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.

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.

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.

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.

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.

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.

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.

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.

Docosahexaenoic Acid Reduces Palmitic Acid-Induced Endoplasmic Reticulum Stress in Pancreatic Β Cells.

Endoplasmic reticulum (ER) stress leads to peripheral insulin resistance and the progression of pancreatic beta cell failure in type 2 diabetes. Although ER stress plays an important role in the pathogenesis of diabetes, it is indispensable for cellular activity. Therefore, when assessing the pathological significance of ER stress, it is important to monitor and quantify ER stress levels. Here, we have established a novel system to monitor ER stress levels quickly and sensitively, and using this method, we have clarified the effect of differences in glucose concentration and various fatty acids on the ER of pancreatic ß cells. First, we developed a cell system that secretes Gaussia luciferase in culture medium depending on the activation of the GRP78 promoter. This system could sensitively monitor ER stress levels that could not be detected with real-time RT-PCR and immunoblotting. This system revealed that hyperglycemia does not induce unfolded protein response (UPR) in a short period of time in MIN6 cells, a mouse pancreatic ß cell line. Physiological concentrations of palmitic acid, a saturated fatty acid, induced ER stress quickly, while physiological concentrations of oleic acid, an unsaturated fatty acid, did not. Docosahexaenoic acid, an n-3 unsaturated fatty acid, inhibited palmitic acid-induced ER stress. In this study, we have established a system that can sensitively detect ER stress levels of living cells in a short period of time. This system can be used to monitor the state of the ER in living cells and lead to the investigation of the significance of physiological or pathological ER stress levels.

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.

Islet protection and amelioration of type 2 diabetes mellitus by treatment with quercetin from the flowers of Edgeworthia gardneri.

BACKGROUND AND PURPOSE: The traditional Chinese medicine - the flower of Edgeworthia gardneri - is reported as an effective therapeutic for type 2 diabetes mellitus (T2DM). Nevertheless, most constituents of the flowers of E. gardneri have not yet been studied. This study was conducted to investigate the effect of quercetin extracted from the flowers of E. gardneri on islet protection and amelioration in T2DM and explore its mechanism. METHOD: Quercetin was extracted from the flowers of E. gardneri and verified by high-performance liquid chromatography. Quercetin or crude extract's effect on insulin secretion was investigated. ERK1/2 and phospho-ERK1/2 were detected by Western blot analysis, and fluo-3 AM was used to detect intracellular Ca2+. The anti-apoptosis effect of quercetin or crude extract on MIN-6 cells was investigated by thiazolyl blue tetrazolium bromide (MTT) assay and flow cytometry analysis. Activation of caspases and expression of Bcl-2 and BAX were tested by Western blot analysis. In addition, the mitochondrial membrane potential was determined by JC-1 probe. Moreover, in vivo activity was also tested in db/db mice. RESULTS: A quercetin level of >10 µmol/L could induce insulin secretion. Intracellular Ca2+ and ERK1/2 were involved in the signaling pathway of quercetin-induced insulin secretion. We also observed that quercetin could inhibit palmitic acid-induced cell apoptosis via suppressing the activation of caspase-3, -9, -12; increasing the ratio of Bcl-2/BAX and reversing the impaired mitochondrial membrane potential. Crude extract's effect on insulin secretion was similar to that of pure extracted quercetin, while it possessed higher anti-apoptosis activity. Additionally, intraperitoneal glucose tolerance, plasma insulin level, hepatic triglyceride, hepatic glycogen and the pathological histology of both pancreatic islet and liver in db/db mice were significantly improved by the administration of the extracted quercetin. CONCLUSION: Our study indicated that quercetin extracted from the flowers of E. gardneri exerted excellent properties in islet protection and amelioration.

Docosahexaenoic acid antagonizes the boosting effect of palmitic acid on LPS inflammatory signaling by inhibiting gene transcription and ceramide synthesis.

It is well known that saturated fatty acids (SFAs) and unsaturated fatty acid, in particular omega-3 polyunsaturated fatty acids (n-3 PUFAs), have different effects on inflammatory signaling: SFAs are pro-inflammatory but n-3 PUFAs have strong anti-inflammatory properties. We have reported that palmitic acid (PA), a saturated fatty acid, robustly amplifies lipopolysaccharide (LPS) signaling to upregulate proinflammatory gene expression in macrophages. We also reported that the increased production of ceramide (CER) via sphingomyelin (SM) hydrolysis and CER de novo synthesis plays a key role in the synergistic effect of LPS and PA on proinflammatory gene expression. However, it remains unclear if n-3 PUFAs are capable of antagonizing the synergistic effect of LPS and PA on gene expression and CER production. In this study, we employed the above macrophage culture system and lipidomical analysis to assess the effect of n-3 PUFAs on proinflammatory gene expression and CER production stimulated by LPS and PA. Results showed that DHA strongly inhibited the synergistic effect of LPS and PA on proinflammatory gene expression by targeting nuclear factor kappa B (NFκB)-dependent gene transcription. Results also showed that DHA inhibited the cooperative effect of LPS and PA on CER production by targeting CER de novo synthesis, but not SM hydrolysis. Furthermore, results showed that myriocin, a specific inhibitor of serine palmitoyltransferase, strongly inhibited both LPS-PA-stimulated CER synthesis and proinflammatory gene expression, indicating that CER synthesis is associated with proinflammatory gene expression and that inhibition of CER synthesis contributes to DHA-inhibited proinflammatory gene expression. Taken together, this study demonstrates that DHA antagonizes the boosting effect of PA on LPS signaling on proinflammatory gene expression by targeting both NFκB-dependent transcription and CER de novo synthesis in macrophages.

BPN, a marine-derived PTP1B inhibitor, activates insulin signaling and improves insulin resistance in C2C12 myotubes.

Insulin resistance is a key feature of type 2 diabetes mellitus (T2DM) and is characterized by defects in insulin signaling. Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of insulin signaling cascade and has attracted intensive investigation in recent T2DM therapy study. BPN, a marine-derived bromophenol compound, was isolated from the red alga Rhodomela confervoides. This study investigated the effects of BPN on the insulin signaling pathway in insulin-resistant C2C12 myotubes by inhibiting PTP1B. Molecular docking study and analysis of small- molecule interaction with PTP1B all showed BPN inhibited PTP1B activity via binding to the catalytic site through hydrogen bonds. We then found that BPN permeated into C2C12 myotubes, on the one hand, activated insulin signaling in an insulin-independent manner in C2C12 cells; on the other hand, ameliorated palmitate-induced insulin resistance through augmenting insulin sensitivity. Moreover, our studies also showed that PTP1B inhibition by BPN increased glucose uptake in normal and insulin-resistant C2C12 myotubes through glucose transporter 4 (GLUT4) translocation. Taken together, BPN activates insulin signaling and alleviates insulin resistance and represents a potential candidate for further development as an antidiabetic agent.

Dihydromyricetin inhibits NLRP3 inflammasome-dependent pyroptosis by activating the Nrf2 signaling pathway in vascular endothelial cells.

Increasing evidence demonstrates that pyroptosis, pro-inflammatory programmed cell death, is linked to atherosclerosis; however, the underlying mechanisms remain to be elucidated. Dihydromyricetin (DHM), a natural flavonoid, was reported to exert anti-oxidative and anti-inflammatory bioactivities. However, the effect of DHM on atherosclerosis-related pyroptosis has not been studied. In the present study, palmitic acid (PA) treatment led to pyroptosis in human umbilical vein endothelial cells (HUVECs), as evidenced by caspase-1 activation, LDH release, and propidium iodide-positive staining; enhanced the maturation and release of proinflammatory cytokine IL-1ß and activation of the NLRP3 inflammasome; and markedly increased intracellular reactive oxygen species (ROS) and mitochondrial ROS (mtROS) levels. Moreover, NLRP3 siRNA transfection or treatment with inhibitors efficiently suppressed PA-induced pyroptosis, and pretreatment with total ROS scavenger or mtROS scavenger attenuated PA-induced NLRP3 inflammasome activation and subsequent pyroptosis. However, DHM pretreatment inhibited PA-induced pyroptotic cell death by increasing cell viability, decreasing LDH and IL-1ß release, improving cell membrane integrity, and abolishing caspase-1 cleavage and subsequent IL-1ß maturation. We also found that DHM pre-treatment remarkably reduced the levels of intracellular ROS and mtROS and activated the Nrf2 signaling pathway. Moreover, knockdown of Nrf2 by siRNA abrogated the inhibitory effects of DHM on ROS generation and subsequent PA-induced pyroptosis. Together, these results indicate that the Nrf2 signaling pathway plays a role, as least in part, in the DHM-mediated improvement in PA-induced pyroptosis in vascular endothelial cells, which implies the underlying medicinal value of DHM targeting immune/inflammatory-related diseases, such as atherosclerosis. © 2017 BioFactors, 44(2):123-136, 2018.

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.

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.

Silymarin protects against renal injury through normalization of lipid metabolism and mitochondrial biogenesis in high fat-fed mice.

Obesity is associated with an increased risk of chronic kidney diseases and the conventional treatment with renin-angiotensin-aldosterone system (RAAS) inhibitors is not enough to prevent renal injury and prolong the progression of disease. Recently, silymarin has shown protective effects on renal tissue injury, but the underlying mechanisms remain elusive. The goal of this study was to investigate the potential capacity of silymarin to prevent renal injury during obesity induced by high fat diet (HFD) in mice. In vivo, male C57BL/6 mice received HFD (60% of total calories) for 12 weeks, randomized and treated orally with vehicle saline or silymarin (30mg/kg body weight/d) for 4 weeks. In vitro, human proximal tubular epithelial cells (HK2) were exposed to 300µM palmitic acid (PA) for 36h followed by silymarin administration at different concentrations. The administration of silymarin significantly ameliorated HFD induced glucose metabolic disorders, oxidative stress and pathological alterations in the kidney. Silymarin significantly mitigated renal lipid accumulation, fatty acid ß-oxidation and mitochondrial biogenesis in HFD mice and PA treated HK2 cells. Furthermore, silymarin partly restored mitochondrial membrane potential of HK2 cells after PA exposure. In conclusion, silymarin can improve oxidative stress and preserve mitochondrial dysfunction in the kidney, potentially via preventing accumulation of renal lipids and fatty acid ß-oxidation.

Diet-induced obesity impairs spermatogenesis: a potential role for autophagy.

Autophagy is an evolutionarily conserved process that plays a crucial role in maintaining a series of cellular functions. It has been found that autophagy is closely involved in the physiological process of spermatogenesis and the regulation of sperm survival and motility. However, the role of autophagy in high-fat diet (HFD)-induced impaired spermatogenesis remains unknown. This study was designed to investigate the role of autophagy in HFD-induced spermatogenesis deficiency and employed chloroquine (CQ) to inhibit autophagy and rapamycin (RAP) to induce autophagy. 3-methyladenine (3-MA) and CQ were administered via intratesticular injection in vivo. The effects of CQ and 3-MA on the parameters of spermatozoa co-cultured with palmitic acid (PA) in vitro were also investigated. Human semen samples from obese, subfertile male patients were also collected to examine the level of autophagy. The results suggested that HFD mice subjected to CQ showed improved spermatogenesis. Inhibiting autophagy with CQ improved the decreased fertility of HFD male mice. Moreover, the in vivo and in vitro results indicated that both CQ and 3-MA could suppress the pathological changes in spermatozoa caused by HFD or PA treatment. Additionally, the excessive activation of autophagy was also observed in sperm samples from obese, subfertile male patients.