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PNPLA3 variant M148 causes resistance to starvation-mediated lipid droplet autophagy in human hepatocytes

Negoita, Florentina LU ; Blomdahl, Julia; Wasserstrom, Sebastian LU ; Winberg, Martin E.; Osmark, Peter LU ; Larsson, Sara LU ; Stenkula, Karin G. LU ; Ekstedt, Mattias; Kechagias, Stergios and Holm, Cecilia LU , et al. (2019) In Journal of Cellular Biochemistry 120(1). p.343-356
Abstract

The mechanism of how patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant M148 is associated with increased risk of development of hepatic steatosis is still debated. Here, we propose a novel role of PNPLA3 as a key player during autophagosome formation in the process of lipophagy. A human hepatocyte cell line, HepG2 cells, expressing recombinant I148 or 148M, was used to study lipophagy under energy deprived conditions, and lipid droplet morphology was investigated using florescence microscopy, image analysis and biochemical assays. Autophagic flux was studied using the golden-standard of LC3-II turnover in combination with the well characterized GFP-RFP-LC3 vector. To discriminate between, perturbed autophagic... (More)

The mechanism of how patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant M148 is associated with increased risk of development of hepatic steatosis is still debated. Here, we propose a novel role of PNPLA3 as a key player during autophagosome formation in the process of lipophagy. A human hepatocyte cell line, HepG2 cells, expressing recombinant I148 or 148M, was used to study lipophagy under energy deprived conditions, and lipid droplet morphology was investigated using florescence microscopy, image analysis and biochemical assays. Autophagic flux was studied using the golden-standard of LC3-II turnover in combination with the well characterized GFP-RFP-LC3 vector. To discriminate between, perturbed autophagic initiation and lysosome functionality, lysosomes were characterized by Lysotracker staining and LAMP1 protein levels as well as activity and activation of cathepsin B. For validation, human liver biopsies genotyped for I148 and 148M were analyzed for the presence of LC3-II and PNPLA3 on lipid droplets. We show that the M148-PNPLA3 variant is associated with lipid droplets that are resistant to starvation-mediated degradation. M148 expressing hepatocytes reveal decreased autophagic flux and reduced lipophagy. Both I148-PNPLA3 and M148-PNPLA3 colocalize and interact with LC3-II, but the M148-PNPLA3 variant has lower ability to bind LC3-II. Together, our data indicate that PNPLA3 might play an essential role in lipophagy in hepatocytes and furthermore that the M148-PNPLA3 variant appears to display a loss in this activity, leading to decreased lipophagy.

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published
subject
keywords
adiponutrin, lipophagy, liver, steatosis
in
Journal of Cellular Biochemistry
volume
120
issue
1
pages
343 - 356
publisher
Wiley-Blackwell
external identifiers
  • scopus:85052837166
ISSN
0730-2312
DOI
10.1002/jcb.27378
language
English
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yes
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5507fd38-29de-44c0-b02a-3019513a59dd
date added to LUP
2018-10-05 08:20:01
date last changed
2019-11-05 05:03:02
@article{5507fd38-29de-44c0-b02a-3019513a59dd,
  abstract     = {<p>The mechanism of how patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant M148 is associated with increased risk of development of hepatic steatosis is still debated. Here, we propose a novel role of PNPLA3 as a key player during autophagosome formation in the process of lipophagy. A human hepatocyte cell line, HepG2 cells, expressing recombinant I148 or 148M, was used to study lipophagy under energy deprived conditions, and lipid droplet morphology was investigated using florescence microscopy, image analysis and biochemical assays. Autophagic flux was studied using the golden-standard of LC3-II turnover in combination with the well characterized GFP-RFP-LC3 vector. To discriminate between, perturbed autophagic initiation and lysosome functionality, lysosomes were characterized by Lysotracker staining and LAMP1 protein levels as well as activity and activation of cathepsin B. For validation, human liver biopsies genotyped for I148 and 148M were analyzed for the presence of LC3-II and PNPLA3 on lipid droplets. We show that the M148-PNPLA3 variant is associated with lipid droplets that are resistant to starvation-mediated degradation. M148 expressing hepatocytes reveal decreased autophagic flux and reduced lipophagy. Both I148-PNPLA3 and M148-PNPLA3 colocalize and interact with LC3-II, but the M148-PNPLA3 variant has lower ability to bind LC3-II. Together, our data indicate that PNPLA3 might play an essential role in lipophagy in hepatocytes and furthermore that the M148-PNPLA3 variant appears to display a loss in this activity, leading to decreased lipophagy.</p>},
  author       = {Negoita, Florentina and Blomdahl, Julia and Wasserstrom, Sebastian and Winberg, Martin E. and Osmark, Peter and Larsson, Sara and Stenkula, Karin G. and Ekstedt, Mattias and Kechagias, Stergios and Holm, Cecilia and Jones, Helena A.},
  issn         = {0730-2312},
  keyword      = {adiponutrin,lipophagy,liver,steatosis},
  language     = {eng},
  number       = {1},
  pages        = {343--356},
  publisher    = {Wiley-Blackwell},
  series       = {Journal of Cellular Biochemistry},
  title        = {PNPLA3 variant M148 causes resistance to starvation-mediated lipid droplet autophagy in human hepatocytes},
  url          = {http://dx.doi.org/10.1002/jcb.27378},
  volume       = {120},
  year         = {2019},
}