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

Abstract

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 hepatomito-gen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) caused an increase in hepato-cyte 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 m l b (Foxmlb) 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. (HEPATOLOGY 2004;40:981-988.) he liver plays a central role in the metabolic functions of the body, carrying out over 5,000 func-T tions, with a remarkable capacity to adapt to metabolic perturbance. Liver regeneration after partial hepatectomy (PH) typifies one such capacity: the organ Abbreviations: PH, partial hepatectomy; Foxmlb, Forkhead Box mlb; TCPOBOP, 1,4-bis[2-(3,5-dicbloropyridyloxy)]benzene; CAR, constitutive an-drostane receptor; TNF-a tumor necrosis factor (Y IL-6, interleukin 6; mRNA, messenger RNA; B r d u bromodeoyuridine; LI, labeling index; kb, kilobase; cDNA, complementary DNA; P A , R N h e protection assay; SDS, sodium dodecyl suFate. changes instantly from an essentially quiescent state to a rapidly growing one. Experimental models of regenera-tion have been the target of many studies that probed the control mechanisms of liver growth.2-5 This type of re-generative liver growth, termed "compensatory regenera-tion" or "compensatory hyperplasia," also occurs after liver cell necrosis due to chemical, nutritional, vascular, or viral injury. Several studies on the regenerative response of the liver that follows two-thirds PH have shown a striking difference in both the magnitude of the peak response in DNA synthesis and the time at which the maximal DNA synthesis occurs between young (4-8 weeks) and aged (12-15 month...

Global gene expression profile of normal and regenerating liver in young and old mice

“…In particular, the finding that aged hepatocytes thoroughly maintain their replicative capacity following mitogenic stimuli (Ledda- Columbano et al 2004) suggests that the depressed replicative response observed in aged rodent liver after PH results from lowered levels of extra-cellular factors, such as growth factors and/or hormones, rather than from intrinsic changes within the cell. In agreement with this, our present work found that the expression of several early changes, considered to be essential for liver regeneration (Yamada et al 1997;Cressman et al 1996;Michalopoulos and DeFrances 1997), is essentially similar in regenerating (young mouse) and non-regenerating (old mouse) liver.…”

“…Western blot analysis Total cell extracts were prepared from frozen livers as described previously (Ledda- Columbano et al 2004). For cyclin D1, D2, D3, and YAP, 100 mg of total extracts was prepared.…”

Potential utility of xenobiotic mitogens in the context of liver regeneration in the elderly and living-related transplantation

“…19,24 The probe for b-actin was prepared from total RNA using SuperScriptt IIIOne-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA). Following PCR primers were used for b-actin: forward AGC CATGTACGTAGCCATCC and reverse AAGGGTGTAAAAC GCAGCTC.…”

“…Notably, it was shown that hepatocyte proliferation induced by TCPOBOP, a ligand of the constitutive androstane receptor (CAR), is age independent, suggesting that hepatocytes retain their proliferative capacity in old age despite impaired liver regeneration. 19 On the basis of these observations, the first aim of this study was to investigate whether pretreatment with xenobiotic mitogens, for example TCPOBOP, may allow the growth of the liver in conditions associated with a reduced regenerative capacity, such as the elderly. In particular, we address the question whether nuclear receptor-mediated signal transduction pathways could be capable of bypassing the growth-constrained environment of an aged liver, thus improving its proliferative capacity after 70% PH.…”

Nuclear receptors CAR and PXR in the regulation of hepatic metabolism

“…CAR is able to regulate a set of genes common to the promotion of cellular growth. After a short treatment with TCPOBOP, the mice showed increased BrdU staining as well as rapid induction of cell cycle genes including cyclin D1 and cdk2 (Ledda-Columbano et al 2000;Ledda-Columbano et al 2004). Gadd45β, a gene involved in the repression of apoptosis, is also up-regulated in response to CAR activators (Columbano et al 2005).…”