Connexin-36 gap junctions regulate in vivo first- and second-phase insulin secretion dynamics and glucose tolerance in the conscious mouse

Abstract

Insulin is secreted from the islets of Langerhans in coordinated pulses. These pulses are thought to lead to plasma insulin oscillations, which are putatively more effective in lowering blood glucose than continuous levels of insulin. Gap-junction coupling of β-cells by connexin-36 coordinates intracellular free calcium oscillations and pulsatile insulin release in isolated islets, however a role in vivo has not been shown. We test whether loss of gap-junction coupling disrupts plasma insulin oscillations and whether this impacts glucose tolerance. We characterized the connexin-36 knockout (Cx36(-/-)) mouse phenotype and performed hyperglycemic clamps with rapid sampling of insulin in Cx36(-/-) and control mice. Our results show that Cx36(-/-) mice are glucose intolerant, despite normal plasma insulin levels and insulin sensitivity. However, Cx36(-/-) mice exhibit reduced insulin pulse amplitudes and a reduction in first-phase insulin secretion. These changes are similarly found in isolated Cx36(-/-) islets. We conclude that Cx36 gap junctions regulate the in vivo dynamics of insulin secretion, which in turn is important for glucose homeostasis. Coordinated pulsatility of individual islets enhances the first-phase elevation and second-phase pulses of insulin. Because these dynamics are disrupted in the early stages of type 2 diabetes, dysregulation of gap-junction coupling could be an important factor in the development of this disease.

FIG. 1.

Phenotype of Cx36^−/− mice. A: Intraperitoneal (i.p.) glucose tolerance test (IPGTT) on male Cx36^+/+ (■) and Cx36^−/− mice (◇), each 16 weeks of age, after 2 g/kg body weight (b.w.) i.p. glucose injection. n = 6 littermate mice in each group. B: IPGTT on female mice 16 weeks of age, as in A. n = 8 littermate mice in each group. C: Oral GTT on male mice 16 weeks of age after 2 g/kg b.w. oral gavage. n = 6 littermate mice in each group. D: Area under the curve (AUC) of the glucose excursion during IPGTT after i.p. injection of variable amounts of glucose. n = 9 age-matched mice studied in parallel in each group. E: Plasma insulin measurements in male mice aged 16 weeks before (0) and 30 min after 3 g/kg b.w. i.p. glucose injection (black bars, Cx36^+/+ mice; white bars, Cx36^−/− mice). n = 8 littermate mice in each group. F: Plasma glucagon measurements in male mice aged 16 weeks, before (0) and 30 min after i.p. glucose injection, as in D. n = 7 littermate mice in each group. G: Insulin tolerance test after 0.75 units/kg i.p. insulin injection (see arrow). n = 10 littermate mice in each group. *, significant difference (P < 0.05, two-tailed Student t test) at each time point comparing measurements in Cx36^+/+ and Cx36^−/− mice.

FIG. 2.

Measuring plasma insulin dynamics. A: Three representative time courses of rapid sampling blood glucose (Glucose, mg/dL) and plasma insulin levels (Insulin, ng/mL) from Cx36^+/+ mice during hyperglycemic clamp. Sampling rate is one per minute. The x-axis indicates the time after glucose infusion is started. #, center of each pulse identified during pulse analysis. B: Three representative time courses of rapid sampling glucose and plasma insulin levels from Cx36^−/− mice during hyperglycemic clamp, as in A. C: Time-averaged blood glucose in male littermate Cx36^+/+ mice (black bars) and Cx36^−/− mice (white bars) at 16–17 weeks of age before and during the hyperglycemic clamp, 40 min after the start of glucose infusion. n = 10 and 8 mice in Cx36^+/+ and Cx36^−/− groups, respectively. D: Time-averaged plasma insulin levels corresponding to measurements made in C. E: Time-averaged glucose infusion rate required to establish glucose clamp in C. F: Norepinephrine levels at the end point of hyperglycemic clamp combined from both T0 and T40 groups, averaged over n = 8 mice in each experimental group. *, significant difference (P < 0.05, Student t test) comparing each measurement in Cx36^+/+ and Cx36^−/− mice.

FIG. 3.

Analysis of plasma insulin oscillations. A: Representative time courses of plasma insulin levels from a Cx36^+/+ mouse (trace iii in Fig. 2A) together with pulse analysis, which is offset for clarity. B: Representative time courses of plasma insulin levels from a Cx36^−/− mouse (trace iii in Fig. 2B) together with pulse analysis, as in A. C: Mean time interval between consecutive pulses for Cx36^+/+ and Cx36^−/− mice, 0 (T0) or 40 min (T40) after glucose infusion. n = 5 and 10 Cx36^+/+ mice and 5 and 8 Cx36^−/− mice in the T0 and T40 groups, respectively. D: Pulse regularity, defined by the SD of pulse interval in each time course for Cx36^+/+ and Cx36^−/− mice, as in C. E: Mean pulse area for Cx36^+/+ and Cx36^−/− mice, as in C. F: Mean pulse amplitude above basal levels for Cx36^+/+ and Cx36^−/− mice, as in C. *, significant difference (P ≤ 0.05, Student t test) comparing each measurement in Cx36^+/+ and Cx36^−/− mice.

FIG. 4.

First-phase insulin secretion in vivo and ex vivo. A: Mean time course of plasma insulin levels from Cx36^+/+ (■) and Cx36^−/− mice (◇) before and immediately after glucose infusion, as well as between 45 and 55 min after glucose infusion. Sampling rate is one per minute. n = 5 littermate mice in each group. B: Mean time course of insulin secretion levels from isolated Cx36^+/+ and Cx36^−/− islets (per 100 IEQ) after elevated glucose levels. Arrow indicates glucose step from 2.8 to 11.1 mM. Sampling rate is 0.5 per minute until t = 20, then 0.25 per minute thereafter. n = 6 sets of islets from individual mice in each group. *, significant reduction; †, significant elevation (P < 0.05, paired Student t test) in Cx36^−/− islets compared with Cx36^+/+ islets. C: Summary of mean plasma insulin levels prior to glucose infusion (basal), peak plasma insulin levels 1–3 min after glucose infusion (first phase), and mean plasma insulin levels 45–55 min after glucose infusion (second phase). D: Summary of mean insulin secretion levels during perifusion, prior to glucose elevation (basal), peak insulin secretion at 10 min after glucose elevation (first phase), and mean insulin secretion 40 min after glucose elevation (second phase). E: Mean time from first-phase peak of insulin secretion to first minimum of insulin secretion. F: Mean time-integrated insulin secretion from initial elevation at t = 5 min to first minimum of insulin secretion between t = 10 and 20 min. *, significant difference (P < 0.05, Student t test); NS, no significant difference (P > 0.1, Student t test), comparing each measurement in Cx36^+/+ and Cx36^−/− mice or islets.