Magnesium Isoglycyrrhizinate Reduces Hepatic Lipotoxicity through Regulating Metabolic Abnormalities.

The excessive accumulation of lipids in hepatocytes induces a type of cytotoxicity called hepatic lipotoxicity, which is a fundamental contributor to liver metabolic diseases (such as NAFLD). Magnesium isoglycyrrhizinate (MGIG), a magnesium salt of the stereoisomer of natural glycyrrhizic acid, is widely used as a safe and effective liver protectant. However, the mechanism by which MGIG protects against NAFLD remains unknown. Based on the significant correlation between NAFLD and the reprogramming of liver metabolism, we aimed to explore the beneficial effects of MGIG from a metabolic viewpoint in this paper. We treated HepaRG cells with palmitic acid (PA, a saturated fatty acid of C16:0) to induce lipotoxicity and then evaluated the antagonistic effect of MGIG on lipotoxicity by investigating the cell survival rate, DNA proliferation rate, organelle damage, and endoplasmic reticulum stress (ERS). Metabolomics, lipidomics, and isotope tracing were used to investigate changes in the metabolite profile, lipid profile, and lipid flux in HepaRG cells under different intervention conditions. The results showed that MGIG can indeed protect hepatocytes against PA-induced cytotoxicity and ERS. In response to the metabolic abnormality of lipotoxicity, MGIG curtailed the metabolic activation of lipids induced by PA. The content of total lipids and saturated lipids containing C16:0 chains increased significantly after PA stimulation and then decreased significantly or even returned to normal levels after MGIG intervention. Lipidomic data show that glycerides and glycerophospholipids were the two most affected lipids. For excessive lipid accumulation in hepatocytes, MGIG can downregulate the expression of the metabolic enzymes (GPATs and DAGTs) involved in triglyceride biosynthesis. In conclusion, MGIG has a positive regulatory effect on the metabolic disorders that occur in hepatocytes under lipotoxicity, and the main mechanisms of this effect are in lipid metabolism, including reducing the total lipid content, reducing lipid saturation, inhibiting glyceride and glycerophospholipid metabolism, and downregulating the expression of metabolic enzymes in lipid synthesis.

5-ALA Attenuates the Palmitic Acid-Induced ER Stress and Apoptosis in Bovine Mammary Epithelial Cells.

The conservation of mammary gland physiology by maintaining the maximum number of mammary epithelial cells (MECs) is of the utmost importance for the optimum amount of milk production. In a state of negative energy balance, palmitic acid (PA) reduces the number of bovine MECs. However, there is no effective strategy against PA-induced apoptosis of MECs. In the present study, 5-aminolevulinic acid (5-ALA) was established as a remedial agent against PA-induced apoptosis of MAC-T cells (an established line of bovine MECs). In PA-treated cells, the apoptosis-related genes BCL2 and BAX were down- and upregulated, respectively. The elevated expression of major genes of the unfolded protein response (UPR), such as CHOP, a proapoptotic marker (C/EBP homologous protein), reduced the viability of PA-treated MAC-T cells. In contrast, 5-ALA pretreatment increased and decreased BCL2 and BAX expression, respectively. Moreover, cleaved caspase-3 protein expression was significantly reduced in the 5-ALA-pretreated group in comparison with the PA group. The downregulation of major UPR-related genes, including CHOP, extended the viability of MAC-T cells pretreated with 5-ALA and also reduced the enhanced intensity of the PA-induced expression of phospho-protein kinase R-like ER kinase. Moreover, the enhanced expression of HO-1 (antioxidant gene heme oxygenase) by 5-ALA reduced PA-induced oxidative stress (OxS). HO-1 is not only protective against OxS but also effective against ER stress. Collectively, these findings offer new insights into the protective effects of 5-ALA against PA-induced apoptosis of bovine MECs.

MKP-5 Relieves Lipotoxicity-Induced Islet ß-Cell Dysfunction and Apoptosis via Regulation of Autophagy.

Mitogen-activated protein kinase phosphatase-5 (MKP-5) is a regulator of extracellular signaling that is known to regulate lipid metabolism. In this study, we found that obesity caused by a high-fat diet (HFD) decreased the expression of MKP-5 in the pancreas and primary islet cells derived from mice. Then, we further investigated the role of MKP-5 in the protection of islet cells from lipotoxicity by modulating MKP-5 expression. As a critical inducer of lipotoxicity, palmitic acid (PA) was used to treat islet ß-cells. We found that MKP-5 overexpression restored PA-mediated autophagy inhibition in Rin-m5f cells and protected these cells from PA-induced apoptosis and dysfunction. Consistently, a lack of MKP-5 aggravated the adverse effects of lipotoxicity. Islet cells from HFD-fed mice were infected using recombinant adenovirus expressing MKP-5 (Ad-MKP-5), and we found that Ad-MKP-5 was able to alleviate HFD-induced apoptotic protein activation and relieve the HFD-mediated inhibition of functional proteins. Notably, HFD-mediated impairments in autophagic flux were restored by Ad-MKP-5 transduction. Furthermore, the autophagy inhibitor 3-methyladenine (3-MA) was used to treat Rin-m5f cells, confirming that the MKP-5 overexpression suppressed apoptosis, dysfunction, inflammatory response, and oxidative stress induced by PA via improving autophagic signaling. Lastly, employing c-Jun amino-terminal kinas (JNK), P38, or extracellular-regulated kinase (ERK) inhibitors, we established that the JNK and P38 MAPK pathways were involved in the MKP-5-mediated apoptosis, dysfunction, and autophagic inhibition observed in islet ß cells in response to lipotoxicity.

A Novel Role for Somatostatin in the Survival of Mouse Pancreatic Beta Cells.

BACKGROUND/AIMS: Cross-talk between different pancreatic islet cell types regulates islet function and somatostatin (SST) released from pancreatic delta cells inhibits insulin secretion from pancreatic beta cells. In other tissues SST exhibits both protective and pro-apoptotic properties in a tissue-specific manner, but little is known about the impact of the peptide on beta cell survival. Here we investigate the specific role of SST in the regulation of beta cell survival in response to physiologically relevant inducers of cellular stress including palmitate, cytokines and glucose. METHODS: Pancreatic MIN6 beta cells and primary mouse islet cells were pre-treated with SST with or without the Gi/o signalling inhibitor, pertussis toxin, and exposed to different cellular stress factors. Apoptosis and proliferation were assessed by measurement of caspase 3/7 activity, TUNEL and BrdU incorporation, respectively, and expression of target genes was measured by qPCR. RESULTS: SST partly alleviated upregulation of cellular stress markers (Hspa1a and Ddit3) and beta cell apoptosis in response to factors such as lipotoxicity (palmitate), pro-inflammatory cytokines (IL1ß and TNFα) and low glucose levels. This effect was mediated via a Gi/o protein-dependent pathway, but did not modify transcriptional upregulation of the specific NFκB-dependent genes, Nos2 and Ccl2, nor was it associated with transcriptional changes in SST receptor expression. CONCLUSION: Our results suggest an underlying protective effect of SST which modulates the beta cell response to ER stress and apoptosis induced by a range of cellular stressors associated with type 2 diabetes.

κ-Opioid receptor stimulation reduces palmitate-induced apoptosis via Akt/eNOS signaling pathway.

BACKGROUND: This study was designed to test the hypothesis that κ-opioid receptor (κ-OR) stimulation reduces palmitate-induced HUVECs apoptosis and to investigate its mechanisms. METHODS: HUVECs were subjected to sodium palmitate, apoptosis and cell viability were determined, HUVECs were treated with specific inhibitors to PI3K, Akt, eNOS and siRNAs targeting κ-OR and Akt. Groups were divided as follows: the control group, the sodium palmitate group, the sodium palmitate+U50,488H (a selective κ-OR agonist) group and the sodium palmitate+U50,488H + nor-BNI (a selective κ-OR antagonist) group. RESULTS: Treatment with sodium palmitate significantly reduced cell viability and increased apoptosis rate which were significantly alleviated by pretreatment with U50,488H, the effect of U50,488H was abolished by nor-BNI. Phosphorylation of Akt and eNOS, as well as NO production were attenuated and accompanied by an increased expression of caspase 3 when HUVECs were subjected to sodium palmitate, and all these changes were restored by pretreatment with U50,488H, the effects of U50,488H were abolished by nor-BNI, and specific inhibitors to PI3K, Akt, eNOS, respectively. SiRNAs targeting κ-OR or Akt abolished the effects of U50,488H on phosphorylation of Akt and eNOS as well as the expressions of caspase 3, Bax and Bcl-2. SiRNAs targeting Akt elicited no effect on the expression of κ-OR. CONCLUSION: This study provides the evidence for the first time that κ-OR stimulation possesses anti-palmitate-induced apoptosis effect, which is mediated by PI3K/Akt/eNOS signaling pathway.

Therapeutic effect and autophagy regulation of myriocin in nonalcoholic steatohepatitis.

BACKGROUND: Ceramide plays pathogenic roles in nonalcoholic fatty liver disease (NAFLD) via multiple mechanisms, and as such inhibition of ceramide de novo synthesis in the liver may be of therapeutically beneficial in patients with NAFLD. In this study, we aimed to explore whether inhibition of ceramide signaling by myriocin is beneficial in animal model of NAFLD via regulating autophagy. METHODS: Sprague Dawley rats were randomly divided into three groups: standard chow (n = 10), high-fat diet (HFD) (n = 10) or HFD combined with oral administration of myriocin (0.3 mg/kg on alternate days for 8 weeks) (n = 10). Liver histology and autophagy function were measured. HepG2 cells were incubated with fatty acid with or without myriocin treatment. Lipid accumulation and autophagy markers in the HepG2 cells were analyzed. Serum ceramide changes were studied in 104 subjects consisting healthy adults, liver biopsy-proven patients with NAFLD and liver biopsy-proven patients with chronic hepatitis B (CHB). RESULTS: Myriocin reversed the elevated body weight and serum transaminases and alleviated dyslipidemia in HFD fed rats. Myriocin treatment significantly attenuated liver pathology including steatosis, lobular inflammation and ballooning. By qPCR analysis, it was revealed that myriocin corrected the expression pattern of fatty acid metabolism associated genes including Fabp1, Pparα, Cpt-1α and Acox-2. Further, myriocin also restored the impaired hepatic autophagy function in rats with HFD-induced NASH, and this has been verified in HepG2 cells. Among the sphingolipid species that we screened in lipidomic profiles, significantly increased ceramide was observed in NASH patients as compared to the controls and non-NASH patients, regardless of whether or not they have active CHB. CONCLUSIONS: Ceramide may play an important regulatory role in the autophagy function in the pathogenesis of NASH. Hence, blockade of ceramide signaling by myriocin may be of therapeutically beneficial in NASH. TRIAL REGISTRATION: Registration ID: ChiCTR-DDT-13003983 . Data of registration: 13 May, 2013, retrospectively registered.

Resveratrol Maintains Lipid Metabolism Homeostasis via One of the Mechanisms Associated with the Key Circadian Regulator Bmal1.

Resveratrol (RES) possesses anti-inflammatory and anti-oxidant activities, and it can prevent liver lipid metabolism disorders in obese and diabetic individuals. This study elucidated the mechanisms of brain and muscle Arnt-like protein-1 (Bmal1) in the protective effects of RES against liver lipid metabolism disorders. The results indicated that RES ameliorated free fatty acid (FFA)-induced (oleic acid (OA): palmitic acid (PA) = 2:1) glycolipid metabolic disorders in hepatocytes. Simultaneously, RES partially reverted the relatively shallow daily oscillations of FFA-induced circadian clock gene transcription and protein expression in HepG2 cells. RES also attenuated FFA-triggered reactive oxygen species (ROS) secretion and restored mitochondrial membrane potential consumption, as well as the restoration of mitochondrial respiratory complex expression. This study provides compelling evidence that RES controls intracellular lipid metabolic imbalance in a Bmal1-dependent manner. Overall, RES may serve as a promising natural nutraceutical for the regulation of lipid metabolic disorders relevant to the circadian clock.

Combination of Chymostatin and Aliskiren attenuates ER stress induced by lipid overload in kidney tubular cells.

BACKGROUND: Lipotoxicity plays an important role in the pathogenesis of kidney injury. Our previous study demonstrated that activation of local renin-angiotensin system (RAS) was involved in saturated free fatty acids palmitic acid (PA)-induced tubular cell injuries. The current study aims to investigate whether suppression of RAS by combination of direct renin inhibitor aliskiren and noncanonical RAS pathway chymase inhibitor chymostatin attenuates PA or cholesterol induced-endoplasmic reticulum stress (ER stress) and apopotosis in cultured human proximal tubular HK2 cells. METHODS: HK2 cells were treated with saturated fatty acid PA (0.6 mM) for 24 h or cholesterol (10 µg/ml) for 6d with or without chymostatin and/or aliskiren. Expressions of the ER stress associated proteins and apoptosis markers were detected by western blotting. The mRNA levels of RAS components were measured by real-time qPCR. RESULTS: Combination treatment of chymostatin and aliskiren markedly suppressed PA or cholesterol-induced ER stress, as reflected by increased BiP, IRE1α, phosphorylated-eIF2α and ATF4 as well as proapoptotic transcription factor CHOP. The ratio of Bax/Bcl-2 and cleaved caspase-3, two markers of apoptosis were upregulated by PA or cholesterol treatment. PA treatment was also associated with increased levels of angiotensinogen and angiotensin type 1 receptor (AT1R) mRNA expression. Combination treatment of chymostatin and aliskiren markedly suppressed PA or cholesterol-induced ER stress and apoptosis. The protective effect of two inhibitors was also observed in primary cultured cortical tubular cells treated with PA. In contrast, chymostatin and/or aliskiren failed to prevent ER stress induced by tunicamycin. CONCLUSIONS: These results suggested that combination treatment of chymostatin and aliskiren attenuates lipid-induced renal tubular cell injury, likely through suppressing activation of intracellular RAS.

Hydrogen Sulphide modulating mitochondrial morphology to promote mitophagy in endothelial cells under high-glucose and high-palmitate.

Endothelial cell dysfunction is one of the main reasons for type II diabetes vascular complications. Hydrogen sulphide (H2 S) has antioxidative effect, but its regulation on mitochondrial dynamics and mitophagy in aortic endothelial cells under hyperglycaemia and hyperlipidaemia is unclear. Rat aortic endothelial cells (RAECs) were treated with 40 mM glucose and 200 µM palmitate to imitate endothelium under hyperglycaemia and hyperlipidaemia, and 100 µM NaHS was used as an exogenous H2 S donor. Firstly, we demonstrated that high glucose and palmitate decreased H2 S production and CSE expression in RAECs. Then, the antioxidative effect of H2 S was proved in RAECs under high glucose and palmitate to reduce mitochondrial ROS level. We also showed that exogenous H2 S inhibited mitochondrial apoptosis in RAECs under high glucose and palmitate. Using Mito Tracker and transmission electron microscopy assay, we revealed that exogenous H2 S decreased mitochondrial fragments and significantly reduced the expression of p-Drp-1/Drp-1 and Fis1 compared to high-glucose and high-palmitate group, whereas it increased mitophagy by transmission electron microscopy assay. We demonstrated that exogenous H2 S facilitated Parkin recruited by PINK1 by immunoprecipitation and immunostaining assays and then ubiquitylated mitofusin 2 (Mfn2), which illuminated the mechanism of exogenous H2 S on mitophagy. Parkin siRNA suppressed the expression of Mfn2, Nix and LC3B, which revealed that it eliminated mitophagy. In summary, exogenous H2 S could protect RAECs against apoptosis under high glucose and palmitate by suppressing oxidative stress, decreasing mitochondrial fragments and promoting mitophagy. Based on these results, we proposed a new mechanism of H2 S on protecting endothelium, which might provide a new strategy for type II diabetes vascular complication.

Oleate ameliorates palmitate-induced reduction of NAMPT activity and NAD levels in primary human hepatocytes and hepatocarcinoma cells.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide adenine dinucleotide (NAD) levels are crucial for liver function. The saturated fatty acid palmitate and the unsaturated fatty acid oleate are the main free fatty acids in adipose tissue and human diet. We asked how these fatty acids affect cell survival, NAMPT and NAD levels in HepG2 cells and primary human hepatocytes. METHODS: HepG2 cells were stimulated with palmitate (0.5mM), oleate (1mM) or a combination of both (0.5mM/1mM) as well as nicotinamide mononucleotide (NMN) (0.5 mM) or the specific NAMPT inhibitor FK866 (10nM). Cell survival was measured by WST-1 assay and Annexin V/propidium iodide staining. NAD levels were determined by NAD/NADH Assay or HPLC. Protein and mRNA levels were analysed by Western blot analyses and qPCR, respectively. NAMPT enzyme activity was measured using radiolabelled 14C-nicotinamide. Lipids were stained by Oil red O staining. RESULTS: Palmitate significantly reduced cell survival and induced apoptosis at physiological doses. NAMPT activity and NAD levels significantly declined after 48h of palmitate. In addition, NAMPT mRNA expression was enhanced which was associated with increased NAMPT release into the supernatant, while intracellular NAMPT protein levels remained stable. Oleate alone did not influence cell viability and NAMPT activity but ameliorated the negative impact of palmitate on cell survival, NAMPT activity and NAD levels, as well as the increased NAMPT mRNA expression and secretion. NMN was able to normalize intracellular NAD levels but did not ameliorate cell viability after co-stimulation with palmitate. FK866, a specific NAMPT inhibitor did not influence lipid accumulation after oleate-treatment. CONCLUSIONS: Palmitate targets NAMPT activity with a consequent cellular depletion of NAD. Oleate protects from palmitate-induced apoptosis and variation of NAMPT and NAD levels. Palmitate-induced cell stress leads to an increase of NAMPT mRNA and accumulation in the supernatant. However, the proapoptotic action of palmitate seems not to be mediated by decreased NAD levels.

Protective Effect of 2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione, Isolated from Averrhoa Carambola L., Against Palmitic Acid-Induced Inflammation and Apoptosis in Min6 Cells by Inhibiting the TLR4-MyD88-NF-κB Signaling Pathway.

BACKGROUND/AIMS: Studies have demonstrated that 2-dodecyl-6-methoxycyclohexa-2, 5-diene-1, 4-dione (DMDD), isolated from the roots of Averrhoa carambola L., has significant therapeutic potential for the treatment of diabetes. However, the protective effect of DMDD against pancreatic beta cell dysfunction has never been reported. We investigated whether DMDD protected against palmitic acid-induced dysfunction in pancreatic ß-cell line Min6 cells by attenuating the inflammatory response and apoptosis and to shed light on its possible mechanism. METHODS: Cell viability was assessed by CCK-8. Glucose-stimulated insulin secretion levels and inflammatory cytokines levels were examined by ELISA. Apoptosis was assessed by Annexin V-FITC/PI Flow cytometry assay, Hoechst 33342/PI double-staining assay, and Transmission electron microscopy assay. Relative quantitative real-time PCR and western blot were used to determine the expressions of genes and proteins. RESULTS: Cell viability and glucose-stimulated insulin secretion levels were increased in DMDD-pretreated Min6 cells. DMDD inhibited inflammatory cytokines IL-6, TNF-α and MCP-1 generations in palmitic acid (PA)-induced Min6 cells. Moreover, DMDD protected against PA-induced Min6 cells apoptosis and the expression of Cleaved-Caspase-3, -8 and -9 were down-regulated and the Bcl-2/Bax ratio was increased in DMDD-pretreated Min6 cells. In addition, the expression of TLR4, MyD88 and NF-κB were down-regulated in DMDD-pretreated Min6 cells and TAK-242-pretreated group cells. CONCLUSIONS: DMDD protected Min6 cells against PA-induced dysfunction by attenuating the inflammatory response and apoptosis, and its mechanism of this protection was associated with inhibiting the TLR4-MyD88-NF-κB signaling pathway.

Oleate prevents palmitate-induced mitochondrial dysfunction, insulin resistance and inflammatory signaling in neuronal cells.

Elevated circulating levels of saturated free fatty acids (sFFAs; e.g. palmitate) are known to provoke inflammatory responses and cause insulin resistance in peripheral tissue. By contrast, mono- or poly-unsaturated FFAs are protective against sFFAs. An excess of sFFAs in the brain circulation may also trigger neuroinflammation and insulin resistance, however the underlying signaling changes have not been clarified in neuronal cells. In the present study, we examined the effects of palmitate on mitochondrial function and viability as well as on intracellular insulin and nuclear factor-κB (NF-κB) signaling pathways in Neuro-2a and primary rat cortical neurons. We next tested whether oleate preconditioning has a protective effect against palmitate-induced toxicity. Palmitate induced both mitochondrial dysfunction and insulin resistance while promoting the phosphorylation of mitogen-activated protein kinases and nuclear translocation of NF-κB p65. Oleate pre-exposure and then removal was sufficient to completely block subsequent palmitate-induced intracellular signaling and metabolic derangements. Oleate also prevented ceramide-induced insulin resistance. Moreover, oleate stimulated ATP while decreasing mitochondrial superoxide productions. The latter were associated with increased levels of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Inhibition of protein kinase A (PKA) attenuated the protective effect of oleate against palmitate, implicating PKA in the mechanism of oleate action. Oleate increased triglyceride and blocked palmitate-induced diacylglycerol accumulations. Oleate preconditioning was superior to docosahexaenoic acid (DHA) or linoleate in the protection of neuronal cells against palmitate- or ceramide-induced cytotoxicity. We conclude that oleate has beneficial properties against sFFA and ceramide models of insulin resistance-associated damage to neuronal cells.

Epidermal fatty acid-binding protein protects nerve growth factor-differentiated PC12 cells from lipotoxic injury.

Epidermal fatty acid-binding protein (E-FABP/FABP5/DA11) binds and transport long-chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E-FABP protects nerve growth factor-differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM-induced lipotoxicity (PAM-LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E-FABP. Antioxidants MCI-186 and N-acetyl cysteine prevented E-FABP's induction in expression by PAM-LTx, while tert-butyl hydroperoxide increased ROS and E-FABP expression. Non-metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E-FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE-FABP showed reduced E-FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E-FABP cellular levels by pre-loading the cells with recombinant E-FABP diminished the PAM-induced ROS and cell death. Finally, agonists for PPARß (GW0742) or PPARγ (GW1929) increased E-FABP expression and enhanced the resistance of NGFDPC12 cells to PAM-LTx. We conclude that E-FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS. Epidermal fatty acid-binding protein (E-FABP) may protect nerve cells from the damaging exposure to high levels of free fatty acids (FA). We show that E-FABP can neutralize the effects of reactive oxygen species (ROS) generated by the high levels of FA in the cell and protect PC12 cells from lipotoxic injuries common in Type 2 diabetes neuropathy. Potentially, E-FABP gene up-regulation may be mediated through the NFkB pathway and future studies are needed to further evaluate this proposition.

Antagonism Between Saturated and Unsaturated Fatty Acids in ROS Mediated Lipotoxicity in Rat Insulin-Producing Cells.

BACKGROUND/AIMS: Elevated levels of non-esterified fatty acids (NEFAs) are under suspicion to mediate ß-cell dysfunction and ß-cell loss in type 2 diabetes, a phenomenon known as lipotoxicity. Whereas saturated fatty acids show a strong cytotoxic effect upon insulin-producing cells, unsaturated fatty acids are not toxic and can even prevent toxicity. Experimental evidence suggests that oxidative stress mediates lipotoxicity and there is evidence that the subcellular site of ROS formation is the peroxisome. However, the interaction between unsaturated and saturated NEFAs in this process is unclear. METHODS: Toxicity of rat insulin-producing cells after NEFA incubation was measured by MTT and caspase assays. NEFA induced H2O2 formation was quantified by organelle specific expression of the H2O2 specific fluorescence sensor protein HyPer. RESULTS: The saturated NEFA palmitic acid had a significant toxic effect on the viability of rat insulin-producing cells. Unsaturated NEFAs with carbon chain lengths >14 showed, irrespective of the number of double bonds, a pronounced protection against palmitic acid induced toxicity. Palmitic acid induced H2O2 formation in the peroxisomes of insulin-producing cells. Oleic acid incubation led to lipid droplet formation, but in contrast to palmitic acid induced neither an ER stress response nor peroxisomal H2O2 generation. Furthermore, oleic acid prevented palmitic acid induced H2O2 production in the peroxisomes. CONCLUSION: Thus unsaturated NEFAs prevent deleterious hydrogen peroxide generation during peroxisomal ß-oxidation of long-chain saturated NEFAs in rat insulin-producing cells.

Bactericidal activity of the human skin fatty acid cis-6-hexadecanoic acid on Staphylococcus aureus.

Human skin fatty acids are a potent aspect of our innate defenses, giving surface protection against potentially invasive organisms. They provide an important parameter in determining the ecology of the skin microflora, and alterations can lead to increased colonization by pathogens such as Staphylococcus aureus. Harnessing skin fatty acids may also give a new avenue of exploration in the generation of control measures against drug-resistant organisms. Despite their importance, the mechanism(s) whereby skin fatty acids kill bacteria has remained largely elusive. Here, we describe an analysis of the bactericidal effects of the major human skin fatty acid cis-6-hexadecenoic acid (C6H) on the human commensal and pathogen S. aureus. Several C6H concentration-dependent mechanisms were found. At high concentrations, C6H swiftly kills cells associated with a general loss of membrane integrity. However, C6H still kills at lower concentrations, acting through disruption of the proton motive force, an increase in membrane fluidity, and its effects on electron transfer. The design of analogues with altered bactericidal effects has begun to determine the structural constraints on activity and paves the way for the rational design of new antistaphylococcal agents.

The effects of palmitic acid on nitric oxide production by rat skeletal muscle: mechanism via superoxide and iNOS activation.

BACKGROUND: Increased plasma concentrations of free fatty acids (FFA) can lead to insulin resistance in skeletal muscle, impaired effects on mitochondrial function, including uncoupling of oxidative phosphorylation and decrease of endogenous antioxidant defenses. Nitric oxide (NO) is a highly diffusible gas that presents a half-life of 5-10 seconds and is involved in several physiological and pathological conditions. The effects of palmitic acid on nitric oxide (NO) production by rat skeletal muscle cells and the possible mechanism involved were investigated. METHODS: Primary cultured rat skeletal muscle cells were treated with palmitic acid and NO production was assessed by nitrite measurement (Griess method) and 4,5-diaminofluorescein diacetate (DAF-2-DA) assay. Nuclear factor-kappa B (NF-ĸB) activation was evaluated by electrophoretic mobility shift assay and iNOS protein content by western blotting. RESULTS: Palmitic acid treatment increased nitric oxide production. This effect was abolished by treatment with NOS inhibitors, L-nitro-arginine (LNA) and L-nitro-arginine methyl esther (L-NAME). NF-ĸB activation and iNOS content were increased due to palmitic acid treatment. The participation of superoxide on nitric oxide production was investigated by incubating the cells with DAF-2-DA in the presence or absence of palmitic acid, a superoxide generator system (X-XO), a mixture of NOS inhibitors and SOD-PEG (superoxide dismutase linked to polyethylene glycol). Palmitic acid and X-XO system increased NO production and this effect was abolished when cells were treated with NOS inhibitors and also with SOD-PEG. CONCLUSIONS: In summary, palmitic acid stimulates NO production in cultured skeletal muscle cells through production of superoxide, nuclear factor-kappa B activation and increase of iNOS protein content.

Black mulberry (Morus nigra L.) polysaccharide ameliorates palmitate-induced lipotoxicity in hepatocytes by activating Nrf2 signaling pathway.

Black mulberry (Morus nigra L.) has shown health benefits against metabolic disorders. Lipotoxicity is considered as a potentially cause of metabolic syndrome, and there is no effective treatment. However, the protective effect and its mechanism of black mulberry against lipotoxicity are unclear. In this study, three polysaccharide fractions (BP1, BP2, BP3) were isolated from black mulberry by stepwise precipitation with 30%, 60%, and 90% of ethanol and analyzed by GPC, HPLC and FT-IR methods. BP1 exhibited a better protective effect than BP2 and BP3 on palmitic acid (PA)-induced lipotoxicity in HepG2 cells. BP1 effectively reduced PA-induced lipotoxicity by eliminating accumulation of ROS, improving mitochondrial function, reversing glutathione depletion and enhancing antioxidant enzyme activities. Mechanistically, BP1 activated the Nrf2 signaling pathway, a master regulator of the antioxidant defense system, through increasing Nrf2 nuclear translocation and phosphorylation. Collectively, these results demonstrate that BP1 has the great potential for applications in lipid disorders.

[Protective effect of fibroblast growth factors-21 and rosiglitazone sodium on palmitic acid-induced apoptosis in HIT-T15 cells].

OBJECTIVE: To investigate the protective effect of fibroblast growth factors-21 (FGF-21) and rosiglitazone sodium (RO) on palmitic acid-induced apoptosis in HIT-T15 cells. METHODS: (1) HIT-T15 cells were treated with 0.25 mmol/L, 0.50 mmol/L and 1.00 mol/L of palmitic acid for 24 hours. (2) FGF-21 [12.50 nmol/L (A), 25.00 nmol/L(B), 50.00 nmol/L(C)], or 1 micromol/L of RO, or a combination of the two were added to the cells treated with 0.50 mmol/L of palmitic acid. Cell apoptosis was measured by flow cytometer. Phosphorylation-c-Jun N-terminal kinases (p-JNK) was detected by immuocytochemistry and Western blot. RESULTS: (1) The cells treated with palmitic acid has significantly higher apoptosis rates than controls (P < 0.05). (2) FGF-21 reduced apoptosis rates induced by palmitic acid (P < 0.05). But the apoptosis rates remained higher than controls (P < 0.05). A combination of FGF-21 and RO further reduced the apoptosis rates compared with FGF-21 alone (P < 0.05). (3) The cells treated with palmitic acid had higher expression of p-JNK than controls (P < 0.05). FGF-21 and a combination of FGF-21 and RO reduced the expression of p-JNK in cells treated with palmitic acid (P < 0.05). CONCLUSION: (1) Palmitic acid induces apoptosis in HIT-T15 cells in a dose dependent way. (2) The expression of p-JNK induced by palimitic acid is reduced by FGF-21 and the combination of FGF-21 and RO.

Discovery and biological evaluation of vinylsulfonamide derivatives as highly potent, covalent TEAD autopalmitoylation inhibitors.

Transcriptional enhancer associated domain family members (TEADs) are the most important downstream effectors that play the pivotal role in the development, regeneration and tissue homeostasis. Recent biochemical studies have demonstrated that TEADs could undergo autopalmitoylation that is indispensable for its function making the lipid-binding pocket an attractive target for chemical intervention. Herein, through structure-based virtual screen and rational medicinal chemistry optimization, we identified DC-TEADin02 as the most potent, selective, covalent TEAD autopalmitoylation inhibitor with the IC50 value of 197 ±â€¯19 nM while it showed minimal effect on TEAD-YAP interaction. Further biochemical counter-screens demonstrate the specific thiol reactivity and selectivity of DC-TEADin02 over the kinase family, lipid-binding proteins and epigenetic targets. Notably, DC-TEADin02 inhibited TEADs transcription activity leading to downregulation of YAP-related downstream gene expression. Taken together, our findings proved the validity of modulating transcriptional output in the Hippo signaling pathway through irreversible chemical interventions of TEADs autopalmitoylation activity, which may serve as a qualified chemical tool for TEADs palmitoylation-related studies in the future.

Plasmodium palmitoylation machinery engineered in E. coli for high-throughput screening of palmitoyl acyl-transferase inhibitors.

Lipid-based palmitoylation is a post-translation modification (PTM) which acts as a biological rheostat in life cycle progression of a deadly human malaria parasite, Plasmodium falciparum. P. falciparum palmitoylation is catalyzed by 12 putative palmitoyl acyl-transferase enzymes containing the conserved DHHC-CRD (DHHC motif within a cysteine-rich domain) which can serve as a druggable target. However, the paucity of high-throughput assays has impeded the design of drugs targeting palmitoylation. We have developed a novel strategy which involves engineering of Escherichia coli, a PTM-null system, to enforce ectopic expression of palmitoyl acyl-transferase in order to study Plasmodium-specific palmitoylation and screening of inhibitors. In this study, we have developed three synthetic E. coli strains expressing Plasmodium-specific DHHC proteins (PfDHHC7/8/9). These cells were used for validating acyl-transferase activity via acyl-biotin exchange (ABE) and clickable chemistry methods. E. coli proteome was found to be palmitoylated in PfDHHC-expressing clones, suggesting that plasmodium DHHC can catalyze palmitoylation of E. coli proteins. Upon treatment with generic inhibitor 2-bromopalmitate (2-BMP), a predominant reduction in palmitic acid incorporation is detected. Overall, these findings suggest that synthetic E. coli strains expressing PfDHHCs can enforce global palmitoylation in the E. coli proteome. Interestingly, this finding was corroborated by our in silico palmitoylome profiling, which revealed that out of the total E. coli proteome, 108 proteins were predicted to be palmitoylated as represented by the presence of three cysteine consensus motifs (cluster type I, II, III). In summary, our study reports a proof of concept for screening of chemotherapeutics targeting the palmitoylation machinery using a high-throughput screening platform.