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

Mukai E, Fujimoto S, Sato H, Oneyama C, Kominato R, Sato Y, Sasaki M, Nishi Y, Okada M, Inagaki N - Diabetes (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Diabetes and Clinical Nutrition, Kyoto University, Japan.

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.

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Related in: MedlinePlus

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.
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Figure 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.

Mentions: We then investigated whether the decrease in ROS production by exendin-4 is dependent on PKA. As shown in Fig. 5A, decreased ROS production by exendin-4 or forskolin was not affected by 10 μmol/l H-89 or PKI, a PKA inhibitor, indicating that the effect is PKA independent. Not only dibutyryl cAMP, a general cAMP analog, but also 8CPT-2Me-cAMP, an Epac-specific cAMP analog, decreased ROS production (Fig. 5C). Epac possesses guanine nucleotide exchange factor activity toward Rap1, a member of the Ras superfamily of small GTPases. Epac2 and Rap1 proteins were expressed similarly in both Wistar and GK islets (Fig. 5B). To determine involvement of Epac in Src activation, Src phosphorylation was examined. Src pY416 was significantly decreased by 8CPT-2Me-cAMP (Fig. 5D).


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

Mukai E, Fujimoto S, Sato H, Oneyama C, Kominato R, Sato Y, Sasaki M, Nishi Y, Okada M, Inagaki N - Diabetes (2010)

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.
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Related In: Results  -  Collection

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Figure 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.
Mentions: We then investigated whether the decrease in ROS production by exendin-4 is dependent on PKA. As shown in Fig. 5A, decreased ROS production by exendin-4 or forskolin was not affected by 10 μmol/l H-89 or PKI, a PKA inhibitor, indicating that the effect is PKA independent. Not only dibutyryl cAMP, a general cAMP analog, but also 8CPT-2Me-cAMP, an Epac-specific cAMP analog, decreased ROS production (Fig. 5C). Epac possesses guanine nucleotide exchange factor activity toward Rap1, a member of the Ras superfamily of small GTPases. Epac2 and Rap1 proteins were expressed similarly in both Wistar and GK islets (Fig. 5B). To determine involvement of Epac in Src activation, Src phosphorylation was examined. Src pY416 was significantly decreased by 8CPT-2Me-cAMP (Fig. 5D).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Diabetes and Clinical Nutrition, Kyoto University, Japan.

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.

Show MeSH
Related in: MedlinePlus