Amar Bahadur Singh

High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway

Long-chain acyl-CoA synthetases (ACSL) play key roles in fatty acid metabolism in liver and other metabolic tissues in an isozyme-specific manner. In this study, we examined the effects of a fructose-enriched diet on expressions of ACSL isoforms in the liver of hamsters. We showed that the fructose diet markedly reduced the mRNA and protein expressions of ACSL3 in hamster liver without significant effects on other ACSLs. The decrease in ACSL3 abundance was accompanied by a reduction in ACSL-catalyzed synthesis of arachidonyl-CoA and oleoyl-CoA in liver homogenates of hamsters fed the fructose diet as opposed to normal diet. We further showed that fructose diet specifically reduced expressions of three key components of the LXR signaling pathway, namely, liver X receptor (LXR)α, LXRβ, and retinoid X receptor (RXR)β. Exogenous expression and activation of LXRα/β increased hamster ACSL3 promoter activities in a LXR-responsive element (LXRE)-dependent fashion. Finally, we showed that treating hamsters with LXR agonist GW3965 increased hepatic ACSL3 expression without affecting other ACSL isoforms. Furthermore, the ligand-induced increases of ACSL3 expression were accompanied with the reduction of hepatic triglyceride levels in GW3965-treated hamster liver. Altogether, our studies demonstrate that fructose diet has a negative impact on LXR signaling pathway in liver tissue and reduction of ACSL3 expression/activity could be a causal factor for fructose-induced hepatic steatosis.

High-fructose feeding promotes accelerated degradation of hepatic LDL receptor and hypercholesterolemia in hamsters via elevated circulating PCSK9 levels

BACKGROUND High fructose diet (HFD) induces dyslipidemia and insulin resistance in experimental animals and humans with incomplete mechanistic understanding. By utilizing mice and hamsters as in vivo models, we investigated whether high fructose consumption affects serum PCSK9 and liver LDL receptor (LDLR) protein levels. RESULTS Feeding mice with an HFD increased serum cholesterol and reduced serum PCSK9 levels as compared with the mice fed a normal chow diet (NCD). In contrast to the inverse relationship in mice, serum PCSK9 and cholesterol levels were co-elevated in HFD-fed hamsters. Liver tissue analysis revealed that PCSK9 mRNA and protein levels were both reduced in mice and hamsters by HFD feeding, however, liver LDLR protein levels were markedly reduced by HFD in hamsters but not in mice. We further showed that circulating PCSK9 clearance rates were significantly lower in hamsters fed an HFD as compared with the hamsters fed NCD, providing additional evidence for the reduced hepatic LDLR function by HFD consumption. The majority of PCSK9 in hamster serum was detected as a 53 kDa N-terminus cleaved protein. By conducting in vitro studies, we demonstrate that this 53 kDa truncated hamster PCSK9 is functionally active in promoting hepatic LDLR degradation. CONCLUSION Our studies for the first time demonstrate that high fructose consumption increases serum PCSK9 concentrations and reduces liver LDLR protein levels in hyperlipidemic hamsters. The positive correlation between circulating cholesterol and PCSK9 and the reduction of liver LDLR protein in HFD-fed hamsters suggest that hamster is a better animal model than mouse to study the modulation of PCSK9/LDLR pathway by atherogenic diets.

Arachidonic acid downregulates acyl-CoA synthetase 4 expression by promoting its ubiquitination and proteasomal degradation

ACSL4 is a member of the long-chain acyl-CoA synthetase (ACSL) family with a marked preference for arachidonic acid (AA) as its substrate. Although an association between elevated levels of ACSL4 and hepatosteatosis has been reported, the function of ACSL4 in hepatic FA metabolism and the regulation of its functional expression in the liver remain poorly defined. Here we provide evidence that AA selectively downregulates ACSL4 protein expression in hepatic cells. AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis. The inhibitory action of AA on ACSL4 protein stability could not be prevented by rosiglitazone or inhibitors that interfere with the cellular pathways involved in AA metabolism to biologically active compounds. In contrast, treatment of cells with inhibitors specific for the proteasomal degradation pathway largely prevented the AA-induced ACSL4 degradation. We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination. Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

A novel posttranscriptional mechanism for dietary cholesterol-mediated suppression of liver LDL receptor expression

It is well-established that over-accumulation of dietary cholesterol in the liver inhibits sterol-regulatory element binding protein (SREBP)-mediated LDL receptor (LDLR) gene transcription leading to a reduced hepatic LDLR mRNA level in hypercholesterolemic animals. However, it is unknown whether elevated cholesterol levels can elicit a cellular response to increase LDLR mRNA turnover to further repress LDLR expression in liver tissue. In the current study, we examined the effect of a high cholesterol diet on the hepatic expression of LDLR mRNA binding proteins in three different animal models and in cultured hepatic cells. Our results demonstrate that high cholesterol feeding specifically elevates the hepatic expression of LDLR mRNA decay promoting factor heterogeneous nuclear ribonucleoprotein (HNRNP)D without affecting expressions of other LDLR mRNA binding proteins in vivo and in vitro. Employing the approach of adenovirus-mediated gene knockdown, we further show that depletion of HNRNPD in the liver results in a marked reduction of serum LDL-cholesterol and a substantial increase in liver LDLR expression in hyperlipidemic mice. Additional studies of gene knockdown in albumin-luciferase-untranslated region (UTR) transgenic mice provide strong evidence supporting the essential role of 3′UTR in HNRNPD-mediated LDLR mRNA degradation in liver tissue. Altogether, this work identifies a novel posttranscriptional regulatory mechanism by which dietary cholesterol inhibits liver LDLR expression via inducing HNRNPD to accelerate LDLR mRNA degradation.

Regulation of lipid metabolism by obeticholic acid in hyperlipidemic hamsters

The farnesoid X receptor (FXR) plays critical roles in plasma cholesterol metabolism, in particular HDL-cholesterol (HDL-C) homeostasis. Obeticholic acid (OCA) is a FXR agonist being developed for treating various chronic liver diseases. Previous studies reported inconsistent effects of OCA on regulating plasma cholesterol levels in different animal models and in different patient populations. The mechanisms underlying its divergent effects have not yet been thoroughly investigated. The scavenger receptor class B type I (SR-BI) is a FXR-modulated gene and the major receptor for HDL-C. We investigated the effects of OCA on hepatic SR-BI expression and correlated such effects with plasma HDL-C levels and hepatic cholesterol efflux in hyperlipidemic hamsters. We demonstrated that OCA induced a time-dependent reduction in serum HDL-C levels after 14 days of treatment, which was accompanied by a significant reduction of liver cholesterol content and increases in fecal cholesterol in OCA-treated hamsters. Importantly, hepatic SR-BI mRNA and protein levels in hamsters were increased to 1.9- and 1.8-fold of control by OCA treatment. Further investigations in normolipidemic hamsters did not reveal OCA-induced changes in serum HDL-C levels or hepatic SR-BI expression. We conclude that OCA reduces plasma HDL-C levels and promotes transhepatic cholesterol efflux in hyperlipidemic hamsters via a mechanism involving upregulation of hepatic SR-BI.

PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro

The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells.

Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated By Peroxisome Proliferator-activated Receptor δ.

Peroxisome proliferator-activated receptor δ (PPARδ) regulates many genes involved in lipid metabolism. Hepatic lysophosphatidylcholine acyltransferase 3 (LPCAT3) has critical functions in triglycerides transport and endoplasmic reticulum stress response due to its unique ability to catalyze the incorporation of polyunsaturated fatty acids into phospholipids. Previous studies identified liver X receptor as the transcription factor controlling LPCAT3 expression in mouse liver tissue. Here we show that the hepatic LPCAT3 gene is transcriptionally regulated by PPARδ. Adenovirus-mediated knockdown of PPARδ in cultured hepatic cells and liver tissue reduced LPCAT3 mRNA levels, and exogenous overexpression of PPARδ increased LPCAT3 mRNA expression. Activation of PPARδ in HepG2, Huh7, and Hepa 1-6 cells with its specific agonists increased LPCAT3 mRNA levels in all three hepatic cell lines. Through conducting sequence analysis, LPCAT3 promoter assays, and direct DNA binding assays, we have mapped the functional PPAR-responsive element to a proximal region from −135 to −123 of the LPCAT3 promoter that plays an essential role in mediating PPARδ-induced transactivation of the LPCAT3 gene. Finally, we have provided in vivo evidence showing that activation of PPARδ by agonist L165041 in mice increased hepatic LPCAT3 mRNA abundance and LPCAT enzymatic activity, which is associated with increased incorporations of arachidonate into liver phosphatidylcholine and phosphatidylethanolamine. Furthermore, transient liver-specific knockdown of LPCAT3 in mice affected PPARδ-mediated activation of several hepatic genes involving in FA metabolism. Altogether, our new findings identify LPCAT3 as a direct PPARδ target gene and suggest a novel function of PPARδ in regulation of phospholipid metabolism through LPCAT3.

A novel peroxisome proliferator response element modulates hepatic low-density lipoprotein receptor gene transcription in response to PPARδ activation.

The hepatic expression of low-density lipoprotein (LDL) receptor (LDLR) gene is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein 2 (SREBP2). However whether additional cis-regulatory elements contribute to LDLR transcription has not been fully explored. We investigated the function of a putative peroxisome proliferator-activated receptor (PPAR)-response element (PPRE) sequence motif located at -768 to -752 bases upstream of the transcription start site of human LDLR gene in response to PPARδ activation. Promoter luciferase reporter analyses showed that treating HepG2 cells with PPARδ agonist L165041 markedly increased the activity of a full-length LDLR promoter construct (pLDLR-1192) without any effects on the shorter promoter reporter pLDLR-234 that contains only the core regulatory elements SRE-1 and SP1 sites. Importantly, mutation of the PPRE sequence greatly attenuated the induction of the full-length LDLR promoter activity by L165041 without affecting rosuvastatin (RSV)-mediated transactivation. EMSA and ChIP assay further confirmed the binding of PPARδ to the LDLR-PPRE site. Treating HepG2 cells with L165041 elevated the mRNA and protein expressions of LDLR without affecting the LDLR mRNA decay rate. The induction of LDLR expression by PPARδ agonist was further observed in liver tissue of mice and hamsters treated with L165041. Altogether, our studies identify a novel PPRE-mediated regulatory mechanism for LDLR transcription and suggest that combined treatment of statin with PPARδ agonists may have advantageous effects on LDLR expression.