Sustained effect of glucagon on body weight and blood glucose: Assessed by continuous glucose monitoring in diabetic rats

Abstract

Insulin is a vital part of diabetes treatment, whereas glucagon is primarily used to treat insulin-induced hypoglycemia. However, glucagon is suggested to have a central role in the regulation of body weight, which would be beneficial for diabetic patients. Since the glucagon effect on blood glucose is known to be transient, it is relevant to investigate the pharmacodynamics of glucagon after repeated dosing. In the present study, we used telemetry to continuously measure blood glucose in streptozotocin induced diabetic Sprague-Dawley rats. This allowed for a more detailed analysis of glucose regulation compared to intermittent blood sampling. In particular, we evaluated the blood glucose-lowering effect of different insulin doses alone, and in combination with a long acting glucagon analog (LAG). We showed how the effect of the LAG accumulated and persisted over time. Furthermore, we found that addition of the LAG decreased body weight without affecting food intake. In a subsequent study, we focused on the glucagon effect on body weight and food intake during equal glycemic control. In order to obtain comparable maximum blood glucose lowering effect to insulin alone, the insulin dose had to be increased four times in combination with 1 nmol/kg of the LAG. In this set-up the LAG prevented further increase in body weight despite the four times higher insulin-dose. However, the body composition was changed. The insulin group increased both lean and fat mass, whereas the group receiving four times insulin in combination with the LAG only significantly increased the fat mass. No differences were observed in food intake, suggesting a direct effect on energy expenditure by glucagon. Surprisingly, we observed decreased levels of FGF21 in plasma compared to insulin treatment alone. With the combination of insulin and the LAG the blood glucose-lowering effect of insulin was prolonged, which could potentially be beneficial in diabetes treatment.

Fig 1. Changes in blood glucose during the two experimental periods as assessed by glucose telemetry.

Solid lines represent dosing during the light periods, and dotted lines represent dosing during the dark periods. Top panel: Low insulin dose period (86 nmol/kg insulin (light blue) and 86 nmol/kg insulin + 1 nmol/kg LAG (light green)). Bottom panel: High insulin dose period (120 nmol/kg insulin (dark blue) and 120 nmol/kg insulin + 1 nmol/kg LAG (dark green)). Shaded background highlights the period where the LAG was added to the insulin treatment. Data are expressed as means ± SEM.

Fig 2. The change in blood glucose after insulin dosing during dark and light periods.

The blood glucose lowering effect of 86 nmol/kg insulin compared to 120 nmol/kg insulin during light (A and B) and dark period (C and D), respectively. The change in blood glucose plotted as a function of time (A and C). The maximum change from baseline (B and D). Data are expressed as means of 5 days (86 nmol/kg) and 2 days (120 nmol/kg), respectively ± SEM; n = 7–8. A significant difference between 86 and 120 nmol/kg insulin during the light period and dark period is indicated by ** (p = 0.001) and * (p = 0.03), respectively. (analyzed using a two-tailed paired t-test).

Fig 3. Mean activity during light and dark periods.

Mean activity from 0–8 h. after dosing of 86 nmol/kg insulin (A) and 120 nmol/kg insulin (B) during both light and dark periods. Data are expressed as means of 5 days (86 nmol/kg) and 2 days (120 nmol/kg), respectively ± SEM; n = 7–8. The significant differences in activity between the light and the dark period are indicated by **** (p < 0.0001) and *** (p = 0.0003), respectively (analyzed using a two-tailed paired t-test).

Fig 4. Sustained glucagon effect on blood glucose regulation.

The blood glucose lowering effect of 86 nmol/kg (A and B) and 120 nmol/kg (C and D) insulin ± 1 nmol/kg LAG during the light period. The change in blood glucose plotted as a function of time (A and C). The maximum change from baseline (B and D). Data are expressed as means of 5 days (86 nmol/kg insulin) and 2 days (86 nmol/kg insulin + 1nmol/kg LAG, 120 nmol/kg ± 1nmol/kg LAG), respectively ± SEM; n = 4–8. A significant effect of adding 1 nmol/kg LAG to both 86 nmol/kg and 120 nmol/kg insulin is indicated by ** (p < 0.003) (analyzed using a two-tailed paired t-test).

Fig 5. Glucagon effect on body weight and food intake.

Top panel: the change in body weight after twice daily dosing for two days with insulin alone or four days in combination with the LAG. A) 86 nmol/kg insulin ± 1nmol/kg LAG. B) 120 nmol/ kg insulin ± 1nmol/kg LAG. Data are expressed as means ± SEM; n = 20. A significant change in body weight compared to insulin alone is indicated by ** (p = 0.002) and **** (p < 0.0001) asterisks (analyzed using two-tailed paired t-test). Bottom panel: The average food intake per day per animal during insulin treatment alone or in combination with the LAG. C) 86 nmol/kg insulin ± 1nmol/kg LAG. D) 120 nmol/ kg insulin ± 1nmol/kg LAG. Data are expressed as means of 2 days ± SEM; n = 10. A significant effect of adding 1 nmol/kg LAG to 120 nmol/kg insulin is indicated by ** (p = 0.001) (analyzed using a two-tailed paired t-test).

Fig 6. Glucagon effect on activity during light period.

Mean activity from 0–8 h. after dosing of 86 nmol/kg insulin ± 1 nmol/kg LAG (A) and 120 nmol/kg insulin ± 1nmol/kg LAG (B) during the light period. Data are expressed as means of 5 days (86 nmol/kg insulin) and 2 days (86 nmol/kg insulin + 1nmol/kg LAG, 120 nmol/kg ± 1nmol/kg LAG), respectively ± SEM; n = 4–8. A significant difference in activity between 120 nmol/kg insulin and 120 nmol/kg insulin + 1 nmol/kg LAG is indicated by * (p = 0.026) (analyzed using a two-tailed paired t-test).

Fig 7. Glucagon effect on blood glucose and glycogen content in the liver.

A) Blood glucose (mM) plotted as function of time. At t = 0 min. the animals were dosed sc. with insulin (103 nmol/kg), insulin (103 nmol/kg) + LAG (1 nmol/kg) or 4 x insulin (412 nmol/kg) + LAG (1 nmol/kg). Blood glucose was measured in tail tip blood for 6 hours. Significant lower blood glucose levels with 4 x insulin + LAG after 300 and 360 min are indicated by **** (p < 0.0001), (analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test). B) The lowest blood glucose levels (mM) obtained during the 6-hour experiment. C) Liver glycogen content (μmol/g tissue) 6 hours after sc. dosing. Data are expressed as means ± SEM; n = 12. A significant impaired blood glucose lowering effect of 103 nmol/kg insulin + 1 nmol/kg LAG (B), and a significant increased glycogen content in the liver (C) are indicated by **** (p < 0.0001), (analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test).

Fig 8. Glucagon effect on body weight, food intake and FGF21.

A) The change in body weight after twice daily dosing for two weeks with insulin (103 nmol/kg), insulin (103 nmol/kg) + LAG (1 nmol/kg) or 4 x insulin (412 nmol/kg) + LAG (1 nmol/kg). B) Average food intake per animal. C) Plasma FGF21(pg/ml). Data are expressed as means ± SEM; n = 12. The significant effects of the LAG in combination with insulin or 4 x insulin, are indicated by **** (p < 0.0001), ** (p = 0.009), * (p = 0.03), respectively (analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test).

Fig 9. Triglycerides in plasma and liver tissue.

Triglycerides in plasma (A) and liver tissue (B) after twice daily dosing for two weeks with insulin (103 nmol/kg), insulin (103 nmol/kg) + LAG (1 nmol/kg) or 4 x insulin (412 nmol/kg) + LAG (1 nmol/kg). Data are expressed as means ± SEM; n = 12. *** (p = 0.0002) indicate significant effect of the 4 x insulin + LAG compared to insulin alone (analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test).