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A High-Calorie Diet Aggravates Mitochondrial Dysfunction and Triggers Severe Liver Damage in Wilson Disease Rats

Open AccessPublished:December 23, 2018DOI:https://doi.org/10.1016/j.jcmgh.2018.12.005

      Background & Aims

      In Wilson disease, ATP7B mutations impair copper excretion into bile. Hepatic copper accumulation may induce mild to moderate chronic liver damage or even acute liver failure. Etiologic factors for this heterogeneous phenotype remain enigmatic. Liver steatosis is a frequent finding in Wilson disease patients, suggesting that impaired copper homeostasis is linked with liver steatosis. Hepatic mitochondrial function is affected negatively both by copper overload and steatosis. Therefore, we addressed the question of whether a steatosis-promoting high-calorie diet aggravates liver damage in Wilson disease via amplified mitochondrial damage.

      Methods

      Control Atp7b+/- and Wilson disease Atp7b-/- rats were fed either a high-calorie diet (HCD) or a normal diet. Copper chelation using the high-affinity peptide methanobactin was used in HCD-fed Atp7b-/- rats to test for therapeutic reversal of mitochondrial copper damage.

      Results

      In comparison with a normal diet, HCD feeding of Atp7b-/- rats resulted in a markedly earlier onset of clinically apparent hepatic injury. Strongly increased mitochondrial copper accumulation was observed in HCD-fed Atp7b-/- rats, correlating with severe liver injury. Mitochondria presented with massive structural damage, increased H2O2 emergence, and dysfunctional adenosine triphosphate production. Hepatocellular injury presumably was augmented as a result of oxidative stress. Reduction of mitochondrial copper by methanobactin significantly reduced mitochondrial impairment and ameliorated liver damage.

      Conclusions

      A high-calorie diet severely aggravates hepatic mitochondrial and hepatocellular damage in Wilson disease rats, causing an earlier onset of the disease and enhanced disease progression.

      Graphical abstract

      Keywords

      Abbreviations used in this paper:

      Acetyl-CoA (acetyl coenzyme A), ADP (adenosine diphosphate), AST (aspartate aminotransferase), ATP (adenosine triphosphate), BSA (bovine serum albumin), Cp (ceruloplasmin), CS (citrate synthase), EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid), F1FO (adenosine triphosphate synthase), HAI (Histologic Activity Index), HCD (high-calorie diet), ICP (Inductively Coupled Plasma), MB (methanobactin), MS (mass spectrometry), NADH (reduced nicotinamide adenine dinucleotide), NAFLD (nonalcoholic fatty liver disease), NAS (nonalcoholic fatty liver disease activity score), ND (normal diet), ROS (reactive oxygen species), TRIS (tris-(hydroxymethyl)aminomethane), WD (Wilson disease)
      See editorial on page 684.
      A high-calorie diet severely aggravates hepatic mitochondrial and hepatocellular damage in Wilson disease rats. A toxic triad of adenosine triphosphate depletion, increased reactive oxygen species, and increased bile salts lead to an earlier onset of the disease and to enhanced disease progression.
      Wilson disease (WD) is an autosomal-recessively inherited disorder of copper metabolism caused by ATP7B gene mutations, resulting in impaired biliary copper excretion. Subsequent hepatic copper accumulation induces a heterogeneous phenotype that lacks a clear genotype correlation.
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      Results

      An HCD Severely Aggravates and Strongly Accelerates Liver Damage in WD Rats

      An HCD significantly increased the visceral fat mass (Figure 1A) and liver triglyceride levels (Figure 1B) within a few weeks of feeding in Atp7b+/- control rats, compared with their ND-fed counterparts. This coincided with the presence of abundant macrosteatosis in liver histology (Figure 1D) and an increased NAFLD Activity Score (NAS) of 3–5 (Figure 1C, Table 1). Despite steatosis, however, constant body and liver weights were encountered in the HCD-fed control Atp7b+/- rats (Figure 1E–G, Table 2). HCD- vs ND-fed Atp7b-/- rats (ie, WD rats) also had tendentiously increased visceral fat mass, significantly increased liver triglyceride levels, liver steatosis, and equal body but increased liver weight (Figures 1A–G, Table 2).
      Figure thumbnail gr1
      Figure 1An HCD induces liver steatosis in Atp7b+/- and Atp7b-/- rats. (A–C) An HCD increases (A) visceral fat mass (N = 6–12), (B) liver triglyceride content (N = 6–7), and (C) NAFLD activity (NAS, N = 6–8) in Atp7b+/- and Atp7b-/- rats. (D) Liver histology (H&E stain; scale bar: 100 μm) from ND-fed vs HCD-fed Atp7b+/- and Atp7b-/- rats presents macrosteatosis (open asterisk), ballooned hepatocytes (open arrow), inflammatory infiltrations (black arrow), and some signs of fibrosis (black asterisk), apoptosis (open arrowhead), or necrosis (black arrowhead). (E and F) Comparable body weights of either male (E, N = 2–7) or female (F, N = 3–5) ND- or HCD-fed Atp7b+/- and Atp7b-/- rats. (G) Relative liver weight (% of body weight [bw]) of HCD-fed Atp7b-/- rats increased compared with ND-fed Atp7b-/- and Atp7b+/- rats (N = 6–11). One-way analysis of variance with (A, B, E–G) the Tukey multiple comparisons test, or (C) the nonparametric Kruskal–Wallis test. N, number of analyzed animals. *Significant to Atp7b+/- ND. #Significant to Atp7b+/- HCD. Significant to Atp7b-/- ND. ∗,#,†P < .05; ∗∗,##,††P < .01; ∗∗∗,###,†††P < .001; ∗∗∗∗,####,††††P < .0001. TG, triglyceride.
      Table 1Histologic Assessment of Steatosis, Lobular Inflammation, and Hepatocyte Ballooning (NAS) in Livers of ND- and HCD-Fed Atp7b+/- and Atp7b-/- Rats
      NASGenotype+/-+/-+/-+/-+/-+/-+/-+/-+/-+/-+/-+/-+/-+/--/--/--/--/--/--/--/--/--/--/--/--/-
      ChowNDNDNDNDNDNDNDNDHCDHCDHCDHCDHCDHCDNDNDNDNDNDNDHCDHCDHCDHCDHCDHCD
      Animal IDRat 3Rat 15Rat 1Rat 2Rat 14Rat 16Rat 17Rat 20Rat 5Rat 4Rat 6Rat 24Rat 23Rat 27Rat 31Rat 8Rat 7Rat 9Rat 29Rat 30Rat 11Rat 10Rat 12Rat 35Rat 36Rat 37
      Steatosis grade<5%000000000000000
      5%–33%111111111
      >33% to 66%22222
      >66%3
      Lobular inflammationNo foci00
      <2 foci per 200× field11111111111111
      2–4 foci per 200× field222222222
      >4 foci per 200× field33333
      BallooningNone00000
      Few balloon cells1111111111111111
      Many cells/ prominent ballooning22222222
      Diagnostic classification (NAS)
       Not steatohepatitis<3221121222221
       Possible/ borderline3–4343443
       Definite steatohepatitis≥555665666
      Table 2An HCD Accelerates Disease Progression in Atp7b-/- Rats
      Animal IDGenotypeChowAge, daysSexBody weight, gAST, U/LBilirubin, mg/dL
      Rat 1+/-ND67M206181<0.5
      Rat 2+/-ND73F132146<0.5
      Rat 3+/-ND75F155137<0.5
      Rat 4+/-HCD67F148108<0.5
      Rat 5+/-HCD73F140125<0.5
      Rat 6+/-HCD75F167127<0.5
      Rat 7-/-ND67M214152<0.5
      Rat 8-/-ND73F148181<0.5
      Rat 9-/-ND75F145130<0.5
      Rat 10-/-HCD67F144122<0.5
      Rat 11-/-HCD73F128318
      Animals are considered as having clinically apparent WD if AST level is greater than 200 U/L and/or bilirubin level is greater than 0.5 mg/dL.
      <0.5
      Rat 12-/-HCD75F144298
      Animals are considered as having clinically apparent WD if AST level is greater than 200 U/L and/or bilirubin level is greater than 0.5 mg/dL.
      <0.5
      Rat 13+/-ND79M25993.5<0.5
      Rat 14+/-ND79F13793.1<0.5
      Rat 15+/-ND80F13995.8<0.5
      Rat 16+/-ND80F16573.6<0.5
      Rat 17+/-ND81M27278.5<0.5
      Rat 18+/-ND81M21996.5<0.5
      Rat 19+/-ND81M248222<0.5
      Rat 20+/-ND82M263156<0.5
      Rat 21+/-ND82M226123<0.5
      Rat 22+/-HCD79M28590.4<0.5
      Rat 23+/-HCD80F16865.2<0.5
      Rat 24+/-HCD81M28169.9<0.5
      Rat 25+/-HCD81M27277.6<0.5
      Rat 26+/-HCD81M244123<0.5
      Rat 27+/-HCD82M275139<0.5
      Rat 28+/-HCD82M243125<0.5
      Rat 29-/-ND80F16282.2<0.5
      Rat 30-/-ND81M27180.5<0.5
      Rat 31-/-ND82M263115<0.5
      Rat 32-/-ND82M246141<0.5
      Rat 33-/-ND82M26490.2<0.5
      Rat 34-/-ND82M262117<0.5
      Rat 35-/-HCD80F167460
      Animals are considered as having clinically apparent WD if AST level is greater than 200 U/L and/or bilirubin level is greater than 0.5 mg/dL.
      4.4
      Animals are considered as having clinically apparent WD if AST level is greater than 200 U/L and/or bilirubin level is greater than 0.5 mg/dL.
      Rat 36-/-HCD81M250486
      Animals are considered as having clinically apparent WD if AST level is greater than 200 U/L and/or bilirubin level is greater than 0.5 mg/dL.
      <0.5
      Rat 37-/-HCD82M282610
      Animals are considered as having clinically apparent WD if AST level is greater than 200 U/L and/or bilirubin level is greater than 0.5 mg/dL.
      1.8
      Animals are considered as having clinically apparent WD if AST level is greater than 200 U/L and/or bilirubin level is greater than 0.5 mg/dL.
      F, female; M, male.
      a Animals are considered as having clinically apparent WD if AST level is greater than 200 U/L and/or bilirubin level is greater than 0.5 mg/dL.
      Importantly, clinically apparent liver injury (ie, serum aspartate aminotransferase [AST] level, >200 U/L) was present only in age-matched HCD-fed Atp7b-/- rats, but not in ND-fed Atp7b-/- or in HCD-fed Atp7b+/- rats (Figure 2A, Table 2). Gross liver damage was further detectable by histologic analyses only in HCD-fed Atp7b-/-, but not in the other groups (Figures 1D, 2C and D, Table 3). Although in the other groups, single necrosis and apoptosis, low signs of inflammation or fibrosis, occasionally were observed, these features became severely apparent and increased significantly (vs ND-fed controls) only in HCD-fed Atp7b-/- rats (Figure 2C and D, Tables 1 and 3). Summation of these parameters resulted in low NAS and Histologic Activity Index (HAI) scores for the other rats, but showed strongly and significantly (vs both ND-fed controls and ND-fed Atp7b-/- rats) increased NAS and HAI scores for HCD-fed Atp7b-/- rats (Figures 1C and 2C). Because steatosis, hepatocyte ballooning, and inflammation are the hallmarks of nonalcoholic steatohepatitis,
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      such nonalcoholic steatohepatitis was present in all 6 HCD-fed Atp7b-/- rats, but in only 2 of 6 HCD-fed Atp7b+/- rats (Table 1).
      Figure thumbnail gr2
      Figure 2An HCD causes severe liver damage in Atp7b-/- rats. (A) Serum AST is increased specifically in HCD-fed Atp7b-/- rats (N = 6–12). (B) After disease progression, animals are considered as having clinically apparent WD if AST level is greater than 200 U/L (blue dashed line). HCD causes an earlier disease onset in Atp7b-/- rats compared with ND-fed animals (HCD, N = 6; ND, N = 18). (C) Total hepatic injury score (HAI, N = 6–8) as well as the (D) HAI score parameters of necrosis, apoptosis, liver cell injury, and fibrosis (N = 6–8) increase significantly only in HCD-fed Atp7b-/- rats. One-way analysis of variance with the (A) Tukey multiple comparisons test, (B) nonlinear curve fitting, or (C and D) nonparametric Kruskal–Wallis test. N, number of analyzed animals. *Significant to Atp7b+/- ND. #Significant to Atp7b+/- HCD. Significant to Atp7b-/- ND. ∗,#,†P < .05; ∗∗,##,††P < .01; ∗∗∗,###,†††P < .001; ∗∗∗∗,####,††††P < .0001.
      Table 3Histologic Assessment of Hepatic Injury (HAI Score) in Livers of ND- and HCD-Fed Atp7b+/- and Atp7b-/- Rats
      Hepatic injury scoreGenotype+/-+/-+/-+/-+/-+/-+/-+/-+/-+/-+/-+/-+/-+/--/--/--/--/--/--/--/--/--/--/--/--/-
      ChowNDNDNDNDNDNDNDNDHCDHCDHCDHCDHCDHCDNDNDNDNDNDNDHCDHCDHCDHCDHCDHCD
      Animal IDRat 3Rat 15Rat 1Rat 2Rat 14Rat 16Rat 17Rat 20Rat 5Rat 4Rat 6Rat 24Rat 23Rat 27Rat 31Rat 8Rat 7Rat 9Rat 29Rat 30Rat 11Rat 10Rat 12Rat 35Rat 36Rat 37
      SteatosisLocationZone 300000000000000000
      Zone 1111111
      Azonal222222
      Panacinar3
      Microvesicular steatosisAbsent0000000000000000000
      Present111111111
      InflammationMicrogranulomasAbsent0
      Present111111111111111111111111111
      Large lipogranulomasAbsent000000000000000000000000000
      Present1
      Portal inflammationNone to minimal0000000000000000000
      More than minimal111111111
      Liver cell injuryAcidophil bodiesNone to rare0000000000000000
      Many111111111111
      Pigmented macrophagesNone to rare0000000000000000000000000
      Many111
      MegamitochondriaNone to rare000000000000000000000000000
      Many1
      Necrosis (periportal/ periseptal interface hepatitis)Piecemeal necrosisAbsent0000000000000000
      MildFocal, few portal areas1111111111
      Mild–moderateFocal, most portal areas222
      ModerateContinuous, approximately <50% tracts/septa3
      SevereContinuous, approximately >50% tracts/septa4
      Confluent necrosisAbsent000000000000000000000000000
      Focal confluent necrosis1
      Zone 3 necrosis-some areas2
      Zone 3 necrosis-most areas3
      Zone 3 necrosis+ occasional bridging (p-c)4
      Zone 3 necrosis+ multiple bridging (portal-central)5
      Panacinar/multiacinar necrosis6
      Lytic necrosis/apoptosis/focal inflammationNo foci0000
      ≤1 foci per 100× field1111111111111
      2–4 foci per 100× field222222222
      5–10 foci per 100× field3333
      >10 foci per 200× field4
      FibrosisStageNone000000000000
      Perisunusoidal or periportal11111111111
      Mild, zone 31A1
      Moderate, zone 31B
      Portal/periportal1C1
      Perisinusoidal and periportal2222
      Bridging fibrosis3
      Cirrhosis4
      Other findingsMallory's hyalineNone to rare0000000000000000000
      Many111111111
      Total42315222278978265332101111111311
      With respect to WD, we further tested for a difference in disease progression in HCD- vs ND-fed Atp7b-/- rats. As can be seen in Figure 2B, HCD feeding led to a earlier disease onset by approximately 20 days in HCD- vs ND-fed Atp7b-/- rats. This is a remarkable disease acceleration, because earlier we had determined a median survival of 106 days in ND-fed Atp7b-/- rats.
      • Zischka H.
      • Lichtmannegger J.
      • Schmitt S.
      • Jagemann N.
      • Schulz S.
      • Wartini D.
      • Jennen L.
      • Rust C.
      • Larochette N.
      • Galluzzi L.
      • Chajes V.
      • Bandow N.
      • Gilles V.S.
      • DiSpirito A.A.
      • Esposito I.
      • Goettlicher M.
      • Summer K.H.
      • Kroemer G.
      Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease.
      In addition, a noticeable steeper slope of the trend curve for liver damage was observed in HCD- vs ND-fed Atp7b-/- rats (Figure 2B). Thus, HCD feeding causes severely aggravated liver damage that appears much earlier and progresses faster in HCD- vs ND-fed Atp7b-/- rats.

      An HCD Increases Serum and Mitochondrial Copper Load in Atp7b-/- Rats

      In WD livers, copper-loading of ceruloplasmin (Cp) is impaired because of ATP7B mutations.
      • Ferenci P.C.A.
      • Stremmel W.
      • Houwen R.
      • Rosenberg W.
      • Schilsky M.
      • Jansen P.
      • Moradpour D.
      EASL clinical practice guidelines: Wilson's disease.
      Consequently, Cp oxidase activity and copper concentrations in peripheral blood typically are reduced.
      • Tao T.Y.
      • Gitlin J.D.
      Hepatic copper metabolism: insights from genetic disease.
      Accordingly, ND-fed Atp7b-/- rats presented almost no Cp oxidase activity and significantly decreased plasma copper levels compared with Atp7b+/- controls (Figure 3A and B). Of note, HCD feeding of Atp7b+/- rats resulted in significantly lower serum copper and Cp oxidase activity compared with ND-fed controls (Figure 3A and B). This is in line with observations in NAFLD patients, in whom reduced Cp oxidase activity was found to be associated strongly with hepatocyte ballooning or liver steatosis and therefore was suggested as a potential marker of liver dysfunction in fatty liver injury.
      • Nobili V.
      • Siotto M.
      • Bedogni G.
      • Rava L.
      • Pietrobattista A.
      • Panera N.
      • Alisi A.
      • Squitti R.
      Levels of serum ceruloplasmin associate with pediatric nonalcoholic fatty liver disease.
      • Aigner E.
      • Strasser M.
      • Haufe H.
      • Sonnweber T.
      • Hohla F.
      • Stadlmayr A.
      • Solioz M.
      • Tilg H.
      • Patsch W.
      • Weiss G.
      • Stickel F.
      • Datz C.
      A role for low hepatic copper concentrations in nonalcoholic fatty liver disease.
      In contrast to Atp7b+/- control rats, HCD feeding of Atp7b-/- rats still resulted in low Cp oxidase activity (Figure 3A), but in a significant increase of serum copper if compared with ND-fed Atp7b-/- rats (Figure 3B). Such increased non-CP bound serum copper is a hallmark of overt liver damage in WD
      • Ferenci P.C.A.
      • Stremmel W.
      • Houwen R.
      • Rosenberg W.
      • Schilsky M.
      • Jansen P.
      • Moradpour D.
      EASL clinical practice guidelines: Wilson's disease.
      that may arise from disintegrated hepatocytes, because necrosis was largely present in the HCD-fed Atp7b-/- rats (Figure 2D).
      Figure thumbnail gr3
      Figure 3An HCD increases the serum and mitochondrial copper load in Atp7b-/- rats. (A) Serum Cp activity is depleted in Atp7b-/- rats. (B) Serum copper is decreased in Atp7b-/- rats compared with Atp7b+/- rats, but increases upon HCD in Atp7b-/- rats. (C and D) Equally increased copper load in (C) whole-liver homogenate and (D) hepatic cytosol in either HCD- or ND-fed Atp7b-/- rats. (E) Massive mitochondrial copper load in HCD-fed Atp7b-/- rats. (F) The mitochondrial copper load significantly correlates with NAS and HAI score (N = 31). One-way analysis of variance with the (A–E) Tukey multiple comparisons test (N = 6–12), or (F) Spearman correlation. *Significant to Atp7b+/- ND. #Significant to Atp7b+/- HCD. Significant to Atp7b-/- ND. ∗,#,†P < .05; ∗∗,##,††P < .01; ∗∗∗,###,†††P < .001; ∗∗∗∗,####,††††P < .0001. Cu, copper.
      WD Atp7b-/- rats, either ND- or HCD-fed, accumulated comparable copper amounts in whole liver homogenates and liver cytosol (Figure 3C and D). This finding shows a comparable copper intake via the ND/tap water diet vs the HCD/sugar water diet, respectively. In contrast, however, an excessive increase in copper was found in mitochondria isolated from HCD- vs ND-fed Atp7b-/- rats (Figure 3E). Of note, a significant increase in mitochondrial copper also was determined in HCD- vs ND-fed control Atp7b+/- rats. Importantly, such increased mitochondrial copper significantly correlated with a higher NAS and a progressive HAI score (Figure 3F).

      An HCD Strongly Amplifies Hepatic Mitochondrial Damage in Atp7b-/- Rats

      Structural and functional alterations in liver mitochondria are early key features in WD patients
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      • Sternlieb I.
      Mitochondrial and fatty changes in hepatocytes of patients with Wilson's disease.
      • Sternlieb I.
      Electron microscopy of mitochondria and peroxisomes of human hepatocytes.
      and related animal models.
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      • Roberts E.A.
      • Robinson B.H.
      • Yang S.
      Mitochondrial structure and function in the untreated Jackson toxic milk (tx-j) mouse, a model for Wilson disease.
      Mitochondrial alterations also are prominent features in NAFLD patients
      • Cortez-Pinto H.
      • Chatham J.
      • Chacko V.P.
      • Arnold C.
      • Rashid A.
      • Diehl A.M.
      Alterations in liver ATP homeostasis in human nonalcoholic steatohepatitis: a pilot study.
      • Caldwell S.H.
      • Swerdlow R.H.
      • Khan E.M.
      • Iezzoni J.C.
      • Hespenheide E.E.
      • Parks J.K.
      • Parker Jr., W.D.
      Mitochondrial abnormalities in non-alcoholic steatohepatitis.
      and related animal models.
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Mitochondrial adaptation in steatotic mice.
      • Serviddio G.
      • Bellanti F.
      • Tamborra R.
      • Rollo T.
      • Romano A.D.
      • Giudetti A.M.
      • Capitanio N.
      • Petrella A.
      • Vendemiale G.
      • Altomare E.
      Alterations of hepatic ATP homeostasis and respiratory chain during development of non-alcoholic steatohepatitis in a rodent model.
      We therefore hypothesized that the combined effect of genetically driven copper accumulation owing to the Atp7b knockout and metabolic disturbance induced by the high-calorie nutrition may accelerate the deterioration of hepatic mitochondria.
      In full agreement with this supposition, feeding Atp7b-/- rats with an HCD severely affected their hepatic mitochondria (Figure 4). Compared with mitochondria from ND-fed Atp7b-/- rats and from control (Atp7b+/-) rats, mitochondria from HCD-fed Atp7b-/- rats appeared with detached inner and outer membranes, prominent matrix condensations, and ballooned cristae (Figure 4A and B, arrows). Such typical WD features were partly observed in mitochondria from age-matched ND-fed Atp7b-/- rats, albeit to a significantly lower extent (Figure 4B, and quantification in 4C). In contrast to mitochondria from ND-fed Atp7b+/- rats, HCD-fed Atp7b+/- mitochondria had partly rounded vesicular cristae that also were abundantly present in HCD-fed Atp7b-/- mitochondria (Figure 4A and B, asterisks). Thus, hepatic mitochondria are affected in structure by both copper deposition and HCD, and their combination resulted in a most severe mitochondrial phenotype.
      Figure thumbnail gr4
      Figure 4An HCD amplifies hepatic mitochondrial damage in WD Atp7b-/- rats. (A and B) Mitochondria either (A) in situ (scale bar: 250 nm) or (B) isolated (scale bar: 1 μm) from HCD-fed Atp7b-/- rats presenting with severe structural alterations, including detachments of the mitochondrial inner and outer membranes (arrows) or matrix condensations together with ballooned cristae (asterisk). (C) Quantification of structurally altered mitochondria from the 4 animal groups. One-way analysis of variance with the Tukey multiple comparisons test (N = 2–3, 350–750 mitochondria per group of animal). *Significant to Atp7b+/- ND. #Significant to Atp7b+/- HCD. Significant to Atp7b-/- ND. ∗,#,†P < .05; ∗∗,##,††P < .01; ∗∗∗,###,†††P < .001; ∗∗∗∗,####,††††P < .0001.
      These structural impairments were paralleled by remarkable mitochondrial functional deficits. The capacity to produce ATP was significantly lower in mitochondria from either HCD-fed Atp7b+/- or ND-fed Atp7b-/- rats in comparison with those from ND-fed Atp7b+/- controls (Figure 5A). However, the strongest decrease in ATP production capacity was determined in mitochondria from HCD-fed Atp7b-/- rats, in which ATP production capacity was significantly lower than in all other tested mitochondrial populations (Figure 5A). In addition, the lowest, albeit not significantly decreased, mean ATP synthase (F1FO) activity was found in mitochondria from HCD-fed Atp7b-/- rats (Figure 5B). In contrast, HCD feeding hardly affected mitochondrial oxygen consumption (Figure 5C) and did not change respiratory control ratios (Figure 5D) because only nonsignificant tendencies for increased succinate-linked, leak, and maximum oxygen consumption rates were observed in the restricted number of investigated HCD- vs ND-fed animals. It thus remains for future studies to evaluate whether these alterations do or do not contribute to mitochondrial dysfunction in HCD-fed Atp7b-/- rats.
      Figure thumbnail gr5
      Figure 5An HCD severely impairs mitochondrial function in Atp7b-/- rats. (A) HCD feeding significantly reduces mitochondrial ATP production capacity in Atp7b-/- rats (N = 5–7). (B) Tendentiously lowest F1FO activity (ATP synthase, normalized to CS activity) in mitochondria from HCD-fed Atp7b-/- rats (N = 5). HCD feeding (C) hardly affected mitochondrial oxygen consumption and (D) did not change respiratory control ratios (RCR; N = 3). (E and F) Strongly increased H2O2 emergence from mitochondria of HCD-fed Atp7b-/- rats using either (E) succinate/rotenone and ADP or (F) glutamate/malate as substrates (N = 5). One-way analysis of variance with the Tukey multiple comparisons test. *Significant to Atp7b+/- ND. #Significant to Atp7b+/- HCD. Significant to Atp7b-/- ND. ∗,#,†P < .05; ∗∗,##,††P < .01; ∗∗∗,###,†††P < .001; ∗∗∗∗,####,††††P < .0001. CII-linked_P, succinate-linked phosphorylation; LEAK, oxygen consumption upon oligomycin treatment; ETS, electron transfer system capacity in a noncoupled carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP)-treated state.
      Importantly, mitochondria from HCD-fed Atp7b-/- rats appeared with 2 highly cell-toxic features that were changed significantly in comparison with the mitochondrial populations from all other rats. The first was a significantly lower ATP production (Figure 5A), and the second was that only mitochondria from HCD-fed Atp7b-/- rats showed significantly enhanced mitochondrial H2O2 emergence, whether tested with either respiratory complex II–linked succinate (Figure 5E) or with respiratory complex I–linked glutamate/malate (Figure 5F) as substrates. Appreciable mitochondrial reactive oxygen species (ROS) were neither emerging from mitochondria from (still) healthy ND-fed WD rats nor from HCD-fed control rats, but were exclusive features of severely damaged mitochondria in HCD-fed Atp7b-/- rats.

      An HCD Increases Enzyme Abundancies of Hepatic Lipid and Bile Salt Synthesis in Atp7b-/- Rats

      How does the combined challenge of decreased copper excretion (owing to Atp7b deletion) and increased fatty acid intake (via the HCD) alter hepatic lipid metabolism? To address this question, we subjected liver homogenates of Atp7b+/- and Atp7b-/- rats, either ND- or HCD-fed, to a quantitative proteomic comparison (Table 4, Table 5, Table 6, Table 7). This analysis provided evidence for a strongly increased mitochondrial ß-oxidation in HCD-fed Atp7b-/- rats (Table 4). This finding agrees well with our earlier results observed in wild-type mice fed an HCD for a prolonged time, and may be an adaptive response to the increased nutritive fatty acid supply.
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Mitochondrial adaptation in steatotic mice.
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Data on chow, liver tissue and mitochondrial fatty acid compositions as well as mitochondrial proteome changes after feeding mice a western diet for 6-24 weeks.
      In further agreement, we also observed higher levels of lipid biosynthesis enzymes, partly in HCD-fed Atp7b+/- control rats, but very prominently in HCD-fed Atp7b-/- rats (Table 5). In line with these observations, increased triglyceride levels were observed in livers from HCD- vs ND-fed rats (Figure 1B), but not in serum (Figure 6A), and only mildly nonsignificantly increased levels of serum nonesterified free fatty acids (Figure 6B). Thus, the highly increased supply of fatty acids via the HCD vs ND
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Data on chow, liver tissue and mitochondrial fatty acid compositions as well as mitochondrial proteome changes after feeding mice a western diet for 6-24 weeks.
      plausibly causes a 2-fold adaptation in hepatocytes: first, their increased degradation in mitochondria via ß-oxidation, and, second, their esterification to triglycerides, which preferentially are stored in cytosol.
      Table 4Quantitative Proteome Comparisons of Liver Homogenate Proteins of the -/- ND, +/- HCD, and -/- HCD Groups Vs +/- ND Group: Fatty Acid Degradation (ß-Oxidation)
      SymbolDescription-/- ND vs +/- ND+/- HCD vs +/- ND-/- HCD vs +/- NDCellular localization
      Acot13Acyl-CoA thioesterase 130.871.123.12
      Fold-changes greater than 2 compared with +/-ND control.
      Mitochondrion
      Echdc2Enoyl-CoA hydratase domain containing 21.081.002.39
      Fold-changes greater than 2 compared with +/-ND control.
      Mitochondrion
      Echdc3Enoyl-CoA hydratase domain containing 31.150.902.11
      Fold-changes greater than 2 compared with +/-ND control.
      Mitochondrion
      EtfdhElectron-transferring-flavoprotein dehydrogenase1.160.842.09
      Fold-changes greater than 2 compared with +/-ND control.
      Mitochondrion
      IvdIsovaleryl-CoA dehydrogenase1.261.333.03
      Fold-changes greater than 2 compared with +/-ND control.
      Mitochondrion
      MceeMethylmalonyl CoA epimerase0.941.142.68
      Fold-changes greater than 2 compared with +/-ND control.
      Mitochondrion
      AcadsbAcyl-CoA dehydrogenase, short/branched chain0.931.73
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      1.90
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      Decr12,4-Dienoyl CoA reductase 1, mitochondrial0.990.60
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      1.52
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      Acaa2Acetyl-CoA acyltransferase 20.990.931.65
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      AcadsAcyl-CoA dehydrogenase, C-2 to C-3 short chain0.990.951.56
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      AuhAdenosine-uridine RNA binding protein/enoyl-CoA hydratase1.091.031.58
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      Echs1Enoyl-CoA hydratase, short chain, 1, mitochondrial0.901.282.00
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      EtfaElectron-transfer-flavoprotein, α polypeptide0.981.131.98
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      EtfbElectron-transfer-flavoprotein, β polypeptide1.081.211.79
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      GcdhGlutaryl-CoA dehydrogenase0.861.091.91
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      Hacl12-Hydroxyacyl-CoA lyase 11.240.751.92
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      HadhHydroxyacyl-CoA dehydrogenase0.960.751.81
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      MutMethylmalonyl CoA mutase1.091.291.67
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      PccaPropionyl-CoA carboxylase, α polypeptide1.081.191.81
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      PccbPropionyl-CoA carboxylase, β polypeptide1.001.111.96
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      Acot2Acyl-CoA thioesterase 21.100.40
      Fold-changes greater than 2 compared with +/-ND control.
      0.65
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      Acad9Acyl-CoA dehydrogenase family, member 90.760.960.63
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      MmaaMethylmalonic aciduria (cobalamin deficiency) cblA type1.101.330.55
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
      Eci1Enoyl-CoA delta isomerase 10.890.50
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      0.70Mitochondrion
      EhhadhEnoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase1.200.65
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      0.99Mitochondrion
      HadhaHydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), α subunit0.860.62
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      1.07Mitochondrion
      Acad10Acyl-CoA dehydrogenase family, member 100.860.901.30Mitochondrion
      Acad11Acyl-CoA dehydrogenase family, member 110.910.900.75Mitochondrion
      Acad8Acyl-CoA dehydrogenase family, member 80.971.261.02Mitochondrion
      AcadlAcyl-CoA dehydrogenase, long chain1.070.791.26Mitochondrion
      AcadmAcyl-CoA dehydrogenase, C-4 to C-12 straight chain0.951.040.99Mitochondrion
      AcadvlAcyl-CoA dehydrogenase, very long chain0.870.720.98Mitochondrion
      Cpt1aCarnitine palmitoyltransferase 1a, liver1.100.680.94Mitochondrion
      Cpt2Carnitine palmitoyltransferase 20.870.811.01Mitochondrion
      McatMalonyl-CoA-acyl carrier protein transacylase (mitochondrial)0.710.741.12Mitochondrion
      MecrMitochondrial trans-2-enoyl-CoA reductase0.931.061.32Mitochondrion
      Acox2Acyl-CoA oxidase 2, branched chain1.080.832.54
      Fold-changes greater than 2 compared with +/-ND control.
      Peroxisome, mitochondrion
      PhyhPhytanoyl-CoA 2-hydroxylase1.201.482.39
      Fold-changes greater than 2 compared with +/-ND control.
      Peroxisome, mitochondrion
      Abcd3ATP-binding cassette, subfamily D, member 30.950.64
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      0.77Peroxisome, mitochondrion
      Ech1Enoyl-CoA hydratase 1, peroxisomal1.010.66
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      1.06Peroxisome, mitochondrion
      Acox3Acyl-CoA oxidase 3, pristanoyl1.000.751.12Peroxisome, mitochondrion
      Eci3Enoyl-CoA delta isomerase 30.910.911.34Peroxisome, mitochondrion
      Eci3Enoyl-CoA delta isomerase 30.770.910.85Peroxisome, mitochondrion
      Slc27a2Solute carrier family 27 (fatty acid transporter), member 21.030.821.07ER, mitochondrion
      Hacl12-Hydroxyacyl-CoA lyase 11.240.751.92
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Peroxisome
      Acot4Acyl-CoA thioesterase 41.100.48
      Fold-changes greater than 2 compared with +/-ND control.
      0.44
      Fold-changes greater than 2 compared with +/-ND control.
      Peroxisome
      Acot8Acyl-CoA thioesterase 81.050.910.49
      Fold-changes greater than 2 compared with +/-ND control.
      Peroxisome, cytoplasm
      Acox1Acyl-CoA oxidase 1, palmitoyl1.090.62
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      1.17Peroxisome
      Acaa1aAcetyl-CoA acyltransferase 11.011.000.82Peroxisome
      Acot12Acyl-CoA thioesterase 121.040.860.99Cytoplasm
      Acox1Acyl-CoA oxidase 1, palmitoyl0.870.811.49Peroxisome
      Decr22,4-dienoyl CoA reductase 2, peroxisomal1.080.800.84Peroxisome
      HadhbHydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), β subunit0.920.710.97Peroxisome
      Hsd17b4Hydroxysteroid (17-β) dehydrogenase 41.040.851.01Peroxisome
      NOTE. Data are presented as mean ratios from 3 age-matched animals (age, 80–82 days).
      ER, endoplasmic reticulum.
      a Fold-changes greater than 2 compared with +/-ND control.
      b Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Table 5Quantitative Proteome Comparisons of Liver Homogenate Proteins of the -/- ND, +/- HCD, and -/- HCD Groups Vs +/- ND Group: Fatty Acid and Triglyceride Synthesis
      SymbolDescription-/- ND vs +/- ND+/- HCD vs +/- ND-/- HCD vs +/- NDCellular localization
      Fatty acid synthesis
       Acsm3Acyl-CoA synthetase medium-chain family member 30.842.79
      Fold-changes greater than 2 compared with +/-ND control.
      4.25
      Fold-changes greater than 2 compared with +/-ND control.
      Mitochondrion
       Acss2Acyl-CoA synthetase short-chain family member 21.221.56
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      2.09
      Fold-changes greater than 2 compared with +/-ND control.
      Cytoplasm, nucleus
       Elovl5Elongation of very long chain fatty acids protein 51.64
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      0.892.12
      Fold-changes greater than 2 compared with +/-ND control.
      ER
       TecrTrans-2,3-enoyl-CoA reductase1.421.012.85
      Fold-changes greater than 2 compared with +/-ND control.
      ER
       Acsl1Acyl-CoA synthetase long-chain family member 11.140.833.46
      Fold-changes greater than 2 compared with +/-ND control.
      ER, mitochondrion
       Acsm1Acyl-CoA synthetase medium-chain family member 10.921.062.35
      Fold-changes greater than 2 compared with +/-ND control.
      Mitochondrion
       Acss3Acyl-CoA synthetase short-chain family member 30.901.262.39
      Fold-changes greater than 2 compared with +/-ND control.
      Mitochondrion
       Fads2Fatty acid desaturase 20.873.25
      Fold-changes greater than 2 compared with +/-ND control.
      1.62
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      ER
       Hsd17b8Hydroxysteroid (17-β) dehydrogenase 80.950.981.83
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
       LOC683884Similar to acyl carrier protein, mitochondrial precursor1.161.031.52
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
       AcacbAcetyl-CoA carboxylase β1.301.371.89
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion, nucleus
       PecrPeroxisomal trans-2-enoyl-CoA reductase0.980.971.77
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Peroxisome, mitochondrion
       Acsm1Acyl-CoA synthetase medium-chain family member 10.673.43
      Fold-changes greater than 2 compared with +/-ND control.
      0.18
      Fold-changes greater than 2 compared with +/-ND control.
      Mitochondrion
       Acsf2Acyl-CoA synthetase family member 20.681.66
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      0.56
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
       Acsm5Acyl-CoA synthetase medium-chain family member 50.59
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      0.56
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      0.53
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
       FasnFatty acid synthase1.080.26
      Fold-changes greater than 2 compared with +/-ND control.
      0.77Cytoplasm
       AclyATP citrate lyase1.060.34
      Fold-changes greater than 2 compared with +/-ND control.
      0.97Nucleus
       AcacaAcetyl-CoA carboxylase α1.180.55
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      0.83Mitochondrion
       Acsl4Acyl-CoA synthetase long-chain family member 41.170.65
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      1.29Mitochondrion
       Hacd23-hydroxyacyl-CoA dehydratase 21.001.311.45ER
       Hsd17b12Hydroxysteroid (17-β) dehydrogenase 121.030.881.11ER
       Hacd33-hydroxyacyl-CoA dehydratase 31.321.161.10ER, mitochondrion
       Ppt1Palmitoyl-protein thioesterase 11.051.060.81Lysosome
       Acsf3Acyl-CoA synthetase family member 30.931.221.03Mitochondrion
       Acsl5Acyl-CoA synthetase long-chain family member 51.270.681.16Mitochondrion
       Cbr4Carbonyl reductase 40.890.831.04Mitochondrion
       McatMalonyl-CoA-acyl carrier protein transacylase0.710.741.12Mitochondrion
       MecrMitochondrial trans-2-enoyl-CoA reductase0.931.061.32Mitochondrion
       Slc25a1Solute carrier family 25 (mitochondrial carrier, citrate transporter), member 11.201.031.30Mitochondrion
      Triglyceride synthesis
       GkGlycerol kinase1.300.752.16
      Fold-changes greater than 2 compared with +/-ND control.
      Mitochondrion
       GpamGlycerol-3-phosphate acyltransferase1.160.941.97
      Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Mitochondrion
       AgmoAlkylglycerol monooxygenase0.820.690.75ER
       Dgat1Diacylglycerol O-acyltransferase 11.091.131.24ER
      NOTE. Data are presented as mean ratios from 3 age-matched animals (age, 80–82 days).
      ER, endoplasmic reticulum.
      a Fold-changes greater than 2 compared with +/-ND control.
      b Fold-changes between 1.5 and 2.0 compared with +/-ND control.
      Table 6Quantitative Proteome Comparisons of Liver Homogenate Proteins of the -/- ND, +/- HCD, and -/- HCD Groups Vs +/- ND Group: Ketone Body and Cholesterol Synthesis
      SymbolDescription-/- ND vs +/- ND+/- HCD vs +/- ND-/- HCD vs +/- NDCellular localization
      Ketone body synthesis
       Acss3Acyl-CoA synthetase short-chain family member 30.901.262.39
      Fold-changes greater than 2 compared with +/- ND control.
      Mitochondrion
       Bdh13-Hydroxybutyrate dehydrogenase, type 10.950.823.20
      Fold-changes greater than 2 compared with +/- ND control.
      Mitochondrion
       Hmgcs23-Hydroxy-3-methylglutaryl-CoA synthase 21.040.58
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      1.95
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      Mitochondrion
       Acat1Acetyl-CoA acetyltransferase 10.920.821.30Mitochondrion
       Hmgcl3-Hydroxymethyl-3-methylglutaryl-CoA lyase0.810.871.43Mitochondrion
      Cholesterol synthesis
       Acat2Acetyl-CoA acetyltransferase 21.73
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      2.67
      Fold-changes greater than 2 compared with +/- ND control.
      8.79
      Fold-changes greater than 2 compared with +/- ND control.
      Nucleus
       Hsd17b7Hydroxysteroid (17-β) dehydrogenase 71.57
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      2.03
      Fold-changes greater than 2 compared with +/- ND control.
      6.52
      Fold-changes greater than 2 compared with +/- ND control.
      Cell membrane
       Idi1Isopentenyl-diphosphate delta isomerase 11.63
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      4.08
      Fold-changes greater than 2 compared with +/- ND control.
      3.39
      Fold-changes greater than 2 compared with +/- ND control.
      Peroxisome
       Msmo1Methylsterol monooxygenase 11.70
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      3.25
      Fold-changes greater than 2 compared with +/- ND control.
      4.16
      Fold-changes greater than 2 compared with +/- ND control.
      ER
       Cyp51Cytochrome P450, family 51, lanosterol 14-α demethylase1.312.60
      Fold-changes greater than 2 compared with +/- ND control.
      4.75
      Fold-changes greater than 2 compared with +/- ND control.
      Unknown
       Hmgcs13-Hydroxy-3-methylglutaryl-CoA synthase 1 (soluble)1.082.60
      Fold-changes greater than 2 compared with +/- ND control.
      3.74
      Fold-changes greater than 2 compared with +/- ND control.
      Cytoplasm
       MvkMevalonate kinase1.212.21
      Fold-changes greater than 2 compared with +/- ND control.
      4.12
      Fold-changes greater than 2 compared with +/- ND control.
      Peroxisome
       SqleSqualene epoxidase1.473.62
      Fold-changes greater than 2 compared with +/- ND control.
      4.05
      Fold-changes greater than 2 compared with +/- ND control.
      ER
       EbpEmopamil binding protein (sterol isomerase)1.66
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      1.68
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      2.71
      Fold-changes greater than 2 compared with +/- ND control.
      ER
       Dhcr77-Dehydrocholesterol reductase1.371.77
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      2.42
      Fold-changes greater than 2 compared with +/- ND control.
      ER
       LssLanosterol synthase (2,3-oxidosqualene-lanosterol cyclase)1.401.98
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      4.01
      Fold-changes greater than 2 compared with +/- ND control.
      ER
       PmvkPhosphomevalonate kinase1.221.73
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      4.47
      Fold-changes greater than 2 compared with +/- ND control.
      Peroxisome
       Tm7sf2Transmembrane 7 superfamily member 21.171.88
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      3.84
      Fold-changes greater than 2 compared with +/- ND control.
      ER
       FdpsFarnesyl diphosphate synthase1.491.303.99
      Fold-changes greater than 2 compared with +/- ND control.
      Cytoplasm
       MvdMevalonate (diphospho) decarboxylase1.012.00
      Fold-changes greater than 2 compared with +/- ND control.
      1.94
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      Peroxisome
       Fdft1Farnesyl diphosphate farnesyl transferase 11.221.99
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      1.80
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      ER
       Hmgcs23-Hydroxy-3-methylglutaryl-CoA synthase 21.040.58
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      1.95
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      Mitochondrion
       Acaa2Acetyl-CoA acyltransferase 20.990.931.65
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      Mitochondrion
       Dhcr2424-Dehydrocholesterol reductase1.171.87
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      1.35ER, Golgi
       Acat1Acetyl-CoA acetyltransferase 10.920.821.30Mitochondrion
       Hmgcl3-Hydroxymethyl-3-methylglutaryl-CoA lyase0.810.871.43Mitochondrion
       NsdhlNAD(P)-dependent steroid dehydrogenase-like1.060.921.00ER
      NOTE. Data are presented as mean ratios from 3 age-matched animals (age, 80–82 days).
      ER, endoplasmic reticulum.
      a Fold-changes greater than 2 compared with +/- ND control.
      b Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      Table 7Quantitative Proteome Comparisons of Liver Homogenate Proteins of the -/- ND, +/- HCD, and -/- HCD Groups Vs +/- ND Group: Bile Acid Synthesis and Transport
      SymbolDescription-/- ND vs +/- ND+/- HCD vs +/- ND-/- HCD vs +/- NDCellular localization
      Bile synthesis
       Acox2Acyl-CoA oxidase 2, branched chain1.080.832.54
      Fold-changes greater than 2 compared with +/- ND control.
      Mitochondrion, peroxisome
       Amacrα-Methylacyl-CoA racemase1.070.992.34
      Fold-changes greater than 2 compared with +/- ND control.
      Mitochondrion, peroxisome
       Cyp8b1Cytochrome P450, family 8, subfamily b, polypeptide 10.970.992.09
      Fold-changes greater than 2 compared with +/- ND control.
      ER
       Acot8Acyl-CoA thioesterase 81.050.910.49
      Fold-changes greater than 2 compared with +/- ND control.
      Mitochondrion, peroxisome
      Bile transport and secretion
       Abcc2ATP-binding cassette, subfamily C, member 20.971.343.51
      Fold-changes greater than 2 compared with +/- ND control.
      Membrane
       Slc10a1Solute carrier family 10 (sodium/bile acid cotransporter), member 11.69
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      1.283.49
      Fold-changes greater than 2 compared with +/- ND control.
      Cell membrane
       Slc22a7Solute carrier family 22 (organic anion transporter), member 71.170.983.85
      Fold-changes greater than 2 compared with +/- ND control.
      Cell membrane
       Slc22a1Solute carrier family 22 (organic cation transporter), member 11.160.952.25
      Fold-changes greater than 2 compared with +/- ND control.
      Cell membrane
       Slc27a5Solute carrier family 27 (fatty acid transporter), member 51.360.984.37
      Fold-changes greater than 2 compared with +/- ND control.
      ER
       Slco1a1Solute carrier organic anion transporter family, member 1a10.940.866.44
      Fold-changes greater than 2 compared with +/- ND control.
      Cell membrane
       Slco1a4Solute carrier organic anion transporter family, member 1a41.302.45
      Fold-changes greater than 2 compared with +/- ND control.
      10.96
      Fold-changes greater than 2 compared with +/- ND control.
      Cell membrane
       Slco1b2Solute carrier organic anion transporter family, member 1B21.350.882.41
      Fold-changes greater than 2 compared with +/- ND control.
      Cell membrane
       Stard10Steroidogenic acute regulatory protein-related lipid transfer domain containing 101.381.142.58
      Fold-changes greater than 2 compared with +/- ND control.
      Cell membrane
       Sult2a1Sulfotransferase family 2A, dehydroepiandrosterone–preferring, member 11.53
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      1.066.69
      Fold-changes greater than 2 compared with +/- ND control.
      Cytoplasm
       Sult2a1Sulfotransferase family 2A, dehydroepiandrosterone–preferring, member 12.09
      Fold-changes greater than 2 compared with +/- ND control.
      1.004.40
      Fold-changes greater than 2 compared with +/- ND control.
      Cytoplasm
       Abcb1aATP-binding cassette, subfamily B, member 1A0.55
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      1.340.34
      Fold-changes greater than 2 compared with +/- ND control.
      Cell membrane
       Abcc3ATP-binding cassette, subfamily C, member 30.65
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      0.57
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      0.08
      Fold-changes greater than 2 compared with +/- ND control.
      Cell membrane
       Ephx1Epoxide hydrolase 1, microsomal (xenobiotic)0.771.230.35
      Fold-changes greater than 2 compared with +/- ND control.
      ER
       LdlrLow-density lipoprotein receptor0.990.65
      Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      0.49
      Fold-changes greater than 2 compared with +/- ND control.
      Cell membrane, Golgi
       Abcb11ATP-binding cassette, subfamily B, member 111.001.141.06Cell membrane
      NOTE. Data are presented as mean ratios from 3 age-matched animals (age, 80–82 days).
      ER, endoplasmic reticulum.
      a Fold-changes greater than 2 compared with +/- ND control.
      b Fold-changes between 1.5 and 2.0 compared with +/- ND control.
      Figure thumbnail gr6
      Figure 6An HCD increases total serum bile salts in Atp7b-/- rats. (A) Serum triglycerides, (B) serum nonesterified fatty acids (NEFAs), and (C) total serum cholesterol do not differ between ND and HCD groups (N = 4–5). (D) Total serum bile salts increase in HCD-fed Atp7b-/- rats (N = 3–5). One-way analysis of variance with the Tukey multiple comparisons test. *Significant to Atp7b+/- ND. #Significant to Atp7b+/- HCD. Significant to Atp7b-/- ND. ∗,#,†P < .05; ∗∗,##,††P < .01; ∗∗∗,###,†††P < .001; ∗∗∗∗,####,††††P < .0001.
      How would hepatocytes deal with an increasing acetyl coenzyme A (acetyl-CoA) amount resulting from increased mitochondrial ß-oxidation of fatty acids? One response is increased lipid biosynthesis and storage. However, acetyl-CoA also is the precursor in hepatic ketogenesis and cholesterol biosynthesis.
      • McGarry J.D.
      • Foster D.W.
      Ketogenesis and cholesterol synthesis in normal and neoplastic tissues of the rat.
      The proteomic comparison showed only slightly increased to doubled levels of the ketogenic mitochondrial enzymes (Table 6), and first analyses of serum levels indicated comparable 3-hydroxybutyrate levels in HCD- vs ND-fed Atp7b-/- rats (3.25 ± 0.07 vs 3.58 ± 1.23 mg/dL, respectively), indicating minor effects on ketogenesis. In contrast, approximately 4-fold increased enzyme levels (in comparison with ND-fed control rats) were found for nearly the whole cholesterol biosynthesis pathway (Table 6). Moreover, in HCD-fed Atp7b-/- rats, strong increases were found in enzymes responsible for cholesterol excretion via bile acid biosynthesis and bile excretion (Table 7). These data indicated an increased synthesis of cholesterol and plausibly, consequently elevated bile salts in HCD-fed Atp7b-/- rats. In fact, although unchanged cholesterol levels were determined in serum (Figure 6C) of HCD- vs ND-fed rats, significantly increased bile salt levels were determined only in HCD-fed Atp7b-/- rat serum (Figure 6D).

      The High-Affinity Copper Binding Peptide MB Efficiently Reverses HCD-Induced Mitochondrial Dysfunction and Improves Liver Integrity in Atp7b-/- Rats

      The bacteria-derived peptide MB prevents disease progression in Atp7b-/- rats.
      • Zischka H.
      • Lichtmannegger J.
      • Schmitt S.
      • Jagemann N.
      • Schulz S.
      • Wartini D.
      • Jennen L.
      • Rust C.
      • Larochette N.
      • Galluzzi L.
      • Chajes V.
      • Bandow N.
      • Gilles V.S.
      • DiSpirito A.A.
      • Esposito I.
      • Goettlicher M.
      • Summer K.H.
      • Kroemer G.
      Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease.
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      This therapeutic effect is largely owing to efficient hepatic de-coppering (Figure 7A), most prominently in the mitochondrial compartment restoring their structure and function.
      • Zischka H.
      • Lichtmannegger J.
      • Schmitt S.
      • Jagemann N.
      • Schulz S.
      • Wartini D.
      • Jennen L.
      • Rust C.
      • Larochette N.
      • Galluzzi L.
      • Chajes V.
      • Bandow N.
      • Gilles V.S.
      • DiSpirito A.A.
      • Esposito I.
      • Goettlicher M.
      • Summer K.H.
      • Kroemer G.
      Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease.
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      Because we aimed here to improve the mitochondrial status in HCD-fed Atp7b-/- rats as fast as possible, together with a low stress level due to therapy, we chose a once-daily treatment for 5 consecutive days only. We previously observed that such short-term treatments already resulted in mitochondrial improvement, whereas more intense treatments were needed to rescue diseased animals.
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      Consequently, we assessed here whether the beneficial effect of MB treatment still would hold in HCD-fed Atp7b-/- rats. Animals were treated once daily for 5 consecutive days with MB, starting at day 75 (ie, at an age when HCD-fed Atp7b-/- rats presented with marked liver damage) (Figure 2A and B).
      Figure thumbnail gr7
      Figure 7MB rescues HCD-induced mitochondrial dysfunction in Atp7b-/- rats. (A) Laser ablation ICP-MS distribution maps (lower panels) show lower copper concentrations in liver samples from ND-fed, MB-treated Atp7b-/- rats compared with untreated ND-fed Atp7b-/- controls (copper concentration range, 0–500 μg/g; laser spot size, 25 μm; scan speed, 50 μm/s). (B) Electron micrographs of mitochondria either in situ (upper panel, scale bar: 500 nm) or isolated (lower panel, scale bar: 500 nm) show mitochondrial structure normalization upon MB treatment (right panels) vs untreated (left panels). (C) Quantification of isolated mitochondria with altered structure (N = 3, 700–750 mitochondria in each animal group). (D–F) Methanobactin treatment (D) decreases mitochondrial copper load (N = 5–6), (E) increases mitochondrial ATP production (N = 5), and (F) decreases mitochondrial H2O2 emergence (substrates, glutamate/malate; N = 5). Unpaired t test, significant if ∗∗P < .01; ∗∗∗∗P < .0001.
      MB treatment clearly improved the mitochondrial structure, as evidenced either in situ or at the level of isolated mitochondria (Figure 7B). A significantly lower number of isolated mitochondria from MB-treated HCD-fed Atp7b-/- rats presented with cristae detachments and matrix condensations in comparison with mitochondria from untreated HCD-fed Atp7b-/- rats (Figure 7B, and quantification shown in 7C). This structural normalization was paralleled by a 50% reduction in mitochondrial copper load (Figure 7D, Table 8), highly comparable with our previous results for this treatment regimen (ie, 32%–62% mitochondrial copper depletion using 5 single consecutive MB injections).
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      Moreover, strongly enforced mitochondrial ATP production capacity (Figure 7E) and significantly decreased mitochondrial H2O2 emergence was observed (Figure 7F). Thus, a 5-day-only MB treatment efficiently ameliorated copper-induced structural and functional deficits in liver mitochondria from HCD-fed Atp7b-/- rats.
      Table 8MB Treatment (Intraperitoneally, Once Daily for 5 Days) Reduces Liver Damage and Copper Overload in Serum, Kidney, and Livers of Female HCD-Fed Atp7b-/- Rats
      Animal IDRat 38Rat 39Rat 40Rat 41Rat 42-/- HCD + MB, mean-/- HCD, mean
      SexFemaleFemaleFemaleFemaleFemale
      Age, day797980808080
      Values are shown as medians.
      74
      Values are shown as medians.
      AST, U/L
       Before227330419368545378300
       After172123196136136191nd
      Bilirubin, mg/dL
       Before<0.5<0.5<0.5<0.51.2<0.5
      Values are shown as medians.
      <0.5
      Values are shown as medians.
       After<0.5<0.5<0.5<0.5<0.5<0.5
      Values are shown as medians.
      nd
      Body weight, g
       Before121141128122102123146
       After126149134132113131nd
      Spleen weight, g0.230.150.250.230.280.230.28
      Visceral fat, g1.932.392.661.992.042.202.53
      Serum Cu, ng/mLn.d.455296224525375640
      Kidney Cu, μg/g wet weight364062441005699
      Liver homogenate Cu, μg/g wet weight318237247296284276392
      Mitochondrial Cu, ng/mg protein538384386445467444893
      Mitochondrial copper depletion relative to the mean mitochondrial copper value of female untreated Atp7b-/- rats.
      Mitochondrial Cu depletion, %
      Mitochondrial copper depletion relative to the mean mitochondrial copper value of female untreated Atp7b-/- rats.
      39.857.056.750.147.7500
      NAS454555a6
      Values are shown as medians.
      HAI8108121210
      Values are shown as medians.
      11
      Values are shown as medians.
      NOTE. Data on AST, bilirubin, and body weight are present before and after 5 days of MB treatment. NAFLD activity score was as follows: ≤2, no nonalcoholic steatohepatitis; 3–4, borderline nonalcoholic steatohepatitis; ≥5 definite nonalcoholic steatohepatitis.
      Cu, copper; nd, not determined.
      a Values are shown as medians.
      b Mitochondrial copper depletion relative to the mean mitochondrial copper value of female untreated Atp7b-/- rats.
      Besides the mitochondrial status, we next investigated effects on overt liver damage upon treatment. All animals experienced a significant decrease in AST serum levels after the MB treatment vs pretreatment levels (Figure 8A, Table 8). One animal that additionally showed increased serum bilirubin levels before treatment returned to levels below the detection limit, and all animals regained weight, showing a positive therapeutic effect (Table 8). In addition, if compared with untreated HCD-fed Atp7b-/- rats, we found significantly lower serum copper levels and a massive decrease in total serum bile salts (Figure 8B and C). Thus, overt liver damage was rescued by this short-term MB treatment. At a histologic level, however, liver damage of HCD-fed Atp7b-/- rats only partially was resolved (Figures 8D–F), resulting in a mild but significant decrease in NAS (Figures 8F), while liver triglyceride levels were unchanged (Figure 8G).
      Figure thumbnail gr8
      Figure 8MB rescues overt/acute liver damage in HCD-fed Atp7b-/- rats. (A–C) Short-term MB treatment (A) significantly reduces the liver damage marker AST (comparison of HCD-fed Atp7b-/- rats before and after MB treatment), and (B and C) significantly decreases serum copper and bile salts (comparison of HCD-fed Atp7b-/- rats with vs without MB treatment). (D) Liver sections (scale bar: 100 μm) from MB-treated (right panel) vs untreated HCD-fed Atp7b-/- rats (left panel) showed no significant reduction of lobular inflammation (black arrow), apoptosis (open arrowhead), necrosis (black arrowhead), fibrosis (black asterisk), or steatosis (open asterisk), as also evidenced by only slightly lower (E) HAI score and (F) NAS. (G) Liver triglyceride content was unchanged after MB treatment. (A–C, and G) Unpaired t test (N = 3–6), significant if *P < .05, means ± SD; (E and F) nonparametric Mann–Whitney test (N = 5–6), significant if *P < .05, medians ± range.

      Discussion

      In WD, ATP7B malfunction impairs hepatic copper excretion. This leads to a progressive copper burden in mitochondria.
      • Zischka H.
      • Lichtmannegger J.
      • Schmitt S.
      • Jagemann N.
      • Schulz S.
      • Wartini D.
      • Jennen L.
      • Rust C.
      • Larochette N.
      • Galluzzi L.
      • Chajes V.
      • Bandow N.
      • Gilles V.S.
      • DiSpirito A.A.
      • Esposito I.
      • Goettlicher M.
      • Summer K.H.
      • Kroemer G.
      Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease.
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      • Sternlieb I.
      Mitochondrial and fatty changes in hepatocytes of patients with Wilson's disease.
      • Roberts E.A.
      • Robinson B.H.
      • Yang S.
      Mitochondrial structure and function in the untreated Jackson toxic milk (tx-j) mouse, a model for Wilson disease.
      Copper ultimately causes mitochondrial destruction, hepatocyte death, liver failure, and decease of WD Atp7b-/- rats.
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      We previously reported that efficient depletion of mitochondrial copper load with the high-affinity copper chelator MB leads to full recovery from even severe states of liver damage.
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      Upon pausing still, the rate of mitochondrial copper re-accumulation determines the rate of re-occurring liver damage.
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      These findings show that hepatic mitochondria are exceptionally susceptible to liver copper overload, thereby being a key organelle in WD pathogenesis. However, mitochondria also readily respond to environmental changes other than increased copper by metabolic adaptations and can balance imposed challenges to different extents for a long time.
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Mitochondrial adaptation in steatotic mice.
      • Koliaki C.
      • Szendroedi J.
      • Kaul K.
      • Jelenik T.
      • Nowotny P.
      • Jankowiak F.
      • Herder C.
      • Carstensen M.
      • Krausch M.
      • Knoefel W.T.
      • Schlensak M.
      • Roden M.
      Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis.
      We reasoned that this mitochondrial flexibility might contribute to the high variability of the clinical presentation of WD.
      • Ferenci P.
      Phenotype-genotype correlations in patients with Wilson's disease.
      The ATP7B genotype is not clearly predictive for the age of onset, the disease presentation or progression, or for the response to treatment.
      • Ferenci P.
      Phenotype-genotype correlations in patients with Wilson's disease.
      • Bandmann O.
      • Weiss K.H.
      • Kaler S.G.
      Wilson's disease and other neurological copper disorders.
      This absence of a genotype–phenotype correlation may be best exemplified by studies on genetically identical WD twins, whose clinical appearance ranged from presymptomatic phenotypes to liver failure.
      • Kegley K.M.
      • Sellers M.A.
      • Ferber M.J.
      • Johnson M.W.
      • Joelson D.W.
      • Shrestha R.
      Fulminant Wilson's disease requiring liver transplantation in one monozygotic twin despite identical genetic mutation.
      • Czlonkowska A.
      • Gromadzka G.
      • Chabik G.
      Monozygotic female twins discordant for phenotype of Wilson's disease.
      Consequently, it has been suggested that the WD phenotype may be highly attributable to environmental factors.
      • Czlonkowska A.
      • Gromadzka G.
      • Chabik G.
      Monozygotic female twins discordant for phenotype of Wilson's disease.
      • Medici V.
      • Weiss K.H.
      Genetic and environmental modifiers of Wilson disease.
      Steatosis is a frequently observed early characteristic in livers of WD patients,
      • Sternlieb I.
      Mitochondrial and fatty changes in hepatocytes of patients with Wilson's disease.
      • Stattermayer A.F.
      • Traussnigg S.
      • Dienes H.P.
      • Aigner E.
      • Stauber R.
      • Lackner K.
      • Hofer H.
      • Stift J.
      • Wrba F.
      • Stadlmayr A.
      • Datz C.
      • Strasser M.
      • Maieron A.
      • Trauner M.
      • Ferenci P.
      Hepatic steatosis in Wilson disease--role of copper and PNPLA3 mutations.
      and mutations in the ε3 and ε4 isoforms of the apolipoprotein E gene are associated with WD onset.
      • Schiefermeier M.
      • Kollegger H.
      • Madl C.
      • Polli C.
      • Oder W.
      • Kuhn H.
      • Berr F.
      • Ferenci P.
      The impact of apolipoprotein E genotypes on age at onset of symptoms and phenotypic expression in Wilson's disease.
      • Litwin T.
      • Gromadzka G.
      • Czlonkowska A.
      Apolipoprotein E gene (APOE) genotype in Wilson's disease: impact on clinical presentation.
      Moreover, mutations in the lipase gene PNALP3 are linked to the percentage of liver steatosis in patients with WD.
      • Stattermayer A.F.
      • Traussnigg S.
      • Dienes H.P.
      • Aigner E.
      • Stauber R.
      • Lackner K.
      • Hofer H.
      • Stift J.
      • Wrba F.
      • Stadlmayr A.
      • Datz C.
      • Strasser M.
      • Maieron A.
      • Trauner M.
      • Ferenci P.
      Hepatic steatosis in Wilson disease--role of copper and PNPLA3 mutations.
      Furthermore, lower serum cholesterol levels were observed in WD patients with hepatic symptoms,
      • Rodo M.
      • Czonkowska A.
      • Pulawska M.
      • Swiderska M.
      • Tarnacka B.
      • Wehr H.
      The level of serum lipids, vitamin E and low density lipoprotein oxidation in Wilson's disease patients.
      • Seessle J.
      • Gohdes A.
      • Gotthardt D.N.
      • Pfeiffenberger J.
      • Eckert N.
      • Stremmel W.
      • Reuner U.
      • Weiss K.H.
      Alterations of lipid metabolism in Wilson disease.
      and WD animal models, such as Atp7b-/- mice, present a down-regulation of enzymes involved in cholesterol and lipid metabolism,
      • Huster D.
      • Purnat T.D.
      • Burkhead J.L.
      • Ralle M.
      • Fiehn O.
      • Stuckert F.
      • Olson N.E.
      • Teupser D.
      • Lutsenko S.
      High copper selectively alters lipid metabolism and cell cycle machinery in the mouse model of Wilson disease.
      • Ralle M.
      • Huster D.
      • Vogt S.
      • Schirrmeister W.
      • Burkhead J.L.
      • Capps T.R.
      • Gray L.
      • Lai B.
      • Maryon E.
      • Lutsenko S.
      Wilson disease at a single cell level: intracellular copper trafficking activates compartment-specific responses in hepatocytes.
      whereas Long-Evans Cinnamon rats show lower serum but higher liver cholesterol and triglyceride levels.
      • Levy E.
      • Brunet S.
      • Alvarez F.
      • Seidman E.
      • Bouchard G.
      • Escobar E.
      • Martin S.
      Abnormal hepatobiliary and circulating lipid metabolism in the Long-Evans Cinnamon rat model of Wilson's disease.
      These studies clearly indicate a link between the lipid/cholesterol metabolism and WD pathophysiology. We therefore asked whether a steatosis-promoting diet would influence WD-related liver damage, and especially mitochondrial damage, in Atp7b-/- rats. The rationale was that both enriched copper and fatty acids cause bioenergetic defects and therefore synergistically and detrimentally may coincide on hepatic mitochondria.
      An HCD caused strongly increased and accelerated liver damage, evidenced by serum markers of liver damage (Figure 2A and B) and histologic assessment (HAI score and NAS). In HCD- vs ND-fed Atp7b-/- rats, a drastically increased copper load was found in mitochondria, but equal cytosolic or overall hepatic copper contents. Compared with mitochondria from either HCD- or ND-fed control rats or from ND-fed Atp7b-/- rats, mitochondria from HCD-fed Atp7b-/- rats appeared to have the most severe mitochondrial structural alterations, a significantly lower ATP production, and a significantly enhanced mitochondrial ROS emergence. Hence, the combination of an HCD with an increasing copper load caused severe structural and functional mitochondrial impairments, whereas mitochondrial copper overload strongly correlated with progressive liver damage (Figure 2, Figure 3, Figure 4, Figure 5).
      Thus, a simple change in nutrition from a normal diet to an HCD severely aggravated and accelerated WD pathophysiology in HCD-fed Atp7b-/- rats. This could be the result of 2 not mutually exclusive reasons: enhanced copper uptake and/or an additional metabolic burden imposed on the hepatocytes and their mitochondria because the later organelles are both the prime site for cellular copper utilization (via complex IV of the respiratory chain) and for fatty acid degradation. Based on reports that ND-fed control animals consumed similar amounts of food and water as their HCD-fed counterparts,
      • Tetri L.H.
      • Basaranoglu M.
      • Brunt E.M.
      • Yerian L.M.
      • Neuschwander-Tetri B.A.
      Severe NAFLD with hepatic necroinflammatory changes in mice fed trans fats and a high-fructose corn syrup equivalent.
      we adjusted for an equal copper supply by the 2 diets (see the Materials and Methods section). In subsequent testing, however, we found that the rats consumed less HCD than ND food, but more fructose water instead of water, respectively. Upon recalculating the supplied copper amounts, we found that the HCD/syrup diet provided approximately 18% more copper. Interestingly, this slightly increased copper supply, however, did not result in correspondingly increased liver copper levels (Figure 3) because we observed equal copper loads in total liver homogenates and in liver cytosol. To the contrary, mitochondria from HCD- vs ND-fed control animals had drastically and significantly increased copper levels (Figure 3). This may indicate that the distribution of hepatocyte copper changes upon HCD feeding. In the HCD-fed Atp7b-/- rat liver mitochondria this copper overload caused severe structural and functional mitochondrial deficits (Figures 4 and 5), paralleled by cell death (Figure 2). We recently reported that progressive mitochondrial copper accumulation causes a steady reduction of their capacity to produce ATP.
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      This is owing to the direct impact of copper on the protein complexes involved in ATP production, but also ATP delivery to the cytosol.
      • Zischka H.
      • Lichtmannegger J.
      • Schmitt S.
      • Jagemann N.
      • Schulz S.
      • Wartini D.
      • Jennen L.
      • Rust C.
      • Larochette N.
      • Galluzzi L.
      • Chajes V.
      • Bandow N.
      • Gilles V.S.
      • DiSpirito A.A.
      • Esposito I.
      • Goettlicher M.
      • Summer K.H.
      • Kroemer G.
      Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease.
      This bioenergetic deficit matches the clinical presentation of liver damage in Atp7b-/- rats. A decrease in the ATP production capacity to 70% in comparison with mitochondria from Atp7b+/- control rats was found to be critical for the onset of clinically apparent liver damage (ie, AST levels > 200 U/L).
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      In the present study, a mitochondrial ATP production capacity of 80% was preserved in young ND-fed Atp7b-/- rats (Figure 5A). In agreement with our earlier study, these rats still were healthy (Figure 2). In a mouse study, we reported that the increased supply of fatty acids via an HCD causes lipidomic alterations in the membranes of liver mitochondria that also reduce their ATP production capacity,
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Mitochondrial adaptation in steatotic mice.
      along with only mild signs of liver impairment.
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Mitochondrial adaptation in steatotic mice.
      In agreement with this study, we determined a reduced mitochondrial ATP production capacity but comparatively mild signs of apparent liver damage in HCD-fed Atp7b+/- control vs Atp7b-/- rats (steatohepatitis was present in only 2 of 6 HCD-fed Atp7b+/- rats) (Figures 1C, 2, and 5A, Tables 1 and 3). This situation changed when an increasing copper load coincided with steatosis: a decreased ATP production capacity to less than 40% was paralleled by severe structural impairments and strongly increased ROS emergence in mitochondria from HCD-fed Atp7b-/- rats, in comparison with mitochondria from all other rats, whether HCD- or ND-fed controls or ND-fed Atp7b-/- rats (Figures 4 and 5). One interesting question for future experiments is why mitochondrial turnover and renewal via mitophagy was incapable to rescue the detrimental effects of copper and steatosis coinciding on mitochondria. Either such pathways were efficiently inactivated (possibly involving activated mechanistic target of rapamycin (mTOR) pathways), blocked by copper excess, negatively affected by bioenergetic deficits that were too strong, or simply overwhelmed. As a result, however, such mitochondrial damage strongly challenges hepatocytes, and cell death (both necrosis and apoptosis) was extensive (Figure 2D), paralleled by severe liver damage in HCD-fed Atp7b-/- rats (Figure 2, Table 1, Table 2, Table 3). We therefore conclude that WD pathophysiology in HCD-fed Atp7b-/- rats is aggravated because of a highly detrimental combination of massive copper- and fatty acid–induced impacts on liver mitochondria.
      Further support for the decisive role of mitochondrial damage in the pathophysiology of HCD-fed Atp7b-/- rats comes from the results of the applied short-term treatment with the copper chelator MB (Figures 7 and 8). We chose this drug, and applied it once daily for 5 consecutive days, to test for a fast mitochondrial recovery in HCD-fed Atp7b-/- rats, as a potential remedy against acute and overt liver damage. In comparison with our earlier reports, such treatments are relatively mild because we also had applied MB 2 or 3 times daily or for increased time periods of up to 1 month.
      • Zischka H.
      • Lichtmannegger J.
      • Schmitt S.
      • Jagemann N.
      • Schulz S.
      • Wartini D.
      • Jennen L.
      • Rust C.
      • Larochette N.
      • Galluzzi L.
      • Chajes V.
      • Bandow N.
      • Gilles V.S.
      • DiSpirito A.A.
      • Esposito I.
      • Goettlicher M.
      • Summer K.H.
      • Kroemer G.
      Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease.
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      Nevertheless, this short-term MB treatment significantly reduced mitochondrial structural damage and improved mitochondrial ATP production with a concomitant decrease in ROS emergence (Figure 7). This mitochondrial amelioration was paralleled by a rescue of overt/acute liver damage in all treated animals, as serum AST levels decreased, bilirubin levels were below detection and animals regained body weight (Table 8). In addition, compared with untreated HCD-fed Atp7b-/- rats, serum copper and bile salt levels, plausibly increased upon hepatocyte disintegration, significantly decreased in MB-treated HCD-fed Atp7b-/- rats (Figure 8B and C).
      Despite this highly beneficial effect of the applied short-term MB-treatment, mitochondrial and liver rescue was not complete. Although the mitochondrial copper content decreased to 50% (Figure 7D), in absolute terms, the remaining copper load still was a borderline burden (Table 8) and comparable with the values of ND-fed Atp7b-/- rats (Figure 3E) that were about to develop hepatitis within days (Figure 2B). This borderline status may explain the noticeable but limited improvements in liver histology (Figure 8), and it remains for future studies to test whether prolonged/intensified MB treatments would result in a more complete reversal of mitochondrial and liver damage in HCD-fed Atp7b-/- rats.
      HCD feeding caused steatosis in all HCD-fed animals. In contrast, steatohepatitis was present in only 2 of 6 HCD-fed Atp7b+/- rats, but in all 6 HCD-fed Atp7b-/- rats (Figure 1, Table 1). Accordingly, tendentious lower levels of visceral fat (Figure 1A) and lower levels of liver triglycerides (Figure 1B) were found in HCD-fed Atp7b-/- vs Atp7b+/- rats, indicating a comparatively higher energy turnover in Atp7b-/- livers. Indeed, mitochondrial enzymes involved in fatty acid degradation were enriched in livers of HCD-fed Atp7b+/- rats, but especially in HCD-fed Atp7b-/- rat livers (Table 4). Interestingly, we also observed higher abundancies of lipid biosynthesis enzymes, partly in HCD-fed Atp7b+/- control rats, but very prominently in HCD-fed Atp7b-/- rats (Table 5). Although these increased enzyme abundancies do not necessarily mean an enhanced flux via these pathways, the highly increased supply of fatty acids via the HCD vs ND
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Data on chow, liver tissue and mitochondrial fatty acid compositions as well as mitochondrial proteome changes after feeding mice a western diet for 6-24 weeks.
      indicates a 2-fold adaptation in hepatocytes: first, their increased degradation in mitochondria via ß-oxidation, and, second, their esterification to triglycerides that are preferentially stored in cytosol. Future measurements have to validate such enhanced metabolic fluxes and/or specific metabolites from these pathways. Furthermore, besides the mere levels of enzyme abundancies, metabolic enzyme activities may be modulated further via post-translational modifications such as acetylation and succinylation,
      • Newman J.C.
      • He W.
      • Verdin E.
      Mitochondrial protein acylation and intermediary metabolism: regulation by sirtuins and implications for metabolic disease.
      which were not assessed in this study. Indeed, increased acetyl-CoA levels from augmented ß-oxidation may result in increased acetylation
      • Menzies K.J.
      • Zhang H.
      • Katsyuba E.
      • Auwerx J.
      Protein acetylation in metabolism - metabolites and cofactors.
      of mitochondrial enzymes involved in the tricarboxylic acid cycle, fatty acid oxidation, amino acid and carbohydrate metabolism, ketone body synthesis, and the urea cycle.
      • Newman J.C.
      • He W.
      • Verdin E.
      Mitochondrial protein acylation and intermediary metabolism: regulation by sirtuins and implications for metabolic disease.
      • Hirschey M.D.
      • Shimazu T.
      • Jing E.
      • Grueter C.A.
      • Collins A.M.
      • Aouizerat B.
      • Stancakova A.
      • Goetzman E.
      • Lam M.M.
      • Schwer B.
      • Stevens R.D.
      • Muehlbauer M.J.
      • Kakar S.
      • Bass N.M.
      • Kuusisto J.
      • Laakso M.
      • Alt F.W.
      • Newgard C.B.
      • Farese Jr., R.V.
      • Kahn C.R.
      • Verdin E.
      SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome.
      Moreover, an increasing acetyl-CoA amount would influence not only these pathways, but also possibly would cause increased cholesterol levels because acetyl-CoA is the precursor in hepatic cholesterol biosynthesis.
      • McGarry J.D.
      • Foster D.W.
      Ketogenesis and cholesterol synthesis in normal and neoplastic tissues of the rat.
      Such hepatic cholesterol accumulation has been reported in NAFLD patients and rodents
      • Arguello G.
      • Balboa E.
      • Arrese M.
      • Zanlungo S.
      Recent insights on the role of cholesterol in non-alcoholic fatty liver disease.
      and correlated with histologic severity of the disease and thus seems to be associated with HCD malnutrition. In fact, increased enzyme abundancies for nearly the whole cholesterol biosynthesis pathway were found in HCD-fed Atp7b-/- rat livers (Table 6). Unexpectedly, however, we did not observe specifically increased cholesterol levels in these animals (Figure 6C). This may have been prevented by an increased routing of cholesterol into bile salts because we determined 2- to 11-fold increases in abundancies of enzymes involved in bile salt synthesis and bile excretion in HCD-fed Atp7b-/- rat livers (Table 7). In agreement, we found increased serum bile salts only in serum of HCD-fed Atp7b-/- rats (Figure 6D), which were reduced significantly upon MB treatment (Figure 8C). Clearly, such increased bile salt synthesis may be a further detrimental impact in HCD-fed Atp7b-/- rat livers because accumulating bile salts are hepatotoxic (especially to hepatic mitochondria).
      • Schulz S.
      • Schmitt S.
      • Wimmer R.
      • Aichler M.
      • Eisenhofer S.
      • Lichtmannegger J.
      • Eberhagen C.
      • Artmann R.
      • Tookos F.
      • Walch A.
      • Krappmann D.
      • Brenner C.
      • Rust C.
      • Zischka H.
      Progressive stages of mitochondrial destruction caused by cell toxic bile salts.
      • Hohenester S.
      • Gates A.
      • Wimmer R.
      • Beuers U.
      • Anwer M.S.
      • Rust C.
      • Webster C.R.L.
      Phosphatidylinositol-3-kinase p110γ contributes to bile salt-induced apoptosis in primary rat hepatocytes and human hepatoma cells.
      In conclusion, the combination of accumulating copper with an HCD is highly detrimental to hepatic mitochondria. A toxic triad of ATP depletion, massively increased ROS, and bile salts seals the fate of affected hepatocytes. This indicates that a high- vs normal-calorie nutrition may have a tremendous impact on WD progression and severity and may contribute to the striking phenotype–genotype discrepancies encountered in WD patients, in agreement with a recent review article that indicated the importance of lifestyle modifications in WD.
      • Kieffer D.A.
      • Medici V.
      Wilson disease: at the crossroads between genetics and epigenetics-a review of the evidence.
      We therefore suggest monitoring such aspects of nutrition in much more detail in the future, to establish whether dietary counseling of WD patients may be of therapeutic benefit.

      Materials and Methods

      Animal Studies

      Animals were maintained under the Guidelines for the Care and Use of Laboratory Animals of the Helmholtz Center Munich. Animal experiments were approved by the government authorities of the Regierung von Oberbayern, Munich, Germany.
      Control Atp7b+/- and WD Atp7b-/- rats of both sexes (Table 2; strain name, LPP crossbreed between Long Evans cinnamon rats and Piebald Virol Glaxo rats; bred in-house, provided by Borjigin) were used because we currently have no indication for a sex-dependent altered WD phenotype in these animals.
      • Ahmed S.
      • Deng J.
      • Borjigin J.
      A new strain of rat for functional analysis of PINA.
      Animals were fed ad libitum either on an ND (1314; Altromin Spezialfutter GmbH, Seelenkamp, Germany; copper content, 13.9 mg/kg; metabolic energy, 3301 kcal/kg; 14% kcal from fat) and tap water (copper content, <0.2 mg/L) or on an HCD (Altromin Spezialfutter GmbH; copper content, 9.3 mg/kg; metabolic energy, 4523 kcal/kg; 45% kcal from fat) and fructose syrup (metabolic energy, 722 kcal/L) in drinking water supplemented with 3.1 mg/L copper.
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Data on chow, liver tissue and mitochondrial fatty acid compositions as well as mitochondrial proteome changes after feeding mice a western diet for 6-24 weeks.
      • Tetri L.H.
      • Basaranoglu M.
      • Brunt E.M.
      • Yerian L.M.
      • Neuschwander-Tetri B.A.
      Severe NAFLD with hepatic necroinflammatory changes in mice fed trans fats and a high-fructose corn syrup equivalent.
      Rats were fed an HCD starting at an age of 46–50 days until an age of 79–82 days. To additionally test for a difference in the age of onset and rate of liver damage progression on the 2 diets (HCD vs ND), a subset of animals was analyzed in parallel at an age of 67, 73, and 75 days (Table 2). Daily consumption values for rats were estimated from the literature to be approximately 20 g chow and 30 mL water, respectively.

      Weiss J. Haus- und versuchstierpflege: 80 Tabellen: Enke, Stuttgart, Germany 2003.

      In subsequent control measurements in Atp7b+/- rats, we determined an average uptake of 16.1 ± 1.7 g ND food and 31 ± 3 mL water per day (ie, approximately 230 μg copper/day), and 12.5 ± 1.7 g HCD food and 50 ± 15 mL fructose syrup (ie, approximately 272 μg copper/day). Thus, the HCD/sugar-water diet supplied slightly more copper (approximately 18%) compared with the ND/tap water diet, but nevertheless resulted in almost equal liver homogenate copper contents in both Atp7b+/- rats and Atp7b-/- rats (Figure 3C). MB treatment of HCD-fed Atp7b-/- rats was performed once daily for 5 consecutive days starting at an age of 74–75 days, as recently described (150 mg/kg body weight intraperitoneally).
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.

      Chemicals

      Chemicals were mostly obtained from Sigma–Aldrich (Taufkirchen, Germany). Nitric acid, K2HPO4, KCl, malate, iodacetamide, multi-element standard IV, copper (II) sulfate pentahydrate, ethanol, and xylene were purchased from Merck (Darmstadt, Germany). Acetyl-CoA, reduced nicotinamide adenine dinucleotide (NADH), phosphoenolpyruvate, pyruvate kinase and lactate dehydrogenase were obtained from Roche Diagnostics (Mannheim, Germany). Bovine serum albumin (BSA) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Carl-Roth (Karlsruhe, Germany). Tris-(hydroxymethyl)aminomethane (TRIS) was obtained from VWR International GmbH (Ismaning, Germany). Gelatin was purchased from Grüssing (Filsum, Germany). Rhodium Inductively Coupled Plasma (ICP) standard solution was purchased from SCP Science (Baie D’Urfé, Canada). Osmium tetroxide and uranyl-less contrasting agent were obtained from Science Services GmbH (Munich, Germany). Propylene oxide and epoxy resin were purchased from SERVA Electrophoresis GmbH (Heidelberg, Germany). Lead citrate was purchased from Leica Biosystems (Wetzlar, Germany).

      Liver Examination

      Serum AST and bilirubin levels were measured with the Reflotron system (Roche Diagnostics, Penzberg, Germany) and liver damage in animals was considered clinically apparent if the serum AST level was greater than 200 U/L and/or the bilirubin level was greater than 0.5 mg/dL.
      • Lichtmannegger J.
      • Leitzinger C.
      • Wimmer R.
      • Schmitt S.
      • Schulz S.
      • Kabiri Y.
      • Eberhagen C.
      • Rieder T.
      • Janik D.
      • Neff F.
      • Straub B.K.
      • Schirmacher P.
      • DiSpirito A.A.
      • Bandow N.
      • Baral B.S.
      • Flatley A.
      • Kremmer E.
      • Denk G.
      • Reiter F.P.
      • Hohenester S.
      • Eckardt-Schupp F.
      • Dencher N.A.
      • Adamski J.
      • Sauer V.
      • Niemietz C.
      • Schmidt H.H.
      • Merle U.
      • Gotthardt D.N.
      • Kroemer G.
      • Weiss K.H.
      • Zischka H.
      Methanobactin reverses acute liver failure in a rat model of Wilson disease.
      Serum cholesterol nonesterified fatty acids and serum triglycerides were analyzed with Respons910 (Diasys Greiner GmbH, Flacht, Germany) according to the manufacturer's guidelines. Serum ceruloplasmin activity was measured as described elsewhere.
      • Schosinsky K.H.
      • Lehmann H.P.
      • Beeler M.F.
      Measurement of ceruloplasmin from its oxidase activity in serum by use of o-dianisidine dihydrochloride.
      • Erel O.
      Automated measurement of serum ferroxidase activity.
      Total serum bile salt concentrations were quantified in serum samples using the Diazyme total bile salt kit (Diazyme Laboratrories, Poway, CA) according to the manufacturer’s instructions. Histologic evaluation was performed on formalin-fixed, paraffin-embedded H&E-stained liver samples. Morphologic features were summarized as an activity score as recommended for the diagnosis of steatohepatitis in NAFLD (NAS)
      • Kleiner D.E.
      • Brunt E.M.
      • Van Natta M.
      • Behling C.
      • Contos M.J.
      • Cummings O.W.
      • Ferrell L.D.
      • Liu Y.C.
      • Torbenson M.S.
      • Unalp-Arida A.
      • Yeh M.
      • McCullough A.J.
      • Sanyal A.J.
      Nonalcoholic Steatohepatitis Clinical Research Network
      Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
      as well as for hepatitis (HAI score).
      • Ishak K.
      • Baptista A.
      • Bianchi L.
      • Callea F.
      • De Groote J.
      • Gudat F.
      • Denk H.
      • Desmet V.
      • Korb G.
      • MacSween R.N.
      • et al.
      Histological grading and staging of chronic hepatitis.
      For quantification of liver triglycerides, 100 mg/mL liver tissue was sonicated within 5% NP40 solution, heated for 5 minutes at 96°C, and cooled on ice. Homogenates were cleared for 2 minutes at 20,000×g, and supernatants (diluted in 5% NP40 solution 1:1–1:10 as required) were analyzed with Respons910 (Diasys Greiner GmbH).
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Mitochondrial adaptation in steatotic mice.

      Metal Content Determination

      Copper in serum, liver homogenate, cytosol, and mitochondria, as well as kidney homogenate, were analyzed by ICP Optical Emission Spectrometry (Ciros Vision, SPECTRO Analytical Instruments GmbH, Kleve, Germany) after wet ashing of samples with 65% nitric acid.
      • Zischka H.
      • Lichtmannegger J.
      • Schmitt S.
      • Jagemann N.
      • Schulz S.
      • Wartini D.
      • Jennen L.
      • Rust C.
      • Larochette N.
      • Galluzzi L.
      • Chajes V.
      • Bandow N.
      • Gilles V.S.
      • DiSpirito A.A.
      • Esposito I.
      • Goettlicher M.
      • Summer K.H.
      • Kroemer G.
      Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease.

      Preparation of Rat Liver Cytosol and Mitochondria

      Freshly removed liver tissue was homogenized with a Teflon-glass homogenizer (B. Braun Biotech, Melsungen, Germany) in isolation buffer (pH 7.2) containing 300 mmol/L sucrose, 5 mmol/L 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES), 0.2 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 0.1% BSA. Approximately 1 mL of the homogenate was centrifuged at 100,000×g (1 h, 4°C), and the supernatant (liver cytosol) was collected and stored at -80°C.
      For mitochondrial isolation, the remaining homogenate was cleared from debris and nuclei by an 800×g (10 min at 4°C) centrifugation step, and a crude mitochondrial fraction was pelleted at 18,900×g (20 min at 4°C). To purify mitochondria, the pellet was suspended in isolation buffer, loaded on a Nycodenz gradient (Axis-Shield PoC, Oslo, Norway; gradient composition was as follows: 1 mL of 40%, 1 mL of 33%, 3 mL of 28%, 2 mL of 27%, 2 mL of 24% Nycodenz solution; diluted in 10 mmol/L TRIS, pH 7.4), and centrifuged at 74,100×g (1 h, 4°C). The mitochondrial fraction (layer at the 28% gradient phase) was collected and suspended in isolation buffer without BSA and washed 2 times at 18,900×g (10 min at 4°C).
      • Zischka H.
      • Larochette N.
      • Hoffmann F.
      • Hamoller D.
      • Jagemann N.
      • Lichtmannegger J.
      • Jennen L.
      • Muller-Hocker J.
      • Roggel F.
      • Gottlicher M.
      • Vollmar A.M.
      • Kroemer G.
      Electrophoretic analysis of the mitochondrial outer membrane rupture induced by permeability transition.
      • Schmitt S.
      • Saathoff F.
      • Meissner L.
      • Schropp E.M.
      • Lichtmannegger J.
      • Schulz S.
      • Eberhagen C.
      • Borchard S.
      • Aichler M.
      • Adamski J.
      • Plesnila N.
      • Rothenfusser S.
      • Kroemer G.
      • Zischka H.
      A semi-automated method for isolating functionally intact mitochondria from cultured cells and tissue biopsies.
      • Schulz S.
      • Lichtmannegger J.
      • Schmitt S.
      • Leitzinger C.
      • Eberhagen C.
      • Einer C.
      • Kerth J.
      • Aichler M.
      • Zischka H.
      A protocol for the parallel isolation of intact mitochondria from rat liver, kidney, heart, and brain.

      Mitochondrial ATP Production

      ATP production was determined using the ATP Bioluminescence Assay Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's guideline. In detail, 10 μg mitochondria were incubated with 160 μmol/L adenosine diphosphate (ADP) and 5 mmol/L succinate for 30 minutes at room temperature in a buffer containing 0.2 mol/L sucrose, 10 mmol/L 3-(N-morpholino)propanesulfonic acid-TRIS, 1 mmol/L inorganic phosphate, 10 μmol/L EGTA and 2 μmol/L rotenone. As background control, potassium cyanide (2 mmol/L, respiratory chain complex IV inhibitor) was added. Mitochondrial ATP production was calculated in pmol/min/mg protein based on background-corrected luminescence signals and ATP standard curves (Roche Diagnostics, Mannheim, Germany).

      Mitochondrial Respiration

      Mitochondrial respiration was measured with an Oxygraph-2k instrument and processed via DatLab 6.2 software (Oroboros Instruments, Innsbruck, Austria). Per each chamber, 100 μg mitochondria was supplied in a buffer containing 0.25 mol/L sucrose, 1 mmol/L EGTA, 30 mmol/L K2HPO4, 15 mmol/L KCl, 5 mmol/L MgCl2, 25 mmol/L succinate, 1 μmol/L rotenone, and 0.5 mmol/L ADP, and oxygen consumption rates were measured as succinate-linked phosphorylation. To determine leak respiration, 2.5 mmol/L oligomycin was added (final, 2.5 μmol/L) to block ATP synthase. Subsequently, the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone was titrated (2 μL steps from a 20 μmol/L stock solution) to induce maximum oxygen consumption (electron transfer system capacity). The respiratory control ratio was calculated by dividing the succinate-linked phosphorylation oxygen consumption by the oxygen consumption upon oligomycin addition.

      Mitochondrial H2O2 Production

      Mitochondrial H2O2 production was analyzed after resorufin fluorescence
      • Muller F.L.
      • Liu Y.
      • Abdul-Ghani M.A.
      • Lustgarten M.S.
      • Bhattacharya A.
      • Jang Y.C.
      • Van Remmen H.
      High rates of superoxide production in skeletal-muscle mitochondria respiring on both complex I- and complex II-linked substrates.
      (converted from Amplex Red; Molecular Probes, Invitrogen, Karlsruhe, Germany) at λEx. 540/20 nm and λEm. 620/40 nm. The assay was performed with 75 μg mitochondria in 150 μL buffer (pH 7.4) containing 125 mmol/L KCl, 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 5 mmol/L MgCl2, 2 mmol/L K2HPO4, 5 μmol/L MnCl2, and as substrates either 10 mmol/L succinate/2 μmol/L rotenone and 3.2 mmol/L ADP, or 5 mmol/L glutamate/5 mmol/L malate. To start the reaction, 50 μL of a solution containing 320 μmol/L Amplex Red, 2 U/mL horseradish peroxidase, and 60 U/mL superoxide dismutase was added. Resorufin fluorescence was followed up in a plate reader (Synergy 2; BioTek Instruments, Inc, Bad Friedrichshall, Germany) and the Resorufin slope was converted into the rate of H2O2 production in pmol/min/mg with a H2O2 standard curve.
      • Muller F.L.
      • Liu Y.
      • Abdul-Ghani M.A.
      • Lustgarten M.S.
      • Bhattacharya A.
      • Jang Y.C.
      • Van Remmen H.
      High rates of superoxide production in skeletal-muscle mitochondria respiring on both complex I- and complex II-linked substrates.

      F1FO-Activity and Citrate Synthase Activity

      F1FO activity was assessed as described before.
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Mitochondrial adaptation in steatotic mice.
      • Schmitt S.
      • Schulz S.
      • Schropp E.M.
      • Eberhagen C.
      • Simmons A.
      • Beisker W.
      • Aichler M.
      • Zischka H.
      Why to compare absolute numbers of mitochondria.
      In detail, 20 μg mitochondria were incubated in buffer containing either 50 mmol/L TRIS (pH 8.0) or an additional 3 μmol/L oligomycin (F1Fo inhibitor, negative control) for 5 minutes at 37°C in a plate reader (Synergy 2). To start the reaction, a final mixture of 0.5 mmol/L ATP, 3 μmol/L carbonyl cyanide 3-chlorophenylhydrazone, 1 mg/mL BSA, 1 μmol/L antimycin A, 10 mmol/L KCl, 4 mmol/L MgCl2, 0.2 mmol/L NADH, 2 mmol/L phosphoenolpyruvate, 4 U lactate dehydrogenase, and pyruvate kinase were added. The F1FO activity was determined in the reverse direction after ATP hydrolysis and NADH oxidation in parallel to the conversion of pyruvate to lactate. The decrease in NADH absorbance at 340 nm was proportional to the ATPase activity and was calculated in nmol/min/mg protein. The F1FO activity was normalized to the activity of the housekeeping enzyme citrate synthase (CS). The activity of mitochondrial CS was determined according to earlier reports.
      • Saggerson E.D.
      • Carpenter C.A.
      Carnitine palmitoyltransferase in liver and five extrahepatic tissues in the rat. Inhibition by DL-2-bromopalmitoyl-CoA and effect of hypothyroidism.
      • Williams A.J.
      • Coakley J.
      • Christodoulou J.
      Automated analysis of mitochondrial enzymes in cultured skin fibroblasts.
      In brief, 280 μL of a solution containing 2.5% (wt/vol) Triton X-100, 100 μmol/L 5,5’-dithiobis-(2-nitrobenzoic acid), 75 μg acetyl-CoA, and 500 μmol/L oxaloacetate was incubated at 37°C. The reaction was started by adding 20 μg mitochondria and followed at 412 nm for 5 minutes. CS activities were calculated from the linear slopes of the initial rates.
      • Schmitt S.
      • Schulz S.
      • Schropp E.M.
      • Eberhagen C.
      • Simmons A.
      • Beisker W.
      • Aichler M.
      • Zischka H.
      Why to compare absolute numbers of mitochondria.

      Electron Microscopy

      Animal livers and isolated mitochondria were fixed with 2.5% glutaraldehyde (Science Services GmbH), postfixed with 1% osmium tetroxide, dehydrated with ethanol and propylene oxide, and were embedded in epoxy resin. Sixty-nanometer sections were cut using the Leica EM UC7 microtome (Leica Biosystems) or the Reichert-Jung Ultracut E microtome (now Leica Biosystems). Ultrathin sections were negative-stained with uranyl acetate (Uranyless) and lead citrate. Images were acquired using either a FEI Tecnai-12 electron microscope equipped with a VELETTA CCD digital camera (FEI, Eindhoven, The Netherlands) or using a Jeol 1200 EXII electron microscope (Akishima, Tokyo, Japan) equipped with a KeenViewII digital camera (Olympus, Hamburg, Germany) and processed with the iTEM software package (anlySISFive; Olympus).
      For structural analyses, mitochondria were grouped in normally structured mitochondria of the “condensed type”
      • Hackenbrock C.R.
      Ultrastructural bases for metabolically linked mechanical activity in mitochondria. II. Electron transport-linked ultrastructural transformations in mitochondria.
      or in altered mitochondria with marked membrane detachments, matrix condensations, and ballooned cristae. A total of 350–750 mitochondria were included per group of animals.

      Proteome Analysis

      Mass spectrometry (MS) sample preparation

      Liver homogenates were lysed in urea buffer (9 mol/L urea, 6 mol/L thiourea, 65 mmol/L dithiothreitol). A total of 10 μg protein per replicate was proteolytically cleaved by applying a modified filter-aided sample preparation procedure,
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Data on chow, liver tissue and mitochondrial fatty acid compositions as well as mitochondrial proteome changes after feeding mice a western diet for 6-24 weeks.
      including a quenching step using 1 mol/L dithiothreitol to bind unreacted iodacetamide. After elution of peptides, samples were acidified with 0.5% trifluoroacetic acid and analyzed on the OrbitrapXL (Thermo Fisher Scientific, Dreieich, Germany) as described.
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Data on chow, liver tissue and mitochondrial fatty acid compositions as well as mitochondrial proteome changes after feeding mice a western diet for 6-24 weeks.
      • von Toerne C.
      • Kahle M.
      • Schafer A.
      • Ispiryan R.
      • Blindert M.
      • Hrabe De Angelis M.
      • Neschen S.
      • Ueffing M.
      • Hauck S.M.
      Apoe, Mbl2, and Psp plasma protein levels correlate with diabetic phenotype in NZO mice--an optimized rapid workflow for SRM-based quantification.

      Mass spectrometry

      Liquid chromatography tandem-mass spectrometry analysis was performed on a LTQ-Orbitrap XL operated on a nano-high-performance liquid chromatography (UltiMate 3000 RSLCnano System; Thermo Fisher Scientific) as described elsewhere,
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
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      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Data on chow, liver tissue and mitochondrial fatty acid compositions as well as mitochondrial proteome changes after feeding mice a western diet for 6-24 weeks.
      • Obermann J.
      • Priglinger C.S.
      • Merl-Pham J.
      • Geerlof A.
      • Priglinger S.
      • Gotz M.
      • Hauck S.M.
      Proteome-wide identification of glycosylation-dependent interactors of galectin-1 and galectin-3 on mesenchymal retinal pigment epithelial (RPE) cells.
      with the modification of using a nonlinear 300-minute liquid chromatography gradient.

      Protein identification and label-free relative quantification

      Acquired spectra were analyzed using Progenesis QI for proteomics (v2.0; Nonlinear Dynamics, Newcastle upon Tyne, UK), as described previously,
      • Einer C.
      • Hohenester S.
      • Wimmer R.
      • Wottke L.
      • Artmann R.
      • Schulz S.
      • Gosmann C.
      • Simmons A.
      • Leitzinger C.
      • Eberhagen C.
      • Borchard S.
      • Schmitt S.
      • Hauck S.M.
      • von Toerne C.
      • Jastroch M.
      • Walheim E.
      • Rust C.
      • Gerbes A.L.
      • Popper B.
      • Mayr D.
      • Schnurr M.
      • Vollmar A.M.
      • Denk G.
      • Zischka H.
      Data on chow, liver tissue and mitochondrial fatty acid compositions as well as mitochondrial proteome changes after feeding mice a western diet for 6-24 weeks.
      with the following adaptations: spectra were searched using the search engine Mascot (version 2.5.1; Matrix Science, London, UK) against the Ensembl rat database (release 80; 28,609 sequences). The Mascot-integrated decoy database search using the Percolator algorithm was set to a peptide false discovery rate of less than 1.5%. Peptide assignments were imported into Progenesis QI. Normalized abundances of peptides were summed up and allocated to the respective protein.

      Laser Ablation ICP-Mass Spectrometry

      For laser ablation ICP-MS analysis, tissue sections of rat liver samples embedded in paraffin were prepared with a thickness of 5 μm using a microtome HM 355S (Thermo Scientific, Bremen, Germany). To quantify the copper concentration in the tissue samples, matrix-matched standards based on 10% gelatin in aqueous solutions of copper (II) sulfate pentahydrate were prepared as described before.
      • Hachmoller O.
      • Aichler M.
      • Schwamborn K.
      • Lutz L.
      • Werner M.
      • Sperling M.
      • Walch A.
      • Karst U.
      Element bioimaging of liver needle biopsy specimens from patients with Wilson's disease by laser ablation-inductively coupled plasma-mass spectrometry.
      The concentration range for copper was between 10 and 1000 μg/g. To validate the standard concentrations, bulk analysis after digestion with nitric acid was used as described before.
      • Hachmoller O.
      • Aichler M.
      • Schwamborn K.
      • Lutz L.
      • Werner M.
      • Sperling M.
      • Walch A.
      • Karst U.
      Element bioimaging of liver needle biopsy specimens from patients with Wilson's disease by laser ablation-inductively coupled plasma-mass spectrometry.
      A laser ablation system (LSX-213 G2+; Teledyne CETAC Technologies, Omaha, NE) was used. ICP-MS detection was performed with a quadrupole-based iCAP TQ (Thermo Fisher Scientific). The laser ablation and ICP-MS were connected with Tygon tubing Saint-Gobain (Courbevoie, France). The following ICP-MS parameters were applied for all measurements: forward power, 1550 W; cool gas flow, 14 L/min; and auxiliary gas flow, 0.8 L/min. In-house–developed software was used to convert the laser ablation ICP-MS data into 2-dimensional images. The copper concentration was calculated using a linear calibration function derived from the average signal intensities for each standard using Microsoft Excel 2016 (Microsoft Corp, Redmond, WA).

      Miscellaneous

      MB was isolated from the spent media of Methylosinus trichosporium OB3b as previously described.
      • Bandow N.L.
      • Gallagher W.H.
      • Behling L.
      • Choi D.W.
      • Semrau J.D.
      • Hartsel S.C.
      • Gilles V.S.
      • Dispirito A.A.
      Isolation of methanobactin from the spent media of methane-oxidizing bacteria.
      Protein quantification was performed by the Bradford
      • Bradford M.M.
      A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
      or Biuret assay (T1949; Sigma-Aldrich).

      Statistics

      Throughout this study, N refers to the number of analyzed animals. Data are presented as means ± SD. Statistical significance was analyzed using 1-way analysis of variance with the Tukey multiple comparisons test, or the nonparametric Kruskal–Wallis test when comparing 3 or more sample sets (GraphPad Prism 7, GraphPad Software, Inc, San Diego, CA). For 2 group comparisons, the unpaired 2-tailed Student t test was used for parametric data and the Mann–Whitney test was used for nonparametric data, respectively (GraphPad Prism 7).
      All authors had access to the study data and reviewed and approved the final manuscript.

      Acknowledgments

      The authors would like to thank Dr E.E. Rojo for critical reading of the manuscript.

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