Fatty liver in liver transplantation and surgery

Steatosis of the liver is common in Western countries, affecting about 25% of donors for liver transplantation and 20% of patients undergoing liver resection. Transplantation of livers with severe steatosis (> 60%) is associated with a high risk of primary nonfunction, and these livers should not be used for organ donation. In contrast, transplantation with livers containing mild steatosis (< 30%) yields results similar to those of transplantation performed with nonfatty livers. The outcome of livers with moderate steatosis (30 to 60%) are varying, and the use of these organs depends on the existence of additional risk factors. Similarly, liver resection in patients with steatosis is associated with a risk of postoperative mortality when compared with patients with nonfatty livers (14% versus 2%). Although hepatic steatosis is an important risk factor for surgery, little is known about the mechanisms of injury. In animal experiments, steatosis is associated with decreased ATP production and a disturbance of sinusoidal flow. Further contributing factors may include Kupffer cell dysfunction and leukocyte adhesion. Fatty hepatocytes have reduced tolerance against ischemic injury with a predominant necrotic form of cell death. In addition, the ability of hepatocytes to regenerate after major tissue loss is impaired in the steatotic liver. Very few protective strategies are known. Ischemic preconditioning and intermittent clamping protect the human liver against prolonged periods of ischemia. These techniques appear to be particularly protective in the steatotic liver. New insights into the mechanisms of liver failure in steatotic organs are needed to decrease the risk of surgery and increase the pool of organ donors.     

Carbon tetrachloride-induced alterations of hepatic calmodulin and free calcium levels in rats pretreated with chlordecone.

Calmodulin, a low molecular weight Ca2+ binding protein, regulates a large number of cell activities including cell division. Previous studies from our laboratory indicated excessive accumulation of Ca2+ in hepatocytes succeeded by rapid glycogen breakdown and suppressed cell division in rats receiving CCl4 after previous dietary exposure to 10 ppm chlordecone. Since calmodulin plays a major role in Ca2(+)-regulated events and has been reported to be localized in mitotic apparatus during cell division, we have assessed subcellular distribution of calmodulin and estimated cytosolic phosphorylase a to indicate cytosolic free Ca2+ levels in livers of rats fed 0 ppm or 10 ppm (chlordecone) in the diet for 15 days before CCl4 (100 microliters/kg) administration to understand the role of Ca2(+)-calmodulin in chlordecone + CCl4 toxicity. Hepatotoxicity was assessed by determining serum AST and ALT succeeded by histopathological observations of liver sections. Serum aminotransferases were significantly elevated 6 hr after CCl4 administration to normal rats and returned to control level by 24 hr. However, serum AST and ALT elevations were severalfold higher, and progressive increase was observed starting 4 hr after CCl4 administration to chlordecone rats. Histopathological observations of liver sections for necrotic, swollen and lipid-laden cells provided findings commensurate with the serum enzyme data. These data indicate that normal rats do recover from CCl4 hepatotoxicity. However, the CCl4 hepatotoxicity is progressive in chlordecone rats without recovery. In normal rats, CCl4 administration resulted in a slight increase in phosphorylase a starting at 6 hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of ischemic injury are different in the steatotic and normal rat liver

Hepatic steatosis is associated with significant morbidity and mortality after liver resection and transplantation. Although apoptosis is a key mechanism of reperfusion injury in the normal liver, the pathway leading to cell death in steatotic hepatocytes is unknown. A model of hepatic ischemia and reperfusion injury in fatty and lean Zucker rats was used. Fatty animals had increased aspartate aminotransferase (AST) release and decreased survival after 60 minutes of ischemia compared with lean animals. Apoptosis was the predominant form of cell death in the lean rats (82%), whereas necrosis was minimal. In contrast, fatty animals developed only moderate amounts of apoptosis but showed massive necrosis (73%) after 24 hours of reperfusion. Intracellular mediators of apoptosis, such as caspase 8, caspase 3, and cytochrome c, were significantly lower in the steatotic than in the lean liver indicating dysfunction in activation of the apoptotic pathway. The high percentage of necrosis in the steatotic rats was associated with renal acute tubular necrosis after 24 hours of reperfusion in the fatty, but not in lean rats. Caspase inhibition significantly decreased reperfusion injury in lean animals, but was ineffective in fatty animals. The results indicate that the increased susceptibility of fatty livers to reperfusion injury is associated with a change from an apoptotic form of cell death to necrosis. We conclude that new therapeutic strategies are necessary in the fatty liver.     

甘草甜素对肝硬化动物模型肝脏内NF—κB结合活性的抑制作用

目的 从分子水平探讨强力宁生物活性的发生机理。方法 大鼠随机分为正常对照组,模型对照组,强力宁组,后两组给予四氯化碳和乙醇造模处理以诱导肝损伤,强力宁组在造模处理同时予强力宁治疗。各组大鼠在ＣＣｌ４等处理后第９周处死,收集血清和肝脏标本,测定血清ＡＬＴ活性并进行组织学观察。部分肝组织提取细胞核蛋白进行凝胶迟滞实验以观察ＮＦ－κＢ活性。     

Steatotic livers are susceptible to normothermic ischemia-reperfusion injury from mitochondrial Complex-I dysfunction

AIM: To assess the effects of ischemic preconditioning (IPC, 10-min ischemia/10-min reperfusion) on steatotic liver mitochondrial function after normothermic ischemia-reperfusion injury (IRI). METHODS: Sixty male Sprague-Dawley rats were fed 8-wk with either control chow or high-fat/high-sucrose diet inducing > 60% mixed steatosis. Three groups (n = 10/group) for each dietary state were tested: (1) the IRI group underwent 60 min partial hepatic ischemia and 4 h reperfusion; (2) the IPC group underwent IPC prior to same standard IRI; and (3) sham underwent the same surgery without IRI or IPC. Hepatic mitochondrial function was analyzed by oxygraphs. Mitochondrial Complex-I, Complex-II enzyme activity, serum alanine aminotransferase (ALT), and histological injury were measured. RESULTS: Steatotic-IRI livers had a greater increase in ALT (2476 ± 166 vs 1457 ± 103 IU/L, P < 0.01) and histological injury following IRI compared to the lean liver group. Steatotic-IRI demonstrated lower Complex-I activity at baseline [78.4 ± 2.5 vs 116.4 ± 6.0 nmol/(min.mg protein), P < 0.001] and following IRI [28.0 ± 6.2 vs 104.3 ± 12.6 nmol/(min.mg protein), P < 0.001]. Steatotic-IRI also demonstrated impaired Complex-I function post-IRI compared to the lean liver IRI group. Complex-II activity was unaffected by hepatic steatosis or IRI. Lean liver mitochondrial function was unchanged following IRI. IPC normalized ALT and histological injury in steatotic livers but had no effect on overall steatotic liver mitochondrial function or individual mitochondrial complex enzyme activities. CONCLUSION: Warm IRI impairs steatotic liver Complex-I activity and function. The protective effects of IPC in steatotic livers may not be mediated through mitochondria.     

Failure of regeneration of the steatotic rat liver: disruption at two different levels in the regeneration pathway

Hepatic resection or transplantation in patients with fatty liver is associated with increased morbidity and mortality. The regenerative capacity of fatty livers after major tissue loss is unknown. Interleukin 6 (IL-6) is a potent inducer of hepatic regeneration in normal and ischemic livers. Therefore, we studied hepatic regeneration at day 1, day 2, and day 4 in a model of 70% hepatectomy in obese and lean Zucker rats, and obese Zucker rats pretreated with recombinant interleukin 6 (rIL-6). The mitotic cycle in hepatocytes was investigated by 4 different markers of regeneration representing distinct phases of mitosis (proliferating cell nuclear antigen [PCNA] = G(1) phase, bromodeoxy uridine [BrdU] = S phase, mitotic index, and regenerated liver weight = M phase). Obese Zucker rats had significantly decreased regenerative capacity compared with lean Zucker rats (PCNA, BrdU, mitotic index, regenerated liver weight) at days 1 and 2 after surgery. Four days after resection fatty animals showed an increase in the mitotic index indicating a delay of regeneration in steatotic livers. Animal survival after 70% hepatectomy was significantly decreased in obese rats compared with lean animals. Pretreatment of obese animals with rIL-6 normalized PCNA expression (G(1) phase) in steatotic hepatocytes but failed to increase DNA synthesis (BrdU, S phase), mitosis (mitotic index and regenerated liver weight, M phase), and animal survival. These results indicate major impairment of hepatic regeneration in steatotic livers. Two different blockages of regeneration must be present, one rIL-6 sensitive, at the level of IL-6 or upstream, and a second, rIL-6 resistant, at the level of G(1)/S-phase transition.     

Calcium staining by the glyoxal-bis-(2-hydroxyanil)-method in the livers of rats treated with CCl4, diltiazem, and with both agents together.

We studied the histochemistry of Ca in livers treated with CCl4, diltiazem (one of the Ca antagonists), and both agents together to determine whether hepatocytes or other parts of the liver lesions show Ca staining and whether the grade or location of Ca in these injuries varies. For Ca staining, cryostat sections were treated by the glyoxal-bis-(2-hydroxyanil) (GBHA)-method using O.C.T. imbedding compound instead of paraffin. Diltiazem-treated rats showed Ca granules in the bile canaliculi around the terminal hepatic veins and Kupffer cells 6 h after intragastric injection. Rats treated with CCl4 showed fine red granules in the cytoplasm of the hepatocytes around the terminal hepatic veins as soon as 5 min mildly and 2 h moderately after intraperitoneal injection. Hepatocytes around the terminal hepatic veins showed positive Ca granules 6 to 30 h after intragastric injection of CCl4. Hepatocytes stained by Ca showed acidophilic degeneration and coagulative necrosis. The hepatocytes of rats treated with both diltiazem and CCl4 revealed fewer Ca granules than those treated with CCl4 alone. In summary, Ca was stained by the GBHA method from the early stage of liver injury by CCl4 and was closely involved in acidophilic degeneration and coagulative necrosis of hepatocytes. The Ca staining in liver cells in CCl4-treated rats was decreased by diltiazem.     

Little evidence of bone marrow-derived hepatocytes in the replacement of injured liver

We have tested the ability of bone marrow (BM) cells (BMCs) to form hepatocytes in liver injury models. We used three models: (i) carbon tetrachloride (CCl4) treatment, (ii) albumin-urokinase transgenic mouse [TgN(Alb1Plau)], and (iii) hepatitis B transgenic mouse [TgN(Alb1HBV)]. As a nonselective liver injury model, irradiated C57BL/6 (B6) mice were transplanted with BMCs from GFP transgenic mouse [TgN(ActbEGFP)] or beta-galactosidase transgenic mouse [TgN(MtnLacZ)] followed by the administration of CCl4. Irradiated TgN(Alb1HBV) and TgN(Alb1Plau) were also transplanted with BMCs from TgN(ActbEGFP) or TgN(MtnLacZ). Approximately 1.5 x 106 hepatocytes per liver were analyzed for GFP-positive cells, and the whole livers were inspected for beta-galactosidase expression. No GFP-positive hepatocytes and no gross blue staining of the livers with 5-bromo-4-chloro-3-indolyl beta-d-galactoside in any of the 18 recipient mice analyzed were detected. The livers from female animals with gender-mismatched BM transplantation were also tested with Y chromosome fluorescent in situ hybridization analysis to detect donor-derived cells. A total of five isolated hepatocytes were positive for Y chromosome in 4.1 x 105 hepatocytes analyzed. Our results demonstrate that there is little or no contribution of BMCs to the replacement of injured livers in these models. We conclude that BM-derived cells cannot generally lead to a cure of liver damage.     

Interleukin 6 alleviates hepatic steatosis and ischemia/reperfusion injury in mice with fatty liver disease

Fatty liver, formerly associated predominantly with excessive alcohol intake, is now also recognized as a complication of obesity and an important precursor state to more severe forms of liver pathology including ischemia/reperfusion injury. No standard protocol for treating fatty liver exists at this time. We therefore examined the effects of 10 days of interleukin 6 (IL-6) injection in 3 murine models of fatty liver: leptin deficient ob/ob mice, ethanol-fed mice, and mice fed a high-fat diet. In all 3 models, IL-6 injection decreased steatosis and normalized serum aminotransferase. The beneficial effects of IL-6 treatment in vivo resulted in part from an increase in mitochondrial beta oxidation of fatty acid and an increase in hepatic export of triglyceride and cholesterol. However, administration of IL-6 to isolated cultured steatotic hepatocytes failed to decrease lipid contents, suggesting that the beneficial effects of IL-6 in vivo do not result from its effects on hepatocytes alone. IL-6 treatment increased hepatic peroxisome proliferator-activated receptor (PPAR) alpha and decreased liver and serum tumor necrosis factor (TNF) alpha. Finally, 10 days of treatment with IL-6 prevented the susceptibility of fatty livers to warm ischemia/reperfusion injury. In conclusion, long-term IL-6 administration ameliorates fatty livers and protects against warm ischemia/reperfusion fatty liver injury, suggesting the therapeutic potential of IL-6 in treating human fatty liver disease.     

Dietary iron overload inhibits carbon tetrachloride-induced promotion in chemical hepatocarcinogenesis: effects on cell proliferation, apoptosis, and antioxidation

BACKGROUND/AIMS: The aim of this study was to investigate if feeding with carbonyl iron would facilitate the development of preneoplastic lesions initiated by diethylnitrosamine (DEN) and promoted by CCl4-induced liver cirrhosis. METHODS: Male Wistar rats were fed a diet with 1.25%-2.5% carbonyl iron for 23 weeks and received intragastric injections of CCl4 (1.0 or 2.0 ml/kg per week) for 13 weeks, followed by one i.p. injection of DEN (200 mg/kg), after which CCl4 was administered for 8 additional weeks. Animals were killed 48 h after the first CCl4 injection to evaluate liver necrosis, 8 weeks later to evaluate fibrosis, and 9 weeks after DEN to determine formation of glutathione S-transferase 7,7 (GST-7,7) positive foci. RESULTS: Treatment with iron counteracted the increased serum alanine aminotransferase levels and liver necrosis following CCl4 administration. Hepatic levels of reduced Q9 and alpha-tocopherol were elevated in rats treated with CCl4 and decreased in rats treated with iron compared to the controls. Fibrogenesis was not altered by iron treatment. Nine weeks after DEN initiation, the number and volume density of GST-7,7-positive foci in rats treated with CCl4 were significantly increased as compared with controls, but co-treatment with iron inhibited this increase. Apoptotic index was increased in iron-loaded livers, and labelling index (the fraction of S-phase hepatocytes) was decreased by co-treatment with iron in livers exposed to CCl4. CONCLUSION: Carbonyl iron depleted hepatic levels of antioxidants, it decreased CCl4-induced necrosis and cell proliferation, it enhanced apoptosis and did not facilitate fibrogenesis. These effects together may explain the suppression of CCl4-induced promotion after DEN initiation exerted by carbonyl iron in the present study.     

Localization of oxidized phosphatidylcholine in nonalcoholic fatty liver disease: impact on disease progression

Nonalcoholic steatohepatitis/nonalcoholic fatty liver disease is considered to be a hepatic manifestation of various metabolic disorders. However, its precise pathogenic mechanism is obscure. Oxidative stress and consequent lipid peroxidation seem to play a pivotal role in disease progression. In this study, we analyzed the localization of oxidized phosphatidylcholine (oxPC), a lipid peroxide that serves as a ligand for scavenger receptors, in livers of patients with this steatotic disorder. Specimens of non-alcoholic fatty liver disease (15 autopsy livers with simple steatosis and 32 biopsy livers with steatohepatitis) were examined via immunohistochemistry and immunoelectron microscopy using a specific antibody against oxPC. In addition, scavenger receptor expression, hepatocyte apoptosis, iron deposition, and inflammatory cell infiltration in the diseased livers were also assessed. Oxidized phosphatidylcholine was mainly localized to steatotic hepatocytes and some macrophages/Kupffer cells. A few degenerative or apoptotic hepatocytes were also positive for oxPC. Immunoelectron microscopy showed oxPC localized to cytoplasmic/intracytoplasmic membranes including lipid droplets. Steatotic livers showed enhanced expression of scavenger receptors. The number of oxPC cells was correlated with disease severity and the number of myeloperoxidase-positive neutrophils, but not with the degree of iron deposition. In conclusion, distinct localization of oxPC in liver tissues suggest that neutrophil myeloperoxidase-derived oxidative stress may be crucial in the formation of oxPC and the progression of steatotic liver disease.     

Upregulation of calpastatin in regenerating and developing rat liver: role in resistance against hepatotoxicity

Acute liver failure induced by hepatotoxic drugs results from rapid progression of injury. Substantial research has shown that timely liver regeneration can prevent progression of injury leading to a favorable prognosis. However, the mechanism by which compensatory regeneration prevents progression of injury is not known. We have recently reported that calpain released from necrotic hepatocytes mediates progression of liver injury even after the hepatotoxic drug is cleared from the body. By examining expression of calpastatin (CAST), an endogenous inhibitor of calpain in three liver cell division models known to be resistant to hepatotoxicity, we tested the hypothesis that increased CAST in the dividing hepatocytes affords resistance against progression of injury. Liver regeneration that follows CCl(4)-induced liver injury, 70% partial hepatectomy, and postnatal liver development were used. In all three models, CAST was upregulated in the dividing/newly divided hepatocytes and declined to normal levels with the cessation of cell proliferation. To test whether CAST overexpression confers resistance against hepatotoxicity, CAST was overexpressed in the livers of normal SW mice using adenovirus before challenging them with acetaminophen (APAP) overdose. These mice exhibited markedly attenuated progression of liver injury and 57% survival. Whereas APAP-bioactivating enzymes and covalent binding of the APAP-derived reactive metabolites remained unaffected, degradation of calpain specific target substrates such as fodrin was significantly reduced in these mice. In conclusion, CAST overexpression could be used as a therapeutic strategy to prevent progression of liver injury where liver regeneration is severely hampered.     

Hepatocyte growth factor (hepatopoietin A) rapidly increases in plasma before DNA synthesis and liver regeneration stimulated by partial hepatectomy and carbon tetrachloride administration.

An enzyme-linked immunosorbent assay was used to measure the level of hepatocyte growth factor in rat plasma at various times after two-thirds partial hepatectomy or CCl4 administration. An initial 17-fold rise and 13-fold rise in the level of hepatocyte growth factor was observed 2 hr after partial hepatectomy and CCl4 treatment, respectively, well before the onset of DNA synthesis in the liver. The peaks of DNA synthesis in remnant livers and livers exposed to CCl4 occurred at 24 hr and 48 hr, respectively, as determined by 5-bromo-2'-deoxyuridine labeling and [3H]thymidine uptake by the liver. A later peak level (17-fold above control) of hepatocyte growth factor at 24 hr after CCl4 treatment coincided with strong immunostaining of damaged or necrotic hepatocytes around central veins with an antibody to hepatocyte growth factor. This suggests a later intrahepatic origin of the signals for liver regeneration after hepatotoxic injury subsequent to the early extrahepatic production of hepatocyte growth factor at 2 hr after CCl4 administration. The absence of staining in the liver remnants in partially hepatectomized rats implies that the increase in hepatocyte growth factor seen in the plasma is caused by production at extrahepatic site(s). Possible sources include the pancreas, brain, thyroid and salivary glands, and Brunner's glands of the duodenum. Norepinephrine also increases in plasma as early as 2 hr after hepatectomy. In vitro, [3H]thymidine incorporation into hepatocyte DNA in the presence of hepatocyte growth factor is greater if 10(-5) mol/L norepinephrine is also present in the media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cold preservation of fatty liver grafts: prevention of functional and ultrastructural impairments by venous oxygen persufflation

BACKGROUND/AIMS: The incidence of steatosis in livers retrieved for organ transplantation is up to 30%. Due to the shortage of donor organs, many of these livers are accepted for clinical transplantation, although a high rate of graft dysfunction is associated with ischemic preservation of steatotic livers. The present study was intended to reduce the ischemia/reperfusion injury of steatotic grafts by the use of venous systemic oxygen persufflation during cold storage. METHODS: A histologically-documented mild to moderate steatosis was induced in livers of Wistar rats by fasting for 2 days and subsequent feeding of a fat-free diet enriched in carbohydrates. Fatty livers were retrieved and flushed via the portal vein with 60 ml of HTK. In group A, livers were then stored ischemically at 4 degrees C for 24 h. Livers of group B were additionally connected to a gaseous oxygen supply and persufflated with O2 via the venous vascular system during the cold storage period. Viability of the livers was then assessed upon isolated perfusion in vitro with oxygenated Krebs-Henseleit buffer. RESULTS: Venous systemic oxygen sufflation resulted in a relevant and significant reduction of parenchymal (ALT: 132+/-90 vs 434+/-172 U/l; p<0.01) and mitochondrial (GLDH: 116+/-57 vs 633+/-241 U/l; p<0.001) enzyme release during reperfusion. Moreover, Kupffer cell activation, as evaluated from acid phosphatase activity in the perfusate, was reduced to about 1/3 (4.0+/-1.3 vs 11.9+/-5.3 U/l; p<0.01). Electron microscopic analysis revealed that the liver mitochondria and sinusoidal endothelial lining were better preserved after oxygen persufflation, which was in line with the data on enzyme release and the increased portal perfusion pressure in the untreated group, while normal values were found after venous systemic oxygen sufflation. CONCLUSION: Venous oxygen persufflation may thus represent a useful tool for the safe and improved preservation of ischemia-sensitive steatotic livers.     

丹酚酸A抗过氧化鼠肝损伤的作用

目的观察丹酚酸Ａ（ＳＡＡ）抗过氧化肝损伤的作用．方法采用ＣＣｌ４诱导大鼠肝损伤模型,观察ＳＡＡ对肝脏病理、血清Ａｌｂ含量与ＡＬＴ,ＡＳＴ活性,肝组织ＭＤＡ含量的影响．另以ＣＣｌ４熏蒸诱导肝细胞损伤,观察ＳＡＡ对ＡＬＴ,ＡＳＴ,ＧＳＨＰＸ的活性以及对ＭＤＡ,ＧＳＨ含量的变化．结果ＳＡＡ能减轻模型鼠肝脏病理变化,降低ＡＬＴ,ＡＳＴ活性与ＭＤＡ含量;降低损伤肝细胞ＡＬＴ,ＡＳＴ,ＳＯＤ,ＧＳＨＰＸ活性与ＭＤＡ含量,增高ＧＳＨ含量．结论ＳＡＡ具有良好的抗过氧化肝损伤作用．     

Pancreatic-derived factor promotes lipogenesis in the mouse liver: role of the Forkhead box 1 signaling pathway

Pancreatic-derived factor (PANDER) is a pancreatic islet-specific cytokine that cosecretes with insulin and is important for β cell function. Here, we show that PANDER is constitutively expressed in hepatocytes, and its expression is significantly increased in steatotic livers of diabetic insulin-resistant db/db mice and mice fed a high-fat diet. Overexpression of PANDER in the livers of C57Bl/6 mice promoted lipogenesis, with increased Forkhead box 1 (FOXO1) expression, whereas small interfering RNA-mediated knockdown of hepatic PANDER significantly attenuated steatosis, with reduced FOXO1 expression in db/db mice. Hepatic PANDER silencing also attenuated insulin resistance and hyperglycemia in db/db mice. In cultured hepatocytes, PANDER overexpression induced lipid deposition, increased FOXO1 expression, and suppressed insulin-stimulated Akt activation and FOXO1 inactivation. Moreover, FOXO1 overexpression increased PANDER expression in cultured hepatocytes and mouse livers. CONCLUSION: PANDER promotes lipogenesis and compromises insulin signaling in the liver by increasing FOXO1 activity. PANDER may represent a potential therapeutic target for the treatment of fatty liver and insulin resistance.