Hyperglucagonemia precedes a decline in insulin secretion and causes hyperglycemia in chronically glucose-infused rats

Abstract

Islet damage from glucose toxicity is implicated in the pathogenesis of type 2 diabetes, but the sequence of events leading to islet cell dysfunction and hyperglycemia remains unclear. To examine the early stages of islet pathology resulting from increased basal glucose loads, normal awake rats were infused with glucose continuously for 10 days. Plasma glucose and markers of islet and liver function were monitored throughout the infusion. After initial hyperglycemia, rats adapted to the infusion and maintained euglycemia for approximately 4 days. Continued infusion led to worsening hyperglycemia in just 5% of rats after 6 days, but 69% after 8 days and 89% after 10 days, despite unchanged basal and stimulated plasma insulin and C-peptide concentrations. In contrast, plasma glucagon concentrations increased fivefold. Endogenous glucose production (EGP) was appropriately suppressed after 4 days (2.8 ± 0.7 vs. 6.1 ± 0.4 mg·kg(-1)·min(-1) on day 0, P < 0.001) but tripled between days 4 and 8 (9.9 ± 1.7 mg·kg(-1)·min(-1), P < 0.01). Surprisingly, the increase in EGP was accompanied by increased mitochondrial phosphoenolpyruvate carboxykinase expression with appropriate suppression of the cytosolic isoform. Infusion of anti-glucagon antibodies normalized plasma glucose to levels identical to those on day 4 and ∼300 mg/dl lower than controls. This improved glycemia was associated with a 60% reduction in EGP. These data support the novel concept that glucose toxicity may first manifest as α-cell dysfunction prior to any measurable deficit in insulin secretion. Such hyperglucagonemia could lead to excessive glucose production overwhelming the capacity of the β-cell to maintain glucose homeostasis.

Fig. 1.

Caloric intake over the first 8 days of glucose or saline infusion. A: daily caloric intake, including glucose infusion and food consumption. Statistics are for total caloric intake [n = 17 saline infused, 7 chronic, intravenous, glucose-infused (CIGI)-low, and 40 CIGI-high rats]. B: daily carbohydrate intake. ***P < 0.001 vs. saline; 1-way ANOVA with Bonferroni's multiple comparison test was used to determine significance. In this and all of the following figures, data are presented as means ± SE. NS, not significant.

Fig. 2.

Daily, nonfasting glucose and insulin in control and CIGI rats. A: daily plasma glucose concentrations. B: daily plasma insulin concentrations. ○, CIGI-high; ▵, CIGI-low; ●, saline. *P < 0.05, CIGI-high vs. CIGI-low; **P < 0.01, CIGI-high vs. CIGI-low; ***P < 0.001, CIGI-high vs. CIGI-low; §P < 0.05, CIGI-high vs. saline; §§P < 0.01, CIGI-high vs. saline; §§§P < 0.001, CIGI-high vs. saline. One-way ANOVA with Bonferroni's multiple comparison test was performed to determine significance.

Fig. 3.

Fasting parameters of glucose homeostasis in CIGI-high rats. A: fasting plasma glucose; n = 51 (day 0), 26 (day 4), and 42 (day 8). B: fasting plasma insulin; n = 22 (day 0), 19 (day 4), and 27 (day 8). C: fasting plasma C-peptide; n = 16 (day 0), 15 (day 4), and 14 (day 8). D: fasting plasma amylin; n = 12 (day 0), 19 (day 4), and 30 (day 8). E: fasting plasma glucagon; n = 9 (day 0), 9 (day 4), and 32 (day 8). F: endogenous glucose production; n = 10 (day 0), 8 (day 4), and 21 (day 8). *P < 0.05; **P < 0.01; ***P < 0.001. Mixed-model analysis with Bonferroni's multiple comparison test was used in A–D, and the 2-tailed unpaired Student t-test was used in E and F.

Fig. 4.

Intravenous glucose tolerance tests (GTT) in CIGI-high rats on days 0, 4, and 8 of infusion. A: plasma glucose concentrations. B: plasma insulin concentrations. In A and B, ○ = day 0, ● = day 4, and □ = day 8. There were no differences in glucose concentrations at any time point between days 4 and 8. C: insulin area under the curve (AUC) during the first 15 min of the GTT. D: total insulin AUC. There were no significant differences between days 4 and 8. In C and D, closed bars = insulin AUC due to baseline insulin secretion, and open bars = insulin AUC above baseline. There were no significant differences in total AUC, baseline AUC, or AUC above baseline between days 4 and 8. E: total insulin AUC above baseline. *P < 0.05. Data are means ± SE of 19 experiments on day 0, 18 experiments on day 4, and 7 experiments on day 8. Significance was determined by the 2-tailed unpaired Student t-test.

Fig. 5.

Intrahepatic metabolites in CIGI-high rats. A: glycogen (n = 5 each day). There were no significant differences between days 4 and 8 by 2-tailed unpaired Student t-test. B: triglycerides [n = 6 (day 0), 7 (day 4), and 14 (day 8)]. C: acyl-CoA (n = 5 each day). D: diacylglycerol (n = 5 each day). Bars spanning between days 4 and 8 refer to P value summary from 1-way analysis of variance. *P < 0.05, **P < 0.01.

Fig. 6.

Expression and protein of hepatic gluconeogenic enzymes. A: glucose-6-phosphatase (G-6-Pase) expression [n = 7 (day 0), 7 (day 4), and 9 (day 8)]. B: fructose-1,6-bisphosphatase (FBPase) expression [n = 6 (day 0), 6 (day 4), and 10 (day 8)]. C: hepatic cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) mRNA [n = 7 (day 0), 7 (day 4), and 13 (day 8)]. D: hepatic mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) mRNA [n = 6 (day 0), 6 (day 4), and 13 (day 8)]. E: Western blots used to measure PEPCK protein, with GAPDH as a loading control. F: hepatic PEPCK-C protein (n = 3 each day). G: hepatic PEPCK-M protein (n = 3 each day). In A–F, 1-way analysis of variance was calculated (bar spanning from days 0 to 8), with individual comparison of day 0 vs. day 4 and day 0 vs. day 8 by 1-way ANOVA with Bonferroni's multiple comparison test. There were no significant differences between days 4 and 8 by 2-tailed unpaired Student t-test. Statistics over individual bars refer to comparisons vs. day 0 by 1-way ANOVA with Bonferroni's multiple comparison test. *P < 0.05; **P < 0.01.

Fig. 7.

Response to treatment with anti-glucagon antibodies in hyperglycemic CIGI-high rats. A: plasma glucose concentrations before and after antibody treatment. B: plasma insulin concentrations before and after treatment. C: hepatic glucose output 6 h after treatment. In A and B, open bars = control group, and closed bars = anti-glucagon antibody treatment group. In A–C, n = 9 after 6 h and 6 after 24 h in the control group, and n = 7 after 6 h and 7 after 24 h in the anti-glucagon antibody group. Significance was evaluated by 2-tailed unpaired Student t-test. *P < 0.05; ***P < 0.001; ††P < 0.01 vs. control group at baseline; ‡‡‡P < 0.001 vs. anti-glucagon antibody treated group at baseline.

Fig. 8.

Hepatic PEPCK expression after anti-glucagon antibody treatment. A: PEPCK-C expression (n = 6 in each group). B: hepatic PEPCK-M expression (n = 7 in each group). *P < 0.05.