Abstract
Aim
To examine the relationship between plasma glucagon levels and insulin sensitivity and insulin secretion in obese subjects.
Methods
Suppression of plasma glucagon was examined in 275 obese Hispanic Americans with varying glucose tolerance. All subjects received a 2-hour oral glucose tolerance test (OGTT) and a subset (n = 90) had euglycemic hyperinsulinemic clamp. During OGTT, we quantitated suppression of plasma glucagon concentration, Matsuda index of insulin sensitivity, and insulin secretion/insulin resistance (disposition) index. Plasma glucagon suppression was compared between quartiles of insulin sensitivity and beta-cell function.
Results
Fasting plasma glucagon levels were similar in obese subjects with normal glucose tolerance (NGT), prediabetes, and type 2 diabetes (T2D), but the fasting glucagon/insulin ratio decreased progressively from NGT to prediabetes to T2D (9.28 ± 0.66 vs 6.84 ± 0.44 vs 5.84 ± 0.43; P < 0.001). Fasting and 2-hour plasma glucagon levels during OGTT progressively increased and correlated positively with severity of insulin resistance (both Matsuda index and euglycemic hyperinsulinemic clamp). The fasting glucagon/insulin ratio declined with worsening insulin sensitivity and beta-cell function, and correlated with whole-body insulin sensitivity (Matsuda index, r = 0.81; P < 0.001) and beta-cell function (r = 0.35; P < 0.001). The glucagon/insulin ratio also correlated and with beta-cell function during OGTT at 60 and 120 minutes (r = −0.47; P < 0.001 and r = −0.32; P < 0.001).
Conclusion
Insulin-mediated suppression of glucagon secretion in obese subjects is impaired with increasing severity of glucose intolerance and parallels the severity of insulin resistance and beta-cell dysfunction.
Keywords: glucagon, insulin secretion, insulin resistance
Diabetes mellitus is a bihormonal disorder, in which both insulin deficiency and absolute or relative glucagon excess contribute to the metabolic disturbances (1, 2). Insulin resistance and beta-cell dysfunction are core defects in type 2 diabetes mellitus (T2D) (3). Increased glucagon secretion and hyperglucagonemia also contribute to hyperglycemia in T2D patients (2, 4, 5). In the fasting state, plasma glucagon levels in T2D are elevated (1, 6) and fail to suppress normally after a meal or glucose challenge (7-10). Enhanced glucagon response to arginine also has been demonstrated (11). In nonhuman primate models, worsening glycemia is associated not only with decreased beta-cell volume but also relative increase in alpha-cell volume and hyperglucagonemia (12-14).
In individuals with prediabetes, an increased glucagon response to arginine challenge has been shown to predict the development of T2D (15). In nondiabetic (16-18) and T2D (16-20) subjects, impaired suppression of glucagon secretion after glucose ingestion is associated with postprandial hyperglycemia, and hyperglucagonemia has been shown to correlate with insulin resistance (16, 18, 21-23). However, in these studies, subjects with impaired glucose tolerance (IGT) and T2D had a higher body mass index (BMI), and obesity is an insulin-resistant state (24, 25). Dysregulation of glucagon secretion has been shown in obese individuals with and without T2D as compared with lean individuals (26, 27). However, it is not clear whether the altered glucagon secretion in these subjects was secondary to obesity, abnormal glucose tolerance, or the underlying defects in insulin sensitivity and insulin secretion. To further examine the regulation of glucagon secretion by insulin and glucose in obese subjects, we assessed insulin-mediated suppression of plasma glucagon levels and its relationship with insulin resistance and beta-cell function in obese Hispanic Americans with variable levels of glucose tolerance.
Materials and Methods
Subjects
The participants included 275 obese subjects of Hispanic American descent who participated in the Veterans Administration Genetic Epidemiology Study (VAGES) study (28). Obesity was defined as a BMI ≥30 kg/m2. All subjects received a 75-gram 2-hour oral glucose tolerance test (OGTT). Based on American Diabetes Association criteria, individuals were classified as having normal glucose tolerance (NGT) (n = 69, BMI = 32.7 ± 0.6, M/F = 23/46); prediabetes, with fasting plasma glucose (FPG) of 100 to 125 mg/dL and a 2-hour plasma glucose of ≥140 to 199 mg/dL (n = 115, BMI = 33.8 ± 0.5, M/F = 37/78); or T2D (n = 91, BMI = 33.9 ± 0.5, M/F = 38/53). All subjects had normal liver, cardiopulmonary, and kidney function as determined by medical history, physical examination, screening blood tests, electrocardiogram, and urinalysis. NGT and prediabetes subjects were not taking any medication known to affect glucose tolerance. T2D subjects were either on diet or metformin, which was held on the day of study. No subject participated in any regular physical activity program. Body weight was stable (±2 kg) for at least 3 months before study in all participants. The study protocol was approved by the Institutional Review Board of University of Texas Health Science Center at San Antonio, and informed written consent was obtained from each subject before participation.
Oral Glucose Tolerance Test
All OGTT studies were performed at the Bartter Clinical Research Unit (BCRU) in the Audie L. Murphy Memorial VA Hospital at 7:30 to 8:00 am after a 10- to 12-hour overnight fast. Before the start of the OGTT, a small polyethylene catheter was placed into an antecubital vein for blood withdrawal. At time 0, subjects ingested a 75-g glucose solution. Blood samples were collected at −30, −15, 0, 30, 60, 90, and 120 minutes for measurement of plasma glucose, insulin, C-peptide, and glucagon concentrations. On the day of the OGTT, waist and hip circumference was measured with a Gulick tape measure.
Euglycemic Hyperinsulinemic Clamp
After a 10- to 12-hour overnight fast, a subset of participants (26 NGT, 37 prediabetes, 27 T2D) received a 120-minute euglycemic insulin clamp (40 mU/m2•min) (29). Before the start of the euglycemic insulin clamp, a catheter was placed into an antecubital vein for the infusion of all test substances. A second catheter was placed retrogradely into a vein on the dorsum of the hand which was placed in a heated box (60 °C) for arterialized blood withdrawal. During insulin administration, plasma glucose was measured every 5 minutes and a variable glucose infusion was adjusted to maintain each subjects’ plasma glucose at his/her fasting level. In T2D subjects, the plasma glucose concentration was allowed to decline to 100 mg/dL, at which level it was maintained. The coefficient of variation of plasma glucose was less than 5%.
Analytical Methods
Plasma glucose was measured by glucose oxidase reaction (Glucose Analyzer from Beckman, Fullerton, CA). Plasma insulin and C-peptide concentrations were measured by radioimmunoassay (Linco Research, St. Louis, MO). Plasma glucagon concentration was measured by radioimmunoassay (Millipore Sigma, Burlington, MA). The intra-assay coefficient of variation for glucagon was 4.9%.
Calculations
Insulin-mediated suppression of glucagon secretion was determined by plasma glucagon/insulin ratio. Glucagon suppression at 60 minutes during the OGTT was calculated as follows: [1-(glucagon 60 min/glucagon 0 min)] × 100%. The Matsuda index (30) was used to assess whole-body insulin sensitivity, using the plasma glucose and C-peptide (CP) concentrations measured during the 2-hour OGTT. The mean rate of insulin-stimulated glucose metabolism (M value) during the euglycemic hyperinsulinemic clamp was calculated to provide an independent measure of insulin sensitivity. Beta-cell function was calculated using the formula ΔCP/ΔG ÷ IR, where ΔCP/ΔG is the ratio of the incremental area under the curve (AUC) for plasma C-peptide to the incremental AUC for plasma glucose (ΔAUC-CP/ΔAUC-G) during the 2-hour OGTT, and IR is insulin resistance measured by the inverse of the Matsuda index (31, 32). Subjects were stratified into 4 groups based on their insulin sensitivity (Matsuda index and M value), and beta-cell function (insulin secretion/insulin resistance or disposition index). Subjects in Quartile 1 (lowest Matsuda insulin sensitivity index or M value) were the most insulin-resistant and subjects in Quartile 4 were the most insulin-sensitive.
Statistical Analysis
Statistical comparisons were performed using Graph Pad Prism 9 (Graph Pad Software, San Diego, CA, USA) and NCSS (Version 12, Chicago IL). Study participants were categorized into equal quartiles of Matsuda index of insulin sensitivity and insulin secretion (ΔCpep0-120 / ΔGlu0-120). Plasma glucagon levels and suppression of glucagon during OGTT were related to glucose tolerance status and to quartiles of insulin sensitivity and insulin secretion using Spearman’s correlation. Comparison between the quartiles of insulin sensitivity and beta-cell function was performed using 2-way ANOVA with Bonferroni post hoc testing when appropriate. Group comparisons (baseline vs OGTT and baseline vs euglycemic insulin clamp) were performed by Student t test. Data represent the mean ± standard error of the mean. The relationship between plasma glucagon/insulin ratio versus insulin sensitivity and versus insulin secretion were determined by Spearman’s correlation.
Results
The clinical and metabolic characteristics of study participants are shown in Table 1. Of the 115 subjects with prediabetes, 30 had impaired fasting glucose (IFG), 42 had impaired glucose tolerance (IGT) and 43 had combined IFG/IGT. Subjects with NGT were slightly younger than prediabetic and T2D subjects. There was no difference in BMI, waist and hip circumference, or male/female ratio between the 3 groups. The FPG and 2-hour OGTT glucose concentration increased from NGT to prediabetes to T2D (Fig. 1A).
Table 1.
Clinical, Anthropometric, and Laboratory Characteristics of the Normal Glucose Tolerance, Prediabetes, and Type 2 Diabetes Groups
| NGT | Prediabetes | T2D | P value | |
|---|---|---|---|---|
| (IFG/IGT) | ||||
| Number | 69 | 115 | 91 | |
| Male/Female | 23/46 | 37/78 | 38/53 | NS |
| Age (years) | 42 ± 2 | 45 ± 2 | 52 ± 2 | <0.05 |
| HbA1c (%) | 5.0 ± 0.1 | 5.8 ± 0.1 | 7.3 ± 0.2 | <0.001 |
| BMI (kg/m 2 ) | 33 ± 1 | 34 ± 1 | 34 ± 1 | NS |
| Systolic BP (mmHg) | 101 ± 3 | 105 ± 2 | 109 ± 2 | <0.05 |
| Diastolic BP (mmHg) | 87 ± 2 | 90 ± 2 | 93 ± 2 | <0.05 |
| Waist (cm) | 102 ± 2 | 104 ± 2 | 106 ± 1 | NS |
| Hip (cm) | 113 ± 2 | 114 ± 2 | 114 ± 2 | NS |
| FPG (mg/dL) | 92 ± 1 | 102 ± 1 | 162 ± 6 | <0.001 |
| 2-hour PG (mg/dL) | 115 ± 2 | 153 ± 3 | 289 ± 7 | <0.001 |
| Mean PG (mg/dL) | 132 ± 3 | 164 ± 2 | 268 ± 7 | <0.001 |
Data are presented as mean ± standard error of the mean.
P values represent one-way ANOVA.
Abbreviations: BMI, body mass index; BP, blood pressure; FPG, fasting plasma glucose; HbA1c, glycated hemoglobin A1c; IFG, impaired fasting glucose; IGT, impaired glucose tolerance; NGT, normal glucose tolerance; NS, not statistically significant; PG, plasma glucose; T2D, type 2 diabetes.
Figure 1.
Plasma glucose (A), glucagon (B), insulin (C) and C-peptide (D) concentrations in obese normal glucose tolerant (NGT), prediabetic, and type 2 diabetic (T2D) participants. *P < 0.05, **P < 0.01, ***P < 0.001.
Plasma Glucagon, Insulin, and C-Peptide Concentrations During OGTT
There was no difference in fasting plasma glucagon concentration between NGT, prediabetes, and T2D subjects. However, the fasting plasma glucagon in T2D subjects tended to be higher compared with both NGT and prediabetes (Fig. 1B). The mean plasma glucagon concentration during the OGTT at 60 minutes and 120 minutes were similar in NGT and prediabetes groups and slightly, although significantly (P < 0.05), increased in T2D (Fig. 1B). The fasting plasma insulin and C-peptide concentrations rose progressively from NGT to prediabetes to T2D (Fig. 1C and 1D). The mean plasma insulin and C-peptide concentrations during the OGTT at 60 minutes and 120 minutes were significantly increased in prediabetes versus NGT (P < 0.001) and reduced in T2D versus both NGT and prediabetes groups (P < 0.001) (Fig. 1C and 1D). The fasting plasma glucagon/insulin ratio declined progressively from NGT to prediabetes to T2D (P < 0.001) (Fig. 2A). When subtypes of prediabetes, (ie, IFG, IGT, and IFG/IGT) are compared, there was a progressive decline in the fasting glucagon/insulin ratio from IFG to IGT to combined IFG/IGT (Fig. 2B). At 60 minutes (Fig. 2C) and 120 minutes (Fig. 2D) during OGTT, the plasma glucagon/insulin ratio was similar in prediabetes and NGT groups but markedly increased in T2D versus both NGT and prediabetes (P < 0.001). When subtypes of prediabetes, (IFG, IGT, and IFG/IGT) were compared, there was a progressive decline in the glucagon/insulin ratio fasting glucagon/insulin ratio at 120 minutes from IFG to IGT to combined IFG/IGT (Fig. 2E). Suppression of plasma glucagon at 60 minutes during OGTT compared with fasting glucagon was similar in NGT (10.4%) and prediabetes (9.1%) groups, but significantly impaired in T2D individuals (3.3%) (P < 0.05).
Figure 2.
The plasma glucagon/insulin ratio at 0 minutes (A), 60 minutes (C), and 120 minutes (D) in normal glucose tolerant (NGT), impaired glucose tolerant (IGT), and type 2 diabetic (T2D) individuals, and the glucagon/insulin ratio at 0 minutes (B) and 120 minutes (E) in NGT, impaired fasting glucose (IFG), IGT, IFG/IGT, and T2D subjects. *P < 0.05, **P < 0.01, ***P < 0.001.
During the OGTT, the number of subjects in whom the 2-hour plasma glucagon concentration was lower than the fasting plasma glucagon was 53/69 (77%) in NGT, 88/115 (77%) in prediabetes, and 64/91 (71%) in T2D (P = NS). There were no significant differences in age, BMI, FPG, 2-hour glucose, Matsuda insulin sensitivity index, and insulin secretion/insulin sensitivity (disposition) index between subjects with suppressed (2-hour plasma glucagon below fasting plasma glucagon) versus nonsuppressed (2-hour plasma glucagon above fasting plasma glucagon) in the entire group, as well as between NGT, prediabetic, and T2D groups.
Plasma Glucagon in Relation to Insulin Sensitivity
Insulin sensitivity, measured with both the Matsuda index of insulin sensitivity and the rate of insulin-mediated glucose metabolism (M values) during the euglycemic hyperinsulinemic clamp, decreased progressively from NGT to prediabetes to T2D (Fig. 3A and 3B). Subjects were stratified into 4 groups based on their insulin sensitivity; subjects in Quartile 1 were the most insulin-resistant, while those in Quartile 4 were the most insulin-sensitive (Table 2). As expected, the greatest number of NGT subjects were in Quartile 4 (most insulin-sensitive) of Matsuda insulin sensitivity index or the M value, and the largest number of individuals with T2D were in Quartile 1 (most insulin-resistant). The mean plasma glucagon concentration in the fasting state and during the OGTT (0-120 minutes) in relation to quartile of insulin sensitivity is displayed in Fig. 4. Both fasting and 2-hour plasma glucagon concentrations increased progressively with decreasing insulin sensitivity, measured both with the Matsuda index or M value during the euglycemic insulin clamp (Fig. 4A and 4B). The plasma glucagon/insulin ratio progressively declined with worsening insulin resistance (Figs. 5A–5D).
Figure 3.
Whole-body insulin sensitivity (Matsuda index) (A), and insulin sensitivity (M value) (B) during the euglycemic hyperinsulinemic clamp, and beta-cell function (ΔCP0-120/ΔG0-120 ÷ IR, disposition index) (C) during oral glucose tolerance test (OGTT) in obese normal glucose tolerant (NGT), prediabetic, and type 2 diabetic (T2D) subjects. *P < 0.05, **P < 0.01, ***P < 0.001.
Table 2.
Distribution of NGT, Prediabetic, and T2D Subjects Based on Quartiles of Insulin Sensitivity (Matsuda index) and Beta-cell Function (disposition index)
| Quartile | 1 | 2 | 3 | 4 |
| Matsuda index of insulin sensitivity | ||||
| Matsuda index | (0.42-1.47) | (1.48-2.32) | (2.45-3.59) | (3.61-11.34) |
| NGT, n (%) | 8 (11%) | 13 (19%) | 18 (26%) | 30 (48%) |
| Prediabetes, n (%) | 30 (42%) | 30 (43%) | 33 (46%) | 22 (35%) |
| T2D, n (%) | 32 (47%) | 27 (38%) | 17 (28%) | 15 (17%) |
| Disposition index | ||||
| Disposition index | (0.39-9.93) | (10.2-23.64) | (23.90-45.9) | (46.08-159.25) |
| NGT, n (%) | 0 (0%) | 1 (2%) | 22 (32%) | 46 (70%) |
| Prediabetes, n (%) | 6 (8%) | 43 (66%) | 46 (67%) | 20 (30%) |
| T2D, n (%) | 69 (92%) | 21 (32%) | 1 (1%) | 0 (0%) |
Quartile 4 represents the most insulin-sensitive and the best beta-cell function (disposition index). Data are presented as number (%) of subjects.
Figure 4.
Mean plasma glucagon concentration at fasting and 120 minutes during OGTT in relation to the quartile of whole-body insulin sensitivity (Matsuda index) (A, B) and beta-cell function (disposition index) (C, D).
Figure 5.
Plasma glucagon/insulin ratio at fasting and 120 minutes during OGTT in relation to the quartile of insulin sensitivity (M value) (A, B), whole-body insulin sensitivity (Matsuda index) (C, D), and beta-cell function, ΔCP0-120/ΔG0-120 ÷ IR (disposition index) (E, F).
Plasma Glucagon in Relation to Beta-Cell Function
Beta-cell function, measured with insulin secretion/insulin resistance (disposition) index, gradually decreased from NGT to prediabetes to T2D (Fig. 3C). The relationship between plasma glucagon concentration and insulin secretion/insulin resistance (disposition) index is displayed in Fig. 4. As expected, the highest quartile of disposition index (Quartile 4, Table 2) contained the highest percentage of individuals with NGT, while the lowest quartile of disposition index contained the highest percentage of individuals with T2D (Quartile 1, Table 2). Both fasting and 2-hour plasma glucagon levels increased progressively with worsening beta-cell function (Fig. 4C and 4D) and the glucagon/insulin ratio decreased progressively with declining beta-cell function (Fig. 5E and 5F).
Relationship Between Insulin-Mediated Glucagon Suppression Versus Insulin Sensitivity and Beta-Cell Function
The relationship between insulin sensitivity and beta-cell function in NGT, prediabetic, and T2D subjects was examined using the Matsuda index and the insulin secretion/insulin resistance (disposition) index. The ratio between the incremental AUCs of plasma C-peptide and plasma glucose concentrations during the OGTT (0-120 minutes) was used to calculate the disposition index. In the fasting state, the glucagon/insulin ratio was positively correlated with whole-body insulin sensitivity (Matsuda index, r = 0.81; P < 0.001) (Fig. 6A) and beta-cell function (disposition index, r = 0.35; P < 0.001) (Fig. 6B). During OGTT, the glucagon/insulin ratio at 60 minutes (Fig. 6C) and 120 minutes (Fig. 6D) was inversely correlated with beta-cell function (disposition index, r = −0.47; P < 0.001 and r = −0.32; P < 0.001, respectively). Glucagon suppression during the OGTT (r = −0.53; P < 0.001) was inversely correlated with the glycated hemoglobin (HbA1c) in nondiabetic subjects, but not in prediabetic or T2D subjects.
Figure 6.
The relationship between the glucagon/insulin ratio at the fasting state and whole-body insulin sensitivity (Matsuda index) (A). The correlation between the log of beta-cell function (disposition index) and log of the glucagon/insulin ratio at fasting state (B), and 60 (C) and 120 (D) minutes during OGTT.
To identify factors contributing to the impaired suppression of glucagon and fasting hyperglycemia, we performed a multiple regression analysis using glucagon/insulin ratio as a dependent variable and Matsuda insulin sensitivity index, fasting glucose, and insulin secretion (ΔII0-120/ΔG0-120) as independent variables. Together, Matsuda insulin sensitivity index, fasting glucose, and the insulinogenic index explained 61% (r2 = 0.605; P < 0.001) of the variation in fasting glucagon/insulin ratio. Similarly, when the FPG concentration was the dependent variable, and fasting glucagon to insulin ratio, glucagon suppression at 120 minutes, and glucagon to insulin ratio at 60 minutes were independent variables, together they explained 51% of variation in fasting glucose level (r2 = 0.506; P < 0.005).
Discussion
The present results demonstrate that, in a large cohort of obese Hispanic American subjects with a wide range of glucose tolerance, both the fasting and OGTT plasma glucagon concentrations and the glucagon/insulin ratio progressively worsened with increasing insulin resistance and declining beta-cell function. Impaired suppression of glucagon by insulin during the euglycemic hyperinsulinemic clamp also progressively declined with worsening glucose tolerance.
In the present study we focused on obese subjects because: (i) obese subjects are at high risk of developing diabetes; (ii) a recent study (33) suggested that obese subjects have a lower fasting plasma glucagon concentration that results from elevated fasting insulin levels secondary to insulin resistance; and (iii) although several studies (15-17) have reported increased plasma glucagon levels in prediabetes versus NGT subjects, there were significant differences in BMI between the 2 groups (16, 18, 27). Despite similar BMIs between the NGT, prediabetes, and T2D groups, the results clearly show that fasting and 2-hour plasma glucagon levels increase with worsening glucose tolerance as well as with worsening insulin resistance and declining beta-cell function.
The plasma glucagon concentration during the OGTT failed to suppress (ie, return to the fasting level during the OGTT) in 21% to 28% of the participants. Importantly, there were no significant differences in the prevalence of nonsuppressed plasma glucagon levels between NGT, prediabetic, and T2D individuals. However, when stratified by quartiles of insulin sensitivity, there was a marked difference in mean plasma glucagon concentration during the OGTT between subjects in the lowest and highest quartiles of insulin sensitivity (Fig. 4A and 4B). This suggests that the impaired glucagon suppression is more strongly related with insulin resistance than with plasma glucose levels. Our results are consistent with those reported by Færch et al in Danish subjects (16).
Both glucose and insulin are important regulators of the plasma glucagon concentration. Insulin inhibits glucagon secretion both by paracrine mechanisms and elevated plasma levels (34, 35), while glucose inhibits glucagon by increasing cyclic adenosine monophosphate (cAMP) levels in the alpha-cell (36). The current study design does not allow us to determine whether there is resistance to the suppressive effect of glucose or insulin or both. In the current study, plasma glucagon levels are clearly more affected by insulin sensitivity and beta-cell function than by the plasma glucose level per se. The changes in plasma glucagon levels were more pronounced when measured by change in quartiles of insulin sensitivity than changes in plasma glucose. Elevated fasting glucagon levels have been associated with insulin resistance, measured by the euglycemic hyperinsulinemic clamp, in nondiabetic subjects, suggesting insulin resistance at the level of pancreatic alpha-cells (37). In the present study, we show that a similar relationship also exists in individuals with prediabetes and T2D. Among the subtypes of prediabetes, our data suggests that there is a progressive worsening in suppression of glucagon when moving from isolated IFG to combined IFG/IGT, which could be explained by worsening insulin sensitivity and insulin secretion in isolated IFG versus combined IFG/IGT.
Impaired plasma glucagon suppression in response to meals and hypersecretion in response to arginine have previously been shown (11, 38). The present study demonstrates that elevated plasma glucagon levels are present in the fasting state as well as at the 60-minute and 120-minute time points during an oral glucose load. Our results differ from those of Færch et al, who showed that impaired glucagon suppression in T2D patients during an OGTT was most marked at 30 minutes, while a normal decrease was observed at 120 minutes (16). Our results demonstrate that both early and late glucagon suppression are impaired in T2D subjects during the OGTT and contribute to hyperglycemia. This difference could be related to the different ethnic populations that were studied (Mexican Americans versus Caucasians) or the greater severity of insulin resistance in our subjects. Of note, glucagon response to a mixed meal is different from the glucagon response to OGTT. While glucose load leads to suppression of glucagon, following a mixed meal there is an initial increase in glucagon followed by suppression (39, 40). It is possible that the glucagon response to mixed meal in relation to insulin sensitivity could differ from that seen with OGTT.
How insulin sensitivity modulates plasma glucagon levels is not clear. It is possible that insulin resistance is present at the level of the pancreatic alpha-cells, which contain insulin receptors and the insulin signal transduction system (34, 41). Deletion of pancreatic alpha-cell insulin receptors in mice (αIRKO mice) causes impaired glucose tolerance, hyperglycemia, and hyperglucagonemia, demonstrating the important role of the insulin receptor in the regulation of glucagon secretion (34). Our results are consistent with the concept that insulin resistance at the level of pancreatic alpha-cell, as well as impaired insulin secretion, contribute to impaired glucagon suppression in T2D. Consistent with this scenario, insulin has been shown to modulate glucagon secretion by reducing the sensitivity of Katp channels in pancreatic alpha-cells, resulting in decreased phosphatidylinositol 3-kinase (PI3K) and Akt activity (42). An increase in plasma free fatty acid concentrations has been shown to decrease glucagon secretion in humans (42). In contrast, a recent study showed that exposure of human cultured islets to increased fatty acid levels enhanced glucagon secretion (43). Plasma free fatty acid levels were not measured in the present study. In rats, chronic (7 days) exposure to elevated plasma glucose levels was shown to augment glucagon secretion, and this was attributed to glucotoxicity (44). Consistent with these results in rodents, we have shown that mild physiologic hyperglycemia (+50 mg/dL) in healthy NGT individuals for 72 hours markedly impairs suppression of glucagon during euglycemic hyperinsulinemic clamp (45). As expected, a novel human monoclonal antibody and competitive antagonist of the glucagon receptor improved glycemic control, reduced hepatic glucose production, and improved skeletal muscle insulin sensitivity in rodent models of obesity and insulin resistance (46). A glucotoxic effect on pancreatic alpha-cell secretion of glucagon also has been demonstrated in vitro (33), and correction of the hyperglycemia with phlorizin restored the ability of glucose to suppress glucagon secretion (47).
In summary, the present results demonstrate that suppression of glucagon secretion is impaired with increasing severity of glucose intolerance and is closely associated with insulin resistance and to a lesser extent with beta-cell dysfunction in obese Hispanic Americans.
Acknowledgments
This work was supported by National Institutes of Health Grant DK24092, a Veterans Administration Epidemiologic grant, and a General Clinical Research grant. D.T. and X.C. conducted the study. X.C., E.M., and D.T. wrote the manuscript and researched data. R.A.D. contributed to discussion and reviewed/edited the manuscript. Dr. D. Tripathy is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Dr. D. Tripathy’s salary is supported by the South Texas Veterans Health Care System, Audie Murphy Division and the Foundation of Veterans Health and Research (FAVHR) supported the oral presentation at the American Diabetes Association meeting, 2019.
Glossary
Abbreviations
- AUC
area under the curve
- BMI
body mass index
- CP
C-peptide
- FPG
fasting plasma glucose
- G
glucose
- HbA1c
glycated hemoglobin
- IFG
impaired fasting glucose
- IGT
impaired glucose tolerance
- IR
insulin resistance
- M value
rate of insulin-stimulated glucose metabolism
- NGT
normal glucose tolerance
- OGTT
oral glucose tolerance test
- T2D
type 2 diabetes
Additional Information
Disclosures: R.A.D. receives grant support from Janssen, Merck, and Astra Zeneca; is a member of the advisory boards of Astra Zeneca, Janssen Pharmaceuticals, Boehringer Ingelheim, Intarcia, and Novo Nordisk; and is a member of the speakers’ bureaus of Novo Nordisk and Astra Zeneca. No other potential conflicts of interest relevant to this article were reported.
Data Availability
Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
References
- 1. Jiang G, Zhang BB. Glucagon and regulation of glucose metabolism. Am J Physiol Endocrinol Metab. 2003;284(4):E671-E678. [DOI] [PubMed] [Google Scholar]
- 2. Unger RH. Letter: glucagon in pathogenesis of diabetes. Lancet. 1975;1(7914):1036-1032. [DOI] [PubMed] [Google Scholar]
- 3. Defronzo RA. Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes. 2009;58(4):773-795. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Unger RH, Aguilar-Parada E, Müller WA, Eisentraut AM. Studies of pancreatic alpha cell function in normal and diabetic subjects. J Clin Invest. 1970;49(4):837-848. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Unger RH. Glucagon and diabetes mellitus. Adv Metab Disord. 1972;6:73-98. [DOI] [PubMed] [Google Scholar]
- 6. Cherrington AD. Banting Lecture 1997. Control of glucose uptake and release by the liver in vivo. Diabetes. 1999;48(5):1198-1214. [DOI] [PubMed] [Google Scholar]
- 7. Baron AD, Schaeffer L, Shragg P, Kolterman OG. Role of hyperglucagonemia in maintenance of increased rates of hepatic glucose output in type II diabetics. Diabetes. 1987;36(3):274-283. [DOI] [PubMed] [Google Scholar]
- 8. Dimitriadis GD, Pehling GB, Gerich JE. Abnormal glucose modulation of islet A- and B-cell responses to arginine in non-insulin-dependent diabetes mellitus. Diabetes. 1985;34(6):541-547. [DOI] [PubMed] [Google Scholar]
- 9. Müller WA, Faloona GR, Aguilar-Parada E, Unger RH. Abnormal alpha-cell function in diabetes. Response to carbohydrate and protein ingestion. N Engl J Med. 1970;283(3):109-115. [DOI] [PubMed] [Google Scholar]
- 10. Reaven GM, Chen YD, Golay A, Swislocki AL, Jaspan JB. Documentation of hyperglucagonemia throughout the day in nonobese and obese patients with noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab. 1987;64(1):106-110. [DOI] [PubMed] [Google Scholar]
- 11. Larsson H, Ahrén B. Glucose intolerance is predicted by low insulin secretion and high glucagon secretion: outcome of a prospective study in postmenopausal Caucasian women. Diabetologia. 2000;43(2):194-202. [DOI] [PubMed] [Google Scholar]
- 12. Guardado Mendoza R, Perego C, Finzi G, et al. Delta cell death in the islet of Langerhans and the progression from normal glucose tolerance to type 2 diabetes in non-human primates (baboon, Papio hamadryas). Diabetologia. 2015;58(8):1814-1826. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Guardado-Mendoza R, Chávez AO, Jiménez-Ceja LM, et al. Islet amyloid polypeptide response to maximal hyperglycemia and arginine is altered in impaired glucose tolerance and type 2 diabetes mellitus. Acta Diabetol. 2017;54(1):53-61. [DOI] [PubMed] [Google Scholar]
- 14. Guardado-Mendoza R, Davalli AM, Chavez AO, et al. Pancreatic islet amyloidosis, beta-cell apoptosis, and alpha-cell proliferation are determinants of islet remodeling in type-2 diabetic baboons. Proc Natl Acad Sci U S A. 2009;106(33):13992-13997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Robertson RP, Raymond RH, Lee DS, et al. ; Beta Cell Project Team of the Foundation for the NIH Biomarkers Consortium . Arginine is preferred to glucagon for stimulation testing of β-cell function. Am J Physiol Endocrinol Metab. 2014;307(8):E720-E727. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Færch K, Vistisen D, Pacini G, et al. Insulin resistance is accompanied by increased fasting glucagon and delayed glucagon suppression in individuals with normal and impaired glucose regulation. Diabetes. 2016;65(11):3473-3481. [DOI] [PubMed] [Google Scholar]
- 17. 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(2):E283-E290. [DOI] [PubMed] [Google Scholar]
- 18. Wagner R, Hakaste LH, Ahlqvist E, et al. Nonsuppressed glucagon after glucose challenge as a potential predictor for glucose tolerance. Diabetes. 2017;66(5):1373-1379. [DOI] [PubMed] [Google Scholar]
- 19. Henkel E, Gallo S, Siegert G, Koehler C, Hanefeld M. Glucagon as a determinant of fibrinolytic activity in men with different stages of glucose tolerance: impact of glucagon on fibrinolysis. Blood Coagul Fibrinolysis. 2007;18(4):327-334. [DOI] [PubMed] [Google Scholar]
- 20. 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(11):4053-4059. [DOI] [PubMed] [Google Scholar]
- 21. Wewer Albrechtsen NJ, Færch K, Jensen TM, et al. Evidence of a liver-alpha cell axis in humans: hepatic insulin resistance attenuates relationship between fasting plasma glucagon and glucagonotropic amino acids. Diabetologia. 2018;61(3):671-680. [DOI] [PubMed] [Google Scholar]
- 22. Bagger JI, Knop FK, Lund A, Holst JJ, Vilsbøll T. Glucagon responses to increasing oral loads of glucose and corresponding isoglycaemic intravenous glucose infusions in patients with type 2 diabetes and healthy individuals. Diabetologia. 2014;57(8):1720-1725. [DOI] [PubMed] [Google Scholar]
- 23. Suppli MP, Bagger JI, Lund A, et al. Glucagon resistance at the level of amino acid turnover in obese subjects with hepatic steatosis. Diabetes. 2020;69(6):1090-1099. [DOI] [PubMed] [Google Scholar]
- 24. DeFronzo RA. Insulin secretion, insulin resistance, and obesity. Int J Obes. 1982;6(Suppl 1):73-82. [PubMed] [Google Scholar]
- 25. Reaven GM, Moore J, Greenfield M. Quantification of insulin secretion and in vivo insulin action in nonobese and moderately obese individuals with normal glucose tolerance. Diabetes. 1983;32(7):600-604. [DOI] [PubMed] [Google Scholar]
- 26. 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(6):500-510. [DOI] [PubMed] [Google Scholar]
- 27. Stern JH, Smith GI, Chen S, Unger RH, Klein S, Scherer PE. Obesity dysregulates fasting-induced changes in glucagon secretion. J Endocrinol. 2019;243(2):149-160. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Coletta DK, Schneider J, Hu SL, et al. Genome-wide linkage scan for genes influencing plasma triglyceride levels in the Veterans Administration Genetic Epidemiology Study. Diabetes. 2009;58(1):279-284. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol. 1979;237(3):E214-E223. [DOI] [PubMed] [Google Scholar]
- 30. Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care. 1999;22(9):1462-1470. [DOI] [PubMed] [Google Scholar]
- 31. DeFronzo RA, Tripathy D, Abdul-Ghani M, Musi N, Gastaldelli A. The disposition index does not reflect β-cell function in IGT subjects treated with pioglitazone. J Clin Endocrinol Metab. 2014;99(10):3774-3781. [DOI] [PubMed] [Google Scholar]
- 32. Gastaldelli A, Ferrannini E, Miyazaki Y, Matsuda M, DeFronzo RA; San Antonio metabolism study . Beta-cell dysfunction and glucose intolerance: results from the San Antonio metabolism (SAM) study. Diabetologia. 2004;47(1):31-39. [DOI] [PubMed] [Google Scholar]
- 33. Stern JH, Smith GI, Unger RH, Klein S, Scherer PE. Fasting-induced changes in glucagon secretion are dysregulated in obesity. Diabetes. 2018;67:LB71 (271-LB). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Kawamori D, Kurpad AJ, Hu J, et al. Insulin signaling in alpha cells modulates glucagon secretion in vivo. Cell Metab. 2009;9(4):350-361. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35. Maruyama H, Tominaga M, Bolli G, Orci L, Unger RH. The alpha cell response to glucose change during perfusion of anti-insulin serum in pancreas isolated from normal rats. Diabetologia. 1985;28(11):836-840. [DOI] [PubMed] [Google Scholar]
- 36. Zhang Q, Ramracheya R, Lahmann C, et al. Role of KATP channels in glucose-regulated glucagon secretion and impaired counterregulation in type 2 diabetes. Cell Metab. 2013;18(6):871-882. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37. Ferrannini E, Muscelli E, Natali A, et al. ; Relationship between Insulin Sensitivity and Cardiovascular Disease Risk (RISC) Project Investigators . Association of fasting glucagon and proinsulin concentrations with insulin resistance. Diabetologia. 2007;50(11):2342-2347. [DOI] [PubMed] [Google Scholar]
- 38. Larsson H, Ahrén B. Islet dysfunction in insulin resistance involves impaired insulin secretion and increased glucagon secretion in postmenopausal women with impaired glucose tolerance. Diabetes Care. 2000;23(5):650-657. [DOI] [PubMed] [Google Scholar]
- 39. Koopman ADM, Beulens JW, van der Heijden A, et al. A prospective study on glucagon responses to oral glucose and mixed meal and 7-year change in fasting glucose. Clin Endocrinol (Oxf). 2019;91(1):82-86. [DOI] [PubMed] [Google Scholar]
- 40. Rijkelijkhuizen JM, McQuarrie K, Girman CJ, et al. Effects of meal size and composition on incretin, alpha-cell, and beta-cell responses. Metabolism. 2010;59(4):502-511. [DOI] [PubMed] [Google Scholar]
- 41. Diao J, Asghar Z, Chan CB, Wheeler MB. Glucose-regulated glucagon secretion requires insulin receptor expression in pancreatic alpha-cells. J Biol Chem. 2005;280(39):33487-33496. [DOI] [PubMed] [Google Scholar]
- 42. Gerich JE, Langlois M, Schneider V, Karam JH, Noacco C. Effects of alternations of plasma free fatty acid levels on pancreatic glucagon secretion in man. J Clin Invest. 1974;53(5):1284-1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43. Kristinsson H, Sargsyan E, Manell H, Smith DM, Göpel SO, Bergsten P. Basal hypersecretion of glucagon and insulin from palmitate-exposed human islets depends on FFAR1 but not decreased somatostatin secretion. Sci Rep. 2017;7(1):4657. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44. Jamison RA, Stark R, Dong J, et al. Hyperglucagonemia precedes a decline in insulin secretion and causes hyperglycemia in chronically glucose-infused rats. Am J Physiol Endocrinol Metab. 2011;301(6):E1174-E1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45. Tripathy D, Merovci A, Basu R, Abdul-Ghani M, DeFronzo RA. Mild physiologic hyperglycemia induces hepatic insulin resistance in healthy normal glucose-tolerant participants. J Clin Endocrinol Metab. 2019;104(7):2842-2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46. Sharma AX, Quittner-Strom EB, Lee Y, et al. Glucagon receptor antagonism improves glucose metabolism and cardiac function by promoting AMP-mediated protein kinase in diabetic mice. Cell Rep. 2018;22(7):1760-1773. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47. Starke A, Grundy S, McGarry JD, Unger RH. Correction of hyperglycemia with phloridzin restores the glucagon response to glucose in insulin-deficient dogs: implications for human diabetes. Proc Natl Acad Sci U S A. 1985;82(5):1544-1546. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.