Liver Of Aged Mice Research Articles

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.

Circadian mRNA expression of coagulation and fibrinolytic factors is organ-dependently disrupted in aged mice

To evaluate the effects of aging on the circadian gene expression of coagulation and fibrinolytic factors in the mouse tissues, we examined temporal mRNA expression profiles of plasminogen activator inhibitor-1 ( PAI-1), tissue-type plasminogen activator ( tPA), tissue factor ( TF), and thrombomodulin ( TM) genes together with circadian clock genes in the brains, hearts and livers of young (5 weeks old) and aged (15 months old) mice. Cardiac mRNA expression of β-myosin heavy chain ( β-MHC), a molecular marker of cardiac hypertrophy, was obviously increased in the aged mice. Rhythmic expression of the clock genes mPer2 and BMAL1 in these organs was almost identical between young and aged mice, whereas that of PAI-1, TF and TM mRNAs and of clock-controlled genes such as DBP and Dec1 were damped to low levels in the livers of aged mice. Expression levels of tPA mRNA were significantly decreased and those of TF were significantly elevated throughout the day in the brain of aged mice. Expression levels of PAI-1 in the heart of aged mice were continuously elevated over 2-fold the peak levels of young mice throughout the day. However, day/night fluctuations in plasma PAI-1 levels were unaffected by aging. Aging tissue- and time-dependently affects the mRNA expression of coagulation and fibrinolytic factors. Aging-dependent constitutive PAI-1 induction in the heart might be a risk factor for cardiovascular diseases that is independent of plasma PAI-1 levels.

High molecular weight persimmon tannin ameliorates cognition deficits and attenuates oxidative damage in senescent mice induced by d-galactose

Mice were subcutaneously injected with d-galactose (d-gal, 150mg/kg per day) for 6weeks and were administered high molecular weight persimmon condensed tannin (HMWPT) simultaneously. After 6weeks of treatment, the animal behavior was observed in the open field test and water maze test, and the morphology of hippocampus and skin were checked. Meanwhile, the activities of antioxidant enzymes, the levels of non-enzymatic antioxidants, as well as malondialdehyde (MDA) were evaluated. The results indicated that HMWPT markedly inhibited the d-gal induced learning and memory impairment in both open field test and Morris water maze. Biochemical examination revealed that HMWPT significantly increased the decreased activities of superoxide dismutase (SOD), catalase (CAT), elevated the lowered total anti-oxidation capability (T-AOC), glutathione (GSH) and hydroxyproline (Hyp) contents (p<0.01 or p<0.05), and decreased the raised monoamine oxidase (MAO), total cholinesterase (TChE) activities and MDA level (p<0.01) in serum, liver or brain of aging mice induced by d-gal in a dose-dependent fashion. Furthermore, HMWPT significantly and (p<0.01) attenuated the d-gal induced number decrease, neuronal degeneration and karyopycnosis in cells in the hippocampus and decrease of thickness of skin epidermis and dermis.

Resistance to Yersinia pestis infection decreases rapidly and substantially with age in B10.T(6R) mice (162.9)

Abstract We have previously demonstrated that 2-3 month old, female B10.T(6R) mice are highly resistant to a delta pgm strain of Yersinia pestis and by 5 months of age these mice are highly susceptible to Y. pestis infection. In the current study we further characterize this observation in female B10.T(6R) mice. The LD50 of Y. pestis was ~14000 CFU/ml in young (2-3 month) mice while only ~60 CFU/ml in middle aged (&amp;gt;5 month) mice. Significantly elevated bacterial burden was found in both the spleen and liver of middle aged mice while a significant infiltration of immune cells was observed in resistant young mice. The cellular infiltrate in susceptible middle aged was comparable to susceptible young C57BL6/J mice. Sera and spleens from Y. pestis-infected middle aged B10.T(6R) contained elevated levels of KC, MCP-1 (CCL2), MIP-1a (CCL3), G-CSF, IL-6 and Eotaxin. In many cases, cytokine/chemokine levels in middle aged mice increased in an infectious dose-dependent manner and were comparable to Y. pestis infection in C57BL6/J mice with both experimental groups being significantly increased when compared to infected young B10.T(6R) mice with the exception of IL1b that was only increased in both serum and spleen samples from young mice. These findings suggest that IL1b may be critical to prevent Y. pestis infection and may provide insight into defense mechanism(s) necessary to develop resistance to the plague.

Autophagic activity in thymus and liver during aging

Impaired or deficient autophagy is believed to cause or contribute to aging, as well as several age-related pathologies. Thymic epithelial cells had a high constitutive level of autophagy. The autophagic process may play a supporting role or even a crucial role in the presentation of self-Ags in the thymus to shape the T-cell repertoires. Autophagic activity in the liver is important for the balance of energy and nutrients for basic cell functions. The abundance of autophagic structure in both cortical and medullary thymic epithelial cells and liver with mouse age has not been examined in detail. Here, we demonstrated that the architecture of mouse thymus and liver markedly changed with age. We found that the expression of LC3 detected by immunofluorescence and Western blot analysis was greatly decreased in thymus and liver of 12-month-old mice. The same level of reduction was observed in thymus and liver of 24-month-old mice. Ultrastructure analysis by an electron microscope revealed that the number of autophagic structure/vacuole in total thymic epithelial cells and hepatocytes decrease with age. The age-related decrease of autophagic structure in thymic epithelial cells may cause the reduction of immunocompetent T-cell pool in aged mice. The age-related decrease of autophagy in liver may induce accumulation of cellular materials in liver of aged mice.

自著とその周辺 Tetsuji Nagata : Macromolecular synthesis in the livers of aging mice as revealed by electron microscopic radioautography. In, Progress in Histochemistry and Cytochemistry. Vol. 45, No. 1

自著とその周辺 Tetsuji Nagata : Macromolecular synthesis in the livers of aging mice as revealed by electron microscopic radioautography. In, Progress in Histochemistry and Cytochemistry. Vol. 45, No. 1

In Vitro and In Vivo Antioxidant Activity of Bifidobacterium animalis 01 Isolated from Centenarians

Several studies reported the antioxidant activity of bifidobacteria using assays in vitro. In present study, the in vitro and in vivo antioxidant activity of Bifidobacterium animalis 01 was investigated. Culture supernatant, intact cells, and intracellular cell-free extracts of B. animalis 01 were involved in this study. The antioxidant assays in vitro included lipid peroxidation assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, hydroxyl radical ((•)OH) assay and superoxide anion (O₂⁻) assay. The antioxidant assays in vivo were conducted using mice model. Activities of antioxidative enzymes, malondialdehyde (MDA) content in serums and livers of aging mice were evaluated. Monoamine oxidase (MAO) activity and lipofuscin level in brains of aging mice were also characterized. Results showed that culture supernatant, intact cells and intracellular cell-free extracts of B. animalis 01 could effectively scavenge free radicals, significantly enhance mice's activities of antioxidative enzymes and reduce mice's MDA content, lipofuscin level and MAO activity. Our results indicated that B. animalis 01 has the potential to be developed into a dietary antioxidant supplements.

P66Shc has a pivotal function in impaired liver regeneration in aged mice by a redox-dependent mechanism

Liver regeneration involves complicated processes and is affected by various patho-physiological conditions. This study was designed to examine the molecular mechanisms underlying the aging-associated impairment of liver regeneration. Male C57BL/6J mice were used as young and aged mice (<10 weeks and >20 months old, respectively). These mice were subjected to 70% partial hepatectomy (PH). Liver regeneration and liver injury/stresses were evaluated chronologically after PH. Post-hepatectomy liver regeneration was markedly impaired in aged mice. Though the extent of hepatocyte proliferation in the regenerating liver was similar in aged and young mice, cell growth was absent in aged mice. Oxidative stress (OS) was observed immediately after hepatectomy, followed by marked apoptosis in aged mice. Signaling molecules regarding cell proliferation (mitogen-activated protein kinase, STAT3, p46/52Shc) and anti-oxidation (catalase, superoxide dismutase, Ref-1, glutathione peroxidase) were expressed/activated after hepatectomy in livers of both aged and young mice. Akt was not activated in aged-mouse liver, but its expression was similar to that in young mice. p66Shc, known as an age-/oxidant-associated protein, was strongly phosphorylated. By knocking down p66Shc, the impairment of liver regeneration was normalized. OS immediately after hepatectomy induced subsequent liver injury (apoptosis), and deletion of p66Shc suppressed both OS and hepatocyte apoptosis in the regenerating liver of aged mice. Though we need additional data in other animal models to fully understand the mechanism, p66Shc may have a pivotal function in the impairment of liver regeneration in aged mice by triggering OS and subsequent apoptosis. This data may provide a clue to understanding the mechanism underlying the association between aging and the impairment of liver regeneration.

In vitro and in vivo antioxidant activity of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3

The antioxidant activities of exopolysaccharides (EPS) from endophytic bacterium Paenibacillus polymyxa EJS-3 were evaluated by various methods in vitro and in vivo. In antioxidant assays in vitro, both crude EPS and its purified fractions (EPS-1 and EPS-2) were found to have moderate 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, hydrogen peroxide scavenging activity, lipid peroxidation inhibition effect, and strong ferrous ion chelating activity. And the antioxidant activities in vitro of EPS decreased in the order of crude EPS > EPS-2 > EPS-1. In antioxidant assays in vivo, mice were subcutaneously injected with d-galactose ( d-Gal) for 6 weeks and administered EPS-1 via gavage simultaneously. As a result, administration of EPS-1 significantly increased the thymus and spleen indices of d-Gal induced aging mice. Moreover, EPS-1 administration significantly enhanced the activities of antioxidant enzymes and total antioxidant capacity and decreased the levels of malondialdehyde in both serums and livers of aging mice. These results suggested that EPS had potent antioxidant activities and could be explored as novel natural antioxidant.

Energy Restriction Does Not Compensate for the Reduced Expression of Hepatic Drug-Processing Genes in Mice with Aging

Liver is the major organ that eliminates xenobiotics from the body, a process that is accomplished by a series of drug-processing genes (DPGs). These genes encode transporters on both basolateral and apical membranes of hepatocytes, as well as phase I and II enzymes. The current study compares the expression of hepatic DPGs in adult and aged mouse livers and explores the potential effects of energy restriction (ER) on these genes during aging. Of 79 quantified hepatic DPGs, 52 were expressed lower in 24-month-old aged mice than in 12-month-old adult mice. Furthermore, the mRNA expression of multiple xenobiotic-activated transcription factors also decreased with age. Six-month ER exerted less of an effect on the hepatic DPGs than did aging. ER increased the mRNAs of two and decreased the mRNAs of nine DPGs in adult mice. In aged mice, ER increased the mRNAs of 10 and decreased the mRNAs of 5 DPGs. The only mRNA that was increased by both ER and aging was Gstm3. ER increased the mRNAs of Cyp2b10, Ugt1a9, Gsta1, and Oatp1a4 only in adult mice and decreased the mRNAs of Aldh6a1, Pon3, Ugt1a1, Sult1a1, and Atp8b1 only in aged mice. In summary, the reduced mRNA expression of hepatic DPGs in aged mice indicates decreased drug-processing capability, whereas ER did not compensate for the global reduction of hepatic DPG expression in aged mice. The hepatic transcription factors are likely to mediate the changes in hepatic DPG expression during aging and ER.

Abstract A41: Regulation of Nrf2-dependent gene expression in aging

Abstract Nrf2 is a transcription factor which controls induction of antioxidant and detoxification enzymes, and, therefore, plays an essential role in protection against chemical carcinogens. Here, we reported that expression of Nrf2 and its dependent genes was affected by age and became compromised in aged mouse livers. Detail analysis revealed that age-dependent Nrf2 expression was regulated primarily at transcriptional level. Accordingly, we cloned and characterized the promoter region of mouse Nrf2 gene. We identified multiple Sp1 binding sites and confirmed their binding activity by gel-shift and chromatin immunoprecipitation assay. We also showed that the identified Sp1 sites were functional since knockdown of Sp1 expression impaired Nrf2 promoter activity as well as expression of Nrf2-dependent genes. We then compared Sp1 expression and its binding activity to Nrf2 promoter in young and aged mouse liver tissues. While no significant change in Sp1 expression was found, Sp1 binding activity was severely impaired in aged tissues. Given that Sp1-binding sites are GC rich elements, we analyzed their methylation status in Nrf2 promoter, and found that Sp1 sites were highly methylated in aged tissues. In support of the role of methylation, treatment of a mouse hepatoma cell line with demethylase inhibitors indeed reduced Sp1 binding to Nrf2 promoter. We also cloned and analyzed human Nrf2 promoter. Multiple Sp1 binding sites were identified and shown to be equally functional as in mouse Nrf2 promoter. Thus, we propose that age-dependent expression of Nrf2 and its downstream genes is regulated by methylation. A strategy that targets methylation would help to restore Nrf2-mediated protection against oxidative stress or chemical carcinogens in aged tissues. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A41.

Aging Promotes Neutrophil-Induced Mortality by Augmenting IL-17 Production during Viral Infection

Morbidity and mortality associated with viral infections increase with age, although the underlying mechanisms are unclear. Here, we investigated whether aging alters inflammatory responses during systemic viral infection and thereby contributes to virus-induced death. We found that infection of aged mice with systemic herpes viruses led to rapid increases in serum IL-17, neutrophil activation, and mortality due to hepatocyte necrosis. In contrast, all young mice survived infection, displaying weaker IL-17 induction and neutrophil activation. Natural killer T (NKT) cells isolated from the livers of aged mice produced more IL-17 than did young cells, and adoptively transferred aged NKT cells induced liver injury in young mice impaired in viral control. Importantly, IL-17 neutralization or neutrophil depletion during viral infection reduced liver damage and prevented death of aged mice. These results demonstrate that, during systemic viral infection, aging alters the host-pathogen interaction to overproduce IL-17, contributing to liver injury and death.

Comparative Study of Anti-hepatitis B Virus RNA Interference by Double-stranded Adeno-associated Virus Serotypes 7, 8, and 9

Using a hepatitis B virus (HBV) transgenic mouse model, we previously showed that a single dose of double-stranded adeno-associated virus (dsAAV) vector serotype 8 carrying a small hairpin RNA (shRNA) effectively reduces HBV replication and gene expression, but the effect gradually decreases with time. In this report, we compared the anti-HBV RNA interference (RNAi) effect of dsAAV8 with those of dsAAV7 and dsAAV9, two other hepatotropic AAV vectors, and examined whether the sequential use of these heterologous AAV vectors could prolong the anti-HBV effect. Our results showed that shRNA delivered by each of the three dsAAV vectors profoundly reduced the serum HBV titer and liver HBV mRNA and DNA levels in the transgenic mice for up to 22 weeks, with dsAAV8 having the greatest inhibitory effect, followed by dsAAV9 and dsAAV7. The potency of dsAAV8 correlated with the presence of higher levels of vector DNA and anti-HBV shRNA in the liver. An in vivo cross-administration experiment showed that preexisting anti-AAV8 antibody completely blocked the anti-HBV RNAi effect of dsAAV8, but had no effect on the potency of dsAAV7 and dsAAV9. Moreover, we demonstrated that a longer anti-HBV effect could be achieved by the sequential use of dsAAV8 and dsAAV9. These results indicate that effective and persistent HBV suppression might be achieved by a combination of the power of RNAi silencing effect and multiple treatments with different AAV serotypes.Molecular Therapy (2009) 17 2, 352-359 doi:10.1038/mt.2008.245.

Decreased enzyme activities of chaperones PDI and BiP in aged mouse livers

The endoplasmic reticulum (ER) is a target for endogenously generated reactive oxygen species (ROS) during aging. We have previously shown that the ER chaperones, protein disulfide isomerase (PDI) and immunoglobulin heavy chain binding protein (BiP), are oxidatively modified within the livers of aged mice. In this study we assess the functional consequences of the age-dependent oxidation of these two proteins. Specific activity measurements, performed on purified protein samples obtained from young and aged mouse livers, show definitive decreases in BiP ATPase activity and dramatic reductions in PDI enzymatic activity with age. Overall, these results suggest that protein folding and other activities mediated through PDI and BiP are diminished during aging. Furthermore, the relative loss of these chaperone-like activities could directly contribute to the age-dependent accumulation of misfolded proteins, a characteristic of the aging phenotype.

Impaired Genome Maintenance Suppresses the Growth Hormone–Insulin-Like Growth Factor 1 Axis in Mice with Cockayne Syndrome

Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria that is caused by a defect in the transcription-coupled repair subpathway of nucleotide excision repair (NER). Here, complete inactivation of NER in Csbm/m/Xpa−/− mutants causes a phenotype that reliably mimics the human progeroid CS syndrome. Newborn Csbm/m/Xpa−/− mice display attenuated growth, progressive neurological dysfunction, retinal degeneration, cachexia, kyphosis, and die before weaning. Mouse liver transcriptome analysis and several physiological endpoints revealed systemic suppression of the growth hormone/insulin-like growth factor 1 (GH/IGF1) somatotroph axis and oxidative metabolism, increased antioxidant responses, and hypoglycemia together with hepatic glycogen and fat accumulation. Broad genome-wide parallels between Csbm/m/Xpa−/− and naturally aged mouse liver transcriptomes suggested that these changes are intrinsic to natural ageing and the DNA repair–deficient mice. Importantly, wild-type mice exposed to a low dose of chronic genotoxic stress recapitulated this response, thereby pointing to a novel link between genome instability and the age-related decline of the somatotroph axis.

Thioredoxin‐ASK1 complex levels regulate ROS‐mediated p38 MAPK pathway activity in livers of aged and long‐lived Snell dwarf mice

We have proposed that the age-associated increase of reactive oxygen species (ROS) by electron transport chain (ETC) dysfunction may cause the elevated basal level of p38 MAPK stress response pathway activity. However, the mechanism by which ROS activates this pathway is not clear. Here we propose that activation of the p38 MAPK pathway by complex I (CI) generated ROS, in response to rotenone (ROT) treatment, is based on the ability of reduced Trx to bind to and inhibit ASK 1 and its release from the complex upon oxidation. This balance of free vs. bound ASK1 regulates the level of p38 MAPK pathway activity. To support this mechanism we demonstrate that the production of ROS by ROT treated AML12 hepatocyte cells dissociates the Trx-ASK1 complex, thereby increasing p38 MAPK pathway activity. This mechanism is supported by the ability of N-acetyl cysteine (NAC) to prevent dissociation of Trx-ASK1 and activation of the p38 MAPK pathway. We also demonstrated that the ratio of ASK1/Trx-ASK1 increases in aged mouse livers and that this correlates with the increased basal activity of the p38 MAPK pathway. The longevity of Snell dwarf mice has been attributed to their resistance to oxidative stress. A comparison of the levels of Trx-ASK1 in young and aged dwarfs showed a higher abundance of the complex than in their age-matched controls. These results, which are indicative of a decreased level of oxidative stress, suggest that increased ROS production in aged liver may alter the ratio of ASK1 and Trx-ASK1, thereby increasing the age-associated basal level of p38 MAPK pathway activity.

991. Adenovirus-Mediated IL-12 Gene Transfer Increase Virus Specific CD8 T Cell Response in Aged Mice

Top of pageAbstract Respiratory viral infection is a major cause of morbidity and mortality in the elderly population. IL-12 and other Th1 cytokines can prevent the age-related defect in the cytotoxic T lymphocyte (CTL) response by blocking the age-associated down modulation of CD28 and immune senescence of CD8+ T cells. Gene therapy is one method to restore defective Th1 cytokines and reverse immune senescence. However, the effect of IL-12 on the generation and effector function of the viral epitope-specific cytotoxic CD8 T cells (CTLs) after delivery by an adenovirus vector has been difficult to dissect due to the lack of appropriate tetramers capable of identifying the virus-specific CTL response. This limitation can now be overcome by the use of our recently developed MHC class I tetramer containing the adenovirus (Ad) epitope peptide E1Bp, referred to as Db-E1Bp, in combination with the use of an in vivo killing assay to analyze the virus specific CD8 T cell response in young and old mice after Ad administration. To determine the effect of IL-12 on the generation and effector function of the virus-specific CTLs, we utilized an recombinant Ad vector to deliver the IL-12 gene into aged (16-mo-old) and young (2-mo-old) C57BL/6 (B6; H-2b) mice before infected with a wild-type Ad5. The primary CTL response was assayed by the Db-E1Bp tetramer and E1Bp-specific in vivo killing assay. Our results indicate that there was a 58% decrease in the production of virus-specific Db-E1Bp+CD8+ T cells in aged mice at day 8 after Ad5 infection, compared with that from young mice (p<0.01; Table). This was associated with a 30% decrease in the specific killing activity by these CTLs in aged mice (p<0.01). In vivo pretreatment of aged mice with Ad-IL12 resulted in an increase in serum IL-12, from 18|[plusmn]|7 ng/ml before pretreatment to 96|[plusmn]|12 ng/ml at the time of Ad5 infection 3 days later. Ad-IL12 pretreatment also induced strong IFNg production of 34|[plusmn]|11 ng/ml at the time of Ad5 injection compared to the base line of 0.5|[plusmn]|0.2 ng/ml. There was only a moderate increase of these cytokines in young mice. However, the serum IL-12 correlated with IFNg production in both old (r2=0.889; p<0.001) and young (r2=0.946; p<0.001) mice. At day 8 after Ad5 infection, both the Db-E1Bp+CD8+ /total CD8+ T cells and E1Bp specific lysis efficiency in the spleen and liver of aged mice increased to levels comparable to those of young mice (Table). This was associated with an increased thymocyte proliferative response mediated through IL-2 and IL-7 in aged mice. Our results indicate that aged mice have a defect in the CTL response to Ad infection, and that Ad-IL12 treatment significantly enhances the Ad-specific CTL response in aged mice, partially by induction of IFN|[gamma]|.

Gene expression of drug metabolizing enzymes in adult and aged mouse liver: A modulation by immobilization stress

The role of stress in the regulation of enzymatic systems involved in the biotransformation of xenobiotics, as well as endogenous substrates in the liver was investigated using single immobilization stress as a model. Adult (3 months of age) and aged (26 months) C3H/a male mice were used. Cytochrome P450 1A1 and 1A2 (CYP1A1 and CYP1A2), glutathione S-transferase M1 (GSTM1), aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT) and catechol- O-methyltransferase (COMT) mRNA levels in the mouse liver were measured by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Excluding CYP1A1, experiments revealed significant differences in the expression of these genes between adult- and aged-control animals. The influence of stress on the expression of genes studied was shown to be higher in adult mice than in aged ones. Our results clearly demonstrate the lack of response or even the attenuation of gene expression in aged animals that may play an important role in age-related pathologies and diseases.

Aging does not reduce the hepatocyte proliferative response of mice to the primary mitogen TCPOBOP.

It has been shown that the magnitude of DNA synthesis and the time at which maximal DNA synthesis occurs after two-thirds partial hepatectomy (PH) is greatly reduced in the liver of aged rodents compared to young animals. This reduction could represent an intrinsic defect in proliferation or a more specialized change in the response to PH. We therefore evaluated the proliferative capacity of hepatocytes in aged animals, following treatment with primary liver mitogens. We show that treatment of 12-month-old CD-1 mice with the hepatomitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) caused an increase in hepatocyte proliferation similar to that seen in young (8-week-old) mice. The labeling index was 82% in the livers of aged mice versus 76% in young animals. Histological observation demonstrated that the number of hepatocytes entering mitoses was similar in both groups; the mitotic indices were 2.5 per thousand and 2.7 per thousand, respectively. Additional experiments showed that the timing of DNA synthesis and M phase were nearly identical in both aged and young mice. Stimulation of hepatocyte DNA synthesis was associated with increased expression of several cell cycle-associated proteins (cyclin D1, cyclin A, cyclin B1, E2F, pRb, and p107); all were comparable in aged mice and young mice. TCPOBOP treatment also increased expression of the Forkhead Box transcription factor m1b (Foxm1b) to a similar degree in both groups. In conclusion, hepatocytes retain their proliferative capacity in old age despite impaired liver regeneration. These findings suggest that therapeutic use of mitogens would alleviate the reduction in hepatocyte proliferation observed in the elderly.

Carbonylation of ER chaperone proteins in aged mouse liver

Progressive accumulation of oxidative damage to macromolecules in aged tissues is thought to contribute to the decline in tissue function characteristic of the aged phenotype. Mitochondria are a major intracellular source of reactive oxygen species (ROS); however, other organelles are also endogenous sources of oxyradicals and oxidants, which can damage macromolecules. We, therefore, sought to examine the relationship between aging and oxidative damage to ER resident proteins, which exist in a strongly oxidizing environment necessary for disulfide bond formation. In these studies, we have fractionated young and aged liver homogenates, resolved the proteins by 2D gel electrophoresis, assayed for oxidative damage as indicated by protein carbonylation, and identified BiP/Grp78, protein disulfide isomerase (PDI), and calreticulin as exhibiting an age-associated increase in oxidative damage. Increased carbonylation of these key proteins in aged liver suggests an age-associated impairment in protein folding, disulfide crosslinking, and glycosylation in the aged mouse liver.