A new quorum-sensing system (TprA/PhrA) for Streptococcus pneumoniae†D39 that regulates a lantibiotic biosynthesis gene cluster.

The Phr peptides of the Bacillus species mediate quorum sensing, but their identification and function in other species of bacteria have not been determined. We have identified a Phr peptide quorum-sensing system (TprA/PhrA) that controls the expression of a lantibiotic gene cluster in the Gram-positive human pathogen, Streptococcus pneumoniae. Lantibiotics are highly modified peptides that are part of the bacteriocin family of antimicrobial peptides. We have characterized the basic mechanism for a Phr-peptide signaling system in S. pneumoniae and found that it induces the expression of the lantibiotic genes when pneumococcal cells are at high density in the presence of galactose, a main sugar of the human nasopharynx, a highly competitive microbial environment. Activity of the Phr peptide system is not seen when pneumococcal cells are grown with glucose, the preferred carbon source and the most prevalent sugar encountered by S. pneumoniae during invasive disease. Thus, the lantibiotic genes are expressed under the control of both cell density signals via the Phr peptide system and nutritional signals from the carbon source present, suggesting that quorum sensing and the lantibiotic machinery may help pneumococcal cells compete for space and resources during colonization of the nasopharynx.

Contribution of brown adipose tissue activity to the control of energy balance by GLP-1 receptor signalling in mice.

AIMS/HYPOTHESIS: We assessed the contribution of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) signalling to thermogenesis induced by high-fat diet (HFD) consumption. Furthermore, we determined whether brown adipose tissue (BAT) activity contributes to weight loss induced by chronic subcutaneous treatment with the GLP-1R agonist, liraglutide, in a model of diet-induced obesity. METHODS: Metabolic phenotyping was performed using indirect calorimetry in wild-type (WT) and Glp1r-knockout (KO) mice during chow and HFD feeding at room temperature and at thermoneutrality. In a separate study, we investigated the contribution of BAT thermogenic capacity to the weight lowering effect induced by GLP-1 mimetics by administering liraglutide (10 or 30 nmol kg(-1) day(-1) s.c.) to diet-induced obese (DIO) mice for 6 or 4 weeks, respectively. In both studies, animals were subjected to a noradrenaline (norepinephrine)-stimulated oxygen consumption [Formula: see text] test. RESULTS: At thermoneutrality, HFD-fed Glp1r-KO mice had similar energy expenditure (EE) compared with HFD-fed WT controls. However, HFD-fed Glp1r-KO mice exhibited relatively less EE when housed at a cooler standard room temperature, and had relatively lower [Formula: see text] in response to a noradrenaline challenge, which is consistent with impaired BAT thermogenic capacity. In contrast to the loss of function model, chronic peripheral liraglutide treatment did not increase BAT activity as determined by noradrenaline-stimulated [Formula: see text] and BAT gene expression. CONCLUSIONS/INTERPRETATION: These data suggest that although endogenous GLP-1R signalling contributes to increased BAT thermogenesis, this mechanism does not play a significant role in the food intake-independent body weight lowering effect of the GLP-1 mimetic liraglutide in DIO mice.

Intrinsic Size Parameters for Palmitoylated and Carboxyamidomethylated Peptides.

Cross sections for 61 palmitoylated peptides and 73 cysteine-unmodified peptides are determined and used together with a previously obtained tryptic peptide library to derive a set of intrinsic size parameters (ISPs) for the palmitoyl (Pal) group (1.26 ± 0.04), carboxyamidomethyl (Am) group (0.92 ± 0.04), and the 20 amino acid residues to assess the influence of Pal- and Am-modification on cysteine and other amino acid residues. These values highlight the influence of the intrinsic hydrophobic and hydrophilic nature of these modifications on the overall cross sections. As a part of this analysis, we find that ISPs derived from a database of a modifier on one amino acid residue (CysPal) can be applied on the same modification group on different amino acid residues (SerPal and TyrPal). Using these ISP values, we are able to calculate peptide cross sections to within ± 2% of experimental values for 83% of Pal-modified peptide ions and 63% of Am-modified peptide ions. We propose that modification groups should be treated as individual contribution factors, instead of treating the combination of the particular group and the amino acid residue they are on as a whole when considering their effects on the peptide ion mobility features.

Glucagon-like peptide-1 receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic ß-cells.

Glucagon-like peptide-1 receptor (GLP-1R) plays a major role in promoting glucose-stimulated insulin secretion in pancreatic ß-cells. In the present study, we synthesized a novel functional analog of GLP-1 conjugated to tetramethyl rhodamine to monitor the internalization of the receptor. Our data show that after being internalized the receptor is sorted to lysosomes. In endosomes, receptor-ligand complex is found to be colocalized with adenylate cyclase. Pharmacological inhibition of endocytosis attenuates GLP-1R-mediated cAMP generation and consequent downstream protein kinase A substrate phosphorylation and glucose-stimulated insulin secretion. Our study underlines a paradigm shift in GLP-1R signaling and trafficking. The receptor ligand complex triggers cAMP generation both in plasma membrane and in endosomes, which has implications for receptor-mediated regulation of insulin secretion.

Ghrelin-induced adiposity is independent of orexigenic effects.

Ghrelin is a hormone produced predominantly by the stomach that targets a number of specific areas in the central nervous system to promote a positive energy balance by increasing food intake and energy storage. In that respect, similarities exist with the effects of consuming a high-fat diet (HFD), which also increases caloric intake and the amount of stored calories. We determined whether the effects of ghrelin on feeding and adiposity are influenced by the exposure to an HFD. Chronic intracerebroventricular ghrelin (2.5 nmol/d) increased feeding in lean rats fed a low-fat control diet (CD) [192 ± 5 g (ghrelin+CD) vs. 152 ± 5 g (control i.c.v. saline+CD), P<0.001], but the combination of ghrelin plus HFD did not result in significantly greater hyperphagia [150 ± 7 g (ghrelin+HFD) vs. 136 ± 4 g (saline+HFD)]. Despite failing to increase food intake in rats fed the HFD, ghrelin nonetheless increased adiposity [fat mass increase of 14 ± 2 g (ghrelin+HFD) vs. 1 ± 1 g (saline+HFD), P<0.001] up-regulating the gene expression of lipogenic enzymes in white adipose tissue. Our findings demonstrate that factors associated with high-fat feeding functionally interact with pathways regulating the effect of ghrelin on food intake. We conclude that ghrelin's central effects on nutrient intake and nutrient partitioning can be separated and suggest an opportunity to identify respective independent neuronal pathways.

A new glucagon and GLP-1 co-agonist eliminates obesity in rodents.

We report the efficacy of a new peptide with agonism at the glucagon and GLP-1 receptors that has potent, sustained satiation-inducing and lipolytic effects. Selective chemical modification to glucagon resulted in a loss of specificity, with minimal change to inherent activity. The structural basis for the co-agonism appears to be a combination of local positional interactions and a change in secondary structure. Two co-agonist peptides differing from each other only in their level of glucagon receptor agonism were studied in rodent obesity models. Administration of PEGylated peptides once per week normalized adiposity and glucose tolerance in diet-induced obese mice. Reduction of body weight was achieved by a loss of body fat resulting from decreased food intake and increased energy expenditure. These preclinical studies indicate that when full GLP-1 agonism is augmented with an appropriate degree of glucagon receptor activation, body fat reduction can be substantially enhanced without any overt adverse effects.

Addition of Sialic Acid to Insulin Confers Superior Physical Properties and Bioequivalence.

Native insulin is susceptible to biophysical aggregation and fibril formation, promoted by manual agitation and elevated temperatures. The safety of the drug and its application to alternative forms of administration could be enhanced through the identification of chemical modifications that strengthen its physical stability without compromising its biological properties. Complex polysialic acids (PSAs) exist naturally and provide a means to enhance the physical properties of peptide therapeutics. A set of insulin analogues site-specifically derivatized with sialic acid were prepared in an overall yield of 50-60%. Addition of a single or multiple sialic acids conferred remarkable enhancement to the biophysical stability of human insulin while maintaining its potency. The time to the onset of fibrillation was extended by more than 10-fold relative to that of the native hormone. These results demonstrate that simplified sialic acid conjugates represent a viable alternative to complex natural PSAs in increasing the stability of therapeutic peptides.

Crystallization and preliminary x-ray analysis of anti-obesity peptide hormone oxyntomodulin.

Oxyntomodulin is a proglucagon-derived gut hormone that reduces food intake and body weight, thus represents a potential therapy for obesity. We synthesized and crystallized oxyntomodulin. The crystal diffracts x-ray to 2.4 A resolution and belongs to space group P2(1)3 with unit-cell parameters a=b=c= 48.44 A, alpha=beta=gamma=90 degrees . Preliminary analysis indicates a trimer packing in one asymmetric unit.

Design, synthesis and crystallization of a novel glucagon analog as a therapeutic agent.

Glucagon and glucagon-like peptide 1 (GLP-1) are drugs or drug candidates for the treatment of metabolic diseases such as diabetes and obesity. The native hormones have pharmacological deficiencies such as short half-life and poor solubility. A novel glucagon receptor agonist named glucagon-Cex has been designed, synthesized and crystallized. This peptide was highly soluble under physiological conditions and crystallized readily. The crystal diffracted X-rays to 2.2 A resolution and the diffraction was consistent with space group P23, with unit-cell parameters a = b = c = 48.20 A, alpha = beta = gamma = 90.0 degrees. The crystals were suitable for a full structural determination to reveal the conformational differences between glucagon-Cex and the native hormone.

A novel and selective beta-melanocyte-stimulating hormone-derived peptide agonist for melanocortin 4 receptor potently decreased food intake and body weight gain in diet-induced obese rats.

alphaMSH has generally been accepted as the endogenous ligand for melanocortin 4 receptor (MC4R), which plays a major role in energy homeostasis. Targeting MC4R to develop antiobesity agents, many investigators have performed a structure-activity relationship (SAR) studies based on alphaMSH structure. In this report, we performed a SAR study using human betaMSH (5 - 22) (DEGPYRMEHFRWGSPPKD, peptide 1) as a lead sequence to develop potent and selective agonists for MC4R and MC3R. The SAR study was begun with a truncation of N terminus of betaMSH (5 - 22) together with acetylation of the N terminus and amidation of the C terminus of the peptide. Introduction of a cyclic disulfide constrain and replacement of L-Phe with D-Phe afforded a super potent agonist (peptide 5). Furthermore truncation at the C terminus generated a small and potent MC4R and MC3R agonist (Ac-YRcyclo[CEHdFRWC]amide, peptide 6), which exhibited no MC5R and greatly reduced MC1R activity. Molecular modeling of Ac-YRcyclo[CEHdFRWC]amide (peptide 6) revealed that Arg2 in the peptide formed a salt bridge with Glu4. Subcutaneous or intracerebroventricular administration of peptide 6 in rats showed potent in vivo efficacy as evidenced by its effects in reducing energy balance, increasing fat use, and decreasing weight gain in both acute and chronic rat metabolic studies. Furthermore, the antiobesity effect by peptide 6 was manifested only in wild-type but not MC4R-deficient mice, indicating that antiobesity effects of the peptide were attributed largely through MC4R but not MC3R agonist activity of the peptide.

Discovery of a beta-MSH-derived MC-4R selective agonist.

A series of novel, disulfide-constrained human beta-melanocyte stimulating hormone (beta-MSH)-derived peptides were optimized for in vitro melanocortin-4 receptor (MC-4R) binding affinity, agonist efficacy, and selectivity. The most promising of these, analogue 18, was further studied in vivo using chronic rat food intake and body weight models.

Potent peptide agonists for human melanocortin 3 and 4 receptors derived from enzymatic cleavages of human beta-MSH(5-22) by dipeptidyl peptidase I and dipeptidyl peptidase IV.

Human beta-MSH(1-22) was first isolated from human pituitary as a 22-amino acid (aa) peptide derived from a precursor protein, pro-opiomelanocortin (POMC). However, Bertagna et al. demonstrated that a shorter human beta-MSH(5-22), (DEGPYRMEHFRWGSPPKD), is a true endogenous peptide produced in human hypothalamus. In this report, we demonstrated that in vitro enzymatic cleavage of native human beta-MSH(5-22) with two ubiquitous dipeptidyl peptidases (DPP), DPP-I and DPP-IV, generated two potent MC3/4R peptide analogues, beta-MSH(7-22) (GPYRMEHFRWGSPPKD) and beta-MSH(9-22) (YRMEHFRWGSPPKD). In fact, the MC4R binding affinity and functional potency of beta-MSH(7-22) (Ki=4.6 nM, EC50=0.6 nM) and beta-MSH(9-22) (Ki=5.7 nM, EC50=0.6 nM) are almost an order of magnitude greater than those of their parent peptide, beta-MSH(5-22) (MC4R, Ki=23 nM, EC50= 3nM). Furthermore, the DPP-I/DPP-IV cleaved peptide, beta-MSH(9-22), when administered intracerebroventricularly (ICV) at a dose of 3 nmol/rat, potently induced an acute negative energy balance in a diet-induced obese rat model, while its parent molecule, beta-MSH(5-22), administered at the same dose did not have any effect. These data suggest that DPP-I and DPP-IV may play a role in converting the endogenous beta-MSH(5-22) to more potent peptides that regulate energy homeostasis in the hypothalamus.

A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents.

We report the discovery of a new monomeric peptide that reduces body weight and diabetic complications in rodent models of obesity by acting as an agonist at three key metabolically-related peptide hormone receptors: glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors. This triple agonist demonstrates supraphysiological potency and equally aligned constituent activities at each receptor, all without cross-reactivity at other related receptors. Such balanced unimolecular triple agonism proved superior to any existing dual coagonists and best-in-class monoagonists to reduce body weight, enhance glycemic control and reverse hepatic steatosis in relevant rodent models. Various loss-of-function models, including genetic knockout, pharmacological blockade and selective chemical knockout, confirmed contributions of each constituent activity in vivo. We demonstrate that these individual constituent activities harmonize to govern the overall metabolic efficacy, which predominantly results from synergistic glucagon action to increase energy expenditure, GLP-1 action to reduce caloric intake and improve glucose control, and GIP action to potentiate the incretin effect and buffer against the diabetogenic effect of inherent glucagon activity. These preclinical studies suggest that, so far, this unimolecular, polypharmaceutical strategy has potential to be the most effective pharmacological approach to reversing obesity and related metabolic disorders.

Neuropeptide Y-Y2 receptors mediate anxiety in the amygdala.

The behavioral effects of direct injection of the neuropeptide Y (NPY) Y2 receptor agonist C2-NPY into the basolateral nucleus of the amygdala (BLA) was assessed in rats utilizing the social interaction test (SI). C2-NPY decreased SI time in a dose-dependent manner with a significant change observed at a dose of 80 pmol/100 nl. The anxiogenic effects produced by intra-amygdalar C2-NPY injections were reversed with intraperitoneal administration of alprazolam (1 mg/kg), a known anxiolytic. These findings support the hypothesis that Y2 receptors are involved in the regulation of the anxiety response.

A glucagon analog chemically stabilized for immediate treatment of life-threatening hypoglycemia.

For more than half a century glucagon has been used as a critical care medicine in the treatment of life-threatening hypoglycemia. It is commercially supplied as a lyophilized powder intended to be solubilized in dilute aqueous hydrochloric acid immediately prior to administration. We have envisioned a "ready-to-use" glucagon as a drug of more immediate and likely use. Through a series of iterative changes in the native sequence we have identified glucagon analogs of appreciably enhanced aqueous solubility at physiological pH, and of chemical stability suitable for routine medicinal use. The superior biophysical properties were achieved in part through adjustment of the isoelectric point by use of a C-terminal Asp-Glu dipeptide. The native glutamines at positions 3, 20 and 24 as well as the methionine at 27 were substituted with amino acids of enhanced chemical stability, as directed by a full alanine scan of the native hormone. Of utmost additional importance was the dramatically enhanced stability of the peptide when Ser16 was substituted with alpha,aminoisobutyric acid (Aib), a substitution that stabilizes peptide secondary structure. The collective set of changes yield glucagon analogs of comparable in vitro and in vivo biological character to native hormone but with biophysical properties much more suitable for clinical use.

Both acyl and des-acyl ghrelin regulate adiposity and glucose metabolism via central nervous system ghrelin receptors.

Growth hormone secretagogue receptors (GHSRs) in the central nervous system (CNS) mediate hyperphagia and adiposity induced by acyl ghrelin (AG). Evidence suggests that des-AG (dAG) has biological activity through GHSR-independent mechanisms. We combined in vitro and in vivo approaches to test possible GHSR-mediated biological activity of dAG. Both AG (100 nmol/L) and dAG (100 nmol/L) significantly increased inositol triphosphate formation in human embryonic kidney-293 cells transfected with human GHSR. As expected, intracerebroventricular infusion of AG in mice increased fat mass (FM), in comparison with the saline-infused controls. Intracerebroventricular dAG also increased FM at the highest dose tested (5 nmol/day). Chronic intracerebroventricular infusion of AG or dAG increased glucose-stimulated insulin secretion (GSIS). Subcutaneously infused AG regulated FM and GSIS in comparison with saline-infused control mice, whereas dAG failed to regulate these parameters even with doses that were efficacious when delivered intracerebroventricularly. Furthermore, intracerebroventricular dAG failed to regulate FM and induce hyperinsulinemia in GHSR-deficient (Ghsr(-/-)) mice. In addition, a hyperinsulinemic-euglycemic clamp suggests that intracerebroventricular dAG impairs glucose clearance without affecting endogenous glucose production. Together, these data demonstrate that dAG is an agonist of GHSR and regulates body adiposity and peripheral glucose metabolism through a CNS GHSR-dependent mechanism.

Unimolecular dual incretins maximize metabolic benefits in rodents, monkeys, and humans.

We report the discovery and translational therapeutic efficacy of a peptide with potent, balanced co-agonism at both of the receptors for the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). This unimolecular dual incretin is derived from an intermixed sequence of GLP-1 and GIP, and demonstrated enhanced antihyperglycemic and insulinotropic efficacy relative to selective GLP-1 agonists. Notably, this superior efficacy translated across rodent models of obesity and diabetes, including db/db mice and ZDF rats, to primates (cynomolgus monkeys and humans). Furthermore, this co-agonist exhibited synergism in reducing fat mass in obese rodents, whereas a selective GIP agonist demonstrated negligible weight-lowering efficacy. The unimolecular dual incretins corrected two causal mechanisms of diabesity, adiposity-induced insulin resistance and pancreatic insulin deficiency, more effectively than did selective mono-agonists. The duration of action of the unimolecular dual incretins was refined through site-specific lipidation or PEGylation to support less frequent administration. These peptides provide comparable pharmacology to the native peptides and enhanced efficacy relative to similarly modified selective GLP-1 agonists. The pharmacokinetic enhancement lessened peak drug exposure and, in combination with less dependence on GLP-1-mediated pharmacology, avoided the adverse gastrointestinal effects that typify selective GLP-1-based agonists. This discovery and validation of a balanced and high-potency dual incretin agonist enables a more physiological approach to management of diseases associated with impaired glucose tolerance.

Fibroblast growth factor 21 mediates specific glucagon actions.

Glucagon, an essential regulator of glucose homeostasis, also modulates lipid metabolism and promotes weight loss, as reflected by the wasting observed in glucagonoma patients. Recently, coagonist peptides that include glucagon agonism have emerged as promising therapeutic candidates for the treatment of obesity and diabetes. We developed a novel stable and soluble glucagon receptor (GcgR) agonist, which allowed for in vivo dissection of glucagon action. As expected, chronic GcgR agonism in mice resulted in hyperglycemia and lower body fat and plasma cholesterol. Notably, GcgR activation also raised hepatic expression and circulating levels of fibroblast growth factor 21 (FGF21). This effect was retained in isolated primary hepatocytes from wild-type (WT) mice, but not GcgR knockout mice. We confirmed this link in healthy human volunteers, where injection of natural glucagon increased plasma FGF21 within hours. Functional relevance was evidenced in mice with genetic deletion of FGF21, where GcgR activation failed to induce the body weight loss and lipid metabolism changes observed in WT mice. Taken together, these data reveal for the first time that glucagon controls glucose, energy, and lipid metabolism at least in part via FGF21-dependent pathways.

The orphan receptor Gpr83 regulates systemic energy metabolism via ghrelin-dependent and ghrelin-independent mechanisms.

The G protein-coupled receptor 83 (Gpr83) is widely expressed in brain regions regulating energy metabolism. Here we report that hypothalamic expression of Gpr83 is regulated in response to nutrient availability and is decreased in obese mice compared with lean mice. In the arcuate nucleus, Gpr83 colocalizes with the ghrelin receptor (Ghsr1a) and the agouti-related protein. In vitro analyses show heterodimerization of Gpr83 with Ghsr1a diminishes activation of Ghsr1a by acyl-ghrelin. The orexigenic and adipogenic effect of ghrelin is accordingly potentiated in Gpr83-deficient mice. Interestingly, Gpr83 knock-out mice have normal body weight and glucose tolerance when fed a regular chow diet, but are protected from obesity and glucose intolerance when challenged with a high-fat diet, despite hyperphagia and increased hypothalamic expression of agouti-related protein, Npy, Hcrt and Ghsr1a. Together, our data suggest that Gpr83 modulates ghrelin action but also indicate that Gpr83 regulates systemic metabolism through other ghrelin-independent pathways.

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.