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We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.

We report a series of potent and selective MC4R agonists based on spiroindane amide privileged structures for potential treatments of obesity. Among the synthetic methods used, Method C allows rapid synthesis of the analogs. The series of compounds can afford high potency on MC4R as well as good rodent pharmacokinetic profiles. Compound 1r (MK-0489) demonstrates MC4R mediated reduction of food intake and body weight in mouse models. Compound 1r is efficacious in 14-day diet-induced obese (DIO) rat models.

We report the design, synthesis and properties of spiroindane based compound 1, a potent, selective, orally bioavailable, non-peptide melanocortin subtype-4 receptor agonist. Compound 1 shows excellent erectogenic activity in the rodent models.

We report SAR studies on a novel non-peptidic bombesin receptor subtype-3 (BRS-3) agonist lead series derived from high-throughput screening hit RY-337. This effort led to the discovery of compound 22e with significantly improved potency at both rodent and human BRS-3.

A novel isoquinuclidine containing selective melanocortin subtype-4 receptor small molecule agonist, 3 (RY764), is reported. Its in vivo characterization revealed mechanism-based food intake reduction and erectile activity augmentation in rodents.

Objective: To examine the effect of a high-fat diet on gene expression in adipose tissues and to determine induction kinetics of adipose monocyte chemoattractant protein-1 and −3 (MCP-1 and MCP-3) in diet-induced obesity (DIO) and the effect of a lack of MCP-1 signaling on DIO susceptibility and macrophage recruitment into adipose tissue. Research Methods and Procedures: Obese and lean adipose tissues were profiled for expression changes. The time-course of MCP-1 and MCP-3 expression was examined by reverse transcriptase-polymerase chain reaction. Plasma MCP-1 levels were determined by enzyme-linked immunosorbent assay (ELISA). Chemokine receptor-2 (CCR2) knockout mice were placed on the high-fat diet to determine DIO susceptibility. Macrophage infiltration in adipose tissue was examined by immunohistochemistry with F4/80 antibody. Results: DIO elevated adipose expression of many inflammatory genes, including MCP-1 and MCP-3. Adipose MCP-1 and MCP-3 mRNA levels increased within 7 days of starting a high-fat diet, with elevation of plasma MCP-1 detected after 4 weeks on the diet. The induction of MCP-1 and MCP-3 expression preceded that of tumor necrosis factor-α. The elevated plasma MCP-1 concentration in obese mice was partially reversed by treatment with AM251. No change in DIO susceptibility and macrophage accumulation in adipose tissue were observed in CCR2 knockout mice, which lack the MCP-1 receptor CCR2. Discussion: A high-fat diet elevated adipose expression of inflammatory genes, including early induction of MCP-1 and MCP-3, supporting the view that obese adipose tissues contribute to systemic inflammation. However, despite increased MCP-1 in obesity, disruption of MCP-1 signaling did not confer resistance to DIO in mice or reduce adipose tissue macrophage infiltration.

THE MELANOCORTIN (MC) system is composed of peptides that are cleaved from the polypeptide precursor proopiomelanocortin (POMC). Central MC peptides are produced by neurons within the hypothalamic arcuate nucleus and the medulla (Dores et al., 1986; Jacobowitz and O’Donohue, 1978; O’Donohue and Dorsa, 1982). These peptides include adrenocorticotropic hormone, α-melanocyte–stimulating hormone (α-MSH), β-MSH, and γ-MSH (Hadley and Haskell-Luevano, 1999). Genetic and pharmacological evidence reveals that MC receptor (MCR) signaling is involved in grooming behavior (Gispen et al., 1975), antipyretic (Murphy et al., 1983) and anti-inflammatory (Macaluso et al., 1994) responses, learning (Zhao et al., 1995), reproductive function (Schioth and Watanobe, 2002), and regulation of appetite and energy homeostasis (Chen et al., 2000; Fan et al., 1997; Huszar et al., 1997; Marsh et al., 1999; Schwartz and Wisse, 2000). There are several observations that suggest that the MC system is a prime candidate for regulating drug-seeking behavior. MCRs are expressed in brain regions thought to mediate drug self-administration, including the nucleus accumbens (NAcc), the hypothalamus, and the ventral tegmental area (VTA) (Alvaro et al., 1996; Griffon et al., 1994; Mountjoy et al., 1994; Roselli-Rehfuss et al., 1993). Interestingly, α-MSH administered into the VTA increases dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the NAcc (Lindblom et al., 2001), and chronic intracerebroventricular (ICV) infusion of the nonselective MCR agonist melanotan II (MTII) to rats increases dopamine D1 receptor binding in the NAcc and dopamine D2 receptor binding in the VTA, suggesting that MTII alters dopamine signaling in these regions (Lindblom et al., 2002a). Chronic treatment of a high dose of morphine decreases MC4 receptor (MC4R) mRNA expression in the NAcc, the periaqueductal gray, and neostriatum (Alvaro et al., 1996), brain regions that modulate drug reward, opiate tolerance, and psychomotor stimulation, respectively (Kalivas and Stewart, 1991; Koob and Bloom, 1988; Wise and Bozarth, 1987). On the other hand, chronic treatment with low doses of morphine or cocaine increases MC4R mRNA in the striatum and hippocampus (Alvaro et al., 2003). Consistent with a role in drug self-administration, central infusion of an MCR agonist decreases the acquisition of heroin self-administration in rats (van Ree et al., 1981). Ethanol has direct effects on central POMC and α-MSH activity (Angelogianni and Gianoulakis, 1993; Rainero et al., 1990). It is therefore possible that MC neuropeptides modulate neurobiological responses to ethanol and participate in the control of voluntary ethanol consumption. Several observations are consistent with this hypothesis. First, α-MSH is expressed in brain regions involved with neurobiological responses to ethanol, including the striatum, NAcc, VTA, amygdala, hippocampus, and hypothalamus (Bloch et al., 1979; Dube et al., 1978; Jacobowitz and O’Donohue, 1978; O’Donohue and Jacobowitz, 1980; O’Donohue et al., 1979; Yamazoe et al., 1984). Second, rats selectively bred for high ethanol drinking (AA [Alko, alcohol]) have low levels of MC3R in the shell of the NAcc but have high levels of MC3R and MC4R in various regions of the hypothalamus, when compared with low–ethanol-drinking rats (Lindblom et al., 2002b). Third, central infusion of MTII significantly reduces voluntary ethanol drinking in AA rats with an established history of ethanol intake (Ploj et al., 2002). Recently, MTII-induced reduction of ethanol consumption was shown to be receptor mediated and not associated with alterations of ethanol metabolism in C57BL/6 mice (Navarro et al., 2003). One objective of the present report was to study the role of selected MCRs in the modulation of MCR agonist–induced reduction of ethanol intake. In rodents, MC peptides act through five receptors (MC1R–MC5R) (Hadley and Haskell-Luevano, 1999). MC3R and MC4R are expressed at high levels in the brain (Alvaro et al., 1997), whereas MC1R and MC5R are detected at low levels and in only limited brain regions (Adan and Gispen, 1997; Barrett et al., 1994; Xia et al., 1995). MTII binds, with varying affinity, to all centrally expressed MCRs (Haskell-Luevano et al., 1997; Schioth et al., 1997). Thus, it is unclear which MCRs are important for modulating MTII-induced reductions of ethanol consumption (Navarro et al., 2003; Ploj et al., 2002). To assess the contribution of MC3R, we examined MTII-induced alteration of ethanol drinking in Mc3r–knock-out (Mc3r−/−) and wild-type (Mc3r+/+) mice. To assess the role of MC4R, we studied ethanol intake by C57BL/6J mice after central infusion of the highly selective MC4R agonist cyclo(NH-CH2-CH2-CO-His-d-Phe-Arg-Trp-Glu)-NH2. A second objective was to determine whether central administration of AgRP-(83-132) would increase ethanol consumption by mice. In vivo, AgRP-(83-132) is a potent and nonselective MCR antagonist (Quillan et al., 1998). Such results would strengthen the argument that MCR signaling plays an important role in the modulation of ethanol self-administration. Because MCR agonists reduce and MCR antagonists increase feeding (Hagan et al., 2000; Hohmann et al., 2000; Hollopeter et al., 1998; Marsh et al., 1999; Thiele et al., 1998), food intake was concurrently assessed in most studies described in the present report.

The enzyme 11β–hydroxysteroid dehydrogenase (HSD) type 1 converts inactive cortisone into active cortisol in cells, thereby raising the effective glucocorticoid (GC) tone above serum levels. We report that pharmacologic inhibition of 11β-HSD1 has a therapeutic effect in mouse models of metabolic syndrome. Administration of a selective, potent 11β-HSD1 inhibitor lowered body weight, insulin, fasting glucose, triglycerides, and cholesterol in diet-induced obese mice and lowered fasting glucose, insulin, glucagon, triglycerides, and free fatty acids, as well as improved glucose tolerance, in a mouse model of type 2 diabetes. Most importantly, inhibition of 11β-HSD1 slowed plaque progression in a murine model of atherosclerosis, the key clinical sequela of metabolic syndrome. Mice with a targeted deletion of apolipoprotein E exhibited 84% less accumulation of aortic total cholesterol, as well as lower serum cholesterol and triglycerides, when treated with an 11β-HSD1 inhibitor. These data provide the first evidence that pharmacologic inhibition of intracellular GC activation can effectively treat atherosclerosis, the key clinical consequence of metabolic syndrome, in addition to its salutary effect on multiple aspects of the metabolic syndrome itself.

Somatostatin (SRIF) regulates pancreatic insulin and glucagon secretion. In the present study we describe the generation of SRIF receptor subtype 5 knockout (sst(5) KO) mice to examine the role of SRIF receptor subtypes (sst) in regulating insulin secretion and glucose homeostasis. Mice deficient in sst(5) were viable, fertile, appeared healthy, and displayed no obvious phenotypic abnormalities. Pancreatic islets isolated from sst(5) KO mice displayed increased total insulin content as compared with islets obtained from wild-type (WT) mice. Somatostatin-28 (SRIF-28) and the sst(5)/sst(1)-selective agonist compound 5/1 potently inhibited glucose-stimulated insulin secretion from WT islets. SRIF-28 inhibited insulin secretion from sst(5) KO islets with 16-fold less potency while the maximal effect of compound 5/1 was markedly diminished when compared with its effects in WT islets. sst(5) KO mice exhibited decreased blood glucose and plasma insulin levels and increased leptin and glucagon concentrations compared with WT mice. Furthermore, sst(5) KO mice displayed decreased susceptibility to high fat diet-induced insulin resistance. The results of these studies suggest sst(5) mediates SRIF inhibition of pancreatic insulin secretion and contributes to the regulation of glucose homeostasis and insulin sensitivity. Our findings suggest a potential beneficial role of sst(5) antagonists for alleviating metabolic abnormalities associated with obesity and insulin resistance.

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp(-/-)) mice to examine the physiological role of AgRP. Agrp(-/-) mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp(-/-) mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp(-/-) mice. As AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, we generated AgRP and NPY double-knockout (Agrp(-/-);Npy(-/-)) mice to determine whether NPY or AgRP plays a compensatory role in Agrp(-/-) or NPY-deficient (Npy(-/-)) mice, respectively. Similarly to mice deficient in either AgRP or NPY, Agrp(-/-);Npy(-/-) mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. Our results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY.

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

Genetic and pharmacological studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiological function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r(-/-)) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4-6-month Mc3r-/- mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. (Feed efficiency is the ratio of weight gain to food intake.) Consistent with increased fat mass, Mc3r(-/-) mice are hyperleptinaemic and male Mc3r(-/-) mice develop mild hyperinsulinaemia. Mc3r(-/-) mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r(-/-) mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.

We evaluated the role of the melanocortin-4 receptor (MC-4R) in the control of metabolic rate and food intake in mice. Intraperitoneal administration of the non-selective MC-R agonist melanotan II (MT-II; a cyclic heptapeptide) increases metabolic rate in wildtype mice, while MC-4R knockout mice are insensitive to the effects of MT-II on metabolic rate. MC-4R knockout mice are also insensitive to the effects of MT-II on reducing food intake. We conclude that MC-4R can mediate control of both metabolic rate and food intake in mice. We infer that a role for MC-3R in mediating the acute effects of MT-II on basal metabolic rate and food intake in wildtype mice seems limited.

It has been well established that the spiroindoline sulfonamide MK-0677 stimulates GH secretion from the pituitary both in vitro and in vivo. MK-0677 has also been shown to increase serum insulin-like growth factor I (IGF-I) and cortisol levels in vivo; these increases are assumed to be driven by the increased serum GH and ACTH levels, respectively. However, such increases could also be due to a direct stimulatory action of MK-0677 at the level of the liver and adrenal cortex. To address this possibility, we investigated whether MK-0677 increased IGF-I and cortisol levels in hypophysectomized dogs. Baseline GH, IGF-I, and cortisol responses to MK-0677 (1 mg/kg, orally) were initially determined. Hypophysectomy (hypox; n = 7) or sham surgery (sham; n = 5) was then carried out. Six days postsurgery, the GH and cortisol responses to MK-0677 were reevaluated in each dog. In addition, each dog was treated with porcine GH (PST; 0.1 IU/kg, s.c.) to confirm the responsiveness of the GH-IGF-I axis. The mean peak GH increases in response to MK-0677 in the presham dogs (83.7 +/- 19.2 ng/ml), post-sham dogs (108 +/- 26.2 ng/ml), and pre-hypox dogs (121.2 +/- 13.6 ng/ml) were not significantly different. Mean peak GH levels were unchanged after MK-0677 administration in the hypox dogs (2.3 +/- 0.7 ng/ml). Before surgery, serum IGF-I levels increased to 243 +/- 27 and 224 +/- 47 ng/ml in the sham and hypox groups, respectively, after MK-0677 administration. Surgery was associated with a marked (> or =50%) decrease in serum IGF-I levels. MK-0677 administration increased IGF-I levels in the sham dogs from 78 +/- 14 to 187 +/- 31 ng/ml, whereas IGF-I levels remained unchanged (17.7 +/- 2.4 ng/ml) in the-hypox dogs. PST treatment increased IGF-I levels in the sham dogs from 162 +/- 30 to 325 +/- 32 ng/ml. In the hypox dogs PST treatment restored IGF-I to physiological levels (from 17.7 +/- 2.4 to 199 +/- 41 ng/ml). Cortisol was increased after MK-0677 administration 3.7-fold in the pre-sham, 3.6-fold in the post-sham, and 3.6-fold in the pre-hypox dogs, but no increase was seen in the post-hypox dogs. ACTH GEL administration (2.2 U/kg, i.m.) to hypox dogs returned cortisol to normal physiological levels, demonstrating the functional integrity of the adrenal cortex. This study demonstrates that the GH secretagogue MK-0677 does not directly stimulate an increase in serum IGF-I or cortisol levels, but depends upon the presence of an intact pituitary.

The process of evaluating the in vivo efficacy of non–peptidyl receptor antagonists in animal models is frequently complicated by failure of compounds displaying high affinity against the human receptors to show measurable affinity at the corresponding rodent receptors. In order to generate a suitable animal model in which to evaluate the in vivo activity of non–peptidyl glucagon receptor antagonists, we have utilized a direct targeting approach to replace the murine glucagon receptor with the human glucagon receptor gene by homologous recombination. Specific expression of the human glucagon receptor (GR) in the livers of transgenic mice was confirmed with an RNase protection assay, and the pharmacology of the human GRs expressed in the livers of these mice parallels that of human GR in a recombinant CHO cell line with respect to both binding of 125I–glucagon and the ability of glucagon to stimulate cAMP production. L–168,049, a non–peptidyl GR antagonist selective for the human GR shows a 3.5 fold higher affinity for liver membrane preparations of human GR expressing mice (IC50=172±98nM) in the presence of MgCl2 in marked contrast to the measured affinity of the murine receptor (IC50=611±97nM) for this non–peptidyl antagonist. The human receptors expressed are functional as measured by the ability of glucagon to stimulate cAMP production and the selectivity of this antagonist for the human receptor is further verified by its ability to block glucagon–stimulated cyclase activity with 5 fold higher potency (IC50=97.2±13.9nM) than for the murine receptor (IC50=504±247nM). Thus we have developed a novel animal model for evaluating GR antagonists in vivo. These mice offer the advantage that the regulatory sequences which direct tissue specific and temporal expression of the GR have been unaltered and thus expression of the human gene in these mice remains in the normal chromosomal context.

Transgenic mutation assays utilizing bacterial target genes display a high frequency of spontaneous mutation at CpG sequences. This is believed to result from the fact that: (1) the prokaryotic genes currently being used as transgenic mutation targets have a high CpG content and (2) these sequences are methylated by mammalian cells to produce 5-methylcytosine (5MC), a known promutagenic base. To study the effect of CpG content on the frequency and type of spontaneous mutation, we have synthesized an analogue of the bacterial lacI target gene ( mrkII ) that contains a reduced number of CpG sequences. This gene was inserted into a lambda vector and used to construct trangenic mice that undergo vector rescue from genomic DNA upon in vitro packaging. Results on spontaneous mutation frequency and spectrum have been collected and compared to those observed at the lacI gene in Big Blue™ transgenic mice. Spontaneous mutations at the mrkII gene occurred at a frequency in the mid-10 −5 range and were predominantly base pair substitutions, similar to results seen in Big Blue™. However, mrkII mutations were distributed toward the carboxyl end of the gene instead of the bias toward the amino terminus seen in lacI . Unexpectedly, 23% of the spontaneous mrkII mutations were GC → AT transitions at CpG sequences (compared to 32% in lacI ), despite the reduction in CpG number from 95 in lacI to only 13 in mrkII . Nine of the CpG bases undergoing transition mutations in mrkII have not been recorded previously as spontaneous sites in Big Blue™. Therefore, substantial reduction of the number of CpG sequences in the lacI transgene did not significantly reduce the rate of spontaneous mutation or alter the contribution of CpG-related events. This suggests that other factors are also operating to establish frequency and composition of spontaneous mutations in transgenic targets.

The purpose of this study was to evaluate the pharmacological and toxicological effects of exogenous GH administration in normal adult dogs. Because porcine GH (pGH) is structurally identical to canine GH, pGH was selected for a 14-wk study in dogs. Thirtytwo dogs (

Objective It has long been proposed that stromelysin is one of the major degradative matrix metalloproteinases responsible for the loss of cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA). This hypothesis was tested by examining the arthritic paws of stromelysin 1 (SLN1)-deficient mice for loss of cartilage and for generation of neoepitopes that would be indicative of aggrecan cleavage. Methods The SLN1 gene was inactivated in murine embryonic stem cells, and knockout mice deficient in SLN1 activity were bred onto the B10.RIII background. The incidence and severity of collagen-induced arthritis (CIA) were compared in wild-type and knockout mice. Paws from mice with CIA were examined for loss of cartilage and for proteoglycan staining, as well as for the generation of the neoepitope FVDIPEN341. Results SLN1-deficient mice developed CIA, as did the wild-type N2 mice. Histologic analyses demonstrated no significant differences among the B10.RIII, wild-type, and knockout mice in loss of articular cartilage and proteoglycan staining. No decrease in the FVDIPEN341 epitope was observed in the SLN1-deficient mice. Conclusion Disruption of the SLN1 gene neither prevents nor reduces the cartilage destruction associated with CIA. Moreover, SLN1 depletion does not prevent the cleavage of the aggrecan Asn341-Phe342 bond.

We have tested the vasoactive effects of kinins in addition to various other endothelium-dependent or independent agonists in the arterial and venous perfused mesenteric circuits of the mouse. Bradykinin (0.1 pmol–100 nmol), but not des–Arg 9 –bradykinin (10 nmol) induced a dose-dependent vasodilation of the precontracted arterial and venous mesenteric vasculature of the mouse. Furthermore, acetylcholine (2.5 nmol) also induced a marked arterial vasodilation but was without effect on the venous side. Other endothelium-dependent vasodilators, such as platelet-activating factor (PAF) (1 nmol), tachykinin NK 1 selective agonist ([Sar 9 ,Met(O 2 ) 11 ]substance P) (0.5 nmol) and adenosine diphosphate (5 nmol), were without effect on either side of the mesenteric bed of the mouse. The bradykinin B 2 receptor selective antagonist (HOE 140) abolished the arterial and venous vasodilation induced by bradykinin without affecting that of acetylcholine or sodium nitroprusside. In addition, the bradykinin B 1 receptor antagonist des–Arg 9 –[Leu 8 ]bradykinin was without effect on the responses induced by bradykinin. A nitric oxide synthase inhibitor N ω -nitro- l -arginine methyl ester ( l -NAME) markedly reduced, whereas removal of the endothelium with 3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) abolished dilatation to bradykinin and acetylcholine (arterial side only) without affecting that induced by sodium nitroprusside in the mouse arterial and venous mesenteric circuits. In the same two circuits of transgenic B 2 knockout mice, the vasodilatory responses to bradykinin were absent, whereas the arterial circuit still responded to acetylcholine by a l -NAME-sensitive vasodilation. Our results suggest the exclusive contribution of B 2 receptors located on the endothelium in the vasodilatory effects of bradykinin in the arterial and venous mesenteric circuits of the mouse.

Approximately 10% of cases of Alzheimer's disease are familial and associated with autosomal dominant inheritance of mutations in genes encoding the amyloid precursor protein1, presenilin 1 (PS1)2 and presenilin 2 (PS2)3,4. Mutations in PS1 are linked to about 25% of cases of early-onset familial Alzheimer's disease5. PS1, which is endoproteolytically processed in vivo6, is a multipass transmembrane protein and is a functional homologue of SEL-12 (ref. 7), a Caenorhabditis elegans protein that facilitates signalling mediated by the Notch/LIN-12 family of receptors8,9. To examine potential roles for PS1 in facilitating Notch-mediated signalling during mammalian embryogenesis, we generated mice with targeted disruptions of PS1 alleles (PS1 –/– mice). PS1 –/–embryos exhibited abnormal patterning of the axial skeleton and spinal ganglia, phenotypes traced to defects in somite segmentation and differentiation. Moreover, expression of mRNA encoding Notch 1 and DII 1 (delta-like gene I)10, a vertebrate Notch ligand, is markedly reduced in the presomitic mesoderm of PS1 –/– embryos compared to controls. Hence, PS1 is required for the spatiotemporal expression of Notch 1 and Dll 1, which are essential for somite segmentation and maintenance of somite borders11–13.

We have reported that MK-0677 is a novel, orally active GH secretagogue that stimulates an immediate and longlasting increase in serum GH levels in dogs. Significant elevations in IGF-I levels were associated with the increased GH secretion. Cortisol secretion was also increased following MK-0677 administration. In the current study, we determined the effect of repeat oral administration of MK-0677 on GH, IGF-I and cortisol levels; we also investigated if the GH and cortisol responses to MK-0677 are influenced by circulating IGF-I concentrations. Following the initial oral administration of MK-0677, GH secretion (area under the time–response curve (AUC) ng/ml per h) was increased 7·9- to 9·8-fold (1·0 mg/kg), 5·6-fold (0·5 mg/kg) or 3·9-fold (0·25 mg/kg). With repeat MK-0677 administration, the GH response was decreased by 41–77%; GH concentrations remained significantly above control in the 0·5 mg/kg and 1·0 mg/kg groups. Individual beagle GH profiles indicated that the increased GH concentration was associated with an amplified GH pulsatile profile. Serum IGF-I levels were significantly increased over control levels at all dosage levels by 480 min on the first day of MK-0677 administration. With repeated administration, IGF-I levels were increased up to 126% and remained elevated through 14 days, the longest treatment period evaluated. While daily MK-0677 administration appeared to increase IGF-I levels over 24 h, as evidenced by significant increases in the pretreatment IGF-I levels on days 4–14, no such increase was noted with alternate day MK-0677 administration; thus the dosage regimen modulated circulating IGF-I levels. MK-0677 stimulated increases in cortisol secretion (AUC μg/dl per h) on the first day of treatment. A decreased cortisol response was observed following repeated daily treatment with MK-0677; in contrast, with alternate day treatment, no decrease in cortisol response to MK-0677 occurred. A marked increase in circulating IGF-I concentrations following administration of exogenous GH resulted in a significant decrease in both the GH and cortisol response to MK-0677 compared with control animals. Our findings suggested, therefore, that circulating IGF-I concentrations regulate GH and cortisol response to MK-0677. In summary, chronic oral administration of MK-0677 was associated with significant increases in GH and IGF-I levels that were maintained for the duration of the treatment. The GH profile following MK-0677 administration consisted of episodic increases above control. Compared with day 1, repeated daily treatment with MK-0677 resulted in an attenuated GH response that was associated with an increase in circulating IGF-I levels. The cortisol response was similarly reduced during chronic MK-0677 treatment, suggesting that IGF-I mediated negative feedback on both the GH and cortisol axes. The fact that similar attenuation of the GH and cortisol responses to MK-0677 on day 1 was observed if IGF-I levels were increased by treating animals with exogenous GH suggested that the attenuated response to MK-0677 that occurred during chronic treatment was mediated by increases in IGF-I rather than desensitization to MK-0677. Thus, a regulatory feedback loop apparently prevents hyperstimulation of the GH axis by MK-0677. We conclude that MK-0677 offers the potential of an orally active GH secretagogue that can maintain elevated IGF-I levels when administered chronically. Journal of Endocrinology (1997) 152, 183–192

To investigate the effect of hypophyseal transection (HST) on GH secretagogue activity of the non-peptidyl GH secretagogue L-692,585 in the conscious pig, male castrated swine were randomly assigned to either a hypophyseal stalk transection group (HST; n=3) or to a sham-operated control group (SOC; n=3). Treatments administered were L-692,585 (100 γg/kg), human GH-releasing factor(1–29)NH2 (GRF; 20 γg/kg) or L-692,585 (100 γg/kg) + GRF (20 γg/kg) on days −7 to −3 before surgery and days +3 to +8 after surgery. To evaluate the integrity of the pituitary gland, the animals were challenged with corticotropin-releasing hormone (CRH; 150 γg) or GnRH (150 ng/kg) both before and after surgery. Blood was collected from −60 to +180 min post treatment and assayed for GH, cortisol and LH. Before surgery, no significant difference (P>0·05) in peak GH response (ng/ml) was present between the two groups (SOC vs HST) in response to L-692,585 (101 ± 12 vs 71 ± 9) or L-692,585 + GRF (171 ± 21 vs 174 ± 21). Only two out of three SOC vs three out of three HST pigs responded to GRF (13 ± 2 vs 25 ± 3) resulting in a significant difference between groups. Following surgery, significant differences were present in peak GH response (ng/ml) between SOC and HST groups following L-692,585 (79 ± 6 vs 13·8 ± 1·0); however, the response to L-692,585 + GRF was similar (115 ± 8 vs 94 ± 7). All animals responded to GRF; however, a significant difference was present between groups due to the magnitude of the responses. Whereas the cortisol responses (ng/ml) to L-692,585 in the SOC and HST groups were similar before surgery, a significant difference was present after surgery (44·4 ± 6·4 vs 14·6 ± 2·1). No significant difference was noted between the HST and SOC groups in response to CRH or GnRH either before or after surgery. These results indicated that L-692,585 induced an immediate GH response in the intact animal in contrast to GRF where the GH release was variable. L-692,585 also stimulated an immediate increase in cortisol levels. Transection of the hypophyseal stalk dramatically decreased but did not ablate the GH or cortisol response to L-692,585. Co-administration of L-692,585 + GRF induced an immediate GH response of similar magnitude in the intact and HST animal. We conclude that L-692,585 has a direct but limited action at the level of the pituitary and that an intact hypophyseal stalk is required for a maximal GH and cortisol response. L-692,585 acts with GRF at the level of the pituitary to induce a maximal GH response. These findings suggest that L-692,585 stimulates GH secretion by acting in combination with GRF and interrupting the inhibitory tone of somatostatin on the somatotroph. Journal of Endocrinology (1996) 148, 371–380

To understand the in vivo function of the amyloid precursor protein (APP) we generated an APP null mutation in mice by homologous recombination in embryonic stem (ES) cells. We show here that homozygous APP deficient mice were produced at expected frequencies. Neither APP mRNA nor protein could be detected in these animals. Yet the homozygous APP mutant mice are fertile and do not show overt abnormalities at up to 12 weeks of age. Neuroanatomical studies of the brain did not reveal significant differences in the knockout mice as compared to the wild-type controls. These results argue against an essential function of APP in mouse embryonic and early neuronal development.

Mice deficient in inducible nitric oxide synthase (iNOS) were generated to test the idea that !NOS defends the host against infectious agents and tumor cells at the risk of contributing to tissue damage and shock. iNOS -I- mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Bacterial endotoxic lipopolysaccharide (LPS) caused shock and death in anesthetized wild-type mice, but in iNOS -I- mice, the fall in central arterial blood pressure was markedly attenuated and early death averted. However, unanesthetized iNOS -I- mice suffered as much LPS-induced liver damage as wild type, and when primed with Propionobacterium acnes and challenged with LPS, they succumbed at the same rate as wild type. Thus, there exist both iNOS-dependent and iNOS-independent routes to LPS-Induced hypotension and death.

L-700,653 is a potent nonpeptidyl GH secretagogue consisting of a benzolactam structure: (4'-[[3(R)-[[3-[(2(S),3-dihydroxypropyl) amino]3-methyl-1-oxobutyl]amino]2,3,4,5-tetrahydro-2-oxo-1H-1-benzaze pin- 1-yl]methyl][1,1'-biphenyl]2-carboxamide hydrochloride). When administered sc by a Medi-Jector device at 0, 0.003, 0.01, 0.03, and 0.1 mg/kg BW to male castrated swine (approximately 50 kg BW), L-700,653 stimulated dose-related increases in peak plasma GH concentrations by 20% (P = NS), 150% (P = NS), 250% (P < 0.05), and 340% (P < 0.05), respectively, over the saline vehicle control value (11.3 +/- 6.5 ng/ml) and stimulated increases in GH areas under the curve (AUCs) by 10% (P = NS), 30% (P = NS), 90% (P < 0.05), and 100% (P < 0.01), respectively, over the saline vehicle control value (799 +/- 145 ng/min.ml). After sc administration of L-700, 653, there were no significant changes in plasma LH levels. Subcutaneous dose of 0.03 or 0.1 mg/kg increased plasma cortisol AUCs by 60% (P = NS) and 150% (P < 0.03) over the control value (2461 +/- 935 ng/min.ml) and increased cortisol peaks by 80% (P = NS) and 200% (P < 0.01), respectively, over the control value (38.3 +/- 12.3 ng/ml). Repeated sc administration of L-700,653 (0.03 or 0.1 mg/kg) at 0800, 1400, and 2000 h daily over 3 days consistently increased mean GH peak and GH AUC at each treatment period, with minimal and maximal increases of 40% and 190% in GH peak level at the 0.03 mg/kg dose and 100% and 400% increases in GH peak level at the 0.01 mg/kg dose, respectively. Continuous i.v. infusion of L-700,653, at either 0.01 or 0.1 mg/kg BW.h over a 180-min period, increased GH AUCs by 60% (P = NS) or 470% (P < 0.01) and GH peaks by 190% (P = NS) or 1520% (P < 0.01), respectively, over the control value (589 +/- 313 ng/min.ml; 7.0 +/- 11.1 ng/ml). After a 180- to 300-min saline infusion, an iv bolus dose of 0.1 mg/kg L-700,653 resulted in GH responses inversely proportional to the previous infusion dose, i.e. 0, 0.01, or 0.1 mg/kg.(ABSTRACT TRUNCATED AT 400 WORDS)

L-692,585 is a 2-hydroxypropyl derivative of L-692,429, both novel non-peptidyl growth hormone (GH) secretagogues. The effects of single and repeated intravenous administration of L-692,585 on serum or plasma GH and other hormones in beagles were evaluated. In a balanced 8-dog dose-ranging study, compared to the saline control with a mean (± s.e.m.) after-dose serum GH peak of 6·1 ± 1·3 ng/ml, L-692,585 significantly increased (P These studies demonstrated that L-692,585 is a potent GH secretagogue that induces an acute, transient increase in GH levels. The magnitude of the response is not down-regulated following repeated daily administration for 14 days. IGF-1 levels are increased for an extended period following each treatment. Journal of Endocrinology (1994) 143, 399–406

Design, synthesis, and SAR of a series of 3H-spiro[isobenzofuran-1,4'-piperidine] based compounds as potent, selective and orally bioavailable melanocortin subtype-4 receptor (MC4R) agonists are disclosed.

We report an SAR study of MC4R analogs containing spiroindane heterocyclic privileged structures. Compound 26 with N-Me-1,2,4-triazole moiety possesses exceptional potency at MC4R and potent anti-obesity efficacy in a mouse model. However, the efficacy is not completely mediated through MC4R. Additional SAR studies led to the discovery of compound 32, which is more potent at MC4R. Compound 32 demonstrates MC4R mediated anti-obesity efficacy in rodent models.

Design, syntheses and structure-activity relationships of N-acetylated piperazine privileged structures containing MC4R agonist compounds were described. The most potent derivatives were low nM MC4R selective full agonists. Several compounds from the series had modest pharmacokinetic properties.

Biologically active replication-competent (subgroups A, B, and C) and replication-defective Rous sarcoma virus-derived vectors containing the cDNA encoding firefly luciferase as a reporter gene were constructed. In these retroviral vectors, luciferase is expressed from a spliced subgenomic mRNA. A biologically active replication-defective UR2 virus-derived vector expressing the reporter gene as a gag -luciferase fusion protein from an unspliced genomic mRNA was also constructed. The luciferase reporter gene was used because it lacks homology with chicken genomic sequences and because a rapid and sensitive direct enzymatic assay is available to monitor luciferase expression in retrovirus-infected cells. The levels of luciferase expression in luciferase recombinant retrovirus-infected chicken embryo fibroblasts are >10 3 higher than that detected in uninfected cells or in cells infected with retroviral vectors carrying other genes. Endpoint dilution titration experiments demonstrated that one infected cell can be detected in a background of 10 3 uninfected cells. The vectors are stable in tissue culture and high level expression of the unselected luciferase reporter gene is maintained. The vectors were used to express luciferase in chicken embryos, demonstrating the potential utility of luciferase as a reporter in vivo .

Histamine H3 receptors (H3Rs) are located on the presynaptic membranes and cell soma of histamine neurons, where they negatively regulate the synthesis and release of histamine. In addition, H3Rs are also located on nonhistaminergic neurons, acting as heteroreceptors to regulate the releases of other amines such as dopamine, serotonin, and norepinephrine. The present study investigated the effects of H3R ligands on appetite and body-weight regulation by using WT and H3R-deficient mice (H3RKO), because brain histamine plays a pivotal role in energy homeostasis. The results showed that thioperamide, an H3R inverse agonist, increases, whereas imetit, an H3R agonist, decreases appetite and body weight in diet-induced obese (DiO) WT mice. Moreover, in DiO WT mice, but not in DiO H3RKO mice, imetit reduced fat mass, plasma concentrations of leptin and insulin, and hepatic triglyceride content. The anorexigenic effects of imetit were associated with a reduction in histamine release, but a comparable reduction in histamine release with α-fluoromethylhistidine, an inhibitor of histamine synthesis, increased appetite. Moreover, the anorexigenic effects of imetit were independent of the melanocortin system, because imetit comparably reduced appetite in melanocortin 3 and 4 receptor-deficient mice. The results provide roles of H3Rs in energy homeostasis and suggest a therapeutic potential for H3R agonists in the treatment of obesity and diabetes mellitus.

The adipose tissue-derived hormone adiponectin improves insulin sensitivity and its circulating levels are decreased in obesity-induced insulin resistance. Here, we report the generation of a mouse line with a genomic disruption of the adiponectin locus. We aimed to identify whether these mice develop insulin resistance and which are the primary target tissues affected in this model. Using euglycemic/insulin clamp studies, we demonstrate that these mice display severe hepatic but not peripheral insulin resistance. Furthermore, we wanted to test whether the lack of adiponectin magnifies the impairments of glucose homeostasis in the context of a dietary challenge. When exposed to high fat diet, adiponectin null mice rapidly develop glucose intolerance. Specific PPARgamma agonists such as thiazolidinediones (TZDs) improve insulin sensitivity by mechanisms largely unknown. Circulating adiponectin levels are significantly up-regulated in vivo upon activation of PPARgamma. Both TZDs and adiponectin have been shown to activate AMP-activated protein kinase (AMPK) in the same target tissues. We wanted to address whether the ability of TZDs to improve glucose tolerance is dependent on adiponectin and whether this improvement involved AMPK activation. We demonstrate that the ability of PPARgamma agonists to improve glucose tolerance in ob/ob mice lacking adiponectin is diminished. Adiponectin is required for the activation of AMPK upon TZD administration in both liver and muscle. In summary, adiponectin is an important contributor to PPARgamma-mediated improvements in glucose tolerance through mechanisms that involve the activation of the AMPK pathway.

Ghrelin, a stomach-derived orexigenic hormone, has stimulated great interest as a potential target for obesity control. Pharmacological evidence indicates that ghrelin's effects on food intake are mediated by neuropeptide Y (NPY) and agouti-related protein (AgRP) in the central nervous system. These include intracerebroventricular application of antibodies to neutralize NPY and AgRP, and the application of an NPY Y1 receptor antagonist, which blocks some of the orexigenic effects of ghrelin. Here we describe treatment of Agrp(-/-);Npy(-/-) and Mc3r(-/-);Mc4r(-/-) double knockout mice as well as Npy(-/-) and Agrp(-/-) single knockout mice with either ghrelin or an orally active nonpeptide ghrelin agonist. The data demonstrate that NPY and AgRP are required for the orexigenic effects of ghrelin, as well as the involvement of the melanocortin pathway in ghrelin signaling. Our results outline a functional interaction between the NPY and AgRP pathways. Although deletion of either NPY or AgRP caused only a modest or nondetectable effect, ablation of both ligands completely abolished the orexigenic action of ghrelin. Our results establish an in vivo orexigenic function for NPY and AgRP, mediating the effect of ghrelin.

To study the effect of NPY deletion on the regulation of its receptors in the NPY knockout (NPY KO) mice, the expression and binding of NPY receptors were investigated by in situ hybridization and receptor autoradiography using 125I-[Leu31,Pro34]PYY and 125I-PYY3-36 as radioligands. A 6-fold increase in Y2 receptor mRNA was observed in the CA1 region of the hippocampus in NPY KO mice, but a significant change could not be detected for Y1, Y4, Y5 and y6 receptors. Receptor binding reveals a 60–400% increase of Y2 receptor binding in multiple brain areas. A similar increase in Y1 receptor binding was seen only in the hypothalamus. These results demonstrate the NPY receptor expression is altered in mice deficient for its natural ligand.

Basement membrane proteins are targeted in a variety of pathologic autoimmune responses, yet little is known regarding the origins and regulation of this subset of pathogenic lymphocytes. To examine the generation and fate of B cells reactive with a matrix autoantigen, nonautoimmune C57BL/6 mice were rendered transgenic for a nephrotropic lupus anti-laminin immunoglobulin (Ig) H chain, termed LamH-Cmu. We previously reported recovery of two distinct phenotypes among LamH-Cmu-transgenic mice: progeny of founders M6 and M29 contained abundant transgene-expressing B cells but little anti-laminin Ig, whereas spontaneous autoreactivity was readily recovered from the M7 lineage that expressed minimal B-cell mIgM. To explore the spectrum of autoreactivity generated in vivo by different LamH-Cmu-endogenous L-chain combinations, we determined in vitro and in vivo antigen reactivity and L-chain V-region sequences of 17 LamH-Cmu-transgenic anti-laminin Igs. The results reveal a heterogeneous population of anti-laminin Igs with different fine specificities encoded by diverse endogenous L chains, encompassing nine different Vk gene families, 11 Vk genes, and three Jk genes. Many of the L chains are identical to known or putative unmutated germline Vk genes used to encode Igs reactive with self and foreign antigens in nonautoimmune and genetically autoimmune-prone mouse strains. These observations confirm that the LamH-Cmu H chain plays a dominant role in determining anti-laminin reactivity, and indicate that nonautoimmune B6 mice are fully capable of generating a diverse pool of basement-membrane-reactive B cells using unmutated Ig genes. When interpreted in the context of the divergent M6/M29 and M7 transgenic mouse phenotypes, our findings further suggest that these matrix-reactive lymphocytes are not spontaneously activated in vivo under normal circumstances.

Voltage-gated L-type Ca2+ channels (LTCCs) containing a pore-forming alpha1D subunit (D-LTCCs) are expressed in neurons and neuroendocrine cells. Their relative contribution to total L-type Ca2+ currents and their physiological role and significance as a drug target remain unknown. Therefore, we generated D-LTCC deficient mice (alpha1D-/-) that were viable with no major disturbances of glucose metabolism. alpha1D-/-mice were deaf due to the complete absence of L-type currents in cochlear inner hair cells and degeneration of outer and inner hair cells. In wild-type controls, D-LTCC-mediated currents showed low activation thresholds and slow inactivation kinetics. Electrocardiogram recordings revealed sinoatrial node dysfunction (bradycardia and arrhythmia) in alpha1D-/- mice. We conclude that alpha1D can form LTCCs with negative activation thresholds essential for normal auditory function and control of cardiac pacemaker activity.

Mutations in the beta-amyloid precursor protein are strongly associated with some cases of familial Alzheimer's disease. The normal physiological role of beta-amyloid precursor protein in the brain was evaluated in a cross-sectional analysis of mice deficient in beta-amyloid precursor protein. Compared with wild-type control mice the beta-amyloid precursor protein-null mice developed age-dependent deficits in cognitive function and also had impairments in long-term potentiation. In addition, the brains of the beta-amyloid precursor protein-null mice had marked reactive gliosis in many areas, especially in the cortex and hippocampus. A subpopulation of mice (n = 15) died prematurely (between three and 18 months of age). Analysis of another six mice from the same population that were showing weight loss and hypolocomotor activity exhibited a marked reactive gliosis as detected by immunoreactivity for glial fibrillary acidic protein and a profound loss of immunoreactivities for the presynaptic terminal vesicle marker proteins synaptophysin and synapsin and the dendritic marker microtubule-associated protein-2 in many brain areas, but most predominantly in the cortex and hippocampus. These results suggest that normal beta-amyloid precursor protein may serve an essential role in the maintenance of synaptic function during ageing. A compromise of this function of the beta-amyloid precursor protein may contribute to the progression of the memory decline and the neurodegenerative changes seen in Alzheimer's disease.

Alterations of hypothalamic neuropeptide Y(NPY) and melanocortinergic functions in diet-induced obese (DIO) C57BL/6J mice were investigated by in situ hybridization. Compared with controls, the DIO mice displayed a profound induction (approximately 40-fold) of NPY expression in the dorsomedial (DMH) and ventromedial (VMH) hypothalamic nuclei, whereas the level of NPY mRNA in the arcuate nucleus (ARC) was reduced by 44%. The expression of pro-opiomelanocortin (POMC) and agouti-related protein was not significantly altered in the ARC of obese mice. Both excess body weight gain and altered hypothalamic NPY expression were reversible. We propose that the highly induced NPY expression in DMH and/or VMH may be a contributing etiological factor for the development of obesity and leptin resistance in the DIO mice.

Amyloid precursor protein (APP) is a member of a larger gene family including amyloid precursor-like proteins (APLP), APLP2 and APLP1. To examine the function of APLP2 in vivo, we generated APLP2 knockout (KO) mice. They are of normal size, fertile, and appear healthy up to 22 months of age. We observed no impaired axonal outgrowth of olfactory sensory neurons following bulbectomy, suggesting against an important role for APLP2 alone in this process. Because APLP2 and APP are highly homologous and may serve similar functions in vivo, we generated mice with targeted APLP2 and APP alleles. Approximately 80% of double KO mice die within the first week after birth, suggesting that APLP2 and APP are required for early postnatal development. The surviving approximately 20% of double KO mice are 20-30% reduced in weight and show difficulty in righting, ataxia, spinning behavior, and a head tilt, suggesting a deficit in balance and/or strength. Adult double KO mice mate poorly, despite apparent normal ovarian and testicular development. Otherwise, double KO mice appear healthy up to 13 months of age. We conclude, that APLP2 and APP can substitute for each other functionally.

Neither the normal functions of presenilins nor the mechanism(s) by which familial Alzheimer’s disease (FAD)-linked mutations cause AD have been defined. Presenilin 1 (PS1) is a polytopic membrane protein that is subject to endoproteolytic processing in vivo; PS1 derivatives accumulate to saturable levels and to ~ 1:1 stoichiometry by mechanism(s) that are not fully defined. We show here that the two fragments coassemble. Moreover, we have detected neither interactions between PS1/PS2 and amyloid precursor protein (APP) nor influences of presenilin expression on APP maturation/secretion. To examine the in vivo function(s) of PS1, we developed mice with functionally inactivated PS1 alleles. These animals die before birth and exhibit several developmental defects, including a poorly differentiated vertebral column, a phenotype traced to abnormal segmentation of somites. Whole mount in situ hybridization analyses reveal that specification of somitic cell lineages is apparently unaffected, despite the clear disruption in somite segmentation. However, notable differences in expression of Notch1 and Dll1 mRNAs were observed in PS −/− embryos; in contrast to wild-type embryos in which abundant expression of Notch1 and Dll1 mRNAs are observed in the presomitic mesoderm, the expression of these genes is nearly abolished in the PSl −/− embryos. Hence, PS1 serves to regulate the spatiotemporal expression of Notch1 and Dll1 in the paraxial mesoderm. Finally, we failed to detect any differences in the levels of Aβ42 and Aβ40 in brains of mice heterozygous for PS1 relative to wild-type litter mates. Thus, mutations in PS1 probably cause AD not by the loss but rather by the gain of deleterious function of mutant polypeptides.

The pulsatile nature of GH release is apparently regulated by alternating sequential changes in two hypothalamic hormones, GH releasing hormone (GHRH) and somatostatin. Entrainment of this pulsatility appears to involve GH-mediated negative feedback. Recently a new receptor involved in GH release was cloned. Activation of this receptor by GH-releasing peptides and MK-0677 initiates and amplifies GH pulsatility and is associated with increased Fos immunoreactivity and electrical activity in GHRH containing arcuate neurons. We show that pretreating mice with GH blocks activation of these neurons by MK-0677. Similarly, octreotide inhibited the action of MK-0677. To determine whether this GH-mediated negative feedback on GHRH neurons was direct, or by GH stimulation of somatostatin release from periventricular neurons, we selectively inactivated the gene for one of the five specific somatostatin receptor subtypes (subtype 2). In the knockout mice, both GH and octreotide failed to inhibit MK-0677 activation of arcuate neurons. GH did, however, increase Fos immunoreactivity in the periventricular nucleus, consistent with GH stimulation of somatostatin release from periventricular neurons. Thus, GH-mediated negative feedback involves signaling between periventricular and arcuate neurons with the signal being transduced specifically through somatostatin subtype 2 receptors.

Growing evidence suggests that recombinatorial events prior to antigen contact can generate pathogenic autoantibodies in the nonautoimmune individual, thus providing potential disease mediators if conditions arise that permit bypass of tolerance and activation of autoreactive lymphocytes. To examine the disease potential of selected germline antibody genes, Ig were created de novo by in vitro recombination of Ig H and L chains. H chain loss variant (i.e., L-chain only) cell lines were transfected with a DNA construct encoding the variable region and regulatory sequences (LamH) of a nephrotropic murine lupus anti-laminin Ig, and the resultant Ig were examined for in vitro antigen reactivity and in vivo glomerular immune deposition. The results indicate that two light chains, LamL (Vk8, Jk5) and 238L (Vk4, Jk5), expressing unrelated germline V1 genes, combine with LamH to generate Ig that bind basement membrane laminin in vitro, diverge in their capacity to bind ssDNA, and produce two distinct patterns of glomerular immune deposits in vivo: dense mesangial matrix (LamH/LamL) and dramatic linear glomerular basement membrane (LamH/238L) deposits. The Ig genes used by both LamH and 238L are present in nonautoimmune mice as well as in lupus-prone strains. We conclude that certain unmutated Ig genes can contribute to multiple distinct disease associated specificities, including binding to intrinsic kidney antigens, and that mutation is not essential to generate these Ig. Collectively, these observations suggest that pathogenic autoantibodies can be generated in the normal preimmune repertoire by random recombinatorial and somatic events in the absence of mutation.

Theβ-amyloid peptide (Aβ), the major component of the neuritic plaques characterizing Alzheimer’s disease (AD), is a 39 to 43 amino acid peptide derived from proteolytic cleavage of a larger β-amyloid precursor protein (APP). Mutations in the APP gene have been identified which cause familial, early onset AD, suggesting that APP metabolism is a central event in AD progression (Mullan & Crawford, 1993). APP is an abundant protein in the brain and Aβ is produced not only in AD patients, but also in cerebrospinal fluid (CSF) of normal individuals (Selkoe, 1994). APP has been implicated in numerous activities based on in vitro studies, such as regulation of cell adhesion, neurite outgrowth and intraneuronal calcium (Selkoe, 1994). However, the in vivo function of APP remains unclear.

MK-0677, a spiroindoline sulfonamide, is a novel, orally active GH secretagogue. The effects of MK-0677 on serum GH and other hormones after oral and iv single dose administrations in beagles were evaluated. After oral administration in a balanced eight-dog crossover study, treatment with MK-0677 significantly increased peak GH concentrations, with a 5.3-fold increase (mean +/- SEM, 10.5 +/- 1.9 ng/ml) at the 0.25 mg/kg dose, a 9.0-fold increase (18.0 +/- 3.3 ng/ml) at the 0.50 mg/kg dose, and a 15.8-fold increase (31.6 +/- 5.8 ng/ml) at the 1.0 mg/kg dose. Total GH release, expressed as the area under the curve, showed similar significant increases over the effect of the water placebo. A single oral 1 mg/kg dose in three dogs induced a mean GH peak of 27.6 +/- 1.5 ng/ml at 120 min, and GH levels remained elevated up to 360 min after treatment. Insulin-like growth factor I (IGF-I) levels were significantly increased by 30% at 480 min after treatment. Cortisol levels were increased 2.4-fold over pretreatment levels. After i.v. administration, compared to the saline control group which had a mean (+/- SEM) serum GH peak of 3.8 +/- 0.7 ng/ml, MK-0677 at 0.25 mg/kg significantly increased (P < 0.05) peak GH concentrations 20.4-fold (77.4 +/- 13.7 ng/ml). Total GH release, expressed as the area under the curve, showed a similar increase. The mean peak GH level was recorded 10 min after treatment, with GH levels elevated up to 180 min after treatment. IGF-I levels were significantly elevated by 25% 360 min after the administration of MK-0677. Cortisol levels were increased 2.3-fold over pretreatment levels. Insulin and glucose levels were higher, LH and PRL levels were unaltered, and T4 levels were marginally lower; the levels of each of these hormones remained within the normal ranges for dogs throughout the experiment. In summary, MK-0677 is a potent GH secretagogue that induces an immediate, large, long lasting increase in GH levels when administered orally or i.v. In contrast to GH-releasing peptide-6 and benzolactam secretagogues, GH levels were elevated up to 360 min after treatment, and this was associated with a significant increase in IGF-I levels. Cortisol levels were increased; however, the increases were modest compared to those in GH.

The leptin receptor (OB-R) bears homology to members of the class I cytokine receptor family. We demonstrate that leptin binding to OB-R stimulates formation of STAT-1 and STAT-3 complexes, thereby defining transcriptional motifs for genes that are under leptin control. Transfected fa OB-R bound leptin with equal affinity to that of wild type OB-R. fa OB-R abundance was about 7 fold reduced compared to control cells. Surprisingly, the low level of fa OB-R is fully capable of activating the STAT signal transduction pathway. We discuss plausible explanations for the obese phenotype in Zucker fatty rats.

Growth hormone (GH) releasing hexapeptide, His-DTrp-Ala-Trp-DPhe- LysNH2 (GHRP-6), was first described as a potent GH-specific secretagogue by Bowers, et al. (2). The first described nonpeptidyl GH secretagogue, L-692,429 (3), has been shown to elicit GH release in many species including swine (4), rats (5), beagles (6), and man (7), while the more potent analogue L-692,585 has also been shown to elicit GH release in beagles (8) and swine (9). L-692,585, a novel nonpeptidyl GH secretagogue analogue of L-692,429, was found to be approximately 20-fold more potent than L-692,429 in rat pituitary cell assays (10) and in beagles in vivo (8). The GH secretagogue activity of L-692,585 has also been reported in swine (9). A weak substance P antagonist, H2N-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D- Trp-Phe-D-Trp-Leu-Leu-NH2 (SPA), has been shown to inhibit GHRP-6 binding in membrane preparations of rat pituitary and hypothalamus (11) and to inhibit both GHRP-6- and L-692,429-stimulated GH responses in rat pituitary cell cultures and in rats in vivo (1). The objective of this study was to determine if SPA could inhibit the GH response elicited by L-692,585 in swine.

We have studied by in situ hybridization histochemistry the mRNA expression of the c-fos immediate early gene in the brains of wild type and dwarf(dw/dw) and little(lit/lit) mutant mice after systemic injections of the synthetic GH secretagogues GHRP-6 and L-163,191. Both GH secretagogues induced a marked c-fos mRNA expression in the arcuate-ventromedial hypothalamus (ARC-VMH) of both control and mutant mice indicating a possible action on growth hormone releasing hormone (GHRH) neurones in the ARC-VMH. Both dw/dw and lit/lit mice showed a 5-fold elevation in GHRH mRNA expression in the ARC-VMH compared with control animals under basal conditions. Since lit/lit mice have a reduced ability to secrete GH and lack a functional GHRH receptor while dw/dw mice lack both GH and presumably GHRH receptors, the GH-secretagogue-induced c-fos mRNA in the brain of these mutants are unlikely to be mediated by an indirect action of GH or a interaction of the synthetic GH-secretagogue with the GHRH receptor.

We used gene targeting in embryonic stem cells to introduce an IL-1 beta null allele in mice. The IL-1 beta-deficient mice develop normally and are apparently healthy and fertile. The IL-1 beta null mice responded normally in models of contact and delayed-type hypersensitivity or following bacterial endotoxin LPS-induced inflammation. The IL-1 beta-deficient mice showed equivalent resistance to Listeria monocytogenes compared with wild-type controls. In contrast, when challenged with turpentine, which causes localized inflammation and tissue injury, the IL-1 beta mutant mice exhibited an impaired acute-phase inflammatory response and were completely resistant to fever development and anorexia. These results highlight a central role for IL-1 beta as a pyrogen and a mediator of the acute-phase response in a subset of inflammatory disease models, and support the notion that blocking the action of a single key cytokine can alter the course of specific immune and inflammatory responses.

Mice that are homozygous for the targeted disruption of the gene encoding the B2 bradykinin receptor have been generated. The gene disruption results in a deletion of the entire coding sequence for the B2 receptor. The disruption of the B2 receptor gene has been confirmed by genetic, biochemical, and pharmacological analyses. Mice that are homozygous for the disruption of the B2 receptor gene are fertile and indistinguishable from their littermates by visual inspection. Bradykinin fails to produce responses in pharmacological preparations from ileum, uterus, and the superior cervical ganglia from these mice. Therefore, expression of a single gene appears to be responsible for conferring responsiveness to bradykinin in these tissues.

In several pedigrees of early onset familial Alzheimer's disease (FAD), point mutations in the β-amyloid precursor protein (APP) gene are genetically linked to the disease. This finding implicates APP in the pathogenesis of Alzheimer's disease in these individuals. To understand the in vivo function of APP and its processing, we have generated an APP-null mutation in mice. Homozygous APP-deficient mice were viable and fertile. However, the mutant animals weighed 15%-20% less than age-matched wild-type controls. Neurological evaluation showed that the APP-deficient mice exhibited a decreased locomotor activity and forelimb grip strength, indicating a compromised neuronal or muscular function. In addition, four out of six homozygous mice showed reactive gliosis at 14 weeks of age, suggesting an impaired neuronal function as a result of the APP-null mutation.