Defective insulin secretion and increased susceptibility to experimental diabetes are induced by reduced Akt activity in pancreatic islet beta cells

Abstract

The insulin and IGF signaling pathways are critical for development and maintenance of pancreatic beta cell mass and function. The serine-threonine kinase Akt is one of several mediators regulated by these pathways. We have studied the role of Akt in pancreatic beta cell physiology by generating transgenic mice expressing a kinase-dead mutant of this enzyme in beta cells. Reduction of Akt activity in transgenic animals resulted in impaired glucose tolerance due to defective insulin secretion. The mechanisms involved in dysregulation of secretion in these mice lie at the level of insulin exocytosis and are not the result of abnormalities in glucose signaling or function of voltage-gated Ca2+ channels. Therefore, transgenic mice showed increased susceptibility to developing glucose intolerance and diabetes following fat feeding. These observations suggest that Akt plays a novel and important role in the regulation of distal components of the secretory pathway and that this enzyme represents a therapeutic target for improvement of beta cell function in diabetes.

Figure 1

Overexpression of kdAkt inhibits Akt activity and phosphorylation of Akt targets in RIP-kdAkt islets. (A) Total pancreatic islet lysates from RIP-kdAkt and WT mice were immunoblotted with Ab’s against HA, Akt, or actin. Semiquantitation of total Akt protein level in islets from RIP-kdAkt and WT mice was adjusted to actin as loading control (n = 6). (B) Total in vitro Akt kinase activity from 400 μg of islet lysates. Upper blot: Immunoblotting for Akt in the immunoprecipitate. Middle blot and bar graph: In vitro Akt kinase activity assayed by immunoblotting with anti_phospho-GSK3 Ab’s (p-GSK3) with semiquantitative analysis (n = 3). Lower blot: Immunoblotting for Akt in the post-immunoprecipitation supernatant (Post-IP sup). (C and D) Phosphorylation status in islet lysates and semiquantitative analysis of band intensities for phospho-GSK3 (Ser9; n = 5) (C) and phospho-S6K (Thr389; n = 6) (D) in transgenics and WT mice. (E) Immunoblotting for phospho-Foxo1 (Ser256) and actin in lysates from RIP-kdAkt and WT islets. The data are representative of 3 independent experiments done in duplicate. Data for phospho-GSK3 and phospho-S6K were adjusted to actin and are presented as mean ± SE. *P < 0.05.

Figure 2

Expression of kdAkt in β cells results in impaired glucose tolerance and defective insulin secretion. (A andB) Intraperitoneal glucose tolerance tests were performed on 6- to 8-week-old (A) and 6-month-old (B) RIP-kdAkt and WT male mice fasted for 15_18 hours (n ≥ 5). (C and D) Insulin secretion in vivo after intraperitoneal glucose (3 g/kg) in 6- to 8-week-old (C) and 6-month-old (D) RIP-kdAkt and WT male mice (n ≥ 5). Data are presented as mean ± SE. *P < 0.05.

Figure 3

RIP-kdAkt mice exhibit abnormal insulin secretion in vitro. (A) Assessment of insulin secretion by islet perifusion experiments with low (2 mM) and high (20 mM) glucose in islets from RIP-kdAkt and WT mice (n = 3). Net insulin-release response, quantified by integration of the area under the curve (AUC) during the treatment period, is shown in the inset. (B) Assessment of insulin secretion by islet perifusion experiments after stimulation with KCl (30 mM) in islets from RIP-kdAkt and WT mice (n = 3). (C) Exposure of RIP-kdAkt and WT islets to step increases in ionomycin concentration in perifusion experiments (n = 3). Data are presented as mean ± SE. *P < 0.05.

Figure 4

Insulin secretory defect in RIP-kdAkt mice is independent of alterations in islet morphology and β cell mass. (A) Immunofluorescence staining for insulin (green) and a mixture of anti_glucagon, anti_pancreatic polypeptide, and anti_somatostatin Ab’s (red) in RIP-kdAkt and WT mice (n = 3). (B) Islet β cell mass in 4-month-old RIP-kdAkt and control males (n = 6). (C) Islet size distribution obtained from animals used for β cell mass measurements. Data are presented as mean ± SE.

Figure 5

RIP-kdAkt mouse islets exhibit similar gene expression, normal intracellular calcium responses to glucose and KCl, and comparable secretory protein levels. (A) Gene expression by real-time RT-PCR in islets from RIP-kdAkt and WT mice (n = 3). (B and C) Measurements of whole-islet Ca²⁺ induced by a progressive increase in glucose concentration from 2 to 26 mM over 50 minutes followed by an increase in KCl to 20 mM in isolated islets from RIP-kdAkt (B) and WT (C) mice. (D and E) Measurements of intracytosolic Ca²⁺ induced by 10 mM glucose and 20 mM KCl in single β cells from RIP-kdAkt (D) and WT (E) islets. Representative tracing is depicted in red (RIP-kdAkt) and blue (WT), and average cytosolic Ca²⁺ responses are observed in gray (n = 10_15). (F) Secretory protein expression by Western blotting in RIP-kdAkt and WT islets. The experiment is representative of 3 independent experiments done in duplicate.

Figure 6

Deterioration of the glucose intolerance associated with insulin resistance induced by fat feeding in RIP-kdAkt mice. (A) Intraperitoneal glucose tolerance test in 12-week-old RIP-kdAkt and WT mice after 12 hours of fasting (n = 7). (B) Islet area after 14 weeks of fat feeding in RIP-kdAkt and WT mice (n = 6). (C) Islet size distribution obtained from mice used for β cell mass measurements. Data are presented as mean ± SE. *P ≤ 0.05.

Figure 7

Assessment of islet apoptosis in RIP-kdAkt and WT mice. (A) Frequency of activated caspase-positive cells in islets from RIP-kdAkt and WT mice (n = 3). (B) DNA laddering in RIP-kdAkt and WT islets after 48 hours of incubation in RPMI containing 10 mM glucose, 10% serum (control medium), and 1 μM thapsigargin. (C) Apoptosis assessment by DNA laddering in RIP-kdAkt and WT islets cultured for 7 days in control medium and in serum-free RPMI with 10 mM glucose. Data are representative of at least 3 experiments in duplicate.