(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 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.
CpsI ()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) .
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, 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) .
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
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) . (
)
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 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.
In this report, we present the novel finding that reduction of
dietary calories by 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.
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 20 proteins clearly
visible in the stained gel, CpsI is the only protein consistently
altered by diet.
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 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.
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 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.
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 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.
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 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.