Energy Depletion of Bovine Mammary Epithelial Cells Activates AMPK and Suppresses Protein Synthesis Through Inhibition of mTORC1 Signaling

S. A. Burgos; J.J. M. Kim; M. Dai; J. P. Cant

doi:10.1055/s-0032-1323742

Hormone and Metabolic Research, Table of Contents

Horm Metab Res 2013; 45(03): 183-189
DOI: 10.1055/s-0032-1323742

Original Basic

Energy Depletion of Bovine Mammary Epithelial Cells Activates AMPK and Suppresses Protein Synthesis Through Inhibition of mTORC1 Signaling

Authors

S. A. Burgos

¹Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada
J.J. M. Kim

¹Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada
M. Dai

¹Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada
J. P. Cant

¹Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada

Abstract

The molecular mechanisms by which cellular energy status regulates global protein synthesis in mammary epithelial cells have not been characterized. The objective of this study was to examine the effect of AMP-activated protein kinase (AMPK) activation by 2-deoxyglucose on protein synthesis and the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in bovine mammary epithelial cells. Phosphorylation of AMPK at Thr172 increased by 1.4-fold within 5 min, and remained elevated throughout a 30-min time course, in response to 2-deoxyglucose. Global rates of protein synthesis declined by 78% of control values. The decline in protein synthesis was associated with repression of mTORC1 signaling, as indicated by reduced phosphorylation of ribosomal protein S6 kinase 1 and eIF4E binding protein-1 (4E-BP1). Phosphorylation of ER-stress marker eIF2α was also increased but only at 30 min of 2-deoxyglucose exposure. 2-Deoxyglucose increased phosphorylation of tuberous sclerosis complex 2 (TSC2) on AMPK consensus sites but did not change the amount of TSC1 bound to TSC2. Activation of AMPK did not result in changes in the amount of raptor bound to mTOR. The inhibitory effects of AMPK activation on mTORC1 signaling were associated with a marked increase in Ser792 phosphorylation on raptor. Collectively, the results suggest that activation of AMPK represses global protein synthesis in mammary epithelial cells through inhibition of mTORC1 signaling.

Key words

2-deoxyglucose - mTOR - tuberous sclerosis complex - eIF2

Full Text

References

References
1 Buttgereit F, Brand MD. A hierarchy of ATP-consuming processes in mammalian cells. Biochem J 1995; 312: 163-167
2 Villa-Trevino S, Shull KH, Farber E. Role of adenosine triphosphate deficiency in ethionine-induced inhibition of protein synthesis. J Biol Chem 1963; 238: 1757-1763
3 Crozier SJ, Vary TC, Kimball SR, Jefferson LS. Cellular energy status modulates translational control mechanisms in ischemic-reperfused rat hearts. Am J Physiol 2005; 289: H1242-H1250
4 Rose AJ, Richter EA. Regulatory mechanisms of skeletal muscle protein turnover during exercise. J Appl Physiol 2009; 106: 1702-1711
5 Bolster DR, Crozier SJ, Kimball SR, Jefferson LS LS. AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling. J Biol Chem 2002; 277: 23977-23980
6 Reiter AK, Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. Repression of protein synthesis and mTOR signaling in rat liver mediated by the AMPK activator aminoimidazole carboxamide ribonucleoside. Am J Physiol 2005; 288: E980-E988
7 Ma XM, Blenis J. Molecular mechanisms of mTOR-mediated translational control. Nature Rev Mol Cell Biol 2009; 10: 307-318
8 Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE, Shaw RJ. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 2008; 30: 214-226
9 Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 2005; 1: 15-25
10 Hardie DG. AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nature Rev Mol Cell Biol 2007; 8: 774-785
11 Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell 2003; 115: 577-590
12 Baracos VE, Brunbellut J, Marie M. Tissue protein synthesis in lactating and dry goats. Br J Nutr 1991; 66: 451-465
13 Hanigan MD, France J, Mabjeesh SJ, McNabb WC, Bequette BJ. High rates of mammary tissue protein turnover in lactating goats are energetically costly. J Nutr 2009; 139: 1118-1127
14 Davis SR, Farr VC, Stelwagen K. Regulation of yield loss and milk composition during once-daily milking: a review. Livest Prod Sci 1999; 59: 77-94
15 Toerien CA, Cant JP. Duration of a severe feed restriction required to reversibly decrease milk production in the high-producing dairy cow. Can J Anim Sci 2007; 87: 455-458
16 Stewart KW, Cooper GJS, Davis SR. Coordination of mammary metabolism and blood flow after refeeding in rats. J Dairy Sci 2009; 92: 1543-1553
17 Hanigan MD, Baldwin RL. A mechanistic model of mammary gland metabolism in the lactating cow. Agric Syst 1994; 45: 369-419
18 Prosser CG, Davis SR, Farr VC, Lacasse P. Regulation of blood flow in the mammary microvasculature. J Dairy Sci 1996; 79: 1184-1197
19 Verhoeven AJM, Woods A, Brennan CH, Hawley SA, Hardie DG, Scott J, Beri RK, Carling D. The AMP-activated protein-kinase gene is highly expressed in rat skeletal muscle – alternative splicing and tissue distribution of the messenger RNA. Eur J Biochem 1995; 228: 236-243
20 McFadden JW, Corl BA. Activation of AMP-activated protein kinase (AMPK) inhibits fatty acid synthesis in bovine mammary epithelial cells. Biochem Biophys Res Commun 2009; 390: 388-393
21 Jiang W, Zhu ZJ, Thompson HJ. Dietary energy restriction modulates the activity of AMP-activated protein kinase, Akt, and mammalian target of rapamycin in mammary carcinomas, mammary gland, and liver. Cancer Res 2008; 68: 5492-5499
22 Moshel Y, Rhoads RE, Barash I. Role of amino acids in translational mechanisms governing milk protein synthesis in murine and ruminant mammary epithelial cells. J Cell Biochem 2006; 98: 685-700
23 Sobolewska A, Gajewska M, Zarzynska J, Gajkowska B, Motyl T. IGF-I, EGF, and sex steroids regulate autophagy in bovine mammary epithelial cells via the mTOR pathway. Eur J Cell Biol 2009; 88: 117-130
24 Burgos SA, Cant JP. IGF-I stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells. Domest Anim Endocrinol 2010; 38: 211-221
25 Jiang W, Zhu Z, Thompson HJ. Modulation of the activities of AMP-activated protein kinase, protein kinase B, and mammalian target of rapamycin by limiting energy availability with 2-deoxyglucose. Mol Carcinogen 2008; 47: 616-628
26 Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, Spooner E, Carr SA, Sabatini DM. PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol Cell 2007; 25: 903-915
27 Hawley SA, Davison M, Woods A, Davies SP, Beri RK, Carling D, Hardie DG. Characterization of the AMP-activated protein kinase kinase from rat liver, and identification of threonine-172 as the major site at which it phosphorylates and activates AMP-activated protein kinase. J Biol Chem 1996; 271: 27879-27887
28 Ha J, Daniel S, Broyles SS, Kim KH. Critical phosphorylation sites for acetyl-CoA carboxylase activity. J Biol Chem 1994; 269: 22162-22168
29 Hara K, Yonezawa K, Kozlowski MT, Sugimoto T, Andrabi K, Weng QP, Kasuga M, Nishimoto I, Avruch J. Regulation of eIF-4E BP1 phosphorylation by mTOR. J Biol Chem 1997; 272: 26457-26463
30 Burnett PE, Barrow RK, Cohen NA, Snyder SH, Sabatini DM. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci USA 1998; 95: 1432-1437
31 Haghighat A, Mader S, Pause A, Sonenberg N. Repression of cap-dependent translation by 4E-binding protein 1: Competition with p220 for binding to eukaryotic initiation factor-4E. EMBO J 1995; 14: 5701-5709
32 Xi H, Kertoglu M, Liu H, Wangpaichitr M, You M, Liu X, Savaraj N, Lampidis TJ. 2-Deoxy-D-glucose activates autophagy via endoplasmic reticulum stress rather than ATP depletion. Cancer Chemother Pharmacol 2011; 67: 899-910
33 Proud CG. eIF2 and the control of cell physiology. Semin Cell Dev Biol 2005; 16: 3-12
34 Williamson DL, Bolster DR, Kimball SR, Jefferson LS. Time course changes in signaling pathways and protein synthesis in C2C12 myotubes following AMPK activation by AICAR. Am J Physiol 2006; 291: E80-E89
35 Miyamoto T, Oshiro N, Yoshino K, Nakashima A, Eguchi S, Takahashi M, Ono Y, Kikkawa U, Yonezawa K. AMP activated protein kinase phosphorylates Golgi-specific brefeldin A resistance factor 1 at Thr1337 to induce disassembly of Golgi apparatus. J Biol Chem 2008; 283: 4430-4438
36 Eguchi S, Oshiro N, Miyamoto T, Yoshino K, Okamoto S, Ono T, Kikkawa U, Yonezawa K. AMP activated protein kinase phosphorylates glutamine: fructose-6-phosphate amidotransferase 1 at Ser243 to modulate its enzymatic activity. Genes Cells 2009; 14: 179-189
37 Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell 2002; 10: 151-162
38 Roux PP, Ballif BA, Anjum R, Gygi SP, Blenis J. Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase. Proc Natl Acad Sci USA 2004; 101: 13489-13494
39 Sanders MJ, Grondin PO, Hegarty BD, Snowden MA, Carling D. Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem J 2007; 403: 139-148
40 Stahmann N, Woods A, Spengler K, Heslegrave A, Bauer R, Krause S, Viollet B, Carling D, Heller R. Activation of AMP-activated protein kinase by vascular endothelial growth factor mediates endothelial angiogenesis independently of nitric-oxide synthase. J Biol Chem 2010; 285: 10638-10652
41 Herrero-Martin G, Høyer-Hansen M, Garcia-Garcia C, Fumarola C, Farkas T, Lopez-Rivas A, Jäättelä M. TAK1 activates AMPK-dependent cytoprotective autophagy in TRAIL-treated epithelial cells. EMBO J 2009; 28: 677-685
42 Pruznak AM, Kazi AA, Frost RA, Vary TC, Lang CH. Activation of AMP-activated protein kinase by 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside prevents leucine-stimulated protein synthesis in rat skeletal muscle. J Nutr 2008; 138: 1887-1894
43 Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, Hidayat S, Tokunaga C, Avruch J, Yonezawa K. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 2002; 110: 177-189
44 Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 2002; 110: 163-175
45 Nojima H, Tokunaga C, Eguchi S, Oshiro N, Hidayat S, Yoshino K, Hara K, Tanaka N, Avruch J, Yonezawa K. The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. J Biol Chem 2003; 278: 15461-15464