Fig 1: GRP75 phospho-mutants suppress EtOH-induced MCC complex formation and mitochondrial Ca2+ accumulation.a GRP75 phosphorylation and its interaction with IP3R1 and VDAC1 in AML12 cells overexpressing WT and phospho-mutant GRP75 treated with 100 mM EtOH for 24 h were analyzed by co-IP using HA antibody and immunoblotting. Quantifications relative to control are provided below each blot. b Analysis of IP3R1-VDAC1 interaction by in situ PLA in AML12 cells overexpressing WT and phospho-mutant GRP75 treated with EtOH (Scale bars, 20 µm). c Quantification of in situ PLA blobs of (b) (WT Ctrl, n = 18; WT/A120/A266/A267 + EtOH, n = 12 microscopic fields with >500 cells from 3 independent experiments). d Measurement of mitochondrial Ca2+ flux in AML12 cells (stably expressing 4mitD3-CPV) transfected with phospho-mutant GRP75 constructs and treated 100 mM EtOH for 24 h. The YFP and CFP fluorescence was recorded every 10 s and 90 s later 100 µM ATP was injected. e, f Quantification of basal (e) and ATP-stimulated delta-peak from the basal (f) values of (d) (n = 10 biological replicates from 3 independent experiments). g Analysis of Mitochondrial ROS level (mean fluorescence intensity; MFI) in AML12 cells overexpressing WT or phospho-mutant GRP75 and treated with 100 mM EtOH for 24 h using MitoSOX dye (n = 9 biological replicates from 3 independent experiments). h Intracellular neutral lipid accumulation was analyzed using the LipidTox dye in AML12 cells overexpressing WT or phospho-mutant GRP75 and treated with 100 mM EtOH for 24 h (Scale bars, 20 µm). i Quantification of fluorescence intensity of (h) (n = 10 microscopic fields with >500 cells from three independent experiments). All quantifications are represented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (Ordinary one-way ANOVA, Dunnett’s multiple comparisons test).
Fig 2: PDK4 is highly induced in human and mouse model of ALD.a Gene expression of 97 known genes associated with MAM (Black dots) was analyzed from RNA-Seq (GEO155830) datasets of CD and ED-fed mice liver and represented as Volcano-plot. NS: Non-significant (Gray dots), UP: Significantly upregulated (Red dots) and DOWN: Significantly downregulated genes (Blue dots). b Heatmap showing the upregulated and downregulated MAM-associated genes in ED-fed mice compared to CD-fed mice. c–e mRNA (n = 5 mice/group) (c) and protein (n = 3 mice/group) (d-e) expression of PDK isoenzymes in CD and ED-fed mice liver. f–h protein (f, g) and mRNA (h) expression of PDKs in healthy subjects (HS) and alcohol-associated liver disease (ALD) human liver specimens (n = 5 each). i Representative image of immunohistochemical (IHC) analysis of PDK4 expression in the liver sections of HS and patients with ALD (n = 5 each) (Scale bars, 100 µm). j Immunoblot analysis of MCC complex proteins in the subcellular fractions isolated from CD and ED-fed mice liver. TL: tissue lysate, Mito: pure mitochondria, MAM: mitochondria-associated ER membrane and ER: endoplasmic reticulum. k MCC complex formation was evaluated by co-immunoprecipitation (co-IP) using GRP75 antibody and immunoblotting of the indicated proteins (left), quantifications relative to CD are provided below each blot and inputs (right). l Representative confocal microscope images (left) and quantification (right) of in situ PLA showing the IP3R1-VDAC1 interactions in primary mouse hepatocytes treated with or without 100 mM EtOH for 24 h (Scale bars, 20 µm) (n = 20 microscopic fields each with >500 cells from three independent experiments). m Representative confocal microscope images (left) and quantification (right) of in situ PLA showing the IP3R1-VDAC1 interactions in HS and ALD human liver tissue sections (Scale bars, 20 µm) (HS, n = 29; ALD, n = 40 microscopic fields with >500 nuclei from 5 HS and ALD patient liver specimens). All values are represented as mean ± SEM, *p < 0.05; **p < 0.01; ****p < 0.0001 (Two-tailed unpaired t-test).
Fig 3: Graphical illustration of PDK4-mediated Ca2+-channeling complex formation at the ER-mitochondria contact site promotes mitochondrial dysfunction in alcohol-associated liver disease.Ca2+-channeling complex formation in MAM is augmented in alcohol-associated liver disease (ALD). An increase in PDK4 expression enhances the MAM Ca2+-channeling complex formation via the phosphorylation of GRP75 to promote mitochondrial Ca2+ accumulation and dysfunction in ALD. ROS: Reactive Oxygen Species, OCR: Oxygen Consumption Rate, MMP: Mitochondrial Membrane Potential.
Fig 4: PDK4 deficiency suppresses EtOH-induced MAM formation and mitochondrial dysfunction.a Analysis of interaction between MCC complex proteins in Pdk4+/+ and Pdk4-/- primary mouse hepatocytes treated with or without 100 mM EtOH for 24 h by in situ PLA (Scale bars, 20 µm). b–d Quantification of (a) [IP3R1-GRP75 (b), n = 14; VDAC1-GRP75 (c), n = 14; IP3R1-VDAC1 (d), n = 15 microscopic fields with >300 cells from 3 independent experiments]. e Immunofluorescence analysis of ER and mitochondria using PDI (Green) and TOM20 (Red) antibodies in Pdk4+/+ and Pdk4-/- primary mouse hepatocytes treated with or without EtOH for 24 h. Colocalized portion of ER and Mitochondria is shown in white (Scale bars, 20 µm). f Quantification of TOM20-PDI colocalization of (e) (Ctrl Pdk4+/+, n = 28; Ctrl Pdk4-/-, n = 24; EtOH Pdk4+/+ and EtOH Pdk4-/-, n = 30 microscopic fields with >300 cells from 3 independent experiments). g Measurement of mitochondrial Ca2+ level in Pdk4+/+ and Pdk4-/- primary mouse hepatocytes transduced with 4mitD3 adenovirus and treated with or without EtOH for 24 h (n = 8 biological replicates from three independent experiments). h, i Mitochondria ROS (mean fluorescence intensity; MFI) (n = 6 biological replicates) (h) and membrane potential (n = 18 biological replicates) (i) were measured using MitoSOX and the JC1 dye, respectively, in Pdk4+/+ and Pdk4-/- primary mouse hepatocytes treated with EtOH for 24 h (three independent experiments were performed). j Oxygen consumption rate (OCR) was measured by XF-analyzer. Rot: Rotenone, AA: Antimycin A. k Quantification of (j) (n = 13 biological replicates from three independent experiments). All quantifications are represented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (Two-way ANOVA, Tukey’s multiple comparisons test).
Fig 5: Genetic ablation of PDK4 prevents alcohol-induced MAM formation in vivo.a GRP75 phosphorylation in CD/ED-fed mice liver isolated from Pdk4+/+ and Pdk4-/- mice was evaluated by IP with p-S/T antibody (representative blot of n = 3 mice/group). Quantifications relative to control are provided below each blot. b–e Immunoblot analysis of the MCC complex proteins in MAM fractions isolated from CD/ED-fed Pdk4+/+ and Pdk4-/- mice livers (b), and quantifications (c–e) (CD Pdk4+/+/Pdk4-/-, n = 2; ED Pdk4+/+/Pdk4-/-, n = 3 mice/group). f MAM formation in the perivenous region of CD/ED-fed Pdk4+/+ and Pdk4-/- mice liver sections were analyzed by TEM imaging [Scale bars, 2 µm (top panel) and 1 µm (magnified)]. The ER and mitochondria in the TEM images were reconstructed graphically to visualize MAM formation. Mito: Mitochondria (red), ER: Endoplasmic Reticulum (green), Nucleus (blue). g Quantification of MAM length to mitochondrial perimeter ratio, CD Pdk4+/+, n = 8 (with 250 mitochondria); CD Pdk4-/-, n = 14 (with 670 mitochondria); ED Pdk4+/+, n = 22 (with 990 mitochondria); EtOH Pdk4-/-, n = 25 (with 800 mitochondria) microscopic fields from 3 mice/group. All quantifications are represented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001 (Two-way ANOVA, Tukey’s multiple comparisons test).
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