Lxralpha deficiency hampers the hepatic adaptive response to fasting in mice

Besides its well established role in control of cellular cholesterol homeostasis, the liver X receptor (LXR) has been implicated in the regulation of hepatic gluconeogenesis. We investigated the role of the major hepatic LXR isoform in hepatic glucose metabolism during the feeding-to-fasting transition in vivo. In addition, we explored hepatic glucose sensing by LXR during carbohydrate refeeding. Lxralpha(-/-) mice and their wild-type littermates were subjected to a fasting-refeeding protocol and hepatic carbohydrate fluxes as well as whole body insulin sensitivity were determined in vivo by stable isotope procedures. Lxralpha(-/-) mice showed an impaired response to fasting in terms of hepatic glycogen depletion and triglyceride accumulation. Hepatic glucose 6-phosphate turnover was reduced in 9-h fasted Lxralpha(-/-) mice as compared with controls. Although hepatic gluconeogenic gene expression was increased in 9-h fasted Lxralpha(-/-) mice compared with wild-type controls, the actual gluconeogenic flux was not affected by Lxralpha deficiency. Hepatic and peripheral insulin sensitivity were similar in Lxralpha(-/-) and wild-type mice. Compared with wild-type controls, the induction of hepatic lipogenic gene expression was blunted in carbohydrate-refed Lxralpha(-/-) mice, which was associated with lower plasma triglyceride concentrations. Yet, expression of "classic" LXR target genes Abca1, Abcg5, and Abcg8 was not affected by Lxralpha deficiency in carbohydrate-refed mice. In summary, these studies identify LXRalpha as a physiologically relevant mediator of the hepatic response to fasting. However, the data do not support a role for LXR in hepatic glucose sensing.     

The phospholipid transfer protein gene is a liver X receptor target expressed by macrophages in atherosclerotic lesions

The liver X receptors (LXRs) are members of the nuclear receptor superfamily that are activated by oxysterols. In response to ligand binding, LXRs regulate a variety of genes involved in the catabolism, transport, and uptake of cholesterol and its metabolites. Here we demonstrate that LXRs also regulate plasma lipoprotein metabolism through control of the phospholipid transfer protein (PLTP) gene. LXR ligands induce the expression of PLTP in cultured HepG2 cells and mouse liver in vivo in a coordinate manner with known LXR target genes. Moreover, plasma phospholipid transfer activity is increased in mice treated with the synthetic LXR ligand GW3965. Unexpectedly, PLTP expression was also highly inducible by LXR in macrophages, a cell type not previously recognized to express this enzyme. The ability of synthetic and oxysterol ligands to regulate PLTP mRNA in macrophages and liver is lost in animals lacking both LXRalpha and LXRbeta, confirming the critical role of these receptors. We further demonstrate that the PLTP promoter contains a high-affinity LXR response element that is bound by LXR/RXR heterodimers in vitro and is activated by LXR/RXR in transient-transfection studies. Finally, immunohistochemistry studies reveal that PLTP is highly expressed by macrophages within human atherosclerotic lesions, suggesting a potential role for this enzyme in lipid-loaded macrophages. These studies outline a novel pathway whereby LXR and its ligands may modulate lipoprotein metabolism.     

LXR is crucial in lipid metabolism

Liver X receptors (LXRalpha and LXRbeta) are members of the nuclear receptor superfamily and are activated by oxysterols and intermediates in the cholesterol synthetic pathway. The pivotal role of LXRs in the metabolic conversion of cholesterol to bile acids is well established. Analysis of gene expression in LXRalpha and LXRbeta deficient mice have confirmed that LXR regulates a number of target genes involved in both cholesterol and fatty acid metabolism in liver, macrophages and intestine. The observation that LXRalpha is responsive to fatty acids and is expressed in metabolic tissues suggests that it also plays a general role in lipid metabolism. Adipose tissue is the main storage site for fat in the body and plays a crucial role in overall lipid handling. Both LXRalpha and LXRbeta are expressed and activated by endogenous and synthetic ligands, which lead to lipid accumulation into adipocytes. This indicates an important regulatory role of LXR in several metabolic signaling pathways in the adipose tissue, such as glucose uptake and de novo fatty acid synthesis. Here, we review recent studies that provide new insights into the mechanisms by which LXRs act to influence fatty acid synthesis in liver and adipose tissue.     

On the role of liver X receptors in lipid accumulation in adipocytes

The pivotal role of liver X receptors (LXRs) in the metabolic conversion of cholesterol to bile acids in mice is well established. More recently, the LXRalpha promoter has been shown to be under tight regulation by peroxisome proliferator-activated receptors (PPARs), implying a role for LXRalpha in mediating the interplay between cholesterol and fatty acid metabolism. We have studied the role of LXR in fat cells and demonstrate that LXR is regulated during adipogenesis and augments fat accumulation in mature adipocytes. LXRalpha expression in murine 3T3-L1 adipocytes as well as in human adipocytes was up-regulated in response to PPARgamma agonists. Administration of a PPARgamma agonist to obese Zucker rats also led to increased LXRalpha mRNA expression in adipose tissue in vivo. LXR agonist treatment of differentiating adipocytes led to increased lipid accumulation. An increase of the expression of the LXR target genes, sterol regulatory binding protein-1 and fatty acid synthase, was observed both in vivo and in vitro after treatment with LXR agonists for 24 h. Finally, we demonstrate that fat depots in LXRalpha/beta-deficient mice are smaller than in age-matched wild-type littermates. These findings imply a role for LXR in controlling lipid storage capacity in mature adipocytes and point to an intriguing physiological interplay between LXR and PPARgamma in controlling pathways in lipid handling.     

The LXR ligand T0901317 induces severe lipogenesis in the db/db diabetic mouse

Liver X receptor (LXR) ligands are currently being evaluated as potential therapeutic agents for the treatment of low HDL. The LXR ligand T0901317 elevates ATP binding cassette transporter A1 (ABCA1) and HDL levels in animal models and induces moderate lipogenesis through upregulation of sterol regulatory element binding protein 1c (SREBP1c). Because insulin may also regulate lipogenesis through SREBP1c and fatty acid synthase (FAS), we investigated the effect of an LXR ligand in hyperinsulinemic mice. Administration of T0901317 to male db/db mice for 12 days resulted in a more severe hypertriacylglycerolemia and hepatic triacylglycerol accumulation than observed in nondiabetic mice. The LXR target genes ABCA1, SREBP1c, FAS, and stearoyl-CoA desaturase 1 were upregulated by T0901317 treatment in both diabetic db/db and nondiabetic C57BLKS mice. Changes in lipogenic gene expression were independent of mouse strain, indicating that the severe lipogenesis observed in LXR ligand-treated db/db mice was not due to additive effects of insulin on lipogenic gene expression. Phosphoenolpyruvate carboxykinase expression was suppressed, suggesting that a shift from gluconeogenesis toward lipogenesis could partially explain our observations in db/db mice. Our data suggest that LXR ligands that have effects on both fatty acid and carbohydrate metabolism should be carefully evaluated in obesity, insulin, and leptin resistance.     

C1q/TNF-related protein-3 (CTRP3), a novel adipokine that regulates hepatic glucose output

Adipose tissue-derived adipokines play important roles in controlling systemic insulin sensitivity and energy balance. Our recent efforts to identify novel metabolic mediators produced by adipose tissue have led to the discovery of a highly conserved family of secreted proteins, designated as C1q/TNF-related proteins 1-10 (CTRP1 to -10). However, physiological functions regulated by CTRPs are largely unknown. Here we provide the first in vivo functional characterization of CTRP3. We show that circulating levels of CTRP3 are inversely correlated with leptin levels; CTRP3 increases with fasting, decreases in diet-induced obese mice with high leptin levels, and increases in leptin-deficient ob/ob mice. A modest 3-fold elevation of plasma CTRP3 levels by recombinant protein administration is sufficient to lower glucose levels in normal and insulin-resistant ob/ob mice, without altering insulin or adiponectin levels. The glucose-lowering effect in mice is linked to activation of the Akt signaling pathway in liver and a marked suppression of hepatic gluconeogenic gene expression. Consistent with its effects in mice, CTRP3 acts directly and independently of insulin to regulate gluconeogenesis in cultured hepatocytes. In humans, alternative splicing generates two circulating CTRP3 isoforms differing in size and glycosylation pattern. The two human proteins form hetero-oligomers, an association that does not require interdisulfide bond formation and appears to protect the longer isoform from proteolytic cleavage. Recombinant human CTRP3 also reduces glucose output in hepatocytes by suppressing gluconeogenic enzyme expression. This study provides the first functional evidence linking CTRP3 to hepatic glucose metabolism and establishes CTRP3 as a novel adipokine.     

Suppression of hepatic glucose production by human neutrophil alpha-defensins through a signaling pathway distinct from insulin

In this study, we tested the hypothesis that human neutrophil alpha-defensins (HNPs) inhibit hepatic glucose production through a signaling pathway distinct from insulin. The effect of HNP-1 on fasting blood glucose levels and the expression of hepatic gluconeogenic genes was first examined. Using hyperinsulinemic-euglycemic clamps, we determined the effect of HNP-1 on endogenous glucose production, hepatic expression of key gluconeogenic genes and glucose uptake in skeletal muscle in Zucker diabetic fatty rats. In isolated primary hepatocytes, we studied the effect of HNP-1 and -2 on glucose production, expression of gluconeogenic genes, and phosphorylation of Akt, c-Src, and FoxO1. Our results show that HNP-1 reduced blood glucose levels of both normal mice and Zucker diabetic fatty rats predominantly through suppression of hepatic glucose production. HNPs inhibited glycogenolysis and gluconeogenesis in isolated hepatocytes. HNPs also suppressed expression of key gluconeogenic genes including phosphoenoylpyruvate carboxyl kinase and glucose-6-phosphatase. To investigate the mechanism, we found that HNPs stimulated phosphorylation of Akt and FoxO1 without activating IRS1. Nevertheless, HNPs activated c-Src. Blockade of c-Src activity with either a chemical inhibitor PP2 or an alternative inhibitor CSK prevented the inhibitory effect of HNPs on gluconeogenesis. Together, our results support the hypothesis that HNPs can suppress hepatic glucose production through an intracellular mechanism distinct from the classical insulin signaling pathway.     

Induction of intestinal ATP-binding cassette transporters by a phytosterol-derived liver X receptor agonist

The nuclear receptors liver X receptor (LXR) alpha and LXRbeta serve as oxysterol receptors and regulate the expression of genes involved in lipid metabolism. LXR activation induces the expression of ATP-binding cassette (ABC) transporters, such as ABCG5 and ABCG8, which inhibit intestinal absorption of cholesterol and phytosterols. Although several synthetic LXR agonists have been generated, these compounds have limited clinical application, because they cause hypertriglycemia by inducing the expression of lipogenic genes in the liver. We synthesized derivatives of phytosterols and found some of them to act as LXR agonists. Among them, YT-32 [(22E)-ergost-22-ene-1alpha,3beta-diol], which is related to ergosterol and brassicasterol, is the most potent LXR agonist. YT-32 directly bound to LXRalpha and LXRbeta and induced the interaction of LXRalpha with cofactors, such as steroid receptor coactivator-1, as effectively as the natural ligands, 22(R)-hydroxycholesterol and 24(S),25-epoxycholesterol. Although the nonsteroidal synthetic LXR agonist T0901317 induced the expression of intestinal ABC transporters and liver lipogenic genes, oral administration of YT-32 selectively activated intestinal ABC transporters in mice. Unlike T0901317 treatment, YT-32 inhibited intestinal cholesterol absorption without increasing plasma triglyceride levels. The phytosterol-derived LXR agonist YT-32 might selectively modulate intestinal cholesterol metabolism.     

The human myeloperoxidase gene is regulated by LXR and PPARalpha ligands

Myeloperoxidase (MPO) is an oxidant-generating enzyme expressed in macrophages and implicated in atherosclerosis and cholesterol homeostasis. LXRalpha and PPARalpha regulate genes involved in cholesterol metabolism and the inflammatory response in macrophages. Here, we examine the effect of LXR and PPARalpha ligands on MPO expression. LXR and PPARalpha, as heterodimers with RXR, are shown to bind overlapping sites in an Alu receptor response element (AluRRE) in the MPO promoter. The LXR ligand T0901317 suppresses MPO mRNA expression in primary human macrophages, and in bone marrow cells and macrophages from huMPO transgenic mice. The PPARalpha ligand GW9578 downregulates MPO expression in GMCSF-macrophages, while upregulating in MCSF-macrophages. In contrast, the mouse MPO gene, which lacks the primate-specific AluRRE, is not regulated by LXR or PPARalpha ligands. These findings identify human MPO as a novel LXR and PPARalpha target gene, consistent with the role of these receptors in regulation of proinflammatory genes in macrophages.     

Activation of liver X receptors prevents statin-induced death of 3T3-L1 preadipocytes

The biological functions of liver X receptors (LXRs) alpha and beta have primarily been linked to pathways involved in fatty acid and cholesterol homeostasis. Here we report a novel role of LXR activation in protecting cells from statin-induced death. When 3T3-L1 preadipocytes were induced to differentiate by standard isobutylmethylxanthine/dexamethasone/insulin treatment in the presence of statins, they failed to differentiate and underwent massive apoptosis. The simultaneous addition of selective LXR agonists prevented the statin-induced apoptosis. By using mouse embryo fibroblasts from wild-type (LXRalpha+/+/LXRbeta+/+), LXRalpha knock-out mice (LXRalpha(-/-)/LXRbeta+/+), LXRbeta knock-out mice (LXRalpha+/-/LXRbeta(-/-)), and LXR double knock-out mice (LXRalpha(-/-)/LXRbeta(-/-)) as well as 3T3-L1 cells transduced with retroviruses expressing either wild-type LXRalpha or a dominant negative version of LXRalpha, we demonstrate that the response to LXR agonists is LXR-dependent. Interestingly, LXR-mediated rescue of statin-induced apoptosis was not related to up-regulation of genes previously shown to be involved in the antiapoptotic action of LXR. Furthermore, forced expression of Bcl-2 did not prevent statin-induced apoptosis; nor did LXR action depend on protein kinase B, whose activation by insulin was impaired in statin-treated cells. Rather, LXR-dependent rescue of statin-induced apoptosis in 3T3-L1 preadipocytes required NF-kappaB activity, since expression of a dominant negative version of IkappaBalpha prevented LXR agonist-dependent rescue of statin-induced apoptosis. Thus, the results presented in this paper provide novel insight into the action of statins on and LXR-dependent inhibition of apoptosis.