Nuclear transcription factors and lipid homeostasis in liver

Abstract

The liver plays a major role in the regulation of glucose, lipid and energy metabolism. Increased hepatic fat deposit is a very common feature in obese, insulin-resistant patients, in metabolic syndrome, alcoholic steatohepatitis (ASH) and nonalchoholic fatty liver disaseas (NAFLD). As a central organ for whole body lipid metabolism, disruption of the normal mechanisms for synthesis, transport and removal/metabolism of long-chain fatty acids and triglycerides are the basis for the development of fatty liver. The exact mechanisms that link hepatic lipid accumulation, impaired glucose metabolism, and insulin resistance are unknown, but increasing evidence suggest that nuclear transcription factors play important roles. Members of the nuclear receptor superfamily, especially the peroxisome proliferator-activated receptors (PPARs) and the liver X receptor (LXR), other factors such as sterol regulatory element binding proteins (SREBPs), carbohydrate-response element-binding protein (ChREBP), and nuclear transcription fator-κB (NF-κB) have emerged as dominant regulators of these processes, but the relative role of each of these factors in fatty liver disease is still undefined. This review will focus on the regulation of these transcription factors and their role in the control of hepatic lipid homeostasis. Peroxisome proliferator-activated receptors (PPARs) PPARs belong to a ligand-activated nuclear hormone receptor superfamily and are known to regulate the expression of numerous genes involved in fatty acid metabolism and adipocyte differentiation.1,2 PPARs were originally identified as factors that mediate transcriptional responses to peroxisome proliferators, a broad class of xenobiotic chemicals that include fibrate, hypolipidemic drugs, and other nongenotoxic rodent hepatocarcinogens.3,4 Subsequently, PPARs were shown to be differentially activated by a variety of saturated or unsaturated long chain fatty acids and lipid-like compounds,5-9 suggesting that fatty acids or fatty acid derivatives serve as physiological activators. PPARs have three isoforms (PPARα, PPARβ/δ and PPARγ) which have different patterns of tissue expression and functional activity. PPARα is primarily expressed in the liver, in which it has been shown to promote β-oxidation of fatty acids.1 The hepatic expression of PPARα is nutritionally activated by fasting. Fatty acids serve as ligands for PPARα, and when fatty acid levels increase, activation of PPARα induces a battery of fatty acid-metabolizing enzymes to restore fatty acid levels to normal. PPARα-null mice exposed to prolonged fasting develop fatty liver and hypoglycaemia. This suggests that inadequate PPAR-mediated responses may contribute to abnormal fatty acid metabolism in alcoholic and nonalcoholic steatohepatitis (ASH/NASH). Lipid accumulation in hepatocytes is one of the cardinal features of NAFLD. Studying different animal models is providing insights into the contribution of a variety of genetic and host factors to NAFLD. A number of animal studies have linked fatty acid oxidation with NAFLD.10 One of the challenges based on these studies is to determine the role of PPARα and fatty acid oxidation on the development of hepatic steatosis and liver injury in patients with NAFLD. PPARα agonists bind to PPARα, resulting in peroxisomal proliferation and increased expression of enzymes involved in fatty acid oxidation, including the rate-limiting enzyme for peroxisomal β-oxidation, acyl-CoA oxidase (AOX).11 Hepatic fatty acid levels are increased during ethanol consumption. However, results of in vitro studies showed that ethanol metabolism inhibited the ability of PPARα to bind DNA and activate reporter genes.12 This observation has been further studied in mice. Four weeks of ethanol feeding of C57BL/6J mice also impairs fatty acid catabolism in liver by blocking PPARα-mediated responses.13 Ethanol feeding decreased the level of retinoid X receptor alpha (RXRα) as well as the ability of PPARα/RXR in liver nuclear extracts to bind its consensus sequence, and decreased the levels of mRNAs for several PPARα-regulated genes, such as long-chain acyl-CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase, liver carnitine palmitoyl-CoA transferase I, very long-chain acyl-CoA synthetase and very long-chain acyl-CoA dehydrogenase. Consistent with this finding, ethanol feeding did not induce the rate of fatty acid β-oxidation, as assayed in liver homogenates. Inclusion of WY14 643, a PPARα agonist, in the diet restored the DNA-binding activity of PPARα/RXR, induced mRNA levels of several PPARα. target genes, stimulated the rate of fatty acid β-oxidation in liver homogenates, and prevented fatty liver in ethanol-fed animals. Blockade of PPARα. function during ethanol consumption contributes to the development of alcoholic fatty liver, which can be overcome by WY14 643.14 PPARγ is mainly expressed in adipose tissue,15 where it has been shown to be an essential component of adipocyte differentiation program;16 and in macrophages, where it modulates differentiation and cytokine production.17-19 The functional role of PPARδ is less well characterized but it is also involved in fatty acid metabolism.11,20,21 Sterol regulatory element binding proteins (SREBPs) SREBPs are a family of transcription factors that regulate lipid homeostasis by controlling the expression of a range of enzymes required for endogenous cholesterol, fatty acid (FA), triglyceride and phospholipid synthesis. SREBP-1a is highly expressed in cell lines and tissues with a high capacity for cell proliferation, such as spleen and intestine. SREBP-1c and SREBP-2 are the predominant isoform expressed in most of the tissues of mice and humans, with especially high levels in the liver, white adipose tissue, skeletal muscle, adrenal gland and brain.22 Overexpression of SREBP-1a in mice liver23,24 markedly increases the expression of genes involved in cholesterol synthesis (for example, HMG-CoA synthase, HMG-CoA reductase, squalene synthase) and FA synthesis (acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD-1)) and to cause corresponding accumulation of both cholesterol and triglyceride.23,25 Mice overexpressing hepatic SREBP-1c demonstrate a selective induction of lipogenic genes, with no effect on genes of cholesterol synthesis.26 Foretz et al27 have shown in isolated hepatocytes that over-expression of SREBP-1c isoform induces not only lipogenic genes but also glucokinase (GK) which is necessary for the utilization of glucose in the liver and is regulated by insulin at the transcriptional level.27 The hepatic over-expression of SREBP-2 isoform in mice causes a preferential induction of genes involved in cholesterol biosynthesis, although a moderate induction of genes involved in FA synthesis is also observed. Importantly, in addition to genes encoding enzymes of these pathways, SREBPs augment the expression of genes involved in the generation of NADPH, an obligatory co-factor for lipid synthesis. Using luciferase-reporter gene assays in HepG2 cells, Amemiya-Kudo et al28 showed that the selectivity of the SREBP isoforms for cholesterogenic and/or lipogenic genes is at least partly due to their differing affinities for the various consensus sequences in the target gene DNA. SREBP transcription factors are regulated at three major levels: (1) transcription, (2) proteolytic cleavage of SREBP precursors, and (3) post-translational modification of SREBPs. Although there is some overlap of regulatory mechanisms across SREBP isoforms, significant differences do exist. In particular, whereas SREBP-1a and SREBP-2 appear to be primarily regulated at the level of precursor cleavage, evidence suggests that SREBP-1c is mainly regulated at the transcriptional level. SREBP transcription factors are synthetized as inactive precursors bound to the endoplasmic reticulum (ER) membranes. Upon activation, the precursor undergoes a sequential two-step cleavage process in the golgi and then release the NH2-terminal active domain to the nucleus (designated nSREBPs). nSREBPs processing is mainly controlled by cellular sterol content. When sterol levels decrease, the precursor is cleaved to activate cholesterogenic genes and maintain cholesterol homeostasis. This sterol-sensitive process appears to be a major point of regulation for the SREBP-1a and SREBP-2 isoforms but not for SREBP-1c. Moreover, the SREBP-1c isoform seems to be mainly regulated at the transcriptional level by insulin. The unique regulation and activation properties of each SREBP isoform facilitate the co-ordinate regulation of lipid metabolism; however, further studies are needed to understand the detailed regulation pathways that specifically regulate each SREBP isoform. The role of SREBPs in NAFLD was suggested by investigators on the basis of results of a number of studies. Transgenic mice overexpressing SREBP-1a or SREBP-1c produced massive fatty livers owing to increased accumulation of cholesteryl esters and triglycerides.23 Findings of studies further demonstrate that the fatty liver of obese (ob/ob) mice with insulin resistance is caused by elevated SREBP-1c levels, thereby increasing lipogenic gene expression, enhances fatty acid synthesis, and accelerates triglyceride accumulation in the liver.29-31 In addition, increasing evidence has demonstrated that chronic inflammation could promote NAFLD. The role of SREBPs in alcoholic fatty liver was investigated by examining the effect of ethanol on SREBP activation and transcriptional function in cultured hepatoma cells, as well as in mice fed ethanol-containing diets.32 The effect of ethanol on a sterol regulatory element (SRE)-regulated promoter activity was tested in rat hepatoma and a nonhepatic cell lines. Ethanol significantly increased transcription of SRE-regulated promoter activity in rat hepatoma cells, but not in nonhepatic cells which do not express alcohol dehydrogenase, suggesting that acetaldehyde generated from ethanol metabolism might be required for this effect. Further study showed that ethanol or acetaldehyde caused a marked increase in the amount of mature SREBP-1 in hepatic cells. This increase was associated with an acetaldehyde-stimulated de novo cholesterol synthesis.33 Consistent with the in vitro findings, consumption of a low-fat diet with ethanol by mice led to the development of fatty liver. Addition of ethanol to the low-fat diet resulted in a substantial increase in the amount of mature SREBP-1 protein in the liver.32 Activation of SREBP-1 by ethanol feeding was associated with the accumulation of triglyceride in the livers, as well as with increased expression of several hepatic lipogenic genes known to be controlled by SREBP-1. These included FAS, a major enzyme of fatty acid synthesis; malic enzyme and ATP citrate lyase, two enzymes that supply reduced form of nicotinamide adenine dinucleotide phosphate and acetyl-CoA for fatty acid synthesis; and stearoyl-CoA desaturase (SCD), the enzyme responsible for conversion of palmitate and stearate to palmitoleate and oleate.32 It has been demonstrated that ethanol feeding also induced the level of ACC mRNA by 2.5-fold.13 ACC is generally regarded as the ratelimiting enzyme in fatty acid biosynthesis in liver. The product of ACC, malonyl-CoA, is both a precursor for the biosynthesis of fatty acids and a potent inhibitor of mitochondrial fatty acid beta-oxidation. Taken together, these findings support the idea that metabolism of ethanol increased hepatic lipogenesis by activating SREBP-1, and this effect of ethanol may contribute to the development of alcoholic fatty liver. Results of a study demonstrated that the anti-diabetic drug metformin lowers the lipid accumulation in livers of insulin resistant ob/ob mice, partly through reduced hepatic SREBP-1 levels, therefore, reverses fatty liver.34 Metformin activates adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK), a protein kinase that inhibits lipid synthesis through phosphorylation and inactivation of key lipogenic enzymes such as ACC.35,36 Further mechanistic studies led to the discovery that, in both rat hepatocytes and liver, metformin treatment-induced activation of AMPK leads to decreased mRNA and protein expression of SREBP-1c.36 Moreover, findings of another study provide evidence that activation of AMPK by a fat-derived hormone, adiponectin, resulted in dramatic reduction of the expression of hepatic SREBP-1c in the liver.37 It has been shown that adiponectin treatment alleviated alcoholic and nonalcoholic fatty liver diseases in mice, partly by increasing activity of AMPK in the liver, which, in turn, phosphorylates ACC and attenuates the activity of this enzyme.38 Findings of these studies establish a direct link between AMPK action and SREBP transcriptional activity. In summary, fatty liver induced by chronic ethanol consumption may involve two potential signaling pathways. First, the inhibition of AMPK by ethanol feeding causes an increase in SREBP-1 activation. As a result, known target lipogenic genes for SREBP-1 are up-regulated in liver, thereby contributing to ethanol-induced hepatic lipid synthesis and fatty liver. Second, AMPK inhibition by ethanol feeding results in increased activity of ACC, and it could cause a reduced rate of fatty acid oxidation through increased malonyl-CoA concentration in the liver, and further contribute to development of alcoholic fatty liver. Adiponectin seems to have the ability in reversing all these and inflammation. Liver X receptor (LXR) LXRs belong to a subclass of nuclear hormone receptors that form obligate heterodimers with RXR and are activated by oxysterols.39-42 There have been two subtypes of LXRs identified: LXRα and LXRβ. LXRα is expressed in liver, spleen, kidney, adipose, and small intestine,43 whereas LXRβis ubiquitously expressed.44 The two forms appear to respond to the same natural and synthetic ligands. Natural LXR ligands include physiologic concentrations of sterol metabolites such as 22(R)-, 24(S)-, 27-, and 24(S), 25-hydroxycholestero.40,41,45 LXR/RXR heterodimers are constitutively nuclear, bound to LXR response elements that contain two canonical hexamer sequences, AGGTCA, separated by four nucleotides (a DR-4 recognition sequence). A large body of evidence supports the theory that LXRs function as whole-body cholesterol sensors.46 Many target genes for LXR are integral parts of the cholesterol and fatty acid metabolic pathways. One of the first identified targets was the cytochrome P450 family member, CYP7A1, which is the rate-limiting enzyme in hepatic bile synthesis. LXRα-null mice cannot induce CYP7A1 in response to a high-cholesterol diet and accumulate massive amounts of cholesterol in their livers.47 Mice lacking both LXR isoforms have an even more severe hepatic phenotype on the high-cholesterol diet, but LXRβ-null mice are normal.48 This suggests that LXRα is the prime mover of hepatic lipid metabolism. LXRα-null mice show reduced expression of the major lipogenic regulators in the liver: SREBP-1c, FAS, and SCD-1.47 Giving synthetic LXR ligands to mice triggers the lipogenic program and results in elevated plasma and hepatic triglyceride levels.49 Other major cholesterol-related targets of LXR include the ABC family of transporters. ABCA1, which modulates cholesterol efflux and mediates reverse cholesterol transport from peripheral tissues, is the most established one; other target ABC transporters include ABCG1 for cholesterol efflux50 and ABCG5/ABCG8 for hepatic excretion of cholesterol into bile.51 Genes that encode lipid-remodeling enzymes such as LPL, CETP, and phospholipid transfer protein (PLTP) are also LXR targets. These enzymes participate in the peripheral modification of lipoprotein particles, especially HDL. Carbohydrate-response element-binding protein (ChREBP) The transcription factor ChREBP is translocated to the nucleus and activated in response to high glucose concentrations in the liver, independently of insulin. Current knowledge on the molecular actions of ChREBP is limited. As the name suggests, it was first identified by its ability to bind the carbohydrate-response element of the gene encoding liver pyruvate kinase (L-PK). L-PK catalyses the conversion of phosphoenolpyruvate to pyruvate, which enters the Krebs cycle to generate citrate, the principal source of acetyl-CoA used for fatty acid synthesis.52 ChREBP has recently been shown to play a pivotal role in the control of lipogenesis through the transcriptional regulation of lipogenic genes, including ACC and FAS. ChREBP binds to its functional heterodimeric partner, Max-like protein X, and induces the transcription of lipogenic and glycolytic genes containing a carbohydrate response element, such as those encoding ACC, FAS and L-PK.53 Nuclear transcription fator-κB (NF-κB) Recent experimental and clinical evidence suggest that inflammation is an aggravating factor in lipid-mediated tissue injury. A low-level, chronic inflammatory state may induce insulin resistance and endothelial dysfunction and thus link the latter phenomena with obesity, fatty liver, cardiovascular disease. The inflammatory marker C-reactive protein (CRP) is elevated concentrations in metabolic syndrome,54 and prospective association with the development type 2 diabetes and fatty liver.55 We have also demonstrated in HepG2 cells that inflammatory cytokines disrupted LDL receptor feedback regulation via increasing SREBP mRNA level, allowing unregulated uptake of cholesterol in liver cells which causes intracellular accumulation of lipid (published in this issue). Our studies provide a potential mechanism for abnormal intracellular cholesterol level that contributes to changes in hepatocytes cholesterol homeostasis during inflammatory states. NF-κB is a transcription factor that induces inflammatory cytokines and anti-apoptotic proteins. Apoptosis is recognized as common in liver injury and may also contribute to tissue inflammation, fibrogenesis, and development of cirrhosis. Liver injury in NASH/ASH is associated with increased hepatocyte apoptosis mediated by death receptors. Further, apoptosis, degrees of inflammation and fibrosis correlated with active NF-kB expression. Patients with ASH showed a remarkable expression of active NF-κB.56 Methionine and choline deficient (MCD)-fed mice developed steatohepatitis accompanied by dramatic accumulation of hepatic lipoperoxides, activation of NF-κB and induction of pro-inflammatory ICAM-1, COX-2, MCP-1 and CINC mRNA. The NF-κB pathway is one among several possible signalling pathways by which inflammation is recruited in experimental steatohepatitis.57 Nuclear transcription factors cross-talks in regulation of lipid metabolism LXR have been identified as a dominant activator of SREBP-1c promoter.58,59 The promoter of the SREBP-1c gene contains a regulatory element for LXRα, which strongly induces its transcription. In turn, activated SREBP-1c stimulates the transcription of genes involved in de novo lipogenesis, such as ACC and FAS, and interacts with regulatory elements in the promoters of various insulin-regulated genes.60 In mice lacking the LXR genes, the basal expression level of liver SREBP-1c mRNA is significantly reduced. In addition, feeding diets containing high cholesterol, rexinoids or LXR agonists results in an increase in SREBP-1c mRNA and protein expression in wild-type mice, but not in LXRα/β−/−. mice. These studies establish mouse SREBP-1c as a target gene of the oxysterol receptor, LXR, and suggest a novel convergence of homeostatic mechanisms for cholesterol and fatty acid metabolism, implicating a new link between cholesterol and fatty acid metabolism. SREBP-1c has also been is inhibited by activation of AMPK, a major cellular regulator of lipid and glucose metabolism. Fatty acid metabolism in liver is transcriptionally regulated by two reciprocal systems: PPARαcontrols fatty acid degradation, whereas SREBP-1c activated by LXR regulates fatty acid synthesis. The roles of these transcription factors in whole body physiology and metabolism can be best illustrated by comparing two opposite nutritional states: fasted and refed states. In the fasted liver, fatty acids are oxidized to acetyl-CoA and subsequently to ketone bodies. PPARα, plays a major role in both processes, which was confirmed by observations in PPARα-null mice.61,62 In contrast, expression of SREBP-1c is reduced during fasting. In the refed state, lipogenesis is induced through increased amount of SREBP-1, whereas PPARα is decreased. This coordinated reciprocal regulation of the two transcription factors is a key to nutritional regulation of fatty acids and triglycerides as energy storage system and implicates the presence of a cross-talk between these factors. In addition, the reciprocal modulation of thyroid hormone and peroxisome proliferator-responsive genes through cross-talk between thyroid hormone receptors (TRs) and PPARs has been demonstrated.63,64 In addition, LXRα expression was shown to be regulated by PPAR.65-67 PPAR enhances cholesterol efflux in macrophages via PPAR-LXRα-ABCA1 pathway, and a combination treatment with both PPAR and LXR agonists result in a further induction in ABCA1 mRNA in macrophages65,66 Furthermore, PPARs activation can suppress LXR-SREBP-1c pathway through reduction of LXR/RXR formation, proposing a novel transcription factor cross-talk between LXR and PPARα in hepatic lipid homeostasis. PPARα suppress SREBP-1c promoter activity through LXR response elements (LXREs) and inhibit LXRα/RXR binding to LXRE of SREBP-1c promoter.68 In contrast, overexpression of LXRα or βsuppressed PPARα-induced peroxisome proliferator response element (PPRE) activity in a dose-dependent manner. LXRs suppress lipid degradation gene promoters through inhibition of binding of PPARα/RXRα to PPRE.69 Fatty liver is highly sensitive to inflammatory activation. NF-κB is a primary mediator of inflammation in experimental steatohepatitis, but PPAR have anti-inflammatory effects and regulate lipid metabolism in the fatty liver. In the MCD dietary model of steatohepatitis, NF-κB is activated early and is an important pro-inflammatory mediator of lesion development.70 Thus, an increase of NF-κB-mediated pro-inflammatory signals or decrease of PPAR-mediated anti-inflammatory mechanism may promote inflammation, thereby promote fatty liver formation. On the other hand, some of the proinflammatory cytokines generated through NF-kB activation also produce adaptor proteins which are involved in generating survival factors which might help liver regeneration and recovery. Taken together, the presence of an intricate network of nutritional transcription factors with mutual interactions, results in efficient reciprocal regulation of lipid degradation and lipogenesis. Cross-talk of these nuclear factors could play crucial roles in lipid metabolism. However, as recent studies show that there could be a global and complex network of nutritional transcriptional factors, further studies are needed to evaluate the physiological relevance of the cross-talk between these and other nuclear transcription factors to harvest the net beneficial effects.