Hiroshi Uchinami

Differential requirement for c-Jun NH2-terminal kinase in TNFα- and Fas-mediated apoptosis in hepatocytes

The c‐Jun NH2‐terminal kinase (JNK) is involved in the regulation of cell death, but its role in tumor necrosis factor (TNF)‐α‐and Fas‐mediated apoptosis in primary cells is not well defined. In primary rat hepatocytes expressing an IκB superrepressor, the JNK inhibitor SP600125 strongly decreased TNF‐α‐induced cell death, caspase 3 activation, and DNA laddering. In contrast, SP600125 did not rescue mouse hepatocytes from Fas‐induced apoptosis. Apoptosis in mouse hepatocytes, induced by human TNF‐α, was blocked by SP600125, indicating that TNF‐receptor (TNF‐R) 1‐mediated JNK activation is important for TNF‐α‐induced death. However, mouse TNF‐α was more efficient than human TNF‐α in activating JNK and killing mouse hepatocytes, suggesting that TNF‐R1 and TNF‐R2 cooperate in JNK activation and apoptosis. SP600125 rescued actinomycin D‐pretreated hepatocytes and hepatocytes expressing a dominant negative c‐Jun from TNF‐α, indicating that JNK exerts its proapoptotic effect independently of transcription and c‐Jun. SP600125 delayed the mitochondrial permeability transition, inhibited cytochrome c release and prevented bid degradation after TNF‐α, suggesting that JNK‐regulated proapoptotic factors act upstream of the mitochondria. Moreover, overexpression of JNK1 activated a mitochondrial death pathway in hepatocytes, albeit less efficiently than TNF‐α . This study demonstrates that JNK augments TNF‐α‐induced apoptosis in hepatocytes through a signaling pathway that is distinct from the pathway by which it regulates proliferation.

Roles for C16-ceramide and sphingosine-1-phosphate in regulating hepatocyte apoptosis in response to TNF-α

Tumor necrosis factor (TNF)-α signals cell death and simultaneously induces the generation of ceramide, which is metabolized to sphingosine and sphingosine 1-phosphate (S1P) by ceramidase (CDase) and sphingosine kinase. Because the dynamic balance between the intracellular levels of ceramide and S1P (the “ceramide/S1P rheostat”) may determine cell survival, we investigated these sphingolipid signaling pathways in TNF-α-induced apoptosis of primary hepatocytes. Endogenous C16-ceramide was elevated during TNF-α-induced apoptosis in both rat and mouse primary hepatocytes. The putative acid sphingomyelinase (ASMase) inhibitor imipramine inhibited TNF-α-induced apoptosis and C16-ceramide increase as did the knock out of ASMase. Overexpression of neutral CDase (NCDase) inhibited the TNF-α-induced increase of C16-ceramide and apoptosis in rat primary hepatocytes. Moreover, NCDase inhibited liver injury and hepatocyte apoptosis in mice treated with d-galactosamine plus TNF-α. This protective effect was abrogated by the sphingosine kinase inhibitor N,N-demethylsphingosine, suggesting that the survival effect of NCDase is due to not only C16-ceramide reduction but also S1P formation. Administration of S1P or overexpression of NCDase activated the pro-survival kinase AKT, and overexpression of dominant negative AKT blocked the survival effect of NCDase. In conclusion, activation of ASMase and generation of C16-ceramide contributed to TNF-α-induced hepatocyte apoptosis. NCDase prevented apoptosis both by reducing C16-ceramide and by activation of AKT through S1P formation. Therefore, the cross-talk between sphingolipids and AKT pathway may determine hepatocyte apoptosis by TNF-α.

Fatty Acid Amide Hydrolase Determines Anandamide-induced Cell Death in the Liver

The endocannabinoid anandamide (AEA) induces cell death in many cell types, but determinants of AEA-induced cell death remain unknown. In this study, we investigated the role of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in AEA-induced cell death in the liver. Primary hepatocytes expressed high levels of FAAH and were completely resistant to AEA-induced cell death, whereas primary hepatic stellate cells (HSCs) expressed low levels of FAAH and were highly sensitive to AEA-induced cell death. Hepatocytes that were pretreated or with the FAAH inhibitor URB597 isolated from FAAH-/- mice displayed increased AEA-induced reactive oxygen species (ROS) formation and were susceptible to AEA-mediated death. Conversely, overexpression of FAAH in HSCs prevented AEA-induced death. Since FAAH inhibition conferred only partial AEA sensitivity in hepatocytes, we analyzed additional factors that might regulate AEA-induced death. Hepatocytes contained significantly higher levels of glutathione (GSH) than HSCs. Glutathione depletion by dl-buthionine-(S,R)-sulfoximine rendered hepatocytes susceptible to AEA-mediated ROS production and cell death, whereas GSH ethyl ester prevented ROS production and cell death in HSCs. FAAH inhibition and GSH depletion had additive effects on AEA-mediated hepatocyte cell death resulting in almost 70% death after 24 h at 50 μm AEA and lowering the threshold for cell death to 500 nm. Following bile duct ligation, FAAH-/- mice displayed increased hepatocellular injury, suggesting that FAAH protects hepatocytes from AEA-induced cell death in vivo. In conclusion, FAAH and GSH are determinants of AEA-mediated cell death in the liver.

High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers

Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, thus allowing the study of fibrogenesis by genetic approaches in transgenic mice. However, mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal and fibrotic livers via subsequent flow cytometric sorting, thus providing a validated method to determine gene expression changes during HSC activation devoid of cell culture artifacts or contamination with other cells. The described isolation procedure takes ∼4 h to complete.

Nrf2 Activation Protects the Liver From Ischemia/Reperfusion Injury in Mice

Objective:To investigate the role of Nrf2 in the pathogenesis of hepatic ischemia-reperfusion (I/R) injury. Background:Hepatic I/R injury is a serious complication that leads to liver failure after liver surgery. NF-E2-related factor 2 (Nrf2) is a transcription factor that plays a critical role in protecting cells against oxidative stress. Therefore, it is suggested that Nrf2 activation protects the liver from I/R injury. Methods:Wild-type and Nrf2-deficient mice were treated with 15-deoxy-&Dgr;12,14–prostaglandin J2 (15d-PGJ2), or a vehicle. Subsequently, these mice were subjected to 60-minute hepatic 70% ischemia, followed by reperfusion. Liver and blood samples were collected to evaluate liver injury and mRNA expressions. Results:After hepatic I/R, Nrf2-deficient livers exhibited enhanced tissue damage; impaired GSTm1, NQO1, and GCLc inductions; disturbed redox state; and aggravated tumor necrosis factor &agr; mRNA expression in comparison with wild-type livers. 15d-PGJ2 treatment protected the livers of wild-type mice from I/R injury via increased expressions of GSTm1, NQO1, and GCLc; maintained redox status; and decreased tumor necrosis factor &agr; induction. These effects induced by 15d-PGJ2 were not seen in the livers of Nrf2−/− mice and were not annulled by peroxisome proliferator-activated receptor &ggr; antagonist in Nrf2+/+ mice, suggesting that the protective effect of 15d-PGJ2 is mediated by Nrf2-dependent antioxidant response. Conclusions:Nrf2 plays a critical role in the mechanism of hepatic I/R injury and would be a new therapeutic target for preventing hepatic I/R injury during liver surgery.

Changes in the fatty acid composition of the liver with the administration of N-3 polyunsaturated fatty acids and the effects on warm ischemia/reperfusion injury in the rat liver.

Prostanoids play a pivotal role among the inflammatory mediators associated with I/R injury. The aim of this study was to determine the effects of oral supplementation of n-3 polyunsaturated fatty acids (PUFA)-rich oil on inflammatory reactions and microcirculatory disorders caused by a hepatic warm I/R in rats. The rats were orally supplemented with n-3 PUFA-rich oil, n-6 PUFA-rich oil, or the same volume of water for 7 days. The PUFA concentration in the blood and liver tissues were evaluated, and the effects on I/R injury of the liver were assessed. The n-3 PUFA supplementation elevated the n-3/n-6 ratio in the blood and liver tissues. After reperfusion, thromboxane B2 in the blood and prostaglandin E2 in the liver were significantly suppressed in the n-3 PUFA-treated rats. Hepatic microcirculation was well maintained from the early phase (30 min) of reperfusion, and the serum concentrations of TNF-&agr; and IL-6 were significantly lower in this group. The transaminase blood levels were also suppressed in the n-3 PUFA-treated rats. Expression of COX-2 mRNA was increased in all groups at 2 h after reperfusion but there were no differences among three groups. In conclusion, preoperative n-3/n-6 ratio augmentation in the blood and in the liver can result in a successful alleviation of hepatic I/R injury.ABBREVIATIONS-AA-arachidonic acid; &agr;-LA-&agr;-linolenic acid; ALT-alanine aminotransferase; AST-aspartate aminotransferase; COX-cyclooxygenase; DHA-docosahexaenoic acid; DPA-docosapentaenoic acid; ELISA-enzyme-linked immunosorbent assay; EPA-eicosapentaenoic acid; ICU-intensive care unit; LA-linoleic acid; LDH-lactic dehydrogenase; MPO-myeloperoxidase; PG-prostaglandin; PUFA-polyunsaturated fatty acids; TX-thromboxane

A newly designed magnet‐retracting forceps for laparoscopic cholecystectomy in a swine model

We designed a method for remote‐controlled endoscopic surgery using magnet‐retracting forceps. To evaluate the feasibility of this technique, laparoscopic cholecystectomy was attempted in a swine model. This method takes advantage of the attractive force between two magnets, one inserted into the peritoneal cavity and the other located outside the abdominal wall. An intra‐peritoneal magnet was fixed to the fundus of the gallbladder using an endovascular clip. Laparoscopic cholecystectomy was accomplished by magnetic retraction of the gallbladder. This magnet‐retracting forceps provided port‐less access to the abdominal cavity. Since the direction and range of retraction were unrestricted by the location of access‐ports fixed on the abdominal wall, surgery could be less invasive. In addition, this procedure provided surgeons with excellent endoscopic views, as retraction force was supplied without any shaft device in the abdomen. This operation system using magnetic retraction appears promising.

Role of preferential cyclooxygenase-2 inhibition by meloxicam in ischemia/reperfusion injury of the rat liver.

Background: Ischemia/reperfusion injury (IRI) is one of the major clinical problems in liver and transplant surgery. Livers subjected to warm ischemia in vivo often show a severe dysfunction and the release of numerous inflammatory cytokines and arachidonic acid metabolites. Cyclooxygenase (COX)-2 is the inducible isoform of an intracellular enzyme that converts arachidonic acid into prostaglandins. The aim of the study was to evaluate the effect of COX-2 inhibition and the role of Kupffer cells in IRI of the liver. Methods: Male Wistar rats [250- 280 g body weight (BW)] were anesthetized and subjected to 30-min warm ischemia of the liver (Pringles maneuver) and 60-min reperfusion after median laparotomy. The I/R group received no additional treatment. In the COX-2 inhibitor (COX-2I) group, the animals received 1 mg/kg BW meloxicam prior to operation. Gadolinium chloride (GdCl3) (10 mg/kg BW) was given 24 h prior to operation in the GdCl3 and GdCl3 + COX-2I groups for the selective depletion of Kupffer cells. The GdCl3 + COX-2I group received both GdCl3 and meloxicam treatment prior to operation. Blood and liver samples were obtained at the end of the experiments for further investigations. Results: After 30 min of warm ischemia in vivo, severe hepatocellular damage was observed in the I/R group. These impairments could be significantly prevented by the selective COX-2 inhibition and the depletion of Kupffer cells. Alanine aminotransferase was significantly reduced upon meloxicam and GdCl3 treatment compared to the I/R group: I/R, 3,240 ± 1,262 U/l versus COX-2I, 973 ± 649 U/l, p < 0.001; I/R versus GdCl3, 1,611 ± 600 U/l, p < 0.05, and I/R versus GdCl3 + COX-2I, 1,511 ± 575 U/l, p < 0.01. Plasma levels of tumor necrosis factor alpha (TNF-α) were significantly reduced in the COX-2I treatment group compared to I/R (3.5 ± 1.5 vs. 16.3 ± 11.7 pg/ml, respectively; p < 0.05). Similarly, the amount of TxB2, a marker for COX-2 metabolism, was significantly reduced in the meloxicam treatment groups compared to the I/R group: I/R, 22,500 ± 5,210 pg/ml versus COX-2I, 1,822 ± 938 pg/ml, p < 0.001, and I/R versus GdCl3 + COX-2I, 1,530 ± 907 pg/ml, p < 0.001. All values are given as mean ± SD (n = 6). Conclusion: These results suggest that the inhibition of COX-2 suppressed the initiation of an inflammatory cascade by attenuating the release of TNF-α, which is an initiator of the inflammatory reaction in hepatic IRI. Therefore, we conclude that preferential inhibition of COX-2 is a possible therapeutic approach against warm IRI of the liver.

The roles of interferon regulatory factors 1 and 2 in the progression of human pancreatic cancer.

Objective Pancreatic cancer is one of the most malignant diseases worldwide. Interferon regulatory factor (IRF) 1 and IRF2 function as a tumor suppressor and oncoprotein, respectively, in several types of cancers. We investigated whether IRF1 and IRF2 are involved in the progression of pancreatic cancer. Methods We examined the expressions of IRF1 and IRF2 in pancreatic cancer specimens and analyzed the association with clinicopathologic features. We evaluated the biological effects of IRF1 and IRF2 using a pancreatic cancer cell line. Results The expression levels of IRF1 and IRF2 were decreased and increased, respectively, in the pancreatic cancer cells compared with those observed in the paired normal areas. A higher expression of IRF1 was associated with better features of tumor differentiation, infiltration depth, tumor size, and survival, whereas that of IRF2 was associated with a worse feature of tumor infiltration depth. Interferon regulatory factor 2–overexpressing PANC-1 cells exhibited an increase in cell growth, less apoptotic features, and chemoresistance to gemcitabine treatment. In contrast, IRF1-overexpressing cells exhibited the opposite characteristics. Conclusions Interferon regulatory factors 1 and 2 may regulate the progression of pancreatic cancer by functioning as an antioncoprotein and oncoprotein, respectively. These molecules may serve as potential targets of therapy.

Enhancement of liver regeneration by adenosine triphosphate-sensitive K⁺ channel opener (diazoxide) after partial hepatectomy.

Background Enhancement of liver regeneration is a matter of importance after partial liver transplantation including small-for-size grafting. Mitochondrial adenosine triphosphate (ATP)-sensitive K+ (mitoKATP) channel plays an important role in mitochondrial bioenergetics, which is a prerequisite for liver regeneration. However, the ATP-sensitive K+ (KATP) channel in hepatocytes is incompletely understood. We investigated the KATP channel in hepatocytes and examined the effects of diazoxide, a potent KATP channel opener, on liver regeneration using a rat model. Methods Using rat primary hepatocytes, expression and localization of KATP channel subunits, Kir6.x and sulfonylurea receptor (SUR)x, were studied by polymerase chain reaction, Western blotting, and immunostaining. To investigate the role of KATP channel openers in liver regeneration, we allocated rats into four groups: control (vehicle) (n=24), diazoxide (n=24), vehicle plus channel blocker (n=6), and diazoxide plus channel blocker (n=6) groups. After 70% partial hepatectomy, hepatic tissue ATP levels, liver-to-body weight ratio, and proliferation rate of hepatocytes were examined. Results KATP channel subunits, Kir6.1 and SUR1, were detected on hepatic mitochondria. During liver regeneration, liver-to-body weight ratio, proliferation rate of hepatocytes, and the hepatic ATP level were significantly higher in the diazoxide group than the control group at 2 days after partial hepatectomy. These effects of diazoxide were neutralized by a KATP channel blocker. Conclusions We demonstrated the existence of a mitoKATP channel in hepatocytes composed of Kir6.1 and SUR1. Diazoxide could enhance liver regeneration by keeping a higher ATP content of the liver tissue. These results suggest that diazoxide will sustain the mitochondrial energetics through the mitoKATP channel opening.