Effects of caloric restriction and aging on erythrocyte membrane Ca2+-ATPase activity in specific pathogen-free Fischer 344 rats

Effects of Caloric Restriction and Aging on Erythrocyte Membrane Ca’+-ATPase Activity in Specific Pathogen-Free Fischer 344 Rats Faith 6. Davis, Mark R. Deziel, Judith B. Van Liew, Paul J. Davis, Lee L. Bernardis,

and Susan D. Blas

Dietary caloric restriction extends life span in the Fischer 344 rat. The interaction of aging and caloric restriction was examined at the level of the plasma membrane transport-associated enzymes, Ca2+-adenosine triphosphatase (ATPase) and Na,KATPase, in the Fischer rat. Animals were in four age groups, ranging from 6.1 to 25.0 months, and were specific pathogen-free (SPF, barrier-raised). Results from male and female animals raised on an ad libitum diet were compared with those from rats that received 60% of the age-specific caloric intake of their ad lib littermates. The responses of erythrocyte membrane Ca*+-ATPase activity in vitro to thyroid hormone (L-thyroxine [T,]; 3.53’.triiodothyronine (TJ and to purified calmodulin, a Ca*+-binding protein activator of Ca’+ -ATPase, were measured. Erythrocyte membrane Na,K-ATPase was also compared in the two diet groups, as was plasma glucose. Plasma membrane Ca ‘+-ATPase activity in the absence of added thyroid hormone and calmodulin was significantly reduced in calorically restricted rats (-39%, P < .OOl), compared with ad lib-fed animals, and the response was similar in the four age groups aged 6.1, 12.7, 17.0, and 25.0 months. In contrast, pooled (all ages) Ca’+-ATPase response in vitro to T, and to T, in calorically restricted animals was enhanced compared with the ad lib group (+62% and +58%, P < ,001, respectively). Calmodulin responsiveness of the enzyme was increased by 45% (P < .OOl) in calorie-deprived animals, similar to the change in T, and T, responsiveness. Analyzed on an age-specific basis, the diet-related difference in calmodulin responsiveness of Ca’+-ATPase activity was found to be greatest in young (6 months) animals; the diet effect was lessened in the 13-, 17. and 25.month-old rats. Diet-conditioned heightening of T, and T, stimulation of Ca’+-ATPase activity was similar in all age groups. There were no sex-, age-, or diet-dependent effects on red blood cell (RBC) Na,K-ATPase activity. There was no significant difference in plasma glucose levels between the ad lib and calorie-restricted animals. Thus, chronic caloric restriction in Fischer 344 SPF rats reduces RBC membrane Ca*+-ATPase, but not Na,K-ATPase, activity in animals ranging in age from 6 to 25 months. In contrast, the restricted diet was associated with significantly increased Ca*+-ATPase response in vitro to T,, T,, and calmodulin. The enhanced response to calmodulin was most marked in the g-month age group. Copyright 0 1991 by W.B. Saunders Company

D

IETARY CALORIC restriction extends longevity in a variety of animal models.‘.2 We have previously shown that the response in vitro of human erythrocyte membrane Ca’+-adenosine triphosphatase (ATPase) activity to thyroid hormone declines as a function of age of the red blood cell (RBC) donor-’ and that carbohydrate intake can acutely suppress RBC Cal+-ATPase activity.“ Na,K-ATPase activity, in contrast, is transiently increased by carbohydrate intake.4 The availability to us of specific pathogen-free (SPF; barrier-raised) Fischer 344 rats’ of varying ages reduces the possibility of contributions by “secondary aging” due, for example, to acute or chronic infection. We have studied in these animals the interaction of aging and chronic caloric restriction on RBC membrane Ca”-ATPase activity and its response in vitro to L-thyroxine (TJ, 3,5,3’-t.-triiodothyronine (T,), and calmodulin, the soluble Ca”-binding activator protein for Ca’+-ATPase. RBC Na,KATPase activity was also monitored, and the possible correlation of plasma glucose concentrations with enzymatic changes was investigated,

flow cages for 1 week before they were killed. Each shipment of animals included 16 to 20 rats in four age groups, and males and females maintained on ad lib or chronic calorically restricted diets. There were no animal losses in shipment or during local barrier maintenance. Animals were on a 12-hour light-dark cycle throughout their lives. Erythrocyte

Membranes

Animals were killed by decapitation and carotid arterial blood collected in heparinized containers. The RBC were immediately pelleted by centrifugation, washed, and hypotonically lysed as previously described.6 The ghosts qere washed twice and stored in 10 mmol/L Tris at -70°C until assayed within 72 hours. RBC membrane samples from 38 animals on the calorie-restricted, and 35 animals on the ad lib diet, were suitable for study. Ca”-ATPase

Activity

MATERIALS AND METHODS

RBC membrane Ca2+-ATPase activity was measured by adenosine triphosphate (ATP) hydrolysis6 as the difference in inorganic phosphate (P,) generation in the presence and absence of 20 u,mol/L free Ca”. The enzyme assay was performed for 60 minutes at 37°C and enzyme activity expressed as kmol P, per mg membrane protein per 60-min assay time. Membrane protein was measured by the Lowry method,’ using bovine serum albumin as

SPF Fischer 344 rats were bred and raised at the National Center for Toxicology Research (NCTR) for the Biomarkers of Aging program of the National Institute of Aging, National Institutes of Health. The animals were maintained on autoclaved NIH Diet and littermates were randomized at 16 weeks at the NCTR to either ad libitum feeding or a chronic diet consisting of 60% of the caloric intake of the age-specific ad lib diet.5 Calorierestricted animals received a vitamin-mineral supplement, which provided this group with vitamin and mineral intake equal to that of the ad lib group. Rats were shipped in barrier containers to the State University of New York at Buffalo and acclimated in laminar

From the Endocrinology Division, Department of Medicine, State Universityof New York at Buffalo School of Medicine and Biomedical Sciences, Veterans Administration Medical Center and Erie County Medical Center, Buffalo, NY. Supported by National Institutes of Health Research Gram No. AGO7736 Address reprint requests to Paul J. Davis, MD, Department of Medicine, Albany Medical College, Albany, NYI2208. Copyright 0 1991 by W B. Saunders Company 0026-0495/91/4008-0009$03.0010

Animals

Metabolism, Vol40, No 8 (August), 1991: pp 819-824

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standard, and inorganic phosphate measured by the malachite green technique.’ Membrane samples were studied in duplicate in two separate enzyme assays. Intraassay and interassay coefficients of variation were, respectively, 2% and 3%. To determine the responsiveness of membrane Ca’+-ATPase in vitro to T,, T,, and calmodulin, aliquots of membranes were incubated for 30 minutes at 37°C with lo-“’ mol/L T, or T,. or 6 x lOmymol/L purified bovine calmodulin, immediately before enzyme assay. Control samples included membranes and diluent for iodothyronines or calmodulin. These hormone and calmodulin concentrations are those which have been shown to be optimal for enzyme stimulation in our assay system.‘,“’

Na, K-A TPase Activig

Hormones, Reagents T,. ‘Ti. and Na,ATP were obtained from Sigma (St Louis. MO). Bovine brain calmodulin was extracted and purified in our laboratory by the method of Charbonneau et al. using phenothiazine affinity chromatography.” Levels of total plasma T, were measured with the “Coat-a-Count” radioimmunoassay kit from Diagnostic Products (Los Angeles, CA).

Statistical Analysis Significance of hormone and calmodulin etfects. and of differences between diet and age groups. was determined by one-way ANOVA. Linear regression analysis was used to study correlation between plasma glucose levels and enzyme activities,.

Erythrocyte Na,K-ATPase activity was determined as previously reported,’ and measured as the difference in hydrolysis of Na,ATP in the presence and absence of 10e3 mol/L ouabain.

RESULTS

Etythrocyte Ca’ ‘-ATPase Activity in the Absence of Added Thyroid Hormone and Calmodulin: Diet Effect

Plasma Glucose Concentration Plasma glucose levels were measured in tail vein samples of blood obtained 1 hour before death in 35 and 33 animals on the restricted and ad lib diets, respectively; these animals represented all age groups. Plasma glucose was measured with the Glucose-HK kit, obtained from Boehringer Mannheim Diagnostics (Indianapolis. IN). Each analysis was accompanied by a sample blank to correct for nonspecific color present in the plasma samples.

Shown as pooled analysis of all data from both sexes and all age groups in Fig IA, chronic calorically restricted animals had significantly less RBC membrane basal Ca’+ATPase activity than ad lib-fed rats (mean decrease in enzyme activity, 39%: P < ,001). The term basal refers to enzyme activity measured in the absence of added T,, T,, or calmodulin. Results from male and female rats were compa-

0.3

0.8

Ad Lib Feeding

n

40% Restriction

•j

P
*

0.6 +

0.2

0.1

C 10 -lo M T4

Basal Activity

10 -lo M T3

6x10.’

M CaM

Thyroid Hormone- and CalmodulinStimulated Activity

Fig 1. Levels of basal, thyroid hormone-, and calmodulin-stimulated rat erythrocyte membrane Ca”-ATPase activity in ad lib-fed animals, compared with animals receiving a diet restricted to 60% of the ad lib calorie content. Thirty-eight animals received the restricted diet, and 35 animals the ad lib diet. Results shown in this and subsequent figures are mean z SE of pooled data from two assays run in duplicate on membranes from each animal. IA) The ordinate shows basal enzyme activity measured in the absence of thyroid hormone or calmodulin. In membranes from animals on the restricted diet, basal enzyme activity was reduced by 39% (P < ,001, ANOVA) compared with enzyme activity from ad lib-fed animals. 16) The increase in enzyme activity above basal activity with the addition of T,, T,, or calmodulin. Note the change in scale compared with (A). T4-, T,-. and calmodulin-stimulated Ca*+-ATPase was enhanced in the restricted group compared with the ad lib group.

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DIET, AGING, AND Ca2+-ATPASE

rable (data not shown) and subsequent derived from animals of both sexes.

results are means

\

Ca”-ATPase Response In Vitro to Thyroid Hormone and Calmodulin: Diet Effect

Pooled data derived from rats of all ages showed that there was significant stimulation in vitro of Ca*‘-ATPase activity by thyroid hormones and purified calmodulin. Stimulation of enzyme activity by T,, T,, and calmodulin in membranes from calorie-restricted animals, shown in Fig lB, was significantly greater than that measured in RBC membranes from ad lib-fed animals (P < .OOl, ANOVA). In the ad lib animals, for example, T,-stimulated enzyme activity was 0.124 kmol PJmg/60 min, which was a 17% increase over the basal activity of 0.718 wmol P,. This degree of stimulation is comparable to that seen with human RBC and rabbit reticulocyte membranesY,‘” In contrast, erythrocyte membranes from chronic calorie-restricted animals showed enhanced enzyme response to T,, T,, and calmodulin; for example, T,-stimulated activity was 0.201 kmol P,, which was a 46% increase in activity over the basal activity of 0.436 kmol Pi. Total levels of enzyme activity achieved, in membranes from ad lib animals, with the addition of T,, T,, and calmodulin (basal activity plus hormone- or calmodulinstimulated activity), were 0.842, 0.848, and 0.855 p,mol Pi, respectively. In membranes from the chronic calorierestricted animals, the total enzyme activities with T,, T,, and calmodulin were 0.637, 0.641, and 0.634 pmol P,, respectively, indicating a suppressed maximal enzyme response. Age-Specific Analysis of Diet-Conditioned Ca”‘-ATPase Activity Responses In vitro to Calmodulin and Thyroid Hormone

The suppressive effect of chronic caloric restriction on basal enzyme activity was similar in all age groups (Fig 2A). There was a slight downward trend in basal activity in the oldest ad lib animals (mean age, 25.0 months), which was not statistically significant. Basal activity did not change in the restricted animals over the range of ages studied. As indicated above, and in contrast to the diet effect on basal Ca*‘-ATPase activity, calmodulin-responsiveness of RBC enzyme activity was enhanced by chronic caloric restriction. However, age-specific analysis (Fig 2B), indicates this effect of diet was limited to the 6-month-old animals; 13-, 17-, and 25month-old rats did not exhibit a significant diet effect with regard to in vitro calmodulin stimulability. This age-dependent effect reflects a modest decrease in the heightened enzyme response to diet restriction in older animals and an increased response to the Ca*‘-binding protein in older animals on the ad lib diet, which was significant over the entire age range (P < ,025, ANOVA). Analyzed on an age-specific basis, the heightened T, and T, stimulation of membrane Ca’+-ATPase activity due to caloric restriction was similar in all age groups (Fig 3). Although there was a decrease in hormone response of the enzyme in the 13-month restricted diet membranes, the

Ad Lib Feeding

Basal Activity

1 .a

40%

17, T

Restriction

n q

0.8

0.6

.g

.;

0.4

-2 o a.

0 (0

0.2

%F I?-G CL&’

d

c

JCalmodulin

0.:

Stimulated Activity

Gi 0.2

0.1

( 6.1

(51 7.2 )

17.0

12.7

( 11.114.1 ) Age

25.0

1 (16.6-173) (228 26.5

( months )

Fig 2. Basal and calmodulin-stimulated RBC membrane Ca’+ATPase activity in four age groups of rats fed ad lib or calorierestricted diets. The mean age and range of ages within each group are indicated below Fig 28. Numbers in parentheses indicate the number of animals in each group. (A) There was no significant change in basal enzyme activity from 6 to 26 months in either diet group, and basal activity remained consistently lower in the restricted animals. (6) Calmodulin-stimulated activity did not change significantly over the range of ages studied in animals on the restricted diet. There was a significant increase in calmodulin responsiveness with increasing age in animals on the ad lib diet (P < .025, ANOVA). There was no significant difference between the two diet groups at 13, 17, and 26 months, but at 6 months, there was a significant difference between the two diet groups (P < ,001, ANOVA). Note that the ordinate scale in (A) shows basal enzyme activity, whereas the ordinate scale in (B) shows the amount of increase in enzyme activity with the addition of celmodulin, 6 x 10e9 mol/L.

age-related change overall was not significant. Hormone stimulation in the ad lib membranes did not change significantly over the age range studied. Na,K-ATPase Activity

There were no diet or age effects on rat RBC membrane Na,K-ATPase activity (Fig 4). We did not measure the effect of thyroid hormone on this enzyme, as we have previously shown that erythrocyte Na,K-ATPase is not responsive in vitro to physiologic levels of T,.4 Plasma Glucose Concentrations

The plasma glucose levels in the ad lib and restricted animals were 103.8 f 3.9 (mean r SE) and 95.9 r 3.1 mg/dL, respectively, a difference that was not significant by ANOVA. There was no difference between sexes in glucose levels. In the 17-month-old animals, there was a significant difference between glucose levels in the two diet groups

DAVIS ET AL

822

I

T,

Stimulated

Ad LibFeedmg

Activity

basal, hormone-stimulable, ATPase activities.

m

q

40% Restrlctlon

'21

or calmodulin-stimulable

Ca”-

Plasma T, Concerltrations Plasma total T, levels were measured on equal pooled aliquots of plasma from three or four animals in each of eight groups according to age and diet. The mean T; levels were 3.9 ? 0.5 and 2.8 ? 0.2 &dL in the ad lib and chronic calorie-restricted groups, respectively, levels not significantly different by one-way ANOVA. A diminution in T; level with age was observed: in the 6-. 13.. 17-. and 25month-old animals, the T, levels were 4.7, 4.0, 4.3, and 2.5 pg/dL, respectively, in the ad lib group, and 3.1. 3.2. 2.5, and 2.3 @dL in the chronic calorie-restricted group. This decrease in T, level with age has also been reported in aging barrier-raised Fischer rats by Waner and Nyska.”

I

I3-

T3 Stimulated ,121

Activity

7

Effect I-

O-

6.1 ,51 721

12.7 (ll1.141)

Age

17.0 1166~17%

25.0 1228~265)

Fig 3. T,- and T,-stimulated Ca’+-ATPase activity in RBC membranes from rats of four age groups on two diets. Hormone concentration was lo-” mol/L. There was a consistent increase in hormone response of the enzyme in animals on the calorie-restricted diet, compared with the ad lib diet group. There was no age-specific change in this affect.

(124 2 8 mg/dL, ad lib, v 87 r 3, restricted, P < ,001) while in the other age groups there was no significant difference between animals on the two diets. There were no correlations between plasma glucose levels and levels of

0.8

40% Restriction

on Enzyme Act&in

Because of our previously reported finding that recent carbohydrate ingestion leads to transient elevation of RBC Na,K-ATPase and suppression of basal and hormoneresponsive Ca”-ATPase activities,’ we altered the feeding schedule of the ad lib animals in one half of the six shipment groups. These animals were placed in fresh sterile cages without food 16 hours before they were killed. The animals on restricted diets, because they consume their food immediately after morning feedings, had been fasting for 20 hours for all experiments. Basal, thyroid hormonestimulablc. and calmodulin-stimulable enzyme activities in the ad lib animals subjected to a 16.hour fast were not different from those of the ad lib animals permitted to eat immediately before death. Therefore, results from the fasted and fed ad lib groups were pooled in the data presented above. The mean plasma glucose level in the ad lib animals was 113.4 t 4.6 mg/dL when the animals were permitted to eat shortly before death. and 90.8 2 5.3 when

( months )

Ad Lib Feeding

of Feeding Time

l q

0.6

”

6.1

( 5.1 - 7.2)

12.7

(11.1

14.1

17.0

)

(16.6173)

Age ( months )

25.0

( 22.8 - 26.5 )

Fig 4. Na,K-ATPase activity in RBC membranes from animals in four age groups and on ad lib and calorie-restricted diets. There was no age- or diet-related difference in enzyme activity.

DIET, AGING, AND Ca2’-ATPASE

they were fasted for 16 hours (P < .OOl, ANOVA), but this difference was not associated with changes in enzyme activities. DISCUSSION

The mechanism by which dietary caloric restriction extends longevity in animal models of aging is not known. The significance of the observation rests, at least in part, on the issue of preservation of function with extension of life span. We had previously shown in man that RBC membrane Ca’+-ATPase activity in the healthy elderly is less responsive in vitro to physiological concentrations of thyroid hormone3; response of the human enzyme to calmodulin, the Ca”-ATPase activator protein, declined, but the change was of borderline statistical significance. The present studies examined the possible interaction of aging and caloric restriction at the level of rat erythrocyte Cal+ATPase and its in vitro regulation by thyroid hormone and calmodulin. Analyzed independently of animal age, erythrocyte membrane Ca’+-ATPase activity was substantially decreased in animals receiving the chronic diet calorically restricted to 60% of the intake of ad lib-fed rats (Fig 1). In contrast, the response of enzyme in calorically restricted rats to regulatory influences, namely, thyroid hormone and the calmodulin-Ca” complex, was significantly heightened (Fig 1). The response of Ca*‘-ATPase to T3 was comparable to that obtained with T, and with calmodulin. We have previously shown in the human RBC that Ca”-ATPase is similarly responsive to both thyroid hormone analogues.9,‘3 Starvation in man and animal models has been shown by others to alter the metabolism of thyroid hormone, particularly the extrathyroidal conversion of T, to T3 by tissue 5’-monodeiodinase,14 leading to reduction of circulating levels of T, and supporting the plasma concentration of T,. However, the reduction in serum T, concentration induced by a 60% calorie-restricted diet in Fischer 344 rats is trivial; Herlihy et al reported a decrease in mean 24-hour plasma T, concentrations from 95 to 87 ng/dL by caloric restriction, but the fasting values were identical to those of ad lib-fed animals.” These investigators reported no difference in plasma T, levels, but studied only animals aged 5.5 to 6.5 months.” That a change in thyroid hormone levels did not contribute to the results we report is also suggested by the fact that T, and T, are equipotent in the Ca’+-ATPase model. Thus, a decrease in endogenous plasma T, concentration alone cannot be expected to lower basal activity of the enzyme, as shown in Fig 1. In addition, we have reported that hypothyroid human subjects with low plasma T, and T, levels have decreased basal erythrocyte Ca’+ATPase activity, but also have diminished enzyme response in vitro to T3 and T,.” This finding contrasts with the present study, in which basal activity is decreased, but in vitro hormone responsiveness increased, by chronic caloric restriction. It should be noted that membrane Na,K-ATPase activity was not affected by diet (Fig 4), so that the diet effect on Ca”-ATPase is not part of a generalized membrane response. Because the endogenous calmodulin content of

823

RBC cytoplasm is many-fold greater than that bound to the erythrocyte membrane,91’7 the possibility of contribution, by a diet-conditioned change in RBC calmodulin concentration, to the decrease in Ca2’-ATPase appears remote. As interesting as the reduction in basal enzyme activity with calorie restriction is the enhanced response of Ca’+ATPase in vitro in membranes from calorie-restricted animals to T,, T,, and calmodulin. Thus, despite downregulation of overall enzyme activity, the immediate response of Ca”-ATPase to factors believed to modulate enzyme activity endogenously is very well preserved. Teleologically, this could represent an alternative set of controls for enzyme activity, which is invoked in anticipation of variable food intake. However, we have examined 16-hour fasting in ad lib-fed animals and have found no change, compared with recently fed ad lib animals, in basal enzyme activity or response of Ca*‘-ATPase in vitro to T,, T,, and calmodulin. Studies of hormone- and calmodulin-binding to membranes in ad lib-fed and calorie-restricted animals are needed, as are direct measurements of CaZt pumping by these RBC membranes to determine whether the coupling of ATP hydrolysis and Ca2+ transport is similar in animals on the two diets. Studied on an animal age-specific basis, the effect of the two diets on basal activity of RBC Ca’+-ATPase was comparable in all age groups (6 to 25 months) (Fig 2). The decline in enzyme activity in ad lib-fed rats 25 months of age was not significant. On the other hand, age-specific analysis of calmodulin responsiveness showed a loss with increasing age of the difference between the two diets (Fig 2). That is, differences in calmodulin-stimulability of Ca”-ATPase in restricted and ad lib-fed rats were not significant at ages 13, 17, and 25 months. The data on calmodulin-responsiveness presented in Fig 1 thus reflect the striking influence of data collected in young (6 months) animals. The loss of the diet effect in older rats is complex, apparently resulting from a modest increase in susceptibility of membranes from ad lib animals to calmodulin stimulation and a decline in calmodulin response in calorie-restricted rats. The diet-induced difference in T,- and T,-stimulated enzyme activity remained intact over the animals’ life span (Fig 3) although a small age-dependent decline in thyroid hormone response of the enzyme in restricted-diet animals was seen at age 13 months. One can speculate that we did not see an agedependent decline in hormone responsiveness in these animals because they were raised in a pathogen-free environment, in contrast to the elderly subjects, allegedly healthy, who contributed to our earlier study of human RBC membranes showing a decrease in hormone stimulation of Cal+-ATPase with aging.’ The possibility that recent food ingestion or ambient plasma glucose levels at the time of death account for any of the enzyme changes we have observed seems unlikely. This is not surprising, as we have demonstrated suppression of basal Ca’+-ATPase, but enhanced hormone and calmodulin effects, in animals on the restricted diet. In our prior studies examining in vivo and in vitro effects of physiologic elevations in glucose levels, 4 in contrast, we found that an increase in plasma glucose was associated with transient,

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DAVIS ET AL

uniform suppression of basal, hormone-, and calmodulinstimulated RBC membrane Ca”-ATPase activity, and elevation of Na,K-ATPase activity. Our finding that plasma glucose levels were not significantly different overall in the two diet groups is in contrast with that of Masoro et al,18who found a significant decrease in plasma glucose in 4- to 6-month-old Fischer rats fed a calorie-restricted diet for 2.5 to 4.5 months. We did find a difference in glucose levels in the two diet groups, but only in the 17-month-old animals. The timing of blood-drawing relative to feeding, as well as the age range of animals studied, was different in our study as compared with that of Masoro et al. Although we did demonstrate a difference in plasma glucose concentrations between ad lib animals recently fed and those fasted overnight, these differences were not correlated with any changes in enzyme activity. We and others have shown that the behavior of membrane Ca”-ATPase is importantly determined by its microdomain, particularly the lipid environment within the membrane. Specific long-chain fatty acids, for example, render the enzyme more or less responsive to calmodulin”~‘” and to

thyroid hormone.‘” Activity of the plasma membrane phosphatidylinositol pathway may also modulate the response of Ca”-ATPase to various factors.” .” Preliminary evidence from our laboratories has indicated a significant difference in plasma triglyceride levels in the ad lib and calorierestricted groups: 220 * 10 1’ 103 * 8 mg/dL, respectively (Van Liew JB, manuscript in preparation). This change in triglyceride levels, as well as alterations in membrane fatty acid composition, may contribute to the change in thyroid hormone responsiveness of Ca”-ATPase,” but is not likely to underlie the diet-related change in basal Ca”-ATPase activity that we observed.

ACKNOWLEDGMENT The excellent secretarial assistance of Alice M. Seres and Margaret A. Thompson is much appreciated. The very capable support of Edward H. Halsted and his staff in the State University of New York at Buffalo Animal Care Facility is gratefully acknowledged. We thank Dr Margaret MacGillivray and her laboratory staff for the performance of the plasma T, determinations.

REFERENCES 1. Yu BP. Masoro EJ, McMahan CA: Nutritional influences on aging of Fischer 344 rats: I. Physical, metabolic and longevity characteristics. J Gerontol40:657-670, 1985 2. Weindruch R. Walford RL: Dietary restriction in mice beginning at one year of age: Effect on lifespan and spontaneous cancer incidence. Science 215:1415-1418.1982 3. Davis PJ, Davis FB, Blas SD, et al: Donor age-dependent decline in response of human red cell Ca”-ATPase activity to thyroid hormone in vitro. J Clin Endocrinol Metab 64:921-925. 1987 4. Davis FB, Davis PJ, Nat G, et al: The effect of in vivo glucose administration on human erythrocyte Ca’+-ATPase activity and on enzyme responsiveness in vitro to thyroid hormone and calmodulin. Diabetes 34:639-646. 1985 5. DufTy PH. Feuers RJ, Leakey JA, et al: Effect of chronic caloric restriction on physiological variables related to energy metabolism in the male Fischer 344 rat. Mech Ageing Dev 48:117-133. 1989 6. Davis PJ. Blas SD: In vitro stimulation of human red blood cell Ca”-ATPase by thyroid hormone. Biochem Biophys Res Commun 99:1073-1080,198l 7. Lowry OH, Rosebrough NJ. Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 8. Chan K-M. Delfert D. Junger KD: A direct calorimetric assay for Ca”-stimulated ATPase activity. Anal Biochem 157:375-380. 1986 9. Davis FB, Davis PJ, Blas SD: Role of calmodulin in thyroid hormone stimulation in vitro of human erythrocyte Cal+-ATPase activity. J Clin Invest 71:579-586, 1983 10. Lawrence WD, Davis PJ, Blas SD, et al: Interaction of thyroid hormone and sex steroids at the rabbit reticulocyte membrane in vitro: Control by 17p-estradiol and testosterone of thyroid hormone-responsive Ca*‘-ATPase activity. Arch Biochem Biophys 235:78-85, 1984 11. Charbonneau H, Hice R, Hart RC. et al: Purification of calmodulin by Cal+-dependent affinity chromatography. Methods Enzymol 102:17-39.1983 12. Waner T. Nyska A: Thyroxine (Td) and triiodothyronine (T,) levels in the Fischer 344 inbred rat. Lab Anim 22:276-280, 1988

13. Davis FB, Cody V. Davis PJ, et al: Stimulation by thyroid hormone analogues of red blood cell Ca”-ATPase activity in vitro. J Biol Chem 258:12373-12377.1983 14. Vagenakis AG. Portnay GI. O’Brian JT. et al: Effect of starvation on the production and metabolism of thyroxine and triiodothyronine in euthyroid obese patients. J Clin Endocrinol Metab 45:1305-1309. 1977 15. Herlihy JT. Stacy C, Bertrand HA: Long-term tion depresses serum thyroid hormone concentrations Mech Ageing Dev 53:9-16, 1990

food restricin the rat.

16. Dube MP. Davis FB. Davis PJ, et al: Effects of hyperthyroidism and hypothyroidism on human red blood cell Ca”-ATPase activity. J Clin Endocrinol Metab 62:253-257, 1986 17. Nieman LK. Davis FB, Davis PJ, et al: Effect of end-stage renal disease on responsiveness to calmodulin and thyroid hormone of calcium-ATPase in human red blood cells. Kidney Int 24:Sl67-S170, 1983 IX. Masoro EJ. Katz MS, McMahan CA: Evidence glycation hypothesis of aging from the food-restricted model. J Gerontol44:B20-B22, 1989

for the rodent

19. Davis FB, Davis PJ. Blas SD. et al: Action of long-chain fatty acids in vitro on Ca’*-stimulatable, Mg’+-dependent ATPase activity in human red cell membranes. Biochem J 248:51 l-516. 1987 20. Niggli V. Adunyah ES, Carafoli E: Acidic phospholipids. unsaturated fatty acids, and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Ca”-ATPase. J Biol Chem 256:8588-8592. 1981 21. Choquette D. Hakim G. Filoteo AC;, et al: Regulation of plasma membrane Ca”-ATPases by lipids of the phosphatidylinositol cycle. Biochem Biophys Res Commun 125:908-915. 1984 22. Davis PJ, Davis FB. Blas SD: Components of the phosphatidylinositol cycle modulate thyroid hormone stimulation of human red cell Ca”-ATPase activity. Program of the 68th Annual Meeting of the Endocrine Society, Anaheim, CA, June 1986 (abstr 114) 23. Galo MG, Unares LE. Farias RN: Effect of membrane fatty acid composition on the action of thyroid hormones on (Ca” + Mg’+) adenosine triphosphatase from rat erythrocyte. J Biol Chem256:7113-7114. 1981