Pasquale P. Vicario

Peroxisome Proliferator-Activated Receptors γ and α Mediate in Vivo Regulation of Uncoupling Protein (UCP-1, UCP-2, UCP-3) Gene Expression

A role for peroxisome proliferator-activated receptors, PPAR gamma and PPAR alpha, as regulators of energy homeostasis and lipid metabolism, has been suggested. Recently, three distinct uncoupling protein isoforms, UCP-1, UCP-2, and UCP-3, have also been identified and implicated as mediators of thermogenesis. Here, we examined whether in vivo PPAR gamma or PPAR alpha activation regulates the expression of all three UCP isoforms. Rats or lean and db/db mice were treated with PPAR gamma [thiazolidinedione (TZD)] or PPAR alpha (WY-14643) agonists, followed by measurement of messenger RNAs (mRNAs) for UCP-1, UCP-2, and UCP-3 in selected tissues where they are expressed. TZD treatment (AD 5075 at 5 mg/kg x day) of rats (14 days) increased brown adipose tissue (BAT) depot size and induced the expression of each UCP mRNA (3x control levels for UCP-1 and UCP-2, 2.5x control for UCP-3). In contrast, UCP-2 and UCP-3 mRNA levels were not affected in white adipose tissue or skeletal muscle. Chronic (30 days) low-dose (0.3 mg/kg x day) TZD treatment induced UCP-1 mRNA and protein in BAT (2.5x control). In contrast, chronic TZD treatment (30 mg/kg x day) suppressed UCP-1 mRNA (>80%) and protein (50%) expression in BAT. This was associated with further induction of UCP-2 expression (>10-fold) and an increase in the size of lipid vacuoles, a decrease in the number of lipid vacuoles in each adipocyte, and an increase in the size of the adipocytes. TZD treatment of db/db mice (BRL 49653 at 10 mg/kg x day for 10 days) also induced UCP-1 and UCP-3 (but not UCP-2) expression in BAT. PPAR alpha is present in BAT, as well as liver. Treatment of rats or db/db mice with WY-14643 did not affect expression of UCP-1, -2, or -3 in BAT. Hepatic UCP-2 mRNA was increased (4x control level) in db/db and lean mice, although this effect was not observed in rats. Thus, in vivo PPAR gamma activation can induce expression of UCP-1, -2, and -3 in BAT; whereas chronic-intense PPAR gamma activation may cause BAT to assume white adipose tissue-like phenotype with increased UCP-2 levels. PPAR alpha activation in mice is sufficient to induce liver UCP-2 expression.

Characterization of a Novel, Non-peptidyl Antagonist of the Human Glucagon Receptor

We have identified a series of potent, orally bioavailable, non-peptidyl, triarylimidazole and triarylpyrrole glucagon receptor antagonists. 2-(4-Pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)pyrrole (L-168,049), a prototypical member of this series, inhibits binding of labeled glucagon to the human glucagon receptor with an IC50 = 3.7 ± 3.4 nm(n = 7) but does not inhibit binding of labeled glucagon-like peptide to the highly homologous human glucagon-like peptide receptor at concentrations up to 10 μm. The binding affinity of L-168,049 for the human glucagon receptor is decreased 24-fold by the inclusion of divalent cations (5 mm). L-168,049 increases the apparent EC50 for glucagon stimulation of adenylyl cyclase in Chinese hamster ovary cells expressing the human glucagon receptor and decreases the maximal glucagon stimulation observed, with aK b (concentration of antagonist that shifts the agonist dose-response 2-fold) of 25 nm. These data suggest that L-168,049 is a noncompetitive antagonist of glucagon action. Inclusion of L-168,049 increases the rate of dissociation of labeled glucagon from the receptor 4-fold, confirming that the compound is a noncompetitive glucagon antagonist. In addition, we have identified two putative transmembrane domain residues, phenylalanine 184 in transmembrane domain 2 and tyrosine 239 in transmembrane domain 3, for which substitution by alanine reduces the affinity of L-168,049 46- and 4.5-fold, respectively. These mutations do not alter the binding of labeled glucagon, suggesting that the binding sites for glucagon and L-168,049 are distinct.

Alterations in Receptor Activation and Divalent Cation Activation of Agonist Binding by Deletion of Intracellular Domains of the Glucagon Receptor

Deletion of residues 252-259 within the putative second intracellular loop of the human glucagon receptor results in a protein with high affinity for glucagon but with attenuated agonist activation of adenylyl cyclase. The Δ252-259 mutant has 4-fold higher affinity for glucagon than does the wild type receptor. The nonhydrolyzable GTP analog, guanosine 5′-(β,γ-imido)triphosphate (Gpp(NH)p), inhibits binding of 125I-glucagon to the wild type receptor but not to the Δ252-259 mutant. Divalent cations such as MgCl2 and CaCl2 stimulate the binding of 125I-glucagon to the wild type receptor by increasing glucagon affinity. The rate of dissociation of 125I-glucagon is decreased 4-fold by MgCl2 and increased 6-fold by Gpp(NH)p. However, divalent cations do not affect the binding of 125I-glucagon to the Δ252-259 mutant. The rate of dissociation of 125I-glucagon from the Δ252-259 mutant protein is equivalent to the rate of dissociation from the wild type receptor in the presence of MgCl2. These data suggest that at least three conformations of the glucagon receptor can exist in the membrane based on their differing affinities for 125I-glucagon. Deletion of residues 252-259 appears to lock the protein in the conformation promoted by divalent cations and prevents the protein from normal coupling to Gs.

3-Pyridyloxypropanolamine agonists of the β3 adrenergic receptor with improved pharmacokinetic properties

Pyridyloxypropanolamines L-749,372 (8, beta 3 EC50 = 3.6 nM) and L-750,355 (29, beta 3 EC50 = 13 nM) are selective partial agonists of the human receptor, with 33% and 49% activation, respectively. Both stimulate lipolysis in rhesus monkeys (ED50 = 2 and 0.8 mg/kg, respectively), with minimal effects on heart rate. Oral bioavailability in dogs, 41% for L-749,372 and 47% for L-750,355, is improved relative to phenol analogs.

Design, synthesis, and structure-activity relationship of novel CCR2 antagonists.

A series of novel 1-aminocyclopentyl-3-carboxyamides incorporating substituted tetrahydropyran moieties have been synthesized and subsequently evaluated for their antagonistic activity against the human CCR2 receptor. Among them analog 59 was found to posses potent antagonistic activity.

Insulin receptor tyrosine kinase activity is unaltered in ob/ob and db/db mouse skeletal muscle membranes.

Insulin binding and insulin receptor tyrosine kinase activity were examined in two rodent models with genetic insulin resistance using partially-purified skeletal muscle membrane preparations. Insulin binding activity was decreased about 50% in both 12-week (219 +/- 184 vs 1255 +/- 158 fmoles/mg, p less than 0.01) and 24-week old (2120 +/- 60 vs 1081 +/- 60 fmoles/mg, p less than 0.01) ob/ob mice. In contrast, insulin binding to membrane derived from 24-week old db/db mice was not significantly different from lean controls (1371 +/- 212 vs 1253 +/- 247 fmoles/mg). Insulin-associated tyrosine kinase activity of membranes from ob/ob skeletal muscle was decreased, compared to its normal lean littermate, when compared on a per mg of protein basis in both 12-week (37 +/- 3 vs 21 +/- 3 pmoles/min/mg, p less than 0.05) and 24-week old (71 +/- 5 vs 37 +/- 6 pmoles/min/mg, p less than 0.01) mice. However, no significant differences in kinase activities were observed when the data were normalized and compared on a per fmole of insulin-binding activity basis for the 12-week (12 +/- 1 vs 11 +/- 2) and 24-week (27 +/- 2 vs 20 +/- 3) age groups. Insulin receptor tyrosine kinase activity of db/db skeletal muscle membranes was not different than its normal lean littermate whether expressed on a protein (34 +/- 7 vs 30 +/- 3) or fmole of insulin-binding activity (21 +/- 4 vs 18 +/- 4) basis. These data suggest that insulin receptor tyrosine kinase is not associated with the insulin resistance observed in ob/ob and db/db mice and demonstrate differences in receptor regulation between both animal models.

Potent heteroarylpiperidine and carboxyphenylpiperidine 1-alkyl-cyclopentane carboxamide CCR2 antagonists.

This report describes replacement of the 4-(4-fluorophenyl)piperidine moiety in our CCR2 antagonists with 4-heteroaryl piperidine and 4-(carboxyphenyl)-piperidine subunits. Some of the resulting analogs retained potency in our CCR2 binding assay and had improved selectivity versus the I(Kr) channel; poor selectivity against I(Kr) had been a liability of earlier analogs in this series.

Isolation and Structures of Novel Fungal Metabolites as Chemokine Receptor (CCR2) Antagonists

The chemokine receptor, CCR2, is predominantly expressed on monocytes/macrophages, and on a subset of memory T cells. It binds to several CC type chemokines of the monocyte chemoattractant protein (MCP) family of which MCP-1 exhibits the highest affinity. CCR2/MCP-1 expression/association in monocyte/macrophage/T cells has been associated with inflammatory processes such as rheumatoid arthritis, multiple sclerosis and atherosclerosis. Neutralization of CCR2 with either a peptide or receptor antagonist results in the prevention of joint swelling in rodent models of arthritis. In this paper, bioassay-guided discovery of CCR2 receptor antagonists derived from natural product extracts are reported. These antagonists belong to two main classes exemplified by bis-thiodiketopiperazines and cytochalasins. Six compounds, including emestrin, two new emestrin analogs, and chaetomin represent the first group of compounds. These compounds inhibited the binding of MCP-1 to CCR2 (CHO membrane) with IC50 values of 0.8 to 9 µM and exhibited good activity in a whole cell assay using MCP-1 and human monocytes with IC50′S ranging from 4∼9 µM. Cytochalasins A and B represented the second group and inhibited the binding activity with IC50 values of 5 and 188 µM, respectively. This is the first report of natural product antagonists of the CCR2 receptor.

Conformational studies of 3-amino-1-alkyl-cyclopentane carboxamide CCR2 antagonists leading to new spirocyclic antagonists.

In an effort to shed light on the active binding conformation of our 3-amino-1-alkyl-cyclopentane carboxamide CCR2 antagonists, we prepared several conformationally constrained analogs resulting from backbone cyclization. Evaluation of CCR2 binding affinities for these analogs gave insight into the optimal relative positions of the piperidine and benzylamide moieties while simultaneously leading to the discovery of a new, potent lead type based upon a spirocyclic acetal scaffold.

Impaired insulin-like growth factor I-mediated stimulation of glucose incorporation into glycogen in vivo in the ob/ob mouse

SummaryThe ability of insulin to modulate glucose metabolism is impaired in insulin resistant ob/ob mice. It has been shown that insulin-like growth factor I stimulates the uptake and metabolism of glucose in muscle through the insulin-like growth factor receptor not the insulin receptor. Thus, we have compared the abilities of insulin-like growth factor I and insulin to stimulate the in vivo incorporation of [14C]-glucose into glycogen in the diaphragm of ob/ob mice and their lean littermates. The animals used in these studies were 12–14 weeks old and the serum insulin levels of the ob/ob mice were 16-fold higher than in their lean littermates. There were no differences in the serum levels of glucose or insulin-like growth factor I. Both insulin and insulin-like growth factor I stimulate the incorporation of [14C]-glucose into glycogen in lean mice. Significant stimulation occurs at doses as low as 1 μg/kg of either peptide. The effective doses of insulin and insulin-like growth factor I are quite similar, which indicates that the effect of insulin-like growth factor I is mediated by the insulin-like growth factor receptor and not the insulin receptor. In contrast, greater than 100 μg/kg of insulin-like growth factor I is required to stimulate [14C]-glucose incorporation into glycogen in the diaphragm of ob/ob mice. Thus, ob/ob mice are resistant to the action of both insulin and insulin-like growth factor I. In contrast to the decrease in the number of insulin receptors which occurs in ob/ob mice, there is no significant difference in the number of type 1 insulin-like growth factor receptors or in their affinity for insulin-like growth factor I in muscle membranes prepared from lean and ob/ob mice. In addition, the ability of insulin-like growth factor I to stimulate the catalysis of Val5-angiotensin II phosphorylation by the partially purified muscle type 1 insulin-like growth factor receptor is not decreased in ob/ob mice as compared with their lean littermates. These data indicate that the loss in sensitivity of the ob/ob mouse of both insulin and insulin-like growth factor I is most likely mediated by a post-receptor defect in metabolism and not by receptor down-regulation or desensitisation.