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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 56:B259-B267 (2001)
© 2001 The Gerontological Society of America

Aging Lowers Steady-State Antioxidant Enzyme and Stress Protein Expression in Primary Hepatocytes

David M. Halla, Gerald L. Sattlerc, Carol A. Sattlerc, Hannah J. Zhanga, Larry W. Oberleyb, Henry C. Pitotc and Kevin C. Kregela

a Department of Exercise Science, The University of Iowa, Iowa City
b Free Radical and Radiation Biology Program, The University of Iowa, Iowa City
c McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison

Kevin C. Kregel, Department of Exercise Science, 532 FH, The University of Iowa, Iowa City, IA 52242 E-mail: kevin-kregel{at}uiowa.edu.

Decision Editor: John A. Faulkner, PhD


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
It has been reported that the isolation and culture of primary hepatocytes can compromise cellular ability to constituitively express antioxidant enzyme (AE) genes, making it difficult to study their regulation ex vivo. In the present study, the steady-state expression of manganese-containing superoxide dismutase, copper- and zinc-containing superoxide dismutase, catalase, and glutathione peroxidase was assessed in primary hepatocytes isolated from young and senescent rats and cultured in Matrigel. There was no change in steady-state superoxide dismutase protein or activity levels in cells collected from young animals and cultured for 7 days. Catalase expression was initially increased, and then it declined 30%. In contrast, superoxide dismutase expression declined 60% and catalase expression declined 50% in cells from senescent animals. Constitutive and inducible 70-kDa heat shock protein expression increased coincident with declining AE levels in the young cells but not senescent cells. For both age groups, electron micrographs showed rounded hepatocytes with abundant rough endoplasmic reticulum, mitochondria, and peroxisomes. Hepatocytes were organized into clusters of 6–12 cells surrounding a large central lumen devoid of microvilli. Each cluster also contained smaller microvilli-lined lumens between adjacent hepatocytes that resembled canniculi. The plasma membranes of these lumens were sealed from the extracellular space by junctional complexes. Gap junctions in the plasma membrane suggest that hepatocytes were capable of intercellular communication. We conclude that the Matrigel system can be used to study AE regulation in primary hepatocytes from young and senescent animals, provided that experiments can be conducted within a time frame of 5–7 days in culture. These data also support the hypothesis that aging compromises hepatocellular ability to maintain AE status and upregulate stress protein expression.

AGING impairs eukaryotic ability to upregulate stress protein expression in response to environmental challenges such as heat stress (1)(2), exercise (3), and viral infection (4). In particular, aged organisms have a reduced capacity to rapidly synthesize protective cellular stress proteins such as heat shock proteins (2)(5)(6) and antioxidant enzymes (1). Mechanisms responsible for these critical age-related deficiencies in the cellular stress response are unclear, but experimental evidence implicates an elevation in steady state cellular electromotive force (7)(8) and poor transcriptional regulation of redox sensitive acute response (stress) genes (5)(9)(10).

The primary antioxidant enzymes (AEs) manganese-containing superoxide dismutase (MnSOD), copper- and zinc-containing SOD (CuZnSOD), catalase, and glutathione peroxidase (GPx) are an important subset of the stress proteins that can protect cells against the damaging effects of hyperthermia (11), viral infection (4), and ionizing radiation (12). Because of its ability to detoxify oxidants directly during episodic stress, the AE system is an important determinant of cellular electromotive force (8), which in turn influences cell signaling events (13), transcription factor activation (14), and ultimately, gene expression (15). We have recently observed that aging lowers the capacity of the liver to induce AEs in response to environmental heat stress (1) and significantly increases morbidity (16). Morbidity in the aged animals was closely linked with an increased generation of radical biomarkers of cellular oxidative stress. Although these are important in vivo observations that appear to link hyperthermia and cellular oxidative stress, the mechanisms responsible for age-related deficiencies in AE expression are unknown. Therefore, we have adopted a reductionist approach to the problem by establishing a primary hepatocyte culture system that will permit us to address mechanistic questions related to age-associated changes in cell function.

Differentiated primary cells hold many advantages over immortalized cell lines in aging research. However, several research groups have reported that liver-specific functions are compromised (17)(18)(19) and that nuclear transcription of the AE genes is significantly lowered (20) following the isolation and culture of hepatocytes. Recent advances in primary cell culture environments have shown that liver-specific functions (e.g., cytochrome-P450 activity, albumin production) can be maintained at near-normal levels for up to 21 days in culture (21)(22). However, no group has revisited the issue of steady-state AE expression in cultures of primary cells subsequent to these developments.

Using the hepatocyte growth matrix Matrigel (Becton Dickinson Labware, Bedford, MA), the present study assessed the steady-state levels of MnSOD, CuZnSOD, catalase, and GPx as well as the expression of the constitutive (Hsc70) and inducible (Hsp70) isoforms of the 70-kDa heat shock protein in cultures of hepatocytes isolated from young and senescent rats. We hypothesized that the stresses associated with hepatocyte isolation and culture (e.g., disruption of the liver, exposure to 20% oxygen) would stimulate adaptive increases in cellular Hsp70 and AE expression. We further postulated that cells collected from aged rats would lag behind their younger counterparts in adapting to culture conditions.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Isolation and Culture of Hepatocytes
All procedures for handling rats were approved by the University of Wisconsin and The University of Iowa Institutional Animal Care and Use Committees. Hepatocytes for primary culture were isolated from 6- and 24-month-old male Fischer 344 rats that were obtained from Harlan Labs (Harlan, Madison, WI). Briefly, hepatocytes were isolated by using the Seglen (23) in situ collagenase perfusion method with the following modifications.

The liver was perfused via the inferior vena cava with Ca2+, Mg+-free Hanks balanced salt solution (phenol red free, pH 7.4) supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), insulin (5 µg/ml), and glucose (1.5 mg/ml; all from Sigma Chemical Co., St. Louis, MO). After the initial blanching of the liver, the perfusion was continued with the same buffer plus 2 mg/ml of Type 1 collagenase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and 0.15 mg/ml of soybean trypsin inhibitor (Sigma). Dissociated cells were washed twice in cold L15 medium supplemented with penicillin, streptomycin, insulin, and glucose as above, and then they were purified by Percoll isodensity centrifugation (24). This procedure yielded a nearly pure population of hepatocytes with 95% or greater viability. One control aliquot of cells was prepared for immediate assessment of AE, Hsc70, and Hsp70 protein levels. A second control aliquot was incubated in a spinner flask at 4°C for 3 hours followed by determination of AE, Hsc70, and Hsp70 protein levels. A third control consisted of freshly isolated liver tissue collected from a littermate. There were no differences in the respective protein levels among these three controls; therefore, hepatocytes from two rats were pooled and incubated in spinner flasks for 3 hours prior to plating. This allowed us to obtain enough cells for the multiple time points in these experiments.

The purified and washed cells were suspended in HepatoSTIM (Becton Dickinson) medium supplemented with penicillin–streptomycin, glutamine (2 mM), and epidermal growth factor (10 ng/ml) and then plated in either six-well or 100-mm tissue culture dishes that had been precoated with Matrigel (Becton Dickinson). Plating density was 1.25 x 106 cells/well for the six-well dishes and 12–14 x 106 cells/100-mm dish. The culture medium was changed every 2 days per manufacturer's instruction, and cells were harvested for assay at the indicated times by using Matrisperse (Becton Dickinson) to depolarize the Matrigel.

Protein Immunoblots
Cells for protein immunoblots were homogenized in 0.05M of phosphate buffer (pH 7.8) and then briefly spun at 4°C in a clinical microcentrifuge. The supernatants were sonicated on ice and protein content was quantified by the Bradford assay (BioRad Laboratories, Richmond, CA). Equal protein amounts of cell homogenate were separated on a one-dimensional 12.5% polyacrylamide gel under standard denaturing conditions according to the method of Laemmli (25). After separation by electrophoresis, proteins were transferred to nitrocellulose membranes (100 V, 4°C, 1 hour). The membranes were blocked overnight at 4°C with 5% dry milk and 2% bovine serum albumin in TBST, which is 10 mM of tris(hydroxymethyl) aminomethane (Tris), 150 mM of NaCl, pH 8.0, and 0.1% Tween 20. The nitrocellulose membranes were incubated for 1 hour at room temperature with antibodies specific for MnSOD (1:2000 dilution), CuZnSOD (1:200 dilution), catalase (1:500 dilution), Hsc70 (1:500 dilution), and Hsp70 (1:1000 dilution). The AE blots were probed with a goat antirabbit IgG peroxidase antibody (Boehringer Mannheim) for 1 hour at room temperature (1:10,000 dilution for MnSOD and 1:5000 dilution for CuZnSOD and catalase). The Hsc70 blot was probed with rabbit antirat IgG peroxidase antibody (1:5000; StressGen Biotechnologies, Victoria, BC, Canada), and the Hsp70 blot was probed with goat antimouse IgG peroxidase antibody (1:1000; Sigma Chemical). Blots were developed by using the ECL-Western blot detection kit (Amersham Life Science, Piscataway, NJ) and BioMaxMR film (Kodak, Rochester, NY).

Antioxidant Enzyme Activity Gels
SOD, catalase, and GPx activities were analyzed by using native polyacrylamide gel electrophoresis (PAGE) gels. A 10% gel was used for evaluating SOD activity and an 8% gel was used for determining catalase and GPx activities. Protein concentrations were measured by the Bradford method (BioRad) and proteins (75 µg for SOD and 50 µg for catalase and GPx) were separated under nondenaturing conditions. SOD activity was visualized by staining gels in 2.43 mM of Nitro Blue Tetrazolium (NBT) in distilled water for 20 minutes, then incubating the gels for 15 minutes in 50 mM of phosphate buffer (pH 7.8) containing 2.8 x 10-5 M riboflavin and 28 mM of tetramethylethylenediamine (TEMED) (26). So that only MnSOD activity would be observed; 0.75 mM of NaCN (which inhibits CuZnSOD activity) was added to both the NBT and riboflavin–TEMED solutions. However, rat MnSOD is not visualized in these activity gels; therefore, SOD activity results represent only CuZnSOD activity. All incubations were performed at room temperature in the dark. Superoxide dismutase bands were observed by exposing gels to fluorescent light.

Catalase activity was measured by incubating gels in 0.003% H2O2 for 10 minutes (27). GPx activity was measured by incubating gels in 0.003% cumene hydroperoxide in 1 mM of glutathione (27). Gels were rinsed with distilled water and then stained with 2% potassium ferricyanide and 2% ferric chloride until bands were visible. Achromatic bands indicate removal of peroxides and thus are a direct assessment of catalase or GPx activity. Activity gels were thoroughly washed following staining procedures, and photographic images were obtained by using the Digital Imaging and Analysis System (Alpha Innotech Corporation, San Leandro, CA).

Data Analysis
Densitometries were performed on immunblots by using the National Institutes of Health Image 1.61 software package (NIH, Bethesda, MD).

Transmission Electron Microscopy
Primary hepatocytes isolated from senescent rats and cultured in six-well plates were processed for transmission electron microscopy on days 1, 3, 5, 14, and 21 in culture. Individual cultures were gently washed with phosphate-buffered saline (PBS) and fixed at room temperature in 3% glutaraldehyde in 0.1M of cacodylate buffer, pH 7.4 for 45 minutes. Cultures were then washed in three changes of 0.1M cacodylate and stored overnight at 4°C. Following a 45-minute postfixation on ice with 2% osmium tetroxide in 0.1M cacodylate (pH 7.4), cultures were washed three times with cold distilled water. A scalpel was used to detach the Matrigel "wafer" from the edges of each well, and one cut was made through the center of the wafer. The thick Matrigel layer containing clusters of fixed hepatocytes was separated from the dish with a plastic Swiss spatula and transferred to tissue culture dishes containing distilled water, where they were cut into strips approximately 3–4 mm long and 1.5 mm wide. Samples were then transferred to tissue carrier cylinders (Ted Pella, Redding, CA), dehydrated through a series of graded alcohols, and then placed in small glass vials with propylene oxide followed by 1 hour infiltration with 1:1 propylene oxide:Eponate resin (Ted Pella). After overnight infiltration with 100% Eponate, samples were imbedded in freshly made Eponate in flat embedding molds. Thin sections were cut on a Reichert Ultracut E3 ultramicrotome (Reichert, Austria) equipped with a diamond knife, and they were stained with uranyl acetate and lead citrate before examination in a Hitachi H-7000 electron microscope (Hitachi Scientific Instruments, San Jose, CA) operated at 75 kV.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Antioxidant Enzymes and Heat Shock Proteins
We measured a time course of AE protein and enzymatic activity levels in hepatocyte cultures to gain insight into the effects of isolation and culture on steady-state cellular antioxidant capacity. Fig. 1Fig. 2Fig. 3 show that hepatocytes derived from young rats maintained steady-state AE expression at or near control levels for 7 days in culture. MnSOD (Fig. 1) and CuZnSOD (Fig. 2) expression were unaltered through 7 days in culture and only marginally diminished at days 10–14. Steady-state catalase levels (Fig. 3) were initially increased on days 1–3, and then slowly declined to ~65% of control by day 14. Activity gels specific for CuZnSOD, catalase, and GPx (Fig. 4) demonstrate that SOD activity was maintained for 14 days in culture, whereas peroxide-removing capacity was sharply lower on days 10 and 14.



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  		Figure 1. Time course of steady-state manganese-containing superoxide dismutase (MnSOD) protein expression in hepatocyte cultures derived from young and senescent rats. Primary hepatocytes were isolated from A, young (6-month-old), and B, senescent (24-month-old), Fischer 344 rats and cultured for 14 days as described in Methods. Cultures were harvested at the time indicated and MnSOD protein was quantitated by using Western immunoblots and densitometry analyses. Hepatocytes isolated from young rats maintained steady-state MnSOD expression at or near control levels for 10 days in culture with only minor decrements in steady-state protein levels at day 14. In contrast, cultures derived from senescent animals maintained MnSOD expression at control levels through 5 days in culture followed by an approximate 60% decline in steady-state protein expression at day 14.

 


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  		Figure 2. Time-course of steady-state copper- and zinc-containing superoxide dismutase (CuZnSOD) protein expression in hepatocyte cultures derived from young and senescent rats. Primary hepatocytes were isolated from A, young (6-month-old), and B, senescent (24-month-old) Fischer 344 rats and cultured for 14 days as described in Methods. Cultures were harvested at the time indicated and CuZnSOD protein was quantitated by using Western immunoblots and densitometry analyses. Hepatocytes isolated from young rats maintained steady-state CuZnSOD expression at or near control levels for 14 days in culture. In contrast, cells collected from senescent animals presented lower CuZnSOD protein on day 3 followed by progressively increased protein levels on days 5–10. CuZnSOD expression declined 75% between days 10 and 14.

 


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  		Figure 3. Time course of steady-state catalase protein expression in hepatocyte cultures derived from A, young, and B, senescent rats. Hepatocyte cultures derived from both young and senescent animals showed an initial increase in catalase expression followed by declining steady-state catalase expression at days 5–14. Catalase expression fell 35% in the young cultures and 50% in the old.

 


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  		Figure 4. Antioxidant enzyme activities in hepatocyte cultures derived from A, young, and B, senescent rats. Hepatocytes isolated from young rats maintained steady-state copper- and zinc-containing superoxide dismutase (CuZnSOD) and catalase activity at or near control levels for 14 days in culture, with glutathione peroxidase (GPx) activity declining at 7–10 days. In contrast, the cultures of cells from older rats showed a loss of catalase activity by day 5 and declining GPx and CuZnSOD activities at day 7. By day 14, CuZnSOD activity was extremely low and GPx activity was below the detection limits of the system.

 
In contrast to cells derived from young rats, hepatocytes collected from old animals maintained AE expression at or near control levels for 5 days in culture followed by progressively declining AE protein and activity at days 7–14. Both MnSOD (Fig. 1) and catalase (Fig. 3) expression were initially increased above control on days 1–3, followed by a progressive decline in steady-state protein levels to ~40% of control by day 14. Interestingly, CuZnSOD expression appeared to be elevated in the senescent cells on days 5–10 (Fig. 2), which coincided with declining MnSOD and catalase expression. Increased CuZnSOD expression on days 7–10 was followed by a sharp 80% decline in CuZnSOD levels at day 14 (Fig. 2).

Catalase activity was well preserved in the senescent cells through 10 days in culture (Fig. 4), and catalase activity gels closely matched the level of immunoreactive protein measured by immunoblot (compare Fig. 3 and Fig. 4). However, CuZnSOD activity was declining on days 5, 7, and 10 while protein levels were increasing, suggesting that a portion of the new protein was inactive.

Steady-state Hsc70 and Hsp70 protein levels were measured as indicators of the magnitude of cell stress and the cellular ability to respond to stress. Fig. 5 shows that Hsc70 expression was acutely depressed in hepatocytes from both age groups following 1 day in culture. However, young cells rapidly recovered the ability to synthesize Hsc70, whereas cells from senescent animals did not. Steady-state Hsc70 expression was back to control levels by day 3 in the young cohort, and it was maintained at or near control levels through 14 days. A slight elevation in Hsc70 expression was observed at day 10 in the young cells (Fig. 5), which was concurrent with the decline in cellular AE activity (Fig. 4). In contrast, steady-state Hsc70 expression fell to ~50% of control on day 1 in the senescent group, and to <25% of control on days 3–10. By day 14, Hsc70 levels were barely above the limit of detection on immunoblots.



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  		Figure 5. Aging reduces steady-state Hsc70 protein expression in primary hepatocytes from A, young, and B, senescent rats. Hepatocytes isolated from young rats showed an initial decrease in Hsc70 expression at day 1 in culture, followed by a return to control levels at days 3–14. In contrast, steady-state Hsc70 expression was reduced by ~50% at day 1 in culture and ~75% at day 3 in cells collected from senescent animals. Steady-state levels of Hsc70 protein continued to decline in the senescent cells. By day 14, Hsc70 protein was less than 10% of that observed in controls.

 
A similar trend was observed with Hsp70 (Fig. 6). Steady-state Hsp70 protein expression was maintained at or near control levels for 7 days in culture in the young group. On days 10–14 the young cells responded to declining AE activity with an increase in Hsp70 expression. These results demonstrate that young cells maintained the capacity to constitutively express both Hsc70 and Hsp70, and to respond to declining cellular AE levels with an induction of both proteins. In contrast, Hsp70 expression was not elevated above control levels in the senescent cells at any time point in the study (Fig. 6).



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  		Figure 6. Aging reduces hepatocellular capacity to express Hsp70 in A, young, and B, senescent rats. Hepatocytes isolated from young rats maintained steady-state Hsp70 expression at or near control levels through 10 days in culture. Hsp70 expression was elevated at days 10–14. In contrast, steady-state Hsp70 expression was not increased in cells collected from senescent animals at any time point in the study.

 
Cell Morphology and Cell-to-Cell Interaction
All micrographs presented (Fig. 7Fig. 8Fig. 9Fig. 10Fig. 11) are from old cells. Cells from the young groups had similar morphology (micrographs not presented). Fig. 7 shows light micrographs depicting the typical organization of hepatocytes within the Matrigel matrix. Hepatocytes maintained a rounded morphology that is typical of differentiated cells and formed progressively larger clusters of spherical cells.



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  		Figure 7. Light micrographs of hepatocyte cultures derived from senescent rats. Hepatocytes maintained a rounded morphology and formed progressively larger clusters of spherical cells. A, day 1; B, day 3; C, day 5; and D, day 14 in culture.

 


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  		Figure 8. Electron micrographs of hepatocytes from senescent rats after 1 day in culture. Hepatocytes formed small aggregates of two to three cells. Peripheral plasma membranes had numerous cytoplasmic projections; microvilli-lined lumens were located between cells. Cytoplasmic lipid (L) deposits occurred near the cell periphery. Rough endoplasmic reticulum (RER) was prominent and intact mitochondria (M) and peroxisomes (P) were numerous; magnification 3600x.

 


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  		Figure 9. Electron micrographs of hepatocytes from senescent rats after 3 days in culture. Day 3 cultures displayed many clusters of six to eight hepatocytes with small lumens (SL) between adjacent hepatocytes. Junctional complexes (JC) occurred between the luminal and the lateral plasma membranes of these cells. Individual cells displayed abundant rough endoplasmic reticulum (RER) in lamellar arrays and in association with mitochondria (M); magnification 3600x. Insert: high-magnification gap junction (GJ) in plasma membranes between two hepatocytes; magnification 54,000x.

 


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  		Figure 10. Electron micrographs of hepatocytes from senescent rats following 5 days in culture. Large hepatocyte clusters (>10 cells) were evident. Small lumens (SL) between cells contained microvilli; large lumens (LL) lacked microvilli. Individual cells displayed abundant rough endoplasmic reticulum (RER) often associated with mitochondria (M); magnification 2850x. Insert: high magnification of the plasma membrane between adjacent hepatocytes, illustrating two desmosomes flanking a gap junction (GJ); magnification 37,500x.

 


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  		Figure 11. Electron micrographs of hepatocytes from senescent rats following 14 days in culture. This figure depicts two adjacent clusters of hepatocytes containing large lumens (LL) devoid of microvilli and small lumens (SL) with microvilli; junctional complexes (JC) bordered the small lumens. Autophagic vacuoles (AV) were large and numerous in many of the cells, but the cells still possessed lamellar rough endoplasmic reticulum (RER) and numerous mitochondria; magnification 2700x. Insert: high magnification of a gap junction (GJ) in day 14 culture; magnification 38,000x.

 
Electron micrographs of cells cultured for 1 day (Fig. 8) showed many clusters of two or three hepatocytes possessing cytoplasmic organelles typical of normally functioning cells, including well-defined rough endoplasmic reticulum (RER), mitochondria, and peroxisomes. By day 3 (Fig. 9), hepatocytes were organized into clusters of 6–12 cells surrounding a central lumen devoid of microvilli. Each cluster also contained small microvilli-lined lumens between adjacent hepatocytes that resembled canniculi. The plasma membranes of these lumens were sealed from the extracellular space by junctional complexes, and gap junctions were present in the lateral plasma membranes between adjacent cells (Fig. 9 insert), suggesting that hepatocytes were capable of intercellular communication. Individual cells displayed abundant RER in lamellar arrays, often in association with mitochondria.

At days 5–14 (Fig. 10 and Fig. 11), cells were organized into clusters of 10 or more hepatocytes. Hepatocytes in the day 5 cultures (Fig. 10) contained numerous intact mitochondria, lamellar arrays of RER associated with mitochondria, golgi arrays, and peroxisomes. Lateral plasma membranes displayed desmosomes and gap junctions (Fig. 10 insert) as well as small microvilli-lined lumens. By day 14, very large lumens devoid of microvilli were observed (Fig. 11), and cells contained numerous autophagic vacuoles. However, day 14 cultures still exhibited gap junctions (Fig. 11 insert), RER, intact mitochondria, and peroxisomes. These morphological data are consistent with the biochemical assays (Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6), indicating that basic cell function was maintained during the culture period.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The main findings of the present study indicate that primary hepatocytes collected from young and senescent rats and cultured using Matrigel adopt a differentiated phenotype, establish complex cell-to-cell interactions, and maintain the ability to constituitively express primary AE genes through 14 days in culture. These results are in contrast to previously published data reporting that primary hepatocyte cultures rapidly lose the ability to express AE proteins ex vivo (20).

Our results also suggest that hepatocytes collected from senescent rats may be less competent than cells isolated from younger animals at withstanding the environmental insults associated with disruption of the liver and long-term culture. Hepatocyte cultures from senescent animals were morphologically similar to cultures of younger cells, but they maintained SOD and catalase expression at control levels for 3–5 days as opposed to 7–10 days for the younger cells. The resulting decrement in catalase expression was not offset by a compensatory increase in GPx activity. We speculate that an early imbalance in the SOD/peroxidase ratio may have exposed the senescent cultures to higher intracellular fluxes of H2O2 or lipid peroxides. Progressively increasing numbers of cellular autophagic vacuoles in the senescent cultures support this interpretation.

In addition to declining AE expression, Hsc70 levels fell to ~25% of control in the senescent cells after only 3 days in culture. Neither Hsc70 nor Hsp70 expression was elevated above control levels in the senescent cultures at any time point in the study. In contrast, cells from younger rats increased both Hsc70 and Hsp70 expression coincident with declining cellular AE levels. We interpret these results as evidence that aging reduces cellular capacity to express heat shock protein genes ex vivo. These results are consistent with previous reports suggesting that hepatocytes become less responsive to epidermal growth factor stimulation with age (28), exhibit a lowered proliferative capacity following growth factor stimulation (29), and are less capable of transcriptional activation and expression of the heat shock protein genes (9)(30). On the basis of our previous work (1) and the present data, we speculate that transcriptional activation and expression of the AE genes may also be compromised with age, potentially magnifying the exposure of senescent cells to oxidants and blunting their ability to mount an effective stress response.

It should also be noted that detectable levels of the inducible Hsp70 were observed in the control condition for both young and old cells, which may suggest that hepatocytes cultured in the Matrigel system are under a low level of stress. Culturing primary cells is likely a stressful process, and every attempt was made to minimize stress during each step of the collection and plating process. Once hepatocytes were within the Matrigel matrix, individual cells migrated toward one another and organized themselves into complex multicellular structures requiring cell-to-cell interaction and communication. These observations indicate that cells retained the ability to respond to the rigors of collection and culture. In addition, Hsp70 levels, although detectable, were still quite low in control conditions.

Multiple groups have examined the stress response in aged animals to determine if a deficit in 70-kDa heat shock protein synthesis could explain the loss of stress tolerance in older populations. Heat shock proteins act as molecular chaperones and are intimately involved in cellular protein–protein interactions (31). Constitutive Hsc70 is ubiquitously expressed throughout the cell and is essential for normal cell function (32). The inducible Hsp70 is expressed at low levels under putative nonstress conditions and can be highly induced by environmental stresses such as hyperthermia (16)(33) and exercise (33)(34). Studies using hepatocytes and splenocytes (35), alveolar macrophages (36), and fibroblasts (37) collected from young and old animals have shown that aging lowers heat-inducible Hsp70 mRNA and protein expression. The mechanism responsible for declining Hsp expression appears to involve an age-related loss of transcriptional activation of the heat shock gene by heat shock factor (9)(35)(36). In vivo studies from our laboratory contrasting heat-inducible Hsp70 accumulation in tissues collected from young and senescent heat-stressed Fischer 344 rats have shown that aging significantly blunts Hsp70 expression in the liver (16)(33). Therefore, the present results are consistent with data from many laboratories suggesting that aging compromises cellular ability to upregulate stress protein gene expression.

It is noteworthy that in addition to decrements in Hsp70 expression, senescent hepatocytes showed a marked decline in CuZnSOD activity that appeared to occur independent of decrements in CuZnSOD protein expression. This phenomenon was not observed in cultures derived from young animals, suggesting that aging may not compromise cellular ability to express CuZnSOD, but that a portion of the protein may be inactive. A recent study by Yoo and colleagues (38) reported that cellular expression of CuZnSOD and the 70-kDa heat shock proteins could be induced by heat shock, hydrogen peroxide (H2O2), and by paraquat, an agent known to stimulate intracellular reactive oxygen species production. In addition, these investigators demonstrated that both oxidative stress and heat shock response elements on the promoters of the CuZnSOD and Hsp70 genes were responsive to heat shock, H2O2, and paraquat. These data suggest that CuZnSOD is a stress protein that can be regulated in a manner similar to the heat shock proteins. From the present data and our previous in vivo work (1), we postulate that in addition to lowering cellular capacity to express heat shock protein genes, aging also reduces cellular capacity to increase CuZnSOD activity and thus antioxidant capacity.

In summary, the main findings of the present investigation establish that primary hepatocytes isolated from young and senescent animals and cultured with Matrigel adopt a differentiated phenotype and maintain the ability to express AEs. Although our results suggest that Matrigel may provide primary hepatocytes with an environment that promotes cellular organization closer to that observed in vivo, changes in cellular AE and heat shock protein expression were evident at time points after 7 days ex vivo. Accordingly, we can conclude that the Matrigel system minimizes, but does not eliminate, environmental factors that lead to altered gene expression in cultured cells. However, it is important to note that both AE and heat shock protein expression were maintained at or near control levels for the first 7 days ex vivo, suggesting that an adequate window of time exists for interventional studies using this preparation. With this outcome in mind, we find that the present data show that cells derived from young animals maintained steady-state AE expression at or near control levels longer than cells derived from senescent rats. Moreover, 70-kDa heat shock protein expression is relatively well maintained in hepatocyte cultures from young rats, whereas cultures derived from senescent animals rapidly lose the ability to express the 70-kDa heat shock proteins. These results are consistent with the hypothesis that age-related deficiencies in cellular AE and heat shock protein regulation contribute to declining stress tolerance in the elderly population.


   Acknowledgments
 
This research was supported by National Institutes of Health Grants AG-12350, AG-14687, CA-66081, CA-81090, and T32 AG-00214 (Interdisciplinary Research Training Program on Aging Fellowship to D.M. Hall).

The authors gratefully acknowledge the technical support of Victoria J. Drake, Linjing Xu, and Alana T. Brennan, and the kind gift of Biocoat reagents from Becton Dickinson Labware.

Received July 7, 2000

Accepted January 12, 2001


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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