Liver Of Old Mice Research Articles

Flavonoid 4,4′-dimethoxychalcone selectively eliminates senescent cells via activating ferritinophagy

Flavonoids are bioactive natural polyphenolic compounds with health benefits, including anti-tumor, anti-inflammatory and anti-aging effects. Our previous studies revealed that a flavonoid 4,4′-dimethoxychalcone (DMC) induced ferroptosis via inhibiting ferrochelatase (FECH). However, the effect of DMC on cellular senescence is unknown. In the present study, we found that DMC treatment selectively eliminated senescent cells, and DMC alone or a combination of DMC and quercetin or dasatinib showed high efficiency in the clearance of senescent cells. We identified FECH was highly expressed in senescent cells compared to non-senescent cells. Mechanistically, we found that DMC inhibited FECH and induced ferritinophagy, which led to an increase of labile iron pool, triggering ferroptosis of senescent cells. Importantly, we found that DMC treatment prevented hair loss, improved motor coordination, and reduced the expression of several senescence-associated secretory phenotype factors (IL-6, IL-1β, CXCL-10, and MMP12) in the liver of old mice. Collectively, we revealed that, through the induction of ferroptosis, DMC holds the promise as a new senolytics to prevent age-related pathologies.

Suppression of FOXO1 attenuates inflamm-aging and improves liver function during aging.

The liver is a key metabolic organ that maintains whole-body nutrient homeostasis. Aging-induced liver function alterations contribute to systemic susceptibility to aging-related diseases. However, the molecular mechanisms of liver aging remain insufficiently understood. In this study, we performed bulk RNA-Seq and single-cell RNA-Seq analyses to investigate the underlying mechanisms of the aging-induced liver function changes. We found that liver inflammation, glucose intolerance, and liver fat deposition were aggravated in old mice. Aging significantly increased pro-inflammation in hepatic macrophages. Furthermore, we found that Kupffer cells (KCs) were the major driver to induce pro-inflammation in hepatic macrophages during aging. In KCs, aging significantly increased pro-inflammatory levels; in monocyte-derived macrophages (MDMs), aging had a limited effect on pro-inflammation but led to a functional quiescence in antigen presentation and phagosome process. In addition, we identified an aging-responsive KC-specific (ARKC) gene set that potentially mediates aging-induced pro-inflammation in KCs. Interestingly, FOXO1 activity was significantly increased in the liver of old mice. FOXO1 inhibition by AS1842856 significantly alleviated glucose intolerance, hepatic steatosis, and systemic inflammation in old mice. FOXO1 inhibition significantly attenuated aging-induced pro-inflammation in KCs partially through downregulation of ARKC genes. However, FOXO1 inhibition had a limited effect on aging-induced functional quiescence in MDMs. These results indicate that aging induces pro-inflammation in liver mainly through targeting KCs and FOXO1 is a key player in aging-induced pro-inflammation in KCs. Thus, FOXO1 could be a potential therapeutic target for the treatment of age-associated chronic diseases.

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.

Necroptosis contributes to chronic inflammation and fibrosis in aging liver.

Inflammaging, characterized by an increase in low‐grade chronic inflammation with age, is a hallmark of aging and is strongly associated with various age‐related diseases, including chronic liver disease (CLD) and hepatocellular carcinoma (HCC). Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we tested the hypothesis that age‐associated increase in necroptosis contributes to chronic inflammation in aging liver. Phosphorylation of MLKL and MLKL oligomers, markers of necroptosis, as well as phosphorylation of RIPK3 and RIPK1 were significantly upregulated in the livers of old mice relative to young mice and this increase occurred in the later half of life (i.e., after 18 months of age). Markers of M1 macrophages, expression of pro‐inflammatory cytokines (TNFα, IL6 and IL1β), and markers of fibrosis were all significantly upregulated in the liver with age and the change in necroptosis paralleled the changes in inflammation and fibrosis. Hepatocytes and liver macrophages isolated from old mice showed elevated levels of necroptosis markers as well as increased expression of pro‐inflammatory cytokines relative to young mice. Short‐term treatment with the necroptosis inhibitor, necrostatin‐1s (Nec‐1s), reduced necroptosis, markers of M1 macrophages, fibrosis, and cell senescence as well as reducing the expression of pro‐inflammatory cytokines in the livers of old mice. Thus, our data show for the first time that liver aging is associated with increased necroptosis and necroptosis contributes to chronic inflammation in the liver, which in turn appears to contribute to liver fibrosis and possibly CLD.

Aging Reduces Insulin Clearance in Mice.

Hyperinsulinemia is frequently associated with aging and may cause insulin resistance in elderly. Since insulin secretion and clearance decline with age, hyperinsulinemia seems to be maintained, primarily, due to a decrease in the insulin clearance. To investigate these aging effects, 3- and 18-month-old male C57BL/6 mice were subjected to intraperitoneal glucose and insulin tolerance tests (ipGTT and ipITT) and, during the ipGTT, plasma c-peptide and insulin were measure to evaluate in vivo insulin clearance. Glucose-stimulated insulin secretion in isolated pancreatic islets was also assessed, and liver samples were collected for molecular analyses (western blot). Although insulin sensitivity was not altered in the old mice, glucose tolerance, paradoxically, seems to be increased, accompanied by higher plasma insulin, during ipGTT. While insulin secretion did not increase, insulin clearance was reduced in the old mice, as suggested by the lower c-peptide:insulin ratio, observed during ipGTT. Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) and insulin-degrading enzyme (IDE), as well as the activity of this enzyme, were reduced in the liver of old mice, justifying the decreased insulin clearance observed in these mice. Therefore, loss of hepatic CEACAM1 and IDE function may be directly related to the decline in insulin clearance during aging.

Aging-induced aberrant RAGE/PPARα axis promotes hepatic steatosis via dysfunctional mitochondrial β oxidation.

Non‐alcoholic fatty liver disease (NAFLD), characterized by an increase in hepatic triglyceride (TG) content, is the most common liver disease worldwide. Aging has been shown to increase susceptibility to NAFLD; however, the underlying molecular mechanism remains poorly understood. In the present study, we examined hepatic TG content and gene expression profiles in body weight‐matched young (3 months old), middle‐aged (10 months old), and old (20 months old) C57BL/6 mice and found that TGs were markedly accumulated while mitochondrial β‐oxidation‐related genes, including PPARα, were downregulated in the liver of old mice. In addition, advanced glycation end product receptor (RAGE), a key regulator of glucose metabolism, was upregulated in the old mice. Mechanistically, suppression of RAGE upregulated PPARα and its downstream target genes, which in turn led to reduced TG retention. Finally, we found that hepatic RAGE expression was increased in aging patients, a finding that correlated with decreased PPARα levels. Taken together, our findings demonstrate that the upregulation of RAGE may play a critical role in aging‐associated liver steatosis.

Effect of senolytic treatment on expression of senescence‐associated miR‐126 expression in young and old female mice.

Cellular senescence represents one of the major risk factors for chronic liver disease and contributes to morbidity, mortality, and increased healthcare spending. A relevant strategy for understanding the role of senescent cells in liver pathogenesis is to use genetic or pharmacological strategies to eliminate senescent cells. The combination of the senolytic drugs, Dasatinib and Quercetin (D+Q), effectively reduces senescent cell burden in multiple tissues, extending both healthspan and lifespan in mice, and alliviating obesity and age‐associated hepatic steatosis. The aim of this study was to investigate effects of D+Q treatment on livers of young and old female mice. A total of 40 mice, 20 young females (3‐months) and 20 old females (18‐months), was divided into 4 groups: Young (Y) mice treated with either D+Q or placebo and old (O) mice treated with D+Q or placebo. The treatment was performed for 3 consecutive days every 2 weeks over 10 weeks. Comparative analysis of hepatic miRNAs indicated that D+Q treatment significantly decreased levels of senescence‐specific miR‐126 expression in livers of old mice treated with D+Q compared to vehicle‐treated old mice (p=0.025), As expected, there was no effect of D+Q treatment in young animals. Analysis of miR‐126 target genes indicated that PI3K gene expression was significantly increased in response to D+Q treatment (p=0.003). Additionally, Akt1 and mTOR mRNA levels were increased in D+Q‐treated mice (p=0.008 and p=0.02, respectively). In summary, our data suggest that the mechanism of PI3K signaling pathway activation upon elimination of senescent cells by treatment with D+Q is mediated through downregulation of senescence‐specific miR‐126.Support or Funding InformationThis work is supported by National Institutes of Health (NIH) (R56AG061414, R15AG059190 and R03AG059846)

Effect of young exosomes injected in aged mice.

IntroductionExosomes, nanosized extracellular vesicles, are known to circulate through the blood stream to transfer molecular signals from tissue to tissue.MethodsTo determine whether exosomes affect aging in animals, we primarily identified the changes in exosomal miRNA contents during the aging process. In exosomes from 12-month-old mice, mmu-miR-126-5p and mmu-miR-466c-5p levels were decreased and mmu-miR-184-3p and mmu-miR-200b-5p levels were increased significantly compared with those of 3-month-old mice. Their levels in exosomes were partially correlated with those in tissues: levels of only mmu-miR-126-5p and mmu-miR-466c-5p in lungs and/or liver were decreased, but those of mmu-miR-184-3p and mmu-miR-200b-5p in tissues did not coincide with those of exosomes.Results and discussionIn the aged tissues injected with young exosomes isolated from serum, mmu-miR-126b-5p levels were reversed in the lungs and liver. Expression changes in aging-associated molecules in young exosome-injected mice were obvious: p16Ink4A, MTOR, and IGF1R were significantly downregulated in the lungs and/or liver of old mice. In addition, telomerase-related genes such as Men1, Mre11a, Tep1, Terf2, Tert, and Tnks were significantly upregulated in the liver of old mice after injection of young exosomes.ConclusionThese results indicate that exosomes from young mice could reverse the expression pattern of aging-associated molecules in aged mice. Eventually, exosomes may be used as a novel approach for the treatment and diagnosis of aging animals.

YIA-3 - p38α deficiency and oxidative stress cause cytokinesis failure in hepatocytes

Cytokinesis is the last step in mitosis and it implies re-organization of the actin cytoskeleton. Its failure is one of the major mechanisms of polyploidy and binucleation in mammals. Our aims were 1) to assess the role of redox-sensitive p38α MAPK in cytokinesis by studying the liver of wild type mice or liver-specific p38α knock-out mice; 2) to assess the role of oxidative stress associated with hepatocyte isolation on cytokinesis. When p38α was down-regulated in hepatocytes, MK2 phosphorylation on threonine 334 was completely abrogated. Activation of MNK-1, required for abscission of the intercellular bridge, was diminished. Key proteins of the RhoA pathway (phospho-PRK2, nuclear phosphorylated cofilin, and cytosolic p27) were assessed confirming the impairment of this pathway. The absence of p38α in aging liver also led to a decrease in HSP27 phosphorylation, which is required for actin polymerization. Indeed, a severe impairment in the F-actin filamentous structure was found in the liver of old mice upon p38α deficiency. Consequently, long-term p38α MAPK down-regulation markedly affects the RhoA pathway and actin cytoskeleton dynamics inducing actin disassembly and cytokinesis failure upon aging. On the other hand, hepatocyte isolation caused marked glutathione depletion, increased generation of reactive oxygen species, and activated cell cycle entry. Addition of N-acetyl cysteine to isolation media prevented glutathione depletion, restrained the cell cycle entry, and abrogated defective cytokinesis and the formation of binucleated hepatocytes during isolation. Our results show that hepatocytes do enter into S phase but they do not complete cell division with age upon p38α deficiency or upon oxidative stress associated with isolation leading in both cases to cytokinesis failure and binucleation.

P91 - Long term p38-a deficiency up-regulates antioxidant enzymes through compensatory NF-?B activation

p38a MAPK may function as a mediator of reactive oxygen species signaling and thus p38a is considered a sensor of oxidative stress. In liver-specific p38a knock-out (KO) adult mice we previously found glutathione depletion and down-regulation of antioxidant enzymes. Our aim was to assess the influence of long-term p38a deficiency on oxidative stress and on the regulation of antioxidant enzymes in liver of old mice. To this end, wild type or liver-specific KO mice after weaning, at 4-6 months of age, or at 24 months of age were used. Reduced glutathione (GSH) and oxidized glutathione levels were determined by mass spectrometry, gene expression of antioxidant enzymes was determined by RT-PCR, and induction of NRF-2 and PGC-1a as well as activation of NF-?B were assessed by western blotting. We report that GSH levels decreased upon aging only in liver of wild-type mice, but not in p38a KO mice. The mRNA expression of glutathione peroxidase, Cu-Zn superoxide dismutase, Mn-superoxide dismutase, and glutamate cysteine ligase was up-regulated in adult wild-type in comparison with mice after weaning, but their gene expression was down-regulated in old wild-type mice. Although the mRNA expression of glutathione peroxidase, Cu-Zn superoxide dismutase, Mn-superoxide dismutase, and glutamate cysteine ligase was down-regulated in adult KO mice vs KO mice after weaning, their gene expression was up-regulated in old KO mice. This up-regulation was not associated with nuclear translocation of NRF-2, which decreased upon aging in KO mice, nor with up-regulation of PGC-1a. However, phosphorylation of p65 was markedly increased in old KO mice as an index of NF-?B activation. In conclusion, long term deficiency of p38a in the liver causes compensatory activation of NF?B that is likely to be responsible for the up-regulation of antioxidant enzymes upon aging, independently of Nrf-2 and PGC-1a.

Daumone fed late in life improves survival and reduces hepatic inflammation and fibrosis in mice.

The liver is one of the most susceptible organs to aging, and hepatic inflammation and fibrosis increase with age. Chronic inflammation has been proposed as the major molecular mechanism underlying aging and age-related diseases, whereas calorie restriction has been shown to be the most effective in extending mammalian lifespan and to have anti-aging effects through its anti-inflammatory action. Thus, it is necessary to develop effective calorie restriction mimetics. Daumone [(2)-(6R)-(3,5-dihydroxy-6-methyltetrahydropyran-2-yloxy)heptanoic acid], a pheromone secreted by Caenorhabditis elegans, forces them to enter the dauer stage when facing inadequate conditions. Because Caenorhabditis elegans live longer during the dauer stage under energy deprivation, it was hypothesized that daumone may improve survival in mammals by mimicking calorie restriction. Daumone (2 mg kg−1 day−1) was administered orally for 5 months to 24-month-old male C57BL/6J mice. Daumone was found to reduce the risk of death by 48% compared with age-matched control mice, and the increased plasma insulin normally presented in old mice was significantly reduced by daumone. The increased hepatic hypertrophy, senescence-associated β-galactosidase activity, insulin resistance, lipid accumulation, inflammation, oxidative stress, and fibrosis in old mice were significantly attenuated by daumone. From a mechanistic view, daumone reduced the phosphorylation of the IκBα and upregulation of Rela and Nfkbia mRNA in the livers of old mice. The anti-inflammatory effect of daumone was confirmed in lipopolysaccharide-induced liver injury model. Oral administration of daumone improves survival in mice and delivers anti-aging effects to the aged liver by modulating chronic inflammation, indicating that daumone could be developed as an anti-aging compound.

Autophagy Suppresses Age-Dependent Ischemia and Reperfusion Injury in Livers of Mice

As life expectancy increases, there are greater numbers of patients with liver diseases who require surgery or transplantation. Livers of older patients have significantly less reparative capacity following ischemia and reperfusion (I/R) injury, which occurs during these operations. There are no strategies to reduce the age-dependent I/R injury. We investigated the role of autophagy in the age dependence of sensitivity to I/R injury. Hepatocytes and livers from 3- and 26-month-old mice were subjected to in vitro and in vivo I/R, respectively. We analyzed changes in autophagy-related proteins (Atg). Mitochondrial dysfunction was visualized using confocal and intravital multi-photon microscopy of isolated hepatocytes and livers from anesthetized mice, respectively. Immunoblot, autophagic flux, genetic, and imaging analyses all associated the increase in sensitivity to I/R injury with age with decreased autophagy and subsequent mitochondrial dysfunction due to calpain-mediated loss of Atg4B. Overexpression of either Atg4B or Beclin-1 recovered Atg4B, increased autophagy, blocked the onset of the mitochondrial permeability transition, and suppressed cell death after I/R in old hepatocytes. Coimmunoprecipitation analysis of hepatocytes and Atg3-knockout cells showed an interaction between Beclin-1 and Atg3, a protein required for autophagosome formation. Intravital multi-photon imaging revealed that overexpression of Beclin-1 or Atg4B attenuated autophagic defects and mitochondrial dysfunction in livers of older mice after I/R. Loss of Atg4B in livers of old mice increases their sensitivity to I/R injury. Increasing autophagy might ameliorate liver damage and restore mitochondrial function after I/R.

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.

Linking Klotho, Nrf2, MAP kinases and aging

Oxidative damage of DNA, proteins, lipids and other molecules is one of the oldest mechanisms proposed to explain the phenomenon of aging [1,2]. Reactive oxygen species (ROS) are generated in the mitchondria as by-products of normal aerobic respiration and in the cytosol by oxidases such as NADPH oxidase. It has been proposed that a decline in mitochondrial function leads to aging because of higher ROS production and increased damage to macromolecules [1]. This is debatable, however, because age-dependent mitochondrial DNA mutations can promote aging without causing increased ROS production [2]. High ROS levels can also regulate signaling via the stress activated-kinases, p38 and JNK. These pathways are often activated in aged tissues and contribute to the inflammation, apoptosis and senescence that is associated with old age. For example, JNK is activated in the liver of old rats and p38 is activated in the liver and brain of old rats as well as in the liver of old mice [3,4]. In the September issue of Aging, C-C Hseih et al. report that the p38 kinase is activated in Klotho -/- mice and suppressed by Klotho overexpression [5]. This is dependent on the oxidation and dissociation of Ask-1 bound thioredoxin to generate the active Ask1-signalosome which, as previously shown, is an activator of p38 [6]. Klotho overexpressing mice live longer and are characterized by their resistance to oxidative stress [7]. Previous studies by C-C Hseih et al. have shown that the ASK1-signalosome-p38 pathway is suppressed in the long-lived and stress-resistant Ames and Snell dwarf mice [6,8]. The current study demonstrates that a similar suppression of p38 signaling occurs in Klotho overexpressing cells and provides further evidence that oxidative stress, p38 activity and lifespan are closely linked. Interestingly, this study also reveals that the stress-protective transcription factor Nrf2 localizes to the nucleus in Klotho overexpressing cells. Nrf2 is activated by oxidative stress and enhances transcription of genes containing an antioxidant response element (ARE). It is also believed to play an important role in some of the protective effects caused by caloric restriction and by GH/IGF-I deficiencies [9,10]. Also, thioredoxin which is an inhibitor of Ask1, is a target of Nrf2 [11]. This implies that Nrf2 can regulate the expression of thioredoxin in Klotho mice thereby inhibiting the Ask1-signalosome-p38 pathway. Thus it is possible that the beneficial effects of Klotho are mediated, at least partially, by inactivation of p38 signaling. The premature aging phenotype of Klotho -/- mice has been attributed to the absence of Klotho mediated suppression of the Insulin/IGF-I pathway [7,12]. Klotho can activate the transcription factor FOXO, causing up-regulation of the mitochondrial MnSOD [7]. Because many of these cellular phenotypes are also promoted by reduced IGF-I signaling, the new study by C-C Hseih et al. not only confirms the major overlap between the pro-aging effects of Klotho -/- and IGF-I but also adds new players that can help identify common downstream anti-aging mechanisms.

Epigenetic changes play critical role in age‐associated dysfunctions of the liver

CCAAT/Enhancer Binding Proteins family proteins are important regulators of liver functions. Here, we show the critical role of C/EBPα-mediated chromatin remodeling in the age-associated dysfunctions of the liver and in the maintenance of physiological homeostasis. Because ph-S193 isoform of C/EBPα is increased in livers of old mice, we have generated C/EBPα-S193D knockin mice, which mimic the ph-S193 isoform of C/EBPα. Analyses of these mice showed that the S193D mutation causes chromatin remodeling leading to histological appearance of 'foci-like' nodules, which are also observed in livers of old mice. These 'foci-like' structures contain K9 trimethylated histone H3, a marker of heterochromatin. The increase of heterochromatin regions in S193D mice correlates with the elevation of S193D-C/EBPα-HDAC1 complexes and with dys-regulation of gene expression including epigenetic silencing of cyclin D1 and D2 promoters and the inhibition of liver proliferation. The elimination of C/EBPα-HDAC1 complexes in S193D mice by inhibition of HDAC1 corrects chromatin structure and normalizes expression of cyclin D1 and D2. We found that epigenetic dys-regulation is also associated with the elevation of C/EBPβ and with the increase of C/EBPα/β heterodimers in S193D mice. The C/EBPα/β heterodimers activate transcription of Glut4 and increase the levels of Glut4. As the result, S193D livers have accelerated uptake of glucose and accumulation of glycogen in the liver. Thus, this study demonstrates that the phosphorylation of C/EBPα at S193 leads to the appearance of heterochromatin regions, which correlates with the development of age-related dysfunctions of the liver.

The Age-Associated Decline of Glycogen Synthase Kinase 3β Plays a Critical Role in the Inhibition of Liver Regeneration

Aging reduces the regenerative capacities of many tissues. In this paper, we show a critical role of the glycogen synthase kinase 3beta (GSK3beta)-cyclin D3 pathway in the loss of the regenerative capacity of the liver. In young animals, high levels of growth hormone (GH) increase expression of GSK3beta, which associates with cyclin D3 and triggers degradation of cyclin D3. In livers of old mice, the GSK3beta promoter is repressed by C/EBPbeta-histone deacetylase 1 (HDAC1) complexes, leading to the reduction of GSK3beta. The treatment of old mice with GH increases expression of GSK3beta via removal of the C/EBPbeta-HDAC1 complexes from the GSK3beta promoter. We found that the GSK3beta-cyclin D3 pathway is also altered in young GH-deficient Little mice and that treatment of Little mice with GH corrects the GSK3beta-cyclin D3 pathway. We present evidence that GSK3beta regulates liver proliferation by controlling growth-inhibitory activity of C/EBPalpha. The downregulation of GSK3beta in young mice inhibits liver proliferation after partial hepatectomy via the cyclin D3-C/EBPalpha pathway, while the elevation of GSK3beta in old mice accelerates liver proliferation. Thus, this paper shows that GSK3beta is a critical regulator of liver proliferation and that the reduction of GSK3beta with age causes the loss of regenerative capacities of the liver.

GSK3β and aging liver

The loss of regenerative capacity of tissues is one of the major characteristics of aging. Liver represents a powerful system for investigations of mechanisms by which aging reduces regenerative capacity of tissues. The studies within last five years revealed critical role of epigenetic silencing in the inhibition of liver proliferation in old mice. These studies have shown that a number of cell cycle proteins are silenced in livers of old mice by C/EBPalpha-HDAC1-Brm complex and that old liver fails to reduce the complex and activate these genes in response to proliferative stimulus such as partial hepatectomy. The complex modifies histone H3 on the promoters of c-myc and FoxM1B in the manner which prevents expression of these genes. Despite this progress, little is known about mechanisms by which aging causes this epigenetic silencing. We have recently discovered signal transduction pathways which operate upstream of the C/EBPalpha-HDAC1-Brm complex. These pathways involve communications of growth hormone, GSK3beta and cyclin D3. In addition to the liver, GH-GSK3beta-cyclin D3 pathway is also changed with age in lung, brain and adipose tissues. We suggest that other age-associated alterations in these tissues might be mediated by the reduced levels of GSK3beta and by elevation of cyclin D3. In this review, we summarize these new data and discuss the role of such alterations in the development of aging phenotype in the liver and in other tissues.

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.

Lymphoid neogenesis and immune infiltration in aged liver.

Immune dysregulation and inflammation play a major role in the pathology of age-related disorders. In an earlier study, the microarray data from our laboratory indicated an increase in inflammation-related gene expression in the liver with age. We further investigated immune-related changes in the aged liver and found that the levels of inflammatory cytokines, chemokines, and inflammatory genes were higher in aged animals. Immunohistochemical studies showed that immune cells formed clusters or foci in the livers of old mice, preferentially near the perivascular regions. Further analysis revealed an enrichment of macrophages, T cells, B cells, natural killer cells, and neutrophils in old liver. Characterization of the immune clusters showed the presence of shared markers of tertiary lymphoid neogenesis. Levels of lymph node homing cytokines were elevated. Expression of immunoglobulin and recombinase gene transcripts was also higher, indicating the presence of ectopic lymphoid structures in the aged liver. Aged liver exhibits a marked inflammatory status accompanied by increased immune cell infiltration. Inflammation and ectopic lymphoid structures have previously been shown to be associated with carcinogenesis, a condition that becomes more prevalent with age. Thus, further study of inflammation-related changes in the microenvironment of the aged liver could provide insights into these disorders.

Age-associated perturbations in glutathione synthesis in mouse liver

The nature of the mechanisms underlying the age-related decline in glutathione (GSH) synthetic capacity is at present unclear. Steady-state kinetic parameters of mouse liver GCL (glutamate-cysteine ligase), the rate-limiting enzyme in GSH synthesis, and levels of hepatic GSH synthesis precursors from the trans-sulfuration pathway, such as homocysteine, cystathionine and cysteine, were compared between young and old C57BL/6 mice (6- and 24-month-old respectively). There were no agerelated differences in GCL V(max), but the apparent K(m) for its substrates, cysteine and glutamate, was higher in the old mice compared with the young mice (approximately 800 compared with approximately 300 microM, and approximately 710 compared with 450 microM, P<0.05 for cysteine and glutamate in young and old mice respectively). Amounts of cysteine, cystathionine and Cys-Gly increased with age by 91, 24 and 28% respectively. Glutathione (GSH) levels remained unchanged with age, whereas GSSG content showed an 84% increase, suggesting a significant pro-oxidizing shift in the 2GSH/GSSG ratio. The amount of the toxic trans-sulfuration/glutathione biosynthetic pathway intermediate, homocysteine, was 154% higher (P<0.005) in the liver of old mice compared with young mice. The conversion of homocysteine into cystathionine, a rate-limiting step in trans-sulfuration catalysed by cystathionine beta-synthase, was comparatively less efficient in the old mice, as indicated by cystathionine/homocysteine ratios. Incubation of tissue homogenates with physiological concentrations of homocysteine caused an up to 4.4-fold increase in the apparent K(m) of GCL for its glutamate substrate, but had no effect on V(max). The results suggest that perturbation of the catalytic efficiency of GCL and accumulation of homocysteine from the trans-sulfuration pathway may adversely affect de novo GSH synthesis during aging.