The glutamate receptor GluK2 contributes to the regulation of glucose homeostasis and its deterioration during aging

Abstract

Objective: Islets secrete neurotransmitters including glutamate which participate in fine regulation of islet function. The excitatory ionotropic glutamate receptor GluK2 of the kainate receptor family is widely expressed in brain and also found in islets, mainly in α and γ cells. α cells co-release glucagon and glutamate and the latter increases glucagon release via ionotropic glutamate receptors. However, neither the precise nature of the ionotropic glutamate receptor involved nor its role in glucose homeostasis is known. As isoform specific pharmacology is not available, we investigated this question in constitutive GluK2 knock-out mice (GluK2^-/-) using adult and middle-aged animals to also gain insight in a potential role during aging.

Methods: We compared wild-type GluK2^+/+ and knock-out GluK2^-/- mice using adult (14-20 weeks) and middle-aged animals (40-52 weeks). Glucose (oral OGTT and intraperitoneal IPGTT) and insulin tolerance as well as pyruvate challenge tests were performed according to standard procedures. Parasympathetic activity, which stimulates hormones secretion, was measured by electrophysiology in vivo. Isolated islets were used in vitro to determine islet β-cell electrical activity on multi-electrode arrays and dynamic secretion of insulin as well as glucagon was determined by ELISA.

Results: Adult GluK2^-/- mice exhibit an improved glucose tolerance (OGTT and IPGTT), and this was also apparent in middle-aged mice, whereas the outcome of pyruvate challenge was slightly improved only in middle-aged GluK2^-/- mice. Similarly, insulin sensitivity was markedly enhanced in middle-aged GluK2^-/- animals. Basal and glucose-induced insulin secretion in vivo was slightly lower in GluK2^-/- mice, whereas fasting glucagonemia was strongly reduced. In vivo recordings of parasympathetic activity showed an increase in basal activity in GluK2^-/- mice which represents most likely an adaptive mechanism to counteract hypoglucagonemia rather than altered neuronal mechanism. In vitro recording demonstrated an improvement of glucose-induced electrical activity of β-cells in islets obtained from GluK2^-/- mice at both ages. Finally, glucose-induced insulin secretion in vitro was increased in GluK2^-/- islets, whereas glucagon secretion at 2 mmol/l of glucose was considerably reduced.

Conclusions: These observations indicate a general role for kainate receptors in glucose homeostasis and specifically suggest a negative effect of GluK2 on glucose homeostasis and preservation of islet function during aging. Our observations raise the possibility that blockade of GluK2 may provide benefits in glucose homeostasis especially during aging.

Keywords: Aging; GRIK2; GluK2; Islets; Kainate receptor; Microelectrode array.

Figure 1

Improved glucose tolerance, pyruvate challenge outcome and insulin sensitivity in GluK2^−/− mice. Given are time courses and the area under the curve (AUC). (a, b) Intraperitoneal Glucose Tolerance Test (c, d), Oral Glucose Tolerance Test (OGTT) and (e,f) Intraperitoneal Pyruvate Challenge Test were performed in WT and KO mice, in both adult and middle-aged animals. (g) Insulin Tolerance Test (0.5 U/kg) was performed in WT (open circles) and KO mice (closed red circles), both in adults and middle-aged. (h) Area under the curves of g (AUC). Means ± SEM; a, c, e and g, t-test; b, d, f and h, ANOVA followed by Tukey post-hoc test; *p < 0.05, **p < 0.01, ***p < 0.001 or by Holm-Sidak post-hoc test, ^#p < 0.05; N = 7 except for g and h (N = 6).

Figure 2

Middle-aged GluK2^−/− mice have reduced basal and glucose-stimulated insulinemia as wells reduced fasting glucagonemia. (a) Insulin levels in vivo were lower in KO mice. Means ± SEM; N = 7. (b) Fasting glucagonemia is significantly reduced in middle-aged KO mice. Means ± SEM; N = 7. Mann–Whitney; * 2p < 0.05, ** 2p < 0.01.

Figure 3

Middle-aged GluK2^−/− mice have an increased electrical activity of the parasympathetic nerve in the fasted state. (a) Electrical signals of in vivo recordings during fasting state and after an intraperitoneal injection of glucose (2 mg/kg) in middle-aged GluK2^+/+ (WT) or GluK2^−/− (KO) mice. (b) Statistics. Means ± SEM; ANOVA and Tukey's post-hoc analysis; *2p < 0.05, ***p < 0.001; N = 3–5.

Figure 4

Increased glucose-induced islet β-cell activity in adult and middle aged GluK2^−/− mice. (a–c) Electrical activity of islets was recorded in vitro on microelectrode arrays at 3 and 8.2 mM glucose. (a, b) Given are means and SEM of average slow potential frequency per mice (GluK2^+/+, WT; GluK2^−/−, KO; N = 4 independent preparations of mouse islets for each condition in adult (a) or middle aged (b) mice). (c, d) Areas under the curve (AUC) for first phase (5–15 min) and second phase (25–50 min). Given are means ± SD, N = 4 independent islet preparations and assays representing analysis of 126–183 islets for each condition (WT and KO, adult (c) or middle aged (d) mice). Mann–Whitney test; ****, 2p < 0.0001. (e) Glucose perfusion pattern (as used in f and h); initial perfusion at 11 mM glucose was reduced to 2 mM and subsequently raised again to 11 mM. (f) Insulin release (as glucose induced insulin secretion in regard to the value at 3 mM glucose at 60 min) for middle aged GluK2^+/+ (WT) and GluK2^−/− (KO) mice. (g) Area under the curves for insulin (AUC, t = 82–110 min). Means ± SEM. (h) Glucagon release (as fold stimulation in regard to value at 11 mM glucose at 50 min) for middle-aged GluK2^+/+ (WT) and GluK2^−/− (KO) mice. (k) Area under the curves for glucagon (AUC, t = 55–80 min). (f–k) Means ± SEM; N = 3 independent islet preparations and assays; t-test; * 2p < 0.05; ** 2p < 0.01. Hormone contents did not differ significantly between islets from WT and KO animals (see Supplemental Fig. S3).