Pamela M. Rogers

Identification of heme as the ligand for the orphan nuclear receptors REV-ERBα and REV-ERBβ

The nuclear receptors REV-ERBα (encoded by NR1D1) and REV-ERBβ (NR1D2) have remained orphans owing to the lack of identified physiological ligands. Here we show that heme is a physiological ligand of both receptors. Heme associates with the ligand-binding domains of the REV-ERB receptors with a 1:1 stoichiometry and enhances the thermal stability of the proteins. Results from experiments of heme depletion in mammalian cells indicate that heme binding to REV-ERB causes the recruitment of the co-repressor NCoR, leading to repression of target genes including BMAL1 (official symbol ARNTL), an essential component of the circadian oscillator. Heme extends the known types of ligands used by the human nuclear receptor family beyond the endocrine hormones and dietary lipids described so far. Our results further indicate that heme regulation of REV-ERBs may link the control of metabolism and the mammalian clock.

Identification of Adropin as a Secreted Factor Linking Dietary Macronutrient Intake with Energy Homeostasis and Lipid Metabolism

Obesity and nutrient homeostasis are linked by mechanisms that are not fully elucidated. Here we describe a secreted protein, adropin, encoded by a gene, Energy Homeostasis Associated (Enho), expressed in liver and brain. Liver Enho expression is regulated by nutrition: lean C57BL/6J mice fed high-fat diet (HFD) exhibited a rapid increase, while fasting reduced expression compared to controls. However, liver Enho expression declines with diet-induced obesity (DIO) associated with 3 months of HFD or with genetically induced obesity, suggesting an association with metabolic disorders in the obese state. In DIO mice, transgenic overexpression or systemic adropin treatment attenuated hepatosteatosis and insulin resistance independently of effects on adiposity or food intake. Adropin regulated expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor gamma, a major regulator of lipogenesis. Adropin may therefore be a factor governing glucose and lipid homeostasis, which protects against hepatosteatosis and hyperinsulinemia associated with obesity.

Regulation of Cholesterologenesis by the Oxysterol Receptor, LXRα

Cholesterol is required for normal cellular and physiological function, yet dysregulation of cholesterol metabolism is associated with diseases such as atherosclerosis. Cholesterol biosynthesis is regulated by end product negative feedback inhibition where the levels of sterols and oxysterols regulate the expression of cholesterologenic enzymes. Sterol regulatory element-binding protein-2 is responsive to both sterols and oxysterols and has been shown to mediate the transcriptional response of the cholesterologenic enzymes to these lipids. Here, we show that the nuclear hormone receptor for oxysterols, the liver X receptor α (LXRα), regulates cholesterol biosynthesis by directly silencing the expression of two key cholesterologenic enzymes (lanosterol 14α-demethylase (CYP51A1), and squalene synthase (farnesyl diphosphate farnesyl transferase 1)) via novel negative LXR DNA response elements (nLXREs) located in each of these genes. Examination of the CYP51A1 gene revealed that both the SRE and nLXRE are required for normal oxysterol-dependent repression of this gene. Thus, these data suggest that LXRα plays an important role in the regulation of cholesterol biosynthesis.

Regulation of Adipogenesis by Natural and Synthetic REV-ERB Ligands

The nuclear hormone receptor, REV-ERB, plays an essential role in adipogenesis. Rev-erbalpha expression is induced in 3T3-L1 cells during adipogenesis, and overexpression of this receptor leads to expression of adipogenic genes. We recently demonstrated that the porphyrin heme functions as a ligand for REV-ERB, and binding of heme is required for the receptors activity. We therefore hypothesized that REV-ERB ligands may play a role in regulation of adipogenesis. We detected an increase intracellular heme levels during 3T3-L1 adipogenesis that correlated with induction of aminolevulinic acid synthase 1 (Alas1) expression, the rate-limiting enzyme in heme biosynthesis. If the increase in Alas1 expression was blocked, adipogenesis was severely attenuated, indicating that induction of expression of Alas1 and the increase in heme synthesis is critical for differentiation. Inhibition of heme synthesis during adipogenesis leads to decreased recruitment of nuclear receptor corepressor to the promoter of a REV-ERB target gene, suggesting alteration of REV-ERB activity. Treatment of 3T3-L1 cells with a synthetic REV-ERB ligand, SR6452, resulted in induction of adipocyte differentiation to a similar extent as treatment with the peroxisomal proliferator-activated receptor-gamma agonist, rosiglitazone. Combination of SR6452 and rosiglitazone had an additive effect on stimulation of adipocyte differentiation. These results suggest that heme, functioning as a REV-ERB ligand, is an important signaling molecule for induction of adipogenesis. Moreover, synthetic small molecule ligands for REV-ERB are effective modulators of adipogenesis and may be useful for treatment of metabolic diseases.

Human Adenovirus Type 36 Enhances Glucose Uptake in Diabetic and Nondiabetic Human Skeletal Muscle Cells Independent of Insulin Signaling

OBJECTIVE—Human adenovirus type 36 (Ad-36) increases adiposity but improves insulin sensitivity in experimentally infected animals. We determined the ability of Ad-36 to increase glucose uptake by human primary skeletal muscle (HSKM) cells. RESEARCH DESIGN AND METHODS—The effect of Ad-36 on glucose uptake and cell signaling was determined in HSKM cells obtained from type 2 diabetic and healthy lean subjects. Ad-2, another human adenovirus, was used as a negative control. Gene expression and proteins of GLUT1 and GLUT4 were measured by real-time PCR and Western blotting. Role of insulin and Ras signaling pathways was determined in Ad-36–infected HSKM cells. RESULTS—Ad-36 and Ad-2 infections were confirmed by the presence of respective viral mRNA and protein expressions. In a dose-dependent manner, Ad-36 significantly increased glucose uptake in diabetic and nondiabetic HSKM cells. Ad-36 increased gene expression and protein abundance of GLUT1 and GLUT4, GLUT4 translocation to plasma membrane, and phosphatidylinositol 3-kinase (PI 3-kinase) activity in an insulin-independent manner. In fact, Ad-36 decreased insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and IRS-1–and IRS-2–associated PI 3-kinase activities. On the other hand, Ad-36 increased Ras gene expression and protein abundance, and Ras siRNA abrogated Ad-36–induced PI 3-kinase activation, GLUT4 protein abundance, and glucose uptake. These effects were not observed with Ad-2 infection. CONCLUSIONS—Ad-36 infection increases glucose uptake in HSKM cells via Ras-activated PI 3-kinase pathway in an insulin-independent manner. These findings may provide impetus to exploit the role of Ad-36 proteins as novel therapeutic targets for improving glucose handling.

Metabolically Favorable Remodeling of Human Adipose Tissue by Human Adenovirus Type 36

OBJECTIVE—Experimental infection of rats with human adenovirus type 36 (Ad-36) promotes adipogenesis and improves insulin sensitivity in a manner reminiscent of the pharmacologic effect of thiozolinediones. To exploit the potential of the viral proteins as a therapeutic target for treating insulin resistance, this study investigated the ability of Ad-36 to induce metabolically favorable changes in human adipose tissue. RESEARCH DESIGN AND METHODS—We determined whether Ad-36 increases glucose uptake in human adipose tissue explants. Cell-signaling pathways targeted by Ad-36 to increase glucose uptake were determined in the explants and human adipose–derived stem cells. Ad-2, a nonadipogenic human adenovirus, was used as a negative control. As a proof of concept, nondiabetic and diabetic subjects were screened for the presence of Ad-36 antibodies to ascertain if natural Ad-36 infection predicted improved glycemic control. RESULTS—Ad-36 increased glucose uptake by adipose tissue explants obtained from nondiabetic and diabetic subjects. Without insulin stimulation, Ad-36 upregulated expressions of several proadipogenic genes, adiponectin, and fatty acid synthase and reduced the expression of inflammatory cytokine macrophage chemoattractant protein-1 in a phosphotidylinositol 3-kinase (PI3K)-dependent manner. In turn, the activation of PI3K by Ad-36 was independent of insulin receptor signaling but dependent on Ras signaling recruited by Ad-36. Ad-2 was nonadipogenic and did not increase glucose uptake. Natural Ad-36 infection in nondiabetic and diabetic subjects was associated with significantly lower fasting glucose levels and A1C, respectively. CONCLUSIONS—Ad-36 proteins may provide novel therapeutic targets that remodel human adipose tissue to a more metabolically favorable profile.

Relationship between circadian oscillations of Rev-erbα expression and intracellular levels of its ligand, heme

The nuclear hormone receptors, REV-ERBalpha [NR1D1] and REV-ERBbeta [NR1D1], were recently demonstrated to be receptors for the porphyrin, heme. Heme regulates the ability of these receptors to repress transcription of their target genes via modulation of the affinity of the receptors ligand binding domain for the corepressor, NCoR. The REV-ERBs function as critical components of the mammalian clock and their expression oscillates in a circadian manner. Here, we show that in NIH3T3 cells intracellular heme levels also oscillate in a circadian fashion. These data are the first to show the temporal relationship of intracellular heme levels to the expression of its receptor, Rev-erbalpha, and suggest that the rapid oscillations in heme levels may an important component regulating REV-ERB transcriptional activity.

Adipogenic Cascade Can Be Induced Without Adipogenic Media by a Human Adenovirus

Several metabolic abnormalities are associated with relative excess or deficiency of adipose tissue. Identifying the regulators of adipogenic differentiation is critical for its successful manipulation. Ad36, a human adenovirus, is a novel factor that promotes adipogenesis. We exploited the adipogenic potential of Ad36 to reveal exogenous modifiers of adipogenesis in rodent preadipocyte cell line in the presence or absence of differentiation inducers methyl‐isobutyl‐xanthine, dexamethasone, and insulin (M, D, and I; MDI). A nonadipogenic human adenovirus Ad2 was used as a negative control for viral infection. First, we confirmed that, Ad36, but not Ad2, increases lipid accumulation in the presence or absence of MDI. Time‐course studies for expression of key genes of adipogenic cascade showed that it is Ad36, but not Ad2, which downregulated preadipocyte marker gene Wnt10b, and upregulated expression of early (C/EBPΔ and C/EBPβ), intermediate (PPARγ2), and late genes (aP2 and G3PDH) of adipogenic cascade even in the absence of MDI. In the presence of MDI, onset of expression of adipogenic genes coincided for Ad36 and control groups, but the expressions were significantly greater for the Ad36 group. Next, we observed that attenuation of Ad36 mRNA expression by an antiadenoviral agent reduced 3T3‐L1 differentiation, indicating that viral mRNA expression is required for the process. Furthermore, with or without MDI or its components, Ad36 significantly increased lipid accumulation in 3T3‐L1 cells. Cell confluency at the time of Ad36 infection positively influenced lipid accumulation. The results reveal that Ad36 is an MDI‐independent exogenous regulator of the adipogenic process. Elucidating the molecular pathways involved may reveal novel regulatory controls of adipogenesis.

The Selective Alzheimer's Disease Indicator-1 Gene (Seladin-1/DHCR24) Is a Liver X Receptor Target Gene

The nuclear hormone receptors liver X receptor α (LXRα) and LXRβ function as physiological receptors for oxidized cholesterol metabolites (oxysterols) and regulate several aspects of cholesterol and lipid metabolism. Seladin-1 was originally identified as a gene whose expression was down-regulated in regions of the brain associated with Alzheimers disease. Seladin-1 has been demonstrated to be neuroprotective and was later characterized as 3β-hydroxysterol-Δ24 reductase (DHCR24), a key enzyme in the cholesterologenic pathway. Seladin-1 has also been shown to regulate lipid raft formation. In a whole genome screen for direct LXRα target genes, we identified an LXRα occupancy site within the second intron of the Seladin-1/DHCR24 gene. We characterized a novel LXR response element within the second intron of this gene that is able to confer LXR-specific ligand responsiveness to reporter gene in both HepG2 and human embryonic kidney 293 cells. Furthermore, we found that Seladin-1/DHCR24 gene expression is significantly decreased in skin isolated from LXRβ-null mice. Our data suggest that Seladin-1/DHCR24 is an LXR target gene and that LXR may regulate lipid raft formation.

REGULATION OF HUMAN 3α-HYDROXYSTEROID DEHYDROGENASE (AKR1C4) EXPRESSION BY THE LIVER X RECEPTOR α

Type I human hepatic 3α-hydroxysteroid dehydrogenase (AKR1C4) plays a significant role in bile acid biosynthesis, steroid hormone metabolism, and xenobiotic metabolism. Utilization of a hidden Markov model for predictive modeling of nuclear hormone receptor response elements coupled with chromatin immunoprecipitation/microarray technology revealed a putative binding site in the AKR1C4 promoter for the nuclear hormone receptor known as liver X receptor α, (LXRα [NR1H3]), which is the physiological receptor for oxidized cholesterol metabolites. The putative LXRα response element (LXRE), identified by chromatin immunoprecipitation, was ∼1.5 kilobase pairs upstream of the transcription start site. LXRα was shown to bind specifically to this LXRE and mediate transcriptional activation of the AKR1C4 gene, leading to increased AKR1C4 protein expression. These data suggest that LXRα may modulate the bile acid biosynthetic pathway at a unique site downstream of CYP7A1 and may also modulate the metabolism of steroid hormones and certain xenobiotics.