Canagliflozin or acarbose versus placebo to ameliorate post-bariatric hypoglycaemia - The HypoBar I randomized clinical trial protocol.

INTRODUCTION: Post-bariatric hypoglycaemia (PBH) is a rare yet disabling clinical condition, mostly reported after Roux-en-Y gastric bypass (RYGB) surgery. RYGB is one of the most widely used and effective bariatric procedures. The pathophysiology of PBH remains unclear, and treatment options are limited in effectiveness and/or carry significant side effects. Acarbose slows carbohydrates digestion and absorption and is generally considered first-line pharmacological treatment for PBH but its gastrointestinal side effects limit patient compliance. Canagliflozin inhibits intestinal and renal sodium-dependent glucose absorption and reduces postprandial excursions of glucose, insulin and incretins after RYGB - effects that could be beneficial in ameliorating PBH. AIMS: The trial aims to investigate how blood glucose levels are affected during daily living in subjects with PBH during treatment with canagliflozin or acarbose compared with placebo, and to study the meal-induced entero-endocrine mechanisms implied in the treatment responses. METHODS: In a double-blinded, randomized, crossover clinical trial, HypoBar I will investigate the effectiveness in reducing the risk of PBH, safety, ambulatory glucose profile and entero-endocrine responses when PBH is treated with canagliflozin 300 mg twice daily during a 4-week intervention period, compared with acarbose 50 mg thrice daily or placebo. ETHICS AND DISSEMINATION: HypoBar I is approved by the Local regulatory entities. Results will be published in peer-reviewed journals. CONCLUSION: If effective, well-tolerated and safe, canagliflozin could be a novel treatment for people with PBH. HypoBar I might also unravel new mechanisms underlying PBH, potentially identifying new treatment targets. TRIAL REGISTRATION: EudraCT number 2022-000157-87.

A randomized, double-blind, crossover study of the effect of the fluoroquinolone moxifloxacin on glucose levels and insulin sensitivity in young men and women.

AIM: The voltage-gated potassium channel Kv 11.1 is important for repolarizing the membrane potential in excitable cells such as myocytes, pancreatic α- and ß-cells. Moxifloxacin blocks the Kv 11.1 channel and increases the risk of hypoglycaemia in patients with diabetes. We investigated glucose regulation and secretion of glucoregulatory hormones in young people with and without moxifloxacin, a drug known to block the Kv 11.1 channel. MATERIALS AND METHODS: The effect of moxifloxacin (800 mg/day for 4 days) or placebo on glucose regulation was assessed in a randomized, double-blind, crossover study of young men and women (age 20-40 years and body mass index 18.5-27.5 kg/m2 ) without chronic disease, using 6-h oral glucose tolerance tests and continuous glucose monitoring. RESULTS: Thirty-eight participants completed the study. Moxifloxacin prolonged the QTcF interval and increased heart rate. Hypoglycaemia was more frequently observed with moxifloxacin, both during the 8 days of continuous glucose monitoring and during the oral glucose tolerance tests. Hypoglycaemia questionnaire scores were higher after intake of moxifloxacin. Moxifloxacin reduced the early plasma-glucose response (AUC0-30 min ) by 7% (95% CI: -9% to -4%, p < .01), and overall insulin response (AUC0-360 min ) decreased by 18% (95% CI: -24% to -11%, p < .01) and plasma glucagon increased by 17% (95% CI: 4%-33%, p = .03). Insulin sensitivity calculated as the Matsuda index increased by 11%, and MISI, an index of muscle insulin sensitivity, increased by 34%. CONCLUSIONS: In young men and women, moxifloxacin, a drug known to block the Kv 11.1 channel, increased QT interval, decreased glucose levels and was associated with increased muscle insulin sensitivity and more frequent episodes of hypoglycaemia.

GIP and GLP-2 together improve bone turnover in humans supporting GIPR-GLP-2R co-agonists as future osteoporosis treatment.

The intestinal hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are key regulators of postprandial bone turnover in humans. We hypothesized that GIP and GLP-2 co-administration would provide stronger effect on bone turnover than administration of the hormones separately, and tested this using subcutaneous injections of GIP and GLP-2 alone or in combination in humans. Guided by these findings, we designed series of GIPR-GLP-2R co-agonists as template for new osteoporosis treatment. The clinical experiment was a randomized cross-over design including 10 healthy men administered subcutaneous injections of GIP and GLP-2 alone or in combination. The GIPR-GLP-2R co-agonists were characterized in terms of binding and activation profiles on human and rodent GIP and GLP-2 receptors, and their pharmacokinetic (PK) profiles were improved by dipeptidyl peptidase-4 protection and site-directed lipidation. Co-administration of GIP and GLP-2 in humans resulted in an additive reduction in bone resorption superior to each hormone individually. The GIPR-GLP-2R co-agonists, designed by combining regions of importance for cognate receptor activation, obtained similar efficacies as the two native hormones and nanomolar potencies on both human receptors. The PK-improved co-agonists maintained receptor activity along with their prolonged half-lives. Finally, we found that the GIPR-GLP-2R co-agonists optimized toward the human receptors for bone remodeling are not feasible for use in rodent models. The successful development of potent and efficacious GIPR-GLP-2R co-agonists, combined with the improved effect on bone metabolism in humans by co-administration, support these co-agonists as a future osteoporosis treatment.

On measurements of glucagon secretion in healthy, obese, and Roux-en-Y gastric bypass operated individuals using sandwich ELISA.

Glucagon is a key regulator of metabolism and is used in the diagnostic of neuroendocrine tumors. Accurate measurement of glucagon requires both extreme sensitivity and specificity since several peptides are derived from the same proglucagon precursor encoding part of the glucagon sequence and given that glucagon circulates in picomolar concentrations. A sandwich ELISA was recently developed and extensively evaluated; however, this method may not be accurate when measuring glucagon in patients with an enhanced production of proglucagon-derived peptides as seen after Roux-en-Y gastric bypass (RYGB). To overcome this, a modified version of the ELISA was developed. In this study, we evaluate an unmodified and a modified version of the ELISA in healthy individuals, individuals with obesity, and finally in two cohorts of patients following RYGB surgery using different nutrient stimuli to assess glucagon dynamics. Finally, in vitro spike-in recoveries using native glucagon and proglucagon-derived peptides were performed in buffer and in plasma. Our data support that both versions of the ELISA accurately capture endogenous and exogenous glucagon in healthy individuals and in individuals with obesity. However, the unmodified version of the assay may overestimate glucagon levels in patients following RYGB in line with minimal but consistent cross-reactivity to oxyntomodulin and glicentin that both are 50-fold increased after RYGB. Importantly, we did not find any changes between the two protocols at fasted conditions and therefore diagnostics of glucagonomas is not affected by the choice of assay procedure nor the surgical history of the patient (RYGB).

GLP-1-induced renal vasodilation in rodents depends exclusively on the known GLP-1 receptor and is lost in prehypertensive rats.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone known to stimulate postprandial insulin release. However, GLP-1 also exerts extrapancreatic effects, including renal effects. Some of these renal effects are attenuated in hypertensive rats, where renal expression of GLP-1 receptors is reduced. Here, we assessed the expression and vascular function of GLP-1 receptors in kidneys from young prehypertensive rats. We also examined GLP-1-induced vasodilation in the renal vasculature in wild-type (WT) and GLP-1 receptor knockout mice using wire and pressure myography and the isolated perfused juxtamedullary nephron preparation. We investigated whether GLP-1 and the metabolite GLP-1(9-36)amide had renal vascular effects independent of the known GLP-1 receptor. We hypothesized that hypertension decreased expression of renal GLP-1 receptors. We also hypothesized that GLP-1-induced renal vasodilatation depended on expression of the known GLP-1 receptor. In contrast to normotensive rats, no immunohistochemical staining or vasodilatory function of GLP-1 receptors was found in kidneys from prehypertensive rats. In WT mice, GLP-1 induced renal vasodilation and reduced the renal autoregulatory response. The GLP-1 receptor antagonist exendin 9-39 inhibited relaxation, and GLP-1(9-36)amide had no vasodilatory effect. In GLP-1 receptor knockout mice, no relaxation induced by GLP-1 or GLP-1(9-36)amide was found, the autoregulatory response in afferent arterioles was normal, and no GLP-1-induced reduction of autoregulation was found. We conclude that in prehypertensive kidneys, expression and function of GLP-1 receptors is lost. The renal vasodilatory effect of GLP-1 is mediated exclusively by the known GLP-1 receptor. GLP-1(9-36)amide has no renal vasodilatory effect. GLP-1 attenuates renal autoregulation by reducing the myogenic response.

The effect of acute dual SGLT1/SGLT2 inhibition on incretin release and glucose metabolism after gastric bypass surgery.

Enhanced meal-related enteroendocrine secretion, particularly of glucagon-like peptide-1 (GLP-1), contributes to weight-loss and improved glycemia after Roux-en-Y gastric bypass (RYGB). Dietary glucose drives GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) secretion postoperatively. Understanding how glucose triggers incretin secretion following RYGB could lead to new treatments of diabetes and obesity. In vitro, incretin release depends on glucose absorption via sodium-glucose cotransporter 1 (SGLT1). We investigated the importance of SGLT1/SGLT2 for enteropancreatic hormone concentrations and glucose metabolism after RYGB in a randomized, controlled, crossover study. Ten RYGB-operated patients ingested 50 g of oral glucose with and without acute pretreatment with 600 mg of the SGLT1/SGLT2-inhibitor canagliflozin. Paracetamol and 3-O-methyl-d-glucopyranose (3-OMG) were added to the glucose drink to evaluate rates of intestinal entry and absorption of glucose, respectively. Blood samples were collected for 4 h. The primary outcome was 4-h plasma GLP-1 (incremental area-under the curve, iAUC). Secondary outcomes included glucose, GIP, insulin, and glucagon. Canagliflozin delayed glucose absorption (time-to-peak 3-OMG: 50 vs. 132 min, P < 0.01) but did not reduce iAUC GLP-1 (6,067 vs. 7,273·min·pmol-1·L-1, P = 0.23), although peak GLP-1 concentrations were lowered (-28%, P = 0.03). Canagliflozin reduced GIP (iAUC -28%, P = 0.01; peak concentrations -57%, P < 0.01), insulin, and glucose excursions, whereas plasma glucagon (AUC 3,216 vs. 4,160 min·pmol·L-1, P = 0.02) and amino acids were increased. In conclusion, acute SGLT1/SGLT2-inhibition during glucose ingestion did not reduce 4-h plasma GLP-1 responses in RYGB-patients but attenuated the early rise in GLP-1, GIP, and insulin, whereas late glucagon concentrations were increased. The results suggest that SGLT1-mediated glucose absorption contributes to incretin hormone secretion after RYGB.

Mechanism of glucose-lowering by metformin in type 2 diabetes: Role of bile acids.

Type 2 diabetes mellitus (T2DM) is an increasingly prevalent chronic condition, characterized by abnormally elevated blood glucose concentrations and, as a consequence, increased risk of micro- and macrovascular complications. Metformin is usually the first-line glucose-lowering medication in T2DM; however, despite being used for more than 60 years, the mechanism underlying the glucose-lowering action of metformin remains incompletely understood. Although metformin reduces hepatic glucose production, there is persuasive evidence that the gastrointestinal tract is crucial in mediating this effect, particularly via secretion of the incretin hormone glucagon-like peptide 1 (GLP-1). It is now well recognized that bile acids, in addition to their established function in fat digestion and absorption, are important regulators of glucose metabolism. Exposure of the small and large intestine to bile acids induces GLP-1 secretion, modulates the composition of the gut microbiota, and reduces postprandial blood glucose excursions in humans with and without T2DM. Metformin reduces intestinal bile acid resorption substantially, such that intraluminal bile acids may, at least in part, account for its glucose-lowering effect. The present review focuses on the conceptual shift in our understanding as to how metformin lowers blood glucose in T2DM, with a particular emphasis on the role of intestinal bile acids.

Gut hormone release after gastric bypass depends on the length of the biliopancreatic limb.

BACKGROUND/OBJECTIVES: Changes in gut hormone secretion are important for the anti-diabetic effects of bariatric surgery. Roux-en-Y gastric bypass (RYGB) with extended biliopancreatic limb (BPL) length may improve the metabolic outcomes when compared to the classical procedure. The purpose of this study was to compare the gut hormone responses to a liquid mixed meal after RYGB with one of two different BPL lengths. SUBJECTS/METHODS: Non-diabetic weight-stable individuals previously submitted to classical RYGB (n = 9; BPL length: 87.8 ± 20.5 cm) or long BPL RYGB (n = 11; BPL length: 200 cm) underwent a liquid mixed-meal tolerance test (MMTT). Blood was sampled at baseline and 15, 30, 45, 60, 90 and 120 min later for measurement of plasma glucose, enteropancreatic hormones and total bile acids (TBA). RESULTS: Plasma glucose excursion curves were similar in the two groups. The long BPL RYGB group displayed significantly higher fasting and post-prandial GLP-1 (t = 0 min, p = 0.01 and t = 45 min, p < 0.05; tAUC: 11,205 ± 3399 vs 7889 ± 1686 pmol/L × min, p = 0.02) and neurotensin (t = 0 min, p = 0.02; t = 45 min, p < 0.05 and t = 60 min, p < 0.01; tAUC: 18,392 ± 7066 vs 11,437 ± 3658 pmol/L × min, p = 0.02) levels, while responses of GIP (t = 15 min, p < 0.01), insulin and C-peptide (t = 30 min, p < 0.001) were lower as compared to classical RYGB. There were no differences in glucagon, PP, PYY and TBA between the groups. CONCLUSIONS: RYGB with a longer BPL results in a distinctive post-prandial hormone profile with augmented GLP-1 and neurotensin responses that could be beneficial for the metabolic outcomes of the surgery.

A sandwich ELISA for measurement of the primary glucagon-like peptide-1 metabolite.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from the gastrointestinal tract. It is best known for its glucose-dependent insulinotropic effects. GLP-1 is secreted in its intact (active) form (7-36NH2) but is rapidly degraded by the dipeptidyl peptidase 4 (DPP-4) enzyme, converting >90% to the primary metabolite (9-36NH2) before reaching the targets via the circulation. Although originally thought to be inactive or antagonistic, GLP-1 9-36NH2 may have independent actions, and it is therefore relevant to be able to measure it. Because reliable assays were not available, we developed a sandwich ELISA recognizing both GLP-1 9-36NH2 and nonamidated GLP-1 9-37. The ELISA was validated using analytical assay validation guidelines and by comparing it to a subtraction-based method, hitherto employed for estimation of GLP-1 9-36NH2 Its accuracy was evaluated from measurements of plasma obtained during intravenous infusions (1.5 pmol × kg-1 × min-1) of GLP-1 7-36NH2 in healthy subjects and patients with type 2 diabetes. Plasma levels of the endogenous GLP-1 metabolite increased during a meal challenge in patients with type 2 diabetes, and treatment with a DPP-4 inhibitor fully blocked its formation. Accurate measurements of the GLP-1 metabolite may contribute to understanding its physiology and role of GLP-1 in diabetes.

Cephalic phase secretion of insulin and other enteropancreatic hormones in humans.

Enteropancreatic hormone secretion is thought to include a cephalic phase, but the evidence in humans is ambiguous. We studied vagally induced gut hormone responses with and without muscarinic blockade in 10 glucose-clamped healthy men (age: 24.5 ± 0.6 yr, means ± SE; body mass index: 24.0 ± 0.5 kg/m(2); HbA1c: 5.1 ± 0.1%/31.4 ± 0.5 mmol/mol). Cephalic activation was elicited by modified sham feeding (MSF, aka "chew and spit") with or without atropine (1 mg bolus 45 min before MSF + 80 ng·kg(-1)·min(-1) for 2 h). To mimic incipient prandial glucose excursions, glucose levels were clamped at 6 mmol/l on all days. The meal stimulus for the MSF consisted of an appetizing breakfast. Participants (9/10) also had a 6 mmol/l glucose clamp without MSF. Pancreatic polypeptide (PP) levels rose from 6.3 ± 1.1 to 19.9 ± 6.8 pmol/l (means ± SE) in response to MSF and atropine lowered basal PP levels and abolished the MSF response. Neither insulin, C-peptide, glucose-dependent insulinotropic polypeptide (GIP), nor glucagon-like peptide-1 (GLP-1) levels changed in response to MSF or atropine. Glucagon and ghrelin levels were markedly attenuated by atropine prior to and during the clamp: at t = 105 min on the atropine (ATR) + clamp (CLA) + MSF compared with the saline (SAL) + CLA and SAL + CLA + MSF days; baseline-subtracted glucagon levels were -10.7 ± 1.1 vs. -4.0 ± 1.1 and -4.7 ± 1.9 pmol/l (means ± SE), P < 0.0001, respectively; corresponding baseline-subtracted ghrelin levels were 303 ± 36 vs. 39 ± 38 and 3.7 ± 21 pg/ml (means ± SE), P < 0.0001. Glucagon and ghrelin levels were unaffected by MSF. Despite adequate PP responses, a cephalic phase response was absent for insulin, glucagon, GLP-1, GIP, and ghrelin.

The insulinotropic effect of exogenous glucagon-like peptide-1 is not affected by acute vagotomy in anaesthetized pigs.

What is the central question of this study? We investigated whether intestinal vagal afferents are necessary for the insulinotropic effect of glucagon-like peptide-1 (GLP-1) infused into a mesenteric artery or a peripheral vein before and after acute truncal vagotomy. What is the main finding and its importance? We found no effect of truncal vagotomy on the insulinotropic effect of exogenous GLP-1 and speculate that high circulating concentrations of GLP-1 after i.v. and i.a. infusion might have overshadowed any neural signalling component. We propose that further investigations into the possible vagal afferent signalling of GLP-1 would best be pursued using enteral stimuli to provide high subepithelial levels of endogenous GLP-1. Glucagon-like peptide 1 (GLP-1) is secreted from the gut in response to luminal stimuli and stimulates insulin secretion in a glucose-dependent manner. As a result of rapid enzymatic degradation of GLP-1 by dipeptidyl peptidase-4, a signalling pathway involving activation of intestinal vagal afferents has been proposed. We conducted two series of experiments in α-chloralose-anaesthetized pigs. In protocol I, pigs (n = 14) were allocated for either i.v. or i.a. (mesenteric) GLP-1 infusions (1 and 2 pmol kg(-1)  min(-1) , 30 min) while maintaining permissive glucose concentrations at 6 mmol l(-1) by i.v. glucose infusion. The GLP-1 infusions were repeated after acute truncal vagotomy. In protocol II, pigs (n = 27) were allocated into six groups. Glucagon-like peptide 1 was infused i.v. or i.a. (mesenteric) for 1 h at 3 or 30 pmol kg(-1)  min(-1) . During the steady state (21 min into the GLP-1 infusion), glucose (0.2 g kg(-1) , i.v.) was administered over 9 min to stimulate ß-cell secretion. Thirty minutes after the glucose infusion, GLP-1 infusions were discontinued. Following a washout period, the vagal trunks were severed in four of six groups (vagal trunks were left intact in two of six groups), whereupon all infusions were repeated. We found no effect of vagotomy on insulin or glucagon secretion during administration of exogenous GLP-1 in any experiment. We speculate that the effect of exogenous GLP-1 overshadowed any effect occurring via the vagus. Within dosage groups, total GLP-1 concentrations were similar, but intact GLP-1 concentrations were much lower when infused via the mesenteric artery because of extensive degradation of GLP-1 in the splanchnic bed. This demonstrates the effectiveness with which intestinal capillary dipeptidyl peptidase-4 protects the systemic circulation from intact GLP-1, consistent with a local role for GLP-1 involving activation of vagal pathways.

The role of efferent cholinergic transmission for the insulinotropic and glucagonostatic effects of GLP-1.

The importance of vagal efferent signaling for the insulinotropic and glucagonostatic effects of glucagon-like peptide-1 (GLP-1) was investigated in a randomized single-blinded study. Healthy male participants (n = 10) received atropine to block vagal cholinergic transmission or saline infusions on separate occasions. At t = 15 min, plasma glucose was clamped at 6 mmol/l. GLP-1 was infused at a low dose (0.3 pmol·kg(-1)·min(-1)) from t = 45-95 min and at a higher dose (1 pmol·kg(-1)·min(-1)) from t = 95-145 min. Atropine blocked muscarinic, cholinergic transmission, as evidenced by an increase in heart rate [peak: 70 ± 2 (saline) vs. 90 ± 2 (atropine) beats/min, P < 0.002] and suppression of pancreatic polypeptide levels [area under the curve during the GLP-1 infusions (AUC45-145): 492 ± 85 (saline) vs. 247 ± 59 (atropine) pmol/l × min, P < 0.0001]. More glucose was needed to maintain the clamp during the high-dose GLP-1 infusion steady-state period on the atropine day [6.4 ± 0.9 (saline) vs. 8.7 ± 0.8 (atropine) mg·kg(-1)·min(-1), P < 0.0023]. GLP-1 dose-dependently increased insulin secretion on both days. The insulinotropic effect of GLP-1 was not impaired by atropine [C-peptide AUCs45-145: 99 ± 8 (saline) vs. 113 ± 13 (atropine) nmol/l × min, P = 0.19]. Atropine suppressed glucagon levels additively with GLP-1 [AUC45-145: 469 ± 70 (saline) vs. 265 ± 50 (atropine) pmol/l × min, P = 0.018], resulting in hypoglycemia when infusions were suspended [3.6 ± 0.2 (saline) vs. 2.7 ± 0.2 (atropine) mmol/l, P < 0.0001]. To ascertain whether atropine could independently suppress glucagon levels, control experiments (n = 5) were carried out without GLP-1 infusions [AUC45-145: 558 ± 103 (saline) vs. 382 ± 76 (atropine) pmol/l × min, P = 0.06]. Our results suggest that efferent muscarinic activity is not required for the insulinotropic effect of exogenous GLP-1 but that blocking efferent muscarinic activity independently suppresses glucagon secretion. In combination, GLP-1 and muscarinic blockade strongly affect glucose turnover.

Hyperglucagonaemia analysed by glucagon sandwich ELISA: nonspecific interference or truly elevated levels?

AIM/HYPOTHESIS: Hyperglucagonaemia is a characteristic of several clinical conditions (e.g. end-stage renal disease (ESRD), type 2 diabetes, obesity before and after Roux-en-Y gastric bypass (RYGB) and vagotomy with pyloroplasty), but the molecular nature of 'immunoreactive' glucagon is poorly characterised. The specific determination of fully processed, intact glucagon requires a 'sandwich' assay employing a combination of antibodies directed against both N- and C-termini. We compared a novel assay for intact glucagon with a highly sensitive C-terminal RIA (hitherto considered specific) to determine the extent to which the hyperglucagonaemia measured in clinical samples was caused by authentic glucagon. METHODS: We examined the performance of three commercial glucagon 'sandwich' ELISAs. The ELISA with the best overall performance was selected to compare glucagon measurements in clinical samples with an established glucagon RIA. RESULTS: The first assay performed poorly: there was high cross-reactivity with glicentin (22%) and a lack of sensitivity for glucagon. The second and third assays showed minor cross-reactivity (1-5%) with oxyntomodulin and glicentin; however, the second assay had insufficient sensitivity for glucagon in plasma (>10-20 pmol/l). Thus, only the third assay was suitable for measuring glucagon concentrations in clinical samples. The ELISA and RIA measured similar glucagon levels in healthy individuals. Measurements of samples from individuals with abnormally high (type 2 diabetes or obese) or very elevated (post vagotomy with pyloroplasty, post-RYGB) glucagon levels were also similar in both assays. However, glucagon levels in participants with ESRD were much lower when measured by ELISA than by RIA, indicating that the apparent hyperglucagonaemia is not caused by fully processed intact glucagon. CONCLUSIONS/INTERPRETATION: For most purposes, sensitive C-terminal glucagon RIAs are accurate. However, measurements may be spuriously high, at least in patients with renal disease. Trial Registration Samples from type 2 diabetic and normoglucose-tolerant patients before and 1 year after RYGB were from a study by Bojsen-Møller et al (trial registration number NCT 01202526). Samples from vagotomised and control individuals were from a study by Plamboeck et al (NCT01176890). Samples from ESRD patients were from a study by Idorn et al (NCT01327378).

Intraduodenal calcium enhances the effects of L-tryptophan to stimulate gut hormone secretion and suppress energy intake in healthy males: a randomized, cross-over, clinical trial.

BACKGROUND: In humans, intraduodenal infusion of L-tryptophan (Trp) increases plasma concentrations of gastrointestinal hormones and stimulates pyloric pressures, both key determinants of gastric emptying and associated with potent suppression of energy intake. The stimulation of gastrointestinal hormones by Trp has been shown, in preclinical studies, to be enhanced by extracellular calcium and mediated in part by the calcium-sensing receptor. OBJECTIVE: To determine whether intraduodenal calcium can enhance the effects of Trp to stimulate gastrointestinal hormones and pyloric pressures, and if so, whether it is associated with greater suppression of energy intake, in healthy males. METHODS: Fifteen males with normal weight (mean±SD; age: 26±7 years; body mass index: 22±2 kg/m2), received on three separate occasions, 150-min intraduodenal infusions of 0, 500 or 1000 mg calcium (Ca), each combined with Trp (load: 0.1 kcal/min, with submaximal energy intake-suppressant effects) from t=75-150 min, in a randomized, double-blind, cross-over study. Plasma concentrations of GI hormones (gastrin, cholecystokinin, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), peptide tyrosine-tyrosine (PYY)), and Trp, and antropyloroduodenal pressures were measured throughout. Immediately post-infusions (t=150-180 min), energy intake at a standardized buffet-style meal was quantified. RESULTS: In response to calcium alone, both 500-mg and 1000-mg doses stimulated PYY, while only the 1000-mg dose stimulated GLP-1 and pyloric pressures (all P<0.05). The 1000-mg dose also enhanced the effects of Trp to stimulate cholecystokinin and GLP-1, and both doses stimulated PYY, but, surprisingly, reduced the stimulation of GIP (all P<0.05). Both doses substantially and dose-dependently enhanced the effects of Trp to suppress energy intake (kcal; Ca-0+Trp: 1108±70, Ca-500+Trp: 961±90, Ca-1000+Trp: 922±96; P<0.05). CONCLUSIONS: Intraduodenal administration of calcium enhances the effect of Trp to stimulate plasma cholecystokinin, GLP-1 and PYY, and suppress energy intake, in health. These findings have potential implications for novel nutrient-based approaches to energy intake regulation in obesity. CLINICAL TRIAL REGISTRY: The trial was registered with the Australian New Zealand Clinical Trial Registry (www.anzctr.org.au; trial number: ACTRN12620001294943).

GLP-1 increases heart rate by a direct action on the sinus node.

AIMS: Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are increasingly used to treat type 2 diabetes and obesity. Albeit cardiovascular outcomes generally improve, treatment with GLP-1 RAs is associated with increased heart rate, the mechanism of which is unclear. METHODS AND RESULTS: We employed a large animal model, the female landrace pig, and used multiple in-vivo and ex-vivo approaches including pharmacological challenges, electrophysiology and high-resolution mass spectrometry to explore how GLP-1 elicits an increase in heart rate. In anaesthetized pigs, neither cervical vagotomy, adrenergic blockers (alpha, beta or combined alpha-beta blockade), ganglionic blockade (hexamethonium) nor inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (ivabradine) abolished the marked chronotropic effect of GLP-1. GLP-1 administration to isolated perfused pig hearts also increased heart rate, which was abolished by GLP-1 receptor blockade. Electrophysiological characterization of GLP-1 effects in vivo and in isolated perfused hearts localized electrical modulation to the atria and conduction system. In isolated sinus nodes, GLP-1 administration shortened action potential cycle length of pacemaker cells and shifted the site of earliest activation. The effect was independent of HCN blockade. Collectively, these data support a direct effect of GLP-1 on GLP-1 receptors within the heart. Consistently, single nucleus RNA sequencing (snRNAseq) showed GLP-1 receptor expression in porcine pacemaker cells. Quantitative phosphoproteomics analyses of sinus node samples revealed that GLP-1 administration leads to phosphorylation changes of calcium cycling proteins of the sarcoplasmic reticulum, known to regulate heart rate. CONCLUSION: GLP-1 has direct chronotropic effects on the heart mediated by GLP-1 receptors in pacemaker cells of the sinus node, inducing changes in action potential morphology and the leading pacemaker site through a calcium signaling response characterized by PKA-dependent phosphorylation of Ca2+ cycling proteins involved in pace making. Targeting the pacemaker calcium clock may be a strategy to lower heart rate in GLP-1 RA recipients.

The effect of exogenous GLP-1 on food intake is lost in male truncally vagotomized subjects with pyloroplasty.

Rapid degradation of glucagon-like peptide-1 (GLP-1) by dipeptidyl peptidase-4 suggests that endogenous GLP-1 may act locally before being degraded. Signaling via the vagus nerve was investigated in 20 truncally vagotomized subjects with pyloroplasty and 10 matched healthy controls. Subjects received GLP-1 (7-36 amide) or saline infusions during and after a standardized liquid mixed meal and a subsequent ad libitum meal. Despite no effect on appetite sensations, GLP-1 significantly reduced ad libitum food intake in the control group but had no effect in the vagotomized group. Gastric emptying was accelerated in vagotomized subjects and was decreased by GLP-1 in controls but not in vagotomized subjects. Postprandial glucose levels were reduced by the same percentage by GLP-1 in both groups. Peak postprandial GLP-1 levels were approximately fivefold higher in the vagotomized subjects. Insulin secretion was unaffected by exogenous GLP-1 in vagotomized subjects but was suppressed in controls. GLP-1 significantly reduced glucagon secretion in both groups, but levels were approximately twofold higher and were nonsuppressible in the early phase of the meal in vagotomized subjects. Our results demonstrate that vagotomy with pyloroplasty impairs the effects of exogenous GLP-1 on food intake, gastric emptying, and insulin and glucagon secretion, suggesting that intact vagal innervation may be important for GLP-1's actions.

Entero-Pancreatic Hormone Secretion, Gastric Emptying, and Glucose Absorption After Frequently Sampled Meal Tests.

CONTEXT: Entero-pancreatic hormone secretion has been reported during the pre-absorptive cephalic and gastric meal phases, but never with a blood sampling frequency providing a temporal resolution that allows close scrutiny and correlations with gastric emptying and glucose absorption. OBJECTIVE: We hypothesized that entero-pancreatic hormone secretion after nutrient ingestion would be rapid and correlate with gastric emptying and glucose absorption. METHODS: During 2 visits in a clinical research facility, 10 healthy young men ingested a 75-g glucose drink (OG) and a liquid mixed meal (LMM) (t = 0-2 minutes) on separate days. Acetaminophen and 3-O-methyl-D-glucopyranose (3-OMG) were added to the drinks to evaluate gastric emptying and glucose absorption, respectively. Arterialized venous blood was sampled (t = -30, -20, -18, -16, -14, -12, -10, -8, -6, -4, -2, 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30 minutes). Plasma glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), gastrin, cholecystokinin (CCK), glucagon, pancreatic polypeptide (PP), 3-OMG, and glucose were measured, as were serum insulin, C-peptide, and acetaminophen. RESULTS: Acetaminophen increased 8 minutes after OG (P < 0.001) and LMM (P < 0.05); 3-OMG, 8 minutes after LMM (P < 0.0001), 10 minutes after OG (P = 0.04); PP, 4 minutes after LMM (P < 0.03); gastrin, 6 minutes after LMM (P < 0.003) and OG (P < 0.003); CCK, 6 minutes after LMM (P = 0.0001); GIP, 8 minutes after OG (P < 0.05) and LMM (P < 0.03); glucose, 8 minutes after OG (P < 0.001); 12 minutes after LMM (P < 0.02); GLP-1, 12 minutes after OG (P < 0.01), 10 minutes after LMM (P < 0.01); insulin, 12 minutes after LMM (P = 0.02) and OG (P = 0.002); C-peptide, 12 minutes after OG (P = 0.002) and LMM (P = 0.04). CONCLUSION: Early postprandial hormone responses show characteristic differences with regard to timing and amplitude but also great individual differences. This should be considered when interpreting mean responses and designing study protocols.

Colonic Lactulose Fermentation Has No Impact on Glucagon-like Peptide-1 and Peptide-YY Secretion in Healthy Young Men.

CONTEXT: The colon houses most of humans' gut microbiota, which ferments indigestible carbohydrates. The products of fermentation have been proposed to influence the secretion of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) from the many endocrine cells in the colonic epithelium. However, little is known about the colonic contribution to fasting or postprandial plasma levels of L-cell products. OBJECTIVE: To determine the impact of colonic lactulose fermentation on gut peptide secretion and to evaluate whether colonic endocrine secretion contributes to gut hormone concentrations measurable in the fasting state. METHODS: Ten healthy young men were studied on 3 occasions after an overnight fast. On 2 study days, lactulose (20 g) was given orally and compared to water intake on a third study day. For 1 of the lactulose visits, participants underwent a full colonic evacuation. Over a 6-h study protocol, lactulose fermentation was assessed by measuring exhaled hydrogen, and gut peptide secretion, paracetamol, and short-chain fatty acid levels were measured in plasma. RESULTS: Colonic evacuation markedly reduced hydrogen exhalation after lactulose intake (P = 0.013). Our analysis suggests that the colon does not account for the measurable amounts of GLP-1 and PYY present in the circulation during fasting and that fermentation and peptide secretion are not acutely related. CONCLUSION: Whether colonic luminal contents affect colonic L-cell secretion sufficiently to influence circulating concentrations requires further investigation. Colonic evacuation markedly reduced lactulose fermentation, but hormone releases were unchanged in the present study.

Neurotensin secretion after Roux-en-Y gastric bypass, sleeve gastrectomy, and truncal vagotomy with pyloroplasty.

OBJECTIVE: Neurotensin (NT) is released from enteroendocrine cells and lowers food intake in rodents. We evaluated postprandial NT secretion in humans after surgeries associated with accelerated small intestinal nutrient delivery, and after Roux-en-Y gastric bypass (RYGB) when glucagon-like peptide-1 (GLP-1) signalling and dipeptidyl peptidase 4 (DPP-4) were inhibited, and during pharmacological treatments influencing entero-pancreatic functions. METHODS: We measured NT concentrations in plasma from meal studies: (I) after truncal vagotomy with pyloroplasty (TVP), cardia resection +TVP (CTVP), and matched controls (n = 10); (II) after RYGB, sleeve gastrectomy (SG), and in matched controls (n = 12); (III) after RYGB (n = 11) with antagonism of GLP-1 signalling using exendin(9-39) and DPP-4 inhibition using sitagliptin; (IV) after RYGB (n = 11) during a run-in period and subsequent treatment with, sitagliptin, liraglutide (GLP-1 receptor agonist), verapamil (calcium antagonist), acarbose (alpha glucosidase inhibitor), and pasireotide (somatostatin analogue), respectively. RESULTS: (I) NT secretion was similar after TVP/CTVP (p = 0.9), but increased vs. controls (p < 0.0001). (II) NT secretion was increased after RYGB vs. SG and controls (p < 0.0001). NT responses were similar in SG and controls (p = 0.3), but early postprandial NT concentrations were higher after SG (p < 0.05). (III) Exendin (9-39) and sitagliptin did not change NT responses vs placebo (p > 0.2), but responses were lower during sitagliptin vs. exendin(9-39) (p = 0.03). (IV) Pasireotide suppressed NT secretion (p = 0.004). Sitagliptin tended to lower NT secretion (p = 0.08). Liraglutide, verapamil, and acarbose had no effect (p > 0.9). CONCLUSION: Neurotensin secretion is increased after surgeries associated with accelerated gastric emptying and lowered by pasireotide.