Essential Role Of High Glucose-Induced Overexpression Of PKCβ And PKCδ In GLP-1 Resistance In Rodent Cardiomyocytes

Figure 1. Impaired cardioprotective effects in response to GLP-1 in diabetic mice after myocardial IRI. (A) Elevated circulating GLP-1 level induced by myocardial IRI. *P<0.01 vs 0 hr+NDM+IRI group and 0 hr + DM + IRI group; #P<0.05 vs 0 hr + NDM + IRI group. (B) Representative echocardiography and LV fractional shortening change of each group. *P<0.05 vs NDM + IRI group. (C) QRT-PCR analysis of Bcl-2 and BAX. *P<0.05 vs NDM + IRI group. (D) Caspase-3 activity assay. *P<0.05 vs NDM + IRI group. (E) QRT-PCR analysis of NOX4 and p22-phox. *P<0.05 vs NDM + IRI group. (F) ROS measurement. *P<0.05 vs NDM + IRI group. Abbreviations: DM, diabetes mellitus; NDM, non-diabetes mellitus; IRI, ischemia/reperfusion injury; LV, left ventricle; ROS, reactive oxygen species.

Figure 2. Overexpression of PKCβ induced by high glucose downregulated GLP-1R expression in H9C2 cells. (A) High glucose-induced downregulation of GLP-1R and impact of myocardial IRI on GLP-1R expression. *P<0.05 vs NDM group and NDM + IRI group; (B) high glucose-induced overexpression of PKC. *P<0.01 vs NG group; (C) impact of PMA and Go 6983 on GLP-1R expression in H9C2 cells. *P<0.01 vs HG group, #P<0.05 vs HG group and HG + PMA + Go6983 group; (D) high glucose-induced overexpression of PKCα, -β, -γ and –δ. *P<0.01 vs NG group; (E) transfection efficiency of PKCα (70.3±2.1%), -β (71.5±3.2%), -γ (68.3±2.6%) and –δ (66.4±3.1%) siRNA; (F) change of GLP-1R expression by knockdown of PKCα, -β, -γ and –δ. *P<0.01 vs HG group and HG + UNC siRNA group; (G) impact of RBX and PKCβ siRNA on GLP-1R expression in H9C2 cells, *P<0.05 vs HG group and HG + UNC siRNA group, #P<0.05 vs HG + RBX group. Abbreviations: NDM, non-diabetes mellitus; DM, diabetes mellitus; NG, normal glucose; HG, high glucose; PMA, phorbol-12-myristate-13-acetate; RBX, ruboxistaurin; UNC, universal negative control.

Figure 3. Restoration of GLP-1R expression by knockdown of PKCβ recovered anti-oxidative stress effects of GLP-1R in H9C2 cells cultured by high glucose. (A) Western blot analysis confirmed the restoration of GLP-1R expression by knockdown of PKCβ in H9C2 cells cultured by high glucose. *P<0.01 vs HG, HG + H/R and HG + H/R + UNC siRNA group. (B) Restored anti-oxidative stress effects of GLP-1R. *P<0.05 vs HG + H/R + Ex-4 group. Abbreviations: HG, high glucose; Ex-4, exendin-4. H/R, hypoxia/re-oxygenation; UNC, universal negative control.

Figure 4. Restoration of GLP-1R expression by knockdown of PKCβ insignificantly recovers anti-apoptotic effects of GLP-1R in H9C2 cells cultured by high glucose. (A) QRT-PCR analysis of Bcl-2 and BAX. (B) Caspase-3 activity. (C) Representative TUNEL images and quantitative analysis of the TUNEL results (shown as apoptosis index). Abbreviations: HG, high glucose; Ex-4, exendin-4; H/R, hypoxia/re-oxygenation; UNC, universal negative control.

Figure 5. Overexpression of PKCδ induced by high glucose-impaired post-receptor anti-apoptotic signaling pathways of GLP-1 in H9C2 cells by inhibition of Akt phosphorylation. (A) Impact of PKCα, -β, -γ, -δ siRNA and UNC siRNA on Bcl-2 and BAX mRNA level after H/R in H9C2 cells cultured by high glucose. *P<0.01 vs HG + H/R + Ex-4 group and HG + H/R + Ex-4 + UNC siRNA group; (B) impact of PKCα, -β, -γ, -δ siRNA and UNC siRNA on caspase-3 activity level after H/R in H9C2 cells cultured by high glucose. *P<0.01 vs HG + H/R + Ex-4 group and HG + H/R + Ex-4 + UNC siRNA group; (C) knockdown of PKCδ increased phosphorylation level of Akt in H9C2 cells. *P<0.05 vs HG + H/R group; #P<0.01 vs HG + H/R + Ex-4 group and HG + H/R + Ex-4 + UNC siRNA group; (D) impact of activation and inhibition of both PKCδ and PI3K on each other. Abbreviations: Con, control; HG, high glucose; H/R, hypoxia/re-oxygenation; IGF-1, insulin growth factor-1; UNC, universal negative control; Ex-4, exendin-4.

Figure 6. Knockdown of PKCβ but not PKCδ restored anti-oxidative stress effects of GLP-1R in H9C2 cells cultured by high glucose. *P<0.05 vs HG + H/R + Ex-4 group and HG + H/G + Ex-4 + UNC siRNA group. Abbreviations: HG, high glucose; H/R, hypoxia/re-oxygenation; Ex-4, exendin-4; UNC, universal negative control.

Figure 7. Knockdown of both PKCβ and PKCδ restored anti-apoptotic effects of GLP-1 analog significantly in H9C2 cells cultured by high glucose. (A) QRT-PCR analysis of Bcl-2 and BAX mRNA. (B) Caspase-3 activity analysis. (C) Representative images of TUNEL staining and corresponding apoptosis index. *P<0.05 vs HG + H/R + Ex-4 group, HG + H/R + Ex-4 + PKCβ siRNA + PKCδ siRNA group and HG + H/R + Ex-4 + UNC siRNA group. #P<0.01 vs HG + H/R + Ex-4 group and HG + H/R + Ex-4 + UNC siRNA group. Abbreviations: HG, high glucose; H/R, hypoxia/re-oxygenation; Ex-4, exendin-4; UNC, universal negative control.

Figure 8. Essential role of PKCβ and PKCδ in GLP-1 resistance in diabetic cardiomyocytes.