Dual-agonist incretin peptides from fish with potential for obesity-related Type 2 diabetes therapy - A review.

The long-acting glucagon-like peptide-1 receptor (GLP1R) agonist, semaglutide and the unimolecular glucose-dependent insulinotropic polypeptide receptor (GIPR)/GLP1R dual-agonist, tirzepatide have been successfully introduced as therapeutic options for patients with Type-2 diabetes (T2DM) and obesity. Proglucagon-derived peptides from phylogenetically ancient fish act as naturally occurring dual agonists at the GLP1R and the glucagon receptor (GCGR) with lamprey GLP-1 and paddlefish glucagon being the most potent and effective in stimulating insulin release from BRIN-BD11 clonal ß-cells. These peptides were also the most effective in lowering blood glucose and elevating plasma insulin concentrations when administered intraperitoneally to overnight-fasted mice together with a glucose load. Zebrafish GIP acts as a dual agonist at the GIPR and GLP1R receptors. Studies with the high fat-fed mouse, an animal model with obesity, impaired glucose-tolerance and insulin-resistance, have shown that twice-daily administration of the long-acting analogs [D-Ala2]palmitoyl-lamprey GLP-1 and [D-Ser2]palmitoyl-paddlefish glucagon over 21 days improves glucose tolerance and insulin sensitivity. This was associated with ß-cell proliferation, protection of ß-cells against apoptosis, decreased pancreatic glucagon content, improved lipid profile, reduced food intake and selective alteration in the expression of genes involved in ß-cell stimulus-secretion coupling. In insulin-deficient GluCreERT2;ROSA26-eYFP transgenic mice, the peptides promoted an increase in ß-cell mass with positive effects on transdifferentiation of glucagon-producing to insulin-producing cells. Naturally occurring fish dual agonist peptides, particularly lamprey GLP-1 and paddlefish glucagon, provide templates for development into therapeutic agents for obesity-related T2DM.

A Facile Procedure for One-Pot Stable Conjugation of Two Proglucagon Cysteine-Containing Peptide Analogs.

Optimization of peptides for therapeutic purposes often includes chemical conjugation or modification with substituents that serve to broaden pharmacology or improve pharmacokinetics. We report a convenient and rapid procedure for one-pot, site-specific conjugation of two cysteine-containing peptides that utilizes a bivalent linker comprising maleimide and iodoacetyl functional groups. Following maleimide-mediated peptide conjugation the linker was converted from an unstable thiosuccinimide to a stable thioether bond suitable for biological study by mild aqueous hydrolysis. The procedure is exemplified by peptide-peptide, peptide-small molecule, and peptide-fatty acid conjugations. The method provides a facile approach to search for enhanced biological outcomes through additive and sustained peptide pharmacology unencumbered by the prospect of chemical rearrangement in the course of biological study.

Mutational Landscape of the Proglucagon-Derived Peptides.

Strong efforts have been placed on understanding the physiological roles and therapeutic potential of the proglucagon peptide hormones including glucagon, GLP-1 and GLP-2. However, little is known about the extent and magnitude of variability in the amino acid composition of the proglucagon precursor and its mature peptides. Here, we identified 184 unique missense variants in the human proglucagon gene GCG obtained from exome and whole-genome sequencing of more than 450,000 individuals across diverse sub-populations. This provides an unprecedented source of population-wide genetic variation data on missense mutations and insights into the evolutionary constraint spectrum of proglucagon-derived peptides. We show that the stereotypical peptides glucagon, GLP-1 and GLP-2 display fewer evolutionary alterations and are more likely to be functionally affected by genetic variation compared to the rest of the gene products. Elucidating the spectrum of genetic variations and estimating the impact of how a peptide variant may influence human physiology and pathophysiology through changes in ligand binding and/or receptor signalling, are vital and serve as the first important step in understanding variability in glucose homeostasis, amino acid metabolism, intestinal epithelial growth, bone strength, appetite regulation, and other key physiological parameters controlled by these hormones.

The Enigmatic N-Terminal Domain of Proglucagon; A Historical Perspective.

Enteroglucagon refers to the predominant peptide with glucagon-like immunoreactivity (GLI) that is released by the intestine into the circulation in response to nutrients. Development of a radioimmunoassay for glucagon revealed issues that were not apparent in applications of the insulin radioimmunoassay. The fact that some antisera raised against glucagon recognized glucagon-related peptides in extracts of both pancreas and gut whereas others recognized only components in the pancreas remained a mystery until it was realized that the "gut GLI cross-reactive" antisera were directed against an epitope in the N-terminal to central region of glucagon whereas the "pancreatic glucagon specific" antisera were directed against an epitope in the C-terminal region. Unlike the cross-reactive antisera, the glucagon specific antisera did not recognize components in which glucagon was extended from its C-terminus by additional amino acids. Initial attempts to purify enteroglucagon from porcine ileum led to the erroneous conclusion that enteroglucagon comprised 100 amino acids with an apparent molecular mass of 12,000 Da and was consequently given the name glicentin. Subsequent work established that the peptide constituted residues (1-69) of proglucagon (Mr 8128). In the 40 years since the structural characterization of glicentin, attempts to establish an unambiguous physiological function for enteroglucagon have not been successful. Unlike the oxyntomodulin domain at the C-terminus of enteroglucagon, the primary structure of the N-terminal domain (glicentin-related pancreatic peptide) has been poorly conserved among mammals. Consequently, most investigations of the bioactivity of porcine glicentin may have been carried out in inappropriate animal models. Enteroglucagon may simply represent an inactive peptide that ensures that the intestine does not release equimolar amounts of a hyperglycemic agent (glucagon) and a hypoglycemic agent (GLP-1) after ingestion of nutrients.

The islet's bridesmaid becomes the bride: Proglucagon-derived peptides deliver transformative therapies.

The 2021 Gairdner Prize is awarded to Daniel Drucker, Joel Habener, and Jens Juul Holst for the discovery of novel peptides encoded in the proglucagon sequence and the establishment of their physiological roles. These discoveries underpinned the development of therapeutics that are now benefiting patients with type 2 diabetes and other disorders worldwide.

Role of Proglucagon Peptides in Osmoregulation.

Glucagon-like peptide-1 (GLP-1), a product of partial proteolysis of proglucagon, is involved not only in regulation of carbohydrates, but also in water-salt metabolism. The study examined the role of proglucagon derivatives GLP-1, GLP-2, and oxyntomodulin in rat osmoregulation. Of them, only blood plasma GLP-1 increased in response to water load (20 ml/kg). Administration of glucose (1.5 g/kg) elevated GLP-1 and oxyntomodulin but did not change the level of GLP-2. GLP-1 accelerated excretion of excess water during hyperhydration, whereas GLP-2 decreased this parameter. No physiological effects of oxyntomodulin in the kidneys were revealed. Probably, the blood levels of proglucagon derivatives are independently regulated for each peptide. In contrast to GLP-2 and oxyntomodulin, GLP-1 is involved in osmoregulation.

Proglucagon-Derived Peptides, Glucose-Dependent Insulinotropic Polypeptide, and Dipeptidyl Peptidase-4-Mechanisms of Action in Adipose Tissue.

Proglucagon-derived peptides (PGDPs) and related gut hormones exemplified by glucose-dependent insulinotropic polypeptide (GIP) regulate energy disposal and storage through actions on metabolically sensitive organs, including adipose tissue. The actions of glucagon, glucagon-like peptide (GLP)-1, GLP-2, GIP, and their rate-limiting enzyme dipeptidyl peptidase-4, include direct and indirect regulation of islet hormone secretion, food intake, body weight, all contributing to control of white and brown adipose tissue activity. Moreover, agents mimicking actions of these peptides are in use for the therapy of metabolic disorders with disordered energy homeostasis such as diabetes, obesity, and intestinal failure. Here we highlight current concepts and mechanisms for direct and indirect actions of these peptides on adipose tissue depots. The available data highlight the importance of indirect peptide actions for control of adipose tissue biology, consistent with the very low level of endogenous peptide receptor expression within white and brown adipose tissue depots. Finally, we discuss limitations and challenges for the interpretation of available experimental observations, coupled to identification of enduring concepts supported by more robust evidence.

Oxyntomodulin: Actions and role in diabetes.

Oxyntomodulin is a product of the glucagon precursor, proglucagon, produced and released from the endocrine L-cells of the gut after enzymatic processing by the precursor prohormone convertase 1/3. It corresponds to the proglucagon sequence 33-69 and thus contains the entire glucagon sequence plus a C-terminal octapeptide, comprising in total 37 amino acids. As might have been expected, it has glucagon-like bioactivity, but also and more surprisingly also activates the receptor for GLP-1. This has given the molecule an interesting status as a glucagon-GLP-1 co-agonist, which is currently attracting considerable interest for its potential in the treatment of diabetes and obesity. Here, we provide an update on oxyntomodulin with a focus on its potential role in metabolic diseases.

Proglucagons in vertebrates: Expression and processing of multiple genes in a bony fish.

In contrast to mammals, where a single proglucagon (PG) gene encodes three peptides: glucagon, glucagon-like peptide 1 and glucagon-like peptide 2 (GLP-1; GLP-2), many non-mammalian vertebrates carry multiple PG genes. Here, we investigate proglucagon mRNA sequences, their tissue expression and processing in a diploid bony fish. Copper rockfish (Sebastes caurinus) express two independent genes coding for distinct proglucagon sequences (PG I, PG II), with PG II lacking the GLP-2 sequence. These genes are differentially transcribed in the endocrine pancreas, the brain, and the gastrointestinal tract. Alternative splicing identified in rockfish is only one part of this complex regulation of the PG transcripts: the system has the potential to produce two glucagons, four GLP-1s and a single GLP-2, or any combination of these peptides. Mass spectrometric analysis of partially purified PG-derived peptides in endocrine pancreas confirms translation of both PG transcripts and differential processing of the resulting peptides. The complex differential regulation of the two PG genes and their continued presence in this extant teleostean fish strongly suggests unique and, as yet largely unidentified, roles for the peptide products encoded in each gene.

Peptide production and secretion in GLUTag, NCI-H716, and STC-1 cells: a comparison to native L-cells.

GLUTag, NCI-H716, and STC-1 are cell lines that are widely used to study mechanisms underlying secretion of glucagon-like peptide-1 (GLP-1), but the extent to which they resemble native L-cells is unknown. We used validated immunoassays for 14 different hormones to analyze peptide content (lysis samples; n = 9 from different passage numbers) or peptide secretion in response to buffer (baseline), and after stimulation with 50 mM KCl or 10 mM glucose + 10 µM forskolin/3-isobutyl-1-methylxanthine (n = 6 also different passage numbers). All cell lines produced and processed proglucagon into GLP-1, GLP-2, glicentin, and oxyntomodulin in a pattern (prohormone convertase (PC)1/3 dependent) similar to that described for human gut. All three cell lines showed basal secretion of GLP-1 and GLP-2, which increased after stimulation. In contrast to freshly isolated murine L-cells, all cell lines also expressed PC2 and secreted large amounts of pancreatic glucagon. Neurotensin and somatostatin storage was low and secretion was not consistently increased by stimulation. STC-1 cells released more glucose-dependent insulinotropic polypeptide than GLP-1 at baseline (P < 0.01) and KCl elevated its secretion (P < 0.05). Peptide YY, which normally co-localizes with GLP-1 in distal L-cells, was not detected in any of the cell lines. GLUTag and STC-1 cells also expressed vasoactive intestinal peptide, but none expressed pancreatic polypeptide or insulin. GLUTag contained and secreted large amounts of CCK, while NCI-H716 did not store this peptide and STC-1 contained low amounts. Our results show that hormone production in cell line models of the L-cell has limited similarity to the natural L-cells.

Two dipolar α-helices within hormone-encoding regions of proglucagon are sorting signals to the regulated secretory pathway.

Proglucagon is expressed in pancreatic α cells, intestinal L cells, and some hypothalamic and brainstem neurons. Tissue-specific processing of proglucagon yields three major peptide hormones as follows: glucagon in the α cells and glucagon-like peptides (GLP)-1 and -2 in the L cells and neurons. Efficient sorting and packaging into the secretory granules of the regulated secretory pathway in each cell type are required for nutrient-regulated secretion of these proglucagon-derived peptides. Our previous work suggested that proglucagon is directed into granules by intrinsic sorting signals after initial processing to glicentin and major proglucagon fragment (McGirr, R., Guizzetti, L., and Dhanvantari, S. (2013) J. Endocrinol. 217, 229-240), leading to the hypothesis that sorting signals may be present in multiple domains. In the present study, we show that the α-helices within glucagon and GLP-1, but not GLP-2, act as sorting signals by efficiently directing a heterologous secretory protein to the regulated secretory pathway. Biophysical characterization of these peptides revealed that glucagon and GLP-1 each encode a nonamphipathic, dipolar α-helix, whereas the helix in GLP-2 is not dipolar. Surprisingly, glicentin and major proglucagon fragment were sorted with different efficiencies, thus providing evidence that proglucagon is first sorted to granules prior to processing. In contrast to many other prohormones in which sorting is directed by ordered prodomains, the sorting determinants of proglucagon lie within the ordered hormone domains of glucagon and GLP-1, illustrating that each prohormone has its own sorting "signature."

Fertility and pregnancy-associated ß-cell proliferation in mice deficient in proglucagon-derived peptides.

Proglucagon, which is encoded by the glucagon gene (Gcg), is the precursor of several peptide hormones, including glucagon and glucagon-like peptide 1 (GLP-1). Whereas glucagon stimulates hepatic glycogenolysis and gluconeogenesis, GLP-1 stimulates insulin secretion to lower blood glucose and also supports ß-cell proliferation and protection from apoptotic stimuli. Pregnancy is a strong inducer of change in islet function, however the roles of proglucagon-derived peptides in pregnancy are only partially understood. In the present study, we analyzed fertility and pregnancy-associated changes in homozygous glucagon-green fluorescent protein (gfp) knock-in mice (Gcg(gfp/gfp)), which lack all the peptides derived from proglucagon. Female Gcg(gfp/gfp) mice could deliver and raise Gcg(gfp/gfp) pups to weaning and Gcg(gfp/gfp) pups from Gcg(gfp/gfp) dams were viable and fertile. Pregnancy induced ß-cell proliferation in Gcg(gfp/gfp) mice as well as in control mice. However, serum insulin levels in pregnant Gcg(gfp/gfp) females were lower than those in control pregnant females under ad libitum feeding, and blood glucose levels in pregnant Gcg(gfp/gfp) females were higher after gestational day 12. Gcg(gfp/gfp) females showed a decreased pregnancy rate and smaller litter size. The rate of successful breeding was significantly lower in Gcg(gfp/gfp) females and was not improved by experience of breeding. Taken together, proglucagon-derived peptides are not required for pregnancy-associated ß-cell proliferation, however, are required for regulation of blood glucose levels and normal reproductive capacity. Gcg(gfp/gfp) mice may serve as a novel model to analyze the effect of mild hyperglycemia during late gestational periods.

Remodeling of hepatic metabolism and hyperaminoacidemia in mice deficient in proglucagon-derived peptides.

Glucagon is believed to be one of the most important peptides for upregulating blood glucose levels. However, homozygous glucagon-green fluorescent protein (gfp) knock-in mice (Gcg(gfp/gfp): GCGKO) are normoglycemic despite the absence of proglucagon-derived peptides, including glucagon. To characterize metabolism in the GCGKO mice, we analyzed gene expression and metabolome in the liver. The expression of genes encoding rate-limiting enzymes for gluconeogenesis was only marginally altered. On the other hand, genes encoding enzymes involved in conversion of amino acids to metabolites available for the tricarboxylic acid cycle and/or gluconeogenesis showed lower expression in the GCGKO liver. The expression of genes involved in the metabolism of fatty acids and nicotinamide was also altered. Concentrations of the metabolites in the GCGKO liver were altered in manners concordant with alteration in the gene expression patterns, and the plasma concentrations of amino acids were elevated in the GCGKO mice. The insulin concentration in serum and phosphorylation of Akt protein kinase in liver were reduced in GCGKO mice. These results indicated that proglucagon-derived peptides should play important roles in regulating various metabolic pathways, especially that of amino acids. Serum insulin concentration is lowered to compensate the impacts of absent proglucagon-derived peptide on glucose metabolism. On the other hand, impacts on other metabolic pathways are only partially compensated by reduced insulin action.

Neuropeptides of the islets of Langerhans: a peptidomics study.

Neuropeptides from the endocrine pancreas (the islets of Langerhans) play an important role in the regulation of blood glucose levels. Therefore, our aim is to identify the "peptidome" (the in vivo peptide profile at a certain time) of the pancreatic islets, which is beneficial for medical progress related to the treatment of diabetes. So far, there are few neuropeptides isolated and sequenced from the endocrine pancreas and mainly in situ hybridisation and immunocytochemical techniques have been used to demonstrate the occurrence of peptides in the pancreas. These techniques do not allow for unequivocal identification of peptides. In contrary, mass spectrometry identifies peptides unambiguously. We have analysed the peptidome of the islets using peptidomics, i.e. a combination of liquid chromatography, mass spectrometry and bioinformatics. We are able to identify the peptidome of islets extracts. We not only confirm the presence of peptides with a well-known effect on blood glucose levels, but also identify new peptides, which are unknown to affect blood glucose levels.