Thyroid hormone receptor-ß agonists prevent hepatic steatosis in fat-fed rats but impair insulin sensitivity via discrete pathways.

Liver-specific thyroid hormone receptor-ß (TRß)-specific agonists are potent lipid-lowering drugs that also hold promise for treating nonalcoholic fatty liver disease and hepatic insulin resistance. We investigated the effect of two TRß agonists (GC-1 and KB-2115) in high-fat-fed male Sprague-Dawley rats treated for 10 days. GC-1 treatment reduced hepatic triglyceride content by 75%, but the rats developed fasting hyperglycemia and hyperinsulinemia, attributable to increased endogenous glucose production (EGP) and diminished hepatic insulin sensitivity. GC-1 also increased white adipose tissue lipolysis; the resulting increase in glycerol flux may have contributed to the increase in EGP. KB-2115, a more TRß- and liver-specific thyromimetic, also prevented hepatic steatosis but did not induce fasting hyperglycemia, increase basal EGP rate, or diminish hepatic insulin sensitivity. Surprisingly, insulin-stimulated peripheral glucose disposal was diminished because of a decrease in insulin-stimulated skeletal muscle glucose uptake. Skeletal muscle insulin signaling was unaffected. Instead, KB-2115 treatment was associated with a decrease in GLUT4 protein content. Thus, although both GC-1 and KB-2115 potently treat hepatic steatosis in fat-fed rats, they each worsen insulin action via specific and discrete mechanisms. The development of future TRß agonists must consider the potential adverse effects on insulin sensitivity.

Sitagliptin reduces hyperglycemia and increases satiety hormone secretion more effectively when used with a novel polysaccharide in obese Zucker rats.

The novel polysaccharide (NPS) PolyGlycopleX (PGX) has been shown to reduce glycemia. Pharmacological treatment with sitagliptin, a dipeptidyl peptidase 4 (DPP4) inhibitor, also reduces glycemia by increasing glucagon-like peptide-1 (GLP-1). Our objective was to determine if using NPS in combination with sitagliptin reduces hyperglycemia in Zucker diabetic fatty (ZDF) rats more so than either treatment alone. Male ZDF rats were randomized to: 1) cellulose/vehicle [control (C)]; 2) NPS (5% wt:wt)/vehicle (NPS); 3) cellulose/sitagliptin [10 mg/(kg · d) (S)]; or 4) NPS (5%) + S [10 mg/(kg · d) (NPS+S)]. Glucose tolerance, adiposity, satiety hormones, and mechanisms related to DPP4 activity and hepatic and pancreatic histology were examined. A clinically relevant reduction in hyperglycemia occurred in the rats treated with NPS+S (P = 0.001) compared with NPS and S alone. Blood glucose, measured weekly in fed and feed-deprived rats and during an oral glucose tolerance test, was lower in the NPS+S group compared with all other groups (all P = 0.001). At wk 6, glycated hemoglobin was lower in the NPS+S group than in the C and S (P = 0.001) and NPS (P = 0.06) groups. PGX (P = 0.001) and S (P = 0.014) contributed to increased lean mass. Active GLP-1 was increased by S (P = 0.001) and GIP was increased by NPS (P = 0.001). Plasma DPP4 activity was lower in the NPS+S and S groups than in the NPS and C groups (P = 0.007). Insulin secretion and ß-cell mass was increased with NPS (P < 0.05). NPS alone reduced LDL cholesterol and hepatic steatosis (P < 0.01). Independently, NPS and S improve several metabolic outcomes in ZDF rats, but combined, their ability to markedly reduce glycemia suggests they may be a promising dietary/pharmacological co-therapy for type 2 diabetes management.

Energetic signalling in the control of mitochondrial F1F0 ATP synthase activity in health and disease.

The mitochondrial F1F0 ATP synthase is a critical enzyme that works by coupling the proton motive force generated by the electron transport chain via proton transfer through the F0 or proton-pore forming domain of this enzyme to release ATP from the catalytic F1 domain. This enzyme is regulated by calcium, ADP, and inorganic phosphate as well as increased transcription through several pathways. This enzyme is also an ATP hydrolase under ischemic conditions. This "inefficient" hydrolysis of ATP consumes 90% of ATP consumed during ischemia as shown with non-selective ATPase inhibitors oligomycin and Aurovertin B. A benzopyran analog, BMS-199264, selectively inhibits F1F0 ATP hydrolase activity with no effect on ATP synthase activity. BMS-199264 had no effect on ATP before ischemia, but reduced the decline in ATP during ischemia. Selective hydrolase inhibition seen with the small molecule BMS-199264 suggests a conformational change in the F1F0 ATPase enzyme when switching from synthase to hydrolase activity.

Anti-obesity, anti-diabetic, and lipid lowering effects of the thyroid receptor beta subtype selective agonist KB-141.

Selective thyroid hormone receptor subtype-beta (TRbeta) agonists have received attention as potential treatments for hypercholesterolemia and obesity, but have received less attention as treatments for diabetes, partly because this condition is not improved in thyroid hormone excess states. The TRbeta selective agonist KB-141 induces 5-10% increases in metabolic rate and lowering of plasma cholesterol levels without tachycardia in lean rats, unlike the major active thyroid hormone, T3. In the current study, we determined whether KB-141 promotes weight loss in obese animals and whether it exhibits anti-diabetogenic effects. Body weight, adiposity (DEXA), and lipid levels were examined following p.o. administration of KB-141 to obese Zucker fa/fa rats at 0.00547-0.547 mg/kg/day for 21 days, and in ob/ob mice at 0.5mg/kg/day KB-141 for 7 days. In rats, KB-141 reduced body weight by 6 and 8%, respectively, at 0.167 and 0.0547 mg/kg/day without tachycardia and adiposity was reduced at 0.167 mg/kg/day (5-6%). In ob/ob mice, KB-141 lowered serum cholesterol (35%), triacylglycerols (35%) and both serum and hepatic free fatty acids (18-20%) without tachycardia. Treatment of ob/ob mice with KB-141 (0.0547 or 0.328 mg/kg/day over 2 weeks) improved glucose tolerance and insulin sensitivity in a dose-dependent manner with no effect on heart rate. Thus, KB-141 elicits anti-obesity, lipid lowering and anti-diabetic effects without tachycardia suggesting that selective TRbeta activation may be useful strategy to attenuate features of the metabolic syndrome.

Pharmacological and x-ray structural characterization of a novel selective androgen receptor modulator: potent hyperanabolic stimulation of skeletal muscle with hypostimulation of prostate in rats.

A novel, highly potent, orally active, nonsteroidal tissue selective androgen receptor (AR) modulator (BMS-564929) has been identified, and this compound has been advanced to clinical trials for the treatment of age-related functional decline. BMS-564929 is a subnanomolar AR agonist in vitro, is highly selective for the AR vs. other steroid hormone receptors, and exhibits no significant interactions with SHBG or aromatase. Dose response studies in castrated male rats show that BMS-564929 is substantially more potent than testosterone (T) in stimulating the growth of the levator ani muscle, and unlike T, highly selective for muscle vs. prostate. Key differences in the binding interactions of BMS-564929 with the AR relative to the native hormones were revealed through x-ray crystallography, including several unique contacts located in specific helices of the ligand binding domain important for coregulatory protein recruitment. Results from additional pharmacological studies effectively exclude alternative mechanistic contributions to the observed tissue selectivity of this unique, orally active androgen. Because concerns regarding the potential hyperstimulatory effects on prostate and an inconvenient route of administration are major drawbacks that limit the clinical use of T, the potent oral activity and tissue selectivity exhibited by BMS-564929 are expected to yield a clinical profile that provides the demonstrated beneficial effects of T in muscle and other tissues with a more favorable safety window.

Discovery of potent and muscle selective androgen receptor modulators through scaffold modifications.

A novel series of imidazolin-2-ones were designed and synthesized as highly potent, orally active and muscle selective androgen receptor modulators (SARMs), with most of the compounds exhibiting low nM in vitro potency in androgen receptor (AR) binding and functional assays. Once daily oral treatment with the lead compound 11a (AR Ki = 0.9 nM, EC50 = 1.8 nM) for 14 days induced muscle growth with an ED50 of 0.09 mg/kg, providing approximately 50-fold selectivity over prostate growth in an orchidectomized rat model. Pharmacokinetic studies in rats demonstrated that the lead compound 11a had oral bioavailability of 65% and a plasma half-life of 5.5 h. On the basis of their preclinical profiles, the SARMs in this series are expected to provide beneficial anabolic effects on muscle with minimal androgenic effects on prostate tissue.

Therapeutic potential for thyroid hormone receptor-beta selective agonists for treating obesity, hyperlipidemia and diabetes.

Obesity and metabolic syndrome are increasing dramatically worldwide, contributing to cardiovascular morbidity and mortality. There are currently few safe and efficacious therapeutics for obesity and most strategies are focused on appetite suppression. Thyroid hormones reduce adiposity via increased metabolic rate, but unfortunately they cause large changes in metabolic rate and direct cardiac acceleration, making them useless for treating obesity. Thyroid hormone receptors (TRs) work as transcription factors and two subtypes exist: TRalpha and TRbeta. TRalpha mediates tachycardia and much of the metabolic rate effect, while TRbeta mediates cholesterol and TSH lowering effects of thyroid hormones. TRbeta activation modestly increases metabolic rate such that a therapeutic window of 5-10 fold increases in metabolic rate can be seen without tachycardia. This was initially studied in TRalpha(1)(-/-) mice. Recent structure activity work has resulted in the discovery of several TRbeta selective thyromimetics such as KB-141. Studies with KB-141 show that it has a 10-fold window in which therapeutic increases in metabolic rate are seen without tachycardia or cardiac hypertrophy. This agent lowers cholesterol in rats and primates. In primates, KB-141 causes significant weight and cholesterol reduction in addition to the independent risk factor Lp(a). These effects were seen without any effect on heart rate, unlike thyroid hormone (T(3)). Further work with TRbeta selective agents is warranted and recent work suggests the possibility of developing compounds that selectively penetrate different tissues which may have an even more desirable therapeutic window. Selective thyromimetics, therefore, may be useful as adjunctive therapy to appetite suppressants along with exercise and diet restriction.

The isolated, perfused pseudo-working heart model.

Increased interest in cardiac safety and renewed interest in drugs for treating myocardial ischemia and congestive heart failure have led to increased use of cardiovascular models. Unfortunately, many molecular or cell-based screens are not perfect predictors of activity in vivo. One rapid means to test "proof of principle" in hearts is the isolated or Langendorff perfused heart system. This allows direct measurement of cardiac contractile function and coronary flow without interference from changes in the systemic circulation. The setup basically is a heart whose coronary arteries are retrogradely perfused through the aortic root with heated and oxygenated buffer solutions. We will describe the constant pressure technique as this is the most commonly used and allows us to directly measure changes in coronary flow without changes in turgor noted with constant flow methods. Instead of a classical working heart preparation, we describe the use of a balloon properly fitted into the left ventricle to measure end diastolic and peak systolic pressure making this a much simpler and less expensive technique without loss of scientific quality. Such a setup can be used to measure effects of compounds or treatments on coronary flow, contractile function either in the normal or pathological states.

Thyroid receptor ligands. 6. A high affinity "direct antagonist" selective for the thyroid hormone receptor.

A new high-affinity thyroid hormone antagonist 6 with druglike properties was designed and synthesized. The compound behaved as an antagonist in a cell transactivation assay, and in a first in vivo experiment in rats.

Discovery of potent, orally-active, and muscle-selective androgen receptor modulators based on an N-aryl-hydroxybicyclohydantoin scaffold.

A novel, N-aryl-bicyclohydantoin selective androgen receptor modulator scaffold was discovered through structure-guided modifications of androgen receptor antagonists. A prototype compound (7R,7aS)-10b from this series is a potent and highly tissue-selective agonist of the androgen receptor. After oral dosing in a rat atrophied levator ani muscle model, (7R,7aS)-10b demonstrated efficacy at restoring levator ani muscle mass to that of intact controls and exhibited >50-fold selectivity for muscle over prostate.

Reduction of skeletal muscle atrophy by a proteasome inhibitor in a rat model of denervation.

The ubiquitin-proteasome system is the primary proteolytic pathway implicated in skeletal muscle atrophy under catabolic conditions. Although several studies showed that proteasome inhibitors reduced proteolysis under catabolic conditions, few studies have demonstrated the ability of these inhibitors to preserve skeletal muscle mass and architecture in vivo. To explore this, we studied the effect of the proteasome inhibitor Velcade (also known as PS-341 and bortezomib) in denervated skeletal muscle in rats. Rats were given vehicle or Velcade (3 mg/kg po) daily for 7 days beginning immediately after induction of muscle atrophy by crushing the sciatic nerve. At the end of the study, the rats were euthanized and the soleus and extensor digitorum longus (EDL) muscles were harvested. In vehicle-treated rats, denervation caused a 33.5 +/- 2.8% and 16.2 +/- 2.7% decrease in the soleus and EDL muscle wet weights (% atrophy), respectively, compared to muscles from the contralateral (innervated) limb. Velcade significantly reduced denervation-induced atrophy to 17.1 +/- 3.3% in the soleus (P < 0.01), a 51.6% reduction in atrophy associated with denervation, with little effect on the EDL (9.8 +/- 3.2% atrophy). Histology showed a preservation of muscle mass and preservation of normal cellular architecture after Velcade treatment. Ubiquitin mRNA levels in denervated soleus muscle at the end of the study were significantly elevated 120 +/- 25% above sham control levels and were reduced to control levels by Velcade. In contrast, testosterone proprionate (3 mg/kg sc) did not alleviate denervation-induced skeletal muscle atrophy but did prevent castration-induced levator ani atrophy, while Velcade was without effect. These results show that proteasome inhibition attenuates denervation-induced muscle atrophy in vivo in soleus muscles. However, this mechanism may not be operative in all types of atrophy.

Cardioselective sulfonylthiourea HMR 1098 blocks mitochondrial uncoupling induced by a KATP channel opener, P-1075, in beating rat hearts.

We investigated effects of blockade of cardiac ATP-sensitive potassium channels (KATP) with a novel cardioselective sulfonylthiourea, HMR 1098, on metabolic uncoupling caused by a potent KATP opener, P-1075, in Langendorff-perfused rat hearts. We used (1) 87Rb-NMR to detect activation-deactivation of sarcolemmal KATP, (2) 31P-NMR to monitor high-energy phosphates, (3) oxygen uptake measurements to monitor cellular respiration, and (4) myocardial optical absorbance measurements at 603 nm to follow changes in cytochrome c oxidase redox state. Activation of sarcolemmal KATP by P-1075 (5 microM) and a mitochondrial uncoupler 2,4-dinitrophenol (DNP) (50 microM) stimulated Rb+ efflux from the hearts by 130% and 60%, respectively. HMR 1098 (5 and 30 microM) blocked activation of sarcolemmal KATP in situ. HMR 1098 also prevented cardiac arrest and mitochondrial uncoupling induced by P-1075, such as (a) depletion of phosphocreatine and ATP by 40%, (b) two-fold decrease in venous oxygen, and (c) reduction of cytochrome c oxidase (demonstrated by an increase in 603 nm optical absorbance). The metabolic effects of P-1075 can be readily explained by activation of putative mitochondrial KATP. We concluded that blockade of mitochondrial uncoupling by HMR 1098 included an inhibiting effect of HMR 1098 on sarcolemmal and mitochondrial KATP in beating rat hearts.

Sarcolemmal and mitochondrial effects of a KATP opener, P-1075, in "polarized" and "depolarized" Langendorff-perfused rat hearts.

We investigated consequences of cardiac arrest on sarcolemmal and mitochondrial effects of ATP-sensitive potassium channel (KATP) opener, P-1075, in Langendorff-perfused rat hearts. Depolarised cardiac arrest (24.7 mM KCl) did not affect glibenclamide-sensitive twofold activation of rubidium efflux by P-1075 (5 microM) from rubidium-loaded hearts, but eliminated uncoupling produced by P-1075 in beating hearts: 40% depletion of phosphocreatine and ATP, 50% increase in oxygen consumption, and reduction of cytochrome c oxidase. Depolarized cardiac arrest by calcium channel blocker, verapamil (5 microM), also prevented uncoupling. Lack of P-1075 mitochondrial effects in depolarized hearts was not due to changes in phosphorylation potential, because 2,4-dintrophenol (10 microM) reversed the [PCr]/[Cr] increase and Pi decrease, characteristic of KCl-arrest, but did not restore uncoupling. In agreement with this conclusion, pyruvate (5 mM) increased [PCr]/[Cr] and decreased Pi, but did not prevent uncoupling in beating hearts. A decrease in mean [Ca2+] in KCl-arrested hearts could not account for lack of P-1075 mitochondrial effects, because calcium channel opener, S-(-)-Bay K8644 (50 nM), and beta-agonist, isoproterenol (0.5 microM), did not facilitate uncoupling. In contrast, in adenosine (1 mM)-arrested hearts (polarized arrest), P-1075 caused 40% phosphocreatine and ATP depletion. In isolated rat liver mitochondria, P-1075 (20 microM) decreased mitochondrial membrane potential (DeltaPsi) by approximately 14 mV (demonstrated by redistribution of DeltaPsi-sensitive dye, rhodamine 800) in a glibenclamide-sensitive manner. We concluded that cell membrane depolarization does not prevent activation of sarcolemmal KATP by P-1075, but it plays a role in mitochondrial uncoupling effects of P-1075.

Effects of mitochondrial K(ATP) modulators on cardioprotection induced by chronic high altitude hypoxia in rats.

OBJECTIVES: Adaptation of rats to intermittent high altitude hypoxia increases the tolerance of their hearts to acute ischemia/reperfusion injury. Our aim was to examine the role of mitochondrial ATP-sensitive potassium channels (K(ATP)) in this form of protection. METHODS: Adult male Wistar rats were exposed to hypoxia of 5000 m in a barochamber for 8 h/day, 5 days a week; the total number of exposures was 24-32. A control group was kept under normoxic conditions (200 m). Infarct size (tetrazolium staining) was measured in anesthetized open-chest animals subjected to 20-min regional ischemia (coronary artery occlusion) and 4-h reperfusion. Isolated perfused hearts were used to assess the recovery of contractile function following 20-min global ischemia and 40-min reperfusion. In the open-chest study, a selective mitochondrial K(ATP) blocker, 5-hydroxydecanoate (5 mg/kg), or openers, diazoxide (10 mg/kg) or BMS-191095 (10 mg/kg), were administered into the jugular vein 5 and 10 min before occlusion, respectively. In the isolated heart study, 5-hydroxydecanoate (250 micromol/l) or diazoxide (50 micromol/l) were added to the perfusion medium 5 or 10 min before ischemia, respectively. RESULTS: In the control normoxic group, infarct size occupied 62.2+/-2.0% of the area at risk as compared with 52.7+/-2.5% in the chronically hypoxic group (P<0.05). Post-ischemic recovery of contractile function (dP/dt) reached 60.0+/-3.9% of the pre-ischemic value and it was improved to 72.4+/-1.2% by adaptation to hypoxia (P<0.05). While 5-hydroxydecanoate completely abolished these protective effects of chronic hypoxia, it had no appreciable influence in normoxic groups. In contrast, diazoxide significantly increased the recovery of contractile function and reduced infarct size in normoxic groups only. The later effect was also observed following treatment with BMS-191095. CONCLUSION: The results suggest that opening of mitochondrial K(ATP) channels is involved in the cardioprotective mechanism conferred by long-term adaptation to intermittent high altitude hypoxia.

Effects of the thyroid hormone receptor agonist GC-1 on metabolic rate and cholesterol in rats and primates: selective actions relative to 3,5,3'-triiodo-L-thyronine.

Current drug therapies for obesity are ineffective, and existing treatments for lipid disorders can be further improved. Thyroid hormones affect both conditions, although currently available nonselective thyromimetics are not clinically useful for such treatment due to cardiac side effects. Recent studies suggest that thyroid hormone receptor subtype beta (TRbeta) selective agonists have a profile in which cholesterol can be reduced with minimal tachycardia. The purpose of this study was to determine whether modest (5-10%) increases in metabolic rate could also be observed with minimal tachycardia after TRbeta stimulation. For these studies, the TRbeta selective agonist, GC-1, was used to assess selectivity for lipid-lowering and metabolic rate changes relative to tachycardia. Studies in cholesterol-fed rats (7 d treatment) showed that GC-1 reduced cholesterol (ED(50) = 190 nmol/kg x d) approximately 30 times more potently than it induced tachycardia (ED(15) = 5451 nmol/kg x d). T(3) showed no potency difference between cholesterol lowering and tachycardia. GC-1 showed approximately 10-fold selectivity for increasing metabolic rate (ED(5) = 477 nmol/kg x d) relative to tachycardia compared with T(3), which showed no selectivity. In cynomolgus monkeys treated for 7 d, significant cholesterol-lowering and lipoprotein (a) reduction was noted for both T(3) and GC-1, whereas no tachycardia was observed for GC-1, unlike T(3). T(3) and GC-1 caused a significant (approximately 4%) reduction in body weight in these animals. Therefore, selective TRbeta activation may be a potentially usefully treatment for obesity and reduction of low density lipoprotein cholesterol and reduction of the atherogenic risk factor lipoprotein (a).

Small molecule mitochondrial F1F0 ATPase hydrolase inhibitors as cardioprotective agents. Identification of 4-(N-arylimidazole)-substituted benzopyran derivatives as selective hydrolase inhibitors.

In this paper we show that 4-aryl-CH2-imidazole-substituted benzopyran compounds with 3S,4R-stereochemistry are cardioprotective by inhibiting the F1F0 mitochondrial ATP hydrolase. Compounds (e.g., 13) with 3R,4S-stereochemistry act as mitochondrial KATP openers. This resulted from an inversion of stereochemistry for the F1F0 mitochondrial ATP hydrolase vs mitochondrial KATP. Structure-activity relationships for the inhibition of mitochondrial ATP hydrolase are also delineated. It is not clear how 13 (3R,4S) can selectively inhibit the hydrolytic activity of the F1F0 mitochondrial enzyme without interfering with the synthase activity.

The mitochondrial K(ATP) channel opener BMS-191095 induces neuronal preconditioning.

BMS-191095, reportedly a selective mitoK(ATP) channel opener which is free from the known side effects of the prototype mitoK(ATP) channel opener diazoxide, induced acute and delayed preconditioning against glutamate excitotoxicity and delayed preconditioning against oxygen-glucose deprivation in primary cultures of rat cortical neurons. BMS-191095 dose dependently depolarized the mitochondria, increased the phosphorylation of PKC isoforms, but had no detectable effects on the activation of MAP kinases and did not influence the expressions of HSP70 and Mn-SOD. In BMS-191095-preconditioned neurons the glutamate-induced free-radical production was abolished. Our data give the first evidence that selective opening of mitoK(ATP) channels with BMS-191095 leads to remarkable neuroprotection via mechanisms that involve mitochondrial depolarization, PKC activation and attenuated free radical production during neuronal stress.

Combining sitagliptin/metformin with a functional fiber delays diabetes progression in Zucker rats.

Our primary objective was to determine whether administering the viscous and fermentable polysaccharide PolyGlycopleX (PGX) with metformin (MET) or sitagliptin/metformin (S/MET) reduces hyperglycemia in Zucker diabetic fatty (ZDF) rats more so than monotherapy of each. Glucose tolerance, adiposity, satiety hormones and mechanisms related to dipeptidyl peptidase 4 activity, gut microbiota and, hepatic and pancreatic histology were examined. Male ZDF rats (9-10 weeks of age) were randomized to: i) cellulose/vehicle (control, C); ii) PGX (5% wt/wt)/vehicle (PGX); iii) cellulose/metformin (200  mg/kg) (MET); iv) cellulose/S/MET (10  mg/kg+200  mg/kg) (S/MET); v) PGX (5%)+MET (200  mg/kg) (PGX+MET); vi) cellulose/sitagliptin/MET (5%)+(10  mg/kg+200  mg/kg) (PGX+S/MET) for 6 weeks. PGX+MET and PGX+S/MET reduced glycemia compared with C and singular treatments (P=0.001). Weekly fasted and fed blood glucose levels were lower in PGX+MET and PGX+S/MET compared with all other groups at weeks 4, 5, and 6 (P=0.001). HbA1c was lower in PGX+S/MET than C, MET, S/MET, and PGX at week 6 (P=0.001). Fat mass was lower and GLP1 was higher in PGX+S/MET compared with all other groups (P=0.001). ß-cell mass was highest and islet degeneration lowest in PGX+S/MET. Hepatic lipidosis was significantly lower in PGX+S/MET compared with PGX or S/MET alone. When combined with PGX, both MET and S/MET markedly reduce glycemia; however, PGX+S/MET appears advantageous over PGX+MET in terms of increased ß-cell mass and reduced adiposity. Both combination treatments attenuated diabetes in the obese Zucker rat.

Bacterial biosensors for screening isoform-selective ligands for human thyroid receptors α-1 and ß-1.

Subtype-selective thyromimetics have potential as new pharmaceuticals for the prevention or treatment of heart disease, high LDL cholesterol and obesity, but there are only a few methods that can detect agonistic behavior of TR-active compounds. Among these are the rat pituitary GH3 cell assay and transcriptional activation assays in engineered yeast and mammalian cells. We report the construction and validation of a newly designed TRα-1 bacterial biosensor, which indicates the presence of thyroid active compounds through their impacts on the growth of an engineered Escherichia coli strain in a simple defined medium. This biosensor couples the configuration of a hormone receptor ligand-binding domain to the activity of a thymidylate synthase reporter enzyme through an engineered allosteric fusion protein. The result is a hormone-dependent growth phenotype in the expressing E. coli cells. This sensor can be combined with our previously published TRß-1 biosensor to detect potentially therapeutic subtype-selective compounds such as GC-1 and KB-141. To demonstrate this capability, we determined the half-maximal effective concentration (EC50) for the compounds T3, Triac, GC-1 and KB-141 using our biosensors, and determined their relative potency in each biosensor strain. Our results are similar to those reported by mammalian cell reporter gene assays, confirming the utility of our assay in identifying TR subtype-selective therapeutics. This biosensor thus provides a high-throughput, receptor-specific, and economical method (less than US$ 0.10 per well at laboratory scale) for identifying important therapeutics against these targets.

Thyroid hormone suppression of ß-amyloid precursor protein gene expression in the brain involves multiple epigenetic regulatory events.

Thyroid hormone (T3) suppresses cerebral gene expression of the ß-amyloid precursor protein (APP), an integral membrane protein that plays a key role in the onset and progression of Alzheimer's disease. However, the mechanisms by which T3 signaling pathways inhibit APP gene transcription in the brain remain unclear. By carrying out chromatin immunoprecipitation with neuroblastoma cells and primary rat brain tissue, we show for the first time that thyroid hormone receptors (TRs) directly bind at the APP gene in vivo at a promoter region containing a negative T3-response element. We further show that T3 treatment decreases both histone H3 acetylation and histone H3 lysine 4 methylation at the APP promoter and that chemical inhibitors of histone deacetylases and histone lysine demethylase abrogate T3-dependent APP silencing. Our findings thus suggest that TRs actively facilitate T3-dependent silencing of APP gene expression via the recruitment of distinct histone modifying enzymes associated with transcriptional repression.