It was evidenced that saturated fatty acids (FAs) have a detrimental effect on pancreatic β-cells function and survival, leading to endoplasmic reticulum (ER) calcium release, ER stress, and apoptosis. In the present study, we have tested the effect of three calcium influx inhibitors, i.e., diazoxide, nifedipine, and verapamil, on the apoptosis-inducing effect of saturated stearic acid (SA) in the human pancreatic β-cell lines NES2Y and 1.1B4. We have demonstrated that the application of all three calcium influx inhibitors tested has no inhibitory effect on SA-induced ER stress and apoptosis in both tested cell lines. Moreover, these inhibitors have pro-apoptotic potential per se at higher concentrations. Interestingly, these findings are in contradiction with those obtained with rodent cell lines and islets. Thus our data obtained with human β-cell lines suggest that the prospective usage of calcium channel blockers for prevention and therapy of type 2 diabetes mellitus, developed with the contribution of the saturated FA-induced apoptosis of β-cells, seems rather unlikely.

Department of Biochemistry Cell and Molecular Biology and Center for Research of Diabetes Metabolism and Nutrition 3rd Faculty of Medicine Charles University Prague Czech Republic

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Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, Santangelo C, Patane G, Boggi U, Piro S, Anello M, et al. Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes. 2002;51:1437–1442. doi: 10.2337/diabetes.51.5.1437. PubMed DOI

Azevedo-Martins AK, Monteiro AP, Lima CL, Lenzen S, Curi R. Fatty acid-induced toxicity and neutral lipid accumulation in insulin-producing RINm5F cells. Toxicol In Vitro. 2006;20:1106–1113. doi: 10.1016/j.tiv.2006.02.007. PubMed DOI

Furstova V, Kopska T, James RF, Kovar J. Comparison of the effect of individual saturated and unsaturated fatty acids on cell growth and death induction in the human pancreatic beta-cell line NES2Y. Life Sci. 2008;82:684–691. doi: 10.1016/j.lfs.2007.12.023. PubMed DOI

Maedler K, Oberholzer J, Bucher P, Spinas GA, Donath MY. Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic beta-cell turnover and function. Diabetes. 2003;52:726–733. doi: 10.2337/diabetes.52.3.726. PubMed DOI

Welters HJ, Diakogiannaki E, Mordue JM, Tadayyon M, Smith SA, Morgan NG. Differential protective effects of palmitoleic acid and cAMP on caspase activation and cell viability in pancreatic beta-cells exposed to palmitate. Apoptosis. 2006;11:1231–1238. doi: 10.1007/s10495-006-7450-7. PubMed DOI

Diakogiannaki E, Welters HJ, Morgan NG. Differential regulation of the endoplasmic reticulum stress response in pancreatic beta-cells exposed to long-chain saturated and monounsaturated fatty acids. J Endocrinol. 2008;197:553–563. doi: 10.1677/JOE-08-0041. PubMed DOI

Nemcova-Furstova V, Balusikova K, Sramek J, James RF, Kovar J. Caspase-2 and JNK activated by saturated fatty acids are not involved in apoptosis induction but modulate ER stress in human pancreatic beta-cells. Cell Physiol Biochem. 2013;31:277–289. doi: 10.1159/000343367. PubMed DOI

Sramek J, Nemcova-Furstova V, Pavlikova N, Kovar J. Effect of saturated stearic acid on MAP kinase and ER stress signaling pathways during apoptosis induction in human pancreatic beta-cells is inhibited by unsaturated oleic acid. Int J Mol Sci. 2017;18:2313. PubMed PMC

Welters HJ, Tadayyon M, Scarpello JHB, Smith SA, Morgan NG. Mono-unsaturated fatty acids protect against β-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure. FEBS Lett. 2004;560:103–108. doi: 10.1016/S0014-5793(04)00079-1. PubMed DOI

Sabatini PV, Speckmann T, Lynn FC. Friend and foe: beta-cell Ca(2+) signaling and the development of diabetes. Mol Metab. 2019;21:1–12. doi: 10.1016/j.molmet.2018.12.007. PubMed DOI PMC

Gwiazda KS, Yang TL, Lin Y, Johnson JD. Effects of palmitate on ER and cytosolic Ca2+ homeostasis in beta-cells. Am J Physiol Endocrinol Metab. 2009;296:E690–701. doi: 10.1152/ajpendo.90525.2008. PubMed DOI

Ly LD, Ly DD, Nguyen NT, Kim JH, Yoo H, Chung J, Lee MS, Cha SK, Park KS. Mitochondrial Ca(2+) uptake relieves palmitate-induced cytosolic Ca(2+) overload in MIN6 cells. Mol Cells. 2020;43:66–75. PubMed PMC

Remizov O, Jakubov R, Dufer M, Krippeit Drews P, Drews G, Waring M, Brabant G, Wienbergen A, Rustenbeck I, Schofl C. Palmitate-induced Ca2+-signaling in pancreatic beta-cells. Mol Cell Endocrinol. 2003;212:1–9. doi: 10.1016/j.mce.2003.09.026. PubMed DOI

Shu J, Gambardella J, Sorriento D, Santulli G. Mechanistic role of IP3R calcium release channel in pancreatic beta-cell function. Diabetes. 2018;67:313. doi: 10.2337/db18-313-LB. DOI

Egnatchik RA, Leamy AK, Jacobson DA, Shiota M, Young JD. ER calcium release promotes mitochondrial dysfunction and hepatic cell lipotoxicity in response to palmitate overload. Mol Metab. 2014;3:544–553. doi: 10.1016/j.molmet.2014.05.004. PubMed DOI PMC

Dror V, Kalynyak TB, Bychkivska Y, Frey MH, Tee M, Jeffrey KD, Nguyen V, Luciani DS, Johnson JD. Glucose and endoplasmic reticulum calcium channels regulate HIF-1beta via presenilin in pancreatic beta-cells. J Biol Chem. 2008;283:9909–9916. doi: 10.1074/jbc.M710601200. PubMed DOI

Back SH, Kaufman RJ. Endoplasmic reticulum stress and type 2 diabetes. Annu Rev Biochem. 2012;81:767–793. doi: 10.1146/annurev-biochem-072909-095555. PubMed DOI PMC

Cnop M, Ladriere L, Igoillo-Esteve M, Moura RF, Cunha DA. Causes and cures for endoplasmic reticulum stress in lipotoxic beta-cell dysfunction. Diabetes Obes Metab. 2010;12(Suppl 2):76–82. doi: 10.1111/j.1463-1326.2010.01279.x. PubMed DOI

Cunha DA, Hekerman P, Ladriere L, Bazarra-Castro A, Ortis F, Wakeham MC, Moore F, Rasschaert J, Cardozo AK, Bellomo E, et al. Initiation and execution of lipotoxic ER stress in pancreatic beta-cells. J Cell Sci. 2008;121:2308–2318. doi: 10.1242/jcs.026062. PubMed DOI PMC

Karaskov E, Scott C, Zhang L, Teodoro T, Ravazzola M, Volchuk A. Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress, which may contribute to INS-1 pancreatic beta-cell apoptosis. Endocrinology. 2006;147:3398–3407. doi: 10.1210/en.2005-1494. PubMed DOI

Tabas I, Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol. 2011;13:184–190. doi: 10.1038/ncb0311-184. PubMed DOI PMC

Choi SE, Kim HE, Shin HC, Jang HJ, Lee KW, Kim Y, Kang SS, Chun J, Kang Y. Involvement of Ca2+-mediated apoptotic signals in palmitate-induced MIN6N8a beta cell death. Mol Cell Endocrinol. 2007;272:50–62. doi: 10.1016/j.mce.2007.04.004. PubMed DOI

Rorsman P, Arkhammar P, Bokvist K, Hellerström C, Nilsson T, Welsh M, Welsh N, Berggren PO. Failure of glucose to elicit a normal secretory response in fetal pancreatic beta cells results from glucose insensitivity of the ATP-regulated K+ channels. Proc Natl Acad Sci USA. 1989;86:4505–4509. doi: 10.1073/pnas.86.12.4505. PubMed DOI PMC

Sargsyan E, Ortsater H, Thorn K, Bergsten P. Diazoxide-induced beta-cell rest reduces endoplasmic reticulum stress in lipotoxic beta-cells. J Endocrinol. 2008;199:41–50. doi: 10.1677/JOE-08-0251. PubMed DOI

Vogel J, Yin J, Su L, Wang SX, Zessis R, Fowler S, Chiu CH, Wilson AC, Chen A, Zecri F, et al. A phenotypic screen identifies calcium overload as a key mechanism of beta-cell glucolipotoxicity. Diabetes. 2020;69:1032–1041. doi: 10.2337/db19-0813. PubMed DOI

Xu G, Chen J, Jing G, Shalev A. Preventing β-cell loss and diabetes with calcium channel blockers. Diabetes. 2012;61:848–856. doi: 10.2337/db11-0955. PubMed DOI PMC

Zhou Y, Sun P, Wang T, Chen K, Zhu W, Wang H. Inhibition of calcium influx reduces dysfunction and apoptosis in lipotoxic pancreatic beta-cells via regulation of endoplasmic reticulum stress. PLoS ONE. 2015;10:e0132411. doi: 10.1371/journal.pone.0132411. PubMed DOI PMC

Huang Q, Bu S, Yu Y, Guo Z, Ghatnekar G, Bu M, Yang L, Lu B, Feng Z, Liu S, Wang F. Diazoxide prevents diabetes through inhibiting pancreatic beta-cells from apoptosis via Bcl-2/Bax rate and p38-beta mitogen-activated protein kinase. Endocrinology. 2007;148:81–91. doi: 10.1210/en.2006-0738. PubMed DOI

Guldstrand M, Grill V, Björklund A, Lins PE, Adamson U. Improved beta cell function after short-term treatment with diazoxide in obese subjects with type 2 diabetes. Diabetes Metab. 2002;28:448–456. PubMed

Doyle ME, Egan JM. Pharmacological agents that directly modulate insulin secretion. Pharmacol Rev. 2003;55:105–131. doi: 10.1124/pr.55.1.7. PubMed DOI

Striessnig J, Ortner NJ, Pinggera A. Pharmacology of L-type calcium channels: novel drugs for old targets? Curr Mol Pharmacol. 2015;8:110–122. doi: 10.2174/1874467208666150507105845. PubMed DOI PMC

Elmslie KS. Calcium channel blockers in the treatment of disease. J Neurosci Res. 2004;75:733–741. doi: 10.1002/jnr.10872. PubMed DOI

Genazzani AA, Carafoli E, Guerini D. Calcineurin controls inositol 1,4,5-trisphosphate type 1 receptor expression in neurons. Proc Natl Acad Sci USA. 1999;96:5797–5801. doi: 10.1073/pnas.96.10.5797. PubMed DOI PMC

Abou-Saleh H, Pathan AR, Daalis A, Hubrack S, Abou-Jassoum H, Al-Naeimi H, Rusch NJ, Machaca K. Inositol 1,4,5-trisphosphate (IP3) receptor up-regulation in hypertension is associated with sensitization of Ca2+ release and vascular smooth muscle contractility. J Biol Chem. 2013;288:32941–32951. doi: 10.1074/jbc.M113.496802. PubMed DOI PMC

Macfarlane WM, Cragg H, Docherty HM, Read ML, James RF, Aynsley-Green A, Docherty K. Impaired expression of transcription factor IUF1 in a pancreatic beta-cell line derived from a patient with persistent hyperinsulinaemic hypoglycaemia of infancy (nesidioblastosis) FEBS Lett. 1997;413:304–308. doi: 10.1016/S0014-5793(97)00874-0. PubMed DOI

McCluskey JT, Hamid M, Guo-Parke H, McClenaghan NH, Gomis R, Flatt PR. Development and functional characterization of insulin-releasing human pancreatic beta cell lines produced by electrofusion. J Biol Chem. 2011;286:21982–21992. doi: 10.1074/jbc.M111.226795. PubMed DOI PMC

Musílková J, Kovár J. Additive stimulatory effect of extracellular calcium and potassium on non-transferrin ferric iron uptake by HeLa and K562 cells. Biochim Biophys Acta. 2001;1514:117–126. doi: 10.1016/S0005-2736(01)00367-4. PubMed DOI

Abdelmagid SA, Clarke SE, Nielsen DE, Badawi A, El-Sohemy A, Mutch DM, Ma DW. Comprehensive profiling of plasma fatty acid concentrations in young healthy Canadian adults. PLoS ONE. 2015;10:e0116195. doi: 10.1371/journal.pone.0116195. PubMed DOI PMC

Lagerstedt SA, Hinrichs DR, Batt SM, Magera MJ, Rinaldo P, McConnell JP. Quantitative determination of plasma c8–c26 total fatty acids for the biochemical diagnosis of nutritional and metabolic disorders. Mol Genet Metab. 2001;73:38–45. doi: 10.1006/mgme.2001.3170. PubMed DOI

Cnop M, Hannaert JC, Hoorens A, Eizirik DL, Pipeleers DG. Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. Diabetes. 2001;50:1771–1777. doi: 10.2337/diabetes.50.8.1771. PubMed DOI

Kovar J, Franek F. Growth-stimulating effect of transferrin on a hybridoma cell line: relation to transferrin iron-transporting function. Exp Cell Res. 1989;182:358–369. doi: 10.1016/0014-4827(89)90241-3. PubMed DOI

Nemcova-Furstova V, James RFL, Kovar J. Inhibitory effect of unsaturated fatty acids on saturated fatty acid-induced apoptosis in human pancreatic beta-cells: activation of caspases and ER stress induction. Cell Physiol Biochem. 2011;27:525–538. doi: 10.1159/000329954. PubMed DOI

Sramek J, Nemcova-Furstova V, Balusikova K, Daniel P, Jelinek M, James RF, Kovar J. p38 MAPK is activated but does not play a key role during apoptosis induction by saturated fatty acid in human pancreatic beta-cells. Int J Mol Sci. 2016;17:159. doi: 10.3390/ijms17020159. PubMed DOI PMC

Lin N, Chen H, Zhang H, Wan X, Su Q. Mitochondrial reactive oxygen species (ROS) inhibition ameliorates palmitate-induced INS-1 beta cell death. Endocrine. 2012;42:107–17. PubMed

Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150:76–85. doi: 10.1016/0003-2697(85)90442-7. PubMed DOI

Ehrlichová M, Koc M, Truksa J, Naldová Z, Václavíková R, Kovárr J. Cell death induced by taxanes in breast cancer cells: cytochrome C is released in resistant but not in sensitive cells. Anticancer Res. 2005;25:4215–4224. PubMed

Voborilova J, Nemcova-Furstova V, Neubauerova J, Ojima I, Zanardi I, Gut I, Kovar J. Cell death induced by novel fluorinated taxanes in drug-sensitive and drug-resistant cancer cells. Invest New Drugs. 2011;29:411–423. doi: 10.1007/s10637-009-9368-8. PubMed DOI PMC

Feng XT, Duan HM, Li SL. Protective role of Pollen Typhae total flavone against the palmitic acid-induced impairment of glucose-stimulated insulin secretion involving GPR40 signaling in INS-1 cells. Int J Mol Med. 2017;40:922–930. doi: 10.3892/ijmm.2017.3070. PubMed DOI

Ogilvie RI, Nadeau JH, Sitar DS. Diazoxide concentration-response relation in hypertension. Hypertension. 1982;4:167–173. doi: 10.1161/01.HYP.4.1.167. PubMed DOI

Schütz E, Ha HR, Bühler FR, Follath F. Serum concentration and antihypertensive effect of slow-release verapamil. J Cardiovasc Pharmacol. 1982;4(Suppl 3):S346–349. PubMed

Bacracheva N, Thuermann P, Rietbrock N. Dose adjustment of nifedipine in hypertensive patients. Eur J Clin Pharmacol. 1990;38:17–20. doi: 10.1007/BF00314796. PubMed DOI