The feasibility of distant wavelength conversion in photonic crystal fiber was explored. The one-pump parametric architecture was used to demonstrate translation from the conven- tional (1550 nm) to visible (500 nm) band. Experimental results are reported, which demonstrate the conventional-to-visible con- version of single- and multiple-channel signals encoded in the nonreturn-to-zero format.

A novel class of spiro-ureas has been discovered as potent human glucagon receptor antagonists in both binding and functional assays. Preliminary studies have revealed that compound 15 is an orally active human glucagon receptor antagonist in a transgenic murine pharmacodynamic model at 10 and 30 mpk. Compound 15 is orally bioavailable in several preclinical species and shows selectivity toward cardiac ion channels and other family B receptors, such as hGIP1 and hGLP.

A novel class of antagonists of the human glucagon receptor (hGCGR) has been discovered. Systematic modification of the lead compound identified substituents that were essential for activity and those that were amenable to further optimization. This SAR exploration resulted in the synthesis of 13, which exhibited good potency as an hGCGR functional antagonist (IC50 = 34 nM) and moderate bioavailability (36% in mice).

Glucagon maintains glucose homeostasis during the fasting state by promoting hepatic gluconeogenesis and glycogenolysis. Hyperglucagonemia and/or an elevated glucagon-to-insulin ratio have been reported in diabetic patients and animals. Antagonizing the glucagon receptor is expected to result in reduced hepatic glucose overproduction, leading to overall glycemic control. Here we report the discovery and characterization of compound 1 (Cpd 1), a compound that inhibits binding of 125I-labeled glucagon to the human glucagon receptor with a half-maximal inhibitory concentration value of 181 +/- 10 nmol/l. In CHO cells overexpressing the human glucagon receptor, Cpd 1 increased the half-maximal effect for glucagon stimulation of adenylyl cyclase with a KDB of 81 +/- 11 nmol/l. In addition, Cpd 1 blocked glucagon-mediated glycogenolysis in primary human hepatocytes. In contrast, a structurally related analog (Cpd 2) was not effective in blocking glucagon-mediated biological effects. Real-time measurement of glycogen synthesis and breakdown in perfused mouse liver showed that Cpd 1 is capable of blocking glucagon-induced glycogenolysis in a dosage-dependent manner. Finally, when dosed in humanized mice, Cpd 1 blocked the rise of glucose levels observed after intraperitoneal administration of exogenous glucagon. Taken together, these data suggest that Cpd 1 is a potent glucagon receptor antagonist that has the capability to block the effects of glucagon in vivo.

Glucagon receptor antagonists have been actively pursued as potential therapeutics for the treatment of type 2 diabetes. Peptidyl and non-peptidyl glucagon receptor antagonists have been shown to block glucagon-induced blood glucose elevation in both animals and humans. How the antagonists and the glucagon receptor interact in vivo has not been reported and is the subject of the current study. Using 125I-labeled glucagon as a radiotracer, we developed an in vivo glucagon receptor occupancy assay in mice expressing a human glucagon receptor in place of the endogenous mouse glucagon receptor (hGCGR mice). Using this assay, we first showed that the glucagon receptor is expressed predominantly in liver, to a much lesser extent in kidney, and is below detection in several other tissues/organs in the mice. We subsequently showed that, at 2 mg/kg body weight (mg/pk) dosed intraperitoneally (i.p.), peptidyl glucagon receptor antagonist des-His-glucagon binds to ∼78% of the hepatic glucagon receptor and blocks an exogenous glucagon-induced blood glucose elevation in the mice. Finally, we also showed that, at 10 and 30 mg/kg dosed orally (p.o.), compound A, a non-peptidyl small molecule glucagon receptor antagonist, occupied 65–70% of the hepatic glucagon receptor, and significantly diminished exogenous glucagon-induced blood glucose elevation in the mice. At 3 mg/kg, however, compound A occupied only ∼39% of the hepatic glucagon receptor and did not affect exogenous glucagon-induced blood glucose elevation in the mice. Taken together, the results confirmed previous reports that glucagon receptors are present predominantly in the liver, and provide the first direct evidence that peptidyl and non-peptidyl glucagon receptor antagonists bind to the hepatic glucagon receptor in vivo, and that at least 60% receptor occupancy correlates with the glucose lowering efficacy by the antagonists in vivo.

A new class of diacid analogues that binds at the AMP site not only are very potent but have approximately 10-fold selectivity in liver versus muscle glycogen phosphorylase (GP) in the in vitro assay. The synthesis, structure, and in vitro and in vivo biological evaluation of these liver selective glycogen phosphorylase inhibitors are discussed.

The effects of two beta(3)-adrenergic receptor agonists, (R)-4-[4-(3-cyclopentylpropyl)-4,5-dihydro-5-oxo-1H-tetrazol-1-yl]-N-[4-[2-[[2-hydroxy-2-(3-pyridinyl)ethyl]amino]ethyl]phenyl]benzenesulfonamide and (R)-N-[4-[2-[[2-hydroxy-2-(3-pyridinyl)- ethyl]amino]ethyl]phenyl]-1-(4-octylthiazol-2-yl)-5-indolinesulfonamide, on indices of metabolic and cardiovascular function were studied in anesthetized rhesus monkeys. Both compounds are potent and specific agonists at human and rhesus beta(3)-adrenergic receptors. Intravenous administration of either compound produced dose-dependent lipolysis, increase in metabolic rate, peripheral vasodilatation, and tachycardia with no effects on mean arterial pressure. The increase in heart rate in response to either compound was biphasic with an initial rapid component coincident with the evoked peripheral vasodilatation and a second more slowly developing phase contemporaneous with the evoked increase in metabolic rate. Because both compounds exhibited weak binding to and activation of rhesus beta(1)-adrenergic receptors in vitro, it was hypothesized that the increase in heart rate may be reflexogenic in origin and proximally mediated via release of endogenous norepinephrine acting at cardiac beta(1)-adrenergic receptors. This hypothesis was confirmed by determining that beta(3)-adrenergic receptor agonist-evoked tachycardia was attenuated in the presence of propranolol and in ganglion-blocked animals, under which conditions there was no reduction in the evoked vasodilatation, lipolysis, or increase in metabolic rate. It is not certain whether the beta(3)-adrenergic receptor-evoked vasodilatation is a direct effect of compounds at beta(3)-adrenergic receptors in the peripheral vasculature or is secondary to the release or generation of an endogenous vasodilator. Peripheral vasodilatation in response to beta(3)-adrenergic receptor agonist administration was not attenuated in animals administered mepyramine, indomethacin, or calcitonin gene-related peptide(8-37). These findings are consistent with a direct vasodilator effect of beta(3)-adrenergic receptor agonists.

We recently described the identification of a non-peptidyl fungal metabolite (l-783,281, compound 1), which induced activation of human insulin receptor (IR) tyrosine kinase and mediated insulin-like effects in cells, as well as decreased blood glucose levels in murine models of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T., Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. & Moller, D. E. (1999) Science 284, 974–977). Here we report the characterization of an active analog (compound 2) with enhanced IR kinase activation potency and selectivity over related receptors (insulin-like growth factor I receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor). The IR activators stimulated tyrosine kinase activity of partially purified native IR and recombinant IR tyrosine kinase domain. Administration of the IR activators to mice was associated with increased IR tyrosine kinase activity in liver.In vivo oral treatment with compound 2 resulted in significant glucose lowering in several rodent models of diabetes. In db/db mice, oral administration of compound 2 elicited significant correction of hyperglycemia. In a streptozotocin-induced diabetic mouse model, compound 2 potentiated the glucose-lowering effect of insulin. In normal rats, compound 2 improved oral glucose tolerance with significant reduction in insulin release following glucose challenge. A structurally related inactive analog (compound 3) was not effective on insulin receptor activation or glucose lowering in db/db mice. Thus, small molecule IR activators exert insulin mimetic and sensitizing effects in cells and in animal models of diabetes. These results have implications for the future development of new therapies for diabetes mellitus.

The beta3-adrenergic receptor is an integral membrane protein consisting of seven transmembrane domains. Unlike the beta1 and beta2 receptors, this subtype lacks the consensus phosphorylation sites required for desensitization by serine kinases. Using the rodent specific beta3 agonist BRL 35135, our initial data indicated that beta3 receptor-mediated glycerol levels progressively decreased following daily oral doses of 5 mg/kg. Therefore, we initiated studies designed to delineate the possible mechanism(s) for this decreased response. Within 3 hours following a single oral dose of BRL 35135, serum glycerol levels and UCP (uncoupling protein) RNA levels were significantly increased whereas beta3 RNA levels were significantly decreased. Rats were dosed daily for 5 days with either vehicle or BRL 35135 (5 mg/kg, p.o.) and blood samples were collected for glycerol analysis. Adipose tissue was excised for lipolysis and adenyl cyclase measurements. In addition, UCP and beta3 receptor RNA levels were assessed. No effect on adipocyte BRL 37344-stimulated adenylyl cyclase activity was observed 3 hours following the initial dose of BRL 35135. Although a slight decrease (approximately 25%) in adenylyl cyclase activity could be observed 24 hours following the initial dose, it wasn't until day 4 of dosing that a significant decrease (50%) was observed. In contrast, beta3- stimulated lipolysis in adipocytes from BRL 35135-treated rats was decreased 85% within 24 hours and this decrease persisted through four days of treatment. These data indicate that the lipolytic response to beta3 receptor activation is decreased after only a single oral dose of BRL 35135, whereas receptor-mediated adenylyl cyclase activation, although initially unaffected, also desensitizes by day four of treatment.

Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.

The thiazolidinediones are novel insulin sensitizers that serve as orally active antidiabetic agents, in rodents, nonhuman primates, and man. We have examined the effects of 4-week oral administration of three thiazolidinediones (AD-5075, BRL 49653, and CS-045) on plasma glucose and triglyceride concentrations in obese hyperglycemic db/db mice. All three agents lower plasma glucose and triglyceride concentrations. Normal levels of glucose are achieved after treatment with AD-5075 (> 1.7 mg/kg) or BRL 49653 (> or = 30 mg/kg), whereas CS-045 (100 or 300 mg/kg) produces only modest reductions in either parameter. Although the thiazolidinediones have demonstrated insulin-sensitizing activities both in vivo and in vitro, their primary molecular target has been unclear. We have compared the in vivo antidiabetic actions described above with the in vitro activities on peroxisomal proliferator-activated receptor-gamma (PPAR gamma). Hamster PPAR gamma 1 was transiently expressed in COS-1 cells to study the binding of [3H]AD-5075. The concentrations of compounds needed to displace radiolabeled AD-5075 from PPAR gamma correlate with their in vivo potency; the Ki values for displacement by cold AD-5075, BRL 49653, and CS-045 are 22, 68, and 1600 nM, respectively. To examine activation of the receptor, it was transiently cotransfected into COS-1 cells with a reporter plasmid containing two copies of a peroxisome proliferator response element. The EC50 values for activation are 2, 6, and 140 nM for AD-5075, BRL 49653, and CS-045, respectively. We have also analyzed limited proteolytic digests of in vitro translated hamster PPAR gamma. The thiazolidinediones produce a conformational change in PPAR gamma analogous to those produced by agonists of other nuclear hormone receptors. In the presence of saturating concentrations of either AD-5075 or BRL 49653, a receptor fragment of 27 kDa is protected from proteolysis by trypsin. These data support the conclusion that the antidiabetic actions of the thiazolidinediones are directly mediated through binding to PPAR gamma and the resulting active conformation of the receptor. Therefore, binding and transactivation assays using PPAR gamma should serve to identify other novel therapeutic agents with potential antidiabetic activities.

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 approximately 50 mumol/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 mumol/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 beta-cell-specific PDE isoforms may reveal novel PDEs as targets for the development of therapeutically useful glucose-dependent insulin secretagogues.

Depolarizing concentrations of glucose produce characteristic alterations of intracellular free Ca2+ ([Ca2+]i) in pancreatic β-cells. The effects of the proposed incretin, glucagon-like peptide-1(7-36amide) (GLP-1a) on [Ca2+]i were determined from Fura-2 fluorescence ratio imaging of cultured obob mouse pancreatic β-cells. In control cells, [Ca2+]i is low in 3 mM glucose; increasing [glucose] to 8–12 mM results in an initial dip in [Ca2+]i followed by slow oscillating increases in [Ca2+]i. GLP-1a (0.03–10 000 pM) does not alter [Ca2+]i in 3 mM glucose, but does change the response to elevated glucose (8–12 mM). The time integral of the initial dip is reduced ([GLP-1a] 10–100 pM), and the integral of the [Ca2+]i signal is increased ([GLP-1a] ≥ 1 pM). GLP-1a increases the frequency of sustained, stable plateau responses to elevated glucose, and the frequency of large, rapid spikes of increased [Ca2+]i associated with either plateaus, or oscillations. Application of a cAMP analog mimics most of the actions of GLP-1a. Activation of the GLP-1a receptor, or application of cAMP alters pancreatic β-cell [Ca2+]i only when [glucose] is high.

In testing and refining our model of the receptor-bound conformation of the potent small-ring somatostatin analog cyclo-(Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11), we have investigated structures constrained within bicyclic systems. Specifically, we have incorporated the 8-membered -Cys-Cys- unit in place of the -Phe11-Pro6-segment, thus achieving two aims: 1) constraint of the 11- > 6 amide bond to the cis geometry established for the cyclic hexapeptide; 2) positioning of the disulfide in place of the position-11 phenyl group, to act as surrogate for phenyl in receptor binding. Synthetic methodology which provides ready access to this class of compounds is presented, along with results of NMR spectral studies of the bicyclic systems. Biological assays show retention of high potency, in confirmation of our view of cyclo-cystine as a good mimetic for cis amide. Other reported mimetics for the cis amide bond are reviewed from the perspectives of comparative ease of accessibility and approximation of various amide bond parameters.

Adenylate cyclase activity was examined as a measure of inhibitory guanine nucleotide binding protein (Gi) function in liver plasma membranes from rats made chemically diabetic by streptozotocin (STZ) treatment. Clonidine activation of the alpha 2 adrenergic receptor, which activates Gi, inhibited forskolin--stimulated adenylate cyclase activity in control membranes. However, there was no effect on adenylate cyclase activity in membranes from STZ diabetic animals. Also, a polyclonal antipeptide antibody was raised to a highly conserved segment of the Gi alpha 2 subunit. This antibody specifically recognizes a 41 kilodalton protein, is blocked by an excess of peptide, does not recognize the alpha-subunit of transducin, and immunoprecipitates a 41 kilodalton protein which was ADP-ribosylated by pertussis toxin. Immunoblots using this antibody detect no difference between normal and STZ diabetic animals in the level of liver plasma membrane Gi expression. Therefore, STZ-induced diabetes altered the function of Gi but had no effect on Gi expression.