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.

Exercise-induced increase in muscle insulin sensitivity in men is amplified when assessed using a meal test.

AIMS/HYPOTHESIS: Exercise has a profound effect on insulin sensitivity in skeletal muscle. The euglycaemic-hyperinsulinaemic clamp (EHC) is the gold standard for assessment of insulin sensitivity but it does not reflect the hyperglycaemia that occurs after eating a meal. In previous EHC investigations, it has been shown that the interstitial glucose concentration in muscle is decreased to a larger extent in previously exercised muscle than in rested muscle. This suggests that previously exercised muscle may increase its glucose uptake more than rested muscle if glucose supply is increased by hyperglycaemia. Therefore, we hypothesised that the exercise-induced increase in muscle insulin sensitivity would appear greater after eating a meal than previously observed with the EHC. METHODS: Ten recreationally active men performed dynamic one-legged knee extensor exercise for 1 h. Following this, both femoral veins and one femoral artery were cannulated. Subsequently, 4 h after exercise, a solid meal followed by two liquid meals were ingested over 1 h and glucose uptake in the two legs was measured for 3 h. Muscle biopsies from both legs were obtained before the meal test and 90 min after the meal test was initiated. Data obtained in previous studies using the EHC (n=106 participants from 13 EHC studies) were used for comparison with the meal-test data obtained in this study. RESULTS: Plasma glucose and insulin peaked 45 min after initiation of the meal test. Following the meal test, leg glucose uptake and glucose clearance increased twice as much in the exercised leg than in the rested leg; this difference is twice as big as that observed in previous investigations using EHCs. Glucose uptake in the rested leg plateaued after 15 min, alongside elevated muscle glucose 6-phosphate levels, suggestive of compromised muscle glucose metabolism. In contrast, glucose uptake in the exercised leg plateaued 45 min after initiation of the meal test and there were no signs of compromised glucose metabolism. Phosphorylation of the TBC1 domain family member 4 (TBC1D4; p-TBC1D4Ser704) and glycogen synthase activity were greater in the exercised leg compared with the rested leg. Muscle interstitial glucose concentration increased with ingestion of meals, although it was 16% lower in the exercised leg than in the rested leg. CONCLUSIONS/INTERPRETATION: Hyperglycaemia after meal ingestion results in larger differences in muscle glucose uptake between rested and exercised muscle than previously observed during EHCs. These findings indicate that the ability of exercise to increase insulin-stimulated muscle glucose uptake is even greater when evaluated with a meal test than has previously been shown with EHCs.

Indole-3-carboxyaldehyde does not reverse the intestinal effects of fiber-free diet in mice.

Objective: Fiber-free diet impairs intestinal and colonic health in mice, in parallel with a reduction in glucagon like peptide-1 (GLP-1) levels. Endogenous GLP-1 is important for intestinal growth and maintenance of the intestinal integrity. We aimed to investigate whether fiber-free diet reduces luminal content of metabolites which, upon supplementation, could increase GLP-1 secretion and restore the adverse effects of fiber-free diet. Methods: Untargeted metabolomics (LC-MS) was performed on colonic content of mice fed a fiber-free diet, identifying a metabolite of particular interest: indole-3-carboxyaldehyde (I3A). We exposed cultured GLUTag cells to I3A, and measured cumulative GLP-1 secretion. Isolated colon perfusions were performed in male C57BL/6JRj mice and Wistar rats. I3A was administered luminally or vascularly, and GLP-1 was measured in portal vein effluent. Finally, female C57BL/6JRJ mice were fed chow or fiber-free diet, with I3A or vehicle by oral gavage. After 10 days, plasma GLP-1 (ELISA) and intestinal permeability (FITC-dextran) were measured, animals were sacrificed and organs removed for histology. Results: Mice fed a fiber-free diet had significantly lower I3A in their colonic content compared to a control diet (7883 ± 3375 AU, p=0.04). GLP-1 secretion from GLUTag cells was unchanged after five minutes of exposure to I3A. However, GLP-1 levels increased after 120 minutes of exposure to 1 mM (60% increase, p=0.016) and 5 mM (89% increase, p=0.0025) I3A. In contrast, 48 h exposure to 1 mM decreased GLP-1 secretion (51% decrease, p<0.001) and viability. In isolated perfused mouse and rat colon, I3A applied into the luminal or vascular side did not affect GLP-1 secretion. Mice fed a fiber-free diet tended to weigh less compared to chow fed mice; and the small intestine and colon were significantly smaller. No differences were seen in crypt depth, villus length, mucosal area, and intestinal permeability. Supplementing I3A did not affect body weight, morphology or plasma GLP-1 levels. Conclusions: Fiber-free diet lowered colonic content of I3A in mice. I3A stimulates GLP-1 secretion in vitro, but not in animal studies. Moreover, it has no evident beneficial effect on intestinal health when administered in vivo.

The association of cardiometabolic, diet and lifestyle parameters with plasma glucagon-like peptide-1: An IMI DIRECT study.

CONTEXT: The role of glucagon-like peptide-1(GLP-1) in Type 2 diabetes (T2D) and obesity is not fully understood. OBJECTIVE: We investigate the association of cardiometabolic, diet and lifestyle parameters on fasting and postprandial GLP-1 in people at risk of, or living with, T2D. METHOD: We analysed cross-sectional data from the two Innovative Medicines Initiative (IMI) Diabetes Research on Patient Stratification (DIRECT) cohorts, cohort 1(n=2127) individuals at risk of diabetes; cohort 2 (n=789) individuals with new-onset of T2D. RESULTS: Our multiple regression analysis reveals that fasting total GLP-1 is associated with an insulin resistant phenotype and observe a strong independent relationship with male sex, increased adiposity and liver fat particularly in the prediabetes population. In contrast, we showed that incremental GLP-1 decreases with worsening glycaemia, higher adiposity, liver fat, male sex and reduced insulin sensitivity in the prediabetes cohort. Higher fasting total GLP-1 was associated with a low intake of wholegrain, fruit and vegetables inpeople with prediabetes, and with a high intake of red meat and alcohol in people with diabetes. CONCLUSION: These studies provide novel insights into the association between fasting and incremental GLP-1, metabolic traits of diabetes and obesity, and dietary intake and raise intriguing questions regarding the relevance of fasting GLP-1 in the pathophysiology T2D.

Glucagon augments the secretion of FGF21 and GDF15 in MASLD by indirect mechanisms.

INTRODUCTION: Glucagon receptor agonism is currently explored for the treatment of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). The metabolic effects of glucagon receptor agonism may in part be mediated by increases in circulating levels of Fibroblast Growth Factor 21 (FGF21) and Growth Differentiation Factor 15 (GDF15). The effect of glucagon agonism on FGF21 and GDF15 levels remains uncertain, especially in the context of elevated insulin levels commonly observed in metabolic diseases. METHODS: We investigated the effect of a single bolus of glucagon and a continuous infusion of glucagon on plasma concentrations of FGF21 and GDF15 in conditions of endogenous low or high insulin levels. The studies included individuals with overweight with and without MASLD, healthy controls (CON) and individuals with type 1 diabetes (T1D). The direct effect of glucagon on FGF21 and GDF15 was evaluated using our in-house developed isolated perfused mouse liver model. RESULTS: FGF21 and GDF15 correlated with plasma levels of insulin, but not glucagon, and their secretion were highly increased in MASLD compared with CON and T1D. Furthermore, FGF21 levels in individuals with overweight with or without MASLD did not increase after glucagon stimulation when insulin levels were kept constant. FGF21 and GDF15 levels were unaffected by direct stimulation with glucagon in the isolated perfused mouse liver. CONCLUSION: The glucagon-induced secretion of FGF21 and GDF15 are augmented in MASLD and may depend on insulin. Thus, glucagon receptor agonism may augment its metabolic benefits in patients with MASLD through enhanced secretion of FGF21 and GDF15.

Colesevelam has no acute effect on postprandial GLP-1 levels but abolishes gallbladder refilling.

OBJECTIVE: Colesevelam, a bile acid sequestrant approved for the treatment of hypercholesterolaemia, improves glycaemic control in type 2 diabetes. We hypothesised that single-dose colesevelam increases postprandial GLP-1 secretion, thus, reducing postprandial glucose excursions in individuals with type 2 diabetes. Further, we explored the effects of single-dose colesevelam on ultrasonography-assessed postprandial gallbladder motility, paracetamol absorption (proxy for gastric emptying), and circulating factors known to affect gallbladder motility. METHODS: In a randomised, double-blind, placebo-controlled crossover study, 12 individuals with type 2 diabetes (mean ± SD: age 61 ± 8.8 years; body mass index 29.8 ± 3.0 kg/m2) were subjected to 4 mixed meal tests on separate days; 2 with orally administered colesevelam (3.75 g) and 2 with placebo, with intravenous infusion of the GLP-1 receptor antagonist exendin(9-39)NH2 or saline. RESULTS: Single-dose colesevelam had no effect on postprandial concentrations of glucose (P = .786), C-peptide (P = .440), or GLP-1 (P = .729), and exendin(9-39)NH2 administration revealed no GLP-1-mediated effects of colesevelam. Colesevelam did not affect gallbladder emptying but abolished gallbladder refilling (P = .001), increased postprandial cholecystokinin (CCK) secretion (P = .010), and decreased postprandial serum concentrations of fibroblast growth factor 19 (FGF19) (P = .035) and bile acids (P = .043). CONCLUSION: Single-dose colesevelam had no effect on postprandial GLP-1 responses or glucose tolerance but disrupted postprandial gallbladder refilling by increasing CCK secretion and reducing circulating concentrations of FGF19 and bile acids. These findings leave the antidiabetic actions of colesevelam unresolved but provide mechanistic insights into its effect on gallbladder motility.

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.

Counterregulatory hormone and symptom responses to hypoglycaemia in people with type 1 diabetes, insulin-treated type 2 diabetes or without diabetes: the Hypo-RESOLVE hypoglycaemic clamp study.

AIM: The sympathetic nervous and hormonal counterregulatory responses to hypoglycaemia differ between people with type 1 and type 2 diabetes and may change along the course of diabetes, but have not been directly compared. We aimed to compare counterregulatory hormone and symptom responses to hypoglycaemia between people with type 1 diabetes, insulin-treated type 2 diabetes and controls without diabetes, using a standardised hyperinsulinaemic-hypoglycaemic clamp. MATERIALS: We included 47 people with type 1 diabetes, 15 with insulin-treated type 2 diabetes, and 32 controls without diabetes. Controls were matched according to age and sex to the people with type 1 diabetes or with type 2 diabetes. All participants underwent a hyperinsulinaemic-euglycaemic-(5.2 ± 0.4 mmol/L)-hypoglycaemic-(2.8 ± 0.13 mmol/L)-clamp. RESULTS: The glucagon response was lower in people with type 1 diabetes (9.4 ± 0.8 pmol/L, 8.0 [7.0-10.0]) compared to type 2 diabetes (23.7 ± 3.7 pmol/L, 18.0 [12.0-28.0], p < 0.001) and controls (30.6 ± 4.7, 25.5 [17.8-35.8] pmol/L, p < 0.001). The adrenaline response was lower in type 1 diabetes (1.7 ± 0.2, 1.6 [1.3-5.2] nmol/L) compared to type 2 diabetes (3.4 ± 0.7, 2.6 [1.3-5.2] nmol/L, p = 0.001) and controls (2.7 ± 0.4, 2.8 [1.4-3.9] nmol/L, p = 0.012). Growth hormone was lower in people with type 2 diabetes than in type 1 diabetes, at baseline (3.4 ± 1.6 vs 7.7 ± 1.3 mU/L, p = 0.042) and during hypoglycaemia (24.7 ± 7.1 vs 62.4 ± 5.8 mU/L, p = 0.001). People with 1 diabetes had lower overall symptom responses than people with type 2 diabetes (45.3 ± 2.7 vs 58.7 ± 6.4, p = 0.018), driven by a lower neuroglycopenic score (27.4 ± 1.8 vs 36.7 ± 4.2, p = 0.012). CONCLUSION: Acute counterregulatory hormone and symptom responses to experimental hypoglycaemia are lower in people with type 1 diabetes than in those with long-standing insulin-treated type 2 diabetes and controls.

Effect of a 6-Week Carbohydrate-Reduced High-Protein Diet on Levels of FGF21 and GDF15 in People With Type 2 Diabetes.

Context: Fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) are increased in type 2 diabetes and are potential regulators of metabolism. The effect of changes in caloric intake and macronutrient composition on their circulating levels in patients with type 2 diabetes are unknown. Objective: To explore the effects of a carbohydrate-reduced high-protein diet with and without a clinically significant weight loss on circulating levels of FGF21 and GDF15 in patients with type 2 diabetes. Methods: We measured circulating FGF21 and GDF15 in patients with type 2 diabetes who completed 2 previously published diet interventions. Study 1 randomized 28 subjects to an isocaloric diet in a 6 + 6-week crossover trial consisting of, in random order, a carbohydrate-reduced high-protein (CRHP) or a conventional diabetes (CD) diet. Study 2 randomized 72 subjects to a 6-week hypocaloric diet aiming at a ∼6% weight loss induced by either a CRHP or a CD diet. Fasting plasma FGF21 and GDF15 were measured before and after the interventions in a subset of samples (n = 24 in study 1, n = 66 in study 2). Results: Plasma levels of FGF21 were reduced by 54% in the isocaloric study (P < .05) and 18% in the hypocaloric study (P < .05) in CRHP-treated individuals only. Circulating GDF15 levels increased by 18% (P < .05) following weight loss in combination with a CRHP diet but only in those treated with metformin. Conclusion: The CRHP diet significantly reduced FGF21 in people with type 2 diabetes independent of weight loss, supporting the role of FGF21 as a "nutrient sensor." Combining metformin treatment with carbohydrate restriction and weight loss may provide additional metabolic improvements due to the rise in circulating GDF15.

The effect of exogenous glucagon on circulating amino acids in individuals with and without type 2 diabetes and obesity.

Objective: In obesity and type 2 diabetes, hyperglucagonaemia may be caused by elevated levels of glucagonotropic amino acids due to hepatic glucagon resistance at the level of amino acid turnover. Here, we investigated the effect of exogenous glucagon on circulating amino acids in obese and non-obese individuals with and without type 2 diabetes. Design: This was a post hoc analysis in a glucagon infusion study performed in individuals with type 2 diabetes (n = 16) and in age, sex, and body mass index-matched control individuals without diabetes (n = 16). Each group comprised two subgroups of eight individuals with and without obesity, respectively. Methods: All participants received a 1-h glucagon infusion (4 ng/kg/min) in the overnight fasted state. Plasma amino acid concentrations were measured with frequent intervals. Results: Compared to the control subgroup without obesity, baseline total amino acid levels were elevated in the control subgroup with obesity and in the type 2 diabetes subgroup without obesity. In all subgroups, amino acid levels decreased by up to 20% in response to glucagon infusion, which resulted in high physiological steady-state glucagon levels (mean concentration: 74 pmol/L, 95% CI [68;79] pmol/L). Following correction for multiple testing, no intergroup differences in changes in amino acid levels reached significance. Conclusion: Obesity and type 2 diabetes status was associated with elevated fasting levels of total amino acids. The glucagon infusion decreased circulating amino acid levels similarly in all subgroups, without significant differences in the response to exogenous glucagon between individuals with and without obesity and type 2 diabetes. Significance statement: The hormone glucagon stimulates glucose production from the liver, which may promote hyperglycaemia if glucagon levels are abnormally elevated, as is often seen in type 2 diabetes and obesity. Glucagon levels are closely linked to, and influenced by, the levels of circulating amino acids. To further investigate this link, we measured amino acid levels in individuals with and without obesity and type 2 diabetes before and during an infusion of glucagon. We found that circulating amino acid levels were higher in type 2 diabetes and obesity, and that glucagon infusion decreased amino acid levels in both individuals with and without type 2 diabetes and obesity. The study adds novel information to the link between circulating levels of glucagon and amino acids.

In Vivo Inhibition of Dipeptidyl Peptidase 4 Allows Measurement of GLP-1 Secretion in Mice.

Dipeptidyl peptidase 4 (DPP-4) and neprilysin (NEP) rapidly degrade glucagon-like peptide 1 (GLP-1) in mice. Commercially available sandwich ELISA kits may not accurately detect the degradation products, leading to potentially misleading results. We aimed to stabilize GLP-1 in mice, allowing reliable measurement with sensitive commercially available ELISA kits. Nonanesthetized male C57Bl/6JRj mice were subjected to an oral glucose tolerance test (OGTT; 2 g/kg glucose), and plasma total and intact GLP-1 were measured (Mercodia and Alpco ELISA kits, respectively). No GLP-1 increases were seen in samples taken beyond 15 min after the glucose load. Samples taken at 5 and 10 min after the OGTT showed a minor increase in total, but not intact, GLP-1. We then administered saline (control), or a DPP-4 inhibitor (valine pyrrolidide or sitagliptin) with or without an NEP-inhibitor (sacubitril), 30 min before the OGTT. In the inhibitor groups only, intact GLP-1 increased significantly during the OGTT. After injecting male C57Bl/6JRj mice with a known dose of GLP-1(7-36)NH2, peak GLP-1 levels were barely detectable after saline but were 5- to 10-fold higher during sitagliptin and the combination of sitagliptin/sacubitril. The half-life of the GLP-1 plasma disappearance increased up to sevenfold during inhibitor treatment. We conclude that reliable measurement of GLP-1 secretion is not possible in mice in vivo with commercially available sandwich ELISA kits, unless degradation is prevented by inhibition of DPP-4 and perhaps NEP. The described approach allows improved estimates of GLP-1 secretion for future studies, although it is a limitation that these inhibitors additionally influence levels of insulin and glucagon.

Effects of exogenous GIP and GLP-2 on bone turnover in individuals with type 2 diabetes.

CONTEXT: Individuals with type 2 diabetes (T2D) have an increased risk of bone fractures despite normal or increased bone mineral density (BMD). The underlying causes are not well understood but may include disturbances in the gut-bone axis, in which both glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are regulators of bone turnover. Thus, in healthy fasting participants, both exogenous GIP and GLP-2 acutely reduce bone resorption. OBJECTIVE: The objective of this study was to investigate the acute effects of subcutaneously administered GIP and GLP-2 on bone turnover in individuals with T2D. METHODS: We included 10 men with T2D. Participants met fasting in the morning on three separate test days and were injected subcutaneously with GIP, GLP-2, or placebo in a randomized crossover design. Blood samples were drawn at baseline and regularly after injections. Bone turnover was estimated by circulating levels of collagen type 1 C-terminal telopeptide (CTX), procollagen type 1 N-terminal propeptide (P1NP), sclerostin, and PTH. RESULTS: GIP and GLP-2 significantly reduced CTX to (mean ± SEM) 66 ± 7.8% and 74 ± 5.9% of baseline, respectively, compared with after placebo (p = 0.001). In addition, P1NP and sclerostin increased acutely after GIP whereas a decrease in P1NP was seen after GLP-2. PTH levels decreased to 67 ± 2.5% of baseline after GLP-2 and to only 86 ± 3.4% after GIP. CONCLUSION: Subcutaneous GIP and GLP-2 affect CTX and P1NP in individuals with T2D to the same extent as previously demonstrated in healthy individuals.

An extended minimal model of OGTT: estimation of α- and ß-cell dysfunction, insulin resistance, and the incretin effect.

Loss of insulin sensitivity, α- and ß-cell dysfunction, and impairment in incretin effect have all been implicated in the pathophysiology of type 2 diabetes (T2D). Parsimonious mathematical models are useful in quantifying parameters related to the pathophysiology of T2D. Here, we extend the minimum model developed to describe the glucose-insulin-glucagon dynamics in the isoglycemic intravenous glucose infusion (IIGI) experiment to the oral glucose tolerance test (OGTT). The extended model describes glucose and hormone dynamics in OGTT including the contribution of the incretin hormones, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1), to insulin secretion. A new function describing glucose arrival from the gut is introduced. The model is fitted to OGTT data from eight individuals with T2D and eight weight-matched controls (CS) without diabetes to obtain parameters related to insulin sensitivity, ß- and α-cell function. The parameters, i.e., measures of insulin sensitivity, a1, suppression of glucagon secretion, k1, magnitude of glucagon secretion, γ2, and incretin-dependent insulin secretion, γ3, were found to be different between CS and T2D with P values < 0.002, <0.017, <0.009, <0.004, respectively. A new rubric for estimating the incretin effect directly from modeling the OGTT is presented. The average incretin effect correlated well with the experimentally determined incretin effect with a Spearman rank test correlation coefficient of 0.67 (P < 0.012). The average incretin effect was found to be different between CS and T2D (P < 0.032). The developed model is shown to be effective in quantifying the factors relevant to T2D pathophysiology.NEW & NOTEWORTHY A new extended model of oral glucose tolerance test (OGTT) has been developed that includes glucagon dynamics and incretin contribution to insulin secretion. The model allows the estimation of parameters related to α- and ß-cell dysfunction, insulin sensitivity, and incretin action. A new function describing the influx of glucose from the gut has been introduced. A new rubric for estimating the incretin effect directly from the OGTT experiment has been developed. The effect of glucose dose was also investigated.

Empagliflozin Normalizes Fasting Hyperglycemia and Improves Postprandial Glucose Tolerance in Totally Pancreatectomized Patients: A Randomized, Double-Blind, Placebo-Controlled Crossover Study.

OBJECTIVE: Insulin remains the only glucose-lowering treatment modality recommended for totally pancreatectomized patients. We investigated the effects of the sodium-glucose cotransporter 2 inhibitor empagliflozin on fasting and postprandial glucose concentrations in pancreatectomized patients and matched healthy control participants. RESEARCH DESIGN AND METHODS: In a randomized, double-blind, placebo-controlled crossover study, 10 pancreatectomized patients and 10 matched control participants underwent two 3-h liquid mixed meal tests preceded by two doses of 25 mg empagliflozin (administered the night before and in the morning of the meal test) or placebo, respectively. Basal insulin was administered as usual, but bolus insulin was omitted before the meal test during experimental days. RESULTS: Compared with placebo, empagliflozin lowered fasting plasma glucose (5.0 ± 0.4 vs. 7.9 ± 0.9 mmol/L [mean ± SEM], P = 0.007) and postprandial plasma glucose excursions as assessed by baseline-subtracted area under the curve (1,080 [733; 1,231] vs. 1,169 [1,036; 1,417] pmol/L × min [median (25th and 75th percentiles)], P = 0.014) in the pancreatectomized patients. In the control participants, empagliflozin lowered fasting plasma glucose compared with placebo (5.1 ± 0.1 vs. 5.5 ± 0.1 mmol/L, P = 0.008) without affecting postprandial glucose excursions significantly. The pancreatomy group exhibited greater postprandial glucagon excursions compared with the control group on both experimental days (P ≤ 0.015); no within-group differences between days were observed. CONCLUSIONS: Empagliflozin administered the day before and immediately before a standardized liquid mixed meal test normalized fasting hyperglycemia and improved postprandial glucose tolerance in pancreatectomized patients.

Elucidating the glucose-lowering effect of the bile acid sequestrant sevelamer.

AIM: Bile acid sequestrants are cholesterol-lowering drugs, which also improve glycaemic control in people with type 2 diabetes. The mechanism behind the glucose-lowering effect is unknown but has been proposed to be mediated by increased glucagon-like peptide-1 (GLP-1) secretion. Here, we investigated the glucose-lowering effects of sevelamer including any contribution from GLP-1 in people with type 2 diabetes. MATERIALS AND METHODS: In a randomized, double-blind, placebo-controlled, crossover study, 15 people with type 2 diabetes on metformin monotherapy underwent two 17-day treatment periods with the bile acid sequestrant sevelamer and placebo, respectively, in a randomized order and with an interposed wash-out period of minimum 6 weeks. On days 15 and 17 of each treatment period, participants underwent experimental days with 4-h liquid meal tests and application of concomitant infusion of exendin(9-39)NH2 or saline. RESULTS: Compared with placebo, sevelamer improved insulin sensitivity (assessed by homeostatic model assessment of insulin resistance) and beta-cell sensitivity to glucose and lowered fasting and postprandial plasma glucose concentrations. In both treatment periods, exendin(9-39)NH2 increased postprandial glucose excursions compared with saline but without absolute or relative difference between the two treatment periods. In contrast, exendin(9-39)NH2 abolished the sevelamer-induced improvement in beta-cell glucose sensitivity. CONCLUSIONS: The bile acid sequestrant sevelamer improved insulin sensitivity and beta-cell sensitivity to glucose, but using the GLP-1 receptor antagonist exendin(9-39)NH2 we were not able to detect a GLP-1-mediated glucose-lowering effect of sevelamer in individuals with type 2 diabetes. Nevertheless, the sevelamer-induced improvement of beta-cell sensitivity to glucose was shown to be GLP-1-dependent.

Hyperglucagonaemia and amino acid alterations in individuals with type 2 diabetes and non-alcoholic fatty liver disease.

Aims: Hyperglucagonaemia contributes to the pathophysiology in type 2 diabetes (T2D), but the mechanisms behind the inappropriate glucagon secretion are not fully understood. Glucagon and amino acids are regulated in a feedback loop referred to as the liver-α cell axis. Individuals with non-alcoholic fatty liver disease (NAFLD) appear to be glucagon resistant, disrupting the liver-α cell axis resulting in hyperglucagonaemia and hyperaminoacidaemia. We investigated the associations between circulating glucagon, amino acids, and liver fat content in a cohort of individuals with T2D. Methods: We included 110 individuals with T2D in this cross-sectional study. Liver fat content was quantified using 1H magnetic resonance spectroscopy (MRS). Associations between liver fat content and plasma glucagon and amino acids, respectively, were estimated in multivariate linear regression analyses. Results: Individuals with NAFLD (n = 52) had higher plasma glucagon concentrations than individuals without NAFLD (n = 58). The positive association between plasma glucagon concentrations and liver fat content was confirmed in the multivariable regression analyses. Plasma concentrations of isoleucine and glutamate were increased, and glycine and serine concentrations were decreased in individuals with NAFLD. Concentrations of other amino acids were similar between individuals with and without NAFLD, and no clear association was seen between liver fat content and amino acids in the regression analyses. Conclusion: MRS-diagnosed NAFLD in T2D is associated with hyperglucagonaemia and elevated plasma concentrations of isoleucine and glutamate and low plasma concentrations of glycine and serine. Whether NAFLD and glucagon resistance per se induce these changes remains to be elucidated.

Liraglutide treatment for the prevention of glucose tolerance deterioration in women with prior gestational diabetes mellitus: A 52-week randomized controlled clinical trial.

AIM: We investigated the effect of 52-week treatment with liraglutide, a glucagon-like peptide 1 receptor agonist, on glucose tolerance and incretin effect in women with previous gestational diabetes mellitus (pGDM). MATERIALS AND METHODS: Women with overweight/obesity and pGDM were randomized to once daily subcutaneous liraglutide 1.8 mg or placebo for 52 weeks. Participants underwent oral glucose tolerance test (OGTT) and isoglycaemic intravenous glucose infusion at baseline and at 52 weeks, and an additional OGTT after the drug wash-out. RESULTS: In total, 104 women [age: mean ± SD, 38 ± 5 years; fasting plasma glucose (FPG): 5.5 ± 0.4 mmol/L; glycated haemoglobin (HbA1c): 33 ± 4 mmol/mol, bodyweight: 88.2 ± 14.8 kg, body mass index: 31.1 ± 4.3 kg/m2 ] were assigned to liraglutide (n = 49) or placebo (n = 55). Estimated treatment difference (ETD) for area under curve during OGTT was -173 (95% confidence interval -250 to -97) mmol/L × min, p < .0001, but after wash-out the difference disappeared [ETD 58 (-30 to 146) mmol/L × min, p = .536]. Liraglutide reduced FPG [ETD -0.2 (-0.4 to -0.1) mmol/L, p = .018], HbA1c [-2.2 (-3.5 to -0.8) mmol/mol, p = .018] and bodyweight [-3.9 (-6.2 to -1.6) kg, p = .012]. No change in the incretin effect was observed. The number of women with prediabetes was reduced from 64% to 10% with liraglutide vs. 50% with placebo [adjusted odds ratio 0.10 (0.03-0.32), p = .002]. CONCLUSIONS: Treatment with liraglutide for 52 weeks improved glucose tolerance, FPG, HbA1c and bodyweight in women with overweight/obesity and pGDM. Progression to prediabetes while on drug was markedly reduced, but after a 1-week drug wash-out, the effect was lost.

Preanalytical impact on the accuracy of measurements of glucagon, GLP-1 and GIP in clinical trials.

BACKGROUND: Plasma concentrations of glucagon, GLP-1 and GIP are reported in numerous clinical trials as outcome measures but preanalytical guidelines are lacking. We addressed the impact of commonly used blood containers in metabolic research on measurements of glucagon, GLP-1 and GIP in humans. METHODS: Seventeen overweight individuals were subjected to an overnight fast followed by an intravenous infusion of amino acids to stimulate hormonal secretion. Blood was sampled into five containers: EDTA-coated tubes supplemented with DMSO (control), a neprilysin inhibitor, aprotinin (a kallikrein inhibitor) or a DPP-4 inhibitor, and P800 tubes. Plasma was kept on ice before and after centrifugation and stored at -80 Celsius until batch analysis using validated sandwich ELISAs or radioimmunoassays (RIA). RESULTS: Measures of fasting plasma glucagon did not depend on sampling containers, whether measured by ELISA or RIA. Amino acid-induced hyperglucagonemia was numerically higher when blood was collected into P800 tubes or tubes with aprotinin. The use of p800 tubes resulted in higher concentrations of GLP-1 by RIA compared to control tubes but not for measurements with sandwich ELISA. Plasma concentrations of GIP measured by ELISA were higher in control tubes and negatively affected by P800 and the addition of aprotinin. CONCLUSIONS: The choice of blood containers impacts on measurements of plasma concentrations of glucagon, GLP-1 and GIP, and based on this study, we recommend using EDTA-coated tubes without protease inhibitors or P800 tubes for measurements of glucagon, GLP-1 and GIP in clinical trials.

Oral glucose has little or no effect on appetite and satiety sensations despite a significant gastrointestinal response.

OBJECTIVE: The effect of oral glucose-induced release of gastrointestinal hormones on satiety and appetite independently of prevailing plasma glucose excursions is unknown. The objective is to investigate the effect of oral glucose on appetite and satiety sensations as compared to isoglycemic IV glucose infusion (IIGI) in healthy volunteers. DESIGN: A crossover study involving two study days for each participant. PARTICIPANTS: Nineteen healthy participants (6 women, mean age 55.1 [SD 14.2] years; mean body mass index 26.7 [SD 2.2] kg/m2). INTERVENTIONS: Each participant underwent a 3-h 50-g oral glucose tolerance test (OGTT) and, on a subsequent study day, an IIGI mimicking the glucose excursions from the OGTT. On both study days, appetite and satiety were indicated regularly on visual analog scale (VAS), and blood was drawn regularly for measurement of pancreatic and gut hormones. PRIMARY OUTCOMES: Difference in appetite and satiety sensations during OGTT and IIGI. RESULTS: Circulating concentrations of glucose-dependent insulinotropic polypeptide (P < .0001), glucagon-like peptide 1 (P < .0001), insulin (P < .0001), C-peptide (P < .0001), and neurotensin (P = .003) increased significantly during the OGTT as compared to the IIGI, whereas glucagon responses were similarly suppressed (P = .991). Visual analog scale-assessed ratings of hunger, satiety, fullness, thirst, well-being, and nausea, respectively, were similar during OGTT and IIGI whether assessed as mean 0-3-h values or area under the curves. For both groups, a similar, slow increase in appetite and decrease in satiation were observed. Area under the curve, for prospective food consumption (P = .049) and overall appetite score (P = .044) were slightly lower during OGTT compared to IIGI, whereas mean 0-3-h values were statistically similar for prospective food consumption (P = .053) and overall appetite score (P = .063). CONCLUSIONS: Despite eliciting robust responses of appetite-reducing and/or satiety-promoting gut hormones, we found that oral glucose administration has little or no effect on appetite and satiety as compared to an IIGI, not affecting the release of appetite-modulating hormones. TRIAL REGISTRY NO: ClinicalTrials.gov: NCT01492283 and NCT06064084.

Neprilysin activity is increased in metabolic dysfunction-associated steatotic liver disease and normalizes after bariatric surgery or GLP-1 therapy.

Inhibitors of neprilysin improve glycemia in patients with heart failure and type 2 diabetes (T2D). The effect of weight loss by diet, surgery, or pharmacotherapy on neprilysin activity (NEPa) is unknown. We investigated circulating NEPa and neprilysin protein concentrations in obesity, T2D, metabolic dysfunction-associated steatotic liver disease (MASLD), and following bariatric surgery, or GLP-1-receptor-agonist therapy. NEPa, but not neprilysin protein, was enhanced in obesity, T2D, and MASLD. Notably, MASLD associated with NEPa independently of BMI and HbA1c. NEPa decreased after bariatric surgery with a concurrent increase in OGTT-stimulated GLP-1. Diet-induced weight loss did not affect NEPa, but individuals randomized to 52-week weight maintenance with liraglutide (1.2 mg/day) decreased NEPa, consistent with another study following 6-week liraglutide (3 mg/day). A 90-min GLP-1 infusion did not alter NEPa. Thus, MASLD may drive exaggerated NEPa, and lowered NEPa following bariatric surgery or liraglutide therapy may contribute to the reported improved cardiometabolic effects.