Chronic Hyperinsulinaemic Hypoglycaemia in Rats Is Accompanied by Increased Body Weight, Hyperleptinaemia, and Decreased Neuronal Glucose Transporter Levels in the Brain

Abstract

The brain is vulnerable to hypoglycaemia due to a continuous need of energy substrates to meet its high metabolic demands. Studies have shown that severe acute insulin-induced hypoglycaemia results in oxidative stress in the rat brain, when neuroglycopenia cannot be evaded despite increased levels of cerebral glucose transporters. Compensatory measures in the brain during chronic insulin-induced hypoglycaemia are less well understood. The present study investigated how the brain of nondiabetic rats copes with chronic insulin-induced hypoglycaemia for up to eight weeks. Brain level of different substrate transporters and redox homeostasis was evaluated. Hyperinsulinaemia for 8 weeks consistently lowered blood glucose levels by 30-50% (4-6 mM versus 7-9 mM in controls). The animals had increased food consumption, body weights, and hyperleptinaemia. During infusion, protein levels of the brain neuronal glucose transporter were decreased, whereas levels of lipid peroxidation products were unchanged. Discontinued infusion was followed by transient systemic hyperglycaemia and decreased food consumption and body weight. After 4 weeks, plasma levels of lipid peroxidation products were increased, possibly as a consequence of hyperglycaemia-induced oxidative stress. The present data suggests that chronic moderate hyperinsulinaemic hypoglycaemia causes increased body weight and hyperleptinaemia. This is accompanied by decreased neuronal glucose transporter levels, which may be leptin-induced.

Figure 1

(a) Body weight, group mean + SD. (b) Food consumption, group mean + SD. Grey areas indicate infusion period. Days −7 to 29: n = 32 − 38/group per time point, days 32–57: n = 20 − 28/group per time point (except for days 53 and 57 for group HI-M, where n = 15 − 17/time point), and days R4–R29: n = 5 − 10/group per time point. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus controls.

Figure 2

Blood glucose levels. Plasma profile Day 1 (a), Day 25 (b), Day 53 (c), and Day R1 (d). Individual (symbols) and mean (lines) values, n = 1 − 2/time point. Infusion start was defined as time point zero on day 1. Time point zero on day R1 corresponds to the time point when infusion was stopped. (e) Whole blood mean values + SD. Grey area indicates infusion period. During infusion: days 2–23: n = 28 − 38/group per time point, days 26–51: n = 22 − 28/group per time point, and day 54: n = 12 − 17/group per time point. During infusion-free period (recovery): n = 5 − 10/group per time point. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 for HI-M versus CTRL-M; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 for HI-F versus CTRL-F. Levels in the HI-M and HI-F groups were only significantly different on two time points: day 5 (p < 0.05) and day R12 (p < 0.05).

Figure 3

Relative protein levels of substrate transporters at the two different time points during infusion. Mean + SD. (a) GLUT1, each day depicted separately, (b) GLUT3, each day depicted separately, (c) GLUT3, overall effect of HI-dosing, sex pooled. (d) SGLT1 and (e) MCT1, each day depicted separately. (f) Representative pictures of the western blots. CTRL-M: n = 7 − 10, HI-M: n = 4 − 8, CTRL-F: n = 6 − 9, and HI-F: n = 6 − 10. OD: optical density, AU: arbitrary units. Actin levels were used as an internal reference. ^∗p < 0.05 for effect of treatment; ^#p < 0.05 for effect of sex.

Figure 4

MDA concentration in brain tissue, mean + SD. CTRL-M: n = 7 − 10, HI-M: n = 5 − 7, CTRL-F: n = 8 − 9, and HI-F: n = 6 − 9. Statistical analysis showed no effect of HI-dosing or sex at any of the time points.

Figure 5

Plasma levels of lipoperoxidation products, mean + SD. (a) MDA, day 56: n = 4 − 10 and day R29: n = 5 − 10. (b) 8-ISO, day 56: n = 4 − 10 and day R29: n = 5 − 10. ^∗p < 0.05 and ^∗∗∗p < 0.001 for effect of HI-dosing. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 for effect of sex.

Figure 6

Plasma leptin levels, mean + SD. CTRL-M: n = 7 − 10, HI-M: n = 4 − 8, CTRL-F: n = 8 − 10, and HI-F: n = 6 − 9. At all three time points, males had significantly higher leptin levels compared to females (p = 0.0325, p < 0.0001, and p = 0.0182). ^∗∗∗p < 0.001.