Regulation of AMP-activated protein kinase by cAMP in adipocytes: roles for phosphodiesterases, protein kinase B, protein kinase A, Epac and lipolysis

Abstract

AMP-activated protein kinase (AMPK) is an important regulator of cellular energy status. In adipocytes, stimuli that increase intracellular cyclic AMP (cAMP) have also been shown to increase the activity of AMPK. The precise molecular mechanisms responsible for cAMP-induced AMPK activation are not clear. Phosphodiesterase 3B (PDE3B) is a critical regulator of cAMP signaling in adipocytes. Here we investigated the roles of PDE3B, PDE4, protein kinase B (PKB) and the exchange protein activated by cAMP 1 (Epac1), as well as lipolysis, in the regulation of AMPK in primary rat adipocytes. We demonstrate that the increase in phosphorylation of AMPK at T172 induced by the adrenergic agonist isoproterenol can be diminished by co-incubation with insulin. The diminishing effect of insulin on AMPK activation was reversed upon treatment with the PDE3B specific inhibitor OPC3911 but not with the PDE4 inhibitor Rolipram. Adenovirus-mediated overexpression of PDE3B and constitutively active PKB both resulted in greatly reduced isoproterenol-induced phosphorylation of AMPK at T172. Co-incubation of adipocytes with isoproterenol and the PKA inhibitor H89 resulted in a total ablation of lipolysis and a reduction in AMPK phosphorylation/activation. Stimulation of adipocytes with the Epac1 agonist 8-pCPT-2'O-Me-cAMP led to increased phosphorylation of AMPK at T172. The general lipase inhibitor Orlistat decreased isoproterenol-induced phosphorylation of AMPK at T172. This decrease corresponded to a reduction of lipolysis from adipocytes. Taken together, these data suggest that PDE3B and PDE4 regulate cAMP pools that affect the activation/phosphorylation state of AMPK and that the effects of cyclic AMP on AMPK involve Epac1, PKA and lipolysis.

Fig. 1

PDE3B and PDE4 are involved in the regulation of AMPK Primary adipocytes were stimulated with 30 nM isoproterenol (ISO) alone or in combination with 1nM insulin (INS) for 10 minutes. Adipocytes were pre-treated for 10 minutes with OPC3911 (OPC) or Rolipram (RO) prior to stimulation with hormones as indicated. Representative immunoblots for A) phospho-AMPK T172, phospho-ACC S79 and total ACC and B) for phospho-PKA substrates are shown. Data is presented as the mean ± SEM of the quantified phosphorylation relative to the isoproterenol stimulated sample. n = 3-7. ** p<0.01, *** p<0.001.

Fig. 1

PDE3B and PDE4 are involved in the regulation of AMPK Primary adipocytes were stimulated with 30 nM isoproterenol (ISO) alone or in combination with 1nM insulin (INS) for 10 minutes. Adipocytes were pre-treated for 10 minutes with OPC3911 (OPC) or Rolipram (RO) prior to stimulation with hormones as indicated. Representative immunoblots for A) phospho-AMPK T172, phospho-ACC S79 and total ACC and B) for phospho-PKA substrates are shown. Data is presented as the mean ± SEM of the quantified phosphorylation relative to the isoproterenol stimulated sample. n = 3-7. ** p<0.01, *** p<0.001.

Fig. 2

Overexpression of PDE3B and constitutively active PKB results in reduced isoproterenol-induced AMPK activation Primary adipocytes were infected overnight with adenoviruses containing β-Galactosidase (Ad-β-Gal) and A) FLAG-PDE3B (Ad-PDE3B) or B) myristoylated PKB (Ad-PKBmyr) adenoviral vectors. Infected adipocytes were stimulated with 3, 10, 30 or 100 nM isoproterenol (ISO) for 30 minutes prior to homogenization. Aliquots of homogenates were used for western blotting. Representative western blots for phosphor-AMPK T172, phosphor-ACC S79, total ACC and PDE3B or total PKB are shown. Data is presented as mean ± SEM relative to the unstimulated sample in each group. n = 3-4. * p<0.05, *** p<0.001.

Fig. 2

Overexpression of PDE3B and constitutively active PKB results in reduced isoproterenol-induced AMPK activation Primary adipocytes were infected overnight with adenoviruses containing β-Galactosidase (Ad-β-Gal) and A) FLAG-PDE3B (Ad-PDE3B) or B) myristoylated PKB (Ad-PKBmyr) adenoviral vectors. Infected adipocytes were stimulated with 3, 10, 30 or 100 nM isoproterenol (ISO) for 30 minutes prior to homogenization. Aliquots of homogenates were used for western blotting. Representative western blots for phosphor-AMPK T172, phosphor-ACC S79, total ACC and PDE3B or total PKB are shown. Data is presented as mean ± SEM relative to the unstimulated sample in each group. n = 3-4. * p<0.05, *** p<0.001.

Fig. 3

Activation of AMPK by isoproterenol is partially mediated by PKA. Adipocytes were pre-treated with vehicle (DMSO) or H89 at the indicated concentrations for 30 minutes followed by stimulation with 30nM isoproterenol for 10 minutes. Aliquots of homogenates were used for A) western blotting and B) AMPK activity assays. C) Prior to homogenizations, the incubation media was collected for measurement of glycerol content. Data is presented as the mean ± SEM relative to the isoproterenol stimulated sample unless indicated. n = 4. * p<0.05, *** p<0.001.

Fig. 3

Activation of AMPK by isoproterenol is partially mediated by PKA. Adipocytes were pre-treated with vehicle (DMSO) or H89 at the indicated concentrations for 30 minutes followed by stimulation with 30nM isoproterenol for 10 minutes. Aliquots of homogenates were used for A) western blotting and B) AMPK activity assays. C) Prior to homogenizations, the incubation media was collected for measurement of glycerol content. Data is presented as the mean ± SEM relative to the isoproterenol stimulated sample unless indicated. n = 4. * p<0.05, *** p<0.001.

Fig. 3

Activation of AMPK by isoproterenol is partially mediated by PKA. Adipocytes were pre-treated with vehicle (DMSO) or H89 at the indicated concentrations for 30 minutes followed by stimulation with 30nM isoproterenol for 10 minutes. Aliquots of homogenates were used for A) western blotting and B) AMPK activity assays. C) Prior to homogenizations, the incubation media was collected for measurement of glycerol content. Data is presented as the mean ± SEM relative to the isoproterenol stimulated sample unless indicated. n = 4. * p<0.05, *** p<0.001.

Fig. 4

Activation of Epac1 results in increased AMPK phosphorylation and activity. Adipocytes were incubated in the absence or presence of 10 μM of the Epac agonist 8-pCPT-2’-O-Me-cAMP (Epac Ag) for 40 minutes. As a control untreated adipocytes were incubated for 30 minutes followed by 10 minutes of stimulation with 30 nM isoproterenol. Prior to homogenizations, the incubation media were collected for measurement of glycerol content. Data is presented as the mean ± SEM. n = 7. * p<0.05, *** p<0.001.

Fig. 4

Activation of Epac1 results in increased AMPK phosphorylation and activity. Adipocytes were incubated in the absence or presence of 10 μM of the Epac agonist 8-pCPT-2’-O-Me-cAMP (Epac Ag) for 40 minutes. As a control untreated adipocytes were incubated for 30 minutes followed by 10 minutes of stimulation with 30 nM isoproterenol. Prior to homogenizations, the incubation media were collected for measurement of glycerol content. Data is presented as the mean ± SEM. n = 7. * p<0.05, *** p<0.001.

Fig. 4

Activation of Epac1 results in increased AMPK phosphorylation and activity. Adipocytes were incubated in the absence or presence of 10 μM of the Epac agonist 8-pCPT-2’-O-Me-cAMP (Epac Ag) for 40 minutes. As a control untreated adipocytes were incubated for 30 minutes followed by 10 minutes of stimulation with 30 nM isoproterenol. Prior to homogenizations, the incubation media were collected for measurement of glycerol content. Data is presented as the mean ± SEM. n = 7. * p<0.05, *** p<0.001.

Fig. 5

The general lipase inhibitor Orlistat reduces isoproterenol-mediated AMPK activation in primary rat adipocytes. A) Primary adipocytes were pre-treated with either vehicle (60% DMSO, 40% ethanol) or 100 μM Orlistat for 30 minutes followed by stimulation with 0, 10, 30 or 100 nM isoproterenol for 10 minutes. Aliquots of homogenates were used for western blotting. B) Prior to homogenizations, the incubation media was collected for measurement of glycerol content. Data is presented as mean ± SEM relative to the unstimulated sample. n = 4. * p<0.05, ** p<0.01.

Fig. 5

The general lipase inhibitor Orlistat reduces isoproterenol-mediated AMPK activation in primary rat adipocytes. A) Primary adipocytes were pre-treated with either vehicle (60% DMSO, 40% ethanol) or 100 μM Orlistat for 30 minutes followed by stimulation with 0, 10, 30 or 100 nM isoproterenol for 10 minutes. Aliquots of homogenates were used for western blotting. B) Prior to homogenizations, the incubation media was collected for measurement of glycerol content. Data is presented as mean ± SEM relative to the unstimulated sample. n = 4. * p<0.05, ** p<0.01.