Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body weight and energy stores. Using a mouse model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we found that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue in a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Protein Data Bank
Lippard, S.J. & Berg, J.M. Principles of Bioinorganic Chemistry (University Science Books, Mill Valley, California, USA, 1994).
Barnham, K.J., Masters, C.L. & Bush, A.I. Neurodegenerative diseases and oxidative stress. Nat. Rev. Drug Discov. 3, 205–214 (2004).
Rae, T.D., Schmidt, P.J., Pufahl, R.A., Culotta, V.C. & O'Halloran, T.V. Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science 284, 805–808 (1999).
Robinson, N.J. & Winge, D.R. Copper metallochaperones. Annu. Rev. Biochem. 79, 537–562 (2010).
Banci, L. et al. Affinity gradients drive copper to cellular destinations. Nature 465, 645–648 (2010).
Boal, A.K. & Rosenzweig, A.C. Structural biology of copper trafficking. Chem. Rev. 109, 4760–4779 (2009).
Brady, D.C. et al. Copper is required for oncogenic BRAF signalling and tumorigenesis. Nature 509, 492–496 (2014).
Turski, M.L. et al. A novel role for copper in Ras/mitogen-activated protein kinase signaling. Mol. Cell. Biol. 32, 1284–1295 (2012).
Dodani, S.C. et al. Calcium-dependent copper redistributions in neuronal cells revealed by a fluorescent copper sensor and X-ray fluorescence microscopy. Proc. Natl. Acad. Sci. USA 108, 5980–5985 (2011).
Dodani, S.C. et al. Copper is an endogenous modulator of neural circuit spontaneous activity. Proc. Natl. Acad. Sci. USA 111, 16280–16285 (2014).
Cotruvo, J.A. Jr., Aron, A.T., Ramos-Torres, K.M. & Chang, C.J. Synthetic fluorescent probes for studying copper in biological systems. Chem. Soc. Rev. 44, 4400–4414 (2015).
Chang, C.J. Searching for harmony in transition-metal signaling. Nat. Chem. Biol. 11, 744–747 (2015).
Aron, A.T., Ramos-Torres, K.M., Cotruvo, J.A. Jr. & Chang, C.J. Recognition- and reactivity-based fluorescent probes for studying transition metal signaling in living systems. Acc. Chem. Res. 48, 2434–2442 (2015).
Kahn, S.E., Hull, R.L. & Utzschneider, K.M. Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature 444, 840–846 (2006).
Khandekar, M.J., Cohen, P. & Spiegelman, B.M. Molecular mechanisms of cancer development in obesity. Nat. Rev. Cancer 11, 886–895 (2011).
Van Gaal, L.F., Mertens, I.L. & De Block, C.E. Mechanisms linking obesity with cardiovascular disease. Nature 444, 875–880 (2006).
Burkhead, J.L. & Lutsenko, S. The role of copper as a modifier of lipid metabolism. in Lipid Metabolism (ed. Baez, R.V.) (InTech 2013).
Engle, T.E. Copper and lipid metabolism in beef cattle: a review. J. Anim. Sci. 89, 591–596 (2011).
al-Othman, A.A., Rosenstein, F. & Lei, K.Y. Copper deficiency alters plasma pool size, percent composition and concentration of lipoprotein components in rats. J. Nutr. 122, 1199–1204 (1992).
Carr, T.P. & Lei, K.Y. High-density lipoprotein cholesteryl ester and protein catabolism in hypercholesterolemic rats induced by copper deficiency. Metabolism 39, 518–524 (1990).
Huster, D. et al. High copper selectively alters lipid metabolism and cell cycle machinery in the mouse model of Wilson disease. J. Biol. Chem. 282, 8343–8355 (2007).
Huster, D. & Lutsenko, S. Wilson disease: not just a copper disorder. Analysis of a Wilson disease model demonstrates the link between copper and lipid metabolism. Mol. Biosyst. 3, 816–824 (2007).
Seessle, J. et al. Alterations of lipid metabolism in Wilson disease. Lipids Health Dis. 10, 83 (2011).
Huster, D. et al. Consequences of copper accumulation in the livers of the Atp7b−/− (Wilson disease gene) knockout mice. Am. J. Pathol. 168, 423–434 (2006).
Lutsenko, S. Atp7b−/− mice as a model for studies of Wilson's disease. Biochem. Soc. Trans. 36, 1233–1238 (2008).
Buiakova, O.I. et al. Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation. Hum. Mol. Genet. 8, 1665–1671 (1999).
Gerbasi, V., Lutsenko, S. & Lewis, E.J. A mutation in the ATP7B copper transporter causes reduced dopamine beta-hydroxylase and norepinephrine in mouse adrenal. Neurochem. Res. 28, 867–873 (2003).
Aigner, E. et al. A role for low hepatic copper concentrations in nonalcoholic fatty liver disease. Am. J. Gastroenterol. 105, 1978–1985 (2010).
Kawamura, M. et al. Hormone-sensitive lipase in differentiated 3T3-L1 cells and its activation by cyclic AMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 78, 732–736 (1981).
Kim, B.E. et al. Cardiac copper deficiency activates a systemic signaling mechanism that communicates with the copper acquisition and storage organs. Cell Metab. 11, 353–363 (2010).
Duncan, R.E., Ahmadian, M., Jaworski, K., Sarkadi-Nagy, E. & Sul, H.S. Regulation of lipolysis in adipocytes. Annu. Rev. Nutr. 27, 79–101 (2007).
Chijiwa, T. et al. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J. Biol. Chem. 265, 5267–5272 (1990).
Greenberg, A.S. et al. Stimulation of lipolysis and hormone-sensitive lipase via the extracellular signal-regulated kinase pathway. J. Biol. Chem. 276, 45456–45461 (2001).
Xue, B., Greenberg, A.G., Kraemer, F.B. & Zemel, M.B. Mechanism of intracellular calcium ([Ca2+]i) inhibition of lipolysis in human adipocytes. FASEB J. 15, 2527–2529 (2001).
Zhou, L. et al. Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes. Biochim. Biophys. Acta 1812, 527–535 (2011).
Zhou, D. et al. CD36 level and trafficking are determinants of lipolysis in adipocytes. FASEB J. 26, 4733–4742 (2012).
Snyder, P.B., Esselstyn, J.M., Loughney, K., Wolda, S.L. & Florio, V.A. The role of cyclic nucleotide phosphodiesterases in the regulation of adipocyte lipolysis. J. Lipid Res. 46, 494–503 (2005).
Ahmad, F. et al. Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB. Biochem. J. 404, 257–268 (2007).
Varnerin, J.P. et al. Expression, refolding, and purification of recombinant human phosphodiesterase 3B: definition of the N-terminus of the catalytic core. Protein Expr. Purif. 35, 225–236 (2004).
McMillin, D.R. The origin of the intense absorption in azurin. Bioinorg. Chem. 8, 179–184 (1978).
Pountney, D.L., Schauwecker, I., Zarn, J. & Vasák, M. Formation of mammalian Cu8-metallothionein in vitro: evidence for the existence of two Cu(I)4-thiolate clusters. Biochemistry 33, 9699–9705 (1994).
Angeletti, B. et al. BACE1 cytoplasmic domain interacts with the copper chaperone for superoxide dismutase-1 and binds copper. J. Biol. Chem. 280, 17930–17937 (2005).
Omburo, G.A., Brickus, T., Ghazaleh, F.A. & Colman, R.W. Divalent metal cation requirement and possible classification of cGMP-inhibited phosphodiesterase as a metallohydrolase. Arch. Biochem. Biophys. 323, 1–5 (1995).
Scapin, G. et al. Crystal structure of human phosphodiesterase 3B: atomic basis for substrate and inhibitor specificity. Biochemistry 43, 6091–6100 (2004).
Degerman, E., Belfrage, P. & Manganiello, V.C. Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3). J. Biol. Chem. 272, 6823–6826 (1997).
Hung, S.H. et al. New insights from the structure-function analysis of the catalytic region of human platelet phosphodiesterase 3A: a role for the unique 44-amino acid insert. J. Biol. Chem. 281, 29236–29244 (2006).
Tang, K.M., Jang, E.K. & Haslam, R.J. Expression and mutagenesis of the catalytic domain of cGMP-inhibited phosphodiesterase (PDE3) cloned from human platelets. Biochem. J. 323, 217–224 (1997).
Ovadia, H. et al. Increased adipocyte S-nitrosylation targets anti-lipolytic action of insulin: relevance to adipose tissue dysfunction in obesity. J. Biol. Chem. 286, 30433–30443 (2011).
Maurice, D.H. et al. Advances in targeting cyclic nucleotide phosphodiesterases. Nat. Rev. Drug Discov. 13, 290–314 (2014).
Chung, Y.W. et al. Targeted disruption of PDE3B, but not PDE3A, protects murine heart from ischemia/reperfusion injury. Proc. Natl. Acad. Sci. USA 112, E2253–E2262 (2015).
Gray, N.E. et al. Angiopoietin-like 4 (Angptl4) protein is a physiological mediator of intracellular lipolysis in murine adipocytes. J. Biol. Chem. 287, 8444–8456 (2012).
Stephens, J.M., Lee, J. & Pilch, P.F. Tumor necrosis factor-alpha-induced insulin resistance in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor substrate-1 and GLUT4 expression without a loss of insulin receptor-mediated signal transduction. J. Biol. Chem. 272, 971–976 (1997).
Hong-Hermesdorf, A. et al. Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas. Nat. Chem. Biol. 10, 1034–1042 (2014).
Kenan, Y., Murata, T., Shakur, Y., Degerman, E. & Manganiello, V.C. Functions of the N-terminal region of cyclic nucleotide phosphodiesterase 3 (PDE 3) isoforms. J. Biol. Chem. 275, 12331–12338 (2000).
Cotruvo, J.A. Jr. & Stubbe, J. NrdI, a flavodoxin involved in maintenance of the diferric-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase. Proc. Natl. Acad. Sci. USA 105, 14383–14388 (2008).
Leonard, W.R., Romine, J.L. & Meyers, A.I. A rapid and efficient synthesis of chiral 2-hydro-2-oxazolines. J. Org. Chem. 56, 1961–1963 (1991).
Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
Cobine, P.A. et al. Stoichiometry of complex formation between copper(I) and the N-terminal domain of the Menkes protein. Biochemistry 39, 6857–6863 (2000).
Xiao, Z. et al. Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins: detection probes and affinity standards. J. Biol. Chem. 286, 11047–11055 (2011).
Dworkin, M. & Keller, K.H. Solubility and diffusion coefficient of adenosine 3′:5′-monophosphate. J. Biol. Chem. 252, 864–865 (1977).
We thank the US National Institutes of Health (NIH) (GM 79465 to C.J.C., GM067166 and GM101502 to S.L.) for providing funding for this work. C.J.C. and L.K. are supported by the Howard Hughes Medical Institute. J.A.C. is supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research. J.C. was supported by a postdoctoral fellowship from the Human Frontiers Science Program. A.T.A. was supported by a National Science Foundation Graduate Research Fellowship. C.M.A. was supported by a Hertz Foundation Graduate Fellowship. A.T.A. and C.M.A. were partially supported by Chemical Biology Training Grant T32 GM066698 from the NIH. L.P.S. was supported by the German National Academic Foundation with an international scholarship. S.L.F. was supported by scholarships from Amgen and Merage Foundation for the American Dream Scholarship. E.J.N. was supported by a fellowship from the Royal Commission for the Exhibition of 1851. We thank members of the UCB Cell Culture Facility (A. Fischer, X. Zhang, A. Killilea, C. Tosta), which is funded by the University of California Berkeley, for 3T3-L1 cultures; J. Larsen and C. Mangels for expert technical assistance; V. Manganiello (Laboratory of Biochemical Physiology, NIH) for mPDE3B plasmids; and M. Uhm for advice regarding PDE3B overexpression.
The authors declare no competing financial interests.
Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–33 (PDF 7084 kb)
Synthetic procedures (PDF 725 kb)
3T3-L1 cells were incubated with 2 μM CSR1 at 37 °C in DMEM. After removal of the dye-containing solution, cells were treated with vehicle control or 500 μM TEMEA on-stage and imaged every 5 min for 60 min. (AVI 6886 kb)
3T3-L1 cells were incubated with 2 μM CSR1 at 37 °C in DMEM. After removal of the dye-containing solution, cells were treated with 50 μM CuCl2 on-stage and imaged every 5 min for 120 min. (AVI 19462 kb)
3T3-L1 cells were incubated with 2 μM Ctrl-CSR1 at 37 °C in DMEM. After removal of the dye-containing solution, cells were treated with vehicle control or 500 μM TEMEA on-stage and imaged every 5 min for 60 min. (AVI 7301 kb)
3T3-L1 cells were incubated with 2 μM Ctrl-CSR1 at 37 °C in DMEM. After removal of the dye-containing solution, cells were treated with 50 μM CuCl2 on-stage and imaged every 5 min for 120 min. (AVI 4770 kb)
3T3-L1 cells were incubated with 2 μM CSR1 at 37 °C in DMEM. After removal of the dye-containing solution, cells were treated with 100 nM Iso on-stage and imaged every 5 min for 60 min. (AVI 5151 kb)
3T3-L1 cells were incubated with 2 μM Ctrl-CSR1 at 37 °C in DMEM. After removal of the dye-containing solution, cells were treated with 100 nM Iso on-stage and imaged every 5 min for 60 min. (AVI 4088 kb)
About this article
Cite this article
Krishnamoorthy, L., Cotruvo, J., Chan, J. et al. Copper regulates cyclic-AMP-dependent lipolysis. Nat Chem Biol 12, 586–592 (2016). https://doi.org/10.1038/nchembio.2098
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research (2021)
Dietary Copper Supplementation Increases Growth Performance by Increasing Feed Intake, Digestibility, and Antioxidant Activity in Rex Rabbits
Biological Trace Element Research (2021)
Zn Induces Lipophagy via the Deacetylation of Beclin1 and Alleviates Cu-Induced Lipotoxicity at Their Environmentally Relevant Concentrations
Environmental Science & Technology (2021)
Nature Communications (2021)
Nature Biotechnology (2021)