APPL1 potentiates insulin secretion in pancreatic β cells by enhancing protein kinase Akt-dependent expression of SNARE proteins in mice

Abstract

Insulin resistance and defective insulin secretion are the two major features of type 2 diabetes. The adapter protein APPL1 is an obligatory molecule in regulating peripheral insulin sensitivity, but its role in insulin secretion remains elusive. Here, we show that APPL1 expression in pancreatic β cells is markedly decreased in several mouse models of obesity and diabetes. APPL1 knockout mice exhibit glucose intolerance and impaired glucose-stimulated insulin secretion (GSIS), whereas transgenic expression of APPL1 prevents high-fat diet (HFD)-induced glucose intolerance partly by enhancing GSIS. In both pancreatic islets and rat β cells, APPL1 deficiency causes a marked reduction in expression of the exocytotic machinery SNARE proteins (syntaxin-1, synaptosomal-associated protein 25, and vesicle-associated membrane protein 2) and an obvious decrease in the number of exocytotic events. Such changes are accompanied by diminished insulin-stimulated Akt activation. Furthermore, the defective GSIS and reduced expression of SNARE proteins in APPL1-deficient β cells can be rescued by adenovirus-mediated expression of APPL1 or constitutively active Akt. These findings demonstrate that APPL1 couples insulin-stimulated Akt activation to GSIS by promoting the expression of the core exocytotic machinery involved in exocytosis and also suggest that reduced APPL1 expression in pancreatic islets may serve as a pathological link that couples insulin resistance to β-cell dysfunction in type 2 diabetes.

Fig. 1.

Decreased APPL1 expression in pancreatic islets and impaired GSIS in both dietary and genetic obese mice. (A) Immunofluorescence staining of APPL1 (green) and insulin (red) in pancreas sections of 12-wk-old male C57BL/6J (BL6) lean mice on standard chow, or high-fat diet (HFD), or C57BKS db/+ lean mice or C57BKS (BKS) db/db obese/diabetic mice. (B) Western blot analysis of APPL1 in nonislet (exocrine cells) and islet fractions isolated from the pancreas of C57BL/6J lean, or HFD-fed obese mice, or C57BKS db/+ lean or db/db mice. (C) Densitometric analysis for the relative abundance of APPL1 as in B. (D) Static GSIS in islets isolated from different mouse models as specified. *P < 0.05 (n = 5). NS, not significant.

Fig. 2.

Effects of APPL1 deficiency on glucose tolerance and insulin secretion in mice. (A) GTT in 20-wk-old male APPL1 KO and WT littermates fed with HFD or STC. (B) In vivo GSIS in APPL1 KO mice and WT littermates fed with STC or HFD (fold change over basal insulin level). (C) Glucose- and KCl-stimulated static insulin secretion in islets isolated from 20-wk-old male APPL1 KO mice and WT controls on STC. Insulin concentration was measured at 30 min after stimulation. (D) Dynamic insulin secretion of pancreatic islets isolated from APPL1 KO and WT mice in response to glucose (25 mM) using a perifusion system. (E) Area under curve (AUC) for the first phase (6–18 min) and second phase (18–42 min) of insulin secretion during the perifusion experiment as in D. *P < 0.05 (n = 6–8). NS, not significant.

Fig. 3.

Transgenic expression of APPL1 improves glucose tolerance and insulin sensitivity and enhances insulin secretion in HFD-fed obese mice. (A) Western blot analysis for APPL1 expression in isolated pancreatic islets of 12-wk-old male APPL1 transgenic (Tg) mice and WT littermates. (B) GTT in 16-wk-old APPL1 Tg mice and WT littermates fed with either STC or HFD. (C) In vivo GSIS in 16-wk-old male mice, and plasma insulin level was expressed as fold change over the baseline. (D and E) Static insulin secretion in response to glucose and KCl stimulation in islets from 20-wk-old APPL1 Tg and WT littermates on STC or HFD, respectively. (F) ITT in 20-wk-old APPL1 Tg mice and WT littermates fed with either STC or HFD. *P < 0.05 (n = 6–7).

Fig. 4.

APPL1 deficiency decreases the expression of SNARE proteins and reduces the number of docked insulin granules and exocytotic events in pancreatic islets. (A) Real-time PCR analysis for mRNA expression levels of the three SNARE proteins (SNAP25, STX1, and VAMP2) in pancreatic islets isolated from 20-wk-old male APPL1 KO mice or WT controls on STC. (B) Western blot analysis for the SNARE proteins in the isolated islets. (C) Densitometric analysis for the relative abundance of SNARE proteins as in B. (D) Representative electron microscopic images of docked insulin granules (Left, denoted with arrows), and quantification of the number of docked granules (Right) in the isolated islets. (E) Distribution of exocytotic events in isolated islets of APPL1 KO mice and WT controls as measured by two-photon excitation imaging. White dots represent sites where exocytotic events were observed during 5 min of glucose (20 mM)-stimulated insulin secretion. The underlying red image is sulforhodamine B fluorescence. (F) Average number of glucose-stimulated exocytotic events in a cell surface area of 1,000 μm². (G) Latency (in seconds) for the onset of staining with sulforhodamine B relative to that of staining with 10 kDa dextran. *P < 0.05 (n = 5–6). NS, not significant.

Fig. 5.

Adenovirus-mediated expression of APPL1 enhances SNARE protein expression and insulin secretion in the islets of APPL1 KO mice. (A) Islets isolated from 20-wk-old male APPL1 KO mice on STC were infected with recombinant adenovirus expressing APPL1 or luciferase (Luci) for 36 h, followed by Western blot analysis to detect the expression levels of the three SNARE proteins. Bar chart on the Right is the densitometric analysis for the relative abundance of the three SNARE proteins in the infected islets. (B) Static insulin secretion in the infected islets measured after stimulation with glucose or KCl for 30 min. (C) First phase (6–18 min) and second phase (18–42 min) of GSIS of the infected islets using the perifusion system. (D) AUC for the first and second phase of insulin secretion during the perifusion experiment as in C. *P < 0.05 (n = 8). NS, not significant.

Fig. 6.

APPL1 induces SNARE protein expression and insulin secretion via Akt activation. (A) Islets from 20-wk-old male APPL1 KO mice and WT controls on STC were treated with insulin (50 nM) for various time points as indicated, followed by Western blot analysis using antitotal or phospho-FoxO1 (Ser-256), antitotal or phospho-Akt (Ser-473), antitotal or phospho-ERK1/2 (Thr-202/Tyr-204), or anti-GAPDH antibody as a loading control. Bar chart on the Right represents the relative fold change of phosphorylation as quantified by densitometry. (B) Real-time PCR analysis for mRNA expression levels of the three SNARE proteins in the islets infected with adenovirus encoding the constitutively active form of Akt (CA-Akt) or luciferase (Luci) for 48 h. (C) Western blot analysis for protein expression levels of the three SNARE proteins of the infected islets. Note that CA-Akt is tagged with a Myc epitope at the NH2 terminus and can be detected by an anti-Myc antibody. (D) Densitometric analysis for the relative abundance of the three SNARE proteins in the infected islets as in C. (E) Static GSIS in the infected islets (n = 4). *P < 0.05 (n = 6). NS, not significant.