Defective hepatic regeneration after partial hepatectomy in leptin-deficient mice is not rescued by exogenous leptin

Liver regeneration after partial hepatectomy (PH) is impaired in leptin-deficient ob/ob mice. Here, we tested whether exogenous leptin and/or correction of the obese phenotype (by food restriction or long-term leptin administration) would rescue hepatocyte proliferation and whether the hepatic progenitor cell compartment was activated in leptin-deficient ob/ob livers after PH. Because of the high mortality following 70% PH to ob/ob mice, we performed a less extensive (55%) resection. Compared to lean mice, liver regeneration after 55% PH was deeply impaired and delayed in ob/ob mice. Administration of exogenous leptin to ob/ob mice at doses that restored circulating leptin levels during the surgery and postsurgery period or for 3 weeks prior to the surgical procedure did not rescue defective liver regeneration. Moreover, correction of obesity, metabolic syndrome and hepatic steatosis by prolonged administration of leptin or food restriction (with or without leptin replacement at the time of PH) did not improve liver regeneration in ob/ob mice. The hepatic progenitor cell compartment was increased in ob/ob mice. However, after PH, the number of progenitor cells decreased and signs of proliferation were absent from this cell compartment. In this study, we have conclusively shown that neither leptin replacement nor amelioration of the metabolic syndrome, obese phenotype and hepatic steatosis, with or without restitution of normal circulating levels of leptin, was able to restore replicative competence to ob/ob livers after PH. Thus, leptin does not directly signal to liver cells to promote hepatocyte proliferation, and the obese phenotype is not solely responsible for impaired regeneration.     

Limited therapeutic efficacy of thrombopoietin on the regeneration of steatotic livers

Liver regeneration after partial hepatectomy is impaired in steatotic livers of leptin-deficient ob/ob mice. Previous studies have shown that thrombopoietin (TPO) promotes liver regeneration and improves liver cirrhosis by an increase of platelet counts and the expansion of hepatic progenitor cells. Herein we studied whether TPO exerts pro-proliferative and hepatoprotective effects and thereby improves the regenerative capacity of steatotic livers. For this purpose, we studied hepatic regeneration at day 2, 3, 7 and 10 in a model of 55% hepatectomy in obese (ob/ob) and non-obese (C57BL/6J) mice. Liver function and injury, platelet counts, weight of the regenerated liver, proliferating liver cells as well as the number of hepatic (CK19-positive) oval cells were quantified by biochemical and immunohistochemical analysis. As expected, obese mice had a markedly decreased regenerative capacity of livers compared with lean animals. Pretreatment of mice with recombinant TPO (12.5 μg/kg) had no evident effect on regeneration of fatty livers, but ameliorated acute liver damage in obese mice, as indicated by decreased liver enzyme release early after resection. TPO was unable to enhance hepatocyte proliferation, but increased proliferation of non-parenchymal cells, including CK19-positive oval cells, at later observation time points after resection. Interestingly, TPO completely inhibited the resection-induced increase of plasma triglycerides immediately after resection in non-obese mice. In conclusion, TPO slightly prevents acute liver damage after resection in obese mice, but fails to significantly enhance regeneration of fatty livers.     

Endoplasmic reticulum stress, hepatocyte CD1d and NKT cell abnormalities in murine fatty livers

The liver regulates lipid homeostasis and is enriched with natural killer T (NKT) cells that respond to lipid antigens. Optimal maturation and activation of NKT cells requires their interaction with lipid antigens that are presented by cluster of differentiation-1 (CD-1) molecules on antigen-presenting cells. Hepatocytes express CD1d and present lipid antigens to NKT cells. Depletion and dysregulation of hepatic NKT cells occurs in mice with fatty livers. Herein, we assess whether reduced CD1d content on steatotic hepatocytes contributes to fatty liver-associated NKT cell abnormalities. We show that despite expressing normal levels of CD1d mRNA, fatty hepatocytes from ob/ob mice have significantly less CD1d on their plasma membranes than normal hepatocytes. This has functional significance because ob/ob hepatocytes are less able to activate CD1d-restricted T-cell responses in vitro, and CD1d-reactive NKT cells are reduced in ob/ob livers. Events in the endoplasmic reticulum (ER) normally regulate CD1d trafficking to plasma membranes. Hepatic steatosis has been associated with ER stress. To determine if ER stress reduces CD-1 accumulation on hepatocytes, we evaluated hepatic ER stress in ob/ob mice and treated cultured hepatocytes and lean mice with tunicamycin to induce ER stress. Lipid accumulation and ER stress occurred in the livers of both ob/ob and tunicamycin-treated mice. Tunicamycin caused dose-dependent decreases in hepatocyte CD1d, inhibited hepatocyte activation of CD1d-restricted T-cell responses, depleted liver populations of CD1d-reactive NKT cells and promoted Th-1 polarization of hepatic cytokine production. In conclusion, ER stress-related decreases in hepatocyte CD1d contribute to NKT cell dysregulation in fatty livers.     

Impaired proliferation of non-parenchymal cells participates in an impairment of liver regeneration in db/db mice

In this study, we examined the possibility that impaired proliferation of non-parenchymal cells affects in an impairment of liver regeneration in db/db mice, which are congenitally deficient in receptors for leptin. Liver regeneration after a two thirds partial hepatectomy (2/3 PH) was impaired in 10-week-old female db/db mice. The proliferation of both hepatocytes and non-parenchymal cells estimated from a bromodeoxyuridine (BrdU) labeling index was suppressed, and the protein expression of vascular endothelial growth factor was blocked in db/db mice. Although the extent of fatty change and the level of epidermal growth factor receptor protein expression in the liver were improved in 5-week-old db/db mice, the regeneration of liver was impaired after 2/3 PH in both 5- and 10-week-old db/db mice. These results suggested that suppressed proliferation of non-parenchymal cells contributes to the impairment of liver regeneration in db/db mice. As leptin has also the angiogenic effect, the angiogenic inhibitor FR-118487 was administered to ICR mice to examine liver regeneration after 2/3 PH, and the rate of regeneration was affected. In conclusion, it is suggested that the suppressed proliferation of non-parenchymal cells contributes to the impairment of liver regeneration probably through a disrupted angiogenesis in db/db mice.     

Role of IL-6 in the resolution of pancreatitis in obese mice

Obesity increases severity of acute pancreatitis and risk of pancreatic cancer. Pancreatitis and obesity are associated with elevated IL-6, a cytokine involved in inflammation and tumorigenesis. We studied the role of IL-6 in the response of lean and obese mice to pancreatitis induced by IL-12 + IL-18. Lean and diet-induced obese (DIO) WT and IL-6 KO mice and ob/ob mice pretreated with anti-IL-6 antibodies were evaluated at Days 1, 7, and 15 after induction of pancreatitis. Prolonged elevation of IL-6 in serum and visceral adipose tissue was observed in DIO versus lean WT mice, whereas circulating sIL-6R declined in DIO but not lean mice with pancreatitis. The severe inflammation and lethality of DIO mice were also observed in IL-6 KO mice. However, the delayed resolution of neutrophil infiltration; sustained production of CXCL1, CXCL2, and CCL2; prolonged activation of STAT-3; and induction of MMP-7 in the pancreas, as well as heightened induction of serum amylase A of DIO mice, were blunted significantly in DIO IL-6 KO mice. In DIO mice, production of OPN and TIMP-1 was increased for a prolonged period, and this was mediated by IL-6 in the liver but not the pancreas. Results obtained in IL-6 KO mice were confirmed in ob/ob mice pretreated with anti-IL-6 antibodies. In conclusion, IL-6 does not contribute to the increased severity of pancreatitis of obese mice but participates in delayed recovery from acute inflammation and may favor development of a protumorigenic environment through prolonged activation of STAT-3, induction of MMP-7, and sustained production of chemokines.     

Expression of epidermal growth factor receptor protein in the liver of db/db mice after partial hepatectomy

Liver regeneration was impaired after partial hepatectomy (PH) in leptin receptor-deficient db/db mice with severe liver steatosis. In the present study, we analyzed the mode of epidermal growth factor receptor (EGFR) protein expression in the liver of 5- and 10-week-old db/db and age-matched control mice. In 5-week-old db/db mice, neither the expression of EGFR protein in the intact liver nor the rate of liver regeneration after PH was significantly different from that in age-matched control mice. However, in 10-week-old db/db mice, the level of EGFR protein expression was very low and liver regeneration was prominently suppressed. Histopathologically, much severer fatty change was observed in the liver of 10-week-old db/db mice than 5-week-old db/db mice. These results suggest that the down-regulation of EGFR protein expression is associated with an impairment of liver regeneration in db/db mice and that the severity of hepatic steatosis plays an indirect role in the impairment of liver regeneration by modifying EGFR expression.     

Participation of liver progenitor cells in liver regeneration: lack of evidence in the AAF/PH rat model

When hepatocyte proliferation is impaired, liver progenitor cells (LPC) are activated to participate in liver regeneration. We used the 2-acetaminofluorene/partial hepatectomy (AAF/PH) model to evaluate the contribution of LPC to liver cell replacement and function restoration. Fischer rats subjected to AAF/PH (or PH alone) were investigated 7, 10 and 14 days post-hepatectomy. Liver mass recovery (LMR) was estimated, and the liver mass to body weight ratio calculated. We used serum albumin and bilirubin levels, and liver albumin mRNA levels to assess the liver function. LPC expansion was analyzed by cytokeratin 19 (CK19), glutathione S-transferase protein (GSTp) immunohistochemistry and by CK19, CD133, transforming growth factor-β1 and hepatocyte growth factor mRNA expression in livers. Cell proliferation was evaluated by Ki67 and BrdU immunostaining. Compared with PH alone where LMR was ～100% 14 days post-PH, LMR was defective in AAF/PH rats (64.1±15.5%, P=0.0004). LPC expansion was scarce in PH livers (0.5±0.4% of CK19(+) area), but significant in AAF/PH livers (8.5±7.2% of CK19(+)), and inversely correlated to LMR (r(2)=0.63, P<0.0001). A quarter of AAF/PH animals presented liver failure (low serum albumin and high serum bilirubin) 14 days post-PH. Compared with animals with preserved function, this was associated with a lower LMR (50±6.8 vs 74.6±9.4%, P=0.0005), a decreased liver to body weight ratio (2±0.3 vs 3.5±0.6%, P=0.001), and a larger LPC expansion such as proliferating Ki67(+) LPC covered 17.4±4.2% of the liver parenchyma vs 3.1±1.5%, (P<0.0001). Amongst those, rare LPC with an intermediate hepatocyte-like phenotype were seen. Also, less than 2% of hepatocytes were engaged into the cell cycle (Ki67(+)), while more numerous (～25% of hepatocytes) in the livers with preserved function. These observations suggest that, in this model, the efficient recovery of the liver function was ensured rather by the proliferation of mature hepatocytes than by the LPC expansion and differentiation into hepatocytes.     

Up-regulated expression of HGF in rat liver cells after experimental endotoxemia: a potential pathway for enhancement of liver regeneration

A lipopolysaccharide (LPS)-induced inflammation prior to an hepatic resection has been shown to enhance liver regeneration in rat. The aim of the present study was to investigate the expression of hepatocyte growth factor (HGF) and its c-Met receptor under such experimental conditions. Animals were submitted to a two-third hepatectomy or a LPS challenge carried out 12 h prior to resection. Non parenchymal and parenchymal cells were isolated from livers obtained at various times post-hepatectomy. Quantitative RT-PCR for HGF and c-Met mRNAs were performed from total liver or purified cell fractions and HGF mRNA was also analyzed by in situ RT-PCR on liver sections. A LPS challenge alone induced a marked up-regulation of HGF mRNA level in whole liver and isolated hepatocytes. Furthermore, when partial hepatectomy (PH) was preceded by a LPS challenge, an increase of HGF mRNA level was seen in whole liver and contrasted with a decreased level in non parenchymal cells. These results were confirmed by in situ RT-PCR. In isolated hepatocytes from endotoxemic rats, the mRNA level for the LPS-specific membranous receptor mCD14 was markedly up-regulated and even more so when LPS was followed by PH. Moreover, a TNFalpha challenge alone induced an up-regulation of HGF mRNA in hepatocytes and a down-regulation in non parenchymal cells (NPCs). Overall, when a LPS challenge is given prior to PH the major source of hepatic HGF appears to be the hepatocyte itself rather than NPCs. An autocrine HGF/c-Met loop which promotes the proliferative potential of the hepatic parenchymal cell and participates in liver regeneration is postulated.     

Protection of hepatotoxic and lethal effects of CCl4 by partial hepatectomy

CCl4 is a hepatotoxic haloalkane, capable of producing hepatocellular fatty degeneration and centrilobular necrosis. Previous reports indicate induction of liver regeneration after 36-48 hr of CCl4 treatment, which is considered as a secondary effect. The present investigation was undertaken to evaluate the primary effects of CCl4 on hepatic DNA synthesis and to correlate liver regeneration with CCl4 toxicity. These studies were conducted in normal and actively regenerating livers using male Sprague-Dawley rats undergoing sham operation (SH), or partial (70%) hepatectomy (PH). Incorporation of 3H-thymidine (3H-T) in hepatocellular nuclear DNA and autoradiographic analyses of liver sections served as indices for hepatocellular regeneration. Initial experiments established that peak regeneration occurs at 2 days post-PH (PH2) and liver regeneration phases out by 7 days post-PH (PH7). SH and PH rats were challenged with a single ip dose of either corn oil vehicle or CCl4 at either 0.1 ml/kg (to represent subtoxic dose) or 2.5 ml/kg (to represent toxic dose). The low dose of CCl4 was not toxic and did not alter 3H-T incorporation and percentage labelled cells at 6 or 24 hours after administration to SH, PH2 or PH7 groups, indicating that there was no interference with PH-stimulated hepatocellular regeneration. The high dose of CCl4 was significantly hepatotoxic and lethal in SH rats, while in PH2 rats both hepatotoxic and lethal effects were significantly decreased. 3H-T incorporation as well as percentage labelled cells, highly stimulated by PH, were significantly decreased by high dose of CCl4. However, hepatocellular regeneration in PH2 rats treated with high dose of CCl4 was still significantly higher than SH or PH7 groups by virtue of the stronger stimulatory effect of PH. In PH7 rats, where hepatocellular regeneration had returned to the SH level, the hepatotoxic and lethal effects of the large dose of CCl4 were also restored. These findings show that the progressive phase of a single high dose of CCl4 injury which normally culminates in hepatotoxic and lethal effects is significantly mitigated by previously stimulated hepatocellular regeneration. High dose of CCl4 suppresses hepatocellular regeneration at early time points after administration in contrast to the smaller subtoxic dose of CCl4. By virtue of the much stronger stimulatory effect, PH results in the protection against the hepatotoxic and lethal effects of CCl4 despite the obtunding effects of the high dose on hepatocellular regeneration.     

Cathepsin B knockout mice are resistant to tumor necrosis factor-alpha-mediated hepatocyte apoptosis and liver injury: implications for therapeutic applications.

Tumor necrosis factor-alpha (TNF-alpha) contributes to liver injury by inducing hepatocyte apoptosis. Recent evidence suggests that cathepsin B (cat B) contributes to TNF-alpha-induced apoptosis in vitro. The aim of the present study was to determine whether cat B contributes to TNF-alpha-induced hepatocyte apoptosis and liver injury in vivo. Cat B knockout (catB(-/-)) and wild-type (catB(+/+)) mice were first infected with the adenovirus Ad5I kappa B expressing the I kappa B superrepressor to inhibit nuclear factor-kappa B-induced survival signals and then treated with murine recombinant TNF-alpha. Massive hepatocyte apoptosis with mitochondrial release of cytochrome c and activation of caspases 9 and 3 was detected in catB(+/+) mice 2 hours after the injection of TNF-alpha. In contrast, significantly less hepatocyte apoptosis and no detectable release of cytochrome c or caspase activation occurred in the livers of catB(-/-) mice. By 4 hours after TNF-alpha injection, only 20% of the catB(+/+) mice were alive as compared to 85% of catB(-/-) mice. Pharmacological inhibition of cat B in catB(+/+) mice with L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl-L-isoleucyl-L-proline (CA-074 Me) also reduced TNF-alpha-induced liver damage. The present data demonstrate that a cat B-mitochondrial apoptotic pathway plays a pivotal role in TNF-alpha-induced hepatocyte apoptosis and liver injury.     

Priming of hepatocytes for cell culture by partial hepatectomy prior to cell isolation

The combination of ex vivo gene transfer and a sufficient transplant model for hepatocytes may permit treatment of single enzyme-based metabolic liver diseases. Induction of replicative potential (priming) in hepatocyte cultures may enhance the efficiency of gene transfer under stable in vitro conditions. It is known that hepatocyte replication is increased in vivo after partial hepatectomy. We investigated the effect of partial hepatectomy prior to cell isolation on hepatocytes in vitro. Male Lewis rats served as donors. Hepatocytes were isolated by collagenase digestion from either intact livers or from livers 48 h after 70% hepatectomy (PH). Cells were seeded on collagen-coated culture dishes with hormone-supplemented culture media. Hepatocyte morphology, number, albumin secretion rate, and mono-ethyl-glycin-xylidid (MEGX)-biotransformation capacity were assessed on days 1, 3, and 5 in culture. PH significantly increased hepatocyte number and albumin secretion of cultured hepatocytes over the whole observation period. In contrast, MEGX-biotransformation capacity was significantly decreased. Morphology of cultured hepatocytes was not affected by PH prior to hepatocyte isolation. These results suggest a prolonged and complex response of hepatocytes to PH in vitro. Hepatocyte priming by PH is a promising approach toward stable cultures of proliferating hepatocytes and may provide a model for in vitro studies of hepatic regeneration mechanisms. Further research on hepatocyte priming toward an application in ex vivo gene transfer and hepatic tissue engineering seems justified.     

A novel therapy of inducing hepatocyte hypertrophy by activating Akt/mTORC1 pathway in acute liver failure

As a severe clinical syndrome, acute liver failure (ALF) is associated with high morbidity and mortality and liver transplantation is the best treatment of choice in most critically-ill cases, which yet suffers from obvious drawbacks including lack of resources and unnecessary surgery for those who can recover spontaneously. In ALF, massive cell deaths occur whereas liver regeneration via hepatocyte proliferation is largely inhibited. The serine/threonine kinase Akt serves as a central role in regulating signaling of growth factors, cytokines and other cellular stimuli within cell and thus correlates with diverse cellular functions, including the pro-survival pathway by inducing hypertrophy. Physiologically, cellular hypertrophy is an adaption in response to increased functional demand or other stresses. It has been demonstrated recently that Akt/mTORC1-controlled switch of hyperplasia to hypertrophy in pregnancy restored the regenerative capacity of aged liver and pharmacological activation of this pathway is sufficient to induce cell growth in the liver of nonpregnant aged mice. We hypothesized, therefore, that pharmacological therapy targeting to induce hepatocyte hypertrophy by activating the Akt/mTORC1 pathway might be a novel and promising option in the early management of patients with ALF as an alternative way to impaired process of cell proliferation.