Reducing Glucokinase Activity Restores Endogenous Pulsatility and Enhances Insulin Secretion in Islets From db/db Mice

Abstract

An early sign of islet failure in type 2 diabetes (T2D) is the loss of normal patterns of pulsatile insulin release. Disruptions in pulsatility are associated with a left shift in glucose sensing that can cause excessive insulin release in low glucose (relative hyperinsulinemia, a hallmark of early T2D) and β-cell exhaustion, leading to inadequate insulin release during hyperglycemia. Our hypothesis was that reducing excessive glucokinase activity in diabetic islets would improve their function. Isolated mouse islets were exposed to glucose and varying concentrations of the glucokinase inhibitor d-mannoheptulose (MH) to examine changes in intracellular calcium ([Ca2+]i) and insulin secretion. Acutely exposing islets from control CD-1 mice to MH in high glucose (20 mM) dose dependently reduced the size of [Ca2+]i oscillations detected by fura-2 acetoxymethyl. Glucokinase activation in low glucose (3 mM) had the opposite effect. We then treated islets from male and female db/db mice (age, 4 to 8 weeks) and heterozygous controls overnight with 0 to 10 mM MH to determine that 1 mM MH produced optimal oscillations. We then used 1 mM MH overnight to measure [Ca2+]i and insulin simultaneously in db/db islets. MH restored oscillations and increased insulin secretion. Insulin secretion rates correlated with MH-induced increases in amplitude of [Ca2+]i oscillations (R2 = 0.57, P < 0.01, n = 10) but not with mean [Ca2+]i levels in islets (R2 = 0.05, not significant). Our findings show that correcting glucose sensing can restore proper pulsatility to diabetic islets and improved pulsatility correlates with enhanced insulin secretion.

Figure 1.

[Ca²⁺]_i levels depend on glucokinase activity. (A) In high glucose (20 mM), acute exposure with a 10-mM concentration of the glucokinase inhibitor MH caused a rapid decrease in [Ca²⁺]_i to levels typically observed in very low glucose (n = 17 islets, ***P < 0.001). (B) Stimulation of glucokinase activity with 500 nM Ro-28-1675 in low glucose (3 mM) led to constitutively high [Ca²⁺]_i (n = 10 islets, ***P < 0.001).

Figure 2.

Islet pulsatility depends on glucokinase activity. (A) Representative [Ca²⁺]_i trace showing increasing doses of the glucokinase inhibitor MH in high (20 mM) glucose causes constitutively high [Ca²⁺]_i to go through a range of pulsatile patterns to basal [Ca²⁺]_i. (B) Mean amplitude ± SEM of [Ca²⁺]_i oscillations for each MH concentration (n = 12 islets). (C) Stimulation of glucokinase activity by Ro-28-1675 led to a dose-dependent increase in [Ca²⁺]_i pulsatility to constitutive calcium influx at the highest Ro-28-1675 concentration (n = 12 islets). (D) Mean amplitude ± SEM of [Ca²⁺]_i oscillations for each Ro-28-1675 concentration.

Figure 3.

Overnight MH exposure does not substantially alter [Ca²⁺]_i patterns. Islets from db/db mice were acutely exposed to (A) 1 mM or (B) 2 mM MH and recorded for changes in [Ca²⁺]_i. In a minority of cases [(A) 18%; (B) 21%], short-term MH exposure was sufficient to induce larger amplitude oscillations. (C) In ~80% of cases [(A) 82%; (B) 79%], no change in [Ca²⁺]_i patterns was observed. (D) For islets from heterozygous (het) controls, acute 1 mM MH was sufficient to inhibit calcium influx in 100% of cases.

Figure 4.

The glucose-stimulated [Ca²⁺]_i response was left shifted in islets from newly diabetic db/db mice, which was reversed by MH treatment. (A) Example of islet traces from untreated db/db islets (blue), MH-treated db/db islets (orange), and heterozygous (het) controls (black) that were stimulated from 0 to 8 to 16 mM glucose. Other islets were similarly stimulated from 4 to 12 to 20 mM glucose. (B) Mean [Ca²⁺]_i response to glucose for each treatment group averaged from three trials. (C) EC50 for glucose stimulation calculated by sigmoidal curve fitting of each of three trials for all conditions. (D) Dose–response curves after overnight treatment with 0.5, 1, or 2 mM MH. MH showed a clear dose-dependent right shift and reduction of maximal [Ca²⁺]_i response to glucose. ^#P < 0.10; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5.

Overnight MH treatment produced a dose-dependent inverted U-shaped curve in islets from newly diabetic db/db mice. Islets from (A) db/db mice or (B) heterozygous (het) control mice were exposed to 0.5, 1, 2, or 5 mM MH overnight. (C) Percentage of islets displaying oscillations for each MH dose produced an inverted U-shaped curve for db/db and a dose-dependent decrease for het controls. (D) Mean amplitude of oscillations for each MH dose produced a pattern similar to that shown in (C). At least five islets were included for each condition for each of five trials. *P < 0.05, ^#P < 0.10 by paired t test.

Figure 6.

Overnight MH treatment enhanced ATP content and glucose-stimulated NAD(P)H flux in diabetic islets. (A) Islets from db/db mice and heterozygous (het) controls were incubated overnight in RPMI media with 11 mM glucose with or without 1 mM MH. After washout and additional incubation, islets were collected and lysed to detect ATP by luminescence assay; 15 to 20 islets per condition that had been size matched for islet mass were used for each of 8 replicates. (B) Islets were incubated with or without MH as described in (A). Islets were then imaged to detect changes in NAD(P)H from 3 to 20 mM glucose stimulation. The mean change in NAD(P)H is listed for each group of islets (n = five to nine islets for each treatment condition). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7.

(A,B) Examples of a 30-min [Ca²⁺]_i pattern from (top) db/db or (bottom) heterozygous (het) islets (A) untreated or (B) after overnight MH exposure. Sets of 30 islets per condition were used to collect perifusate during the [Ca²⁺]_i recording. The perfusate was then assayed for insulin release. After MH exposure, both the percentage of islets displaying (C) oscillations and (D) insulin secretion decreased for islets from het mice (n = 5) but increased for islets from db/db mice (n = 10). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8.

Insulin secretion correlated with pulsatility but not [Ca²⁺]_i levels. (A,B) Scatter plots of MH-induced differences in insulin secretion (x-axis) plotted against MH-induced differences in the (A) amplitude of oscillations or (B) mean [Ca²⁺]_i level (y-axis) for islets from db/db mice. Each data point represents the secretion of a set of 30 islets paired with [Ca²⁺]_i imaging data recorded simultaneously for each individual islet in 11 mM glucose for 30 min. These findings support the hypothesis that reducing excessive glycolytic activity restores glucose sensing and normal islet function.

Figure 9.

Model of the range of blood glucose concentrations promoting insulin pulsatility. Normal nonfasted blood glucose range (for mice) boxed in green. Dashed blue lines indicate the range of pulsatile insulin release. Normal insulin release (black curve within the green box) is largely within the pulsatile range (between blue dashed lines). In early T2D, the curve (blue) is shifted: pulsatile insulin occurs at lower than normal glucose levels to the left of the green box, but maximum secretory capacity is still intact. However, this shift in glucose sensing results in excess insulin release with low glucose and a loss of pulsatility in normal postprandial glucose levels, which might further contribute to hepatic insulin resistance. Loss-of-function glucokinase mutations such as MODY2 in the most severe cases result in the opposite effect. Insulin pulsatility begins for MODY2 (red curve) in hyperglycemic conditions to the right of the green box. Chronic T2D (gray curve) results in substantial deterioration of insulin secretory capacity across all glucose levels. Note that the blood glucose concentrations are more closely related to the mouse; however, the curves also apply conceptually to humans.