Douglas L. Schmucker

Age-related changes in liver structure and function: Implications for disease ?

The geriatric populations of many countries are growing rapidly and they present major problems to healthcare infrastructures from both medical and economic perspectives. The elderly are predisposed to a variety of diseases, which contribute to a marked increase in morbidity in this subpopulation. The incidence of liver disease increases in the elderly, but the cellular and subcellular perturbations that underlie this suspected predisposition to pathology remain unresolved. Several age-related changes have been documented, including (a) a decline in liver volume, (b) an increase in the hepatic dense body compartment (lipofuscin), (c) moderate declines in the Phase I metabolism of certain drugs, (d) shifts in the expression of a variety of proteins and (e) diminished hepatobiliary functions. Other more subtle changes (e.g., muted responses to oxidative stress, reduced expression of growth regulatory genes, diminished rates of DNA repair, telomere shortening) may contribute to reduced hepatic regenerative capacity, shorter post-liver transplant survival and increased susceptibility to certain liver diseases in the elderly.

Effects of age and gender on in vitro properties of human liver microsomal monooxygenases

Aging in humans is associated with marked declines in the disposition of numerous drugs and other xenobiotics that require hepatic biotransformation before elimination. Considerable pharmacokinetic evidence in humans, coupled with data on in vitro liver microsomal monooxygenase functions generated in inbred male rodent models, has implicated impaired liver phase I drug metabolism (i.e., diminished efficacy of microsomal monooxygenases) in reduced drug clearance in the elderly. This study (1) assessed the in vitro activities and amounts of liver microsomal monooxygenases as a function of donor age and gender in healthy humans and (2) provides the most extensive and comprehensive data to date demonstrating the absence of significant age‐ and gender‐dependent differences in the activities and contents of human liver monooxygenases.

Impact of aging on gastrointestinal mucosal immunity

There is considerable evidence that the mucosal or secretory immune response in the gastrointestinal tract is compromised by aging. The generation of a mucosal immune response is an extremely complex process that involves antigenic stimulation of a specific subpopulation of immunologically competent cells in the Peyers patches, differentiation and migration of these cells to the small intestinal lamina propria, initiation and regulation of local antibody production in the intestinal wall, and mucosal epithelial cell receptor-mediated transport of antibodies to the intestinal lumen. Available data suggest that gastrointestinal mucosal immunosenescence reflects deficits in: (1) the differentiation and/or migration (homing) of immunoglobulin A immunoblasts to the intestinal lamina propria, and (2) the initiation and/or regulation of local antibody production. The significant age-related increases in the incidence and severity of gastrointestinal infectious diseases, coupled with the potential for immunopharmacologic manipulation of the mucosal immune compartment, substantiate the merit of studies designed to resolve the etiology of mucosal immunodeficiency in the elderly.

Morphometric Analysis of Rat Hepatocytes After Total Biliary Obstruction

Using light and electron microscopic morphometric techniques, the effects of 48 hr of extrahepatic biliary obstruction on hepatocyte structure were examined in the rat. Liver cells near the portal area were compared to those in the centrilobular regions of the hepatic lobule. Observations on the normal animals confirm earlier evidence of quantitative differences in the surface density of organelles in hepatocytes located within different regions of the lobule. A striking difference in the quantity of the Golgi complex in the two areas of the lobule was noted for the first time, with the portal cells containing a significantly greater quantity of this organelle than centrolobular hepatocytes. After 48 hr of total obstruction, most of the previously reported qualitative changes in the canalicular and pericanalicular regions were confirmed. Morphometric analysis at the light-microscopic level showed an increase in the number of cells and a decrease in cell size in those cells near the portal area were compared to those in the centrolobular regions of the helar level demonstrated a significant decrease in both rough and smooth surfaced endoplasmic reticulum in cells of both zones, a finding in marked contrast to the hypertrophy of smooth endoplasmic reticulum suggested by other investigators on the basis of qualitative assessments. There was also a striking decrease in the amount of the Golgi complex, limited to cells in the portal regions. In addition, in all zones a decrease in the volume density of mitochondria and lysosomes was noted, whereas the volume of microbodies was increased. It is suggested that this loss in total membrane material within the cell may be secondary to the degranulation and decrease in total surface area of rough surfaced endoplasmic reticulum, an organelle thought to be responsible in part for the synthesis of new cellular membranes. These observations suggest that present concepts concerning the pathogenesis of cholestatic liver disease require reappraisal.

Liver Regeneration and Aging: A Current Perspective

Many organ systems exhibit significant age-related deficits, but, based on studies in old rodents and elderly humans, the liver appears to be relatively protected from such changes. A remarkable feature of the liver is its capacity to regenerate its mass following partial hepatectomy. Reports suggests that aging compromises the livers regenerative capacity, both in the rate and to the extent the organs original volume is restored. There has been modest definitive information as to which cellular and molecular mechanisms regulating hepatic regeneration are affected by aging. Changes in hepatic sensitivity to growth factors, for example, epidermal growth factor (EGF), appear to influence regeneration in old animals. Studies have demonstrated (a) a 60% decline in EGF binding to hepatocyte plasma membranes, (b) reduced expression of the hepatic high affinity EGF receptor and (c) a block between G1 and S-phases of the cell cycle in old rats following EGF stimulation. Recent studies suggest that reduced phosphorylation and dimerization of the EGF receptor, critical steps in the activation of the extracellular signal-regulated kinase pathway and subsequent cell proliferation are responsible. Other studies have demonstrated that aging affects the upregulation of a Forkhead Box transcription factor, FoxM1B, which is essential for growth hormone-stimulated liver regeneration in hepatectomized mice. Aging appears to compromise liver regeneration by influencing several pathways, the result of which is a reduction in the rate of regeneration, but not in the capacity to restore the organ to its original volume.

Effects of aging and phenobarbital on the rat liver microsomal drug-metabolizing system.

Significant declines in the non-induced activities of liver microsomal drug-metabolizing enzymes and in the amount of cytochrome P-450 occur between maturity (16 months) and senescence (27 months) in male Fischer 344 rats, whereas there are essentially no differences between very young (1 month) and mature animals. Several hepatic responses to chronic phenobarbital administration also demonstrate marked age-dependent changes. The livers of young and mature animals exhibit: (1) greater hepatomegaly; (2) faster rates of induction and post-induction recovery of microsomal mixed function oxidase enzyme activities and hemoprotein concentration; and (3) higher maximally induced levels of these components in comparison to senescent rats. When considered with information from previous studies, the present data suggest that the age-related decline in liver drug metabolism may be due to qualitative and/or quantitative changes in the structural and/or functional components of the hepatic microsomal mixed function oxidase system.

Quantifying dense bodies and lipofuscin during aging: a morphologist's perspective

Secondary lysosomes, residual or dense bodies containing lipofuscin or age pigment accumulate in post-mitotic and inter-mitotic cells during aging. The consensus is that the accumulation of this auto-fluorescent material is an index of cellular senescence. Biochemical and morphological studies have independently demonstrated marked age-related increases in the cell and tissue contents of lipofuscin. Most morphological studies on aging have been qualitative, have included only two or three age groups and have not yielded data that are easily correlated with biochemical analyses. One of the best documented age-related changes in hepatocytes and cardiac myocytes is the accumulation of dense bodies and lipofuscin inclusions. Independent stereologic studies reported two- to eightfold age-related increases in the dense body volume fraction of rat hepatocytes. Furthermore, we reported a fourfold increase in the dense body volume fraction of cardiac myocytes in rats between 6 and 30 months of age. These and other studies confirm the use of quantitative morphology to estimate the increases in dense body and lipofuscin inclusions as indices of age. Whether or not the accumulated lipofuscin compromises cell functions in senescent animals has not been adequately addressed. On the one hand, there is little evidence that several-fold increases in this subcellular compartment impair the functional capacities of either hepatocytes or cardiac myocytes. On the other hand, the age-related accumulation of immunoprecipitable, but catalytically inactive, lysosomal enzymes in both liver and heart muscle may be a reflection of increased lipofuscin deposits in the dense bodies.

Basis for the Age-related Decline in Intestinal Mucosal Immunity

The elderly are characterized by mucosal immunosenescence and high rates of morbidity and mortality associated with infectious diseases of the intestinal tract. Little is known about how the differentiation of immunoglobulin A (IgA) plasma cells in Peyers patches (PPs) and their subsequent homing to the small intestinal lamina propria (LP) is affected by aging. Quantitative immunohistochemical analyses demonstrated a 2-fold increase in the number of IgA+ cells in the PPs, coupled with significant declines in the numbers of IgA+ and antibody-positive cells in the intestinal LP of senescent rats compared to young adult animals. These data suggest that aging diminishes the emigration of IgA immunoblasts from these lymphoid aggregates, as well as their migration to the intestinal LP. Flow cytometry and lymphocyte adoptive transfer studies showed 3- to 4-fold age-related declines in the homing of antibody-containing cells and mesenteric lymph node lymphocytes to the small intestines of rhesus macaques and rats, respectively. The number of peripheral blood IgA immunoblasts expressing the homing molecule α4β7 declined 30% in senescent rats. This was accompanied by a >17% decrease in the areal density of LP blood vessels staining positive for the cell adhesion molecule MAdCAM-1. Cumulatively, declines in expression of these homing molecules constitute a substantial age-related diminution of IgA immunoblast homing potential. In vitro antibody secretion by LP plasma cells, i.e. antibody secreted per antibody-positive cell, remains unchanged as a function of donor age. Intestinal mucosal immunosenescence is a consequence of reduced homing of IgA plasma cells to the intestinal LP as a result of declines in homing molecule expression.

Aging impairs intestinal immunity.

The elderly are characterized by immunosenescence accompanied by high rates of morbidity and mortality associated with infectious diseases. Despite suggestions that the mucosal immune compartment is relatively unaffected by aging, there are marked deficits in the intestinal mucosal immune responses of old animals and elderly humans. Little is known about the mechanism(s) whereby aging disrupts intestinal immunity. However, several events in the genesis of the intestinal immune response may be perturbed during aging. The first step is the uptake of antigens by specialized epithelial cells (M cells) that overlie the domes of Peyers patches. We are unaware of any studies on the efficacy of antigen uptake in the intestine as a function of age. The effects of aging on the next step, antigen presentation by dendritic cells and lymphocyte isotype switching, have not been resolved. The third event is the maturation of immunoglobulin A (IgA) immunoblasts and their migration from the Peyers patches to the intestinal mucosa. Quantitative immunohistochemical analyses suggest that the migration of these putative plasma cells to the intestinal effector site is compromised in old animals. Local antibody production by mature IgA plasma cells in the intestinal mucosa constitutes the fourth step. We recently reported that in vitro IgA antibody secretion by intestinal lamina propria lymphocytes from young and senescent rats is equivalent. The last event is the transport of IgA antibodies across the epithelial cells via receptor-mediated vesicular translocation onto the mucosal surface of the intestine. Receptor-binding assays did not detect age-associated declines in receptor number or binding affinity in either rodent or primate enterocytes as a function of donor age. Efforts to identify the mechanism(s) responsible for the age-related decline in intestinal mucosal immune responsiveness may benefit by focusing on the homing of IgA immunoblasts to the effector site.

Age-dependent alterations in rat liver microsomal NADPH-cytochrome c (P-450) reductase: A qualitative and quantitative analysis

The hepatic drug-metabolizing capacities of rodents and man exhibit age-dependent declines. The extensive use of medication in geriatric patients demonstrates the need to characterize the mechanism(s) responsible for this reduced liver function. An analysis of microsomal NADPH-cytochrome c (P-450) reductase in young adult (3 months), mature (9 months) and senescent (27 months) male Fischer 344 rats revealed specific age-related qualitative and quantitative changes in this enzyme. The specific activity of the purified enzyme from young animals was two-fold higher than that recovered from the other age groups. In addition, there was: (1) no change in molecular weight, (2) alterations in heat inactivation profiles, (3) an apparent reduction in substrate affinity, and (4) a two-fold loss of enzyme activity per unit of immunoprecipitable material as a function of animal age. Our data suggest that this important liver drug-metabolizing enzyme undergoes post-translational modifications in its conformation which are reflected in the above parameters and which, ultimately, affect its efficacy.