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(Received for publication, August 17, 1995; and in revised form, November 13,
1995) From the
Dietary calorie restriction (CR) delays age-related physiologic
changes, increases maximum life span, and reduces cancer incidence.
Here, we present the novel finding that chronic reduction of dietary
calories by 50% without changing the intake of dietary protein induced
the activity of mouse hepatic carbamyl phosphate synthetase I (CpsI)
5-fold. In liver, CpsI protein, mRNA, and gene transcription were each
stimulated by CpsI ( The enzyme is coded for by
a single copy nuclear gene(5, 6) . The gene is
regulated cell-type specifically, developmentally, nutritionally, and
hormonally. In the liver, the enzyme and its mRNA vary with the level
of dietary protein(7, 8) . In rats, cpsI precursor RNA and mRNA are induced 3-fold by isocaloric diets
containing 20% versus 4% protein(9) . Increased plasma
glucagon concentrations (increased intracellular cAMP) have been shown
to directly induce the level of cpsI mRNA(10, 11, 12, 13) .
Glucocorticoids also stimulate cpsI mRNA in the liver (13, 14) . This glucocorticoid response is reduced
about 50% by insulin in hepatoma cells in culture(15) .
Epinephrine reduces the rate of CpsI synthesis in isolated rat
hepatocytes(16) . An attractive aspect of cpsI for
studying the effects of nutrition on life-span is that enzyme activity
and protein content do not vary with age in rodents, simplifying the
analysis(17) . Intestinal expression of the gene is not
nutritionally or hormonally regulated, making possible cell
type-specific studies of its regulation(11) . The
transcription factors and cis elements mediating the cell-specific,
hormonal, and nutritional regulation of cpsI expression are
not well characterized. Six sequence elements proximal to the
transcription initiation site are specifically bound by liver nuclear
factors and by bacterially expressed C/EBP CR delays most
age-related physiologic changes and increases both mean and maximum
life span in every model system tested(21) . It is the only
method known for extending life span in homeothermic vertebrates and
the most effective means known for reducing cancer incidence and
increasing the mean age of onset of age-related diseases and tumors (21, 22) . CR reduces sustained plasma glucose
concentrations, and this leads to reduced intracellular glucose
concentrations in hepatocytes (23, 24, 25, 26) . Liver glucose
transport is mediated mostly by the GLUT2 glucose
transporter(27) . It is present in hepatocytes, pancreatic
Although calorie-restricted mice (CR mice) have lowered
plasma glucose concentrations, they are not stressed. They are
healthier than mice fed ad libitum(21) . CR mice are
not starving. The diet is formulated so that the animals are not
limited for any nutrient(26) . Glucose transporter 1 mRNA is
induced In this report, we present the novel finding that reduction of
dietary calories by
Figure 1:
CR induced CpsI protein levels and
enzyme activity in liver. Panel A, total SDS soluble mouse
liver protein resolved by SDS-polyacrylamide gel electrophoresis. Odd-numbered lanes represent proteins from AL mice. Even-numbered lanes represent protein from CR mice. The arrow labeled CPSI indicates the position of the
protein, and the numbers to the left of the figure
indicate the positions of standards in kilodaltons. Panel B,
quantitation of the relative level of CpsI protein present. The means
and standard deviations are shown for samples from four AL and four CR
mice. Panel C, CpsI activity present in livers from each
dietary group. The means and standard deviations are shown for four AL
and four CR mice.
Hepatic CpsI levels in AL and CR mice (odd- and even-numbered lanes, respectively) are
shown in Fig. 1A. Because CpsI is a highly abundant
protein and of an unusually large size, it can be visualized directly
by dye binding. The differences in the level of CpsI cannot be
accounted for by differences in the amount of protein loaded in each
lane. When the level of CpsI was quantified and corrected for the total
amount of protein present in each lane, CpsI was induced approximately
3-fold (p < 0.01; Fig. 1B). The staining of
CpsI was linear with respect to protein over the range of
concentrations present in this study (data not shown). The data
shown in Fig. 1A also illustrate that the effect of CR
on CpsI levels is highly specific. Of the
Figure 2:
CR induced hepatic cpsI mRNA. Panel A, hepatic cpsI mRNA is induced by CR. Total
RNA isolated from AL (lanes 1-4) and CR (lanes
5-8) mice was subjected to Northern blot analysis. The
results of probing the blot with radiolabeled cpsI cDNA
sequences are shown. The arrow labeled CPSI indicates
the position of the 6.2-kb mRNA, and the numbers to the left of the figure indicate the positions of RNA molecular
weight standards in kilobases. Panel B, the level of 18 S rRNA
present in each lane of the blot shown in panel A. Panel
C, the levels of transcription factor S-II mRNA present in each
lane of the blot in panel A. Panel D, the means and
standard deviations of the cpsI mRNA levels in livers of four
mice from each dietary group are illustrated. To control for RNA
loading and transfer, cpsI mRNA was normalized to the level of
18 S rRNA.
Figure 3:
CR did not change the stability of hepatic cpsI mRNA. Shown is the level of hepatic cpsI mRNA
present at various times after treating animals with actinomycin D. The
results obtained using CR (open symbols) and AL (closed symbols) mice
are displayed. cpsI mRNA levels were determined by dot blot
analysis and normalized to the level of 18 S rRNA present. Each point
and error bar represents the mean and standard deviation obtained using
3 animals. For the zero time point, the error bar pointing up is for AL
mice, and the bar pointing down for CR
mice.
Figure 4:
Intestinal CpsI activity but not mRNA
responded to CR. CpsI activity and mRNA were measured in the livers and
small intestines of AL and CR mice. Each panel represents the
mean and standard deviation of determinations from three animals in
each dietary group. Panel A, intestinal CpsI activity levels
are shown. Panel B, hepatic CpsI activity levels are shown. Panel C, intestinal cpsI mRNA levels are shown. Panel D, liver cpsI mRNA levels are
shown.
To determine whether the increase in CpsI activity
in the intestine is accompanied by a change in cpsI mRNA, dot
blots were used to quantify intestinal and liver cpsI mRNA in
CR and AL mice (Fig. 4, C and D). cpsI mRNA levels were 3- and 6-fold lower in intestine than in liver of
CR and AL mice, respectively. There was no statistically significant
change in the level of intestinal cpsI mRNA, while the level
of cpsI mRNA in the liver was stimulated approximately
2.5-fold in these mice (Fig. 4D). These results suggest
that the enzyme is translationally regulated or post-translationally
modified to a more active form in the small intestine of CR mice.
To
investigate the possibility that the 40% difference in protein
consumption induced the gene, another study was performed. Two groups
of mice of approximately equal weights were fed ad libitum and
CR. After only 1 week, cpsI mRNA levels were twice as high in
the CR mice (p < 0.001), even though protein consumption
per gram (body weight) was 10% lower in the CR group (Table 2).
These results are consistent with those of the long term diet studies,
suggesting that cpsI gene expression is induced by reduction
of dietary calories and not by changes in the amount of protein
consumed. Thus, protein metabolism and cpsI gene expression
adjust rapidly to shifts in the amount of calories consumed. In the studies reported here, we present the novel finding
that chronic 50% reduction in dietary calories, without a change in
dietary protein, led to a specific, statistically significant 3-fold
induction of a 160,000 molecular weight hepatic protein we have
identified as CpsI. To better understand the basis for nutritional
regulation of gene expression, we investigated the effects of CR on key
steps in the expression of the cpsI gene. The increased level
of CpsI was accompanied by a statistically significant 5-fold induction
of the enzyme activity. Hepatic cpsI mRNA levels and
transcription were both induced by The mechanism by which CR
regulates cpsI transcription is not known yet. cpsI mRNA levels are induced by glucagon and glucocorticoids,
suggesting that CR-induced changes in the levels of one or both of
these hormones might be responsible. However, serum glucagon
concentrations are not altered by CR in rats or
mice(21, 46) . Thus, glucagon regulation of
intracellular cAMP levels is not a likely source of the change in the
rate of cpsI transcription. The effects of CR on
glucocorticoid levels are more complex(47) . The mean 24-h
plasma total corticosterone concentrations of AL and CR rats are
similar in younger animals. As the animals age, there is a modest rise
in the mean 24-h plasma total corticosterone concentrations in AL mice.
However, there is also a decline with age in the level of
corticosterone binding globulin in CR animals, resulting in a gradual
increase with age in mean 24-h free corticosterone concentrations and
in the daily circadian peaks of free corticosterone. The possible
effects of free, total, mean, and circadian peak concentrations of
glucocorticoids on cpsI gene transcription are unclear.
However, it is difficult to see how small changes such as a 25%
increase in mean 24-h plasma corticosterone concentrations in AL mice
could result in 3-fold inhibition of cpsI transcription. Growth hormone suppresses CpsI activity in vivo, and serum
growth hormone concentrations are reduced Insulin and epinephrine both suppress CpsI
synthesis in primary cultured hepatocytes and Reuber hepatoma H-35
cells, and their effects are additive(16) . Blood epinephrine
levels do not change with CR in rats(50) . Insulin may act by
suppressing glucocorticoid stimulation of cpsI mRNA by
50%(15) . CR does decrease serum insulin
levels(46, 51, 52) . However, it is not clear
whether this decrease in insulin could produce the 3-fold induction of cpsI transcription found in the studies reported here. Thus,
at this time we are unable to suggest a known regulatory signal that is
likely to be responsible for the change in the rate of cpsI transcription found in CR mice. Isocaloric diets containing 20% versus 4% protein increase hepatic cpsI precursor RNA
and mRNA by The relative levels of cpsI mRNA in liver and intestine were similar to those
reported by others(2) . Also in agreement with others, we found
no regulation of cpsI mRNA levels in the small
intestine(11) . However, we did find induction of CpsI activity
in the small intestine of CR mice. This result is novel, and it
suggests that the CpsI is translationally regulated or the specific
activity of the enzyme is enhanced post-translationally. We
consistently find that cpsI mRNA and protein are increased
3-fold by CR in liver, while the activity of the enzyme increases
5-6-fold ( Fig. 1and Fig. 4). Thus, hepatic CpsI
appears to be post-translationally modified and we believe that CpsI is
regulated similarly in the small intestine. The increase may be a
response to higher ammonia production by the luminal bacteria of the
intestine. These bacteria may catabolize more protein due to the lower
carbohydrate intake of CR mice. The total protein synthetic activity
of liver and other tissues decreases with age in organisms as diverse
as insects and humans (reviewed in (31) ), but the rate of
synthesis of some proteins remains unchanged while the synthesis of
others even increases. The effects of aging on protein degradation are
not as well described. However, since protein synthetic activity
decreases with age while the total protein content of cells and tissues
remains constant, the rate of protein degradation is thought to decline
with age. The ability of cells to degrade structurally aberrant
proteins appears to decrease with age(31) . Dietary restriction
reduces this age-related decline, increasing the rate of protein
turnover(54, 55, 56, 57) . Enhanced
protein turnover leads to increased levels of metabolic nitrogen. Thus,
the increase in hepatic CpsI activity in CR animals is likely to result
from both increased catabolism of dietary protein for the generation of
metabolic energy and enhanced turnover of cellular proteins. The
ammonia produced by protein catabolism is highly neurotoxic, playing a
role in pathologies such as hepatic encephalopathy and perhaps
Alzheimer's disease(58, 59) . The 5-fold
induction in CpsI activity during CR is likely to reduce the level of
free ammonia in blood and therefore to decrease the level of brain
ammonia. However, it is presently unclear how the increased metabolic
capacity for ammonia detoxification compares to the increase in ammonia
production from protein catabolism. The increase in CpsI found in CR
animals suggests an increase in urea production and the ability to
handle ammonia and HCO
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3500-3506
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
3-fold. Thus, CR increased both the rate of gene
transcription and the specific activity of the enzyme. Short-term
feeding studies demonstrated that higher cpsI expression was
due to CR and not consumption of more dietary protein. Intestinal CpsI
activity was stimulated 2-fold, while its mRNA level did not change,
suggesting enzyme activity or translation efficiency was stimulated.
CpsI catalyzes the conversion of metabolic ammonia to carbamyl
phosphate, the rate-limiting step in urea biosynthesis. cpsI induction suggests there is a shift in the metabolism of
calorie-restricted animals toward protein catabolism. CpsI induction
likely facilitates metabolic detoxification of ammonia, a strong
neurotoxin. Enhanced protein turnover and metabolic detoxification may
extend life span. Physiologic similarities between calorie-restricted
and hibernating animals suggest the effects of CR may be part of a
spectrum of adaptive responses that include hibernation.
)is specifically expressed in hepatocytes and
epithelial cells of the intestinal mucosa. It is localized in
mitochondria, where it catalyzes the condensation of metabolic ammonia
and HCO
to carbamyl phosphate, the first
step in the urea cycle in the liver(1, 2) . CpsI is an
abundant protein, comprising approximately 4% of liver
protein(3, 4) . CpsI levels are approximately 10 times
lower in the small intestine(2) .
, and one of these sites
is required for activation of the promoter by overexpressed C/EBP
in transfected cells(18) . Three of the six sites appear to be
bound by liver-specific factors(19) . More recently, four sites
were shown to play roles in the expression of the gene. A direct repeat
adjacent to the TATA homology activates transcription, while two other
sites repress this activation. A fourth site appears to obviate the
effects of the two negative sites(20) .
cells, and specialized regions of the plasma membrane of a few
other cell types. Because transport through GLUT2 is symmetric, the
flux of glucose is directly proportional to extracellular and
intracellular glucose concentrations(28, 29) . The
decrease in intracellular glucose concentration is likely to affect the
expression of some hepatic genes. We have found that CR results in the
liver-specific, negative, post-transcriptional regulation of the gene
for glucose-regulated protein 78(26, 63) . (
)3-fold in rat hepatocytes by starvation(30) . We
have found that this mRNA is not regulated by CR in the liver, adipose,
brain, heart, kidney, lung, muscle, or intestine of mice.
50%, without changing the intake of dietary
protein, induced the level of hepatic CpsI activity, protein, mRNA, and
gene transcription by 3-5-fold while having no detectable effect
on the stability of the mRNA. Our results suggest that CR increases
protein catabolism, probably for gluconeogenesis. The change in cpsI mRNA also occurred with a short-term shift from CR to ad libitum feeding, indicating that the change in metabolism
responsible for gene induction is relatively rapid. Ammonia is a toxic
end product of protein catabolism, and CpsI is the rate-limiting enzyme
for the metabolic detoxification of ammonia. Whether enhanced protein
catabolism and ammonia detoxification are related to the many
beneficial effects of CR is unclear at present. However, enhanced
protein turnover and enhanced metabolic detoxification have been
postulated to have roles in life span
extension(31, 32) . There are also physiologic
similarities between CR and hibernating animals which suggest that the
effects of CR are part of the adaptive responses that include
hibernation.
Animals
Females of the long-lived F hybrid strain C3B10RF have been used by us previously
for studies of CR (e.g.(26) ). The mice are bred from
C57BL10.RIII and C3H.Sw/Sn lines obtained from Jackson Laboratories.
Mice were weaned and started on the diets at 28 days of age. They were
housed individually and subjected to one of the two diet regimens
described below. Mice were maintained at 20-24 °C and
50-60% humidity with lights on from 0600 to 1800 hours. Animals
had free access to water. Sentinel mice were kept in the same room as
the experimental mice, and serum samples were screened every 6 months
for titers against 11 common pathogens. No positive titers were found
during these studies. Three cohorts of mice were utilized. A cohort
consisting of eight 21-month-old mice in each dietary group was
utilized for hepatic transcription run-on assays, mRNA analysis, and
determining CpsI protein levels. Another cohort, consisting of ten
6-month-old CR mice, was used for a study of short-term dietary
effects. Five CR mice were fed ad libitum for 1 week while
five remained calorie restricted. A third cohort, consisting of twelve
24-month-old mice in each dietary group, was utilized for studying cpsI mRNA half-life using actinomycin D. Mice were killed by
cervical dislocation, and their livers and small intestines were
removed. The intestines were gently flushed with phosphate-buffered
saline before use (Flow Laboratories, McLean, VA).Diets
The composition of the diets was as
described(26) . The mice were fed and maintained as
described(33) . Both dietary groups ate a purified diet
containing all protein, fat, vitamins, and minerals. The ad
libitum-fed mice (AL mice) consumed between 100 and 110 kcal per
week, and the 50% CR mice consumed 49 kcal per week.Determination of CpsI Levels
Approximately 25 mg
of liver tissue was sonicated for 8 s at a setting of 3 (Branson model
350 sonifier cell disrupter) in 300 µl of 50 mM Tris (pH
6.8), 5% -mercaptoethanol, 2% SDS, and 10% glycerol. Debris was
removed by centrifugation in a Beckman microfuge at 16,000 
g, and protein concentrations were determined(34) .
Each sample (15 µg) was subjected to SDS-polyacrylamide gel
electrophoresis on a 5% gel(35) . The level of the 160,000
molecular weight CpsI band was quantified by scanning the dried gel
with a light densitometer (E-C Apparatus Corp., St. Petersburg, FL).
Measurement of CpsI Activity
Approximately 20 mg
of liver tissue was sonicated three times for 1 s each with cooling in
an ice bath, at a setting of 3 (Branson model 350 sonifier cell
disrupter) in 380 µl of ice-cold water. Debris was removed by
centrifugation in a Beckman microfuge at 16,000 g for
10 min at 4 °C. CpsI activity assays were performed and units were
calculated as described (36) . The assay was linear with
respect to input liver homogenate over the range of activities
measured.RNA Isolation and Visualization
Approximately 0.2
g of frozen liver tissue was homogenized for 30 s in 4 ml of TRI
reagent (Molecular Research Center, Inc., Cincinnati, OH) using a
Tekmar Tissuemizer (Tekmar Co., Cincinnati, OH) at a setting of 55. RNA
was isolated using TRI reagent as described by the supplier. RNA was
resuspended in FORMAzol (Molecular Research Center, Inc.). Northern and
dot blots were performed as described using 20 and 10 µg of RNA,
respectively(37, 38) . To quantify specific mRNA,
blots were serially probed. cDNA probes were labeled with
[-P]ATP to a specific activity of 1
10
cpm/µg by using a multiprime labeling kit (Pharmacia
Biotech Inc.). The cpsI probe was a 1.2 kb PstI and EcoRI fragment excised from pHN3491 (ATCC/National Institutes
of Health Repository), a plasmid vector containing the rat cpsI cDNA(5) . Mouse transcription factor S-II cDNA was
purified from the vector in a similar manner(39) . Blots were
also probed with a synthetic oligonucleotide complementary to mouse 18
S rRNA (40) and with oligo(dT) (Pharmacia Biotech Inc.) 5`-end
labeled with [-
P]ATP and T
polynucleotide kinase (New England Biolabs, Beverly, MA) to a
specific activity of 4 10
cpm/pmol. Blots were
subjected to autoradiography using two intensifying screens, and
hybridization was quantified using a phosphorimager (Molecular
Dynamics).Nuclear Run-on Assays
Frozen liver tissue
(0.25-0.3 g) from three or four different animals fed ad
libitum was pooled, and an equivalent amount of liver tissue from
four different 50% CR animals was pooled separately. Samples were
homogenized in 10 ml of ice-cold NA buffer (10 mM Tris (pH
8.0), 2.5 mM magnesium acetate, 0.3 M sucrose, and
0.25% Triton X-100) with five strokes at 2000 rpm using a motor-driven
Potter-Elvehjem tissue grinder (Wheaton, Millville, NJ). 10 ml of NB
buffer (10 mM Tris (pH 8.0), 2.5 mM magnesium
acetate, 2.4 M sucrose, and 0.1% Triton X-100) was added, and
the homogenate was layered onto 15 ml of NB buffer. The homogenate was
centrifuged at 113,000 g for 1 h at 4 °C in a
Beckman SW28 rotor. The supernatant was discarded, and the nuclei were
washed in 0.75 ml of 50 mM HEPES (pH 7.5), 0.1 mM EDTA, 5.0 mM dithiothreitol, and 10% glycerol. Nuclear
transcription reactions were performed as described (41) .
After transcription, a volume of TRI reagent equal to 10 times the
volume of the transcription reaction was added, and RNA was isolated as
described by the manufacturer. Unincorporated nucleotides were removed
by gel filtration. The total P-labeled RNA was diluted to
100 µl in a final concentration of 50 mM HEPES (pH 7.0),
500 mM NaCl, 8 mM EDTA, 0.4% SDS, 35% formamide, and
1500 cpm of
H-labeled cpsI cRNA. The radiolabeled
RNAs were hybridized at 42 °C for 17 h to denatured DNA fixed to 38
mm nitrocellulose filters. The filters had 5 µg of
pBR322 DNA or 5 µg of rat cpsI cDNA (pHN3491; (5) ) bound to them. Filters were washed, and the amount of
hybrid was determined as described(41) .In Vitro Transcription of
To synthesize cpsI cRNA for use as an internal
hybridization standard for nuclear run-on assays, plasmid SP6-cpsI was constructed. SP6-cpsI contains the 1.2-kb PstI to EcoRI fragment of the cpsI cDNA
present in pHN3491 linked to the SP6 promoter in poLUC(42) .
SP6-cpsI was linearized with EcoRI and used as
template in a transcription reaction with SP6 RNA polymerase and 0.5
mM GTP, UTP, and ATP and 13 µM [H-Labeled cpsI
cRNAH]CTP (21 Ci/mmol). The cRNA was labeled to
a specific activity of 3000 cpm/fmol.Actinomycin D Treatment of Mice
Animals were given
intraperitoneal injections of 4 mg/kg, body weight, actinomycin D
(Sigma) in phosphate-buffered saline. At the indicated times, livers
were removed, and RNA was prepared as described above. cpsI mRNA levels determined using dot and Northern blots were
normalized to the level of 18 S rRNA, since rRNA is much more stable
than mRNA, and its level does not change with diet(26) .Statistical Analysis
Statistical significance was
determined using Student's unpaired t test. A 95% level
of confidence was considered significant.
CpsI Levels Were Induced by CR
We found that a
protein with an apparent molecular weight of 160,000 on an
SDS-polyacrylamide gel was induced about 3-fold in the liver of CR mice (Fig. 1). This band was identified as CpsI by 4 criteria:
molecular weight, tissue distribution, abundance, and N-terminal amino
acid sequence. The molecular weight of the mature form of rat CpsI is
160,000(5) . The band was well separated from other
proteins and appeared to be composed of a single protein, representing
approximately 4% of the total protein present. The band was absent from
all other tissues examined (brain, heart, kidney, fat, muscle) with the
exception of intestine, where it was present at less than 10% of the
level in liver. This matches the tissue distribution and abundance
reported for CpsI(2) . Microsequencing of the N-terminal 10
amino acids of the 160,000 molecular weight protein by the
Biotechnology Instrumentation Facility (University of California,
Riverside) yielded a sequence that matched 7 out of 10 of the
N-terminal amino acids of the mature form of rat CpsI, as predicted
from its cDNA sequence(5, 43) . The sequence of the
mouse cDNA is not known yet.
20 proteins clearly
visible in the stained gel, CpsI is the only protein consistently
altered by diet.
Hepatic CpsI Enzyme Activity Was Induced by
CR
Studies were conducted to determine whether the induction of
CpsI was accompanied by an increase in the activity of the enzyme.
Analyzing liver homogenates from animals in each dietary group revealed
a 5-fold induction of CpsI activity in CR mice (p < 0.01; Fig. 1C). Therefore, the 3-fold induction of CpsI was
accompanied by a roughly 5-fold increase in the activity of the enzyme.
These results suggest that in addition to an increase in the amount of
enzyme in CR mice, the specific activity of the enzyme itself may
increase.The Induction of CpsI Was Accompanied by an Increase in
cpsI mRNA
The level of hepatic cpsI mRNA was determined
using livers from the studies analyzing CpsI protein and enzyme
activity (Fig. 2A). A single species of mRNA was
detected, with an apparent size of 6.2 kb. After correction for loading
and transfer of total RNA using the level of 18 S rRNA present in each
lane (Fig. 2B) or the level of transcription factor
S-II mRNA present (Fig. 2C), hepatic cpsI mRNA
levels were induced just over 3-fold in CR mice (Fig. 2D). This increase was statistically significant (p < 0.01). We have shown that S-II mRNA does not change
with respect to polyadenylated RNA or rRNA in CR or AL
mice(44) . The increase in cpsI mRNA was specific. We
have examined the expression of many other mRNA, including S-II mRNA,
and none of these changed with CR ( (26) and data not shown).
Thus, hepatic cpsI mRNA is specifically induced by CR, and
this induction closely parallels the increase in hepatic CpsI protein
and enzyme activity. These results indicate that CR acts at an early
step in gene expression and does not influence the translation or
stability of CpsI protein.
cpsI mRNA Stability Is Not Altered by CR
Because
it is possible that CR might affect the stability of cpsI mRNA, a study of its stability was conducted (Fig. 3). In
this study, the level of expression in the control mice, injected with
the vehicle, was 0.5 ± 0.07 and 1.1 ± 0.23 for AL and CR
mice, respectively (Fig. 3, zero time point; p <
0.02). During the first 6 h of actinomycin D treatment, there was a
transient superinduction of cpsI mRNA in both AL and CR mice.
Superinduction of other genes has been reported after actinomycin D
treatment(45) . This increase was greater in AL mice,
suggesting that the transcription of the gene may be repressed
differentially in AL mice by a relatively unstable RNA or protein. In
these same animals we found that glucose-regulated protein 78 precursor
RNA levels decreased by 6 h to about 10% the level found before
actinomycin D treatment, suggesting that transcription is strongly
inhibited by that time (data not shown). The decay rates of cpsI mRNA between 6 and 18 h after drug treatment indicate that the
half-life of cpsI mRNA was 12 h in both dietary groups.
Thus, no change in stability of hepatic cpsI mRNA was
detected.
CR Induced the Transcription of cpsI
To determine
whether CR directly induced the transcription of cpsI, two
independent transcription run-on studies were performed (Table 1). In these experiments, the rate of cpsI transcription was enhanced an average of 3-fold in CR mice.
These results are shown in Table 1. The rate of transcription of
the glucose-regulated protein 78 gene also was determined in these
hybridization reactions. It does not change with CR(63) . The
magnitude of the increase in cpsI transcription can account
for the increase in cpsI mRNA and protein levels.
CR Increased CpsI Activity but Not mRNA in the Small
Intestine
Because cpsI expression in the small
intestine is not altered by glucocorticoids or glucagon (cAMP), we
investigated the effects of CR on intestinal CpsI activity (Fig. 4). Consistent with other reports, the level of CpsI
activity was approximately ten times lower in intestine than in liver (2) . Surprisingly, the activity of intestinal CpsI responded
to CR. CpsI activity almost doubled in the small intestine of CR mice (Fig. 4A), while it was enhanced approximately 6-fold
in liver (Fig. 4B). The increases were statistically
significant in both intestine (p < 0.05) and liver (p < 0.03).
cpsI mRNA Is Induced by the Reduction in Calories, Not by
the Increase in Protein Consumption
cpsI gene
expression is induced by high protein consumption. For example, rat
hepatic cpsI mRNA increased 3-fold when the composition of
isocaloric diets was shifted from 4 to 20% protein content, a 500%
increase(9) . For these reasons, we investigated the
possibility that cpsI was induced in CR mice by higher protein
consumption. Total protein intake is similar in both AL and CR mice (Table 2). However, long term calorie restriction resulted in a
40% difference in body weight (Table 2). Therefore, CR mice
consumed 40% more calories from protein per gram (body weight). The
increase in the protein consumed by CR mice is much smaller than the
500% increase used in studies such as those cited above.
3-fold. Thus, CR increases both
the rate of gene transcription and the specific activity of the enzyme.
Short-term feeding studies demonstrated that higher cpsI expression in CR mice was due to reduced consumption of dietary
calories and not to consumption of more dietary protein. The change in cpsI mRNA occurred with a short-term shift from CR to ad
libitum feeding, indicating that the change in metabolism
responsible for gene induction is relatively rapid. In intestine, CR
led to a roughly 2-fold induction in intestinal CpsI activity, without
a change in the level of cpsI mRNA. These results suggest that
CR increases the specific activity or rate of translation of CpsI in
intestine. Together, our results suggest that CR increases protein
catabolism, probably for gluconeogenesis.
50% by CR in
rats(48, 49) . However, growth hormone decreases CpsI
activity by decreasing the intracellular level of N-acetyl-L-glutamate, an allosteric activator of
CpsI. The activator is present in excess in our in vitro assays and therefore cannot be responsible for the differences in
activity reported here.
3-fold in rats (9) . In the study reported
here, hepatic cpsI mRNA and gene transcription were induced
3-fold by a 50% reduction of dietary calories without any change
in dietary protein. In both kinds of studies, cpsI mRNA and
gene transcription rates are high when calories derived from
carbohydrates are low. Since it is not clear whether glucagon, insulin,
or glucocorticoids are responsible for cpsI regulation in CR
animals, it is possible that the gene responds directly to blood
glucose concentrations. CR decreases blood glucose levels by 43% under
the conditions used in this study(26) . Because the
transcription factors and cis elements mediating the hormonal and
nutritional regulation of cpsI expression are poorly
characterized, it is possible that the gene contains carbohydrate
response elements or other genetic elements mediating responsiveness to
ammonia. A cis element has been described for the rat S14 gene, which
appears to mediate responsiveness to carbohydrate
concentrations(53) . The six specific binding sites for
bacterially expressed C/EBP
located proximal to the cpsI transcription initiation site could be involved in carbohydrate
regulation of the gene. Gadd153, a CCAAT/enhancer-binding protein
(C/EBP) which lacks a DNA binding domain and heterodimerizes with other
C/EBPs, is induced by glucose deprivation in at least two cultured cell
lines(40) . Thus, it is possible that regulation of the level
or activity of this or another C/EBP by dietary calories could
influence the expression of cpsI. as ``end
products'' of metabolism. To our knowledge, CpsI has not been
measured in hibernating animals. However, a major problem during
hibernation is the accumulation of ammonia and
HCO
(60) . Other similarities
exist between CR and hibernating animals. These include reduced body
temperature, lower serum T
, lower blood glucose, and a
substantial increase in protein synthesis and
turnover(21, 31, 60) . It is well established
that a mild reduction in environmental temperature may greatly extend
the life span of poikilothermic vertebrates (61) and that in
homeotherms, hibernation affects ``biological
time''(60) . The reduction in body temperature during
hibernation need not be severe. The body temperature of bears, for
example, falls by only about 2 degrees during hibernation, and
hibernation extends their life span. A comparable reduction in body
temperature has been observed in CR mice(62) . We suggest on
the basis of these various parallels that the calorie restriction
paradigm may be part of a broad spectrum of adaptive responses that
include hibernation. This ``hibernation hypothesis'' suggests
lines of inquiry for future studies into the mechanisms by which CR
affects life span and metabolism.
), triiodothyronine.)
-We thank Nancy Luck for illustrations and Dr.
Gary Hathaway (PPMAF, California Institute of Technology) for reviewing
the protein sequencing data.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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