Abstract
Objective
Type 2 diabetes (T2D) pathophysiology includes fasting and postprandial hyperglucagonemia, which has been linked to hyperglycemia via increased endogenous glucose production (EGP). We used a glucagon receptor antagonist (LY2409021) and stable isotope tracer infusions to investigate the consequences of hyperglucagonemia in T2D.
Design
A double-blinded, randomized, placebo-controlled crossover study was conducted.
Methods
Ten patients with T2D and ten matched non-diabetic controls underwent two liquid mixed meal tests preceded by single-dose administration of LY2409021 (100 mg) or placebo. Double-tracer technique was used to quantify EGP. Antagonist selectivity toward related incretin receptors was determined in vitro.
Results
Compared to placebo, LY2409021 lowered the fasting plasma glucose (FPG) from 9.1 to 7.1 mmol/L in patients and from 5.6 to 5.0 mmol/L in controls (both P < 0.001) by mechanisms involving reduction of EGP. Postprandial plasma glucose excursions (baseline-subtracted area under the curve) were unaffected by LY2409021 in patients and increased in controls compared to placebo. Glucagon concentrations more than doubled during glucagon receptor antagonism. The antagonist interfered with both glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide receptors, complicating the interpretation of the postprandial data.
Conclusions
LY2409021 lowered FPG concentrations but did not improve postprandial glucose tolerance after a meal in patients with T2D and controls. The metabolic consequences of postprandial hyperglucagonemia are difficult to evaluate using LY2409021 because of its antagonizing effects on the incretin receptors.
Introduction
Type 2 diabetes (T2D) is a multifactorial disorder and involves among other pathophysiological mechanisms impaired insulin secretion in combination with relative hyperglucagonemia (1). Inappropriately, high glucagon concentrations are well-defined in T2D, and several glucose-lowering treatment strategies target hyperglucagonemia (2, 3). Hyperglucagonemia has been proposed to result from impaired glucose sensing and/or insulin resistance of the diabetic alpha cells combined with reduced insulin secretion (4). This has been met with rebuttal (5, 6) and postprandial hyperglucagonemia in T2D might reflect a gut-dependent phenomenon, either due to secretion of glucagonotropic factors from the gut (e.g. glucose-dependent insulinotropic polypeptide (GIP)) or secretion of glucagon from the gut, in agreement with recent findings in totally pancreatectomized patients (7, 8).
Hyperglucagonemia is thought to elicit increased endogenous glucose production (EGP) (9, 10, 11) and thus contributes to hyperglycemia in T2D (12, 13). Previous attempts to determine the role of hyperglucagonemia have involved infusions of glucagon and somatostatin clamps (12, 13). These experimental settings are not optimal due to potential extrapancreatic effects of somatostatin and because of significant differences between peripheral and portal concentrations of substituted hormones; most importantly insulin (14). Glucagon receptor (GCGR) antagonists, including LY2409021, lower the fasting plasma glucose (FPG) and glycated hemoglobin A1c (HbA1c) efficiently (15, 16, 17); however, the effect of LY2409021 on postprandial glucose excursions has not been thoroughly investigated.
We used the GCGR antagonist LY2409021 as a tool to elucidate the role of glucagon in fasting and postprandial glucose homeostasis in patients with T2D and non-diabetic controls during a mixed meal test (MMT) with the use of the double-glucose tracer technique to quantify glucose disposal and EGP. We hypothesized that LY2409021 would lower FPG concentrations and postprandial glucose excursions by mechanisms involving reduction in EGP.
Subjects and methods
Ethical approval
The study (clinical trial no. NCT02669524) was conducted in accordance with regulatory standards of good clinical practice and the Declaration of Helsinki, and all applicable local regulations. Written consent was obtained from each participant after full explanation of the purpose and nature of all procedures used. The study protocol was approved by the ethical board of The Capital Region of Denmark (H-15007312).
Participants
Ten patients with T2D and ten non-diabetic controls were included (Table 1). Inclusion criteria were Caucasians in the age range of 35–80 years with T2D or normal glucose tolerance defined according to the World Health Organization diagnostic criteria (18). Key exclusion criteria were glucose-lowering medication other than metformin, pregnancy, breastfeeding, inflammatory bowel disease, intestinal resections, family history of pancreatic cancers or diabetes (controls), nephropathy or abnormal blood biochemistry.
Clinical characteristics of the participants. Data are presented as mean ± s.d.
| Patients with T2D | Controls | |
|---|---|---|
| n (male/female) | 10 (5/5) | 10 (5/5) |
| Age (years) | 57.1 ± 12.9 | 57.4 ± 12.6 |
| BMI (kg/m2) | 33.0 ± 5.4 | 31.7 ± 4.2 |
| HbA1c (%) | 6.4 ± 0.6* | 5.3± 0.3* |
| HbA1c (mmol/mol) | 46.2 ± 6.1* | 33.9 ± 3.0* |
| Fasting plasma glucose (mmol/L) | 8.1 ± 1.0* | 5.6 ± 0.3* |
| Diabetes duration (years) | 5.4 ± 3.6 | – |
*Statistically significant difference between the groups (i.e. P < 0.05 tested with unpaired t-test and adjusted for false discovery rate).
HbA1c, glycosylated hemoglobin.
Glucagon receptor antagonist
LY2409021 (a gift from Eli Lilly and Company) is a selective and potent (inhibitory constant (Ki) of 6.66 nmol/L), orally administered, competitive, small-molecule GCGR antagonist that binds the human GCGR with a median time for maximum drug concentration (Tmax) of 4–8 h and a mean half-life (T½) of ~55 h (15, 16).
Primary outcome
The primary outcome was the difference in postprandial glucose excursions corrected for FPG (baseline-subtracted area under the curve (bsAUC)) between days with and without LY2409021.
Study design, randomization and blinding
This study was part of an investigator-initiated, randomized, double-blind, single-dose, crossover, placebo-controlled trial consisting of 6 experimental days: two MMTs (reported here), two oral glucose tolerance tests and two isoglycemic IV glucose infusions (8). The study was performed at Gentofte Hospital, University of Copenhagen, Hellerup, Denmark from October 2015 to July 2016. Participants and investigators were blinded for the treatment (LY2409021 or placebo) during study days and assessment of outcomes. The 2 experimental days reported here were separated by a wash-out period of minimum 2 weeks. Metformin was paused 1 week before each of the experimental days. Participants were instructed to take 100 mg of LY2409021 or placebo tablets 10 h before the beginning of experimental procedures to allow for maximal antagonizing effect during the MMT. On each experimental day, participants were met in the morning after 10 h fast; participants were placed in a recumbent position and a cannula was inserted in a cubital vein for infusion of stable isotopes and another in a contralateral cubital vein for collection of arterialized blood with the forearm wrapped in a heating pad at ~45°C. At −120 min, a primed constant infusion of the stable isotopes (Cambridge Isotope Laboratories, Tewksbury, MA, USA) dissolved in saline was started ([6,6-2H2] glucose (rate of 0.6 µmol kg−1 min−1 and priming dose of 17.5 µmol kg−1 × FPG/5) and [1,1,2,3,3-D5] glycerol (rate of 0.1 µmol kg−1 min−1 and priming dose of 2 µmol kg−1)). The standardized liquid MMT (47.2 g anhydrous glucose, 15.2 g whey protein powder and 14.1 g rapeseed oil (200 mL, 394 kcal, 50%E carbohydrates, 15%E protein and 35%E fat) mixed in 150 mL of water) was ingested at time 0 min. Acetaminophen (1.5 g) was dissolved in the meal for evaluation of acetaminophen absorption (proxy for gastric emptying rate (20, 21)), and 2.8 g [U-13C6] glucose was used for tracing of ingested glucose (22, 23). To conclude the experimental day, an pasta Bolognese meal was served ad libitum (energy content per 100 g: 147 kcal; 5.9 g fat; 17.4 g carbohydrates; 5.6 g protein), and participants were instructed to eat until pleasantly satiated.
In vivo data collection and sample procedures
Blood was collected and immediately centrifuged (30 s, 7500 g, room temperature) before bedside analysis of plasma glucose (PG) using the glucose oxidase method (YSI Model 2300 Stat Plus and 2900 Biochemistry Analyzers, Yellow Springs, OH, USA). For measurements of glucagon, GIP, GLP-1 and isotope enrichment, blood was collected in pre-chilled EDTA tubes containing dipeptidyl peptidase 4 inhibitor (valine pyrrolidide, 0.01 mmol/L) to preserve the hormone levels. Plasma glucagon concentrations were measured by an in-house RIA (antibody 4305) directed against the C-terminal (24), total GIP with antiserum 867 (25) and total GLP-1 with antiserum 89390 (26) directed against the C-terminal. Plasma enrichment of [U-13C6] glucose, [6,6-2H2] glucose and [1,1,2,3,3-D5] glycerol and corresponding PG and glycerol concentrations were measured by liquid chromatography-tandem mass spectrometry (27). Plasma acetaminophen (lithium heparin tubes) was measured by reflectance photometry at 670 nm (Vitros 5.1 FS, Ortho-Clinical Diagnostics, Neckargemünd, Germany). Serum C-peptide/insulin (dry tubes with silica particles) concentrations were measured with a two-site sandwich immunoassays using direct chemiluminescent technology (ADVIA Centaur XP, Siemens Healthcare A/S). All tubes were centrifuged for 15 min (2900g at 4°C). Visual analog scales (VAS) were marked by the participants for evaluation of appetite sensations, wellbeing and nausea (28), and for evaluation of palatability of the ad libitum meal. Energy intake during the ad libitum meal was calculated by weighing the served meal subtracting leftovers.
In vitro functionality and selectivity of LY2409021
The ability of LY2409021 to inhibit GCGR signaling was determined in COS-7 cells transiently transfected with the human GCGR (29). The selectivity of LY2409021 to the GLP-1 receptor (GLP1R) and the GIP receptor (GIPR) was evaluated in cells expressing these receptors (29). The COS-7 cells were cultured at 10% CO2 and 37°C in Dulbecco’s modified Eagles medium 1885 supplemented with 10% FBS, 2 mmol/L glutamine, 180 U/mL penicillin and 45 g/mL streptomycin. Transient transfection was performed using the calcium phosphate precipitation method (30). The transfected cells were seeded in 96-well plates 1 day after transfection (35 000 cells/well), washed twice with HEPES-buffered saline (HBS) after 2 days and incubated with HBS and 1 mmol/L 3-isobutyl-1-methylxanthine for 30 min at 37°C. Increasing concentrations of LY2409021 were added to the cells expressing one of the three receptors (GCGR, GLP1R or GIPR) in the absence or presence of a constant concentration of its cognate endogenous agonist (glucagon, GLP-1 or GIP, respectively) corresponding to 50–80% of their maximum effect (Emax), determined from dose–response curves for each of the endogenous agonists. After ligand addition and 20 min incubation, measurements of cAMP were performed using the HitHunterTM cAMP XS assay (DiscoveRx, Herlev, Denmark) according to the manufacturer’s instructions.
Statistical analysis
Summary statistics are shown as mean with lower and upper 95% CI limits except for log-normally distributed data which are geometric means. Outcomes that were not normally distributed are displayed as median and interquartile ranges and transformed for analyses (31). The power of the study (1-β) was set to 80%, where β (20%) is the risk of accepting a null hypothesis that is false, with a significance level (α) = 5%. The calculation was performed for all 6 experimental days together and was based on a previous study on the incretin effect of 16 patients with T2D with a 4.2% s.e.m. of gastrointestinal-mediated glucose disposal (32). We defined a minimal relevant difference of 20% as reasonable for our protocol, and the calculated number of participants needed in each group was six. Since data on bsAUC after MMT during GCGR antagonism were limited, we pre-defined a needed number of ten participants in each group to ensure adequate power and to avoid type 2 errors. Area under the curve (AUC) was calculated using the trapezoidal rule. In vitro pharmacological evaluation was carried out with GraphPad Prism 8 (GraphPad Software). Statistical comparisons were performed with SAS Enterprise Guide 7.1 (SAS Institute, Cary, NC, US) with a linear mixed model including experimental day (treatment), group and the interaction between them as fixed effects and with an unstructured covariance pattern to account for correlation between repeated measurements on the same individuals (31). To avoid false positives due to multiple testing, all P values were adjusted using the method of Benjamini and Hochberg which controls the false discovery rate (33), that is an adjusted (adj.) P value ≤0.05 means that the reported significance is ≤5%, likely to be a false positive. P values for within group comparisons and between group comparisons were adjusted separately. Insulin secretion rate (ISR) was calculated by deconvolution of C-peptide and C-peptide kinetics (34, 35). Isotope tracer data are displayed as rate of appearance (Ra) and rate of disappearance (Rd) calculated from changes in enrichment using Steele’s one-compartment, fixed-volume, non-steady-state model for use with stable isotopes and a fixed pool fraction of 70 mL kg−1 (36, 37). Metabolic clearance rate (MCR) was calculated as Rd/glucose concentration. For VAS (resulting in values from 1 to 100 for each of the four parameters recorded) a composite appetite score (CAS) was calculated, as ((hunger + prospective food consumption + (100 – satiety) + (100 – fullness)) / 4).
Results
Participant baseline characteristics
Eleven patients with T2D and 12 controls were randomized, of whom 10 in each group completed both study days (Table 1). Discontinuations were due to participants’ decisions (adverse events (general discomfort and mild headache, not drug-related), n = 1, and personal reasons, n = 1) and one investigator-based exclusion due to prediabetic glucose concentrations during the oral glucose tolerance test in a control individual (n = 1). Clinical characteristics were statistically comparable between groups (Table 1).
Glucose concentration and kinetics
Following a single dose of LY2409021 (100 mg), the median FPG was reduced in patients with T2D and controls (Fig. 1A and Table 2). Likewise, Ra in the fasted state (equals EGP) was lowered by LY2409021 compared to placebo in patients (P < 0.001, adj. P = 0.010), but not significantly in controls after adjusting for multiple comparisons (P = 0.032, adj. P = 0.114) (Fig. 1B). Noteworthy, EGP in the fasted state was reduced with LY2409021 in patients with T2D to levels not different from those of controls (P = 0.573) (Fig. 1E). Postprandially, AUC of glucose was not lower with LY2409021 compared to placebo in controls and after adjusting for multiple comparisons in patients with T2D (P = 0.023, adj. P = 0.100) (Table 2). However, when AUC was corrected for the FPG (bsAUC), it was increased by LY2409021 in controls and remained unchanged in patients with T2D (Fig. 1A, J and Table 2). LY2409021 did not affect the increase from fasting to peak PG (Δ glucose), peak PG (Table 2) or postprandial oral glucose Ra (Fig. 1D and M). Meal intake reduced EGP compared to the fasting EGP in all participants, but the reduction in EGP (bsAUC) was greater with placebo compared to LY2409021 in patients with T2D (Fig. 1E and N). However, compared to placebo, the EGP during the 4-hour MMT (AUC) was substantially lower with LY2409021 in patients with T2D (mean (lower; upper 95% CI): 178 (146; 211) mmol vs 133 (116; 149) mmol, P = 0.004, adj. P = 0.030) and controls (132 (105; 159) mmol vs 116 (92; 140) mmol, P = 0.001, adj. P = 0.010). Glucose Rd during the MMT was lower with LY2409021 compared to placebo in controls (P < 0.001, adj. P = 0.003) and less convincingly in patients (P = 0.017, adj. P = 0.073). MCR of glucose in the fasted state was not affected by LY2409021 compared to placebo in patients with T2D (1.74 (1.49; 1.98) (mL kg−1 min−1) vs 1.73 (1.50; 1.97) (mL kg−1 min−1), P = 0.98) nor in controls (2.25 (2.07; 2.42) vs 2.21 (1.96; 2.46), P = 0.63) (Fig. 1I). Postprandially, MCR was unchanged in patients (30.7 (27; 34) vs 30.9 (26; 35) (mL kg−1), P = 0.98), but LY2409021 lowered the AUC of MCR compared to placebo in controls (47.9 (41; 5)] vs 56.5 (50; 63), P = 0.004 (adj. P = 0.03)) (Fig. 1I). Glucose excretion in urine was not affected by LY2409021 compared to placebo in patients (0.5 (−0.1; 1.1) g vs 2.2 (−0.5; 4.8) g glucose, P = 0.091) nor in controls (0.02 (0.01; 0.03) g vs 0.02 (0.01; 0.02) g glucose, P = 0.915).
Plasma glucose and glycerol concentrations and kinetics with baseline-subtracted AUC. Plasma glucose (A) and glycerol (F) concentrations after a standardized liquid mixed meal, with corresponding rate of appearances (Ra) (B and G), rate of disappearances (Rd) (C and H) and baseline-subtracted area under the curve (bsAUC) (J, K and L) for plasma glucose and glycerol in patients with T2D (red) and non-diabetic controls (blue). Ra and Rd were adjusted for the participants’ body weight (K, L, M and N). Metabolic clearance rate (MCR) calculated by glucose Rd divided by the mass effect of the glucose gradient (glucose concentration) (I). Measurements were performed by liquid chromatography-tandem mass spectrometry. Symbols/columns are mean values ± s.e.m. Glucagon receptor antagonist (LY2409021) (closed symbols/full-color columns) and placebo (open symbols/striped columns). P values were calculated using linear mixed models and have been corrected for multiple comparisons by false discovery rate. *P ≤ 0.05, **P ≤ 0.01.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
Plasma glucose and glycerol concentrations and kinetics with baseline-subtracted AUC. Plasma glucose (A) and glycerol (F) concentrations after a standardized liquid mixed meal, with corresponding rate of appearances (Ra) (B and G), rate of disappearances (Rd) (C and H) and baseline-subtracted area under the curve (bsAUC) (J, K and L) for plasma glucose and glycerol in patients with T2D (red) and non-diabetic controls (blue). Ra and Rd were adjusted for the participants’ body weight (K, L, M and N). Metabolic clearance rate (MCR) calculated by glucose Rd divided by the mass effect of the glucose gradient (glucose concentration) (I). Measurements were performed by liquid chromatography-tandem mass spectrometry. Symbols/columns are mean values ± s.e.m. Glucagon receptor antagonist (LY2409021) (closed symbols/full-color columns) and placebo (open symbols/striped columns). P values were calculated using linear mixed models and have been corrected for multiple comparisons by false discovery rate. *P ≤ 0.05, **P ≤ 0.01.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
Plasma glucose and glycerol concentrations and kinetics with baseline-subtracted AUC. Plasma glucose (A) and glycerol (F) concentrations after a standardized liquid mixed meal, with corresponding rate of appearances (Ra) (B and G), rate of disappearances (Rd) (C and H) and baseline-subtracted area under the curve (bsAUC) (J, K and L) for plasma glucose and glycerol in patients with T2D (red) and non-diabetic controls (blue). Ra and Rd were adjusted for the participants’ body weight (K, L, M and N). Metabolic clearance rate (MCR) calculated by glucose Rd divided by the mass effect of the glucose gradient (glucose concentration) (I). Measurements were performed by liquid chromatography-tandem mass spectrometry. Symbols/columns are mean values ± s.e.m. Glucagon receptor antagonist (LY2409021) (closed symbols/full-color columns) and placebo (open symbols/striped columns). P values were calculated using linear mixed models and have been corrected for multiple comparisons by false discovery rate. *P ≤ 0.05, **P ≤ 0.01.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
Fasting plasma glucose and postprandial AUCs of glucose, hormones and acetaminophen pharmacokinetics. Estimates for placebo are means with lower and upper 95% CI limits for normally distributed data, except from geometric means for log-normally distributed data. Similarly, effect estimates for LY2409021 are mean difference and median relative difference compared to placebo and estimates for T2D vs control are difference in effect and relative difference in effect. P values were adjusted for multiple testing to control the false discovery rate.
| Patients with T2D | Controls | Effect of LY2409021 for patients with T2D vs controls | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Placebo, n = 10 | LY2409021, n = 10 | Placebo, n = 10 | LY2409021, n = 10 | ||||||||
| LSM estimate (95% CI) |
LSM Diff estimate (95% CI) | P value | adj. P value | LSM estimate (95% CI) |
LSM Diff estimate (95% CI) | P value | adj. P value | LSM Diff estimate (95% CI) | P value | adj. P value | |
| Glucose | |||||||||||
| Fasting (mmol/L)* Max peak (mmol/L) Δ (max–min) (mmol/L) AUC (mmol/L min) bsAUC (mmol/L min) |
9.1 (7.6; 10.9)* 15.3 (12.7; 17.9) 5.92 (4.84; 7.00) 2873 (2369; 3378) 625 (476; 774) |
−1.3* (−1.2; −1.5)* −2.3 (−4.4; −0.2) 0.17 (−0.90; 1.25) −491 (−898; −85) 99 (−83; 280) |
<0.001 0.038 0.726 0.023 0.249 |
0.010 0.132 0.902 0.096 0.495 |
5.5 (5.4; 5.6) 9.0 (8.5; 9.6) 3.53 (2.98; 4.09) 1494 (1444; 1544) 177 (112; 242) |
−1.10* (−1.06; −1.13)* −0.1 (−1.0; 0.8) 0.32 (−0.46; 1.25) 76 (−66; 218) 193 (61; 325) |
<0.001 0.823 0.321 0.260 0.009 |
0.010 0.914 0.565 0.500 0.044 |
−1.2* (−1.1; 1.4)* −2.2 (−4.4; 0.03) −0.22 (−1.50; 1.06) −567 (−985; −149) −95 (−304; 115) |
0.007 0.053 0.717 0.012 0.354 |
0.306 0.541 0.923 0.306 0.741 |
| C−peptide | |||||||||||
| Fasting (pmol/L) AUC (nmol/L min) bsAUC (nmol/L min) |
1025 (763; 1288) 518 (390; 646) 272 (189; 355) |
−253 (−410; −96) −4.2 (−97; 89) 56 (−12; 125) |
0.005 0.920 0.096 |
0.032 0.966 0.292 |
641 (482; 799) 425 (340; 510) 271 (206; 336) |
−148 (−244; −52) 4.3 (−38; 46) 40 (0.8; 79) |
0.007 0.822 0.046 |
0.038 0.914 0.153 |
−105 (−278; 69) −8.6 (−106; 89) 17 (−58; 91) |
0.218 0.853 0.642 |
0.619 0.923 0.923 |
| C−peptide:glucose | |||||||||||
| Fasting (pmol/mmol) AUC (nmol/mmol min) bsAUC (nmol/mmol min) |
115 (69; 213) 45 (21; 84) 18 (4.9; 36) |
−2.8 (−27; 21) 5.6 (−2.1; 13) 6.3 (1.7; 11) |
0.795 0.135 0.013 |
0.914 0.365 0.059 |
117 (75; 212) 66 (39; 97) 38 (18; 65) |
−18 (−34; 1.9) −3.8 (−10; 2.4) 0.5 (−5.4; 6.4) |
0.032 0.198 0.861 |
0.114 0.459 0.931 |
15 (−12; 42) 9.4 (0.2; 19) 5.8 (−1.2; 13) |
0.255 0.046 0.098 |
0.619 0.541 0.553 |
| Insulin | |||||||||||
| Fasting (pmol/L) AUC (nmol/L min) bsAUC (nmol/L min) |
121 (91; 152) 92 (59; 125) 63 (36; 90) |
−39 (−62; −17) −18 (−47; 11) −8.7 (−34; 16) |
0.004 0.188 0.447 |
0.030 0.443 0.709 |
82 (50; 113) 87 (58; 116) 67 (43; 92) |
−22 (−45; 0.2) −12 (−32; 8.3) −6.7 (−24; 11) |
0.052 0.214 0.412 |
0.169 0.473 0.684 |
−17 (−47; 13) −6.2 (−39; 27) −2.1 (−31; 26) |
0.242 0.697 0.880 |
0.619 0.923 0.923 |
| Insulin:glucose | |||||||||||
| Fasting (pmol/mmol) AUC (nmol/mmol min) bsAUC (nmol/L min) |
13.6 (9.2; 18.1) 7.7 (4.1; 11.4) 4.5 (1.8; 7.2) |
−1.8 (−5.6; 1.9) −0.6 (−3.0; 1.7) −0.2 (−1.9; 1.5) |
0.300 0.547 0.794 |
0.545 0.783 0.914 |
14.9 (9.0; 20.8) 11.8 (8.2; 15.5) 8.3 (5.6; 10.9) |
−3.0 (−7.0; 1.0) −0.8 (−3.9; 2.4) −0.04 (−2.7; 2.6) |
0.128 0.605 0.977 |
0.365 0.914 0.991 |
1.1 (−4.0; 6.2) 0.1 (−3.6; 3.8) −0.2 (−3.2; 2.8) |
0.649 0.958 0.905 |
0.923 0.958 0.923 |
| Insulin secretion rate | |||||||||||
| Fasting (pmol kg−1 min−1) AUC (pmol kg−1 min−1) min bsAUC (pmol kg−1 min−1) min |
2.8 (2.3; 3.4) 1444 (1102; 1786) 769 (490; 1048) |
−0.7 (−1.1; −0.3) −5.7 (−272; 261) 170 (−68; 407) |
0.002 0.962 0.140 |
0.018 0.991 0.365 |
1.8 (1.3; 2.2) 1096 (845; 1347) 673 (492; 854) |
−0.4 (−0.7; −0.1) −29 (−181; 122) 66 (−67; 200) |
0.009 0.670 0.292 |
0.044 0.858 0.545 |
−0.3 (−0.8; 0.1) 24 (−266; 314) 104 (−154; 362) |
0.130 0.864 0.403 |
0.553 0.923 0.791 |
| Insulin secretion rate:glucose | |||||||||||
| Fasting ((pmol kg−1 min−1) / (mmol L−1)) AUC ((pmol kg−1 min−1) / (mmol L−1)) min bsAUC ((pmol kg−1 min−1) / (mmol L−1)) min |
0.31 (0.24; 0.39) 123 (86; 161) 48 (22; 74) |
−0.01 (−0.08; 0.05) 12 (−9; 33) 15 (−3.1; 34) |
0.659 0.235 0.094 |
0.852 0.494 0.292 |
0.32 (0.24; 0.41) 170 (131; 209) 93 (67; 118) |
−0.05 (−0.09; −0.002) −11 (−34; 11) −0.04 (−20; 20) |
0.041 0.280 0.997 |
0.139 0.531 0.997 |
0.03 (−0.04; 0.11) 23 (−5.3; 52) 15 (−10; 41) |
0.363 0.104 0.223 |
0.741 0.553 0.619 |
| GIP | |||||||||||
| Fasting (pmol/L) AUC (pmol/L min) bsAUC (pmol/L min) |
7.6 (4.4; 10.8) 9627 (8257; 10997) 7795 (6569; 9021) |
2.2 (−2.3; 6.6) 716 (251; 1181) 196 (−1128; 1520) |
0.297 0.007 0.745 |
0.545 0.038 0.914 |
5.0 (3.1; 6.9) 9078 (7761; 10395) 7878 (6632; 9125) |
3.9 (1.4; 6.5) 825 (−298; 1948) −119 (−1294; 1056) |
0.007 0.131 0.824 |
0.038 0.365 0.914 |
−1.8 (−6.6; 3.1) −109 (−1280; 1062) 315 (−1331; 1961) |
0.446 0.843 0.692 |
0.842 0.923 0.923 |
| GLP−1 | |||||||||||
| Fasting (pmol/L) AUC (pmol/L min) bsAUC (pmol/L min) |
12.9 (10.0; 15.8) 4514 (3540; 5488) 1426 (772; 2081) |
5.4 (2.1; 8.7) 907 (597; 1217) −381 (−1176; 413) |
0.005 <0.001 0.306 |
0.032 0.010 0.545 |
9.8 (6.1; 13.5) 3626 (2852; 4401) 1282 (433; 2131) |
4.9 (0.5; 9.2) 423 (−148; 995) −745 (−1851; 361) |
0.032 0.128 0.162 |
0.114 0.365 0.406 |
0.5 (−4.6; 5.6) 484 (−134; 1101) 364 (−911; 1638) |
0.838 0.115 0.554 |
0.923 0.553 0.923 |
| Glucagon | |||||||||||
| Fasting (pmol/L)* AUC (pmol/L min)* bsAUC (pmol/L min) |
6.4 (2.8; 8.2) 1987 (909; 4344) 582 (177; 986) |
3.0* (2.2; 4.2)* 2.7* (1.9; 3.9)* −133 (−652; 386) |
<0.001 <0.001 0.576 |
0.010 0.010 0.809 |
3.7 (1.6; 8.2) 988 (549; 1777) −88 (−620; 443) |
5.0* (2.5; 9.9)* 3.5* (2.9; 4.2)* −746 (−1995; 502) |
<0.001 <0.001 0.209 |
0.010 0.010 0.472 |
−1.7* (−3.4; 1.2)* −1.3* (−1.9; 1.2)* 613 (−689; 1915) |
0.156 0.195 0.325 |
0.612 0.619 0.721 |
| Acetaminophen | |||||||||||
| Max peak (μmol/L) Tmax (min) AUC (mmol/L min) |
99 (81; 117) 117 (101; 133) 15.5 (13.0; 18.1) |
−2.4 (−14; 8.8) 8 (−25; 41) 0.5 (−0.9; 1.9) |
0.640 0.600 0.465 |
0.849 0.826 0.730 |
91 (73; 108) 115 (81; 149) 14.8 (11.9; 17.7) |
0.05 (−8; 8) 11 (−19; 41) 0.6 (−0.7; 1.9) |
0.990 0.430 0.330 |
0.997 0.698 0.574 |
−2.4 (−15; 11) −3.0 (−45; 37) −0.1 (−1.9; 1.7) |
0.695 0.881 0.894 |
0.923 0.923 0.923 |
P values <0.05 are marked in bold.
*Log-normally distributed data.
adj., adjusted P values; AUC, area under the curve; bsAUC, baseline-subtracted AUC; GIP, glucose-dependent insulinotropic polypeptide; T2D, type 2 diabetes; Tmax, time to reach maximum (peak) plasma concentration.
Glucagon, insulin, C-peptide, ISR, GIP and GLP-1
Glucagon concentrations more than doubled with LY2409021 during fasting conditions and postprandially (AUC) in patients with T2D and in controls (Fig. 2A and Table 2). Corrected for fasting concentrations (bsAUC), postprandial glucagon concentrations were not affected by LY2409021 (Table 2). Fasting insulin, C-peptide and ISR were lower with LY2409021 compared to placebo in patients and controls (insulin not significant in controls). However, when the insulin:glucose, C-peptide:glucose and ISR:glucose ratios were calculated, no differences between placebo and LY2409021 were evident in the fasted state (Table 2). Postprandial insulin, and C-peptide and ISR responses (AUC and bsAUC) were not altered by LY2409021 (Fig. 2E, G, Fand Table 2). Fasting GIP concentrations tended to be increased with LY2409021 compared to placebo (significantly in controls) (Table 2) and the postprandial GIP response (AUC) was statistically higher in patients with LY2409021 but not in controls (Fig. 2C and Table 2). Compared to placebo, LY2409021 increased plasma GLP-1 concentrations in the fasted state as well as postprandially (AUC) in patients (Fig. 2B and Table 2); and similar (insignificant) tendencies were seen for controls (Fig. 2B and Table 2).
Circulating glucagon, GLP-1, GIP, acetaminophen, C-peptide, insulin and insulin secretion rate. Circulating mean concentrations of glucagon (A), glucagon-like peptide 1 (GLP-1) (B), glucose-dependent insulinotropic polypeptide (GIP) (C), acetaminophen (gastric emptying) (D), C-peptide (E), insulin secretion rate calculated by ISEC (F) and insulin (G). Symbols are mean values ± s.e.m. Days with glucagon receptor antagonism (LY2409021) are illustrated with closed circles and placebo with open squares. T2D patients are marked in red and non-diabetic controls in blue.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
Circulating glucagon, GLP-1, GIP, acetaminophen, C-peptide, insulin and insulin secretion rate. Circulating mean concentrations of glucagon (A), glucagon-like peptide 1 (GLP-1) (B), glucose-dependent insulinotropic polypeptide (GIP) (C), acetaminophen (gastric emptying) (D), C-peptide (E), insulin secretion rate calculated by ISEC (F) and insulin (G). Symbols are mean values ± s.e.m. Days with glucagon receptor antagonism (LY2409021) are illustrated with closed circles and placebo with open squares. T2D patients are marked in red and non-diabetic controls in blue.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
Circulating glucagon, GLP-1, GIP, acetaminophen, C-peptide, insulin and insulin secretion rate. Circulating mean concentrations of glucagon (A), glucagon-like peptide 1 (GLP-1) (B), glucose-dependent insulinotropic polypeptide (GIP) (C), acetaminophen (gastric emptying) (D), C-peptide (E), insulin secretion rate calculated by ISEC (F) and insulin (G). Symbols are mean values ± s.e.m. Days with glucagon receptor antagonism (LY2409021) are illustrated with closed circles and placebo with open squares. T2D patients are marked in red and non-diabetic controls in blue.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
Glycerol concentrations and kinetics
Compared to placebo, LY2409021 did not affect fasting plasma glycerol, fasting glycerol Ra (whole body lipolytic rate) and fasting glycerol Rd (glycerol clearance from the circulation) in any of the groups (Fig. 1F, G and H). Likewise, LY2409021 did not affect postprandial plasma glycerol (bsAUC) or glycerol Ra and Rd (Fig. 1F, G, H, J, K and L).
Gastric emptying, appetite and food intake
Compared to placebo, LY2409021 did not affect gastric emptying rate assessed by the acetaminophen absorption test (Fig. 2E and Table 2), underlined by similar oral glucose Ra (Fig. 1D and M). LY2409021 did not affect the fasting appetite measures nor the postprandial VAS scores in patients with T2D (Fig. 3 and Table 3), but mean VAS scores of satiety and fullness during the MMT were lower (feeling less satiated and less full) and CAS were higher with LY2409021 in controls compared to placebo. However, the effect of LY2409021 was not significantly different in patients with T2D compared to controls (Table 3), and LY2409021 did not affect ad libitum food intake or the palatability of the meal compared to placebo in patients with T2D nor in controls (Table 4).
Visual analogue scale questionnaires of appetite, well-being and nausea. Visual analog scale questionnaires of appetite (hunger (100 mm = never been more hungry), satiety (100 mm = I cannot eat another bite), fullness (100 mm = totally full), prospective food consumption (PFC) (100 mm = I think I can eat a lot), wellbeing (100 mm = feel really good), nausea (100 mm = high level of nausea) and thirst (100 mm = feel very thirsty)). Composite appetite score is calculated from (hunger + PFC + (100 −satiety) + (100 − fullness))/4. Symbols are mean values ± s.e.m. Days with glucagon receptor antagonism (LY2409021) are illustrated with closed circles and placebo with open squares. T2D patients are marked in red and non-diabetic controls in blue.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
Visual analogue scale questionnaires of appetite, well-being and nausea. Visual analog scale questionnaires of appetite (hunger (100 mm = never been more hungry), satiety (100 mm = I cannot eat another bite), fullness (100 mm = totally full), prospective food consumption (PFC) (100 mm = I think I can eat a lot), wellbeing (100 mm = feel really good), nausea (100 mm = high level of nausea) and thirst (100 mm = feel very thirsty)). Composite appetite score is calculated from (hunger + PFC + (100 −satiety) + (100 − fullness))/4. Symbols are mean values ± s.e.m. Days with glucagon receptor antagonism (LY2409021) are illustrated with closed circles and placebo with open squares. T2D patients are marked in red and non-diabetic controls in blue.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
Visual analogue scale questionnaires of appetite, well-being and nausea. Visual analog scale questionnaires of appetite (hunger (100 mm = never been more hungry), satiety (100 mm = I cannot eat another bite), fullness (100 mm = totally full), prospective food consumption (PFC) (100 mm = I think I can eat a lot), wellbeing (100 mm = feel really good), nausea (100 mm = high level of nausea) and thirst (100 mm = feel very thirsty)). Composite appetite score is calculated from (hunger + PFC + (100 −satiety) + (100 − fullness))/4. Symbols are mean values ± s.e.m. Days with glucagon receptor antagonism (LY2409021) are illustrated with closed circles and placebo with open squares. T2D patients are marked in red and non-diabetic controls in blue.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
Visual analogue scale (VAS) measures of appetite, comfort, nausea and thirst. VAS questionnaires of appetite, comfort, nausea, and thirst performed on a 100-mm long line. Mean is the average of VAS scores at times 30, 60, 90, 120, 150, 180 and 240 min. Composite appetite score (CAS) is calculated from (hunger + prospective food consumption + (100 − satiety) + (100 − fullness))/4. Values are means with lower and upper 95% CI. Effect estimates for LY2409021 are mean difference compared to placebo and estimates for T2D vs controls are difference in effect. P values were adjusted (adj.) for multiple testing to control the false discovery rate.
| Patients with T2D | Controls | Effect of LY2409021 for patients with T2D vs controls | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Placebo | LY2409021 | Placebo | LY2409021 | ||||||||
| LSM estimate (95% CI) |
LSM Diff estimate (95% CI) | P value | adj. P value | LSM estimate (95% CI) |
LSM Diff estimate (95% CI) | P value | adj. P value | LSM Diff estimate (95% CI) | P value | adj. P value | |
| Hunger (100 = never been hungrier) | |||||||||||
| Baseline Mean |
47 (26; 68) 49 (31; 66) |
−6.1 (−23; 11) −5.5 (−17; 5.9) |
0.45 0.30 |
0.71 0.55 |
58 (46; 69) 57 (51; 63) |
−7.6 (−19; 3.5) −1.0 (−10; 8.1) |
0.16 0.82 |
0.40 0.91 |
1.5 (−18; 21) −4.6 (−18; 9.0) |
0.87 0.49 |
0.92 0.89 |
| Satiety (100 = cannot eat another bite) | |||||||||||
| Baseline Mean |
46 (31; 62) 48 (32; 64) |
2.9 (−9.6; 15) −0.9 (−8.6; 6.9) |
0.61 0.80 |
0.84 0.91 |
44 (35; 53) 41 (35; 46) |
−5.6 (−16; 4.7) −5.1 (−8.5; −1.7) |
0.25 0.01 |
0.50 0.04 |
8.5 (−6.6; 24) 4.2 (−3.9; 12) |
0.25 0.28 |
0.62 0.65 |
| Fullness (100 = totally full) | |||||||||||
| Baseline Mean |
28 (15; 41) 38 (23; 52) |
9.4 (−6.3; 25) 1.9 (−8.4; 12) |
0.21 0.69 |
0.47 0.87 |
46 (31; 60) 40 (33; 47) |
−7.1 (−20; 5.6) −6.0 (−9.9; −2.1) |
0.24 0.01 |
0.49 0.04 |
17 (−2.4; 35) 7.9 (−2.8; 19) |
0.08 0.13 |
0.55 0.55 |
| Prospective food consumption (100 = can eat a lot) | |||||||||||
| Baseline Mean |
51 (35; 67) 50 (33; 67) |
−0.8 (−7.4; 5.7) −1.0 (−6.3; 4.4) |
0.76 0.69 |
0.91 0.75 |
55 (43; 67) 57 (51; 63) |
7.0 (−5.3; 19) 7.4 (1.1; 14) |
0.23 0.03 |
0.49 0.11 |
−7.8 (−21; 5.4) −8.4 (−16; −0.7) |
0.22 0.04 |
0.62 0.54 |
| Comfort (100 = very comfortable) | |||||||||||
| Baseline Mean |
88 (75; 100) 87 (75; 100) |
1.3 (−10; 12) 3.9 (−7.5; 15) |
0.80 0.46 |
0.91 0.72 |
79 (63; 94) 76 (62; 90) |
0.3 (−4.4; 5.0) 5.6 (−2.7; 14) |
0.90 0.16 |
0.91 0.41 |
1.0 (−10; 13) −1.7 (−15; 12) |
0.85 0.79 |
0.92 0.92 |
| Nausea (100 = high level of nausea) | |||||||||||
| Baseline Mean |
99 (97; 100) 98 (96; 100) |
0.6 (−0.8; 2.0) −1.3 (−4.9; 2.2) |
0.37 0.42 |
0.63 0.69 |
96 (90; 100) 96 (90; 100) |
0.4 (−2.5; 3.3) −0.7 (−2.8; 1.5) |
0.76 0.51 |
0.91 0.75 |
0.2 (−2.9; 3.3) −0.7 (−4.6; 3.3) |
0.89 0.72 |
0.92 0.92 |
| Thirst (100 = very thirsty) | |||||||||||
| Baseline Mean |
55 (37; 74) 52 (34; 70) |
0.3 (−8.9; 9.5) 4.0 (−3.2; 11) |
0.94 0.24 |
0.98 0.49 |
54 (37; 71) 58 (42; 73) |
−2.2 (−13; 8.4) 5.9 (−2.3; 14) |
0.65 0.14 |
0.85 0.37 |
2.5 (−11; 16) −1.9 (−12; 8.2) |
0.69 0.70 |
0.92 0.92 |
| Composite Appetite Score (CAS) (100 = high appetite) | |||||||||||
| Baseline Mean |
56 (42; 70) 53 (39; 68) |
−5.1 (−14; 4.3) −2.4 (−10; 5.2) |
0.25 0.50 |
0.50 0.75 |
56 (46; 65) 58 (54; 63) |
3.0 (−6.4; 12) 4.4 (1.8; 6.9) |
0.49 <0.01 |
0.75 0.03 |
−8.1 (−20; 4.3) −6.7 (−14; 1.0) |
0.19 0.08 |
0.62 0.55 |
P values <0.05 are marked in bold.
T2D, type 2 diabetes.
Energy consumed and palatability of the ad libitum meal. Energy intake of the ad libitum meal (pasta Bolognese) measured as g consumed multiplied by energy content (616 kJ/100 g or 147 kcal/100 g). Visual analog scale questionnaires of overall palatability, appearance, smell, taste and after taste performed on a line 100 mm long. Values are means with lower and upper 95% CI limits. Effect estimates for LY2409021 are mean difference compared to placebo. P values were adjusted (adj.) for multiple testing to control the false discovery rate.
| Patients with T2D | Controls | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Placebo | LY2409021 | Placebo | LY2409021 | ||||||
| LSM estimate (95% CI) |
LSM Diff estimate (95% CI) | P value | adj. P value | LSM estimate (95% CI) |
LSM Diff estimate (95% CI) | P value | adj. P value | ||
| Energy consumed ad libitum meal | |||||||||
| Grams kJ kcal |
331 (209; 452) 486 (308; 665) 2,038 (1,290; 2,787) |
6.4 (−57; 70) 9.4 (−84; 102) 39 (−350; 429) |
0.82 0.82 0.82 |
0.91 0.91 0.91 |
505 (373; 637) 742 (548; 936) 3,110 (2,296; 3,923) |
−6.2 (−105; 93) −9.1 (−155; 136) −38 (−648; 571) |
0.89 0.89 0.89 |
0.96 0.96 0.96 |
|
| Overall palatability (100 = unappetizing) | |||||||||
| 45 (28; 62) | −1.3 (−17; 15) | 0.86 | 0.93 | 45 (33; 58) | 0.1 (−5.4; 5.6) | 0.96 | 0.99 | ||
| Appearance (100 = unappealing) | |||||||||
| 44 (28; 60) | −3.1 (−18; 12) | 0.65 | 0.85) | 43 (30; 55) | 7.4 (−0.9; 16) | 0.08 | 0.24 | ||
| Smell (100 = unappetizing) | |||||||||
| 40 (23; 56) | −5.5 (−18; 7.1) | 0.35 | 0.60 | 49 (36; 61) | 2.4 (−1.4; 6.2) | 0.19 | 0.44 | ||
| Taste (100 = bad) | |||||||||
| 41 (26; 56) | −2.9 (−21; 15) | 0.73 | 0.90 | 47 (36; 59) | −1.7 (−7.1; 3.7) | 0.50 | 0.74 | ||
| Aftertaste (100 = no aftertaste) | |||||||||
| 87 (77; 97) | 2.6 (−2.1; 7.3) | 0.54 | 0.78 | 72 (53; 91) | 2.1 (−2.1; 7.3) | 0.24 | 0.49 | ||
In vitro functionality and selectivity of LY2409021
COS-7 cells were successfully transfected with the GCGR, GIPR and GLP1R, respectively, and yielded dose–response curves of the cognate endogenous agonists with potencies in the expected range (29) with half maximal effective concentration (EC50) values of 104 pmol/L for glucagon, 7.1 pmol/L for GIP and 34 pmol/L for GLP-1 on their respective receptors (Fig. 4A, C and E ). At the GCGR, LY2409021 inhibited the glucagon-induced cAMP accumulation potently with a half maximal inhibitory concentration (IC50) value of 28 nmol/L (95% CI: 16–46 nmol/L) (Fig. 4B). At a concentration of 1 µmol/L, LY2409021 inhibited the GIP-induced activation weakly (~35%) of the GIPR, and a stronger inhibition was observed at a concentration of 10 µmol/L (~85%), revealing an estimated IC50 value of 2.0 µmol/L (Fig. 4D). LY2409021 had weak antagonistic properties on the GLP1R with ~25% inhibition at a concentration of 1 µmol/L and ~80% inhibition at 10 µmol/L, yielding an estimated IC50 value of 3.4 µmol/L (Fig. 4F). In the absence of endogenous agonists, LY2409021 alone (0.1–10 µmol/L) did not stimulate any of the receptors (Fig. 4A, C and E ).
In vitro functionality and selectivity of LY2409021. cAMP accumulation assay (% of maximal induced activity of the endogenous hormone) in COS-7 cells transiently transfected with the human glucagon receptor (A and B), glucose-dependent insulinotropic polypeptide (GIP) receptor (C and D) and glucagon-like peptide 1 (GLP-1) receptor (E and F). Receptor activation for the cognate endogenous hormones (black circles) and LY2409021 (open squares) are shown in A, C and E and the inhibitory effect of LY2409021 of activation by the cognate hormones in B, D and F (black triangles). Symbols are mean ± s.e.m.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
In vitro functionality and selectivity of LY2409021. cAMP accumulation assay (% of maximal induced activity of the endogenous hormone) in COS-7 cells transiently transfected with the human glucagon receptor (A and B), glucose-dependent insulinotropic polypeptide (GIP) receptor (C and D) and glucagon-like peptide 1 (GLP-1) receptor (E and F). Receptor activation for the cognate endogenous hormones (black circles) and LY2409021 (open squares) are shown in A, C and E and the inhibitory effect of LY2409021 of activation by the cognate hormones in B, D and F (black triangles). Symbols are mean ± s.e.m.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
In vitro functionality and selectivity of LY2409021. cAMP accumulation assay (% of maximal induced activity of the endogenous hormone) in COS-7 cells transiently transfected with the human glucagon receptor (A and B), glucose-dependent insulinotropic polypeptide (GIP) receptor (C and D) and glucagon-like peptide 1 (GLP-1) receptor (E and F). Receptor activation for the cognate endogenous hormones (black circles) and LY2409021 (open squares) are shown in A, C and E and the inhibitory effect of LY2409021 of activation by the cognate hormones in B, D and F (black triangles). Symbols are mean ± s.e.m.
Citation: European Journal of Endocrinology 186, 2; 10.1530/EJE-21-0865
Discussion
We investigated the glucose-lowering effects of a single-dose (100 mg) of the GCGR antagonist LY2409021. We confirm previous findings of an efficient FPG-lowering effect of LY2409021 in patients with T2D and non-diabetic controls, albeit far less in controls; and show that (i) postprandial glucose excursions (bsAUC) are not lowered by LY2409021 in patients with T2D and are increased in controls and (ii) LY2409021 lowers EGP in the fasted state in both patients and controls, whereas it increases EGP postprandially in patients with T2D.
Plasma glucose and endogenous glucose production
Previous studies have found reduced FPG concentrations with single (15) and once-daily administration of LY2409021 (16), but measurements of postprandial glucose tolerance (2-h postmeal glucose concentration, glucose excursions and incremental glucose AUC) have been inconsistent (15). A dose-dependent tendency of increased postprandial Δglucose values and glucose bsAUCs was reported (15). In the present study, we reproduced a clear reduction of FPG in patients with T2D and demonstrate no effect on postprandial glucose excursions (bsAUC) during GCGR antagonism in these patients. A reduction of FPG was also observed in controls, however, quantitatively far less compared to the patients with T2D but comparable to previous data (15). We demonstrate that the LY2409021-induced reduction of FPG involves decreased fasting EGP in T2D and probably also in controls (P = 0.032, adj. P = 0.114). The missing effect of LY2409021 on postprandial baseline-corrected glucose excursions in our patients with T2D may in part be due to a smaller meal-induced reduction of EGP, shown here. A previous study in healthy individuals showed that LY2409021 attenuated EGP in a dose-dependent manner by 15–84% during a hyperglucagonemic clamp (38). It is well known that insulin is a major regulator of EGP (39), and in our study, baseline insulin was higher during placebo compared to LY2409021 treatment in patients with T2D. Thus, the possible postprandial effect of hyperglucagonemia (intact GCGR activity with placebo) in T2D might in this experimental setting be masked by insulin and its strong inhibitory effect on EGP. MCR, taking the mass effect of the glucose gradient into account, was however not affected by LY2409021 in patients with T2D, but peripheral insulin actions might be affected postprandially in control participants, demonstrated by lower MCR postprandially (AUC). Whether differences in meal composition between this and previous studies or different dosing regimens (repeated vs single dose of LY2409021) could contribute to the observed differences is not clear nor possible to conclude from this study. In a clinical perspective, however, the lack of reduction of bsAUC with LY2409021 does not seem to impose concerns given the relatively little difference combined with the beneficial reduction in fasting glucose. If GCGR antagonists were a treatment option, insulin secretagogues could probably counteract the postprandial glucose rise.
Selectivity of the glucagon receptor antagonist
The possibility of LY2409021 activation or inhibition of GIPR and GLP1R is possible because the GCGR, GIPR and GLP1R belong to the same class B (secretin-like) subclass of closely related G protein-coupled receptors (40). Cross-reactivity between the endogenous agonists has been reported for several of these receptors. Glucagon, for example, activates the GLP1R (41) and glucagon-like peptide 2 may activate the GIPR (42). Our in vitro studies showed that LY2409021 to some degree antagonizes the GLP1R and GIPR with inhibition of GLP-1 and GIP-induced cAMP accumulation with an estimated IC50 of 3.4 and 2 µmol/L, respectively. Based on previous results (15), we expect a circulating concentration of LY2409021 of about 5–7 µmol/L after a single dose of 100 mg in this population, that is a concentration close to the IC50. Thus, together with the long t½ (~55 h), LY2409021 could possibly antagonize the GLP-1 and GIPR at this dose in vivo, and LY2409021-induced GLP1R and GIPR antagonism may explain part of the absent reduction in postprandial glucose concentrations in this study.
Glucagon, GLP-1 and GIP
Fasting and postprandial hyperglucagonemia occurred in patients with T2D compared to controls as previously shown (1, 2, 3, 5). The approximately three times higher glucagon concentrations after LY2409021 is a mechanism-related effect of the antagonists seen in pre-clinical and clinical studies (43, 44). GLP-1 concentrations were higher with LY2409021, most evident in patients with T2D. Since glucagon and GLP-1 are derived from the same proglucagon precursor, our findings corroborate the idea of a disrupted negative feedback loop by glucagon antagonism or, alternatively, GLP-1 is increased due to the inhibition of GLP1R, as with the competitive antagonist of GLP-1, exendin(9-39)NH2 (45, 46). The higher GLP-1 concentrations do not seem to improve glucose tolerance with the GCGR antagonists because reports show no change in active GLP-1 despite the higher total GLP-1 concentration (15, 16, 17). Our GLP-1 data comply with this seeing that the higher total GLP-1 concentrations are not insulinotropic. Recent intra-islet research highlights the insulinotropic potential for glucagon in vivo (41, 47) and that interrupting the dynamic alpha and beta cell feedback regulation by inhibiting GCGR on beta cells acutely destabilizes the glycemic set point (48). With our data in mind, this explains how GCGR antagonism is not as simple as limiting hyperglucagonemia, but it affects delicate feedback mechanisms possibly explaining our postprandial increased (or no change in) glucose tolerance with LY2409021 compared to placebo.
Gastric emptying, appetite and food intake
Exogenous glucagon has been shown to reduce gastrointestinal motility and slow gastric emptying when studied with a barium meal fluoroscopy method (49). In the present study, LY2409021 did not affect gastric emptying assessed by the acetaminophen absorption test (or glucose kinetics) suggesting that endogenous glucagon has little or no role in controlling gastric emptying in the context of a liquid meal and under the limitations of the acetaminophen absorption test. Exogenous glucagon has been shown to decrease food intake and promote weight loss in several species including humans through suppressed feeding (50); and in rats, a neutralizing glucagon antibody increased food intake (51). In our study, LY2409021 decreased satiety and increased appetite sensations in the controls. The effects on appetite did however not lead to increased food intake ad libitum, and LY2409021 did not affect appetite sensations or food intake in patients with T2D. Statistically tested, we did not find a difference in effect of LY2409021 on these parameters of appetite between patients with T2D and controls. These endpoints may however be limited by lack of power and it would be interesting to explore in a larger sample size.
Strengths and limitations
The study is strengthened by its double-blinded, placebo-controlled, crossover design reducing the influence of inter-individual confounding factors and limiting carry-over effects by randomizing experimental days. The main endpoint, bsAUC for glucose, in this physiological setting may have its limitations, since it may be affected by the glucose fasting baseline itself and, thus, may complicate the read-out of the results. Here, we support the glucose concentration read-outs with EGP by isotope tracer enrichments.
Glucagon receptor antagonists and antibodies
Several small-molecule GCGR antagonists (52, 53, 54) have shown dose-dependent reductions in HbA1c and improved FPG in patients with T2D. The most common adverse effects associated with the GCGR antagonists have been mild increases in liver transaminases, hepatic steatosis, dyslipidemia and blood pressure in patients with T2D which have prevented further development of all these compounds so far (15, 17, 52, 53, 55, 56, 57). However, volagidemab (a GCGR antibody previously known as REMD-477) has shown promising results as add-on treatment to insulin in patients with type 1 diabetes with improvements in HbA1c, time in glycemic range and reduction of insulin dose in single-dose and 12-week studies (58, 59). In patients with type 1 diabetes, intermittent increases in blood pressure and transaminases were observed as well, thus further studies will elucidate whether side effects will outweigh the benefits in this group of patients.
Conclusion
GCGR antagonism with LY2409021 lowered FPG but did not improve postprandial glucose tolerance after a mixed meal in patients with T2D or in non-diabetic controls. The complex interactions of the antagonist with incretin receptors as well as possible intra-islet effects renders the interpretation of glucagon antagonism and the contribution of hyperglucagonemia to postprandial glucose excursions difficult. It can be foreseen that therapeutic application of GCGR antagonism may be similarly challenging.
Declaration of interest
A L has received lecture fees from Novo Nordisk and AstraZeneca. F K K has served on scientific advisory panels and/or been part of speaker’s bureaus for, served as a consultant to and/or received research support from Amgen, AstraZeneca, Bayer Boehringer Ingelheim, Carmot Therapeutics, Eli Lilly, Gubra, MedImmune, MSD/Merck, Mundipharma, Norgine, Novo Nordisk, Sanofi and Zealand Pharma. M M R is a minority shareholder of and consultant for Antag Therapeutics, Bainan Biotech and Synklino. J J H is currently receiving speaker honoraria from Novo Nordisk and MSD and is on advisory boards for Novo Nordisk. T V has served on scientific advisory panels, been part of speaker’s bureaus for, served as a consultant to and/or received research support from Amgen, AstraZeneca, Boehringer Ingelheim, Eli Lilly, Gilead, Mundipharma, MSD/Merck, Novo Nordisk, Sanofi and Sun Pharmaceuticals. LSG, JJH and FKK are minority shareholders of Antag Therapeutics. Other authors declare that there is no duality of interest associated with their contribution to this manuscript. All authors declare that the study was conducted in absence of any financial or conflicts of interest.
Funding
The study was funded by the Danish Diabetes Academy supported by the Novo Nordisk Foundation, Eli Lilly and Company as an investigator-initiated study and the A.P. Møller Foundation for the Advancement of Medical Science.
Author contribution statement
S H, A L, F K K and T V designed the study and wrote the study protocol. S H, H M and E N H performed the study. M M R carried out the in vitro analysis and L S G wrote the corresponding manuscript part. G v H and J J H generated the data. S H, A L, F K K and T V wrote the manuscript. All authors critically edited the manuscript and approved the final version. S H and T V are the guarantors of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Acknowledgements
The authors gratefully thank the study participants for their contribution. The authors thank Sisse M Schmidt, Inass A Nachar and Lene Albæk for laboratory assistance.
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