Triglyceride Assay Research Articles

Targeting Galanin Receptor Signaling as a Novel Therapeutic Strategy for the Treatment of Fatty Liver Disease

BackgroundNon‐alcoholic fatty liver disease (NAFLD) is a common liver disease in the United States and is associated with dysregulated lipid metabolism and fat accumulation in hepatocytes (i.e. steatosis) leading to inflammation and fibrosis and cirrhosis. Galanin (Gal) is a neuropeptide with a role in food intake behavior and chronic upregulation of Gal is associated with increases in serum triglycerides, hyperinsulinemia, impaired glucose tolerance and hepatic steatosis. Targeting Gal receptor (GalR) signaling has been shown to have antifibrotic in models of cholestatic liver disease although the effects in NAFLD is unknown. The aim of this study was to evaluate the Gal/GalR axis in NAFLD and determined the effects of Gal Inhibition on the pathogenesis of NAFLD.MethodsC57Bl6 mice were fed a western diet (WD) consisting of high fat/high sugar/high cholesterol for a total of 16 weeks. At the 4 week time point, mice were treated with the GalR1/GalR2 antagonist, M40 (1 nmol, 3X per week for the duration of the experiment), or corresponding vehicle control. Serum and liver assays of Gal, triglycerides, and cholesterol were performed using commercially available kits. The expression of Gal, GalR1, GalR2, markers of steatosis (perilipin 2, PLIN2), biliary hyperplasia (cytokeratin 19, CK19), fibrosis (collagen type 1A1, Col1A1; α‐smooth muscle actin, α‐SMA) and inflammation (chemokine C‐C motif ligand 2, CCL2; tumor necrosis factor‐α, TNFα) by qPCR and immunostaining. Liver macrophages CD11b+ and CD68+ were quantified by immunohistochemistry (IHC). Liver lipid droplets (LD) were stained with Oil Red O. In vitro, HepG2 cells were used to assess the effects of M40 on palmitate (PA)‐induced LD formation.ResultsGal was significantly increased in the serum and liver of WD‐fed mice, with a corresponding increase in GalR1 and GalR2 expression. WD‐mice exhibited significant increase in serum transaminases, triglycerides and cholesterol compared to control mice, and M40 significantly reduced these indicators of liver injury. A drastic decrease in liver steatosis was also observed in M40‐treated WD‐mice compared to vehicle‐treated WD‐mice. M40 treatment also ameliorated WD‐induced increase in ductular reaction, fibrosis and liver inflammation in NAFLD mice. In vitro experiments confirmed that M40 decreased PA‐induced steatosis of HepG2.ConclusionsThe expression of Gal and its receptors GalR1 and GalR2, is enhanced in livers of mice on a WD. Treatment of WD‐fed mice with M40 reduces steatosis, intrahepatic biliary mass, fibrosis and inflammation. Therefore, targeting GalR1 and GalR2 may be a novel therapeutic option for the treatment of NAFLD.

Anti‐obesity effect of Premature Citrus Extract in high‐fat diet induced obesity mice

BackgroundObesity is a worldwide health threat. Premature citrus extract (PCE) are known to contain high concentrations of flavonoids, polyphenols, and organic acids with health functions. The current study aimed to investigate the anti‐obesity effect of PCE on a high‐fat diet (HFD)‐induced obesity mice.MethodMice were fed on chow or HFD with or without Orlistat (ORL), Hesperidin (HES), and PCE treatment for 10 weeks. Triglyceride (TG) content and total cholesterol (TC), and low‐density lipoprotein cholesterol (LDL‐c), and high‐density lipoprotein cholesterol (HDL‐c) levels were determined by TG, HDL and LDL Assay Kit (BioAssay System). The phosphorylation levels of AMP‐activated protein kinase (AMPK) and acetyl Co‐A carboxylase (ACC); the expression of sterol regulatory element binding protein‐2 (SREBP‐2), 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGCR), Acyl‐coenzyme A: cholesterol acyltransferases (ACAT‐1) and mitochondrial uncoupling proteins (UCP‐3) were observed via western blot analysis.ResultsPCE and HES administered group significantly reduced the weights of body, liver, and white adipose tissue compared to the HFD group. Supplementation with PCE and HES significantly decreased the levels of TG, TC, and (LDL‐c), and increased (HDL‐c) levels in serum compared to the HFD group. Furthermore, treatment with PCE and HES significantly decreased the levels of TG and TC in the liver whereas increased the levels of TG and TC in feces compared to the HFD group. Western blot analysis revealed that PCE and HES increased the phosphorylation levels of both AMPK and ACC and significantly decreased the expression levels of SREBP‐2, HMGCR, and ACAT‐1 in the liver compared to the HFD group. In addition, both PCE and HES increased the expression level of UCP‐3 in skeletal muscle tissue.ConclusionOverall results suggest that PCE and HES exerts an anti‐obesity effect via activation of AMPK and associated signaling cascades related to lipid metabolism in HFD‐induced obesity mice

Preventive Effects of Ilex Cornuta Aqueous Extract on High-Fat Diet-Induced Fatty Liver of Mice.

Objective To investigate the preventive effects of Ilex cornuta aqueous extract (ICAE) on high-fat diet (HFD)-induced fatty liver of mice and its mechanisms. Materials and Methods Twenty-six male KM (Kunming) mice were divided into 3 groups, including the control group (n = 9), fed with normal diet; HFD group (n = 9), fed with HFD; ICAE + HFD group (n = 8), fed with HFD and administered with ICAE (3 g·kg−1·d−1) at the same time for 10 weeks. Body weight, liver weight, intra-abdominal and subcutaneous fat weight, serum triglyceride (TG), total cholesterol (TC), and blood glucose were determined to evaluate the preventive effects of ICAE on obesity. The average 24 h food consumption of the mice was monitored for 5 times in the 9th week of the experiment to investigate the effects of ICAE on food intake. Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) were assayed to observe the influences of HFD and ICAE on liver function. HE staining was adopted to observe the influence of ICAE on the morphology of adipose tissue and liver tissue. Hepatic TG and TC content assay and oil red O staining were used to evaluate the influences of ICAE on HFD-induced fatty liver, and the protein expression of peroxisome proliferator-activated receptors γ (PPARγ) and adipose differentiation-related protein (ADRP) in liver were examined by immunoblotting. Results ICAE treatment significantly reduced the increase of body weight, intra-abdominal, and subcutaneous fat and liver weight induced by HFD (P < 0.001), but has no influence on food intake; ICAE treatment attenuated the elevation of serum TG, TC, and glucose, as well as serum ALT and AST (P < 0.01, P < 0.05, P < 0.001) and dramatically decreased the content of TG in liver (P < 0.01), but has no influence on hepatic TC content. HE staining and oil red O staining showed that ICAE significantly reduced HFD-induced white adipocyte hypertrophy and significantly inhibited lipid accumulation in liver. Immunoblotting showed that the protein levels of PPARγ and ADRP were significantly increased by HFD induction, which can be dramatically reduced by ICAE treatment (P < 0.05, P < 0.0001). Conclusion ICAE has preventive effects on HFD-induced obesity and fatty liver in mice, exerted beneficial effects upon HFD-induced hepatic injury. The preventive effects of ICAE on fatty liver are concerned with the downregulation of PPARγ and ADRP protein expression in liver.

Blood group and its correlation with cardiovascular risk factors: A community survey

Cardiovascular disease is a leading cause of morbidity and mortality globally. Non-O blood groups have been shown to confer a high risk of coronary heart disease. There is paucity of data on the relationship between blood group and left ventricular hypertrophy, among other cardiovascular risk factors. A community-based study was done to evaluate the blood groups and their associations with conventional cardiovascular risk factors in Ejigbo. Anthropometry, blood pressure and other clinical variables were measured. Blood samples were taken for biochemical analysis for blood group typing, serum cholesterol and triglyceride assay. A 12- lead electrocardiogram was performed. The analysis was done using SPSS version 20 to determine possible relationships between the variables. Two hundred and six cases were recruited and analyzed. The prevalence of the various groups was as follows: A - 23%; B-31.4%; AB-4.4%; O-41.2%; rhesus-positive-92.7% and rhesus-negative-7.3%. Blood group B had similar systolic blood pressure (136.0±23.9 vs 137.3±22.3; p=0.726), higher Sokolow- Lyon voltage sum (3.3±1.1 vs 2.9±0.9; p=0.025) and serum triglycerides [14(21.9%) vs7(8.4%), p=0.021] than those with blood group O. Those with Rhesus Positive status had longer PR interval (169.3±25.4 vs 154.2±19.1; p value=0.055) and QRS duration (83.8±12.8 vs 78.4±7.6; p value=0.043) than those with Rhesus negative blood groups. Binary logistic regression revealed blood group B as an independent determinant of left ventricular hypertrophy( LVH) (OR: 3.028; p=0.012; 95% CI:1.275-7.192). Blood group B is a determinant of LVH. Rhesus positive status is associated with delayed electrical conduction through the myocardium.

LncRNA MEG3 up-regulates SIRT6 by ubiquitinating EZH2 and alleviates nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a global health threat. Here, we presented the significant role of a novel signaling axis comprising long non-coding RNA maternally expressed gene 3 (MEG3), enhancer of zeste homolog 2 (EZH2), and sirtuin 6 (SIRT6) in controlling lipid accumulation, inflammation, and the progression of NAFLD. Mice fed with high-fat diet (HFD) were established as in vitro and in vivo NAFLD models, respectively. Lipid accumulation was measured by oil red O staining and assays for triglycerides or cholesterol. Inflammation was examined by ELISA for pro-inflammatory cytokines. Gene expressions were examined by RT-qPCR or Western blot. Interactions between key signaling molecules were examined by combining expressional analysis, RNA immunoprecipitation, cycloheximide stability assay, co-immunoprecipitation, and chromatin immunoprecipitation. MEG3 level was reduced in FFA-challenged hepatocytes or liver from HFD-fed mice, and the reduction paralleled the severity of NAFLD in clinic. Overexpressing MEG3 suppressed FFA-induced lipid accumulation or inflammation in hepatocytes. By promoting the ubiquitination and degradation of EZH2, MEG3 upregulated SIRT6, an EZH2 target. SIRT6 essentially mediated the protective effects of MEG3 in hepatocytes. Consistently, overexpressing MEG3 alleviated HFD-induced NAFLD in vivo. By controlling the expressions of genes involved in lipid metabolism and inflammation, the MEG3/EZH2/SIRT6 axis significantly suppressed lipid accumulation and inflammation in vitro, and NAFLD development in vivo. Therefore, boosting MEG3 level may benefit the treatment of NAFLD.

Dulaglutide Ameliorates Palmitic Acid-Induced Hepatic Steatosis by Activating FAM3A Signaling Pathway.

BackgroundDulaglutide, a long-acting glucagon-like peptide-1 receptor agonist (GLP-1RA), has been shown to reduce body weight and liver fat content in patients with type 2 diabetes. Family with sequence similarity 3 member A (FAM3A) plays a vital role in regulating glucose and lipid metabolism. The aim of this study was to determine the mechanisms by which dulaglutide protects against hepatic steatosis in HepG2 cells treated with palmitic acid (PA).MethodsHepG2 cells were pretreated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM dulaglutide for 24 hours. Hepatic lipid accumulation was determined using Oil red O staining and triglyceride (TG) assay, and the expression of lipid metabolism-associated factor was analyzed using quantitative real time polymerase chain reaction and Western blotting.ResultsDulaglutide significantly decreased hepatic lipid accumulation and reduced the expression of genes associated with lipid droplet binding proteins, de novo lipogenesis, and TG synthesis in PA-treated HepG2 cells. Dulaglutide also increased the expression of proteins associated with lipolysis and fatty acid oxidation and FAM3A in PA-treated cells. However, exendin-(9–39), a GLP-1R antagonist, reversed the expression of FAM3A, and fatty acid oxidation-associated factors increased due to dulaglutide. In addition, inhibition of FAM3A by siRNA attenuated the reducing effect of dulaglutide on TG content and its increasing effect on regulation of fatty acid oxidation.ConclusionThese results suggest that dulaglutide could be used therapeutically for improving nonalcoholic fatty liver disease, and its effect could be mediated in part via upregulation of FAM3A expression through a GLP-1R-dependent pathway.

Anti-adipogenic effect of the flavonoids through the activation of AMPK in palmitate (PA)-treated HepG2 cells.

BackgroundFlavonoids are natural polyphenols found widely in citrus fruit and peel that possess anti-adipogenic effects. On the other hand, the detailed mechanisms for the anti-adipogenic effects of flavonoids are unclear.ObjectivesThe present study observed the anti-adipogenic effects of five major citrus flavonoids, including hesperidin (HES), narirutin (NAR), nobiletin (NOB), sinensetin (SIN), and tangeretin (TAN), on AMP-activated protein kinase (AMPK) activation in palmitate (PA)-treated HepG2 cells.MethodsThe intracellular lipid accumulation and triglyceride (TG) contents were quantified by Oil-red O staining and TG assay, respectively. The glucose uptake was assessed using 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) assay. The levels of AMPK, acetyl-CoA carboxylase (ACC), and glycogen synthase kinase 3 beta (GSK3β) phosphorylation, and levels of sterol regulatory element-binding protein 2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression were analyzed by Western blot analysis. The potential interaction between the flavonoids and the γ-subunit of AMPK was investigated by molecular docking analysis.ResultsThe flavonoid treatment reduced both intracellular lipid accumulation and TG content in PA-treated HepG2 cells significantly. In addition, the flavonoids showed increased 2-NBDG uptake in an insulin-independent manner in PA-treated HepG2 cells. The flavonoids increased the AMPK, ACC, and GSK3β phosphorylation levels and decreased the SREBP-2 and HMGCR expression levels in PA-treated HepG2 cells. Molecular docking analysis showed that the flavonoids bind to the CBS domains in the regulatory γ-subunit of AMPK with high binding affinities and could serve as potential AMPK activators.ConclusionThe overall results suggest that the anti-adipogenic effect of flavonoids on PA-treated HepG2 cells results from the activation of AMPK by flavonoids.

Network pharmacology approach to evaluate the therapeutic effects of mulberry leaf components for obesity.

Obesity is a chronic condition that has become a serious public health challenge globally due to the association with a high incidence of complications. Mulberry leaf is one of the most commonly used medicinal and herbal medicines that has been reported to ameliorate obesity and hyperlipidemia. However, the mechanism remains unclear. In the present study, a network pharmacology approach was used to explore the potential mechanism underlying the effects of mulberry leaf extract on obesity. First, the potential targets of mulberry leaf and obesity were predicted using SwissTargetPrediction, Online Mendelian Inheritance in Man, GeneCards and Comparative Toxicogenomics Database databases, which were then used to construct the protein-protein interaction networks. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyzes were performed using R version 3.6.3. Finally, results of this network analysis were verified by using the mulberry leaf extract to treat high-fat diet-induced obese mice. In total, 24 target genes associated with obesity that could potentially be affected by mulberry leaf treatment were predicted by network pharmacology, using which top seven related pathways were determined by KEGG enrichment analysis. Triglyceride (TG) and total cholesterol (TC) levels in mice serum were detected using TG and TC assay kits. Hepatic fat accumulation was detected by H&E staining whereas liver lipid droplets were detected by Oil red O staining in mice tissues. The expression of IL-1β, NF-κB inhibitor α, inducible nitric oxide synthase, AMP-activated protein kinase (AMPK), sterol regulatory element-binding proteins and fatty acid synthase in the visceral white adipose tissues of mice was analyzed by western blotting. The expression of TNF-α, peroxisome proliferator activated receptor (PPAR)D, PPARG, fatty acid amide hydrolase (FAAH) and hydroxysteroid 11-β dehydrogenase 1 (HSD11B1) in the visceral white adipose tissues of mice was detected by reverse transcription-quantitative PCR. Mulberry leaf extract was found to reduce fat accumulation and hepatic lipid droplet formation. Mulberry leaf also alleviated inflammation and lipogenesis whilst promoting lipid catabolism and fatty acid oxidation by promoting the AMPK signaling pathway. The possible anti-obesity effects of mulberry leaf on the mice may be due to the downregulation of TNF-α, PPARD and PPARG and the upregulation of FAAH and HSD11B1. These results were consistent with the GO enrichment analysis and suggested that mulberry leaf may regulate lipid metabolism and catabolism, fatty acid metabolism and biosynthesis and the inflammatory response to reduce obesity.

Quinizarin suppresses the differentiation of adipocytes and lipogenesis in vitro and in vivo via downregulation of C/EBP-beta/SREBP pathway

AimsPotential anti-obesity effects of quinizarin, a plant anthraquinone, were investigated using 3 T3-L1 preadipocyte cells and high-fat diet (HD)-induced obese mice. Main methodCell viability was determined using the MTT assay. Triglyceride (TG) and lipid accumulation were determined using a TG assay kit and Oil Red O staining, respectively. Adipogenic, lipogenic, and lipolytic gene and protein expression was measured by RT–PCR or Western blot. Serum biochemical indices, including cholesterol and blood glucose, in HD-fed obese mice were determined using corresponding assay kits. Histological analysis was performed with haematoxylin and eosin (H&E) staining. ResultsQuinizarin (0–10 μM) significantly reduced intracellular TG and lipid droplets during the differentiation of preadipocytes. Quinizarin significantly suppressed the expression of adipocyte differentiation marker proteins, such as CCAAT/enhancer-binding protein β (C/EBP-β), C/EBP-α, PPAR-γ, and aP2, and lipogenic marker proteins, including SREBP1c, SREBP2, fatty acid synthase (FAS), and acetyl-CoA carboxylase 1 (ACC1), reduced ACC2 expression and increased carnitine palmitoyltransferase 1 (CPT1) expression. Oral administration of quinizarin (15–30 mg/kg/day) to HD-fed mice for 6 weeks reduced the body weight gain and size of liver adipocytes and epididymal fat tissues, with significant reductions in liver TG and serum total cholesterol, blood glucose, LDL, and HDL levels. SignificanceThe results of this study indicated that quinizarin exerts anti-obesity effects by inhibiting both adipogenesis and lipogenesis and stimulating lipolysis in vitro and in vivo mainly by downregulating the SREBP signalling pathway; thus, it might be a potent candidate as a health-beneficial food or therapeutic agent to prevent or treat obesity.

Selection of candidate genes for differences in fat metabolism between cattle subcutaneous and perirenal adipose tissue based on RNA-seq

The site of fat deposition plays an important role in meat quality and body health. Biologically, the perirenal visceral fat (PF) and back subcutaneous fat (BF) are distinct. Angus and Simmental cattle (Bos taurus) were used as models. HE staining, triglyceride assay kit and RNA-seq were used to analyze the differences in tissue morphology and lipid accumulation, co-genes, and differentially expressed genes (DEGs) between the two tissues. According to the findings, BF has a smaller cell area and greater lipid deposition ability than PF. RNA-seq generated approximately 10.99 Gb of data in each library, and 23,472 genes were identified. The genes FABP4, ADIRF, and SCD that are related to adipose deposition were highly expressed in four tissues. There were 1678 DEGs and 1955 DEGs between BF and PF in Angus and Simmental cattle respectively. Gene Ontology function analysis identified several DEGs involved in metabolism. KEGG pathway analysis showed that four pathways related to fat metabolism were enriched. In the BF, seven genes (COL1A1, COL1A2, COL3A1, COL2A1, RXRA, C1QTNF7, and MOGAT2) were up-regulated. Five genes (ADRB3, ABHD5, CPT1B, CD36, LPIN1) were down-regulated. This study identified candidate genes that led to differences in fat metabolism, which could be useful in cattle breeding.

Anti-obesity effect of sulforaphane in broccoli leaf extract on 3T3-L1 adipocytes and ob/ob mice

The present study evaluated the anti-obesity effect of sulforaphane (SFN) and glucoraphanin (GRN) in broccoli leaf extract (BLE) on 3T3-L1 adipocytes and ob/ob mice. Based on Oil Red O staining and triglyceride (TG) assay, SFN and BLE significantly reduced (P<.05) both lipid accumulation and TG content in the differentiated 3T3-L1 adipocytes. SFN and BLE increased 2-NBDG uptake by 3T3-L1 adipocytes in a dose-dependent manner. Western blot analysis confirmed that SFN and BLE increased the phosphorylation levels of both AMPK (Thr172) and ACC (Ser79), and reduced the expression of HMGCR in liver and white adipose tissues of ob/ob mice. Histological analysis revealed that SFN and BLE ameliorated hepatic steatosis, and reduced the size of adipocyte in ob/ob mice. Treatment with SFN and BLE significantly reduced (P<.05) TG content, low-density lipoprotein (LDL) cholesterol, total cholesterol (TC), and glucose in the serum of ob/ob mice. RNA sequencing analysis showed that up- or down-regulation of 32 genes related to lipid metabolism was restored to control level in both SFN and BLE-treated ob/ob mice groups. A protein-protein interaction (PPI) network was constructed via STRING analysis, and Srebf2, Pla2g2c, Elovl5, Plb1, Ctp1a, Lipin1, Fgfr1, and Plcg1 were located in the functional hubs of the PPI network of lipid metabolism. Overall results suggest that the SFN content in BLE exerts a potential anti-obesity effect by normalizing the expression of genes related to lipid metabolism, which are up- or down-regulated in ob/ob mice.

Low-intensity pulsed ultrasound induces metabolic lipolysis of adipocytes

We used low-intensity pulsed ultrasound (LIPUS) to uncover underlying lipolysis mechanism of 3T3-L1 adipocyte. Ultrasound pulses with the center frequency of 2 MHz were applied for 10 min/day to promote the lipolysis of white adipocyte. Lipolysis is the process of breaking down lipids and the metabolic pathway through which lipid triglycerides are hydrolyzed into a glycerol and three fatty acids. The proliferation of 3T3-L1 adipocytes was detected by cell viability XXT assay, RT-PCR and Western blot. The Triglyceride Assay quantitatively measures triglycerides levels in differentiated adipocytes. LIPUS reduced the triglycerol content significantly in adipocytes. The differences between transcriptional genes and metabolites were analyzed by transcript analysis and metabolomic profiling experiments. The results of RT-PCR, and Western blot after LIPUS showed the increased lipolysis. The expression of genes related to lipolysis-related factors, ATGLand HSL, was up-regulated. In addition, cellular RNA-sequencing (RNA-Seq) showed an increase in lipolysis and adaptive immune cells under ultrasound stimulation. The algorithm and additional analysis reveals a gene network that is predicted to be associated and potentially drive adipocyte lipolysis and heterogeneity. LIPUS can promote the lipolytic capacity of 3T3-L1 cells in the differentiated state. This study provides an important experimental and theoretical basis for the clinical application of LIPUS in promoting the lipolysis of 3T3-L1 adipocytes.

Functions of Monocytes and Macrophages and the Associated Effective Molecules and Mechanisms at the Early Stage of Atherosclerosis.

This study aimed to explore the functions and possible underlying regulatory molecules and mechanisms of monocytes and macrophages under early atherosclerotic conditions. THP-1-derived monocytes or macrophages were induced by 50 μg/ml oxidized low density lipoprotein (ox-LDL) for 24 hours, and the degree of lipid metabolism and inflammation were determined. In addition, we identified differentially expressed genes, noncoding ribonucleic acids (RNAs), pathways and mechanisms by RNA sequencing, and performed further correlation analysis and molecular expression verification. Monocytes could not form foam cells with oil red O staining directly and had low levels of lipids as determined by total cholesterol and triglycerides assays, cholesterol uptake molecules CD36, the class A macrophage scavenger receptor and lectin-like oxidized low-density lipoprotein receptor-1 and cholesterol efflux molecules ATP binding cassette transporter A1, ATP binding cassette transporter G1 and liver X receptor α, and inflammatory factors, which were markedly different from those in macrophages. Additionally, sequencing data showed obviously differentially expressed genes, microRNAs and long noncoding RNAs in the atherosclerotic group. We identified 15 upregulated and downregulated genes, and 10 biological processes and pathways involved in atherosclerosis. Specifically, fatty acid desaturase 2 and apolipoprotein A1 in the peroxisome proliferator-activated receptor signaling pathway were differentially expressed in stimulated macrophages, whereas no changes were observed in the monocyte groups. Furthermore, correlation analysis showed differential expressed lncRNAs targeting miRNAs and mRNAs, and 24 competing endogenous RNA (ceRNA) networks of long noncoding RNA-microRNA-messenger RNA in early oxidative macrophages. Monocytes did not directly participate in lipid metabolism before differentiation into macrophages at the early stage in vitro. Furthermore, noncoding RNAs and ceRNA networks might play important roles in regulating the lipid metabolism of macrophages at the early stage of atherosclerosis.

Bisphenol S promotes fat storage in multiple generations of Caenorhabditis elegans in a daf-16/nhr-49 dependent manner

Bisphenol S (BPS) has been gradually used in all kinds of productions. Our previous study has demonstrated that BPS increases the obesogenic effects of a high-glucose diet through regulating lipid metabolism in Caenorhabditis elegans (C. elegans). Whether the effects pass on to the next generations remains uncovered. In the present study, C. elegans was selected as the model organism to investigate the effects of BPS on lipid metabolism in multiple generations. Oil Red O staining and triglyceride assays showed that multi-generational exposure to BPS in C. elegans significantly increased the fat accumulation in wild type worms, while not in the daf-16 gene-deficient worms. In addition, BPS affected the expressions of fat-7 and acs-2 in four generations of C. elegans. Furthermore, BPS promotes fat storage in C. elegans of multiple generations by the daf-16/nhr-49-mediated signaling pathway.

Topical Delivery of Nanosized Hydroxycitric Acid Enriched Extract-Loaded Ethosomes for Suppression of Lipid Droplet Deposition.

The aim of this study was to improve the skin accumulation of hydroxycitric acid by using ethosomes with nanosize. We fabricated nanosized ethosome for the topical delivery of hydrophilic hydroxycitric acid and evaluated their physical properties and furthermore cytotoxicity. As results, in cell-based experiments, the use of ethosomes encapsulating hydroxycitric acid extract reduced the lipid droplet deposition in differentiated adipocytes, which was visualized by Oil Red O staining assay and also quantitatively measured by a triglyceride assay. The observed reduction in lipid droplet deposition occurred in a hydroxycitric acid extract concentration-dependent manner. In addition, the high accumulation of hydroxycitric acid in murine skin (66.28%) was observed following treatment with hydroxycitric acid extract-loaded ethosomes compared with treatment with hydroxycitric acid alone (1.19%) without ethosome as a nanocarrier. Based on these results, our findings showed that nanosized ethosomes improved the topical delivery of hydroxycitric acid and thus reduced lipid droplet deposition in adipocytes.

Triclosan down-regulates fatty acid synthase through microRNAs in HepG2 cells

Triclosan is a promising candidate of fatty acid synthase (FASN) inhibitor by blocking FASN activity, but its effect on FASN expression and the underling epigenetic mechanism remain elusive. In this study, the effect of triclosan on FASN mRNA and protein expressions in human HepG2 cells and the regulatory role of microRNAs (miRNAs) in the downregulation of FASN induced by triclosan were explored through experiments and bioinformatics analysis. The results showed that triclosan not only directly inhibited FASN activity, but also significantly decreased FASN mRNA and protein levels in human liver HepG2 cells. Nine miRNAs targeting FASN mRNA degradation were identified by miRNA prediction tools, and the expression levels of these nine miRNAs were then detected by real-time quantitative PCR. Triclosan significantly increased the expressions of the six miRNAs, namely miR-15a, miR-107, miR-195, miR-424, miR-497 and miR-503, leading to the downregulation of FASN. Further investigation revealed that the six triclosan-upregulated miRNAs played an important regulatory role in lipid metabolism and cell cycle by gene ontology annotations and pathway analysis. Consistent with the results of bioinformatics analyses, triclosan significantly reduced the intracellular lipid content by triglyceride assay, oil red O, BODIPY 493/503 and Nile Red staining, thereby inhibiting the growth of HepG2 cells through apoptosis. Taken together, our study reveals that triclosan downregulates FASN expression through a variety of miRNAs, providing new insight for triclosan as a FASN inhibitor candidate to regulate lipid metabolism in human hepatoma cells.

Berteroin ameliorates lipid accumulation through AMPK-mediated regulation of hepatic lipid metabolism and inhibition of adipocyte differentiation

AimsBerteroin (5-methylthiopentyl isothiocyanate) is a naturally occurring sulforaphane analog containing a non-oxidized sulfur atom in cruciferous vegetables. The objectives of the present study were to determine the effects of berteroin on lipid metabolism in hepatocytes and adipocytes and to elucidate the mechanisms involved. Main methodsThe effect of berteroin on lipid metabolism were evaluated in liver X receptor α agonist-stimulated HepG2 cells and adipocyte differentiation-induced 3T3-L1 cells using MTT assay, western blot, real time polymerase chain reaction, oil red O staining, and triglyceride assay. Key findingsT0901317 treatment increased the expression of sterol regulatory element binding protein (SREBP)-1c, a major transcription factor that mediates lipogenesis, and berteroin pretreatment significantly inhibited the expressions of T0901317-induced SREBP-1c and lipogenic genes. Especially, berteroin had a greater inhibitory effect on T0901317-induced SREBP-1c activation than sulforaphane, AICAR, or metformin. Berteroin also markedly suppressed lipid droplet formations and triglyceride accumulations caused by both T0901317 stimulation in HepG2 hepatocytes and differentiation induction in 3T3-L1 preadipocytes. However, berteroin significantly increased the expression of mitochondrial fatty acid oxidation-related genes (carnitine palmitoyltransferase 1 (CPT-1) and peroxisome proliferator-activated receptor gamma coactivator-1α) and the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in HepG2 cells. Interestingly, effects of berteroin on the expressions of SREBP-1c protein and CPT-1 mRNA were remarkably prevented by compound C (an AMPK inhibitor). SignificanceOur results suggest berteroin-inhibited hepatic lipid accumulation and adipocyte differentiation might be mediated by AMPK activation and that berteroin might be useful for the prevention, amelioration, and treatment of metabolic diseases, including hepatic steatosis.

튀니지 식물 11종 추출물의 HepG2 세포에서 산화적 스트레스 및 지질 축적 억제 효과

This study was undertaken to evaluate the antioxidative and lipid accumulation inhibitory effects in HepG2 cell of 11 Tunisian plants extracts. Total phenolics contents (TPC), and total flavonoid contents (TFC) of 11 plants extracts were measured, and antioxidative activities was analyzed using DPPH, ABTS, FRAP, ORAC and TBA assay. Inhibitory effect of oxidative stress was evaluated by cell viability and lipid peroxidation level in H2O2-treated HepG2 cells. Lipid accumulation inhibitory effect was determined by Oil-Red-O staining and intracellular triglyceride assay in HepG2 cell. M. communis L. (156.73 mgGAE/g) and N. glauca Graham (108.81 mgNAE/g) were the highest TPC and TFC, respectively, among 11 plants. M. communis L. were the highest antioxidant activity in DPPH and ABTS. FRAP and ORAC results revealed that antioxidant activity in 10 species were higher than the positive control. Among the 11 species, 5 species with the lowest malondialdehyde level were selected and HPLC analysis revealed that plants contain caffeic acid, quercetin, and rutin. 5 plants treatment inhibited lipid peroxidation level and protected HepG2 cells from oxidative stress. Moreover 5 plants significantly inhibited the lipid accumulation and triglyceride content. These results imply scientific evidence for the development of functional foods using 11 plants from Tunisia which has oxidative stress and lipid accumulation reduction effects.

Chlorogenic acid and caffeine combination attenuates adipogenesis by regulating fat metabolism and inhibiting adipocyte differentiation in 3T3-L1 cells.

Obesity is a complex disease spreading in the world. In our previous studies, chlorogenic acid (CGA) and caffeine had ever been reported to reduce the body weight gain and fat accumulation in mice. This study investigated the anti-obesity effect of CGA and caffeine on 3T3-L1 cells. According to triglyceride (TG) assay and Oil-Red O staining, 40μg/ml CGA and 160μg/ml caffeine reduced TG content. Moreover, CGA+caffeine inhibited the mRNA expression of major adipogenic markers, PPAR-γ2, and C/EBPα in the metaphase and anaphase stages of differentiation induction (Day 2 and 4). CGA+caffeine improved P-AMPK/AMPK accompanied by decreasing the expression of GPDH and FAS to depress the lipid synthesis, increasing the mRNA expression of ACO and CAT to promote fatty acid oxidation and up-regulated the expression of hydrolysis-related enzyme adipose TG lipase (ATGL) and P-HSL/HSL. Furthermore, CGA+caffeine improved the expression of Glut4 which promoted the glucose transport. Taken together, these data demonstrated CGA+caffeine inhibited 3T3-L1 cells differentiation in the middle and late stages and reduced the fat accumulation through AMPK pathway by regulating the fat metabolism-related enzyme in 3T3-L1 cells to attenuates adipogenesis. PRACTICAL APPLICATIONS: The aim of this study was to elucidate the potential role of chlorogenic acid and caffeine in the treatment of obesity.

Long Noncoding RNA MALAT1 Regulates the Progression of Atherosclerosis by miR-330-5p/NF-κB Signal Pathway.

Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was reported to be related to atherosclerosis (AS) progression. However, the underlying mechanism of MALAT1 in AS remains unknown. Quantitative real-time polymerase chain reaction was performed to detect the expression of MALAT1 and miR-330-5p. Western blot was applied to assess the protein levels of cluster of differentiation 36, interleukin-1β, interleukin-6 and tumor necrosis factor-α, phosphorylation of nuclear factor kappa-B inhibitor alpha and phosphorylation of p65. Flow cytometry assay, cell counting kit 8 assay, triglyceride, and total cholesterol detection assays were used to detect the apoptosis, viability, and lipid indexes of THP-1 macrophages-derived foam cells. Online database starbasev2.0 was used to predict the binding sequences between MALAT1 and miR-330-5p and it was verified by dual-luciferase reporter system and RNA immunoprecipitation assay. Besides, an AS mice model was used to evaluate the effect of MALAT1 in vivo. As a result, MALAT1 was overexpressed, whereas miR-330-5p was downregulated in THP-1 macrophages-derived foam cells. MiR-330-5p was a target of MALAT1. MALAT1 depletion inhibited cell formation, apoptosis, and inflammation in THP-1 macrophages-derived foam cells. Besides, MALAT1 overexpression promoted the inflammation in AS mice model, which promoted the pathogenesis of AS. Furthermore, miR-330-5p regulated the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) pathway in THP-1 macrophages-derived foam cells. Moreover, MALAT1 regulated NF-κB signal pathway to mediate the pathogenesis of AS by sponging miR-330-5p. MALAT1 sponges miR-330-5p to activate NF-κB signal pathway in THP-1 macrophages-derived foam cells. This finding may provide a novel biomarker for AS diagnosis.