Nancy S. Hayes

NOVEL PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) GAMMA AND PPARDELTA LIGANDS PRODUCE DISTINCT BIOLOGICAL EFFECTS

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARα, PPARδ, and PPARγ. PPARγ has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPARγ and PPARδ that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPARγ and PPARδ directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARγ agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabeticdb/db mice all PPARγ agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selectivein vivo activation of PPARδ did not significantly affect these parameters. In vivo PPARα activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPARγ and PPARδ; 2) ligand-dependent activation of PPARδ involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARγ activation (but not PPARδ or PPARα activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARγ agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARα activation is sufficient to affect triglyceride metabolism, PPARδ activation does not appear to modulate glucose or triglyceride levels.

A potent synthetic LXR agonist is more effective than cholesterol-loading at inducing ABCA1 mRNA and stimulating cholesterol efflux

The LXR nuclear receptors are intracellular sensors of cholesterol excess and are activated by various oxysterols. LXRs have been shown to regulate multiple genes of lipid metabolism, including ABCA1 (formerly known asABC1). ABCA1 is a lipid pump that effluxes cholesterol and phospholipid out of cells. ABCA1 deficiency causes extremely low high density lipoprotein (HDL) levels, demonstrating the importance of ABCA1 in the formation of HDL. The present work shows that the acetyl-podocarpic dimer (APD) is a potent, selective agonist for both LXRα (NR1H3) and LXRβ (NR1H2). In transient transactivation assays, APD was ∼1000-fold more potent, and yielded ∼6-fold greater maximal stimulation, than the widely used LXR agonist 22-(R)-hydroxycholesterol. APD induced ABCA1mRNA levels, and increased efflux of both cholesterol and phospholipid, from multiple cell types. Gas chromatography-mass spectrometry measurements demonstrated that APD stimulated efflux of endogenous cholesterol, eliminating any possible artifacts of cholesterol labeling. For both mRNA induction and stimulation of cholesterol efflux, APD was found to be more effective than was cholesterol loading. Taken together, these data show that APD is a more effective LXR agonist than endogenous oxysterols. LXR agonists may therefore be useful for the prevention and treatment of atherosclerosis, especially in the context of low HDL levels.

A Novel Liver X Receptor Agonist Establishes Species Differences in the Regulation of Cholesterol 7α-Hydroxylase (CYP7a)

The liver X receptors, LXRα and LXRβ, are members of the nuclear receptor superfamily. Originally identified as orphans, both receptor subtypes have since been shown to be activated by naturally occurring oxysterols. LXRα knockout mice fail to regulate cyp7a mRNA levels upon cholesterol feeding, implicating the role of this receptor in cholesterol homeostasis. LXR activation also induces the expression of the lipid pump involved in cholesterol efflux, the gene encoding ATP binding cassette protein A1 (ABCA1). Therefore, LXR is believed to be a sensor of cholesterol levels and a potential therapeutic target for atherosclerosis. Here we describe a synthetic molecule named F3MethylAA [3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenyl acetic acid] that is more potent than 22(R)-hydroxycholesterol in LXR in vitro assays. F3MethylAA is capable not only of inducing ABCA1 mRNA levels, but also increasing cholesterol efflux from THP-1 macrophages. In rat hepatocytes, F3MethylAA induce...

Expression, purification and characterization of recombinant human insulin-like growth factor I in yeast

Insulin-like growth factor I (IGF-I) is a 70 amino acid (aa) protein that is structurally similar and functionally related to insulin. We have inserted a synthetic gene coding for human IGF-I into a Saccharomyces cerevisiae expression vector utilizing the MF alpha 1 promoter and pre-pro leader peptide. This vector directs the expression and secretion of native, biologically active growth factor. Cleavage of the pre-pro alpha factor leader sequence in vivo results in the secretion of a 70-aa recombinant IGF-I molecule with the native N-terminal glycine residue. Human IGF-I purified from yeast culture supernatant is equipotent to serum-derived IGF-I in inhibiting [125I]IGF-I binding to type-I IGF receptors and crude human serum-binding proteins. Recombinant IGF-I is also equipotent to human IGF-I in the stimulation of DNA synthesis in rat aortic smooth-muscle cells. In contrast, yeast recombinant IGF-I is less potent than serum-derived IGF-I in binding to type-2 IGF receptors. The ability to produce native, biologically active IGF-I in yeast will allow the elucidation of binding domains through the expression and characterization of specific structural analogs.

A PPARγ mutant serves as a dominant negative inhibitor of PPAR signaling and is localized in the nucleus

The peroxisomal proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that act as ligand-activated transcription factors. PPARgamma plays a critical role in regulating adipocyte differentiation and lipid metabolism. Recently, thiazolidinedione (TZD) and select non-TZD antidiabetic agents have been identified as PPARgamma agonists. To further characterize this receptor subclass, a mutant hPPARgamma lacking five carboxyl-terminal amino acids was produced (hPPARgamma2Delta500). In COS-1 cells transfected with PPAR-responsive reporter constructs, the mutant receptor could not be activated by a potent PPARgamma agonist. When cotransfected with hPPARgamma2 or hPPARalpha, hPPARgamma2Delta500 abrogated wild-type receptor activity in a dose-responsive manner. hPPARgamma2Delta500 was also impaired with respect to binding of a high-affinity radioligand. In addition, its conformation was unaffected by normally saturating concentrations of PPARgamma agonist as determined by protease protection experiments. Electrophoretic mobility shift assays demonstrated that hPPARgamma2Delta500 and hPPARgamma2 both formed heterodimeric complexes with human retinoidxreceptor alpha (hRXRalpha) and could bind a peroxisome proliferator-responsive element (PPRE) with similar affinity. Therefore, hPPARgamma2Delta500 appears to repress PPAR activity by competing with wild type receptor to dimerize with RXR and bind the PPRE. In addition, the mutant receptor may titrate out factors required for PPAR-regulated transcriptional activation. Both hPPARgamma2 and hPPARgamma2Delta500 localized to the nucleus of transiently transfected COS-1 cells as determined by immunofluorescence using a PPARgamma-specific antibody. Thus, nuclear localization of PPARgamma occurs independently of its activation state. The dominant negative mutant, hPPARgamma2Delta500, may prove useful in further studies to characterize PPAR functions both in vitro and in vivo

A Novel Insulin Secretagogue Is a Phosphodiesterase Inhibitor

The arylpiperazine L-686,398 was described as an oral hypoglycemic agent and is shown to be an insulin secretagogue in vitro. The characteristics of its activity were similar to those of the incretin glucagon-like peptide I (GLP-I). We demonstrate that both the peptide and L-686,398 increase the accumulation of cAMP in isolated ob/ob mouse pancreatic islet cells, but by different mechanisms. Although GLP-I activates adenylate cyclase, the arylpiperazine has no effect on this enzyme or on the binding of 125I-labeled GLP-I to its receptor on RINm5F rat insulinoma cell membranes. However, L-686,398 inhibits the total cAMP phosphodiesterase (PDE) activity in homogenates of ob/ob mouse pancreatic islets with an EC50 of ∼ 50 μmol/l. To determine the mechanism of PDE inhibition by the arylpiperazine and to examine its specificity, we studied the kinetics of arylpiperazine inhibition of two recombinant PDEs. The arylpiperazine is a competitive inhibitor of both a human heart type III PDE and a rat type IV-D PDE. Inhibition of the type III and IV isozymes are characterized by Ki values of 27 and 5 μmol/l, respectively. Although not extremely potent, the arylpiperazine does exhibit modest selectivity between these PDEs. The observation that L-686,398 acts as a PDE inhibitor suggests that exploration for β-cell-specific PDE isoforms may reveal novel PDEs as targets for the development of therapeutically useful glucose-dependent insulin secretagogues.

(Thr-59)-insulin-like growth factor I stimulates 2-deoxyglucose transport in BC3H1 myocytes through the insulin-like growth factor receptor, not the insulin receptor

The murine non-fusing muscle cell line contains distinct receptors for insulin and insulin-like growth factors. Pretreatment of myocytes with insulin for 20 h at 37 degrees C inhibits the binding of [125I]iodoinsulin by 60% without affecting the binding of [125I]iodoinsulin-like growth factor I. The ED50 values for down-regulation of the insulin and insulin-like growth factor receptor by their respective ligands are 1 nM and 3 nM, respectively. Insulin, (Thr-59)-insulin-like growth factor I and multiplication-stimulating activity stimulate 2-[3H]deoxyglucose transport in myocytes with ED50 values of 5 nM, 5.6 nM and 33 nM, respectively. In order to determine whether (Thr-59)-insulin-like growth factor I stimulates 2-[3H]deoxyglucose transport in myocytes via its own receptor or the insulin receptor, we determined the activity of these peptides after down-regulation of the insulin receptor. The rate of 2-[3H]deoxyglucose transport in myocytes pretreated with insulin (5 nM) is elevated but returns to control levels by 1 h after the washout of insulin. The dose-response curve for insulin-stimulated 2-[3H]deoxyglucose transport is shifted to the right (ED50 greater than 100 nM) immediately after insulin washout but is normal by 1 h after insulin washout. In contrast, the dose-response curve for (Thr-59)-insulin-like growth factor I is unchanged in insulin-pretreated cells immediately after insulin washout. These data show that (Thr-59)-insulin-like growth factor I stimulates 2-[3H]deoxyglucose transport in myocytes by acting through an insulin-like growth factor receptor and not through the insulin receptor. Since multiplication-stimulating activity is 6-fold less active than (Thr-59)-insulin-like growth factor, they both may be acting through a type 1 insulin-like growth factor receptor.

Steroidal and Triterpenoidal Fungal Metabolites as Ligands of Liver X Receptors

Cholesterol homeostasis is tightly controlled process that involves a variety of regulators including liver X receptors (LXR). Agonists of LXR are expected to increase cholesterol efflux, lower LDL, and raise HDL levels. Screening of a natural product library of microbial extracts using a LXR-scintillation proximity assay (SPA) binding assay and bioassay-guided fractionation of a number of fungal extracts led to the isolation of five ergostane and a cycloartane derivative. These compounds exhibited IC50 value ranging 0.5∼9 µM in the binding assay for α-receptor and a number of these showed in vitro agonist activity in the coactivator association assays but lacked the cell based LXR activation. The isolation and LXR activity of these compounds are described.

Effects of the angiotensin converting enzyme inhibitor, lisinopril, on normal and diabetic rats.

The comparative effects of lisinopril, a third generation angiotensin converting enzyme (ACE) inhibitor, on components of the renin-angiotensin system were assessed in normal and in an animal model of diabetes-related hypertension, the streptozotocin-diabetic rat. Two weeks after injection of streptozotocin the mean systolic blood pressure of diabetic rats was elevated 11% above that of normal rats. This effect was prevented by daily injection of insulin. The mean serum ACE activity was elevated 71% above that of normal rats. Lisinopril reduced systolic blood pressure and inhibited serum ACE activity in both normal and diabetic rats in a dose-response fashion. In normal rats maximum inhibition of blood pressure occurred at a mean dose of 1.0 mg/kg and in the diabetic rat at a mean dose of 5.0 mg/kg. At a mean dose of 5 mg/kg, ACE was inhibited by 100 and 92% in normal and diabetic rats, respectively. Plasma renin activity (PRA) increased sharply in both groups of rats treated with the lower doses of lisinopril, only to decrease at the 5 mg/kg level. At 20 mg/kg, PRA continued to decline in normal animals, but not in diabetic rats. Formation of angiotensin II (Ang II) in both normal and diabetic rats was maximally inhibited at doses of 1.0 and 0.1 mg/kg of lisinopril, respectively without a significantly greater effect at the higher doses of the drug. In separate experiments the effects of chronic treatment with lisinopril at two dosage levels on various physiological parameters of streptozotocin-diabetic rats were compared with the effects of another hypotensive agent, hydralazine, an arteriolar vasodilator.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of endo- and exo-aryl-substitutions and central scaffold modifications on diphenyl substituted alkanes as 5-lipoxygenase activating protein inhibitors.

A search for a suitable replacement for the central norbornyl scaffold presented in the recently disclosed novel FLAP inhibitors is herein described, as well as the SAR study performed on the endo and exo-aryl groups.