Inhibition of Gsk3β activity improves β-cell function in c-KitWv/+ male mice

Abstract

Previous studies have shown that the stem cell marker, c-Kit, is involved in glucose homeostasis. We recently reported that c-Kit(Wv/+) male mice displayed the onset of diabetes at 8 weeks of age; however, the mechanisms by which c-Kit regulates β-cell proliferation and function are unknown. The purpose of this study is to examine if c-Kit(Wv/+) mutation-induced β-cell dysfunction is associated with downregulation of the phospho-Akt/Gsk3β pathway in c-Kit(Wv/+) male mice. Histology and cell signaling were examined in C57BL/6J/Kit(Wv/+) (c-Kit(Wv/+)) and wild-type (c-Kit(+/+)) mice using immunofluorescence and western blotting approaches. The Gsk3β inhibitor, 1-azakenpaullone (1-AKP), was administered to c-Kit(Wv/+) and c-Kit(+/+) mice for 2 weeks, whereby alterations in glucose metabolism were examined and morphometric analyses were performed. A significant reduction in phosphorylated Akt was observed in the islets of c-Kit(Wv/+) mice (P<0.05) along with a decrease in phosphorylated Gsk3β (P<0.05), and cyclin D1 protein level (P<0.01) when compared with c-Kit(+/+) mice. However, c-Kit(Wv/+) mice that received 1-AKP treatment demonstrated normal fasting blood glucose with significantly improved glucose tolerance. 1-AKP-treated c-Kit(Wv/+) mice also showed increased β-catenin, cyclin D1 and Pdx-1 levels in islets, demonstrating that inhibition of Gsk3β activity led to increased β-cell proliferation and insulin secretion. These data suggest that c-Kit(Wv/+) male mice had alterations in the Akt/Gsk3β signaling pathway, which lead to β-cell dysfunction by decreasing Pdx-1 and cyclin D1 levels. Inhibition of Gsk3β could prevent the onset of diabetes by improving glucose tolerance and β-cell function.

Figure 1. Downregulation of phosphorylated Akt/Gsk3β signaling in the pancreas and isolated islet from c-Kit^Wv/+ mice

Western blot analysis of S473-phosphorylated (P) and total (T) Akt (a), phosphorylated (P) and total (T) Gsk3β (b) protein level in the pancreas and isolated islets of c-Kit^+/+ and c-Kit^Wv/+ male mice at 8 week of age. Representative blots are shown. Data are normalized to total protein and expressed as means ± SEM (n=11 pancreas; n=3 isolated islets per experimental group) *P<0.05 vs. c-Kit^+/+ male mice.

Figure 2. Decreased cyclin D1 protein level in isolated islet of c-Kit^Wv/+ mice

(a) Western blot analysis of cyclin D1 in the pancreas and isolated islets of c-Kit^+/+ and c-Kit^Wv/+ male mice at 8 week of age. Representative blots are shown. Data are normalized to loading control calnexin and expressed as means ± SEM (n=4 pancreas; n=6 isolated islets per experimental group) **p<0.01 vs. c-Kit^+/+ male mice. (b) Double immunofluorescence staining for cyclin D1 (green), insulin (red) of c-Kit^+/+ and c-Kit^Wv/+ male mice pancreatic section at 8 week of age. Nuclear counter stained by DAPI (blue). Scale bar: 20μm (inset: 10μm). Arrows indicate cyclin D1/insulin double positive cells. (c) The percent of cyclin D1⁺ cells in β-cells of c-Kit^+/+ and c-Kit^Wv/+ male mice at 8 week of age. Data are expressed as mean percent positive cells over insulin⁺ cells ± SEM (n=6) *P<0.05 vs. c-Kit^+/+ male mice.

Figure 3. Effect of Gsk3β inhibitor on glucose homeostasis of c-Kit^Wv/+/1-AKP mice

Body weight (a) and fasting blood glucose level (b) of c-Kit^+/+/saline, c-Kit^+/+/1-AKP, c-Kit^Wv/+/saline, and c-Kit^Wv/+/1-AKP groups. Intra-peritoneal glucose tolerance test (IPGTT) (c) and insulin tolerance test (IPITT) (d) in c-Kit^+/+/saline, c-Kit^+/+/1-AKP, c-Kit^Wv/+/saline, and c-Kit^Wv/+/1-AKP mouse group. Glucose responsiveness of the corresponding experimental groups is shown as a measurement of area under the curve (AUC) of the IPGTT or IPITT graphs with units of [(mmol/L). min] shown on the Y-axis. Data are expressed as means ± SEM (n=6–8) *P<0.05, **P<0.01 ***P<0.001 vs. c-Kit^Wv/+/saline group.

Figure 4. Effect of Gsk3β inhibitor on β-cell function of c-Kit^Wv/+/1-AKP mice

(a) In vivo glucose-stimulated insulin secretion of c-Kit^+/+/saline, c-Kit^+/+/1-AKP, c-Kit^Wv/+/saline, and c-Kit^Wv/+/1-AKP groups. c-Kit^Wv/+/1-AKP groups demonstrated an increase in plasma insulin release after glucose loading (n=3–8). (b) Insulin secretion is improved in isolated islets from male c-Kit^Wv/+/1-AKP mice in response to 22mM glucose challenge; data are expressed as fold change normalized to basal (2.2mM glucose) secretion (n=4–5). *P<0.05, **P<0.01, ***P< 0.001 vs. c-Kit^Wv/+/saline group.

Figure 5. Effect of Gsk3β inhibitor on pancreatic morphology of c-Kit^Wv/+/1-AKP mice

Morphometric analysis of islet number (a), islet size (b), α-cell mass (c) and β-cell mass (d) in c-Kit^+/+/saline, c-Kit^+/+/1-AKP, c-Kit^Wv/+/saline, and c-Kit^Wv/+/1-AKP groups. (n=6–8). (e) Quantitative RT-PCR analysis of Insulin I and II mRNA in isolated islets of c-Kit^+/+/saline, c-Kit^+/+/1-AKP, c-Kit^Wv/+/saline, and c-Kit^Wv/+/1-AKP mice. Data are expressed as means ± SEM (n=5–8). *P<0.05, **P<0.01, ***P<0.001 was tested by one-way ANOVA analysis; ^#P<0.05 was analyzed by unpaired student t-test.

Figure 6. Inhibition of Gsk3β maintained β-catenin and cyclin D1 protein level in c-Kit^Wv/+/1-AKP mouse islets

Western blot analysis of β-catenin (a) and cyclin D1 (b) in isolated islets from c-Kit^+/+/saline, c-Kit^+/+/1-AKP, c-Kit^Wv/+/saline, and c-Kit^Wv/+/1-AKP mice (left column) and isolated islets from 8 weeks of c-Kit^+/+ and c-Kit^Wv/+mice cultured with or without 1-AKP treatment for 24h (right column). Representative blots are shown. Data are normalized to β-actin and expressed as means ± SEM (n=4–5) *P <0.05, **P<0.01, ***P<0.001 vs. c-Kit^Wv/+/saline groups by one-way ANOVA analysis. ^#P<0.05, ^##P<0.01 was analyzed by unpaired student t-test between c-Kit^Wv/+islets with or without 1-AKP treatment. Quantitative analysis of cyclin D1 (CCND1) mRNA in isolated islets (c) and the number of nuclear cyclin D1⁺ cells in β-cells (d) of c-Kit^+/+/saline, c-Kit^+/+/1-AKP, c-Kit^Wv/+/saline, and c-Kit^Wv/+/1-AKP mice. Data are expressed as means ± SEM (n=5–8). *p<0.05, **p<0.01, ***p<0.001 vs. c-Kit^Wv/+/saline group by one-way ANOVA analysis. ^#P<0.05 was analyzed by unpaired student t-test.

Figure 7. Inhibition of Gsk3β resulted in increased Pdx-1 and Mafa expression, β-cell proliferation in c-Kit^Wv/+/1-AKP mice

Quantitative analysis of Pdx-1 and Mafa mRNA in isolated islets (a) and the number of Pdx-1⁺ (b) and Ki67⁺ (d, right) cells in β-cells of c-Kit^+/+/saline, c-Kit^+/+/1-AKP, c-Kit^Wv/+/saline, and c-Kit^Wv/+/1-AKP mice. (c) Western blot analysis of Pdx-1 protein level in isolated islets from c-Kit^+/+/saline, c-Kit^+/+/1-AKP, c-Kit^Wv/+/saline, and c-Kit^Wv/+/1-AKP mice (left column) and isolated islets from 8 week old c-Kit^+/+ and c-Kit^Wv/+mice cultured with or without 1-AKP treatment for 24h (right column). (d, left) Double immunofluorescence staining of Ki67 (green) with insulin (red), nuclear stained with DAPI (blue). Arrows indicate Ki67/insulin double positive cells. Scale bar: 20 μm. Data are expressed mean ± SEM (n=5–8) *P<0.05, **P<0.01, ***P < 0.001 vs. c-Kit^Wv/+/saline group by one-way ANOVA analysis. ^#P<0.05, ^##P<0.01 was analyzed by unpaired student t-test.

Figure 8. Schematic of proposed model involving c-Kit/Akt/Gsk3β signaling pathways in β-cell survival and function in c-Kit^Wv/+ mice

(a) Mutating the c-Kit receptor at the Wv locus lead to a down-regulation of the PI3K/Akt pathway via reduced phosphorylation of Akt at Ser473, which resulted in enhanced active Gsk3β and increased inhibition of Pdx-1 and cyclin D1 expression. (b) Direct inhibition of active Gsk3β with 1-AKP preserved β-catenin protein level, along with maintained cyclin D1 and Pdx-1 expression that allowed improvement of β-cell proliferation and function and prevented the onset of diabetes in c-Kit ^Wv/+ mice. “+”: enhanced; “−”: reduced; “↓”: stimulation; “⊥” inhibition.