Abstract

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.

  • Compound

    10-(4-((bis(2-((2-(ethylthio)ethyl)thio)ethyl)amino)methyl)-2-methylphenyl)-5,5-dimethyl-7-morpholinodibenzo[b,e]silin-3(5H)-one

  • Compound

    10-(4-((dioctylamino)methyl)-2-methylphenyl)-5,5-dimethyl-7-morpholinodibenzo[b,e]silin-3(5H)-one

  • Compound

    2-(4-bromo-3-methylphenyl)acetic acid

  • Compound

    4-bromo-5-methylbenzylalcohol

  • Compound

    4-bromo-3-methylbenzyl methanesulfonate

  • Compound

    N-(4-bromo-3-methylbenzyl)-3,6,12,15-tetrathia-9-monoazaheptadecane

  • Compound

    3,7-dihydroxy-5,5-dimethyldibenzo[b,e]silin-10(5H)-one

  • Compound

    5,5-dimethyl-10-oxo-5,10-dihydrodibenzo[b,e]siline-diyl bis(trifluoromethanesulfonate)

  • Compound

    10-(4-((bis(2-((2-(ethylthio)ethyl)thio)ethyl)amino)methyl)-2-methylphenyl)-5,5-dimethyl-7-morpholinodibenzo[b,e]silin-3(5H)-one

  • Compound

    10-(4-((dioctylamino)methyl)-2-methylphenyl)-5,5-dimethyl-7-morpholinodibenzo[b,e]silin-3(5H)-one

  • Compound

    2-(4-bromo-3-methylphenyl)acetic acid

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Acknowledgements

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.

Author information

Author notes

    • Jefferson Chan
    •  & Elizabeth J New

    Present addresses: Department of Chemistry, University of Illinois, Urbana, Illinois, USA (J.C.) and School of Chemistry, University of Sydney, New South Wales, Australia (E.J.N.).

    • Lakshmi Krishnamoorthy
    •  & Joseph A Cotruvo Jr

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemistry, University of California, Berkeley, California, USA.

    • Lakshmi Krishnamoorthy
    • , Joseph A Cotruvo Jr
    • , Jefferson Chan
    • , Harini Kaluarachchi
    • , Shang Jia
    • , Allegra T Aron
    • , Cheri M Ackerman
    • , Mark N Vander Wal
    • , Timothy Guan
    • , Lukas P Smaga
    • , Samouil L Farhi
    • , Elizabeth J New
    •  & Christopher J Chang
  2. Howard Hughes Medical Institute, University of California, Berkeley, California, USA.

    • Lakshmi Krishnamoorthy
    •  & Christopher J Chang
  3. Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA.

    • Abigael Muchenditsi
    • , Venkata S Pendyala
    •  & Svetlana Lutsenko
  4. Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.

    • Christopher J Chang
  5. Helen Wills Neuroscience Institute, University of California, Berkeley, California, USA.

    • Christopher J Chang

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Contributions

L.K., J.A.C., S.L. and C.J.C. designed research; L.K. and J.A.C. performed most experiments. J.C., S.J., A.T.A., L.P.S. and S.L.F. synthesized and characterized copper probes. J.C. performed cellular imaging experiments. H.K. purified and characterized PDE3B expressed in insect cells. A.M. and V.S.P. performed the animal experiments. C.M.A. performed ICP-MS experiments. M.N.V.W. synthesized compound A for affinity purification of PDE3B. T.G. assisted with bacterial expression and purification of PDE3B. E.J.N. designed and performed preliminary experiments. L.K., J.A.C. and C.J.C. wrote the paper; S.L., J.C. and S.J. provided valuable input on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Christopher J Chang.

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–33

  2. 2.

    Supplementary Note

    Synthetic procedures

Videos

  1. 1.

    Real-time imaging of copper chelation in 3T3-L1 cells with CSR1.

    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.

  2. 2.

    Real-time imaging of copper-supplemented 3T3-L1 cells with CSR1

    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.

  3. 3.

    Real-time imaging of copper chelation in 3T3-L1 cells with Ctrl-CSR1.

    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.

  4. 4.

    Real-time imaging of copper-supplemented 3T3-L1 cells with Ctrl-CSR1.

    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.

  5. 5.

    Real-time imaging of labile copper with CSR1 in Iso-stimulated 3T3-L1 cells.

    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.

  6. 6.

    Real-time imaging of labile copper with Ctrl-CSR1 in Iso-stimulated 3T3-L1 cells.

    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.

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DOI

https://doi.org/10.1038/nchembio.2098

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