This following article is written for Prof. Abba Kastin’s Festschrift, to add to the tribute to his important role in the advancement of the role of peptides in physiological, as well as pathophysiological processes. There have been many advances during the 35 years of his prominent role in the Peptide field, not only as editor of the journal Peptides, but also as a scientific investigator and editor of two volumes of the Handbook of Biological Active Peptides [146,147]. Similar to the advances with many different peptides, during this 35 year period, there have been much progress made in the understanding of the pharmacology, cell biology and the role of (Bombesin) Bn receptors and their ligands in various disease states, since the original isolation of bombesin from skin of the European frog Bombina bombina in 1970 [76]. This paper will briefly review some of these advances over the time period of Prof Kastin 35 years in the peptide field concentrating on the advances since 2007 when many of the results from earlier studies were summarized [128,129]. It is appropriate to do this because there have been 280 articles published in Peptides during this time on Bombesin-related peptides and it accounts for almost 5% of all publications. Furthermore, 22 Bn publications we have been involved in have been published in either Peptides [14,39,55,58,81,92,93,119,152,216,225,226,231,280,302,309,355,361,362] or in the Prof Kastin’s Handbook of Biological Active Peptides [137,138,331].

It has been suggested that hormones released after nutrient absorption, such as glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 2 (GLP-2), could be responsible for changes in bone resorption. However, information about the role of GLP-1 in this regard is scanty. Diabetes-related bone loss occurs as a consequence of poor control of glucose homeostasis, but the relationship between osteoporosis and type 2 diabetes remains unclear. Since GLP-1 is decreased in the latter condition, we evaluated some bone characteristics in streptozotocin-induced type 2 diabetic (T2D) and fructose-induced insulin-resistant (IR) rat models compared to normal (N) and the effect of GLP-1 or saline (control) treatment (3 days by osmotic pump). Blood was taken before and after treatment for plasma measurements; tibiae and femora were collected for gene expression of bone markers (RT-PCR) and structure (microCT) analysis. Compared to N, plasma glucose and insulin were, respectively, higher and lower in T2D; osteocalcin (OC) and tartrate-resistant alkaline phosphatase 5b were lower; phosphate in IR showed a tendency to be higher; PTH was not different in T2D and IR; all parameters were unchanged after GLP-1 infusion. Bone OC, osteoprotegerin (OPG) and RANKL mRNA were lower in T2D and IR; GLP-1 increased OC and OPG in all groups and RANKL in T2D. Compared to N, trabecular bone parameters showed an increased degree of anisotropy in T2D and IR, which was reduced after GLP-1. These findings show an insulin-independent anabolic effect of GLP-1 and suggest that GLP-1 could be a useful therapeutic agent for improving the deficient bone formation and bone structure associated with glucose intolerance.

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Bombesin-receptor-subtype-3 (BRS-3) is an orphan G-proteincoupled receptor of the bombesin (Bn) family whose natural ligand is unknown and which does not bind any natural Bnpeptide with high affinity. It is present in the central nervous system, peripheral tissues, and tumors; however, its role in normal physiology/pathophysiology is largely unknown because of the lack of selective ligands. Recently, MK-5046and Bantag-1 [Boc-Phe-His-4-amino-5-cyclohexyl-2,4,5-trideoxypentonyl-Leu-(3-dimethylamino) benzylamide N-methylammonium trifluoroacetate], a nonpeptide agonist and a peptide antagonist, respectively, for BRS-3 have been described, but there have been limited studies on their pharmacology. We studied MK-5046 and Bantag-1 interactions with human Bn-receptors-human bombesin receptor subtype-3 (hBRS-3), gastrin-releasing peptide receptor (GRP-R), and neuromedin B receptor (NMB-R)-and compared them with the nonselective, peptide-agonist [D-Tyr6,bAla11,Phe13, Nle14]Bn-(6-14) (peptide #1). Receptor activation was detected by activation of phospholipase C (PLC), mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), paxillin, and Akt. In hBRS-3 cells, the relative affinities were Bantag-1 (1.3 nM) . peptide #1 (2 nM) . MK-5046 (37-160 nM) . GRP, NMB (.10 mM), and the binding-dose-inhibition curves were broad (.4 logs), with Hill coefficients differing significantly from unity. Curve-fitting demonstrated high-affinity (MK-5046, K i 5 0.08 nM) and low-affinity (MK-5046, K i 5 11-29 nM) binding sites. For PLC activation in hBRS-3 cells, the relative potencies were MK-5046 (0.02 nM) . peptide #1 (6 nM) . GRP, NMB, Bantag-1 (.10 mM), and MK-5046 had a biphasic dose response, whereas peptide #1 was monophasic. Bantag-1 was a specific hBRS-3-antagonist. In hBRS-3 cells, MK-5046 was a full agonist for activation of MAPK, FAK, Akt, and paxillin; however, it was a partial agonist for phospholipase A 2 (PLA 2 ) activation. The kinetics of activation/ duration of action for PLC/MAPK activation of MK-5046 and peptide #1 differed, with peptide #1 causing more rapid stimulation; however, MK-5046 had more prolonged activity. Our study finds that MK-5046 and Bantag-1 have high affinity/selectivity for hBRS-3. The nonpeptide MK-5046 and peptide #1 agonists differ markedly in their receptor coupling, ability to activate different signaling cascades, and kinetics/duration of action. These results show that their hBRS-3 receptor activation is not always concordant and could lead to markedly different cellular responses.

Introduction Despite remarkable advances in tumor treatment, many patients still die from common tumors (breast, prostate, lung, CNS, colon, and pancreas), and thus, new approaches are needed. Many of these tumors synthesize bombesin (Bn)-related peptides and over-express their receptors (BnRs), hence functioning as autocrine-growth-factors. Recent studies support the conclusion that Bn-peptides/BnRs are well-positioned for numerous novel antitumor treatments, including interrupting autocrine-growth via the use of over-expressed receptors for imaging and targeting cytotoxic-compounds, either by direct-coupling or combined with nanoparticle-technology. Areas covered The unique ability of common neoplasms to synthesize, secrete, and show a growth/proliferative/differentiating response due to BnR over-expression, is reviewed, both in general and with regard to the most frequently investigated neoplasms (breast, prostate, lung, and CNS). Particular attention is paid to advances in the recent years. Also considered are the possible therapeutic approaches to the growth/differentiation effect of Bn-peptides, as well as the therapeutic implication of the frequent BnR over-expression for tumor-imaging and/or targeted-delivery. Expert opinion Given that Bn-related-peptides/BnRs are so frequently ectopically-expressed by common tumors, which are often malignant and become refractory to conventional treatments, therapeutic interventions using novel approaches to Bn-peptides and receptors are being explored. Of particular interest is the potential of reproducing BnRs in common tumors, such as the recent success of utilizing overexpression of somatostatin-receptors by neuroendocrine-tumors to provide the most sensitive imaging methods and targeted delivery of cytotoxic-compounds.

Glucagon-like peptide 1 (GLP-1) controls glucose metabolism in extrapancreatic tissues through receptors other than the pancreatic cAMP-linked GLP-1 receptor; also, GLP-1 induces an insulin- and PTH-independent bone anabolic action in insulin-resistant and type-2 diabetic rats. Here we searched for the presence and characteristics of GLP-1 receptors in osteoblastic MC3T3-E1 cells. [(125)I]-GLP-1 specific binding to MC3T3-E1 cells was time- and temperature-dependent, reaching maximal value at 30 min at 25 degrees C; in these conditions, [(125)I]-GLP-1 binding was dissociable, and displaced by GLP-1, partially by GLP-2, but not by exendin-4 (Ex-4), exendin-9 (Ex-9), glucagon or insulin; Scatchard analysis of the unlabeled GLP-1 data showed high and low affinity binding sites; cross-linking of GLP-1 binding revealed an estimated 70 kDa band, almost undetectable in the presence of 10(-6) M GLP-1. GLP-1, Ex-9, insulin or glucagon failed to modify cellular cAMP content, while GLP-2 and Ex-4 increased it. However, GLP-1 induced an immediate hydrolysis of glycosylphosphatidylinositols (GPIs) generating short-lived inositolphosphoglycans (IPGs), and an increase in phosphatidylinositol-3 kinase (PI3K) and mitogen activated protein kinase (MAPK) activities; Ex-4 also affected GPIs, but its action was delayed with respect to that of GLP-1. This incretin was found to decrease Runx2 but increased osteocalcin gene expression, without affecting that of osteoprotegerin or the canonical Wnt pathway activity in MC3T3-E1 cells which do not express the pancreatic GLP-1 receptor. Our data demonstrate for the first time that GLP-1 can directly and functionally interact with osteoblastic cells, possibly through a GPI/IPG-coupled receptor.

Increased fat mass contributes to bone deterioration. Glucagon-like peptide 1 (GLP-1) and its related peptide exendin 1-39 amide (Ex-4), two lipid-lowering peptides, exert osteogenic effects in diabetic states. We examined the actions of 3-day administration of GLP-1 or Ex-4 on bone remodeling markers and on bone mass and structure in hyperlipidic (HL) and hypercaloric rats. Wistar rats on a hyperlipidemic diet for 35 days were subcutaneously administered GLP-1 (0 . 86 nmol/kg per h), Ex-4 (0 . 1 nmol/kg per h), or saline (control) by continuous infusion for 3 days. After killing, tibiae were removed for total RNA and protein isolation, as well as femurs and L1-L4 vertebrae for bone mass and quality assessment. Body weight and plasma insulin were unaltered in HL rats, which showed osteopenia (by dual-energy X-ray absorptiometry), associated with hyperglycemia, hypertriglyceridemia, and hypercholesterolemia. GLP-1 or Ex-4 administration decreased the levels of glucose, triglycerides, and total cholesterol in plasma but increased osteocalcin (OC) gene expression and the osteoprotegerin (OPG)/receptor activator of NF-kB ligand (RANKL) ratio -at the expense of an augmented OPG -above corresponding control values in the tibia. Each tested peptide similarly reversed the decreased femoral and vertebral bone mass in these rats, whereas the deteriorated trabecular structure in the vertebrae improved associated with normalization of bone remodeling. These findings demonstrate that GLP-1 and Ex-4 are similarly efficient in reversing the bone alterations in this HL rat model, which has proven to be useful for studying the fat-bone relationships.

Summary Introduction Diabetes mellitus and obesity are important health issues; increasing in prevalence, both in the US and globally. There are only limited pharmacological treatments, and although bariatric surgery is effective, new effective pharmacologic treatments would be of great value. This review covers one area of increasing interest that could yield new novel treatments of obesity/diabetes mellitus. It involves recognition of the central role the G-protein-coupled receptor, bombesin receptor subtype 3(BRS-3) plays in energy/glucose metabolism. Areas covered Since the initial observation that BRS-3-knockout mice develop obesity, hypertension, impaired glucose-metabolism, and hyperphagia, there has been numerous studies of the mechanisms involved and the development of selective BRS-3-agonists/ antagonists which have marked effects on body weight, feeding, and glucose/insulin homeostasis. In this review each of these areas will be briefly reviewed. Expert opinion BRS-3 plays an important role in glucose/energy homeostasis. The development of potent, selective BRS-3 agonists demonstrates promise as a novel approach to treat obesity/diabetic states. One important question that needs to be addressed is whether BRS-3 agonists need to be centrally-acting. This is particular important in light of recent animal and human studies that report transient cardiovascular side-effects with centrally acting oral BRS-agonists.