DEPTOR gene silencing promotes β-cell insulin secretion

doi:10.3969/j.issn.2095-4344.0278

Abstract

Abstract:

BACKGROUND: Mammalian target of rapamycin (mTOR) complexes are a key regulator of pancreatic beta cells mass and function. DEP-domain containing mTOR-interacting protein (DEPTOR) is a common part of mTOR complexes and whether DEPTOR loss in islet β cells affects insulin-secreting function has
never been identified.
OBJECTIVE: To assess the alternation of insulin secretion by silencing DEPTOR gene in pancreatic β cells NIT-1 and to explore the underlying mechanism.
METHODS: Three siRNA sequences for silencing DEPTOR gene were designed and constructed, which were transfected with lipofectamine into NIT-1 cells. There were six groups: blank transfection group (NIT-1 cells plus Lipofectamin), negative control group (NC-FAM), positive control group (GAPDH), siRNA deptor 1 group (siRNA deptor385), siRNA deptor 2 group (siRNA deptor766), and siRNA deptor 3 group (siRNA deptor1275). The transfection efficiency was determined by fluorescence microscope. The relative expression level of DEPTOR mRNA was detected by quantitative-PCR. Insulin secretion in the cell conditioned medium was determined by insulin ELISA kit. The expression level of DEPTOR downstream key protein was detected by western blot assay.
RESULTS AND CONCLUSION: Specific green fluorescence accumulated in a punctated pattern under fluorescence microscope, indicating that the effectiveness of transfection was eligible. Quantitative-PCR results showed two (siDEPTOR385 and siDEPTOR766) of the three siRNA sequences could significantly disrupt the expression of DEPTOR mRNA, which had significant difference with negative control group (P < 0.05). The ELISA results showed that the total amount of insulin secretion in the effective transfected groups was significantly increased (P < 0.05). Western blot assay results showed the grey levels of p-s6 and p-4EBP-1 proteins were significantly elevated, while p-AKT of those former was slightly decreased. These findings suggest that siRNA technology can effectively silence the DEPTOR gene in NIT-1 cells, which improves β-cell insulin secretion in a manner of mTORC1 activation.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Insulin-Secreting Cells, Insulinoma, Gene Silencing

CLC Number:

R318

Qiu Hong, Lai Shu-chang, Pan Dao-yan, Wang Xiao, Shen Jie. DEPTOR gene silencing promotes β-cell insulin secretion[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(16): 2577-2582.

Figures/Tables 2

References

[1] IDF Diabetes Atlas eighth edition. 2017, International Diabetes Federation.

[2] Yang W, Lu J, Weng J,et al.Prevalence of diabetes among men and women in China. N Engl J Med.2010. 362(12): 1090-1101.

[3] Dinesh Shah A, Langenberg C, Rapsomaniki E, et al.Type 2 diabetes and incidence of cardiovascular diseases: a cohort study in 1.9 million people. Lancet Diabetes Endocrinol.2015; 3(2):105-113.

[4] Yagihashi S, Inaba W, Mizukami H. Dynamic pathology of islet endocrine cells in type 2 diabetes: beta-Cell growth, death, regeneration and their clinical implications.J Diabetes Investig. 2016;7(2):155-165.

[5] Peterson TR, Laplante M, Thoreen CC, et al.DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival.Cell.2009;137(5):873-886.

[6] Zhang HR, Chen JM, Zeng ZY, et al.Knockdown of DEPTOR inhibits cell proliferation and increases chemosensitivity to melphalan in human multiple myeloma RPMI-8226 cells via inhibiting PI3K/AKT activity. J Int Med Res.2013;41(3): 584-595.

[7] Proud CG. Dynamic balancing: DEPTOR tips the scales. J Mol Cell Biol.2009;1(2): 61-63.

[8] Lai EY, Chen ZG, Zhou X, et al.DEPTOR expression negatively correlates with mTORC1 activity and tumor progression in colorectal cancer. Asian Pac J Cancer Prev. 2014;15(11):4589-4594.

[9] Meng ZX, Li S, Wang L, Ko HJ, et al.Baf60c drives glycolytic metabolism in the muscle and improves systemic glucose homeostasis through Deptor-mediated Akt activation. Nat Med.2013;19(5):640-645.

[10] Bernal-Mizrachi E, Kulkarni RN, Scott DK, et al.Human beta-cell proliferation and intracellular signaling part 2: still driving in the dark without a road map. Diabetes.2014;63(3): 819-831.

[11] Vergès B, Cariou B. mTOR inhibitors and diabetes. Diabetes Res Clin Pract.2015;110(2):101-108.

[12] Ardestani A, Lupse B, Kido Y, et al.mTORC1 Signaling: A Double-Edged Sword in Diabetic β Cells. Cell Metab. 2018; 27(2):314-331.

[13] Gu Y, Lindner J, Kumar A, et al.Rictor/mTORC2 is essential for maintaining a balance between beta-cell proliferation and cell size. Diabetes.2011;60(3):827-837.

[14] Rachdi L, Balcazar N, Osorio-Duque F, et al.Disruption of Tsc2 in pancreatic beta cells induces beta cell mass expansion and improved glucose tolerance in a TORC1- dependent manner. Proc Natl Acad Sci U S A.2008;105(27): 9250-9255.

[15] Pirollo KF, Chang EH.Targeted delivery of small interfering RNA: approaching effective cancer therapies. Cancer Res. 2008;68(5): 1247-1250.

[16] 袁松,孙会敏,丁丽霞.脂质体物理化学稳定性研究进展[J].中国药事, 2011,25(4):384-388.

[17] 张旭,丁会芹,王冰,等.脂质体介导RNAi的研究[J]. 生物医学工程学杂志, 2012,29(4):722-726.

[18] 许戈阳,刘芬婷,沈哲民,等. mTOR信号通路在糖代谢中作用[J]. 生理科学进展, 2015,46(2): 94-98.

[19] Cota D. Mammalian target of rapamycin complex 1 (mTORC1) signaling in energy balance and obesity. Physiol Behav. 2009;97(5): 520-524.

[20] Le Bacquer O, Petroulakis E, Paglialunga S, et al.Elevated sensitivity to diet-induced obesity and insulin resistance in mice lacking 4E-BP1 and 4E-BP2. J Clin Invest.2007; 117(2): 387-396.

[21] Demidenko ZN, Shtutman M, Blagosklonny MV. Blagosklonny, Pharmacologic inhibition of MEK and PI-3K converges on the mTOR/S6 pathway to decelerate cellular senescence. Cell Cycle.2009；8(12):1896-900.

[22] Fritsche L, Neukamm SS, Lehmann R, et al.Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and mTOR: studies on the function of Ser675 and Ser907.Am J Physiol Endocrinol Metab.2011;300(5):E824-836.

[23] Jacinto E, Loewith R, Schmidt A, et al.Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol.2004;6(11): 1122-1128.

[24] Elghazi L, Balcazar N, Blandino-Rosano M, et al.Decreased IRS signaling impairs beta-cell cycle progression and survival in transgenic mice overexpressing S6K in beta-cells. Diabetes. 2010;59(10): 2390-2399.

[25] Briaud I, Dickson LM, Lingohr MK,et al.Insulin receptor substrate-2 proteasomal degradation mediated by a mammalian target of rapamycin (mTOR)-induced negative feedback down-regulates protein kinase B-mediated signaling pathway in beta-cells. J Biol Chem.2005;280(3): 2282-2293.

[26] Efeyan A, Sabatini DM. mTOR and cancer: many loops in one pathway.Curr Opin Cell Biol. 2010;22(2): 169-176.

[27] Copps KD, White MF.egulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2. Diabetologia.2012; 55(10): 2565-2582.

[28] Chau GC,Im DU,Kang TM,et al.mTOR controls ChREBP transcriptional activity and pancreatic beta cell survival under diabetic stress. J Cell Biol.2017;216(7): 2091-2105.

[29] Koyanagi M,Asahara S,Matsuda T,et al.Ablation of TSC2 enhances insulin secretion by increasing the number of mitochondria through activation of mTORC1. PLoS One. 2011;6(8):e23238.

[30] Gurevitch D,Boura-Halfon S,Isaac R,et al.Elimination of negative feedback control mechanisms along the insulin signaling pathway improves beta-cell function under stress. Diabetes, 2010;59(9):2188-2197.