The Role of Golgi Morphology in Post-Alcohol Recovery of Hepatocytes: Observations in Cellular and Animal Models

Background: In hepatocytes and alcohol-metabolizing cultured cells, Golgi undergoes ethanol (EtOH)-induced disorganization. Periniclear and organized Golgi is important in liver homeostasis, but how the Golgi remains intact is unknown. Work from our laboratories showed that EtOH-altered cellular function could be reversed after alcohol removal; we wanted to determine whether this recovery would apply to Golgi. Methods: We used alcohol-metabolizing HepG2 (VA-13) cells (cultured with or without EtOH for 72 h) and rat hepatocytes (control and EtOH-fed (Lieber-DeCarli diet). For recovery, EtOH was removed and replenished with control medium (48 hours for VA-13 cells) or control diet (10 days for rats). Results: EtOH-induced Golgi disassembly was associated with de-dimerization of the largest Golgi matrix protein giantin, along with impaired transport of selected hepatic proteins. After recovery from EtOH, Golgi regained their compact structure, and alterations in giantin and protein transport were restored. In VA-13 cells, when we knocked down giantin, Rab6a GTPase or non-muscle Myosin IIB, minimal changes were observed in control conditions, but post-EtOH recovery was impaired. Conclusions: These data provide a link between Golgi organization and plasma membrane protein expression and identify several proteins whose expression is important to maintain Golgi structure during the recovery phase after EtOH administration.


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The Golgi apparatus is the central sorting and transportation hub involved in the 36 posttranslational modification and sorting of cargo molecules, and delivering them to appropriate 37 cellular locations or to the exocytic and endocytic pathways [1]. In mammalian cells, Golgi is a

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One protein, giantin, appears to be especially important for Golgi's compact structure. Giantin 54 is the highest molecular weight (376 kDa) Golgi matrix protein. It consists of a short C-terminal 55 domain located in the Golgi lumen [15], where a disulfide bond connects two monomers to form an 56 active homodimer, which is followed by a one-pass trans-membrane domain and then a large (≥350 57 kDa) N-terminal region projecting into the cytoplasm. This unique structure suggests that giantin is 58 the core Golgi protein and therefore could be essential for cross-bridging cisternae during Golgi 59 biogenesis [16]. Giantin dimerization appears to be catalyzed by the chaperone, protein disulfide

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Alterations in Golgi morphology appear to be accompanied by the impaired trafficking and 66 secretion of several essential hepatic glycoproteins. For example, transferrin was found to be 67 retained in the endoplasmic reticulum (ER) and Golgi of the hepatocytes after alcohol administration 68 in both human liver alcoholic cirrhotics and in livers of rats fed with EtOH, causing impairment of 69 its' iron transport function [22]. Similarly, in cellular and rat models of chronic alcohol exposure, we 70 observed the deposition of ASGP-R in cis-medial-Golgi [19]. In addition to this, the activities of 71 different glycosyltransferases are reduced in both ER and Golgi after EtOH administration [23,24].

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Some of these Golgi resident enzymes exhibit altered re-localization due to EtOH-induced 73 impairment of COPI vesicles, which normally deliver these enzymes to appropriate sites within the

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The intensity of obtained bands was analyzed by ImageJ software, and samples with identical

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In this current study, we are examining the link between giantin and EtOH-induced Golgi 186 disorganization. First, we wanted to establish a relevance for these effects in the human condition, so 192 briefly, the lysis of cells was performed under high (5%) and low 1% β-mercaptoethanol (β-ME

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We have shown previously that chronic EtOH administration impairs liver receptor-mediated

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Recently we found that EtOH-induced Golgi disorganization is governed by motor protein

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With this background, we next investigated the distribution of ASGP-R in post-EtOH VA-13 326 cells in absence of either giantin, NMIIB or Rab6a. As anticipated, in control VA-13 cells, the ASGP-R 327 signal was detected at the cell's periphery (Figure 5A, Ctrl, white arrowheads) and Golgi. Treatment

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which, as we shown before, are failed to recover Golgi (Figure 3). Similarly, we found no significant

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As a way to evaluate the trafficking of liver-specific proteins to the cell surface, in addition to 341 ASGP-R, we also measured by W-B the plasma membrane (PM) content of PIGR and transferrin. As 342 shown in Figure 5D, the intensity of bands of all three proteins was reduced in EtOH-treated cells,

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Here, for the first time, we observed the differential impact of depletion of NMIIA and NMIIB-

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In sum, our results confirm the critical role for giantin, Rab6a, and NMIIB in the post-alcohol 432 recovery of Golgi. We believe that the restoration of Golgi is a much more complicated event and 433 requires the active involvement of other players. However, these three proteins appear to be the key 434 regulators of fusion of the nascent Golgi membranes, which is the critical step in Golgi biogenesis.

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Our data support observations of chronic alcohol consumption that indicate the ability of 436 hepatocytes to prompt recovery during alcohol abstinence. However, some parameters require more