Hyperglucagonemia is a well-known contributor to diabetic hyperglycemia, and glucagon-like peptide 1 (GLP-1) suppresses glucagon secretion. Reduced inhibitory effects of glucose and GLP-1 on glucagon secretion may contribute to the hyperglucagonemia in diabetes and influence the success of GLP-1 receptor agonist therapy. We examined the dose-response relationship for GLP-1 on glucose-induced glucagon suppression in healthy individuals and patients with type 2 and type 1 diabetes. In randomized order, 10 healthy individuals with normal glucose tolerance, 10 patients with type 2 diabetes, and 9 C-peptide–negative patients with type 1 diabetes underwent 4 separate stepwise glucose clamps (five 30-min steps from fasting level to 15 mmol/L plasma glucose) during simultaneous intravenous infusions of saline or 0.2, 0.4, or 0.8 pmol GLP-1/kg/min. In healthy individuals and patients with type 2 diabetes, GLP-1 potentiated the glucagon-suppressive effect of intravenous glucose in a dose-dependent manner. In patients with type 1 diabetes, no significant changes in glucagon secretion were observed during the clamps whether with saline or GLP-1 infusions. In conclusion, the glucagonostatic potency of GLP-1 during a stepwise glucose clamp is preserved in patients with type 2 diabetes, whereas our patients with type 1 diabetes were insensitive to the glucagonostatic effects of both glucose and GLP-1.

Inappropriate secretion of glucagon, relative to the prevailing glycemia, contributes significantly to the hyperglycemia of type 1 and type 2 diabetes (13). The gut-derived incretin hormone glucagon-like peptide 1 (GLP-1) suppresses glucagon secretion in addition to its glucose-dependent insulinotropic effect. This glucagon-suppressive effect occurs at plasma glucose levels at or above fasting levels (4,5) and contributes to the glucose-lowering effect of GLP-1 (68). In healthy individuals, exogenous GLP-1 increases glucose clearance and reduces hepatic glucose output (8). The individual contributions of GLP-1’s glucagonostatic and insulinotropic effects to its glucose-lowering effect have been challenging to disentangle. In a pancreatic clamp study in which GLP-1–induced changes in circulating glucagon and insulin concentrations were mimicked separately and together, GLP-1’s glucagonostatic and insulinotropic effects were found to contribute about equally to the glucose-lowering effect of GLP-1 in patients with type 2 diabetes (9). Importantly, the glucagonostatic effect of exogenous GLP-1 is thought to translate into lowering of plasma glucose independently of GLP-1’s insulinotropic effect, as demonstrated in patients with type 1 diabetes (10). In addition, the GLP-1 receptor antagonist exendin(9-39)NH2 has been shown to increase circulating glucagon and glucose levels in patients with type 1 diabetes and no residual β-cell function (11). These studies support a direct—or at least an insulin-independent—suppressive effect of GLP-1 on glucagon secretion. Studies by Kjems et al. (12) established reduced potency of GLP-1’s insulinotropic effect as an important pathophysiological characteristic of type 2 diabetes, but the potency of GLP-1’s glucagonostatic effect in patients with type 1 diabetes and in patients with type 2 diabetes has not been thoroughly investigated. We speculated that reduced potency of GLP-1 to inhibit glucagon secretion while already (inadequately) suppressed by glucose might explain part of the inappropriate secretion of glucagon often observed in patients with type 2 as well as type 1 diabetes.

The study protocol was approved by the Scientific Ethics Committee of The Capital Region of Denmark (H-3–2011–088) and registered at the Danish Data Protection Agency (2011–41–6891) and ClinicalTrials.gov (NCT01507597).

Participants

We recruited 10 individuals with normal glucose tolerance, 10 patients with type 2 diabetes, and 9 patients with type 1 diabetes. Baseline characteristics are displayed in Table 1. Participants with normal glucose tolerance were screened using a 2-h oral glucose tolerance test using 75 g of glucose and were considered glucose tolerant if fasting plasma glucose was <6.1 mmol/L and the 120-min plasma glucose level was <7.8 mmol/L. C-peptide–negative patients with type 1 diabetes were included based on results of an arginine test (5 g of arginine infused intravenously over 1 min with blood sampling at time 0, 6, and 15 min) with C-peptide levels below the detection limit at all time points. In the group with type 1 diabetes, seven patients were treated with basal insulin once or twice daily (three with insulin glargine and four with insulin detemir) combined with mealtime rapid-acting insulin, one patient was treated with isophane insulin twice daily only, and one patient was on insulin pump therapy. In all patients, the basal insulin regimen was continued, and only the morning insulin dose was skipped on study days (four patients used morning basal insulin). The patient on insulin pump therapy continued the infusion on usual basal infusion settings throughout each of the 4 study days. Patients with type 2 diabetes were diagnosed according to the World Health Organization criteria, treated with metformin only, and had a duration of diabetes of >3 months. Patients with type 2 diabetes were instructed not to take metformin 1 week prior to each of the test days.

Table 1

Characteristics of participants

NGT (n = 10)T2D (n = 10)T1D (n = 9)
Sex (women/men) 2/8 2/8 0/9 
Age (years) 56 ± 4 60 ± 3 57 ± 4 
BMI (kg/m226 ± 3 31 ± 1 29 ± 1 
HbA1c (%) 5.4 ± 0.1 6.1 ± 0.2 7.7 ± 0.2 
HbA1c (mmol/mol) 36 ± 1 44 ± 2 61 ± 2 
Fasting plasma glucose (mmol/L) 5.4 ± 0.2 8.3 ± 0.2 7.5 ± 0.2 
Duration of diabetes (years) — 3 ± 1 32 ± 4 
NGT (n = 10)T2D (n = 10)T1D (n = 9)
Sex (women/men) 2/8 2/8 0/9 
Age (years) 56 ± 4 60 ± 3 57 ± 4 
BMI (kg/m226 ± 3 31 ± 1 29 ± 1 
HbA1c (%) 5.4 ± 0.1 6.1 ± 0.2 7.7 ± 0.2 
HbA1c (mmol/mol) 36 ± 1 44 ± 2 61 ± 2 
Fasting plasma glucose (mmol/L) 5.4 ± 0.2 8.3 ± 0.2 7.5 ± 0.2 
Duration of diabetes (years) — 3 ± 1 32 ± 4 

Characteristics of participants with normal glucose tolerance (NGT), participants with type 2 diabetes (T2D), and participants with type 1 diabetes (T1D). Sex is shown as number of female over male patients in each group; otherwise, data are presented as mean ± SEM.

Experimental Procedures

All participants were studied after an overnight (10-h) fast including tobacco and alcohol abstinence on 4 randomized experimental days separated by at least 48 h. On each experimental day, a cannula was inserted into a cubital vein, and the hand and forearm were wrapped in a heating blanket (40–50°C) throughout the experiment for collection of arterialized blood samples. A cannula was inserted into a cubital vein in the contralateral arm for hormone and glucose infusions administered through separate infusion lines. Each experimental day included a 210-min stepwise glucose clamp and a concomitant (blinded) intravenous infusion of either saline or GLP-1 at 0.2, 0.4, and 0.8 pmol/kg/min. The prevailing plasma glucose level was raised stepwise, with bolus injections of glucose (50% glucose) reaching glucose levels of 6, 7, 8, 10, 12, and 15 mmol/L (as outlined in Fig. 1). The glucose levels were kept constant for 30 min between steps using an adjustable glucose infusion. At time 0 min, the test infusion and the glucose clamp were started. Patients with fasting plasma glucose >6 mmol/L were clamped at the individual fasting plasma glucose level until the time for increasing plasma glucose goal according to the protocol was reached. Patients with fasting plasma glucose >10 mmol/L were rescheduled for another appointment. Three patients with type 1 diabetes were admitted on the night before the clamp because of failure to reach fasting glucose <10 mmol/L on their own, for titration of appropriate basal insulin dose, and for observation of potential hypoglycemic episodes. Two blood samples were drawn before infusion start (−15 and 0 min) and thereafter every 15 min throughout the 210-min clamp. Synthetic human GLP-1 (PolyPeptide Laboratories A/S, Hillerød, Denmark) was dissolved in sterilized water containing 2% human albumin (Statens Serum Institut, Copenhagen, Denmark), subjected to sterile filtration (followed by testing for sterility and pyrogens), and dispensed into coded vials at the Central Pharmacy of the Capital Region of Denmark (Herlev, Denmark). These vials were kept frozen (−20°C) until use. During experimental days, arterialized blood was collected in chilled tubes containing EDTA and a dipeptidyl peptidase 4 inhibitor (valine pyrrolidide, final concentration 0.01 mmol/L; a gift from Novo Nordisk A/S, Bagsværd, Denmark) for analyses of GLP-1 and glucagon. Blood for analysis of insulin and C-peptide was collected in dry tubes and left to coagulate for 20 min at room temperature. All samples were centrifuged for 20 min at 1,200g and 4°C. Plasma samples for GLP-1 and glucagon analyses were stored at −20°C, and serum samples for insulin and C-peptide were stored at −80°C until analysis. For bedside measurement of plasma glucose, blood was added to sodium fluoride–coated microtubes and centrifuged immediately at 7,400g for 2 min at room temperature.

Figure 1

Diagram of the experimental protocol. The curve indicates the goals for plasma glucose levels throughout the stepwise glucose clamp.

Figure 1

Diagram of the experimental protocol. The curve indicates the goals for plasma glucose levels throughout the stepwise glucose clamp.

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Analyses

Plasma glucose concentrations were measured bedside using the glucose oxidase method (Model 2300 STAT Plus analyzer; YSI Incorporated, Yellow Springs, OH). Serum insulin and C-peptide concentrations were measured using a two-site electrochemiluminescence immune assay (Roche/Hitachi Modular Analytics; Roche Diagnostics). Plasma concentrations of glucagon and total GLP-1 were measured by radioimmunoassays as previously described (13,14).

Calculations and Statistical Analyses

Area under the curve (AUC) was calculated using the trapezoidal rule. Baseline, peak, and AUC values are expressed as means with 95% CIs. Differences resulting in P values <0.05 were considered significant. Plasma glucose and corresponding plasma glucagon concentrations were plotted, and individual regression lines were drawn. In order to evaluate the slopes of the regression lines, glucagon measurements obtained after reaching the first measurement of glucagon at the detection limit of the glucagon assay were excluded. The slopes of the regression lines (α) were used to evaluate glucose-induced glucagon suppression and the influence of GLP-1 dose using ANCOVA. Linear mixed-effect modeling was used for analysis of longitudinal and repeated measures using statistical software R with the “nlme” package. Data were transformed according to distribution patterns with logarithmic transformation if needed in order to reach normal distribution. We used a top-down modeling strategy, with individual identity as random variable (15), and a homogeneous or heterogeneous residual variance structure was chosen according to likelihood ratios. Results are presented as means and 95% CIs of the estimate.

Data and Resource Availability

The data sets generated during and/or analyzed during the current study are available from the corresponding authors.

GLP-1

Infusions of GLP-1 resulted in dose-dependent increases in plasma levels of GLP-1 reaching steady state after 30 min with no differences between the groups at any level of infusion (Fig. 2).

Figure 2

Plasma concentrations of GLP-1 during stepwise glucose clamps and concomitant infusions of saline (black circles), 0.2 pmol GLP-1/kg/min (white squares), 0.4 pmol GLP-1/kg/min (black triangles), and 0.8 pmol GLP-1/kg/min (white triangles), respectively, in individuals with normal glucose tolerance, patients with type 2 diabetes, and patients with type 1 diabetes.

Figure 2

Plasma concentrations of GLP-1 during stepwise glucose clamps and concomitant infusions of saline (black circles), 0.2 pmol GLP-1/kg/min (white squares), 0.4 pmol GLP-1/kg/min (black triangles), and 0.8 pmol GLP-1/kg/min (white triangles), respectively, in individuals with normal glucose tolerance, patients with type 2 diabetes, and patients with type 1 diabetes.

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Glucose

Plasma glucose was successfully clamped at the desired levels in all groups as depicted in Fig. 3. Fasting plasma glucose was significantly higher in patients with type 2 diabetes (mean 8.5 mmol/L [95% CI 7.5, 9.0]) and type 1 diabetes (7.5 mmol/L [6.5, 8.5]) compared with normal glucose–tolerant individuals (5.5 mmol/L [4.5, 6.0]). With increasing GLP-1 doses, greater amounts of glucose were needed to maintain the clamp in normal glucose–tolerant individuals (P < 0.001) and patients with type 2 diabetes (P < 0.001), whereas negligible amounts or no glucose had to be infused in the C-peptide–negative patients with type 1 diabetes (P = 0.4204) (Fig. 3). The amounts of intravenous glucose required to maintain the clamps were significantly higher in the group with normal glucose tolerance compared with the group with type 2 diabetes (Fig. 3).

Figure 3

Plasma glucose and amount of glucose administered intravenously to maintain the stepwise glucose clamp in individuals with normal glucose tolerance, patients with type 2 diabetes, and patients with type 1 diabetes during intravenous infusion of saline or 0.2, 0.4, or 0.8 pmol GLP-1/kg/min. Curves of mean plasma glucose values with SEM as error bars are related to the left y-axes. In bars, mean amounts of intravenous glucose infused to maintain the stepwise glucose clamp are depicted and related to the right y-axes. Mean values of the total amount of glucose with 95% CIs in parentheses are noted at the top left of each panel.

Figure 3

Plasma glucose and amount of glucose administered intravenously to maintain the stepwise glucose clamp in individuals with normal glucose tolerance, patients with type 2 diabetes, and patients with type 1 diabetes during intravenous infusion of saline or 0.2, 0.4, or 0.8 pmol GLP-1/kg/min. Curves of mean plasma glucose values with SEM as error bars are related to the left y-axes. In bars, mean amounts of intravenous glucose infused to maintain the stepwise glucose clamp are depicted and related to the right y-axes. Mean values of the total amount of glucose with 95% CIs in parentheses are noted at the top left of each panel.

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Glucagon

We observed no differences in fasting levels of glucagon between the experimental days within each group. Fasting plasma glucagon concentrations were significantly higher in patients with type 2 diabetes (mean 8.2 pmol/L [95% CI 6.1, 10.4]) compared with participants with normal glucose tolerance (4.4 pmol/L [2.3, 6.6]; P = 0.017) and patients with type 1 diabetes (5.2 pmol/L [2.9, 7.4]; P = 0.054) with no difference between the groups with normal glucose tolerance and type 1 diabetes (P = 0.6394) (Fig. 4). The glucose clamp with saline induced clear suppression of circulating glucagon in participants with normal glucose tolerance and in patients with type 2 diabetes, whereas patients with type 1 diabetes showed no suppression of plasma glucagon (Fig. 4). In the groups with normal glucose tolerance and type 2 diabetes, GLP-1 infusions potentiated glucose-induced glucagon suppression (as expressed by a steeper mean slope of the regression line from plots of plasma glucose vs. glucagon concentrations) in a dose-dependent manner (ANCOVA P < 0.001 and P = 0.002, respectively). As illustrated in Fig. 5, normal glucose–tolerant participants exhibited numerically steeper slopes during GLP-1 infusions than the patients with type 2 diabetes, but these differences were not statistically significant. In the group with type 1 diabetes, GLP-1 infusions did not appear to suppress the glucagon concentration observed during the clamp (Fig. 5).

Figure 4

Plasma glucagon levels during the stepwise glucose clamp and the four concomitant infusions of saline (black circles), 0.2 pmol GLP-1/kg/min (white squares), 0.4 pmol GLP-1/kg/min (black triangles), and 0.8 pmol GLP-1/kg/min (inverted white triangles), respectively, in individuals with normal glucose tolerance, patients with type 2 diabetes, and patients with type 1 diabetes. The top and middle panels show glucagon levels by time as measured and as baseline-subtracted, respectively. The bottom panel shows the glucagon response as bar plots depicting the area under curve (AUC) as mean values and whiskers as SEM; the horizontal lines indicate significant differences between infusions (P < 0.05).

Figure 4

Plasma glucagon levels during the stepwise glucose clamp and the four concomitant infusions of saline (black circles), 0.2 pmol GLP-1/kg/min (white squares), 0.4 pmol GLP-1/kg/min (black triangles), and 0.8 pmol GLP-1/kg/min (inverted white triangles), respectively, in individuals with normal glucose tolerance, patients with type 2 diabetes, and patients with type 1 diabetes. The top and middle panels show glucagon levels by time as measured and as baseline-subtracted, respectively. The bottom panel shows the glucagon response as bar plots depicting the area under curve (AUC) as mean values and whiskers as SEM; the horizontal lines indicate significant differences between infusions (P < 0.05).

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Figure 5

Plasma glucagon concentrations plotted against concomitant plasma glucose concentrations in individuals with normal glucose tolerance, patients with type 2 diabetes, and patients with type 1 diabetes, respectively, during the infusion of saline or 0.2, 0.4, or 0.8 pmol GLP-1/kg/min. Straight lines represent individual regression lines, and the mean slope with 95% CI is noted at the top right of each panel along with level of significance (P value).

Figure 5

Plasma glucagon concentrations plotted against concomitant plasma glucose concentrations in individuals with normal glucose tolerance, patients with type 2 diabetes, and patients with type 1 diabetes, respectively, during the infusion of saline or 0.2, 0.4, or 0.8 pmol GLP-1/kg/min. Straight lines represent individual regression lines, and the mean slope with 95% CI is noted at the top right of each panel along with level of significance (P value).

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C-Peptide and Insulin

Within each group, fasting serum C-peptide levels were similar on the 4 study days (in the group with type 1 diabetes, serum C-peptide levels were below detection limit as expected) and higher in patients with type 2 diabetes compared with participants with normal glucose tolerance (mean 772 pmol/L [95% CI 717, 828] vs. 546 pmol/L [490, 602]; P < 0.001) (Fig. 6). Baseline serum insulin levels in the group with type 1 diabetes (reflecting basal insulin therapy) were comparable between the experimental days (P = 0.10) (Fig. 6). The stepwise glucose clamp with concomitant saline infusion caused consecutive increments in serum C-peptide levels in both participants with normal glucose tolerance and in patients with type 2 diabetes with greatest responses in the normal glucose–tolerant group (P < 0.001) (Figs. 6 and 7). GLP-1 infusions potentiated glucose-stimulated insulin secretion in a highly dose-dependent manner in both groups (ANCOVA P < 0.001 [normal glucose tolerant] and P < 0.001 [type 2 diabetes]) with significantly greater potency in the normal glucose–tolerant group (P < 0.001 for all three doses). In individuals with type 1 diabetes, we observed minor decreases in circulating basal serum insulin levels along the course of all clamps with increasing plasma glucose (Fig. 7). Furthermore, we observed a minor difference in slope between the clamps in individuals with type 1 diabetes (ANCOVA, P < 0.001), but with no dose dependency (Fig. 7).

Figure 6

Plasma C-peptide and insulin levels during the stepwise glucose clamp and the four concomitant infusions of saline (circles), 0.2 pmol GLP-1/kg/min (white squares), 0.4 pmol GLP-1/kg/min (black triangles), and 0.8 pmol GLP-1/kg/min (inverted white triangles), respectively. In individuals with normal glucose tolerance and patients with type 2 diabetes, plasma C-peptide levels are depicted as mean curves. In patients with type 1 diabetes, insulin levels are depicted as mean curves.

Figure 6

Plasma C-peptide and insulin levels during the stepwise glucose clamp and the four concomitant infusions of saline (circles), 0.2 pmol GLP-1/kg/min (white squares), 0.4 pmol GLP-1/kg/min (black triangles), and 0.8 pmol GLP-1/kg/min (inverted white triangles), respectively. In individuals with normal glucose tolerance and patients with type 2 diabetes, plasma C-peptide levels are depicted as mean curves. In patients with type 1 diabetes, insulin levels are depicted as mean curves.

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Figure 7

Serum C-peptide or insulin levels plotted against plasma glucose levels in individuals with normal glucose tolerance, patients with type 2 diabetes, and patients with type 1 diabetes, respectively, during the infusion of saline or 0.2, 0.4, or 0.8 pmol GLP-1/kg/min. The individual regression lines are shown, and the mean slope and 95% CI of the slope is noted at the top left along the level of significance (P value). C-peptide was measured in individuals with normal glucose tolerance and patients with type 2 diabetes. Serum insulin levels (reflecting basal insulin) were measured in the C-peptide–negative patients with type 1 diabetes.

Figure 7

Serum C-peptide or insulin levels plotted against plasma glucose levels in individuals with normal glucose tolerance, patients with type 2 diabetes, and patients with type 1 diabetes, respectively, during the infusion of saline or 0.2, 0.4, or 0.8 pmol GLP-1/kg/min. The individual regression lines are shown, and the mean slope and 95% CI of the slope is noted at the top left along the level of significance (P value). C-peptide was measured in individuals with normal glucose tolerance and patients with type 2 diabetes. Serum insulin levels (reflecting basal insulin) were measured in the C-peptide–negative patients with type 1 diabetes.

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We show that healthy, glucose-tolerant individuals and patients with type 2 diabetes exhibit increasing suppression of plasma glucagon concentrations during increasing plasma glucose concentrations and that exogenous GLP-1 augments the glucose-induced suppression of glucagon in a dose-dependent fashion in these groups. We also show that patients with type 1 diabetes appear insensitive to the glucagon-suppressive effects of both glucose and GLP-1 under the given circumstances. Lastly, we confirm the dose-dependent stimulatory effect of GLP-1 on glucose-stimulated insulin secretion in individuals with normal glucose tolerance as well as in patients with type 2 diabetes, however with a reduced potency in the latter group (12).

Our findings in participants with normal glucose tolerance and type 2 diabetes reflect a sustained, potent, and dose-dependent inhibition of glucagon by GLP-1 at glucose levels above fasting levels. The glucagon-suppressive potency of exogenous GLP-1 was clearly preserved in our patients with well-controlled type 2 diabetes treated only with metformin. Pathophysiologically, this clearly distinguishes the glucagonostatic effect of GLP-1 from the reduced insulinotropic potency of GLP-1 in type 2 diabetes, probably pointing to impaired β-cell function as the phenomenon underlying reduced GLP-1 action in type 2 diabetes. Moreover, this dissociation between potency of GLP-1 on insulin and glucagon secretion may reflect that α cell and β-cell function are not equally affected in type 2 diabetes. The intraislet hypothesis (16), which states that inhibition of glucagon secretion is secondary to stimulation of β-cells, is not compatible with these findings. Similarly, in a study involving clamping of glucose at high (10.2 mmol/L) and low levels (6.7 mmol/L) in patients with type 2 diabetes (17), stepwise increasing GLP-1 infusions progressively and equally suppressed glucagon secretion despite the much higher insulin levels during high compared with low glucose. GLP-1–stimulated somatostatin secretion from the pancreatic delta cells (7) may play a key role in GLP-1–induced suppression of glucagon secretion, as indicated by studies using the isolated perfused rat pancreas (18). In these studies, glucose levels were kept at 1.5 mmol/L to prevent β-cell secretion. GLP-1 perfusion still stimulated somatostatin secretion and inhibited glucagon secretion concomitantly. Furthermore, somatostatin antibodies as well as a somatostatin receptor antagonist reduced or even abolished the inhibitory effect of GLP-1 on glucagon secretion (18). Perhaps also direct inhibition of the α cell may play a role in GLP-1–induced suppression of glucagon since weak expression of the GLP-1 receptor has been shown in human α cells (19). In cultures of human islets, specific somatostatin receptor 2 antagonism resulted in glucagon secretion, but, nevertheless, permitted a strong suppressive effect of GLP-1 at 1 mmol/L glucose (preventing GLP-1–induced potentiation of glucose-induced insulin secretion) (20). Also, the insulin receptor antagonist S961 did not affect GLP-1–induced α cell secretion, precluding a major involvement of insulin in the inhibitory effect of GLP-1 on the α cell in these cultures (20). The glucagon-suppressive effect of GLP-1 at the low glucose levels used in the two aforementioned studies contrasts the lack of GLP-1–induced glucagon inhibition observed in clinical studies during hypoglycemic conditions (4), and this may be related to the glucose dependency of somatostatin secretion, which, like insulin secretion, is minimal or absent at low glucose levels (21,22). Glucagon secretion may also be regulated by autonomic neuronal mechanisms (23,24), raising the possibility that GLP-1 is ineffective on neurally stimulated glucagon secretion. Glucagon secretion seems to be stimulated by both the cholinergic or adrenergic activation, and both divisions are activated during hypoglycemia (23). This is in contrast to somatostatin secretion, which is inhibited by vagal stimulation; this inhibition might therefore explain the lack of suppression by GLP-1 during hypoglycemia (23,25).

In the current study, the stepwise increase in plasma glucose levels inevitably resulted in high insulin levels, making it hard to discriminate between the effects of GLP-1 and insulin on glucagon secretion. In order to evaluate the effect of GLP-1 on glucagon secretion independently of insulin secretion, we also included a group of C-peptide–negative patients with type 1 diabetes. Surprisingly, we were not able to show any reduction in glucagon secretion at any glucose level or at any GLP-1 dose in this group. Correspondingly, the amount of glucose needed to maintain the clamp was not influenced by GLP-1 at any dose in our C-peptide–negative patients with type 1 diabetes. Previous studies investigating the effect of GLP-1 in C-peptide–negative patients with type 1 diabetes have shown GLP-1–induced suppression of glucagon secretion (10,26). How can these discrepant results be explained? It is well recognized that older age and long disease duration are associated with autonomic neuropathy (27) and reduced hypoglycemia-induced glucagon secretion in patients with type 1 diabetes (28,29). Compared with previous studies using GLP-1 infusions in patients with type 1 diabetes (10,26), the present group of patients with type 1 diabetes had longer duration of diabetes and was older, which may contribute to explain the discrepant results compared with previous studies. Furthermore, fasting glucagon levels in our patients with type 1 diabetes were low compared with the two former studies (10,26). As fasting glucagon levels seem more strongly related to BMI than diabetes (30), the relatively high BMI of our patients with type 1 diabetes would be expected to be accompanied by high fasting levels of glucagon. This was not the case, perhaps explained by a glucagon-suppressive effect of the high circulating concentrations of exogenous insulin in our patients, which may also contribute to the irresponsive levels of glucagon. In several previous studies investigating GLP-1’s effect in C-peptide–negative patients with type 1 diabetes, basal insulin dosing was reduced in order to provoke hyperglucagonemia, which may explain the glucagon-suppressive effect of GLP-1 observed in these studies (10,26).

Despite the lack of GLP-1–induced glucagon suppression in our C-peptide–negative patients with type 1 diabetes, GLP-1 receptor agonist treatment in type 1 diabetes may still provide beneficial effects via its inhibition of gastric emptying (and ensuing reductions in postprandial glucose excursions and insulin dose) (31) and its satiety-promoting effects in obese patients with type 1 diabetes. Also, in the early stages of type 1 diabetes (C-peptide positive), the insulinotropic effect of GLP-1 may contribute to improve glycemic control (32). Nevertheless, until now, clinical trial programs have not resulted in approval of GLP-1 receptor agonism for the treatment of type 1 diabetes (31,3335).

In conclusion, we show that exogenous GLP-1 potentiates glucose-induced suppression of glucagon in a dose-dependent fashion with preserved potency in type 2 diabetes and confirm that type 2 diabetes pathophysiology involves a reduced potency of GLP-1’s glucose-dependent insulinotropic effect. In contrast, patients with long-standing type 1 diabetes in our setting seemed insensitive to the glucagon-suppressive effects of both glucose and GLP-1.

Clinical trial reg. no. NCT01507597, clinicaltrials.gov

Acknowledgments. The authors thank all of the study participants for the invaluable contribution. The authors also thank all involved laboratory technicians for the help with data collection and laboratory assistance.

Funding. The study was supported by unrestricted funding from Gentofte Hospital, University of Copenhagen.

Duality of Interest. No potential conflicts of interest were reported.

Author Contributions. J.I.B. initiated and planned the study, wrote the protocol, conducted the clinical experiments and statistical analyses, interpreted the results, and wrote the manuscript. M.G. conducted the clinical experiments and interpreted the results. A.L. conducted the clinical experiments and interpreted the results. J.J.H. performed analysis of blood samples and interpreted the results. T.V. was involved in planning the study and interpreted the results. F.K.K. conceived and planned the study, interpreted the results, and wrote the manuscript. All authors critically reviewed the manuscript and approved the final version to be submitted. J.I.B. and F.K.K. are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in poster form at the 76th Scientific Sessions of the American Diabetes Association, New Orleans, LA, 10–14 June 2016.

1.
Shah
P
,
Basu
A
,
Basu
R
,
Rizza
R
.
Impact of lack of suppression of glucagon on glucose tolerance in humans
.
Am J Physiol
1999
;
277
:
E283
E290
2.
Shah
P
,
Vella
A
,
Basu
A
,
Basu
R
,
Schwenk
WF
,
Rizza
RA
.
Lack of suppression of glucagon contributes to postprandial hyperglycemia in subjects with type 2 diabetes mellitus
.
J Clin Endocrinol Metab
2000
;
85
:
4053
4059
3.
Mitrakou
A
,
Kelley
D
,
Veneman
T
, et al
.
Contribution of abnormal muscle and liver glucose metabolism to postprandial hyperglycemia in NIDDM
.
Diabetes
1990
;
39
:
1381
1390
4.
Nauck
MA
,
Heimesaat
MM
,
Behle
K
, et al
.
Effects of glucagon-like peptide 1 on counterregulatory hormone responses, cognitive functions, and insulin secretion during hyperinsulinemic, stepped hypoglycemic clamp experiments in healthy volunteers
.
J Clin Endocrinol Metab
2002
;
87
:
1239
1246
5.
Vilsbøll
T
,
Krarup
T
,
Madsbad
S
,
Holst
JJ
.
Both GLP-1 and GIP are insulinotropic at basal and postprandial glucose levels and contribute nearly equally to the incretin effect of a meal in healthy subjects
.
Regul Pept
2003
;
114
:
115
121
6.
Kreymann
B
,
Williams
G
,
Ghatei
MA
,
Bloom
SR
.
Glucagon-like peptide-1 7-36: a physiological incretin in man
.
Lancet
1987
;
2
:
1300
1304
7.
Ørskov
C
,
Holst
JJ
,
Nielsen
OV
.
Effect of truncated glucagon-like peptide-1 [proglucagon-(78-107) amide] on endocrine secretion from pig pancreas, antrum, and nonantral stomach
.
Endocrinology
1988
;
123
:
2009
2013
8.
Hvidberg
A
,
Nielsen
MT
,
Hilsted
J
,
Ørskov
C
,
Holst
JJ
.
Effect of glucagon-like peptide-1 (proglucagon 78-107amide) on hepatic glucose production in healthy man
.
Metabolism
1994
;
43
:
104
108
9.
Hare
KJ
,
Vilsbøll
T
,
Asmar
M
,
Deacon
CF
,
Knop
FK
,
Holst
JJ
.
The glucagonostatic and insulinotropic effects of glucagon-like peptide 1 contribute equally to its glucose-lowering action
.
Diabetes
2010
;
59
:
1765
1770
10.
Creutzfeldt
WOC
,
Kleine
N
,
Willms
B
,
Ørskov
C
,
Holst
JJ
,
Nauck
MA
.
Glucagonostatic actions and reduction of fasting hyperglycemia by exogenous glucagon-like peptide I(7-36) amide in type I diabetic patients
.
Diabetes Care
1996
;
19
:
580
586
11.
Kielgast
U
,
Holst
JJ
,
Madsbad
S
.
Antidiabetic actions of endogenous and exogenous GLP-1 in type 1 diabetic patients with and without residual β-cell function
.
Diabetes
2011
;
60
:
1599
1607
12.
Kjems
LL
,
Holst
JJ
,
Vølund
A
,
Madsbad
S
.
The influence of GLP-1 on glucose-stimulated insulin secretion: effects on beta-cell sensitivity in type 2 and nondiabetic subjects
.
Diabetes
2003
;
52
:
380
386
13.
Ørskov
C
,
Rabenhøj
L
,
Wettergren
A
,
Kofod
H
,
Holst
JJ
.
Tissue and plasma concentrations of amidated and glycine-extended glucagon-like peptide I in humans
.
Diabetes
1994
;
43
:
535
539
14.
Wewer Albrechtsen
NJ
,
Hartmann
B
,
Veedfald
S
, et al
.
Hyperglucagonaemia analysed by glucagon sandwich ELISA: nonspecific interference or truly elevated levels?
Diabetologia
2014
;
57
:
1919
1926
15.
Verbeke
G
,
Molenberghs
G
.
Linear Mixed Models for Longitudinal Data
.
New York
,
Springer
,
2000
16.
Unger
RH
,
Orci
L
.
Glucagon and the A cell: physiology and pathophysiology (first two parts)
.
N Engl J Med
1981
;
304
:
1518
1524
17.
Hare
KJ
,
Knop
FK
,
Asmar
M
, et al
.
Preserved inhibitory potency of GLP-1 on glucagon secretion in type 2 diabetes mellitus
.
J Clin Endocrinol Metab
2009
;
94
:
4679
4687
18.
de Heer
J
,
Rasmussen
C
,
Coy
DH
,
Holst
JJ
.
Glucagon-like peptide-1, but not glucose-dependent insulinotropic peptide, inhibits glucagon secretion via somatostatin (receptor subtype 2) in the perfused rat pancreas
.
Diabetologia
2008
;
51
:
2263
2270
19.
Amisten
S
,
Salehi
A
,
Rorsman
P
,
Jones
PM
,
Persaud
SJ
.
An atlas and functional analysis of G-protein coupled receptors in human islets of Langerhans
.
Pharmacol Ther
2013
;
139
:
359
391
20.
Ramracheya
R
,
Chapman
C
,
Chibalina
M
, et al
.
GLP-1 suppresses glucagon secretion in human pancreatic alpha-cells by inhibition of P/Q-type Ca2+ channels
.
Physiol Rep
2018
;
6
:
e13852
21.
Ørgaard
A
,
Holst
JJ
.
The role of somatostatin in GLP-1-induced inhibition of glucagon secretion in mice
.
Diabetologia
2017
;
60
:
1731
1739
22.
Penman
E
,
Wass
JAH
,
Medbak
S
, et al
.
Response of circulating immunoreactive somatostatin to nutritional stimuli in normal subjects
.
Gastroenterology
1981
;
81
:
692
699
23.
Holst
JJ
,
Schwartz
TW
,
Knuhtsen
S
,
Jensen
SL
,
Nielsen
OV
.
Autonomic nervous control of the endocrine secretion from the isolated, perfused pig pancreas
.
J Auton Nerv Syst
1986
;
17
:
71
84
24.
Holst
JJ
.
The physiology of glucagon-like peptide 1
.
Physiol Rev
2007
;
87
:
1409
1439
25.
Plamboeck
A
,
Veedfald
S
,
Deacon
CF
, et al
.
The role of efferent cholinergic transmission for the insulinotropic and glucagonostatic effects of GLP-1
.
Am J Physiol Regul Integr Comp Physiol
2015
;
309
:
R544
R551
26.
Kielgast
U
,
Asmar
M
,
Madsbad
S
,
Holst
JJ
.
Effect of glucagon-like peptide-1 on alpha- and beta-cell function in C-peptide-negative type 1 diabetic patients
.
J Clin Endocrinol Metab
2010
;
95
:
2492
2496
27.
Dafaalla
MD
,
Nimir
MN
,
Mohammed
MI
,
Ali
OA
,
Hussein
A
.
Risk factors of diabetic cardiac autonomic neuropathy in patients with type 1 diabetes mellitus: a meta-analysis
.
Open Heart
2016
;
3
:
e000336
28.
Siafarikas
A
,
Johnston
RJ
,
Bulsara
MK
,
O’Leary
P
,
Jones
TW
,
Davis
EA
.
Early loss of the glucagon response to hypoglycemia in adolescents with type 1 diabetes
.
Diabetes Care
2012
;
35
:
1757
1762
29.
Bolli
G
,
de Feo
P
,
Compagnucci
P
, et al
.
Abnormal glucose counterregulation in insulin-dependent diabetes mellitus. Interaction of anti-insulin antibodies and impaired glucagon and epinephrine secretion
.
Diabetes
1983
;
32
:
134
141
30.
Knop
FK
,
Aaboe
K
,
Vilsbøll
T
, et al
.
Impaired incretin effect and fasting hyperglucagonaemia characterizing type 2 diabetic subjects are early signs of dysmetabolism in obesity
.
Diabetes Obes Metab
2012
;
14
:
500
510
31.
Johansen
NJ
,
Dejgaard
TF
,
Lund
A
, et al
.
Efficacy and safety of meal-time administration of short-acting exenatide for glycaemic control in type 1 diabetes (MAG1C): a randomised, double-blind, placebo-controlled trial
.
Lancet Diabetes Endocrinol
2020
;
8
:
313
324
32.
Dejgaard
TF
,
Frandsen
C
,
Kielgast
U
, et al
.
Liraglutide preserved insulin secretion in adults with newly diagnosed type 1 diabetes: the NewLira trial
.
Diabetologia
2019
;
62
(
Suppl 1
):
S75
33.
Dejgaard
TF
,
Frandsen
CS
,
Hansen
TS
, et al
.
Efficacy and safety of liraglutide for overweight adult patients with type 1 diabetes and insufficient glycaemic control (Lira-1): a randomised, double-blind, placebo-controlled trial
.
Lancet Diabetes Endocrinol
2016
;
4
:
221
232
34.
Hari Kumar
KVS
,
Shaikh
A
,
Prusty
P
.
Addition of exenatide or sitagliptin to insulin in new onset type 1 diabetes: a randomized, open label study
.
Diabetes Res Clin Pract
2013
;
100
:
e55
e58
35.
Sarkar
G
,
Alattar
M
,
Brown
RJ
,
Quon
MJ
,
Harlan
DM
,
Rother
KI
.
Exenatide treatment for 6 months improves insulin sensitivity in adults with type 1 diabetes
.
Diabetes Care
2014
;
37
:
666
670
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