Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy.

Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C(18)-pyridinium ceramide treatment or endogenous C(18)-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C(18)-ceramide-induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 ß-lipidation, forming LC3B-II, and selective targeting of mitochondria by LC3B-II-containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite-induced mitophagy, and stable LC3B knockdown protected against CerS1- and C(18)-ceramide-dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a new receptor function of ceramide for anchoring LC3B-II autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.

LCL124, a cationic analog of ceramide, selectively induces pancreatic cancer cell death by accumulating in mitochondria.

Treatment of pancreatic cancer that cannot be surgically resected currently relies on minimally beneficial cytotoxic chemotherapy with gemcitabine. As the fourth leading cause of cancer-related death in the United States with dismal survival statistics, pancreatic cancer demands new and more effective treatment approaches. Resistance to gemcitabine is nearly universal and appears to involve defects in the intrinsic/mitochondrial apoptotic pathway. The bioactive sphingolipid ceramide is a critical mediator of apoptosis initiated by a number of therapeutic modalities. It is noteworthy that insufficient ceramide accumulation has been linked to gemcitabine resistance in multiple cancer types, including pancreatic cancer. Taking advantage of the fact that cancer cells frequently have more negatively charged mitochondria, we investigated a means to circumvent resistance to gemcitabine by targeting delivery of a cationic ceramide (l-t-C6-CCPS [LCL124: ((2S,3S,4E)-2-N-[6'-(1″-pyridinium)-hexanoyl-sphingosine bromide)]) to cancer cell mitochondria. LCL124 was effective in initiating apoptosis by causing mitochondrial depolarization in pancreatic cancer cells but demonstrated significantly less activity against nonmalignant pancreatic ductal epithelial cells. Furthermore, we demonstrate that the mitochondrial membrane potentials of the cancer cells were more negative than nonmalignant cells and that dissipation of this potential abrogated cell killing by LCL124, establishing that the effectiveness of this compound is potential-dependent. LCL124 selectively accumulated in and inhibited the growth of xenografts in vivo, confirming the tumor selectivity and therapeutic potential of cationic ceramides in pancreatic cancer. It is noteworthy that gemcitabine-resistant pancreatic cancer cells became more sensitive to subsequent treatment with LCL124, suggesting that this compound may be a uniquely suited to overcome gemcitabine resistance in pancreatic cancer.

Sphingosine kinase-2 inhibition improves mitochondrial function and survival after hepatic ischemia-reperfusion.

BACKGROUND & AIMS: The mitochondrial permeability transition (MPT) and inflammation play important roles in liver injury caused by ischemia-reperfusion (IR). This study investigated the roles of sphingosine kinase-2 (SK2) in mitochondrial dysfunction and inflammation after hepatic IR. METHODS: Mice were gavaged with vehicle or ABC294640 (50 mg/kg), a selective inhibitor of SK2, 1 h before surgery and subjected to 1 h-warm ischemia to ~70% of the liver followed by reperfusion. RESULTS: Following IR, hepatic SK2 mRNA and sphingosine-1-phosphate (S1P) levels increased ~25- and 3-fold, respectively. SK2 inhibition blunted S1P production and liver injury by 54-91%, and increased mouse survival from 28% to 100%. At 2 h after reperfusion, mitochondrial depolarization was observed in 74% of viable hepatocytes, and mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. SK2 inhibition decreased mitochondrial depolarization and prevented MPT onset. Inducible nitric oxide synthase, phosphorylated NFκB-p65, TNFα mRNA, and neutrophil infiltration, all increased markedly after hepatic IR, and these increases were blunted by SK2 inhibition. In cultured hepatocytes, anoxia/re-oxygenation resulted in increases of SK2 mRNA, S1P levels, and cell death. SK2 siRNA and ABC294640 each substantially decreased S1P production and cell death in cultured hepatocytes. CONCLUSIONS: SK2 plays an important role in mitochondrial dysfunction, inflammation responses, hepatocyte death, and survival after hepatic IR and represents a new target for the treatment of IR injury.

Mitochondrial uncoupling protein-2 deficiency protects steatotic mouse hepatocytes from hypoxia/reoxygenation.

Steatotic livers are sensitive to ischemic events and associated ATP depletion. Hepatocellular necrosis following these events may result from mitochondrial uncoupling protein-2 (UCP2) expression. To test this hypothesis, we developed a model of in vitro steatosis using primary hepatocytes from wild-type (WT) and UCP2 knockout (KO) mice and subjected them to hypoxia/reoxygenation (H/R). Using cultured hepatocytes treated with emulsified fatty acids for 24 h, generating a steatotic phenotype (i.e., microvesicular and broad-spectrum fatty acid accumulation), we found that the phenotype of the WT and UCP2 KO were the same; however, cellular viability was increased in the steatotic KO hepatocytes following 4 h of hypoxia and 24 h of reoxygenation; Hepatocellular ATP levels decreased during hypoxia and recovered after reoxygenation in the control and UCP2 KO steatotic hepatocytes but not in the WT steatotic hepatocytes; mitochondrial membrane potential in WT and UCP2 KO steatotic groups was less than control groups but higher than UCP2 KO hepatocytes. Following reoxygenation, lipid peroxidation, as measured by thiobarbituric acid reactive substances, increased in all groups but to a greater extent in the steatotic hepatocytes, regardless of UCP2 expression. These results demonstrate that UCP2 sensitizes steatotic hepatocytes to H/R through mitochondrial depolarization and ATP depletion but not lipid peroxidation.

Minocycline and N-methyl-4-isoleucine cyclosporin (NIM811) mitigate storage/reperfusion injury after rat liver transplantation through suppression of the mitochondrial permeability transition.

UNLABELLED: Graft failure after liver transplantation may involve mitochondrial dysfunction. We examined whether prevention of mitochondrial injury would improve graft function. Orthotopic rat liver transplantation was performed after 18 hours' cold storage in University of Wisconsin solution and treatment with vehicle, minocycline, tetracycline, or N-methyl-4-isoleucine cyclosporin (NIM811) of explants and recipients. Serum alanine aminotransferase (ALT), necrosis, and apoptosis were assessed 6 hours after implantation. Mitochondrial polarization and cell viability were assessed by intravital microscopy. Respiration and the mitochondrial permeability transition (MPT) were assessed in isolated rat liver mitochondria. After transplantation with vehicle or tetracycline, ALT increased to 5242 U/L and 4373 U/L, respectively. Minocycline and NIM811 treatment decreased ALT to 2374 U/L and 2159 U/L, respectively (P < 0.01). Necrosis and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) also decreased from 21.4% and 21 cells/field, respectively, after vehicle to 10.1% and 6 cells/field after minocycline and to 8.7% and 5.2 cells/field after NIM811 (P < 0.05). Additionally, minocycline decreased caspase-3 activity in graft homogenates (P < 0.05). Long-term graft survival was 27% and 33%, respectively, after vehicle and tetracycline treatment, which increased to 60% and 70% after minocycline and NIM811 (P < 0.05). In isolated mitochondria, minocycline and NIM811 but not tetracycline blocked the MPT. Minocycline blocked the MPT by decreasing mitochondrial Ca(2+) uptake, whereas NIM811 blocks by interaction with cyclophilin D. Intravital microscopy showed that minocycline and NIM811 preserved mitochondrial polarization and cell viability after transplantation (P < 0.05). CONCLUSION: Minocycline and NIM811 attenuated graft injury after rat liver transplantation and improved graft survival. Minocycline and/or NIM811 might be useful clinically in hepatic surgery and transplantation.

NIM811 (N-methyl-4-isoleucine cyclosporine), a mitochondrial permeability transition inhibitor, attenuates cholestatic liver injury but not fibrosis in mice.

Cholestasis causes hepatocyte death, possibly because of mitochondrial injury. This study investigated whether NIM811 (N-methyl-4-isoleucine cyclosporine), an inhibitor of the mitochondrial permeability transition (MPT), attenuates cholestatic liver injury in vivo. Cholestasis was induced in mice by bile duct ligation (BDL). NIM811 was gavaged (20 mg/kg) before BDL and daily (10 mg/kg) afterward. Mitochondrial depolarization, cell death, and MPT onset were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. After BDL, serum alanine aminotransferase (ALT), hepatic necrosis, and apoptosis all increased. NIM811 decreased ALT, necrosis, and apoptosis by 60 to 86%. In vehicle-treated mice at 6 h after BDL, viable hepatocytes with depolarized mitochondria were 18/high-power field (hpf), and nonviable cells were approximately 1/hpf, showing that depolarization preceded necrosis. Calcein entered mitochondria after BDL, indicating MPT onset in vivo. NIM811 decreased depolarization by 72%, prevented calcein entry into mitochondria, and blocked release of cytochrome c. Hepatic tumor necrosis factor alpha, transforming growth factor-beta1, procollagen alpha1(I) mRNA, alpha-smooth muscle actin, and Sirius red staining for collagen increased after BDL but were not different in vehicle- and NIM811-treated mice. Taken together, NIM811 decreased cholestatic necrosis and apoptosis but did not block fibrosis, indicating that the MPT plays an important role in cholestatic cell death in vivo.

Ischemic preconditioning prevents free radical production and mitochondrial depolarization in small-for-size rat liver grafts.

BACKGROUND: Ischemic preconditioning (IP) renders tissues more tolerant to subsequent longer episodes of ischemia. This study tested whether IP attenuates injury of small-for-size liver grafts by preventing free radical production and mitochondrial dysfunction. METHODS: IP was induced by clamping the portal vein and hepatic artery for 9 min. Livers were harvested 5 min after releasing the clamp. Mitochondrial polarization and cell death were assessed by intravital confocal/multiphoton microscopy of rhodamine 123 (Rh123) and propidium iodide. Free radicals were trapped with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone and measured using electron spin resonance. RESULTS: After quarter-size liver transplantation, alanine aminotransferase, serum bilirubin, necrosis, and apoptosis all increased. IP blocked these increases by more than 58%. 5-Bromo-2'-deoxyuridine labeling and increases of graft weight were only approximately 3% and 0.2% in quarter-size grafts without IP, respectively, but increased to 32% and 60% in ischemic-preconditioned grafts, indicating better liver regeneration. Eighteen hours after implantation, viable cells with depolarized mitochondria in quarter-size grafts were 15 per high power field, and dead cells were less than 1 per high power field, indicating that depolarization preceded necrosis. A free radical adduct signal was detected in bile from quarter-size grafts. IP decreased this free radical formation and prevented mitochondrial depolarization. IP did not increase heat shock proteins 10, 27, 32, 60, 70, 72, 75 and Cu/Zn-superoxide dismutase (SOD) but increased heat shock protein-90, a chaperone that facilitates protein import into mitochondria, and mitochondrial Mn-SOD. CONCLUSION: Taken together, IP decreases injury and improves regeneration of small-for-size liver grafts, possibly by increasing mitochondrial Mn-SOD, thus protecting against free radical production and mitochondrial dysfunction.

Pinhole shifting lifetime imaging microscopy.

Lifetime imaging microscopy is a powerful tool to probe biological phenomena independent of luminescence intensity and fluorophore concentration. We describe time-resolved imaging of long-lifetime luminescence with an unmodified commercial laser scanning confocal/multiphoton microscope. The principle of the measurement is displacement of the detection pinhole to collect delayed luminescence from a position lagging the rasting laser beam. As proof of principle, luminescence from microspheres containing europium (Eu(3+)), a red emitting probe, was compared to that of short-lifetime green-fluorescing microspheres and/or fluorescein and rhodamine in solution. Using 720-nm two-photon excitation and a pinhole diameter of 1 Airy unit, the short-lifetime fluorescence of fluorescein, rhodamine and green microspheres disappeared much more rapidly than the long-lifetime phosphorescence of Eu(3+) microspheres as the pinhole was repositioned in the lagging direction. In contrast, repositioning of the pinhole in the leading and orthogonal directions caused equal loss of short- and long-lifetime luminescence. From measurements at different lag pinhole positions, a lifetime of 270 micros was estimated for the Eu(3+) microspheres, consistent with independent measurements. This simple adaptation is the basis for quantitative 3-D lifetime imaging microscopy.

Imaging of Mitochondrial pH Using SNARF-1.

Laser scanning confocal microscopy provides the ability to image submicron sections in living cells and tissues. In conjunction with pH-indicating fluorescent probes, confocal microscopy can be used to visualize the distribution of pH inside living cells. Here we describe a confocal microscopic technique to image intracellular pH in living cells using carboxyseminaphthorhodafluor-1 (SNARF-1), a ratiometric pH-indicating fluorescent probe. SNARF-1 is ester-loaded into the cytosol and mitochondria of adult cardiac myocytes or other cell type. Using 568-nm excitation, emitted fluorescence longer and shorter than 595-nm is imaged and then ratioed after background subtraction. Ratio values for each pixel are converted to values of pH using a standard curve (lookup table). Images of the intracellular distribution of pH show cytosolic and nuclear areas to have a pH of ~7.1, but in regions corresponding to mitochondria, pH is 8.0, giving a mitochondrial ΔpH of 0.9. During hypoxia, mitochondrial pH decreases to cytosolic values, signifying the collapse of ΔpH. These results illustrate the ability of laser scanning confocal microscopy to image the intracellular distribution of pH in living cells and to determine mitochondrial ΔpH.

Compartmentation of Mitochondrial and Oxidative Metabolism in Growing Hair Follicles: A Ring of Fire.

Little is known about the energetics of growing hair follicles, particularly in the mitochondrially abundant bulb. Here, mitochondrial and oxidative metabolism was visualized by multiphoton and light sheet microscopy in cultured bovine hair follicles and plucked human hairs. Mitochondrial membrane potential (ΔΨ), cell viability, reactive oxygen species (ROS), and secretory granules were assessed with parameter-indicating fluorophores. In growing follicles, lower bulb epithelial cells had high viability, and mitochondria were polarized. Most epithelially generated ROS co-localized with polarized mitochondria. As the imaging plane captured more central and distal cells, ΔΨ disappeared abruptly at a transition to a nonfluorescent core continuous with the hair shaft. Approaching the transition, ΔΨ and ROS increased, and secretory granules disappeared. ROS and ΔΨ were strongest in a circumferential paraxial ring at putative sites for formation of the outer cortex/cuticle of the hair shaft. By contrast, polarized mitochondria in dermal papillar fibroblasts produced minimal ROS. Plucked hairs showed a similar abrupt transition of degranulation/depolarization near sites of keratin deposition, as well as an ROS-generating paraxial ring of fire. Hair movement out of the follicle appeared to occur independently of follicular bulb bioenergetics by a tractor mechanism involving the inner and outer root sheaths.

Use of light absorbers to alter optical interrogation with epi-illumination and transillumination in three-dimensional cardiac models.

Cardiac optical mapping currently provides 2-D maps of transmembrane voltage-sensitive fluorescence localized near the tissue surface. Methods for interrogation at different depths are required for studies of arrhythmias and the effects of defibrillation shocks in 3-D cardiac tissue. We model the effects of coloading with a dye that absorbs excitation or fluorescence light on the radius and depth of the interrogated region with specific illumination and collection techniques. Results indicate radii and depths of interrogation are larger for transillumination versus epi-illumination, an effect that is more pronounced for broad-field excitation versus laser scanner. Coloading with a fluorescence absorber lessens interrogated depth for epi-illumination and increases it for transillumination, which is confirmed with measurements using transillumination of heart tissue slices. Coloading with an absorber of excitation light consistently decreases the interrogated depths. Transillumination and coloading also decrease the intensities of collected fluorescence. Thus, localization can be modified with wavelength-specific absorbers at the expense of a reduction in fluorescence intensity.

Ethanol and High Cholesterol Diet Causes Severe Steatohepatitis and Early Liver Fibrosis in Mice.

BACKGROUND AND AIM: Because ethanol consumption is commonly associated with a high cholesterol diet, we examined whether combined consumption of ethanol and high cholesterol increases liver injury and fibrosis. METHODS: Male C57BL/6J mice were fed diets containing: 1) 35% of calories from corn oil (CTR), 2) CTR plus 0.5% (w/v) cholesterol (Chol), 3) CTR plus ethanol (27% of calories) (EtOH), or 4) EtOH+Chol for 3 months. RESULTS: In mice fed Chol or EtOH alone, ALT increased to ~160 U/L, moderate hepatic steatosis occurred, and leukocyte infiltration, necrosis, and apoptosis increased modestly, but no observable fibrosis developed. By contrast in mice fed EtOH+Chol, ALT increased to ~270 U/L, steatosis was more extensive and mostly macrovesicular, and expression of proinflammatory molecules (HMGB-1, TLR4, TNFα, ICAM-1) and leukocyte infiltration increased substantially. Necrosis and apoptosis also increased. Trichrome staining and second harmonic generation microscopy revealed hepatic fibrosis. Fibrosis was mostly sinusoidal and/or perivenular, but in some mice bridging fibrosis occurred. Expression of smooth muscle α-actin and TGF-ß1 increased slightly by Chol, moderately by EtOH, and markedly by EtOH+Chol. TGF-ß pseudoreceptor BAMBI increased slightly by Chol, remained unchanged by EtOH and decreased by EtOH+Chol. MicroRNA-33a, which enhances TGF-ß fibrotic effects, and phospho-Smad2/3, the down-stream signal of TGF-ß, also increased more greatly by EtOH+Chol than Chol or EtOH. Metalloproteinase-2 and -9 were decreased only by EtOH+Chol. CONCLUSION: High dietary cholesterol and chronic ethanol consumption synergistically increase liver injury, inflammation, and profibrotic responses and suppress antifibrotic responses, leading to severe steatohepatitis and early fibrosis in mice.

Low Dose Acetaminophen Induces Reversible Mitochondrial Dysfunction Associated with Transient c-Jun N-Terminal Kinase Activation in Mouse Liver.

Acetaminophen (APAP) overdose causes hepatotoxicity involving mitochondrial dysfunction and c-jun N-terminal kinase (JNK) activation. Because the safe limit of APAP dosing is controversial, our aim was to evaluate the role of the mitochondrial permeability transition (MPT) and JNK in mitochondrial dysfunction after APAP dosing considered nontoxic by criteria of serum alanine aminotransferase (ALT) release and histological necrosis in vivo. C57BL/6 mice were given APAP with and without the MPT inhibitor, N-methyl-4-isoleucine cyclosporin (NIM811), or the JNK inhibitor, SP600125. Fat droplet formation, cell viability, and mitochondrial function in vivo were monitored by intravital multiphoton microscopy. Serum ALT, liver histology, total JNK, and activated phospho(p)JNK were also assessed. High APAP (300 mg/kg) caused ALT release, necrosis, irreversible mitochondrial dysfunction, and hepatocellular death. By contrast, lower APAP (150 mg/kg) caused reversible mitochondrial dysfunction and fat droplet formation in hepatocytes without ALT release or necrosis. Mitochondrial protein N-acetyl-p-benzoquinone imine adducts correlated with early JNK activation, but irreversible mitochondrial depolarization and necrosis at high dose were associated with sustained JNK activation and translocation to mitochondria. NIM811 prevented cell death and/or mitochondrial depolarization after both high and low dose APAP. After low dose, SP600125 decreased mitochondrial depolarization. In conclusion, low dose APAP produces reversible MPT-dependent mitochondrial dysfunction and steatosis in hepatocytes without causing ALT release or necrosis, whereas high dose leads to irreversible mitochondrial dysfunction and cell death associated with sustained JNK activation. Thus, nontoxic APAP has the potential to cause transient mitochondrial dysfunction that may synergize with other stresses to promote liver damage and steatosis.

The mitochondria-targeted antioxidant MitoQ attenuates liver fibrosis in mice.

Oxidative stress plays an essential role in liver fibrosis. This study investigated whether MitoQ, an orally active mitochondrial antioxidant, decreases liver fibrosis. Mice were injected with corn oil or carbon tetrachloride (CCl4, 1:3 dilution in corn oil; 1 µl/g, ip) once every 3 days for up to 6 weeks. 4-Hydroxynonenal adducts increased markedly after CCl4 treatment, indicating oxidative stress. MitoQ attenuated oxidative stress after CCl4. Collagen 1α1 mRNA and hydroxyproline increased markedly after CCl4 treatment, indicating increased collagen formation and deposition. CCl4 caused overt pericentral fibrosis as revealed by both the sirius red staining and second harmonic generation microscopy. MitoQ blunted fibrosis after CCl4. Profibrotic transforming growth factor-ß1 (TGF-ß1) mRNA and expression of smooth muscle α-actin, an indicator of hepatic stellate cell (HSC) activation, increased markedly after CCl4 treatment. Smad 2/3, the major mediator of TGF-ß fibrogenic effects, was also activated after CCl4 treatment. MitoQ blunted HSC activation, TGF-ß expression, and Smad2/3 activation after CCl4 treatment. MitoQ also decreased necrosis, apoptosis and inflammation after CCl4 treatment. In cultured HSCs, MitoQ decreased oxidative stress, inhibited HSC activation, TGF-ß1 expression, Smad2/3 activation, and extracellular signal-regulated protein kinase activation. Taken together, these data indicate that mitochondrial reactive oxygen species play an important role in liver fibrosis and that mitochondria-targeted antioxidants are promising potential therapies for prevention and treatment of liver fibrosis.

Spatial localization of cardiac optical mapping with multiphoton excitation.

Depth and radius of regions interrogated by cardiac optical mapping with a laser beam depend on photon travel inside the heart. It would be useful to limit the range of depth and radius interrogated. We modeled the effects of a condensing lens to concentrate laser light at a target depth inside the heart, and near infrared excitation to increase penetration and produce two-photon absorption. A Monte Carlo simulation that incorporated a 0.55-NA lens, and absorption and scattering of 1064- or 488-nm laser light in 3-D cardiac tissue indicated the distribution of excitation fluence inside the tissue. A subsequent simulation incorporating absorption and scattering of transmembrane voltage-sensitive fluorescence (wavelength 669 nm) indicated locations from which fluorescence photons exiting the tissue surface originated. The results indicate that mapping at depths up to 300 microm in hearts can provide significant improvement in localization over existing cardiac optical mapping. The estimated interrogation region is sufficiently small to examine cardiac events at a cellular or subcellular scale and may allow mapping at various depths in the heart.

Acute ethanol causes hepatic mitochondrial depolarization in mice: role of ethanol metabolism.

BACKGROUND/AIMS: An increase of ethanol metabolism and hepatic mitochondrial respiration occurs in vivo after a single binge of alcohol. Here, our aim was to determine how ethanol intake affects hepatic mitochondrial polarization status in vivo in relation to ethanol metabolism and steatosis. METHODS: Hepatic mitochondrial polarization, permeability transition (MPT), and reduce pyridine nucleotides, and steatosis in mice were monitored by intravital confocal/multiphoton microscopy of the fluorescence of rhodamine 123 (Rh123), calcein, NAD(P)H, and BODIPY493/503, respectively, after gavage with ethanol (1-6 g/kg). RESULTS: Mitochondria depolarized in an all-or-nothing fashion in individual hepatocytes as early as 1 h after alcohol. Depolarization was dose- and time-dependent, peaked after 6 to 12 h and maximally affected 94% of hepatocytes. This mitochondrial depolarization was not due to onset of the MPT. After 24 h, mitochondria of most hepatocytes recovered normal polarization and were indistinguishable from untreated after 7 days. Cell death monitored by propidium iodide staining, histology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was low throughout. After alcohol, mitochondrial NAD(P)H autofluorescence increased and decreased, respectively, in hepatocytes with polarized and depolarized mitochondria. Ethanol also caused steatosis mainly in hepatocytes with depolarized mitochondria. Depolarization was linked to ethanol metabolism, since deficiency of alcohol dehydrogenase and cytochrome-P450 2E1 (CYP2E1), the major ethanol-metabolizing enzymes, decreased mitochondrial depolarization by ∼ 70% and ∼ 20%, respectively. Activation of aldehyde dehydrogenase decreased depolarization, whereas inhibition of aldehyde dehydrogenase enhanced depolarization. Activation of aldehyde dehydrogenase also markedly decreased steatosis. CONCLUSIONS: Acute ethanol causes reversible hepatic mitochondrial depolarization in vivo that may contribute to steatosis and increased mitochondrial respiration. Onset of this mitochondrial depolarization is linked, at least in part, to metabolism of ethanol to acetaldehyde.

Imaging of mitochondrial pH using SNARF-1.

Laser scanning confocal microscopy provides the ability to image submicron sections in living cells and tissues. In conjunction with pH-indicating fluorescent probes, confocal microscopy can be used to visualize the distribution of pH inside living cells. Here, we describe a confocal microscopic technique to image intracellular pH in living cells using SNARF-1, a ratiometric pH-indicating fluorescent probe. SNARF-1 is ester-loaded into the cytosol and mitochondria of adult cardiac myocytes. Using 568-nm excitation, emitted fluorescence longer and shorter than 595-nm are imaged and then ratioed after background subtraction. Ratio values for each pixel are converted to values of pH using a standard curve (lookup table). Images of the intracellular distribution of pH show cytosolic and nuclear areas to have a pH of ∼7.1, but in regions corresponding to mitochondria, pH is 8.0, giving a mitochondrial ΔpH of 0.9. During hypoxia, mitochondrial pH decreases to cytosolic values, signifying the collapse of ΔpH. These results illustrate the ability of laser scanning confocal microscopy to image the intracellular distribution of pH in living cells and to determine mitochondrial ΔpH.

Icam-1 upregulation in ethanol-induced Fatty murine livers promotes injury and sinusoidal leukocyte adherence after transplantation.

Background. Transplantation of ethanol-induced steatotic livers causes increased graft injury. We hypothesized that upregulation of hepatic ICAM-1 after ethanol produces increased leukocyte adherence, resulting in increased generation of reactive oxygen species (ROS) and injury after liver transplantation (LT). Methods. C57BL/6 wildtype (WT) and ICAM-1 knockout (KO) mice were gavaged with ethanol (6 g/kg) or water. LT was then performed into WT recipients. Necrosis and apoptosis, 4-hydroxynonenal (4-HNE) immunostaining, and sinusoidal leukocyte movement by intravital microscopy were assessed. Results. Ethanol gavage of WT mice increased hepatic triglycerides 10-fold compared to water treatment (P < 0.05). ICAM-1 also increased, but ALT was normal. At 8 h after LT of WT grafts, ALT increased 2-fold more with ethanol than water treatment (P < 0.05). Compared to ethanol-treated WT grafts, ALT from ethanol-treated KO grafts was 78% less (P < 0.05). Apoptosis also decreased by 75% (P < 0.05), and 4-HNE staining after LT was also decreased in ethanol-treated KO grafts compared to WT. Intravital microscopy demonstrated a 2-fold decrease in leukocyte adhesion in KO grafts compared to WT grafts. Conclusions. Increased ICAM-1 expression in ethanol-treated fatty livers predisposes to leukocyte adherence after LT, which leads to a disturbed microcirculation, oxidative stress and graft injury.

Minocycline decreases liver injury after hemorrhagic shock and resuscitation in mice.

Patients that survive hemorrhage and resuscitation (H/R) may develop a systemic inflammatory response syndrome (SIRS) that leads to dysfunction of vital organs (multiple organ dysfunction syndrome, MODS). SIRS and MODS may involve mitochondrial dysfunction. Under pentobarbital anesthesia, C57BL6 mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer's solution containing minocycline, tetracycline (both 10 mg/kg body weight) or vehicle. Serum alanine aminotransferase (ALT), necrosis, apoptosis and oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization was assessed by intravital microscopy. After H/R with vehicle or tetracycline, ALT increased to 4538 U/L and 3999 U/L, respectively, which minocycline decreased to 1763 U/L (P < 0.01). Necrosis and TUNEL also decreased from 24.5% and 17.7 cells/field, respectively, after vehicle to 8.3% and 8.7 cells/field after minocycline. Tetracycline failed to decrease necrosis (23.3%) but decreased apoptosis to 9 cells/field (P < 0.05). Minocycline and tetracycline also decreased caspase-3 activity in liver homogenates. Minocycline but not tetracycline decreased lipid peroxidation after resuscitation by 70% (P < 0.05). Intravital microscopy showed that minocycline preserved mitochondrial polarization after H/R (P < 0.05). In conclusion, minocycline decreases liver injury and oxidative stress after H/R by preventing mitochondrial dysfunction.

Role of inducible nitric oxide synthase in mitochondrial depolarization and graft injury after transplantation of fatty livers.

This study investigated the role of inducible nitric oxide synthase (iNOS) in failure of ethanol-induced fatty liver grafts. Rat livers were explanted 20 h after gavaging with ethanol (5 g/kg) and storing in UW solution for 24h before implantation. Hepatic oil red O staining-positive areas increased from ∼2 to ∼33% after ethanol treatment, indicating steatosis. iNOS expression increased ∼8-fold after transplantation of lean grafts (LG) and 25-fold in fatty grafts (FG). Alanine aminotransferase release, total bilirubin, hepatic necrosis, TUNEL-positive cells, and cleaved caspase-3 were higher in FG than LG. A specific iNOS inhibitor 1400W (5 µM in the cold-storage solution) blunted these alterations by >42% and increased survival of fatty grafts from 25 to 88%. Serum nitrite/nitrate and hepatic nitrotyrosine adducts increased to a greater extent after transplantation of FG than LG, indicating reactive nitrogen species (RNS) overproduction. Phospho-c-Jun and phospho-c-Jun N-terminal kinase-1/2 (JNK1/2) were higher in FG than in LG, indicating more JNK activation in fatty grafts. RNS formation and JNK activation were blunted by 1400W. Mitochondrial polarization and cell death were visualized by intravital multiphoton microscopy of rhodamine 123 and propidium iodide, respectively. After implantation, viable cells with depolarized mitochondria were 3-fold higher in FG than in LG and 1400W decreased mitochondrial depolarization in FG to the levels of LG. Taken together, iNOS is upregulated after transplantation of FG, leading to excessive RNS formation, JNK activation, mitochondrial dysfunction, and severe graft injury. The iNOS inhibitor 1400W could be an effective therapy for primary nonfunction of fatty liver grafts.