Cholestasis confers resistance to the rat liver mitochondrial permeability transition

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Cholestasis Confers Resistance to the Rat Liver Mitochondrial Permeability Transition MARK J. LIESER,* JOONGWON PARK,‡ SHIHO NATORI,‡ BLAKE A. JONES,‡ STEVEN F. BRONK,‡ and GREGORY J. GORES‡ *Department of Surgery, University of Texas Southwestern Medical Center, Dallas, Texas; and ‡Department of Medicine, Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota

See editorial on page 783. Background & Aims: Bile salts can cause hepatocyte death by inducing the mitochondrial permeability transition (MPT). However, the slow progression of human cholestatic liver diseases suggests that hepatocytes adapt to resist the MPT. Bcl-x, a protein, and increased mitochondrial cardiolipin, a membrane lipid, elevate the threshold for the MPT. Our aims were to determine if liver mitochondria become resistant to the MPT during cholestasis and, if so, if the resistance is mediated by Bcl-x and/or increased cardiolipin. Methods: Hepatocytes and liver mitochondria were obtained from bile duct–ligated (BDL) rats and shamoperated rats (control). Results: After addition of glycochenodeoxycholate (GCDC), the magnitude of the MPT was reduced in mitochondria from BDL rats vs. controls. Although Bcl-xL was not increased, mitochondrial cardiolipin content was significantly greater in BDL rats vs. controls. Cell viability was also increased in hepatocytes from BDL rats vs. controls after treatment with GCDC. Feeding BDL rats a fatty acid–deficient diet prevented the increase in mitochondrial cardiolipin content; mitochondria and hepatocytes from these rats were susceptible to the MPT and hepatocellular death by GCDC. Conclusions: These data suggest that an increase in mitochondria cardiolipin content occurs during cholestasis as an adaptive phenomenon to resist cell death by the MPT.

holestasis, defined physiologically as an impairment in bile flow, occurs in many chronic human liver diseases including primary biliary cirrhosis, primary sclerosing cholangitis, allograft rejection, malignant and iatrogenic obstruction of the bile ducts, and biliary atresia.1 Although the primary insult to the bile ducts is immunologic, toxic, or genetic in many of these diseases, progression of the liver disease seems to be promoted by a secondary chemical damage to hepatocytes by toxic hydrophobic bile salts.2,3 This concept is based on several independent observations. First, failure of bile salt excre-

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tion in cholestasis leads to retention and accumulation of hydrophobic bile salts within the hepatocyte.4,5 Second, hydrophobic bile salts are known to cause hepatocellular death.6–9 Finally, pharmacological administration of ursodeoxycholate, a hydrophilic bile salt that mitigates against injury by hydrophobic bile salts, improves the outcome of human cholestatic liver diseases,2,3 albeit, ursodeoxycholate may have direct cytoprotective effects independent of antagonizing the toxicity of hydrophobic bile salts. Although chronic human cholestatic liver diseases are associated with evidence of mild hepatocyte injury such as apoptosis, feathery degeneration, and bile infarcts, the massive hepatocellular injury associated with short-term bile salt infusion into animals does not occur in human cholestatic liver diseases despite elevated bile salt concentrations. This clinical observation suggests that adaptive responses may occur within hepatocytes, leading to resistance to cell death by toxic bile salts. Understanding the mechanisms of this potential adaptive response may lead to therapeutic insights to further treat these important liver diseases. The ability of the hepatocyte to resist injury during cholestasis is likely multifactorial. Conversion of toxic, hydrophobic bile salts to nontoxic, hydrophilic bile salts by sulfation is a prominent feature of cholestasis in humans and animals.10 Down-regulation of expression of the rat liver Na1/bile acid cotransporter also occurs in extrahepatic cholestasis, likely to prevent influx of toxic bile salts into the hepatocyte.11 However, the hepatocyte seems to have developed additional mechanisms to directly prevent bile salt cytotoxicity. These potentially important adaptive mechanisms to minimize bile salt cytotoxicity have not been investigated. Abbreviations used in this paper: BDL, bile duct–ligated (rats); EGTA, ethylene glycol-bis (b-aminoethyl ether)-N,N,N8,N8-tetraacetic acid; GCDC, glycochenodeoxycholate; MPT, mitochondrial permeability transition; TBS, Tris-buffered saline; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nickend labeling. r 1998 by the American Gastroenterological Association 0016-5085/98/$3.00

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Mitochondrial dysfunction is widely recognized as a key mechanism leading to cell death.12–14 In particular, the mitochondrial permeability transition (MPT), which is characterized by a rapid permeability of mitochondria to low-molecular-weight solutes, mitochondrial swelling, and collapse of the membrane potential, has been strongly implicated as a mechanism of cell death.12–14 We have previously shown that toxic bile salts can induce the MPT and that pharmacological inhibition of the MPT prevents bile salt–induced hepatocyte cytotoxicity.15 Inhibition of the MPT would be a potentially attractive, adaptive mechanism for the hepatocyte to resist cell death by toxic bile salts during cholestasis. The proteins Bcl-x and Bcl-2 and the lipid cardiolipin have been proposed as inhibitors of the MPT.16,17 However, rat hepatocytes do not express Bcl-2.18 Bcl-xL, the long splice variant of Bcl-x, is an anti–cell death member of the Bcl-2 family of proteins.19 Indeed, Bcl-xL is a potent inhibitor of cell death and may replace Bcl-2 in this function in the liver.18 Bcl-xL is localized to mitochondria and has been shown to homodimerize and heterodimerize with other members of the Bcl-2 family of proteins. Recently, this protein was found to regulate the membrane potential and volume homeostasis of mitochondria during a wide variety of cell death insults, thereby providing a mechanism for its antideath properties.20 Cardiolipin or diphosphatidylglycerol is a complex lipid localized predominantly in the inner mitochondrial membrane where it is synthesized.21 It is a negatively charged, major structural lipid of this membrane.17 In particular, increased mitochondrial cardiolipin content of the inner mitochondrial membrane may prevent the development of the MPT,17 and loss of mitochondrial cardiolipin occurs with the onset of the MPT. Thus, this structural lipid, in addition to proteins, also seems to regulate the threshold for the MPT. The specific aims of our study were to investigate if liver mitochondria from bile duct–ligated (BDL) rats is resistant to the MPT and, if so, if the resistance to the MPT is caused by an increase in mitochondrial Bcl-x and/or cardiolipin, using the BDL rat as a model of extrahepatic cholestasis. We chose glycochenodeoxycholate (GCDC) for these studies because it is a toxic, hydrophobic, primary bile salt whose concentration increases in cholestasis.5 Furthermore, we have previously shown that GCDC induces the MPT and causes hepatocyte death.6,7,15

Materials and Methods Bile Duct Ligation, Animal Models, and Cell Isolation The use and the care of the animals for these studies were reviewed and approved by the Institutional Animal Care and Use Committee at the Mayo Clinic. Male Fisher rats

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(200–300 g) were anesthetized with pentobarbital (50 mg/kg body wt intraperitoneally [IP]). The peritoneal cavity was opened, and the common bile duct was double-ligated and cut between the ligatures (BDL rat). Controls underwent a sham operation that consisted of exposure but not ligation of the common bile duct. For some experiments, rats were fed a standard diet (Purina Rodent Chow; Purina, Richmond, IN) or an essential fatty acid–deficient diet (ICN, Costa Mesa, CA) for 14 days before bile duct ligation. The animals underwent bile duct ligation for 10 days before isolation of the mitochondria or hepatocytes. At the time of bile duct ligation or following bile duct ligation, there was no difference in the weights of the two groups of animals. Hepatocyte suspensions were isolated from BDL and sham-operated adult male Fisher rats 10 days after the surgery and cultured as previously described by us.7 The purity of the cultured rat hepatocytes was assessed morphologically by counting hepatocytes vs. nonhepatocytes. The purity of the cultured hepatocytes was 98% 6 2%.

Mitochondrial Isolation Mitochondria were purified as previously described.15 Briefly, the rats were anesthetized with sodium pentobarbital (50 mg/kg IP), the peritoneal cavity was opened, and the liver was perfused in situ via the portal vein with 30–40 mL of ice-cold 0.25 mol/L sucrose containing 1 mmol/L ethylene glycol-bis(b-aminoethyl ether)-N,N,N8,N8-tetraacetic acid (EGTA), pH 7.4. The liver was removed from the animal, and 10–12 g of the liver were minced into small pieces (,1 cm) using scissors. The minced liver was placed in a Teflon glass Potter-Elvehjem homogenizer (Curtin Matheson Scientific, Inc., Houston, TX), and homogenate buffer (70 mmol/L sucrose, 220 mmol/L mannitol, 1 mmol/L EGTA, and 10 mmol/L HEPES, pH 7.4 at 4°C) was added to make a 10% homogenate. The liver tissue was gently homogenized at a speed of 800 rpm using six complete up and down strokes with a wall-mounted and speed-controlled mechanical skill drill and Teflon pestle (Electro-craft Servo Products, Eden Prairie, MN). The homogenate was placed in 50-mL conical plastic tubes and centrifuged at 600g for 10 minutes at 4°C using a Mistral 3000i centrifuge (Curtin Matheson Scientific). The postnuclear supernatant was collected and centrifuged in 50-mL polycarbonate centrifuge tubes (Beckman Instruments, Inc., Palo Alto, CA) at 7000g for 10 minutes using a Beckman Centrifuge Model J2-21 M/E and JA 20 rotor (Beckman Instruments). Mitochondria in the pellet were further purified by sucrosePercoll gradient centrifugation. The mitochondrial pellet was resuspended in 2 mL of homogenate buffer, and 1 mL each of the resuspended pellet was carefully layered on top of 35 mL of the sucrose-Percoll gradient. The sucrose-Percoll solution was prepared by mixing 75 mL of 0.25 mol/L sucrose containing 1 mmol/L EGTA and 25 mL of Percoll (density, 1.129 g/mL); a self-generating gradient was obtained by centrifuging 35 mL of the sucrose-Percoll solution at 43,000g for 20 minutes at 4°C in 50-mL polycarbonate tubes before layering on the mitochondrial suspension.5 The mitochondria were purified by centrifugation at 43,000g for 20 minutes at 4°C using a Beckman Centrifuge model J2-21 M/E and JA 20 rotor. The clear

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supernatant solution was carefully aspirated with a vacuum suction, and the bottom turbid layer was resuspended in 30 mL of wash buffer at 4°C (0.1 mol/L KCl, 5 mmol/L 3-[Nmorpholino]propanesulfonic acid, and 1 mmol/L EGTA at pH 7.4) and centrifuged at 7000g for 10 minutes at 4°C. The resultant pellet was washed by resuspension in wash buffer and repeating the centrifugation procedure twice more. The final pellet was suspended in 2–4 mL of a buffer containing 125 mmol/L sucrose, 50 mmol/L KCl, 5 mmol/L HEPES, and 2 mmol/L KH2PO4. The usual yield of mitochondria was 20–25 mg of protein per gram of liver. The purity of the mitochondria was assessed by electron microscopy as we have previously described in detail.15

Measurement of MPT in Isolated Mitochondria The MPT was measured using a standard spectrophotometric assay as previously described.15 This assay equates the MPT with high amplitude, rapid swelling of mitochondria. An increase in mitochondrial swelling results in a decrease in optical density. Isolated rat liver mitochondria (0.5 mg/mL) were suspended in incubation buffer at 25°C as previously described.15 Although additional calcium was not added to this buffer, there is considerable calcium present in our buffered water. Indeed, as determined by mass spectroscopy, the calcium concentration in our assay buffer was 1.5 µmol/L, a concentration similar to that observed in the cytosol of hepatocytes during bile salt cytotoxicity.7 The optical density was monitored at 540 nm in a Beckman DU 7400 Diode Array Spectrophotometer (Beckman Instruments) at 25°C. The initial mitochondrial absorbance was always 1.9–2.1 and was, therefore, virtually identical in mitochondria isolated from BDL and sham-operated animals.

Immunoblot Analysis for Bcl-x and Cytochrome c Mitochondrial proteins in 53 sample buffer (10% [vol/vol] b-mercaptoethanol, 25% [wt/vol] sodium dodecyl sulfate, 0.25% [wt/vol] bromophenol blue, 50% [vol/vol] glycerol, and 0.31 mol/L Tris-HCl, pH 6.8) were heated to 100°C on a water bath for 3 minutes. Proteins were separated on 14% polyacrylamide gels under reducing conditions and electroblotted to Nitrobind nitrocellulose membrane (Micron Separations, Inc., Westborough, MA). The nitrocellulose membranes were washed with Tris-buffered saline (TBS) (10 mmol/L Tris, pH 8.0, and 150 mmol/L NaCl) and then treated for 30 minutes with a solution consisting of 2% hydrogen peroxide, 20% methanol, and 100 mmol/L Tris, pH 7.4. The membrane was blocked in TBS with 0.05% Tween containing 5% nonfat dry milk, 2% bovine serum albumin, and 1% normal goat serum for 120 minutes at room temperature. Bcl-x was detected by incubating with a 1:40 dilution of a polyclonal rabbit anti–Bcl-x antibody (Calbiochem, Cambridge, MA) for 60 minutes at room temperature. The membrane was washed three times with TBS with 0.05% Tween and then incubated with goat anti-rabbit immunoglobulin peroxidase conjugate at a 1:3000 dilution for 60 minutes at room temperature.

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Cytochrome c was detected by incubating with a 1:500 dilution of a polyclonal mouse anti–cytochrome c antibody (Pharmingen, San Diego, CA) for 45 minutes at room temperature. The membrane was washed as described above and then incubated with goat anti-mouse immunoglobulin peroxidase conjugate at a 1:2000 dilution for 30 minutes at room temperature. For all immunoblots, membranes were washed three times with TBS with 0.05% Tween, once in TBS, and then visualized using the enhanced chemiluminescence kit (Amersham Life Science, Little Chalfont, Buckinghamshire, England) following the manufacturer’s directions.

Measurement of Mitochondrial Cardiolipin Content and Protein Mitochondrial cardiolipin content was quantitated using an established spectrophotometric assay using nonylacridine orange.22 Briefly, rat liver mitochondria (0.25 mg protein/mL) were suspended in 220 mmol/L mannitol, 70 mmol/L sucrose, 10 mmol/L HEPES, and 0.5 mmol/L EDTA, pH 7.4. One hundred fifty microliters of this suspension was added to increasing amounts of nonyl-acridine orange (1–15 µmol/L), and the final volume was adjusted to 1.5 mL with the suspension buffer. The samples were incubated for 5 minutes at room temperature and then centrifuged at 35,000g for 5 minutes using Beckman Centrifuge Model J2-21 M/E and JA18 rotor. The pellets were discarded and the amount of unbound nonyl-acridine orange in the supernatant measured spectrophotometrically at 495 nm. A standard curve was generated using nonyl-acridine orange (1–15 µmol/L) in the absence of mitochondria. The number of moles of nonylacridine orange per milligram of protein was calculated by subtracting the sample absorbance from that of the standard curve. Cardiolipin content was calculated as half of this value due to the 2:1 stoichiometric relationship between nonylacridine orange and cardiolipin. Protein was measured using the Bradford assay.

Measurement of Lipid Peroxidation Thiobarbituric acid–reactive substances were assayed in isolated mitochondria as an index of lipid peroxidation. Mitochondria (1 mg/mL) were suspended in the presence and absence of 400 µmol/L GCDC in an incubation buffer for 15 minutes. A fluorescent thiobarbituric assay was used to measure thiobarbituric acid reactive substances.23 Malondialdehyde bis(dimethylacetal) was used as a standard.

Cytotoxicity Assay Cell viability was determined using calcein acetoxymethyl ester.24 Calcein acetoxymethyl ester is a neutral, membrane-permeant, fluorogenic esterase substrate that is hydrolyzed by intracellular esterases to a polyanionic, membrane-impermeant, green fluorescent product, calcein. Thus, intracellular green fluorescence is an indicator of cell viability because it reflects the presence of an esterase activity and an intact membrane to retain calcein.24 Hepatocytes were loaded with calcein by incubating the cell in the presence of 3 µmol/L calcein acetoxymethyl ester for 30 minutes in media at 37°C.

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Calcein fluorescence was visualized using a phase/fluorescence microscope (Axiovert 35 mol/L; Carl Zeiss, Thornwood, NJ) with excitation and emission wavelengths of 490 nm and 520 nm, respectively. The number of live cells retaining calcein were quantitated and expressed as a percentage of the total number of cells present visualized by phase-contrast microscopy. At least 300 cells per high power field were counted; each experiment was performed in triplicate from three cell isolations.

TUNEL Assay for Determination of Cell Death in Liver Specimens BDL and sham-operated rats were anesthetized with pentobarbital, the peritoneal cavity was opened, the portal vein cannulated, and the liver flushed in situ with phosphatebuffered saline (PBS) for 5 minutes at 22°C. The liver was fixed in situ by perfusion with 200 mL of a freshly prepared 4% paraformaldehyde PBS solution at 22°C. After the perfusion fixation, the whole liver was removed from the carcass and cut into small tissue blocks (5 mm thick). The tissue blocks were further fixed by immersion in 4% paraformaldehyde PBS solution for 24 hours at 4°C. The tissue blocks were embedded in Tissue Path (Curtin Matheson Scientific Inc., Houston, TX). Tissue sections (4 mm) were prepared using a microtome (Reichert Scientific Instruments, Buffalo, NY) and placed on glass slides. The sections were deparaffinized in xylene and dehydrated in ethanol. The sections were incubated with 20 µg/mL of proteinase K (Boehringer Mannheim, Indianapolis, IN) in 10 mmol/L Tris buffer, pH 7.4, containing 5 mmol/L EDTA for 20 minutes at 37°C. After rinsing the specimen twice with double-distilled water, the sections were processed following the instructions of a commercial kit (In Situ Cell Death Detection Kit; Boehringer Mannheim). The specific assay employed uses terminal deoxynucleotidyl transferase to incorporate fluorescein-labeled deoxyuridine triphosphate to free 38-OH DNA ends. The number of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells (i.e., fluorescent nuclei) were counted in 30 random microscopic fields (6303) using a fluorescent microscope equipped with a fluorescein filter set.

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(density, 1.129 g/mL) was obtained from Pharmacia Fine Chemicals (Piscataway, NJ). Collagenase type D was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Dibutyl phthalate was from Fluka Chemicals, and sodium pentobarbital was obtained from Abbott Laboratories (Chicago, IL). HEPES and 3-(N-morpholino)propanesulfonic acid were from Research Organics Inc. (Cleveland, OH). The Bcl-xL antibody was from Calbiochem (Cambridge, MA). The essential fatty acid–deficient diet was obtained from ICN Pharmaceuticals, Inc. (Costa Mesa, CA). All other reagents were of analytical grade from the usual commercial sources.

Results Are liver mitochondria from BDL rats resistant to the MPT? The MPT was initially assessed in liver

mitochondria isolated from BDL and sham-operated rats using a spectrophotometric assay. Although we have previously showed the purity of mitochondria isolated from normal rat livers by Percoll-sucrose gradient centrifugation,15 we had not previously used this technique to obtain mitochondria from BDL rats. Therefore, we used electron microscopy to confirm the purity of the mitochondrial preparation obtained from BDL rats (Figure 1). Having verified the purity of the mitochondrial preparation, we next assayed the MPT in sham-operated vs. BDL rats. The MPT was maximal at a concentration of 400 µmol/L GCDC, but always greater in mitochondria from sham-operated vs. BDL rats (Figure 2). Therefore, we used this concentration of GCDC for the remainder of our experiments. At all time points assessed, the MPT was reduced in rat liver mitochondria from BDL rats vs. sham-operated rats using 400 µmol/L GCDC (Figure 2).

Statistical Analysis All data represent a minimum of three separate experiments and are expressed as means 6 SE unless otherwise indicated. Differences between groups were analyzed using analysis of variance for repeated measures. A post hoc analysis using the Bonferroni test was used to account for multiple comparisons. All statistical analyses were performed using the statistical software package InStat from GraphPAD (San Diego, CA).

Materials KCl, EGTA, EDTA, succinate, rotenone, oligomycin, glutamate, malate, GCDC, chelex-100, sucrose, mannitol, calcium chloride, KH2PO4, and nonyl-acridine orange were all obtained from Sigma Chemical Co. (St. Louis, MO). Percoll

Figure 1. Electron microscopy of mitochondria isolated from a BDL rat. Electron microscopy of mitochondria isolated from rat liver and purified by sucrose-Percoll gradient centrifugation shows minimal contamination by other organelles (original magnification 15,0003).

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Figure 2. Liver mitochondria from BDL rats are resistant to the GCDC-induced MPT. Liver mitochondria were purified from BDL rats and sham-operated rats by sucrose-Percoll gradient centrifugation. Isolated mitochondria (1 mg protein/mL) were suspended in buffer at 25°C. Large-amplitude swelling of suspended mitochondria was measured by monitoring the optical density at 540 nm. At time zero, (A) 400 µmol/L GCDC or (B) 100 µmol/L Ca21 was added, and mitochondrial swelling was monitored for an additional 6 minutes. (A) Concentration-dependence of the GCDC-induced MPT. (B) Time dependence of the 400 µmol/L GCDC-induced MPT.

Indeed, after 7 minutes of incubation, the MPT induced by GCDC was reduced by 57% 6 9% in mitochondria from BDL rats vs. sham-operated rats. The mitochondrial swelling induced by GCDC was inhibited by cyclosporin A plus trifluoperazine in both mitochondria from BDL and sham-operated rats (data not shown) consistent with our previously published observations showing that the GCDC-mediated MPT is inhibitable by these agents.15 These data suggest that liver mitochondria adapt during extrahepatic cholestasis by increasing their threshold for the MPT to prevent cell death by toxic bile salts. Is the resistance of liver mitochondria from BDL rats to the MPT due to an increase in mitochondrial Bcl-x and/or cardiolipin content? Having shown a rela-

tive resistance of BDL rat liver mitochondria to the MPT, we sought to determine the potential biochemical mecha-

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nism(s) responsible for the relative resistance of these mitochondria to the MPT. Although the precise biochemical pathways mediating the MPT remain undefined, both Bcl-xL, a protein, and cardiolipin, a complex phospholipid, have been reported to inhibit the MPT.16,17 Unexpectedly, Bcl-xL was slightly lower in mitochondria isolated from BDL rats compared with sham-operated controls as assessed by immunoblot analysis (Figure 3). Indeed, densitometry analysis of the immunoblots showed a 26% 6 8% decrease in Bcl-xL in liver mitochondria from BDL rats compared with sham-operated animals. These data suggest that Bcl-xL does not mediate the relative resistance of mitochondria from BDL rats to the MPT. In contrast, mitochondria from BDL rats have an increased cardiolipin content vs. sham-operated controls (Figure 4). Liver mitochondrial cardiolipin content was 40% 6 3% greater in BDL rats vs. sham-operated animals. These data show an association between increased mitochondrial cardiolipin content and relative resistance of mitochondrial to the MPT. Cardiolipin is thought to inhibit the MPT by providing an increased pool of negative charges that nonspecifically bind Ca21, thereby preventing Ca21 from binding to protein sites that induce pore opening.17 If cardiolipin is responsible for inhibiting the MPT, it should be possible to saturate the cardiolipin:calcium binding sites and induce the MPT by increasing the Ca21 concentration. Indeed, increasing Ca21 concentrations in the media triggered the MPT in isolated mitochondria from BDL rats, and Ca21 concentrations of .200 µmol/L rapidly induced the MPT (Figure 5). These data provide circumstantial evidence that increased cardiolipin may mediate the resistance of liver mitochondria from BDL rats to the MPT. Does feeding BDL rats a fatty acid–deficient diet prevent the increase in mitochondrial cardiolipin content and restore sensitivity to the GCDC-mediated MPT and cytotoxicity? The above data showing increased

Figure 3. Mitochondria from BDL rats have decreased Bcl-xL compared with sham-operated controls. Liver mitochondria were purified from BDL rats and sham-operated rats by sucrose-Percoll gradient centrifugation. Mitochondrial protein (150 µg/lane) was separated on 12% polyacrylamide gels under reducing conditions and electroblotted to nitrocellulose membranes. Bcl-x polyclonal rabbit anti-rat/mouse antisera was used to identify Bcl-x long. Enhanced chemiluminescence was used to identify the bands. The gel was exposed to x-ray film for 5 minutes.

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Figure 4. Liver mitochondria isolated from BDL rats fed a standard diet, but not a fatty acid–deficient diet, have increased cardiolipin content. Liver mitochondria were purified from BDL rats and shamoperated rats fed a standard diet or an essential fatty acid–deficient diet. Mitochondrial cardiolipin content was determined using a spectrophotometric assay using nonyl-acridine orange. Mitochondrial cardiolipin was statistically greater in BDL rats fed a standard diet (26 6 3 nmol/mg protein in controls fed a standard diet; 40 6 3 nmol/mg protein in the BDL rats fed a standard diet; 25 6 2 nmol/mg protein in controls fed an essential fatty acid–deficient diet; and 26 6 2 nmol/mg protein in BDL rats fed an essential fatty acid–deficient diet; analysis of variance, P , 0.05).

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BDL and sham-operated rats. We first confirmed that an essential fatty acid–deficient diet prevents an increase in cardiolipin content in BDL rat liver mitochondria (Figure 4). In rats fed an essential fatty acid–deficient diet, mitochondrial cardiolipin content did not increase after BDL and was similar to control values (26 6 3 vs. 26 6 4; P 5 NS). Having confirmed that BDL rats fed an essential fatty acid–deficient diet do not increase their mitochondrial cardiolipin content, we next determined the susceptibility of mitochondria from BDL and essential fatty acid–deficient rats to the MPT. At all time points after the addition of GCDC or Ca21, the onset and magnitude of the MPT was nearly identical in both liver mitochondria from BDL and sham-operated rats (Figure 6). Because an essential fatty acid–deficient diet could potentially alter the susceptibility of the mitochondrial membrane to lipid peroxidation, an important variable in assessing the MPT, we also measured thiobarbituric acid–reactive substances as an index of lipid peroxidation in mitochondria from BDL rats fed a standard diet and an

mitochondrial cardiolipin content and an elevated threshold for the MPT in extrahepatic cholestasis do not establish a cause and effect relationship between these two observations. Therefore, we determined if preventing an increase in mitochondrial cardiolipin content during cholestasis restores susceptibility to the MPT. Mitochondrial cardiolipin content was decreased by feeding rats an essential fatty acid–deficient diet for 3 weeks, a wellestablished approach to reducing cardiolipin synthesis.21 After feeding rats the essential fatty acid–deficient diet for 3 weeks, we then repeated our previous experiments in

Figure 5. Increased Ca21 concentrations are required to induce the MPT in mitochondria isolated from BDL rats. Liver mitochondria were purified from BDL rats. Isolated mitochondria (1 mg protein/mL) were suspended in buffer at 25°C. Large-amplitude swelling of suspended mitochondria was measured by monitoring the optical density at 540 nm after addition of varying calcium concentrations. The magnitude of mitochondrial swelling was determined 7 minutes after addition of Ca21.

Figure 6. Liver mitochondria from BDL rats fed an essential fatty acid–deficient diet are not resistant to the MPT. Liver mitochondria were purified from sham-operated rats fed a standard diet and from BDL rats fed an essential fatty acid diet. Isolated mitochondria (1 mg protein/mL) were suspended in buffer at 25°C. Large-amplitude swelling of suspended mitochondria was measured by monitoring the optical density at 540 nm. At time zero, (A) 400 µmol/L GCDC or (B) 100 µmol/L Ca21 was added, and mitochondrial swelling was monitored for an additional 10 minutes.

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essential fatty acid–deficient diet (Table 1). There was no significant difference in thiobarbituric acid–reactive substances between mitochondria isolated from the two groups of animals in the absence or presence of 400 µmol/L GCDC. Thus, the increased susceptibility of mitochondria from BDL rats fed an essential fatty acid–deficient diet cannot be explained by a greater susceptibility of the mitochondria to lipid peroxidation. These composite data suggest that lowering mitochondrial cardiolipin content during extrahepatic cholestasis restores the susceptibility of the mitochondria to the MPT. Are hepatocytes from BDL rats resistant to GCDC-mediated cytotoxicity? The MPT is thought to

be a key mechanism mediating cell death.12 Thus, if hepatocyte mitochondria become relatively resistant to the MPT during extrahepatic cholestasis, the hepatocytes should be less susceptible to bile salt–mediated cytotoxicity. To test this hypothesis, we isolated and cultured hepatocytes from BDL and sham-operated rats and measured loss of cell viability during incubation with 400 µmol/L GCDC (Table 2). After 4 hours of incubation with 400 µmol/L GCDC, the viability of hepatocytes from BDL rats was increased 46% compared with sham-operated controls (78% 6 1% vs. 32% 6 4%; P , 0.01). Consistent with the data on the MPT, hepatocytes isolated from BDL rats fed an essential fatty acid– deficient diet were not resistant to bile salt–mediated cytotoxicity compared with hepatocytes isolated from sham-operated control rats (46% 6 4% vs. 32% 6 4%; P 5 NS). Furthermore, hepatocyte death as assessed by the TUNEL assay was greater in rat livers from BDL rats fed an essential fatty acid–deficient diet compared with Table 1. Lipid Peroxidation in Untreated and GCDC-Treated Mitochondria Thiobarbituric acid–reactive substances

Treatment Control Standard diet (2) EFA diet P value BDL Standard diet (2) EFA diet P value

No treatment

GCDC

P value

20 6 6 30 6 6 NS

21 6 3 14 6 4 NS

NS NS

41 6 15 71 6 13 NS

34 6 6 54 6 16 NS

NS NS

NOTE. Mitochondria were isolated from sham-operated (controls) and BDL rats fed either a standard diet or an essential fatty acid–deficient diet ([2] EFA diet) and suspended in incubation buffer (1 mg/mL). After 15 minutes of incubation at 37°C in the presence or absence of 400 µmol/L GCDC, the mitochondria were quenched by adding trichloroacetic acid (final concentration, 10%). Thiobarbituric acid– reactive substances were measured as an index of lipid peroxidation using a fluorescent assay as described in Materials and Methods.

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Table 2. Hepatocytes Isolated From BDL Rats Fed a Standard Diet, but Not Essential Fatty Acid–Deficient Diet, Are Resistant to GCDC-Induced Cell Death Cell viability (% )

Treatment Control Standard diet (2) EFA diet Difference (% ) P value BDL rats Standard diet (2) EFA diet Difference (% ) P value

Difference (% )

P value

23 6 2 32 6 4 9 NS

59 49

,0.01 ,0.01

78 6 1 46 6 4 29 ,0.01

12 36

NS ,0.01

No treatment

GCDC

82 6 1 81 6 1 1 NS 80 6 1 85 6 2 5 NS

NOTE. Hepatocytes were isolated from sham-operated (controls) and BDL rats fed either a standard diet or an essential fatty acid–deficient diet ([2] EFA diet) and cultured as previously described. After 4 hours in primary culture, the hepatocytes were cultured in media in the absence or presence of 400 µmol/L GCDC. At selected time points, the cells were then loaded with calcein acetoxymethyl ester and cell death was quantitated as described in Materials and Methods.

BDL rats fed a standard diet, 7.8 6 0.7 vs. 1.3 6 0.2 per high-power field; P , 0.01 (Figure 7). Minimal cell death was observed in nonbile duct–ligated animals fed either a standard diet or a fatty acid–deficient diet 0.08% 6 0.4% vs. 0.07% 6 0.02%, respectively (Figure 7). Thus, these data suggest that hepatocytes adapt during cholestasis to resist bile salt cytotoxicity by increasing mitochondrial cardiolipin content to prevent the MPT.

Discussion Our results show that during extrahepatic cholestasis, liver mitochondria have an elevated threshold for the MPT. The MPT is thought to be mediated by a highconductance proteinaceous pore.12 The precise structure of the pore remains unknown, and multiple mechanisms for pore formation may occur. However, one of the current models suggests that the transition pore develops at contact sites of the inner and outer mitochondrial membrane by interactions between the adenine nucleotide translocator of the inner mitochondrial membrane and the voltage-dependent anion channel of the outer mitochondrial membrane.14 Increases of mitochondrial Ca21 concentrations are thought to promote this interaction, and pore formation is strongly Ca21 dependent.12 Bcl-2 and Bcl-xL survival proteins are localized to mitochondria and may inhibit the MPT through their putative channel functions.16 Presumably, the channel functions of these proteins would regulate ion flux and membrane potential to keep the pore closed. However, hepatocytes do not express Bcl-2, and hepatocyte mito-

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Figure 7. Hepatocyte death as assessed by the TUNEL assay is greater in rat livers from BDL rats fed an essential fatty acid– deficient diet ([-] EFA Diet) compared with BDL rats fed a standard diet (Std. Diet). Rats were fed a standard diet or an essential fatty acid–deficient diet for 14 days before bile duct ligation. The animals were BDL for 10 days, and then the livers were per fusion-fixed with 4% paraformaldehyde PBS solution at 22°C. Controls underwent a sham operation and then were fed either a standard diet or an essential fatty acid diet. The TUNEL assay was performed as described in Materials and Methods.

chondrial Bcl-xL actually decreases slightly during cholestasis, making it an unlikely inhibitor of the MPT in this disease model. The membrane lipid composition has also been found to modulate the MPT.17 In particular, increased mitochondrial cardiolipin content of the inner mitochondrial membrane may prevent the development of the MPT.17 Moreover, loss of mitochondrial cardiolipin as assessed by nonyl-acridine orange fluorescence occurs simultaneously with the onset of the MPT in dexamethasone-induced thymocyte apoptosis.25 Cardiolipin has been proposed to mediate its regulation of the MPT by its strongly negatively charged headgroups. This increased pool of negative charges is thought to nonspecifically bind Ca21, preventing it from acting on protein sites that induce pore opening. Several observations from the current study suggest that increased mitochondrial cardiolipin is responsible for the resistance of mitochondria from BDL rats to the MPT. First, mitochondrial cardiolipin is increased in mitochondria from BDL rats. Second, increasing Ca21 concentrations, which would saturate the increased cardiolipin/Ca21 binding sites, induces the MPT. Finally, preventing an increase in mitochondrial cardiolipin by feeding BDL rats an essential fatty acid– deficient diet restores the sensitivity of the mitochondria to the MPT, and increases hepatocyte cell death in vivo in the BDL rat and in vitro during incubation of cultured cells with GCDC. Cardiolipin is the only phospholipid synthesized by the mitochondria. The final step in cardiolipin synthesis is catalyzed by cardiolipin synthase, an enzyme located in the inner mitochondria membrane.21 Our data suggest mitochondria may either alter phospholipid metabolism to enhance the synthesis of cardiolipin or prevent its turnover in extrahepatic cholestasis. A logical extension of our current studies is to evaluate the regulation of

cardiolipin synthase (i.e., rates of transcription and translation and the protein’s half-life) and the availability of its substrates in cholestasis. However, these studies are beyond the scope of the current study. Decreasing substrate availability for cardiolipin synthesis by feeding rats an essential fatty acid–deficient diet did not decrease mitochondrial cardiolipin content below control values. Although this may seem unexpected, our observations are consistent with the experience of others. In the absence of dietary substrates, intracellular salvage pathways are used to help provide substrates for cardiolipin synthesis to maintain the structure and function of mitochondria.21 However, the salvage pathways are insufficient to allow for an increase in cardiolipin synthesis during cholestasis, as shown in our studies. We found that hepatocytes isolated and cultured from BDL rats were relatively resistant to GCDC cytotoxicity. Based on morphological criteria, cell death is thought to occur by either necrosis or apoptosis. However, cell death is better defined by the biochemical and cellular pathways leading to cell demise rather than on the basis of morphological criteria. Indeed, the MPT has been strongly implicated in both necrosis and apoptosis, suggesting that dysregulation of the permeability transition pore is an important biochemical pathway for cell death.12–14 The MPT is thought to cause cell death by causing uncoupling of mitochondria with enhanced formation of superoxide anion and adenosine triphosphate depletion or by releasing pro–cell death effectors such as cytochrome c.14,26,27 The plasma membrane permeability barrier (identified by a loss of calcein from the cells in our current study) is a feature of cell death associated with the MPT.13,27 Therefore, our results are germane to cell death pathways associated with the MPT and loss of the plasma membrane permeability barrier.

September 1998

In conclusion, our working hypothesis is that, during cholestasis, there is retention and accumulation of toxic bile salts within hepatocytes. The resulting increased hepatocellular concentration of these compounds presents a cytotoxic stimulus to the hepatocyte. However, cholestasis is also associated with adaptive phenomenon such as increasing synthesis or reducing degradation of mitochondrial cardiolipin leading to a net increase in mitochondrial cardiolipin content. The increased mitochondrial cardiolipin content increases the mitochondrial threshold for the MPT. The elevated threshold for the MPT renders the hepatocyte less vulnerable to cell death by toxic bile salts and perhaps other pathophysiological disturbances caused by cholestasis. The ability to prevent the MPT and hepatocyte cell death may represent a cytoprotective adaptation that helps permit survival and continued function of hepatocytes and the liver despite cholestatic dysfunction.

CHOLESTASIS, MITOCHONDRIA, AND CELL DEATH

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Received October 17, 1997. Accepted May 11, 1998. Address requests for reprints to: Gregory J. Gores, M.D., Department of Medicine, Mayo Medical School, Clinic, and Foundation, 200 First Street Southwest, Rochester, Minnesota 55905. e-mail: [email protected]; fax: (507) 284-0762. Supported by grants from the National Institutes of Health (DK 41876), by the Gainey Foundation, St. Paul, Minnesota, and by the Mayo Foundation, Rochester, Minnesota. The authors thank Sara Erickson for secretarial assistance.