Calories and aging alter gene expression for gluconeogenic,
glycolytic, and nitrogen-metabolizing enzymes
Joseph M.
Dhahbi1,
Patricia L.
Mote1,
John
Wingo1,
John B.
Tillman1,
Roy L.
Walford2, and
Stephen R.
Spindler1
1 Department of Biochemistry,
University of California, Riverside 92521; and
2 Department of Pathology, School
of Medicine, University of California, Los Angeles, California
90024
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ABSTRACT |
We characterized the
effects of calorie restriction (CR) on the expression of key
glycolytic, gluconeogenic, and nitrogen-metabolizing enzymes in mice.
Of the gluconeogenic enzymes investigated, liver glucose-6-phosphatase
mRNA increased 1.7- and 2.3-fold in young and old CR mice.
Phosphoenolpyruvate carboxykinase mRNA
and activity increased 2.5- and 1.7-fold in old CR mice. Of the key
glycolytic enzymes, pyruvate kinase mRNA and activity decreased ~60%
in CR mice. Hepatic phosphofructokinase-1 and pyruvate dehydrogenase mRNA decreased 10-20% in CR mice. Of the genes that detoxify
ammonia generated from protein catabolism, hepatic glutaminase,
carbamyl phosphate synthase I, and tyrosine aminotransferase mRNAs
increased 2.4-, 1.8-, and 1.8-fold with CR, respectively. Muscle
glutamine synthetase mRNA increased 1.3- and 2.1-fold in young and old
CR mice. Hepatic glutamine synthetase mRNA and activity each decreased 38% in CR mice. These CR-induced changes are consistent with other studies suggesting that CR may decrease enzymatic capacity for glycolysis and increase the enzymatic capacity for hepatic
gluconeogenesis and the disposal of byproducts of muscle protein catabolism.
calorie restriction; enzyme activity; metabolism; mice; mRNA
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INTRODUCTION |
DIETARY CALORIE RESTRICTION (CR) delays most
age-related physiological changes, is the only method known for
extending life span in homeothermic vertebrates, and is the most
effective means known for reducing cancer incidence and increasing the
mean age of onset of age-related diseases and tumors (31, 32). The effects of CR are robust; they have been reported in every species tested (32). Although many of the physiological consequences of CR were
first described more than sixty years ago, no consensus has yet emerged
regarding its mode of action.
In rodents, primates, and humans, CR reduces 24-h and fasting blood
glucose and insulin concentrations, as well as maximum glucose and
insulin concentrations during oral glucose tolerance tests (5, 10, 28).
Several kinds of evidence support a role for glucose and insulin in
many of the pathologies of aging. For example, chronic hyperglycemia is
associated with long-term neurological complications, microvascular
disorders, basement membrane thickening, and impaired cellular immunity
(19). Hyperinsulinemia is associated with coronary heart disease,
hypertension, and atherosclerosis (24). All of the pathologies
associated with elevated glucose levels are reduced or mitigated
entirely by CR. Whether glucose and insulin have a role in determining
the rate of aging itself is unknown. However, there is evidence
suggesting that it may.
There is only one multicellular organism for which life span-regulating
gene systems have been partially elucidated at the molecular level. In
Caenorhabditis elegans, insulin
receptor signaling appears to have a central role in aging. The insulin
receptor homologue of C. elegans,
daf-2, acts on daf-16, a hepatocyte nuclear factor 3 (HNF-3)/forkhead
transcription factor family member, to alter energy metabolism and
development (8). In mammals, insulin may also mediate some of its
actions by altering the activity of HNF-3 (16). Three of the four
well-studied insulin-responsive genes,
phosphoenolpyruvate carboxykinase
(PEPCK), tyrosine aminotransferase (TAT), and insulin-like growth
factor binding protein-1, appear to be regulated by HNF-3 through
insulin-response sequences that also are binding sites for HNF-3 (16).
Thus changes in carbohydrate metabolism may have an important role in
the anti-aging effects of CR. To examine this hypothesis, the mRNA and
in some cases the activities of key glycolytic, gluconeogenic, and
nitrogen-metabolizing enzymes were quantified in hepatic and extrahepatic tissues of CR and control mice. Our studies show that CR
leads to an increase in the mRNA and/or activity of key enzymes of
hepatic gluconeogenesis, decreases the mRNA and/or activity of key
enzymes of hepatic glycolysis, and increases the mRNA and/or activity
of key enzymes responsible for the disposal of nitrogen derived from
muscle protein catabolism for energy production. These CR effects
oppose age-related changes in the mRNA and/or activity of many of these
key metabolic enzymes. The results are consistent with studies in the
literature suggesting that CR enhances the turnover of extrahepatic
protein, extending higher levels of protein turnover into old age.
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MATERIALS AND METHODS |
Mice.
Females of the long-lived F1
hybrid strain C3B10RF1 were used
(22). This is the longest-lived strain of which we are aware, and one
we have used extensively in our studies. Use of an
F1 hybrid avoids the possible
effects of inbreeding-related genetic problems on the data. Mice were
maintained as previously described (2). There were eight mice in each
age and dietary group. To conserve tissue, not all of these animals
were used in each experiment shown. The same six mice from each group
were used in all experiments, with two exceptions. In the pyruvate
kinase (PK) activity assays, only four of the six mice in each group
were utilized. In the PEPCK activity assays, all eight mice were used.
At the time of use, the old CR and control mice weighed 28.9 ± 2.9 and 49.9 ± 5.3 g, respectively, and the young CR and control mice
weighed 20.8 ± 2.1 and 24.9 ± 3.4 g, respectively.
Young mice were 7 mo old, and old mice were 28 mo old.
Diets.
Mice were weaned at 28 days, housed individually, and subjected to one
of the two dietary regimens described below. The composition of the
defined diets and feeding regimen has been described in detail (20).
They are formulated so that dietary groups receive approximately equal
amounts of protein, corn oil, minerals, and vitamins per gram of body
weight. The amount of carbohydrates varied between groups. Control mice
consumed ~105 kcal (~449 kJ) each week. This is approximately the
amount of food required to support normal growth of mice (25). The
control mice were fed daily Monday through Friday. The 50% CR mice
consumed ~52 kcal (225 kJ) per wk and were fed Mondays, Wednesdays,
and Fridays. On Fridays, both groups were given a 3-day allotment of
food. Mice were fed at 0900. Mice had free access to water. For the studies, mice were fed a normal allotment of food Monday morning, and
all the food was eaten within 45 min. They were fasted for ~23 h and
were killed on Tuesday morning.
Nucleic acid hybridization probes.
A mouse phosphofructokinase-1 (PFK-1) 1-kb cDNA probe was excised with
EcoR I and
Xho I from American Type Culture
Collection (ATCC, Rockville, MD) plasmid no. 974547. A 1.4-kb fragment
corresponding to the 3' end of the cDNA of rat
PKF-2/fructose-2,6-bisphosphatase 2 (FBPase-2) was produced by
EcoR I digestion of pEMBL18. The 650-bp EcoR I fragment corresponding
to the 3' end of the rat fructose-1,6-bisphosphatase (Fru
1,6-P2ase) cDNA was excised from pEMBL18. Both PKF-2/FBPase-2 and Fru
1,6-P2ase cDNAs were a gift from
Dr. M. R. El-Maghrabi (State University of New York, Stony Brook, NY).
Mouse glucokinase (GK) cDNA corresponding to exons 2-7 was excised
with BamH I and
EcoR I from mGK2-7 (a gift from Dr. L. Chan, Baylor College of Medicine, Houston, TX). A 1070-bp fragment from nucleotides 730-1820 of murine glucose-6-phosphatase (G-6-Pase) was excised with EcoR I
from pWT28 (a gift from Dr. J. Y. Chou, Department of Health and Human
Services, Bethesda, MD). The rat glutaminase 1.4-kb cDNA fragment was
excised with EcoR I from pGLN2.0 (a
gift from Dr. M. Watford, The State University of New Jersey, New
Brunswick, NJ). The rat CPSI probe was a 1.2-kb Pst I and
EcoR I fragment excised from
pHN3491974547 (ATCC). The mouse pyruvate dehydrogenase (PDH) 2.8-kb
cDNA fragment was excised with EcoR I
from pmIEI-
(a gift from Dr. H. Dahl, The Murdoch Institute,
Melbourne, Australia). The 1.3-kb PEPCK coding fragment was produced by
Sph I followed by
Sal I digestions of pGEM5ZFP (a gift
from Dr. D. K. Granner, Vanderbilt University School of Medicine,
Nashville, TN). Mouse TAT and glutamine synthetase (GS) cDNA were
synthesized with Ready-To-Go PCR beads (Pharmacia Biotech, Piscataway,
NJ) by use of murine liver cDNA. The primers for TAT were complementary
to bases 289 to 320 for the forward primer and bases 1136 to 1166 for
the return primer (Genbank Accession no. X02741). Primers for GS were
complementary to bases 731 to 760 for the forward primer and bases 1889 to 1918 for the return primer (Genbank Accession no. X16314). A murine
muscle PK (MPK) cDNA fragment of 1908 bp was excised with
EcoR I from mPKm2 (a gift from Dr. H. Ariga, Hokkaido University, Sapporo, Japan). Liver PK (LPK) cDNA
was synthesized from the cDNA library described above,
using primers complementary to bases 583 to 610 for the forward primer
and bases 1767 to 1795 for the return (Genbank Accession no.D63764).
These primers were in regions of low homology between the murine LPK
and MPK cDNAs (7). Multiprime labeling of each of the PK cDNAs and
probing of liver and muscle RNA Northern blots showed that there was
little if any cross-hybridization of the muscle probe with LPK mRNA or
of the liver probe with the MPK mRNA. All of the blots probed with the
PK cDNAs received a final wash in 0.1× saline sodium citrate
(SSC) at 55°C for 30 min (15).
RNA isolation and visualization.
Mice were killed by cervical dislocation, and tissues were rapidly
removed. Muscle from the hind legs and back was removed and pooled for
each animal. Tissues were flash-frozen in liquid nitrogen.
Approximately 0.2 g of frozen liver tissue was homogenized for 40 s in
4 ml of TRI Reagent (Molecular Research Center, Cincinnati, OH) by use
of a Tekmar Tissuemizer (Tekmar, Cincinnati, OH) at a setting of 55. RNA was isolated as described by the TRI Reagent supplier. RNA was
resuspended in FORMAzol (Molecular Research Center), and Northern and
dot blots were performed using 20 and 10 µg of RNA, respectively. The
RNA was analyzed using Northern blots to verify its integrity, and
specific mRNA levels were quantified using dot blots. cDNAs were
radioactively labeled with
[
-32P]dCTP to a
specific activity of 1.7 MBq/µg by multiprime labeling with a kit
according to the method of the supplier (Pharmacia, Piscataway, NJ). An
oligonucleotide complementary to 18S rRNA was 5' end-labeled with
[
-32P]ATP and
T4 polynucleotide kinase (New
England Biolabs, Beverly, MA) to a specific activity of 7 KBq/pmol.
Northern and dot blots were hybridized with 22 and 4.2 KBq/ml of the
probes for specific mRNA and 18S rRNA, respectively. Hybridization was
quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Specific mRNA levels were normalized to the level of S-II transcription factor mRNA. S-II and 18S mRNAs are not affected by diet or age (14,
21).
Stability of specific mRNA.
Intraperitoneal injections of 4 mg/kg body weight actinomycin D (Sigma,
St. Louis, MO) in PBS were administered to mice. Sham-injected controls
received identical injections of the PBS vehicle. After 6, 12, and 18 h, livers were removed and RNA was prepared as described in RNA
isolation and visualization. Specific mRNA levels were determined using dot and Northern blots normalized to the level of 18S
rRNA, because rRNA is much more stable than mRNA, and its level does
not change with diet (14). Three 24-mo-old mice from each dietary group
were used for each time point. The control fed mice were 41.6 ± 5.8 g and the CR mice were 27.2 ± 2.5 g. Half-lives were determined by
inspection of semilogarithmic plots of the data. All semilogarithmic
plots were linear.
Enzyme assays.
Approximately 30 mg of tissue were sonicated (Branson model 350 Sonifier Cell Disrupter) in 300 µl of the appropriate homogenization buffer (in mM: 50 Tris, pH 7.4, 1 MnCl2, and 2 dithiothreitol for
PEPCK; 100 Tris, pH 7.5, 10 MgSO4,
150 KCl, and 0.2 EDTA for LPK; 150 potassium phosphate, pH 7.3, 0.2 EDTA, and 0.5% Triton X-100 for MPK; and 50 imidazole, pH 7.0, 0.2 EDTA, 2
-mercaptoethanol, and 0.5% Triton-X 100 for GS). PEPCK
activity was assayed and units were calculated as described (17). The
final reaction mixture contained 50 mM Tris, pH 7.4, 1 mM
MnCl2, 20 mM sodium bicarbonate,
0.5 mM phosphoenolpyruvate (PEP), 0.1 mM NADH, 0.1 mM dGDP, rotenone (2 µg/ml; Sigma), and malate
dehydrogenase (2 units/ml; Boehringer). Reactions were initiated by
addition of 500 µg of the liver homogenate to 300 µl of reaction
mixture in a final volume of 600 µl. PK activity was measured by
modification of the technique of Imamura and Tanaka (6). The final
reaction mixture contained 100 mM Tris, pH 7.5, 10 mM
MgSO4, 150 mM KCl, 2 mM PEP, 2 mM
ADP, 170 µM NADH, 0.5 mM Fru
1,6-P2ase, and 4 units/ml of
lactate dehydrogenase. Reactions were initiated by addition of 150 µg
protein of the liver homogenate or 10 µg protein of the muscle
homogenate to 400 µl of reaction mixture in a final volume of 800 µl. The change in absorbance at 340 nm was determined between
minutes 1 and
2. The units were calculated as
described (6) using a conversion factor of 0.80 A340 units equals 100 µg/ml NADH
(Sigma). GS activity was determined by measuring the conversion of
14C glutamate (Amersham, England)
to 14C glutamine as described
(18). The reaction buffer contained 2.5 mM
14C glutamine (0.5 Ci/mol), 7.5 mM
ATP, 30 mM MgCl2, 25 mM
NH4Cl, and 50 mM imidazole/HCl
buffer, pH 7.0. Reactions were initiated by addition of 20 µg of the
liver homogenate protein or 300 µg of the muscle homogenate protein
to 100 µl of reaction mixture in a final volume of 200 µl. After
incubation for 15 min at 37°C, the reactions were stopped by adding
1 ml of ice-cold 20 mM imidazole/HCl buffer, pH 7.0. A 300-µl aliquot
was then loaded on a 1.2-ml column of AG 1-X8 (chloride form) anion
exchange resin (Bio-Rad) and washed with 1.5 ml imidazole/HCl buffer,
pH 7.0. The 14C glutamine
contained in the effluent was counted by liquid scintillation.
Glycogen determination.
Liver glycogen content was determined as previously described (1).
Briefly, 40- to 50-mg aliquots of liver were sonicated to homogeneity
in three volumes of 30% KOH and heated to 100°C for 30 min. The
homogenates were cooled to room temperature, and ethanol was added to a
final concentration of 66%. After overnight precipitation at 4°C,
centrifugation at 3,000 g, and removal
of supernatant, the pellet was resuspended in 200 µl of 0.6 N NaOH. This suspension was heated to 100°C for 2 h, neutralized, and centrifuged to remove debris. Ten microliters of this solution were
assayed for glucose content in a 0.5-ml reaction by use of a Glucose
(HK) Assay 10 Kit (Sigma).
Statistical analysis.
Student's unpaired t-test was used to
analyze the effect of CR on hepatic PEPCK and PK activity levels in the
28-mo-old animals. The effects of age, diet, and their interaction on
specific mRNA and activity levels were analyzed using a two-factor
ANOVA with age and diet as the factors. There was no significant
difference in the age × diet interaction between control and CR
mice. The significance of differences between any two groups was tested by Tukey's pairwise comparisons. When the means were significantly different, an "a" was assigned to the lowest value. If a value was significantly different from that of group "a," it was
categorized "b." Groups that were not significantly different
from "a" or "b" were labeled "ab." ANOVA computations
were performed using Minitab statistical software (13). A 95% level of
confidence was considered significant.
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RESULTS |
We have investigated the hypothesis that CR alters the expression of
key gluconeogenic, glycolytic, and nitrogen-metabolizing enzymes. The
level of mRNA and, in many cases, the activity of these enzymes were determined.
Hepatic gluconeogenic enzymes.
PEPCK catalyzes the first committed step of gluconeogenesis in the
liver (Fig. 1). Hepatic PEPCK activity was
1.7-fold higher in old CR mice than in control mice
(P = 0.0005; Fig.
2B). The abundance of PEPCK mRNA was 1.9-fold and 2.2-fold greater in the liver
of young and old CR mice, respectively, than it was in control mice of
the same ages (P < 0.0001 and
P = 0.0001; Fig.
2A). There was also a small
age-related decrease in PEPCK mRNA in the liver (P = 0.006). The increase in PEPCK
mRNA and activity is consistent with the idea that CR induces higher
rates of gluconeogenesis in the livers of CR mice.

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Fig. 1.
Summary of effects of age and calorie restriction (CR) on glycolytic
and gluconeogenic pathways of the liver. Glycolytic metabolism in the
liver involves three irreversible, regulated steps. Glucokinase (GK)
initiates glucose metabolism by phosphorylation of C-6, yielding
glucose 6-phosphate (G-6-P). The
committed step in glycolysis, and the second irreversible and regulated
step, is the phosphorylation of fructose 6-phosphate (Fru
6-P) by phosphofructokinase-1
(PFK-1) to produce fructose 1,6-bisphosphate (Fru
1,6-P2). The third irreversible
step controls the outflow of the pathway.
Phosphoenolpyruvate (PEP) and ADP are
utilized by pyruvate kinase (PK) to produce pyruvate (PYR) and ATP.
Pyruvate dehydrogenase (PDH) oxidatively decarboxylates pyruvate to
form acetyl-CoA. Phosphoenolpyruvate
carboxykinase (PEPCK) catalyzes the first committed step in
gluconeogenesis. The main noncarbohydrate precursors for
gluconeogenesis are amino acids from the diet and from muscle protein
breakdown. Other organs also contribute amino acids, but muscle is the
major source. Most of these amino acids are converted to oxaloacetate
(OA), which is metabolized to PEP by PEPCK. In the second regulated and
essentially irreversible step in gluconeogenesis, fructose
1,6-bisphosphatase (Fru 1,6-P2ase)
catalyzes the formation of Fru 6-P
from Fru 1,6-P2. Finally, in the
third essentially irreversible reaction of gluconeogenesis, glucose is
formed by the hydrolysis of G-6-P in a
reaction catalyzed by glucose-6-phosphatase (G-6-Pase). Substrates are
not boxed, enzyme names are in shaded boxes, summaries of experimental
results are in double-bordered boxes, and amino acids are indicated
(AA) in triple-bordered boxes. A down or up arrow indicates a general
decrease or increase in expression of the gene in CR mice. Arrows after
"Age" indicate general results for aged animals in both dietary
groups. NC, no change.
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Fig. 2.
Effects of age and diet on PEPCK mRNA and activity. Liver and muscle
PEPCK mRNA and liver PEPCK activity were quantified.
A: PEPCK mRNA abundance in liver of
young and old, control (open bars) and CR (closed bars) mice (6 animals
from each dietary regimen for each age group).
B: liver PEPCK activity in old control
and CR mice (n = 8 mice of each
dietary group). Student's unpaired
t-test was used to analyze the effect
of CR on hepatic PEPCK in this group of mice. Results are very highly
significant (*** P < 0.001).
C: PEPCK mRNA levels in mixed hind leg
and back muscle of individual mice from group in
A. Significance of differences between
any 2 groups was tested by Tukey's pairwise comparisons. Bars labeled
with different superscript letters are statistically significantly
different (P < 0.05).
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In the muscle, PEPCK is part of the alanine synthetic pathway. Alanine
is derived from the degradation of branched-chain amino acids, which
are unique in that their degradation is mainly initiated in muscle
rather than in liver. Newly synthesized alanine is transported to the
liver to serve as a gluconeogenic precursor. PEPCK mRNA was increased
in the muscle of both young and old CR mice (Fig. 2C; P = 0.0045 and P = 0.0061). This
increase was similar to that found in their liver.
G-6-Pase produces glucose from glucose 6-phosphate
(G-6-P), allowing its release from
the liver into the circulation (Fig. 1). G-6-Pase mRNA was 1.6-fold and
2.3-fold higher in the liver of young and old CR mice than it was in
control mice (P = 0.0004 and
P = 0.0015; Fig.
3). These results are also consistent with the idea that there is an increase in hepatic gluconeogenesis in CR
mice.

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Fig. 3.
Effects of age and diet on G-6-Pase mRNA levels in mouse liver.
G-6-Pase mRNA levels in liver of young and old, control (open bars) and
CR (closed bars) mice were determined. Results are means ± SD; n = 6 mice for each dietary
regimen and age group. Message levels were assessed by 2-factor ANOVA
with age and diet as factors. Statistical analysis of effects of age
and diet is shown as in Fig. 2.
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The level of Fru 1,6-P2ase mRNA
was unaffected by diet, although it did increase with age by ~25% in
control and CR mice (Table 1;
P = 0.0005 and
P = 0.0012).
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Table 1.
Hepatic abundance of GK, PDH, PFK-1, PFK-2/FBPase-2, and Fru
1,6-P2ase mRNA in young and old, control and CR mice
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Liver glycogen.
After 24 h without food, liver glycogen was depleted in both control
and CR mice. Liver glycogen content was 0.80 ± 0.25 and 0.80 ± 0.18 mg/g liver in control and CR mice, respectively.
Tissue specificity.
The kidney is the second major site of gluconeogenesis. Age
significantly decreased expression of both G-6-Pase and PEPCK mRNA in
the kidney (P = 0.001 and
P = 0.009; Table
2), as it does in the liver.
However, unlike the liver, CR had no effect on the expression of
G-6-Pase and PEPCK mRNA in the kidney
(P = 0.30 and
P = 0.56). Thus the effects of diet on
the enzymatic capacity for gluconeogenesis are highly specific to the
liver, whereas the age effect is present in both tissues.
Muscle nitrogen-metabolizing enzymes.
Mice are in the postabsorptive state after 24 h of fasting. The major
source of extrahepatic substrates for gluconeogenesis during fasting is
amino acids derived from muscle (3). Muscle cells also utilize amino
acids derived from protein turnover directly in the tricarboxylic acid
cycle (Fig. 4). Muscle protein catabolism involves two steps collectively called a transdeamination reaction. Transdeamination leads to the liberation of the amino nitrogen as
ammonia. Probably because of its extreme toxicity, this ammonia is
rapidly transferred to glutamate by GS, producing glutamine. This is
the only enzymatic reaction specific to the formation of glutamine in
muscle (9).

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Fig. 4.
Summary of effects of age and diet on muscle and liver nitrogen
metabolism. Transdeamination of amino acids produces tricarboxylic acid
cycle intermediates and ammonia in muscle. Glutamine synthetase
synthesizes glutamine from glutamate and ammonia. Glutamine is
transported to the liver where glutaminase releases ammonia,
regenerating glutamate. CPSI converts this ammonia to carbamyl
phosphate, which is converted to urea in the urea cycle. The amino
group of excess tyrosine is released by tyrosine aminotransferase (TAT)
as ammonia, which is also detoxified beginning with the action of CPSI.
Here substrates are not boxed, enzyme names are in shaded boxes, and
summaries of experimental results are in double-bordered boxes. When
two arrows are given after "CR," they represent degree of change
in young and old mice, respectively. An arrow after "Age" is the
main effect of age. A down arrow indicates the percent decrease, an up
arrow indicates degree of increase. Value given for age is a
combination of both dietary groups. NC, no change.
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CR increased muscle GS mRNA in old mice (2.1-fold;
P = 0.0008; Fig.
5C). It also increased GS mRNA in young
mice, although this increase did not reach statistical significance
(1.3-fold; P = 0.094). There was an
age-related 50% decrease in the expression of muscle GS mRNA in
control mice (P = 0.001; Fig.
5C) but not in CR mice. Repeated
attempts to quantify GS activity in muscle by use of two published
techniques were unsuccessful. The muscle activity was ~50-fold below
that of liver, and there were limited quantities of tissue available.
However, CR clearly induced GS mRNA levels in muscle of old CR mice.

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Fig. 5.
Effects of age and diet on GS mRNA and activity. Quantitation of liver
glutamine synthetase (GS) mRNA (A),
liver GS activity (B), and muscle GS
mRNA (C) of young and old, control
(open bars) and CR (closed bars) mice
(n = 6 mice for each dietary regimen
and age group). Statistical analysis of effects of age and diet is
shown as in Fig. 2.
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Hepatic nitrogen-metabolizing enzymes.
We examined the expression of liver genes essential for the disposal of
the nitrogen derived from amino acid catabolism. In the liver,
glutamine produced in the muscle is metabolized by the enzyme
glutaminase into glutamate and ammonia (Fig. 4). The ammonia derived
from this reaction can be returned to the glutamine pool by liver
glutamine synthetase. However, CR significantly reduced GS activity in
the liver of young (P < 0.0001) and
old (P = 0.021) mice (Fig.
5B). A similar decrease in liver GS
mRNA was found in young and old CR mice
(P < 0.0001 and
P = 0.0006, respectively; Fig.
5A). These changes were the opposite
of those found in muscle (Fig. 5C).
These results suggest that in CR mice the return of glutamate to the
blood glutamine pool is reduced in the liver, likely making glutamate
available as a substrate for hepatic gluconeogenesis (Fig. 4).
With age, there was a significant decrease in liver GS mRNA in control
(P = 0.0005) but not CR
(P = 0.15) mice. This age-related decrease is similar to that found in muscle, suggesting that in control
mice, aging is accompanied by a decrease in the shuttling of nitrogen
and carbon from the muscle to the liver through the glutamine-glutamate
pathway. This decrease might result from the well-described decrease in
muscle protein synthesis and degradation with age (27).
If the changes in GS mRNA and activity in CR mice are related to an
increase in the mobilization of nitrogen and carbon by the muscle for
export to the liver, one might expect to find an increase in the
expression of liver genes associated with nitrogen disposal (Fig. 4).
Glutaminase mRNA was induced ~2.5- and 2.2-fold in the liver of young
and old CR mice, respectively (Fig.
6A). The
effect of diet was significant (P < 0.001), whereas there was no significant effect of age. CPSI mRNA also
was induced ~2-fold in young and old mice by CR
(P = 0.0004 and
P = 0.016, respectively; Fig.
6B). The effects of age and diet
were significant (P = 0.021 and
P < 0.001). We have previously shown
that the induction of CPSI mRNA in CR mice is accompanied by increased
CPSI protein and activity (26). There was a slight (18%) decrease in
CPSI mRNA with age (P = 0.032),
consistent with the age-related decrease in GS mRNA and activity.

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Fig. 6.
Effects of age and diet on glutaminase, CPSI, and TAT mRNA levels in
liver of young and old mice. Dot-blot quantitation of glutaminase
(A), CPSI
(B), and TAT
(C) mRNA in liver of young and old,
control (open bars) and CR (closed bars) mice
(n = 6 animals from each dietary
regimen and age group). Statistical analysis of effects of age and diet
is shown as in Fig. 2.
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TAT is a liver-specific enzyme that provides ketogenic and
gluconeogenic substrates when glucose is limiting and amino acids are
utilized as a major source of energy. TAT mRNA was significantly induced by CR in young and old mice (P = 0.0046 and P = 0.026, respectively;
Fig. 6C). Aging decreased TAT mRNA
in the liver by an average of 37% (P = 0.001), whereas CR returned it to youthful levels.
Hepatic glycolytic enzymes.
PK catalyzes the third irreversible step in glycolysis, the
phosphorylation of ADP to ATP with the utilization of PEP as a high-energy phosphate donor. The reaction produces pyruvate (Fig. 1).
In young and old CR mice, LPK mRNA was reduced to 42 and 34% of the
level present in control mice (Fig.
7A;
P = 0.0016 and P < 0.0001, respectively). Aging
increased hepatic LPK mRNA abundance in control mice
(P = 0.0016). In contrast, the
abundance of MPK mRNA was unchanged by diet or age (data not shown).
Consistent with its effect on mRNA, CR also decreased PK activity by
60% in the liver of old mice (Fig.
7B; P < 0.001). This decrease in PK activity seems likely to slow the rate
of glycolysis in CR mice.

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|
Fig. 7.
Effects of age and diet on pyruvate kinase (PK) mRNA and activity in
liver and muscle of mice. A: liver PK
mRNA levels were quantified in liver from young and old, control (open
bars) and CR (closed bars) mice (n = 6 animals from each dietary regimen for each age group). Statistical
analysis of effects of age and diet are shown as in Fig. 2.
B: PK activity in liver from old
control and CR mice (n = 4 mice in
each dietary group). Student's unpaired
t-test was used to analyze effect of
CR on hepatic PK in this group of mice. Results are very highly
significant (*** P < 0.001).
|
|
The first committed step in glycolysis is the essentially irreversible
phosphorylation of G-6-P by PFK-1 to
produce Fru 1,6-P2 (Fig. 1). PFK-1
mRNA was significantly reduced by an average of 14% in CR mice
(P = 0.02; Table 1). In these studies,
no change was found in the level of GK mRNA with age or diet.
Pyruvate exits glycolysis through oxidative decarboxylation to
acetyl-CoA by the enzyme PDH (Fig. 1). CR significantly reduced PDH
mRNA by ~14% in both young and old mice
(P = 0.01, Table 1). Aging had no
affect on PDH mRNA abundance (P = 0.08; Table 1).
mRNA stability.
To investigate the mechanism for the changes in mRNA abundance
described in the previous paragraphs, the half-lives of a number of the
mRNAs were determined. The levels of specific mRNA were determined at
various times after administration of actinomycin D to CR and control
mice. The decay rates of the specific mRNA were used to estimate their
half-lives (data not shown). The stability of the different mRNA varied
from <2 h, in the case of G-6-Pase, to greater than 24 h for PDH and
LPK. However, we found no diet-related differences in the half-lives of
any of the mRNAs investigated. These data suggest that the diet-related
differences in specific mRNA abundance most likely arise from
differences in the transcription rates of the genes. However, the data
do not exclude other less common means of regulating mRNA abundance,
such as precursor-RNA processing.
 |
DISCUSSION |
We have characterized the expression of the key glycolytic,
gluconeogenic, and nitrogen-metabolizing enzymes in CR and control mice. The pattern of expression in liver and muscle indicates that CR
may reduce the enzymatic capacity of the liver for glycolysis, increase
the enzymatic capacity of the liver for gluconeogenesis, and increase
the enzymatic capacity of liver and muscle for the disposal of nitrogen
derived from protein utilization for energy generation. Together, these
data support the idea that CR enhances protein turnover at all ages and
resists the age-related decline in peripheral tissue protein turnover.
Stimulation and continuation of protein renewal in old animals have
been proposed as one of the key mechanisms for the anti-aging effects
of CR (23, 27).
Enzymes of hepatic gluconeogenesis.
After 24 h without food, dietary sources of carbon for maintaining
blood glucose levels have been long exhausted. The major source at this
time is extrahepatic protein degradation. The only other carbon sources
are minor. They are lactate, produced in the muscle by anaerobic
metabolism, and glycerol, produced from the degradation of
triacylglycerols in adipose tissue.
The doubling of PEPCK mRNA and activity in the liver of CR mice
suggests that CR increases the enzymatic capacity for performing the
first committed step in gluconeogenesis, the conversion of oxaloacetate
to PEP (Figs. 1 and 2). Because our studies are conducted during the
postabsorptive phase, oxaloacetate must be chiefly derived from protein
turnover in peripheral tissues. There are no known allosteric modifiers
of the activity of any PEPCK isoform (4). Therefore, our results are
consistent with the idea that CR increases the utilization of amino
acids from peripheral tissues for liver gluconeogenesis.
The decrease in liver PEPCK mRNA with age is similar to that reported
in hepatocytes isolated from aging rats (33). The results described
here extend these data by showing that the age-related negative
regulation extends to muscle PEPCK mRNA, and that CR attenuates this
age-related decrease (Fig. 2).
Liver glycogen.
Liver glycogen was similarly depleted in control and CR mice after 24 h
of food deprivation. Thus differential glycogen depletion is unlikely
to account for the differences in the enzymatic capacity for
gluconeogenesis found in CR and control mice.
Hepatic glycolysis.
The 60% decrease in PK mRNA and activity in CR mice probably decreases
the rate of hepatic glycolysis, because PK controls the exit of carbon
from the pathway. The statistically significant decrease in liver PFK-1
mRNA in CR mice also may result in decreased enzymatic capacity for
glycolysis. Although GK mRNA was not different in CR and control mice
after 24 h of fasting, in other studies we have found that CR decreased
the induction of GK mRNA by feeding by ~50% (J. M. Dhahbi, P. L. Mote, J. Wingo, S. Cao, B. C. Rowley, R. L. Walford, and S. R. Spindler, unpublished observations). Thus the mRNA and/or activity of
all of the key enzymes of glycolysis were reduced in CR mice. Although
other studies will be required to determine whether the flux of
intermediates through the glycolytic pathway is affected by CR, it
seems likely that CR does decrease the enzymatic capacity of the liver
for glycolysis.
We found a slight but significant decrease in PDH mRNA in the liver of
CR mice. Further work will be required to determine whether this change
in mRNA results in a decrease in enzyme activity. However, it suggests
the possibility that the enzymatic capacity for directing carbon from
glycolysis and gluconeogenesis to acetyl-CoA production also may be
diminished in CR mice (Table 1).
Whole body metabolic rate may not be affected by long-term CR (11).
However, the techniques used in such studies would discern only
substantial changes in energy utilization by major organs. Weindruch
and Sohal (30) have suggested that metabolic rates may differ
organ-specifically in CR and control animals (30). Our results could be
interpreted as support for this idea. We found decreases in the mRNA
and/or activity of key enzymes for glycolysis and acetyl-CoA formation
in the liver, but not in muscle or kidney. Furthermore, MPK activity is
nearly 20 times higher than the same activity in liver on a per weight
basis (data not shown). It is unlikely that even substantial changes in
liver PK activity would be detected in whole body measurements. Thus the possibility for organ-specific changes in energy metabolism should
be investigated further.
Nitrogen metabolism.
Probably because of its extreme toxicity, the ammonia released by
muscle protein catabolism is rapidly transferred to glutamate by GS,
producing glutamine (Fig. 4). Glutamine serves as a carrier of nitrogen
and carbon between tissues. The carbon is utilized by the liver for
gluconeogenesis and the nitrogen for ureagenesis. Hepatic catabolism of
glutamine is initiated in periportal hepatocytes by glutaminase (see
Fig. 4 and Ref. 12). Ammonia production by glutaminase is coupled to
urea synthesis by CPSI.
CR appears to enhance the enzymatic capacity of the liver to dispose of
nitrogen derived from amino acid catabolism in extrahepatic tissues.
Where the levels of glutaminase mRNA and activity have been reported,
changes in the mRNA are always accompanied by equivalent changes in
activity (29, 34). Therefore, the CR-related increase in hepatic
glutaminase mRNA likely leads to a congruent increase in glutaminase
activity. We have reported previously that CR increases hepatic CPSI
mRNA and activity to approximately the same extent (26). Therefore, the
induction of CPSI mRNA reported here in young and old CR mice is very
likely accompanied by induction of CPSI activity (Fig.
6B).
In hepatocytes, nitrogen originating from muscle protein catabolism can
be returned to the glutamine pool by the action of GS (Fig. 4).
Reduction of GS mRNA and activity in the liver of CR mice should reduce
the enzymatic capacity for return of this ammonia and carbon to the
glutamine pool. Reduction of hepatic GS activity would favor disposal
of nitrogen derived from extrahepatic protein catabolism as urea, and
free glutamate for use in gluconeogenesis or protein synthesis.
The induction of GS mRNA in the muscle of old mice is consistent with
our other data suggesting that CR increases the enzymatic capacity to
metabolize the products of protein catabolism for glucose production.
The age-related decrease in muscle GS mRNA in control mice also is
consistent with our other data suggesting that there is a decrease with
age in the enzymes required to catabolize extrahepatic protein for energy.
In conclusion, the data presented here show that aging was accompanied
by a decrease in the expression of genes required for the metabolism of
byproducts of amino acid catabolism for energy production in peripheral
tissues (decreased muscle GS mRNA and liver CPSI and TAT mRNA), and for
gluconeogenesis in the liver (decreased PEPCK and G-6-Pase mRNA). CR
generally had the opposite effect of age. CR enhanced the expression of
genes required for muscle and liver nitrogen disposal (increased muscle
glutamine synthetase mRNA, increased liver glutaminase, CPSI and TAT
mRNA), and decreased the expression of a liver gene essential for
mobilization of carbon and nitrogen by the liver for export to
extrahepatic tissues (liver GS mRNA and activity). CR also decreased
the expression of genes required for glycolysis (reduced PFK-1, PK, and
GK mRNA, and PK activity) and for the exit of carbon from glycolysis
for the biosynthesis of acetyl-CoA (PDH mRNA). The CR-related changes in the activity and/or mRNA of these enzymes suggest that CR enhances protein turnover in mice of all ages, resisting the well-documented decline in peripheral tissue protein turnover with age. Such an effect
on protein renewal may be one of the mechanisms by which CR extends
life span.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: S. R. Spindler,
Dept. of Biochemistry, University of California, Riverside, CA 92521 (E-Mail: spindler{at}ucrac1.ucr.edu).
Received 12 August 1998; accepted in final form 15 April 1999.
 |
REFERENCES |
1.
Chan, T. M.,
and
J. H. Exton.
A rapid method for the determination of glycogen content and radioactivity in small quantities of tissue or isolated hepatocytes.
Anal. Biochem.
71:
96-105,
1976[Medline].
2.
Dhahbi, J. M.,
J. B. Tillman,
S. Cao,
P. L. Mote,
R. L. Walford,
and
S. R. Spindler.
Caloric intake alters the efficiency of catalase mRNA translation in the liver of old female mice.
J. Gerontol.
53A:
B180-B185,
1998.
3.
Felig, P.
Amino acid metabolism in man.
Annu. Rev. Biochem.
44:
933-955,
1975[Medline].
4.
Hanson, R. W.,
and
L. Reshef.
Regulation of phosphoenolpyruvate carboxykinase (GTP) gene.
Annu. Rev. Biochem.
66:
581-611,
1997[Medline].
5.
Harris, S. B.,
M. W. Gunion,
M. J. Rosenthal,
and
R. L. Walford.
Serum glucose, glucose tolerance, corticosterone and free fatty acids during aging in energy restricted mice.
Mech. Ageing Dev.
73:
209-221,
1994[Medline].
6.
Imamura, K.,
and
T. Tanaka.
Pyruvate kinase isozymes from rat.
Methods Enzymol.
90:
150-165,
1982[Medline].
7.
Kanno, H.,
M. Morimoto,
H. Fujii,
T. Tsujimura,
H. Asai,
T. Noguchi,
Y. Kitamura,
and
S. Miwa.
Primary structure of murine red blood cell-type pyruvate kinase (PK) and molecular characterization of PK deficiency identified in the CBA strain.
Blood
86:
3205-3210,
1995[Abstract/Free Full Text].
8.
Kimura, K. D.,
H. A. Tissenbaum,
Y. Liu,
and
G. Ruvkun.
daf-2, An insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans (see comments).
Science
277:
942-946,
1997[Abstract/Free Full Text].
9.
King, P. A.,
L. Goldstein,
and
E. A. Newsholme.
Glutamine synthetase activity of muscle in acidosis.
Biochem. J.
216:
523-525,
1983[Medline].
10.
Lane, M. A.,
S. S. Ball,
D. K. Ingram,
R. G. Cutler,
J. Engel,
V. Read,
and
G. S. Roth.
Diet restriction in rhesus monkeys lowers fasting and glucose-stimulated glucoregulatory end points.
Am. J. Physiol.
268 (Endocrinol. Metab. 31):
E941-E948,
1995[Abstract/Free Full Text].
11.
McCarter, R.,
E. J. Masoro,
and
B. P. Yu.
Does food restriction retard aging by reducing the metabolic rate?
Am. J. Physiol.
248 (Endocrinol. Metab. 11):
E488-E490,
1985[Abstract/Free Full Text].
12.
Meijer, A. J.,
W. H. Lamers,
and
R. A. Chamuleau.
Nitrogen metabolism and ornithine cycle function.
Physiol. Rev.
70:
701-748,
1990[Free Full Text].
13.
Minitab, Inc..
Minitab Statistical Software, Standard Version, Release 7. State College, PA: Minitab, 1992.
14.
Mote, P. L.,
J. M. Grizzle,
R. L. Walford,
and
S. R. Spindler.
Influence of age and caloric restriction on expression of hepatic genes for xenobiotic and oxygen metabolizing enzymes in the mouse.
J. Gerontol.
46:
B95-B100,
1991[Medline].
15.
Noguchi, T.,
H. Inoue,
Y. Nakamura,
H. L. Chen,
K. Matsubara,
and
T. Tanaka.
Molecular cloning of cDNA sequences for rat M2-type pyruvate kinase and regulation of its mRNA.
J. Biol. Chem.
259:
2651-2655,
1984[Abstract/Free Full Text].
16.
O'Brien, R. M.,
and
D. K. Granner.
Diabetes Mellitus, edited by D. LeRoith,
S. I. Taylor,
and J. M. Olefsky. Philadelphia, PA: Lippincott-Raven, 1996, p. 234-241.
17.
Petrescu, I.,
O. Bojan,
M. Saied,
O. Barzu,
F. Schmidt,
and
H. F. Kuhnle.
Determination of phosphoenolpyruvate carboxykinase activity with deoxyguanosine 5'-diphosphate as nucleotide substrate.
Anal. Biochem.
96:
279-281,
1979[Medline].
18.
Prusiner, S.,
and
L. Milner.
A rapid radioactive assay for glutamine synthetase, glutaminase, asparagine synthetase, and asparaginase.
Anal. Biochem.
37:
429-438,
1970[Medline].
19.
Rossetti, L.,
A. Giaccari,
and
R. A. DeFronzo.
Glucose toxicity.
Diabetes Care
13:
610-630,
1990[Abstract].
20.
Spindler, S. R.,
M. D. Crew,
P. L. Mote,
J. M. Grizzle,
and
R. L. Walford.
Dietary energy restriction in mice reduces hepatic expression of glucose-regulated protein 78 (BiP) and 94 mRNA.
J. Nutr.
120:
1412-1417,
1990[Medline].
21.
Spindler, S. R.,
J. M. Grizzle,
R. L. Walford,
and
P. L. Mote.
Aging and restriction of dietary calories increases insulin receptor mRNA, and aging increases glucocorticoid receptor mRNA in the liver of female C3B10RF1 mice.
J. Gerontol.
46:
B233-B237,
1991[Medline].
22.
Staats, J.
Standardized nomenclature for inbred strains of mice: sixth listing.
Cancer Res.
36:
4333-4377,
1976[Abstract].
23.
Stadtman, E. R.
Protein oxidation and aging.
Science
257:
1220-1224,
1992[Medline].
24.
Stout, R. W.
Insulin and atheroma: 20-yr perspective (see comments).
Diabetes Care
13:
631-654,
1990[Abstract].
25.
Subcommittee on Laboratory Animal Nutrition and Committee on Animal Nutrition.
Nutrient requirements of the mouse.
In: Nutrient Requirements of Laboratory Animals: Rat, Mouse, Gerbil, Guinea Pig, Hamster, Vole, Fish. Washington, DC: National Academy of Sciences, 1978, p. 38-50.
26.
Tillman, J. B.,
J. M. Dhahbi,
P. L. Mote,
R. L. Walford,
and
S. R. Spindler.
Dietary calorie restriction in mice induces carbamyl phosphate synthetase I gene transcription tissue specifically.
J. Biol. Chem.
271:
3500-3506,
1996[Abstract/Free Full Text].
27.
Van Remmen, H.,
W. F. Ward,
R. V. Sabia,
and
A. Richardson.
Gene expression and protein degradation.
In: Handbook of Physiology. Aging. Bethesda, MD: Am. Physiol. Soc., 1995, sect. 11, chapt. 9, p. 171-234.
28.
Walford, R. L.,
S. B. Harris,
and
M. W. Gunion.
The calorically restricted low-fat nutrient-dense diet in Biosphere 2 significantly lowers blood glucose, total leukocyte count, cholesterol, and blood pressure in humans.
Proc. Natl. Acad. Sci. USA
89:
11533-11537,
1992[Abstract].
29.
Watford, M.,
N. Vincent,
Z. Zhan,
J. Fannelli,
T. Kowalski,
and
Z. Kovacevic.
Transcriptional control of rat hepatic glutaminase expression by dietary protein level and starvation.
J. Nutr.
124:
493-499,
1994[Medline].
30.
Weindruch, R.,
and
R. S. Sohal.
Seminars in medicine of the Beth Israel Deaconess Medical Center. Caloric intake and aging.
N. Engl. J. Med.
337:
986-994,
1997[Free Full Text].
31.
Weindruch, R.,
and
R. L. Walford.
Dietary restriction in mice beginning at 1 year of age: effects on life-span and spontaneous cancer incidence.
Science
215:
1415-1418,
1982[Medline].
32.
Weindruch, R.,
and
R. L. Walford.
The Retardation of Aging and Disease by Dietary Restriction. Springfield, IL: Thomas, 1988, p. 1-436.
33.
Wimonwatwatee, T.,
A. R. Heydari,
W. T. Wu,
and
A. Richardson.
Effect of age on the expression of phosphoenolpyruvate carboxykinase in rat liver.
Am. J. Physiol.
267 (Gastrointest. Liver Physiol. 30):
G201-G206,
1994[Abstract/Free Full Text].
34.
Zhan, Z.,
N. C. Vincent,
and
M. Watford.
Transcriptional regulation of the hepatic glutaminase gene in the streptozotocin-diabetic rat.
Int. J. Biochem.
26:
263-268,
1994[Medline].
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