Metabolism In HepG2 Cells Research Articles

Simultaneous quantification by HPLC of purines in umami soup stock and evaluation of their effects on extracellular and intracellular purine metabolism

ABSTRACTRibonucleotide flavor enhancers such as inosine monophosphate (IMP) and guanosine monophosphate (GMP) provide umami taste, similarly to glutamine. Japanese cuisine frequently uses soup stocks containing these nucleotides to enhance umami. We quantified 18 types of purines (nucleotides, nucleosides, and purine bases) in three soup stocks (chicken, consommé, and dried bonito soup). IMP was the most abundant purine in all umami soup stocks, followed by hypoxanthine, inosine, and GMP. The IMP content of dried bonito soup was the highest of the three soup stocks. We also evaluated the effects of these purines on extracellular and intracellular purine metabolism in HepG2 cells after adding each umami soup stock to the cells. An increase in inosine and hypoxanthine was evident 1 h and 4 h after soup stock addition, and a low amount of xanthine and guanosine was observed in the extracellular medium. The addition of chicken soup stock resulted in increased intracellular and extracellular levels of uric acid and guanosine. Purine metabolism may be affected by ingredients present in soups.

Exposure to 2,4-dichlorophenoxyacetic acid induced PPARβ-dependent disruption of glucose metabolism in HepG2 cells.

2,4-Dichlorophenoxyacetic acid is one of the most widely used herbicides. Its impact on health is increasingly attracting great attentions. This study aimed to investigate the effect of 2,4-dichlorophenoxyacetic acid on glucose metabolism in HepG2 cells and the underlying mechanism. After 24h exposure to 2,4-dichlorophenoxyacetic acid, glycogen was measured by PAS staining and glucose by ELISA in HepG2 cells. The expression of genes involved in glucose metabolism was measured by real-time PCR, Western blotting, and immunofluorescence. HepG2 cells presented more extracellular glucose consumption and glycogen content after exposed to 2,4-dichlorophenoxyacetic acid. Expression of gluconeogenesis-related genes, FoxO1, and CREB is significantly elevated. Moreover, PPARβ was up-regulated dose-dependently. SiRNA knockdown of PPARβ completely rescued the increase of glycogen accumulation and glucose uptake, and the up-regulation of FOXO1 and CREB expression. Our findings propose novel mechanisms that 2,4-dichlorophenoxyacetic acid causes glucose metabolism dysfunction through PPARβ in HepG2 cells.

Propylene glycol guluronate sulfate (PGGS) reduces lipid accumulation via AMP-activated kinase activation in palmitate-induced HepG2 cells

Cardiovascular disease (CVD) is the No. 1 cause of death worldwide. Hyperlipidemia is one of the major risk factors for CVD. Maintaining lipid homeostasis is an effective way to prevent CVD. We prepared propylene glycol guluronate sulfate (PGGS), a sulfated polysaccharide, and investigated its effect on lipid metabolism in HepG2 cells. We found that total cholesterol (TC) and triglycerides (TG) were significantly decreased in the cells after PGGS treatment. We have also shown that the AMPK signaling is activated after PGGS treatment as evidenced by changes in the expression of many AMPK downstream targets including SREBP-1c, SIRT-1, CPT1, PPARα, and FAS. Our results have demonstrated that PGGS is a potentially novel lipid-lowering agent for CVD prevention.

Lactobacillus acidophilus NS1 attenuates diet-induced obesity and fatty liver.

Obesity is a major threat to public health, and it is strongly associated with insulin resistance and fatty liver disease. Here, we demonstrated that administration of Lactobacillus acidophilus NS1 (LNS1) significantly reduced obesity and hepatic lipid accumulation, with a concomitant improvement in insulin sensitivity, in high-fat diet (HFD)-fed mice. Furthermore, administration of LNS1 inhibited the effect of HFD feeding on the SREBP-1c and PPARα signaling pathways and reduced lipogenesis with an increase in fatty acid oxidation in ex vivo livers from HFD-fed mice. These LNS1 effects were confirmed in HepG2 cells and ex vivo livers by treatment with LNS1 culture supernatant (LNS1-CS). Interestingly, AMPK phosphorylation and activity in the liver of HFD-fed mice were increased by administration of LNS1. Consistently, chemical inhibition of AMPK with compound C, a specific inhibitor of AMPK, dramatically reduced the effect of LNS1-CS on lipid metabolism in HepG2 cells and ex vivo livers by modulating the SREBP-1c and PPARα signaling pathways. Furthermore, administration of LNS1 to HFD-fed mice significantly improved insulin resistance and increased Akt phosphorylation in the liver, white adipose tissue and skeletal muscle. Together, these data suggest that LNS1 may prevent diet-induced obesity and related metabolic disorders by improving lipid metabolism and insulin sensitivity through an AMPK→SREBP-1c/PPARα signaling pathway.

Serum microRNA miR-206 is decreased in hyperthyroidism and mediates thyroid hormone regulation of lipid metabolism in HepG2 human hepatoblastoma cells.

The actions of thyroid hormone (TH) on lipid metabolism in the liver are associated with a number of genes involved in lipogenesis and lipid metabolism; however, the underlying mechanisms through which TH impacts on lipid metabolism remain to be elucidated. The present study aimed to investigate the effects of hyperthyroidism on the serum levels of the microRNA (miR) miR-206 and the role of miR-206 on TH-regulated lipid metabolism in liver cells. Serum was obtained from 12 patients diagnosed with hyperthyroidism and 10 healthy control subjects. Human hepatoblastoma (HepG2) cells were used to study the effects of triiodothyronine (T3) and miR-206 on lipid metabolism. Expression of miR-206 in serum and cells was determined by reverse transcription-quantitative polymerase chain reaction analysis. Lipid accumulation in HepG2 cells was assessed with Oil Red O staining. Suppression or overexpression of miR-206 was performed via transfection with a miR-206 mimic or miR-206 inhibitor. Serum miR-206 was significantly decreased in patients with hyperthyroidism compared with euthyroid controls. Treatment of HepG2 cells with T3 led to reduced total cholesterol (TC) and triglyceride (TG) content, accompanied by reduced miR-206 expression. Inhibition of endogenous miR-206 expression decreased intracellular TG and TC content in HepG2 cells. By contrast, overexpression of miR-206 in HepG2 partially prevented the reduction in TG content induced by treatment with T3. In conclusion, serum miR-206 expression is reduced in patients with hyperthyroidism. In addition, miR-206 is involved in T3-mediated regulation of lipid metabolism in HepG2 cells, indicating a role for miR-206 in thyroid hormone-induced disorders of lipid metabolism in the liver.

YDFYPSSTKDQQS (P3), a peptide from lupin protein, absorbed by Caco-2 cells, modulates cholesterol metabolism in HepG2 cells via SREBP-1 activation

YDFYPSSTKDQQS (P3) is a peptide derived from the hydrolysis of lupin protein with pepsin that may be bioavailable, since it is absorbed in Caco‐2 cells. In the framework of a research aimed at identifying new hypocholesterolemic peptides from plant proteins, this work provides evidence that P3 inhibits in vitro the functionality of 3‐hydroxy‐3‐methylglutaryl CoA reductase (HMGCoAR) and reports the characterization of the molecular mechanism through which this peptide modulates cholesterol metabolism in HepG2 cells. Specifically, through the inhibition of HMGCoAR activity, P3 improves the low density lipoprotein receptor protein levels via SREBP‐1 activation, leading to a better ability of HepG2 cells to uptake extracellular low density lipoproteins with a final in vitro hypocholesterolemic effect. PRACTICAL APPLICATIONS: Plant proteins have many practical applications in human nutrition, since they are environmentally sustainable and provide useful health benefits mostly in the area of hypercholesterolemia prevention. The identification of novel bioavailable hypocholesterolemic peptides and the characterization of their mechanism of action may strengthen the awareness of the role of plant proteins in human health. The mechanism through which P3 exerts its hypocholesterolemic activity is distinctive in respect to other plant peptides. Hypocholesterolemic peptides from plant proteins may be included in dietary supplements.

MiR-26a Potentially Contributes to the Regulation of Fatty Acid and Sterol Metabolism In Vitro Human HepG2 Cell Model of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a metabolic-related disorder ranging from steatosis to steatohepatitis, which may progress to cirrhosis and hepatocellular carcinoma (HCC). This study aimed at assessing the regulatory and protective role of miR-26a on lipid metabolism and progression of NAFLD in human HepG2 cells loaded with free fatty acids (FFA). Lentivirus expressing miR-26a or negative control miR was used to transduce HepG2 cells and to establish stable cell lines. Gain or loss of function using an miR-26a inhibitor was used to compare triglyceride content (TG), total cholesterol level (CL), total antioxidant capacity (TAC), malondialdehyde (MDA) and the level of apoptosis. In addition, quantitative reverse transcription polymerase chain reaction (qPCR) was used to assess the mRNA levels of lipogenesis, TG synthesis, storage genes, inflammatory and fibrogenic markers, and autophagic besides endoplasmic reticulum (ER) stress markers after gaining or losing the function of miR-26a. miR-26a levels decreased in response to FFA in human HepG2 cells. After the establishment of a stable cell line, the upregulation of miR-26a resulted in the downregulation of TG, CL, and MDA levels, through regulating mRNA levels of genes involved in lipid homeostasis, ER stress marker, inflammatory and fibrogenic markers. Nevertheless, there was a marked increment in the mRNA expression of autophagic marker genes. Moreover, miR-26a overexpression protects the cells from apoptosis, whereas inhibition of miR-26a, using an anti-miR-26a oligonucleotide, decreased the expression of miR-26a which potentially contributes to altered lipid metabolism in HepG2 cells loaded with FFA. In conclusion, these findings suggested that miR-26a has a crucial role in regulating fatty acid and cholesterol homeostasis in HepG2 cells, along with the offered protection against the progression of NAFLD in vitro. Hence, miRNAs could receive growing attention as useful noninvasive diagnostic markers to follow the progression of NAFLD and to identify novel therapeutic targets.

Blockade of neuropeptide Y2 receptor regulates gene expression in pathways of lipid metabolism in HepG2cells

Blockade of neuropeptide Y2 receptor regulates gene expression in pathways of lipid metabolism in HepG2cells

Anti-hyperglycemic effect of loquat leaf extract is associated with the redistribution of glucose carbon to its metabolites: a 13C-tracing study in HepG2 cells

Loquat leaf extract (LLE) is commonly used in China for a variety of ailments including diabetes. Several recent reports implicate LLE and a sesquiterpene glycoside, one of its components, as being an anti-hyperglycemic agent. However, the underlying mechanism of action of this anti-hyperglycemic agent has not been reported. We have conducted a tracer-based metabolomics study to investigate the effects of sesquiterpene and loquat extract on the balance of flux of central glucose metabolism in HepG2 cells and to compare with those of “insulin sensitizers”, metformin and rosiglitazone. Human hepatoma HepG2 cells in confluence culture were incubated in Dulbecco’s modified Eagle’s medium containing 50% [1, 2 13C2]-glucose in the presence of rosiglitazone, metformin, LLE or pure sesquiterpene. Cells were harvested in 48 h. Mass isotopomers of metabolites (glycogen, ribose, deoxyribose, glutamate and palmitate) were determined. 13C labeling in metabolic intermediates were summarized in a mass isotopomer matrix. Treatment with loquat extract/sesquiterpene, metformin and rosiglitazone each produced distinctive mass isotopomer patterns reflecting disparate effects on the contribution of glucose to various metabolites production, and on several metabolic flux ratios. The overall effect of LLE and sesquiterpene on glucose metabolism is clearly different from those of the known “insulin sensitizers”. Our study demonstrates the utility of isotopomer matrix in summarizing metabolic actions of LLE on the balance of fluxes occurring within the central glucose metabolism in HepG2 cells. 13C carbon tracing (tracer-based metabolomics) is a useful systems biology tool to elucidate glucose metabolic pathways affected by diabetes and its treatment.

Syringaresinol-4-O-β-d-glucoside alters lipid and glucose metabolism in HepG2 cells and C2C12 myotubes

Syringaresinol-4-O-β-d-glucoside (SSG), a furofuran-type lignan, was found to modulate lipid and glucose metabolism through an activity screen of lipid accumulation and glucose consumption, and was therefore considered as a promising candidate for the prevention and treatment of metabolic disorder, especially in lipid and glucose metabolic homeostasis. In this study, the effects of SSG on lipogenesis and glucose consumption in HepG2 cells and C2C12 myotubes were further investigated. Treatment with SSG significantly inhibited lipid accumulation by oil red O staining and reduced the intracellular contents of total lipid, cholesterol and triglyceride in HepG2 cells. No effect was observed on cell viability in the MTT assay at concentrations of 0.1–10 μmol/L. SSG also increased glucose consumption by HepG2 cells and glucose uptake by C2C12 myotubes. Furthermore, real-time quantitative PCR revealed that the beneficial effects were associated with the down-regulation of sterol regulatory element-binding proteins-1c, -2 (SREBP-1c, -2), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC) and hydroxyl methylglutaryl CoA reductase (HMGR), and up-regulation of peroxisome proliferator-activated receptors alpha and gamma (PPARα and PPARγ). SSG also significantly elevated transcription activity of PPARγ tested by luciferase assay. These results suggest that SSG is an effective regulator of lipogenesis and glucose consumption and might be a candidate for further research in the prevention and treatment of lipid and glucose metabolic diseases.

Galactomannan from Schizolobium amazonicum seed and its sulfated derivatives impair metabolism in HepG2 cells

This study evaluated the effects of native galactomannan from Schizolobium amazonicum seeds and its sulfated forms on certain metabolic parameters of HepG2 cells. Aqueous extraction from S. amazonicum seeds furnished galactomannan with 3.2:1 Man:Gal ratio (SAGM) and molar mass of 4.34×105g/mol. The SAGM fraction was subjected to sulfation using chlorosulfonic acid to obtain SAGMS1 and SAGMS2 with DS of 0.4 and 0.6, respectively. Cytotoxicity of SAGM, SAGMS1, and SAGMS2 was evaluated in human hepatocellular carcinoma cells (HepG2). After 72h, SAGM decreased the viability of HepG2 cells by 50% at 250μg/mL, while SAGMS1 reduced it by 30% at the same concentration. SAGM, SAGMS1, and SAGMS2 promoted a reduction in oxygen consumption and an increase in lactate production in non-permeabilized HepG2 cells after 72h of treatment. These results suggest that SAGM, SAGMS1, and SAGMS2 could be recognized by HepG2 cells and might trigger alterations that impair its survival. These effects could be implicated in the modification of the oxidative phosphorylation process in HepG2 cells and activation of the glycolytic pathway.

Investigations on the hypocholesterolaemic activity of LILPKHSDAD and LTFPGSAED, two peptides from lupin β-conglutin: Focus on LDLR and PCSK9 pathways

P5 (LILPKHSDAD) and P7 (LTFPGSAED) are two peptides from lupin protein that are absorbed in Caco-2 cells. Recent in silico docking studies had suggested that they could potentially function as inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoAR) activity. This paper confirms that both peptides inhibit in vitro the HMGCoAR functionality and it reports the characterisation of the molecular mechanism through which they modulate the cholesterol metabolism in HepG2 cells. Through the inhibition of HMGCoAR activity, P5 and P7 are able to improve the LDLR protein levels leading to a better ability of HepG2 cells to uptake extracellular LDL molecules with a final hypocholesterolaemic effects. The modulation of PCSK9 intracellular processing was also evaluated: only P5 was able to decrease the PCSK9 and HNF1-alpha protein levels leading to a decrease of mature PCSK9 secretion by HepG2 cells.

Effects of Catechol O-Methyl Transferase Inhibition on Anti-Inflammatory Activity of Luteolin Metabolites.

Although luteolin is known to have potent anti-inflammatory activities, much less information has been provided on such activities of its hepatic metabolites. Luteolin was subjected to hepatic metabolism in HepG2 cells either without or with catechol O-methyl transferase (COMT) inhibitor. To identify hepatic metabolites of luteolin without (luteolin metabolites, LMs) or with COMT inhibitor (LMs+CI), metabolites were treated by β-glucuronidase and sulfatase, and found that they were composed of glucuronide and sulfate conjugates of diosmetin in LMs or these conjugates of luteolin in LMs+CI. LMs and LMs+CI were examined for their anti-inflammatory activities on LPS stimulated Raw 264.7 cells. Expression of iNOS and production of nitric oxide and pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 were suppressed more effectively by the treatment with LMs+CI than LMs. Our data provide a new insight on possible improvement in functional properties of luteolin on target cells by modifying their metabolic pathway in hepatocytes.

Upregulation of heat shock protein 70 and the differential protein expression induced by tumor necrosis factor-alpha enhances migration and inhibits apoptosis of hepatocellular carcinoma cell HepG2.

Tumor necrosis factor alpha (TNFα) plays diverse roles in liver damage and hepatocarcinogenesis with its multipotent bioactivity. However, the influence of TNFα on protein expression of hepatocellular carcinoma (HCC) is incompletely understood. Therefore, we aimed to investigate the differential protein expression of HCC in response to TNFα stimulus. We observed that HepG2 cell revealed a higher resistance to TNFα-induced apoptosis as compared to the non-tumorigenic hepatocyte THLE-2. By using a label-free quantitative proteomic analysis, we found that 520 proteins were differentially expressed in the HepG2 cells exposed to TNFα, including 211 up-regulated and 309 down-regulated proteins. We further confirmed several proteins with significant expression change (TNFα/control ratio>2.0 or <0.5) by immunoblotting using specific antibodies. We also analyzed the differential expressed proteins using Gene ontology and KEGG annotations, and the results implicated that TNFα might regulate ribosome, spliceosome, antigen processing and presentation, and energy metabolism in HepG2 cells. Moreover, we demonstrated that upregulation of heat shock protein 70 (HSP70) was involved in both the promoted migration and the inhibited apoptosis of HepG2 cells in response to TNFα. Collectively, these findings indicate that TNFα alters protein expression such as HSP70, which triggering specific molecular processes and signaling cascades that promote migration and inhibit apoptosis of HepG2 cells.

Enhanced pro-protein convertase subtilisin/kexin type 9 expression by C-reactive protein through p38MAPK-HNF1α pathway in HepG2 cells.

Plasma C‐reactive protein (CRP) concentration is associated positively with cardiovascular risk, including dyslipidemia. We suggested a regulating role of CRP on pro‐protein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of low‐density lipoprotein (LDL) metabolism, and demonstrated the PCSK9 as a pathway linking CRP and LDL regulation. Firstly, experiments were carried out in the presence of human CRP on the protein and mRNA expression of PCSK9 and LDL receptor (LDLR) in human hepatoma cell line HepG2 cells. Treatment with CRP (10 μg/ml) enhanced significantly the mRNA and protein expression of PCSK9 and suppressed the expression of LDLR. Of note, a late return of LDLR mRNA levels occurred at 12 hrs, while the LDLR protein continued to decrease at 24 hrs, suggesting that the late decrease in LDLR protein levels was unlikely to be accounted for the decrease in LDL mRNA. Secondly, the role of PCSK9 in CRP‐induced LDLR decrease and the underlying pathways were investigated. As a result, the inhibition of PCSK9 expression by small interfering RNA (siRNA) returned partly the level of LDLR protein and LDL uptake during CRP treatment; CRP‐induced PCSK9 increase was inhibited by the p38MAPK inhibitor, SB203580, resulting in a significant rescue of LDLR protein expression and LDL uptake; the pathway was involved in hepatocyte nuclear factor 1α (HNF1α) but not sterol responsive element‐binding proteins (SREBPs) preceded by the phosphorylation of p38MAPK. These findings indicated that CRP increased PCSK9 expression by activating p38MAPK‐HNF1α pathway, with a certain downstream impairment in LDL metabolism in HepG2 cells.

A multidisciplinary investigation on the bioavailability and activity of peptides from lupin protein

Lupin foods provide useful health benefits. Since tryptic and peptic peptides from lupin protein modulate cholesterol metabolism in HepG2 cells and increase LDL-uptake, this work had the goal of assessing whether these lupin peptides are absorbed by human intestinal Caco-2 cells. Cells were differentiated for 15 days and transport experiments were performed by incubating each lupin peptide mixture from the apical side. After 4 h, basolateral solutions were collected and analysed by HPLC-Chip-MS/MS. Eleven tryptic and eight peptic peptides, deriving from lupin storage proteins, were identified in the basolateral samples. An in vitro assay showed that basolateral peptides maintain their capacity to inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) activity and in silico docking simulations permitted to hypothesise which peptides may bind more efficiently to the HMGCoAR catalytic site. This is the first investigation providing evidence that lupin peptides with specific structures are potentially absorbed in human intestinal cells.

Berberine Attenuates Development of the Hepatic Gluconeogenesis and Lipid Metabolism Disorder in Type 2 Diabetic Mice and in Palmitate-Incubated HepG2 Cells through Suppression of the HNF-4α miR122 Pathway.

Berberine (BBR) has been shown to exhibit protective effects against diabetes and dyslipidemia. Previous studies have indicated that BBR modulates lipid metabolism and inhibits hepatic gluconeogensis by decreasing expression of Hepatocyte Nuclear Factor-4α (HNF-4α). However, the mechanism involved in this process was unknown. In the current study, we examined the mechanism of how BBR attenuates hepatic gluconeogenesis and the lipid metabolism alterations observed in type 2 diabetic (T2D) mice and in palmitate (PA)-incubated HepG2 cells. Treatment with BBR for 4 weeks improve all biochemical parameters compared to T2D mice. Treatment of T2D mice for 4 weeks or treatment of PA-incubated HepG2 cells for 24 h with BBR decreased expression of HNF-4α and the microRNA miR122, the key gluconeogenesis enzymes Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphatase (G6Pase) and the key lipid metabolism proteins Sterol response element binding protein-1 (SREBP-1), Fatty acid synthase-1 (FAS-1) and Acetyl-Coenzyme A carboxylase (ACCα) and increased Carnitine palmitoyltransferase-1(CPT-1) compared to T2D mice or PA-incubated HepG2 cells. Expression of HNF-4α in HepG2 cells increased expression of gluconeogenic and lipid metabolism enzymes and BBR treatment or knock down of miR122 attenuated the effect of HNF-4α expression. In contrast, BBR treatment did not alter expression of gluconeogenic and lipid metabolism enzymes in HepG2 cells with knockdown of HNF-4α. In addition, miR122 mimic increased expression of gluconeogenic and lipid metabolism enzymes in HepG2 cells with knockdown of HNF-4α. These data indicate that miR122 is a critical regulator in the downstream pathway of HNF-4α in the regulation of hepatic gluconeogenesis and lipid metabolism in HepG2 cells. The effect of BBR on hepatic gluconeogenesis and lipid metabolism is mediated through HNF-4α and is regulated downstream of miR122. Our data provide new evidence to support HNF-4α and miR122 regulated hepatic gluconeogenesis and lipid metabolism as promising therapeutic targets for the treatment of T2D.

Development and Characterization of Novel and Selective Inhibitors of Cytochrome P450 CYP26A1, the Human Liver Retinoic Acid Hydroxylase.

Cytochrome P450 CYP26 enzymes are responsible for all-trans-retinoic acid (atRA) clearance. Inhibition of CYP26 enzymes will increase endogenous atRA concentrations and is an attractive therapeutic target. However, the selectivity and potency of the existing atRA metabolism inhibitors toward CYP26A1 and CYP26B1 is unknown, and no selective CYP26A1 or CYP26B1 inhibitors have been developed. Here the synthesis and potent inhibitory activity of the first CYP26A1 selective inhibitors is reported. A series of nonazole CYP26A1 selective inhibitors was identified with low nM potency. The lead compound 3-{4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1,3-dioxolan-2-yl] phenyl}4-propanoic acid (24) had 43-fold selectivity toward CYP26A1 with an IC50 of 340 nM. Compound 24 and its two structural analogues also inhibited atRA metabolism in HepG2 cells, resulting in increased potency of atRA toward RAR activation. The identified compounds have potential to become novel treatments aiming to elevate endogenous atRA concentrations and may be useful as cotreatment with atRA to combat therapy resistance.

Squalene isolated from Schizochytrium mangrovei is a peroxisome proliferator-activated receptor-α agonist that regulates lipid metabolism in HepG2 cells.

Peroxisome proliferator-activated receptor-alpha (PPARα) is a nuclear receptor that has critical roles in the treatment of atherosclerosis, hyperlipidemia and hepatic steatosis. Squalene is a novel nature PPARα agonist, identified from reporter gene assay and qPCR analysis. Cultured hepatocytes stimulated with squalene exhibited significantly decreased cellular triacylglycerols and cholesterol concentrations, while cellular uptake of fatty acids was increased. Quantitative PCR analysis revealed that expression of genes related to fatty acid uptake, fatty acid oxidation, ketogenesis and reverse cholesterol transport metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed in cell treated with squalene. Squalene is hypolipidemic by activation of PPARα via a ligand-mediated mechanism that regulates the expression of lipid metabolism genes in hepatocytes.

Synergetic cholesterol-lowering effects of main alkaloids from Rhizoma Coptidis in HepG2 cells and hypercholesterolemia hamsters

AimsHyperlipidemia contributes to the progression of cardiovascular diseases. Main alkaloids from Rhizoma Coptidis including berberine (BBR), coptisine (COP), palmatine (PAL), epiberberine (EPI) and jatrorrhizine (JAT), improved dyslipidemia in hypercholesterolemic hamsters to a different degree. In this study, HepG2 cells and hypercholesterolemic hamsters were used to investigate the synergetic cholesterol-lowering efficacy of these five main alkaloids. Main methodsThe cellular lipid and cholesterol accumulation and in HepG2 cells were evaluated by Oil Red O staining and HPLC analysis. LDL receptor, 3-Hydroxy-3-methylglutaryl CoA reductase (HMGCR) and cholesterol 7-alpha-hydroxylase (CYP7A1) that involving cholesterol metabolism in HepG2 cells were measured by qRT-PCR, western blot and immunofluorescence analysis. The serum profiles including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c), as well as TC and total bile acids (TBA) of feces in hypercholesterolemic hamsters were also measured. Key findingAs compared to single alkaloids, the combination of five main alkaloids (COM) reduced the lipid and cholesterol accumulation in HepG2 cells more effectively and performed an advantageous effect on controlling TC, TG, LDL-c and HDL-c in hypercholesterolemic hamsters. More effective reduction of TBA and TC levels in feces of hamsters were achieved after the administration of COM. These effects were derived from the up-regulation of LDL receptor and CYP7A1, as well as HMGCR downregulation. SignificanceOur results demonstrated that COM showed a synergetic cholesterol-lowering efficacy, which was better than single alkaloids and it might be considered as a potential therapy for hypercholesterolemia.