1. Background and Aims: Methods: Results: Conclusions:
ASSEMBLY OF COMPLEX I OF THE MITOCHONDRIAL RESPIRATORY CHAIN IN MURINE NON-ALCOHOLIC FATTY LIVER DISEASE AND EFFECTS OF ROSIGLITAZONE THERAPY.
Inmaculada García-Ruiz, Cristina Rodríguez-Juan, Teresa Díaz-Sanjuán,
Daniel Fernández-Moreira, Mercedes Pérez-Carreras, Teresa Muñoz-Yagüe,
José A. Solís-Herruzo
Department of Gastroenterology. Research Center. Hospital “12 de Octubre”
Universidad Complutense. Madrid. Spain
Background and Aims:
2.- Complex I components are decreased in the liver from ob/ob mice. Mitochondrial proteins from control and ob/ob mice were separated in one-dimensional BN-PAGE system and analyzed by Western blot using antibody against the complex I subunit NDUFA9 (p39), complex II subunit 70, complex III subunit core protein 2, and complex IV subunit COX I.
As figure illustrates, fully-assembled complexes I, II, III, and IV were markedly diminished in ob/ob mice relative to control mice. However, treatment of ob/ob mice with 10 mg/Kg/tiw anti-TNF antibody, with 20 mg/day uric acid, or 10 mg/Kg/day MnTBAP, i.p. for 12 weeks normalized the amount of these complexes in mitochondrial preparations of these animals. Treatment of ob/ob mice with IgG1 immunoglobulin, as control of anti-TNF-treated mice, did not cause any significant effect on the amount of complex I. In wild-
Type animals treated with RGZ, fully-assembled complex I displayed
a clearly increase as compared with untreated, control mice. Moreover,
the signal originated by the complex I appears as a double band in lean RGZ-treated mice, suggesting that this drug induces the formation of a lower molecular weight subcomplex. In ob/ob mice treated with RGZ for 12 weeks, the amount of fully-assembled complex I remained low and the band was also duplicated (ob/RGZ), suggesting the presence of a mitochondrial supercomplex.
C57 Ob Ob Ob Ob Ob Ob Ob Ob C57
UA MnTBAP IgG1 aTNF
C57 C57 C57
RGZ RGZ
Ob Ob Ob
RGZ
In previous studies, we have shown that the activity of the mitochondrial respiratory chain (MRC) is decreased in the liver of patients with nonalcoholic steatohepatitis (NASH) (Hepatology, 2003; 38: ). We found the same mitochondrial dysfunction in the liver from ob/ob mice (Hepatology 2006;44: 581). Insulin resistance is present in almost all patients with NASH, and mitochondrial dysfunction is believed to play a critical role in the progression of fatty liver into NASH. However, rosiglitazone (RGZ), an insulin sensitizer drug, did not reverse histological lesions of NASH or improve MRC activity in ob/ob mice. On the contrary, RGZ reduced the activity of complex I of the MRC and increased oxidative stress even more (Hepatology 2007;46:414-23).
In this study we investigate:
(1) The in-gel activity of complex I of the MRC in wild-type and ob/ob mice.
(2) How complex I is assembled in the liver from these animals.
(3) The effects of rosiglitazone treatment in these processes
(4) The in vitro effect of peroxynitrite on the function & assembly of complex I.
Methods:
39 kDa C ob C57+RGZ ob+RGZ
C57 ob
C57 ob/UA
C57 ob/TBAP
C57 ob/aTNF
980
39 kDa
17 kDa
20 kDa
30 kDa
15 kDa MW
kDa
1.17
1.21
1.20
1.02
0.81
0.83
0.82
0.91
0.92
1.33
0.78
1.16
1.98
0.36
0.45
0,62
0,33
0.05
Using this procedure, the most
striking finding is the decrease in
all complex I subunits in ob/ob mice. In none of the subunits tested, is appreciable an accumulation of low-molecular-weight subcomplexes, suggesting that assembly of complex I is not the cause of the reduced amount of this complex found in ob/ob mice. Treatment of ob/ob mice with uric acid, MnTBAP, or anti-TNF antibody results in the recovery of the levels of complex I existing in wild-type mice, while treatment with RGZ elicits the formation of a subcomplex suggesting that this drug impairs the assembly process of this complex.
3.- Amount of complex I subunits of the mitochondrial respiratory chain is markedly reduced in ob/ob mice. To investigate how complex I is assembled in the liver mitochondria of ob/ob mice, this complex was resolved by two dimensional, blue native gel electrophoresis [2D-BN/SDS-PAGE].
Forty-six mice were distributed in six groups: group 1 (wild-type) included 12 C57BL/6J mice. Group 2 (ob/ob) was formed by 16 obese ob/ob mice. Group 3 (MnTBAP) was composed by six ob/ob mice treated with 10 mg/Kg/day of manganese [III] tetrakis (5,10,15,20 benzoic acid) porphyrin (MnTBAP). Group 4 (IgG1) was formed by six ob/ob mice treated with 10 mg/Kg IgG1 immunoglobulin three time week (tiw). Group 5 (anti-TNF) consisted of six ob/ob mice treated with 10 mg/Kg/tiw anti-TNF antibody. Group 6 was made by six ob/ob mice treated with 20 mg/day uric acid (UA). Saline, MnTBAP, IgG1 immunoglobulin, anti-TNF, and UA suspension were administered intraperitoneally for 12 weeks. After killing the animals, 50 mg of liver were homogenized using a tight-fitting glass-Teflon homogenizer in a MOPS sucrose buffer containing protease inhibitors. An enriched mitochondrial fraction was collected by centrifuging at 20,000g for 20 minutes. Mitochondrial pellet was dissolved in a solution of 1 M aminocaproic acid, 50 mM TRIS (pH 7.0), and 1.5-5% dodecyl D-maltose. After centrifugation at 100,000g for 15 min, the supernatant was collected and combined with 5% Serva blue G in 1 M aminocaproic acid.
In-gel activity assay.
To measure complex I in-gel activity, one dimensional gel was incubated overnight at room temperature with a solution containing 2 mM Tris-HCl, pH 7.4, 0.1 mg/ml NADH, and 2.5 mg/ml nitrotetrazolium blue.
One-dimensional Blue Native electrophoresis.
To measure complex I in-gel activity, one-dimensional gel was incubated overnight at room temperature with a solution containing 2mM Tris-HCl, pH 7.4, 0,1 mg/ml NADH, and 2.5 mg/ml nitrotetrazolium blue. Gel was washed in distilled water and photographed.
Two- dimensional Blue-native electrophoresis.
A lane of the one dimensional gel was cut, rotated 90º, placed on a glass plate, and incubated with a dissociated solution for 1 hour at room temperature. After removal of the dissociating solution, a second glass plate was assembled and the second dimension SDS-polyacrylamide gel was prepared as described by Nijtmans et al Mitochondrial proteins were run in the second dimension and transferred to a nitrocellulose membrane.
The membranes were incubated with antibodies against complex I subunits 39 (NDUFA9), 30 (NDUFS3), 20 (MTND6), 17 (NDUFB6), and 15 kDa (NDUFA6), as well as complex III subunits core II (UQCRC1) and FeS (UQCRFS1).
4.- Prohibitin levels are reduced in mitochondrial membrane from ob/ob mice.
Because prohibitin protects mitochondrial complexes from degradation, we investigated whether this protein is decreased in mitochondria from obese mice.
As figure shows, the level of immunoreactive
prohibitin is clearly decreased in protein ex-
tracts obtained from ob/ob mice. The amount of this protein returns to the control level in animals treated with uric acid, MnTBAP or anti-TNF antibody for 12 weeks.
Ctr ob ob ob ob ob ob ob ob Ctr
UA MnTBAP IgG1 aTNF
4.- Peroxynitrite Inhibits Mitochondrial Respiratory Chain Complex I. Preparations of liver mitochondria from lean mice were incubated with 0 to 2600 M peroxynitrite and 3-tyronitrate (3-NT) proteins, complex I enzymatic activity, fully-assembled complex I and prohibitine was measured.
As figure shows, treatment of mitochondrial proteins with 0.8 to 2.8 mM peroxynitrite nitrates mitochondrial proteins, decreases activity of complex I, and degrades complex I and prohibitin in a doses-dependent manner. Low activity of complex I was confirmed when enzymatic activity was measured by espectrophotometry
Complex I
3-NT proteins
Complex I in-gel activity mM
Prohibitin
Results:
As figure A shows, liver mitochondria from untreated ob/ob mice display a strikingly reduced complex I activity (Ob) when compared with control levels (C57). However, activity of this complex returns to the control levels when ob/ob animals are treated for 12 weeks with uric acid, MnTBAP, or anti-TNF,
but not with IgG1. Activity of complex I decreass even more
in both lean (C57/RGZ) and ob/ob (Ob/RGZ) mice treated with
1 mg/Kg/day rosiglitazone for 12 weeks.
1.- In-gel enzyme activity of complex I.
Liver mitochondria isolated from lean, wild-type, and obese, ob/ob, mice untreated and treated with anti-TNF, uric acid, IgG1, or MnTBAP were run under native conditions in a BN-PAGE system and subjected to a complex I in-gel enzyme activity assay.
C57 C57 Ob Ob
RGZ RGZ
C57 Ob Ob Ob Ob Ob Ob Ob Ob C57
UA MnTBAP IgG1 aTNF
Conclusions:
This study confirms that the activity of complex I of the mitochondrial respiratory chain is markedly decreased in ob/ob mice and that treatment of these animals with RGZ reduces even more this activity.
While in ob/ob mice, this defect is caused by a reduction in the amount of all complex I subunits, in RGZ-treated animals it appears to be due to a defect in the assembly process.
Peroxynitrite treatment of mitochondrial proteins elicits inactivation and degradation of complex I.