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Increase in cholesterol intestinal absorption with aging in the rat
Exp. Geront. Vo I. 14, pp. 201-204. ~, Pergamon Press Ltd. 1979. Pri,qted in Great Britain.
0531-5565/79/0801-0201 $02.00[0
INCREASE IN CHOLESTEROL INTESTINAL ABSORPTION WITH AGING IN THE RAT* D. HOLLANDER a n d D. MORGAN Division t~l Gastroenterology, Wayne State University and tlarper Grace ttospitals, Detroit, MI 48201, U.S.A.
(Received 24 July 1978) INTRODUCTION ARTERIOSCt,EROTIC c a r d i o v a s c u l a r disease, a d i s e a s e a s s o c i a t e d w i t h a g i n g , is c a u s e d by e x c e s s i v e a m o u n t s o f c h o l e s t e r o l d e p o s i t s in t h e l i n i n g o f b l o o d vessels. T h e s m a l l i n t e s t i n e is t h e site o f a b s o r p t i o n o f c h o l e s t e r o l f r o m d i e t a r y s o u r c e s ( B o r g s t r o m , 1960). E n d o g e n o u s l y s y n t h e s i z e d c h o l e s t e r o l w h i c h is e x c r e t e d w i t h bile i n t o t h e l u m e n o f t h e d u o d e n u m is a l s o a b s o r b e d b y t h e s m a l l i n t e s t i n e ( T r e a d w e l l et al., 1959). S i n c e a r t e r i o s c l e r o t i c c a r d i o v a s c u l a r d i s e a s e is a d i s e a s e o f a g i n g a n d since t h e s m a l l b o w e l is t h e site o f c h o l e s t e r o l a b s o r p t i o n , we d e c i d e d t o i n v e s t i g a t e t h e r e l a t i o n s h i p b e t w e e n a g i n g a n d i n t e s t i n a l a b s o r p tion of cholesterol. Our studies showed that cholesterol absorption increases linearly with a g i n g , e v e n a t t h e e x t r e m e e n d o f t h e a n i m a l ' s life e x p e c t a n c y . MATERIALS AND METHODS Animal.~ Intestinal absorption of cholesterol was studied in male Sprague-Dawley rats varying in ages from I to 42 months. The rats were allowed free access to regular chow and water and were kept under identical conditions before experimentation. Three to six rats were studied in each age group.
Surgical preparation Each rat was anesthetized with ether, its abdomen was opened, and an inflow cannula was inserted into the lumen of the small bowel 3 cm distal to the entry of the common bile duct into the duodenum. The distal end of the small bowel was cannulated with a glass cannula just prior to the junction of the ileum and cecum. The rat was placed in a restraint cage which allowed mobility but prevented dislodgement of the catheters (Hollander et al., 1977). The animal's normal body temperature was maintained with a forced-air heating device which was activated by a thermostatic temperature controller (Hollander and Muralidhara, 1977). Experimental perfusions The infusate solution was composed of 10 mM sodium taurocholate in a Krebs phosphate buffer at pH 7.4. Inulin, a 14C-labeled nonabsorbable sugar, was added to the perfusate to detect changes in cholesterol concentration secondary to water transport and to correct the final data for this artifact (Miller and Schedl, 1970). SH-la, 2u (n) cholesterol with specific activity of 43 Ci/mmol was used as a tracer compound and was added to the nonradioactive cholesterol in the infusate to a final concentration of 50 taM. A micellar solution was prepared by ultrasound irradiation with a sonicator. The solution was infused into the proximal small bowel at a constant rate of 1 ml/min over a 2 h period and the outflow was collected in 6 separate 20 rain aliquots. Determination of absorption rate The absorption of cholesterol during each 20 rain period was calculated by subtracting the total amount of cholesterol collected out of the outflow cannula from the amount known to have been infused into the proximal small intestine during the same 20 min period. 8H-cholesterol was separated from ~aC-inulin by total combustion of triplicate aliquots of the intestinal perfusate with a tissue oxidizer (Hollander et al., 1976). Radioactivity was than assayed with a beta liquid scintillation counter to an accuracy of ± 1 ° o, The corrected net absorption rate of cholesterol in each 20 min period was calculated and all 6 values for absorption from each animal were pooled with the values obtained from other animals of the same age group, *Mail requests for reprints to: Daniel Hollander, M.D., Department of Medicine, 5901 East 7th St.. Long Beach, CA 90801, U.S.A. 201
202
D. HOLLANDERAND 1). MORGAN
for calculation of the mean i S.E. rate of absorption of cholesterol. Cholesterol absorption by each age group was also expressed in terms of percentage absorption of the amount of cholesterol infused per h. Determination o f intestinal size The animals were killed with an overdose of ether at the completion of perfusion and the perfused intestinal segments were removed. Well-standardized methods (Hollander and Muralidhara, 1977) of stretching and desiccating the bowel were used for measuring the intestinal weight and length in the dry state, so that the segments from the various age groups could be compared.
RESULTS When the rate of cholesterol absorption in each age group was plotted against the age of the animals, a linear relationship was found between the two parameters (Fig. 1). Absorption of cholesterol continued to increase until the extreme end of the animal's life expectancy (42 months) (Hollander, 1976). When cholesterol absorption was expressed as a percentage of the amount infused, 14~o was absorbed at the age of 1 month, as compared to 38 o/ /o at the age of 42 months (Table 1). The body weight and the intestinal length and weight of each group of animals showed a rapid rise during their first 12 months of life (Table 1). However, during the latter 30 months of life the animals showed no further significant increase in these three parameters (Table 1).
I0001
CHOLESTEROL ABSORPTION
900:E
(_)
o
800-
700i.i.i
/ = 4 4 5 ~ Ilx
~. 6oo500-~
I IO
2'0 MONTHS
p
40
FIG. 1. Mean i S.E. absorption rate of cholesterol of groups of 3-6 rats of varying ages. Absorption was measured during 6 separate 20 rain collections in each rat. The regression line was plotted by using the least
squares method. The r-value indicates the fit of the data to the theoretical regression line and the p-value describes the probability of the fit being due to chance alone. DISCUSSION Progressive increase in cholesterol absorption rate was found throughout the entire 42 months of life of the animals (Fig. I, Table I). The increase in cholesterol absorption could not have been caused by increase in intestinal surface area, which correlates with
203
INCREASE IN CHOLESTEROL INTESTINAL ABSORPTION WITH AGING IN THE RAT
intestinal length and intestinal weight (Wilson and Dietschy, 1974). Age-related changes in biliary or pancreatic secretions could not have caused the changes in cholesterol absorption either, since the inflow catheter, which was always positioned beyond the entry site of the common bile duct into the duodenum, eliminated endogenous biliary and pancreatic secretions from the absorbing segment. The positioning of the inflow catheter also prevented endogenously synthesized cholesterol from entering the perfused segment and from diluting the pool of radiolabeled cholesterol. TABLE ], A G I N G : ITS INFLUENCE ON BODY WEIGHT~ INTESTINAL GROWTH AND CHOLESTEROL ABSORPTION IN THE RAT
Age (months) 1 2 12 20 37 42
Animal weight (g) I01 4- 4 254 ~: 5* 627 ~ 27* 707 ± 25 700 ~z 49 763 i 43
Intestinal weight (rag) 900 5: 75 1423 i 36* 2062 i 58* 2125 i 45 2554 i 46 2382 -+- 114
Intestinal length (cm) 92 4-2 100 ~ 5* 109 ± 2* 122 i 3 124 ± 5 125 ~ 3
Absorption per hour (~o) 14 17 18 25* 37* 38
Mean -t- S.E. of observed values from 3-6 animals in each age group. Student's t-test was used to test the significance of the differences between the mean values of each age group as compared to the next lower age. *Significant differences (p < 0.05). Since the intestinal surface area and biliary or pancreatic secretions were eliminated as factors responsible for our observations, either age-related changes in the absorptive cell membrane itself (Dowben, 1969) or alterations in the intracellular intestinal metabolism of cholesterol (Treadwell and Vahouny, 1968) or changes in the transport of cholesterol out of the enterocyte and into the lymphatic circulation (Sylven and Borgstrom, 1968) may account for the increase in cholesterol absorption with aging. Any or all of the above possibilities which could influence cholesterol absorption could be responsible for our present observations. We have shown that aging in the rat is paralleled by increased intestinal absorption of cholesterol. Parallel changes may occur in man as well. Isolating the particular mechanism which is responsible for the age-related increase in cholesterol absorption would be an important step towards the amelioration of arteriosclerotic cardiovascular disease. SUMMARY Intestinal absorption rate of cholesterol was measured in groups of rats varying in age from 1 to 42 months. Cholesterol absorption increased in a linear fashion with aging of the animals. Absorption at the extreme end of the life expectancy of the animals (42 months) was more than twice the amount absorbed at 1 month of age. The increase in absorption of cholesterol with aging may accelerate the development of atherosclerosis with aging.
Acknowledgements--This work was supported by grants AM 17607from the NIH, the Skillman Foundation, and Harper Hospital Medical Staff Fund. Animals were purchased through the auspices of the National Institutes on Aging. C. van der Harst is thanked for technical assistance.
204
D, HOLLANDER AND D. MORGAN
REFERENCES BORGSTROM,B. (1960) I. clin. Invest. 39, 809. DOWBEN, R. M. (1969) Biological Membranes, pp. 1-38. Little & Brown, Boston. HOLLANDER,C. F. (1976) Lab. Anita. Sci. 26, 320. HOLLANDER,D., MURALIDHARA,K. S. and R1M, E. (1976) Am. 1. Physiol. 230, 251. HOLLANDER,D., RIM, E. and MURALIDHARA,K. S. (1977) Am. I. Physiol. 232, E69. HOLLANDER,D. and MURALIDHARA,K. S. (1977) Am. L Phyaiol. 232, E471. MILLER,D. L. and SCHEDL,H. P. 0970) Gastroenterology 58, 40. SYLVEN, C. and BORGSTROM,B. (1968) J. Lipid Res. 9, 596. TREADWELL,C. R., SWELL,L., VAHOUNY,G. V. and FIELD, 1-[., JR. 0959) J. Am. Oil Chem. Soc. 36, 107. TREADWELL,C. R. and VAHOUN¥,G. V. (1968) In: Handbook of Physiology (Edited by C. F. CODE),Vel. 3 p. 1422. American Physiological Society, Washington. WILSON, F. A. and DIEa'SCHY,J. M. (1974) Biochim. biophys. Acta 363, 112.
0531-5565/79/0801-0201 $02.00[0
INCREASE IN CHOLESTEROL INTESTINAL ABSORPTION WITH AGING IN THE RAT* D. HOLLANDER a n d D. MORGAN Division t~l Gastroenterology, Wayne State University and tlarper Grace ttospitals, Detroit, MI 48201, U.S.A.
(Received 24 July 1978) INTRODUCTION ARTERIOSCt,EROTIC c a r d i o v a s c u l a r disease, a d i s e a s e a s s o c i a t e d w i t h a g i n g , is c a u s e d by e x c e s s i v e a m o u n t s o f c h o l e s t e r o l d e p o s i t s in t h e l i n i n g o f b l o o d vessels. T h e s m a l l i n t e s t i n e is t h e site o f a b s o r p t i o n o f c h o l e s t e r o l f r o m d i e t a r y s o u r c e s ( B o r g s t r o m , 1960). E n d o g e n o u s l y s y n t h e s i z e d c h o l e s t e r o l w h i c h is e x c r e t e d w i t h bile i n t o t h e l u m e n o f t h e d u o d e n u m is a l s o a b s o r b e d b y t h e s m a l l i n t e s t i n e ( T r e a d w e l l et al., 1959). S i n c e a r t e r i o s c l e r o t i c c a r d i o v a s c u l a r d i s e a s e is a d i s e a s e o f a g i n g a n d since t h e s m a l l b o w e l is t h e site o f c h o l e s t e r o l a b s o r p t i o n , we d e c i d e d t o i n v e s t i g a t e t h e r e l a t i o n s h i p b e t w e e n a g i n g a n d i n t e s t i n a l a b s o r p tion of cholesterol. Our studies showed that cholesterol absorption increases linearly with a g i n g , e v e n a t t h e e x t r e m e e n d o f t h e a n i m a l ' s life e x p e c t a n c y . MATERIALS AND METHODS Animal.~ Intestinal absorption of cholesterol was studied in male Sprague-Dawley rats varying in ages from I to 42 months. The rats were allowed free access to regular chow and water and were kept under identical conditions before experimentation. Three to six rats were studied in each age group.
Surgical preparation Each rat was anesthetized with ether, its abdomen was opened, and an inflow cannula was inserted into the lumen of the small bowel 3 cm distal to the entry of the common bile duct into the duodenum. The distal end of the small bowel was cannulated with a glass cannula just prior to the junction of the ileum and cecum. The rat was placed in a restraint cage which allowed mobility but prevented dislodgement of the catheters (Hollander et al., 1977). The animal's normal body temperature was maintained with a forced-air heating device which was activated by a thermostatic temperature controller (Hollander and Muralidhara, 1977). Experimental perfusions The infusate solution was composed of 10 mM sodium taurocholate in a Krebs phosphate buffer at pH 7.4. Inulin, a 14C-labeled nonabsorbable sugar, was added to the perfusate to detect changes in cholesterol concentration secondary to water transport and to correct the final data for this artifact (Miller and Schedl, 1970). SH-la, 2u (n) cholesterol with specific activity of 43 Ci/mmol was used as a tracer compound and was added to the nonradioactive cholesterol in the infusate to a final concentration of 50 taM. A micellar solution was prepared by ultrasound irradiation with a sonicator. The solution was infused into the proximal small bowel at a constant rate of 1 ml/min over a 2 h period and the outflow was collected in 6 separate 20 rain aliquots. Determination of absorption rate The absorption of cholesterol during each 20 rain period was calculated by subtracting the total amount of cholesterol collected out of the outflow cannula from the amount known to have been infused into the proximal small intestine during the same 20 min period. 8H-cholesterol was separated from ~aC-inulin by total combustion of triplicate aliquots of the intestinal perfusate with a tissue oxidizer (Hollander et al., 1976). Radioactivity was than assayed with a beta liquid scintillation counter to an accuracy of ± 1 ° o, The corrected net absorption rate of cholesterol in each 20 min period was calculated and all 6 values for absorption from each animal were pooled with the values obtained from other animals of the same age group, *Mail requests for reprints to: Daniel Hollander, M.D., Department of Medicine, 5901 East 7th St.. Long Beach, CA 90801, U.S.A. 201
202
D. HOLLANDERAND 1). MORGAN
for calculation of the mean i S.E. rate of absorption of cholesterol. Cholesterol absorption by each age group was also expressed in terms of percentage absorption of the amount of cholesterol infused per h. Determination o f intestinal size The animals were killed with an overdose of ether at the completion of perfusion and the perfused intestinal segments were removed. Well-standardized methods (Hollander and Muralidhara, 1977) of stretching and desiccating the bowel were used for measuring the intestinal weight and length in the dry state, so that the segments from the various age groups could be compared.
RESULTS When the rate of cholesterol absorption in each age group was plotted against the age of the animals, a linear relationship was found between the two parameters (Fig. 1). Absorption of cholesterol continued to increase until the extreme end of the animal's life expectancy (42 months) (Hollander, 1976). When cholesterol absorption was expressed as a percentage of the amount infused, 14~o was absorbed at the age of 1 month, as compared to 38 o/ /o at the age of 42 months (Table 1). The body weight and the intestinal length and weight of each group of animals showed a rapid rise during their first 12 months of life (Table 1). However, during the latter 30 months of life the animals showed no further significant increase in these three parameters (Table 1).
I0001
CHOLESTEROL ABSORPTION
900:E
(_)
o
800-
700i.i.i
/ = 4 4 5 ~ Ilx
~. 6oo500-~
I IO
2'0 MONTHS
p
40
FIG. 1. Mean i S.E. absorption rate of cholesterol of groups of 3-6 rats of varying ages. Absorption was measured during 6 separate 20 rain collections in each rat. The regression line was plotted by using the least
squares method. The r-value indicates the fit of the data to the theoretical regression line and the p-value describes the probability of the fit being due to chance alone. DISCUSSION Progressive increase in cholesterol absorption rate was found throughout the entire 42 months of life of the animals (Fig. I, Table I). The increase in cholesterol absorption could not have been caused by increase in intestinal surface area, which correlates with
203
INCREASE IN CHOLESTEROL INTESTINAL ABSORPTION WITH AGING IN THE RAT
intestinal length and intestinal weight (Wilson and Dietschy, 1974). Age-related changes in biliary or pancreatic secretions could not have caused the changes in cholesterol absorption either, since the inflow catheter, which was always positioned beyond the entry site of the common bile duct into the duodenum, eliminated endogenous biliary and pancreatic secretions from the absorbing segment. The positioning of the inflow catheter also prevented endogenously synthesized cholesterol from entering the perfused segment and from diluting the pool of radiolabeled cholesterol. TABLE ], A G I N G : ITS INFLUENCE ON BODY WEIGHT~ INTESTINAL GROWTH AND CHOLESTEROL ABSORPTION IN THE RAT
Age (months) 1 2 12 20 37 42
Animal weight (g) I01 4- 4 254 ~: 5* 627 ~ 27* 707 ± 25 700 ~z 49 763 i 43
Intestinal weight (rag) 900 5: 75 1423 i 36* 2062 i 58* 2125 i 45 2554 i 46 2382 -+- 114
Intestinal length (cm) 92 4-2 100 ~ 5* 109 ± 2* 122 i 3 124 ± 5 125 ~ 3
Absorption per hour (~o) 14 17 18 25* 37* 38
Mean -t- S.E. of observed values from 3-6 animals in each age group. Student's t-test was used to test the significance of the differences between the mean values of each age group as compared to the next lower age. *Significant differences (p < 0.05). Since the intestinal surface area and biliary or pancreatic secretions were eliminated as factors responsible for our observations, either age-related changes in the absorptive cell membrane itself (Dowben, 1969) or alterations in the intracellular intestinal metabolism of cholesterol (Treadwell and Vahouny, 1968) or changes in the transport of cholesterol out of the enterocyte and into the lymphatic circulation (Sylven and Borgstrom, 1968) may account for the increase in cholesterol absorption with aging. Any or all of the above possibilities which could influence cholesterol absorption could be responsible for our present observations. We have shown that aging in the rat is paralleled by increased intestinal absorption of cholesterol. Parallel changes may occur in man as well. Isolating the particular mechanism which is responsible for the age-related increase in cholesterol absorption would be an important step towards the amelioration of arteriosclerotic cardiovascular disease. SUMMARY Intestinal absorption rate of cholesterol was measured in groups of rats varying in age from 1 to 42 months. Cholesterol absorption increased in a linear fashion with aging of the animals. Absorption at the extreme end of the life expectancy of the animals (42 months) was more than twice the amount absorbed at 1 month of age. The increase in absorption of cholesterol with aging may accelerate the development of atherosclerosis with aging.
Acknowledgements--This work was supported by grants AM 17607from the NIH, the Skillman Foundation, and Harper Hospital Medical Staff Fund. Animals were purchased through the auspices of the National Institutes on Aging. C. van der Harst is thanked for technical assistance.
204
D, HOLLANDER AND D. MORGAN
REFERENCES BORGSTROM,B. (1960) I. clin. Invest. 39, 809. DOWBEN, R. M. (1969) Biological Membranes, pp. 1-38. Little & Brown, Boston. HOLLANDER,C. F. (1976) Lab. Anita. Sci. 26, 320. HOLLANDER,D., MURALIDHARA,K. S. and R1M, E. (1976) Am. 1. Physiol. 230, 251. HOLLANDER,D., RIM, E. and MURALIDHARA,K. S. (1977) Am. I. Physiol. 232, E69. HOLLANDER,D. and MURALIDHARA,K. S. (1977) Am. L Phyaiol. 232, E471. MILLER,D. L. and SCHEDL,H. P. 0970) Gastroenterology 58, 40. SYLVEN, C. and BORGSTROM,B. (1968) J. Lipid Res. 9, 596. TREADWELL,C. R., SWELL,L., VAHOUNY,G. V. and FIELD, 1-[., JR. 0959) J. Am. Oil Chem. Soc. 36, 107. TREADWELL,C. R. and VAHOUN¥,G. V. (1968) In: Handbook of Physiology (Edited by C. F. CODE),Vel. 3 p. 1422. American Physiological Society, Washington. WILSON, F. A. and DIEa'SCHY,J. M. (1974) Biochim. biophys. Acta 363, 112.