In this report, we detail the discovery of LXRβ as an important potentiating factor in GC-mediated hyperglycemia and hepatic steatosis. As discussed below, these findings are of both physiologic and pharmacologic interest. On the physiologic side, this work reveals an unexpected crosstalk between LXR and GC regulation of glucose and lipid metabolism. GCs potently upregulate hepatic glucose production under conditions of stress to protect the body from excessive nutrient depletion. This outcome is achieved through the upregulation of numerous genes in the gluconeogenic pathway that include PEPCK and G6Pc. PEPCK is considered to be the rate-limiting enzyme in gluconeogenesis and as such the molecular factors controlling the expression of this gene have been extensively studied (25
). PEPCK transcription is strongly activated by GCs and inhibited by insulin (63
). In addition to GR, numerous proteins are bound to accessory factor sites on the PEPCK promoter either directly or indirectly and are essential for maximal GC response including PPARα (64
), FOXO1 (65
), and PGC1α (65
). We found the induction of liver PEPCK, FOXO1, and PGC1α after DEX treatment was significantly attenuated in the Lxr
mice compared with WT mice. In addition, Lxr
mice remained more insulin tolerant than WT mice after chronic DEX treatment determined from basal insulin values and an ITT, and this effect was specifically due to loss of LXRβ. From experiments performed in primary hepatocytes, we found that the selective transcriptional effect of GCs was cell autonomous and required LXRβ despite the fact that the enhanced insulin tolerance of the Lxr
mice would also contribute to protection against GC-induced hyperglycemia. Together, these data suggest that LXRβ in the liver contributes selectively to the regulation of key gluconeogenic enzymes including PEPCK.
Nonalcoholic fatty liver results from an imbalance of fat metabolism in the liver. This can arise if there is excessive fatty acid uptake or synthesis, decreased fatty acid oxidation, or decreased secretion of VLDL particles. GCs are known to contribute to fatty liver production through a combination of increased fatty acid synthesis and decreased fatty acid β oxidation (66
). The effect of GCs on promoting fatty acid synthesis has been shown to require the presence of insulin. In fact, GCs alone, in the absence of insulin, do not promote fatty acid synthesis (53
). However, when combined with insulin, GCs synergistically increase de novo lipogenesis (52
). While extensive studies have been performed to understand the molecular regulation of gluconeogenesis by GCs (61
), the detailed molecular mechanisms by which GCs induce fatty liver have not been well characterized (9
). It was recently reported that downregulation of the transcriptional repressor HES1 by GR is important in promoting fatty liver because reconstitution of HES1 promoted lipolysis through the ectopic expression of pancreatic lipases (67
). These same lipases were recently reported to be upregulated by PPARα activation (68
). Our data suggest that this mechanism is not involved in the resistance to fatty liver seen in the Lxr
mice since the expression of HES1 was not significantly altered by DEX and we were unable to detect the expression of these pancreatic lipases in our liver samples (Supplemental Figure 2 and data not shown). PPARα, a key transcriptional regulator of fatty acid oxidation, has been found to be essential for DEX-mediated induction of hypertension and insulin resistance in Ldlr–/–Ppar
animals, but its role in hepatic steatosis was not analyzed in that study (69
). Here, we found that PPARα expression was unchanged between WT and Lxr
mice and downstream target genes were not dramatically or differentially altered with DEX in WT and Lxr
mice (Supplemental Figure 2). PPARγ has been recently implicated in hepatic steatosis through the regulation of fat-specific protein 27 (FSP27), a direct PPARγ target gene (70
). In our study, PPARγ expression itself was not significantly changed by DEX in both WT and Lxr
mice, whereas FSP27 was induced equally by DEX in WT and Lxr
mice, suggesting this pathway was not involved in selective fatty liver production (Supplemental Figure 2). The transcription factor SREBP-1c has been previously shown to be critical for inducing an overall program of lipogenesis and promoting fatty liver (71
). As expected, due to the known direct regulatory effect of LXRα on SREBP-1c expression (48
), basal expression of SREBP-1c was greatly decreased in Lxr
mice (Supplemental Figure 2). However, DEX actually decreased the expression of SREBP-1c in WT mice, suggesting that this pathway cannot account for the increased fatty liver seen in WT mice treated with DEX. In summary, the mechanism by which the LXRβ and LXRα/β knockout mice are protected from fatty liver appears to be independent of the known pathways affected by GC regulation. This is the first LXRβ-specific role that has been ascribed in the liver, an organ in which LXRα has been recognized as the critical mediator of both cholesterol metabolism and lipogenesis. A plausible hypothesis that is the subject of future studies is that the LXRβ effects on hepatic steatosis may be related to the enhanced glucose tolerance in the Lxr
mice after chronic DEX treatment (Supplemental Figure 1A).
GCs mediate their antiinflammatory effects through the differential tethering of GR to individual transcription factors such as NF-κB and c-Jun (73
). The transrepressive effects of GCs are primarily responsible for the beneficial therapeutic effects on the immune system (for example, repression of IL-6 and TNF-α). Transactivation of GR is believed to be responsible for the negative side effects of therapeutic GCs including hyperglycemia (PEPCK, G6Pc), muscle catabolism (myostatin, glutamine synthetase), and osteoporosis (receptor activator of NF-κB ligand) (75
). As such, the pharmaceutical industry has been interested in developing “dissociated” GR agonists that can separate the transrepression from transactivation activities (76
). This strategy has provided successful novel steroidal candidates in vitro, but the effects have not faithfully translated in vivo. More recent compounds with a nonsteroidal structure have begun to be used in vivo with an enhanced side effect profile compared with steroidal ligands (78
). Complicating the application of the dissociated ligand strategy are genes that are transactivated by GR but critical for its antiinflammatory effects such as MAPK phosphatase 1 (MKP-1) and GC-induced leucine zipper (GILZ) (75
). The finding that LXRβ selectively promotes gene-specific transactivation opens a new avenue for the development of GC-selective ligands that does not depend on separating transactivation from transrepression.
To capitalize on the discovery that LXRβ is potentiating some of the negative metabolic effects of GCs, a detailed molecular mechanism for how selective transactivation occurs must be uncovered. While an improved insulin tolerance in LXRα/β-null mice would provide protection against DEX-induced hyperglycemia, the cell autonomous effect of PEPCK regulation in the primary hepatocytes suggests a more direct mechanism is involved. We have shown that the GC levels in the livers of WT and Lxrα/β–/– mice treated with DEX are similar, yet there is differential regulation of GR target genes that points to a promoter-specific mechanism. To that end, we have shown that GR is differentially recruited to the PEPCK promoter in the absence of LXR. This effect is not due to a general decrease in recruitment of cofactors to the PEPCK promoter in the Lxrα/β–/– mice since 2 other factors important for PEPCK activation (C/EBPβ and SRC-1) were similarly recruited (Supplemental Figure 3). Coimmunoprecipitation studies in HEK293 cells overexpressing GR and Flag-tagged LXRα or LXRβ uncovered an interaction between GR and each isoform of LXR (our unpublished observations). Therefore, this protein-protein interaction is unlikely to account for the LXRβ-specific mechanism demonstrated herein unless competition is occurring between the 2 receptors for binding to GR. Ongoing studies are currently being directed at understanding the basis for the selective recruitment of GR to the PEPCK promoter. In addition, it will be of interest to explore whether other detrimental effects of long-term GCs, including osteoporosis and muscle wasting, show LXR selectivity.
In summary, the discovery that the Lxrα/β–/– mice were hypercorticosteronemic without exhibiting Cushing-like symptoms prompted our investigation into whether the mice were resistant to the effects of GCs. Through the use of the potent synthetic ligand DEX, we discovered that Lxrα/β–/– mice were selectively resistant to some of the effects of GCs — most notably the metabolic effects — but were still sensitive to the immunosuppressive effects (Figure ). Furthermore, we uncovered the liver as a key organ influencing the effect of LXRβ on GC-mediated induction of PEPCK. The data presented herein renew the optimism that a more selective GC agonist can be designed to provide exceptional antiinflammatory action without the development of negative metabolic effects.
Selective activation of the hepatic GC-response pathway.