through AMPK activation in C2C12 myotubes.

BACKGROUND: The fruit of Phyllanthus emblica L., a traditional medicine in China and India, is used to treat diabetes mellitus. Its water extract (WEPE) has demonstrated hypoglycemic effects in diabetic rats, but its mechanisms on glucose utilization and insulin resistance in skeletal muscle remain unclear. Therefore, this study aims to investigate the effects and underlying mechanisms of WEPE on glucose utilization and insulin resistance using C2C12 myotubes. METHODS: Effects of WEPE on glucose uptake, GLUT4 translocation, and AMPK and AKT phosphorylation were investigated in C2C12 myotubes and palmitate-treated myotubes. An AMPK inhibitor and siRNA were used to explore the mechanisms of WEPE. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay, and protein expression and GLUT4 translocation were assessed via western blotting. RESULTS: In normal myotubes, WEPE significantly stimulated glucose uptake and GLUT4 translocation to the plasma membrane at concentrations of 125 and 250 µg/mL. This was accompanied by an increase in the phosphorylation of AMPK and its downstream targets. However, both compound C and AMPK siRNA blocked the WEPE-induced GLUT4 translocation and glucose uptake. Moreover, pretreatment with STO-609, a calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor, inhibited WEPE-induced AMPK phosphorylation and attenuated the WEPE-stimulated glucose uptake and GLUT4 translocation. In myotubes treated with palmitate, WEPE prevented palmitate-induced insulin resistance by enhancing insulin-mediated glucose uptake and AKT phosphorylation. It also restored the insulin-mediated translocation of GLUT4 from cytoplasm to membrane. However, these effects of WEPE on glucose uptake and GLUT4 translocation were blocked by pretreatment with compound C. CONCLUSIONS: WEPE significantly stimulated basal glucose uptake though CaMKKß/AMPK pathway and markedly ameliorated palmitate-induced insulin resistance by activating the AMPK pathway in C2C12 myotubes.

Extracellular vesicles released from microglia after palmitate exposure impact brain function.

Dietary patterns that include an excess of foods rich in saturated fat are associated with brain dysfunction. Although microgliosis has been proposed to play a key role in the development of brain dysfunction in diet-induced obesity (DIO), neuroinflammation with cytokine over-expression is not always observed. Thus, mechanisms by which microglia contribute to brain impairment in DIO are uncertain. Using the BV2 cell model, we investigated the gliosis profile of microglia exposed to palmitate (200 µmol/L), a saturated fatty acid abundant in high-fat diet and in the brain of obese individuals. We observed that microglia respond to a 24-hour palmitate exposure with increased proliferation, and with a metabolic network rearrangement that favors energy production from glycolysis rather than oxidative metabolism, despite stimulated mitochondria biogenesis. In addition, while palmitate did not induce increased cytokine expression, it modified the protein cargo of released extracellular vesicles (EVs). When administered intra-cerebroventricularly to mice, EVs secreted from palmitate-exposed microglia in vitro led to memory impairment, depression-like behavior, and glucose intolerance, when compared to mice receiving EVs from vehicle-treated microglia. We conclude that microglia exposed to palmitate can mediate brain dysfunction through the cargo of shed EVs.

Palmitoylation of NLRP3 Modulates Inflammasome Activation and Inflammatory Bowel Disease Development.

Aberrant activity of NLRP3 has been shown associations with severe diseases. Palmitoylation is a kind of protein post-translational modification, which has been shown to regulate cancer development and the innate immune system. Here, we showed that NLRP3 is palmitoylated at Cys419 and that palmitoyltransferase ZDHHC17 is the predominant enzyme that mediates NLRP3 palmitoylation and promotes NLRP3 activation by interacting with NLRP3 and facilitating NIMA-related kinase 7 (NEK7)-NLRP3 interactions. Blockade of NLRP3 palmitoylation by a palmitoylation inhibitor, 2-bromopalmitate, effectively inhibited NLRP3 activation in vitro. Also, in a dextran sulfate sodium-induced colitis model in mice, 2-bromopalmitate application could attenuate weight loss, improve the survival rate, and rescue pathological changes in the colon of mice. Overall, our study reveals that palmitoylation of NLPR3 modulates inflammasome activation and inflammatory bowel disease development. We propose that drugs targeting NLRP3 palmitoylation could be promising candidates in the treatment of NLRP3-mediated inflammatory diseases.

Design and Development of a Cost-Effective Transnasal Skull Base Bone Wax Applicator: A Technical Note.

Bone wax is an important hemostatic agent used in neurosurgical practice from more than a century. It acts by mechanical tamponade effect to stop the oozing from cancellous bone. Bone wax application over the skull surface over the vertex is easy and can be applied with fingers. In deeper areas, one uses dissector to apply the bone wax; however, it becomes difficult at times to apply in transnasal surgery using the same dissectors. Author designed a cost-effective 20-cm-long different angled bone wax applicator for skull base transnasal surgery. This applicator is cost-effective and not described previously in English literature.

Regulatory Role of Fatty Acid Metabolism on Glucose-Induced Changes in Insulin and Glucagon Secretion by Pancreatic Islet Cells.

A detailed study of palmitate metabolism in pancreatic islets subject to different experimental conditions, like varying concentrations of glucose, as well as fed or starved conditions, has allowed us to explore the interaction between the two main plasma nutrients and its consequences on hormone secretion. Palmitate potentiates glucose-induced insulin secretion in a concentration-dependent manner, in a physiological range of both palmitate (0-2 mM) and glucose (6-20 mM) concentrations; at glucose concentrations lower than 6 mM, no metabolic interaction with palmitate was apparent. Starvation (48 h) increased islet palmitate oxidation two-fold, and the effect was resistant to its inhibition by glucose (6-20 mM). Consequently, labelled palmitate and glucose incorporation into complex lipids were strongly suppressed, as well as glucose-induced insulin secretion and its potentiation by palmitate. 2-bromostearate, a palmitate oxidation inhibitor, fully recovered the synthesis of complex lipids and insulin secretion. We concluded that palmitate potentiation of the insulin response to glucose is not attributable to its catabolic mitochondrial oxidation but to its anabolism to complex lipids: islet lipid biosynthesis is dependent on the uptake of plasma fatty acids and the supply of α-glycerol phosphate from glycolysis. Islet secretion of glucagon and somatostatin showed a similar dependence on palmitate anabolism as insulin. The possible mechanisms implicated in the metabolic coupling between glucose and palmitate were commented on. Moreover, possible mechanisms responsible for islet gluco- or lipotoxicity after a long-term stimulation of insulin secretion were also discussed. Our own data on the simultaneous stimulation of insulin, glucagon, and somatostatin by glucose, as well as their modification by 2-bromostearate in perifused rat islets, give support to the conclusion that increased FFA anabolism, rather than its mitochondrial oxidation, results in a potentiation of their stimulated release. Starvation, besides suppressing glucose stimulation of insulin secretion, also blocks the inhibitory effect of glucose on glucagon secretion: this suggests that glucagon inhibition might be an indirect or direct effect of insulin, but not of glucose. In summary, there seems to exist three mechanisms of glucagon secretion stimulation: 1. glucagon stimulation through the same secretion coupling mechanism as insulin, but in a different range of glucose concentrations (0 to 5 mM). 2. Direct or indirect inhibition by secreted insulin in response to glucose (5-20 mM). 3. Stimulation by increased FFA anabolism in glucose intolerance or diabetes in the context of hyperlipidemia, hyperglycemia, and hypo-insulinemia. These conclusions were discussed and compared with previous published data in the literature. Specially, we discussed the mechanism for inhibition of glucagon release by glucose, which was apparently contradictory with the secretion coupling mechanism of its stimulation.

Palmitate and group B Streptococcus synergistically and differentially induce IL-1ß from human gestational membranes.

Introduction: Rupture of the gestational membranes often precedes major pregnancy complications, including preterm labor and preterm birth. One major cause of inflammation in the gestational membranes, chorioamnionitis (CAM) is often a result of bacterial infection. The commensal bacterium Streptococcus agalactiae, or Group B Streptococcus (GBS) is a leading infectious cause of CAM. Obesity is on the rise worldwide and roughly 1 in 4 pregnancy complications is related to obesity, and individuals with obesity are also more likely to be colonized by GBS. The gestational membranes are comprised of several distinct cell layers which are, from outermost to innermost: maternally-derived decidual stromal cells (DSCs), fetal cytotrophoblasts (CTBs), fetal mesenchymal cells, and fetal amnion epithelial cells (AECs). In addition, the gestational membranes have several immune cell populations; macrophages are the most common phagocyte. Here we characterize the effects of palmitate, the most common long-chain saturated fatty acid, on the inflammatory response of each layer of the gestational membranes when infected with GBS, using human cell lines and primary human tissue. Results: Palmitate itself slightly but significantly augments GBS proliferation. Palmitate and GBS co-stimulation synergized to induce many inflammatory proteins and cytokines, particularly IL-1ß and matrix metalloproteinase 9 from DSCs, CTBs, and macrophages, but not from AECs. Many of these findings are recapitulated when treating cells with palmitate and a TLR2 or TLR4 agonist, suggesting broad applicability of palmitate-pathogen synergy. Co-culture of macrophages with DSCs or CTBs, upon co-stimulation with GBS and palmitate, resulted in increased inflammatory responses, contrary to previous work in the absence of palmitate. In whole gestational membrane biopsies, the amnion layer appeared to dampen immune responses from the DSC and CTB layers (the choriodecidua) to GBS and palmitate co-stimulation. Addition of the monounsaturated fatty acid oleate, the most abundant monounsaturated fatty acid in circulation, dampened the proinflammatory effect of palmitate. Discussion: These studies reveal a complex interplay between the immunological response of the distinct layers of the gestational membrane to GBS infection and that such responses can be altered by exposure to long-chain saturated fatty acids. These data provide insight into how metabolic syndromes such as obesity might contribute to an increased risk for GBS disease during pregnancy.

ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity.

Elevated concentrations of palmitate in serum of obese individuals can impair endothelial function, contributing to development of cardiovascular disease. Although several molecular mechanisms of palmitate-induced endothelial dysfunction have been proposed, there is no consensus on what signaling event is the initial trigger of detrimental palmitate effects. Here we report that inhibitors of ER stress or ceramid synthesis can rescue palmitate-induced autophagy impairment in macro- and microvascular endothelial cells. Furthermore, palmitate-induced cholesterol synthesis was reverted using these inhibitors. Similar to cell culture data, autophagy markers were increased in serum of obese individuals. Subsequent lipidomic analysis revealed that palmitate changed the composition of membrane phospholipids in endothelial cells and that these effects were not reverted upon application of above-mentioned inhibitors. However, ER stress inhibition in palmitate-treated cells enhanced the synthesis of trilglycerides and restored ceramide levels to control condition. Our results suggest that palmitate induces ER-stress presumably by shift in membrane architecture, leading to impaired synthesis of triglycerides and enhanced production of ceramides and cholesterol, which altogether enhances lipotoxicity of palmitate in endothelial cells.

In silico mining and identification of a novel lipase from Paenibacillus larvae: Rational protein design for improving catalytic performance.

Lipases play a vital role in various biological processes, from lipid metabolism to industrial applications. However, the ever-evolving challenges and diverse substrates necessitate the continual exploration of novel high-performance lipases. In this study, we employed an in silico mining approach to search for lipases with potential high sn-1,3 selectivity and catalytic activity. The identified novel lipase, PLL, from Paenibacillus larvae subsp. larvae B-3650 exhibited a specific activity of 111.2 ± 5.5 U/mg towards the substrate p-nitrophenyl palmitate (pNPP) and 6.9 ± 0.8 U/mg towards the substrate olive oil when expressed in Escherichia coli (E. coli). Computational design of cysteine mutations was employed to enhance the catalytic performance of PLL. Superior stability was achieved with the mutant K7C/A386C/H159C/K108C (2M3/2M4), showing an increase in melting temperature (Tm) by 1.9°C, a 2.05-fold prolonged half-life at 45°C, and no decrease in enzyme activity. Another mutant, K7C/A386C/A174C/A243C (2M1/2M3), showed a 4.9-fold enhancement in specific activity without compromising stability. Molecular dynamics simulations were conducted to explore the mechanisms of these two mutants. Mutant 2M3/2M4 forms putative disulfide bonds in the loop region, connecting the N- and C-termini of PLL, thus enhancing overall structural rigidity without impacting catalytic activity. The cysteines introduced in mutant 2M1/2M3 not only form new intramolecular hydrogen bonds but also alter the polarity and volume of the substrate-binding pocket, facilitating the entry of large substrate pNPP. These results highlight an efficient in silico exploration approach for novel lipases, offering a rapid and efficient method for enhancing catalytic performance through rational protein design.

Non-esterified fatty acid palmitate facilitates oxidative endoplasmic reticulum stress and apoptosis of ß-cells by upregulating ERO-1α expression.

Adipose tissue-derived non-esterified saturated long-chain fatty acid palmitate (PA) decisively contributes to ß-cell demise in type 2 diabetes mellitus in part through the excessive generation of hydrogen peroxide (H2O2). The endoplasmic reticulum (ER) as the primary site of oxidative protein folding could represent a significant source of H2O2. Both ER-oxidoreductin-1 (ERO-1) isoenzymes, ERO-1α and ERO-1ß, catalyse oxidative protein folding within the ER, generating equimolar amounts of H2O2 for every disulphide bond formed. However, whether ERO-1-derived H2O2 constitutes a potential source of cytotoxic luminal H2O2 under lipotoxic conditions is still unknown. Here, we demonstrate that both ERO-1 isoforms are expressed in pancreatic ß-cells, but interestingly, PA only significantly induces ERO-1α. Its specific deletion significantly attenuates PA-mediated oxidative ER stress and subsequent ß-cell death by decreasing PA-mediated ER-luminal and mitochondrial H2O2 accumulation, by counteracting the dysregulation of ER Ca2+ homeostasis, and by mitigating the reduction of mitochondrial membrane potential and lowered ATP content. Moreover, ablation of ERO-1α alleviated PA-induced hyperoxidation of the ER redox milieu. Importantly, ablation of ERO-1α did not affect the insulin secretory capacity, the unfolded protein response, or ER redox homeostasis under steady-state conditions. The involvement of ERO-1α-derived H2O2 in PA-mediated ß-cell lipotoxicity was corroborated by the overexpression of a redox-active ERO-1α underscoring the proapoptotic activity of ERO-1α in pancreatic ß-cells. Overall, our findings highlight the critical role of ERO-1α-derived H2O2 in lipotoxic ER stress and ß-cell failure.

Thermal oxidation mechanism of palmitic aicd.

The oxidation and degradation of fats lead to a decrease in the nutritional value of food and pose safety concerns. Saturated fatty acids also hold a significant position in the field of lipid oxidation. In this study, the oxidation products of methyl palmitate were investigated by using gas chromatography mass spectrometry (GC-MS). Seven monohydroperoxides and 72 secondary oxidation products were detected. Combined with density functional theory (DFT) calculations, the formation mechanisms of oxidation products can be summarized into four stages. The initial stage involved the formation of monohydroperoxides and alkanes, followed by the subsequent stage involving methyl x-oxo(hydroxy)hexadecanoates. The third stage involved the formation of methyl ketones, carboxylic acids, and aldehydes, while the final stage involved lactones. Meanwhile, methyl ketones were the most abundant oxidation product, approximately 25 times more abundant than aldehydes; the calculated results agreed well with the experimental results. The establishment of a comprehensive thermal oxidation mechanism for palmitic acid provided a new foundation for future lipid oxidation analyses.

Nigella sativa, active principle thymoquinone, alleviates palmitate-induced cytotoxicity, inflammation and bioenergetic disruptions in macrophages: An invitro study model.

Thymoquinone (TQ) is one of the main phytochemical bioactive ingredients in Nigella sativa, with reported immunity-boosting properties. The current study evaluated the anti-inflammatory effect of TQ against inflammation brought on by free fatty acid Palmitate (PA) using macrophages raw 264.7 cell line. Data revealed that TQ significantly improved the viability of basal and PA stimulated Macrophages at concentrations of 50 and 100 µg/mL. Also, TQ significantly reduced nitric oxide and triglyceride levels in PA-stimulated macrophages at concentrations of 50 and 100 µg/mL. The pro-inflammatory cytokines studies revealed that PA significantly increased the release of the cytokines TNF-α, MHGB-1, IL-1ß, and IL-6. TQ at concentrations 25, 50, and 100 µg/ml significantly decreases the release of the studied cytokines in PA-stimulated macrophages to variable extents with parallel inhibition to their corresponding gene expression. Bioenergetic assays showed that PA significantly decreased cellular ATP, mitochondrial complexes I and III activities and mitochondrial membrane potential with a subsequent significant increase in lactate production. At the same time, TQ can alleviate the effect of PA on macrophages' bioenergetics parameters to variable extent based on TQ concentration. To conclude, TQ could mitigate palmitate-induced inflammation and cytotoxicity in macrophages by improving macrophage viability and controlling cytokine release with improved PA-induced bioenergetics disruption.

Role of Sphingosine Kinase 1 in Glucolipotoxicity-Induced Early Activation of Autophagy in INS-1 Pancreatic ß Cells.

Insulin-producing pancreatic ß cells play a crucial role in the regulation of glucose homeostasis, and their failure is a key event for diabetes development. Prolonged exposure to palmitate in the presence of elevated glucose levels, termed gluco-lipotoxicity, is known to induce ß cell apoptosis. Autophagy has been proposed to be regulated by gluco-lipotoxicity in order to favor ß cell survival. However, the role of palmitate metabolism in gluco-lipotoxcity-induced autophagy is presently unknown. We therefore treated INS-1 cells for 6 and 24 h with palmitate in the presence of low and high glucose concentrations and then monitored autophagy. Gluco-lipotoxicity induces accumulation of LC3-II levels in INS-1 at 6 h which returns to basal levels at 24 h. Using the RFP-GFP-LC3 probe, gluco-lipotoxicity increased both autophagosomes and autolysosmes structures, reflecting early stimulation of an autophagy flux. Triacsin C, a potent inhibitor of the long fatty acid acetyl-coA synthase, completely prevents LC3-II formation and recruitment to autophagosomes, suggesting that autophagic response requires palmitate metabolism. In contrast, etomoxir and bromo-palmitate, inhibitors of fatty acid mitochondrial ß-oxidation, are unable to prevent gluco-lipotoxicity-induced LC3-II accumulation and recruitment to autophagosomes. Moreover, bromo-palmitate and etomoxir potentiate palmitate autophagic response. Even if gluco-lipotoxicity raised ceramide levels in INS-1 cells, ceramide synthase 4 overexpression does not potentiate LC3-II accumulation. Gluco-lipotoxicity also still stimulates an autophagic flux in the presence of an ER stress repressor. Finally, selective inhibition of sphingosine kinase 1 (SphK1) activity precludes gluco-lipotoxicity to induce LC3-II accumulation. Moreover, SphK1 overexpression potentiates autophagic flux induced by gluco-lipotxicity. Altogether, our results indicate that early activation of autophagy by gluco-lipotoxicity is mediated by SphK1, which plays a protective role in ß cells.