The orphan nuclear receptor small heterodimer partner is required for thiazolidinedione effects in leptin-deficient mice

Background

Small heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp^−/−) double mutant mice.

Results

Both ob/ob and double mutant ob/ob;Shp^−/− mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp^−/− mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp^−/− liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp^−/− mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp^−/− mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes.

Conclusions

Our results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.     

Dissociation of diabetes and obesity in mice lacking orphan nuclear receptor small heterodimer partner

Mixed background SHP(-/-) mice are resistant to diet-induced obesity due to increased energy expenditure caused by enhanced PGC-1α expression in brown adipocytes. However, congenic SHP(-/-) mice on the C57BL/6 background showed normal expression of PGC-1α and other genes involved in brown adipose tissue thermogenesis. Thus, we reinvestigated the impact of small heterodimer partner (SHP) deletion on diet-induced obesity and insulin resistance using congenic SHP(-/-) mice. Compared with their C57BL/6 wild-type counterparts, SHP(-/-) mice subjected to a 6 month challenge with a Western diet (WestD) were leaner but more glucose intolerant, showed hepatic insulin resistance despite decreased triglyceride accumulation and increased β-oxidation, exhibited alterations in peripheral tissue uptake of dietary lipids, maintained a higher respiratory quotient, which did not decrease even after WestD feeding, and displayed islet dysfunction. Hepatic mRNA expression analysis revealed that many genes expressed higher in SHP(-/-) mice fed WestD were direct peroxisome proliferator-activated receptor alpha (PPARα) targets. Indeed, transient transfection and chromatin immunoprecipitation verified that SHP strongly repressed PPARα-mediated transactivation. SHP is a pivotal metabolic sensor controlling lipid homeostasis in response to an energy-laden diet through regulating PPARα-mediated transactivation. The resultant hepatic fatty acid oxidation enhancement and dietary fat redistribution protect the mice from diet-induced obesity and hepatic steatosis but accelerate development of type 2 diabetes.     

Loss of small heterodimer partner expression in the liver protects against dyslipidemia

Multiple studies suggest increased conversion of cholesterol to bile acids by cholesterol 7alpha-hydroxylase (CYP7A1) protects against dyslipidemia and atherosclerosis. CYP7A1 expression is repressed by the sequential activity of two nuclear hormone receptors, farnesoid X receptor (FXR) and small heterodimer partner (SHP). Here we demonstrate 129 strain SHP(-/-) mice are protected against hypercholesterolemia resulting from either a cholesterol/cholic acid (chol/CA) diet or from hypothyroidism. In a mixed 129-C57Bl/6 background, LDLR(-/-) and LDLR(-/-)SHP(-/-) mice had nearly identical elevations in hepatic cholesterol content and repression of cholesterol regulated genes when fed a Western diet. However, the LDLR(-/-)SHP(-/-) mice had greatly reduced elevations in serum VLDL and LDL cholesterol levels and triglyceride (TG) levels as compared with LDLR(-/-) mice. Additionally, the hepatic inflammation produced by the Western diet in the LDLR(-/-) mice was abolished in the LDLR(-/-)SHP(-/-) mice. CYP7A1 expression was induced 10-fold by the Western diet in the LDLR(-/-)SHP(-/-) mice but not in the LDLR(-/-) mice. Finally, hepatocyte-specific deletion of SHP expression was also protective against dyslipidemia induced by either a chol/CA diet or by hypothyroidism. While no antagonist ligands have yet been identified for SHP, these results suggest selective inhibition of hepatic SHP expression may provide protection against dyslipidemia.     

Repression of CAR-mediated transactivation of CYP2B genes by the orphan nuclear receptor, short heterodimer partner (SHP)

The induction of CYP2B gene expression by phenobarbital (PB) is mediated by the translocation of the constitutive androstane receptor (CAR) from the cytoplasm to the nucleus. The CAR/RXR heterodimer binds to two DR-4 sites in a complex phenobarbital responsive unit (PBRU) in the CYP2B gene. The short heterodimer partner (SHP), an orphan nuclear receptor that lacks a conventional DNA binding domain, was initially identified by its interaction with CAR. We have examined the role of SHP in CAR-mediated transactivation of the CYP2B gene. Coexpression of SHP inhibited the transactivation of the CYP2B gene by CAR in cultured hepatoma cells and the p160 coactivator GRIP1 reversed the inhibition. The interaction of CAR with SHP was confirmed by GST pulldown experiments. SHP did not block the binding of either CAR/RXR to the PBRU or binding of GRIP1 to the CAR/RXR complex in gel mobility shift assays, but slightly increased CAR/RXR binding and slightly altered the mobility of the CAR/RXR/GRIP1 complex, suggesting an interaction of SHP with these complexes. The presence of SHP in the complexes, however, could not be detected in an antibody supershift assay. Recombinant corepressors mSin3A, SMRT, and HDAC1, but not NCoR1, interacted with GST-SHP but each of these corepressors in liver nuclear extracts bound to GST-SHP. SMRT and NCoR1 inhibited CAR-mediated activation independent of SHP, but mSin3A and HDAC1 had little effect alone, and were additive with SHP. These studies demonstrate that SHP does not inhibit CAR-mediated trans-activation by interfering with DNA binding or by competition with GRIP1. Instead, SHP may either inhibit recruitment of other coactivators by GRIP1 or actively recruit corepressors directly to the CAR/RXR/PBRU complex.     

Differential role of the loop region between helices H6 and H7 within the orphan nuclear receptors small heterodimer partner and DAX-1

The orphan nuclear receptors small heterodimer partner (SHP) and dosage-sensitive sex-reversal adrenal hypoplasia congenital (AHC) critical region on the X chromosome gene 1 (DAX-1) contain extra amino acids between helices H6 and H7 of LBD, and here we investigated a possible role of these additional amino acids. Transient transfection assay demonstrated that, in contrast to wild type, in mutant SHP Delta128-139 deletion of 12 extra amino acids in H6-H7 failed to repress the transactivity of orphan nuclear receptors such as estrogen receptor-related receptor-gamma, hepatocyte nuclear factor 4alpha, and constitutive androstane receptor. Interestingly, yeast two-hybrid and glutathione-S-transferase pull-down assays demonstrated that wild-type and SHP Delta128-139 have similar abilities to interact with estrogen receptor-related receptor-gamma, hepatocyte nuclear factor 4alpha, and constitutive androstane receptor. Unexpectedly, in wild-type DAX-1 and mutant DAX-1 Delta338-362, deletion of 25 extra amino acids in H6-H7 had no significant difference in the interaction and repression of steroidogenic factor 1 transactivation. Mutant SHP that contains DAX-1 extra amino acids or polyalanine stretch in H6-H7 showed indistinguishable pattern of repression from wild-type SHP. Interestingly, the swapped SHP mutant with DAX-1 extra amino acids interacted with EID-1 (E1A-like inhibitor of differentiation 1), which is characterized as an SHP-interacting corepressor. However, interaction between SHP Delta128-139 and EID-1 was significantly diminished. Moreover, SHP-mediated repression of constitutive androstane receptor transactivation was significantly released by down-regulation of EID-1 expression with EID-1 small interfering RNA. The present study suggests that H6-H7 loop regions of SHP and DAX-1 play a different role in the repression of nuclear receptor transactivation.     

The small heterodimer partner interacts with the pregnane X receptor and represses its transcriptional activity

SHP (small heterodimer partner, NR1I0) is an atypical orphan member of the nuclear receptor subfamily in that it lacks a DNA-binding domain. It is mostly expressed in the liver, where it binds to and inhibits the function of nuclear receptors. SHP is up-regulated by primary bile acids, through the activation of their receptor farnesoid X receptor, leading to the repression of cholesterol 7alpha-hydroxylase (CYP7alpha) expression, the rate-limiting enzyme in bile acid production from cholesterol. PXR (pregnane X receptor, NR1I2) is a broad-specificity sensor that recognizes a wide variety of synthetic drugs as well as endogenous compounds such as bile acid precursors. Upon activation, PXR induces CYP3A and inhibits CYP7alpha, suggesting that PXR can act on both bile acid synthesis and elimination. Indeed, CYP7alpha and CYP3A are involved in biochemical pathways leading to cholesterol conversion into primary bile acids, whereas CYP3A is also involved in the detoxification of toxic secondary bile acid derivatives. Here, we show that PXR is a target for SHP. Using pull-down assays, we show that SHP interacts with both murine and human PXR in a ligand-dependent manner. From transient transfection assays, SHP is shown to be a potent repressor of PXR transactivation. Furthermore, we report that chenodeoxycholic acid and cholic acid, two farnesoid X receptor ligands, induce up-regulation of SHP and provoke a repression of PXR-mediated CYP3A induction in human hepatocytes as well as in vivo in mice. These results reveal an elaborate regulatory cascade, tightly controlled by SHP, for both the maintenance of bile acid production and detoxification in the liver.     

DNA-binding mechanism of the monomeric orphan nuclear receptor NGFI-B.

The 2.7 A X-ray crystal structure of the DNA-binding domain (DBD) of the orphan nuclear receptor, nerve growth factor-induced-B (NGFI-B), complexed to its high-affinity DNA target, represents the first structure analysis of a nuclear receptor DBD bound as a monomer to DNA. The structure of the core DBD and its interactions with the major groove of the DNA are similar to previously crystallographically solved DBD-DNA complexes in this superfamily; however, residues C-terminal to this core form a separate and unique substructure that interacts extensively and in a sequence-specific way with the minor groove of its DNA target, in particular with the characteristic 3 A-T base-pair identity element that extends 5' to the usual nuclear receptor half-site (AGGTCA).