Palmitate-induced Inflammation Research Articles

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-1β, 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.

Xanthohumol alleviates palmitate-induced inflammation and prevents osteoarthritis progression by attenuating mitochondria dysfunction/NLRP3 inflammasome axis

Osteoarthritis (OA) is a prevalent chronic degenerative joint disease worldwide. Obesity has been linked to OA, and increased free fatty acid levels (e.g., palmitate) contribute to inflammatory responses and cartilage degradation. Xanthohumol (Xn), a bioactive prenylated chalcone, was shown to exhibit antioxidative, anti-inflammatory, and anti-obesity capacities in multiple diseases. However, a clear description of the preventive effects of Xn on obesity-associated OA is unavailable. This study aimed to assess the chondroprotective function of Xn on obesity-related OA. The in vitro levels of inflammatory and ECM matrix markers in human chondrocytes were assessed after the chondrocytes were treated with PA and Xn. Additionally, in vivo cartilage degeneration was assessed following oral administration of HFD and Xn. This study found that Xn treatment completely reduces the inflammation and extracellular matrix degradation caused by PA. The proposed mechanism involves AMPK signaling pathway activation by Xn, which increases mitochondrial biogenesis, attenuates mitochondrial dysfunction, and inhibits NLRP3 inflammasome and the NF-κB signaling pathway induced by PA. In summary, this study highlights that Xn could decrease inflammation reactions and the degradation of the cartilage matrix induced by PA by inhibiting the NLRP3 inflammasome and attenuating mitochondria dysfunction in human chondrocytes.

Palmitate-Induced Inflammation and Myotube Atrophy in C2C12 Cells Are Prevented by the Whey Bioactive Peptide, Glycomacropeptide

BackgroundMetabolic diseases are often associated with muscle atrophy and heightened inflammation. The whey bioactive compound, glycomacropeptide (GMP), has been shown to exhibit anti-inflammatory properties and therefore may have potential therapeutic efficacy in conditions of skeletal muscle inflammation and atrophy. ObjectivesThe purpose of this study was to determine the role of GMP in preventing lipotoxicity-induced myotube atrophy and inflammation. MethodsC2C12 myoblasts were differentiated to determine the effect of GMP on atrophy and inflammation and to explore its mechanism of action in evaluating various anabolic and catabolic cellular signaling nodes. We also used a lipidomic analysis to evaluate muscle sphingolipid accumulation with the various treatments. Palmitate (0.75 mM) in the presence and absence of GMP (5 μg/mL) was used to induce myotube atrophy and inflammation and cells were collected over a time course of 6–24 h. ResultsAfter 24 h of treatment, GMP prevented the palmitate-induced decrease in the myotube area and myogenic index and the increase in the TLR4-mediated inflammatory genes tumor necrosis factor-α and interleukin 1β. Moreover, phosphorylation of Erk1/2, and gene expression of myostatin, and the E3 ubiquitin ligases, FBXO32, and MuRF1 were decreased with GMP treatment. GMP did not alter palmitate-induced ceramide or diacylglycerol accumulation, muscle insulin resistance, or protein synthesis. ConclusionsIn summary, GMP prevented palmitate-induced inflammation and atrophy in C2C12 myotubes. The GMP protective mechanism of action in muscle cells during lipotoxic stress may be related to targeting catabolic signaling associated with cellular stress and proteolysis but not protein synthesis.

Ginsenoside Rc from Panax Ginseng Ameliorates Palmitate-Induced UB/OC-2 Cochlear Cell Injury.

By 2050, at least 700 million people will require hearing therapy while 2.5 billion are projected to suffer from hearing loss. Sensorineural hearing loss (SNHL) arises from the inability of the inner ear to convert fluid waves into neural electric signals because of injury to cochlear hair cells that has resulted in their death. In addition, systemic chronic inflammation implicated in other pathologies may exacerbate cell death leading to SNHL. Phytochemicals have emerged as a possible solution because of the growing evidence of their anti-inflammatory, antioxidant, and anti-apoptotic properties. Ginseng and its bioactive molecules, ginsenosides, exhibit effects that suppress pro-inflammatory signaling and protect against apoptosis. In the current study, we investigated the effects of ginsenoside Rc (G-Rc) on UB/OC-2 primary murine sensory hair cell survival in response to palmitate-induced injury. G-Rc promoted UB/OC-2 cell survival and cell cycle progression. Additionally, G-Rc enhanced the differentiation of UB/OC-2 cells into functional sensory hair cells and alleviated palmitate-induced inflammation, endoplasmic reticulum stress, and apoptosis. The current study offers novel insights into the effects of G-Rc as a potential adjuvant for SNHL and warrants further studies elucidating the molecular mechanisms.

Developmental endothelial locus-1 attenuates palmitate-induced apoptosis in tenocytes through the AMPK/autophagy-mediated suppression of inflammation and endoplasmic reticulum stress.

Myokine developmental endothelial locus-1 (DEL-1) has been documented to alleviate inflammation and endoplasmic reticulum (ER) stress in various cell types. However, the effects of DEL-1 on inflammation, ER stress, and apoptosis in tenocytes remain unclear. Human primary tenocytes were cultured in palmitate (400 μM) and palmitate plus DEL-1 (0 to 2 μg/ml) conditions for 24 hours. The expression levels of ER stress markers and cleaved caspase 3, as well as phosphorylated 5' adenosine monophosphate-activated protein kinase (AMPK) and autophagy markers, were assessed by Western blotting. Autophagosome formation was measured by staining with monodansylcadaverine, and apoptosis was determined by cell viability assay and caspase 3 activity assay. We found that treatment with DEL-1 suppressed palmitate-induced inflammation, ER stress, and apoptosis in human primary tenocytes. DEL-1 treatment augmented LC3 conversion and p62 degradation as well as AMPK phosphorylation. Moreover, small interfering RNA for AMPK or 3-methyladenine (3-MA), an autophagy inhibitor, abolished the suppressive effects of DEL-1 on inflammation, ER stress, and apoptosis in tenocytes. Similar to DEL-1, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMPK, also attenuated palmitate-induced inflammation, ER stress, and apoptosis in tenocytes, which 3-MA reversed. These results revealed that DEL-1 suppresses inflammation and ER stress, thereby attenuating tenocyte apoptosis through AMPK/autophagy-mediated signalling. Thus, regular exercise or administration of DEL-1 may directly contribute to improving tendinitis exacerbated by obesity and insulin resistance.Cite this article: Bone Joint Res2022;11(12):854-861.

Role of acyl-coenzyme A oxidase 1 (ACOX1) on palmitate-induced inflammation and ROS production of macrophages in large yellow croaker (Larimichthys crocea)

Acyl-coenzyme A oxidase 1 (ACOX1) is the rate-limiting enzyme in peroxisomal β-oxidation, and it plays an essential role in mediating the inflammatory response and reactive oxygen species (ROS) metabolism in mammals. However, the role of ACOX1 in fish has not been completely elucidated. Herein, this study was conducted to investigate the role of large yellow croaker (Larimichthys crocea) ACOX1 (Lc-ACOX1) on palmitate (PA)-induced inflammation and ROS production. In this study, Lc-ACOX1 was cloned and characterized. The full-length CDS of Lc-acox1 was 1986 bp, encoding 661 amino acids. Tissue distribution results showed that the gene expression of Lc-acox1 was the highest in the intestine and the lowest in the spleen. Moreover, results showed that the mRNA expression of Lc-acox1 was upregulated by PA, with elevated pro-inflammatory gene expression, including il-1β, il-6, il-8, tnf-α, cox2 and ifn-γ, as well as ROS content in macrophages of large yellow croaker. Furthermore, the role of Lc-ACOX1 in inflammation induced by PA was investigated by using the ACOX1 inhibitor TDYA. Treatment of macrophages with TDYA reduced the mRNA expression of pro-inflammatory genes induced by PA. Moreover, inhibition of ACOX1 reduced the elevated level of ROS caused by PA and increased the mRNA expression of antioxidant genes. In conclusion, this study first identified that fish ACOX1 was involved in the PA-induced inflammatory response and ROS production.

PDIA4, a novel ER stress chaperone, modulates adiponectin expression and inflammation in adipose tissue.

Increasing evidence supporting a causal link between obesity and endoplasmic reticulum (ER) stress in adipose tissue is being reported. Protein disulfide isomerase 4 (PDIA4) is a novel ER chaperone involved in the pancreatic β-cells pathogenesis in diabetes. However, the role of PDIA4 in obesity progression remains poorly understood. To assess the relationship between PDIA4, adiponectin, and metformin, we used the palmitate-induced inflammation in hypertrophic adipocytes and the high-fat diet-induced obesity mouse model. Our results revealed that palmitate-induced hypertrophic adipocytes exhibit obesity-associated conditions such as increased lipid accumulation, inflammation, and reduced glucose uptake. Pharmacological and genetic inhibition of PDIA4 significantly reverses these obesity-associated conditions in adipocytes. PDIA4 mechanistically promotes obesity progression via adiponectin downregulation. Furthermore, metformin modulates PDIA4 and adiponectin expression and improves obesity-associated conditions in both in vitro adipocytes and in vivo mouse models. Serum PDIA4 concentrations are also associated with body mass index, adiponectin, triglycerides, and inflammatory cytokines in humans. This is the first study demonstrating that PDIA4 modulates adipocytes by downregulating adiponectin. Moreover, metformin may serve as a potential therapeutic for preventing obesity via PDIA4-targeting.

Lysophosphatidylcholine acyltransferase 3 (LPCAT3) mediates palmitate-induced inflammation in macrophages of large yellow croaker (Larimichthys crocea)

LPCAT3, a subtype of lysophosphatidylcholine acyltransferases, is a key enzyme in phosphatidylcholine remodeling pathway and plays a significant role in mediating inflammatory response in mammals. However, its inflammatory function in fish has yet to be discovered. Herein, this study aimed to investigate its role in inflammation in Larimichthys crocea. We analyzed the coding sequence of Larimichthys crocea LPCAT3 (Lc-LPCAT3) and explored the effect of Lc-LPCAT3 on palmitate (PA)-induced inflammation. We found that in macrophage cell line of Larimichthys crocea, the mRNA expression of Lc-lpcat3 was upregulated by PA with the elevated pro-inflammatory genes expression, including il1β, il6, il8, tnfα and ifnγ. Next, the role of Lc-LPCAT3 in inflammation induced by PA was further investigated. Results showed that knockdown of Lc-LPCAT3 mitigated PA-induced pro-inflammatory genes mRNA expression, including il1β, il8, tnfα and ifnγ, in which JNK signaling pathway was involved. In contrast, overexpression of Lc-LPCAT3 induced pro-inflammatory genes expression including il1β, tnfα and ifnγ. Furthermore, several transcription factors with negative regulation of Lc-LPCAT3 promoter activity were discovered including LXRα, RXRα, PPARα, PPARγ, CEBPα, CEBPβ, CEBPδ, SREBP1 and SREBP2, and SREBP1 had the strongest regulatory effect. In conclusion, we first discovered that fish LPCAT3 participated in PA-induced inflammation, and targeting SREBP1 might be an effective coping strategy.

Patchouli alcohol ameliorates skeletal muscle insulin resistance and NAFLD via AMPK/SIRT1-mediated suppression of inflammation

Obesity-induced chronic low-grade inflammation and thus causes various metabolic diseases, such as insulin resistance and non-alcoholic fatty liver disease (NAFLD). Patchouli alcohol (PA), an active component extracted from patchouli, displayed anti-inflammatory effects on different cell types. However, the impact of PA on skeletal muscle insulin signaling and hepatic lipid metabolism remains unclear. This study aimed to investigate whether PA would affect insulin signaling impairment in myocytes and lipid metabolism in hepatocytes. Treatment with PA ameliorated palmitate-induced inflammation and aggravation of insulin signaling in C2C12 myocytes and lipid accumulation in HepG2 hepatocytes. Treatment of C2C12 myocytes and HepG2 cells with PA augmented AMP-activated protein kinase (AMPK) phosphorylation and Sirtuin 1 (SIRT1) expression in a dose-dependent manner. siRNA-mediated suppression of AMPK or SIRT1 mitigated the effects of PA on palmitate-induced inflammation and insulin resistance in C2C12 myocytes and lipid accumulation in HepG2 cells. Animal experiments demonstrated that PA administration increased AMPK phosphorylation and SIRT1 expression, and ameliorated inflammation, thereby attenuating skeletal muscle insulin resistance and hepatic steatosis in high-fat diet-fed mice. These results denote that PA alleviates skeletal muscle insulin resistance and hepatic steatosis through AMPK/SIRT1-dependent signaling. This study might provide a novel therapeutic approach for treating obesity-related insulin resistance and NAFLD.

Valdecoxib improves lipid-induced skeletal muscle insulin resistance via simultaneous suppression of inflammation and endoplasmic reticulum stress

Valdecoxib (VAL), a non-steroidal anti-inflammatory drug, has been widely used for treatment of rheumatoid arthritis, osteoarthritis, and menstrual pain. It is a selective cyclooxygenase-2 inhibitor. The suppressive effects of VAL on cardiovascular diseases and neuroinflammation have been documented; however, its impact on insulin signaling in skeletal muscle has not been studied in detail. The aim of this study was to investigate the effects of VAL on insulin resistance in mouse skeletal muscle. Treatment of C2C12 myocytes with VAL reversed palmitate-induced aggravation of insulin signaling and glucose uptake. Further, VAL attenuated palmitate-induced inflammation and endoplasmic reticulum (ER) stress in a concentration-dependent manner. Treatment with VAL concentration-dependently upregulated AMP-activated protein kinase (AMPK) and heat shock protein beta 1 (HSPB1) expression. In line with in vitro experiments, treatment with VAL augmented AMPK phosphorylation and HSPB1 expression, thereby alleviating high-fat diet-induced insulin resistance along with inflammation and ER stress in mouse skeletal muscle. However, small interfering RNA-mediated inhibition of AMPK abolished the effects of VAL on insulin resistance, inflammation, and ER stress. These results suggest that VAL alleviates insulin resistance through AMPK/HSPB1-mediated inhibition of inflammation and ER stress in skeletal muscle under hyperlipidemic conditions. Hence, VAL could be used as an effective pharmacotherapeutic agent for management of insulin resistance and type 2 diabetes.

Folic acid ameliorates palmitate-induced inflammation through decreasing homocysteine and inhibiting NF-κB pathway in HepG2 cells

Objective Prevention of inflammation is one of the possible remedy procedure for steatohepatitis during NAFLD. In this study, we researched the folic acid (FA) potency to attenuate the inflammation of palmitate-treated HepG2 cells and the related signalling pathways. Methods The molecular mechanisms related to FA anti-inflammatory effect in palmitate and Hcy-treated HepG2 cell line were assessed. Results The results indicated that while palmitate enhances the expression and secretion of TNF-α, IL-6, and IL-1β, and also intracellular ROS level, FA at concentrations of 25, 50, and 75 µg/mL significantly reversed these effects in HepG2 cells. In addition, FA could ameliorate inflammation and decrease ROS production induced by Hcy. Furthermore, FA pre-treatment suppress palmitate -induced (NF-κB) p65 level in palmitate or Hcy stimulated cells. Conclusions Overall, these results suggest that FA reduces inflammation in HepG2 cells through decreasing ROS and Hcy concentration level resulting in inhibiting the NF-κB pathway.

Olive Leaf Extract, from Olea europaea L., Reduces Palmitate-Induced Inflammation via Regulation of Murine Macrophages Polarization.

Olive tree (Olea europaea L.) leaves are an abundant source of bioactive compounds with several beneficial effects for human health. Recently, the effect of olive leaf extract in obesity has been studied. However, the molecular mechanism in preventing obesity-related inflammation has not been elucidated. Obesity is a state of chronic low-grade inflammation and is associated with an increase of pro-inflammatory M1 macrophages infiltration in the adipose tissue. In the current study, we explored Olea europaea L. leaf extract (OLE) anti-inflammatory activity using an in vitro model of obesity-induced inflammation obtained by stimulating murine macrophages RAW 264.7 with high dose of the free fatty acid palmitate. We found that OLE significantly suppressed the induction of pro-inflammatory mediators, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, nitric oxide (NO), prostaglandin E2 (PGE2) and reactive oxygen species (ROS), while it enhanced the anti-inflammatory cytokine, IL-10. Moreover, we demonstrated that OLE reduced the oxidative stress induced by palmitate in macrophages by regulating the NF-E2-related factor 2 (NRF2)−Kelch-like ECH-associated protein 1 (KEAP1) pathway. Finally, we showed that OLE promoted the shift of M1 macrophage toward less inflammatory M2-cells via the modulation of the associated NF-κB and proliferator-activated receptor gamma (PPARγ) signaling pathways. Thereby, our findings shed light on the potential therapeutic feature of OLE in recovering obesity-associated inflammation via regulating M1/M2 status.

1,2-Dilinoleoyl-sn-glycero-3-phosphocholine increases insulin sensitivity in palmitate-treated myotubes and induces lipolysis in adipocytes

Obesity causes the development of insulin resistance and type 2 diabetes. Phosphatidylcholine (PPC) has been reported to increase hepatic insulin sensitivity and lipolysis in adipose tissue to resolve local obesity. In this study, we proposed 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), the main active species of PPC, as an effective substance for the treatment of obesity-mediated disorders such as impaired fat metabolism and insulin resistance. Therefore, we investigated the potential lipolytic effects of DLPC on adipocytes and insulin signaling in muscle cells. In this study, DLPC-treated 3T3-L1 adipocytes showed enhanced tumor necrosis factor α (TNF-α) release. Suppression of TNF-α by short interfering RNA (siRNA) mitigated DLPC-induced lipolysis and apoptosis. DLPC treatment increased peroxisome proliferator-activated receptor α (PPARα) expression levels in C2C12 myocytes. siRNA-mediated suppression of PPARα abrogated the suppressive effects of DLPC on palmitate-induced inflammation and insulin resistance. In conclusion, DLPC enhanced lipolysis and apoptosis via a TNFα-dependent pathway in adipocytes and attenuated palmitate-induced insulin resistance through PPARα-mediated suppression of inflammation in myocytes.

Glycation of HDL blunts its anti-inflammatory and cholesterol efflux capacities in vitro, but has no effect in poorly controlled type 1 diabetes subjects

BackgroundHigh-density lipoproteins (HDL) modified by glycation have been reported to be dysfunctional. Little is known regarding the anti-inflammatory effects on adipocytes of glycated HDL. AimsWe tested whether modification of HDL in vitro by glycolaldehyde (GAD), malondialdehyde (MDA) or glucose affected HDL's anti-inflammatory properties and ability to promote cholesterol efflux. To determine whether similar changes occur in vivo, we examined modifications of apolipoprotein A1 (APOA1) and APOA2 and anti-inflammatory and cholesterol efflux properties of HDL isolated from subjects with type 1 diabetes in poor glycemic control. ResultsIn vitro modification with both GAD and MDA blunted HDL's ability to inhibit palmitate-induced inflammation and cholesterol efflux in adipocytes. Modification of HDL by glucose had little impact on HDL function, like the response using HDL isolated from subjects with diabetes. Mass spectrophotometric analysis revealed that lysine residues in APOA1 and APOA2 of HDL modified by GAD and MDA in vitro differed from those modified by glucose, which resembled that seen with HDL from patients with type1 diabetes. ConclusionsModification of lysine residues in HDL by GAD and MDA in vitro does not mirror the HDL glycation in vivo in patients with diabetes, but resembles HDL modified in vitro by glucose.

Administration of kynurenic acid reduces hyperlipidemia-induced inflammation and insulin resistance in skeletal muscle and adipocytes

Kynurenic acid (KA), an endogenous product of L-tryptophan metabolism in the kynurenine pathway, regulates adipose tissue energy homeostasis and inflammation. However, its role in palmitate-induced insulin resistance and detailed underlying mechanisms in skeletal muscles and adipose tissues are unclear. Herein, we report that KA ameliorated palmitate-induced inflammation and insulin resistance in differentiated C2C12 and 3T3-L1 cell lines as well as soleus skeletal muscle and subcutaneous adipose tissues in mice. Palmitate-induced inflammatory markers, such as nuclear factor κB translocation, inhibitory κBα phosphorylation, pro-inflammatory cytokine expression, and impaired insulin signaling, were markedly attenuated by KA both in vitro and in vivo. KA significantly increased AMP-activated protein kinase (AMPK) phosphorylation and sirtuin 6 (SIRT6) expressions in C2C12 myocytes and 3T3-L1 adipocytes and skeletal muscle and adipose tissues of mice. siRNA-mediated AMPK or SIRT6 inhibition significantly mitigated the suppressive effects of KA on palmitate-induced inflammation and insulin resistance. KA significantly stimulated expression of genes involved in fatty acid oxidation in C2C12 myocytes and skeletal muscle of mice. Moreover, KA inhibits lipogenesis in 3T3-L1 adipocytes. AMPK or SIRT6 siRNA markedly reversed these changes. The siRNA targeting Gpr35 abrogated the effects of KA on AMPK phosphorylation in C2C12 myocytes and 3T3-L1 adipocytes, except SIRT6 expression. It has therefore been shown that KA could potentially alleviate inflammation and insulin resistance in skeletal muscle and adipose tissues through Gpr35/AMPK and SIRT6-mediated pathways.

Tst gene mediates protection against palmitate-induced inflammation in 3T3-L1 adipocytes

Obesity is a disease characterized by chronic inflammation and increases the risk of comorbidities such as type 2 diabetes mellitus, non-alcoholic fatty liver disease and cardiovascular dysfunction. In obese individuals, elevated levels of plasma circulating saturated fatty acids (e.g. palmitate) contribute to subclinical chronic inflammation. Genetic variants play a major role in determining susceptibility or resistance to obesity. Some recently described variant in genetically Lean mice that leads to upregulated expression selectively in adipose tissue of the nuclear encoded mitochondrial gene thiosulfate sulfurtransferase, Tst, has been causally linked to reduced adiposity and insulin sensitisation. Here we show that macrophages derived from the high-TST expressing Lean line of mice have a reduced inflammatory response to TLR-ligand activation than those from the low-TST expressing Fat line of mice. Furthermore, using the mouse clonal 3T3-L1 adipocytes we describe a positive effect of TST upregulation via its substrates, sodium thiosulfate (STS) and the garlic compound diallyl disulfide (DADS), on palmitate-induced inflammation resulting in significant reduction of inflammatory cytokines and inhibition of reactive oxygen species. Additionally, using an IL1β-induced insulin resistance 3T3-L1 cell model, upregulation of TST associated with antidiabetic effects. Therefore, we demonstrate that TST-mediated suppression of the inflammatory response protects in vitro cell model of white adipocytes from insulin resistance-induced inflammation. These results encourage the development of potent medicines in the future that specifically target TST in the fat tissue, which could lead to new ways of treating diabetes associated with obesity.

Fucoxanthin alleviates palmitate-induced inflammation in RAW 264.7 cells through improving lipid metabolism and attenuating mitochondrial dysfunction.

In this study, we aimed to examine the effects of fucoxanthin on inflammation triggered by palmitate in macrophages. Raw 264.7 cells were treated with palmitate with or without fucoxanthin co-treatment. Fucoxanthin greatly alleviated palmitate-induced decrease in cell viability and loss of mitochondrial membrane potential. Fucoxanthin also significantly attenuated the palmitate-induced transcriptional expression of Il-6, Il-1β, Tnfα and Nlrp3 inflammasomes and increased the expression of Tgfb. In addition, fucoxanthin decreased triglyceride accumulation induced by palmitate through enhancing the expression of Cpt1a, Pparg and other lipid metabolism genes. Inhibition of CPT1a by etomoxir attenuated the anti-inflammatory effect of fucoxanthin. Furthermore, fucoxanthin increased AMPK phosphorylation and AMPKa1 knockdown by its specific siRNA diminished protective function. In addition, fucoxanthin restored palmitate-mediated mitochondrial dysfunction and improved mitophagy-related gene expression. These findings suggest that fucoxanthin could attenuate free fatty acid-induced inflammation in macrophages through modulating lipid metabolism and mitigating mitochondrial dysfunction.

Interplay between oxidative stress and autophagy function and its role in inflammatory cytokine expression induced by palmitate in skeletal muscle cells

Autophagy is a cellular process activated in response to various stresses such as starvation, hypoxia, and oxidative stress. Autophagy was reported to modulate the inflammatory pathways. However, whether autophagy is involved in regulation of palmitate-induced inflammation of skeletal muscle C2C12 cells is still unknown. The present study aimed to investigate the autophagic pathway in C2C12 cells treated with 0.5 mM palmitate. The results showed that the protein levels of LC3BII and P62 were increased in C2C12 cells after 12 h palmitate treatment. Besides, inhibition of autophagy by chloroquine or 3-methyladenin and its activation by rapamycin were associated with elevated mRNA and protein levels of IL-6 and TNF-α inflammatory cytokines in C2C12 cells. To study the mechanism by which autophagy impairment leads to activation of inflammatory responses, reactive oxygen species (ROS) levels in palmitate-treated cells were measured. The results showed that while palmitate stimulates ROS production, pretreatment of the cells with N-acetyl cysteine (NAC), a ROS scavenger, reduced inflammatory responses and also improved LC3-BII and P62 protein in the C2C12 cells exposed to palmitate. These findings suggest that palmitate-induced defect of autophagic flux leads to elevated inflammatory cytokine expression in the skeletal muscle cells by regulating the oxidative stress process.

1896-P: Effect of PNPLA3 I148M Genotypes on Palmitate-Induced Hepatic Inflammation and Apoptosis

Background: GWAS has revealed that rs738409 C>G patatin-like phospholipase domain-containing 3(PNPLA3) is strongly associated with nonalcoholic fatty liver disease(NAFLD), but the mechanism remains obscure. Our study aims to investigate differences in mechanism for genotypes of PNPLA3 I148M in palmitate-induced inflammation and apoptosis. Method: We constructed point mutation PNPLA3 148 I/I,148 I/M 148 M/M HepG2 cell lines via CRISPR-associated (Cas) 9 system. Cells were exposed to palmitate to induce lipid accumulation. Oil red O staining was used to explore the lipid deposition. Levels of inflammatory and apoptotic factors were determined by real-time PCR and western blotting. Result: Lipid accumulation was remarkably increased in cells induced by PA, it is remarkably higher in 148M/M cell than in 148I/I HepG2 cell (P<0.05 PA vs. BSA). Expression of apoptotic and inflammatory markers BIP, p-PERK, CHOP, p-JNK increased significantly in 148M/M and 148I/M hepG2 cells (P<0.05). Compared with 148I/I cells, The mRNA expression of TNF-α and IL-8 was higher in 148M/M hepG2 cells induced by palmitate. Conclusion: Different genotypes of PNPLA3 I148M have different effects on PA-induced lipid deposition, PNPLA3 148M may play an important role in pathogenesis of NAFLD through ER stress CHOP and IRE1α-JNK pathway. Disclosure Y. Chen: None. H. Liang: None. X. Yan: None. X. Xu: None.

451-P: SGLT2 Inhibitor Protects Cardiomyocytes against Palmitate-Induced Lipotoxicity by Increasing Activation of AMPK and Lipid Oxidations

Aim: This study was designed to evaluate the effects of SGLT2 inhibitor on palmitate-induced lipotoxicity of cardiomyocytes and the underlying mechanism involved with palmitate. Methods: We checked SGLT2 expression in muscle cells, including H9C2. H9C2 cells were palmitate treated with/without SGLT2 inhibitor. Then, insulin resistance was measured, 2-NBDG uptake, and immunoblotting using insulin signaling pathway antibodies. Inflammation and cardiac metabolism were analyzed several related genes. Cardiomyocyte apoptosis was also detected using DNA fragmentations assay and immunoblotting using cleaved caspase 3. Results: Low doses of palmitates evoked lipotoxicity in cardiomyocytes. Treatment of SGLT2 inhibitor dramatically reduced palmitate-induced expression levels of inflammatory cytokines. Immunoblotting analysis showed treatment of cardiomyocytes, with SGLT2 inhibitor, recovered impaired insulin signaling, such as phospho-IR and phospho-AKT, but total IR and AKT did not change. SGLT2 inhibitor also restored insulin-stimulated glucose uptake, which was destroyed by lipotoxicity. Treatment with SGLT2 inhibitor decreased the level of ER stress markers as well as inflammatory signaling markers. In particular, SGLT2 inhibitor stimulated phosphorylation of AMPK and fatty acid beta-oxidation. Cardiomyocyte exposure to high doses of palmitates evoked apoptotic features such as caspase 3 activation and DNA fragmentations. But pretreatment of SGLT2 inhibitor significantly protected palmitate-induced apoptotic features. Conclusions: This data suggests SGLT2 inhibitors protect palmitate-induced cardiomyocytes inflammation, impaired cardiac metabolism, insulin resistance, and cardiomyocyte apoptosis. Therefore, attempts to use treatment of SGLT2 inhibitor might be a useful strategy for preventing diabetes-associated ventricular remodeling and diabetic cardiac complication. Disclosure H. Lee: None. S. Choi: None. Y. Kang: None. T. Kim: None. N. Lee: None. H. Moon: None. S. Han: None. D. Kim: None. K. Lee: None.