Response to lowering plasma glucose is characterised by decreased oxyntomodulin: Results from a randomised controlled trial.

BACKGROUND: With the prevalence of diabetes reaching an epidemic level, there is a growing interest in the investigation of its remission. Proglucagon-derived peptides (PGDP) have been shown to have a glucose-regulating effect. However, whether they play a role in diabetes remission remains poorly understood. AIM: To investigate changes in plasma levels of PGDP in glycaemic responders versus non-responders. METHODS: The study was a randomised placebo-controlled trial comprising 18 adults with prediabetes (registered at www. CLINICALTRIALS: gov as NCT03889210). Following an overnight fast, participants consumed ketone ß-hydroxybutyrate (KEßHB)-supplemented beverage and placebo beverage in crossover manner. Serial blood samples were collected from baseline to 150 min at 30-min intervals. The endpoints were changes in glucagon-like peptide-1 (GLP-1), glicentin, oxyntomodulin, glucagon, and major proglucagon fragment (MPGF). Participants were stratified into the 'responders' and 'non-responders' subgroups based on their glycaemic changes following the ingestion of KEßHB. The area under the curve (AUC) was calculated to estimate the accumulated changes in the studied PGDP and compared using paired-t test between the KEßHB and placebo beverages. RESULTS: Responders had a significantly greater reduction in plasma glucose compared with non-responders following acute ketosis (p < 0.001). The AUC0-150 for oxyntomodulin was significantly lower following the KEßHB beverage compared with the placebo (p = 0.045) in responders, but not in non-responders (p = 0.512). No significant differences in AUCs0-150 were found for GLP-1, glicentin, glucagon, and MPGF in either responders or non-responders. CONCLUSION: Oxyntomodulin is involved in lowering plasma glucose and may play an important role in diabetes remission.

Determinants of plasma levels of proglucagon and the metabolic impact of glucagon receptor signalling: a UK Biobank study.

AIMS/HYPOTHESES: Glucagon and glucagon-like peptide-1 (GLP-1) are derived from the same precursor; proglucagon, and dual agonists of their receptors are currently being explored for the treatment of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). Elevated levels of endogenous glucagon (hyperglucagonaemia) have been linked with hyperglycaemia in individuals with type 2 diabetes but are also observed in individuals with obesity and MASLD. GLP-1 levels have been reported to be largely unaffected or even reduced in similar conditions. We investigated potential determinants of plasma proglucagon and associations of glucagon receptor signalling with metabolic diseases based on data from the UK Biobank. METHODS: We used exome sequencing data from the UK Biobank for ~410,000 white participants to identify glucagon receptor variants and grouped them based on their known or predicted signalling. Data on plasma levels of proglucagon estimated using Olink technology were available for a subset of the cohort (~40,000). We determined associations of glucagon receptor variants and proglucagon with BMI, type 2 diabetes and liver fat (quantified by liver MRI) and performed survival analyses to investigate if elevated proglucagon predicts type 2 diabetes development. RESULTS: Obesity, MASLD and type 2 diabetes were associated with elevated plasma levels of proglucagon independently of each other. Baseline proglucagon levels were associated with the risk of type 2 diabetes development over a 14 year follow-up period (HR 1.13; 95% CI 1.09, 1.17; n=1562; p=1.3×10-12). This association was of the same magnitude across strata of BMI. Carriers of glucagon receptor variants with reduced cAMP signalling had elevated levels of proglucagon (ß 0.847; 95% CI 0.04, 1.66; n=17; p=0.04), and carriers of variants with a predicted frameshift mutation had higher levels of liver fat compared with the wild-type reference group (ß 0.504; 95% CI 0.03, 0.98; n=11; p=0.04). CONCLUSIONS/INTERPRETATION: Our findings support the suggestion that glucagon receptor signalling is involved in MASLD, that plasma levels of proglucagon are linked to the risk of type 2 diabetes development, and that proglucagon levels are influenced by genetic variation in the glucagon receptor, obesity, type 2 diabetes and MASLD. Determining the molecular signalling pathways downstream of glucagon receptor activation may guide the development of biased GLP-1/glucagon co-agonist with improved metabolic benefits. DATA AVAILABILITY: All coding is available through https://github.com/nicwin98/UK-Biobank-GCG.

Bariatric surgery, through beneficial effects on underlying mechanisms, improves cardiorenal and liver metabolic risk over an average of ten years of observation: A longitudinal and a case-control study.

BACKGROUND: Bariatric surgery has long-term beneficial effects on body weight and metabolic status, but there is an apparent lack of comprehensive cardiometabolic, renal, liver, and metabolomic/lipidomic panels, whereas the underlying mechanisms driving the observed postoperative ameliorations are still poorly investigated. We aimed to study the long-term effects of bariatric surgery on metabolic profile, cardiorenal and liver outcomes in association with underlying postoperative gut hormone adaptations. METHODS: 28 individuals who underwent bariatric surgery [17 sleeve gastrectomy (SG), 11 Roux-en-Y gastric bypass (RYGB)] were followed up 3, 6 and 12 and at 10 years following surgery. Participants at 10 years were cross-sectionally compared with an age-, sex- and adiposity-matched group of non-operated individuals (n = 9) and an age-matched pilot group of normal-weight individuals (n = 4). RESULTS: There were durable effects of surgery on body weight and composition, with an increase of lean mass percentage persisting despite some weight regain 10 years postoperatively. The improvements in metabolic and lipoprotein profiles, cardiometabolic risk markers, echocardiographic and cardiorenal outcomes persisted over the ten-year observation period. The robust improvements in insulin resistance, adipokines, activin/follistatin components and postprandial gastrointestinal peptide levels persisted 10 years postoperatively. These effects were largely independent of surgery type, except for a lasting reduction of ghrelin in the SG subgroup, and more pronounced increases in proglucagon products, mainly glicentin and oxyntomodulin, and in the cardiovascular risk marker Trimethylamine-N-oxide (TMAO) within the RYGB subgroup. Despite similar demographic and clinical features, participants 10 years after surgery showed a more favorable metabolic profile compared with the control group, in conjunction with a dramatic increase of postprandial proglucagon product secretion. CONCLUSIONS: We demonstrate that cardiorenal and metabolic benefits of bariatric surgery remain robust and largely unchanged ten years postoperatively and are associated with durable effects on gastrointestinal- muscle- and adipose tissue-secreted hormones. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04170010.

Low dietary carbohydrate induces structural alterations in enterocytes of the chicken ileum.

We investigated the role of dietary carbohydrates in the maintenance of the enterocyte microvillar structure in the chicken ileum. Male chickens were divided into the control and three experimental groups, and the experimental groups were fed diets containing 50%, 25%, and 0% carbohydrates of the control diet. The structural alterations in enterocytes were examined using transmission electron microscopy and immunofluorescent techniques for ß-actin and villin. Glucagon-like peptide (GLP)-2 and proglucagon mRNA were detected by immunohistochemistry and in situ hybridization, respectively. Fragmentation and wide gap spaces were frequently observed in the microvilli of the 25% and 0% groups. The length, width, and density of microvilli were also decreased in the experimental groups. The experimental groups had shorter terminal web extensions, and there were substantial changes in the mitochondrial density between the control and experimental groups. Intensities of ß-actin and villin immunofluorescence observed on the apical surface of enterocytes were lower in the 0% group. The frequency of GLP-2-immunoreactive and proglucagon mRNA-expressing cells decreased with declining dietary carbohydrate levels. This study revealed that dietary carbohydrates contribute to the structural maintenance of enterocyte microvilli in the chicken ileum. The data from immunohistochemistry and in situ hybridization assays suggest the participation of GLP-2 in this maintenance system.

Expression of the GCG gene and secretion of active glucagon-like peptide-1 varies along the length of intestinal tract in horses.

BACKGROUND: Active glucagon-like peptide-1 (aGLP-1) has been implicated in the pathogenesis of equine insulin dysregulation (ID), but its role is unclear. Cleavage of proglucagon (coded by the GCG gene) produces aGLP-1 in enteral L cells. OBJECTIVES: The aim in vivo was to examine the sequence of the exons of GCG in horses with and without ID, where aGLP-1 was higher in the group with ID. The aims in vitro were to identify and quantify the expression of GCG in the equine intestine (as a marker of L cells) and determine intestinal secretion of aGLP-1. STUDY DESIGN: Genomic studies were case-control studies. Expression and secretion studies in vitro were cross-sectional. METHODS: The GCG gene sequence of the exons was determined using a hybridisation capture protocol. Expression and quantification of GCG in samples of stomach duodenum, jejunum, ileum, caecum and ascending and descending colon was achieved with droplet digital PCR. For secretory studies tissue explants were incubated with 12 mM glucose and aGLP-1 secretion was measured with an ELISA. RESULTS: Although the median [IQR] post-prandial aGLP-1 concentrations were higher (p = 0.03) in animals with ID (10.2 [8.79-15.5]), compared with healthy animals (8.47 [6.12-11.7]), there was 100% pairwise identity of the exons of the GCG sequence for the cohort. The mRNA concentrations of GCG and secretion of aGLP-1 differed (p < 0.001) throughout the intestine. MAIN LIMITATIONS: Only the exons of the GCG gene were sequenced and breeds were not compared. The horses used for the study in vitro were not assessed for ID and different horses were used for the small, and large, intestinal studies. CONCLUSIONS: Differences in post-prandial aGLP-1 concentration were not due to a variant in the exons of the GCG gene sequence in this cohort. Both the large and small intestine are sites of GLP-1 secretion.

Circulating levels of all proglucagon-derived peptides are differentially regulated postprandially by obesity status and in response to high-fat meals vs. high-carbohydrate meals.

BACKGROUND & AIMS: We measured all proglucagon-derived peptides (PGDPs) levels in response to administration of three mixed meal tolerance tests (MMTs), examining differences in postprandial PGDP responses in subjects with leanness and obesity or between high-fat vs. high carbohydrate meals. METHODS: We designed three physiology interventional studies, administering MMTs over a 180-min period to individuals without diabetes after an overnight fast. In Study 1, a 450 kcal MMT was administered to n = 4 normal weight and n = 9 individuals with obesity. In Study 2, a 600 kcal high-fat MMT was administered to n = 15 normal-weight and n = 15 individuals with obesity. In Study 3, n = 32 participants with obesity were assigned to receive a 600-kcal high-fat (n = 15) or an isocaloric high-carbohydrate MMT (n = 17). Fasting and postprandial levels of c-peptide and PGDPs (proglucagon, GLP-1, GLP-2, glicentin, oxyntomodulin, glucagon, major proglucagon fragment [MPGF]) were assessed. RESULTS: In study 1, individuals with normal weight displayed elevated glicentin postprandial secretion compared with people with obesity (p = 0.002). Following a high-fat MMT with 33% higher energy content in study 2, all postprandial PGDPs levels were elevated (p-time<0.001), irrespective of weight status. In study 3, a prolonged postprandial upregulation of PGDPs during the high-fat MMT was observed in contrast with the acute, short-term (max 60 min) PGDP responses to a high-carbohydrate MMT (p-time∗meal<0.001). Across both studies 2 and 3, the postprandial responses of glucagon and MPGF were higher in subjects with male sex whereas glicentin was higher in subjects with female sex. CONCLUSIONS: Fat and carbohydrate content of a meal can substantially affect the postprandial levels of PGDPs. Circulating levels of PGDPs are influenced by the energy content of the meal, and additionally, the presence of leanness or obesity affects circulating levels of select PGDPs. These results, which are to be confirmed by additional studies, expand our understanding of PGDP physiology in leanness and obesity. CLINICALTRIALS: GOV REGISTRATION NUMBERS: (NCT04170010, NCT04430946, NCT04575194).

Circulating levels of proglucagon-derived peptides are differentially regulated by the glucagon-like peptide-1 agonist liraglutide and the centrally acting naltrexone/bupropion and can predict future weight loss and metabolic improvements: A 6-month long interventional study.

AIM: To investigate the changes of circulating levels of all proglucagon-derived peptides (PGDPs) in individuals with overweight or obesity receiving liraglutide (3 mg) or naltrexone/bupropion (32/360 mg), and to explore the association between induced changes in postprandial PGDP levels and body composition, as well as metabolic variables, after 3 and 6 months on treatment. MATERIALS AND METHODS: Seventeen patients with obesity or with overweight and co-morbidities, but without diabetes, were assigned to receive once-daily oral naltrexone/bupropion 32/360 mg (n = 8) or once-daily subcutaneous liraglutide 3 mg (n = 9). Participants were assessed before treatment initiation and after 3 and 6 months on treatment. At the baseline and 3-month visits, participants underwent a 3-hour mixed meal tolerance test to measure fasting and postprandial levels of PGDPs, C-peptide, hunger and satiety. Clinical and biochemical indices of metabolic function, magnetic resonance-assessed liver steatosis and ultrasound-assessed liver stiffness were measured at each visit. RESULTS: Both medications improved body weight and composition, carbohydrate and lipid metabolism, and liver fat and function. Naltrexone/bupropion produced a weight-independent increase in the levels of proglucagon (P < .001) and decreases in glucagon-like peptide-2 (GLP-2), glucagon and the major proglucagon fragment (P ≤ .01), whereas liraglutide markedly upregulated total glucagon-like peptide-1 (GLP-1) levels in a weight-independent manner (P = .04), and similarly downregulated the major proglucagon fragment, GLP-2 and glucagon (P < .01). PGDP levels at the 3-month visit were positively and independently correlated with improvements in fat mass, glycaemia, lipaemia and liver function, and negatively with reductions in fat-free mass, at both the 3- and 6-month visits. CONCLUSIONS: PGDP levels in response to liraglutide and naltrexone/bupropion are associated with improvements in metabolism. Our study provides support for the administration of the downregulated members of the PGDP family as replacement therapy (e.g. glucagon), in addition to the medications currently in use that induced their downregulation (e.g. GLP-1), and future studies should explore whether the addition of other PGDPs (e.g. GLP-2) could offer additional benefits.

Circulating levels of gastrointestinal hormones in prediabetes reversing to normoglycemia or progressing to diabetes in a year-A cross-sectional and prospective analysis.

AIMS: We aimed to compare the concentrations of GLP-1, glucagon and GIP (established regulators of glucose homeostasis) and glicentin (emerging new metabolic marker)during an OGTT in patients with normal glucose tolerance (NGT), prediabetes and diabetes at onset, and one-year before, when all had prediabetes. METHODS: GLP-1, glucagon, GIP and glicentin concentrations were measured and compared with markers of body composition, insulin sensitivity and ß-cell function at a 5-timepoint OGTT in 125 subjects (30 diabetes, 65 prediabetes, 30 NGT) and in 106 of them one-year before, when all had prediabetes. RESULTS: At baseline, when all subjects were in prediabetic state, hormonal levels did not differ between groups. One year later, patients progressing to diabetes had lower postprandial increases of glicentin and GLP-1, lower postprandial decrease of glucagon, and higher levels of fasting GIP compared to patients regressing to NGT. Changes in glicentin and GLP-1 AUC within this year correlated negatively with changes in Glucose AUC of OGTT and with changes in markers of beta cell function. CONCLUSION: Incretins, glucagon and glicentin profiles in prediabetic state cannot predict future glycemic traits, but prediabetes progressing to diabetes is accompanied by deterioration of postprandial increases of GLP-1 and glicentin.

Roseburia intestinalis Modulates PYY Expression in a New a Multicellular Model including Enteroendocrine Cells.

The gut microbiota contributes to human health and disease; however, the mechanisms by which commensal bacteria interact with the host are still unclear. To date, a number of in vitro systems have been designed to investigate the host-microbe interactions. In most of the intestinal models, the enteroendocrine cells, considered as a potential link between gut bacteria and several human diseases, were missing. In the present study, we have generated a new model by adding enteroendocrine cells (ECC) of L-type (NCI-H716) to the one that we have previously described including enterocytes, mucus, and M cells. After 21 days of culture with the other cells, enteroendocrine-differentiated NCI-H716 cells showed neuropods at their basolateral side and expressed their specific genes encoding proglucagon (GCG) and chromogranin A (CHGA). We showed that this model could be stimulated by commensal bacteria playing a key role in health, Roseburia intestinalis and Bacteroides fragilis, but also by a pathogenic strain such as Salmonella Heidelberg. Moreover, using cell-free supernatants of B. fragilis and R. intestinalis, we have shown that R. intestinalis supernatant induced a significant increase in IL-8 and PYY but not in GCG gene expression, while B. fragilis had no impact. Our data indicated that R. intestinalis produced short chain fatty acids (SCFAs) such as butyrate whereas B. fragilis produced more propionate. However, these SCFAs were probably not the only metabolites implicated in PYY expression since butyrate alone had no effect. In conclusion, our new quadricellular model of gut epithelium could be an effective tool to highlight potential beneficial effects of bacteria or their metabolites, in order to develop new classes of probiotics.

Development of a selective, sensitive and robust oxyntomodulin dual monoclonal antibody immunoassay.

Background & Aim: Oxyntomodulin (Oxm) is a proglucagon-derived peptide agonist of both the GLP-1 and glucagon receptors and is a key regulator of gastric acid secretion and energy expenditure. Differential processing from proglucagon hinders assay immunoassay selectivity. Method & results: Antibody engineering was used to develop a sandwich immunoassay that selectively measures endogenous Oxm. The pre- and postprandial levels of Oxm from 19 healthy individuals over the course of 2 h were measured. Postprandial increases in Oxm occurred within minutes and levels significantly correlated with those obtained using previously published mass spectrometry assays. Conclusion: This sandwich immunoassay is appropriately sensitive and selective and is also amenable to high-throughput application for the reliable determination of endogenous levels of intact Oxm from human samples.

Glucagon and other proglucagon-derived peptides in the pathogenesis of obesity.

Because of differential processing of the hormone precursor, proglucagon, numerous peptide products are released from the pancreatic alpha cells and the intestinal L-cells in which the (pro)glucagon gene is expressed. Of particular interest in relation to obesity are glucagon from the pancreas and oxyntomodulin and GLP-1 from the gut, all of which inhibit food intake, but the other products are also briefly discussed, because knowledge about these is required for selection and evaluation of the methods for measurement of the hormones. The distal intestinal L-cells also secrete the appetite-inhibiting hormone PYY. Characteristics of the secretion of the pancreatic and intestinal products are described, and causes of the hypersecretion of glucagon in obesity and type 2 diabetes are discussed. In contrast, the secretion of the products of the L-cells is generally impaired in obesity, raising questions about their role in the development of obesity. It is concluded that the impairment probably is secondary to obesity, but the lower plasma levels may contribute to the development.

Circulating levels of five proglucagon-derived peptides in response to intravenous or oral glucose or lipids and to a mixed-meal in subjects with normal weight, overweight, and obesity.

AIMS: Proglucagon-derived peptides (PGDPs) secreted by the gut and pancreas play a major role in metabolism. We measured concentrations of five PGDPs in response to per os (PO) or intravenous (IV) glucose or lipid intake and a mixed meal test (MMT) consumed by subjects with normal weight, overweight or obesity. MATERIALS AND METHODS: GLP-1, oxyntomodulin and glicentin (gut-secreted PGDPs) and glucagon and MPGF (pancreas-secreted PGDPs) were assessed in: (a) 32 subjects receiving PO or IV glucose, lipids or water over 6 h, (b) 33 subjects with normal weight, overweight or obesity who consumed a MMT. RESULTS: (a) GLP-1, oxyntomodulin, glicentin and glucagon levels increase more profoundly and persistently after lipids PO (2.5  g/kg) than glucose PO (2.5  g/kg) or IV lipids (Intralipid/Liposyn II 20% at 0.35 ml/kg/h and Intralipid/Liposyn II 20% at 0.83  ml/kg/h for 6 h) or IV glucose (10% glucose at 3.6 ml/kg/h for 6 h). Oxyntomodulin and glicentin increased more than GLP-1 in response to lipids PO. MPGF levels decrease in response to glucose PO or IV indicating a shift towards preferential production of gut-secreted peptides. (b) Fasting and postprandial areas under the curve (AUCs) after MMT of GLP-1, MPGF and glucagon levels correlated positively with BMI. The fasting levels of glucagon and MPGF were elevated in obesity and remained elevated after the MMT. CONCLUSION: Circulating levels of PGDPs are differentially regulated by body weight, the type of macronutrients administered and the respective route of administration. Mechanistic studies are needed to define the exact mechanisms underlying this regulation. CLINICAL TRIAL REGISTRATION: Study 1 has the NCT01520454 and the NCT04888325 number in ClinicalTrials.gov. Study 2 has the number NCT01495754 in ClinicalTrials.gov.

Discovery of the GI Effects of GLP-1: An Historical Perspective.

In 1993, my laboratory published an article in Digestive Diseases and Sciences that clearly demonstrated the pronounced effects of the newly discovered intestinal hormone, glucagon-like peptide-1 (GLP-1), on a number of gastrointestinal functions, including gastric emptying rate, gastric acid secretion, and pancreatic enzyme secretion. The gut hormone is released in response to nutrient intake, and in further experiments, its release from the ileum paralleled inhibition of both gastric and pancreatic secretions. Based on these studies, it was concluded that GLP-1 is an important regulator of the so-called ileal brake, a term given for the observation that ileal perfusion of lipids delayed gastric emptying, reduced food intake, and induced satiety Welch et al. (1985), in addition to its functions as an incretin hormone. GLP-1 was subsequently identified as a physiological inhibitor of appetite and food intake, and based on these actions, the GLP-1 receptor agonists are today considered among the most powerful and effective antiobesity and antidiabetic agents available, with the added benefits of reducing the risk of the cardiovascular and renal complications associated with these conditions.

Dietary carbohydrate modifies the density of L cells in the chicken ileum.

Glucagon-like peptides (GLPs) are secreted from intestinal L cells and stimulate various physiological functions in the gastrointestinal tract. The secretion of GLPs is influenced by macronutrient ingestion. This study aims to clarify the effects of dietary carbohydrate (CHO) on L cells in the chicken ileum. Six-week-old, male White Leghorn chickens were divided into three groups: control, low-CHO and CHO-free, with five chickens in each group. Paraffin sections were made from the proximal and distal ileum of each animal and subjected to immunohistochemistry for GLP-1 and GLP-2 peptides and in situ hybridization for proglucagon (PG) mRNA. A significant reduction of GLP-1- and GLP-2-immunoreactive cells was observed in the two experimental groups compared with that in the control. A reduction of cells expressing PG mRNA was observed in the proximal and distal ileum of the CHO-free group compared with that in the control. The ratio of GLP-1-immunoreactive cells showing Ki-67 immunoreactivity was significantly lower in the distal ileum of the CHO-free group than that in the control group. These data suggest that dietary CHO is an effective stimulator for modifying L cell density in the chicken ileum.

Glucagon-like peptide-1: Are its roles as endogenous hormone and therapeutic wizard congruent?

Glucagon-like peptide-1 (GLP-1) is a peptide derived from differential processing of the precursor for the hormone glucagon. It is secreted predominantly by endocrine cells in the gut epithelium in response to nutrient stimulation. Studies from the last 35 years have given us an idea about its physiological functions. On the basis of some of its many actions, it has also been developed into a pharmaceutical agent for the treatment of obesity and type 2 diabetes (T2DM). It is currently positioned as the most effective anti-obesity agent available and is recommended in both national and international guidelines as an effective second-in line treatment for T2DM, in particular in patients with increased cardiovascular risk. In this review, I first discuss whether the processing of proglucagon may also result in GLP-1 formation in the pancreas and in glucagon in the gut. Next, I discuss the relationship between the physiological actions of GLP-1 and the therapeutic effects of the GLP-1 receptor agonists, which are far from being congruent and generally poorly understood. These relationships illustrate both the difficulties and the benefits of bridging results obtained in the laboratory with those emerging from the clinic.

Pathways of Glucagon Secretion and Trafficking in the Pancreatic Alpha Cell: Novel Pathways, Proteins, and Targets for Hyperglucagonemia.

Patients with diabetes mellitus exhibit hyperglucagonemia, or excess glucagon secretion, which may be the underlying cause of the hyperglycemia of diabetes. Defective alpha cell secretory responses to glucose and paracrine effectors in both Type 1 and Type 2 diabetes may drive the development of hyperglucagonemia. Therefore, uncovering the mechanisms that regulate glucagon secretion from the pancreatic alpha cell is critical for developing improved treatments for diabetes. In this review, we focus on aspects of alpha cell biology for possible mechanisms for alpha cell dysfunction in diabetes: proglucagon processing, intrinsic and paracrine control of glucagon secretion, secretory granule dynamics, and alterations in intracellular trafficking. We explore possible clues gleaned from these studies in how inhibition of glucagon secretion can be targeted as a treatment for diabetes mellitus.

Neprilysin Inhibition Increases Glucagon Levels in Humans and Mice With Potential Effects on Amino Acid Metabolism.

CONTEXT: Inhibitors of the protease neprilysin (NEP) are used for treating heart failure, but are also linked to improvements in metabolism. NEP may cleave proglucagon-derived peptides, including the glucose and amino acid (AA)-regulating hormone glucagon. Studies investigating NEP inhibition on glucagon metabolism are warranted. OBJECTIVE: This work aims to investigate whether NEP inhibition increases glucagon levels. METHODS: Plasma concentrations of glucagon and AAs were measured in eight healthy men during a mixed meal with and without a single dose of the NEP inhibitor/angiotensin II type 1 receptor antagonist, sacubitril/valsartan (194 mg/206 mg). Long-term effects of sacubitril/valsartan (8 weeks) were investigated in individuals with obesity (n = 7). Mass spectrometry was used to investigate NEP-induced glucagon degradation, and the derived glucagon fragments were tested pharmacologically in cells transfected with the glucagon receptor (GCGR). Genetic deletion or pharmacological inhibition of NEP with or without concomitant GCGR antagonism was tested in mice to evaluate effects on AA metabolism. RESULTS: In healthy men, a single dose of sacubitril/valsartan significantly increased postprandial concentrations of glucagon by 228%, concomitantly lowering concentrations of AAs including glucagonotropic AAs. Eight-week sacubitril/valsartan treatment increased fasting glucagon concentrations in individuals with obesity. NEP cleaved glucagon into 5 inactive fragments (in vitro). Pharmacological NEP inhibition protected both exogenous and endogenous glucagon in mice after an AA challenge, while NEP-deficient mice showed elevated fasting and AA-stimulated plasma concentrations of glucagon and urea compared to controls. CONCLUSION: NEP cleaves glucagon, and inhibitors of NEP result in hyperglucagonemia and may increase postprandial AA catabolism without affecting glycemia.

Variation in the Evolution and Sequences of Proglucagon and the Receptors for Proglucagon-Derived Peptides in Mammals.

The mammalian proglucagon gene (Gcg) encodes three glucagon like sequences, glucagon, glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 that are of similar length and share sequence similarity, with these hormones having cell surface receptors, glucagon receptor (Gcgr), GLP-1 receptor (Glp1r), and GLP-2 receptor (Glp2r), respectively. Gcgr, Glp1r, and Glp2r are all class B1 G protein-coupled receptors (GPCRs). Despite their sequence and structural similarity, analyses of sequences from rodents have found differences in patterns of sequence conservation and evolution. To determine whether these were rodent-specific traits or general features of these genes in mammals I analyzed coding and protein sequences for proglucagon and the receptors for proglucagon-derived peptides from the genomes of 168 mammalian species. Single copy genes for each gene were found in almost all genomes. In addition to glucagon sequences within Hystricognath rodents (e.g., guinea pig), glucagon sequences from a few other groups (e.g., pangolins and some bats) as well as changes in the proteolytic processing of GLP-1 in some bats are suggested to have functional effects. GLP-2 sequences display increased variability but accepted few substitutions that are predicted to have functional consequences. In parallel, Glp2r sequences display the most rapid protein sequence evolution, and show greater variability in amino acids at sites involved in ligand interaction, however most were not predicted to have a functional consequence. These observations suggest that a greater diversity in biological functions for proglucagon-derived peptides might exist in mammals.

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.