MAIJA H. ZILE, ELIZABETH C. BUNGE ANDHECTOR F. DELUCA 2 Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706 ABSTRACT The influence of vitamin A depletion on tissue composition was studied in rats that were marginally vitamin A deficient, i.e. at their weight plateau stage. The total number of cells ( DNA ) was decreased in most organs as a result of vitamin A depletion. In thymus, spleen and the sublingual glands there was also a dramatic reduction in the number of cells per gram of tissue and in thymus and sublingual glands there was an increase in the protein to DNA ratio as a result of absence of dietary vitamin A. We present the hypothesis that vitamin A stimulates growth by a direct role in cell replication in addition to or instead of stimulating the differenti ation of epithelial and bone cells. J. Nutr. 109: 1787-1796, 1979. INDEXING KEY WORDS vitamin A •growth •DNA replication While the importance of vitamin A to growth of higher animals is well recognized (1, 2), its physiological basis is largely un known and is a matter of debate. Vitamin A utilization in animals is directly related to the rate of growth, i.e. increase in body weight3 ( 3-6 ). This relationship has also been interpreted to mean that the require ment of vitamin A depends on the rate of growth (6, 7). Brown and Morgan (8) examined nitrogen metabolism in young, growing rats during avitaminosis-A and concluded that vitamin A is essential for tissue growth but not for maintenance. Similar conclusions have also been reached in studies with germ-free rats which do not require vitamin A for prolonged survival but exhibit a prompt growth response to vitamin A (9). Growth can result from either a prolif eration of cells or an increase in cell size or both. In the present study we have at tempted to evaluate which of these pro cesses is responsible for the enhancement of growth by vitamin A. If cellular com ponents decrease while DNA remains con stant, a decrease in cell size would account
for the smaller tissue mass associated with vitamin A deficiency. On the other hand, a decrease in total DNA in the vitamin Adeficient tissue would suggest an impair ment in cell division. Lamb et al. ( 10 ) have discussed the lim itations in designing nutritional experi ments for the study of vitamin A. To circumvent these difficulties, we have used vitamin A-deficient animals before they exhibit overt deficiency symptoms. Our re sults demonstrate that vitamin A deficiency causes a decrease in the number of cells of several tissues suggesting a general role of vitamin A in cellular proliferation. MATERIALS
AND METHODS
Animals. Weanling male rats 4 were fed a complete purified diet deficient only in vitamin A. The diet consisted of the folRecelved for publication February 12, 1979. 1 Supported by a program-project grant from the National Institutes of Health No. AM-14881. 2 Author to whom reprint requests should be ad"Ahlinvalia. B. & Bieri. J. G. (1970) Effects of hormones on liver vitamin A. Federation Proc. 29 (Abstr.). p. 564. ' Holtzman Company, Madison, Wisconsin.
1787
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On the Physiological Basis of Vitamin A-Stimulated Growth1
1788
ZILE, BUNGE AND DELUCA
250
200-
150
I
CD 100
50 Food restriction started t IO
Length h 2i
of
20
30
Dietary
31
41
Age of
Treatment, 51
40
Days 61
Rats , Days
Fig. 1 Growth of rats fed experimental and control diets. Closed circles, control rats; open circles, experimental rats (without dietary vitamin A ). The values represent the mean ±SEM ( indi cated by the bars) of 18 rats. The weight differ ences were significant ( P < 0.05 ) when the rats had been fed the experimental diet for 30 days. After that, food intake was controlled.
per nucleus, as established by Enesco and Leblond (12). Statistical analysis. Data were analyzed by the Student's t-test as outlined by Steel and Torrie ( 13). RESULTS
Growth of rats. In the rat there is a rapid increase in cell number (DNA) in all organs, until about the age of 13 to 17 days. From that time until about the age of 44 days the rate of DNA increase varies in different tissues. In the liver, spleen, submaxillary glands, testes and thymus the rapid proliferation of cells proceeds until 5 Cerelose, Corn Products Refining Co., New York. New York. • "Vitamin-free" test casein, Teklad test diets. Madison, Wisconsin. 7 Wesson oil, the Wesson Oil Co.. New Orleans, Louisiana. 8 Celiti flour. Chicago Dietetic Supply House, Chi cago. Illinois.
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lowing: (in %} glucose monohydrate,5 64.2; casein,6 18.0; cystine, 0.2; cotton seed oil,7 10.0; CaCOs, 1.09; equimolar mixture of KH2PO4 and K2HPO4, 0.9; roughage,8 3.0; choline chloride, 0.2; Ca- and P-free salt mixture, 2.0; sodium ascorbate, 0.1; and water soluble vitamins, 0.2. The watersoluble vitamin mixture consisted of: (in % ) thiamin, 0.5; riboflavin, 0.5; pyridoxine, 0.5; calcium pantothenate, 2.8; nicotinamide, 2.0; inositol, 20; folie acid, 0.02; vita min B-12, 0.002; biotin, 0.01 and glucose monohydrate,5 73.7. The Ca- and P-free salt mixture contained: (in %) KC1, 57.7; NaCl, 20.9; MgSO4> 17.9; FeSO4-7H2O, 3.22; CuSO4-5H,O, 0.078; NaF, 0.113; CoCl2-6HoO, 0.004; KI, 0.01; MnSO4-H2O, 0.04; ZnSO4-7H,O, 0.44; and (NH4)6Mo7O24-4H2O, 0.005. In addition, the rats re ceived three times a week a supplement of fat-soluble vitamins in cottonseed oil which supplied 105 /tg of 2-methyl-l,4-naphthoquinone, 875 /¿gof «-tocopherol and 225 IU of vitamin D2 weekly. The rats were divided into two groups, one of which re ceived no vitamin A (experimental group) while the other received 20 /xg of retinol orally every 2 days (control group). The rats were housed in a room with automati cally controlled light and dark cycles. When the rats in the experimental group ceased to gain weight at a constant rate, the growth of the control rats was kept at about the same rate as that of the experi mental group by controlling food intake. The rats were killed when the experimental rats reached a weight plateau stage and had been on it for 2 to 6 days. No distinc tion was made as to the exact number of days on the plateau and the tissues were treated as a group, except in the case of the sublingual glands; this is explained later in the Results section. The experimental group of rats showed no overt vitamin A deficiency signs except for their diminished growth. All rats were fasted for 16 to 18 hours prior to killing. Biochemical assays. Retinol was ex tracted from liver and serum and deter mined by established methods (11). The protein, DNA and RNA content of the tissues was analyzed by previously de scribed procedures (11). Cell number was calculated using the value of 6.2 pg DNA
VITAMIN
1789
A AND GROWTH
TABLE 1 Status of experimental animals
Final body weight, g1 Growth rate during depletion, g/day2 Liver retinol,6 /«g/g3 Liver retinol,5 ¿igtotal3 Serum retinol, >ig/100 ml3
237.1 ±3.5«(18) 6.20±0.12 (18) 24.75±2.36 (9) 253.55±7.89 (9) 54.23 ±3.85 (8)
Experimental 222.4 ±6.1 (18)* 5.74±0.19 (17)' Not detectable Not detectable 16.50±4.18 (7)**
1Weight determined before fasting. 2 Calculated for the first 30 days of depletion period and does not include weight plateau stage. 3Separate groups of rats were used for vitamin A determination. *The values represent the mean±SKMof the number of rats indicated in brackets. * Indicates a significant difference between the two groups at P < 0.05 or ** P < 0.01. 6Total liver extract was hydrolyzed and vitamin A determined as retinol (11).
about the age of 44 days (for thymus, 39 days). After this time, the addition of new cells (DNA) to these organs is very slow. The thymus gland actually begins to atrophy (decrease in DNA) (12, 14). The growth and development of testes is not well understood. When the testes of the rat descend at about the age of 40 days, they are still immature, and some growth and differentiation continues until maturity ( age about 80 days ). At this time the DNA content becomes constant due to the onset of a steady state in cellular population (12). In our studies we raised 21-day old weanling rats on a vitamin A-deficient diet until the age of 9 to 10 weeks. The only source of vitamin A for these rats during the very rapid growth period from wean ing to the age of 7 weeks was their preweaning vitamin A stores. The growth of these experimental rats was somewhat slower than that of the controls (fig. 1), in agreement with our previous observations (11). The smaller overall average weight gain for the experimental group (table 1) resulted from this decreased growth rate that became evident after 21 days ( fig. 1 ) and was statistically significant (P < 0.05) when the rats had been fed the diet for 30 days (51 days old). When the rats were killed (age 63 to 70 days) no vitamin A could be detected in the livers of the ex perimental group and their serum vitamin A had decreased to /a that of their vitamin A-supplemented controls (table 1). Composition of tissues. In assessing tis sue cellularity it is clearly preferable to express the biochemical composition per gram of tissue, rather than on the basis of tissue protein or DNA, because of the great
variability of composition in different tis sues. The effects of vitamin A-deprivation on the final weight and composition of various organs was measured in cellular terms by determining the cell number (total DNA of the tissue), cellularity ( DNA per gram tissue ) and cell size ( pro tein to DNA ratio) (14). Although the enumeration of cell nuclei of tissue homogenates is also an accepted estimate of cellularity, the chemical determination of DNA is a more accurate measure, espe cially for tissues with a high rate of pro liferation ( 15). The composition of various tissues is summarized in tables 2 to 7. The liver from the vitamin A-deprived rats was smaller than that of the respective controls, but the overall composition was not altered by the lack of dietary vitamin A during the period of growth (table 2), in agreement with findings of others ( 16). The testes of the Holtzman rat descend at the age of 40 days and reach full ma turity at 2 to 3 months. We found that the vitamin A-deprived rats had somewhat smaller testes (85% the size of controls) and that the decreased organ weight was associated with a reduced DNA (cell num ber) and protein content (table 3). The thymus gland of the Holtzman rat increases in size until the age of 6 weeks; thereafter a gradual involution occurs at the rate of 6% per week.9 The thymus gland was severely affected by the absence of vitamin A in the diet. Not only was the gland 30% smaller than that of the control, » M. Zile & H. F. DeLuca, unpublished 1976.
observations,
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Control
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ZILE, BUNGE AND DsLUCA
TABLE 2 Effect of vitamin A deficiency on composition of liver
Tissue, wetweight, gTi»ue, bodyweightProtein, % of
(17)2.77 ±0.12'
(36)»2.60 ±0.10
0.05(17)306.65 ±
(36)*317.24 ±0.03
wetweightDNA, mg/g (6)3.56 ±14.95
(14)3.65 ±12.76
wetweightRNA, mg/g (7)17.53 ±0.17
(15)24.12± ±0.12
(6)34.31 ±1.90 tissue(cell mg/ number)Protein/DNA(cell (7)87.00 ±2.64
(14)**88.85 1.13
wetweightDNA, mg/g
size)Control6.49
(14)24.19± 1.66
±7.20 (6)Vitamin
±3.55 (12)
1The values represent the mean ±»EM of the number of rats indicated in brackets. * Indicates a significant difference be tween the two groups at p < 0.01 ; ** p < 0.001.
but a. significant decrease had occurred in DNA and RNA concentration. The de creased thymus mass of the experimental rats was not proportional to the decrease in DNA content, as reflected in the amount of DNA per gram of thymus (table 4). Calculations revealed that each gram of thymus had about one billion fewer cells than the thymus from the corresponding controls. The composition and size of spleen (table 5) was also severely altered by vitamin A depletion. As with the thymus, there was a deficit of about one billion nuclei per gram of the spleen in the vita min A-deprived rat. The submaxillary glands (table 6) did not appear to be significantly affected by the lack of dietary vitamin A during growth after weaning. In contrast, the sublingual glands were markedly altered by the de ficiency (table 7). In this salivary gland there was an extreme change in its compo sition depending on the length of time on the weight plateau stage. At the beginning of the weight plateau, a change in the gland size was accompanied by alterations in RNA and protein composition. A few additional days on the weight plateau re sulted in 507c loss of DNA from the gland, accompanied by even larger (67% ) loss of protein, but causing a 2-fold rise in RNA content.
TABLE 3 Effect of vitamin A deficiency on composition of testes A deficient2.33 gTissue,weight, Tissue, bodyweightProtein, % of
(16)1.16±0.07 ±0.16' (16)116.61
wetweightDNA, mg/g
(32)*1.11 ±0.09 (32)102.19±3.8 ±0.04
(7)3.06 ±4.20
(13)*2.90±0.12
(6)%1.71 ±0.13
(15)1.72
wetweightRNA, mg/g wetweightDNA, mg/g (6)9.82 ±0.37 (cellnumber)Protein/DNA(cell mg/tissue (6)37.44 ±0.42 size)Control2.72
(15)7.41 ±0.09 (15)*36.44 ±0.60
±1.68(6)Vitamin
±1.57(13)
1The values represent the mean ±BEHof the number of rats indicated in brackets. * Indicates a significant difference be tween the two groups at p < 0.05.
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A deficient5.49
DISCUSSION
This study represents an initial survey in which we have attempted to assess the in fluence of lack of dietary vitamin A on the number and size of cells in various tissues of the rat. It is known that during certain critical periods of growth the number as well as the size of cells in organs can be influenced by the state of nutrition (16). When fed a vitamin A-deficient diet, the weanling rat is able to use the vitamin A stored in the liver and will show gross external manifes tations of vitamin A deficiency only when these stores are depleted (17, 18). In our studies here and earlier ( 11 ) we have ob served that during the depletion period, the growth rate of the vitamin A-deprived rats is not entirely comparable to that of the vitamin A-supplemented rats and that early in the depletion period (diet fed for 9 to 14 days) there is decreased food utili zation (19) and alterations in calcium me tabolism (20). Other workers have re ported pathological changes in the thyroid gland (21), epiphyseal cartilage (22) and parotid ducts (23, 24) before vitamin A depletion was complete. Since these ani mals were still growing normally at the time, the abnormal metabolic manifesta tions must be the direct result of the lack of dietary vitamin A even in the presence of liver vitamin A. While there is unrefutable evidence that vitamin A influences growth, examination
1791
VITAMIN A AND GROWTH TABLE 4 Effect of vitamin A deficiency on composition of thymus
Tissue gTissue,weight, weightDry % of body weightLipids, weight, mg/g wet weightProtein, mg/g wet weightDNA, mg/g wet weightRNA, mg/g wet weightDNA, mg/g wet number)Protein/DNA mg/total tissue (cell (cell size)0.434±0.014>
(35)0.18 (35)215.6±0.01 (8)32.12±0.52 ±0.31127.59 ±4.2728.14 (8)5.55±1.00 ±0.2812.65 (8)4.57±0.91 ±0.19
Vitamin A deficient (36)**0.12 (36)**213.0 ±0.01 (8)32.10±8.9 ±0.32133.26 ±4.0722.67 (8)*4.03 ±1.61 ±0.14*6.91 (8)*5.60 ±0.97 (8)0.287±0.020 ±0.19*
1The values represent the mean±SEMof the number of rats indicated in brackets. nificant difference between the two groups at P < 0.01 ; ** P < 0.001.
of the effects of vitamin A deficiency on cell division (mitosis) in individual tissues in vivo (1, 11, 25-28) and in vitro (25, 26, 29, 30) have yielded contradictory results which cannot be reconciled at the present time because of the lack of understanding of the molecular functions of this vitamin. Several workers (6, 31, 32) have sug gested that cell division in the vitamin Adeprived animal is not reduced until after some metabolic alterations have occurred that cause the weight loss. Our animals reached the weight plateau stage of vita min A depletion at least 2 weeks after the end of the proliferative growth period. Thus one would anticipate that the total number of cells per organ in the vitamin A-deprived animal at its weight plateau would be comparable to that of the re spective organ in the vitamin A-supplemented controls. This clearly was not the case in our studies: the decreased weights of all tissues that were examined in the
* Indicates a sig
vitamin A-deprived rats were associated with a definite reduction in total DNA (cell number) (tables 2 to 7). These re sults suggest that the addition of new cells to these tissues during the growing stages after weaning had been significantly im peded by the absence of dietary vitamin A. The possibility exists that a small differ ence in food intake may have occurred prior to food regulation (from the age of 42 days to 51 days, see fig. 1) and could have affected the growth of the tissues at that time. However, in the tissues that we examined the increase in DNA during this period is very small and does not account for the much larger differences we found as a result of vitamin A deprivation at the end of the study. In tissues such as the liver which has a very slow proliferation rate once it has matured and which is generally thought not to be a target tissue for vitamin A action, the DNA content appeared to be
TABLE 5 Composition of spleen during vitamin A depletion Control gTissue, Tissue weight, weightDry % of body weightLipids, weight, mg/g wet weightProtein, mg/g wet weightDNA, mg/g wet weightRNA, mg/g wet weightDNA, mg/g wet number)Protein/DNA mg/total tissue (cell (cell size)0.421
Experimental
(35)0.20 ±0.013' ±0*221.6 (35)218.2±0 (8)59.63±1.6 (8)67.45±9.2 (8)93.56±2.36 (8)78.00±2.80 (8)*13.52 ±3.16 (8)19.22±2.51 (8)*3.27 ±0.36 (8)3.83±1.11 (8)9.68±0.21 (8)4.58±0.29 (8)*5.81 ±0.51 (8)4.57±0.50 ±0.25 (8)0.358±0.058**0.17 ±0.39 (8)
1The values represent the mean±SEMof the number of rats indicated in brackets. nificant difference between the two groups at P < 0.01; ** P < 0.00l.
* Indicates a sig
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Control
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ZILE, BUNGE AND DeLUCA TABLE 6 Effect of vitamin A deficiency on submaxillary gland composition
gTissue, Tissue weight, weightDry % of body weightLipids, weight, mg/g wet weightProtein, mg/g wet weightDNA, mg/g wet weightRNA, mg/g wet weightDNA, mg/g wet number)Protein/DNA mg/total tissue (cell (cell size)0.514±0.087'
Experimental
(35)0.216±0.036' (35)*0.211 ±0.074 (35)235.5 (35)245.1 ±0.035 (6)277.80 ±2.6 (6)84.41 ±3.8 (6)74.87 ±1.51 (6)83.56 ±2.58 (6)9.31 ±0.41 (6)9.24 ±3.44 (6)9.71 ±0.82 (6)10.22 ±0.79 (6)4.81 ±0.31 (6)3.98 ±0.25 (6)8.84 ±0.35 (6)9.41 ±0.30 ±0.48 (6)0.444 ±0.57 (6)
1The calculations of tissue weights represent the mean±SEMof the number of rats indicated in brackets. 2The values for the biochemical components were obtained by pooling the tissues of three rats for each determination ; the number of determinations made is indicated in brackets. * Indicates a significant difference between the two groups at P < 0.001.
directly related to organ size, but due to the absence of dietary supplement of vita min A, the growth of liver reached only 84% the weight of the normal control. However, the unchanged biochemical comEosition of the existing cells suggests that epatic function is normal at this time. Wolbach (1) observed no histological changes in liver even in severely vitamin A-deficient rats. Others (33) have re ported that vitamin A deficiency does not alter liver RNA and protein content, but they observed a loss of glycogen. However, since the food intake of their animals was not regulated, the decrease in the glycogen content is most likely due to simple inani tion of the vitamin A-deficient rats. It is well established that the testes of the rat degenerate in vitamin A deficiency (1, 34). McHowell et al. (35) reported that the first histological lesion in the testes was a loss of spermatids which occurred 1
to 3 weeks after the rats developed the classic signs of vitamin A deficiency. But Bieri et al. (36) found that the levels of vitamin A that maintain body weight were insufficient to maintain testes in a normal condition. This later interpretation is sup ported by our present findings, which sug gest that the growth and development of the testes is already affected during the de pletion period, before the complete exhaus tion of liver vitamin A stores and before the appearance of external vitamin A defi ciency signs. The testes may be particu larly sensitive to the lack of dietary vitamin A as the growth and differentiation of this organ proceed until the age of about 80 days (12). In renewing tissues, such as epithelia of the small intestine, the lining of ducts and the skin, and the erythrocyte- and lympho cyte-forming systems, the production of new cells during the period of growth in-
TABLE 7 Vitamin A depletion and sublingual gland composition Control
Vitamin A-deficient (early plateau)
P
Vitamin A-deficient (late plateau)
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Control
±0.025'0.100±0.00972.59 ±0.0060.089±0.01191.43 gTissue,weight, Tissue ±0.00722.56 weightProtein, % body ±4.123.19 ±2.751.48 ±4.952.91 weightDNA, mg/g wet ±0.442.37 ±0.544.37 ±0.105.38 weightRNA, mg/g wet ±0.760.566 ±0.680.289 ±0.130.704 weightDNA, mg/g wet mg/tissue(cell ±0.6331.07 815.60±0.01 ±0.03626.46 number)Protein/DNA ±3.71(5)(5)(5)(5)(5)(5)(5)0.05
for the smaller tissue mass associated with vitamin A deficiency. On the other hand, a decrease in total DNA in the vitamin Adeficient tissue would suggest an impair ment in cell division. Lamb et al. ( 10 ) have discussed the lim itations in designing nutritional experi ments for the study of vitamin A. To circumvent these difficulties, we have used vitamin A-deficient animals before they exhibit overt deficiency symptoms. Our re sults demonstrate that vitamin A deficiency causes a decrease in the number of cells of several tissues suggesting a general role of vitamin A in cellular proliferation. MATERIALS
AND METHODS
Animals. Weanling male rats 4 were fed a complete purified diet deficient only in vitamin A. The diet consisted of the folRecelved for publication February 12, 1979. 1 Supported by a program-project grant from the National Institutes of Health No. AM-14881. 2 Author to whom reprint requests should be ad"Ahlinvalia. B. & Bieri. J. G. (1970) Effects of hormones on liver vitamin A. Federation Proc. 29 (Abstr.). p. 564. ' Holtzman Company, Madison, Wisconsin.
1787
Downloaded from https://academic.oup.com/jn/article-abstract/109/10/1787/4770639 by Tulane University Medical Library user on 20 January 2019
On the Physiological Basis of Vitamin A-Stimulated Growth1
1788
ZILE, BUNGE AND DELUCA
250
200-
150
I
CD 100
50 Food restriction started t IO
Length h 2i
of
20
30
Dietary
31
41
Age of
Treatment, 51
40
Days 61
Rats , Days
Fig. 1 Growth of rats fed experimental and control diets. Closed circles, control rats; open circles, experimental rats (without dietary vitamin A ). The values represent the mean ±SEM ( indi cated by the bars) of 18 rats. The weight differ ences were significant ( P < 0.05 ) when the rats had been fed the experimental diet for 30 days. After that, food intake was controlled.
per nucleus, as established by Enesco and Leblond (12). Statistical analysis. Data were analyzed by the Student's t-test as outlined by Steel and Torrie ( 13). RESULTS
Growth of rats. In the rat there is a rapid increase in cell number (DNA) in all organs, until about the age of 13 to 17 days. From that time until about the age of 44 days the rate of DNA increase varies in different tissues. In the liver, spleen, submaxillary glands, testes and thymus the rapid proliferation of cells proceeds until 5 Cerelose, Corn Products Refining Co., New York. New York. • "Vitamin-free" test casein, Teklad test diets. Madison, Wisconsin. 7 Wesson oil, the Wesson Oil Co.. New Orleans, Louisiana. 8 Celiti flour. Chicago Dietetic Supply House, Chi cago. Illinois.
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lowing: (in %} glucose monohydrate,5 64.2; casein,6 18.0; cystine, 0.2; cotton seed oil,7 10.0; CaCOs, 1.09; equimolar mixture of KH2PO4 and K2HPO4, 0.9; roughage,8 3.0; choline chloride, 0.2; Ca- and P-free salt mixture, 2.0; sodium ascorbate, 0.1; and water soluble vitamins, 0.2. The watersoluble vitamin mixture consisted of: (in % ) thiamin, 0.5; riboflavin, 0.5; pyridoxine, 0.5; calcium pantothenate, 2.8; nicotinamide, 2.0; inositol, 20; folie acid, 0.02; vita min B-12, 0.002; biotin, 0.01 and glucose monohydrate,5 73.7. The Ca- and P-free salt mixture contained: (in %) KC1, 57.7; NaCl, 20.9; MgSO4> 17.9; FeSO4-7H2O, 3.22; CuSO4-5H,O, 0.078; NaF, 0.113; CoCl2-6HoO, 0.004; KI, 0.01; MnSO4-H2O, 0.04; ZnSO4-7H,O, 0.44; and (NH4)6Mo7O24-4H2O, 0.005. In addition, the rats re ceived three times a week a supplement of fat-soluble vitamins in cottonseed oil which supplied 105 /tg of 2-methyl-l,4-naphthoquinone, 875 /¿gof «-tocopherol and 225 IU of vitamin D2 weekly. The rats were divided into two groups, one of which re ceived no vitamin A (experimental group) while the other received 20 /xg of retinol orally every 2 days (control group). The rats were housed in a room with automati cally controlled light and dark cycles. When the rats in the experimental group ceased to gain weight at a constant rate, the growth of the control rats was kept at about the same rate as that of the experi mental group by controlling food intake. The rats were killed when the experimental rats reached a weight plateau stage and had been on it for 2 to 6 days. No distinc tion was made as to the exact number of days on the plateau and the tissues were treated as a group, except in the case of the sublingual glands; this is explained later in the Results section. The experimental group of rats showed no overt vitamin A deficiency signs except for their diminished growth. All rats were fasted for 16 to 18 hours prior to killing. Biochemical assays. Retinol was ex tracted from liver and serum and deter mined by established methods (11). The protein, DNA and RNA content of the tissues was analyzed by previously de scribed procedures (11). Cell number was calculated using the value of 6.2 pg DNA
VITAMIN
1789
A AND GROWTH
TABLE 1 Status of experimental animals
Final body weight, g1 Growth rate during depletion, g/day2 Liver retinol,6 /«g/g3 Liver retinol,5 ¿igtotal3 Serum retinol, >ig/100 ml3
237.1 ±3.5«(18) 6.20±0.12 (18) 24.75±2.36 (9) 253.55±7.89 (9) 54.23 ±3.85 (8)
Experimental 222.4 ±6.1 (18)* 5.74±0.19 (17)' Not detectable Not detectable 16.50±4.18 (7)**
1Weight determined before fasting. 2 Calculated for the first 30 days of depletion period and does not include weight plateau stage. 3Separate groups of rats were used for vitamin A determination. *The values represent the mean±SKMof the number of rats indicated in brackets. * Indicates a significant difference between the two groups at P < 0.05 or ** P < 0.01. 6Total liver extract was hydrolyzed and vitamin A determined as retinol (11).
about the age of 44 days (for thymus, 39 days). After this time, the addition of new cells (DNA) to these organs is very slow. The thymus gland actually begins to atrophy (decrease in DNA) (12, 14). The growth and development of testes is not well understood. When the testes of the rat descend at about the age of 40 days, they are still immature, and some growth and differentiation continues until maturity ( age about 80 days ). At this time the DNA content becomes constant due to the onset of a steady state in cellular population (12). In our studies we raised 21-day old weanling rats on a vitamin A-deficient diet until the age of 9 to 10 weeks. The only source of vitamin A for these rats during the very rapid growth period from wean ing to the age of 7 weeks was their preweaning vitamin A stores. The growth of these experimental rats was somewhat slower than that of the controls (fig. 1), in agreement with our previous observations (11). The smaller overall average weight gain for the experimental group (table 1) resulted from this decreased growth rate that became evident after 21 days ( fig. 1 ) and was statistically significant (P < 0.05) when the rats had been fed the diet for 30 days (51 days old). When the rats were killed (age 63 to 70 days) no vitamin A could be detected in the livers of the ex perimental group and their serum vitamin A had decreased to /a that of their vitamin A-supplemented controls (table 1). Composition of tissues. In assessing tis sue cellularity it is clearly preferable to express the biochemical composition per gram of tissue, rather than on the basis of tissue protein or DNA, because of the great
variability of composition in different tis sues. The effects of vitamin A-deprivation on the final weight and composition of various organs was measured in cellular terms by determining the cell number (total DNA of the tissue), cellularity ( DNA per gram tissue ) and cell size ( pro tein to DNA ratio) (14). Although the enumeration of cell nuclei of tissue homogenates is also an accepted estimate of cellularity, the chemical determination of DNA is a more accurate measure, espe cially for tissues with a high rate of pro liferation ( 15). The composition of various tissues is summarized in tables 2 to 7. The liver from the vitamin A-deprived rats was smaller than that of the respective controls, but the overall composition was not altered by the lack of dietary vitamin A during the period of growth (table 2), in agreement with findings of others ( 16). The testes of the Holtzman rat descend at the age of 40 days and reach full ma turity at 2 to 3 months. We found that the vitamin A-deprived rats had somewhat smaller testes (85% the size of controls) and that the decreased organ weight was associated with a reduced DNA (cell num ber) and protein content (table 3). The thymus gland of the Holtzman rat increases in size until the age of 6 weeks; thereafter a gradual involution occurs at the rate of 6% per week.9 The thymus gland was severely affected by the absence of vitamin A in the diet. Not only was the gland 30% smaller than that of the control, » M. Zile & H. F. DeLuca, unpublished 1976.
observations,
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Control
1790
ZILE, BUNGE AND DsLUCA
TABLE 2 Effect of vitamin A deficiency on composition of liver
Tissue, wetweight, gTi»ue, bodyweightProtein, % of
(17)2.77 ±0.12'
(36)»2.60 ±0.10
0.05(17)306.65 ±
(36)*317.24 ±0.03
wetweightDNA, mg/g (6)3.56 ±14.95
(14)3.65 ±12.76
wetweightRNA, mg/g (7)17.53 ±0.17
(15)24.12± ±0.12
(6)34.31 ±1.90 tissue(cell mg/ number)Protein/DNA(cell (7)87.00 ±2.64
(14)**88.85 1.13
wetweightDNA, mg/g
size)Control6.49
(14)24.19± 1.66
±7.20 (6)Vitamin
±3.55 (12)
1The values represent the mean ±»EM of the number of rats indicated in brackets. * Indicates a significant difference be tween the two groups at p < 0.01 ; ** p < 0.001.
but a. significant decrease had occurred in DNA and RNA concentration. The de creased thymus mass of the experimental rats was not proportional to the decrease in DNA content, as reflected in the amount of DNA per gram of thymus (table 4). Calculations revealed that each gram of thymus had about one billion fewer cells than the thymus from the corresponding controls. The composition and size of spleen (table 5) was also severely altered by vitamin A depletion. As with the thymus, there was a deficit of about one billion nuclei per gram of the spleen in the vita min A-deprived rat. The submaxillary glands (table 6) did not appear to be significantly affected by the lack of dietary vitamin A during growth after weaning. In contrast, the sublingual glands were markedly altered by the de ficiency (table 7). In this salivary gland there was an extreme change in its compo sition depending on the length of time on the weight plateau stage. At the beginning of the weight plateau, a change in the gland size was accompanied by alterations in RNA and protein composition. A few additional days on the weight plateau re sulted in 507c loss of DNA from the gland, accompanied by even larger (67% ) loss of protein, but causing a 2-fold rise in RNA content.
TABLE 3 Effect of vitamin A deficiency on composition of testes A deficient2.33 gTissue,weight, Tissue, bodyweightProtein, % of
(16)1.16±0.07 ±0.16' (16)116.61
wetweightDNA, mg/g
(32)*1.11 ±0.09 (32)102.19±3.8 ±0.04
(7)3.06 ±4.20
(13)*2.90±0.12
(6)%1.71 ±0.13
(15)1.72
wetweightRNA, mg/g wetweightDNA, mg/g (6)9.82 ±0.37 (cellnumber)Protein/DNA(cell mg/tissue (6)37.44 ±0.42 size)Control2.72
(15)7.41 ±0.09 (15)*36.44 ±0.60
±1.68(6)Vitamin
±1.57(13)
1The values represent the mean ±BEHof the number of rats indicated in brackets. * Indicates a significant difference be tween the two groups at p < 0.05.
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A deficient5.49
DISCUSSION
This study represents an initial survey in which we have attempted to assess the in fluence of lack of dietary vitamin A on the number and size of cells in various tissues of the rat. It is known that during certain critical periods of growth the number as well as the size of cells in organs can be influenced by the state of nutrition (16). When fed a vitamin A-deficient diet, the weanling rat is able to use the vitamin A stored in the liver and will show gross external manifes tations of vitamin A deficiency only when these stores are depleted (17, 18). In our studies here and earlier ( 11 ) we have ob served that during the depletion period, the growth rate of the vitamin A-deprived rats is not entirely comparable to that of the vitamin A-supplemented rats and that early in the depletion period (diet fed for 9 to 14 days) there is decreased food utili zation (19) and alterations in calcium me tabolism (20). Other workers have re ported pathological changes in the thyroid gland (21), epiphyseal cartilage (22) and parotid ducts (23, 24) before vitamin A depletion was complete. Since these ani mals were still growing normally at the time, the abnormal metabolic manifesta tions must be the direct result of the lack of dietary vitamin A even in the presence of liver vitamin A. While there is unrefutable evidence that vitamin A influences growth, examination
1791
VITAMIN A AND GROWTH TABLE 4 Effect of vitamin A deficiency on composition of thymus
Tissue gTissue,weight, weightDry % of body weightLipids, weight, mg/g wet weightProtein, mg/g wet weightDNA, mg/g wet weightRNA, mg/g wet weightDNA, mg/g wet number)Protein/DNA mg/total tissue (cell (cell size)0.434±0.014>
(35)0.18 (35)215.6±0.01 (8)32.12±0.52 ±0.31127.59 ±4.2728.14 (8)5.55±1.00 ±0.2812.65 (8)4.57±0.91 ±0.19
Vitamin A deficient (36)**0.12 (36)**213.0 ±0.01 (8)32.10±8.9 ±0.32133.26 ±4.0722.67 (8)*4.03 ±1.61 ±0.14*6.91 (8)*5.60 ±0.97 (8)0.287±0.020 ±0.19*
1The values represent the mean±SEMof the number of rats indicated in brackets. nificant difference between the two groups at P < 0.01 ; ** P < 0.001.
of the effects of vitamin A deficiency on cell division (mitosis) in individual tissues in vivo (1, 11, 25-28) and in vitro (25, 26, 29, 30) have yielded contradictory results which cannot be reconciled at the present time because of the lack of understanding of the molecular functions of this vitamin. Several workers (6, 31, 32) have sug gested that cell division in the vitamin Adeprived animal is not reduced until after some metabolic alterations have occurred that cause the weight loss. Our animals reached the weight plateau stage of vita min A depletion at least 2 weeks after the end of the proliferative growth period. Thus one would anticipate that the total number of cells per organ in the vitamin A-deprived animal at its weight plateau would be comparable to that of the re spective organ in the vitamin A-supplemented controls. This clearly was not the case in our studies: the decreased weights of all tissues that were examined in the
* Indicates a sig
vitamin A-deprived rats were associated with a definite reduction in total DNA (cell number) (tables 2 to 7). These re sults suggest that the addition of new cells to these tissues during the growing stages after weaning had been significantly im peded by the absence of dietary vitamin A. The possibility exists that a small differ ence in food intake may have occurred prior to food regulation (from the age of 42 days to 51 days, see fig. 1) and could have affected the growth of the tissues at that time. However, in the tissues that we examined the increase in DNA during this period is very small and does not account for the much larger differences we found as a result of vitamin A deprivation at the end of the study. In tissues such as the liver which has a very slow proliferation rate once it has matured and which is generally thought not to be a target tissue for vitamin A action, the DNA content appeared to be
TABLE 5 Composition of spleen during vitamin A depletion Control gTissue, Tissue weight, weightDry % of body weightLipids, weight, mg/g wet weightProtein, mg/g wet weightDNA, mg/g wet weightRNA, mg/g wet weightDNA, mg/g wet number)Protein/DNA mg/total tissue (cell (cell size)0.421
Experimental
(35)0.20 ±0.013' ±0*221.6 (35)218.2±0 (8)59.63±1.6 (8)67.45±9.2 (8)93.56±2.36 (8)78.00±2.80 (8)*13.52 ±3.16 (8)19.22±2.51 (8)*3.27 ±0.36 (8)3.83±1.11 (8)9.68±0.21 (8)4.58±0.29 (8)*5.81 ±0.51 (8)4.57±0.50 ±0.25 (8)0.358±0.058**0.17 ±0.39 (8)
1The values represent the mean±SEMof the number of rats indicated in brackets. nificant difference between the two groups at P < 0.01; ** P < 0.00l.
* Indicates a sig
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Control
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ZILE, BUNGE AND DeLUCA TABLE 6 Effect of vitamin A deficiency on submaxillary gland composition
gTissue, Tissue weight, weightDry % of body weightLipids, weight, mg/g wet weightProtein, mg/g wet weightDNA, mg/g wet weightRNA, mg/g wet weightDNA, mg/g wet number)Protein/DNA mg/total tissue (cell (cell size)0.514±0.087'
Experimental
(35)0.216±0.036' (35)*0.211 ±0.074 (35)235.5 (35)245.1 ±0.035 (6)277.80 ±2.6 (6)84.41 ±3.8 (6)74.87 ±1.51 (6)83.56 ±2.58 (6)9.31 ±0.41 (6)9.24 ±3.44 (6)9.71 ±0.82 (6)10.22 ±0.79 (6)4.81 ±0.31 (6)3.98 ±0.25 (6)8.84 ±0.35 (6)9.41 ±0.30 ±0.48 (6)0.444 ±0.57 (6)
1The calculations of tissue weights represent the mean±SEMof the number of rats indicated in brackets. 2The values for the biochemical components were obtained by pooling the tissues of three rats for each determination ; the number of determinations made is indicated in brackets. * Indicates a significant difference between the two groups at P < 0.001.
directly related to organ size, but due to the absence of dietary supplement of vita min A, the growth of liver reached only 84% the weight of the normal control. However, the unchanged biochemical comEosition of the existing cells suggests that epatic function is normal at this time. Wolbach (1) observed no histological changes in liver even in severely vitamin A-deficient rats. Others (33) have re ported that vitamin A deficiency does not alter liver RNA and protein content, but they observed a loss of glycogen. However, since the food intake of their animals was not regulated, the decrease in the glycogen content is most likely due to simple inani tion of the vitamin A-deficient rats. It is well established that the testes of the rat degenerate in vitamin A deficiency (1, 34). McHowell et al. (35) reported that the first histological lesion in the testes was a loss of spermatids which occurred 1
to 3 weeks after the rats developed the classic signs of vitamin A deficiency. But Bieri et al. (36) found that the levels of vitamin A that maintain body weight were insufficient to maintain testes in a normal condition. This later interpretation is sup ported by our present findings, which sug gest that the growth and development of the testes is already affected during the de pletion period, before the complete exhaus tion of liver vitamin A stores and before the appearance of external vitamin A defi ciency signs. The testes may be particu larly sensitive to the lack of dietary vitamin A as the growth and differentiation of this organ proceed until the age of about 80 days (12). In renewing tissues, such as epithelia of the small intestine, the lining of ducts and the skin, and the erythrocyte- and lympho cyte-forming systems, the production of new cells during the period of growth in-
TABLE 7 Vitamin A depletion and sublingual gland composition Control
Vitamin A-deficient (early plateau)
P
Vitamin A-deficient (late plateau)
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Control
±0.025'0.100±0.00972.59 ±0.0060.089±0.01191.43 gTissue,weight, Tissue ±0.00722.56 weightProtein, % body ±4.123.19 ±2.751.48 ±4.952.91 weightDNA, mg/g wet ±0.442.37 ±0.544.37 ±0.105.38 weightRNA, mg/g wet ±0.760.566 ±0.680.289 ±0.130.704 weightDNA, mg/g wet mg/tissue(cell ±0.6331.07 815.60±0.01 ±0.03626.46 number)Protein/DNA ±3.71(5)(5)(5)(5)(5)(5)(5)0.05
