Saturated Fatty Acid Palmitate Research Articles

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

Adipose tissue-derived non-esterified saturated long-chain fatty acid palmitate (PA) decisively contributes to β-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-1β, 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 β-cells, but interestingly, PA only significantly induces ERO-1α. Its specific deletion significantly attenuates PA-mediated oxidative ER stress and subsequent β-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 β-cell lipotoxicity was corroborated by the overexpression of a redox-active ERO-1α underscoring the proapoptotic activity of ERO-1α in pancreatic β-cells. Overall, our findings highlight the critical role of ERO-1α-derived H2O2 in lipotoxic ER stress and β-cell failure.

Physicochemical composition, centesimal, and phytochemical profile of umbu seed flour (Spondias tuberosa Arruda)

This study aimed to investigate the nutritional potential of umbu (Spondias tuberosa Arruda) seed flours. This was an experimental study. The seeds of the umbu fruit were subjected to an oven-drying process in order to obtain the flour. Subsequently, the analysis of the centesimal composition, physicochemical, mineral, and fatty acid profile, microbiological investigation, and the screening of bioactive phytochemicals of the umbu seed flours in hydroethanolic extracts were carried out. A completely randomized design (CID) was used, with three replications, and the results were expressed as means followed by standard deviation. Analysis of variance (ANOVA) was carried out, and the Tukey’s test was applied at a 5% probability level. It was possible to obtain flour from umbu seeds. It was observed that they contained significant amounts of insoluble dietary fiber (80.9 ± 0.55a g.100-1), protein (6.95 ± 0.10a g.100-1), and lipids (5.54 ± 1.13a g.100-1). The minerals present included zinc (1.87 ± 0.05b mg.100 g-1), manganese (14.36 ± 0.34c mg.100 g-1), and calcium (104.79 ± 0.92c mg.100 g-1). The flours had high levels of oleic monounsaturated fatty acids (33.10 ± 0.13a g.100-1), linoleic polyunsaturated fatty acids (38.06 ± 0.06b g.100-1), and palmitic saturated fatty acids (19.1 ± 0.04b g.100-1), in addition to considerable levels of phenolic compounds (276.21 ± 14.01a mg.g-1 GAE) and flavonoids (891.09 ± 9.01ª mg.g-1 rutin), with satisfactory sanitary conditions for Escherichia coli, Salmonella, Bacillus cereus molds, and yeasts. This study suggests that umbu seed meals can be considered relevant sources of dietary fiber, minerals, and natural antioxidants.

Insufficient TRPM5 Mediates Lipotoxicity-induced Pancreatic β-cell Dysfunction.

While the reduction of transient receptor potential channel subfamily M member 5 (TRPM5) has been reported in islet cells from type 2 diabetic (T2D) mouse models, its role in lipotoxicity-induced pancreatic β-cell dysfunction remains unclear. This study aims to study its role. Pancreas slices were prepared from mice subjected to a high-fat-diet (HFD) at different time points, and TRPM5 expression in the pancreatic β cells was examined using immunofluorescence staining. Glucose-stimulated insulin secretion (GSIS) defects caused by lipotoxicity were mimicked by saturated fatty acid palmitate (Palm). Primary mouse islets and mouse insulinoma MIN6 cells were treated with Palm, and the TRPM5 expression was detected using qRT-PCR and Western blotting. Palm-induced GSIS defects were measured following siRNA-based Trpm5 knockdown. The detrimental effects of Palm on primary mouse islets were also assessed after overexpressing Trpm5 via an adenovirus-derived Trpm5 (Ad-Trpm5). HFD feeding decreased the mRNA levels and protein expression of TRPM5 in mouse pancreatic islets. Palm reduced TRPM5 protein expression in a time- and dose-dependent manner in MIN6 cells. Palm also inhibited TRPM5 expression in primary mouse islets. Knockdown of Trpm5 inhibited insulin secretion upon high glucose stimulation but had little effect on insulin biosynthesis. Overexpression of Trpm5 reversed Palm-induced GSIS defects and the production of functional maturation molecules unique to β cells. Our findings suggest that lipotoxicity inhibits TRPM5 expression in pancreatic β cells both in vivo and in vitro and, in turn, drives β-cell dysfunction.

AMPK Mediates Early Activation of the Unfolded Protein Response through a Positive Feedback Loop in Palmitate-Treated Muscle Cells.

Activation of the unfolded protein response (UPR) is closely related to the pathogenesis of many metabolic disorders. Accumulating evidence also shows that UPR and metabolic signaling pathways are interdependent. The AMP-activated protein kinase (AMPK) signal pathway controls the energy balance of eukaryotes. The aim of this study was therefore to investigate the possible interaction between AMPK signaling and UPR in muscle cells exposed to saturated fatty acids, as well as the potential mechanism. The saturated fatty acid palmitate was used to induce UPR in C2C12 myotubes. Compound C or knockdown of AMPKα with short hairpin RNA (shRNA) were used to inhibit the AMPK signaling pathway in palmitate-treated muscle cells. AMPK signaling in myotubes was activated using 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide (AICAR) or ex229. C2C12 myotubes were pre-treated with taurourdodeoxycholic acid (TUDCA) to inhibit UPR before adding palmitate. Real-time PCR and Western blotting were performed to evaluate the expression of UPR markers and activation of AMPK. Palmitate treatment induced UPR in C2C12 myotubes while activating AMPK signaling. Inhibition of the AMPK pathway with compound C or AMPK shRNA reduced palmitate-induced activation of UPR, while inhibition of UPR with TUDCA reduced palmitate-induced AMPK activation. This indicates a positive feedback loop between UPR and AMPK. Furthermore, activation of the AMPK pathway with AICAR or ex229 caused a dose-dependent upregulation of UPR markers, including activating transcription factor 4 (ATF4), binding immunoglobulin protein (BIP), and growth arrest and DNA damage-inducible 34 (GADD34) protein. These results provide the first evidence that AMPK signaling is involved in the early activation of UPR caused by saturated fatty acids in skeletal muscle. Furthermore, they indicate that physiological or pharmacological activation of the AMPK pathway (e.g., by exercise or phenformin, respectively) can promote muscle health and function, thereby improving the quality of life in individuals with metabolic disorders due to a high-fat diet or obesity.

Palmitate alters miR-2137 and miR-503-5p to induce orexigenic Npy in hypothalamic neuronal cell models: Rescue by oleate and docosahexaenoic acid.

MicroRNAs (miRNAs) are short noncoding RNA implicated in the pathogenesis of obesity. One cause of obesity is excess exposure to the saturated fatty acid palmitate that can alter miRNA levels in the periphery. Palmitate also promotes obesity by acting on the hypothalamus, the central coordinator of energy homeostasis, to dysregulate hypothalamic feeding neuropeptides and induce ER stress and inflammatory signaling. We hypothesized that palmitate would alter hypothalamic miRNAs that control genes involved in energy homeostasis thereby contributing to the obesity-promoting effects of palmitate. We found that palmitate upregulated 20 miRNAs and downregulated six miRNAs in the orexigenic NPY/AgRP-expressing mHypoE-46 cell line. We focused on delineating the roles of miR-2137 and miR-503-5p, as they were strongly up- and downregulated by palmitate, respectively. Overexpression of miR-2137 increased Npy mRNA levels and downregulated Esr1 levels, while increasing C/ebpβ and Atf3 mRNA. Inhibiting miR-2137 had the opposite effect, except on Npy, which was unchanged. The most downregulated miRNA by palmitate, miR-503-5p, negatively regulated Npy mRNA levels. Exposure to the unsaturated fatty acids oleate or docosahexaenoic acid completely or partially blocked the effects of palmitate on miR-2137 and miR-503-5p as well as Npy, Agrp, Esr1, C/ebpβ and Atf3. MicroRNAs may therefore contribute to palmitate actions in dysregulating NPY/AgRP neurons. Effectively combating the deleterious effects of palmitate is crucial to help prevent or reduce the impact of obesity.

Junctional integrity and directional mobility of lymphatic endothelial cell monolayers are disrupted by saturated fatty acids.

The lymphatic circulation regulates transfer of tissue fluid and immune cells toward the venous circulation. While obesity impairs lymphatic vessel function, the contribution of lymphatic endothelial cells (LEC) to metabolic disease phenotypes is poorly understood. LEC of lymphatic microvessels are in direct contact with the interstitial fluid, whose composition changes during the development of obesity, markedly by increases in saturated fatty acids. Palmitate, the most prevalent saturated fatty acid in lymph and blood, is detrimental to metabolism and function of diverse tissues, but its impact on LEC function is relatively unknown. Here, palmitate (but not its unsaturated counterpart palmitoleate) destabilized adherens junctions in human microvascular LEC in culture, visualized as changes in VE-cadherin, α-catenin, and β-catenin localization. Detachment of these proteins from cortical actin filaments was associated with abundant actomyosin stress fibers. The effects were Rho-associated protein kinase (ROCK)- and myosin-dependent, as inhibition with Y27632 or blebbistatin, respectively, prevented stress fiber accumulation and preserved junctions. Without functional junctions, palmitate-treated LEC failed to directionally migrate to close wounds in two dimensions and failed to form endothelial tubes in three dimensions. A reorganization of the lymphatic endothelial actin cytoskeleton may contribute to lymphatic dysfunction in obesity and could be considered as a therapeutic target.

Luminal H2 O2 promotes ER Ca2+ dysregulation and toxicity of palmitate in insulin-secreting INS-1E cells.

The endoplasmic reticulum (ER) lumen is not only the major site for the assembly and folding of newly synthesized proteins but also the main intracellular Ca2+ store. Ca2+ ions are involved in versatile biochemical processes, including posttranslational processing and folding of nascent proteins. Disruption of ER Ca2+ homeostasis is usually accompanied by an ER stress response that can ultimately lead to apoptosis if unresolved. Abnormal ER Ca2+ depletion has been linked to pancreatic β-cell dysfunction and death under lipotoxic conditions. However, the underlying mechanisms how the β-cell toxic saturated free fatty acid palmitate perturbs ER Ca2+ homeostasis and its interplay with other organelles are not fully understood. In the present study, we demonstrate that treatment of insulin-secreting INS-1E cells with palmitate diminished ER Ca2+ levels, elevated cytosolic/mitochondrial Ca2+ content, lowered the mitochondrial membrane potential, and ATP content. In addition, palmitate-pretreated β-cells contained significantly less luminal Ca2+ , revealed a severely impaired ER Ca2+ reuptake rate, and substantially lower insulin content. Importantly, detoxification of luminal H2 O2 by expression of the ER-resident glutathione peroxidase 8 (GPx8) abrogated the lipotoxic effects of palmitate. Moreover, GPx8 supported oxidative protein folding and preserved insulin content under lipotoxic conditions. A direct involvement of luminal H2 O2 in palmitate-mediated ER Ca2+ depletion could be corroborated by the ectopic expression of an ER-luminal active catalase. Our data point to the critical role of luminal H2 O2 in palmitate-mediated depletion of ER Ca2+ through redox-dependent impairment of Ca2+ ATPase pump activity upstream of mitochondrial dysfunction in insulin-secreting INS-1E cells.

Inhibition of Stearoyl-CoA Desaturase Has Anti-Leukemic Properties in Acute Myeloid Leukemia

Metabolic rewiring, a hallmark of acute myeloid leukemia (AML), contributes to the development of resistance to intensive chemotherapy and has been shown to play an essential role in leukemic cell maintenance. Several metabolic alterations have been described in AML, such as dependence on amino acids to fuel oxidative phosphorylation or ability of leukemic cells to switch energy dependence to fatty acid oxidation in response to treatment. However, little is known about the functional significance of the fatty acid synthesis (FAS) pathway in AML. Here we have identified stearoyl-CoA desaturase (SCD), a key enzyme responsible for the conversion of saturated fatty acids (SFA) into monounsaturated fatty acids (MUFA), as a novel therapeutic target in AML. High SCD expression predicts worse survival across several patient datasets, even after correction for gender, age and AML European LeukemiaNet risk category. Moreover, genetic and chemical inhibition of SCD reduces growth and induces cell death in AML cells. Since targeting SCD with currently available inhibitors has been challenging because of their toxicity profile in vivo, we have accessed a novel pharmacological SCD inhibitor with a more favorable toxicity profile, SSI-4 (Modulation Therapeutics Inc., Morgantown, USA). SSI-4 reduces viability in several human and mouse AML models in vitro and in about half of the 36 primary AML samples tested ex vivo. Moreover, SSI-4 displays anti-leukemic effects in AML murine models and patient-derived xenografts (PDX). Since we could not correlate susceptibility to SCD inhibition with any genetic characteristic of AML cells, we aimed to determine the molecular basis of cell sensitivity using phosphoproteomic, proteomic and transcriptomic approaches coupled with metabolic analyses of sensitive and resistant cell lines and primary AML cells. Sensitivity to SCD inhibition directly correlates with rate of de novo FAS, as measured by fatty acid labeling with 13C-glucose. Moreover, modulation of FAS by the AMPK signaling activity dictates sensitivity to SCD inhibition and AMPK activation rescues the anti-leukemic effects of SSI-4. The anti-leukemic effects of SCD inhibition are secondary to the induction of SFA mediated lipotoxicity, as demonstrated by the increased sensitivity of previously resistant cells to SSI-4, when grown in the presence of the SFA palmitate. Conversely, growing cells in the presence of the MUFA oleate, as well as inhibition of FAS by targeting enzymes upstream of SCD, results in resistance to SSI-4, thus confirming that the increased SFA/MUFA ratio drives leukemic cell death. SFA mediated lipotoxicity leads to both increased lipid peroxidation and activation of the unfolded protein response (UPR), namely its PERK branch, which in turn leads to apoptotic cell death. Indeed, inhibition of PERK, but not of other branches of UPR, rescues SCD-mediated cytotoxicity. Despite causing disease debulking in vivo, SSI-4 does not prolong survival in murine AML models as a single agent and the toxic effects of SSI-4 are decreased when AML cells are grown in co-culture with stroma, suggesting a protective role of tumor microenvironment. However, we consistently detect increased lipid peroxidation and DNA damage both in vitro and in vivo following SCD inhibition. We therefore combined SSI-4 with doxorubicin, a known inducer of lipid peroxidation and DNA damage, which is routinely used in AML induction therapy. Indeed, both genetic depletion and pharmacological inhibition of SCD renders AML cells more sensitive to doxorubicin, and addition of SSI-4 to a standard AML chemotherapy protocol consisting of doxorubicin and cytarabine significantly extends survival in AML mouse models. In summary, we identify and validate SCD as a therapeutic target in AML. The novel SCD inhibitor SSI-4 displays anti-leukemic properties both in vitro and in vivo across different AML models and particularly when combined with standard chemotherapy while showing minimal toxicity in vivo. We show that sensitivity to SCD inhibition is driven by metabolic rather than genetic AML features, thus making this therapeutic approach potentially applicable across several AML subtypes, including high risk ones. Finally, the mechanism of toxicity following SCD inhibition suggests that this can be further enhanced by modulating the levels of SFA/MUFA through dietary interventions or by combining it with DNA damaging agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-fat diet and palmitate inhibits FNDC5 expression via AMPK-Zfp57 pathway in mouse muscle cells

Irisin, a muscle-secreted cytokine involved in maintaining glucose homeostasis and improving insulin resistance, is generated from the precursor fibronectin type Ⅲ domain-containing protein 5 (FNDC5) by specific proteases. Zinc-finger protein Zfp57, a transcription factor that maintains the methylation during early embryonic development, is also reported to be associated with diabetes mellitus. However, the association between Zfp57 and FNDC5 is still unclear. In our study, we explored the detailed regulatory effect of Zfp57 on FNDC5 expression. In this study, we found that high-fat diet or saturated fatty acid palmitate increased the Zfp57 expression and decreased FNDC5 expression in muscle tissue or C2C12 myotubes. RNA sequencing analysis disclosed effects of the high-fat diet on genes associated with insulin resistance and the AMP-activated protein kinase (AMPK) signaling pathway in muscle tissue of mice. Chromatin immunoprecipitation experiments revealed that Zfp57 binds the FNDC5 gene promoter at positions −308 to −188. Moreover, Zfp57 overexpression inhibited FNDC5 expression, and Zfp57 knockdown alleviated the inhibitory effect of palmitate on FNDC5 expression in C2C12 myotubes. In addition, in vivo and in vitro studies demonstrated that activation of the AMPK pathway by 5-Aminoimidazole-4-carboxamide riboside (AICAR) or metformin mitigated the inhibitory effect of Zfp57 on FNDC5 expression and improved insulin resistance. These findings collectively suggest that high-fat diet and palmitate inhibit the AMPK pathway to increase Zfp57 expression, which in turn induces FNDC5 inhibition, to further aggravate insulin resistance.

Solvent extraction of oil from three cultivars of Nigerian mango seed kernel: Process modeling, GA - optimization, nonlinear kinetics and comparative characterization

This work investigated the physicochemical and instrumental characterization of oil optimally extracted from three species of mango kernels (Alphonso, Glenn and Fazli) by highlighting their isolated qualities. The effect of temperature (40-58 °C), dosage (0.0033-0.0267 g/mL) and time (0-360 min) was varied at the course of experiment. It was observed that while temperature increment had a corresponding positive impact on mango kernel oil extraction efficiency, the influence of dosage produced a quadratic effect, reaching maximum efficiency at 0.017 g/mL, beyond which an undesirable decline was recorded. Analysis of variance (ANOVA) of process factors gave an F-value of > 80.5 for temperature, indicating that temperature was the most important parameter that influenced the extraction process, while kernel dosage having F-value < 20 was the least influential process parameter. Process optimization by response surface methodology coupled genetic algorithm (RSM-GA) gave an optimum validation yield of 13.62%, 14.7% and 16.1% for Fazli, Glenn and Alphonso, respectively, at temperature of 56.61 °C, dosage of 0.021 g/mL and extraction time of 275 min. Error function indices obtained from comparative kinetic modeling illustrated that the extraction process was best described by pseudo second order model (R2 > 0.96, Adj-R2 > 0.95). Also, Elovich kinetics highlighted the dominance of rate diffusion over washing mechanism in the extraction process. Physicochemical characterization showed that while all the mango kernel oil (MKO) species exhibited stability against oxidative rancidity, Alphonso species having an acid value of 2.74 mg KOH/g and 1.37% free fatty acid value, highlighted its superior physicochemical qualities over Fazli and Glenn mango kernel oil. GC-MS analysis portrayed the dominance of stearic and palmitic saturated fatty acids in all the three species while myristic acid was only detected in Alphonso mango kernel oil (MKO), emphasizing its application in food and cosmetics industries. X-ray florescence (XRF) demonstrated the significant presence of molybdenum (Mo) element in Glenn mango kernel oil, revealing its unique suitability for application in synthesis of special purpose industrial lubricants. For Alphonso mango kernel oil, XRF revealed that while this oil species has potential application in the production of lubricants with high detergent properties, caution must be taken in the utilization due to the presence of sulphur element in the sample.

Glial cell derived neurotrophic factor prevents western diet and palmitate-induced hepatocyte oxidative damage and death through SIRT3

Nonalcoholic fatty liver disease (NAFLD) is associated with increased oxidative stress that leads to hepatocyte and mitochondrial damage. In this study we investigated the mechanisms involved in the induction of oxidative stress and impairment of mitochondrial quality control and mitophagy in hepatocytes by the saturated fatty acid palmitate and Western diet feeding in mice and if their harmful effects could be reversed by the neurotrophic factor glial cell derived neurotrophic factor (GDNF). Western diet (WD)-feeding increased hepatic lipid peroxidation in control mice and, in vitro palmitate induced oxidative stress and impaired the mitophagic clearance of damaged mitochondria in hepatocytes. This was accompanied by reductions in hepatocyte sirtuin 3 (SIRT3) deacetylase activity, gene expression and protein levels as well as in superoxide dismutase enzyme activity. These reductions were reversed in the liver of Western diet fed GDNF transgenic mice and in hepatocytes exposed to palmitate in the presence of GDNF. We demonstrate an important role for Western diet and palmitate in inducing oxidative stress and impairing mitophagy in hepatocytes and an ability of GDNF to prevent this. These findings suggest that GDNF or its agonists may be a potential therapy for the prevention or treatment of NAFLD.

Abstract P1074: High-fat High-sugar Diet Promotes Expression Of The Stress Response Protein REDD1 In Cardiomyocytes By Activation Of A PERK/ATF4 Signaling Axis

Obesity and type 2 diabetes represent a surging public health crisis, impacting nearly half of American adults. Heart failure is a leading cause of mortality and morbidity in patients with diabetes and obesity. Much remains unknown regarding the molecular players that contribute to heart failure, but at the cellular level ER stress and inflammation are considered key hallmarks of cardiomyocyte dysfunction. The stress response protein regulated in development and DNA damage 1 (REDD1) is upregulated as a consequence of ER stress in multiple pathological conditions and was recently shown to promote inflammatory signaling. However, a role for diabetes-induced REDD1 in the context of heart failure has yet to be explored. Herein, we investigated the hypothesis that consumption of a pro-diabetogenic diet enhances REDD1 expression in cardiomyocytes by promoting ER stress. C57BL6/N mice were fed either a control chow or a high-fat high-sugar (HFHS, 42% kcal fat, 34% sucrose by weight) diet for 12 weeks. Mice fed a HFHS diet developed obesity and glucose intolerance. Hearts of mice fed a HFHS diet had an abundance of REDD1 mRNA and exhibited an increase in ER stress and inflammation characterized by phosphorylation of the ER kinase PERK, XBP-1 splicing, and IL-1β expression. We found that exposing both rat H9c2 and human AC16 cardiomyocytes to the saturated fatty acid palmitate and/or hyperglycemic conditions enhanced REDD1 mRNA and protein expression concomitant with an increase in markers of ER stress. PERK inhibition was sufficient to prevent the increase in REDD1 mRNA expression in cardiomyocytes exposed to hyperlipidemic conditions. Similarly, the transcription factor ATF4 was required for the increase in REDD1 mRNA and protein in cells exposed to hyperlipidemic conditions. We also found that REDD1 deletion was sufficient to reduce pro-inflammatory cytokine expression in human cardiomyocytes exposed to hyperglycemic and hyperlipidemic conditions. Overall, the findings provide evidence that HFHS environments contribute to the development of inflammation in cardiomyocytes by promoting REDD1 expression via activation of a PERK/ATF4 signaling axis.

Macrophage-specific MyD88 deletion and pharmacological inhibition prevents liver damage in non-alcoholic fatty liver disease via reducing inflammatory response

Activation of the innate immune system through toll-like receptors (TLRs) has been repeatedly demonstrated in non-alcoholic fatty liver disease (NAFLD) and several TLRs have been shown to contribute. Myeloid differentiation primary response 88 (MyD88) is as an adapter protein for the activation of TLRs and bridges TLRs to NF-κB-mediated inflammation in macrophages. However, whether myeloid cell MyD88 contributes to NAFLD are largely unknown. To test this approach, we generated macrophage-specific MyD88 knockout mice and show that these mice are protected against high-fat diet (HFD)-induced hepatic injury, lipid accumulation, and fibrosis. These protective effects were associated with reduced macrophage numbers in liver tissues and surpassed inflammatory responses. In cultured macrophages, saturated fatty acid palmitate utilizes MyD88 to activate NF-κB and induce inflammatory and fibrogenic factors. In hepatocytes, these factors may cause lipid accumulation and a further elaboration of inflammatory cytokines. In hepatic stellate cells, macrophage-derived factors, especially TGF-β, cause activation and hepatic fibrosis. We further show that pharmacological inhibition of MyD88 is also able to reduce NAFLD injury in HFD-fed mice. Therefore, our study has provided empirical evidence that macrophage MyD88 participates in HFD-induced NAFLD and could be targeted to prevent the development and progression of NAFLD/NASH.

PLAAT1 Exhibits Phosphatidylcholine:Monolysocardiolipin Transacylase Activity.

Tissue-specific cardiolipin fatty acyl profiles are achieved by remodeling of de novo synthesized cardiolipin, and four remodeling enzymes have thus far been identified. We studied the enzyme phospholipase A and acyltransferase 1 (PLAAT1), and we report the discovery that it has phosphatidylcholine (PC):monolysocardiolipin (MLCL) transacylase activity. Subcellular localization was analyzed by differential centrifugation and immunoblotting. Total levels of major phospholipids, and the fatty acyl profile of cardiolipin, were analyzed in HEK293 cells expressing murine PLAAT1 using gas chromatography. Apparent enzyme kinetics of affinity-purified PLAAT1 were calculated using radiochemical enzyme assays. This enzyme was found to localize predominantly to the endoplasmic reticulum (ER) but was detected at low levels in the mitochondria-associated ER matrix. Cells expressing PLAAT1 had higher levels of total cardiolipin, but not other phospholipids, and it was primarily enriched in the saturated fatty acids myristate, palmitate, and stearate, with quantitatively smaller increases in the n-3 polyunsaturated fatty acids linolenate, eicosatrienoate, and eicosapentanoate and the monounsaturated fatty acid erucate. Affinity-purified PLAAT1 did not catalyze the transacylation of MLCL using 1-palmitoyl-2-[14C]-linoleoyl-PC as an acyl donor. However, PLAAT1 had an apparent Vmax of 1.61 μmol/min/mg protein and Km of 126 μM using [9,10-3H]-distearoyl-PC as an acyl donor, and 0.61 μmol/min/mg protein and Km of 16 μM using [9,10-3H]-dioleoyl-PC. PLAAT1 is therefore a novel PC:MLCL transacylase.

Nobiletin ameliorates high fat-induced disruptions in rhythmic glucagon-like peptide-1 secretion

The incretin hormone glucagon-like peptide-1 (GLP-1) is secreted by the intestinal L cell in response to nutrient intake. However, GLP-1 secretion also follows a circadian rhythm which is disrupted by the saturated fatty acid palmitate in vitro and high-fat diet (HFD) feeding in vivo. The flavonoid nobiletin is a clock enhancer which improves metabolic homeostasis. Therefore, the aim of this study was to elucidate whether and how nobiletin mitigates the negative effects of palmitate and HFD-feeding on rhythmic GLP-1 release. Pre-treatment of murine GLUTag L cells with palmitate dampened the GLP-1 secretory response at the normal peak of secretion, while nobiletin co-treatment restored GLP-1 secretion and upregulated the ‘metabolic pathway’ transcriptome. Mice fed a HFD also lost their GLP-1 secretory rhythm in association with markedly increased GLP-1 levels and upregulation of L cell transcriptional pathways related to ‘sensing’ and ‘transducing’ cellular stimuli at the normal peak of GLP-1 release. Nobiletin co-administration reduced GLP-1 levels to more physiological levels and upregulated L cell ‘oxidative metabolism’ transcriptional pathways. Furthermore, nobiletin improved colonic microbial 16S rRNA gene diversity and reduced the levels of Proteobacteria in HFD-fed mice. Collectively, this study establishes that nobiletin improves the normal rhythm in GLP-1 secretion following fat-induced disruption.

Differential routing and disposition of the long-chain saturated fatty acid palmitate in rodent vs human beta-cells

BackgroundRodent and human β-cells are differentially susceptible to the “lipotoxic” effects of long-chain saturated fatty acids (LC-SFA) but the factors accounting for this are unclear. Here, we have studied the intracellular disposition of the LC-SFA palmitate in human vs rodent β–cells and present data that reveal new insights into the factors regulating β-cell lipotoxicity.MethodsThe subcellular distribution of the LC-SFA palmitate was studied in rodent (INS-1E and INS-1 823/13 cells) and human (EndoC-βH1) β-cells using confocal fluorescence and electron microscopy (EM). Protein expression was assessed by Western blotting and cell viability, by vital dye staining.ResultsExposure of INS-1 cells to palmitate for 24 h led to loss of viability, whereas EndoC-βH1 cells remained viable even after 72 h of treatment with a high concentration (1 mM) of palmitate. Use of the fluorescent palmitate analogue BODIPY FL C16 revealed an early localisation of the LC-SFA to the Golgi apparatus in INS-1 cells and this correlated with distention of intracellular membranes, visualised under the EM. Despite this, the PERK-dependent ER stress pathway was not activated under these conditions. By contrast, BODIPY FL C16 did not accumulate in the Golgi apparatus in EndoC-βH1 cells but, rather, co-localised with the lipid droplet-associated protein, PLIN2, suggesting preferential routing into lipid droplets. When INS-1 cells were treated with a combination of palmitate plus oleate, the toxic effects of palmitate were attenuated and BODIPY FL C16 localised primarily with PLIN2 but not with a Golgi marker.ConclusionIn rodent β-cells, palmitate accumulates in the Golgi apparatus at early time points whereas, in EndoC- βH1 cells, it is routed preferentially into lipid droplets. This may account for the differential sensitivity of rodent vs human β-cells to “lipotoxicity” since manoeuvres leading to the incorporation of palmitate into lipid droplets is associated with the maintenance of cell viability in both cell types.

Inductively-Coupled Plasma Mass Spectrometry-Novel Insights From an Old Technology Into Stressed Red Blood Cell Physiology.

BackgroundIon and metal homeostasis are critical to red blood cell physiology and Inductively Coupled Plasma (ICP) is a decades old approach to pursue elemental analysis. Recent evolution of ICP has resulted in its coupling to mass spectrometry (MS) instead of atomic absorption/emission.MethodsHere we performed Inductively-coupled plasma mass spectrometry (ICP-MS) measurements of intra- and extra-cellular Na, K, Ca, Mg, Fe, and Cu in red blood cells undergoing ionic, heat, or starvation stress. Results were correlated with Ca measurements from other common platforms (e.g., fluorescence-based approaches) and extensive measurements of red blood cell metabolism.ResultsAll stresses induced significant intra- and extracellular alterations of all measured elements. In particular, ionomycin treatment or hypertonic stress significantly impacted intracellular sodium and extracellular potassium and magnesium levels. Iron efflux was observed as a function of temperatures, with ionic and heat stress at 40°C causing the maximum decrease in intracellular iron pools and increases in the supernatants. Strong positive correlation was observed between calcium measurements via ICP-MS and fluorescence-based approaches. Correlation analyses with metabolomics data showed a strong positive association between extracellular calcium and intracellular sodium or magnesium levels and intracellular glycolysis. Extracellular potassium or iron were positively correlated with free fatty acids (especially mono-, poly-, and highly-unsaturated or odd-chain fatty acid products of lipid peroxidation). Intracellular iron was instead positively correlated with saturated fatty acids (palmitate, stearate) and negatively with methionine metabolism (methionine, S-adenosylmethionine), phosphatidylserine exposure and glycolysis.ConclusionIn the era of omics approaches, ICP-MS affords a comprehensive characterization of intracellular elements that provide direct insights on red blood cell physiology and represent meaningful covariates for data generated via other omics platforms such as metabolomics.

Protein Lipidation by Palmitate Controls Macrophage Function.

Macrophages are present in all tissues within our body, where they promote tissue homeostasis by responding to microenvironmental triggers, not only through clearance of pathogens and apoptotic cells but also via trophic, regulatory, and repair functions. To accomplish these divergent functions, tremendous dynamic fine-tuning of their physiology is needed. Emerging evidence indicates that S-palmitoylation, a reversible post-translational modification that involves the linkage of the saturated fatty acid palmitate to protein cysteine residues, directs many aspects of macrophage physiology in health and disease. By controlling protein activity, stability, trafficking, and protein–protein interactions, studies identified a key role of S-palmitoylation in endocytosis, inflammatory signaling, chemotaxis, and lysosomal function. Here, we provide an in-depth overview of the impact of S-palmitoylation on these cellular processes in macrophages in health and disease. Findings discussed in this review highlight the therapeutic potential of modulators of S-palmitoylation in immunopathologies, ranging from infectious and chronic inflammatory disorders to metabolic conditions.

Palmitate Enhances the Efficacy of Cisplatin and Doxorubicin against Human Endometrial Carcinoma Cells.

Endometrial cancer is the most common gynecological cancer worldwide. At present there is no effective screening test for its early detection and no curative treatment for women with advanced-stage or recurrent disease. Overexpression of fatty acid synthase is a common molecular feature of a subgroup of sex steroid-related cancers associated with poor prognoses, including endometrial cancers. Disruption of this fatty acid synthesis leads to cell apoptosis, making it a potential therapeutic target. The saturated fatty acid palmitate reportedly induces lipotoxicity and cell death by inducing oxidative stress in many cell types. Here, we explored the effects of palmitate combined with doxorubicin or cisplatin in the HEC-1-A and RL95-2 human endometrial cancer cell lines. The results showed that physiological concentrations of exogenous palmitate significantly increased cell cycle arrest, DNA damage, autophagy, and apoptosis in both RL95-2 and HEC-1-A cells. It also increased the chemosensitivity of both cell types. Notably, we did not observe that palmitate lipotoxicity reflected increased levels of reactive oxygen species, suggesting palmitate acts via a different mechanism in endometrial cancer. This study thus provides a potential therapeutic strategy in which palmitate is used as an adjuvant in the treatment of endometrial cancer.

Interleukin-26 Has Synergistic Catabolic Effects with Palmitate in Human Articular Chondrocytes via the TLR4-ERK1/2-c-Jun Signaling Pathway.

The inflammatory cytokine interleukin-26 (IL-26) is highly expressed in the serum and synovial fluid of patients with inflammatory arthritis. The effect of IL-26 on human articular chondrocytes (HACs) remains unclear. Obesity is associated with disability of patients with rheumatoid arthritis and disease activity in those with ankylosing spondylitis. The saturated free fatty acid palmitate with IL-1β can synergistically induce catabolic effects in HACs. The aim of this study was to evaluate the effects of IL-26 and palmitate in HACs. In this study, palmitate markedly synergizes the IL-26-induced proinflammatory effects and matrix protease, including COX-2, IL-6, and MMP-1, in HACs via the toll-like receptor 4 (TLR4)-ERK1/2-c-Jun signal transduction pathway. The synergistic catabolic effects of palmitate and IL-26 were attenuated by inhibitors of TLR4 (TAK242), ERK1/2 (U0126), or c-Jun (SP600125) in HACs and cartilage matrix. In addition, metformin, a potential inhibitor of TLR4, also decreased expression of COX-2 and IL-6 induced by co-incubation with IL-26 and palmitate. IL-26 and palmitate synergistically induced expression of inflammatory and catabolic mediators, resulting in articular cartilage matrix breakdown. The present study also revealed a possible mechanism and therapeutic targets against articular cartilage degradation by increased saturated fatty acids in patients with inflammatory arthritis.