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. 2018 May:115:273-283.
doi: 10.1016/j.fct.2018.03.014. Epub 2018 Mar 11.

Lipin deactivation after acetaminophen overdose causes phosphatidic acid accumulation in liver and plasma in mice and humans and enhances liver regeneration

Andrew J Lutkewitte  1 , George G Schweitzer  2 , Stefanie Kennon-McGill  3 , Melissa M Clemens  4 , Laura P James  5 , Hartmut Jaeschke  6 , Brian N Finck  7 , Mitchell R McGill  8
Affiliations

Lipin deactivation after acetaminophen overdose causes phosphatidic acid accumulation in liver and plasma in mice and humans and enhances liver regeneration

Andrew J Lutkewitte et al. Food Chem Toxicol. 2018 May.
No abstract available

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Figures

Figure 1
Figure 1. Time course of liver injury and regeneration after acetaminophen overdose
Male C57Bl/6J mice were treated with 300 mg/kg APAP or PBS vehicle and blood and liver tissue were collected at the indicated time points. (A) Plasma ALT activity. (B) H&E-stained liver sections. (C) Proliferating cell nuclear antigen (PCNA) immunoblot. Data are expressed as mean±SEM for n = 3–4. *p<0.05 vs. 0 h and Veh.
Figure 2
Figure 2. Time course of phosphatidic acid after acetaminophen overdose
Male C57Bl/6J mice were treated with 300 mg/kg APAP or PBS vehicle and blood and liver tissue were collected at the indicated time points. (A) Total liver phosphatidic acid (PA) content. (B) Individual PA species in the liver. (C) Total plasma PA content. (D) Individual PA species in the plasma. Data are expressed as mean±SEM for n = 3. *p<0.05 vs. 0 h and Veh.
Figure 3
Figure 3. Phosphatidic acid values in acetaminophen overdose patients
Phosphatidic acid species were measured in plasma or serum from acetaminophen overdose patients and compared between patients that did not develop liver injury (ALT less than 2x the upper limit of normal [ULN]) and those that did (ALT≥2xULN). (A) 16:0/18:1 PA. (B) 18:0/18:2 PA. (C) 18:1/18:2 PA. (D) 18:0/20:4 PA. (E) 18:1/20:4 PA. (F) Total PA. Data are expressed as fold over ALT<2xULN patients. Whiskers show the 5th to 95th percentile, box plots show the 25th to 75th percentile, and lines show the median and mean.
Figure 4
Figure 4. Time course of lipin 1 and 2 expression and activity after APAP overdose
Male C57Bl/6J mice were treated with 300 mg/kg APAP or PBS vehicle and liver tissue was collected at the indicated time points. (A) Lipin 1 mRNA in the liver. (B) Lipin 2 mRNA in the liver. (C) Liver lipin 1 immunoblot. (D) Liver lipin 2 immunoblot. (E) Densitometry for lipin 1 and lipin 2 immunoblots. (F) Lipin-specific Mg2+-dependent PAP activity in the liver. Data are expressed as mean±SEM for n=3–6. *p<0.05 vs. PBS or 0 h. #p = 0.1 vs. PBS.
Figure 5
Figure 5. Expression of genes for phosphatidic acid synthesis enzymes
Male C57Bl/6J mice were treated with 300 mg/kg APAP or PBS vehicle control and liver tissue was collected at the indicated time points. (A) mRNA for the major isoforms of glycerol-3-phosphate acyltransferase (Gpat) in the liver. (B) mRNA for the major isoforms of acylglycerol phosphate acyltransferase (Agpat) in the liver. (C) mRNA for the major isoforms of phospholipase D (Pld) in the liver. (D) mRNA for the major isoforms of diacylglycerol kinase (Dagk) in the liver. Data are expressed as mean±SEM for n = 3–6. *p<0.05 vs. vehicle control (CTRL).
Figure 6
Figure 6. Effect of lipin enzymes on DNA synthesis in vitro
HepG2 cells were untreated or transduced to express GFP, lipin 1, or lipin 2, and DNA synthesis was measured by 3H-thymidine incorporation. (A) Immunoblots for lipin 1 and 2 after adenovirus transduction. (B) Thymidine incorporation. Values are compared to the GFP transduction control. Data are expressed as mean±SEM for n = 4 independent experiments, each performed in triplicate. *p<0.05 vs. GFP control.
Figure 7
Figure 7. Effect of phosphatidic acid synthesis inhibition on liver injury and regeneration after APAP overdose
Male C57Bl/6J mice were treated with 300 mg/kg APAP or PBS vehicle, followed by 20 mg/kg FSG67 at either 2, 24, and 48 h post-APAP (A,B) or at 24 and 48 h post-APAP (C,D). Blood and liver tissue were collected at the indicated time points. (A) Plasma ALT at 6, 24 and 52 h post-APAP. (B) Immunoblot for liver PCNA and associated densitometry at 52 h post-APAP. (C) Plasma ALT at 52 h post-APAP. (D) Immunoblot for liver PCNA and associated densitometry at 52 h post-APAP. (E) Liver PA content at 52 h post-APAP. Data are expressed as mean±SEM for n = 3–5. *p<0.05 vs. PBS treatment. #p<0.05 vs. Veh.
Figure 8
Figure 8. Phosphorylation of mTOR after acetaminophen treatment
Male C57Bl/6J mice were treated with 300 mg/kg APAP or saline vehicle and liver tissue was collected at the indicated time points. (A) Immunoblots for p-mTOR and total mTOR from pooled samples. (B) Densitometry for phosphorylated mTOR (p-mTOR) and total mTOR from non-pooled samples. Data are expressed as mean±SEM for n = 3. *p<0.05 vs. Veh.
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