Hepatocytes Of Old Rats Research Articles

Short-term fenofibrate treatment improves ultrastructure of hepatocytes of old rats.

Fenofibrate (FN) is a hypolipemic drug used for the treatment of mixed dyslipidemia. Since in our previous study FN administration to young and old rats adversely affected the serum activity of liver marker enzymes, we decided to examine the effects of FN on liver ultrastructure of young and old animals. Young and old rats were fed standard rodent chow supplemented with 0.1% FN for 30 days. Liver samples obtained from animals under full anesthesia were processed by routine methods to obtain ultrathin and histological sections for the examination by light microscopy (LM) and transmission electron microscopy (TEM). Furthermore, liver lysates were analyzed by Western blotting for the expression of the autophagy-related proteins LC3A/B and beclin 1. The ultrastructure of hepatocytes in both age groups was well-preserved, with the presence of abundant mitochondria, numerous peroxisomes and lysosomes, glycogen stored in the form of rosettes, and occasionally autolysosomes. However, hepatocytes of old control rats contained less mitochondria and peroxisomes, and more lipid droplets than cells of young animals. The effects of FN on liver ultrastructure were age-depended. FN increased the relative number of mitochondria and peroxisomes in the hepatocytes of old, and did not affect their number in young rats. Moreover, FN decreased and increased the relative number of lipid droplets in the hepatocytes of old and young rats, respectively. At the LM level, Oil Red O staining revealed smaller and larger lipid droplets within hepatocytes and non-parenchymal liver cells. In the livers of young and old rats lipid droplets were distributed mainly in the periportal zones of hepatic lobules. Morphometric analysis confirmed that livers of control old rats contained more lipid-stainable areas than those of young ones; however, no effect of FN was observed either in young or old rats. Despite larger size of autolysosomes and autophagic vacuoles in hepatocytes of old rats, the expression of autophagy-related proteins did not differ in the livers of control and fenofibrate-treated young and old animals. The results of our study suggest that fenofibrate, apart from its hypolipemic action, may have beneficial effect on the energy metabolism in the liver of old individuals by increasing the number of mitochondria and peroxisomes in hepatocytes.

Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

Ceramides inhibit phospholipase D-dependent insulin signaling in liver cells of old rats

Ceramides are a novel class of biologically active molecules involved in the regulation of different signaling pathways. Ceramide is involved in regulation of the phospholipase D (PLD) activity and development of cell resistance to insulin. In this work, we have studied age-related features of insulin regulation of PLD activity and glucose metabolism in intact cells and modeled their resistance to insulin by exogenous ceramide and palmitic acid. Contents of ceramides and of free fatty acids (FFA) are found to increase with age, as well as on incubation of liver cells of young rats in the presence of the ceramide precursor palmitic acid. Under these conditions, the ability of insulin to activate PLD, the cell uptake of glucose, and glycogen synthesis sharply decreased. On incubation of hepatocytes of young animals in the presence of exogenous C2-ceramide, the contents of endogenous ceramides increased but not the contents of FFAs and of neutral lipids. These events were accompanied by suppression of the insulin-induced production of phosphatidylethanol (a result of ethanol transphosphatidylation by PLD), glucose uptake, and glycogen synthesis. Incubation of insulin-resistant liver cells of young rats and also of hepatocytes of old rats in the presence of myriocin (an inhibitor of the de novo synthesis of ceramide) was associated with a decrease in ceramide content in the cells and an increase in the cell sensitivity to insulin. The findings indicate an important role of ceramide in disturbance of insulin signaling due to inhibition of the PLD-dependent link in the liver cells of old animals.

Aging Results in Increased Autophagy of Mitochondria and Protein Nitration in Rat Hepatocytes Following Heat Stress

The natural breakdown of cells, tissues, and organ systems is a significant consequence of aging and is at least partially caused by a decreased ability to tolerate environmental stressors. Based on quantitative ultrastructural analysis using transmission electron microscopy and computer imaging, we show significant differences in hepatocyte morphology between young and old rats during a 48-hr recovery period following a 2-day heat stress protocol. Mitochondrial injury was greater overall in old compared with young rats. Autophagy was observed in both young and old rats, with autophagy greater overall in old compared with young hepatocytes. Lipid peroxidation and protein nitration were evaluated by localization and quantification of 4-hydroxy-2-nonenal (4-HNE)-modified protein adducts and 3-nitrotyrosine (3-NT) levels, respectively. Levels of 3-NT but not 4-HNE-protein adducts were significantly elevated in hepatocytes of old rats in comparison with young at 90 min after heat stress, suggesting a major role for reactive nitrogen species in the pathology observed at this time point. These results show a differential response of hepatocyte mitochondria to heat stress with aging, as well as greater levels of both autophagic and nitrative damage in old vs young hepatocytes. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Direct cell–cell communication: a new approach derived from recent data on the nature and self-organisation of ultradian (circahoralian) intracellular rhythms

Recent data concerning ultradian (circahoralian) intracellular rhythms are used to assess the biochemical mechanisms of direct cell-cell communication. New results and theoretical considerations suggest a fractal nature of ultradian rhythms and their self-organisation. The fundamental and innate nature of these rhythms relates to their self-similarity at different levels of cell and tissue organisation. They can be detected in cell-free systems as well as in cells and organs in vivo. Such rhythms are a means of finding an optimal state of cell function rather than achieving a state of absolute stability. As a consequence, oscillations, being irregular and numerous by the set of periods, are resilient to functional overload and injury. Recent data on the maintenance of their fractal structure and, especially on the selection of optimal periods are discussed. The positive role of chaotic dynamics is stressed. The ultradian rhythm of protein synthesis in hepatocytes in vitro was used as a marker of direct cell-cell communication. The system demonstrates cell cooperation and synchronisation throughout the cell population, and suggests that the ultradian rhythms are self-organised. These observations also led to the detection of mechanisms of direct cell-cell communication in which extracellular factors have an essential role. Experimental evidence indicated the involvement of gangliosides and/or catecholamines in this large-scale synchronisation of protein synthesis. The response of all, or a major part, of the cell population is important; after the initial trigger effect, a periodic pattern is retained for some time. The influence of Ca2+-dependent protein kinases on protein phosphorylation can be a final step in the phase modulation of rhythms during cell-cell synchronisation. The intercellular medium plays an important role in self-synchronisation of ultradian rhythms between individual cells. Low cooperative activity of hepatocytes of old rats resulted from altered composition of the intercellular medium rather than direct effects of animal and cellular ageing. Similarly, in the whole body, changes in levels of gangliosides and catecholamines in the blood serum, a natural intercellular medium, can be critical events in age-dependent changes of the serum and accordingly cell-cell synchronisation. Hepatocytes of old rats exhibit some of the properties of young cells following an increase in blood serum ganglioside level, as well as, in in vitro conditions, after the addition of gangliosides to the culture medium. Together with data on ultradian functional and metabolic rhythms, all the material reviewed here allows us to propose a mechanism of direct cell-cell cooperation via the medium in which the cells exist, that supplements the nervous and hormonal central regulation of organ functions. Ultradian intracellular rhythms may thus provide a finer framework within which the integrated dynamics of respiration, heart rate, brain activity, and even behavioural patterns, are brought to an optimal functional pattern. Innate and direct cell-cell cooperation may have been employed as a means of intercellular regulation during the course of metazoan evolution, that preceded nervous regulation and is presently retained in mammals.

The effects of galactosamine on UTP levels in the livers of young, adult and old rats.

Galactosamine (GalN), a well-known hepatotoxin that depletes the cellular pool of uracil nucleotides, was previously shown to have greater impact on the inhibition of protein synthesis in hepatocytes of old rats as compared with young animals (Kmieć 1994, Ann. N.Y. Ac. Sci. 717, 216-225). In the present study we compared the effects of GalN on the nucleotide content (measured by ion-exchange HPLC) in the livers of young (4 months), adult (12 months), and old (24-26 months old) rats two hours after its intraperitoneal administration. UTP content of the livers of old control rats was significantly lower (by 28%) than that of young animals. GalN administration decreased the UTP content in the livers of young, adult and old rats by, respectively, 55%, 65% and 89%, and increased the content of UDP-sugars by 189%, 175% and 305%. The hepatic content of ATP, ADP, AMP, NAD, GTP except CTP did not differ significantly among the age groups of rats studied, and was not changed by GalN treatment. The content of CTP was significantly higher in old rats (P < 0.03) upon GalN treatment. The lower hepatic content of UTP may partially explain the increased sensitivity of hepatocytes and livers of old rats to the action of galactosamine, and possibly to other hepatotoxic compounds that decrease transcription in the liver.

Hypothalamic mechanisms of aging and anti-aging

A stress-age syndrome is one of the main phenomena related to the aging process. This phenomenon contributes to effective adaptation of an organism under the usual conditions of vital activity, but in the case of the influence of additional stress factors it promotes the development of age-related pathologies. In this respect, age-related modifications of the hypothalamic mechanisms deserve special attention. The electrical activity of the hypothalamic nuclei undergoes age-related modifications; in particular, the parameters of the spike activity of hypothalamic neurons demonstrate specific changes. Hypothalamic stimulation affects the induction of synthesis of a few enzymes (tyrosine aminotransferase, tryptophan pyrolase, glucose-6-phosphatase, and fructose-1,6-diphosphatase) in the liver, and in old rats these effects become weaker. Long-lasting stimulation of the hypothalamus results in an increase in the content of an active chromatin fraction in hepatocytes of old rats; such stimulation intensifies the transcriptive activity of this chromatin and decreases the protein/DNA ratio. In old rats, long-lasting stimulation of the lateral hypothalamic region increases the mean and maximum remaining lifespan of the experimental animals.

Age-related susceptibility to hepatotoxicants

Limited information is available regarding age-associated events that lead to differences in vulnerability to chemicals that injure the liver. For some agents, such as allyl alcohol, alterations in metabolic activation, by liver biotransformation enzymes, are responsible for age-associated changes in severity of liver damage. For other toxicants, such as carbon tetrachloride, there appears to be no relation between changes in activation/detoxification processes and the effects of aging on the extent of liver injury. With diquat, a rise in iron content seems to explain the increased toxicity observed in hepatocytes of old rats compared with those of young-adult rats. Additional research is needed to identify the mechanisms responsible for age-dependent differences in sensitivity to environmental chemicals.

P21waf-1/cip-1/sdi-1Is Expressed at G1 Phase in Primary Culture of Hepatocytes from Old Rats, Presumably Preventing the Cells from Entering the S Phase of the Cell Cycle

To elucidate whether p21waf-1/cip-1/sdi-1expression is associated with loss of growth potential of hepatocytes of old rats, we determined p21waf-1/cip-1/sdi-1expression of hepatocytes from old (30 months) rats during the cell cycle in primary culture. A high level of expression of p21waf-1/cip-1/sdi-1was detected at the G1 phase in old-rat hepatocytes, but after the S phase in young-rat hepatocytes. Consistently, the incidence of the cells positive for p21waf-1/cip-1/sdi-1in nuclei before entering the S phase was significantly higher in old-rat hepatocytes than in young-rat hepatocytes. These results account for the loss of growth potential of old-rat hepatocytesin vitroand the marked retardation of regeneration of liver in old ratsin vivo.

Diquat-Induced Oxidative Damage in BCNU-Pretreated Hepatocytes of Mature and Old Rats

The effects of postmaturational aging on the toxicity of diquat, a redox cycling compound, were investigated in hepatocytes that were isolated from mature (6 months) and old (27 months) male Fischer 344 rats and pretreated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. The hepatocytes were incubated for 2 hr with 0, 0.5, or 2.0 mM diquat dibromide, and samples were taken at various time points for measurements of glutathione, glutathione disulfide, thiobarbituric acid reactive substances, lactate dehydrogenase leakage, protein sulfhydryl groups, and protein carbonyl groups. Diquat cytotoxicity was intensified in hepatocytes of old rats compared with those of mature rats, and the enhanced toxicity was associated with increased lipid peroxidation and protein carbonyl formation. However, the enhanced toxicity in old rat hepatocytes was also accompanied by a decrease in diquat-induced GSH oxidation and there was no difference in protein sulfhydryl loss. Concentrations of total nonheme iron and low-molecular-weight chelatable Fe 2+, measured with ferene as the chromogen, were several times higher in freshly isolated hepatocytes of old rats than in those of mature rats. We hypothesize that the age-associated enhancement of diquat toxicity could be due to an increased availability of iron for reaction with diquat-generated hydrogen peroxide and for stimulation of lipid and protein oxidation.

Plasmic membranes of hepatocytes and adrenocorticocytes in rats of different ages: effect of testosterone

Experiments on adult (6–8-month-old and 26–28-month-old) Wistar rats revealed thef hyperpolarization of plasmic membranes and activation of Na,K-ATPase of adrenocorticocytes in animals of both age groups and of hepatocytes of adult rats. No effect of testosterone was observed on the level of membrane potential and the activity of Na,K-ATPase of hepatocytes of old rats. The effect of testosterone was prevented by inhibitors of protein biosynthesis (actinomycin D and cycloheximide) and a specific inhibitor of Na,K-ATPase (ouabain), but not by K +-channel blocker 2-aminopyridine. Testosterone was assumed to synthesize the specific factor, capable of activating Na,K-ATPase of plasmic membranes. The cytosole of hepatocytes and the blood serum of adult testosterone-treated rats activated the Na,K-ATPase of isolated plasmic membranes of hepatocytes of adult and old intact rats. During aging there was a decrease in the capacity of cells to synthesize the specific factor, which activated Na,K-ATPase of plasmic membranes.