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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-0244
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 8 2910-2916
Copyright © 2007 by The Endocrine Society

Meal-Stimulated Glucagon Release Is Associated with Postprandial Blood Glucose Level and Does Not Interfere with Glycemic Control in Children and Adolescents with New-Onset Type 1 Diabetes

Sven Pörksen, Lotte B. Nielsen, Anne Kaas, Mirjana Kocova, Francesco Chiarelli, Cathrine Ørskov, Jens J. Holst, Kenneth B. Ploug, Philip Hougaard, Lars Hansen, Henrik B. Mortensen the Hvidøre Study Group on Childhood Diabetes

Pediatric Department (S.P., L.B.N., A.K., H.B.M.) and Department of Neurology (K.B.P.), Glostrup University Hospital, DK-2600 Glostrup, Denmark; Pediatric Clinic (M.K.), Department of Endocrinology and Genetics, 91000 Skopje, Republic of Macedonia; Clinica Pediatrica (F.C.), Ospedale Policlinico, 66013 Chieti, Italy; Medical Anatomy (C.Ø.) and Medical Physiology (J.J.H.), The Panum Institute, DK-2200 Copenhagen, Denmark; Statistics (P.H.), University of Southern Denmark, DK-5230 Odense, Denmark; and Development Projects (S.P., L.H.), Novo Nordisk A/S, DK-2880 Bagsværd, Denmark

Address all correspondence and requests for reprints to: Sven Pörksen, Department of Pediatrics, Forskerparken, Glostrup University Hospital, DK-2600 Glostrup, Denmark. E-mail: svepor01{at}glo.regionh.dk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The role of glucagon in hyperglycemia in type 1 diabetes is unresolved, and in vitro studies suggest that increasing blood glucose might stimulate glucagon secretion.

Objective: Our objective was to investigate the relationship between postprandial glucose and glucagon level during the first 12 months after diagnosis of childhood type 1 diabetes.

Design: We conducted a prospective, noninterventional, 12-month follow-up study conducted in 22 centers in 18 countries.

Patients: Patients included 257 children and adolescents less than 16 yr old with newly diagnosed type 1 diabetes; 204 completed the 12-month follow-up.

Setting: The study was conduced at pediatric outpatient clinics.

Main Outcome Measures: We assessed residual ß-cell function (C-peptide), glycosylated hemoglobin (HbA1c), blood glucose, glucagon, and glucagon-like peptide-1 (GLP-1) release in response to a 90-min meal stimulation (Boost) at 1, 6, and 12 months after diagnosis.

Results: Compound symmetric repeated-measurements models including all three visits showed that postprandial glucagon increased by 17% during follow-up (P = 0.001). Glucagon levels were highly associated with postprandial blood glucose levels because a 10 mmol/liter increase in blood glucose corresponded to a 20% increase in glucagon release (P = 0.0003). Glucagon levels were also associated with GLP-1 release because a 10% increase in GLP-1 corresponded to a 2% increase in glucagon release (P = 0.0003). Glucagon levels were not associated (coefficient –0.21, P = 0.07) with HbA1c, adjusted for insulin dose. Immunohistochemical staining confirmed the presence of Kir6.2/SUR1 in human {alpha}-cells.

Conclusion: Our study supports the recent in vitro data showing a stimulation of glucagon secretion by high glucose levels. Postprandial glucagon levels were not associated with HbA1c, adjusted for insulin dose, during the first year after onset of childhood type 1 diabetes.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
GLUCAGON IS THE main counterregulatory hormone to insulin for maintaining glucose homeostasis in humans. Low blood glucose levels result in cessation of insulin secretion and increased glucagon secretion. In the fasting state, glucagon stimulates glycogenolysis and gluconeogenesis and thereby increases hepatic glucose output to maintain euglycemia. Vilsbøll et al. (1) found that across small study groups of normal, obese, type 2 diabetic, and type 1 diabetic subjects, glucagon levels increased after a meal test, and in recent studies, Salehi et al. (2) were able to show that glucose at high concentrations stimulates glucagon secretion in rodents in a dose-dependent manner. Furthermore, Olsen et al. (3) reported that glucose stimulates glucagon secretion in isolated rat {alpha}-cells in a manner similar to glucose-stimulated insulin secretion because glucose, arginine, and tolbutamide were all able to stimulate glucagon release. This glucose sensing-glucagon secretion coupling presumably acts via the KATP channel complex Kir6.2/SUR1, and the existence of this channel complex has previously been shown in rat {alpha}-cells (4). The detailed mechanism of glucagon secretion in vivo is, however, not yet clarified, but we hypothesized that a similar {alpha}-cell glucose sensing might be found in humans as well. Therefore, we investigated the relationship between postprandial blood glucose levels and glucagon release under experimental conditions where glucose-induced insulin secretion is gradually lost, namely in early type 1 diabetes.

Type 1 diabetes develops as a result of absolute insulin deficiency because of autoimmune destruction of the pancreatic ß-cell (5, 6). At diagnosis, there is, however, a clinically significant amount of ß-cell mass left, and after onset of the disease, many patients enter a period of clinical remission with decreased or no insulin needs because they are able to maintain near normoglycemia with their residual ß-cell function (7, 8, 9). During disease progression, the ß-cell destruction continues and usually the ß-cell function reaches clinical insignificance with time. Most children and adolescents with type 1 diabetes are no longer in remission 1 yr after diagnosis, and therefore the period in between, from diagnosis and 12 months ahead, offers ideal conditions for studying postprandial glucagon release in vivo in a physiological model where insulin secretion decreases.

Another important regulator of glucagon secretion is glucagon-like peptide-1 (GLP-1), which inhibits glucagon secretion from the {alpha}-cell. GLP-1 is an incretin hormone released from the L-cells in the small intestine in response to nutrients. It increases glucose-induced insulin secretion but also slows the rate of gastric emptying, together resulting in a significant blunting effect on postprandial glucose excursions in both type 1 and type 2 diabetes (10, 11, 12). However, the relation between physiological stimulation of endogenous GLP-1 and glucagon in response to a meal in the absence of significant endogenous insulin release and without the concomitant administration of exogenous insulin has to our knowledge not been studied.

The aim of our study was to investigate the relationship between meal-stimulated postprandial blood glucose levels and glucagon release in 257 children and adolescents during the first 12 months after diagnosis. We also investigated the relationship between postprandial glucagon levels and glycemic control as well as the relation between postprandial GLP-1 and glucagon. Finally, we studied the colocalization of Kir6.2 and SUR1 proteins with glucagon in human pancreatic islets.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The study was conducted in 18 centers representing 15 countries in Europe and Japan. A total of 257 children and adolescents (144 girls and 131 boys) were followed for 12 months from the diagnosis of type 1 diabetes. Clinical information on demographics, anthropometry, and insulin therapy as well as blood samples for centralized measurement of glycosylated hemoglobin (HbA1c) and meal-stimulated C-peptide, glucagon, and GLP-1 were collected prospectively. Exclusion criteria were non-type-1 diabetes (mature-onset diabetes of the young, secondary diabetes, etc.), decline of enrollment into the study by patients or parents, and patients initially treated outside the centers for more than 5 d. Patients were diagnosed according to the World Health Organization criteria.

The cohort included 131 girls (mean age 9.05 yr) and 126 boys (mean age 9.09 yr), 85% of which were white Caucasian. Age at clinical diagnosis was 9.1 ± 3.7 yr (mean ± SD), age range 0.2–16.8 yr, body mass index 16.5 ± 3.2 kg/m2, and HbA1c 11.2 ± 2.1%. Diabetic ketoacidosis was present in 20.6% of the cases at the time of diagnosis (HCO3 ≤ 15 mmol/liter and/or pH < 7.30).

The study was performed according to the criteria of the Helsinki II Declaration and was approved by the local ethical committee in each center. All patients, their parents, or guardians gave informed consent.

Residual ß-cell function

C-peptide is secreted in equimolar quantities with insulin and therefore reflects insulin release and thereby ß-cell function (13). We used stimulated serum C-peptide as a marker of residual ß-cell function after a disease duration of 1, 6, and 12 months. The patients received the last insulin dose the evening before the study day and reported to the lab after at least 8 h of fasting (no morning insulin or breakfast). Fasting blood glucose levels where measured just before the ingestion of a liquid meal [Boost drink, formerly known as Sustacal (237 ml or 8 fluid ounces containing 33 g carbohydrate, 15 g protein, and 6 g fat, 240 kcal); 6 ml/kg (maximal 360 ml); Novartis Medical Health, Inc., Minneapolis, MN, www.boost.com]. Blood glucose was measured, and blood was drawn 90 min after the ingestion of the drink according to Diabetes Control and Complications Trial standards (14). Serum samples were labeled and frozen at –20 C until shipment on dry ice for the determination of C-peptide, glucagon, and GLP-1. An appropriate dose of insulin was given immediately after the 90-min sample was taken.

Serum C-peptide was analyzed by a fluoroimmunometric assay (AutoDELFIA C-peptide; PerkinElmer Life and Analytical Sciences, Inc., Turku, Finland). The sensitivity was less than 5 pmol/liter, intraassay coefficient of variation less than 6% at 20 pmol/liter, and recovery of standard, added to plasma before extraction, about 100% when corrected for losses inherent in the plasma extraction procedure.

Glucagon and GLP-1

Glucagon and GLP-1 concentrations in plasma sampled at 90 min were measured after extraction of plasma with 70% ethanol (vol/vol, final concentration). The glucagon RIA was directed against the C terminus of the glucagon molecule (antibody code no. 4305) and therefore mainly measures glucagon of pancreatic origin (15). The plasma concentrations of GLP-1 were measured (16) against standards of synthetic GLP-1(7–36) amide using antiserum code no. 89390, which is specific for the amidated C terminus of GLP-1 and therefore mainly reacts with GLP-1 of intestinal origin. The assay reacts equally with intact GLP-1 and with GLP-1(3–36) amide, the primary metabolite. Because of the rapid and intravascular conversion of GLP-1 to their primary metabolites, it is essential to determine both the intact hormone and the metabolite for estimation of the rate of secretion of GLP-1. For both assays, sensitivity was less than 1 pmol/liter, intraassay coefficient of variation less than 6% at 20 pmol/liter, and recovery of standard, added to plasma before extraction, about 100% when corrected for losses inherent in the plasma extraction procedure.

Glycemic control

Glycemic control as assessed by HbA1c was measured at diagnosis and 1, 3, 6, 9, and 12 months after the diagnosis. HbA1c was determined centrally by ion-exchange HPLC (normal reference range 4.1–6.4%) at Steno Diabetes Center, Gentofte, Denmark.

Insulin dose-adjusted HbA1c

We used the glycemic control as a marker of disease severity and investigated whether it is influenced by postprandial glucagon levels. The blood glucose-lowering effect of exogenous insulin has a profound effect on the HbA1c. Therefore, the HbA1c and the insulin dose cannot be considered separately. A unified suggestion, in which both parameters were included, was found by multiple regression analysis with the logarithm of C-peptide as the dependent variable and sex, age, HbA1c, and daily insulin dose (U/kg body weight) as independent variables at 1, 6, and 12 months. The multiple regression analysis showed that there was a negative correlation between stimulated C-peptide and HbA1c and insulin dose. A combined expression of dose-adjusted HbA1c was then formulated: HbA1c (%) + 4 x insulin dose (IU/kg·d). This measure, adjusting for the exogenous insulin, reflects the underlying and theoretically untreated disease and in this setting therefore is superior to the HbA1c alone.

Immunohistochemistry analysis of Kir6.2/SUR1 and glucagon colocalization on human pancreas sections

All immunohistochemistry was carried out on formalin-fixed material. Human pancreatic tissue (n = 6) was obtained from archival material collected during the 1970s according to the contemporary ethical guidelines.

The antibodies used for immunohistochemistry were polyclonal rabbit anti-GLP-1 antibody code GLP2135 diluted 1:10,000 (17), recognizing the large proglucagon peptide, polyclonal goat anti-Kir6.2 code N-18 diluted 1:3000 (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal goat-anti SUR1 code C-16 diluted 1:1000 (Santa Cruz Biotechnology).

Before the immunohistochemical staining, the tissue sections were dewaxed and subjected to antigen retrieval by microwave irradiation in 0.5 mM EGTA buffer (pH 9.0).

For double-labeling experiments with the antibody combination glucagon and Kir6.2 and glucagon and SUR1, the tissue sections were incubated overnight with the primary antibodies at 4 C. Bound goat antibodies were visualized using biotinylated antigoat antibody (1:200; The Binding Site, Birmingham, UK), streptavidin coupled to peroxidase and biotinylated tyramide using the Perkin-Elmer TSA-indirect kit as described by the manufacturer (Perkin-Elmer) and finished by incubation with streptavidin coupled to Texas Red (Amersham, Piscataway, NJ). Bound rabbit antibodies (2135) were visualized using digoxigenin-coupled antirabbit antibody (both 1:25; Chemicon, Temecula, CA) and fluorescein-labeled antidigoxigenin antibody (1:200; ImmunoResearch Laboratories, Suffolk, UK).

Statistical methods

Glucagon, GLP-1, and C-peptide concentrations were all considered on the logarithmic scale. Glucagon was analyzed using multiple regression with age, sex, postprandial glucose, GLP-1, and C-peptide as explanatory variables. HbA1c, adjusted for insulin dose, was analyzed using multiple regression with age, sex, and C-peptide as explanatory variables. To gain power, we performed analyses including all time points (1, 6, and 12 months) using compound symmetric repeated-measurements models. To assure that the assumption of compound symmetry is correct, we performed the analyses with an unstructured variance test confirming that our model of compound symmetry is appropriate. Analyses for single time points were performed using multiple regression analyses only. A P value < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
ß-Cell function

In our study, the children and adolescents with new-onset type 1 diabetes experienced a continuous loss of ß-cell function as meal-stimulated C-peptide declined by approximately 50% from the first visit at 1 month after diagnosis to the 12-month visit (P < 0.0001, Fig. 1AGo). Throughout the study period, the older children had higher residual ß-cell function and overall 10% higher C-peptide levels per year of age (P < 0.0001, not shown).


Figure 1
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  		FIG. 1. Disease progression during 11 months of follow-up. A, Meal-stimulated C-peptide levels (pmol/liter) decreased by 50% (P < 0.0001); B, meal-stimulated blood glucose (BG) levels increased by 6.6 mmol/liter (P < 0.0001); C, meal-stimulated glucagon levels (pmol/liter) increased by 17% (P = 0.001); D, meal-stimulated GLP-1 levels (pmol/liter) increased by 24% (P < 0.0001); E, dose-adjusted HbA1c decreased from onset until the 3-month visit and increased continuously afterward (P < 0.0001). C-peptide, glucagon, and GLP-1 are all considered on the logarithmic scale.

 
We used multiple regression models to evaluate the association between blood glucose levels and stimulated C-peptide (adjusted for sex and age) at 1, 6, and 12 months after diagnosis as shown in Fig. 2AGo. There was no significant association between postprandial blood glucose levels and ß-cell function (P = 0.06) at 1 month. By contrast at 6 and 12 months, blood glucose levels were negatively associated with residual ß-cell function because C-peptide levels declined by 40% at 6 months (P = 0.0006, not shown) and 70% at 12 months (P < 0.0001) per additional 10 mmol/liter increase in stimulated blood glucose values.


Figure 2
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  		FIG. 2. A, The association of meal-stimulated blood glucose (BG) and C-peptide (pmol/liter) levels at 1 (P = 0.06), 6 (P = 0.0006, not shown), and 12 months (P < 0.0001) after diagnosis of type 1 diabetes; B, glucagon (pmol/liter) levels are independent of blood glucose levels 1 month (P = 0.55) after diagnosis of type 1 diabetes but highly associated 6 and 12 months after diagnosis (P = 0.004 at 6 months, not shown; P = 0.03 at 12 months); C, glucagon (pmol/liter) levels are independent of GLP-1 (pmol/liter) levels 1 month after diagnosis (P = 0.22) of type 1 diabetes but highly associated 6 and 12 months after diagnosis (P = 0.004 at 6 months, not shown; P = 0.009 at 12 months). C-peptide, glucagon, and GLP-1 are all considered on the logarithmic scale.

 
{alpha}-Cell function

During the study period, {alpha}-cell function (meal-stimulated glucagon) increased by 17% from the first visit 1 month after diagnosis to the 12-month visit (P = 0.001, Fig. 1CGo). Compound symmetric repeated-measurements models showed that throughout the study period (including all three visits), the increasing levels of glucagon during disease progression were independent of age, sex, residual ß-cell function (meal-stimulated C-peptide), and fasting blood glucose. The postprandial glucagon levels were, however, highly associated with the rise in postprandial blood glucose levels; an increment of 10 mmol/liter in blood glucose corresponded to a 20% increase in glucagon release (P = 0.0003, not shown). We also evaluated the relationship between glucagon release and postprandial blood glucose with multiple regression analyses for each visit at 1, 6, and 12 months alone. We found no statistically significant influence of postprandial blood glucose on the glucagon level 1 month after diagnosis (P = 0.55). However, 6 and 12 months after diagnosis, postprandial blood glucose increments of 10 mmol/liter corresponded to a 20% rise in glucagon release at both time points (P = 0.004, 6 months; P = 0.03, 12 months; Fig. 2BGo). Because of the few blood glucose values above 20 mM at 1 month, the slope of the line at 1 month (Fig. 2BGo) may appear different from the line at 12 months. However, when tested with a compound symmetric repeated-measurements model, including sex and age, we found that the slopes were not statistically different (P = 0.41). This indicates that the association between stimulated blood glucose and glucagon is significant only when the levels are sufficiently high.

Endogenous GLP-1 and islet function

Endogenous, meal-stimulated GLP-1 levels increased by 24% (P < 0.0001) from the first visit at 1 month after diagnosis to the 12-month visit (Fig. 1DGo). GLP-1 levels were independent of age (P = 0.13). At 1 month after diagnosis, multiple regression analysis revealed that postprandial GLP-1 levels were positively associated with ß-cell function (meal-stimulated C-peptide); a relative increment in GLP-1 of 10% was associated with 3% higher meal stimulated C-peptide levels (P = 0.005). However, at 6 and 12 months, postprandial GLP-1 was no longer associated with ß-cell function (P = 0.56; P = 0.19, data not shown).

Likewise, the association of postprandial GLP-1 with glucagon levels was investigated in a repeated-measurements model, adjusted for age, sex, C-peptide, and stimulated blood glucose. Glucagon and GLP-1 were significantly associated; a 10% increase in GLP-1 release corresponded to a 2% increase in glucagon release (P = 0.0003). As in the comparison between glucagon and blood glucose, we evaluated whether the relationships were present at each of the three time points. Therefore we performed multiple regression analyses for each visit at 1, 6, and 12 months alone. We found no statistically significant association between glucagon and GLP-1 1 month after diagnosis (P = 0.21). However, 6 and 12 months after diagnosis, postprandial GLP-1 and glucagon were positively associated because a 10% increase in GLP-1 levels corresponded to a 2% rise in glucagon levels (P = 0.004, 6 months; P = 0.009, 12 months; Fig. 2CGo). When we investigated the direct relationship between stimulated blood glucose and GLP-1, we found the same relationship as for glucagon; GLP-1 was significantly associated with the stimulated blood glucose 6 months (P = 0.0001) and 12 months (P = <0.0001) after diagnosis but not after 1 month (P = 0.19, data not shown). We also investigated the relationship between glucagon and glucose-dependent insulinotropic peptide in a repeated-measurements model but found no association (P = 0.27, data not shown).

Glycemic control

The insulin dose-adjusted HbA1c decreased from onset until the third month after diagnosis. Hereafter the values increased continuously without reaching the level from disease onset (Fig. 1EGo). Throughout the period, the dose-adjusted HbA1c increased with increasing age as evaluated by a compound symmetric repeated-measurements model (0.1% per year, P < 0.0001). Girls had 0.38% (P = 0.049) higher dose-adjusted HbA1c levels than boys. The dose-adjusted HbA1c was highly dependent on meal-stimulated C-peptide levels because a doubling in C-peptide corresponded to a decrease of 0.4% in the insulin dose-adjusted HbA1c (P < 0.0001, not shown). Postprandial glucagon secretion is believed to deteriorate glycemic control in diabetes, but in our study, we found no significant association between dose-adjusted HbA1c and postprandial glucagon levels when all three time points were considered in a compound symmetric repeated-measurements model adjusted for sex, age, and meal-stimulated C-peptide (coefficient –0.21, P = 0.07).

Kir6.2 and SUR1 in human {alpha}-cells

To investigate the presence of the KATP channel complex Kir6.2/SUR1 in human {alpha}-cells we performed immunohistochemical staining of human pancreatic sections. This study showed the colocalization of Kir6.2 and SUR1 in human {alpha}-cells (Fig. 3Go) and confirmed at the anatomical level that a KATP channel complex, identical to that expressed in pancreatic ß-cells, is likely to exist in the human {alpha}-cell as well.


Figure 3
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  		FIG. 3. Detection of Kir6.2 and SUR1 in human pancreatic {alpha}-cells. Immunohistochemical staining shows the colocalization of Kir6.2/SUR1 and glucagon in human pancreatic {alpha}-cells. A, Merged picture showing the bound Texas Red-labeled SUR1 and fluorescein-labeled glucagon antibodies. Red color localizes SUR1 alone, whereas the yellow color shows the colocalization of SUR1 and glucagon. B, Merged picture showing the bound Texas Red-labeled Kir6.2 and fluorescein-labeled glucagon antibodies. Red color localizes Kir6.2 alone, whereas the yellow color shows the colocalization of Kir6.2 and glucagon.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present study, we describe the meal-stimulated {alpha}-cell response in patients with newly diagnosed type 1 diabetes during 12 months of follow-up. At three time points (1, 6, and 12 months after diagnosis) we performed meal stimulation tests to evaluate C-peptide, glucagon, GLP-1, and blood glucose levels in a physiological setting in humans. We found that the decreasing ß-cell function during the first year of type 1 diabetes was associated with increasing postprandial glucagon, GLP-1, and blood glucose. Glucagon levels were highly associated with both blood glucose levels and GLP-1 levels but not with age, sex, and residual ß-cell function (meal-stimulated C-peptide). At the first visit, 1 month after diagnosis, we did not find any association of blood glucose or GLP-1 with glucagon, but this was also the time where the remaining ß-cell function was found to be of clinical significance in most children. Later, at the 6- and 12-month visits, the ß-cell function had declined further, allowing the positive association of blood glucose and GLP-1 with glucagon to become apparent.

The regulation of glucagon secretion is still not fully understood, but there is evidence that insulin and/or other paracrine factors in the pancreatic islet participate in {alpha}-cell regulation (18). Recently, new mechanisms for the regulation of glucagon secretion were identified, which may question the traditionally held concepts regarding the cause and effect relationship between glucagon and postprandial hyperglycemia in patients with insulin-deficient diabetes; Olsen et al. (3) showed that glucose stimulated glucagon secretion in a dose-dependent manner in rat {alpha}-cells when the cells were isolated from their paracrine intra-islet environment. The study also showed that tolbutamide was able to stimulate and diazoxide (a KATP channel opener) was able to inhibit glucagon secretion, clearly indicating a role of the KATP channel complex Kir6.2/SUR1 in pancreatic {alpha}-cells. This evidence is further corroborated by our studies showing expression of the Kir6.2/SUR1 complex in human {alpha}-cells and by studies in type 1 diabetic patients where the KATP channel inhibitor glibenclamide increases glucagon levels, analogous to its effect of increasing the insulin levels in type 2 diabetes (19). In the intact islet, these mechanisms may not be evident because glucagon secretion is influenced by inhibitory paracrine factors secreted by the ß-cell. We found that prevailing postprandial blood glucose levels at 1 month were too low to show a significant association with postprandial glucagon release. However, when postprandial blood glucose increases after 6 and 12 months (due to further decline in residual ß-cell function), the {alpha}-cell might respond to increasing glucose levels by secreting glucagon. Together, this supports the concept that the {alpha}-cells regulate glucagon release with a glucose sensing-secretion coupling mechanism similar to the one present in ß-cells.

Our study also provides new insight into the role of postprandial hyperglucagonemia in glycemic control. We found that throughout the study period (including all three time points), high postprandial glucagon levels were not associated with deterioration in glycemic control, as assessed by insulin dose-adjusted HbA1C, when considered in a repeated-measurements model. Furthermore, fasting blood glucose levels were not associated with increase in postprandial glucagon release, indicating that the high glucagon levels are not primarily involved in hyperglycemia but rather secondary to the postprandial hyperglycemia per se.

The normal pattern of glucagon secretion in response to hypoglycemia together with the findings in our study, where glucagon levels increased with increasing blood glucose levels, support the idea of a U-shaped, bimodal glucagon secretion pattern as recently suggested by Salehi et al. (2) (Fig. 4Go).


Figure 4
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  		FIG. 4. Bimodal distribution of glucose-induced glucagon response. BG, Blood glucose.

 
The defective glucagon response in type 1 diabetes may play a major role in the development of hypoglycemia, and severe nocturnal hypoglycemic episodes are some of the most feared complications as experienced by children and adolescents with type 1 diabetes and their parents (6, 20). The presence of K+ATP channels (Kir6.2/SUR1 complex) in human {alpha}-cells that are responsive to sulfonylureas suggests that the defective glucagon response to low glucose in type 1 diabetic patients could be ameliorated by activation of this pathway/mechanism with a sulfonylurea. This indication, however, requires additional experimental studies before any clinical recommendations can be made.

In our study, we also found that GLP-1 was positively associated with glucagon release in response to the (carbohydrate-enriched) liquid meal. In pharmacological doses, GLP-1 is known to suppress glucagon release in a glucose-dependent manner (9, 10, 21, 22, 23). Experimental studies by Hansen et al. (24) and Gribble et al. (25) also indicate that the L-cell may respond to vascular glucose concentrations, and clinical studies in type 2 diabetic patients found plasma glucose concentrations to correlate with both basal and postprandial GLP-1 levels (24, 25, 26). Another explanation for the positive association could be that GLP-1 may directly stimulate glucagon secretion in insulin-deficient islets. Indeed, Ding et al. (27) demonstrated that GLP-1 stimulates glucagon secretion from isolated {alpha}-cells.

In conclusion, our in vivo data support the recent in vitro data showing a stimulation of glucagon secretion by high glucose. This effect might very well be mediated through a glucose-sensing glucagon-secretion coupling mechanism involving the K+ATP channel complex Kir6.2/SUR1. Postprandial glucagon levels were not associated with impaired glycemic control, as assessed by insulin dose-adjusted HbA1c, during the first year after onset of childhood type 1 diabetes.


   Acknowledgments
 
We thank Novo Nordisk for support throughout this study, with special thanks to Lene Kaa Meier and Stanislav Smirnov. We are also grateful to the technicians Britta Drangsfeldt and Susanne Kjelberg at Steno Diabetes Centre for their assistance. We thank the Danish Diabetes Association for financial support.


   Footnotes
 
Disclosure Statement: S.P., L.B.N., A.K., M.K., F.C., C.Ø., K.B.P., and H.B.M. have nothing to declare. L.H. and P.H. have equity interests in Novo Nordisk A/S. J.J.H. consults for Novo Nordisk A/S and Merck (MSD) and has received lecture fees from Merck (MSD).

First Published Online May 22, 2007

Abbreviations: GLP-1, Glucagon-like peptide-1; HbA1c, glycosylated hemoglobin.

Received February 1, 2007.

Accepted May 14, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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