Delayed liver regeneration after partial hepatectomy in adiponectin knockout mice

We previously demonstrated that adiponectin has anti-fibrogenic and anti-inflammatory effects in the liver of mouse models of various liver diseases. However, its role in liver regeneration remains unclear. The aim of this study was to determine the role of adiponectin in liver regeneration. We assessed liver regeneration after partial hepatectomy in wild-type (WT) and adiponectin knockout (KO) mice. We analyzed DNA replication and various signaling pathways involved in cell proliferation and metabolism. Adiponectin KO mice exhibited delayed DNA replication and increased lipid accumulation in the regenerating liver. The expression levels of peroxisome proliferator-activated receptor (PPAR) α and carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in mitochondrial fatty acid oxidation, were decreased in adiponectin KO mice, suggesting possible contribution of altered fat metabolism to these phenomena. Collectively, the present results highlight a new role for adiponectin in the process of liver regeneration.     

Impaired Ras membrane association and activation in PPARalpha knockout mice after partial hepatectomy

Liver regeneration after partial hepatectomy (PH) involves several signaling mechanisms including activation of the small GTPases Ras and RhoA in response to mitogens leading to DNA synthesis and cell proliferation. Peroxisome proliferator-activated receptor-alpha (PPARalpha) regulates the expression of several key enzymes in isoprenoid synthesis, which are key events for membrane association of Ras and RhoA. Thus the role of PPARalpha in cell proliferation after PH was tested. After PH, an increase in PPARalpha DNA binding was observed in wild-type mice, correlating with an increase in the PPARalpha-regulated enzyme acyl-CoA oxidase. In addition, the PPARalpha-regulated genes farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase were significantly increased in wild-type mice. However, these increases were not observed in PPARalpha knockout (PPARalpha -/-) mice. The peak in DNA synthesis observed 42 h after PH was reduced by approximately 60% in PPARalpha -/- mice, despite increases in TNF-alpha and IL-1. Also, under these conditions, membrane association of Ras was high in wild-type mice after PH but was impaired in PPARalpha -/- mice. Accordingly, Ras was significantly elevated in the cytosol in PPARalpha -/- mice. This observation correlated with lower levels of active GTP-bound Ras after PH in PPARalpha -/- mice compared with wild-type mice. Similar observations were made for RhoA. Moreover, deletion of PPARalpha blunted the activation of cyclin-dependent kinase (cdk)2/cyclin E and cdk4/cyclin D complexes. Collectively, these results support the hypothesis that PPARalpha is necessary for cell cycle progression in regenerating mouse liver via mechanisms involving prenylation of small GTPases Ras and RhoA.     

The ileal FGF15/19 to hepatic FGFR4 axis regulates liver regeneration after partial hepatectomy in mice

Fibroblast growth factor (FGF) has been considered to modulate liver regeneration (LR) after partial hepatectomy (PH) at the tissue level. Previous studies have demonstrated that FGF15 and FGF19 induce the activation of its receptor, FGF receptor 4 (FGFR4), which can promote hepatocellular carcinoma progression and regulate liver lipid metabolism. In this study, we aimed to explore the role of the ileal FGF15/19- hepatic FGFR4 axis in the LR after PH. Male C57BL/6 mice aged 8–12 weeks were partially hepatectomized and assessed for expression of ileal FGF15/19 to hepatic FGFR4 signaling. We used recombinant human FGF19 protein and a small interfering RNA (siRNA) of FGFR4 to regulate expression of the FGF15/19-FGFR4 axis in vitro and in vivo. The proliferation and cell cycle of hepatocytes, the expression levels of FGF15/19-FGFR4 downstream molecules, liver recovery, and lipid metabolism were assessed. We found that both ileal and serum FGF15 expression were upregulated and hepatic FGFR4 was activated after PH in mice. FGF15/19 promoted cell cycle progression, enhanced proliferation, and reduced hepatic lipid accumulation of hepatocytes both in vitro and in vivo. Furthermore, the proliferative effect and lipid regulatory properties of FGF15/19 were dependent on FGFR4 in hepatocytes. In addition, ileal FGF15/19-hepatic FGFR4 transduction during hepatocyte proliferation was regulated by extracellular regulated protein kinase (ERK) 1/2. In conclusion, the ileal FGF15/19 to hepatic FGFR4 axis is activated and promotes LR after PH in mice, supporting the potential of ileal FGF15/19 to hepatic FGFR4 axis-targeted therapy to enhance LR after PH.     

PI3K/Akt activation is critical for early hepatic regeneration after partial hepatectomy

Hepatic resection is associated with rapid proliferation and regeneration of the remnant liver. Phosphatidylinositol 3-kinase (PI3K), composed of a p85alpha regulatory and a p110alpha catalytic subunit, participates in multiple cellular processes, including cell growth and survival; however, the role of PI3K in liver regeneration has not been clearly delineated. In this study, we used the potent PI3K inhibitor wortmannin and small interfering RNA (siRNA) targeting the p85alpha and p110alpha subunits to determine whether total or selective PI3K inhibition would abrogate the proliferative response of the liver after partial hepatectomy in mice. Hepatic resection is associated with an induction in PI3K activity; total PI3K blockade with wortmannin and selective inhibition of p85alpha or p110alpha with siRNA resulted in a significant decrease in hepatocyte proliferation, especially at the earliest time points. Fewer macrophages and Kupffer cells were present in the regenerating liver of mice treated with wortmannin or siRNA to p85alpha or p110alpha, as reflected by a paucity of F4/80-positive cells. Additionally, PI3K inhibition led to an aberrant architecture in the regenerating hepatocytes characterized by vacuolization, lipid deposition, and glycogen accumulation; these changes were not noted in the sham livers. Our data demonstrate that PI3K/Akt pathway activation plays a critical role in the early regenerative response of the liver after resection; inhibition of this pathway markedly abrogates the normal hepatic regenerative response, most likely by inhibiting macrophage infiltration and cytokine elaboration and thus hepatocyte priming for replication.     

The nucleolar protein NML regulates hepatic ATP levels during liver regeneration after partial hepatectomy

We previously identified a novel protein complex, eNoSC, which senses intracellular energy status and epigenetically regulates the rDNA locus by changing the ratio between the numbers of active and silent gene clusters. eNoSC contains a novel nucleolar protein, Nucleomethylin (NML), which has a methyltransferase-like domain and binds to Lys9-dimethylated histone H3 at the rDNA locus, along with the NAD⁺-dependent deacetylase SIRT1 and the histone methyltransferase SUV39H. The aim of this study was to determine the role of NML in liver after partial hepatectomy (PHx). We assessed liver regeneration and lipid metabolism after PHx in wild-type (WT) and NML transgenic (NML-TG) mice. Survival rates of NML-TG mice were reduced after PHx. We found that hepatic triglyceride content in NML-TG mice remained elevated 48 h after PHx, but not delayed liver regeneration. Moreover, hepatic ATP levels in NML-TG mice were higher than that in WT 48 h after PHx. These observations suggest that NML may regulate consumption of hepatic triglyceride in liver regeneration after PHx due to storage of excess ATP. The delayed consumption of hepatic triglyceride may be the cause of reduced survival rate in NML-TG mice.     

Fas engagement accelerates liver regeneration after partial hepatectomy

Fas (CD95) is a receptor involved in induction of apoptotic cell death of Fas-bearing cells, including hepatocytes and T cells. Injection of Fas-specific antibodies into mice leads to fulminant hepatic failure and death. Fas also transduces growth-promoting signals in proliferating T cells, fibroblasts and some tumor cells. Here we show that partial hepatectomy, which triggers the immediate onset of liver regeneration, protected mice against the lethal effects of Fas-specific antibodies and prevented hepatocyte apoptosis in response to Fas engagement in vivo. Furthermore, Fas engagement accelerated liver regeneration after partial hepatectomy. Liver regeneration kinetics were delayed in mutant mice with decreased cell surface Fas expression (lpr mice). In contrast, regeneration was not delayed in lpr-cg mutant mice, which have a Fas mutation that prevents Fas-induced death but not Fas-dependent proliferative stimulation. Our results indicate that Fas engagement on cells in regenerating or healing tissues may promote cell growth.     

Serum alpha-fetoprotein in fulminant hepatitis and hepatic regeneration following partial hepatectomy

The pleomorphism of hepatic regeneration was studied in 10 patients with fulminant hepatitis and 7 with hepatocellular carcinoma, liver cyst, and abscess who underwent partial hepatectomy. Serum AFP levels did not increase significantly following partial hepatectomy. All of four patients who survived fulminant hepatitis had high serum AFP levels with a peak either during or before hepatic encephalopathy. Serum AFP levels decreased rather gradually during the enlargement of the atrophic liver. The observations proposed two kinds of hepatic regeneration, hepatic regeneration following surgical removal of liver and repair of liver damage following virally and probably chemically induced liver deficiency.     

Course of liver regeneration after partial hepatectomy in rats treated with dialysates obtained 17 hours after partial hepatectomy

Various theories have been put forward to explain the regenerative capacity of liver tissue induce by partial hepatectomy (PH). One of them presumes the existence of humoral factors stimulating proliferation of the liver tissue. We evaluated the course of liver regeneration after 65-70% PH as influenced by dialysates (DIA) of the organs of a rat killed 17 h after PH. In addition to kidney DIA, we were particularly interested in the effect of liver and spleen DIA. The experiments were carried out on rats weighting 310-370 g. Kidney, liver or spleen dialysate was administered subcutaneously and the rats were killed 12 or 24 h later by exsanguination from the abdominal aorta. In further rats, PH was performed 24 h after administering DIA and the rat were killed 18, 24, 30, 48 and 72 h after the operation. The initiation of liver regeneration was stimulated by all the given DIA, but especially by liver DIA. The faster onset of liver regeneration 18 h after PH in rats given spleen DIA is interesting. DIA did not greatly affect the hepatocytes of intact liver, but accelerated the initiation of liver regeneration after PH by synchronizing the cell cycle of proliferating hepatocytes. DIA obtained 17 h after PH contained substances which primarily stimulated liver DNA synthesis. From the changes in inhibition of the migration of spleen macrophages in the medium containing liver antigens, and from the circulating immunocomplex values, we conclude that DIA activation of the immune system, a well as the hepatic stimulator substance contained in the DIA, participates in acceleration of the liver regeneration process.     

Chronobiology of the proliferative events related to angiogenesis in mice liver regeneration after partial hepatectomy

In liver regeneration the formation of new capillary blood vessels is a fundamental requirement for cellular proliferation. Vascular endothelial growth factor (VEGF) is involved in the events of angiogenesis, the mRNA of which is expressed in both hepatocytes and non-parenchymal cells. In this experimental design we try to establish if during liver regeneration in mouse, the expression of VEGF is produced before or after the hepatocytes proliferation. C3H/S adult male mice were divided in three groups in order to study: VEGF expression; S-phase index (SI); and mitotic activity (MA) of hepatocytes. The results that were analyzed by ANOVA, show that VEGF expression starts to increase 26 h after PH with a peak at 28 h. Furthermore, the DNA synthesis (DNAs) reaches maximal level 42 h after pH, meanwhile the MA of the hepatocytes shows an increase 8h after the DNAs peak. In conclusion, it could be argued that the chronobiology of the events related to liver regeneration in mice started with a release of VEGF by the hepatocytes, followed by its DNAs and mitosis.     

Role of IL-6 in the induction of hepatic metallothionein in mice after partial hepatectomy

Metallothioneins (MT) are induced upon partial hepatectomy (PH), possibly mediated by various cytokines. In the present study, we studied cytokine-dependent MT synthesis in partially hepatectomized IL-6 gene knock-out (GKO) mice in the remaining lobe of the liver. We focused on IL-6, TNFalpha and IL-1beta, the major cytokines thought to be involved in MT synthesis. The IL-6 GKO mice and B6J129Sv (wild-type control) mice were subjected to 70% PH or laparotomy. We found that MT was significantly decreased in IL-6 GKO mice, although PH induced hepatic MT in both strains of mice. Laparotomy induced MT in the liver of wild-type mice but not in IL-6 GKO mice. Pretreatment of IL-6 GKO mice with rIL-6 (5 microg/mouse) restored hepatic MT synthesis. Serum IL-6 level in wild-type mice was maximal at 6 h after surgery and decreased thereafter. Serum IL-1beta was the same in both strains of mice. Serum TNFalpha basal level in IL-6 GKO mice was higher than in wild-type mice. PH caused an increase in serum TNFalpha level in both strains of mice, and it was two times higher in IL-6 GKO mice than in wild-type mice at 18 h after surgery. We conclude that IL-6 plays a predominant role in hepatic MT synthesis after PH, but that IL-6 GKO mice still reserve the capacity to synthesize MT by an as yet unidentified mechanism.     

Interleukin-1 receptor antagonist modulates the early phase of liver regeneration after partial hepatectomy in mice

Background

Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra) during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration.

Methods

We performed 70%-hepatectomy in wild type (WT) mice, IL-1ra knock-out (KO) mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU) incorporation, proliferating cell nuclear antigen (PCNA) and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes.

Results

At 24h and at 48h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1β and MCP-1) and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPβ expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment.

Conclusion

IL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.     

Impaired male sexual behavior in activin receptor type II knockout mice

Integration of multiple hormonal and neuronal signaling pathways in the medial preoptic area (mPOA) is required for elicitation of male sexual behavior in most vertebrates. Perturbation of nitric oxide synthase (NOS) activity in the mPOA causes significant defects in male sexual behavior. Although activins and their signaling components are highly expressed throughout the brain, including the mPOA, their functional significance in the central nervous system (CNS) is unknown. Here, we demonstrate a neurophysiologic role for activin signaling in male reproductive behavior. Adult activin receptor type II null (Acvr2-/-) male mice display multiple reproductive behavioral deficits, including delayed initiation of copulation, reduced mount, and intromission frequencies, and increased mount, intromission, and ejaculation latencies. These behavioral defects in the adult mice are independent of gonadotropin-releasing hormone (GnRH) homeostasis or mating-induced changes in luteinizing hormone (LH) and testosterone levels. The impairment in behavior can be correlated to the nitric oxide content in the CNS because Acvr2-/- males have decreased NOS activity in the mPOA but not the rest of the hypothalamus or cortex. Olfactory acuity tests confirmed that Acvr2-/- mice have no defects in general odor or pheromone recognition. In addition, motor functions are not impaired and the mutants demonstrate normal neuromuscular coordination and balance. Furthermore, the penile histology in mutant mice appears normal, with no significant differences in the expression of penile differentiation marker genes compared with controls, suggesting the observed behavioral phenotypes are not due to structural defects in the penis. Our studies identify a previously unrecognized role of activin signaling in male sexual behavior and suggest that activins and/or related family members are upstream regulators of NOS activity within the mPOA of the forebrain.