Robert Alan Gadski

Decreased expression of apM1 in omental and subcutaneous adipose tissue of humans with type 2 diabetes.

We have screened a subtracted cDNA library in order to identify differentially expressed genes in omental adipose tissue of human patients with Type 2 diabetes. One clone (#1738) showed a marked reduction in omental adipose tissue from patients with Type 2 diabetes. Sequencing and BLAST analysis revealed clone #1738 was the adipocyte-specific secreted protein gene apM1 (synonyms ACRP30, AdipoQ, GBP28). Consistent with the murine orthologue, apM1 mRNA was expressed in cultured human adipocytes and not in preadipocytes. Using RT-PCR we confirmed that apM1 mRNA levels were significantly reduced in omental adipose tissue of obese patients with Type 2 diabetes compared with lean and obese normoglycemic subjects. Although less pronounced, apM1 mRNA levels were reduced in subcutaneous adipose tissue of Type 2 diabetic patients. Whereas the biological function of apM1 is presently unknown, the tissue specific expression, structural similarities to TNFα and the dysregulated expression observed in obese Type 2 diabetic patients suggest that this factor may play a role in the pathogenesis of insulin resistance and Type 2 diabetes.

[125I]Leu31, Pro34-PYY is a High Affinity Radioligand for Rat PP1/Y4 and Y1 Receptors: Evidence for Heterogeneity in Pancreatic Polypeptide Receptors

Cloned receptors for the PP-fold peptides are subdivided into Y1, Y2, PP1/Y4, Y5 and Y6. NPY and PYY have similar affinity for Y1, Y2, Y5 and Y6 receptors while PP has highest affinity for PP1. Pro34-substituted analogs of NPY and PYY have selectivity for Y1 and Y1-like receptors over Y2 receptors. In the present study, we found the putative Y1-selective radioligand, [125I]Leu31, Pro34-PYY, also binds with high affinity to the rat PP1 receptor in cell lines expressing the receptor. However, in rat brain sections, [125I]Leu31, Pro34-PYY does not appear to bind to the interpeduncular nucleus, a brain region containing a high density of [125I]-bPP binding sites. Therefore, it appears there is additional heterogeneity in receptors recognizing PP.

Characterization of the neuropeptide Y5 receptor in the human hypothalamus: a lack of correlation between Y5 mRNA levels and binding sites

Neuropeptide Y (NPY) is a 36-amino-acid peptide that appears to play a central role in the control of feeding behavior. Recently, a cDNA encoding a novel NPY receptor subtype (Y5) was cloned from the rat and human hypothalamus, and shown to have a pharmacology consistent with NPY-induced feeding. We have subsequently cloned this cDNA from human hypothalamus and stably expressed it in CHO cells. Consistent with earlier reports, hY5 has a high affinity for NPY, [Leu31, Pro34]NPY, and NPY(3-36), but low affinity for larger C-terminal deletions of NPY and BIBP3226. High levels of hY5 mRNA were found in the human testis, brain, spleen and pancreas, with lower levels in several other tissues. In the human brain, hY5 mRNA levels were typically higher than hY2, but lower in comparison to hY1 receptor mRNA. To quantify the relative amounts of hY1, hY2 and hY5 mRNA in the human hypothalamus, we employed competitive RT-PCR. Interestingly, the relative amount of hY5 mRNA was substantially higher than either hY1 or hY2. However, pharmacological characterization of NPY binding sites in human hypothalamus membranes revealed predominantly the hY2 subtype. These data establish that while hY5 mRNA levels are very high in the human hypothalamus, conventional radioligand binding techniques do not detect hY5-like binding site. Whether hY5-like binding sites exist in the other human tissues that express hY5 mRNA (and what function hY5 has in those tissues) awaits future investigation.

Central H3R activation by thioperamide does not affect energy balance

The central histamine 3 receptor (H3R) is a presynaptic autoreceptor that regulates neuronal release and synthesis of histamine, and is thought to play a key role in controlling numerous central nervous system (CNS)-mediated parameters, including energy homeostasis. Thioperamide, the prototypical selective H3R antagonist, was used to examine the role that H3R plays in regulating energy balance in vivo. Thioperamide was administered either intraperitoneally or orally to rats and the pharmacokinetic parameters were examined along with central H3R binding and histaminergic system activation. Food intake and metabolic parameters of either route of thioperamide administration were likewise examined. In a dose-dependent manner, both the intraperitoneal and oral route of administration resulted in similar ex vivo binding curves and tele-methylhistamine dose-response curves despite the route of administration. However, only intraperitoneal administration of 30 mg/kg thioperamide resulted in a significant decrease in 24-h food intake (60% lower than control) and respiratory quotient (RQ), while the oral route of delivery did not. Moreover, the decrease in RQ with the 30 mg/kg ip administration also decreased energy expenditure (EE) thus resulting in an unchanged energy balance. The decrease in food intake and EE was coupled with a conditioned taste aversion with the 30-mg/kg ip administration. These data indicate that the activation of the central H3R system by thioperamide does not play a direct role in decreasing food intake or altering energy homeostasis.

Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes.

Neuropeptide Y (NPY) is a 36 amino acid peptide that is abundant in the brain and peripheral nervous system. NPY has a variety of effects when administered into the brain including a pronounced feeding effect, anxiolysis, regulation of neuroendocrine axes and inhibition of neurotransmitter release. These effects are mediated by up to 6 G protein coupled receptors designated Y1, Y2, Y3, Y4, Y5 and y6. To better understand the phylogeny and pharmacology of NPY in non-human primates, we have cloned and expressed the NPY Y1, Y2 and Y5 receptor subtypes from the Rhesus monkey. No cDNA sequence encoding a Y4 receptor was found suggesting substantial sequence differences when compared to the human sequence. Comparison of these sequences with those from human indicated strong sequence conservation of Y1, Y2 and Y5 between the two species. The displacement of (125)I-PYY binding to the Rhesus monkey and human receptors by various peptides was compared to evaluate the pharmacology of the two species. Similar pharmacologies were noted across the species at the various receptor subtypes. These results indicate the Rhesus monkey and human NPY receptor subtypes have a close amino acid sequence conservation and that the peptide recognition domains are conserved as well.

Structure-Activity Relationships of β -MSH Derived Melanocortin-4 Receptor Peptide Agonists

The recent emergence of obesity as a major health threat in the industrialized world has intensified the search for novel and effective pharmacologic treatment. The proopiomelanocortin (POMC)-melanocortin 4 receptor (MC4R) axis has been shown to regulate food intake and energy homeostasis and is considered among the most promising antiobesity targets. Our initial efforts in this area have focused on affinity and selectivity directed optimization of the native beta-MSH(5-22) sequence and resulted in the discovery of a potent MC4R agonist: Ac-Tyr-Arg-[Cys-Glu-His-D-Phe-Arg-Trp-Cys]-NH(2) (10). Subcutaneous administration of this peptide produced an excellent in vivo efficacy in reducing food intake and increasing fat metabolism. Additionally, suppression of food intake was observed in wild type but not in MC4R deficient mice, suggesting that the effects observed in the wild type mice were mediated through MC4R signaling. Subsequent optimization efforts led to the identification of a novel series of disulfide constrained hexapeptides as exemplified by Ac-[hCys-His-D-Phe-Arg-Trp-Cys]-NH(2) (100). These cyclic hexapeptides showed a further improved potency in binding MC4R and an enhanced selectivity over MC1R. At a dose of 0.07 mg/kg analog 102 reduced food intake by 38% and increased fat utilization by 58% in rats. These cyclic peptides provide novel and enhanced reagents for the elucidation of melanocortin receptors biology and may find applications in the treatment of obesity and related metabolic disorders.

Synthesis and in vitro biological activity of 4α-(2-propenyl)-5α-cholest-24-en-3α,12α-diol, a 12α-hydroxyl analog of 4α-(2-propenyl)-5α-cholest-24-en-3α-ol: The latter is a potent activator of the low-density lipoprotein receptor promoter

Abstract 4α-(2-Propenyl)-5α-cholest-24-en-3α-ol (3) was shown recently in a Chinese hamster ovary (CHO) cell-based low-density lipoprotein receptor/luciferase (LDLR/Luc) assay to be a potent transcriptional activator of the LDL receptor promoter in the presence of 25-hydroxycholesterol. Because of the involvement of 12α-hydroxylation in the metabolism of cholesterol, we are interested in investigating the effect of introducing a 12α-hydroxyl group to 3 on the transcriptional activity of the LDL receptor promoter. Thus 4α-(2-propenyl)-5α-cholest-24-en-3α,12α-diol (14), a 12α-hydroxyl analog of 3, was synthesized from deoxycholic acid via the formation of 12α-[[(tert-butyl)dimethylsilyl]oxy]-4α-(2-propenyl)-5α-cholest-24-en-3-one (11). Test results show that 14 is inactive at concentrations of up to 20 μg/ml, compared to 3 with an EC30 value of 2.6 μM, in the CHO cell-based LDLR/Luc assay. Apparently introduction of a 12α-hydroxyl group abolishes the capability of 3α-sterol 14 to activate the transcription of the LDL receptor promoter. However, in the [1-14C-acetate]cholesterol biosynthesis inhibition assay in CHO cells, 14 at 10 μg/ml (23 μM) is shown to inhibit the cholesterol biosynthesis by 51% relative to the control cells. Our previous studies indicated that 3 showed a 38% inhibition, but 4α-(2-propenyl)-5α-cholestan-3α-ol (1) exhibited no inhibition in the same assay at 10 μg/ml. In summary the results indicate that, in addition to the 24,25-unsaturation, the 12α-hydroxyl group in 14 has also conferred an inhibitory effect on cholesterol biosynthesis in CHO cells; however, the inhibition of cholesterol biosynthesis by 14 does not lead to the transcriptional activation of the LDL receptor promoter.

Synthesis and in vitro biological activity of 4α-(2-propenyl)-5α-cholest-24-en-3α-ol: A 24,25-dehydro analog of the hypocholesterolemic agent 4α-(2-propenyl)-5α-cholestan-3α-ol

4Alpha-(2-propenyl)-5alpha-cholestan-3alpha-ol (LY295427) was previously identified from a Chinese hamster ovary (CHO) cell-based low density lipoprotein receptor/luciferase (LDLR/Luc) assay to be a potent transcriptional activator of the LDL receptor promoter in the presence of 25-hydroxycholesterol. To investigate the effect of the 24,25-unsaturation in the D-ring side chain (desmosterol D-ring side chain) on antagonizing the repressing effect of 25-hydroxycholesterol, 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha-ol (17), a 24,25-dehydro analog of LY295427, was thus synthesized from lithocholic acid via the formation of 3alpha-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4alpha- (2-propenyl)-5alpha-cholan-24-al (15). Test results showed that 17 had an EC30 value of 2.6 microM, comparable to 2.9 microM of LY295427, in the CHO cell-based LDLR/Luc assay in the presence of 25-hydroxycholesterol. Apparently, the built-in 24,25-unsaturation in the D-ring side chain of 17 had added little effect to antagonizing the repressing effect of 25-hydroxycholesterol. In the [1-14C-acetate]cholesterol biosynthesis inhibition assay, 17 at 10 microg/ml (23 microM) has been shown to inhibit the cholesterol biosynthesis in CHO cells by 38% relative to the vehicle control; whereas LY295427 showed no inhibition in the same assay in our previous studies. In contrast to LY295427, the built-in 24,25-unsaturation in the D-ring side chain of 17 has conferred an inhibitory effect on cholesterol biosynthesis in CHO cells. In summary, the observed LDL receptor promoter activity of 17 is related to its ability to prevent 25-hydroxycholesterol from exerting the repressing effect via an undetermined mechanism and, in part, to inhibit the cholesterol biosynthesis.

Synthesis of 4α-(2-propenyl)-5,6-secocholestan-3α-ol, a Novel B-ring Seco Analog of the Hypocholesterolemic Agent 4α-(2-propenyl)-5α-cholestan-3α-ol

Abstract 4α-(2-Propenyl)-5α-cholestan-3α-ol (LY295427) was previously identified from a CHO cell-based assay to be a potent LDL receptor up-regulator and had demonstrated to be an effective agent in lowering plasma cholesterol levels in hypercholesterolemic hamsters. In order to investigate the effect of flexibility of the 3α-hydroxy-bearing A-ring on the activity, 4α-(2-propenyl)-5,6-secocholestan-3α-ol ( 11 ), a B-ring seco analog of LY295427, is thus synthesized from cholest-4-en-3-one. Test results indicate that 11 is not active in the CHO cell-based LDL receptor/luciferase assay at concentrations up to 20 μg/mL. The result underlines the importance of maintaining the A-B-C-D ring rigidity of the 3α-sterols in terms of binding to the putative oxysterol receptor.

Intracellular Binding Site for a Positive Allosteric Modulator of the Dopamine D1 Receptor

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a β2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state β2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.