Intracellular Triglyceride Levels Research Articles

Influence of Radical Generation and Elimination on Sebum Production of Hamster Sebaceous Gland Cells

We focused on the effects of radical induction on cell differentiation and sebum production when antioxidants and oxidants were applied to normal hamster sebaceous gland cells. We also examined the relationship between sebum production and the reactive oxygen species (ROS) scavenging rate in these cells. Eight antioxidants (fullerene, epigallocatechin gallate, α-glucosylrutin, copper (II) gluconate, tannic acid, sodium copper-chlorophyllin, phytic acid, and ascorbyl tocopheryl phosphate) and one oxidant (hydrogen peroxide, H2O2) were used. The number of differentiated cells was determined by counting the viable cells, the intracellular triglyceride (TG) level was determined by separation and quantification by HPTLC, and the superoxide anion radical scavenging rate, nitric oxide scavenging rate, and H2O2 scavenging rate were also investigated. Adding various antioxidants decreased the differentiated cell number and TG content in the hamster sebaceous gland cells. Meanwhile, adding an oxidant (H2O2) increased the differentiated cell number and cellular TG. Pretreatment with antioxidants also prevented the oxidants from increasing the differentiated cell number and TG level. A strong correlation between the intracellular TG content and the H2O2 scavenging rate was identified. These results indicate that radical generation and scavenging are involved in sebum production in hamster sebaceous gland cells, and that the scavenging rate of H2O2 may be particularly important.

Time-Course Strategy Reveals a Dual Potential of Perfluorooctanoic Acid and Its Alternative in Adipocyte Differentiation.

Exposure to perfluorooctanoic acid (PFOA) and hexafluoropropylene oxide dimer acid (HFPO-DA) was associated with adipogenesis. However, potential mechanisms remain to be elucidated. Herein, a 3T3-L1 adipocyte model was used to explore the dynamic changes in adipocyte differentiation (2, 4, and 8 days) under PFOA and HFPO-DA exposure. PFOA and HFPO-DA increased the adipocyte formation rate and intracellular levels of triglycerides (TG). Meanwhile, adipocyte browning was induced by PFOA and HFPO-DA, which was characterized by small lipid droplets, low levels of TG per adipocyte, increased ATP levels, and elevated mitochondrial respiration activities with time-dependent differentiation. The browning potency indexes of PFOA and HFPO-DA were approximately 1.5 times higher than those of the controls. Time-course transcriptomics analysis showed that PFOA and HFPO-DA activated the biological process of adipocyte browning but different gene expression patterns regulated adipocyte browning. Only overexpressed hydroxymethylglutaryl-CoA synthase (Hmgcs2) was shared between PFOA and HFPO-DA groups from 2 to 8 days. Hmgcs2 could regulate adipocyte browning induced by PFOA and HFPO-DA, and this observation was lost when Hmgcs2 was knocked down. Our study suggests that PFOA and HFPO-DA could play dual roles in the differentiation of 3T3-L1 adipocytes, and Hmgcs2 might be a target of PFOA- and HFPO-DA-induced adipocyte browning.

Exploring diabesity pathophysiology through proteomic analysis using Caenorhabditis elegans.

Diabesity, characterized by obesity-driven Type 2 diabetes mellitus (T2DM), arises from intricate genetic and environmental interplays that induce various metabolic disorders. The systemic lipid and glucose homeostasis is controlled by an intricate cross-talk of internal glucose/insulin and fatty acid molecules to maintain a steady state of internal environment. In this study, Caenorhabditis elegans were maintained to achieve glucose concentrations resembling the hyperglycemic conditions in diabetic patients to delve into the mechanistic foundations of diabesity. Various assays were conducted to measure intracellular triglyceride levels, lifespan, pharyngeal pumping rate, oxidative stress indicators, locomotor behavior, and dopamine signaling. Proteomic analysis was also performed to identify differentially regulated proteins and dysregulated KEGG pathways, and microscopy and immunofluorescence staining were employed to assess collagen production and anatomical integrity. Worms raised on diets high in glucose and cholesterol exhibited notably increased intracellular triglyceride levels, a decrease in both mean and maximum lifespan, and reduced pharyngeal pumping. The diabesity condition induced oxidative stress, evident from heightened ROS levels and distinct FT-IR spectroscopy patterns revealing lipid and protein alterations. Furthermore, impaired dopamine signaling and diminished locomotors behavior in diabesity-afflicted worms correlated with reduced motility. Through proteomic analysis, differentially regulated proteins encompassing dysregulated KEGG pathways included insulin signaling, Alzheimer's disease, and nicotinic acetylcholine receptor signaling pathways were observed. Moreover, diabesity led to decreased collagen production, resulting in anatomical disruptions validated through microscopy and immunofluorescence staining. This underscores the impact of diabesity on cellular components and structural integrity in C. elegans, providing insights into diabesity-associated mechanisms.

Withanolides-enriched leaf extract of Withania somnifera exert anti-obesity effects by inducing brown adipocyte-like phenotype via tuning MAP-kinase signaling axis

Present study investigated anti-obesity potential of Withania somnifera (L.) Dunal leaf extract (WSLE). Phytochemical characterization of WSLE was performed by UPLC/MS-QToF and HPLC-based analysis. WSLE was assessed for its effect on lipid metabolism and mitochondrial biogenesis in vitro using differentiated 3T3-L1 adipocytes. WSLE was found to contain 59 phytometabolites with a total of 10.601 μg withanolides per mg of extract. WSLE (30 μg/ml) treatment decreased basal levels of intracellular lipids and triglycerides to 13.85 % and 41.58 %, respectively. WSLE downregulated the expression of PPARγ, C/EBPα, C/EBPβ, and their target genes responsible for lipogenesis dose-dependently. An upregulation in expression of lipolytic (ATGL and HSL), thermogenic (PGC1α, UCP1, and PRDM16), and glucose transporter (GLUT4) genes was also observed. Furthermore, WSLE treatment increased glucose uptake by 1.5-fold. These beneficial effects of WSLE were abolished in presence of AMPK, p38MAPK, and ERK inhibitors. These observations were then validated in vivo using Caenorhabditis elegans as a model organism. Intriguingly, WSLE diminished fat accumulation in wild-type N2 worms as evident from reduced Oil-red-O staining and reduction in GFP expression of fat-5, 6, and 7 in transgenic strains. Overall, these results highlight anti-obesity potential of WSLE exerting its effects via alterations in AMPK/p38MAPK/ERK axis.

A microRNA signature for valproate-induced steatosis in human hepatocytes and its application to predict fatty liver in paediatric epileptic patients on valproate therapy

Valproate (VPA) has been the first-line, most frequently prescribed antiepileptic drug in children over the past 50 years. VPA causes, idiosyncratic hepatotoxicity in some patients, who often presents with hepatic steatosis. Experimental studies also support that VPA has high potential to induce steatosis. However, there is an apparent lack of significant hepatic problems in neuropediatric units, likely because iatrogenic liver steatosis lacks specific biomarkers. Thus, it is possible that a relevant number of children under VPA have asymptomatic fatty liver. Aims1) to demonstrate VPA-induced triglyceride (TG) accumulation in cultured human upcyte hepatocytes, 2) to identify miRNAs that are deregulated by VPA and associated with TG levels in these cells, and 3) to test these miRNAs, as potential non-invasive biomarkers, in plasma of paediatric epileptic patients on VPA, to identify those with a potential risk of liver steatosis.Human upcyte hepatocytes were exposed to subcytotoxic VPA concentrations. Hepatocytes increased intracellular TGs by 27 % and 45 % after 2 and 4 mM VPA for 24 h. The profiling of cellular miRNAs by microarray analysis after 4 mM VPA identified 43 deregulated human miRNAs (fold-change > 1.5 or < −1.5; FDR p<0.05). Some of them (n=11), which were validated by RTqPCR and showed correlation (Pearson r≥ 0.6) with intracellular TG levels, were selected as potential VPA-induced steatosis biomarkers. Next, we investigated the expression of these miRNAs in human plasma and found that 9 of them could be reliably quantified by RTqPCR: miR-485-3p, miR-127-3p, miR-30a-3p, miR-92b-3p, miR-212-3p, miR-182-5p, miR-183-5p, miR-500a-5p and miR-675-5p. Screening of this 9-miRNA signature in 80 paediatric epileptic patients on VPA identified 18 patients (23 %) that clustered separately because of important alterations in the selected plasma miRNAs. These patients were younger and had higher VPA blood concentrations and serum liver enzyme levels.In conclusion, VPA induced both TG accumulation and deregulation of a set of miRNAs in cultured human hepatocytes. Nine of these miRNAs have demonstrated potential as circulating biomarkers to identify VPA-induced steatosis in epileptic patients, which should require closer clinical follow-up.

Quinoa Polyphenol Extract Alleviates Non-Alcoholic Fatty Liver Disease via Inhibiting Lipid Accumulation, Inflammation and Oxidative Stress.

Recently, the incidence of NAFLD has exploded globally, but there are currently no officially approved medications for treating the condition. The regulation of NAFLD through plant-derived active substances has become a new area of interest. Quinoa (Chenopodium quinoa Willd.) has been discovered to contain a large quantity of bioactive compounds. In this study, we established a free fatty acid (FFA)-induced steatosis model and explored the effects of quinoa polyphenol extract (QPE) on the major hallmarks of NAFLD. The results indicated that QPE significantly reduced intracellular triglyceride (TG) and total cholesterol (TC) levels. Additionally, QPE remarkably elevated the levels of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) and lowered levels of malondialdehyde (MDA). Further examination revealed that QPE attenuated intracellular inflammation, which was verified by the reduced levels of pro-inflammatory cytokines. Mechanistically, QPE inhibited fatty acid biosynthesis mainly by targeting de novo lipogenesis (DNL) via the AMPK/SREBP-1c signaling pathway. Moreover, network pharmacology was used to analyze key targets for NAFLD mitigation by ferulic acid (FA), a major component of QPE. Taken together, this study suggests that QPE could ameliorate NAFLD by modulating hepatic lipid metabolism and alleviating oxidative stress and inflammation.

Design, synthesis, and biological evaluation of (E)-3-(3-methoxy-4-substituted phenyl)-acrylic acid derivatives: Role of compound S17 in regulating lipid accumulation via AMPK activation

Non-alcoholic fatty liver disease (NAFLD) is increasingly recognized as a global public health concern characterized by excessive lipid accumulation in the liver. Ferulic acid (FA), a common foodborne phenolic acid, has been shown to reduce lipid accumulation by activating adenosine monophosphate-activated protein kinase (AMPK) and suppressing the expression of sterol regulatory element-binding protein-1 (SREBP-1). In this study, we synthesized a new class of (E)-3-(3-methoxy-4-substituted phenyl)-acrylic acid derivatives as AMPK activators. Among these derivatives, compound S17 demonstrated a more potent inhibitory effect on lipid accumulation compared to its counterparts. Specifically, S17 notably diminished intracellular triglyceride (TG) levels and the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are indicators of liver function. In addition, S17 curtailed intracellular lipid accumulation by activating the AMPK signaling pathway and down-regulating the protein expression of SREBP-1 in free fatty acid (FFA)-induced HepG2 cells. Therefore, findings from this study strongly suggest that compound S17 may serve as a promising therapeutic candidate for the treatment of NAFLD.

The Role of PNPLA3_rs738409 Gene Variant, Lifestyle Factors, and Bioactive Compounds in Nonalcoholic Fatty Liver Disease: A Population-Based and Molecular Approach towards Healthy Nutrition.

This study aimed to investigate the impact of a common non-synonymous gene variant (C>G, rs738409) in patatin-like phospholipase domain-containing 3 (PNPLA3), leading to the substitution of isoleucine with methionine at position 148 (PNPLA3-I148M), on susceptibility to nonalcoholic fatty liver disease (NAFLD) and explore potential therapeutic nutritional strategies targeting PNPLA3. It contributed to understanding sustainable dietary practices for managing NAFLD, recently referred to as metabolic-dysfunction-associated fatty liver. NAFLD had been diagnosed by ultrasound in a metropolitan hospital-based cohort comprising 58,701 middle-aged and older Korean individuals, identifying 2089 NAFLD patients. The interaction between PNPLA3 and lifestyle factors was investigated. In silico analyses, including virtual screening, molecular docking, and molecular dynamics simulations, were conducted to identify bioactive compounds from foods targeting PNPLA3(I148M). Subsequent cellular experiments involved treating oleic acid (OA)-exposed HepG2 cells with selected bioactive compounds, both in the absence and presence of compound C (AMPK inhibitor), targeting PNPLA3 expression. Carriers of the risk allele PNPLA3_rs738409G showed an increased association with NAFLD risk, particularly with adherence to a plant-based diet, avoidance of a Western-style diet, and smoking. Delphinidin 3-caffeoyl-glucoside, pyranocyanin A, delta-viniferin, kaempferol-7-glucoside, and petunidin 3-rutinoside emerged as potential binders to the active site residues of PNPLA3, exhibiting a reduction in binding energy. These compounds demonstrated a dose-dependent reduction in intracellular triglyceride and lipid peroxide levels in HepG2 cells, while pretreatment with compound C showed the opposite trend. Kaempferol-7-glucoside and petunidin-3-rutinoside showed potential as inhibitors of PNPLA3 expression by enhancing AMPK activity, ultimately reducing intrahepatic lipogenesis. In conclusion, there is potential for plant-based diets and specific bioactive compounds to promote sustainable dietary practices to mitigate NAFLD risk, especially in individuals with genetic predispositions.

Abstract 3068: CD146mediates intimate crosstalk between adipocytes and triple-negative breast cancer cells and drives tumor metastasis through lipid metabolic reprogramming

Abstract Background &amp; Aim: In the tumor microenvironment, the adipocytes enhance the invasiveness of breast cancer (BC) by remodeling the lipid metabolism, especially in triple-negative breast cancer (TNBC). The cell adhesion molecule CD146 was highly expressed in TNBC and predicted poor distant metastasis free survival (DMFS) in patients. Meanwhile, CD146 was known to alter the obesity process by regulating the metabolic level of the body. In this study, we aimed to explore the molecular mechanism of CD146 in tumor metastasis mediating by lipid metabolic reprogramming between adipocytes and TNBC cells. Methods: CD146 knockout (KO) or overexpression cell models were constructed by CRISPR/Cas9 or lentivirus. Cells labeled with luciferase were injected in tail vein of Balb/c-nude mice and monitored lung metastasis by IVIS Kinetic system in vivo. Transwell and wound healing assays were analyzed cell aggressiveness in vitro. We synthesized anti-CD146 nanobodies, which significantly degraded CD146 protein and inhibitd its function. RNA-seq and Lipidomics in CD146 knockout models were performed to analyze the role of CD146 in TNBC cell metabolism. Immunofluorescence was used to detect the intracellular content of Lipid Droplet (LD) and protein localization. Finally, we used oleic acid (OA) and adipocyte-conditioned medium (CM) to mimic the tumor microenvironment. Results: CD146 was highly expressed in TNBC and predicted poor DMFS survival. Inhibition of CD146 suppressed tumor aggressiveness and lung metastasis in vitro and in vivo. According to the bioinformatics and RNA-Seq analysis, we showed that CD146 was intimately involved in the lipid metabolic pathway, particularly fatty acid metabolism, and revealed that inhibiting CD146 in TNBC elevated the intracellular triglyceride level through lipidomic assay. Meanwhile, knockout of CD146 promoted LD accumulation and decreased fatty acid oxidation (FAO) and mitochondrial ATP levels. In molecular mechanism, we found that adipocytes secrete high level of IL6 and fatty acid, which activated JAK/STAT3 signaling and FAO mediating by CD146 in TNBC cells. Blocking CD146 using its nanobody inhibited IL6/JAK/STAT3 activities and ACADM, leading to impairment of FAO and lung metastasis suppression in vitro and in vivo. Conclusions: In this study, we demonstrate the crucial role of CD146 in the crosstalk between tumor cells and adipocytes in the tumor microenvironment. CD146 promotes the utilization of fatty acids through fatty acid oxidation and activates the downstream inflammatory pathway response with adipocyte-secreted IL-6, thereby driving BC aggressiveness. Our study has important implications for the development of TNBC therapeutics to block CD146-driven FAO process, inflammatory response and tumor metastasis. Citation Format: Hexing Sun, Kaiyuan Huang, Bingmei Sun, Jiajia Lin, Ruiqian Yang, Jiawei Li, Shimeng Wang, De Zeng, Haoyu Lin, Yuanke Liang. CD146mediates intimate crosstalk between adipocytes and triple-negative breast cancer cells and drives tumor metastasis through lipid metabolic reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3068.

Extracts of tropical green seaweed Caulerpa lentillifera reduce hepatic lipid accumulation by modulating lipid metabolism molecules in HepG2 cells

Seaweed has attracted attention as a bioactive source for preventing different chronic diseases, including liver injury and non-alcoholic fatty liver disease, the leading cause of liver-related mortality. Caulerpa lentillifera is characterized as tropical edible seaweed, currently being investigated for health benefits of its extracts and bioactive substances. This study examined the effects of C. lentillifera extract in ethyl acetate fraction (CLEA) on controlling lipid accumulation and lipid metabolism in HepG2 cells induced with oleic acid through the in vitro hepatic steatosis model. Gas chromatography-mass spectrometry (GC-MS) analysis indicated that CLEA contained diverse organic compounds, including hydrocarbons, amino acids, and carboxylic acids. Docked conformation of dl-2-phenyltryptophane and benzoic acid, two major bioactive CLEA components, showed high affinity binding to SIRT1 and AMPK as target molecules of lipid metabolism. CLEA reduced lipid accumulation and intracellular triglyceride levels in HepG2 cells stimulated with oleic acid. The effect of CLEA on regulating expression of lipid metabolism-related molecules was investigated by qPCR and immunoblotting. CLEA promoted expression of the SIRT1 gene in oleic acid-treated HepG2 cells. CLEA also reduced expression levels of SREBF1, FAS, and ACC genes, which might be related to activation of AMPK signaling in lipid-accumulated HepG2 cells. These findings suggest that CLEA contains bioactive compounds potentially reducing triglyceride accumulation in lipid-accumulated HepG2 hepatocytes by controlling lipid metabolism molecules.

Effect of inhibiting PDHα1 gene expression on the metabolism of fatty liver cells

PDHα1 gene encodes catalytic subunit PDHE1α of pyruvate dehydrogenase (PDH) complex. Based on previous studies, it is hypothesized that inhibition of PDH activity prevents the entry of glycolytic pyruvate into TCA cycle, while promotes fatty acid oxidation and reduces liver triglyceride (TG) level, thereby alleviating nonalcoholic fatty liver disease (NAFLD). In this study, an in vitro model for NAFLD was established with medical fat emulsion; the most effective siRNA for PDHα1 gene was screened out by qPCR technology; the alterations in metabolism of glucose and lipid, and structure & function of mitochondria in the NAFLD cells were primarily evaluated after transfecting PDHα1 siRNA. As the results showed, after inhibiting the expression of PDHα1 gene, glucose level in culture medium was time-dependently increased, and LDH activity in the cells was moderately elevated after 24 h of transfection and then returned to the normal level after 48 h; intracellular TG level was decreased while LPS activity was increased in a time-dependent manner; no significant change in mitochondrial structure was observed with or without siRNA transfection, and ATP content was obviously reduced after 24 h of transfection, followed by restoration after 48 h. It can be concluded that inhibiting PDHα1 gene in fatty liver cells enhances lipid degradation, and represses the utilization of glucose to an extent, thus reducing TG level without impacting energy generation required for cell survival.

Bioactive peptides PDBSN improve mitochondrial function and suppression the oxidative stress in human adiposity cells

ABSTRACT Introduction: Mitochondria are essential for generating cellular energy and are significant in the pathogenesis of obesity. Peptide PDBSN has been demonstrated to inhibit the adipogenic differentiation of adipocytes in vitro and improves metabolic homoeostasis in vivo. Therefore, in this study, we further investigated the effects of PDBSN on the morphology, synthesis, and function of adipocyte mitochondria. Methods: Human visceral and subcutaneous primary preadipocytes (HPA-v and HPA-s) were cultured into mature adipocytes. Intracellular triglyceride content was assessed using oil-red O staining and tissue triglyceride determination. Gene and protein levels associated with mitochondrial synthesis were detected using real-time quantitative polymerase chain reaction and western blotting. Mitochondrial membrane potentials and ROS were detected using fluorescent indicators. Morphological changes were observed by electron microscopy. Results: PDBSN significantly increased mitochondrial membrane potential (MMP), while decreasing intracellular triglyceride (TG) and intracellular reactive oxygen species (ROS) levels. On the other hand, the transcription and protein levels of genetic marker genes PGC1-α and MTFA were significantly up-regulated after PDBSN administration. Further studies showed that transcriptional and protein levels of mitochondrial fusion and fission genetic markers MFN1, MFN2, NRF1, and DRP1 increased. Conclusion: PDBSN significantly reduces intracellular TG and ROS levels and increases MMP. The maximum respiratory capacity in adults significantly increases after PDBSN administration, and ROS levels are significantly reduced. This suggests that PDBSN improves mitochondrial function to some extent, which not only provides an essential basis for the pathophysiology of obesity but also provides insights for the development of new drugs to treat obesity and metabolic diseases.

Apolipoprotein A1 Inhibits Adipogenesis Progression of Human Adipose-Derived Mesenchymal Stem Cells.

According to the reports, the most vital characteristic of obesity is an aberrant accumulation of triglycerides (TG) in the adipocyte. On the other hand, circulating concentrations of apolipoprotein A1 (apoA1) have been demonstrated to be strongly correlated with the prevalence and the pathological development of obesity. Nevertheless, the underlying mechanisms whereby apoA1 modulates the pathogenesis of obesity is still not fully elucidated. Adipose-derived mesenchymal stem cells (AMSCs, isolated from the hospitalized patients were combined with 15 μg/ml recombined human apoA1 protein. The effects of apoA1 on modulating the intracellular levels of TG and the expression contents of adipogenic related cytokines were also analyzed. Furthermore, whether apoA1 modulated the adipogenesis progression via sortilin was also explored in the current research. During the adipogenesis progression, apoA1 could significantly lower the quantity of intracellular lipid droplets (LDs). Meanwhile, apoA1 could decrease the intracellular levels of TG and down-regulate the expression contents of several vital adipogenic related cytokines, such as CCAAT enhancer-binding proteins α/β (C/EBPα/β), fatty acid synthetase (FAS), and fatty acid-binding protein 4 (FABP4). Moreover, the inhibitory effect of apoA1 was further verified to be induced through upregulating the SORT1 gene expression which subsequently increased sortilin protein. Consistent with these findings, silencing the SORT1 gene expression could induce the loss-of-function (LOF) of apoA1 in modulating the adipogenesis progression of AMSCs. In conclusion, apoA1 could suppress the adipogenesis progression of human AMSCs through, at least partly, up-regulating the SORT1 gene expression which subsequently increases the sortilin protein content. Thereby, the present research sheds light on a novel pathogenic mechanism by which apoA1 regulates adipogenesis progression and proposes that apoA1 embraces the function to treat obesity in clinical practice.

Qidan Tiaozhi capsule attenuates metabolic syndrome via activating AMPK/PINK1-Parkin-mediated mitophagy.

Qidan Tiaozhi capsule (QD), a traditional Chinese medicine, has been used to treat metabolic syndrome for over a decade. However, the mechanism of QD in the treatment of metabolic syndrome is still unknown. Growing studies demonstrate that impaired mitophagy is one of the important causes of metabolic syndrome. Thus, this research aims to investigate the mechanism of mitophagy in the QD treatment of metabolic syndrome. Network pharmacology and molecular docking were used to probe the mechanism of QD treatment of metabolic syndrome. In an oleic acid-induced cell model, glucose consumption and uptake capacity, triglyceride (TG), total cholesterol (TC), malonaldehyde (MDA), superoxide dismutase (SOD) and ROS levels, and mitochondrial membrane potential (MMP) were examined. mRFP-GFP-LC3 adenovirus and GFP-LC3 lentivirus were used to examine the effect of QD on mitophagy. The IRS2-PI3K and AMPK/PINK1-Parkin signal pathways were also determined. What's more, the PINK1 gene was silenced to verify the above findings. In a high-fat diet-fed mouse model, body weight, organ indexes, OGTT, ITT, HOMA-IR, insulin sensitivity, serum MDA, SOD, TC, TG, LDL-C and HDL-C, hepatic TC, TG, LDL-C and HDL-C levels, hepatic steatosis, and IRS2-PI3K and AMPK/PINK1-Parkin signal pathways were investigated. Results from network pharmacology and molecular docking suggested that QD might suppress oxidative stress to improve metabolic syndrome. In an oleic acid-induced cell model, compared with the model group, enhanced glucose consumption and uptake ability, inhibited intracellular lipid accumulation, TC, TG, MDA and ROS levels, and increased SOD level and MMP were found in QD groups. And mitophagy levels, IRS2-PI3K and AMPK/PINK1-Parkin signal pathways were promoted. Interestingly, PINK1 silencing reversed the therapeutic action of QD on oleic acid-induced cells. In high-fat diet-fed mice, inhibited body weight, abdominal fat indexes, liver indexes, HOMA-IR, serum and hepatic TC, TG and LDL-C, serum MDA and hepatic steatosis, and increased insulin sensitivity, serum and hepatic HDL-C, serum SOD, and activated IRS2-PI3K and AMPK/PINK1-Parkin signal pathways were found in QD groups. QD activates AMPK/PINK1-Parkin-mediated mitophagy to suppress oxidative stress to treat metabolic syndrome.

Neferine ameliorates nonalcoholic steatohepatitis through regulating AMPK pathway

BackgroundNonalcoholic fatty liver disease (NAFLD), peculiarly nonalcoholic steatohepatitis (NASH), has become the main cause of liver transplantation and liver-related death. However, the US Food and Drug Administration has not approved a specific medication for treating NASH. Neferine (NEF), a natural bisbenzylisoquinoline alkaloid separated from the traditional Chinese medicine Nelumbinis plumula, has a variety of pharmacological properties, especially on metabolic diseases. Nevertheless, the anti-NASH effect and mechanisms of NEF remain unclear. PurposeThis study aimed to investigate the amelioration of NEF on NASH and the potential mechanisms. Study DesignHepG2 cells, hepatic stellate cells (HSCs) and high-fat diet (HFD)+carbon tetrachloride (CCl4) induced C57BL/6 mice were used to observe the effect of NEF against NASH and investigate the engaged mechanism. MethodsHSCs and HepG2 cells stimulated by oleic acid (OA) were treated with NEF. C57BL/6 mice were fed with HFD+CCl4 to induce NASH mouse model and treated with or without NEF (5 mg/kg or 10 mg/kg, once daily, i.p) for 4 weeks. ResultsNEF significantly attenuated the accumulation of lipid droplets, intracellular triglyceride (TG) levels and hepatocytes apoptosis in OA-exposed HepG2 cells. NEF not only enhanced the AMPK and ACC phosphorylation in OA-stimulated HepG2 cells, but also reduced inflammatory response and fibrosis in lipopolysaccharide (LPS)-stimulated HepG2 and in LX-2, respectively. In HFD+CCl4-induced NASH mice, pathological staining confirmed NEF treatment mitigated hepatic lipid deposition, inflammatory cell infiltration as well as hepatic fibrosis. Furthermore, the liver weight, serum and hepatic TG and total cholesterol (TC) and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were decreased compared with the model group. HFD+CCl4 also induced the upregulation of specific proteins and genes associated to inflammation (ILs, TNF-α, NLRP3, ASC, CCL2 and CXCL10) and hepatic fibrosis (collagens, α-SMA, TGF-β and TIPM1), which were also suppressed by NEF treatment. ConclusionOur results demonstrated that NEF played a protective role in hepatic steatosis via the regulation of AMPK pathways, which may serve as an attractive candidate for a potential novel strategy on prevention and treatment of NASH.

Identification of potential bioactive compounds of Passiflora edulis leaf extract against colon adenocarcinoma cells

Plant bioactive compounds such as flavonoids and triterpenes can affect lipid metabolism. Here, we report the cytotoxic and lipid-lowering activities of the ethanolic extract of P. edulis leaves on human colon adenocarcinoma SW480 cells, also the molecular interactions of bioactive compounds present in P. edulis extract on ACC and HMGCR enzymes. The extract reduced cell viability and decreased intracellular triglyceride content by up to 35% and 28% at 24 and 48 h, respectively; whereas the effect was evident on cholesterol only at 24 h. In-silico analysis revealed that luteolin, chlorogenic acid, moupinamide, isoorientin, glucosyl passionflower, cyclopasifloic acid E and saponarin had optimal molecular coupling on Acetyl-CoA Carboxylase 1 and 2 as well as 3-hydroxy-3-methyl-glutaryl-CoA reductase, with possible inhibitory effects. These results show the ability of ethanolic extract to reduce intracellular levels of cholesterol and triglycerides in SW480 cells, which attracts attention for the treatment of colorectal cancer.

Establishment of hyperoxic cell culture system for predicting drug-induced liver injury: reducing accumulated lipids in hepatocytes derived from chimeric mice with humanized liver.

Drug-induced liver injury (DILI) is a major adverse reaction. Species-specific differences between humans and laboratory animals make it difficult to establish evaluation models that can accurately predict DILI in the preclinical phase. Chimeric mice with humanized liver are potential predictive models for understanding DILI. Chimeric mice generated by transplanting human hepatocytes into urokinase-type plasminogen activator/severe combined immunodeficient mice are known to develop fatty liver and show lipid accumulation in isolated hepatocytes. It is speculated that the lipids accumulated in hepatocytes may interfere with DILI assessment. It is known that normal 20% oxygen culture conditions do not meet oxygen demand because oxygen consumption rate is higher than the oxygen supply rate. Therefore, we predicted that hyperoxic cultures could induce hepatocyte function and reduce accumulated lipids. A culture of chimeric mouse hepatocytes in 40% oxygen showed reduced intracellular lipid and triglyceride levels compared to those cultured in 20% oxygen on days 7 and 10. In addition, fatty acid β-oxidation (FAO) activity increased from day 7 under 40% oxygen conditions. On the other hand, FAO activity increased on day 10 under 20% conditions. Microarray and Ingenuity Pathway Analysis showed that lipid metabolism-related pathways were downregulated under 40% oxygen conditions for 7 days, suggesting the involvement of several mechanisms in decreasing lipid levels and increasing FAO. Furthermore, some pathways related to cellular function and maintenance were upregulated under 40% oxygen conditions for 7 days. In conclusion, chimeric mouse hepatocytes cultured under hyperoxic conditions may be useful for predicting DILI.

Inhibitory effect of sulfated polysaccharide from Sargassum thunbergii in PA-induced lipid accumulation in vitro

This study aimed to investigate the In vitro lipid inhibitory effects of sulfated polysaccharide fractions derived from fractional ethanol precipitate 1 of Celluclast assisted hydrolysate from Sargassum thunbergii (STCF1-PSs). Three polysaccharide fractions were obtained, and physical characteristics were determined by SEM, FTIR, and MALS analysis. Monosaccharide analysis indicated that STCF1-PS3 contained high fucose and galactose contents. The intracellular lipid inhibitory effects of STCF1-PS3 were evaluated on PA-treated 3T3-L1 and HepG2 cells and was found to be significant. Furthermore, it suppressed intracellular triglyceride and free fatty acid levels in these cells. Western blot analysis showed that the lipid inhibitory activity was achieved through the regulation of the adipogenesis and lipolysis pathway in PA-treated 3T3-L1 cells and the free fatty acid oxidation pathway in PA-treated HepG2 cells. Overall, sulfated polysaccharide from Sargassum thunbergii has a beneficial effect on intracellular lipid metabolism and it could be used as a functional ingredient in functional food industry.

Binding of Glycerol to Human Galectin-7 Expands Stability and Modulates Its Functions.

Glycerol is seen in biological systems as an intermediate in lipid metabolism. In recent years, glycerol has been reported to act as a chemical chaperone to correct the conformation of proteins. Here, we investigate the role of glycerol in galectin-7 (Gal-7). The thermal shift and CD assays showed that the thermal stability of Gal-7 increased with glycerol concentration but with little secondary structure changes induced by glycerol. In addition, glycerol can inhibit Gal-7-mediated erythrocyte agglutination. We also solved the crystal structures of human Gal-7 in complex with glycerol in two different conditions. Glycerol binds at the carbohydrate-recognition binding sites of Gal-7, which indicates glycerol as a small ligand for Gal-7. Surprisingly, glycerol can bind a new pocket near the N-terminus of Gal-7, which can greatly reduce the flexibility and improve the stability of this region. Moreover, overexpression of Gal-7 decreased the intracellular triglyceride levels and increased mRNA expression of aquaporin-3 (AQP-3) when HeLa cells were incubated with glycerol. These findings indicate that Gal-7 might regulate glycerol metabolism. Overall, our results on human Gal-7 raise the perspective to systematically explore this so far unrecognized phenomenon for Gal-7 in glycerol metabolism.

Reactive oxygen species induce HNF-4α expression via the ASK1-CREB pathway, promoting ChREBP expression and lipogenesis in hepatocytes

AimsThe production of reactive oxygen species (ROS) increases with aging and is associated with liver diseases. ChREBP is involved in lipogenic gene expression in hepatocytes, and we hypothesized that increases in ROS production would induce liver metabolic diseases via regulation of ChREBP expression. Main methodsMechanism studies were conducted using H2O2 induced HepG2 cells and primary hepatocytes from old mouse. Key findingsWe detected increases in ChREBP expression, lipogenic gene (FAS and SCD1) expression, and intracellular triglyceride (TG) levels in H2O2-treated HepG2 cells, which were inhibited by ChREBP knockdown. ChREBP expression and intracellular TG levels were higher in primary hepatocytes from old mice than in those of young mice. Compared with old wild-type mice, intracellular TG levels were significantly reduced in primary hepatocytes from old ChREBP knockout mice. Furthermore, H2O2 stimulation was found to induce HNF-4α expression in HepG2 cells and this expression was higher in primary hepatocytes from old mice. ChIP-qPCR assays indicated that binding of HNF-4α to ChREBP promoter was significantly increased by H2O2 treatment in HepG2 cells. Further study revealed that H2O2-promoted increases in ChREBP expression and intracellular TG levels were significantly inhibited by HNF-4α knockdown. H2O2 treatment also significantly increased ASK1 expression, whereas ASK1 knockdown inhibited H2O2-induced HNF-4α expression. SignificanceCollectively, our data indicate that an increase in ROS production can induce HNF-4α expression via the ASK1 pathway and subsequently enhances the expression of ChREBP, thereby contributing to lipogenesis. This pathway may be involved in the elevated liver lipid levels associated with aging and oxidative stress.