PP2Ac knockdown attenuates lipotoxicity‑induced pancreatic β‑cell dysfunction and apoptosis

Zhang,Zhengwei; Tong,Beier; Liu,Jie; Feng,Jieyuan; Song,Linyang; Wang,Huawei; Ke,Mengting; Xu,Chengkai; Xu,Yancheng

doi:10.3892/etm.2023.12247

PP2Ac knockdown attenuates lipotoxicity‑induced pancreatic β‑cell dysfunction and apoptosis

Authors:
- Zhengwei Zhang
- Beier Tong
- Jie Liu
- Jieyuan Feng
- Linyang Song
- Huawei Wang
- Mengting Ke
- Chengkai Xu
- Yancheng Xu
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Affiliations: Department of Endocrinology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China, Department of Endocrinology, Suizhou Central Hospital, Suizhou, Hubei 441300, P.R. China
Published online on: October 10, 2023 https://doi.org/10.3892/etm.2023.12247
Article Number: 549
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Protein phosphatase 2A (PP2A) is one of the most common serine/threonine phosphatases in mammalian cells, and it primarily functions to regulate cell signaling, glycolipid metabolism and apoptosis. The catalytic subunit of PP2A (PP2Ac) plays an important role in the functions of the protein. However, there are few reports on the regulatory role of PP2Ac in pancreatic β‑cells under lipotoxic conditions. In the present study, mouse insulinoma 6 (MIN6) pancreatic cells were transfected with short hairpin RNAs to generate PP2Ac knockdown cells and incubated with palmitate (PA) to establish a lipotoxicity model. Serine/threonine phosphatase assay system, Cell Counting Kit‑8, flow cytometry, enzyme‑linked immunosorbent assay and western blotting were used to measure PP2A activity, cell viability, apoptosis, oxidative stress and insulin secretion in the cells. In addition, a mouse model of lipotoxicity was established with a high‑fat diet (HFD) and the knockdown of PP2Ac using adeno‑associated viruses to interfere with PP2Ac expression in the pancreatic tissues. The activity of PP2A in the mouse pancreatic tissue and the serum insulin level were measured. Furthermore, the proliferation of mouse pancreatic β‑cells was assessed using pancreatic tissue immunofluorescence. PP2Ac knockdown inhibited lipotoxicity‑induced PP2A hyperactivation, increased the resistance of pancreatic β‑cells to lipotoxicity and attenuated PA‑induced apoptosis in MIN6 cells. It also protected the endoplasmic reticulum and mitochondria, and ameliorated insulin secretion. The results of mRNA sequencing and western blotting analysis suggested that the protective effects of PP2Ac knockdown in MIN6 cells may be mediated via the MAPK pathway. Moreover, the results of the animal experiments suggested that specific knockdown of pancreatic PP2Ac effectively attenuated HFD‑induced insulin resistance and reduced the compensatory proliferation of pancreatic β‑cells in mice. In summary, the present study revealed the effects of interfering with PP2Ac gene expression on pancreatic β‑cells in vivo and in vitro and the underlying mechanisms, which may provide insights for the treatment of type 2 diabetes mellitus in the clinic.

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1	Amanat S, Ghahri S, Dianatinasab A, Fararouei M and Dianatinasab M: Exercise and type 2 diabetes. Adv Exp Med Biol. 1228:91–105. 2020.PubMed/NCBI View Article : Google Scholar
2	Sun H, Saeedi P, Karuranga S, Pinkepank M, Ogurtsova K, Duncan BB, Stein C, Basit A, Chan JCN, Mbanya JC, et al: IDF Diabetes atlas: Global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045. Diabetes Res Clin Pract. 183(109119)2022.PubMed/NCBI View Article : Google Scholar
3	Xiong QY, Yu C, Zhang Y, Ling L, Wang L and Gao JL: Key proteins involved in insulin vesicle exocytosis and secretion. Biomed Rep. 6:134–139. 2017.PubMed/NCBI View Article : Google Scholar
4	Rutter GA, Pullen TJ, Hodson DJ and Martinez-Sanchez A: Pancreatic β-cell identity, glucose sensing and the control of insulin secretion. Biochem J. 466:203–218. 2015.PubMed/NCBI View Article : Google Scholar
5	Zheng Y, Ley SH and Hu FB: Global aetiology and epidemiology of type 2 diabetes mellitus and its complications. Nat Rev Endocrinol. 14:88–98. 2018.PubMed/NCBI View Article : Google Scholar
6	Klein S, Gastaldelli A, Yki-Järvinen H and Scherer PE: Why does obesity cause diabetes? Cell Metab. 34:11–20. 2022.PubMed/NCBI View Article : Google Scholar
7	Oh YS, Bae GD, Baek DJ, Park EY and Jun HS: Fatty acid-induced lipotoxicity in pancreatic beta-cells during development of type 2 diabetes. Front Endocrinol (Lausanne). 9(384)2018.PubMed/NCBI View Article : Google Scholar
8	Ertunc ME and Hotamisligil GS: Lipid signaling and lipotoxicity in metaflammation: Indications for metabolic disease pathogenesis and treatment. J Lipid Res. 57:2099–2114. 2016.PubMed/NCBI View Article : Google Scholar
9	Ron D and Walter P: Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 8:519–529. 2007.PubMed/NCBI View Article : Google Scholar
10	Lin S, Long H, Hou L, Zhang M, Ting J, Lin H, Zheng P, Lei W, Yin K and Zhao G: Crosstalk between endoplasmic reticulum stress and non-coding RNAs in cardiovascular diseases. Wiley Interdiscip Rev RNA. 14(e1767)2022.PubMed/NCBI View Article : Google Scholar
11	Hamacher-Brady A and Brady NR: Mitophagy programs: Mechanisms and physiological implications of mitochondrial targeting by autophagy. Cell Mol Life Sci. 73:775–795. 2016.PubMed/NCBI View Article : Google Scholar
12	Roszczyc-Owsiejczuk K and Zabielski P: Sphingolipids as a culprit of mitochondrial dysfunction in insulin resistance and type 2 diabetes. Front Endocrinol (Lausanne). 12(635175)2021.PubMed/NCBI View Article : Google Scholar
13	Thivolet C, Vial G, Cassel R, Rieusset J and Madec AM: Reduction of endoplasmic reticulum-mitochondria interactions in beta cells from patients with type 2 diabetes. PLoS One. 12(e0182027)2017.PubMed/NCBI View Article : Google Scholar
14	Ježek P, Holendová B, Jabůrek M, Dlasková A and Plecitá-Hlavatá L: Contribution of mitochondria to insulin secretion by various secretagogues. Antioxid Redox Signal. 36:920–952. 2022.PubMed/NCBI View Article : Google Scholar
15	Carta G, Murru E, Banni S and Manca C: Palmitic acid: Physiological role, metabolism and nutritional implications. Front Physiol. 8(902)2017.PubMed/NCBI View Article : Google Scholar
16	Lytrivi M, Castell AL, Poitout V and Cnop M: Recent insights into mechanisms of β-cell Lipo- and glucolipotoxicity in type 2 diabetes. J Mol Biol. 432:1514–1534. 2020.PubMed/NCBI View Article : Google Scholar
17	Raman D and Pervaiz S: Redox inhibition of protein phosphatase PP2A: Potential implications in oncogenesis and its progression. Redox Biol. 27(101105)2019.PubMed/NCBI View Article : Google Scholar
18	Reynhout S and Janssens V: Physiologic functions of PP2A: Lessons from genetically modified mice. Biochim Biophys Acta Mol Cell Res. 1866:31–50. 2019.PubMed/NCBI View Article : Google Scholar
19	Ronk H, Rosenblum JS, Kung T and Zhuang Z: Targeting PP2A for cancer therapeutic modulation. Cancer Biol Med. 19:1428–1439. 2022.PubMed/NCBI View Article : Google Scholar
20	Binder P, Wang S, Radu M, Zin M, Collins L, Khan S, Li Y, Sekeres K, Humphreys N, Swanton E, et al: Pak2 as a novel therapeutic target for cardioprotective endoplasmic reticulum stress response. Circ Res. 124:696–711. 2019.PubMed/NCBI View Article : Google Scholar
21	Yang L, Jin GH and Zhou JY: The role of ceramide in the pathogenesis of alcoholic liver disease. Alcohol Alcohol. 51:251–257. 2016.PubMed/NCBI View Article : Google Scholar
22	Theurey P, Tubbs E, Vial G, Jacquemetton J, Bendridi N, Chauvin MA, Alam MR, Le Romancer M, Vidal H and Rieusset J: Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver. J Mol Cell Biol. 8:129–143. 2016.PubMed/NCBI View Article : Google Scholar
23	Chen X, Chen S, Shen T, Yang W, Chen Q, Zhang P, You Y, Sun X, Xu H, Tang Y, et al: Adropin regulates hepatic glucose production via PP2A/AMPK pathway in insulin-resistant hepatocytes. FASEB J. 34:10056–10072. 2020.PubMed/NCBI View Article : Google Scholar
24	Arora DK, Machhadieh B, Matti A, Wadzinski BE, Ramanadham S and Kowluru A: High glucose exposure promotes activation of protein phosphatase 2A in rodent islets and INS-1 832/13 β-cells by increasing the posttranslational carboxylmethylation of its catalytic subunit. Endocrinology. 155:380–391. 2014.PubMed/NCBI View Article : Google Scholar
25	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar
26	Li Y, Zhang QY, Sun BF, Ma Y, Zhang Y, Wang M, Ma C, Shi H, Sun Z, Chen J, et al: Single-cell transcriptome profiling of the vaginal wall in women with severe anterior vaginal prolapse. Nat Commun. 12(87)2021.PubMed/NCBI View Article : Google Scholar
27	Kanehisa M: Toward understanding the origin and evolution of cellular organisms. Protein Sci. 28:1947–1951. 2019.PubMed/NCBI View Article : Google Scholar
28	Dong WW, Feng Z, Zhang YQ, Ruan ZS and Jiang L: Potential mechanism and key genes involved in mechanical ventilation and lipopolysaccharide-induced acute lung injury. Mol Med Rep. 22:4265–4277. 2020.PubMed/NCBI View Article : Google Scholar
29	Deng S, Yang L, Ma K and Bian W: Astragalus polysaccharide improve the proliferation and insulin secretion of mouse pancreatic β-cells induced by high glucose and palmitic acid partially through promoting miR-136-5p and miR-149-5p expression. Bioengineered. 12:9872–9884. 2021.PubMed/NCBI View Article : Google Scholar
30	Wee J, Pak S, Kim T, Hong GS, Lee JS, Nan J, Kim H, Lee MO, Park KS and Oh U: Tentonin 3/TMEM150C regulates glucose-stimulated insulin secretion in pancreatic β-cells. Cell Rep. 37(110067)2021.PubMed/NCBI View Article : Google Scholar
31	Zhang Y, Yang X, Ge X and Zhang F: Puerarin attenuates neurological deficits via Bcl-2/Bax/cleaved caspase-3 and Sirt3/SOD2 apoptotic pathways in subarachnoid hemorrhage mice. Biomed Pharmacother. 109:726–733. 2019.PubMed/NCBI View Article : Google Scholar
32	Janssens V and Goris J: Protein phosphatase 2A: A highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J. 353:417–439. 2001.PubMed/NCBI View Article : Google Scholar
33	Baskaran R and Velmurugan BK: Protein phosphatase 2A as therapeutic targets in various disease models. Life Sci. 210:40–46. 2018.PubMed/NCBI View Article : Google Scholar
34	Kowluru A and Matti A: Hyperactivation of protein phosphatase 2A in models of glucolipotoxicity and diabetes: Potential mechanisms and functional consequences. Biochem Pharmacol. 84:591–597. 2012.PubMed/NCBI View Article : Google Scholar
35	Kowluru A: Potential roles of PP2A-Rac1 signaling axis in pancreatic β-cell dysfunction under metabolic stress: Progress and promise. Biochem Pharmacol. 180(114138)2020.PubMed/NCBI View Article : Google Scholar
36	Acosta-Montaño P and García-González V: Effects of dietary fatty acids in pancreatic beta cell metabolism, implications in homeostasis. Nutrients. 10(393)2018.PubMed/NCBI View Article : Google Scholar
37	Madec AM, Perrier J, Panthu B and Dingreville F: Role of mitochondria-associated endoplasmic reticulum membrane (MAMs) interactions and calcium exchange in the development of type 2 diabetes. Int Rev Cell Mol Biol. 363:169–202. 2021.PubMed/NCBI View Article : Google Scholar
38	Lee S and Min KT: The interface between ER and mitochondria: Molecular compositions and functions. Mol Cells. 41:1000–1007. 2018.PubMed/NCBI View Article : Google Scholar
39	Szymański J, Janikiewicz J, Michalska B, Patalas-Krawczyk P, Perrone M, Ziółkowski W, Duszyński J, Pinton P, Dobrzyń A and Więckowski MR: Interaction of mitochondria with the endoplasmic reticulum and plasma membrane in calcium homeostasis, lipid trafficking and mitochondrial structure. Int J Mol Sci. 18(1576)2017.PubMed/NCBI View Article : Google Scholar
40	Tubbs E and Rieusset J: Metabolic signaling functions of ER-mitochondria contact sites: Role in metabolic diseases. J Mol Endocrinol. 58:R87–R106. 2017.PubMed/NCBI View Article : Google Scholar
41	Xu D, Liu L, Zhao Y, Yang L, Cheng J, Hua R, Zhang Z and Li Q: Melatonin protects mouse testes from palmitic acid-induced lipotoxicity by attenuating oxidative stress and DNA damage in a SIRT1-dependent manner. J Pineal Res. 69(e12690)2020.PubMed/NCBI View Article : Google Scholar
42	Lan J, Zhong Z, Wang Y, Xiong Y and Ye Q: Endoplasmic reticulum stress induces liver cells apoptosis after brain death by suppressing the phosphorylation of protein phosphatase 2A. Mol Med Rep. 21:567–574. 2020.PubMed/NCBI View Article : Google Scholar
43	Xiong Y, Lan J, Huang K, Zhang Y, Zheng L, Wang Y and Ye Q: PP2Ac upregulates PI3K-Akt signaling and induces hepatocyte apoptosis in liver donor after brain death. Apoptosis. 24:921–933. 2019.PubMed/NCBI View Article : Google Scholar
44	Li N, Frigerio F and Maechler P: The sensitivity of pancreatic beta-cells to mitochondrial injuries triggered by lipotoxicity and oxidative stress. Biochem Soc Trans. 36:930–934. 2008.PubMed/NCBI View Article : Google Scholar
45	Sui X, Kong N, Ye L, Han W, Zhou J, Zhang Q, He C and Pan H: p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents. Cancer Lett. 344:174–179. 2014.PubMed/NCBI View Article : Google Scholar
46	Zheng HY, Shen FJ, Tong YQ and Li Y: PP2A Inhibits cervical cancer cell migration by dephosphorylation of p-JNK, p-p38 and the p-ERK/MAPK signaling pathway. Curr Med Sci. 38:115–123. 2018.PubMed/NCBI View Article : Google Scholar
47	Tao M, Liu L, Shen M, Zhi Q, Gong FR, Zhou BP, Wu Y, Liu H, Chen K, Shen B, et al: Inflammatory stimuli promote growth and invasion of pancreatic cancer cells through NF-κB pathway dependent repression of PP2Ac. Cell Cycle. 15:381–393. 2016.PubMed/NCBI View Article : Google Scholar
48	Jangati GR, Veluthakal R, Susick L, Gruber SA and Kowluru A: Depletion of the catalytic subunit of protein phosphatase-2A (PP2Ac) markedly attenuates glucose-stimulated insulin secretion in pancreatic beta-cells. Endocrine. 31:248–253. 2007.PubMed/NCBI View Article : Google Scholar