Anthony N. Hollenberg

Glucagon-like peptide-1 receptor stimulation increases blood pressure and heart rate and activates autonomic regulatory neurons

Glucagon-like peptide-1 (GLP-1) released from the gut functions as an incretin that stimulates insulin secretion. GLP-1 is also a brain neuropeptide that controls feeding and drinking behavior and gastric emptying and elicits neuroendocrine responses including development of conditioned taste aversion. Although GLP-1 receptor (GLP-1R) agonists are under development for the treatment of diabetes, GLP-1 administration may increase blood pressure and heart rate in vivo. We report here that centrally and peripherally administered GLP-1R agonists dose-dependently increased blood pressure and heart rate. GLP-1R activation induced c-fos expression in the adrenal medulla and neurons in autonomic control sites in the rat brain, including medullary catecholamine neurons providing input to sympathetic preganglionic neurons. Furthermore, GLP-1R agonists rapidly activated tyrosine hydroxylase transcription in brainstem catecholamine neurons. These findings suggest that the central GLP-1 system represents a regulator of sympathetic outflow leading to downstream activation of cardiovascular responses in vivo.

Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling

Starvation causes a rapid reduction in thyroid hormone levels in rodents. This adaptive response is caused by a reduction in thyrotropin-releasing hormone (TRH) expression that can be reversed by the administration of leptin. Here we examined hypothalamic signaling pathways engaged by leptin to upregulate TRH gene expression. As assessed by leptin-induced expression of suppressor of cytokine signaling-3 (SOCS-3) in fasted rats, TRH neurons in the paraventricular nucleus are activated directly by leptin. To a greater degree, they also contain melanocortin-4 receptors (MC4Rs), implying that leptin can act directly or indirectly by increasing the production of the MC4R ligand, alpha-melanocyte stimulating hormone (alpha-MSH), to regulate TRH expression. We further demonstrate that both pathways converge on the TRH promoter. The melanocortin system activates the TRH promoter through the phosphorylation and DNA binding of the cAMP response element binding protein (CREB), and leptin signaling directly regulates the TRH promoter through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). Indeed, a novel Stat-response element in the TRH promoter is necessary for leptins effect. Thus, the TRH promoter is an ideal target for further characterizing the integration of transcriptional pathways through which leptin acts.

Ligand-independent Activation Domain in the N Terminus of Peroxisome Proliferator-activated Receptor γ (PPARγ) DIFFERENTIAL ACTIVITY OF PPARγ1 AND -2 ISOFORMS AND INFLUENCE OF INSULIN

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear hormone receptor superfamily, and is an important regulator of adipogenesis and adipocyte gene expression. PPARγ exists as two isoforms, PPARγ1 and PPARγ2, that differ only in their N termini. Both isoforms are activated by ligands that include the antidiabetic thiazoladinedione drugs and 15-deoxy-Δ12, 14-prostaglandin J2, and potential differences in their function have yet to be described. We report that, in addition to a ligand-activated transcriptional activity, when studied under conditions of ligand depletion, intact PPARγ has a ligand-independent activation domain. To identify the basis for this ligand-independent activation, we used GAL4-PPARγ chimeric expression constructs and UAS-TK-LUC in CV1 cells and isolated rat adipocytes. In both cell systems, isolated PPARγ1 and PPARγ2 N termini have activation domains, and the activation function of PPARγ2 is 5–6-fold greater than that of PPARγ1. Insulin enhances the transcriptional effect mediated by both PPARγ1 and PPARγ2 N-terminal domains. These data demonstrate that 1) PPARγ has an N-terminal (ligand-independent) activation domain; 2) PPARγ1 and PPARγ2 N termini have distinct activation capacities; and 3) insulin can potentiate the activity of the N-terminal domain of PPARγ.

FUNCTIONAL ANTAGONISM BETWEEN CCAAT/ENHANCER BINDING PROTEIN-ALPHA AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-GAMMA ON THE LEPTIN PROMOTER

The ob gene product, leptin, is a major hormonal regulator of appetite and fat cell mass. Recent work has suggested that the antidiabetic agents, the thiazolidinediones (TZ), which are also high affinity ligands of peroxisome proliferator-activated receptor-γ (PPARγ), inhibit leptin expression in rodents. To examine the effects of this class of drug on the leptin gene in adipocytes we performed Northern analysis on primary rat adipocytes cultured in the presence or absence of TZ. TZ reduced leptin mRNA levels by 75%. To determine whether this effect was mediated at the transcriptional level, we isolated 6510 base pairs of 5′-flanking sequence of the leptin promoter and studied reporter constructs in primary rat adipocytes and CV-1 cells. Sequence analysis demonstrated the presence of a consensus direct repeat with a 1-base-pair gap site between −3951 and −3939 as well as a consensus CCAAT/enhancer binding protein (C/EBP) site between −55 and −47. Our functional analysis in transfected primary rat adipocytes demonstrates that, despite the presence of a canonical direct repeat with a 1-base-pair gap site, TZ alone decreases reporter gene expression of leptin promoter constructs ranging from −6510 to +9 to −65 to +9. In CV-1 cells, which contain endogenous PPARγ, TZ treatment alone had little effect on these constructs. However, TZ treatment did inhibit C/EBPα-mediated transactivation of the leptin promoter. This down-regulation of leptin reporter constructs mapped to a −65 to +9 promoter fragment which binds C/EBPα in gel-mobility shift assays but does not bind PPARγ2 alone or as a heterodimer with 9-cis-retinoic acid receptor. Conversely, the promoter (−5400 to +24 base pairs) of the aP2 gene, another adipocyte-specific gene, was induced 7.3-fold by TZ. Co-transfection with C/EBPα minimally stimulated the aP2 promoter from basal levels but notably blocked activation by TZ. These data indicate that PPARγ and C/EBPα can functionally antagonize each other on at least two separate promoters and that this mechanism may explain the down-regulation of leptin expression by thiazolidinediones.

Leptin, nutrition, and the thyroid: the why, the wherefore, and the wiring

The function of the thyroid gland is to produce the thyroid hormones T3 and T4, which regulate gene transcription throughout the body (1). In medical practice, the thyroid becomes an issue when its size or shape becomes abnormal or when it produces too much or too little hormone. Thus, we typically think of the thyroid with reference to the clinical states of goiter, or hyper- or hypothyroidism. But what is the physiology of the thyroid when the gland and the entire hypothalamic-pituitary-thyroid axis are intact? As first year medical students ask each year, Why exactly do we have a thyroid, at all?

Leptin Regulates Prothyrotropin-releasing Hormone Biosynthesis EVIDENCE FOR DIRECT AND INDIRECT PATHWAYS

The hypothalamic-pituitary-thyroid axis is down-regulated during starvation, and falling levels of leptin are a critical signal for this adaptation, acting to suppress preprothyrotropin-releasing hormone (prepro-TRH) mRNA expression in the paraventricular nucleus of the hypothalamus. This study addresses the mechanism for this regulation, using primary cultures of fetal rat hypothalamic neurons as a model system. Leptin dose-dependently stimulated a 10-fold increase in pro-TRH biosynthesis, with a maximum response at 10 nm. TRH release was quantified using immunoprecipitation, followed by isoelectric focusing gel electrophoresis and specific TRH radioimmunoassay. Leptin stimulated TRH release by 7-fold. Immunocytochemistry revealed that a substantial population of cells expressed TRH or leptin receptors and that 8–13% of those expressing leptin receptors coexpressed TRH. Leptin produced a 5-fold induction of luciferase activity in CV-1 cells transfected with a TRH promoter and the long form of the leptin receptor cDNA. Although the above data are consistent with a direct ability of leptin to promote TRH biosynthesis through actions on TRH neurons, addition of α-melanocyte-stimulating hormone produced a 3.5-fold increase in TRH biosynthesis and release, whereas neuropeptide Y treatment suppressed pro-TRH biosynthesis ∼3-fold. Furthermore, the melanocortin-4 receptor antagonist SHU9119 partially inhibited leptin-stimulated TRH release from the neuronal culture. Consequently, our data suggest that leptin regulates the TRH neurons through both direct and indirect pathways.

Regulated Production of a Peroxisome Proliferator-activated Receptor-γ Ligand during an Early Phase of Adipocyte Differentiation in 3T3-L1 Adipocytes

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear hormone receptor that is critical for adipogenesis and insulin sensitivity. Ligands for PPARγ include some polyunsaturated fatty acids and prostanoids and the synthetic high affinity antidiabetic agents thiazolidinediones. However, the identity of a biologically relevant endogenous PPARγ ligand is unknown, and limited insight exists into the factors that may regulate production of endogenous PPARγ ligands during adipocyte development. To address this question, we created a line of 3T3-L1 preadipocytes that carry a β-galactosidase-based PPARγ ligand-sensing vector system. In this system, induction of adipogenesis resulted in elevated β-galactosidase activity that signifies activation of PPARγ via its ligand-binding domain (LBD) and suggests generation and/or accumulation of a ligand moiety. The putative endogenous ligand appeared early in adipogenesis in response to increases in cAMP, accumulated in the medium, and dissipated later in adipogenesis. Organically extracted and high pressure liquid chromatography-fractionated conditioned media from differentiating cells, but not from mature adipocytes, were enriched in this activity. One or more components within the organic extract activated PPARγ through interaction with its LBD, induced lipid accumulation in 3T3-L1 cells as efficiently as the differentiation mixture, and competed for binding of rosiglitazone to the LBD of PPARγ. The active species appears to be different from other PPARγ ligands identified previously. Our findings suggest that a novel biologically relevant PPARγ ligand is transiently produced in 3T3-L1 cells during adipogenesis.

Complex Role of the Vitamin D Receptor and Its Ligand in Adipogenesis in 3T3-L1 Cells

The vitamin D receptor (VDR) and its ligand 1,25-OH2-VD3 (calcitriol) play an essential role in mineral homeostasis in mammals. Interestingly, the VDR is expressed very early in adipogenesis in 3T3-L1 cells, suggesting that the VDR signaling pathway may play a role in adipocyte biology and function. Indeed, it has been known for a number of years that calcitriol is a potent inhibitor of adipogenesis in this model but with no clear mechanism identified. In this study, we have further defined the molecular mechanism by which the unliganded VDR and calcitriol-liganded VDR regulate adipogenesis. In the presence of calcitriol, the VDR blocks adipogenesis by down-regulating both C/EBPβ mRNA expression and C/EBPβ nuclear protein levels at a critical stage of differentiation. In addition, calcitriol allows for the up-regulation of the recently described C/EBPβ corerepressor, ETO, which would further inhibit the action of any remaining C/EBPβ, whose action is required for adipogenesis. In contrast, in the absence of calcitriol, the unliganded VDR appears necessary for lipid accumulation, since knock-down of the VDR using siRNA both delays and prevents this process. Taken together, these data support the notion that the intracellular concentrations of calcitriol can play an important role in either promoting or inhibiting adipogenesis via the VDR and the transcriptional pathways that it targets. Further examination of this hypothesis in vivo may shed new light on the biology of adipogenesis.

DEFECTIVE RELEASE OF COREPRESSOR BY HINGE MUTANTS OF THE THYROID HORMONE RECEPTOR FOUND IN PATIENTS WITH RESISTANCE TO THYROID HORMONE

On positive thyroid hormone response elements (pTREs), thyroid hormone receptor (TR) binding to DNA in the absence of ligand (thyroid hormone, T3) decreases transcription (silencing). Silencing is due to a family of recently described nuclear corepressor proteins (NCoR and SMRT) which bind to the CoR box in the hinge region of TR. Ligand-dependent activation of TR is associated with displacement of corepressors and recruitment of coactivating proteins. Resistance to thyroid hormone (RTH) is due to mutations in the β isoform of the thyroid hormone receptor (TR-β). To date, three RTH mutations reportedly with near-normal T3binding (A234T, R243Q, and R243W) have been described in or near the CoR box. To determine the mechanism of RTH caused by these mutants, the interaction of wild type (wt) and mutant TRs with the corepressor, NCoR, and the coactivator, SRC-1, was tested in gel-shift assays. As expected, NCoR bound wt TR in the absence of T3 and dissociated from TR with increasing T3 concentration. SRC-1 failed to bind wt TR in the absence of T3, but bound to TR with increasing avidity as T3 concentrations rose. At no T3 concentration did both NCoR and SRC-1 bind to wt TR, indicating that their binding to TR was mutually exclusive. Hinge mutants bound NCoR normally in the absence of T3; however, dissociation of NCoR and recruitment of SRC-1 was markedly impaired except at very high T3 concentrations. Importantly, hinge mutant TRs when complexed to DNA bound T3 poorly despite their near-normal T3 binding in solution. These binding studies correlated with functional assays showing defective transactivation of pTREs by hinge mutants except at high T3concentrations. Thus, we describe a novel mechanism of RTH whereby TR hinge mutants selectively affect T3 binding when complexed to DNA, and prevent NCoR dissociation from TR. Our data also suggest that solution T3 binding by RTH mutants may not accurately reflect physiologically relevant T3 binding by TR when bound to DNA.

The Androgen Receptor Recruits Nuclear Receptor CoRepressor (N-CoR) in the Presence of Mifepristone via Its N and C Termini Revealing a Novel Molecular Mechanism for Androgen Receptor Antagonists

The androgen receptor (AR) activates target gene expression in the presence of agonist ligands via the recruitment of transcriptional coactivators, but recent work shows that overexpression of the nuclear corepressors NCoR and SMRT attenuates this agonist-mediated AR activation. Here we demonstrate using NCoR siRNA and chromatin immunoprecipitation that endogenous NCoR is recruited to and represses the dihydrotestosterone (DHT)-liganded AR. Furthermore this study shows that NCoR and coactivators compete for AR in the presence of DHT. AR antagonists such as bicalutamide that are currently in use for prostate cancer treatment can also mediate NCoR recruitment, but mifepristone (RU486) at nanomolar concentrations is unique in its ability to markedly enhance the AR-NCoR interaction. The RU486-liganded AR interacted with a C-terminal fragment of NCoR, and this interaction was mediated by the two most C-terminal nuclear receptor interacting domains (RIDs) present in NCoR. Significantly, in addition to the AR ligand binding domain, the AR N terminus was also required for this interaction. Mutagenesis studies demonstrate that the N-terminal surface of the AR-mediating NCoR recruitment was distinct from tau5 and from the FXXLF motif that mediates agonist-induced N-C-terminal interaction. Taken together these data demonstrate that NCoR is a physiological regulator of the AR and reveal a new mechanism for AR antagonism that may be exploited for the development of more potent AR antagonists.