Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Dec;76(24):4945-4959.
doi: 10.1007/s00018-019-03152-y. Epub 2019 Jun 6.

Regulation of AMPK activity by type 10 adenylyl cyclase: contribution to the mitochondrial biology, cellular redox and energy homeostasis

Vignesh Jayarajan  1   2   3 , Avinash Appukuttan  3 , Muhammad Aslam  4   5 , Peter Reusch  3 , Vera Regitz-Zagrosek  1   2 , Yury Ladilov  6   7
Affiliations

Regulation of AMPK activity by type 10 adenylyl cyclase: contribution to the mitochondrial biology, cellular redox and energy homeostasis

Vignesh Jayarajan et al. Cell Mol Life Sci. 2019 Dec.

Abstract

The downregulation of AMP-activated protein kinase (AMPK) activity contributes to numerous pathologies. Recent reports suggest that the elevation of cellular cAMP promotes AMPK activity. However, the source of the cAMP pool that controls AMPK activity remains unknown. Mammalian cells possess two cAMP sources: membrane-bound adenylyl cyclase (tmAC) and intracellularly localized, type 10 soluble adenylyl cyclase (sAC). Due to the localization of sAC and AMPK in similar intracellular compartments, we hypothesized that sAC may control AMPK activity. In this study, sAC expression and activity were manipulated in H9C2 cells, adult rat cardiomyocytes or endothelial cells. sAC knockdown depleted the cellular cAMP content and decreased AMPK activity in an EPAC-dependent manner. Functionally, sAC knockdown reduced cellular ATP content, increased mitochondrial ROS formation and led to mitochondrial depolarization. Furthermore, sAC downregulation led to EPAC-dependent mitophagy disturbance, indicated by an increased mitochondrial mass and unaffected mitochondrial biogenesis. Consistently, sAC overexpression or stimulation with bicarbonate significantly increased AMPK activity and cellular ATP content. In contrast, tmAC inhibition or stimulation produced no effect on AMPK activity. Therefore, the sAC-EPAC axis may regulate basal and induced AMPK activity and support mitophagy, cellular energy and redox homeostasis. The study argues for sAC as a potential target in treating pathologies associated with AMPK downregulation.

Keywords: ADCY10; AMPK; ATP; Mitophagy; ROS; cAMP.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
sAC knockdown suppressed AMPK activity. Transfection of H9C2 cells with plasmids encoding either sAC-targeted (sAC shRNA) or scramble shRNA significantly reduced sAC expression (a), cellular cAMP content (b) and the phosphorylation of AMPK (c) and its direct target ACC (d). Data are means ± SEM. n = 10 for a, c, d and n = 6 for b. *P < 0.05 vs. scramble
Fig. 2
Fig. 2
Downregulation of sAC expression reduced AMPK activity in adult rat cardiomyocytes and coronary endothelial cells. Expression analyses of sAC and phosphorylated AMPK performed in adult rat cardiomyocytes (a, b) and rat coronary endothelial cells (c, d) transfected with plasmids encoding either sAC-targeted (sAC shRNA) or scramble shRNA (for methodological details see [24]). Data are means ± SEM. n = 8 for b and n = 4 for c, d. *P < 0.05 vs. scramble
Fig. 3
Fig. 3
Activation of EPAC, not PKA, rescued AMPK phosphorylation. Effect of 1 h treatment with 200 µmol/l PKA agonist 6-Bnz-cAMP (a–b) or with 200 µmol/l EPAC agonist 8-CPT-2Me-cAMP (c–d). Treatments were performed in H9C2 cells transfected with plasmids encoding either sAC-targeted (sAC shRNA) or scramble shRNA. Data are means ± SEM. n = 4 for a–c and n = 6 for d. *P < 0.05 vs. scramble control, #P < 0.05 vs. sAC shRNA control
Fig. 4
Fig. 4
sAC knockdown disturbed cellular redox and energy homeostasis and increased mitochondrial mass. Analyses of total cellular ROS formation (DCF fluorescence, a), mitochondrial ROS formation (MitoSox fluorescence, b), mitochondrial membrane potential (c), total cellular ATP (d), and mitochondrial mass (e–f) performed with H9C2 cells. Analyses of mitochondrial ROS formation (g) and mitochondrial mass (h) performed with adult rat coronary endothelial cells (RCEC). Cells were transfected with plasmids encoding either sAC-targeted (sAC shRNA) or scramble shRNA. Data are means ± SEM. n = 6–8. *P < 0.05 vs. scramble. Treatments with H2O2 (1 mmol/l, 1 h), antimycin A (10 µmol/l, 1 h), 1 mmol/l NaCN + 5 mmol/l 2DOG (40 min) and resveratrol (50 µmol/l, 24 h) were used as positive controls
Fig. 5
Fig. 5
sAC knockdown reduced the expression of mitochondria-related transcription factors, but did not affect the expression of mitochondrial genes. Expression analysis of transcription factors PGC1α and TFAM (western blot, a–b), and mitochondria- and nucleus-encoded mitochondrial genes (c) performed in H9C2 cells transfected with plasmids encoding either sAC-targeted (sAC shRNA) or scramble shRNA. Data are means ± SEM. n = 5. *P < 0.05 vs. scramble
Fig. 6
Fig. 6
AMPK activator rescued the expression of mitochondrial proteins under sAC knockdown in H9C2 cells. Treatment with A769662 (A76, 10 µmol/l) for 24 h significantly increased phosphorylation of AMPK (a) and its direct target ACC (b). Analyses of mitochondrial ROS formation (MitoSox fluorescence, c), mitochondrial mass (MitoGreen, d) and expression of mitochondrial proteins TFAM and Sirt3 (e, f) under sAC knockdown with or without treatment with A76 10 µM for 24 h. Data are means ± SEM. n = 3–4 for a, b, e, f and n = 6–8 for c and d. *P < 0.05 vs. control or scramble and #P < 0.05 vs. sAC shRNA
Fig. 7
Fig. 7
sAC overexpression increased AMPK phosphorylation and the cellular ATP content. Analyses of sAC expression (a), AMPK phosphorylation (b), mitochondrial mass (d) and total cellular ATP content (d) performed in H9C2 cells transfected with plasmids encoding either 50-kDa sAC isoform (sAC) or GFP. Data are means ± SEM. n = 4. *P < 0.05 vs. GFP. Treatment with 1 mmol/l NaCN + 5 mmol/l 2DOG (40 min) was used as a positive control
Fig. 8
Fig. 8
Stimulation of sAC, but not tmAC increased AMPK phosphorylation and cellular ATP content. Analyses of AMPK phosphorylation (a, b, d) and total cellular ATP content (c) performed in control, untransfected H9C2 cells, or in cells transfected with plasmids encoding either sAC-targeted (sAC shRNA) or scramble shRNA. Cells were incubated in medium containing 0, 21 or 42 mmol/l bicarbonate for 1 h in (a, b, d) and for 4 h in (c). In d, cells were treated with 1 or 10 µmol/l forskolin, or vehicles (DMSO) in medium containing 21 mmol/l bicarbonate for 1 h. Data are means ± SEM. n = 16 for (a, d), n = 6 for (b) and n = 4 for (c). *P < 0.05 vs. 0 mmol/l HCO3− in (a). *P < 0.05 vs. scramble at 21 mmol/l HCO3− in (b). *P < 0.05 vs. 21 mmol/l HCO3− in (c). *P < 0.05 vs. DMSO in (d). #P < 0.05 vs. 21 mmol/l HCO3− in (a)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jeon S-M. Regulation and function of AMPK in physiology and diseases. Exp Mol Med. 2016;48(7):e245–e245. doi: 10.1038/emm.2016.81. - DOI - PMC - PubMed
    1. Herzig S, Shaw RJ. AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol. 2018;19(2):121–135. doi: 10.1038/nrm.2017.95. - DOI - PMC - PubMed
    1. Fryer LG, Parbu-Patel A, Carling D. The Anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J Biol Chem. 2002;277(28):25226–25232. doi: 10.1074/jbc.M202489200. - DOI - PubMed
    1. Hawley SA, Gadalla AE, Olsen GS, Hardie DG. The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism. Diabetes. 2002;51(8):2420. doi: 10.2337/diabetes.51.8.2420. - DOI - PubMed
    1. Yamauchi T., Kamon J., Minokoshi Y., Ito Y., Waki H., Uchida S., Yamashita S., Noda M., Kita S., Ueki K., Eto K., Akanuma Y., Froguel P., Foufelle F., Ferre P., Carling D., Kimura S., Nagai R., Kahn B.B., Kadowaki T. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nature Medicine. 2002;8(11):1288–1295. doi: 10.1038/nm788. - DOI - PubMed