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. 2009 Nov 26;29(2):469–481. doi: 10.1038/emboj.2009.339

Figure 1.

Figure 1

PKA signalling inhibits Thr-172 phosphorylation of AMPKα. (A) Primary adipocytes were cultivated for 30 min either in the presence of 25 mM glucose (+Glc) (lanes 1–3 and 7–9) or the absence of glucose (−Glc) (lanes 4–6) and treated with 1 mM AICAR (lanes 7–9), 200 nM isoproterenol (Iso) (lanes 2, 5 and 8) or 200 nM isoproterenol in combination with 1 μM H89 (lanes 3, 6 and 9). Protein lysates were prepared and probed with the indicated antibodies. (B) Similar experiment as in (A) except that 20 μM Forskolin (FSK) and 100 μM myristoylated PKi were added instead of isoproterenol and H89, respectively. (C) Primary adipocytes were glucose starved for 60 min (−Glc) and incubated with 20 μM Forskolin (FSK) for indicated times. FSK was either present ab initio (lane 8), added during the last 5, 10, 20, 30 or 45 min of starvation (lanes 3–8) or omitted (lane 2). As control, cells were grown in glucose-rich medium (+Glc) (lane 1). Equal amounts of protein lysates were subjected to immunoblotting with the indicated antibodies. (D) Phosphorylation of HSL by PKA and AMPK at Ser-563 and Ser-565, respectively, is mutually exclusive. In vitro kinase assay of GST-HSL in the presence of PKA (lanes 2, 4 and 5) and constitutively active AMPK(T172D) (lanes 3, 4 and 5). PKA was either added before (lane 4) or after incubation of HSL with AMPK(T172D) (lane 5). Proteins were subjected to immunoblotting with the indicated antibodies. (E) NEFA release in response to PKA signalling. Primary adipocytes were incubated for 30 min with 200 nM isoproterenol alone (Iso) or in combination with 1 μM H89. Furthermore, this treatment was done in the presence (+Glc) or absence (−Glc) of glucose or in the presence of glucose and 1 mM AICAR. NEFA were measured in the incubation medium. Bars represent the mean NEFA release from three independent experiments. (F) Whole-cell extracts of primary mouse adipocytes (WCE, lane 1) were prepared and aliquots were subjected to immunoprecipitation with control IgG (lane 2) or anti-AMPKα1 antibodies (lane 3) and immunoblotted for AMPKα1 and PKAα. Immunoblots are representative of at least three independent experiments.