Adolescents with clinical type 1 diabetes display reduced red blood cell glucose transporter isoform 1 (GLUT1)

Abstract

Type 1 diabetic (T1D) adolescent children on insulin therapy suffer episodes of both hyper- and hypoglycemic episodes. Glucose transporter isoform GLUT1 expressed in blood-brain barrier (BBB) and red blood cells (RBC) compensates for perturbed circulating glucose toward protecting the supply to brain and RBCs. We hypothesized that RBC-GLUT1 concentration, as a surrogate for BBB-GLUT1, is altered in T1D children. To test this hypothesis, we measured RBC-GLUT1 by enzyme-linked immunosorbent assay (ELISA) in T1D children (n = 72; mean age 15.3 ± 0.2 yr) and control children (CON; n = 11; mean age 15.6 ± 0.9 yr) after 12 h of euglycemia and during a hyperinsulinemic-hypoglycemic clamp with a nadir blood glucose of ~3.3 mmol/L for 90 min (clamp I) or ~3 mmol/L for 45 min (clamp II). Reduced baseline RBC-GLUT1 was observed in T1D (2.4 ± 0.17 ng/ng membrane protein); vs. CON (4.2 ± 0.61 ng/ng protein) (p < 0.0001). Additionally, baseline RBC-GLUT1 in T1D negatively correlated with hemoglobin A1c (HbA1c) (R = -0.23, p < 0.05) but not in CON (R = 0.06, p < 0.9). Acute decline in serum glucose to 3.3 mmol/L (90 min) or 3 mmol/L (45 min) did not change baseline RBC-GLUT1 in T1D or CON children. We conclude that reduced RBC-GLUT1 encountered in T1D, with no ability to compensate by increasing during acute hypoglycemia over the durations examined, may demonstrate a vulnerability of impaired RBC glucose transport (serving as a surrogate for BBB), especially in those with the worst control. We speculate that this may contribute to the perturbed cognition seen in T1D adolescents.

Keywords: BBB; RBC glucose transporter 1; T1D; hyperinsulinemic clamp; hypoglycemic clamp.

Figure 1. Standard curve for GLUT1 ELISA

The standard curve (n=6) for ELISA is shown (means ±SEM) ranging from 0.2 to 1 ng of the GLUT1 peptide plotted against the optical density at the 450 nm wave length. (r=0.99, p<0.0001)

Figure 2. Serum glucose and RBC GLUT1 in T1D and CON adolescents

2A: Baseline serum glucose concentration (mmol/L) in CON (n=11) and T1D (n=72) groups, 2 B: HbA_1C in CON and T1D groups, and, 2C: RBC GLUT1 (ng/ng protein) in CON and T1D, *p<0.0014, **p<0.0001 versus CON.

Figure 3. Serum Glucose and RBC GLUT1 in CON and T1D comparing HbA_1C <8% (64 mmol/mol) and HbA_1C >8(64 mmol/mol)

3A:Baseline serum glucose (mmol/L) in CON (n=11), T1D with HbA_1C <8% (<64mmol/mol, n=28)) and HbA_1C >8% (>64 mmol/mol, n=44)), 3B: RBC GLUT1 (ng/ng protein) in CON (n=11), T1D with HbA_1C <8 (<64 mmol/mol, n=28) and HbA_1C >8% (>64 mmol/mol, n=44), 3C: Scatter plot between RBC GLUT1 and HbA_1C in CON (n=11) showing no correlation and 3D: Scatter plot between RBC GLUT1 and HbA_1C in T1D (n=72) showing lower serum HbA_1C levels resulting in higher baseline RBC GLUT1 concentrations in T1D adolescents (n=72) (R=0.23, p<0.05).

Figure 4. Hyperinsulinemic clamp studies in T1D and CON groups

4A: The schematic diagram for clamp 1 and clamp 2 studies. 4B: Hyperinsulinemic clamp I study in CON children (n=8). Left y axis demonstrates the serum glucose concentration in mmol/l and right y-axis indicates the RBC GLUT1 concentrations in ng/ng protein. The x-axis shows time points B, baseline; EU, euglycemia; H45 and H90 min, after 45 and 90 minutes of hypoglycemia; and EU30, after 30 min of return to euglycemia. 4C: Hyperinsulinemic clamp I study in T1D children (n=8). The left y-axis demonstrates serum glucose in mmol/L and right y-axis indicates RBC GLUT1 in ng/ng. B, The x-axis shows time points B, baseline; EU, euglycemia; H45 and H90 min, after 45 and 90 minutes of hypoglycemia; and EU30, after 30 min of return to euglycemia 4D: Hyperinsulinemic clamp II study in T1D children (n=8). Left x-axis demonstrates the serum glucose concentration in mmol/L and right y-axis indicates RBC GLUT1 concentrations in ng/ng protein. The x-axis shows time points : B, baseline; EU1 and EU2, after 60 and 120 minutes of euglycemia; H, during hypoglycemia; and EU30, after 30 min of return to euglycemia., * p<0.0001 Serum glucose values compared to the baseline serum glucose values for each group. α p<0.001 RBC GLUT1 concentration in T1D group compared to the corresponding RBC GLUT1 concentration in the CON group.