Abstract

In liver steatosis (i.e. fatty liver), hepatocytes accumulate many large neutral lipid storage organelles known as lipid droplets (LDs). LDs are important in the maintenance of energy homeostasis, but the signaling mechanisms that stimulate LD metabolism in hepatocytes are poorly defined. In adipocytes, catecholamines target the β-adrenergic (β-AR)/cAMP pathway to activate cytosolic lipases and induce their recruitment to the LD surface. Therefore, the goal of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP-mediated lipolysis in response to β-AR stimulation. Using primary rat hepatocytes and human hepatoma cells, we found that treatment with the β-AR agent isoproterenol caused substantial LD loss via activation of cytosolic lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). β-Adrenergic stimulation rapidly activated PKA, which led to the phosphorylation of ATGL and HSL and their recruitment to the LD surface. To test whether this β-AR-dependent lipolysis pathway was altered in a model of alcoholic fatty liver, primary hepatocytes from rats fed a 6-week EtOH-containing Lieber-DeCarli diet were treated with cAMP agonists. Compared with controls, EtOH-exposed hepatocytes showed a drastic inhibition in β-AR/cAMP-induced LD breakdown and the phosphorylation of PKA substrates, including HSL. This observation was supported in VA-13 cells, an EtOH-metabolizing human hepatoma cell line, which displayed marked defects in both PKA activation and isoproterenol-induced ATGL translocation to the LD periphery. In summary, these findings suggest that β-AR stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic EtOH insult leading to fatty liver.

Highlights

  • In liver steatosis, hepatocytes accumulate many large neutral lipid storage organelles known as lipid droplets (LDs)

  • Using primary rat hepatocytes and human hepatoma cells, we found that treatment with the ␤-AR agent isoproterenol caused substantial LD loss via activation of cytosolic lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). ␤-Adrenergic stimulation rapidly activated PKA, which led to the phosphorylation of ATGL and HSL and their recruitment to the LD surface

  • The central findings of this study support the premise that, like the adipocyte, the hepatocyte is responsive to ␤-AR stimulation that triggers a cAMP-dependent cascade involving cytoplasmic lipases that are recruited to the LD surface to drive lipid catabolism

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Summary

Ethanol blocks hepatocyte lipolysis

We tested the hypothesis that hepatocytes, like adipocytes, utilize a lipolytic machinery that is activated by ␤-adrenergic stimuli to break down and subsequently catabolize LDs. We report that ␤-adrenergic stimulation of hepatocytes using the agonist isoproterenol causes LD loss via the cAMP/PKA pathway. We report that ␤-adrenergic stimulation of hepatocytes using the agonist isoproterenol causes LD loss via the cAMP/PKA pathway This process requires the cytosolic lipases ATGL and HSL, which are phosphorylated and trafficked to LDs within 60 min of stimulation. We find that the activation of this pathway is significantly perturbed in hepatocytes subjected to either chronic or acute EtOH exposure, leading to steatotic cells

Results
Discussion
Cell culture and reagents
Triglyceride efflux measurements
Constructs and transfection
Fluorescence microscopy
Findings
Lipid droplet isolation
Full Text
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