Liver Of Young Mice Research Articles

GRSF1 antagonizes age-associated hypercoagulability via modulation of fibrinogen mRNA stability

Age-associated hypercoagulability is accompanied by the increase of plasma levels of some coagulation factors including fibrinogen which may contribute to the increased risk of cardiovascular, cerebrovascular, and thrombotic diseases in elderly people. However, the underlying mechanism of increased plasma fibrinogen concentration during aging is still elusive. GRSF1 belongs to the heterogeneous nuclear ribonucleoproteins F/H (hnRNP F/H) subfamily. Here, we report that GRSF1 attenuates hypercoagulability via negative modulation of fibrinogen expression. We demonstrated that GRSF1 negatively regulated fibrinogen expression at both mRNA and protein levels. GRSF1 directly interacted with the coding region (CDS) of FGA, FGB, and FGG mRNAs, and decreased their stability thus mitigating fibrinogen expression. We further identified that only a few G-tracts within the Fib C domain of FGA, FGB, and FGG CDS and the qRRM2 domain of GRSF1 were required for their interaction. Moreover, we confirmed hypercoagulability and the decrease of GRSF1 expression level during mice aging. Functionally, GRSF1 overexpression in old mice liver decreased fibrinogen plasma level, reduced hypercoagulability, and mitigated blood coagulation activity, whereas GRSF1 knockdown in young mice liver increased fibrinogen plasma level and promoted blood coagulation activity. Collectively, our findings unveil a novel posttranscriptional regulation of fibrinogen by GRSF1 and uncover a critical role of GRSF1 in regulating blood coagulation activity.

Single-cell RNA sequencing to detect age-associated genes that identify senescent cells in the liver of aged mice

Senescent cells are predicted to occur and increase in animal tissues with aging. However, senescent cells in the tissues of aged animals remain to be identified. We refer to the marker genes to identify senescent cells in tissues as “age-associated genes”. In this study, we searched for age-associated genes to identify senescent cells in the livers of aged animals. We performed single-cell RNA sequencing (scRNA-seq) to screen candidates for age-associated genes using young and aged rat primary hepatocytes. To remove animal species specificity, gene expression analyses in mouse livers were performed, confirming age-associated increases in the mRNA expression levels of Glipr1, Clec12a, and Phlda3. Moreover, the mRNA expression levels of Glipr1 and Phlda3 were increased by stress-induced premature senescence using doxorubicin in primary hepatocytes and livers of young mice. Transcriptome data of aged rat hepatocytes suggested that Glipr1, Clec12a, and Phlda3 were expressed in almost identical cells. Fluorescence in situ hybridization (FISH) confirmed the presence of cells with abundant Glipr1, Clec12a, and Phlda3 mRNA in 27-month-old mouse primary hepatocytes, which are considered to be senescent cells. This study is the first to identify Glipr1, Clec12a, and Phlda3 as age-associated genes in the mouse liver.

Burn-Induced Apoptosis in the Livers of Aged Mice Is Associated With Caspase Cleavage of Bcl-xL

IntroductionOlder adult burn victims have poorer outcomes than younger burn victims. The liver is critical for the recovery of patients with burns. Postburn hepatic apoptosis in young individuals compromises liver integrity; however, this pathway has not yet been studied in older individuals. Because aged animals with burns suffer significant liver damage, we hypothesized that apoptosis is altered in these animals and may affect liver function. Understanding postburn hepatic apoptosis and its effects on liver function in aged animals may help improve outcomes in older patients. MethodsWe compared the protein and gene expression levels in young and aged mice after a 15% total-body-surface-area burn. Liver and serum samples were collected at different time points after injury. ResultsCaspase-9 expression in liver tissue was downregulated by 47% in young animals and upregulated by 62% in aged animals 9 h postburn (P < 0.05). The livers of aged mice showed a Bcl-extra-large (Bcl-xL) transcription increase only after 6 h; however, the livers of young mice exhibited 4.3-fold, 14.4-fold, and 7.8-fold Bcl-xL transcription increases at 3, 6, and 9 h postburn, respectively (P < 0.05). The livers of young mice showed no changes in Caspase-9, Caspase-3, or Bcl-xL protein levels during the early postburn period. In contrast, the livers of aged mice contained cleaved caspase-9, reduced full-length caspase-3, and an accumulation of ΔN-Bcl-x at 6 and 9 h postburn (P < 0.05). p21 expression decreased in aged mice; however, it was significantly increased in the liver tissue of young mice postburn (P < 0.05). Serum amyloid A1 and serum amyloid A2 serum protein levels were 5.2- and 3.1-fold higher in young mice than in aged mice, respectively, at 6 and 9 h postburn (P < 0.05). ConclusionsLivers of aged mice exhibited different apoptotic processes compared to those of young mice early after burn injury. Collectively, burn-induced liver apoptosis in aged mice compromises hepatic serum protein production.

RHODIOLA ROSEA AND FERULIC ACID ACTIVATE EXPRESSION OF GENES RELATED TO AUTOPHAGY AND RESISTANCE TO HEAT SHOCK IN MICE OF DIFFERENT AGE

The aim of our study was testing whether R. rosea extract and ferulic acid activate expression of targets of FoxO, regulators of energy metabolism and autophagy in livers of young and old mice, and to what extent the effects of R. rosea extract and ferulic acid on the genes studied coincide. Methods. . C57BL/6J mice were reared at 22 ± 2 °С, 50-60% humidity, and 12/12 hour light/dark cycle. All groups were reared on a standard chow (4,8% fats, 21,8% protein, and 3,9% fibre). Experimental groups consumed water, supplemented with either sodium ferulate or R. rosea during 12 weeks prior sacrificing. The amounts of ferulate and R. rosea were adjusted to provide 4 mg of phenol-containing substances per 100 g weight, for a mouse, for 24 hours. We tested three-month-old (“young”) and twelve-month-old males (“old”). The levels of messenger ribonucleic acid (mRNA) were assessed using AriaMx real-time polymerase chain reaction (RT-PCR) instrument (Agilent). Ribonucleic acid was purified using the Monarch Miniprep kit (New England BioLabs (NEB), T2010), complementary deoxyribonucleic acid synthesis was performed using the ProtoScript II kit (NEB, E6560), and quantitative RT-PCR (qRT-PCR) was performed using the Luna Universal kit (NEB, E3003). The expression of genes ATG5 (an autophagy marker), HSPB8 (a small heat shock protein, an FoxO target), UCP2 (uncoupling protein 2, a senescence marker), CDKN2 (cell cycle regulator, a senescence marker), PDK2 and PDK4 (pyruvate dehydrogenase kinases 2 and 4, regulators of oxidative metabolism), and TFEB (transcription factor EB, a transcriptional regulator of autophagy) was evaluated. Results. Livers of young mice that consumed food supplemented with either sodium ferulate or R. rosea extract had 3.2-fold and 3.6-fold higher levels of mRNA of the small heat shock protein HspB8 than control mice, respectively. In old mice, the levels of mRNA for this protein were 3.3-fold higher in mice reared on the diet containing R. rosea extract as compared with the control. However, there was no significant difference between control mice and those that consumed ferulate-supplemented food. In young mice, ferulate and R. rosea extract induced synthesis of mRNA of PDK4 by 4.3 and 6.6 times from the control level, respectively. Ferulate and R. rosea extract also affected the levels of mRNA of ATG5 and PDK2 in the livers of old mice. The levels of PDK2 were 1.5-fold higher in the livers of mice that consumed ferulate-supplemented food than in control mice. Conclusions. Both, R. rosea extract and one of its active components – ferulic acid – promote increasing in the levels of mRNA for genes HSPB8 and PDK4, coding for small heat shock protein and pyruvate dehydrogenase kinase 4, respectively. In old mice, R. rosea promote expression of HSPB8, ATG5, PDK2, and PDK4. Thus, ferulic acid and R. rosea exert similar effects on gene expression by supposed activation of heat shock response and autophagy, and concomitant inhibition of mitochondrial metabolism via boosting expression of PDK2 and PDK4.

The protective effect of lncRNA NEAT1/miR-122-5p/Wnt1 axis on hippocampal damage in hepatic ischemic reperfusion young mice

Hepatic ischemic reperfusion (HIR) is a common pathophysiological process in many surgical procedures such as liver transplantation (LT) and hepatectomy. And it is also an important factor leading to perioperative distant organ damage. Children undergoing major liver surgery are more susceptible to various pathophysiological processes, including HIR, since their brains are still developing and the physiological functions are still incomplete, which can lead to brain damage and postoperative cognitive impairment, thus seriously affecting the long-term prognosis of the children. However, the present treatments of mitigating HIR-induced hippocampal damage are not proven to be effective. The important role of microRNAs (miRNAs) in the pathophysiological processes of many diseases and in the normal development of the body has been confirmed in several studies. The current study explored the role of miR-122-5p in HIR-induced hippocampal damage progression. HIR-induced hippocampal damage mouse model was induced by clamping the left and middle lobe vessels of the liver of young mice for 1 h, removing the vessel clamps and re-perfusing them for 6 h. The changes in the level of miR-122-5p in the hippocampal tissues were measured, and its influences on the activity as well as apoptotic rate of neuronal cells were investigated. Short interfering RNA modified with 2’-O-methoxy substitution targeting long-stranded non-coding RNA (lncRNA) nuclear enriched transcript 1 (NEAT1) as well as miR-122-5p antagomir were used to further clarify the role played by the corresponding molecules in hippocampal injury in young mice with HIR. The result obtained in our study was that the expression of miR-122-5p in the hippocampal tissue of young mice receiving HIR is reduced. Upregulated expression of miR-122-5p reduces the viability of neuronal cells and promotes the development of apoptosis, thereby aggravating the damage of hippocampal tissue in HIR young mice. Additionally, in the hippocampal tissue of young mice receiving HIR, lncRNA NEAT1 exerts some anti-apoptotic effects by binding to miR-122-5p, promoting the expression of Wnt1 pathway. An essential observation of this study was the binding of lncRNA NEAT1 to miR-122-5p, which upregulates Wnt1 and inhibits HIR-induced hippocampal damage in young mice.

Remodeling of whole-body lipid metabolism and a diabetic-like phenotype caused by loss of CDK1 and hepatocyte division.

Cell cycle progression and lipid metabolism are well-coordinated processes required for proper cell proliferation. In liver diseases that arise from dysregulated lipid metabolism, proliferation is diminished. To study the outcome of CDK1 loss and blocked hepatocyte proliferation on lipid metabolism and the consequent impact on whole-body physiology, we performed lipidomics, metabolomics, and RNA-seq analyses on a mouse model. We observed reduced triacylglycerides in liver of young mice, caused by oxidative stress that activated FOXO1 to promote expression of Pnpla2/ATGL. Additionally, we discovered that hepatocytes displayed malfunctioning β-oxidation, reflected by increased acylcarnitines (ACs) and reduced β-hydroxybutyrate. This led to elevated plasma free fatty acids (FFAs), which were transported to the adipose tissue for storage and triggered greater insulin secretion. Upon aging, chronic hyperinsulinemia resulted in insulin resistance and hepatic steatosis through activation of LXR. Here, we demonstrate that loss of hepatocyte proliferation is not only an outcome but also possibly a causative factor for liver pathology.

Histological and Physiological Studies on the Long-term Effect of Different Concentrations of Energy Drink (Tiger) on the Renal and Hepatic Systems of Young Mice

The present study aims to investigate the long-term histopathological, and physiological effects of different concentrations of a commercially available energy drink (Tiger) on liver and kidney of young mice. Sixteen Balb/c male mice,6 -week old, were divided into 4 groups (n=4). Two groups consumed the energy drink at a concentration of 28µl energy drink/ml water. One group were killed after 10 days (T1), another group were killed after 20 days (T2). Other group of mice consumed the energy drink at a final concentration of 14µl/ml for 20 days (T3). The last group was provided only with water and served as control. Mice of all groups drank around 3 ml per day. The histopathological study on liver of treated groups showed many changes such as inflammatory cells infiltration and aggregation with hepatocyts necrosis, some of these necrosis replaced by RBCs and inflammatory cells, while the pathohistological changes in kidney of treated groups limited to aggregation of RBCs and inflammatory cells between renal tubules which expressed vacuolar degeneration. These changes based on elevated liver function enzymes (Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvic Transaminase (GPT) and Alkaline phosphatase (ALP)) and blood urea and creatinine. These changes were more in the T2 groups, so it could be concluded that long term of energy drink consuming effect histopathologically and physiologically on kidney and liver of young mice depending on its concentration and period of consuming.

Every-Other-Day Feeding Decreases Glycolytic and Mitochondrial Energy-Producing Potentials in the Brain and Liver of Young Mice.

Intermittent fasting is used to reduce body mass in obese adult humans and animals. However, information on the impact of one type of intermittent fasting (IF) called every-other-day feeding (EODF) on young animals is scarce. In this study, 1-month-old mice of both sexes were subjected to a 4-week regimen of EODF using age-matched counterparts fed ad libitum as controls. At the end of EODF exposure, experimental male and female mice weighed 14 and 13% less than the control counterparts. The EODF regimen resulted in lower liver levels of glycogen, glucose, and lactate, but did not affect lactate level in mouse cerebral cortex of both sexes. Activities of key glycolytic enzymes (hexokinase, phosphofructokinase, and pyruvate kinase) in liver of experimental mice were lower than those in controls. In the cerebral cortex, only hexokinase and pyruvate kinase activities were lower than in controls, but phosphofructokinase activity was not affected in IF females and was higher in IF males as compared with ad libitum fed males. Mitochondria isolated from liver of IF mice had lower respiratory control ratios, but those from the cortex had the same values as control animals. The concentration of β-hydroxybutyrate and the activity of β-hydroxybutyrate dehydrogenase were lower in the IF mouse liver, but not changed or enhanced in the IF cerebral cortex. Thus, animal responses to IF do not depend significantly on sex and are directed to decrease energy metabolism to save resources, and the effects are more pronounced in the liver than in the brain.

Healthy mitochondria inhibit the metastatic melanoma in lungs.

Tumor mitochondria alter their functions to reprogram cell metabolism and then allow tumor cells to rapidly proliferate in the hypoxic and acidic microenvironment. However, roles of normal mitochondria played in tumor progression are still unclear. Here we investigate the normal mitochondrial effect on abnormal metabolism of tumors, and to clarify why the mitochondria have to undergo functional changes in the tumor growth. The mitochondria isolated from healthy mouse livers were intravenously injected into melanoma model mice with lung metastasis, then the tumor growth, animal survival and associated metabolic changes were studied. The results reveal that the mitochondria significantly retard tumor growth and increase survival days of animals. The anti-tumor effect of the mitochondria is related to interfering the tumor cell metabolisms, such as reducing glycolysis and producing an oxidative intracellular environment, all of which are not suitable for tumor cell proliferation. In addition, the mitochondria increases cell apoptosis, necrosis, and mitophagy. These effects are more efficient with the mitochondria isolated from young mouse livers than those from aged mice. Our study not only provides a valuable approach to invest mitochondrial function associated with tumor growth but also offer new insight into tumor therapy through interfering the tumor cell metabolism by healthy mitochondria.

Age-dependent regulation of antioxidant genes by p38α MAPK in the liver

p38α is a redox sensitive MAPK activated by pro-inflammatory cytokines and environmental, genotoxic and endoplasmic reticulum stresses. The aim of this work was to assess whether p38α controls the antioxidant defense in the liver, and if so, to elucidate the mechanism(s) involved and the age-related changes. For this purpose, we used liver-specific p38α-deficient mice at two different ages: young-mice (4 months-old) and old-mice (24 months-old). The liver of young p38α knock-out mice exhibited a decrease in GSH levels and an increase in GSSG/GSH ratio and malondialdehyde levels. However, old mice deficient in p38α had higher hepatic GSH levels and lower GSSG/GSH ratio than young p38α knock-out mice. Liver-specific p38α deficiency triggered a dramatic down-regulation of the mRNAs of the key antioxidant enzymes glutamate cysteine ligase, superoxide dismutase 1, superoxide dismutase 2, and catalase in young mice, which seems mediated by the lack of p65 recruitment to their promoters. Nrf-2 nuclear levels did not change significantly in the liver of young mice upon p38α deficiency, but nuclear levels of phospho-p65 and PGC-1α decreased in these mice. p38α-dependent activation of NF-κB seems to occur through classical IκB Kinase and via ribosomal S6 kinase1 and AKT in young mice. However, unexpectedly the long-term deficiency in p38α triggers a compensatory up-regulation of antioxidant enzymes via NF-κB activation and recruitment of p65 to their promoters. In conclusion, p38α MAPK maintains the expression of antioxidant genes in liver of young animals via NF-κΒ under basal conditions, whereas its long-term deficiency triggers compensatory up-regulation of antioxidant enzymes through NF-κΒ.

Metabolomics of the Wolfram Syndrome 1 Gene (Wfs1) Deficient Mice.

Wolfram syndrome 1 is a rare autosomal recessive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. Mutations in the WFS1 gene encoding the wolframin glycoprotein can lead to endoplasmic reticulum stress and unfolded protein responses in cells, but the pathophysiology at whole organism level is poorly understood. In this study, several organs (heart, liver, kidneys, and pancreas) and bodily fluids (trunk blood and urine) of 2- and 6-month old Wfs1 knockout (KO), heterozygote (HZ), and wild-type (WT) mice were analyzed by untargeted and targeted metabolomics using liquid chromatography-mass spectrometry. The key findings were significant perturbations in the metabolism of pancreas and heart before the onset of related clinical signs such as glycosuria that precedes hyperglycemia and thus implies a kidney dysfunction before the onset of classical diabetic nephropathy. The glucose use and gluconeogenesis in KO mice are intensified in early stages, but later the energetic needs are mainly covered by lipolysis. Furthermore, in young mice liver and trunk blood hypouricemia, which in time turns to hyperuricemia, was detected. In summary, we show that the metabolism in Wfs1-deficient mice markedly differs from the metabolism of WT mice in many aspects and discuss the future biological and clinical relevance of these observations.

METHIONINE RESTRICTION ALTERS HEPATIC MIRNAS INVOLVED IN METABOLISM IN YOUNG, OBESE, AND AGED MICE

Age-associated disorders, such as Type II diabetes and nonalcoholic fatty liver disease, can be regulated by microRNAs. Dietary methionine restriction (MR) in young mice reduces adiposity and increases glucose sensitivity. The aim of this study was to determine whether previously identified miRNAs altered by MR in livers of young mice were similarly regulated in diet-induced obese (DIO) mice and aged mice. Young and aged male (8-week-old and 18-month-old, respectively) C57BL/6J mice were fed either a control (0.86% methionine; CF) or MR (0.12% methionine) diet; DIO mice (12-weeks-old; 40 grams) were fed a high–fat (60%) CF or MR diet for 12 weeks. MR repressed hepatic expression of several miRNAs (miR-455-5p, miR-33-5p, miR-99a-5p, and let-7g-5p) and increased the expression of mRNAs (Scarb1, Abcb11, Irs2, Insr, mTor, and Hadhb) that regulate the synthesis and transport of cholesterol, bile acids, fatty acids, and insulin in both young and DIO mice. However, in aged mice, miR-33-5p and miR-99a-5p, which regulate bile acid transport and mTOR signaling, respectively, were not affected by MR, while let-7g-5p, which regulates insulin signaling, was increased. MiR-455-5p, which can target Scarb1 and regulate cholesterol synthesis, remained repressed by MR. These findings suggest that the benefits of MR on liver function in young and DIO mice appear to be achieved through multiple mechanisms to attenuate a fatty liver condition, and that only some of these mechanisms are still effective in old mice.

DNA Synthesis in Periportal and Perivenous Hepatocytes of Intact and Hepatectomized Young Mice

DNA synthesis of hepatocytes in two areas of Intact and Hepatectomized young mice liver along a circadian period was studied. DNA synthesis was significantly different at all analyzed time points in Intact and Hepatectomized animals. Differences between periportal and perivenous hepatocytes were found in hepatectomized animals at 04/42 and 08/46 hr of day/hour post-hepatectomy. DNAs peak in periportal hepatocytes regenerating liver occurs 4 hr earlier than in perivenous hepatocytes, probably reflecting their shorter G1 phase. Besides, daily mean values of regenerating livers were higher than those observed in Intact animals, as a consequence of surgical removal.

S-3: Age-dependent hepatic lymphoid organization directs immune priming, cytokine production, and successful immunity to Hepatitis B virus

Hepatitis B virus (HBV) is a non-cytopathic hepadnavirus and major human pathogen that causes immune-mediated acute and chronic hepatitis. The immune response to HBV antigens can result in viral clearance by some individuals, or lead to chronic infection and liver disease in others. The age of the individual at the time of infection dictates the likelihood of HBV clearance. We sought mechanisms underpinning the age-dependent outcome of HBV. We now present an animal model that recapitulates key differences in viral clearance during early life and adulthood, and show an unexpected role for T Follicular Helper T cells and IL-21 in determining the effectiveness of the primary hepatic immune response to HBV. Furthermore, we provide evidence that IL-21 is part of an effective immune response to HBV in patients acutely infected with HBV who clear the virus. In addition, we now demonstrate that hepatic macrophages facilitate lymphoid organization and immune priming within the adult liver and promote successful immunity. In contrast, lymphoid organization and immune priming is greatly diminished in the livers of young mice, and of macrophage-depleted adult mice, leading to abrogated HBV immunity. These observations provide a new mechanism to explain the dichotomous, age-dependent outcome of HBV infection in humans, and carry implications for therapeutic augmentation of immune responses to HBV and potentially other persistent liver viruses.

Age-dependent hepatic lymphoid organization directs successful immunity to hepatitis B

Hepatitis B virus (HBV) is a major human pathogen that causes immune-mediated hepatitis. Successful immunity to HBV is age dependent: viral clearance occurs in most adults, whereas neonates and young children usually develop chronic infection. Using a mouse model of HBV infection, we sought mechanisms underpinning the age-dependent outcome of HBV and demonstrated that hepatic macrophages facilitate lymphoid organization and immune priming within the adult liver and promote successful immunity. In contrast, lymphoid organization and immune priming was greatly diminished in the livers of young mice, and of macrophage-depleted adult mice, leading to abrogated HBV immunity. Furthermore, we found that CXCL13, which is involved in B lymphocyte trafficking and lymphoid architecture and development, is expressed in an age-dependent manner in both adult mouse and human hepatic macrophages and plays an integral role in facilitating an effective immune response against HBV. Taken together, these results identify some of the immunological mechanisms necessary for effective control of HBV.

Specific alterations of carbohydrate metabolism are associated with hepatocarcinogenesis in mitochondrially impaired mice

Friedreich's ataxia is an inherited neurodegenerative disease caused by the reduced expression of the mitochondrially active protein frataxin. We have previously shown that mice with a hepatocyte-specific frataxin knockout (AlbFxn(-/-)) develop multiple hepatic tumors in later life. In the present study, hepatic carbohydrate metabolism in AlbFxn(-/-) mice at an early and late life stage was analyzed. In young (5-week-old) AlbFxn(-/-) mice hepatic ATP, glucose-6-phosphate and glycogen levels were found to be reduced by ∼74, 80 and 88%, respectively, when compared with control animals. This pronounced ATP, G6P and glycogen depletion in the livers of young mice reverted in older animals: while half of the mice die before 30 weeks of age, the other half reaches 17 months of age and exhibits glycogen, G6P and ATP levels similar to those in age-matched controls. A key event in this respect seems to be the up-regulation of GLUT1, the predominant glucose transporter in fetal liver parenchyma, which became evident in AlbFxn(-/-) mice being 5-12 weeks of age. The most significant histological findings in animals being 17 or 22 months of age were the appearance of multiple clear cell, mixed cell and basophilic foci throughout the liver parenchyma as well as the development of hepatocellular adenomas and carcinomas. The hepatocarcinogenic process in AlbFxn(-/-) mice shows remarkable differences regarding carbohydrate metabolism alterations when compared with all other chemically and virally driven liver cancer models described up to now.

The reduction of SIRT1 in livers of old mice leads to impaired body homeostasis and to inhibition of liver proliferation

Age declines liver functions, leading to the development of age-associated diseases. A member of the sirtuins family, SIRT1, is involved in the control of glucose homeostasis and fat metabolism. Because aging livers have alterations in glucose and fat metabolism, we examined a possible role of SIRT1 in these alterations. We found that aged livers have a reduced expression of SIRT1 and have lost proper control of the regulation of SIRT1 after partial hepatectomy (PH). Down-regulation of SIRT1 in the liver of old mice is mediated by CCAAT/Enhancer Binding Protein/histone deacetylase 1 (C/EBPβ-HDAC1) complexes, which bind to and repress the SIRT1 promoter. In the livers of young mice, SIRT1 is activated after PH and supports high levels of glucose and triglycerides during liver regeneration. In old mice, however, C/EBPβ-HDAC1-mediated repression of the SIRT1 promoter blocks activation of SIRT1, leading to low levels of glucose and triglycerides during liver regeneration. Down-regulation of SIRT1 in the livers of young mice resulted in alterations similar to those observed in the livers of old mice, whereas the normalization of SIRT1 in the livers of old mice corrects the levels of glucose and triglycerides after PH. The normalization of SIRT1 in old mice also improves liver regeneration via the elimination of the C/EBPα-Brm complex. These studies showed a critical role of the reduction of SIRT1 in age-associated liver dysfunctions and provide a potential tool for the correction of liver functions in old patients after surgical resections.

The difference of glutathione antioxidant system in newly weaned and young mice liver and its involvement in isoline-induced hepatotoxicity

Cellular glutathione antioxidant system plays important roles in counteracting hepatotoxins-induced oxidative stress injury. The present study was designed to observe the differences of this system in newly weaned and young mice liver and its involvement in the susceptibility to isoline-induced liver injury. Our results showed that liver reduced glutathione (GSH) amounts were higher in newly weaned mice than young mice. Glutamate-cysteine ligase (GCL) activity was higher in newly weaned mice due to the higher expression of catalytic subunit of GCL (GCLC) protein and mRNA. However, the activities of glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) were higher in young mice liver, which might be due to the higher expression of GR, GPx-1, and GST-Pi proteins. Next, the results of AST analysis and histopathological evaluation showed that newly weaned mice demonstrated more severe liver injury induced by isoline. Furthermore, liver GSH amounts and the activities of GR, GPx, and GST were all lower in newly weaned mice than young mice after treated with isoline. Depletion of cellular GSH by D,L -buthionine-(S, R)-sulfoximine (BSO) aggravated isoline-induced cytotoxicity, while N-acetyl-l cysteine (NAC) ameliorated such cytotoxicity. Furthermore, the inhibitors of GR, GPx, and GST all aggravated isoline-induced cytotoxicity. In conclusion, our results demonstrated the differences of glutathione antioxidant system between newly weaned and young mice liver. Meanwhile, our results also revealed age-dependent liver injury induced by isoline for the first time, which might be due to the different responses of glutathione antioxidant system to isoline between newly weaned and young mice.

Lack of apoE causes alteration of cytokines expression in young mice liver

To investigate the effect of apolipoprotein E (apoE) on cytokine expression profile of the liver of young mice, quantitative RT-PCR (qRT-PCR) assay and cytokine antibody array for multiplex analysis of 62 cytokines have been used to analyze characteristics of expression of cytokines in the liver of 6-week-old apoE-null (apoE(-/-)) mice. The levels of plasma cytokines were also analyzed. The mRNA level of IL-1 beta, IL-2, IL-6, ICAM-1, VCAM-1, MCP-1, NF-kappaB (p65), IFN-gamma and I kappaB-alpha were increased significantly in apoE(-/-) mice comparative to wild-type (WT) mice. IL-4, IL-10 and GM-CSF, however, were slightly decreased. Compared with WT, levels of 21 cytokines altered twofold or more in apoE(-/-) mice, including 10 cytokines increased and 11 decreased. Expression patterns of IL-1 beta, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma and VCAM-1 showed identical trend between cytokine antibody array and qRT-PCR analysis. Moreover, levels of IL-1 beta, IFN-gamma and IL-6 in the plasma were elevated, while IL-4 was lightly decreased in apoE(-/-) mice compared to those in WT mice. These results implied that promotion of type I immune response in the liver of young apoE(-/-) mice due to alteration of these cytokines, and the phenotypes may be caused by the regulation of NF-kappaB. The inflammation and lipid metabolism dysfunction in the liver cooperated in dysfunction of the liver in young apoE(-/-) mice.

HDAC1 Cooperates with C/EBPα in the Inhibition of Liver Proliferation in Old Mice

Epigenetic control of liver proliferation involves cooperation between transcription factors and chromatin-remodeling proteins. In this work, we found that the levels of HDAC1 (histone deacetylase 1) are increased in quiescent livers of old mice. The elevation of HDAC1 in liver is mediated by the RNA-binding protein CUGBP1. We found that the age-associated CUGBP1-eIF2 complex binds to the 5' region of HDAC1 mRNA and increases translation of HDAC1 in the liver. Further analyses showed that CUGBP1 also increases expression of HDAC1 in cultured cells, in the livers of CUGBP1 transgenic mice, and in the livers of mice injected with cyclin D3, which enhances the formation of the CUGBP1-eIF2 complex. In livers of old mice, HDAC1 interacts with the transcription factor C/EBPalpha and is recruited by this protein to E2F-dependent promoters as a component of high M(r) C/EBPalpha-Brm complexes. The recruitment of HDAC1 to c-Myc and FoxM1B promoters leads to deacetylation of histone H3 at Lys-9 on these E2F-dependent promoters. We show that HDAC1 is an important mediator of growth-inhibitory activity of C/EBPalpha and that small interfering RNA-mediated inhibition of HDAC1 reduces the ability of C/EBPalpha to inhibit cell proliferation. In addition, we have found that both elevation of HDAC1 and interaction of C/EBPalpha with HDAC1 are controlled by cyclin D3-dependent mechanisms. Treatment of old mice with growth hormone, which reduces cyclin D3 levels, leads to the reduction of the CUGBP1-eIF2 complex, normalization of HDAC1 levels, and inhibition of interactions of HDAC1 with C/EBPalpha-Brm complexes. Thus, our data demonstrate that translational elevation of HDAC1 in livers of old mice is involved in the assembly of high M(r) protein-protein complexes that inhibit liver proliferation.