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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

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.
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Figure 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.

Mentions: To examine whether the expression levels of Src in GK islets differ from those in Wistar islets, immunoblotting using fresh islets was performed. As shown in Fig. 1A, the level of Src pY416, which indicates activation of Src, in GK islets was significantly higher than that in Wistar islets. The levels of Src pY527, total Src, and Csk in GK islets were significantly lower than those in Wistar islets. The levels of other Src family kinases (SFKs) were similar in Wistar and GK islets, whereas the expression of Fgr was very low and that of Fyn was undetectable (supplementary Fig. 1 in the online appendix, available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0021/DC1). Results of immunoblotting using islets cultured for 20 h in the presence of 5.5 mmol/l glucose (supplementary Fig. 2) were similar to those shown in Fig. 1A.


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)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3012174&req=5

Figure 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.
Mentions: To examine whether the expression levels of Src in GK islets differ from those in Wistar islets, immunoblotting using fresh islets was performed. As shown in Fig. 1A, the level of Src pY416, which indicates activation of Src, in GK islets was significantly higher than that in Wistar islets. The levels of Src pY527, total Src, and Csk in GK islets were significantly lower than those in Wistar islets. The levels of other Src family kinases (SFKs) were similar in Wistar and GK islets, whereas the expression of Fgr was very low and that of Fyn was undetectable (supplementary Fig. 1 in the online appendix, available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0021/DC1). Results of immunoblotting using islets cultured for 20 h in the presence of 5.5 mmol/l glucose (supplementary Fig. 2) were similar to those shown in Fig. 1A.

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