Lipid droplet (LD) catabolism in hepatocytes is mediated by a combination of lipolysis and a selective autophagic mechanism called lipophagy, but the relative contributions of these seemingly distinct pathways remain unclear. We find that inhibition of lipolysis, lipophagy, or both resulted in similar overall LD content but dramatic differences in LD morphology. Inhibition of the lipolysis enzyme adipose triglyceride lipase (ATGL) resulted in large cytoplasmic LDs, whereas lysosomal inhibition caused the accumulation of numerous small LDs within the cytoplasm and degradative acidic vesicles. Combined inhibition of ATGL and LAL resulted in large LDs, suggesting that lipolysis targets these LDs upstream of lipophagy. Consistent with this, ATGL was enriched in larger-sized LDs, whereas lipophagic vesicles were restricted to small LDs as revealed by immunofluorescence, electron microscopy, and Western blot of size-separated LDs. These findings provide new evidence indicating a synergistic relationship whereby lipolysis targets larger-sized LDs to produce both size-reduced and nascently synthesized small LDs that are amenable for lipophagic internalization.

The liver performs numerous vital functions, including the detoxification of blood before access to the brain while simultaneously secreting and internalizing scores of proteins and lipids to maintain appropriate blood chemistry. Furthermore, the liver also synthesizes and secretes bile to enable the digestion of food. These diverse attributes are all performed by hepatocytes, the parenchymal cells of the liver. As predicted, these cells possess a remarkably well-developed and complex membrane trafficking machinery that is dedicated to moving specific cargos to their correct cellular locations. Importantly, while most epithelial cells secrete nascent proteins directionally toward a single lumen, the hepatocyte secretes both proteins and bile concomitantly at its basolateral and apical domains, respectively. In this Beyond the Cell review, we will detail these central features of the hepatocyte and highlight how membrane transport processes play a key role in healthy liver function and how they are affected by disease.

Fibronectins are adhesive glycoproteins that can be found in tissue matrices and circulating in various fluids of the body. The variable composition of fibronectin molecules facilitates a diversity of interactions with cell surface receptors that suggest a role for these proteins beyond the structural considerations of the extracellular matrix. These interactions implicate fibronectin in the regulation of mechanisms that also determine cell behavior and activity. The two major forms, plasma fibronectin (pFn) and cellular fibronectin (cFn), exist as balanced amounts under normal physiological conditions. However, during injury and/or disease, tissue and circulating levels of cFn become disproportionately elevated. The accumulating cFn, in addition to being a consequence of prolonged tissue damage, may in fact stimulate cellular events that promote further damage. In this review, we summarize what is known regarding such interactions between fibronectin and cells that may influence the biological response to injury. We elaborate on the effects of cFn in the liver, specifically under a condition of chronic alcohol-induced injury. Studies have revealed that chronic alcohol consumption stimulates excess production of cFn by sinusoidal endothelial cells and hepatic stellate cells while impairing its clearance by other cell types resulting in the build up of this glycoprotein throughout the liver and its consequent increased availability to influence cellular activity that could promote the development of alcoholic liver disease. We describe recent findings by our laboratory that support a plausible role for cFn in the promotion of liver injury under a condition of chronic alcohol abuse and the implications of cFn stimulation on the pathogenesis of alcoholic liver disease. These findings suggest an effect of cFn in regulating cell behavior in the alcohol-injured liver that is worth further characterizing not only to gain a more comprehensive understanding of the role this reactive glycoprotein plays in the progression of injury but also for the insight further studies could provide towards the development of novel therapies for alcoholic liver disease.

Chronic ethanol administration alters the process of receptor-mediated endocytosis in isolated rat hepatocytes. Using the asialoglycoprotein receptor (ASGP-R) as a model, we have previously shown decreased binding of asialoglycoproteins to this receptor after as early as 1 week of ethanol administration. In the present study, we further analyzed the mechanism(s) responsible for this impairment by determining the ligand and antibody binding characteristics of the ASGP-R in rats fed ethanol over a 5-week time course. The results presented here demonstrate that ethanol treatment for 4 days significantly impaired total ligand binding without affecting antibody binding. Ethanol administration for a longer period of 1-2 weeks resulted in intermediate impairments in both ligand and antibody binding. After 5 weeks of ethanol exposure, ligand and antibody binding were equally lowered. In contrast to total cellular receptor binding, surface binding of both ligand and antibody were decreased over the entire time course of ethanol administration. Our data indicate that the ASGP-R is initially inactivated during the time course of ethanol exposure and that a redistribution of surface receptors to intracellular compartments occurs. Northern blot analysis showed that there was a significant decrease in receptor mRNA content in the 5-week chronically fed animals but not in the animals fed for 1 week. In addition, after 5 weeks of ethanol feeding, biosynthetic labeling of the ASGP-R was decreased in the ethanol cells, indicating impaired synthesis of the ASGP-R. In summary, an early inactivation of the ASGP-R occurs during ethanol exposure followed by an actual decrease in protein and mRNA content for the receptor.

Lipid droplets (LDs), the organelles central to alcoholic steatosis, are broken down by lipophagy, a specialized form of autophagy. Here, we hypothesize that ethanol administration retards lipophagy by down‐regulating dynamin 2 (Dyn2), a protein that facilitates lysosome re‐formation, contributing to hepatocellular steatosis. Primary hepatocytes were isolated from male Wistar rats fed Lieber–DeCarli control or ethanol (EtOH) liquid diets for 6‐8 weeks. Hepatocytes were incubated in complete medium (fed) or nutrient‐free medium (fasting) with or without the Dyn2 inhibitor dynasore or the Src inhibitor SU6656. Phosphorylated (active) forms of Src and Dyn2 and markers of autophagy were quantified using western blot analysis. Colocalization of LDs with autophagic machinery was determined using confocal microscopy. In hepatocytes from pair‐fed rats, LD breakdown was accelerated during fasting, as judged by smaller LDs and lower triglyceride (TG) content when compared with hepatocytes in complete media. Fasting‐induced TG loss in control hepatocytes was significantly blocked by either SU6656 or Dynasore. Compared with controls, hepatocytes from EtOH‐fed rats had 66% and 40% lower content of phosphorylated Src (pSrc) and phosphorylated Dyn2 (pDyn2), respectively, coupled with a lower rate of fasting‐induced TG loss. This slower rate of fasting‐induced TG loss was blocked in cells coincubated with Dynasore. Microscopic examination of EtOH‐fed rat hepatocytes revealed increased colocalization of the autophagosome marker LC3 on LDs with a concomitant decrease in lysosome marker LAMP1. Whole livers and LD fractions of EtOH‐fed rats exhibited simultaneous increase in LC3II and p62 over that of controls, indicating a block in lipophagy. Conclusion: Chronic ethanol administration slowed the rate of hepatocyte lipophagy, owing in part to lower levels of phosphorylated Src kinase available to activate its substrate, Dyn2, thereby causing depletion of lysosomes for LD breakdown. (Hepatology Communications 2017;1:501–512)