Methanobactin reverses acute liver failure in a rat model of Wilson disease
Josef Lichtmannegger, … , Karl Heinz Weiss, Hans Zischka
Josef Lichtmannegger, … , Karl Heinz Weiss, Hans Zischka
Published June 20, 2016
Citation Information: J Clin Invest. 2016;126(7):2721-2735. https://doi.org/10.1172/JCI85226.
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Research Article Gastroenterology Hepatology

Methanobactin reverses acute liver failure in a rat model of Wilson disease

Abstract

In Wilson disease (WD), functional loss of ATPase copper-transporting β (ATP7B) impairs biliary copper excretion, leading to excessive copper accumulation in the liver and fulminant hepatitis. Current US Food and Drug Administration– and European Medicines Agency–approved pharmacological treatments usually fail to restore copper homeostasis in patients with WD who have progressed to acute liver failure, leaving liver transplantation as the only viable treatment option. Here, we investigated the therapeutic utility of methanobactin (MB), a peptide produced by Methylosinus trichosporium OB3b, which has an exceptionally high affinity for copper. We demonstrated that ATP7B-deficient rats recapitulate WD-associated phenotypes, including hepatic copper accumulation, liver damage, and mitochondrial impairment. Short-term treatment of these rats with MB efficiently reversed mitochondrial impairment and liver damage in the acute stages of liver copper accumulation compared with that seen in untreated ATP7B-deficient rats. This beneficial effect was associated with depletion of copper from hepatocyte mitochondria. Moreover, MB treatment prevented hepatocyte death, subsequent liver failure, and death in the rodent model. These results suggest that MB has potential as a therapeutic agent for the treatment of acute WD.

Authors

Josef Lichtmannegger, Christin Leitzinger, Ralf Wimmer, Sabine Schmitt, Sabine Schulz, Yaschar Kabiri, Carola Eberhagen, Tamara Rieder, Dirk Janik, Frauke Neff, Beate K. Straub, Peter Schirmacher, Alan A. DiSpirito, Nathan Bandow, Bipin S. Baral, Andrew Flatley, Elisabeth Kremmer, Gerald Denk, Florian P. Reiter, Simon Hohenester, Friedericke Eckardt-Schupp, Norbert A. Dencher, Jerzy Adamski, Vanessa Sauer, Christoph Niemietz, Hartmut H.J. Schmidt, Uta Merle, Daniel Nils Gotthardt, Guido Kroemer, Karl Heinz Weiss, Hans Zischka

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Figure 3

MB depletes copper from liver mitochondria, hepatocytes, and whole liver.

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MB depletes copper from liver mitochondria, hepatocytes, and whole liver...
(A) MB, but not D-PA or TETA, extracted copper from Atp7b–/– mitochondria (2 mM each, 30-minute incubation, N = 3). (B) MB was less toxic to the mitochondrial respiratory complex IV than TTM (MB: N = 3, n = 9; TTM: N = 1, n = 3). (C) Copper-preloaded HepG2 (N = 3) and WD patient–derived HLCs (1 of 2 preparations) were de-coppered by MB. (–), untreated control; (+), 24-hour treatment with 300–500 μM MB. (D) Dose-dependent MB uptake into HepG2 cells at 2 and 24 hours (N = 3). (E) Toxicity of MB versus TTM in HepG2 (neutral red assay: N = 3, n = 9). CCCP, positive control. (F) MB-treated (500 μM) HepG2 cells showed only intermediate phases of ΔΨ loss (6 hours, 250 μM CCCP). Nuclei, blue; mitochondria with ΔΨ, orange-red; mitochondria without ΔΨ, green. Arrows indicate cells with low ΔΨ (N = 2). Scale bars: 50 μm. (G) Cumulative copper excretion into bile following a 2-hour Atp7b–/– liver perfusion. Only MB (0.7 μM) forced high copper amounts into bile compared with TTM (0.8 μM), D-PA (2.2 μM), and TETA (1.8 μM). N = 3. Note the different scales for MB (right, blue axis) and buffer control, D-PA, TETA, and TTM (left, black axis). (H) All chelators except TTM transported copper to the perfusate (conditions as in G). N = 2. (I) Only MB markedly reduced liver copper levels during Atp7b–/– liver perfusion, but not D-PA, TETA, or TTM (conditions as in G). N = 3. One-way ANOVA with Tukey’s multiple comparisons test (A, B, D, E, and I); unpaired t test with Welch’s correction (C). *P < 0.05 and ****P < 0.0001 versus control (A–C and E); ####P < 0.0001 versus respective MB concentration (B and E). **P < 0.01 versus 0.05 mM MB; #P < 0.05 versus 0.1 mM MB; and †P < 0.05 versus 0.3 mM MB (D). Co, buffer-treated control.