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. 2016 Sep;240(1):96-107.
doi: 10.1002/path.4760.

Dysregulation of hepatic cAMP levels via altered Pde4b expression plays a critical role in alcohol-induced steatosis

Diana V Avila  1 , David F Barker  2 , JingWen Zhang  2 , Craig J McClain  1   2   3 , Shirish Barve  1   2 , Leila Gobejishvili  1   2
Affiliations

Dysregulation of hepatic cAMP levels via altered Pde4b expression plays a critical role in alcohol-induced steatosis

Diana V Avila et al. J Pathol. 2016 Sep.

Abstract

Alcohol-induced hepatic steatosis is a significant risk factor for progressive liver disease. Cyclic adenosine monophosphate (cAMP) signalling has been shown to significantly regulate lipid metabolism; however, the role of altered cAMP homeostasis in alcohol-mediated hepatic steatosis has never been studied. Our previous work demonstrated that increased expression of hepatic phosphodiesterase 4 (Pde4), which specifically hydrolyses and decreases cAMP levels, plays a pathogenic role in the development of liver inflammation/injury. The aim of this study was to examine the role of PDE4 in alcohol-induced hepatic steatosis. C57BL/6 wild-type and Pde4b knockout (Pde4b(-/-) ) mice were pair-fed control or ethanol liquid diets. One group of wild-type mice received rolipram, a PDE4-specific inhibitor, during alcohol feeding. We demonstrate for the first time that an early increase in PDE4 enzyme expression and a resultant decrease in hepatic cAMP levels are associated with the significant reduction in carnitine palmitoyltransferase 1A (Cpt1a) expression. Notably, alcohol-fed (AF) Pde4b(-/-) mice and AF wild-type mice treated with rolipram had significantly lower hepatic free fatty acid content compared with AF wild-type mice. Importantly, PDE4 inhibition in alcohol-fed mice prevented the decrease in hepatic Cpt1a expression via the Pparα/Sirt1/Pgc1α pathway. These results demonstrate that the alcohol- induced increase in hepatic Pde4, specifically Pde4b expression, and compromised cAMP signalling predispose the liver to impaired fatty acid oxidation and the development of steatosis. Moreover, these data also suggest that hepatic PDE4 may be a clinically relevant therapeutic _target for the treatment of alcohol-induced hepatic steatosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: PDE4; PGC1α, SIRT1, CPT1A; PPARα; alcohol; cAMP; hepatic steatosis.

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Conflict of interest statement

Authors declare no conflicts of interest

Figures

Figure 1
Figure 1
Significant upregulation of hepatic Pde4 expression in alcohol fed mice. (A) mRNA levels of Pde4a, Pde4b, Pde4c, and Pde4d (n=5–7 mice per group). (B) Hepatic Pde4-specific activity after 1 wk of alcohol feeding (n=5–7). (C) Hepatic cAMP levels after 1 and 2 wk of alcohol feeding (n=5–7). (D) cAMP levels in hepatocytes isolated after 2 wk of alcohol feeding (n=3 mice per group). (E) levels of Pde4a, Pde4b, Pde4c, and Pde4d mRNAs in primary rat hepatocytes after 48 h of alcohol exposure (50 mM) (n=3). (F) Representative Western blot images of Pde4a, b, c and d protein levels in primary rat hepatocytes after 48 h of alcohol exposure (50 mM). Data are presented as mean ± S.D. *P < 0.05, **P<0.01compared to PF and UT.
Figure 2
Figure 2
Pde4 inhibition attenuates alcohol induced lipid accumulation in the liver. (A) H&E staining. (B) Oil red O staining. (C) Hepatic free fatty acids (FFA). Data are presented as the mean ± SD, n=5–7 mice per group. *P<0.05, ** P< 0.01.
Figure 3
Figure 3
Effect of Pde4 inhibition on hepatic Cpt1a expression and cAMP/pCREB levels. (A) Immunohistochemical staining with anti-CPT-1A antibody (×20 final magnification). (B) Cpt1a mRNA levels after 4 wk of feeding. (C) Hepatic cAMP levels after 2 wk of alcohol feeding. (D) Nuclear pCREB staining of livers after 4 wk of alcohol feeding. Data are presented as the mean ± SD (n=5–7 mice per group). *P<0.05, ** P< 0.01, *** P< 0.001.
Figure 4
Figure 4
Effect of cAMP signalling on hepatocyte Cpt1a mRNA expression. Rat primary hepatocytes were treated with PKA inhibitor, H89 (10 μM) followed by dbcAMP (250 μM) for 24 h. (A) pCREB levels in primary rat hepatocytes are decreased after H89 treatment. (B) Cpt1a mRNA expression in primary rat hepatocytes. (C) Rat primary hepatocytes were treated with PKA-selective activators N6-Phenyl-cAMP (500 μM) and Sp-5,6-DCl-cBIMPS (100 μM) for 24 h. (D) pCREB levels after 24 h treatment of rat primary hepatocytes with N6-Phenyl-cAMP (500 μM) and Sp-5,6-DCl-cBIMPS (100 μM). Data are presented as mean ± SD from 3 independent experiments. **P<0.01, ***P<0.01.
Figure 5
Figure 5
Effect of Pde4 inhibition on hepatic Pparα and Pgc1α expression after 4 wk of feeding. (A) Western blot analysis of nuclear Pparα protein levels. (B) Pparα mRNA levels were quantified by RT-qPCR. (C) Pgc1α (Ppargc1a) mRNA levels were quantified by RT-qPCR. (D) Western blot analysis of nuclear PGC1α protein levels. Data are presented as mean ± SD (n = 5–7). *P < 0.05, **P < 0.01. Hepatic nuclear lysates from 3 mice/treatment group were resolved using a gradient gel to allow the simultaneous examination of protein levels of Pparα (55kDa) and Pgc1α (90kDa) on the same membrane. Histone 3 (15kDa) served as a loading control for both Pparα and Pgc1α.
Figure 6
Figure 6
Pde4 inhibition increases hepatic Sirt1 expression. (A) After 4 wk of feeding Sirt1 mRNA levels were quantified by RT-qPCR (n=5–7). (B) Western blot analysis of nuclear Sirt1 protein levels after 4 wk of feeding. Western blot membrane probed with Pparα and Pgc1α was stripped and re-probed using Sirt1 (120kDa) antibody. Data are presented as mean ± SD *P < 0.05, ***P<0.001.
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