Diabetes Metab J.  2024 Nov;48(6):1058-1072. 10.4093/dmj.2023.0275.

PDZD8 Augments Endoplasmic Reticulum-Mitochondria Contact and Regulates Ca²⁺ Dynamics and Cypd Expression to Induce Pancreatic β-Cell Death during Diabetes

• Liu Y
• Wei Y
• Jin X
• Cai H
• Chen Q
• Zhang X

• KMID: 2561790
• DOI: http://doi.org/10.4093/dmj.2023.0275

Abstract

Background
Diabetes mellitus (DM) is a chronic metabolic disease that poses serious threats to human physical and mental health worldwide. The PDZ domain-containing 8 (PDZD8) protein mediates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) formation in mammals. We explored the role of PDZD8 in DM and investigated its potential mechanism of action.
Methods
High-fat diet (HFD)- and streptozotocin-induced mouse DM and palmitic acid (PA)-induced insulin 1 (INS-1) cell models were constructed. PDZD8 expression was detected using immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. MAM formation, interactions between voltage-dependent anion-selective channel 1 (VDAC1) and inositol 1,4,5-triphosphate receptor type 1 (IP3R1), pancreatic β-cell apoptosis and proliferation were detected using transmission electron microscopy (TEM), proximity ligation assay (PLA), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, immunofluorescence staining, and Western blotting. The mitochondrial membrane potential, cell apoptosis, cytotoxicity, and subcellular Ca²⁺ localization in INS-1 cells were detected using a JC-1 probe, flow cytometry, and an lactate dehydrogenase kit.
Results
PDZD8 expression was up-regulated in the islets of HFD mice and PA-treated pancreatic β-cells. PDZD8 knockdown markedly shortened MAM perimeter, suppressed the expression of MAM-related proteins IP3R1, glucose-regulated protein 75 (GRP75), and VDAC1, inhibited the interaction between VDAC1 and IP3R1, alleviated mitochondrial dysfunction and ER stress, reduced the expression of ER stress-related proteins, and decreased apoptosis while increased proliferation of pancreatic β-cells. Additionally, PDZD8 knockdown alleviated Ca²⁺ flow into the mitochondria and decreased cyclophilin D (Cypd) expression. Cypd overexpression alleviated the promoting effect of PDZD8 knockdown on the apoptosis of β-cells.
Conclusion
PDZD8 knockdown inhibited pancreatic β-cell death in DM by alleviated ER-mitochondria contact and the flow of Ca²⁺ into the mitochondria.

Keyword

Diabetes mellitus; Endoplasmic reticulum; Mitochondria; Pdzd8 protein, mouse

Figure

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  Fig. 1. Adeno-associated virus 9 (AAV9) delivery of short hairpin PDZ domain-containing 8 (shPDZD8) inhibits mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) formation in the pancreatic islets of diabetic mice. (A) The blood-glucose level, (B) the fasting blood-glucose, and (C) the insulin level in mice. (D, F) The MAM in β-cells was measured using the transmission electron microscopy (TEM). The red arrows point to MAM. (E, G) The interaction between voltage-dependent anion-selective channel 1 (VDAC1) and inositol 1,4,5-triphosphate receptor type 1 (IP3R1) was detected using the proximity ligation assay (PLA). The dots represent the complex of VDAC1 and IP3R1. (H) The protein expression of MAM-related proteins in islet tissues was detected by Western blotting. M, mitochondria; GRP75, glucose-regulated protein 75; PDI, protein disulfide isomerase; COX IV, cytochrome c oxidase 4. aP<0.05 vs. normocaloric diet (NCD) group, bP<0.05 vs. high-fat diet (HFD)+AAV9-shRNA negative control (shNC) group.

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  Fig. 2. Adeno-associated virus 9 (AAV9) delivery of short hairpin PDZ domain-containing 8 (shPDZD8) inhibits mitochondrial dysfunction and pancreatic β-cell death of diabetic mice. The levels of (A) H2O2, (B) 4-hydroxynonenal (HNE), (C) adenosine triphosphate (ATP), and (D) mitochondrial DNA (mtDNA) were measured using corresponding kits or quantitative real-time polymerase chain reaction. (E) The expression of endoplasmic reticulum (ER) stress-related proteins were detected by Western blotting. (F) The apoptosis of β-cells in islet tissues was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. (G) Immunofluorescence of insulin Ki67 was performed for detecting pancreatic β-cell mass and proliferation. (H) Quantitative data of TUNEL-positive β-cells per islet. (I) Quantitative data of Ki67-positive β-cells per islet. p-PERK, phosphorylated protein kinase R (PKR)-like endoplasmic reticulum kinase; p-eIF2α, phosphorylated eukaryotic translation initiation factor 2A; ATF4, activating transcription factor 4; CHOP, C/EBP-homologous protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. aP<0.05 vs. normocaloric diet (NCD) group, bP<0.05 vs. high-fat diet (HFD)+AAV9-shRNA negative control (shNC) group.

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  Fig. 3. PDZ domain-containing 8 (PDZD8) knockdown inhibits palmitic acid (PA)-induced mitochondria-associated endoplasmic reticulum membrane (MAM) formation in insulin 1 (INS-1) cells. (A, B) The MAM in INS-1 cells was measured using the transmission electron microscopy (TEM). The red arrows point to MAM. (C) The interaction between voltage-dependent anionselective channel 1 (VDAC1) and inositol 1,4,5-triphosphate receptor type 1 (IP3R1) was detected using the proximity ligation assay. (D) The protein expression of PDZD8 and MAM-related proteins in INS-1 cells were detected by Western blotting. M, mitochondria; siPDZD8, siRNA PDZ domain-containing 8; GRP75, glucose-regulated protein 75; PDI, protein disulfide isomerase; COX IV, cytochrome c oxidase 4. aP<0.05 vs. control (Con) group, bP<0.05 vs. PA+shRNA negative control (shNC) group.

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  Fig. 4. PDZ domain-containing 8 (PDZD8) knockdown inhibits palmitic acid (PA)-induced mitochondrial dysfunction and endoplasmic reticulum (ER) stress in insulin 1 (INS-1) cells. (A, B) The mitochondrial membrane potential was detected using JC-1 probe. Green fluorescence indicates low mitochondrial membrane potential and red fluorescence indicates high mitochondrial membrane potential. (C) Insulin secretion was detected by enzyme-linked immunosorbent assay (ELISA) induced by 16.7 mM glucose as glucose-stimulated insulin secretion. (D) The copy fold of mitochondrial DNA (mtDNA). (E) The expression of ER stress-related proteins was detected by Western blotting. siPDZD8, siRNA PDZ domain-containing 8; p-PERK, phosphorylated protein kinase R (PKR)-like endoplasmic reticulum kinase; p-eIF2α, phosphorylated eukaryotic translation initiation factor 2A; ATF4, activating transcription factor 4; CHOP, C/EBP-homologous protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. aP<0.05 vs. control (Con) group, bP<0.05 vs. PA+shRNA negative control (shNC) group.

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  Fig. 5. PDZ domain-containing 8 (PDZD8) knockdown inhibits palmitic acid (PA)-induced insulin 1 (INS-1) cell death. (A, B) T he apoptosis of INS-1 cells was detected using the flow cytometry. (C) The cytotoxicity was detected by lactate dehydrogenase (LDH) kit. (D) The expression of mitochondrial apoptosis-related proteins was detected by Western blotting. PE-H, phycoerythrin-H; FITC-H, fluorescein isothiocyanate-H; siPDZD8, siRNA PDZ domain-containing 8; Bcl-2, B cell lymphoma 2; Bax, BCL2 associated X, apoptosis regulator; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. aP<0.05 vs. control (Con) group, bP<0.05 vs. PA+shRNA negative control (shNC) group.

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  Fig. 6. PDZ domain-containing 8 (PDZD8) regulates insulin 1 (INS-1) cell death due to endoplasmic reticulum (ER)-mitochondria Ca2+ transfer and cyclophilin D (Cypd)-dependent mitochondrial permeability transition pore (mPTP) opening. (A, B) The subcellular localization of intracellular Ca2+ was detected using fluorescence probes. (C) The Cypd expression in INS-1 cells was detected by Western blotting. (D) The apoptosis of INS-1 cells was detected using the flow cytometry. ATP, adenosine triphosphate; CPA, cyclopiazonic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. aP<0.05 vs. control (Con) group, bP<0.05 vs. palmitic acid (PA)+shRNA negative control (shNC) group, cP<0.05 vs. PA+siRNA PDZ domain-containing 8 (siPDZD8)+pcDNA3.1 group.

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Reference

1. Pang B, Li QW, Qin YL, Dong GT, Feng S, Wang J, et al. Traditional Chinese medicine for diabetic retinopathy: a systematic review and meta-analysis. Medicine (Baltimore). 2020; 99:e19102.

2. Xu L, Li Y, Dai Y, Peng J. Natural products for the treatment of type 2 diabetes mellitus: pharmacology and mechanisms. Pharmacol Res. 2018; 130:451–65.

3. Tanase DM, Gosav EM, Neculae E, Costea CF, Ciocoiu M, Hurjui LL, et al. Role of gut microbiota on onset and progression of microvascular complications of type 2 diabetes (T2DM). Nutrients. 2020; 12:3719.

4. Inaishi J, Saisho Y. Beta-cell mass in obesity and type 2 diabetes, and its relation to pancreas fat: a mini-review. Nutrients. 2020; 12:3846.

5. Wang JJ, Park KS, Dhimal N, Shen S, Tang X, Qu J, et al. Proteomic analysis of retinal mitochondria-associated ER membranes identified novel proteins of retinal degeneration in longterm diabetes. Cells. 2022; 11:2819.

6. Zhang SX, Sanders E, Fliesler SJ, Wang JJ. Endoplasmic reticulum stress and the unfolded protein responses in retinal degeneration. Exp Eye Res. 2014; 125:30–40.

7. Huang C, Wang JJ, Jing G, Li J, Jin C, Yu Q, et al. Erp29 attenuates cigarette smoke extract-induced endoplasmic reticulum stress and mitigates tight junction damage in retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 2015; 56:6196–207.

8. Csordas G, Renken C, Varnai P, Walter L, Weaver D, Buttle KF, et al. Structural and functional features and significance of the physical linkage between ER and mitochondria. J Cell Biol. 2006; 174:915–21.

9. Lopez Gavilanez E, Johansson H, McCloskey E, Harvey NC, Segale Bajana A, Marriott Blum D, et al. Assessing the risk of osteoporotic fractures: the Ecuadorian FRAX model. Arch Osteoporos. 2019; 14:93.

10. Ma JH, Shen S, Wang JJ, He Z, Poon A, Li J, et al. Comparative proteomic analysis of the mitochondria-associated ER membrane (MAM) in a long-term type 2 diabetic rodent model. Sci Rep. 2017; 7:2062.

11. Schrader M, Godinho LF, Costello JL, Islinger M. The different facets of organelle interplay: an overview of organelle interactions. Front Cell Dev Biol. 2015; 3:56.

12. Betz C, Stracka D, Prescianotto-Baschong C, Frieden M, Demaurex N, Hall MN. Feature article: mTOR complex 2-Akt signaling at mitochondria-associated endoplasmic reticulum membranes (MAM) regulates mitochondrial physiology. Proc Natl Acad Sci U S A. 2013; 110:12526–34.

13. Bononi A, Bonora M, Marchi S, Missiroli S, Poletti F, Giorgi C, et al. Identification of PTEN at the ER and MAMs and its regulation of Ca(2+) signaling and apoptosis in a protein phosphatase-dependent manner. Cell Death Differ. 2013; 20:1631–43.

14. Hirabayashi Y, Kwon SK, Paek H, Pernice WM, Paul MA, Lee J, et al. ER-mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons. Science. 2017; 358:623–30.

15. Jeyasimman D, Saheki Y. SMP domain proteins in membrane lipid dynamics. Biochim Biophys Acta Mol Cell Biol Lipids. 2020; 1865:158447.

16. Shirane M, Wada M, Morita K, Hayashi N, Kunimatsu R, Matsumoto Y, et al. Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation. Nat Commun. 2020; 11:4576.

17. Guillen-Samander A, Bian X, De Camilli P. PDZD8 mediates a Rab7-dependent interaction of the ER with late endosomes and lysosomes. Proc Natl Acad Sci U S A. 2019; 116:22619–23.

18. Khan H, Chen L, Tan L, Im YJ. Structural basis of human PDZD8-Rab7 interaction for the ER-late endosome tethering. Sci Rep. 2021; 11:18859.

19. Gao Y, Xiong J, Chu QZ, Ji WK. PDZD8-mediated lipid transfer at contacts between the ER and late endosomes/lysosomes is required for neurite outgrowth. J Cell Sci. 2022; 135:jcs255026.

20. Wei W, Wang C, Wang L, Zhang J. circ_0020123 promotes cell proliferation and migration in lung adenocarcinoma via PDZD8. Open Med (Wars). 2022; 17:536–49.

21. Hojo Y, Kishi S, Mori S, Fujiwara-Tani R, Sasaki T, Fujii K, et al. Sunitinib and pterostilbene combination treatment exerts antitumor effects in gastric cancer via suppression of PDZD8. Int J Mol Sci. 2022; 23:4002.

22. Henning MS, Stiedl P, Barry DS, McMahon R, Morham SG, Walsh D, et al. PDZD8 is a novel moesin-interacting cytoskeletal regulatory protein that suppresses infection by herpes simplex virus type 1. Virology. 2011; 415:114–21.

23. Szegezdi E, Logue SE, Gorman AM, Samali A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006; 7:880–5.

24. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 2007; 8:519–29.

25. Malhotra JD, Kaufman RJ. Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? Antioxid Redox Signal. 2007; 9:2277–93.

26. Rozpedek W, Pytel D, Mucha B, Leszczynska H, Diehl JA, Majsterek I. The role of the PERK/eIF2α/ATF4/CHOP signaling pathway in tumor progression during endoplasmic reticulum stress. Curr Mol Med. 2016; 16:533–44.

27. Xu J, Zhou Q, Xu W, Cai L. Endoplasmic reticulum stress and diabetic cardiomyopathy. Exp Diabetes Res. 2012; 2012:827971.

28. Thivolet C, Vial G, Cassel R, Rieusset J, Madec AM. Reduction of endoplasmic reticulum-mitochondria interactions in beta cells from patients with type 2 diabetes. PLoS One. 2017; 12:e0182027.

29. Rovira-Llopis S, Banuls C, Diaz-Morales N, Hernandez-Mijares A, Rocha M, Victor VM. Mitochondrial dynamics in type 2 diabetes: pathophysiological implications. Redox Biol. 2017; 11:637–45.

30. Flemming N, Pernoud L, Forbes J, Gallo L. Mitochondrial dysfunction in individuals with diabetic kidney disease: a systematic review. Cells. 2022; 11:2481.

31. Yan C, Duanmu X, Zeng L, Liu B, Song Z. Mitochondrial DNA: distribution, mutations, and elimination. Cells. 2019; 8:379.

32. Wei Y, Rector RS, Thyfault JP, Ibdah JA. Nonalcoholic fatty liver disease and mitochondrial dysfunction. World J Gastroenterol. 2008; 14:193–9.

33. Li Z, Li Y, Zhang HX, Guo JR, Lam CW, Wang CY, et al. Mitochondria-mediated pathogenesis and therapeutics for non-alcoholic fatty liver disease. Mol Nutr Food Res. 2019; 63:e1900043.

34. Boushel R, Gnaiger E, Schjerling P, Skovbro M, Kraunsoe R, Dela F. Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle. Diabetologia. 2007; 50:790–6.

35. Patti ME, Butte AJ, Crunkhorn S, Cusi K, Berria R, Kashyap S, et al. Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: potential role of PGC1 and NRF1. Proc Natl Acad Sci U S A. 2003; 100:8466–71.

36. Vamecq J, Dessein AF, Fontaine M, Briand G, Porchet N, Latruffe N, et al. Mitochondrial dysfunction and lipid homeostasis. Curr Drug Metab. 2012; 13:1388–400.

37. Nishikawa T, Kukidome D, Sonoda K, Fujisawa K, Matsuhisa T, Motoshima H, et al. Impact of mitochondrial ROS production in the pathogenesis of insulin resistance. Diabetes Res Clin Pract. 2007; 77 Suppl 1:S161–4.

38. Molnar MJ, Kovacs GG. Mitochondrial diseases. Handb Clin Neurol. 2017; 145:147–55.

39. Fex M, Nicholas LM, Vishnu N, Medina A, Sharoyko VV, Nicholls DG, et al. The pathogenetic role of β-cell mitochondria in type 2 diabetes. J Endocrinol. 2018; 236:R145–59.

40. Zarkovic N. 4-Hydroxynonenal as a bioactive marker of pathophysiological processes. Mol Aspects Med. 2003; 24:281–91.

41. Xu H, Zhou W, Zhan L, Bi T, Lu X. Liver mitochondria-associated endoplasmic reticulum membrane proteomics for studying the effects of ZiBuPiYin recipe on Zucker diabetic fatty rats after chronic psychological stress. Front Cell Dev Biol. 2022; 10:995732.

42. Cheng H, Gang X, He G, Liu Y, Wang Y, Zhao X, et al. The molecular mechanisms underlying mitochondria-associated endoplasmic reticulum membrane-induced insulin resistance. Front Endocrinol (Lausanne). 2020; 11:592129.

43. Lee S, Min KT. The interface between ER and mitochondria: molecular compositions and functions. Mol Cells. 2018; 41:1000–7.

44. Yang S, Zhou R, Zhang C, He S, Su Z. Mitochondria-associated endoplasmic reticulum membranes in the pathogenesis of type 2 diabetes mellitus. Front Cell Dev Biol. 2020; 8:571554.

45. D’Eletto M, Rossin F, Occhigrossi L, Farrace MG, Faccenda D, Desai R, et al. Transglutaminase type 2 regulates ER-mitochondria contact sites by interacting with GRP75. Cell Rep. 2018; 25:3573–81.

46. Rizzuto R, De Stefani D, Raffaello A, Mammucari C. Mitochondria as sensors and regulators of calcium signalling. Nat Rev Mol Cell Biol. 2012; 13:566–78.

47. Fayaz SM, Raj YV, Krishnamurthy RG. CypD: the key to the death door. CNS Neurol Disord Drug Targets. 2015; 14:654–63.

48. Poitout V, Robertson RP. Glucolipotoxicity: fuel excess and beta-cell dysfunction. Endocr Rev. 2008; 29:351–66.

49. Sivitz WI. Lipotoxicity and glucotoxicity in type 2 diabetes: effects on development and progression. Postgrad Med. 2001; 109:55–64.