TY - JOUR
AU - Bergsten,, Peter
AB - Abstract Context: Proglucagon-derived hormones are important for glucose metabolism, but little is known about them in pediatric obesity and type 2 diabetes mellitus (T2DM). Objective: Fasting and postprandial levels of proglucagon-derived peptides glucagon, GLP-1, and glicentin in adolescents with obesity across the glucose tolerance spectrum were investigated. Design: This was a cross-sectional study with plasma hormone levels quantified at fasting and during an oral glucose tolerance test (OGTT). Setting: This study took place in a pediatric obesity clinic at Uppsala University Hospital, Sweden. Patients and Participants: Adolescents with obesity, age 10–18 years, with normal glucose tolerance (NGT, n = 23), impaired glucose tolerance (IGT, n = 19), or T2DM (n = 4) and age-matched lean adolescents (n = 19) were included. Main Outcome Measures: Outcome measures were fasting and OGTT plasma levels of insulin, glucagon, active GLP-1, and glicentin. Results: Adolescents with obesity and IGT had lower fasting GLP-1 and glicentin levels than those with NGT (0.25 vs 0.53 pM, P < .05; 18.2 vs 23.6 pM, P < .01) and adolescents with obesity and T2DM had higher fasting glucagon levels (18.1 vs 10.1 pM, P < .01) than those with NGT. During OGTT, glicentin/glucagon ratios were lower in adolescents with obesity and NGT than in lean adolescents (P < .01) and even lower in IGT (P < .05) and T2DM (P < .001). Conclusions: Obese adolescents with IGT have lowered fasting GLP-1 and glicentin levels. In T2DM, fasting glucagon levels are elevated, whereas GLP-1 and glicentin levels are maintained low. During OGTT, adolescents with obesity have more products of pancreatically than intestinally cleaved proglucagon (ie, more glucagon and less GLP-1) in the plasma. This shift becomes more pronounced when glucose tolerance deteriorates. The prevalence of childhood obesity has increased worldwide in the past decades, which has been accompanied by a rise in obesity-related comorbidities such as type 2 diabetes mellitus (T2DM) (1). The impact of T2DM on morbidity and mortality underlines the importance of identifying individuals with obesity at risk and creating a better understanding of underlying pathophysiological mechanisms in T2DM development in adolescents with obesity (2). The prevalence of intermediate hyperglycemia, or prediabetes, which is constituted by impaired fasting glucose or impaired glucose tolerance (IGT), varies greatly between populations but is high in pediatric obesity (3). Glucose intolerance in childhood is associated with increased mortality rates in early adulthood and increased risk of early-onset T2DM (4, 5). While impaired fasting glucose is identified by fasting blood sampling, the assessment of glucose tolerance requires a 2-hour oral glucose tolerance test (OGTT). Because an OGTT is time-consuming and inconvenient to use as a routine screening procedure, fasting biomarkers of IGT are needed. The understanding of the importance of proglucagon-derived hormones in glucose metabolism has increased substantially during recent years (6). Proglucagon is processed in a tissue-specific manner, to GLP-1 and glicentin together with other peptides in intestinal endocrine L-cells and to glucagon in α-cells in pancreatic islets of Langerhans (7, 8). Upon nutrient intake, secretion of GLP-1 and glicentin is stimulated (9, 10), whereas glucagon is suppressed by glucose and stimulated by protein intake (11, 12). The most well-known effects of glucagon are on the liver to stimulate glucose output, whereas GLP-1 has competitive actions on the pancreatic islet in the sense that it enhances insulin secretion and lowers glucagon secretion in addition to affecting many other cell types and organs (13). Little is known about glicentin, but reported effects include regulation of gastrointestinal motility (14), gastric acid secretion (15), and insulin secretion (16). Obesity is related to a decreased stimulation of GLP-1 release upon nutrient intake (17), and hyperglucagonemia is well documented in adult obesity and T2DM (18, 19). However, there are few studies of the proglucagon-derived hormones in the context of pediatric obesity and T2DM. The primary aim of the present study was to investigate fasting and postprandial plasma levels of the proglucagon-derived hormones glucagon, glicentin, and active GLP-1, with reported effects on insulin secretion and/or glucose tolerance, in lean adolescents and adolescents with obesity with and without IGT or T2DM. The secondary aim was to evaluate fasting glucagon, glicentin, and active GLP-1 levels as predictors of IGT in adolescents with obesity. Materials and Methods Study population Lean adolescents (n = 19) and adolescents with obesity and normal glucose tolerance (NGT; n = 23), IGT (n = 19), and T2DM (n = 4) matched for age and with glucose data from OGTT available were included. All subjects were part of the Uppsala Longitudinal Study of Childhood Obesity cohort (20) and visited the obesity clinic between June 2012 and October 2014. Inclusion criteria were age 10–18 years, for lean adolescents a Body Mass Index Standard Deviation Score (BMI-SDS) below 1.0 (equivalent to adult BMI 25), and for adolescents with obesity a BMI-SDS of higher than 2 (equivalent to adult BMI 30). Exclusion criteria were ongoing medical treatment with antihyperglycemic agents and/or syndromic obesity. Written informed consent was obtained from study subjects and legal guardians. The study was approved by the local ethics committee (registration number 2012/318). Glucose tolerance was defined according to World Health Organization criteria for plasma glucose and thus the definitions were: T2DM fasting glucose higher than 7.0 mM and/or two-hour glucose higher than 11.1 mM, IGT two-hour glucose higher than 7.8 mM and below 11.1 mM, and NGT fasting glucose below 6.1 mM and two-hour glucose below 7.8 mM. Among the adolescents with obesity and IGT, 11 had normal fasting plasma glucose (ie, below 6.1 mM). Anthropometry Body weight was measured by a digital scale and height by a stadiometer. The age- and gender-adjusted BMI (ie, the BMI-SDS) was calculated according to the World Health Organization 2006–2007 growth reference. Pubertal status was determined by Tanner staging when available and, when not available, by locally derived sex specific cutoffs of testosterone and assessment of growth charts for height. Oral glucose tolerance test After a 12-hour overnight fast, subjects underwent a standard OGTT ingesting glucose (APL) dissolved in water in a dosage of 1.75 g glucose/kg body weight (maximum 75 g), as previously described (20). Blood was drawn from a patent venous catheter inserted after application of a local anesthetic patch (EMLA, AstraZeneca). Blood samples were collected at time points −5, 5, 10, 15, 30, 60, 90, and 120 minutes from glucose intake (0 minutes). Blood samples for determination of GLP-1, glucagon, and glicentin were collected in vacutainer tubes containing a protease inhibitor cocktail including dipeptidyl peptidase-4 inhibitors (P800, Becton Dickinson). Blood samples for determination of insulin were collected in EDTA-containing vacutainer tubes (Becton Dickinson). Tubes were immediately placed on ice and centrifuged at 4 C for 10 minutes. Plasma was stored in a biobank at −80 C until analyses. Biochemical analyses Plasma glucose levels were determined by a glucose oxidation method (Architect c8000 instrument, Abbott Diagnostics). Plasma levels of active GLP-1 were quantified by an electrochemiluminescent enzyme-linked assay (Mesoscale Discoveries) detecting the 7–36-amide GLP-1 as well as cross-reacting 37% to 7–37 GLP-1. The limit of detection of the assay was 0.04 pM and all values were above the limit of detection. The assay has little or no cross-reactivity with other forms of GLP-1, including the predominant inactive form 9–37 GLP-1. No cross-reactivity to other proglucagon-derived peptides has been detected. Plasma levels of insulin, glucagon, and glicentin were quantified by ELISA (Mercodia). The glucagon assay uses one C-terminal and one N-terminal antibody to eliminate cross-reactivity to any other proglucagon-derived peptides. The glicentin ELISA uses two side-viewing antibodies, one toward the glucagon sequence and the other toward the glicentin-related poly-peptide sequence. It has no reported detectable cross-reactivity with glucagon, oxyntomodulin, mini-glucagon, GLP-1, or GLP-2. The insulin assay has a cross-reactivity with c-peptide and proinsulin of less than 0.01%. Calculations and statistical analysis β-cell function was estimated by the oral disposition index, calculated as the product of 1/insulin0 and the insulinogenic index (ie, insulinΔ30/glucoseΔ30), as previously validated in obese youth (21). Hormone levels during OGTT was determined by area under curve (AUC), calculated by the trapezoid rule with 0 as baseline. One subject had missing 120-minute values, which for AUC calculations were estimated by the last value carried forward. Early-phase hormone response during OGTT was estimated by a stimulation index, calculated as the level at 30 minutes divided by fasting level. To study changes in pancreatically vs intestinally cleaved plasma proglucagon during OGTT, ratios between glicentin and glucagon were calculated. Data were tested for normal distribution by the Shapiro-Wilk test and for equal variance by Levene's test. In case of violation of the normality and/or equal variance assumption, differences between groups were analyzed on log-transformed data. For reasons of clarity, all data are presented as nontransformed. Differences between groups in fasting levels, AUC, or early-phase response were analyzed by one-way ANOVA with post hoc Fischer's least significant different test comparing all groups to the adolescents with obesity and NGT. Logistic regression analysis was performed to assess the ability of fasting insulin, glucose, and proglucagon-derived hormones to predict IGT among adolescents with obesity and normal fasting glucose, with P < .05 being considered significant. For significant predictors, receiver operating characteristic curve analysis was performed and cut-off points were determined from the receiver operating characteristic curve coordinates by Youden's index (22). Statistical analysis was done by GraphPad Prism 6.0 (GraphPad Software Inc.) and SPSS, version 20.0 (IBM Corp.). Level of significance was set to P < .05. Results Study population characteristics There were no significant age or gender differences between the study groups (Table 1). Furthermore, mean BMI-SDS did not differ between groups of adolescents with obesity. Distribution of pubertal stage of the study subjects did not differ between groups; however, the adolescents with obesity and T2DM were all pubertal or postpubertal. β-cell function was lower in adolescents with obesity and T2DM than in adolescents with obesity and NGT. Table 1. Clinical Characteristics and Fasting Hormone Levels of the Study Population of Lean Adolescents and Adolescents With Obesity and NGT, IGT, and T2DM . Lean (n = 19) . Obesity NGT (n = 23) . Obesity IGT (n = 19) . Obesity T2DM (n = 4) . Age (y) 14.0 ± 2.3 13.7 ± 2.1 14.0 ± 2.1 14.8 ± 2.5 Sex (n)     Male 8 14 9 1     Female 11 9 10 3 BMI 18.4 (16.3–20.4)a 32.6 (29.5–36.0) 35.7 (32.7–38.9) 34.5 (32.5–48.1) BMI-SDS −0.1 (−1.7 to 0.5)a 3.0 (2.7–3.2) 3.1 (3.0–3.5) 3.1 (2.9–4) Puberty (n)     Prepubertal 4 7 4 0     Pubertal 8 9 7 2     Postpubertal 7 7 8 2 Glucose (mM)     Fasting 5.4 ± 0.3 5.5 ± 0.3 5.9 ± 0.5b 6.9 ± 1.5a     2 h 5.8 ± 1.2c 6.6 ± 0.7 8.8 ± 0.7a 11.5 ± 2.2a     AUCOGTT 800 ± 23 858 ± 19 1018 ± 21a 1380 ± 125a Fasting insulin (pM) 42.0 ± 3.3a 119.2 ± 7.9 143.7 ± 19.2 423.7 ± 90.2a Fasting glucagon (pM) 7.6 (6.6–10.1)c 10.1 (8.2–13.7) 12.1 (8.5–14.1) 18.1 (15.7–21.4)b Fasting active GLP-1 (pM) 0.32 (0.18–0.53)c 0.53 (0.32–0.75) 0.25 (0.23–0.44)c 0.20 (0.13–0.43)c Fasting glicentin (pM) 17.6 (13.0–25.0) 23.6 (17.7–32.8) 18.2 (8.6–21.8)b 15.1 (13.3–18.0) β-cell function     Oral disposition index 3.4 (2.6–5.3)c 2.6 (1.8–3.4) 2.0 (1.3–2.8) 0.7 (0.5–1.2)a . Lean (n = 19) . Obesity NGT (n = 23) . Obesity IGT (n = 19) . Obesity T2DM (n = 4) . Age (y) 14.0 ± 2.3 13.7 ± 2.1 14.0 ± 2.1 14.8 ± 2.5 Sex (n)     Male 8 14 9 1     Female 11 9 10 3 BMI 18.4 (16.3–20.4)a 32.6 (29.5–36.0) 35.7 (32.7–38.9) 34.5 (32.5–48.1) BMI-SDS −0.1 (−1.7 to 0.5)a 3.0 (2.7–3.2) 3.1 (3.0–3.5) 3.1 (2.9–4) Puberty (n)     Prepubertal 4 7 4 0     Pubertal 8 9 7 2     Postpubertal 7 7 8 2 Glucose (mM)     Fasting 5.4 ± 0.3 5.5 ± 0.3 5.9 ± 0.5b 6.9 ± 1.5a     2 h 5.8 ± 1.2c 6.6 ± 0.7 8.8 ± 0.7a 11.5 ± 2.2a     AUCOGTT 800 ± 23 858 ± 19 1018 ± 21a 1380 ± 125a Fasting insulin (pM) 42.0 ± 3.3a 119.2 ± 7.9 143.7 ± 19.2 423.7 ± 90.2a Fasting glucagon (pM) 7.6 (6.6–10.1)c 10.1 (8.2–13.7) 12.1 (8.5–14.1) 18.1 (15.7–21.4)b Fasting active GLP-1 (pM) 0.32 (0.18–0.53)c 0.53 (0.32–0.75) 0.25 (0.23–0.44)c 0.20 (0.13–0.43)c Fasting glicentin (pM) 17.6 (13.0–25.0) 23.6 (17.7–32.8) 18.2 (8.6–21.8)b 15.1 (13.3–18.0) β-cell function     Oral disposition index 3.4 (2.6–5.3)c 2.6 (1.8–3.4) 2.0 (1.3–2.8) 0.7 (0.5–1.2)a Values are means ± sem for normally distributed variables and median and interquartile range for non-normally distributed variables. a P < .001, b P < .01, and c P < .05 and vs obesity NGT. Open in new tab Table 1. Clinical Characteristics and Fasting Hormone Levels of the Study Population of Lean Adolescents and Adolescents With Obesity and NGT, IGT, and T2DM . Lean (n = 19) . Obesity NGT (n = 23) . Obesity IGT (n = 19) . Obesity T2DM (n = 4) . Age (y) 14.0 ± 2.3 13.7 ± 2.1 14.0 ± 2.1 14.8 ± 2.5 Sex (n)     Male 8 14 9 1     Female 11 9 10 3 BMI 18.4 (16.3–20.4)a 32.6 (29.5–36.0) 35.7 (32.7–38.9) 34.5 (32.5–48.1) BMI-SDS −0.1 (−1.7 to 0.5)a 3.0 (2.7–3.2) 3.1 (3.0–3.5) 3.1 (2.9–4) Puberty (n)     Prepubertal 4 7 4 0     Pubertal 8 9 7 2     Postpubertal 7 7 8 2 Glucose (mM)     Fasting 5.4 ± 0.3 5.5 ± 0.3 5.9 ± 0.5b 6.9 ± 1.5a     2 h 5.8 ± 1.2c 6.6 ± 0.7 8.8 ± 0.7a 11.5 ± 2.2a     AUCOGTT 800 ± 23 858 ± 19 1018 ± 21a 1380 ± 125a Fasting insulin (pM) 42.0 ± 3.3a 119.2 ± 7.9 143.7 ± 19.2 423.7 ± 90.2a Fasting glucagon (pM) 7.6 (6.6–10.1)c 10.1 (8.2–13.7) 12.1 (8.5–14.1) 18.1 (15.7–21.4)b Fasting active GLP-1 (pM) 0.32 (0.18–0.53)c 0.53 (0.32–0.75) 0.25 (0.23–0.44)c 0.20 (0.13–0.43)c Fasting glicentin (pM) 17.6 (13.0–25.0) 23.6 (17.7–32.8) 18.2 (8.6–21.8)b 15.1 (13.3–18.0) β-cell function     Oral disposition index 3.4 (2.6–5.3)c 2.6 (1.8–3.4) 2.0 (1.3–2.8) 0.7 (0.5–1.2)a . Lean (n = 19) . Obesity NGT (n = 23) . Obesity IGT (n = 19) . Obesity T2DM (n = 4) . Age (y) 14.0 ± 2.3 13.7 ± 2.1 14.0 ± 2.1 14.8 ± 2.5 Sex (n)     Male 8 14 9 1     Female 11 9 10 3 BMI 18.4 (16.3–20.4)a 32.6 (29.5–36.0) 35.7 (32.7–38.9) 34.5 (32.5–48.1) BMI-SDS −0.1 (−1.7 to 0.5)a 3.0 (2.7–3.2) 3.1 (3.0–3.5) 3.1 (2.9–4) Puberty (n)     Prepubertal 4 7 4 0     Pubertal 8 9 7 2     Postpubertal 7 7 8 2 Glucose (mM)     Fasting 5.4 ± 0.3 5.5 ± 0.3 5.9 ± 0.5b 6.9 ± 1.5a     2 h 5.8 ± 1.2c 6.6 ± 0.7 8.8 ± 0.7a 11.5 ± 2.2a     AUCOGTT 800 ± 23 858 ± 19 1018 ± 21a 1380 ± 125a Fasting insulin (pM) 42.0 ± 3.3a 119.2 ± 7.9 143.7 ± 19.2 423.7 ± 90.2a Fasting glucagon (pM) 7.6 (6.6–10.1)c 10.1 (8.2–13.7) 12.1 (8.5–14.1) 18.1 (15.7–21.4)b Fasting active GLP-1 (pM) 0.32 (0.18–0.53)c 0.53 (0.32–0.75) 0.25 (0.23–0.44)c 0.20 (0.13–0.43)c Fasting glicentin (pM) 17.6 (13.0–25.0) 23.6 (17.7–32.8) 18.2 (8.6–21.8)b 15.1 (13.3–18.0) β-cell function     Oral disposition index 3.4 (2.6–5.3)c 2.6 (1.8–3.4) 2.0 (1.3–2.8) 0.7 (0.5–1.2)a Values are means ± sem for normally distributed variables and median and interquartile range for non-normally distributed variables. a P < .001, b P < .01, and c P < .05 and vs obesity NGT. Open in new tab Insulin, glucagon, active GLP-1, and glicentin levels at fasting Adolescents with obesity and NGT had 3-fold higher insulin, 30% higher glucagon, and 70% higher active GLP-1 levels than lean adolescents (Table 1). Glicentin levels did not differ between lean adolescents and adolescents with obesity and NGT. With a decline in glucose tolerance in adolescents with obesity, fasting insulin and glucagon levels increased, but GLP-1 and glicentin levels decreased (Table 1). In adolescents with obesity, the T2DM group had more than 3-fold higher fasting insulin and almost doubled fasting glucagon compared to the NGT group. However, there were no differences in fasting insulin or glucagon between the IGT and NGT groups. On the contrary, in adolescents with obesity and IGT, GLP-1 levels were 50% lower and glicentin levels 20% lower than in adolescents with obesity and NGT. Insulin and glucagon during OGTT In lean adolescents and adolescents with obesity and NGT, insulin levels peaked at 30 minutes (Figure 1A, left panel). Adolescents with obesity and IGT had a biphasic insulin curve with one peak at 30 minutes and a second peak at 120 minutes. In adolescents with obesity and T2DM, insulin levels peaked at 90–120 minutes. Adolescents with obesity and NGT had 2.5-fold higher postprandial insulin levels than lean adolescents (Figure 2A, left panel). In adolescents with obesity, the T2DM group had 2.8-fold higher postprandial insulin levels than the NGT group, whereas there was no difference between the IGT and NGT groups. Compared to lean adolescents, the early-phase insulin response was lower in adolescents with obesity and declined with progression of glucose intolerance (Figure 2A, right panel). Figure 1. Open in new tabDownload slide Plasma levels of insulin (A), glucagon (B), active GLP-1 (C), and glicentin (D) during an OGTT in lean adolescents (open circles, n = 19) and adolescents with obesity and normal glucose tolerance (closed circles, n = 23), impaired glucose tolerance (squares, n = 19), and type 2 diabetes (triangles, n = 4). Values are means ± sem. Figure 1. Open in new tabDownload slide Plasma levels of insulin (A), glucagon (B), active GLP-1 (C), and glicentin (D) during an OGTT in lean adolescents (open circles, n = 19) and adolescents with obesity and normal glucose tolerance (closed circles, n = 23), impaired glucose tolerance (squares, n = 19), and type 2 diabetes (triangles, n = 4). Values are means ± sem. Figure 2. Open in new tabDownload slide Insulin (A), glucagon (B), active GLP-1 (C), and glicentin (D) area under the curve (AUC) during OGTT (left panel) and level at 30 minutes into the OGTT relative to fasting (right panel). Lean adolescents (n = 19) and adolescents with obesity and NGT (n = 23), IGT (n = 19), and T2DM (n = 4). *P < .05, **P < .01, and ***P < .001 vs obesity NGT. Figure 2. Open in new tabDownload slide Insulin (A), glucagon (B), active GLP-1 (C), and glicentin (D) area under the curve (AUC) during OGTT (left panel) and level at 30 minutes into the OGTT relative to fasting (right panel). Lean adolescents (n = 19) and adolescents with obesity and NGT (n = 23), IGT (n = 19), and T2DM (n = 4). *P < .05, **P < .01, and ***P < .001 vs obesity NGT. Glucagon levels in lean adolescents started to decrease between 5 and 10 minutes with suppression close to the observed maximum suppression after 30 minutes (Figure 1B). In adolescents with obesity and NGT or IGT, glucagon levels tended to increase during the first 5 minutes of the OGTT, with no lowering below fasting levels until 30 minutes. In adolescents with obesity and T2DM, glucagon levels increased during the initial 15 minutes, with no reduction below baseline until 60 minutes. To understand the hyperglucagonemic response, correlation analyses were performed between the changes in levels at 15 minutes (level15 min–level0 min). Neither insulin, GLP-1, nor glicentin response was correlated to the glucagon response at 15 minutes (data not shown). Total OGTT glucagon was 1.6-fold higher in adolescents with obesity and NGT than in lean adolescents and 2.4-fold higher in adolescents with obesity and T2DM than in those with NGT (Figure 2B, left panel). Glucagon levels at 30 minutes relative to fasting were lower in lean adolescents than in obese adolescents with NGT and tended to be highest in the obesity and T2DM group (Figure 2B, right panel). Active GLP-1 and glicentin during OGTT Active GLP-1 levels increased after 5 minutes and peaked 15 minutes into the OGTT in lean adolescents (Figure 1C). In adolescents with obesity, active GLP-1 levels similarly increased after 5 minutes, peaking after 15 minutes irrespective of glucose tolerance. Total postprandial active GLP-1 levels tended to be lower in adolescents with obesity and to decline with progression of glucose intolerance with a 50% reduction in the T2DM group compared to the NGT group (Figure 2C, left panel). Early-phase active GLP-1 response was lower in adolescents with obesity than in lean adolescents without differences across the glucose tolerance spectrum (Figure 2C, right panel). Glicentin levels increased after 5 minutes and peaked at 30 minutes in lean adolescents with a similar pattern in adolescents with obesity and NGT (Figure 1D). Adolescents with obesity and IGT had an earlier peak at 15 minutes and adolescents with obesity and T2DM had a later peak at 60 minutes. Postprandial glicentin levels were 20% lower in adolescents with obesity and IGT than in those with NGT (Figure 2D, left panel). Early-phase glicentin response was lower in adolescents with obesity than in lean adolescents without differences across the glucose tolerance spectrum (Figure 2D, right panel). Pancreatically vs intestinally cleaved proglucagon during OGTT To compare the postprandial plasma pool of proglucagon-derived peptides cleaved by prohormone convertase 2 yielding glucagon, preferentially occurring in the α-cells of the endocrine pancreas, and prohormone convertase 1, preferentially occurring in the L-cells of the intestine, glicentin/glucagon and GLP-1/glucagon ratios were calculated. In fasting, obese adolescents with IGT and T2DM had a lower glicentin/glucagon and GLP-1/glucagon ratio than obese adolescents with NGT (Figure 3A). The glicentin/glucagon and GLP-1/glucagon ratio increased more rapidly during OGTT in lean adolescents than in obese adolescents (Figure 3B). The glicentin/glucagon and GLP-1/glucagon ratio during OGTT was higher in lean adolescents than in obese adolescents with NGT (Figure 3C). Furthermore, obese adolescents with IGT and T2DM had lower glicentin/glucagon and GLP-1/glucagon ratios during OGTT than obese adolescents with NGT. Figure 3. Open in new tabDownload slide Ratios of active GLP-1 and glucagon (left panel) and glicentin and glucagon (right panel) at fasting (A) and during OGTT (B) and ratios of AUC during OGTT (C). Lean adolescents (open circles, n = 19) and adolescents with obesity and NGT (closed circles, n = 23), IGT (squares, n = 19), and T2DM (triangles, n = 4) *P < .05, **P < .01, and ***P < .001 vs obesity NGT. Figure 3. Open in new tabDownload slide Ratios of active GLP-1 and glucagon (left panel) and glicentin and glucagon (right panel) at fasting (A) and during OGTT (B) and ratios of AUC during OGTT (C). Lean adolescents (open circles, n = 19) and adolescents with obesity and NGT (closed circles, n = 23), IGT (squares, n = 19), and T2DM (triangles, n = 4) *P < .05, **P < .01, and ***P < .001 vs obesity NGT. Prediction of IGT Fasting plasma GLP-1 and glicentin but not glucose, insulin, or glucagon could predict IGT among adolescents with obesity and normal fasting glucose. The cutoff point with the highest combined sensitivity and specificity were obtained from fasting glicentin, which at a cutoff of 22.05 pM predicted IGT with a 100% sensitivity and 56% specificity (Figure 4). Fasting active GLP-1 with a cutoff at 0.25 pM predicted IGT with 62% sensitivity and 87% specificity. For both hormones, lower levels indicated IGT. Figure 4. Open in new tabDownload slide Received operating characteristic curves for cutoffs of fasting plasma active GLP-1 (dashed line; receiver operating characteristic area under curve = 0.73) and glicentin (solid line; receiver operating characteristic area under curve = 0.81) as predictors of IGT in obese adolescents with normal fasting glucose. Arrows at optimal cutoffs as defined by Youden's index. GLP-1 cutoff at 0.245 pM and glicentin cutoff at 22.05 pM; for both hormones, a lower value indicate IGT. Figure 4. Open in new tabDownload slide Received operating characteristic curves for cutoffs of fasting plasma active GLP-1 (dashed line; receiver operating characteristic area under curve = 0.73) and glicentin (solid line; receiver operating characteristic area under curve = 0.81) as predictors of IGT in obese adolescents with normal fasting glucose. Arrows at optimal cutoffs as defined by Youden's index. GLP-1 cutoff at 0.245 pM and glicentin cutoff at 22.05 pM; for both hormones, a lower value indicate IGT. Discussion In the present study, we found elevated glucagon levels in adolescents with obesity and T2DM and lower levels of active GLP-1 and glicentin in adolescents with obesity and prediabetes. Furthermore, we found that the early hyperglucagonemic response to OGTT observed in adults with T2DM is present also in pediatric obesity and T2DM. Hyperglucagonemia is present in adult obesity and diabetes (18, 19), and previous pediatric studies are in agreement with this, reporting relative hyperglucagonemia in fasting and during OGTT (23) and mixed-meal test (24). In the present study, insulin and glucagon was elevated in normoglycemic obesity, whereas there was no difference in insulin and glucagon between the NGT and IGT groups. However, adolescents with T2DM had higher levels than the NGT group, suggesting that an elevation of both basal insulin and glucagon beyond what is seen in obesity alone is a characteristic of the most severe impairment of glucose metabolism in adolescents with obesity. Hyperinsulinemia, associated with both obesity and T2DM, would be expected to lower glucagon secretion. However, disproportionately elevated glucagon in the presence of high insulin has been observed both in vitro and in vivo and α-cell insulin resistance is a potential mechanism (25, 26). The finding that insulin resistant adolescents with obesity have inadequate suppression of glucagon in hyperinsulinemic euglycemia supports this (27). There was an increase in glucagon levels during the first 15 minutes after glucose intake in adolescents with obesity and T2DM and to a lesser extent in those with IGT. Although this has been described in adults with T2DM (28), the present study reveals that this inverted glucagon response occurs also in adolescents with obesity and T2DM. The mechanism behind this phenomenon is not clear, but in studies of the hyperglucagonemic response by the isoglycemic IV glucose infusion approach, glucose or insulin levels were found unlikely to be responsible (28). Rather, the involvement of gut-dependent mechanisms has been suggested, either through gut-derived glucagon secretion or as a result of altered glucagonotropic response to glucose-dependent insulinotropic polypeptide (GIP) (11). Considering the glucagonostatic effect of GLP-1 (13), one might anticipate a blunted GLP-1 response to be connected to the hyperglucagonemic response. However, neither the early GLP-1 response nor the early insulin response correlated to the early glucagon response in the present study. The present finding of lower GLP-1 response in adolescents with obesity regardless of glucose tolerance is in agreement with studies in adults (17). One study in adolescent boys reported a decreased GLP-1 response to a mixed meal, although the aspect of glucose tolerance was not studied (29). Pediatric studies of GLP-1 are scarce, but a recent study found a lower incretin effect in adolescents with obesity and IGT or T2DM with no reduction of total plasma GLP-1 levels (23). Another study found reduced fasting total GLP-1 in adolescent girls with obesity (30). Thus, one might also expect levels of active GLP-1 to be reduced in adolescents with obesity; however, in the present study, fasting levels of active GLP-1 were higher in adolescents with obesity and NGT than in all other groups, including lean adolescents. The relative importance of circulating active GLP-1 conveying an endocrine signal to islet β-cells is a matter of debate (31). Indeed, circulating plasma active GLP-1 is low due to rapid metabolism by dipeptidyl peptidase-4 and as much as 70% is metabolized locally in the gut circulation (32). Furthermore, experimental data suggest that the effect of GLP-1 is, at least in part, mediated by GLP-1 receptor activation in vagal afferent nerves (33, 34). However, rescued β-cell GLP-1 receptor expression in GLP-1 receptor knockout mice restored oral glucose tolerance (35), and β-cell specific knockdown of the GLP-1 receptor resulted in impaired ip glucose tolerance (36). Thus, the elevated basal plasma levels of active GLP-1 observed in normoglycemic adolescents with obesity in the present study might constitute a protective mechanism, which is absent in adolescents with obesity who develop IGT and T2DM. The physiological role of glicentin is unclear and little is known about glicentin levels in context of obesity and T2DM. In a study of gastrectomized patients, glicentin levels increased after surgery (10). Furthermore, hypoglycemia that correlated to the relative glicentin response was seen in these patients after an OGTT (37). Also, in vitro studies have shown stimulation of insulin secretion by glicentin (16). Although these observations suggest a potential role for glicentin in glucose metabolism, no correlation was found between fasting glicentin and fasting blood glucose in T2DM patients; however, the aspect of obesity was not studied (10). In the present study, glicentin levels were lower in IGT than in NGT both at fasting and postprandially. It is important to note that the present study does not add to the knowledge on the function of glicentin and it is not possible to tell whether the reduced glicentin levels in IGT bears any pathophysiological relevance. Nevertheless, in adolescents with obesity and normal fasting glucose, fasting glicentin was a better predictor of glucose intolerance than fasting glucose, insulin, glucagon, or active GLP-1. However, the assessment of the clinical usefulness of glicentin as a fasting predictor of IGT requires further study in larger, less selected patient groups. Taken together, the results reveal a change toward more pancreatically cleaved (ie, by prohormone convertase 2) and less intestinally cleaved (ie, by prohormone convertase 1) proglucagon in the plasma of adolescents with T2DM. Similar results were found in adults with T2DM after a mixed meal (38). In addition, the present results show that this occurs also in normoglycemic obesity. Given the effects of intestinally cleaved proglucagon to lower blood glucose through augmenting insulin secretion and the effect of pancreatically cleaved proglucagon to elevate blood glucose, such a change is likely to be important in blood glucose regulation during an OGTT. The present study does not, however, provide clues as to whether there is a change in proglucagon processing or altered secretion or elimination of proglucagon-derived peptides from different cell types. There is indeed overlap between intestine and pancreas in proglucagon processing because pancreatic α-cells can also produce GLP-1 and do so increasingly in states of hyperglycemia (39). Similar results were found with GLP-1 and glicentin as numerator, which is not surprising given that both are secreted from intestinal L cells. Thus, given the short and variable half-life of GLP-1, glicentin might be a good alternative for studying the intestinal cleaving of proglucagon. The results need to be considered in the light of the following limitations. First, the sample size is small, especially in the T2DM group, limiting the power to detect differences in hormone levels in this group. Nevertheless, we believe that the inclusion of even a small number of adolescents with T2DM contributes to increase the overall knowledge of the T2DM disease progression in pediatric obesity. Our inability to include more subjects with T2DM is most likely the result of a low prevalence of T2DM in children and adolescents in Sweden, despite a high prevalence of prediabetes (40). Second, the lack of Tanner staging in some of the adolescents with obesity and in most of the lean adolescents for determining pubertal status is a limitation. However, subjects were well matched with regard to age, and the estimation of pubertal status from testosterone levels and growth charts indicated no major differences in distribution of pubertal status across groups. Furthermore, the cross-sectional nature of this study is a limitation, and longitudinal studies of the role of proglucagon-derived hormones in the development of T2DM in childhood obesity are warranted. In conclusion, IGT in adolescents with obesity is associated with lowered levels of active GLP-1 and glicentin, indicating that this is an early-stage abnormality in obesity-related glucose dysregulation. However, the relative GLP-1 and glicentin response to oral glucose is lower in obesity with no worsening across the spectrum of glucose tolerance. In adolescents with obesity, insulin and glucagon levels are elevated and the progression to T2DM is related to a further increase of these hormones as well as an early-phase hyperglucagonemic response to OGTT. Collectively, this reveals a change in the composition of the plasma pool of proglucagon-derived peptides toward more pancreatically cleaved and less intestinally cleaved proglucagon in pediatric obesity and T2DM. This suggests that elevating incretin hormones and suppressing glucagon are treatment strategies worth exploring in this patient group. Acknowledgments We thank the children and adolescents of the Uppsala Longitudinal Study of Childhood Obesity cohort and their families. The authors also acknowledge the contribution by the staff at the obesity clinic at Uppsala University Hospital and especially thank Marie Dahlbom and Malte Lidström for their contributions to this work. This work was supported by the Swedish Governmental Agency for Innovation Systems-VINNOVA, the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement number 279153 (Beta-JUDO), Swedish Diabetes Association (grant number DIA 2013-043), Family Ernfors Foundation (grant number 150430), Uppsala-Örebro Regional Research Council and Gillbergska Foundation. Disclosure Summary: The authors have nothing to disclose. Abbreviations AUC area under the curve BMI-SDS Body Mass Index Standard Deviation Score IGT impaired glucose tolerance NGT normal glucose tolerance OGTT oral glucose tolerance test T2DM type 2 diabetes mellitus. 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TI - Altered Plasma Levels of Glucagon, GLP-1 and Glicentin During OGTT in Adolescents With Obesity and Type 2 Diabetes
JF - Journal of Clinical Endocrinology and Metabolism
DO - 10.1210/jc.2015-3885
DA - 2016-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/altered-plasma-levels-of-glucagon-glp-1-and-glicentin-during-ogtt-in-fu18KUPIzR
SP - 1181
VL - 101
IS - 3
DP - DeepDyve
ER -