Liver X Receptors as Insulin-mediating Factors in Fatty Acid and Cholesterol Biosynthesis*

The nuclear receptor liver X receptor (LXR) α, an important regulator of cholesterol and bile acid metabolism, was analyzed after insulin stimulation in liver in vitro andin vivo. A time- and dose-dependent increase in LXRα steady-state mRNA level was seen after insulin stimulation of primary rat hepatocytes in culture. A maximal induction of 10-fold was obtained when hepatocytes were exposed to 400 nminsulin for 24 h. Cycloheximide, a potent inhibitor of protein synthesis, prevented induction of LXRα mRNA expression by insulin, indicating that the induction is dependent on de novo synthesis of proteins. Stabilization studies using actinomycin D indicated that insulin stimulation increased the half-life of LXRα transcripts in cultured primary hepatocytes. Complementary studies where rats and mice were injected with insulin induced LXRα mRNA levels and confirmed our in vitrostudies. Furthermore, deletion of both the LXRα and LXRβ genes (double knockout) in mice markedly suppressed insulin-mediated induction of an entire class of enzymes involved in both fatty acid and cholesterol metabolism. The discovery of insulin regulation of LXR in hepatic tissue as well as gene targeting studies in mice provide strong evidence that LXRs plays a central role not only in cholesterol homeostasis, but also in fatty acid metabolism. Furthermore, LXRs appear to be important insulin-mediating factors in regulation of lipogenesis.

Insulin plays a major role in the regulation of carbohydrate and lipid metabolism in the liver, adipose tissue, and muscle. Hepatic fatty acid oxidation, lipogenesis, and glycerolipid synthesis are subject to regulation by insulin (for review, see Ref. 1). Control of lipid synthesis is especially important in the liver, which synthesizes lipids from glucose as precursor, for its own uses but also for export into plasma as lipoproteins. Hepatic fatty acid synthesis is elevated when plasma insulin rises, as in states of obesity and non-insulin-dependent diabetes mellitus. The fatty acids are exported from the liver in lipoproteins and reach extrahepatic organs in which they are either utilized or stored.
The factors mediating the insulin regulation of lipid metabolism have for a long time been unknown, but recently several reports have identified sterol regulatory element-binding protein-1c (SREBP-1c) 1 as a necessary transcription factor activating fatty acid synthesis in response to insulin (2). SREBP-1c activates transcription of the major genes of fatty acid synthesis including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase-1 (3)(4)(5).
The liver X receptors (LXRs) belong to a subclass of nuclear hormone receptors that form obligate heterodimers with retinoid X receptors and are bound and activated by oxysterols. During recent years the LXRs have been proposed to act as sterol sensors that function to help the organism adapt so it can cope with the effects of high free cholesterol levels in blood (6 -8). LXR␣ has a relatively restricted expression pattern (liver, kidney, intestine, adipose tissue, and adrenals) (also termed NR1H3 per the Nuclear Receptors Nomenclature Committee) (9,10), whereas LXR␤ is ubiquitously expressed (NR1H2) (9,(11)(12)(13)(14). LXR␣ and LXR␤ share a high degree of amino acid similarity (78%) and have thus been proposed to be paralogues (15).
The first responsive element for LXR was found in the promoter of cholesterol 7␣-hydroxylase (Cyp7a), an essential enzyme constituting the initial and rate-limiting step in the conversion of cholesterol to bile acids (16). Consistent with these in vitro observations, LXR␣ knockout mice lose the capacity to regulate catabolism of excess dietary cholesterol in liver resulting in a rapid accumulation of hepatic cholesteryl esters that eventually leads to liver failure (17). This effect cannot be compensated for by the related isoform, LXR␤ (17,18). Additional LXR target genes involved in lipid metabolism include the human cholesteryl ester transfer protein, which translocates cholesteryl esters between lipoprotein fractions (19), and the ATP-binding cassette transporters, ABC1 and ABC8, which are implicated in the efflux of cellular free cholesterol (20 -22). In addition, SREBP-1c is regulated by LXRs and has LXR regulatory elements in its promoter (23,24).
In view of the important cross-regulation between hormonal signaling pathways and lipid homeostasis, the principal aim of this study was to analyze whether LXR␣ expression is under the control of insulin. We show that, indeed, insulin regulates LXR␣ in primary cultures of hepatocytes as well as in vivo in liver of rodents injected with insulin. Furthermore, we show that LXRs are necessary for insulin regulation of several lipogenic enzymes, indicating a key role for LXR in mediating insulin effects on lipid metabolism.
Animals-All animal use was approved and registered by the Norwegian Animal Research Authority in Norway and the regional ethical committee for animal experiments in Sweden, respectively. Male Wistar or Sprague-Dawley rats (B & K Universal Ltd, Norway) of ϳ200 -250 g and mice were kept in cages at a constant temperature (22°C) with a fixed 12-h light/dark cycle with free access to water and a standard low fat diet. Rats were given 2 units of Actrapid insulin (subcutaneous) and 2 units of Insulatard insulin (intraperitoneal), whereas mice were given a single 0.2-unit injection (intraperitoneal) of Actrapid insulin (insulin from Novo Nordisk, Bagsvaerd, Denmark). Control animals received vehicle (phosphate-buffered saline (PBS)). All animals were killed 90 min after the insulin injection.
Wild type or LXR␣/␤ Ϫ/Ϫ (double knockout) mice on a mixed genetic background based on C57BL/6 and SV129 strains were used. 2 Rats were anesthetized using Hypnorm Dormicum, and blood was collected from the abdominal aorta, whereas blood from the mice was collected by cardiac puncture under light methoxy fluorane anesthesia. Livers were rapidly frozen in liquid nitrogen and stored at Ϫ70°C until isolation of RNA. The blood was collected in vials containing EDTA, and serum was collected by centrifugation. Plasma insulin was analyzed using a rat insulin radioimmunoassay kit (RI-13K, Linco, St. Louis, MO), and triglycerides and glucose were analyzed spectrophotometrically on the Cobas Mira plus (Hoffman-La Roche, Basel, Switzerland) using Calibrator Human (07 3718 6, Roche, Basel, Switzerland) as calibrant. For triglycerides the enzymatic kit "Triglycerides/Glycerol Blanking" (450032, Roche Molecular Biochemicals) was used, and for glucose "Glucose HK" (07 3672 4, Roche).
Cell Culture-Hepatocytes were isolated using the method of Berry and Friend (25) with modifications according to Seglen (26). The culture conditions were as described previously (27). Insulin was added as described in legends to figures, and 2.5 g/ml actinomycin D was used.
RNA Extraction and Northern Blot Analysis-Total RNA from cultured hepatocytes was extracted by the guanidinium thiocyanate method (28), whereas total RNA from liver tissue was extracted by TRIzol reagent for total RNA extraction (Invitrogen). Northern blot analysis of RNA was essentially performed as described previously (29). The cDNAs for rat LXR␣ (11) and human ribosomal protein L27 (ATCC catalog no. 107385) were used as probes in the hybridizations. Hybridization with the ribosomal protein L27 or ethidium bromide staining of the agarose gel was used as a control to show that the treatments did not cause a general alteration in gene expression. The remaining cDNA probes were generated by reverse transcription-PCR with mouse liver RNA as described previously (18) or using the following primers: malic enzyme (ME), 5Ј-ATGACGCCTTCCTGGATGAGTT-3Ј and 5Ј-TTTGC-TGGTCGGATTGCTCA-3Ј; glucokinase (GK), 5Ј-TGGCCCAGTGAAAT-CCAGGT-3Ј and 5Ј-TGGGAGGGTTCATCCCAGAA-3Ј; and Spot-14 (S14), 5Ј-GTCCTGTCAATCTGCTGTCTGCTCAA-3Ј and 5Ј-CTTCCAT-AGAGTCGAAGACCTACAGG-3Ј. The [␣-32 P]dCTP-labeled cDNA probes were prepared using a standard Multiprime DNA-labeling kit (Amersham Biosciences, Inc., RPN 1601Y). Specific activities from 2 to 6 ϫ 10 8 cpm/g DNA were obtained. The sizes of the mRNA transcripts were calculated on the basis of the migration of the 18 and 28 S rRNA, which were visualized by ethidium bromide. Semiquantitative results were obtained from scanning of autoradiograms using XRS 3sc scanner and the Bio Image System from Millipore, showing linear increments within the working range used (5-30 g of RNA), or by a Phosphor-Imager (Molecular Dynamics, Amersham Biosciences, Inc.). After autoradiography the RNA filters were washed using 50% formamide and 10 mM sodium phosphate, pH 6.5, for 1 h at 65°C and reprobed.
Immunoblotting-Cultured primary hepatocytes were lysed in 0.1% Triton X-100 in PBS including protease inhibitors (Complete, Roche Molecular Biochemicals). Liver tissue protein lysate was prepared by homogenization of 100 mg tissue in PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and the same protease inhibitor as above. Total protein lysates were obtained after centrifugation, and protein concentration was determined with the Bio-Rad colorimetric assay system. Proteins (150 g) was separated in a 10% SDS-polyacrylamide gel, and transferred to nitrocellulose filters (Hybond-C Extra, Amersham Biosciences, Inc.). The LXR␣ protein was immunochemically detected using commercially available antibodies (SC-1206, Santa Cruz), and signal detection was achieved using ECL chemiluminescence (Amersham Biosciences, Inc.) according to the manufacturer's instructions.
Densitometry and Statistics-Semiquantitative results were obtained by scanning of autoradiograms using an XRS 3sc scanner and the Bio Image System from Millipore, or by a PhosphorImager (Molecular Dynamics, Amersham Biosciences, Inc.). For statistical analysis of the mRNA results, the mean control values were set to unity and variation within the group calculated accordingly. Corresponding relative values (expressed as mean Ϯ S.E.) were calculated for the experimental groups.

Effects of Insulin on LXR␣ mRNA Expression in Hepatocytes
in Culture-A dose-dependent increase in LXR␣ mRNA expression was seen after 24 h insulin stimulation of primary rat hepatocytes in culture. The maximum increase (10-fold) was obtained with 100 nM insulin (Fig. 1A). The induction of LXR␣ mRNA levels became evident as early as 6 h after addition of insulin, with maximal levels obtained after 12 h (Fig. 1B). Insulin also induced LXR␣ protein levels; this induction was first evident 24 h after addition of hormone (Fig. 1C).
We have previously shown that maximal effects of insulin on gene expression were obtained with hormone concentrations above 250 nM. Because 400 nM insulin did not result in reduced cell viability, as judged from the trypan blue exclusion test (0.4% trypan blue), this concentration was used in the subsequent experiments (27, 29 -31).
In supplementing experiments we have also studied the effect of the synthetic glucocorticoid dexamethasone on LXR␣ mRNA expression. Dexamethasone had a minor effect on LXR␣ mRNA expression, but antagonized the effect of insulin (data not shown). We have also studied the effect of insulin on LXR␣ mRNA expression under serum-free conditions and we observed an effect of insulin similar to that we observed under the serum conditions used in this work (data not shown).
Effect of the Protein Synthesis Inhibitor Cycloheximide on LXR␣ Steady-state mRNA Expression-To investigate whether the insulin induction of LXR␣ mRNA was dependent on ongoing protein synthesis, primary hepatocytes were treated with the translation inhibitor cycloheximide, prior to insulin treatment (Fig. 2). Treatment of cultured hepatocytes with cycloheximide for 2 h almost abolished LXR␣ mRNA expression, showing that LXR␣ mRNA synthesis is indeed dependent on de novo protein synthesis and indicating a short turnover time of this protein(s). Insulin, coincubated for 6 and 12 h, was incapable of reestablishing or inducing LXR␣ mRNA. These results suggests that LXR␣ mRNA transcription requires de novo protein synthesis, and that insulin might exert its effect, at least partly, through stabilization of LXR␣ mRNA.
Effect of the Transcription Inhibitor Actinomycin D on LXR␣ Steady-state mRNA Expression-To further define the mechanism underlying the elevated LXR␣ steady-state mRNA level observed after insulin administration, we tested whether insulin treatment affected the stability of LXR␣ mRNAs by using the RNA polymerase II inhibitor, actinomycin D. Primary hepatocytes were treated with 400 nM insulin for 24 h before addition of actinomycin D (2.5 g/ml), and cells were then harvested at different time points within a period of 12 h. At 6 h, actinomycin D increased the LXR␣ mRNA level by itself, as also observed for other genes (30). Insulin added together with actinomycin D induced LXR␣ mRNA in an additive or synergistic manner at both 6 and 12 h (Fig. 3A), indicating that the increased LXR␣ mRNA level following addition of insulin is not dependent on continuous transcription of the LXR␣ gene. Thus, at least part of the stimulatory effect of insulin on LXR␣ mRNA could be caused by stabilization of the transcript. The absolute values of the LXR␣ mRNA transcripts were plotted in a time curve, and the half-lives of the transcripts were estimated by extrapolation of the linear part of the mRNA time curve (Fig.  3B). The LXR␣ mRNA half-life increased from 6 to 9 h after treatment with insulin.
Effect of Insulin Injections in Sprague-Dawley Rats-Next, we investigated whether the LXR␣ gene is regulated by insulin in a physiological setting by giving rats injections of insulin. Sprague-Dawley rats were given 2 units of Actrapid insulin (intraperitoneal) and 2 units of Insulatard insulin (subcutaneous) (insulin from Novo Nordisk). Control animals were given vehicle (PBS). Table I shows plasma values of triglycerides, glucose, and insulin after 90 min of insulin administration. There were no variations in triglyceride levels in the two groups, but the plasma concentration of insulin was 20 times higher in the insulin-treated than in the control group, and, accordingly, the glucose level in the insulin group decreased to ϳ30% of that in the control group. After 90 min of insulin stimulation, liver LXR␣ mRNA was increased by 1.7-fold. In comparison, another known insulin-responsive gene, SREBP1-c (3,5,32), was increased by 1.9-fold (Fig. 4). The cDNA probe used recognizes both the SREBP-1c and SREBP-1a isoforms. In rodent liver, however, the expression of SREBP-1c predominates over that of SREBP-1a by a ratio of 9:1 (33). We have therefore concluded that the signal obtained in Northern blots was representative of SREBP-1c expression.
Effect of Insulin Injections in LXR␣/␤ Double Knockout (LXR␣/␤ Ϫ/Ϫ) Mice-A mouse line where both the LXR␣ and the LXR␤ genes have been deleted (LXR␣/␤ Ϫ/Ϫ) mice) 2 was used to investigate whether LXRs were involved in insulin signaling in lipid metabolism. LXR␣/␤ Ϫ/Ϫ mice and corresponding wild type mice were given one injection of insulin (0.2 units of Actrapid insulin, Novo Nordisk); 90 min later blood was collected and livers excised for mRNA analysis. Table II shows plasma values of triglycerides, glucose, and insulin after 90 min of insulin administration. Interestingly, the basal levels of triglycerides, glucose, and insulin were lower in LXR␣/␤ double knockout mice than in wild type animals. Following insulin administration, triglyceride levels in plasma did not change either in wild type or in LXR␣/␤ Ϫ/Ϫ mice, as also seen in rats (Table I). As expected, both wild type and LXR␣/␤ Ϫ/Ϫ mice had a higher level of plasma insulin after insulin treatment (2.7-and 12-fold, respectively), and, in line with this, the concentration of plasma glucose values in both normal and knockout animals was lower after insulin treatment (Table II).
As shown earlier, we detected a truncated LXR␣ transcript in LXR␣/␤ Ϫ/Ϫ mice, corresponding to a deletion of exons 4 and FIG. 1. Effects of insulin on LXR␣ mRNA expression and protein in cultured rat hepatocytes. The steady-state mRNA level of LXR␣ was measured using Northern analysis (see "Experimental Procedures") of total RNA (20 g) from control cells and after treatment with 400 nM insulin for 24 h. A, dose-response curve showing results from treatment of hepatocytes with 0, 10, 100, and 400 nM insulin for 24 h. Average result from three independent experiments is given relative to control. B, the time-response curve was obtained for insulin after treatment (400 nM) for up to 24 h, presented as an average result from three separate experiments. The values are presented relative to control (control ϭ 1) and given as the mean Ϯ S.E. C, immunoblotting was performed using protein lysates from primary hepatocytes treated with 400 nM insulin for 24 h.

FIG. 2. Effects of cycloheximide on insulin induction of LXR␣ mRNA.
Hepatocytes were pre-treated with cycloheximide (CHX, 5 g/ ml) for 2 h, and during 6 or 12 h together with insulin (400 nM) in comparison to control cells (C) and cells treated with insulin only. The LXR␣ steady-state mRNA levels were measured using Northern analysis (see "Experimental Procedures"). The ethidium bromide staining of the ribosomal RNA, 18 S rRNA, is shown to verify equal loading on the gel. 5 (Fig. 5, indicated by an arrow) (18). After 90 min of insulin administration, the LXR␣ mRNA level in liver was increased by 1.8-fold in wild type mice compared with mice given vehicle only (Fig. 5, Table III). Furthermore, SREBP-1c mRNA was strongly induced (4.5-fold) following insulin administration. Remarkably, these inductions following insulin administration did not occur in LXR␣/␤ Ϫ/Ϫ mice. This strongly implicates LXRs as intermediary components in at least parts of the insulin signaling pathways; however, the intact glucose lowering effect of insulin in LXR␣/␤ Ϫ/Ϫ mice (Table III) probably reflects that some insulin signaling pathway(s) are LXR-independent.
In light of the several reports on the importance of SREBP-1c on transcriptional regulation of hepatic lipogenic enzymes, and the fact that SREBP-1c is an LXR target gene, we wanted to investigate whether insulin induction of lipogenic enzymes was affected in LXR␣/␤ Ϫ/Ϫ mice. Fig. 5 shows Northern blots for enzymes involved in fatty acid synthesis (ACC, FAS), cholesterol synthesis (HMG-CoA synthase, HMG-CoA reductase, squalene synthase, farnesyl pyrophosphate synthase), and other branches of the lipogenic program (GK, ME, S14). As seen in Fig. 5, and as expected, several known insulin regulated genes in normal mice were induced by the hormone (FAS, ACC, GK, ME, S14, HMG-CoA reductase, and squalene synthase), but as seen for SREBP-1c, this induction by insulin was abolished or significantly impaired in LXR␣/␤ Ϫ/Ϫ mice. HMG-CoA synthase and farnesyl pyrophosphate synthase were not in-duced by insulin. Interestingly, the basal mRNA level for genes involved in fatty acid synthesis (FAS, ME) was repressed in LXR␣/␤ Ϫ/Ϫ mice, whereas expression of cholesterogenic enzyme genes (HMG-CoA synthase and HMG-CoA reductase) was increased in LXR␣/␤ Ϫ/Ϫ mice as compared with wild type mice (Fig. 5, Table III). DISCUSSION This study clearly demonstrates that insulin induces LXR␣ mRNA levels in liver, leading to an increase in the steadystate mRNA level of LXR target genes, such as SREBP-1c as well as many genes encoding enzymes in fatty acid and cholesterol biosynthesis. Furthermore, gene targeting studies in mice (LXR␣/␤ double knockout mice), where insulin-mediated induction of an entire class of lipogenic and cholesterogenic enzymes was suppressed, provide strong evidence that LXR plays a central role as an insulin sensor in hepatic lipid homeostasis.
Lipogenesis encompasses fatty acid synthesis and subsequent triglyceride synthesis, and takes place in both liver and adipose tissue. Insulin is probably the most important hormonal factor influencing lipogenesis. Evidence gathered over the past few years clearly indicates that the effects of insulin on the expression of lipogenic genes are mediated by SREBP-1c (2, 4, 5, 16, 32, 34). However, there are reports indicating that SREBP-1c is not the only transcription factor involved. In starved and refed SREBP-1c Ϫ/Ϫ mice, expression of some of the genes encoding hepatic lipogenic enzymes was completely abolished, whereas that of others was only partially suppressed, indicating that factors other than SREBP-1c might be involved in the refeeding response (35). Additionally, the rapid effect of insulin on transcription of genes such as glucokinase does not require an increased amount of the mature form of SREBP-1c in the nucleus, suggesting that additional actions of insulin are necessary (3,36). We demonstrate here that SREBP-1c is not the only transcription factor mediating the effects of insulin on gene expression. We show in this study that LXR␣ mRNA and protein are stimulated by insulin to the same extent as SREBP-1c in the liver. Interestingly, the upregulation of SREBP-1c was abolished in LXR␣/␤ double knockout mice, indicating a cross-regulation between LXR and SREBP-1c in response to insulin. Cross-regulation between LXR and transcription factors in lipid metabolism has also recently been shown in other studies. Schultz et al. (37) identified an LXR binding site in the SREBP-1c promoter, and disruption of this binding site abolished the response to T0901317, a non-sterol synthetic ligand of LXR. Furthermore, unsaturated fatty acids inhibit transcription of the SREBP-1c gene by antagonizing ligand-dependent activation of the LXR (38). Recently, we demonstrated in liver and adipocytes that LXR␣ is a target gene for PPAR␣ and PPAR␥, respectively (39). 3 We further analyzed the physiological role of LXR and the transcriptional regulation by insulin on hepatic lipogenic enzymes (GK, ME, ACC, and FAS) in normal and LXR␣/␤ double knockout mice. Whereas the expression of these genes was induced in normal mice injected with insulin, the basal mRNA levels of lipogenic enzymes in LXR␣/␤ double knockout mice were repressed and insensitive to insulin compared with wild type animals. Additionally, the expression of genes in cholesterol biosynthesis (HMG-CoA reductase, squalene synthase) was induced by insulin in normal mice. In contrast to the down-regulation of basal mRNA levels of genes involved in lipogenesis, the expression of genes in cholesterol biosynthesis (HMG-CoA synthase and HMG-CoA reductase) was markedly up-regulated in LXR␣/␤ double knockout mice compared with wild type animals. Again, these genes like the genes encoding enzymes in lipogenesis were insensitive to insulin, indicating a suppressive role of LXR for genes in cholesterol synthesis.  5. Effects of insulin on lipogenic enzyme genes in LXR␣/␤ double knockout (LXR␣/␤ ؊/؊) mice. LXR␣/␤ Ϫ/Ϫ mice and corresponding wild type mice were given 0.2 units of Actrapid insulin (intraperitoneal) (Novo Nordisk), and control animals were given vehicle (PBS). After 90 min of insulin stimulation, livers were rapidly taken out and frozen in liquid nitrogen until RNA extraction was performed and assayed by Northern blot analysis. Asterisk indicates that these hybridizations were performed on a separate membrane compared with the others. This membrane contained three lanes of the LXR␣/␤ Ϫ/Ϫ mice as control groups, whereas the others contained four lanes. Down-regulation of the basal mRNA levels of lipogenic enzymes as well as of cholesterogenic enzymes in LXR␣ knockout mice has been reported previously (17). However, in another study involving both LXR␣ and LXR␤ single knockout mice, no major differences were shown between wild type and knockout animals in the basal expression levels of these enzyme genes (18), possibly indicating a synergistic/redundant action of LXR␣ and LXR␤ in maintaining expression of these genes in hepatic lipid metabolism. This coordinated regulation of fatty acid and cholesterol biosynthesis might constitute a way for the body to protect itself against elevated levels of cholesterol by esterifying fatty acids into cholesteryl esters (23). Anyway, our data strongly suggest that LXR is an obligatory intermediary component in insulin regulation of cholesterol and fatty acid biosynthesis. The mechanisms by which insulin stimulates the transcriptional activity of LXR are presently unknown. Regulation of transcriptional activity of LXR by insulin could be through, e.g., mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways (1). The phosphatidylinositol 3-kinase pathway has earlier been shown to account for the metabolic action of insulin because it is involved in the stimulation of glycogen synthesis and glucose transport (GLUT4) by the hormone (3, 40 -42). Furthermore, several cis-acting elements in genes that mediate the effect of insulin on gene transcription have recently been defined. These are referred to as insulin response sequences or elements (43). In vitro studies have clearly established the importance of the upstream stimulatory factors in the insulin regulation of the fatty acid synthetase promoter through insulin response elements consisting of E boxes of CANNTG sequence (44). The SREBP-1c-mediated insulin regulation of genes also occurs by binding to E-boxes; however, the effects of upstream stimulatory factors and SREBP-1c seem to be additive and independent (45). The LXR␣ induction by insulin is dose-and time-dependent, and dependent on ongoing protein synthesis. This up-regulation could, at least partly, be the result of stabilization of the transcripts. The robust insulin regulation of LXR␣ suggests that upstream factors, as yet undefined, are influenced by insulin at the transcriptional and/or post-transcriptional levels. In line with this notion, we have found that LXR␣ mRNA regulation by insulin is dependent on de novo synthesis of proteins. Further studies of the LXR␣ promoter will be required to understand the mechanisms of this regulation and the corresponding effects on cholesterol and lipid metabolism. Similarly, changes in the levels of PPAR␣ and 9-cis-retinoic acid receptor ␣ (RXR␣) as well as products of PPAR␣ target genes in rat liver cells by insulin and glucocorticoids are the result of a major effect on steady-state mRNA levels giving rise to corresponding alterations in protein levels explained by changes in mRNA stability and/or translation efficiency (27,29,30). Further studies are needed to clarify the mechanisms by which insulin stimulates the transcriptional activity of LXR in the liver. We have previously shown a PPAR␣-dependent fatty acid up-regulation of LXR␣ mRNA and protein (39), and PPAR␣ has previously been shown to be phosphorylated in response to insulin, resulting in stimulation of basal as well as ligand-dependent transcriptional activity of PPAR␣ (46,47). PPAR␣ could therefore be an upstream factor mediating the insulin effect on LXR␣.
We have previously shown in rats that liver LXR␣ is upregulated during fasting, and down-regulated during refeeding (39). This is in contrast to the typical insulin effect on gene regulation, where the general trend is down-regulation when energy supply is low, and up-regulation during refeeding. We have also shown fatty acid induction of LXR␣, which could explain the induction of LXR␣ seen during fasting, a state characterized by a high level of free fatty acids. Further studies are required to understand the interplay between insulin and fatty acids in the regulation of LXRs and the effect this might have on target genes. Furthermore, the present study opens up new perspectives regarding cellular mechanisms involved in insulin regulation of gene expression in lipid metabolism. Taken together, our results show that LXR functions as an essential regulatory component in insulin regulation of both cholesterol homeostasis and triglyceride metabolism.