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. 2017 May 18;7(1):2062.
doi: 10.1038/s41598-017-02213-1.

Comparative Proteomic Analysis of the Mitochondria-associated ER Membrane (MAM) in a Long-term Type 2 Diabetic Rodent Model

Jacey Hongjie Ma  1   2   3 , Shichen Shen  4   5 , Joshua J Wang  1   2 , Zhanwen He  4   6 , Amanda Poon  4 , Jun Li  5   7 , Jun Qu  5   7 , Sarah X Zhang  8   9   10
Affiliations

Comparative Proteomic Analysis of the Mitochondria-associated ER Membrane (MAM) in a Long-term Type 2 Diabetic Rodent Model

Jacey Hongjie Ma et al. Sci Rep. .

Abstract

The mitochondria-associated ER membrane (MAM) plays a critical role in cellular energetics and calcium homeostasis; however, how MAM is affected under diabetic condition remains elusive. This study presented a comprehensive proteome profiling of isolated brain MAM from long-term type 2 diabetic mice vs. non-diabetic controls. MAM protein was extracted efficiently by a surfactant-aided precipitation/on-pellet digestion (SOD) method, and MAM proteome was quantified by an ion-current-based MS1 method combined with nanoLC-MS/MS. A total of 1,313 non-redundant proteins of MAM were identified, among which 144 proteins were found significantly altered by diabetes. In-depth IPA analysis identified multiple disease-relevant signaling pathways associated with the MAM proteome changes in diabetes, most significantly the unfolded protein response (UPR), p53, hypoxia-related transcription factors, and methyl CpG binding protein 2. Using immunofluorescence labeling we confirmed the activation of three UPR branches and increased ERp29 and calreticulin in diabetic retinas. Moreover, we found GRP75, a key MAM tethering protein, was drastically reduced by long-term diabetes. In vitro, acute high glucose treatment reduces ER-mitochondrial contact in retinal endothelial cells. This study provides first insight into the significant alterations in MAM proteome associated with activation of the UPR in diabetes, which may serve as novel benchmarks for the future studies of diabetic complications.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Schematic diagram of MAM isolation and confirmation of MAM associated proteins by western blotting. (A) MAM was isolated from mouse brain by applying differential centrifugations and a self-forming Percoll gradient centrifugation. Other cell organelles, as crude mitochondria, pure mitochondria and ER, were also obtained following the multiple centrifuge steps. (B,C) Western blot analysis of organelle markers in isolated MAM from the brain (B) and retina (C) were enriched for KDEL, and free from tubulin and cytochrome-C contamination. H: homogenate, Mp: pure mitochondria, Mc: crude mitochondria, ER: endoplasmic reticulum, MAM: ER mitochondria-associated membrane, C: cytosol, Mc after percoll: crude mitochondria after percoll gradient centrifuge.
Figure 2
Figure 2
Scheme of proteomics strategy applied to analysis of mouse brain MAM samples from db/db mice vs. age- and gender-matched db/+ mice. A highly reproducible and extensive ion-current-based quantification method, known as long gradient nano-reverse-phase liquid chromatography/mass spectrometry, was applied in the analysis of the 5 biological replicates. David bioinformatics database (6.7, NIAID/NIH) and Ingenuity Pathway Analysis (IPA) were used for the function annotation of the identified proteins. The pathways and proteins of high interests were verified by immunohistochemical studies in the retina.
Figure 3
Figure 3
Localization and biological relevance of the mouse brain MAM proteins identified by proteomics. (A) Organelle association for MAM proteins determined by annotation. (B) According to GO biological processes analysis, 4 major clusters cellular activities including 21 catalogues of biological processes are shown. Processes with a p value less than 0.01 were considered significant.
Figure 4
Figure 4
Functional annotation of the MAM proteins that are differentially expressed in db/db mice vs. db/+ controls. (A) The major locations of the MAM proteins that are altered in diabetes. (B) Bar graph showing the major categories reflecting the key biological processes pertaining to the pathophysiology of diabetic complications.
Figure 5
Figure 5
Loss of retinal ganglion cells and decreased retinal GRP75 expression in db/db mice. (A) Retinal whole mounts were stained for Brn3a to visualize the retinal ganglion cells (RGCs) and examined by confocal microscope. The density of RGCs was decreased in db/db mice. Scale bar = 50 μm. Data were shown as mean ± SD, n = 3. *P < 0.05. Student’s t test. (B–D) Immunostaining showing decreased GRP75 (B), ERp29 (C), and calreticulin (D) in db/db mouse retinas and controls. Scale bar = 50 μm for (B–D). Images represent results from 3 individual mice in each group. GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. Fluorescence intensity was quantified by Image J software and expressed as fold of change relative to control (mean ± SD, n = 3). *P < 0.05. **P < 0.01, Student’s t test.
Figure 6
Figure 6
Activation of the UPR and increased level of ER chaperones in the retina of db/db mice. (A–C) Immunostaining of retinal sections from db/db mice and db/+ controls of for p-PERK (A), XBP1s (B), and ATF6 (C). Scale bar = 50 μm for (A–C). Images represent results from 3 individual mice in each group. GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. (D) Fluorescence intensity was quantified by Image J software and expressed as fold of change relative to control (mean ± SD, n = 3). *P < 0.05. **P < 0.01, Student’s t test.
Figure 7
Figure 7
Acute high glucose treatment reduces ER-mitochondrial contact in retinal endothelial cells. (A) Representative confocal images of human retinal endothelial cells (HRECs) treated with 25 mmol/ml glucose (HG) for 0 to 24 hours, stained with MitoTracker (green) and ERTracker (red). Scale bars: 10 μm. (B) Quantification of the Manders’ coefficient M1 (fraction of mitochondria overlapping with the ER). Mean ± SD; n = 10–15 cells per group). *P < 0.05; one-way ANOVA with Tukey’s post hoc test.
Figure 8
Figure 8
Schematic summary of signaling pathways associated with MAM dysfunction in the pathogenesis of diabetic complications.
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