Exendin-4 suppresses SRC activation and reactive oxygen species production in diabetic Goto-Kakizaki rat islets in an Epac-dependent manner

Abstract

Objective: Reactive oxygen species (ROS) is one of most important factors in impaired metabolism secretion coupling in pancreatic β-cells. We recently reported that elevated ROS production and impaired ATP production at high glucose in diabetic Goto-Kakizaki (GK) rat islets are effectively ameliorated by Src inhibition, suggesting that Src activity is upregulated. In the present study, we investigated whether the glucagon-like peptide-1 signal regulates Src activity and ameliorates endogenous ROS production and ATP production in GK islets using exendin-4.

Research design and methods: Isolated islets from GK and control Wistar rats were used for immunoblotting analyses and measurements of ROS production and ATP content. Src activity was examined by immunoprecipitation of islet lysates followed by immunoblotting. ROS production was measured with a fluorescent probe using dispersed islet cells.

Results: Exendin-4 significantly decreased phosphorylation of Src Tyr416, which indicates Src activation, in GK islets under 16.7 mmol/l glucose exposure. Glucose-induced ROS production (16.7 mmol/l) in GK islet cells was significantly decreased by coexposure of exendin-4 as well as PP2, a Src inhibitor. The Src kinase-negative mutant expression in GK islets significantly decreased ROS production induced by high glucose. Exendin-4, as well as PP2, significantly increased impaired ATP elevation by high glucose in GK islets. The decrease in ROS production by exendin-4 was not affected by H-89, a PKA inhibitor, and an Epac-specific cAMP analog (8CPT-2Me-cAMP) significantly decreased Src Tyr416 phosphorylation and ROS production.

Conclusions: Exendin-4 decreases endogenous ROS production and increases ATP production in diabetic GK rat islets through suppression of Src activation, dependently on Epac.

FIG. 1.

Comparison of expression of Src between fresh Wistar and GK islets. Fresh islets were lysated and subjected to immunoblot analyses. Blots (50 μg of protein) were probed with anti–phospho-Src (Tyr⁴¹⁶), anti–phospho-Src (Tyr⁵²⁷), anti-Src, or anti-Csk. The same blots were stripped and reprobed with anti–β-actin, respectively. Intensities of the bands were quantified with densitometric imager. The bar graphs are expressed relative to Wistar islet value corrected by β-actin level (means ± SE). *P < 0.05; †P < 0.01; ‡P < 0.001. Representative blot panels of three to five independent experiments are shown.

FIG. 2.

Exendin-4 suppresses Src activity at high glucose in GK islets. Effects of exendin-4 on Src activity at high glucose in GK (A) and Wistar (B) islets. After preincubation in the presence of 2.8 mmol/l glucose for 30 min, islets were incubated in the presence of 16.7 mmol/l glucose with or without 100 nmol/l exendin-4 for 10 min. Islet lysates (∼2 mg of protein) were immunoprecipitated with anti-Src antibody and subjected to immunoblot analyses. Blots were probed with anti–phospho-Src (Tyr⁴¹⁶), anti–phospho-Src (Tyr⁵²⁷), or anti-Src by stripping and reprobing of the same blots. Intensities of the bands were quantified with densitometric imager. The bar graphs are expressed relative to control value corrected by Src level (means ± SE). *P < 0.05; †P < 0.01. Representative blot panels of four (A) or three (B) independent experiments are shown.

FIG. 3.

Exendin-4 decreases ROS production at high glucose in GK islet cells. A: Time course of high-glucose–induced ROS production with or without 100 nmol/l exendin-4 in GK islet cells. After preincubation in the presence of 2.8 mmol/l glucose and 10 μmol/l CM-H₂DCFDA for 20 min, dispersed islet cells were incubated in the presence of 16.7 mmol/l glucose with (●) or without (○) 100 nmol/l exedin-4 for 60 min. Fluorescence is represented as fold increases against the value at time zero. Data are expressed as means ± SE (n = 5–7). *P < 0.05 vs. control. B: Effects of exendin-4 and PP2 on high-glucose–induced ROS production at 60 min in GK islet cells. Data are expressed as means ± SE (n = 4–6). *P < 0.05. C: Effects of exendin-4 and forskolin on high-glucose–induced ROS production at 60 min in GK islet cells. Data are expressed as means ± SE (n = 5–6). *P < 0.05; †P < 0.01. D: Effects of exendin-4 and forskolin on high-glucose–induced ROS production at 60 min in Wistar islet cells. Data are expressed as means ± SE (n = 3–4). E: Effects of PP3 on high-glucose–induced ROS production at 60 min in GK islet cells. Data are expressed as means ± SE (n = 3). F: Effect of Src-KN on high-glucose–induced ROS production at 60 min in GK islet cells. Retroviral (empty vector and Src-KN vector)-mediated gene transfer to islets was carried out by in vivo gene transduction method, as described in research design and methods. Data are expressed as means ± SE (n = 3). ‡P < 0.001.

FIG. 4.

Exendin-4 increases ATP content at high glucose in GK islets. Effects of exendin-4 and PP2 on ATP content in the presence of high glucose for 30 min in GK (A) and Wistar (B) islets. After preincubation in the presence of 2.8 mmol/l glucose for 30 min, islets were incubated in the presence of 2.8 or 16.7 mmol/l glucose with or without 100 nmol/l exendin-4, 10 μmol/l PP2, or both for 30 min. Data are expressed as means ± SE (n = 7–8). *P < 0.05; †P < 0.01.

FIG. 5.

The effects of exendin-4 are dependent not on PKA but on Epac. A: Effects of H-89 or PKI on the decrease in high-glucose–induced ROS production by exendin-4 or forskolin at 60 min in GK islet cells. After preincubation in the presence of 2.8 mmol/l glucose and 10 μmol/l CM-H₂DCFDA for 20 min, dispersed islet cells were incubated in the presence of 16.7 mmol/l glucose with or without 100 nmol/l exendin-4 or 10 μmol/l forskolin with or without 10 μmol/l H-89 or 10 μmol/l PKI for 60 min. Fluorescence is represented as fold increases against the value at time zero. Data are expressed as means ± SE (n = 3). †P < 0.01; ‡P < 0.001. B: Expression of Epac2 and Rap1 in Wistar and GK islets. Fresh islets were lysated and subjected to immunoblot analyses. Blots (50 μg of protein) were probed with anti-Epac2 or anti-Rap1. The same blots were stripped and reprobed with anti–β-actin, respectively. Representative blot panels of three independent experiments are shown. C: Effects of cAMP analogs on high-glucose–induced ROS production at 60 min in GK islet cells. Data are expressed as means ± SE (n = 3–4). ‡P < 0.001. D: Epac-specific cAMP analog suppresses Src activity at high glucose in GK islets. After preincubation in the presence of 2.8 mmol/l glucose for 30 min, islets were incubated in the presence of 16.7 mmol/l glucose with or without 0.1 mmol/l 8CPT-2Me-cAMP for 8 min. Islet lysates (∼2 mg of protein) were immunoprecipitated with anti-Src antibody and subjected to immunoblot analyses. Blots were probed with anti–phospho-Src (Tyr⁴¹⁶), anti–phospho-Src (Tyr⁵²⁷), or anti-Src by stripping and reprobing of the same blots. Intensities of the bands were quantified with densitometric imager. The bar graphs are expressed relative to control value corrected by Src level (means ± SE). †P < 0.01. Representative blot panels of four independent experiments are shown.

FIG. 6.

The downstream pathway of Src is involved in PI3K/Akt signaling. A: Effects of LY294002 or wortmannin on high-glucose–induced ROS production at 60 min in GK islet cells. After preincubation in the presence of 2.8 mmol/l glucose and 10 μmol/l CM-H₂DCFDA for 20 min, dispersed islet cells were incubated in the presence of 16.7 mmol/l glucose with or without 50 μmol/l LY294002 or 0.5 μmol/l wortmannin for 60 min. Fluorescence was represented as fold increases against the value at time zero. Data are expressed as means ± SE (n = 3–5). †P < 0.01. B: Akt phosphorylation in the presence of high glucose with or without exendin-4 or PP2 in GK islets. After preincubation in the presence of 2.8 mmol/l glucose for 30 min, islets were incubated in the presence of 16.7 mmol/l glucose with or without 10 μmol/l forskolin or 10 μmol/l PP2 for 10 min. Islets were lysated and subjected to immunoblot analyses. Blots (50 μg of protein) were probed with anti–phospho-Akt or anti-Akt by stripping and reprobing of the same blots. Intensities of the bands were quantified with densitometric imager. The bar graphs are expressed relative to control value corrected by Akt level (means ± SE). ‡P < 0.001. Representative blot panels of five independent experiments are shown. C: Akt phosphorylation in the presence of high glucose with or without exendin-4 or PP2 in Wistar islets. Representative blot panels of three independent experiments are shown. D: Effects of PD98059 on high-glucose–induced ROS production at 60 min in GK islet cells. Data are expressed as means ± SE (n = 4). E: ERK phosphorylation in the presence of high glucose with or without exendin-4 or PP2 in GK islets. Blots (50 μg of protein) were probed with anti–phospho-ERK or anti-ERK by stripping and reprobing of the same blots. The bar graphs are expressed relative to control value corrected by ERK level (means ± SE). Representative blot panels of three independent experiments are shown. F: Effects of AG1478 on high-glucose–induced ROS production at 60 min in GK islet cells. Data are expressed as means ± SE (n = 5). ‡P < 0.001.