Gary G. Chicchi

Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice

OBJECTIVE Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist. RESEARCH DESIGN AND METHODS We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in diet-induced obese (DIO) mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r−/− and Gcgr−/− mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG. RESULTS Peptide DualAG exhibits superior weight loss, lipid-lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The antiobesity effects of DualAG require activation of both GLP1R and GCGR. CONCLUSIONS Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity.

Identification, localization and receptor characterization of novel mammalian substance P-like peptides

Hemokinin-1 (HK-1) is a novel substance P (SP)-like peptide that is encoded by the preprotachykinin C (PPT-C) gene recently identified in mouse B cells and shown to be a potentially important regulator of B cell development (Nat. Immunol. 1 (2000) 392). We have now isolated and characterized the human and rat orthologs of PPT-C and examined activities of human and mouse HK-1 on the three tachykinin receptors, neurokinin-1-3 (NK1-3). The rat PPT-C polypeptide is highly homologous to mouse PPT-C and contains the same processing sites to generate predicted HK-1. The human PPT-C polypeptide is also homologous to mouse PPT-C, however, it contains two potential monobasic cleavage sites rather than a single dibasic cleavage site at the amino-terminal end of the predicted HK-1 peptide. Thus, human PPT-C has the potential to generate full length predicted HK-1 as well as a truncated version (HK-1(4-11)). Polymerase chain reaction analysis revealed that both human and mouse PPT-C were expressed in a variety of tissues with strong signals detected in the skin of both species and in the mouse brain. Binding and functional analysis indicated that human and mouse HK-1 peptides were nearly identical to SP in their overall activity profile on the three NK receptors with the most potent affinity for the NK1 receptor. The results indicate that PPT-C encodes another high affinity ligand of the NK1 receptor which may play an important role in mediating some of the physiological roles previously assigned to the NK1 receptor.

A comparative autoradiographic study of the distributions of substance P and eledoisin binding sites in rat brain.

The relative potencies of tachykinin peptide analogs competing for binding of [125I]Bolton Hunter-conjugated substance P ([125I]BH-SP) or [125I]Bolton Hunter-conjugated eledoisin ([125I]BH-ED) in slide-mounted rat brain sections are very different, indicating the presence of two distinct tachykinin binding sites. The structure-activity profiles resemble those described in peripheral bioassay studies in which two tachykinin receptors have been postulated. Autoradiography of the two iodinated ligands bound with selective and one-site in vitro incubation conditions shows two discrete and distinctly different distribution patterns in brain. Binding sites for [125I]BH-ED are densely distributed in the accessory olfactory bulb, intermediate layers of the cerebral neocortex, portions of the hippocampal CA fields, hypothalamic supraoptic and paraventricular nuclei, central portions of the interpeduncular nucleus, sphenoid nucleus, medial subdivision of the solitary tract complex, and the substantia gelatinosa of the spinal cord. Binding sites for [125I]BH-SP are present in many of these same structures, but the densities and distribution patterns are different. In addition, [125I]BH-SP binds in numerous structures not labeled by [125I]BH-ED. Neither pattern matches the locations of terminations of endogenous tachykinin pathways marked by immunohistochemistry. The results suggest that it would be inappropriate to name brain tachykinin receptors according to the endogenous ligand which binds with highest affinity.

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

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

The Glucagon Receptor Is Involved in Mediating the Body Weight-Lowering Effects of Oxyntomodulin

Oxyntomodulin (OXM) is a peptide secreted postprandially from the L‐cells of the gut that has a weak affinity for both the glucagon‐like peptide‐1 receptor (GLP1R) and the glucagon receptor (GCGR). Peripheral administration of OXM in humans and rodents causes weight loss reducing food intake and increasing energy expenditure. It has been suggested that OXM modulates energy intake solely through GLP1R agonism. Because glucagon decreases food intake in rodents and humans, we examined whether activation of the GCGR is involved in the body weight‐lowering effects of OXM. We identified an equipotent GLP1R‐selective peptide agonist that differs from OXM by only one residue (Q3→E, OXMQ3E), but has no significant GCGR agonist activity in vitro and ∼100‐fold reduced ability to stimulate liver glycogenolysis. Chronic treatment of obese mice with OXM and OXMQ3E demonstrated that OXM exhibits superior weight loss and lipid‐lowering efficacy, and antihyperglycemic activity that is comparable to the corresponding GLP1R‐selective agonist. Studies in Glp1r−/− mice and coadministration of OXM and a GCGR antagonist revealed that the antiobesity effect of OXM requires activation of both GLP1R and GCGR. Our data provide new insight into the mechanism of action of OXM and suggest that activation of GCGR is involved in the body weight‐lowering action of OXM.

Optimization of co‐agonism at GLP‐1 and glucagon receptors to safely maximize weight reduction in DIO‐rodents

The ratio of GLP‐1/glucagon receptor (GLP1R/GCGR) co‐agonism that achieves maximal weight loss without evidence of hyperglycemia was determined in diet‐induced obese (DIO) mice chronically treated with GLP1R/GCGR co‐agonist peptides differing in their relative receptor agonism. Using glucagon‐based peptides, a spectrum of receptor selectivity was achieved by a combination of selective incorporation of GLP‐1 sequences, C‐terminal modification, backbone lactam stapling to stabilize helical structure, and unnatural amino acid substitutions at the N‐terminal dipeptide. In addition to α‐amino‐isobutyric acid (Aib) substitution at position two, we show that α,α′‐dimethyl imidazole acetic acid (Dmia) can serve as a potent replacement for the highly conserved histidine at position one. Selective site‐specific pegylation was used to further minimize enzymatic degradation and provide uniform, extended in vivo duration of action. Maximal weight loss devoid of any sign of hyperglycemia was achieved with a co‐agonist comparably balanced for in vitro potency at murine GLP1R and GCGR. This peptide exhibited superior weight loss and glucose lowering compared to a structurally matched pure GLP1R agonist, and to co‐agonists of relatively reduced GCGR tone. Any further enhancement of the relative GCGR agonist potency yielded increased weight loss but at the expense of elevated blood glucose. We conclude that GCGR agonism concomitant with GLP1R agonism constitutes a promising approach to treatment of the metabolic syndrome. However, the relative ratio of GLP1R/GCGR co‐agonism needs to be carefully chosen for each species to maximize weight loss efficacy and minimize hyperglycemia.

Chronic substance P (NK1) receptor antagonist and conventional antidepressant treatment increases burst firing of monoamine neurones in the locus coeruleus

The mechanism of action of conventional antidepressants (e.g. imipramine) has been linked to modulation of central monoamine systems. Substance P (NK1) receptor antagonists may have antidepressant and anxiolytic effects in patients with major depressive disorder and high anxiety but, unlike conventional antidepressants, are independent of activity at monoamine reuptake sites, transporters, receptors, or monoamine oxidase. To investigate the possibility that substance P receptor antagonists influence central monoamine systems indirectly, we have compared the effects of chronic administration of imipramine with that of the substance P receptor antagonist L-760735 on the spontaneous firing activity of locus coeruleus neurones. Electrophysiological recordings were made from brain slices prepared from guinea-pigs that had been dosed orally every day for 4 weeks with either L-760735 (3 mg/kg), imipramine (10 mg/kg), or vehicle (water), or naive animals. Chronic, but not acute, treatment with the substance P receptor antagonist L-760735, induced burst firing of neurones in the locus coeruleus. This effect resembles that of the conventional antidepressant imipramine. However, their effects are dissociable since, in contrast to chronic imipramine treatment, chronic L-760735 treatment does not cause functional desensitisation of somatic alpha2 adrenoceptors. The mechanism by which chronic substance P receptor antagonist or conventional antidepressant treatment influences the pattern of firing activity of norepinephrine neurones remains to be elucidated. However, an indirect action in the periphery or distant brain nuclei has been excluded by the use of the in vitro slice preparation, suggesting a local site of action in the locus coeruleus.

2-Aryl Indole NK1 receptor antagonists: optimisation of indole substitution

The synthesis and biological evaluation of a series of 2-aryl indoles with high affinity for the human neurokinin-1 (hNK1) receptor are reported, concentrating on optimisation of the indole substitution.

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

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

Modulation of basal and stress‐induced amygdaloid substance P release by the potent and selective NK1 receptor antagonist L‐822429

It has been shown that anxiety and stress responses are modulated by substance P (SP) released within the amygdala. However, there is an important gap in our knowledge concerning the mechanisms regulating extracellular SP in this brain region. To study a possible self‐regulating role of SP, we used a selective neurokinin‐1 (NK1) receptor antagonist to investigate whether blockade of NK1 receptors results in altered basal and/or stress‐evoked SP release in the medial amygdala (MeA), a critical brain area for a functional involvement of SP transmission in enhanced anxiety responses induced by stressor exposure. In vitro binding and functional receptor assays revealed that L‐822429 represents a potent and selective rat NK1 receptor antagonist. Intra‐amygdaloid administration of L‐822429 via inverse microdialysis enhanced basal, but attenuated swim stress‐induced SP release, while the low‐affinity enantiomer of L‐822429 had no effect. Using light and electron microscopy, synaptic contacts between SP‐containing fibres and dendrites expressing NK1 receptors was demonstrated in the medial amygdala. Our findings suggest self‐regulatory capacity of SP‐mediated neurotransmission that differs in the effect on basal and stress‐induced release of SP. Under basal conditions endogenous SP can serve as a signal that tonically inhibits its own release via a NK1 receptor‐mediated negative feedback action, while under stress conditions SP release is further facilitated by activation of NK1 receptors, likely leading to high local levels of SP and activation of receptors to which SP binds with lower affinity.