Copper regulates cyclic-AMP-dependent lipolysis

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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.

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Figure 1: Genetically induced copper misregulation affects lipid metabolism in vivo.
Figure 2: Labile copper pools alter cAMP-dependent lipolysis.
Figure 3: Molecular imaging reveals that lipolysis alters labile copper.
Figure 4: Labile copper pools alter cAMP levels by regulating activity of PDE3B.
Figure 5: The catalytic domain of PDE3B binds Cu+ at a conserved Cys residue that is implicated in the cellular copper-dependent lipolytic phenotype.

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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

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.

Correspondence to Christopher J Chang.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–33 (PDF 7084 kb)

Supplementary Note

Synthetic procedures (PDF 725 kb)

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. (AVI 6886 kb)

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. (AVI 19462 kb)

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. (AVI 7301 kb)

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. (AVI 4770 kb)

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. (AVI 5151 kb)

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. (AVI 4088 kb)

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Krishnamoorthy, L., Cotruvo, J., Chan, J. et al. Copper regulates cyclic-AMP-dependent lipolysis. Nat Chem Biol 12, 586–592 (2016) doi:10.1038/nchembio.2098

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