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,
* Department of Exercise Science,
The Free Radical Research Institute, and
Radiation Research Laboratory, The University of Iowa, Iowa City, Iowa 52242, USA;
§ Department of Internal Medicine, University of New Mexico, New Mexico 87131, USA;
|| Pathology and Laboratory Medicine Service, VA Hospital, Madison, and Department of Pathology, University of Wisconsin Medical School, Madison, Wisconsin 53705, USA; and
Department of Medicine, University of Wisconsin and Veterans Administration Geriatric Research, Education and Clinical Center, Madison, Wisconsin 53705, USA
1Correspondence: Integrative Physiology Laboratory, 532 FH, The University of Iowa, Iowa City, IA, 52242. E-mail: kevin-kregel{at}uiowa.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: free radicals • oxidative stress • stress proteins • HSP70 • aging • antioxidant enzymes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. The
mechanisms underlying these alterations with senescence are not well
defined but likely include modifications in transcriptional regulation
of stress-inducible genes (4)
, inappropriate production of
inflammatory cytokines (5)
, blunted synthesis of
protective acute-phase proteins such as heat shock proteins (1
, 3
, 6)
and, at a more integrated level, multi-organ system
failure (2
, 4)
. One clinically important expression of
this age-associated loss of function is the increase in heat-related
morbidity and mortality rates reported during periodic heat waves in
urban areas (7
8
9)
.
Although mechanisms underlying age-related alterations in stress
responses are not well defined, accumulating evidence supports the
validity of the oxidative stress hypothesis of aging, which suggests
that cellular accumulation of oxidative injury contributes to the
lowered functional capacity in aged organisms (10
, 11)
.
One proposed mechanism for these observations is that the increase in
oxidative stress and subsequent biomolecular damage associated with
aging are the result of an increased rate of reactive oxygen species
(ROS) generation and a greater susceptibility of tissues to oxidative
injury. However, it is not known whether the increased generation of
radicals typically associated with the application of a stress
(12
13
14)
is exaggerated with advancing age and whether
these radicals play a role in the reduced thermotolerance and increased
mortality observed with senescence.
One intervention that has been demonstrated to offset the
age-associated accrual of oxidative injury is caloric restriction (CR)
(11
, 15)
. CR, which involves a reduction in calorie intake
while maintaining adequate nutrition, preserves the activities of
antioxidant enzymes in post-mitotic tissues, maintains organ function,
opposes the development of spontaneous diseases, and prolongs maximum
life span in laboratory rodents (11
, 16
17
18)
. It has been
proposed that reductions in ROS production and cellular oxidative
injury are central to the positive effects of CR. Although extensive
data support this tenet, these experiments were largely conducted in
isolated tissues from nonstressed animals such that little information
exists regarding the ability of CR animals to respond to an acute
stress or adapt to repeated challenges.
We have been investigating the impact of aging on physiological control
mechanisms using stressors such as environmental heating and have found
that the ability to generate protective intracellular stress proteins
is blunted in old compared with young rats (3)
. Consistent
with this observation, we have discovered that with repeated heat
challenge, older rats are markedly less thermotolerant and have
significantly higher mortality rates than their younger counterparts
(2)
, thereby providing an excellent in vivo
model for the high morbidity and mortality rates observed in older
humans (7
8
9)
. Also, we have established that heat stress
increases radical generation and radical-mediated tissue injury in the
splanchnic region of rats (19)
. Low concentrations of ROS
can induce antioxidant enzyme gene expression while high levels can
inactivate antioxidant enzymes and subsequently induce stress protein
gene expression (20)
. The degree to which antioxidant
enzyme and stress protein systems can withstand and respond to stress
is an important determinant of stress tolerance (21)
.
Therefore, the aim of the present study was to determine the effects of
heat stress on mortality rates, radical generation, antioxidant enzyme
responsiveness, and stress protein accumulation in old rats subjected
to CR. Experiments were focused on the liver because it shows
age-dependent evidence of increased cellular ROS production
(22)
and is a prime target of tissue injury during
physiological challenges such as heat stress (23)
and
ischemia-reperfusion (13)
. We hypothesized that long-term
intervention with CR would improve heat tolerance in older animals by
reducing cellular oxidative stress in a critical splanchnic tissue such
as the liver.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Semi-purified diets were obtained from Teklad (Madison, Wis.). Compared
with the diet fed to CON rats, the diet fed the CR rats was enriched in
content of protein (casein), vitamins, and minerals in order to provide
adequate amounts of these nutrients. The compositions of these diets
were previously reported (24)
. The powdered diets were
suspended (1:1) in a 1% agar solution, mixed thoroughly, and allowed
to gel. Initial feedings were 65 kcal/day to CON rats and 51.4 kcal/day
to CR rats (24)
. Over the next 1.5 months, the diets were
gradually reduced to 45.5 kcal/day (CON rats) and to 30.4 kcal/day (CR
rats) such that the CR rats ate 33% fewer calories than the CON rats.
Thereafter, the diets were fed on Tuesdays and Thursdays; each feeding
provided 91.0 kcal/day (CON) or 60.8 kcal/day (CR). On Saturday, each
rat was given either 136.0 kcal (CON) or 91.2 kcal (CR).
At the age of 22–24 months, rats were moved from Madison, Wis., to Iowa City, Iowa, and housed in The University of Iowa Animal Care facility for a minimum of 7 days before experimentation. Rats from each cohort, CON and CR, were fed as in Madison and randomly assigned to heat stress (n=8) or sham (n=5) groups. An additional cohort of normally-fed Lobund-Wistar rats (4 months old, n=5) was used as young, euthermic controls. Animals were handled daily and familiarized with a colonic temperature (Tco) probe before testing. All procedures and experiments were performed in accordance with institutional animal care guidelines.
Experimental procedures
Experiments were performed at midday between 1000 and 1400,
48 h after the last feeding. Rats were fitted with a thermistor
temperature probe (Yellow Springs Instruments, Yellow Springs, Ohio)
inserted 6–7 cm into the colon and then placed in plastic cages
(45x25x20 cm), conscious and unrestrained. Tco
was monitored continuously on a digital display and an analog output.
After a control period of 30 min, in which baseline
Tco was similar between the groups (37–38°C),
CON and CR rats were heat stressed as described previously
(6)
. An infrared lamp was placed ~40 cm above the animal
and raised or lowered to achieve a constant rate of rise in
Tco. Ambient temperature
(Ta) ranged from 38 to 44°C inside the cage.
The infrared lamp was switched off when Tco
reached 41°C. Tco was then maintained at 41°C
for 30 min by periodically resuming heating. At the end of this period
heat exposure was terminated and the rat was placed in a clean cage
(Ta of 25°C) for recovery. Rats subsequently
underwent a second identical heat exposure and recovery protocol
24 h later. During the heating protocol, heating rates
(~0.04°C/min) and time to reach 41°C (~80 min) were matched for
each heating trial and between heating trials on day 1 vs. day 2 within
each group. Weight-matched euthermic controls (shams) from the CR and
CON groups were handled identically to their heat-stressed counterparts
except that the sham rats were maintained at a Ta
of 24–26°C during the experimental periods. The young normally fed
euthermic control group underwent an equivalent 2-day sham protocol.
Forty-eight hours after the second heat exposure (i.e., day 4), mortality was recorded and surviving rats were administered an overdose of sodium pentobarbital (100 mg/kg i.p.) followed by transcardial perfusion with cold phosphate buffered saline (PBS). Liver biopsies (lobi caudatus) were then collected and immediately placed in ice-cold PBS. Portions of each tissue were processed for quantitation of the 70 kDa heat shock protein (HSP70), assessment of radical species via low temperature electron paramagnetic resonance (EPR) spectroscopy, light microscopy characterization and grading of injury severity, and immunohistochemical quantitation of immunoreactive antioxidant enzyme expression and subcellular localization. The same process was followed on death of a rat that did not survive the 4-day experimental protocol. These rats were closely monitored throughout the protocol and tissue samples were obtained before death.
HSP70 protein immunoblots
Liver and intestinal samples were assayed for constitutive plus
inducible HSP70 protein as described previously (3)
.
Proteins were quantitated by the Bradford method, then serial dilutions
of the proteins were separated by 1-dimensional gel electrophoresis,
transferred to nitrocellulose, and probed with a monoclonal antibody
specific to both the constitutive (HSP73) and inducible (HSP72) form of
HSP70 (N27, Stressgen, Victoria, BC, Canada). Protein immunoblots were
scanned with a flatbed scanner and analyzed with commercially available
software (NIMH Image 1.35). To ensure uniformity, density
determinations were only made among bands on the same immunoblot.
EPR spectroscopy
Biopsies from the liver were cleaned of blood, placed in a 2.5
mm length of TeflonTM tubing (3 mm i.d.), and stored in liquid
nitrogen. To evaluate radical content, a sample was removed from the
tubing and placed in a Dewar of liquid nitrogen in the spectrometer
cavity. EPR spectra were recorded with a Bruker ESP 300 EPR
spectrometer (Bruker Instruments, Karlsruhe, Germany) equipped with an
ER-036M gaussmeter, ER4111VT variable temperature unit, and EIP-625A
microwave frequency counter as described previously (19)
.
Signal averaging (multiple scans of the same sample) was used to
improve the signal-to-noise ratio. Sample volume and geometry were kept
constant to allow direct comparisons of relative radical concentration
among samples. All spectra were collected at 77 K with data reported as
the normalized average of 20 scans. EPR settings included a receiver
gain of 5.00 x 105, a modulation frequency
of 100 kHz, a modulation amplitude of 4.0 G, a microwave frequency of
9.43 GHz, a microwave power of 10 mW, and a scan rate of 6.2 G.5.
Immunohistochemistry and histology
Liver and intestine samples were prepared for
immunohistochemical and histological evaluation as described previously
(25)
. Triplicate slides were prepared for each stain.
Normal rabbit serum or preimmune serum controls were run for each
antibody tested. These controls were uniformly negative.
Histology sections were analyzed microscopically for evidence of
injury. In addition, sections were incubated with antibodies specific
for manganese-containing superoxide dismutase (MnSOD), copper-zinc SOD
(CuZnSOD), and catalase to assess immunoreactive protein levels. The
specificity of these antioxidant enzyme antibodies has been previously
described (25)
. All sections were graded by the same
reviewer, who was blinded to their origin. Liver injury was graded
using scales modified from Chui et al. (26
, 27)
.
Immunoreactive protein levels were graded using an intensity of
staining scale developed by Oberley et al. (28)
ranging
from 0 (negative) to 4+ (strongly positive).
Data analyses
Results were presented as means ± SE.
Statistical comparisons between Tco values,
heating rates, and liver HSP70 levels were determined with an analysis
of variance for two factors (i.e., experimental intervention and
treatment group) design. Post hoc comparisons were made with
Duncan’s multiple range test. The effect of treatment (CR vs. CON) on
survival was tested statistically by performing multiple linear
regression with a computer spreadsheet program (Microsoft Excel). Dummy
variables were used as independent variables to encode the treatment
effect (diet) and to account for inter-individual variability among
rats (29)
. Differences were considered significant at the
P < 0.05 level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HSP accumulation
Densitometric analyses of immunoblots from tissues probed for
constitutive plus inducible HSP70 demonstrated that HSP70 accumulation
in the liver of heat-stressed animals was markedly influenced by the CR
intervention (Fig. 1
). In the CON group, heat stress stimulated a 142% increase in liver
HSP70 levels vs. sham controls (123±6 vs. 51±6 units,
P<0.001). In contrast, there was no change in liver HSP70
accumulation in heat-stressed compared with sham CR rats (71±5 vs.
59±3 units). Comparing both heat-stressed groups, HSP70 levels were
74% higher in CON vs. CR rats (P<0.005). HSP70 levels were
similar in the CON and CR sham groups.
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EPR
Low temperature (77 K) EPR spectra collected from liver of rats
from all groups showed evidence of multiple species, including both
transition metal and organic radicals (Fig. 2
). A major component of these spectra is a strong six-line feature
centered at g = 2.03 with a hyperfine splitting
constant of 
90 G that is consistent with immobilized manganese(II).
For comparison, we show a protein-manganese(II) spectrum collected from
E.coli MnSOD treated with sodium hydrodisulfite, a reducing
agent (Fig. 2F
). Note the similarities between
spectrum F and spectra A–D; however, superimposed on the manganese(II)
spectrum are additional features at g = 2.25, 2.005,
and 1.935. The relatively narrow line at g = 2.005
(
Hpp 10–15 G) is highly responsive to microwave power; the
intensity of this line decreases relative to all other spectral lines
with increasing microwave power (data not shown). This response,
coupled with the g value and line width, is consistent with
the g = 2.005 species being an organic radical, such as
a semiquinone radical (30)
. The broad features at
g = 2.25 and 1.935 are indicative of iron(III),
possibly from mitochondrial succinate dehydrogenase (31)
.
|
Contrasting the EPR signals from liver (Fig. 2)
of young
(A), CR (B), and CON (D) groups under
nonstress conditions showed remarkable similarity between the young and
CR animals (A vs. B) but elevated radical levels,
both metal and organic, in the old CON cohort (B vs.
D). Heat stress marginally increased the concentration of
semiquinone radical in liver from old CON rats and did not alter
transition metal levels. In contrast, heat stress and recovery markedly
reduced the level of both metal and organic radicals in liver from CR
animals.
Histology and immunoreactive antioxidant enzyme expression in the
liver: euthermic controls
Under euthermic conditions, hepatocellular antioxidant enzyme
expression and injury profiles were similar for young and CR rats. In
contrast, CON animals showed clear evidence of injury and increased
antioxidant enzyme expression prior to heat stress. Hematoxylin and
eosin stained liver sections from young sham and old CR sham rats
showed normal morphology. Similar samples from old sham CON rats
contained extensive evidence of diffuse hepatic fatty changes and mild
cell injury that was expressed as pyknotic nuclei and cytoplasmic
vacuolization (grade 1; data not shown).
Figure 3
depicts immunoperoxidase staining for catalase of liver sections from
representative rats. Immunohistochemical determination of
hepatocellular MnSOD, CuZnSOD, and catalase protein expression and
subcellular localization in euthermic control animals (Fig. 3E
) showed striking evidence of gradients of
antioxidant enzyme expression within hepatic acini of rats from all
groups. The intensities of staining for immunoreactive MnSOD, CuZnSOD,
and catalase were high (3+)
in central vein regions (zone
3 hepatocytes) and lower in centrilobular areas (1+/2+) in zone 1 and
zone 2 cells (Table 1
).
|
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CuZnSOD and MnSOD levels and cellular distributions were similar in
young control and CR rats, but MnSOD and CuZnSOD expression was
increased in acinus zone 2 and zone 3 hepatocytes of CON rats (Table 1)
. In addition, moderate (2+/3+) nuclear expression of both CuZnSOD
and catalase was observed in zone 2 and 3 hepatocytes in the CON group
(Table 1
, Fig. 3A
), while nuclear CuZnSOD and catalase
expression was not seen in the young group and only rarely observed in
zone 3 hepatocytes from CR rats.
Histology and immunoreactive antioxidant enzyme expression in the
liver: heat stress
Histological evaluation of sections from heat-stressed CR rats
revealed normal to mild hepatocyte damage that was expressed as mild
hepatocyte vacuolization (grade 1). In contrast, moderate to severe
injury (grade 3) was observed in samples from heat-stressed CON rats
that included widespread cytoplasmic hepatocyte vacuolization with
pyknotic nuclei, evidence of membrane loss, and sinusoidal congestion
(data not shown).
Both CR and CON rats showed evidence of hepatocellular MnSOD, CuZnSOD,
and catalase induction in zone 2 and zone 3 hepatocytes during recovery
from heat stress (Table 1)
. However, induction of protein was
heterogenous in CON tissues and cells showed no consistent change from
euthermic control levels. In contrast, uniform induction of antioxidant
proteins combined with striking evidence of nuclear catalase expression
was observed in zone 2 and zone 3 hepatocytes of the CR group (Fig. 3D
; Table 1
). Nuclear catalase staining intensity was 4+ in
all 8 CR animals after heat stress, suggesting a consistently large
increase in protein concentration. In contrast, there was no
heating-induced increase in nuclear catalase protein above euthermic
sham conditions for CON rats.
Cytoplasmic CuZnSOD levels were elevated in zone 3 hepatocytes and
nuclear CuZnSOD expression was prominent in zone 2 and 3 cells of
heat-stressed CON animals. Conversely, cytoplasmic levels of CuZnSOD
were increased only in zone 3 cells, and nuclear expression of CuZnSOD
was not observed in CR rats (Table 1)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CR slows the rate of progression of senescence and alters the
phenotypic changes of aging such that oxidative injury is reduced,
suggesting that lowered cellular oxidative stress may cause the
positive effects of CR (11
, 15)
. Consistent with this
concept, liver morphology and cellular antioxidant enzyme profiles of
sham (i.e., euthermic) CR rats in the present study were remarkably
similar to that of young animals. There was consistent evidence of
uniform gradients of antioxidant enzyme protein expression within
hepatic acini in both groups. In addition, we observed only limited
evidence of nuclear catalase expression in the CR group, and neither
young nor CR rats showed significant evidence of hepatocellular injury
or stress under sham conditions. In contrast, liver biopsies from CON
rats showed widespread centrilobular necrosis and hepatocellular
injury. Moreover, gradients of antioxidant enzyme proteins were much
less evident as centrilobular antioxidant enzyme expression was higher
in CON than CR rats. We also observed widespread centrilobular nuclear
CuZnSOD and catalase expression in hepatocytes adjacent to injured
tissue. Taken together, these observations establish that long-term CR
protects against age-related liver pathology and suggest that chronic
cellular oxidative stress may be an important mediator of
hepatocellular injury.
After heat stress, MnSOD and catalase were strongly induced in central vein and centrilobular regions of CR rats. Extremely high levels of nuclear catalase were also observed in a large majority of hepatocytes in these areas. Not surprisingly, liver radical content was lower in the CR group. In contrast, the magnitude of zone 2 and zone 3 cellular antioxidant enzyme induction was reduced in CON rats and there was no increase in nuclear catalase expression above sham levels. Radical levels were increased in liver samples of CON rats in recovery from heat stress. We interpret these data as evidence that CR blunted cellular stress and injury by preserving the capacity to respond to heat challenge with appropriate antioxidant enzyme expression. Although we cannot discount a CR-mediated decrease in ROS production, the fact that we observed such a strong induction of antioxidant enzymes suggests that levels of their respective ROS substrates are also increased.
We have previously reported that heat stress stimulates the
generation of both ROS and reactive nitrogen species in intact animals
(19)
. In the present study, EPR spectra from liver showed
the presence of iron(III), manganese(II), and semiquinone. Heat stress
marginally increased semiquinone radical levels in the CON group but
reduced the concentration of EPR-detected transition metals and
semiquinone radical in the CR rats. The Mn2+
oxidation state is the most prevalent for manganese in biological
systems (32)
. The concentration of
Mn2+ in rat hepatocytes ranges from 0.25 to 0.70
µM (33)
, and hepatic tissues have high levels of the
manganese-containing metalloenzymes MnSOD, arginase, and
serine/threonine protein phosphatase-1 (34)
. Thus, the
strong Mn2+ EPR signal obtained in liver samples
in the current study was not unexpected. However, the observation that
heat stress reduced metal and organic radical production in CR animals
is a provocative result worthy of further study. One explanation is
that metal-catalyzed oxidative stress is important in the hepatic
pathology that we observed after stress (12)
.
Alternatively, the CR group may have responded to the stress of
hyperthermia earlier in the post-heating period such that this
adaptation was not measurable at the 48 h point.
Another interesting result from this project is the observation
that heat stress produced strong nuclear catalase expression in the CR
rats but stimulated no additional increase above euthermic levels in
the CON animals. It has previously been reported that hyperthermia
induces CuZnSOD activity in vitro and in vivo in
rat lung (35)
. In addition, induction of MnSOD activity
has been observed in E. coli B exposed to heat shock
(36)
. However, our current results are the first
demonstration in a eukaryotic system of increased total cellular
catalase levels following a hyperthermic challenge as well as the first
evidence in any system of stress-induced nuclear catalase
translocation. These data are of interest in light of the proposal that
the age-related increase in cellular oxidative stress and injury could
be in part a result of declining antioxidant enzyme activities
(10)
. Though some studies do not support this idea
(37)
, the experimental approaches that have been used to
date have not examined compartmentalized alterations in cellular
antioxidant status with aging, nor have they investigated
stress-inducible antioxidant enzyme expression. The present data show
clear age-related and diet-dependent differences in hepatic antioxidant
enzyme expression and subcellular compartmentalization. For example, in
the CR group, catalase expression was prominent in a majority of
hepatocyte nuclei, including both the centrilobular and periportal
regions, 48 h after the second heat exposure. Concomitantly, liver
damage in these same animals was negligible. In contrast, the CON group
appeared unable to mount an appropriate adaptive antioxidant enzyme
response after heat stress as evidenced by the unchanged nuclear
catalase expression from euthermic sham conditions at the 48 h
time point. Massive, widespread liver injury and significant mortality
were also observed in CON rats. We postulate that this pattern of
antioxidant enzyme expression and translocation plays a critical role
in determining survival of senescent animals during a chronic physical
challenge.
In support of our findings that the increases in cell stress produced
with heating in the CON group were sharply reduced in CR animals, HSP70
accumulation in the liver also differed between these two groups. Data
from several laboratories, including our own (3
, 6)
, show
that HSP70 levels increase in response to heating, suggesting that this
protein may serve as an index of the magnitude of cellular stress.
Consistent with past results, the current data support the concept that
the hyperthermic challenge used in these experiments produced less
cellular stress in rats that had undergone CR than their CON
counterparts. Thus, mortality, ROS, injury, antioxidant enzyme, and
stress protein outcomes provide strong evidence at the whole organism
and cellular levels supporting the conclusion that CR attenuates the
age-related decline in heat tolerance.
In summary, the present data show that long-term CR in rats protects the liver from the pathogenesis of normal age-related injury, as well as providing significant protection from the cytotoxic effects of heat stress. Increased thermotolerance of CR animals was associated with robust stress-inducible primary antioxidant enzyme expression and nuclear translocation of catalase, suggesting that the ability to synthesize antioxidant enzymes in response to stress in an important adaptive response to an environmental challenge such as heat stress.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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D. M. Hall, G. L. Sattler, C. A. Sattler, H. J. Zhang, L. W. Oberley, H. C. Pitot, and K. C. Kregel Aging Lowers Steady-State Antioxidant Enzyme and Stress Protein Expression in Primary Hepatocytes J. Gerontol. A Biol. Sci. Med. Sci., June 1, 2001; 56(6): B259 - B267. [Abstract] [Full Text] [PDF]
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D. M. Hall, G. R. Buettner, L. W. Oberley, L. Xu, R. D. Matthes, and C. V. Gisolfi Mechanisms of circulatory and intestinal barrier dysfunction during whole body hyperthermia Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H509 - H521. [Abstract] [Full Text] [PDF]
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D. M. Hall, L. Xu, V. J. Drake, L. W. Oberley, T. D. Oberley, P. L. Moseley, and K. C. Kregel Aging reduces adaptive capacity and stress protein expression in the liver after heat stress J Appl Physiol, August 1, 2000; 89(2): 749 - 759. [Abstract] [Full Text] [PDF]
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