Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (17): 3429-3438.doi: 10.3864/j.issn.0578-1752.2017.17.017

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles    

Effect of Melatonin on Insulin and Gαi/o Expression in Rat Insulinoma Cell Line

ZHAO YiWen1, ZHAO Jia2, PANG QuanHai1   

  1. 1College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi; 2 Institute of Molecular Medicine, Peking University, Beijing 100871
  • Received:2017-03-21 Online:2017-09-01 Published:2017-09-01

Abstract: 【Objective】To demonstrate the role of melatonin in the circadian secretion of insulin, the effect of melatonin on insulin and Gαi/o expression in rat insulinoma cell line (INS-1) and molecular mechanism were studied. As melatonin can regulate insulin secretion in a circadian manner, maintaining a homeostasis of blood glucose by regulating diurnal glucose metabolism, thus study on the effect of melatonin on expression of insulin may provide a basis for regulation of melatonin in circadian secretion of insulin.【Method】The cryopreserved INS-1 cells were passaged for at least three generations. After the passage of cells, INS-1 cells were cultured in RPMI1640 medium for about 24-48 h. When the cell confluency reached 60%-70%, washed with RPMI1640 without serum and glucose for two times. INS-1 cells were cultured in media supplemented with different concentrations of glucose(0, 10, 20, 30, and 50 mmol·L-1) and 100 nmol·L-1 melatonin for 12 h treatment, then observation of morphological changes of INS-1 cells and statistical analysis were performed. The total RNA extracted from INS-1 cells by Easypure RNA kit, measuring the cells total RNA concentration and purity, then the quality of total RNA was measured by formaldehyde denaturing agarose gel electrophoresis. TranScript One-step Removal and Synthesis Supermix were used to synthesize cDNA by reverse transcription. Primer Premier 5.0 primer design software, reference gene sequence of GenBank were used to design primers. qRT-PCR was used to detect the changes of Insulin1, Gαi1, Gαi2, Gαo, PKA and PKCα inmRNA level.【Result】After incubation of INS-1 cells with different concentrations of glucose, it was observed that the growth of cell synapse first increased and then decreased along with the increase of the concentration of glucose in the culture medium. In the 20 mmol·L-1 glucose treatment group, the synaptic growth of INS-1 cells was increased significantly; in the 20 mmol·L-1glucose and melatonin co-treatment group, the area of INS-1 cells was increased, but the synaptic growth was not obvious. In the glucose treatment group, the mRNA level of Insulin1 showed a trend of increase and then reduction, especially was increased at 20 mmol·L-1 (P<0.05). The mRNA level of Gαi1 showed a trend of reduction and then increase, especially was reduced at 20 mmol·L-1 (P<0.05). The mRNA level of Gαi2 and Gαo was not significantly reduced (P>0.05). Thus, Gαi1 but not Gαi2 and Gαo showed a negative correlation with Insulin1 mRNA level. Compared with 20 mmol·L-1 glucose treatment group, the mRNA level of Gαi1 was significantly increased (P<0.05) at 20 mmol·L-1 glucose and melatonin co-treatment group, the mRNA level of Insulin1 and PKCα was significantly reduced (P<0.05), whereas the mRNA level of PKA was not significantly reduced (P>0.05).【Conclusion】Melatonin inhibits the expression of Insulin1 in INS-1 cells and reduces insulin secretion resulting in the adaptive hyperplasia of INS-1 cells. The binding of melatonin to its receptor can promote the increase of Gαi1 mRNA levels and inhibit the expression of PKCα, which leads to the inhibited expression of Insulin1 and decreased insulin secretion, so that blood glucose remains homeostatic during the night.

Key words: melatonin, INS-1 cells, glucose, insulin, Gαi/o

[1]    高超, 田秀芝, 张璐, 徐静, 汪锋, 卓志勇, 戴蕴平, 刘国世.外源褪黑素对牛卵母细胞体外成熟的影响. 中国农业科学, 2011, 44(17): 3634-3640.
GAO C, TIAN X Z, ZHANG L, XU J, WANG F, ZHUO Z Y, DAI Y P, LIU G S. Effect of exogenous melatonin (MT) in bovine oocyte in vitro maturation. Scientia Agricultura Sinica, 2011, 44(17):3634-3640. (in Chinese)
[2]    BODEN G, RUIZ J, URBAIN J L, CHEN X. Evidence for a circadian rhythm of insulin secretion. American Journal of Physiology - Endocrinology and Metabolism, 1996, 271(2):E246-E252.
[3]    GUARDIOLA-LEMAITRE B. Toxicology of melatonin. Journal of Biological Rhythms, 1997, 12(6):697-706.
[4]    CARDINALI D P, CANO P, JIMENEZ-ORTEGA V, ESQUIFINO A I. Melatonin and the metabolic syndrome: physiopathologic and therapeutical implications. Neuroendocrinology, 2011, 93(3):133-142.
[5]    PICINATO M C, HABER E P, CIPOLLA-NETO J, CURI R, CARVALHO C R D O, CARPINELLI A R. Melatonin inhibits insulin secretion and decreases PKA levels without interfering with glucose metabolism in rat pancreatic islets. Pineal Research, 2002, 33(3): 156-160.
[6]    STUMPF I, MUHLBAUER E, PESCHKE E. Involvement of the cGMP pathway in mediating the insulin-inhibitory effect of melatonin in pancreatic beta-cells. Pineal Research, 2008, 45(3):318-327.
[7]    STUMPF I, BAZWINSKY I, PESCHKE E. Modulation of the cGMP signaling pathway by melatonin in pancreatic beta-cells. Pineal Research, 2009, 46(2):140-147.
[8]    SHARP G W. Mechanisms of inhibition of insulin release. American Journal of Physiology-Cell Physiology, 1996, 271(6): C1781-C1799.
[9]    STRAUB S G, SHARP G W G. Evolving insights regarding mechanisms for the inhibition of insulin release by norepinephrine and heterotrimeric G proteins. American Journal of Physiology-Cell Physiology, 2012, 302(12): C1687-C1698.
[10]   VAN CAUTER E, POLONSKY K S, SCHEEN A J. Roles of circadian rhythmicity and sleep in human glucose regulation 1. Endocrine Reviews, 1997, 18(5): 716-738.
[11]   SHEA S A, HILTON M F, ORLOVA C, TIMOTHY AYERS R, MANTZOROS C S. Independent circadian and sleep/wake regulation of adipokines and glucose in humans. The Journal of Clinical Endocrinology & Metabolism, 2005, 90(5): 2537-2544.
[12]   QIAN J, SCHEER F A J L. Circadian system and glucose metabolism: Implications for physiology and disease. Trends in Endocrinology & Metabolism, 2016, 27(5): 282-293.
[13]   MORRIS C J, YANG J N, SCHEER F A J L. The impact of the circadian timing system on cardiovascular and metabolic function. Progress in Brain Research, 2012, 199: 337.
[14]   MORRIS C J, YANG J N, GARCIA J I, MYERSA S, BOZZIA I, WANGA W, BUXTONA O M, SHEAA S A, SCHEER F A J L. Endogenous circadian system and circadian misalignment impact glucose tolerance via separate mechanisms in humans. Proceedings of the National Academy of Sciences of the USA, 2015, 112(17): E2225-E2234.
[15]   SCHEER F A, HILTON M F, MANTZOROS C S, SHEA S A. Adverse metabolic and cardiovascular consequences of circadian misalignment. Proceedings of the National Academy of Sciences of the USA, 2009, 106(11):4453-4458.
[16]   PESCHKE E, BÄHR I, MÜHLBAUER E. Experimental and clinical aspects of melatonin and clock genes in diabetes. Journal of Pineal Research, 2015, 59(1): 1-23.
[17]   PESCHKE E. Melatonin, endocrine pancreas and diabetes. Pineal Research, 2008, 44: 26-40.
[18]   SRINIVASAN V, OHTA Y, ESPINO J, PARIENTE J A, RODRIGUEZ A B, MOHAMED M, ZAKARIA R. Metabolic syndrome, its pathophysiology and the role of melatonin. Recent Patents on Endocrine, Metabolic & Immune Drug Discovery, 2013, 7(1): 11-25.
[19]   ZANUTO R, SIQUEIRA-FILHO M A, CAPERUTO L C, BACURAU R F, HIRATA E, PELICIARI-GARCIA R A, DO AMARAL F G, MARÇAL A C, RIBEIRO L M, CAMPOREZ J P, CARPINELLI A R, BORDIN S, CIPOLLA-NETO J, CARVALHO C R. Melatonin improves insulin sensitivity independently of weight loss in old obese rats. Pineal Research, 2013, 55(2):156-165.
[20]   BONNEFOND A, CLÉMENT N, FAWCETT K, YENGO L, VAILLANT E, GUILLAUME J L, DECHAUME A, PAYNE F, ROUSSEL R, CZERNICHOW S, HERCBERG S, HADJADJ S, BALKAU B, MARRE M, LANTIERI O, LANGENBERG C, BOUATIA-NAJI N. Rare MTNR1B variants impairing melatonin receptor 1B function contribute to type 2 diabetes. Nature Genetics, 2012, 44(3): 297-301.
[21]   GAULTON K J, FERREIRA T, LEE Y, Raimiondo A, Mägi R. Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci. Nature Genetics, 2015, 47(12): 1415-1425.
[22]   COSTES S, BOSS M, THOMAS A P, MATVEYENKO A V. Activation of melatonin signaling promotes β-cell survival and function. Molecular Endocrinology, 2015, 29(5): 682-692.
[23]   RUBIO-SASTRE P, SCHEER F A, GÓMEZ-ABELLÁN P, MADRID J A, GARAULET M. Acute melatonin administration in humans impairs glucose tolerance in both the morning and evening. Sleep, 2014, 37(10): 1715-1719.
[24]   MORRIS C J, AESCHBACH D, SCHEER F A J L. Circadian system, sleep and endocrinology. Molecular and Cellular Endocrinology, 2012, 349(1): 91-104.
[25]   MÜHLBAUER E, ALBRECHT E, HOFMANN K, BAZWINSKY- WUTSCHKE I, PESCHKE E. Melatonin inhibits insulin secretion in rat insulinoma β-cells (INS-1) heterologously expressing the human melatonin receptor isoform MT2. Journal of Pineal Research, 2011, 51(3): 361-372.
[26]   ZHANG Z, LI J, JIANG X, LEI Y, LEI L, DE H C, HUA Z, HONG  C. GLP-1 ameliorates the proliferation activity of INS-1 cells inhibited by intermittent high glucose concentrations through the regulation of cyclins. Molecular Medicine Reports, 2014, 10(2): 683-688.
[27]   TUOMI T, NAGORNY C L F, SINGH P, ET A L. Increased melatonin signaling is a risk factor for type 2 diabetes. Cell Metabolism, 2016, 23: 1067-1077.
[28]   HULL R L, KODAMA K, UTZSCHNEIDER K M, CARR D B, PRIGEON R L, KAHN S E. Dietary-fat-induced obesity in mice results in beta cell hyperplasia but not increased insulin release: evidence for specificity of impaired beta cell adaptation. Diabetologia, 2005, 48(7): 1350-1358.
[29]   HELLMAN B, GRAPENGIESSER E. Glucose-induced inhibition of insulin secretion. Acta Physiologica, 2014, 210(3): 479-488.
[30]   SHARMA S, SINGH H, AHMAD N, PRIYANKA M, ARCHANA T. The role of melatonin in diabetes: therapeutic implications. Archives of Endocrinology and Metabolism, 2015, 59(5): 391-397.
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