Bioactive conformation of luteinizing hormone-releasing hormone: evidence from a conformationally constrained analog.

An analog of luteinizing hormone-releasing hormone containing a gamma-lactam as a conformational constraint has been prepared with the use of a novel cyclization of a methionine sulfonium salt. The analog is more active as a luteinizing hormone-releasing hormone agonist that the parent hormone, and provides evidence for a bioactive conformation containing a beta-turn.

A selective human beta3 adrenergic receptor agonist increases metabolic rate in rhesus monkeys.

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.

Processing advantages of linear chirped fiber Bragg gratings in the time domain realization of optical frequency-domain reflectometry.

The inclusion of a linear chirped fiber Bragg grating for short pulse dispersion is shown to enhance the time domain realization of optical frequency-domain reflectometry. A low resolution demonstrator is constructed with single surface scans containing 140 resolvable spots. The system dynamic range meets that shown in earlier demonstrations without digital post-processing for signal linearization. Using a conjugate pair of chirped pulses created by the fiber grating, ranging is performed with position and velocity information decoupled. Additionally, by probing the target with short pulses and introducing grating dispersion just before photodetection, velocity immune ranging is demonstrated.

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 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.

Glucose-dependent alterations of intracellular free calcium by glucagon-like peptide-1(7-36amide) in individual ob/ob mouse beta-cells.

Depolarizing concentrations of glucose produce characteristic alterations of intracellular free Ca2+ ([Ca2+]i) in pancreatic beta-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 ob/ob mouse pancreatic beta-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] > or = 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 beta-cell [Ca2+]i only when [glucose] is high.

Thyrotropin releasing hormone (TRH) in pineal and hypothalamus of the frog: effect of season and illumination.

The influence of photo-illumination and season on the pineal and hypothalamic content of TRH in the leopard frog (Rana pipiens) was studied. Animals (4-6 in each group) were exposed to constant light or darkness for 72 h and then sacrificed. The pineal and hypothalamus from each frog were extracted for TRH measurement by radioimmunoassay. The experiment was performed in spring, autumn, early winter and mid-winter. In early winter, mid-winter and spring the pineal content of TRH ranged from 0.14-0.70 ng. Significant differences between groups due to season and illumination were recorded. In autumn, mean levels of TRH were 12.95 ng (dark exposed) and 4.19 ng (light exposed)-values 10-20 times higher than at other times of the year (P less than 0.001). The hypothalamic content of TRH ranged from 11.4 ng in spring to 19.5 ng in autumn. Seasonal differences were present, but no effect of light or darkness was found. There was no definite relation between the TRH levels in hypothalamus and pineal. The alteration in pineal and hypothalamic TRH produced by season, and the effect of illumination on pineal TRH content, support the view that TRH has a neuronal function in vertebrates, possibly as a neurotransmitter.

The insulin-mimetic effect of vanadate is not correlated with insulin receptor tyrosine kinase activity nor phosphorylation in mouse diaphragm in vivo.

The in vivo administration of sodium orthovanadate stimulated the incorporation of [14C]glucose into [14C] glycogen, in a dose- and time-dependent manner, in mouse diaphragm. Activation of diaphragm insulin receptor was measured by exogenous tyrosine kinase activity and an antibody that recognizes a conformational change in the receptor beta-subunit upon autophosphorylation. Neither method detected insulin receptor activation by in vivo vanadate administration, suggesting that vanadate's insulin-mimetic effect on mouse diaphragm glycogenesis occurs at a site distal to the insulin receptor.

Inhibition of luteinizing hormone (LH)-releasing hormone-induced secretion of LH in rat anterior pituitary cell culture by testosterone without conversion to 5 alpha-dihydrotestosterone.

The role of 5 alpha-reduction of testosterone in the inhibition of LH secretion was investigated in rat anterior pituitary cell cultures. Pituitary cells were preincubated with testosterone or dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for 17 h and then with LHRH for an additional 4 h. Dihydrotestosterone was 6-fold more potent than testosterone in the inhibition of LHRH-induced LH release. Basal LH secretion was not affected by either androgen. The inhibition curves of testosterone and dihydrotestosterone were not shifted by the presence of the 5 alpha-reductase inhibitors 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA) and 17 beta-N,N-diisopropylcarbamoyl-4-aza-androstan-3-one (DIPA). Neither 4-MA nor DIPA alone had an effect on either basal or LHRH-induced LH release. When pituitary cells were incubated with [3H]testosterone for 17 h, the radioactivities were found to be unmetabolized testosterone (66.9 +/- 2.4%), dihydrotestosterone (13.3 +/- 0.5%), androstenedione (15.9 +/- 1.3%), 5 alpha-androstane-3,17-dione (2.8 +/- 0.3%), and 3 alpha (beta), 17 beta-androstanediol (less than 1%). In the presence of 4-MA or DIPA, 5 alpha-reduction of testosterone was completely inhibited; androstenedione was the only metabolite. Androstenedione was only 12% as potent as testosterone in the inhibition of LHRH stimulation of LH release, and conversion of [3H]androstenedione to testosterone and dihydrotestosterone did occur in these cells. When [3H]dihydrotestosterone was incubated with pituitary cells, the radioactivities were dihydrotestosterone (64.4 +/- 0%), 5 alpha-androstanedione (19.3 +/- 1%), 3 alpha (beta), 17 beta-androstanediol (7.7 +/- 1.7%), and unknown polar metabolites. 4-MA and DIPA had no effect on the metabolism of dihydrotestosterone. These results indicate that both testosterone and dihydrotestosterone inhibit LHRH-induced LH release and that this activity of testosterone does not depend on its 5 alpha-reduction.

The function but not the expression of rat liver inhibitory guanine nucleotide binding protein is altered in streptozotocin-induced diabetes.

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.

Serum half-life and biological activity of mutants of human insulin-like growth factor I which do not bind to serum binding proteins.

We have characterized the biological properties of two mutants of human insulin-like growth factor I (IGF-I) which, as we have shown previously, have normal affinity for the type I IGF receptor, but drastically reduced affinity for the acid-stable components of human serum binding proteins. [Phe-1,Val1,Asn2,Gln3,His4,Ser8,His9,Glu12 ,Tyr15,Leu16]IGF I (B-chain mutant) and [Gln3,Ala4,Tyr15,Leu16]IGF I have 1000 and 500 times lower affinity than IGF-I for the native 150K binding protein in adult rat serum. Like IGF-I, these two peptides migrate as monomers during size exclusion chromatography on TSK 125. [125I]IGF-I, [125I]B-chain mutant, and [125I] [Gln3,Ala4,Tyr15,Leu16]IGF-I have in vivo serum half-lives of 100, 27.5, and 26.9 min, respectively, after iv injection. These data suggest that serum binding protein-bound peptide is cleared from the serum more slowly than free peptide. The tissue distributions of [125I]IGF-I and [125I]B-chain mutant are similar 10 min after dosing, with more than 80% of the tissue-sequestered intact radioactive peptides in the kidney. Despite decreased serum half-lives, the B-chain mutant and [Gln3,Ala4,Tyr15,Leu16]IGF-I are, respectively, 4 times and twice as active as IGF-I in stimulating the incorporation of [14C]glucose into glycogen in rat diaphragm in vivo. This effect of IGF-I is thought to be mediated by the type 1 IGF receptor in muscle, since the same doses of peptide that stimulated glycogen synthesis more than 30-fold did not stimulate the incorporation of [14C]glucose into total lipid in adipose tissue, an effect known to be mediated by the insulin receptor. These data support the hypothesis that serum- or tissue-derived binding proteins impair the ability of IGF-I to exert its effects through the type 1 IGF receptor in vivo.

Thiazolidinediones produce a conformational change in peroxisomal proliferator-activated receptor-gamma: binding and activation correlate with antidiabetic actions in db/db mice.

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.

Vanadate treatment of streptozotocin diabetic rats restores expression of the insulin-responsive glucose transporter in skeletal muscle.

Streptozotocin-treated rats were diabetic, as assessed by blood glucose and plasma insulin values, while vanadate treatment restored blood glucose values to normal. Immunoblot analysis using a monoclonal antibody to the insulin-responsive glucose transporter demonstrated a 70% decline in transporter expression in skeletal muscle of diabetic rats. Subsequent treatment of diabetic animals with vanadate resulted in renewed expression of the transporter to 87% of control levels. Northern blot analysis of total skeletal muscle RNA from diabetic animals revealed a 55% decline in the steady state level of muscle glucose transporter mRNA, while vanadate treatment led to a 187% increase in transporter mRNA over normal levels. These results support the conclusion that vanadate acts to relieve diabetic hyperglycemia by inducing expression of the insulin-responsive glucose transporter at the pretranslational level.

Synthesis and hypoglycemic activity of substituted 8-(1-piperazinyl)imidazo[1,2-a]pyrazines.

A series of alkyl- and halo-substituted 8-(1-piperazinyl)imidazo[1,2-a]pyrazines were prepared using two approaches, the condensation of alpha-halocarbonyl derivatives with an aminopyrazine or the oxidation-dehydration of a [(beta-hydroxyalkyl)amino]pyrazine. These imidazo[1,2-a]pyrazines were evaluated for their binding affinity to the alpha 1, alpha 2, beta 1, and beta 2 adrenergic receptors as well as their ability to lower blood glucose in insulin resistant hyperglycemic ob/ob mice. Modifications on 8-(1-piperazinyl)imidazo[1,2-a]pyrazine (4) reduced alpha 2 binding, lowered hypoglycemic potency, and showed variations in binding to the alpha 1, beta 1, and beta 2 adrenergic receptors. In addition to 4, the 2-methyl, 3-methyl, and 5-methyl 8-(1-piperazinyl)imidazo[1,2-a]pyrazines (16k, 25m, and 16f, respectively) displayed high affinity for the alpha 2 receptor and were potent hypoglycemic agents when compared to 2-amino-7,8-dihydro-4-(1-piperazinyl)-6H-thiopyrano[3,2- d]pyrimidine (MTP-1403, 2). Receptor binding was modified by use of a 4-methylpiperazine moiety which reduced alpha 1 and beta 1 binding while retaining some hypoglycemic activity. The structure-activity relationship for heterocyclic alkyl and halo substitution on biological activity is discussed.

Effects of an alpha 2-adrenoceptor antagonist on glucose tolerance in the genetically obese mouse (C57BL/6J ob/ob).

This study examines the effects of a relatively selective alpha 2-adrenoceptor antagonist, 8-(L-piperazinyl)imado-[1,2-alpha] pyrazine (compound A), and the preferential alpha 2-agonist clonidine on blood glucose, glucose tolerance, and plasma insulin levels in the C57BL/6J ob/ob mouse and its lean littermate. While clonidine raised blood glucose levels and impaired glucose tolerance, oral administration of compound A resulted in decreased blood glucose levels, as well as improved glucose tolerance in ob/ob mice. Insulin levels in ob/ob mice treated with clonidine were significantly reduced, while compound A raised insulin levels threefold and blocked the effects of clonidine when co-administered to the same animals. Clonidine-induced hyperglycemia in lean littermates was not accompanied by a decrease in insulin levels, while a small but significant increase in insulin levels was observed by compound A administration. Glycogen synthesis in diaphragm of ob/ob mice was enhanced after oral administration of compound A and was accompanied by an increase in plasma insulin levels. Concomitant treatment with a potent somatostatin analog to inhibit insulin release blocked the effects of the alpha 2-adrenoceptor antagonist, compound A. These observations suggest that the alpha 2-antagonist studied, increased plasma insulin levels with an accompanying reduction in blood glucose and an improvement in glucose tolerance in a genetic model of insulin resistance. Differential sensitivity to alpha 2-agonist in these genetically obese mice, ob/ob, was demonstrated by decreased insulin levels due to clonidine administration.

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.

A super active cyclic hexapeptide analog of somatostatin.

The cyclic hexapeptide, cyclo (Pro-Phe-D-Trp-Lys-Thr-Phe), I, has been shown to have the biological properties of somatostatin. We now report structure-activity studies which optimize the potency of this cyclic hexapeptide series with the synthesis of cyclo (N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe), II, which is 50-100 times more potent than somatostatin for the inhibition of insulin, glucagon and growth hormone release. The hydroxyl group of tyrosine is seen to lend a 10-fold enhancement to the potency. Potency also is found to be correlated with hydrophobicity. II is found to improve the control of postprandial hyperglycemia in diabetic animals when given in combination with insulin. The analog is found to be quite stable in the blood and in the gastrointestinal tract, but the bioavailability after oral administration is only 1-3%. The biological properties and long duration of II should allow clinical evaluation of the inhibition of glucagon release as an adjunct to insulin in the treatment of patients with diabetes.

Desensitization of beta3-adrenergic receptor-stimulated adenylyl cyclase activity and lipolysis in rats.

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.

Role of divalent metals in the activation and regulation of insulin receptor tyrosine kinase.

Multiple equilibrium equations were solved to separate the individual effects of ionic divalent metals, free nucleotides and their chelated species on insulin receptor tyrosine kinase (IRTK). Basal IRTK is activated by divalent metal cations when present in excess of that required for substrate formation, indicating the presence of a divalent cation-dependent regulatory site on the kinase. The activatory order for basal activity was Mn2+ greater than Co2+ greater than Mg2+ and Ca2+ = 0. The insulin-dependent activation of IRTK was minimal in the absence of excess free divalent metal, even when the concentration of MnATP or MgATP substrate present exceeded the apparent Km of the kinase. The activatory order for insulin-dependent activation of IRTK changed to Mg2+ greater than Mn2+ and Co2+ = 0. The titration of the MnCl2 saturation response at several concentrations of MgCl2 revealed that the insulin-dependent response of IRTK increases as a function of increasing MgCl2, while basal activity was unaffected. This enhancement of the responsiveness to insulin in the presence of both cations was not due to differing affinities of the kinase for substrate, as evidenced by nearly identical apparent Km values for MnATP and MgATP. The Mg2+-dependent increase in the response of the kinase to insulin may be due to Mg2+ inducing a stronger coupling between receptor and kinase than that observed with Mn2+ alone. The plotting of the effect of several concentrations of free divalent metals on substrate saturation curves revealed that an increase in either of the reactants increased the affinity of the insulin-activated kinase for the other respective reactant. Accordingly, free divalent metal and metal-ATP substrate interact with IRTK in a mutually inclusive manner. CaCl2 saturation curves in the presence of constant MnCl2 and increasing MgCl2 showed that the affinity of IRTK for Ca2+ decreases and the affinity for CaATP increased with increasing Mg2+. Our data suggests that IRTK contains three sites for interaction with divalent metal cations: a MeATP (active) site, a regulatory site, and a metal-dependent site acting to couple the receptor with the kinase.

The effects of somatostatin and selected analogs on lipid absorption in animals.

Based upon the clinical finding that a Merck somatostatin-14 (S-14) analog induced steatorrhea in man, we sought to develop animal models to study the effects of S-14 and a series of synthetic analogs on absorption. Rats were trained to eat a diet (preweighed) containing 15% fat. Following the feeding period, the remaining diet was removed and the amount consumed recorded. This food conditioning of the rats was continued until the rats consumed approximately 15 g of the diet per day. Feces were collected and weighed prior to feeding periods. On test days, S-14 or analogs were administered sc to rats immediately prior to feeding. For each compound tested, fat absorption decreased in dose-dependent fashion. For example, S-14 at 0.5 mg/kg did not increase % of dietary fat in feces (% DFF). At 1.0 mg/kg, S-14 increased % DFF from 7.9 to 10.2 (p less than 0.01, pretest day vs test day), and at 10 mg/kg S-14, % DFF increased from 9.1 to 12.8 (p less than 0.001). For each analog, the subcutaneous dose required to decrease fat absorption in rats was several orders of magnitude higher than the intravenous dose required to inhibit insulin and glucagon. Moreover, the threshold for production of statistically significant increases in fecal fat differed among analogs when compared to their endocrine potencies. One analog administered in the model for 14 days was shown to produce consistent fat malabsorption throughout the entire test period; however, this lipid malabsorption was substantially more pronounced on the first three days of the treatment period. When the compound was not administered on day 15, the % DFF significantly decreased. In an attempt to develop a system more suitable for rapid screening, pancreatic secretagogues such as secretin or cholecystokinin, were administered intravenously to anesthetized rats whose duodena had been cannulated and perfused to enable collection of pancreatic secretions. Total amylase, lipase, and protein were determined in single animals in response to a secretagogue, both before and after iv pretreatment by S-14 or an analog. Pancreatic enzyme secretion in response to sequential secretagogue-stimulation was found to be reproducible for up to three injections and behaved in a dose-dependent fashion. In general, secretagogue-induced increases in amylase, lipase, and total protein were comparable. Pretreatment with the S-14 analogs substantially inhibited secretagogue-induced pancreatic exocrine secretion and was dose-dependent.(ABSTRACT TRUNCATED AT 400 WORDS)