Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis

Defective Hepatic Mitochondrial Respiratory Chain in Patients With Nonalcoholic Steatohepatitis Mercedes Pe´ rez-Carreras,1 Pilar Del Hoyo,2 Miguel A. Martı´n,2 Juan C. Rubio,2 Ana Martı´n,1 Gregorio Castellano,1 Francisco Colina,3 Joaquı´n Arenas,2 and Jose´ A. Solis-Herruzo1 Mitochondrial dysfunction might play a central role in the pathogenesis of nonalcoholic steatohepatitis (NASH). The aims of this study were to evaluate whether free fatty acid (FFA) transport into the mitochondria or the activity of mitochondria respiratory chain (MRC) complexes are impaired in NASH. In patients with NASH and control subjects, we measured free carnitine, short-chain acylcarnitine (SCAC) and long-chain acylcarnitine (LCAC) esters, carnitine palmitoyltransferase (CPT) activity, and MRC enzyme activity in liver tissue as well as serum concentration of tumor necrosis factor ␣ (TNF-␣), homeostatic metabolic assessment of insulin resistance (HOMAIR), and body mass index (BMI). In patients with NASH, the LCAC/free carnitine ratio was significantly increased and the SCAC/free carnitine ratio was decreased. In patients with NASH, the activity of the MRC complexes was decreased to 63% ⴞ 20% (complex I), 58.5% ⴞ 16.7% (complex II), 70.6% ⴞ 10.3% (complex III), 62.5% ⴞ 13% (complex IV), and 42.4% ⴞ 9.1% (adenosine triphosphate synthase) of the corresponding control values. Activity of these complexes correlated significantly with serum TNF-␣ and HOMAIR. Serum TNF-␣ (36.3 ⴞ 23.1 pg/mL), HOMAIR (4.5 ⴞ 2.38), and BMI (29.9 ⴞ 3.5 kg/m2) values were significantly increased in patients with NASH. In conclusion, activities of MRC complexes were decreased in liver tissue of patients with NASH. This dysfunction correlated with serum TNF-␣, insulin resistance, and BMI values. (HEPATOLOGY 2003;38:999-1007.)

N

onalcoholic steatohepatitis (NASH) is a clinicopathologic condition characterized by histologic features of alcoholic liver disease that occurs in patients who do not consume significant amounts of alcohol.1 At this moment, NASH is considered part of a large spectrum of nonalcoholic fatty liver disease that also includes pure fatty liver (hepatic steatosis), hepatic steatoAbbreviations: NASH, nonalcoholic steatohepatitis; FFA, free fatty acid; ROS, reactive oxygen species; MRC, mitochondrial respiratory chain; SCAC, short-chain acylcarnitine; LCAC, long-chain acylcarnitine; CoA, coenzyme A; CPT, carnitine palmitoyltransferase; CS, citrate synthase; NADH, reduced nicotinamide dinucleotide phosphate; TNF, tumor necrosis factor; BMI, body mass index; HOMAIR, homeostatic metabolic assessment of insulin resistance; FADH2, flavin adenine dinucleotide dehydrogenase. From the Departments of 1Gastroenterology and 3Pathology and 2Unit for the Study of Mitochondrial Myopathies and Medical Research Center, Hospital Universitario “Doce de Octubre,” Madrid, Spain. Received February 26, 2003; accepted July 1, 2003. Supported in part by “Fondo de Investigacio´n Sanitaria” grants 98/1253, 01/ 1447, and 01/1426 and grant 03/015 from the “Red Tema´tica de Investigacio´n Cooperativa (Instituto Carlos III),” Spain. Address reprint requests to: Mercedes Pe´rez-Carreras, Ph.D., M.D., Servicio de Medicina Aparato Digestivo, Hospital Universitario “12 de Octubre,” Carretera de Andalucı´a, Km. 5,400, 28041 Madrid, Spain. E-mail: [email protected]. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3804-0025$30.00/0 doi:10.1053/jhep.2003.50398

sis with lobular inflammation, ballooning degeneration, sinusoidal fibrosis or Mallory bodies (NASH), and cirrhosis.2,3 Nonalcoholic fatty liver disease is an emerging worldwide common problem that represents the most frequent histologic finding in patients with unexplained abnormalities of liver test results. In some western countries, the prevalence of nonalcoholic fatty liver disease in the general population is approximately 20% and the prevalence of NASH ranges between 1.2% and 4.8%.4 NASH has been found to be associated with a large number of metabolic, surgical, and toxic conditions. However, the pathogenesis of NASH is not well understood. A growing body of evidence suggests that development of NASH requires a double “hit.” The “first hit” involves the accumulation of fat in the liver, and the “second hit” includes oxidative stress resulting in lipid peroxidation, the production of malondialdehyde, 4-hydroxynonenal, proinflammatory cytokines, stellate cell activation, and fibrogenesis.5,6 Mitochondrial dysfunction might play a central role in the induction of both “hits,” because mitochondria are involved in the ␤-oxidation of free fatty acids (FFAs) and are the most important cellular source of reactive oxygen species (ROS) (Fig. 1).7-11 999

1000

PE´ REZ-CARRERAS ET AL.

HEPATOLOGY, October 2003

Fig. 1. Mitochondrial fat metabolism and energy production. Mitochondrial ␤-oxidation of FFAs involves 3 successive steps. The first step is entry of long-chain FFAs into the mitochondria. This step is dependent on CPT-I, an enzyme located in the external mitochondrial membrane. The second step is successive ␤-oxidation of FFAs leading to the formation of short-chain acyl-CoA and acetyl-CoA and the conversion of oxidized nicotinamide adenine dinucleotide and flavin adenine dinucleotide into NADH and FADH2. The third step is reoxidation of NADH and FADH2 into oxidized nicotinamide adenine dinucleotide and flavin adenine dinucleotide, respectively, by the MRC. Electrons from NADH and FADH2 are transferred to this enzymatic chain, where they are finally combined with oxygen and protons to form water. This transfer of electrons along the MRC is coupled with the exit of protons from the mitochondrial matrix into the intermembrane space, creating an electrochemical potential across the inner membrane. Protons return to the matrix through adenosine triphosphate synthase, causing the conversion of adenosine diphosphate into adenosine triphosphate. In normal conditions, a small fraction of electrons flowing along the MRC react with oxygen to form ROS. Impaired MRC function results in increased ROS formation and diminished reoxidation of NADH and FADH2, which in turn interferes with the ␤-oxidation of FFAs. ADP, adenosine diphosphate; ATP, adenosine triphosphate; Cyt. a-a3 oxidase, cytochrome oxidase complex; translocase, carnitine/acylcarnitine translocase.

The aims of the present study were to evaluate whether FFA transport into the mitochondria is impaired in patients with NASH and to assess the activity of the mitochondrial respiratory chain (MRC) enzymatic complexes in these patients.

Patients and Methods Forty-three patients with a clinicopathologic diagnosis of NASH seen consecutively at our gastroenterology unit from January 1995 to October 1999 were prospectively studied. Laboratory features of controls and patients included in this study are summarized in Table 1. Thirteen patients were women (30.2%), and age ranged from 20 to 64 years (mean age, 44 ⫾ 11 years). All patients under-

went percutaneous liver biopsy, and the diagnosis of NASH was established by the presence of pericentral macrovesicular steatosis, ballooning degeneration of the liver cells with or without Mallory bodies or fibrosis, and lobular and portal inflammation in the absence of other causes of liver disease (viral, drugs, toxin, autoimmune, metabolic). Significant alcohol consumption (⬎40 g/wk) was excluded by careful questioning of the patients and confirmed by close family members and measurement of serum carbohydrate-deficient transferrin. Other less specific laboratory markers commonly used to assess alcohol abuse, such as serum alanine aminotransferase/aspartate aminotransferase ratio, ␥-glutamyltransferase, and mean corpuscular volume of red blood cells, were also evaluated.

PE´ REZ-CARRERAS ET AL.

HEPATOLOGY, Vol. 38, No. 4, 2003

1001

Table 1. Characteristics and Laboratory Features of Patients With NASH

BMI (kg/m2) Hemoglobin (g/dL) White cells (⫻109/L) Platelets (⫻103/␮L) Prothrombin time (%) Albumin (g/dL) Total bilirubin (mg/dL) AST (U/L) ALT (U/L) AST/ALT ␥-glutamyltransferase (U/L) Alkaline phosphatase (U/L) Glucose (mg/dL) Cholesterol (mg/dL) Triglyceride (mg/dL) Ferritin (ng/dL) Transferrin saturation (%) Median corpuscular volume (fl) Carbohydrate-deficient transferrin (U/L)

Normal Range

Control Subjects, Mean ⴞ SD (n ⴝ 16)

Patients With NASH, Mean ⴞ SD (n ⴝ 43)

No. Abnormal (%)

⬍25 12.0-17.2 4,000-10,300 140-450 75-125 3.2-5.5 0.20-1.00 5-45 5-45 ⬍2 3-52 98-295 70-110 150-200 50-170 ⬎200 women, ⬎300 men ⬎45% 80.7-100 ⬍24

25.6 ⫾ 0.66 14 ⫾ 1.4 6,500 ⫾ 1,369 289 ⫾ 89 95.9 ⫾ 13 4.9 ⫾ 0.3 0.7 ⫾ 0.2 16.5 ⫾ 6.3 24.4 ⫾ 10 0.6 ⫾ 0.2 36 ⫾ 11 167.3 ⫾ 50.4 92.5 ⫾ 8.2 188 ⫾ 15 123 ⫾ 34 113 ⫾ 57.4 36.6 ⫾ 6.8 88.7 ⫾ 5 13.5 ⫾ 5

29.9 ⫾ 3.5 15.4 ⫾ 1.0 7,134 ⫾ 1,625 224 ⫾ 55 96.4 ⫾ 9.3 4.9 ⫾ 0.4 0.7 ⫾ 0.3 45.4 ⫾ 28.6 90.3 ⫾ 58.4 0.5 ⫾ 0.2 42 ⫾ 36 171.3 ⫾ 74.6 109 ⫾ 36 211 ⫾ 49 158 ⫾ 82 273.7 ⫾ 208.2 30.5 ⫾ 10.7 89.3 ⫾ 5 14.6 ⫾ 4

95.3 5 5 7 10 0 10 28 91 0 21 5 20 18 47 37 6 2 0

Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase.

The control group consisted of 16 subjects undergoing elective abdominal surgery with no history of alcohol abuse and normal liver enzyme levels. The male/female ratio was 2.2:1, and the age of this group of patients ranged from 23 to 59 years (mean age, 41 ⫾ 13 years). None of these patients had a history of liver disease. Liver biopsy specimens were obtained during laparotomy and were histologically normal (Table 2). A portion of the liver tissue from both patients with NASH and controls was routinely processed, and sections were stained with hematoxylin-eosin, Masson’s trichrome, Wilder’s impregnation for reticulin fibers, periodic acid– Schiff stain with diastase digestion, and Perls’ Prussian blue. Another portion of tissue was immediately frozen in liquid nitrogen and stored at ⫺70°C for later analysis. Informed consent for the study was obtained from each patient and control subject after a detailed description of the procedure was provided, and the study was approved by the ethics committee of the hospital. At the time of liver biopsy, venous blood samples were drawn to determine serum levels of alanine aminotransferase, aspartate aminotransferase, bilirubin, albumin, alkaline phosphatase, ceruloplasmin, ␣1-antitrypsin, glucose, iron, ferritin, and transferrin. All of these biochemical tests were performed using a conventional automated analyzer. Carbohydrate-deficient transferrin was determined by enzyme-linked immunosorbent assay (Cdtect; Pharmacia & Upjohn, S.A., Diagnostics, Uppsala, Sweden). Each patient was also tested for antibodies to hepatitis C virus by second-generation enzyme-linked immunosorbent assay (Ortho Diagnostic Systems, Inc., Raritan,

Table 2. Histologic Findings in 43 Patients With NASH and 16 Controls Patients With NASH (n ⴝ 43)

Steatosis Macrovesicular Microvesicular Macrovesicular-microvesicular Degree 0 (0%-10%) 1 (11%-25%) 2 (26%-50%) 3 (⬎50%) Inflammation (grade)* Lobular L0 L1 L2 L3 L4 Portal-periportal P0 P1 P2 P3 P4 Fibrosis (stage)† F0 F1 F2 F3 F4

Control Subjects (n ⴝ 16)

No.

%

No.

%

43 13 0 30

100 30.2 0 69.8

0 0 0 0

0 0 0 0

6 11 12 14

13.9 25.6 27.9 32.6

0 0 0 0

0 0 0 0

1 23 15 2 2

2.3 53.5 28.3 4.6 4.6

16 0 0 0 0

100 0 0 0 0

18 17 5 3 0

41.8 39.5 11.6 7.0 0

16 0 0 0 0

100 0 0 0 0

15 21 4 3 0

34.9 48.8 9.3 7 0

16 0 0 0 0

100 0 0 0 0

*Grade was scored according to the Scheuer classification for viral hepatitis.12 †Fibrosis stage was scored according to the Gerber and Popper classification for alcoholic liver disease13: F0, absent; F1, perisinusoidal fibrosis; F2, periportal fibrosis; F3, bridging fibrosis; F4, cirrhosis.

1002

PE´ REZ-CARRERAS ET AL.

NJ) and for hepatitis B surface antigen by a commercially available radioimmunoassay (Abbot Laboratories, North Chicago, IL). Smooth muscle antibodies, antinuclear antibodies, and antibodies to liver/kidney microsome type I were determined by indirect immunofluorescence on kidney tissue sections. Liver Histology A single pathologist who was blinded to the clinical data analyzed each biopsy specimen. Histologic grade was scored using the classification of Scheuer for viral hepatitis,12 and fibrosis stage was evaluated according to the classification of Gerber and Popper for alcoholic liver disease13 (Table 2). Laboratory Studies Determination of Carnitine. The amount of free carnitine and short-chain acylcarnitine (SCAC) and longchain acylcarnitine (LCAC) esters was determined in 5 to 10 mg liver tissue from 26 patients with NASH and 16 controls after homogenization in 0.6N perchloric acid using a radiochemical procedure as described by Di Donato et al.14 Briefly, after 1.5-minute centrifugation at 8,000 rpm, we extracted free carnitine and SCAC in the acid-soluble fraction and LCAC in the acid-insoluble fraction. The difference between total carnitine and free carnitine in the acid-soluble fraction corresponded to SCAC. The assay used a radioenzymatic method in which the sample was incubated with [1-14C]acetyl-CoA (coenzyme A) together with carnitine acetyltransferase, HEPES buffer, and N-ethylmaleimide as a trap for free CoA-SH. The [1-14C]acetylcarnitine formed in the reaction mixture was separated using a Dowex 2-X8 anion-exchange column (Dowes; Sigma Chemical Co., St. Louis, MO), counted, and related to standard curves of L-carnitine and acetylcarnitine. Liver carnitine values were expressed in nanomoles per milligram of noncollagenous protein as estimated by Lowry’s procedure.15 Hepatic Carnitine Palmitoyltransferase. Carnitine palmitoyltransferase (CPT) activity was measured in 5 to 10 mg liver tissue from 26 patients with NASH and 16 controls by the isotope exchange assay described by Di Mauro and Di Mauro,16 which measures the palmitoyl[14C]carnitine formation from palmitoyl-CoA and L-[methyl-14C]carnitine. CPT activity was expressed as nanomoles of palmitoylcarnitine obtained during 1 minute per gram of tissue and related to milligrams of noncollagenous protein content. All measurements were performed in duplicate. We added 0.2 mmol/L malonyl CoA, which is a specific inhibitor of CPT I but not of CPT II, to liver homogenates from 13 patients with NASH and 11 controls. Thus, the malonyl-CoA–sensi-

HEPATOLOGY, October 2003

tive fraction represented the activity of CPT I, whereas the residual fraction indicated the activity of CPT II. MRC Enzyme Activity Assays. Frozen liver tissues (15-20 mg) from 18 patients with NASH and 11 controls were homogenized with 15 vol of 20 mmol/L KP buffer, pH 7.4, and centrifuged at 800g for 10 minutes. Respiratory chain enzymes and citrate synthase (CS) activities were measured in a DU-650 spectrophotometer (Beckman Instruments, Palo Alto, CA). Incubation temperatures were 30°C for complex I (rotenone-sensitive reduced nicotinamide dinucleotide phosphate [NADH] coenzyme Q1 reductase), complex II (succinate dehydrogenase), complex III (antimycin A–sensitive ubiquinol-2 cytochrome c reductase), complex V (oligomycin-sensitive F1–adenosine triphosphatase), and CS and 38°C for complex IV (cytochrome c oxidase). Enzyme activities were performed in supernatants as described elsewhere,17 expressed in nanomoles of substrate used per minute per milligram of protein and, to correct for the hepatic content of mitochondria, referred as a percentage of the specific activity of CS. Enzyme assays were performed in triplicate. Serum Tumor Necrosis Factor ␣. Serum tumor necrosis factor ␣ (TNF-␣) levels were measured using a commercially purchased radioimmunoassay (MedgenixTNF-␣/IRMA, Nivelles, Belgium) in all patients with NASH and control subjects. Body Mass Index. Body mass index (BMI) was calculated by the Quetlet index18: weight (kg)/height (m2). Obesity and overweight were defined by a BMI greater than or equal to 30 kg/m2 and 25 to 29.9 kg/m2, respectively. Insulin Resistance. Insulin resistance was calculated by the homeostatic metabolic assessment (HOMAIR) method as follows: HOMAIR ⫽ fasting serum insulin ⫻ fasting serum glucose/22.5, where insulin is expressed in micro-international units per milliliter and glucose in millimoles per milliliter.19 Serum insulin levels were measured with an enzyme immunoassay (AxSYM Insulin; Abbott Laboratories, Diagnostic Division, Wiesbaden, Germany). Statistical Analysis Statistical analysis was aided by using SPSS statistical software for Windows, version 9 (SPSS Inc., Chicago, IL). The unpaired t test was used to assess the significance of differences between means. All results were expressed as mean ⫾ SD unless otherwise mentioned. Pearson’s correlation coefficient was used for correlation analysis between variables. P values less than .05 were considered significant.

HEPATOLOGY, Vol. 38, No. 4, 2003

PE´ REZ-CARRERAS ET AL.

1003

activity in the liver tissue of 26 patients with NASH and 16 healthy controls. Total CPT activity was similar in patients with NASH (5.69 ⫾ 1.36 nmol/min/mg protein) and in control subjects (5.54 ⫾ 1.38 nmol/min/mg protein). Moreover, both CPT I and CPT II activities were rather similar in 13 patients and 11 controls (1.98 ⫾ 0.41 vs. 1.94 ⫾ 0.44 nmol/min/mg protein, respectively, for CPT I and 3.71 ⫾ 0.95 vs. 3.60⫾0.94 nmol/min/mg protein, respectively, for CPT II).

Fig. 2. Carnitine and carnitine ester hepatic content: total liver carnitine (TC), free carnitine (FC), LCAC, and SCAC in liver specimens of 16 control subjects and 26 patients with NASH. NS, not significant.

Results Because carnitine plays a pivotal role in the transport of fatty acids, particularly of the long-chain fatty acids, into the matrix of mitochondria, we studied the free and total carnitine as well as the LCAC and SCAC content in the liver of control subjects and patients with NASH. Mean values of total and free carnitine in liver tissue were not significantly different in 26 subjects with NASH (8.73 ⫾ 2.15 nmol/mg protein and 5.57 ⫾ 1.97 nmol/mg protein, respectively) from those in 16 healthy controls (10.5 ⫾ 3 nmol/mg protein and 6.16 ⫾ 2.9 nmol/mg protein, respectively). The mean levels of LCAC were significantly greater in patients with NASH (0.61 ⫾ 0.3 nmol/mg protein) than in controls (0.38 ⫾ 0.22 nmol/mg protein; P ⬍ .05). Likewise, the ratio of LCAC/ free carnitine was significantly increased in patients with NASH (0.11 ⫾ 0.06; P ⬍ .01) compared with controls (0.06 ⫾ 0.01). Conversely, the mean SCAC levels were significantly lower in patients with NASH (2.52 ⫾ 1.28 nmol/mg protein) than in controls (3.81 ⫾ 1.25 nmol/mg protein; P ⬍ .01) (Fig. 2). The ratio of SCAC/ free carnitine was 0.45 ⫾ 0.14 in patients with NASH and 0.62 ⫾ 0.19 in controls (P ⬍ .01). The hepatic levels of LCAC/free carnitine in the liver of patients with NASH correlated significantly with the BMI (r ⫽ 0.42; P ⬍ .05). On the other hand, hepatic SCAC/free carnitine ratio correlated significantly with the BMI (r ⫽ 0.43; P ⬍ .05) and serum TNF-␣ concentrations (r ⫽ 0.62; P ⬍ .05). The transport of LCAC is mediated by the activity of both CPT and the carnitine-acylcarnitine translocase. Although deficiencies in these mitochondrial enzymatic systems are very rare in liver pathology, we measured CPT

Activity of the MRC Because the MRC plays a critical role in the conversion of NADH and flavin adenine dinucleotide dehydrogenase (FADH2) into nicotinamide adenine dinucleotide and flavin adenine dinucleotide, respectively, and in the generation of adenosine triphosphate from adenosine diphosphate (Fig. 1),9,10 we measured the activity of the MRC complexes in both groups of subjects (11 controls and 18 patients with NASH). The activity of complex I in liver tissue, which accepts electrons from NADH and transfers them to ubiquinone, was only 63% ⫾ 20% of the corresponding control values. Thus, whereas the activity of this complex was 36.3 ⫾ 6.2 (nmol ⫻ min⫺1 ⫻ mg protein⫺1/nmol ⫻ min⫺1 ⫻ mg protein⫺1 CS) ⫻ 100 ([complex I/CS] ⫻ 100) in control subjects, this activity was decreased to 22.6 ⫾ 7.6 (complex I/CS) ⫻ 100 in patients with NASH (P ⬍ .001) (Fig. 3). The activity of this complex in patients with NASH correlated with the serum levels of TNF-␣ (r ⫽ ⫺0.82; P ⬍ .01), BMI (r ⫽ ⫺0.58; P ⬍ .05), and HOMAIR index (r ⫽ ⫺0.61; P ⬍ .01). The activity of complex II (succinate dehydrogenase complex), which passes electrons directly to ubiquinone

Fig. 3. Activity of the MRC complexes in liver specimens of 11 control subjects and 18 patients with NASH. Enzyme activities are expressed as nmol ⫻ min⫺1 ⫻ mg protein⫺1 ⫻ 100/nmol ⫻ min⫺1 ⫻ mg protein⫺1 CS ([complex/CS] ⫻ 100).

1004

PE´ REZ-CARRERAS ET AL.

HEPATOLOGY, October 2003

Table 3. MRC Enzyme Complexes Activity in 18 Patients With NASH According to Fibrosis Stage Mitochondrial Respiratory Complexes* I

Fibrosis stage F0–F1 F2–F4 P

62.3 ⫾ 34 57.8 ⫾ 17 NS

II

65.8 ⫾ 18 57 ⫾ 16.1 NS

III

IV

V

81 ⫾ 33 58.3 ⫾ 15 NS

68.2 ⫾ 17 54.2 ⫾ 9.3 NS

46.6 ⫾ 12.5 40.7 ⫾ 7.8 NS

*Activities are expressed as percentage of control activity. Abbreviation: NS, not significant.

(Fig. 1), was also significantly reduced to 58.5% ⫾ 16.7% of control activity in patients with NASH (controls, 36.8 ⫾ 8.8 [complex II/CS] ⫻ 100; NASH, 21.5 ⫾ 6.1 [complex II/CS] ⫻ 100; P ⬍ .001) (Fig. 3). This decreased activity correlated with serum levels of TNF-␣ (r ⫽ ⫺0.63; P ⬍ .05), BMI (r ⫽ ⫺0.77; P ⬍ .01), and HOMAIR index (r ⫽ ⫺0.46; P ⬍ .05). Ubiquinone passes electrons from complexes I and II to the b-c1 complex (complex III), which transfers them to cytochrome c (Fig. 1). The activity of complex III was decreased to 70.6% ⫾ 10.3% of control values in patients with NASH (34.6 ⫾ 5 [complex III/CS] ⫻ 100) when compared with control subjects (49.1 ⫾ 11 [complex III/ CS] ⫻ 100; P ⬍ .01) (Fig. 3). The activity of this complex in patients with NASH correlated significantly with serum levels of TNF-␣ (r ⫽ ⫺0.63; P ⬍ .01), BMI (r ⫽ ⫺0.46; P ⬍ .05), and HOMAIR index (r ⫽ ⫺0.58; P ⬍ .05). Cytochrome c is involved in carrying electrons from the b-c1 complex to the cytochrome oxidase complex (complex IV), which finally transfers these electrons to oxygen (Fig. 1). Measurement of the activity of this complex in the liver tissue of patients with NASH showed that it was decreased to 62.5% ⫾ 13% of the control activity (controls, 35.4 ⫾ 11 [complex IV/CS] ⫻ 100; NASH, 22.7 ⫾ 5.1 [complex IV/CS] ⫻ 100; P ⬍ .01) (Fig. 3). The activity of this complex correlated significantly with serum levels of TNF-␣ (r ⫽ ⫺0.63; P ⬍ .01) and BMI (r ⫽ ⫺0.56; P ⬍ .05). Transport of electrons through the MRC is coupled to the pumping of protons from the mitochondrial matrix to the intermembrane space. Complex V converts adenosine diphosphate in adenosine triphosphate when protons flow back from the intermembrane space into the matrix (Fig. 1). The activity of complex V (adenosine triphosphate synthase) was markedly reduced to 42.4% ⫾ 9.1% of the control activity. Thus, while normal complex V activity in the liver tissue was 133 ⫾ 34.2 (complex V/CS) ⫻ 100, this activity was only 48 ⫾ 11.3 (complex V/CS) ⫻ 100 (P ⬍ .001) in patients with NASH. Complex V activity correlated significantly with serum TNF-␣

levels (r ⫽ ⫺0.61; P ⬍ .05), BMI (r ⫽ ⫺0.71; P ⬍ .01), and HOMAIR index (r ⫽ ⫺0.54; P ⬍ .05). Specific activities of CS were 140 ⫾ 28 nmol/min/mg protein for controls and 151 ⫾ 35 nmol/min/mg protein for patients, indicating no proliferation of mitochondria in patients with NASH. There were no significant differences between both values. The decreased activity of all complexes was more marked in patients with stages F2 to F3 of fibrosis than in patients with stage F0 or F1. However, differences did not reach significant levels (Table 3). TNF-␣ Serum levels of TNF-␣ were significantly higher in 43 patients with NASH (36.3 ⫾ 23.1 pg/mL) than in 16 control subjects (6.5 ⫾ 4.3 pg/mL; P ⬍ .001). These levels were above the normal range in 39 patients with NASH (90.7%) and correlated with the BMI (r ⫽ 0.81; P ⬍ .001), HOMAIR index (r ⫽ 0.72; P ⬍ .001), fasting glucose concentrations (r ⫽ 0.66; P ⬍ .01), and the activity of the MRC enzyme complexes. BMI The BMI was significantly higher in patients with NASH (29.9 ⫾ 3.5 kg/m2) than in controls (25.6 ⫾ 0.66 kg/m2; P ⬍ .001). Nineteen patients with NASH were overweight and 22 obese. The BMI was greater than 35 kg/m2 in only 5 of these obese patients. In 2 patients, BMI was less than 25 kg/m2. BMI correlated significantly with the LCAC/free carnitine ratio, SCAC/free carnitine ratio, serum TNF-␣, HOMAIR index (r ⫽ 0.65; P ⬍ .05), and activity of MRC enzyme complexes. HOMAIR Index HOMAIR index was 4.5 ⫾ 2.38 in patients with NASH and 1.41 ⫾ 0.71 (P ⬍ .001) in control subjects. This index was normal (⬍2) in only 2 patients and also correlated significantly with serum TNF-␣ and BMI.

Discussion Carnitine is required in mammalian tissue to transfer long-chain acyl CoAs across the inner mitochondrial

HEPATOLOGY, Vol. 38, No. 4, 2003

membrane for ␤-oxidation. Furthermore, intramitochondrial carnitine can react with short- and mediumchain acyl CoAs to produce SCAC, which can be shuttled out of mitochondria.20 Acquired carnitine deficiency has been suggested to be involved in some cases of steatosis and steatohepatitis.21-23 However, our results do not support a role of carnitine in the pathogenesis of NASH, because the liver content in total and free carnitine was normal in patients with NASH (Fig. 2). These results concur with those reported by Harper et al.23 in obese patients and De Sousa et al.24 in alcoholic subjects with fatty liver. Our study also shows that LCAC and the LCAC/free carnitine ratio were increased in patients with NASH and SCAC and SCAC/free carnitine ratio were decreased in the same patients. The reasons for these changes are uncertain. However, both changes can be ascribed to an impaired ability to ␤-oxidize long-chain acyl CoAs to medium- and short-chain acyl CoAs.20,25 Very little information is available on the mitochondrial ␤-oxidation in NASH. Some investigators have suggested that it is impaired in these patients,5,26,27 which concurs with the finding of microvesicular and macrovesicular steatosis in 76.7% of our patients with NASH (Table 2). Microvesicular hepatic steatosis is generally attributed to severe impairment of mitochondrial ␤-oxidation.26 Alternatively, the higher LCAC and LCAC/free carnitine ratio in liver content found in patients with NASH may also be a consequence of an enhanced fatty acid delivery to the liver. This mechanism is supported by the fact that LCAC and LCAC/free carnitine ratio correlated significantly with BMI. Medium- and short-chain fatty acids are formed in the mitochondria as a result of the ␤-oxidation of long-chain fatty acids. Reduced activity of the ␤-oxidation may result in a decrease in these fatty acids. Our study shows that these changes correlated with serum levels of TNF-␣ and BMI. These correlations may be ascribed to the effects of TNF-␣ on the MRC. Besides carnitine, CPT is also required to transfer long-chain fatty acids into the mitochondria and is considered a rate-limiting step for mitochondrial fatty acid ␤-oxidation. Low activity in hepatic CPT has been reported in children with some gene mutations.28 However, the normal values found in patients with NASH indicate that a deficiency in this enzyme does not seem to play a role in the pathogenesis of this disease (Fig. 2). NADH and FADH2 generated during the oxidation of fatty acids and other fuels are reoxidized by transferring their electrons to the MRC. No information is available on the activity of these complexes in the liver of patients with NASH. Caldwell et al.29 found normal activity of complex I and complex III in platelet-derived mitochon-

PE´ REZ-CARRERAS ET AL.

1005

dria from patients with NASH, and Sanyal et al.30 failed to find any defect in the MRC enzyme expression in the muscle of a patient with NASH. These results led these investigators to exclude a systemically expressed MRC dysfunction in NASH. However, Haque et al.31 recently reported that NASH is associated with decreased cytochrome c oxidase activity. Our study first shows that the activity of all enzyme complexes of the MRC is decreased in patients with NASH by 30% to 50% of control activity. This defect involves both mitochondrial (complexes I, III, IV, and V) and nuclear (complex II) DNA-encoded complexes. The cause of this defect is unknown, although it is not apparently related to differences in the hepatic content in mitochondria between patients and controls, as indicated by the similar CS levels observed in both groups of subjects. On the contrary, we found a significant inverse correlation between the serum levels of TNF-␣ and the activity of these complexes. A growing body of evidence indicates that TNF-␣ can interfere with the mitochondrial function.32 Thus, impaired electron flow at the level of complex I33 and complex III34 has been detected in cells treated with TNF-␣. Under physiologic conditions, small quantities (1%5%) of the oxygen consumed in the mitochondria are released as ROS (Fig. 1).35 However, the mitochondrial ROS production increases markedly when the electron flow in the MRC is impaired.36 In a previous study, we showed that TNF-␣ causes electrons to be retained along cytochrome b, which may be transferred to molecular oxygen, leading to the generation of an elevated amount of ROS.34 ROS production exceeding cellular antioxidant defense capabilities can result in severe metabolic dysfunction, including peroxidation of lipid membrane,34 mitochondrial DNA damage,33,34,37 and direct damage of mitochondrial enzymes containing iron-sulfur clusters.38 Furthermore, ␤-oxidation of long-chain fatty acids can be interfered by the impaired reoxidation of NADH and FADH2 to nicotinamide adenine dinucleotide and flavin adenine dinucleotide. This oxidative stress can initiate several vicious circles, which lead to greater mitochondrial DNA damage, which in turn can decrease the synthesis of mitochondrial DNA– encoded enzymes and the electron flow along the MRC.39 Although in vitro studies indicate that TNF-␣ interferes with the flow of electrons at the level of complex III, these vicious circles may extend this defect to other complexes. Evidence supporting a role for TNF-␣ in the pathogenesis of NASH comes from animal models of NASH and human studies40 (reviewed by Crespo et al.40 and Tilg and Diehl41). Mitochondrial abnormalities have been observed in patients with NASH and in those treated with drugs known to cause NASH. In these patients, mito-

1006

PE´ REZ-CARRERAS ET AL.

chondria are large, swollen, and multilamellar, with loss of cristae and often with paracrystalline inclusion bodies.29,30 These inclusions have also been found in a variety of mitochondrial myopathies42 associated with deficient expression of MRC enzymes. In a previous study, we showed that TNF-␣ induces significant changes in mitochondrial appearance in target cells.34 After 8 hours of incubation, mitochondria showed a swollen and rounded appearance with a clear matrix, fragmented cristae, and breaks in their external membrane. In addition to these changes, other investigators found onion-like structures inside the matrix.43 Our study also shows that the activity of MRC enzyme complexes correlated significantly with BMI and the HOMAIR index. The correlation between MRC activity and BMI may be ascribed to the fact that adipose tissue is likely a major source of TNF-␣.44 This tissue is capable of transcribing and secreting TNF-␣,40,44 and adipose tissue TNF-␣ messenger RNA and serum levels of TNF-␣ correlate with BMI.45 In our study, we confirm that BMI and TNF-␣ serum levels were closely correlated (r ⫽ 0.805; P ⬍ .001). The correlation with the insulin resistance index can also be ascribed to the TNF-␣ effects, because this cytokine has been shown to have the ability to induce insulin resistance in animal models and in adipocytes, hepatoma cells, and other cell lines.46,47 Moreover, neutralization of circulating TNF-␣ in the insulin-resistant mouse led to a significant increase in insulin sensitivity.48 In conclusion, our study shows for the first time that activity of MRC enzyme complexes is decreased in the liver of patients with NASH. The cause of this defect was not clarified in this study. However, TNF-␣ might play a role, because mitochondrial dysfunction was associated with increased serum TNF-␣ levels. Hepatic carnitine content and CPT activity were normal in patients with NASH and do not seem to play any role in this disease.

HEPATOLOGY, October 2003

8.

9.

10. 11. 12. 13.

14.

15. 16. 17.

18. 19. 20. 21.

22.

23. 24.

25.

References 1. Ludwig J, Viggiano RT, McGill DB. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc 1980; 55:342-348. 2. Matteoni CA, Younossi ZM, Gramlich T, Boparal N, Liu YC, McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 1999;116:1413-1419. 3. Brunt EM, Janney CG, Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 1999;94:2467-2474. 4. Younossi ZM, Diehl AM, Ong JP. Nonalcoholic fatty liver disease: an agenda for clinical research. HEPATOLOGY 2002;35:746-752. 5. Chittury S, Farrell GC. Etiopathogenesis of nonalcoholic steatohepatitis. Semin Liver Dis 2001;21:27-41. 6. James O, Day CP. Non-alcoholic steatohepatitis: another disease of affluence. Lancet 1999;353:1634-1636. 7. Berson A, De Beco V, Lette´ron P, Robin MA, Moreau C, Kahwaji J, Verthier N, et al. Steatohepatitis-inducing drugs cause mitochondrial dys-

26.

27.

28.

29.

30.

function and lipid peroxidation in rat hepatocytes. Gastroenterology 1998; 114:764-774. Fromenty B, Berson A, Pessayre D. Microvesicular steatosis and steatohepatitis: role of mitochondrial dysfunction and lipid peroxidation. J Hepatol 1997;26:13-22. Pessayre D, Mansouri A, Fromenty B. Nonalcoholic steatosis and steatohepatitis V. Mitochondrial dysfunction in steatohepatitis. Am J Physiol 2002;282:G193-G199. Morris A. Mitochondrial respiratory chain disorders and the liver. Liver 1999;19:357-368. Halliwell B. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 1994;344:721-724. Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol 1991;13:372-274. Gerber MA, Popper H. Relation between central canals and portal tracts in alcoholic hepatitis. A contribution to pathogenesis of cirrhosis in alcoholics. Hum Pathol 1972;3:199-207. Di Donato S, Rimoldi M, Garavaglia B, Uziel G. Propionil carnitine excretion in propionic and methylmalonic acidurias: a cause of carnitine deficiency. Clin Chim Acta 1984;139:13-21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-271. Di Mauro S, Di Mauro PM. Muscle carnitine palmityl transferase deficiency and myoglobinuria. Science 1973;182:929-931. Martinez B, Del Hoyos P, Martin MA, Arenas J, Perez-Castillo A, Santos A. Thyroid hormone regulates oxidative phosphorylation in the cerebral cortex and striatum of neonatal rats. J Neurochemistry 2001;78:10541063. Willett WC, Dietz WH, Colditz GA. Guidelines for healthy weight. N Engl J Med 1999;341:427-434. Haffner SM, Miettinen H, Stern MP. The homeostasis model in the San Antonio heart study. Diabetes Care 1997;20:1087-1092. Bremer J. Carnitine: metabolism and functions. Physiol Rev 1983;63: 1420-1480. Bowyer BA, Miles JM, Haymond MW, Fleming CR. L-Carnitine therapy in home parenteral nutrition patients with abnormal liver tests and low plasma carnitine concentration. Gastroenterology 1988;94:434-438. Kra¨henbu¨ hl S, Mang G, Kupferschmidt H, Meier PJ, Krause M. Plasma and hepatic carnitine and coenzyme A pools in a patient with fatal, valproate induced hepatotoxicity. Gut 1995;37:140-143. Harper P, Wadstro¨ m C, Backman L, Cederblad G. Increased liver carnitine content in obese women. Am J Clin Nutr 1995;61:18-25. De Sousa C, Leung NWY, Chalmers RA, Peters TJ. Free and total carnitine and acylcarnitine content of plasma, urine, liver and muscle of alcoholics. Clin Sci 1988;75:437-440. Campos Y, Huertas R, Lorenzo G, Bautista J, Gutie´rrez E, Aparicio M, Alesso L, et al. Plasma carnitine insufficiency and effectiveness of L-carnitine therapy in patients with mitochondrial myopathy. Muscle Nerve 1993;16:150-153. Fre´neaux E, Labbe G, Lette´ron P, Le Dinh T, Deggot C, Gene´ve J, Larey D, et al. Inhibition of the mitochondrial oxidation of fatty acids by tetracycline in mice and in man: possible role in microvesicular steatosis induced by this antibiotic. HEPATOLOGY 1988;8:1056-1062. Deschamps D, De Beco V, Fisch C, Fromenty B, Guillouzo A, Pessayre D. Inhibition by perhexiline of oxidative phosphorylation and the ␤– oxidation of fatty acids: possible role in pseudoalcoholic liver lesions. HEPATOLOGY 1994;19:948-961. Yamamoto S, Abe H, Kohgo T, Ogawa A, Ohtake A, Hayashibe H, Sakuraba H, et al. Two novel gene mutations (Glu 74Lys, Phe 383Tyr) causing the “hepatic” form of carnitine palmitoyltransferase II deficiency. Hum Genet 1996;98:116-118. Caldwell SH, Swerdlow RH, Khan EM, Iezzoni JC, Hespenheide EE, Parks JK, Parker WD Jr. Mitochondrial abnormalities in non-alcoholic steatohepatitis. J Hepatol 1999;31:430-434. Sanyal AJ, Campbell-Sargent C, Mirshahi F, Rizzo WB, Contos MJ, Sterling RK, Luketic VA, et al. Nonalcoholic steatohepatitis: association of

PE´ REZ-CARRERAS ET AL.

HEPATOLOGY, Vol. 38, No. 4, 2003

31.

32.

33.

34.

35. 36. 37.

38.

39.

insulin resistance and mitochondrial abnormalities. Gastroenterology 2001;120:1183-1192. Haque M, Mirshahi F, Campbell-Sargent C, Sterling RK, Luketic VA, Shiffman ML, Stravitz RT, et al. Nonalcoholic steatohepatitis (NASH) is associated with hepatocyte mitochondrial depletion [Abstract]. HEPATOLOGY 2002;36:430A. Lancaster JR, Laster SM, Gooding LR. Inhibition of target cell mitochondrial electron transfer by tumor necrosis factor. FEBS Lett 1989;248:169174. Higuchi M, Proske RJ, Yeh ET. Inhibition of mitochondrial respiratory chain complex I by TNF results in cytochrome c release, membrane permeability transition, and apoptosis. Oncogene 1998;17:2515-2524. Sanchez-Alcazar JA, Schneider E, Martinez MA, Carmona P, HernandezMunoz I, Siles E, De la Torre P, et al. Tumor necrosis factor-␣ increases the steady state reduction of cytochrome b of the mitochondrial respiratory chain in metabolically inhibited L929 cells. J Biol Chem 2000;275:1335313361. Wallace DC. Mitochondrial disease in man and mouse. Science 1999;283: 1482-1488. Fromenty B, Pessayre D. Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity. Pharmacol Ther 1995;67:101-154. Cadet J, Delatour T, Douki T, Gasparutto D, Pouget JP, Ravanat JL, Sauvaigo S. Hydroxyl radicals and DNA base damage. Mutat Res 1999; 424:9-21. Paradies G, Petrosillo G, Pistolese M, Ruggiero FM. The effect of reactive oxygen species generated from the mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles. FEBS Lett 2000;466:323-326. Ide T, Tsutsui H, Hayashidani S, Kang D, Suematsu N, Nakamura K, Utsumi H, et al. Mitochondrial DNA damage and dysfunction associated

40.

41. 42. 43. 44.

45.

46.

47.

48.

1007

with oxidative stress in failing hearts after myocardial infarction. Circ Res 2001;88:529-535. Crespo J, Cayo´ n A, Ferna´ndez-Gil P, Herna´ndez-Guerra M, Mayorga M, Domı´nguez-Dı´az A, Ferna´ndez-Escalante JC, et al. Gene expression of tumor necrosis factor ␣ and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients. HEPATOLOGY 2001;34:1158-1163. Tilg H, Diehl AM. Cytokines in alcoholic and nonalcoholic steatohepatitis. N Engl J Med 2000;343:1467-1476. Zeviani M, Tiranti V, Piantadosi C. Mitochondrial disorders. Medicine 1998;77:59-72. Collins AR. Oxidative DNA damage, antioxidants, and cancer. Bioessays 1999;21:238-246. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor alpha: direct role in obesity linked insulin resistance. Science 1993;259:87-91. Kern PA, Saghizadeh M, Ong JM, Bosch RJ, Deem R, Simsolo RB. The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss and relationship to lipoprotein lipase. J Clin Invest 1995;95:2111-2119. Peraldi P, Hotamisligil GS, Buurman WA, White MF, Spiegelman BM. Tumor necrosis factor (TNF)-alpha inhibits insulin signaling through stimulation of the p55TNF receptor and activation of sphingomyelinase. J Biol Chem 1996;271:13018-13022. Teoman Uysal K, Wiesbrock SM, Marino MW, Hotamisligil GS. Protection from obesity-induced insulin resistance in mice lacking TNF␣ function. Nature 1997;389:610-614. Li Z, Peraldi P, Yang S, Lin H, Huang J, Watkins PA, Moster AB, et al. Probiotics and antibodies to TNF inhibit inflammatory activity and improve nonalcoholic fatty liver disease. HEPATOLOGY 2003;37:343-350.