Fatty acid β‐oxidation and mitochondrial fusion are involved in cardiac microvascular endothelial cell protection induced by glucagon receptor antagonism in diabetic mice

Figures

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  FIGURE 1. Echocardiography and histological analysis in db/db mice treated with GCGR mAb or IgG (as control) for 4 weeks. Age‐matched db/m mice served as the normal controls. (A) E/A; (B) E/e′; (C) EF; (D) FS; and (E) MPI as determined by echocardiography. (F) Immunofluorescent staining of CD31 to determine the cardiac microvascular density in the heart tissues. Scale bar = 50 μm. (G) Quantification of the microvascular density as measured by the CD31‐positive cells in (F). n = 13 mice per group in (A–E) and n = 4 mice per group in (F and G ). Data are shown as mean ± S.D. Statistical analysis was conducted by one‐way analysis of variance followed by the post hoc Tukey–Kramer test. *p < .05 vs. db/m; # p < .05 vs. db/db Ctrl. A, the peak flow velocities during late diastole; Ctrl, control; DAPI, 4′, 6‐diamidino‐2‐phenylindole; E, the peak flow velocities during early diastole; e′, early‐diastolic peak velocity; EF, ejection fraction; FS, fractional shortening; GCGR, glucagon receptor; IgG, immunoglobulin G; mAb, monoclonal antibody; MPI, myocardial performance index; MVD, microvascular density.

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  FIGURE 2. Identification of differentially expressed proteins (DEPs). (A) Volcano plot showing the quantitative protein expression between db/db mice and db/m mice. (B) Volcano plot exhibiting the quantitative protein expression between GCGR mAb‐treated db/db mice and db/db mice. (C) Venn diagram showing the overlapped DEPs in db/db mice vs. db/m mice and in GCGR mAb‐treated db/db mice vs. db/db mice. (D) The heatmap of hierarchical clustering analysis on the DEPs. Rows represent proteins and columns represent different samples. The proteins in CMECs isolated from every three mice were used as an independent sample. n = 3 samples per group. CMECs, cardiac microvascular endothelial cells; Ctrl, control; GCGR, glucagon receptor; mAb, monoclonal antibody.

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  FIGURE 3. Bioinformatic analysis of differentially expressed proteins (DEPs). All the overlapped 199 DEPs, which were upregulated in db/db mice vs. db/m mice and downregulated in GCGR mAb‐treated db/db mice vs. db/db mice, underwent the enrichment analyses of COG/KOG functional classification (A), KEGG signaling pathway (B), and GO annotation of biological processes (C), molecular function (D), and cellular component (E). COG/KOG, clusters of orthologous groups of proteins; Ctrl, control; GCGR, glucagon receptor; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; mAb, monoclonal antibody.

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  FIGURE 4. Selection and identification of differentially expressed proteins (DEPs). (A) Heatmaps depicting relative expression levels of DEPs involved in fat uptake and oxidation. (B) Heatmaps showing relative expression levels of DEPs involved in tricarboxylic acid (TCA) cycle. (C–E) Mass spectrometry intensities of CPT1B, OPA1, and MFN1. (F–K) Quantification (F–H) and image (I–K) of western blot analysis for the protein levels of CPT1B, OPA1, and MFN1 in db/m mice, db/db mice and GCGR mAb‐treated db/db mice. (L and M) The level of 4‐hydroxynonenal (4‐HNE) modified protein, an oxidative stress marker, was detected by western blot. The proteins in CMECs isolated from every three mice were used as an independent sample. n = 3 samples per group. Data are shown as mean ± SD. Statistical analysis was conducted by one‐way analysis of variance followed by the post hoc Tukey–Kramer test. *p < .05 vs. db/m; # p < .05 vs. db/db Ctrl. CMECs, cardiac microvascular endothelial cells; CPT1B, carnitine palmitoyltransferase 1B; Ctrl, control; GCGR, glucagon receptor; mAb, monoclonal antibody; MFN1, mitofusin‐1; OPA1, optic atrophy type 1.

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  FIGURE 5. Central illustration. The mitochondrial damage secondary to an increased fatty acid β‐oxidation and mitochondrial fusion can be found in the CMECs of type 2 diabetic mice. REMD 2.59, an antagonistic GCGR mAb, reverses these abnormalities in CMECs and has cardioprotective effects by downregulating the protein levels of CPT1B, OPA1, and MFN1. CMECs, cardiac microvascular endothelial cells; CPT1B, carnitine palmitoyltransferase 1B; GCGR, glucagon receptor; mAb, monoclonal antibody; MFN1, mitofusin‐1; OPA1, optic atrophy type 1.