Peter A. Crawford

Nuclear Receptor DAX-1 Recruits Nuclear Receptor Corepressor N-CoR to Steroidogenic Factor 1

ABSTRACT The orphan nuclear receptor steroidogenic factor 1 (SF-1) is a critical developmental regulator in the urogenital ridge, because mice targeted for disruption of the SF-1 gene lack adrenal glands and gonads. SF-1 was recently shown to interact with DAX-1, another orphan receptor whose tissue distribution overlaps that of SF-1. Naturally occurring loss-of-function mutations of the DAX-1 gene cause the human disorder X-linked adrenal hypoplasia congenita (AHC), which resembles the phenotype of SF-1-deficient mice. Paradoxically, however, DAX-1 represses the transcriptional activity of SF-1, and AHC mutants of DAX-1 lose repression function. To further investigate these findings, we characterized the interaction between SF-1 and DAX-1 and found that their interaction indeed occurs through a repressive domain within the carboxy terminus of SF-1. Furthermore, we demonstrate that DAX-1 recruits the nuclear receptor corepressor N-CoR to SF-1, whereas naturally occurring AHC mutations of DAX-1 permit the SF-1–DAX-1 interaction, but markedly diminish corepressor recruitment. Finally, the interaction between DAX-1 and N-CoR shares similarities with that of the nuclear receptor RevErb and N-CoR, because the related corepressor SMRT was not efficiently recruited by DAX-1. Therefore, DAX-1 can serve as an adapter molecule that recruits nuclear receptor corepressors to DNA-bound nuclear receptors like SF-1, thereby extending the range of corepressor action.

The ketone metabolite β-hydroxybutyrate blocks NLRP3 inflammasome–mediated inflammatory disease

The ketone bodies β-hydroxybutyrate (BHB) and acetoacetate (AcAc) support mammalian survival during states of energy deficit by serving as alternative sources of ATP. BHB levels are elevated by starvation, caloric restriction, high-intensity exercise, or the low-carbohydrate ketogenic diet. Prolonged fasting reduces inflammation; however, the impact that ketones and other alternative metabolic fuels produced during energy deficits have on the innate immune response is unknown. We report that BHB, but neither AcAc nor the structurally related short-chain fatty acids butyrate and acetate, suppresses activation of the NLRP3 inflammasome in response to urate crystals, ATP and lipotoxic fatty acids. BHB did not inhibit caspase-1 activation in response to pathogens that activate the NLR family, CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome and did not affect non-canonical caspase-11, inflammasome activation. Mechanistically, BHB inhibits the NLRP3 inflammasome by preventing K+ efflux and reducing ASC oligomerization and speck formation. The inhibitory effects of BHB on NLRP3 are not dependent on chirality or starvation-regulated mechanisms like AMP-activated protein kinase (AMPK), reactive oxygen species (ROS), autophagy or glycolytic inhibition. BHB blocks the NLRP3 inflammasome without undergoing oxidation in the TCA cycle, and independently of uncoupling protein-2 (UCP2), sirtuin-2 (SIRT2), the G protein–coupled receptor GPR109A or hydrocaboxylic acid receptor 2 (HCAR2). BHB reduces NLRP3 inflammasome–mediated interleukin (IL)-1β and IL-18 production in human monocytes. In vivo, BHB or a ketogenic diet attenuates caspase-1 activation and IL-1β secretion in mouse models of NLRP3-mediated diseases such as Muckle–Wells syndrome, familial cold autoinflammatory syndrome and urate crystal–induced peritonitis. Our findings suggest that the anti-inflammatory effects of caloric restriction or ketogenic diets may be linked to BHB-mediated inhibition of the NLRP3 inflammasome.

Activation of Luteinizing Hormone β Gene by Gonadotropin-releasing Hormone Requires the Synergy of Early Growth Response-1 and Steroidogenic Factor-1

We have previously shown that early growth response (Egr) 1-deficient mice exhibit female infertility, reflecting a luteinizing hormone (LH) β deficiency. Egr-1 activates the LHβ gene in vitro through synergy with steroidogenic factor-1 (SF-1), a protein required for gonadotrope function. To test if this synergy is essential for gonadotropin-releasing hormone (GnRH) stimulation of LHβ, we examined the activity of the LHβ promoter in the gonadotrope cell line LβT2. GnRH markedly stimulated the LHβ promoter (15-fold). Mutation of either Egr-1 or SF-1 elements within the LHβ promoter attenuated this stimulation, whereas mutation of both promoter elements abrogated GnRH induction of the LHβ promoter. Furthermore, GnRH stimulated Egr-1 but not SF-1 expression in LβT2 cells. Importantly, overexpression of Egr-1 alone was sufficient to enhance LHβ expression. Although other Egr proteins are expressed in LβT2 cells and are capable of interacting with SF-1, GnRH stimulation of Egr-1 was the most robust. We also found that the nuclear receptor DAX-1, a repressor of SF-1 activity, reduced Egr-1–SF-1 synergy and diminished GnRH stimulation of the LHβ promoter. We conclude that the synergy between Egr-1 and SF-1 is essential for GnRH stimulation of the LHβ gene and plays a central role in the dynamic regulation of LHβ expression.

Regulation of myocardial ketone body metabolism by the gut microbiota during nutrient deprivation.

Studies in mice indicate that the gut microbiota promotes energy harvest and storage from components of the diet when these components are plentiful. Here we examine how the microbiota shapes host metabolic and physiologic adaptations to periods of nutrient deprivation. Germ-free (GF) mice and mice who had received a gut microbiota transplant from conventionally raised donors were compared in the fed and fasted states by using functional genomic, biochemical, and physiologic assays. A 24-h fast produces a marked change in gut microbial ecology. Short-chain fatty acids generated from microbial fermentation of available glycans are maintained at higher levels compared with GF controls. During fasting, a microbiota-dependent, Pparα-regulated increase in hepatic ketogenesis occurs, and myocardial metabolism is directed to ketone body utilization. Analyses of heart rate, hydraulic work, and output, mitochondrial morphology, number, and respiration, plus ketone body, fatty acid, and glucose oxidation in isolated perfused working hearts from GF and colonized animals (combined with in vivo assessments of myocardial physiology) revealed that the fasted GF heart is able to sustain its performance by increasing glucose utilization, but heart weight, measured echocardiographically or as wet mass and normalized to tibial length or lean body weight, is significantly reduced in both fasted and fed mice. This myocardial-mass phenotype is completely reversed in GF mice by consumption of a ketogenic diet. Together, these results illustrate benefits provided by the gut microbiota during periods of nutrient deprivation, and emphasize the importance of further exploring the relationship between gut microbes and cardiovascular health.

Adrenocortical Function and Regulation of the Steroid 21-Hydroxylase Gene in NGFI-B-Deficient Mice

The immediate-early gene NGFI-B encodes an orphan nuclear receptor that binds DNA as a monomer and activates transcription through a canonical response element (NBRE). NGFI-B is expressed under basal conditions and in response to external stimuli in many mammalian tissues. In particular, NGFI-B expression is dramatically elevated in the adrenal cortex in response to stress and in Y1 adrenocortical cells in response to adrenocorticotropin. NGFI-B activates transcription through an NBRE of the gene encoding 21-hydroxylase (P450c21) in Y1 cells. Steroidogenic factor 1 (SF-1), a homolog of NGFI-B, also activates the P450c21 promoter. To examine the influence of these factors on P450c21 expression in vivo and the function of the hypothalamic-pituitary-adrenocortical axis as a whole, we generated NGFI-B (-/-) mice. These mice thrive and reproduce normally and maintain normal basal adrenocorticotropin, corticosterone, and P450c21 mRNA levels. In response to increases in adrenocorticotropin, NGFI-B (-/-) and wild-type mice demonstrated equivalent increases in serum corticosterone levels. Furthermore, and in contrast to in vitro results, no increases in P450c21 mRNA levels were observed in response to increases in adrenocorticotropin in NGFI-B (-/-) or wild-type mice. While SF-1 mRNA levels were not increased with increased steroidogenic demand, adrenal expression of Nurr1, a close homolog of NGFI-B, was induced to a greater extent by lipopolysaccharide in NGFI-B (-/-) mice than in wild-type mice. Finally, when the administration of dexamethasone for suppression was stopped, P450c21 mRNA and serum corticosterone levels recovered at the same rate in wild-type and NGFI-B (-/-) mice. Thus, while NGFI-B appears poised to affect the structure and function of the adrenal gland, the gland functions normally in its absence, suggesting that other factors, including Nurr1 and SF-1, are sufficient to drive P450c21 expression in mice and maintain normal steroidogenesis.

Ketone body metabolism and cardiovascular disease

Ketone bodies are metabolized through evolutionarily conserved pathways that support bioenergetic homeostasis, particularly in brain, heart, and skeletal muscle when carbohydrates are in short supply. The metabolism of ketone bodies interfaces with the tricarboxylic acid cycle, β-oxidation of fatty acids, de novo lipogenesis, sterol biosynthesis, glucose metabolism, the mitochondrial electron transport chain, hormonal signaling, intracellular signal transduction pathways, and the microbiome. Here we review the mechanisms through which ketone bodies are metabolized and how their signals are transmitted. We focus on the roles this metabolic pathway may play in cardiovascular disease states, the bioenergetic benefits of myocardial ketone body oxidation, and prospective interactions among ketone body metabolism, obesity, metabolic syndrome, and atherosclerosis. Ketone body metabolism is noninvasively quantifiable in humans and is responsive to nutritional interventions. Therefore, further investigation of this pathway in disease models and in humans may ultimately yield tailored diagnostic strategies and therapies for specific pathological states.

The Failing Heart Relies on Ketone Bodies as a Fuel

Background— Significant evidence indicates that the failing heart is energy starved. During the development of heart failure, the capacity of the heart to utilize fatty acids, the chief fuel, is diminished. Identification of alternate pathways for myocardial fuel oxidation could unveil novel strategies to treat heart failure. Methods and Results— Quantitative mitochondrial proteomics was used to identify energy metabolic derangements that occur during the development of cardiac hypertrophy and heart failure in well-defined mouse models. As expected, the amounts of proteins involved in fatty acid utilization were downregulated in myocardial samples from the failing heart. Conversely, expression of &bgr;-hydroxybutyrate dehydrogenase 1, a key enzyme in the ketone oxidation pathway, was increased in the heart failure samples. Studies of relative oxidation in an isolated heart preparation using ex vivo nuclear magnetic resonance combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids. Distinct myocardial metabolomic signatures of ketone oxidation were identified. Conclusions— These results indicate that the hypertrophied and failing heart shifts to ketone bodies as a significant fuel source for oxidative ATP production. Specific metabolite biosignatures of in vivo cardiac ketone utilization were identified. Future studies aimed at determining whether this fuel shift is adaptive or maladaptive could unveil new therapeutic strategies for heart failure.

Nuclear receptor steroidogenic factor 1 directs embryonic stem cells toward the steroidogenic lineage.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is expressed in the adrenal gland and gonads and is an important regulator of the expression of cytochrome P-450 steroidogenic enzymes in cultured cells. Targeted disruption of the SF-1 gene in mice shows that it is a critical participant in the genetic program that promotes the development of urogenital mesoderm into the adrenal gland and gonads. To assess the ability of SF-1 to regulate this differentiation pathway, we ectopically expressed SF-1 in murine embryonic stem (ES) cells. We found that stable expression of SF-1 is sufficient to alter ES cell morphology, permit cyclic AMP (cAMP) and retinoic acid-induced expression of the endogenous side chain cleavage enzyme gene, and consequently, promote steroidogenesis. While steroid production is dependent upon SF-1, cAMP induction of steroidogenesis does not enhance the responsiveness of an SF-1-specific reporter. Furthermore, the activity of a P450SCC promoter/luciferase reporter construct, which is induced by cAMP in steroidogenic cells and ES cells converted by stable expression of SF-1, is not induced by cAMP in wild-type ES cells transiently transfected with SF-1, suggesting that the induction of downstream gene products is required before steroidogenesis can occur. We demonstrate that mutants which disrupt the DNA binding domain or the AF2 transcriptional activation domain of SF-1 do not confer the steroidogenic phenotype to ES cells. Interestingly, however, AF2 mutants fused to the VP16 activation domain do confer the steroidogenic phenotype to ES cells, but only in the presence of a portion of the ligand binding domain. These studies extend the role of SF-1 in steroidogenic tissues to that of a dominant regulator of the steroidogenic cell phenotype.

Postnatal lymphatic partitioning from the blood vasculature in the small intestine requires fasting-induced adipose factor

Lymphatic vessels develop from specialized venous endothelial cells. Using knockout mice, we found that fasting-induced adipose factor (Fiaf) is required for functional partitioning of postnatal intestinal lymphatic and blood vessels. In wild-type animals, levels of intestinal Fiaf expression rise during the first postnatal day and peak at day 2, which coincides with the onset of the lymphatico-venous partitioning abnormality in Fiaf−/− mutants on a mixed 129/SvJ:C57BL/6 genetic background. Fiaf deficiency is not associated with disruption of the blood vasculature or with lymphatic endothelial recruitment of smooth muscle cells. We identified Prox1, a critical regulator of lymphangiogenesis, as a downstream target for Fiaf signaling in the intestinal lymphatic endothelium. This organ-specific lymphovascular abnormality can be rescued by allowing embryonic Fiaf−/− intestinal isografts to develop in Fiaf+/+ recipients.

X13CMS: Global Tracking of Isotopic Labels in Untargeted Metabolomics

Studies of isotopically labeled compounds have been fundamental to understanding metabolic pathways and fluxes. They have traditionally, however, been used in conjunction with targeted analyses that identify and quantify a limited number of labeled downstream metabolites. Here we describe an alternative workflow that leverages recent advances in untargeted metabolomic technologies to track the fates of isotopically labeled metabolites in a global, unbiased manner. This untargeted approach can be applied to discover novel biochemical pathways and characterize changes in the fates of labeled metabolites as a function of altered biological conditions such as disease. To facilitate the data analysis, we introduce X13CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS. X13CMS uses the XCMS platform to detect metabolite peaks and perform retention-time alignment in liquid chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, the program then identifies isotopologue groups that correspond to isotopically labeled compounds. The retrieval of these groups is done without any a priori knowledge besides the following input parameters: (i) the mass difference between the unlabeled and labeled isotopes, (ii) the mass accuracy of the instrument used in the analysis, and (iii) the estimated retention-time reproducibility of the chromatographic method. Despite its name, X13CMS can be used to track any isotopic label. Additionally, it detects differential labeling patterns in biological samples collected from parallel control and experimental conditions. We validated the ability of X13CMS to accurately retrieve labeled metabolites from complex biological matrices both with targeted LC/MS/MS analysis of a subset of the hits identified by the program and with labeled standards spiked into cell extracts. We demonstrate the full functionality of X13CMS with an analysis of cultured rat astrocytes treated with uniformly labeled (U-)13C-glucose during lipopolysaccharide (LPS) challenge. Our results show that out of 223 isotopologue groups enriched from U-13C-glucose, 95 have statistically significant differential labeling patterns in astrocytes challenged with LPS compared to unchallenged control cells. Only two of these groups overlap with the 32 differentially regulated peaks identified by XCMS, indicating that X13CMS uncovers different and complementary information from untargeted metabolomic studies. Like XCMS, X13CMS is implemented in R. It is available from our laboratory website at http://pattilab.wustl.edu/x13cms.php.