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In utero exposure to maternal low protein diets induces hypertension in weanling rats, independently of maternal blood pressure changes
Clinical Nutrition (1994) 13: 319-324 0 Longman Group Ltd 1994
In utero exposure to maternal low protein diets induces hypertension in weanling rats, independently of maternal blood pressure changes S. C. LANGLEY-EVANS,
G. J. PHILLIPS and A. A. JACKSON
Department of Human Nutrition, University 3TU, UK (Correspondence to SCL-EI
of Southampton,
Bassett Crescent fast,
Southampton
SO9
ABSTRACT-The association between maternal nutrition, fetal growth and the later development of hypertension was investigated in the rat. Animals were habituated to diets containing 18% (control) or 9% (low) protein by weight. The rats were mated and maintained on the diets until the end of pregnancy. Lactating dams were transferred onto standard chow diet. Systolic blood pressure was determined in male and female weanling offspring, using an indirect tail-cuff method. To assess the direct effects of low protein diets upon blood pressure of adult animals, a group of male and female rats were fed 18% or 9% protein for 14 days. Blood pressure was determined at the beginning and end of the feeding period. Blood pressure was additionally assessed over 14 days in pregnant rats fed control or low protein diets. Low protein diets did not alter systolic blood pressure in adult male or female rats. The blood pressures of pregnant females fed 18% or 9% protein diets did not significantly differ at any stage of pregnancy. Rats fed 9% protein diets gave birth to significantly smaller pups. Litter sizes were unaltered, and no differences in perinatal mortality were observed. Pups exposed to maternal low protein in utero had higher systolic blood pressure at the age of 4 weeks, when compared to control pups. The phenomenon was observed in both male and female offspring. Blood pressures at 4 weeks of age were strongly associated with maternal protein intake (r = -0.55). Associations were also noted between blood pressure and maternal weight at mating (r = 0.48), and weight gain in pregnancy (r = -0.30). Fetal exposure to maternal low protein diets induces hypertension in rats. The phenomenon is observed early in life and is independent of sex and the influence of maternal blood pressure. The low protein diet itself did not produce an increase in the blood pressure of adult rats.
fetus and the full-grown offspring resulting from the pregnancy (12, 13, 15-17). Most recently it has been demonstrated, in our laboratory, that rats exposed to maternal diets containing between 12% and 6% protein in utero develop significant hypertension relative to control rats exposed to 18% protein (1.5, 18, 19). These hypertensive rats have elevated systolic blood pressures associated with increased pulmonary angiotensin converting enzyme (ACE) activity (15), and their raised blood pressure is responsive to captopril treatment (20). The effects of ACE inhibitor therapy are long-lasting and strongly resemble observations in spontaneously hypertensive rats (21-23). This may suggest that, as in the spontaneously hypertensive strain, rats with maternal-diet-induced
Introduction Recent epidemiological evidence suggests that there is a significant association between the development of non-communicable diseases of adulthood and maternal nutrition (1, 2). Lower birthweightiplacental weight ratios increase risk of coronary heart disease (3-5), hypertension (6, 7), and diabetes (8, 9) in later life. Poor fetal growth has been associated with less than optimal maternal nutritional status in pregnancy (1, 2, 10, 1l), and animal studies suggest effects of maternal diet upon placental and fetal development (12-14). In the rat the feeding of diets low in protein during pregnancy has a number of effects upon the placenta, 319
320 MATERNAL NUTRITION AND HYPERTENSION IN WEANLING RATS
hypertension may have altered vascular structure (21-23). The findings of initial studies with the maternal low protein diet-induced model of hypertension in the rat (15) are of considerable importance. The Health of the Nation initiative requires a reduction of the incidence of hypertension and related cardiovascular disease in the UK population (24), and our findings, if confirmed, may influence future public health planning. However, the early findings from our laboratory were considered inconclusive (25). The use of only female offspring for blood pressure determinations was perhaps selective, and it was suggested that high blood pressures in the offspring of rats fed low protein diets may have represented a survival mechanism (25). Additionally the blood pressures of the parents remained an outstanding question. In the present paper we have addressed the majority of the criticisms directed at our preliminary studies. The new data will hopefully clear the way forward, allowing detailed examination of the molecular mechanisms underlying these important observations.
Methods 14 virgin, female Wistar rats bred in the Southampton University Animal Facility (initial weight 200-225 g) were housed individually in wire mesh cages maintained at 24°C on a 12 h light cycle. Rats were provided with synthetic diets containing 18% or 9% protein, as previously described (15). Protein was provided in the form of casein, with supplements (50 g/kg) of D,L-methionine to avoid sulphur deficiency. The diets differed principally in terms of protein content (90 g/kg difference), although the 9% casein diet contained 60 g/kg more starch and 30 g/kg more sucrose than the 18% casein diet, to equalise the energy content. All rats had free access to water. A period of 14 days was allowed to habituate the rats to the diets prior to mating. The feeding regime was maintained throughout the mating period (l-4 days), and pregnancy. The appearance of a mucous plug was taken to indicate that conception had occurred (pregnancy day 0). Within an hour of birth the mothers were all transferred to CRMX chow (SDS Ltd, Cambs.), which was fed throughout suckling, and was the diet onto which the pups were weaned at 4 weeks of age. At birth all litters of over 8 pups were thinned to 8, with the excess being killed by cervical dislocation. Birthweight was recorded for 48 pups removed from their dams. Other pups were not handled until 3 weeks old. Two dams in each
dietary group failed to carry their pregnancies to full term. Pups were finally removed from their mothers at 4 weeks old, after which they were housed in groups of 2 or 3. One to two days after this separation the systolic blood pressure of all the pups was determined by an indirect tail-cuff method, as previously described (15). Arterial pulses were detected using a light sensor, and recorded by an IITC Blood Pressure Monitor (Linton Instruments, Diss, UK). A cuff was placed over the tail and inflated to 300 mm Hg to occlude blood flow. The cuff was allowed to deflate at 3 mm Hg per second, and systolic blood pressure taken as the pressure at which tail pulses reappeared. Care was taken to ensure that the cuff was located at the top of the tail. All pups were of similar size, so any artefact of tail size would be considered unlikely. The effects of low protein diet on the blood pressures of adult rats were determined in two separate studies. These animals included 18 females used in a breeding programme identical to that described above, and 12 males of a similar weight (200-225 g). All animals had been reared on CRMX chow throughout prenatal and postnatal life. Blood pressures were determined in males before and after 2 weeks of feeding on 18% casein or 9% casein. Similarly, the blood pressures of the females were assessed at the start of feeding, and then at weekly intervals, until day 14 of pregnancy. The pregnant females included the 14 rats used to generate the weanling offspring described above. These studies allowed the assessment of whether the low protein diet itself may directly raise blood pressure in adult rats, and, furthermore, whether the blood pressures observed in the offspring of rats fed the diets during pregnancy, might be accounted for by either an effect of the diets on maternal blood pressure during pregnancy, or genetic factors. Statistical analysis
Data are presented as means f SEM. Analyses were performed using one or two way analysis of variance (ANOVA) as appropriate. Where significant interactions were noted a Tukey test was performed as a secondary test. A probability of 5% or less was accepted as statistically significant.
Results The effects of the different dietary regimens upon systolic blood pressures of adult male and female rats
CLINICAL NUTRITION
Table 1 The effects of feeding low protein diet upon systolic blood pressure in adult male and female rats
Diet
n
18% casein 9% casein
15 1.5
Systolic blood pressure (mm Hg) Day 0 Day 14 133f5 136f4
138f5 143f6
Adult male and female rats (200-225 g) were fed the different diets for 14 days. Blood pressure was determined on day 0 and 14. Data are mean _+SEM for n observations. ANOVA indicated no effects of diet at either time point.
Table 2 The effects of feeding low protein diet upon systolic blood pressure in adult female rats during pregnancy
Diet
n
Systolic blood pressure (mm Hg) Day 0 Day 7 Day 14
18% casein 9% casein
11 6
139 * 10 141 f 8
130+ 11 126+6
124 f 5+ 120 + 7’
Adult female rats (200-225 g) were fed the different diets for 14 days, prior to mating and during pregnancy. Blood pressure was determined on days 0 and 14 of the pre-pregnancy period and on days 0,7 and 14 of pregnancy. Data are mean + SEM for n observations. ANOVA indicated an interaction of pregnancy and time (F = 5.41 p < 0.005). +indicates significantly different to pregnancy day 0 (P < 0.05).
are shown in Table 1. In males and females, feeding 18% casein or 9% casein over 2 weeks had no significant effects upon blood pressure. Pregnancy (Table 2) had no significant effects upon blood pressure, but during the first 2 weeks of gestation, significant falls in blood pressure were noted in both the 18% and 9% casein groups. Pregnant rat dams fed the 9% protein diet consumed significantly less protein than the 18% protein fed controls (2.52 f 0.25 g protein/day vs 4.89 + 0.10 g protein/day, F = 46.59, P < 0.0001). Food intake was similar in both groups. At mating, rats habituated to 9% casein over 14 days were significantly heavier than 18% casein fed controls (18% casein: 250 + 3 g; 9% casein: 274 f 3 g, P < 0.05). At the time of birth all rats were of similar weight (18% casein: 391 + 8 g; 9% casein 401 + 7 g). Weight gains during pregnancy were similar in both groups (Table 3), as were the resulting litter sizes. Immediately following delivery all litters were thinned to a maximum of 8 pups, and the body weights of the randomly removed pups recorded. Significant effects of maternal diet upon birthweight were observed (Table 3). Pups delivered by rats on the 9% casein diet were significantly smaller than those delivered by dams in the 18% protein control group. Survival in the perinatal period was similar in both groups.
321
Table 3 Effects of maternal diet on outcome of pregnancy and maternal growth Diet
18% casein n 9% casein n
Food intake g/day
Weight gain in pregnancy g
Litter size no pups
Birth weight g
27.0 f 1.5 5 28.0 f 1.O 5
141?sll 5 120+7 5
12.2+2.1 5 13.3 f 1.8 5
5.250.1 22 4.7 * 0.1+ 26
Virgin female rats were habituated to the dietary regimes over 14 days before mating. Food intake and weight gain were recorded through pregnancy. Litter size and birthweight of pups were assessed within an hour of birth. Data are means f SEM for n observations. “indicated significantly different to 18% casein group, P < 0.05. ANOVA indicated significant effects of diet on birthweight, (F = 34, 82. P < 0.0001).
Table 4 The effects of maternal diet upon systolic blood pressure of offspring at 4 weeks of age Systolic blood pressure (mm Hg) Diet 18% casein Male Female 9% casein Male Female
;o 16 21 20
91 f5 90+5 119fS 120 f 4’
Blood pressure was determined at 4 weeks of age. Data are mean & SEM for n observations. ANOVA indicated a significant effect of diet, (F = 8.48, P < 0.001) but no effect of sex. tindicates significantly different to 18% casein group (P < 0.01).
The systolic blood pressures of the pups were assessed at the age of 4 weeks (Table 4), when the animals were fully weaned. Maternal diet significantly influenced the blood pressure of the offspring (P < O.OOl),irrespective of sex. Blood pressures were significantly elevated in 9% casein exposed animals relative to the 18% casein group (18% casein; 91 + 5 mm Hg, 9% casein; 119 f 5 mm Hg, for combined males and females). All 77 pups surviving to 4 weeks of age were included in this study. Linear regression analyses revealed significant relationships between systolic blood pressures of the offspring at 4 weeks old, and aspects of maternal nutrition and weight gain. A striking inverse correlation between blood pressure and maternal protein intake was observed (r = -0.55, P < O.OOOl),with a positive relationship with maternal weight at the time of mating (P c 0.48, P < 0.0001). Mating weight and maternal protein intake were related variables (r = -0.67, P c 0.0001). Weight gain in pregnancy was inversely related to systolic blood pressure in the offspring (r = -0.30, P < 0.05).
322 MATERNALNUTRITIONANDHYPERTENSIONINWEANLINGRATS
Discussion Observations suggesting that the development of coronary heart disease and hypertension may be programmed in utero by maternal nutrition (1, 4-7), have provoked considerable interest and controversy in recent years. The lack of any defined molecular mechanism underlying the association between noncommunicable disease of adulthood and maternal nutrition, and the multiplicity of confounding factors involved in human studies, has prompted the development of an animal model. Preliminary studies in our laboratory (15, 18-20) have described such a model, with hypertension being induced in the offspring of rats fed low protein diets before, and during pregnancy. These preliminary findings have raised significant questions (25), which need to be resolved before any consideration of mechanistic issues may begin. The present study addresses a number of the limitations of the previous work, and serves to validate the rat model of maternal-diet-induced hypertension by including all surviving offspring of both sexes in the analysis. Hypertension, relative to the 18% casein exposed control group, was observed in the 9% casein exposed offspring at the age of 4 weeks, when the animals weighed between 70 and 100 g. The appearance of the hypertension has, thus, been defined at a much younger age than in our previous studies, in which blood pressures were first determined at the age of 9 weeks (15). The observed elevation of systolic blood pressure in the 9% casein exposed group was of a similar magnitude to that previously reported (20-30 mmHg). This would suggest that the mechanism(s) underlying the development of hypertension in the offspring operate in very early life, and possibly in utero. The full hypertensive syndrome would appear to be present at the age of weaning. The aetiology of hypertension is proposed to have two components (26). An initiating process raises the blood pressure, and a second component amplifies the effect throughout life. In humans it would appear that hypertension may be initiated in utero. Raised systolic blood pressure, associated with low birthweight is observable as early as 1 week old (7). The present data is strikingly similar, and our previous data show that hypertension in animals exposed to low protein diets in utero, persists into adulthood (15). No long term studies to examine a possible amplifier effect have yet been performed. It is proposed in the human studies that initiation of hypertension stems from changes in vascular structure, promoted by alterations to placental and
fetal blood flow (7). We have noted similarities between maternal-diet-induced hypertensive rats and spontaneously hypertensive rats following ACE inhibitor treatment (20-23), which, particularly given the early onset of the hypertension, may suggest alterations to vascular structure in utero. The basic principle suggested by the epidemiological studies of Barker et al (1, 3-7, 9) is that the risk of hypertension is increased in individuals with a low birthweight relative to placental weight. In the present study we have, importantly, demonstrated lower birthweight in the 9% casein exposed group, relative to 18% casein exposed controls, and the associated development of hypertension. Significantly larger placentas are observed in rats fed 9% protein diets, relative to 18% protein fed controls (12). The rat model is, therefore consistent with epidemiological evidence suggesting that lower birthweight, and a larger placenta increase the risk of developing hypertension (6). As in previous investigations (15) a strong relationship was noted between maternal protein intake and the systolic blood pressure of the offspring. A correlation between maternal protein intake and weight at mating was also observed. Those dams that were heavier at the time of mating tended to gain less weight during pregnancy (r = -0.47, P < 0.0001). Thus the strongest association between maternal protein intake and the blood pressure of the offspring was in those dams which were heavier at conception, and which gained least weight during pregnancy. Human studies show a similar relationship, with poor weight gain in the second trimester of pregnancy being associated with higher blood pressure in children aged 10-12 years (2). The present study suggests, as do human studies, that the plane of maternal nutrition prior to conception may be highly significant in determining the outcome for the fetus. The basic requirements of the rat for dietary protein alter with pregnancy. Non-pregnant animals have a minimum requirement for 9% protein in the diet, whilst pregnant rats require 12% protein (27). Animals fed the 9% casein diet were, therefore, protein deficient in pregnancy. Previous studies have shown this is not a prerequisite for the induction of hypertension in the offspring, with restriction to 12% protein being sufficient to exert the effect (15). In the present study no effect of low protein diets upon the systolic blood pressures of adult male and female rats were observed. It has been reported that increasing protein intake has no effect upon blood pressure in the pregnant rat (28), but there are no reports of the effects of low protein. The data would
CLINICAL NUTRITION
suggest that to exert an effect upon the blood pressure of the offspring, some fetal programming of control mechanisms is required, and that the blood pressure of the offspring does not simply track that of the mother. No effects of the diets upon blood pressure were observed in pregnancy, beyond a progressive fall of pressure in both groups of animals. This fall may be attributable to the high corn oil content of the diets. Diets containing 10% corn oil are rich in linoleic acid and have a hypotensive effect in rats (Langley-Evans et al, unpublished observations). Recent work has proposed that hypertension associated with poor maternal nutrition may be a consequence of increased fetal exposure to maternal glucocorticoids (29,30). Reduced activity of placental 11p hydroxysteroid dehydrogenase is associated with increased placental weight and lower birthweight. Lower activity of the enzyme exposes the fetus to maternal steroids. Preliminary data obtained in collaboration with Edwards et al suggests that rats fed low protein diets have lower placental activity of 1lp hydroxysteroid dehydrogenase (unpublished data). This finding may provide a molecular basis for alterations to the fetus in utero, induced by changes to maternal diet. The precise course of events linking fetal exposure to maternal glucocorticoids to the development of hypertension remains to be evaluated.
7.
8.
9.
10.
11. 12.
13.
14.
1.5.
16.
17.
Acknowledgement
18.
Work funded by Wessex Medical Trust.
19.
References
20.
1. Godfrey K M, Redman C W G, Barker D J P, Osmond C. The effect of maternal anaemia and iron deficiency on the ratio of fetal weight to placental weight. Br J Obst Gynaecol 1991; 98: 886-891. 2. Godfrey K M, Forrester T, Barker D J P, Jackson A A, Landman J P, Hall J St E. The relation of maternal nutritional status during pregnancy to blood pressure in childhood. Br J Obst Gynaecol 1994; (in press). 3. Barker D J P, Osmond C, Golding J, Kuh D, Wadsworth M E J. Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease. BMJ 1989; 298: 564-567. 4. Barker D J P, Winter P D, Osmond C, Margetts B, Simmonds S J. Weight in infancy and death from ischaemic heart disease. Lancet 1989; 9 Sept: 577-582. 5. Barker D J P, Gluckman P D, Godfrey K M, Harding J E, Owens J A, Robinson J S. Fetal nutrition and cardiovascular disease in adult life. Lancet 1993; 341: 938-941. 6. Barker D J P, Bull A R, Osmond C, Simmonds S J. Fetal and
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placental size and risk of hypertension in adult life. BMJ 1990; 301: 259-262. Law C M, de Swiet M, Osmond C, Fayers P M, Barker D J P, Cmddas A M. Initiation of hypertension in utero and its amplification throughout life. BMJ 1993; 306: 24-27. Hales C N. Barker D J P, Clark P M S. Cox L J. Fall C. Osmond C. Fetal and infant growth and impaired glucose tolerance at age 64 years. BMJ 1991; 303: 1019-1022. Barker D J P, Hales C N, Fall C H D, Osmond C. Phipps K. Clark P M S. Type 2 (non-insulin dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth. Diabetologia 1993; 36: 62-67. Hinchcliffe S A, Lynch M R J, Sargent P H, Howard C V, van Zelzen D. The effect of intrauterine growth retardation on the development of renal nephrons. Br J Obst Gynaecol 1992; 99: 296-301. Miller H C, Jekel J F. Malnutrition and growth retardation in newborn infants. Pediatr 1989; 83: 443-444. Levy L, Jackson A A. Modest restriction of dietary protein during pregnancy in the rat: fetal and placental growth. J Dev Physiol 1993; 19: 113-l 18. Hastings-Roberts M M, Zeman F J. Effects of protein deficiency, pair-feeding, or diet supplementation on maternal, fetal and placental growth in rats. J Nutr 1977; 107: 973-982. Mellor D J. Nutritional and placental determinants of foetal growth rate in sheep and consequences for the newborn lamb. B Vet J 1983; 139: 307-324. Langley S C, Jackson A A. Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diet. Clin Sci 1994; 86: 217-222. Langley S C, Seakins M, Grimble R F, Jackson A A. The acute phase response of adult rats is altered by in utero exposure to maternal low protein diets. J Nutr 1994; (in press) Dahri S, Snoeck A, Reusens-Billen B, Remacle C, Hoet J J. Islet function in offspring of mothers on low-protein diet during gestation. Diabetes 1991; 40 (Suppl. 2): 115-120. Langley S C, Jackson A A. Fetal exposure to maternal low protein diets induces hypertension in adult rats, Proc Nutr Sot 1993; 52: 274A. Langley S C, Jackson A A. Induction of hypertension in adult rats. mediated by exposure to maternal low protein diets in utero. J Hypertension 1993; 11: 1148. Langley S C, Jackson A A. Captopril normalises systolic blood pressure in rats with hypertension induced by fetal exposure to maternal low protein diets, CBP 1994; (submitted) Adams M A, Bobik A, Komer P I. Enalapril can prevent vascular amplifier development in spontaneously hypertensive rats. Hypertension 1990; 16: 252-260. Morton J J, Beattie E C, MacPherson F. Angiotensin II receptor antagonist losartan has persistent effects on blood pressure in the young spontaneously hypertensive rat: lack of relation to vascular structure. J Vast Res 1992: 29: 264-269. Freslon J L. Giudicelli J F. Compared myocardial and vascular effects of captopril and dihydralazine during hypertension development in spontaneously hypertensive rats. Br J Pharmacol 1983; 80: 533-543. Department of Health. The Health of the Nation. (HMSO publication. Dd8398601 7192 Cl50 51-1075.) London: HMSO, 1992. Anonymous. Comment. Clin Sci 1994; 86: 121. Folkow B. Cardiovascular structural adaptation; its role in the initiation and maintenance of primary hypertension. Clin Sci Mol Med 1978; 55: 3s-22s.
324 MATERNALNUTRITIONANDHYPERTENSIONINWEANLINGRATS 27. National Research Council. Nutrient requirements of the rat. In: Nutrient requirements of domestic animals. Washington DC: National Academy of Sciences, 1978. 28. Barbour C J, Stonier C, Aber G M. Influence of dietary protein on glomerular binding of angiotensin II in virgin and pregnant normotensive and spontaneously hypertensive rats. Hypertension F’reg 1993; 12: 103-111.
Submission date: 8 April 1994; Accepted after revision: 9 June 1994
29. Benediktsson R, Lindsay R S, Noble .I, Seckl J R, Edwards C R W. Glucocorticoid exposure in utero: new model for adult hypertension. Lancet 1993; 341: 339-341. 30. Edwards C R W, Benediktsson R, Lindsay R S, Se&J .I R. Dysfunction of placental glucocorticoid barrier: link between fetal environment and adult hypertension. Lancet 1993; 341: 35.5-357.
In utero exposure to maternal low protein diets induces hypertension in weanling rats, independently of maternal blood pressure changes S. C. LANGLEY-EVANS,
G. J. PHILLIPS and A. A. JACKSON
Department of Human Nutrition, University 3TU, UK (Correspondence to SCL-EI
of Southampton,
Bassett Crescent fast,
Southampton
SO9
ABSTRACT-The association between maternal nutrition, fetal growth and the later development of hypertension was investigated in the rat. Animals were habituated to diets containing 18% (control) or 9% (low) protein by weight. The rats were mated and maintained on the diets until the end of pregnancy. Lactating dams were transferred onto standard chow diet. Systolic blood pressure was determined in male and female weanling offspring, using an indirect tail-cuff method. To assess the direct effects of low protein diets upon blood pressure of adult animals, a group of male and female rats were fed 18% or 9% protein for 14 days. Blood pressure was determined at the beginning and end of the feeding period. Blood pressure was additionally assessed over 14 days in pregnant rats fed control or low protein diets. Low protein diets did not alter systolic blood pressure in adult male or female rats. The blood pressures of pregnant females fed 18% or 9% protein diets did not significantly differ at any stage of pregnancy. Rats fed 9% protein diets gave birth to significantly smaller pups. Litter sizes were unaltered, and no differences in perinatal mortality were observed. Pups exposed to maternal low protein in utero had higher systolic blood pressure at the age of 4 weeks, when compared to control pups. The phenomenon was observed in both male and female offspring. Blood pressures at 4 weeks of age were strongly associated with maternal protein intake (r = -0.55). Associations were also noted between blood pressure and maternal weight at mating (r = 0.48), and weight gain in pregnancy (r = -0.30). Fetal exposure to maternal low protein diets induces hypertension in rats. The phenomenon is observed early in life and is independent of sex and the influence of maternal blood pressure. The low protein diet itself did not produce an increase in the blood pressure of adult rats.
fetus and the full-grown offspring resulting from the pregnancy (12, 13, 15-17). Most recently it has been demonstrated, in our laboratory, that rats exposed to maternal diets containing between 12% and 6% protein in utero develop significant hypertension relative to control rats exposed to 18% protein (1.5, 18, 19). These hypertensive rats have elevated systolic blood pressures associated with increased pulmonary angiotensin converting enzyme (ACE) activity (15), and their raised blood pressure is responsive to captopril treatment (20). The effects of ACE inhibitor therapy are long-lasting and strongly resemble observations in spontaneously hypertensive rats (21-23). This may suggest that, as in the spontaneously hypertensive strain, rats with maternal-diet-induced
Introduction Recent epidemiological evidence suggests that there is a significant association between the development of non-communicable diseases of adulthood and maternal nutrition (1, 2). Lower birthweightiplacental weight ratios increase risk of coronary heart disease (3-5), hypertension (6, 7), and diabetes (8, 9) in later life. Poor fetal growth has been associated with less than optimal maternal nutritional status in pregnancy (1, 2, 10, 1l), and animal studies suggest effects of maternal diet upon placental and fetal development (12-14). In the rat the feeding of diets low in protein during pregnancy has a number of effects upon the placenta, 319
320 MATERNAL NUTRITION AND HYPERTENSION IN WEANLING RATS
hypertension may have altered vascular structure (21-23). The findings of initial studies with the maternal low protein diet-induced model of hypertension in the rat (15) are of considerable importance. The Health of the Nation initiative requires a reduction of the incidence of hypertension and related cardiovascular disease in the UK population (24), and our findings, if confirmed, may influence future public health planning. However, the early findings from our laboratory were considered inconclusive (25). The use of only female offspring for blood pressure determinations was perhaps selective, and it was suggested that high blood pressures in the offspring of rats fed low protein diets may have represented a survival mechanism (25). Additionally the blood pressures of the parents remained an outstanding question. In the present paper we have addressed the majority of the criticisms directed at our preliminary studies. The new data will hopefully clear the way forward, allowing detailed examination of the molecular mechanisms underlying these important observations.
Methods 14 virgin, female Wistar rats bred in the Southampton University Animal Facility (initial weight 200-225 g) were housed individually in wire mesh cages maintained at 24°C on a 12 h light cycle. Rats were provided with synthetic diets containing 18% or 9% protein, as previously described (15). Protein was provided in the form of casein, with supplements (50 g/kg) of D,L-methionine to avoid sulphur deficiency. The diets differed principally in terms of protein content (90 g/kg difference), although the 9% casein diet contained 60 g/kg more starch and 30 g/kg more sucrose than the 18% casein diet, to equalise the energy content. All rats had free access to water. A period of 14 days was allowed to habituate the rats to the diets prior to mating. The feeding regime was maintained throughout the mating period (l-4 days), and pregnancy. The appearance of a mucous plug was taken to indicate that conception had occurred (pregnancy day 0). Within an hour of birth the mothers were all transferred to CRMX chow (SDS Ltd, Cambs.), which was fed throughout suckling, and was the diet onto which the pups were weaned at 4 weeks of age. At birth all litters of over 8 pups were thinned to 8, with the excess being killed by cervical dislocation. Birthweight was recorded for 48 pups removed from their dams. Other pups were not handled until 3 weeks old. Two dams in each
dietary group failed to carry their pregnancies to full term. Pups were finally removed from their mothers at 4 weeks old, after which they were housed in groups of 2 or 3. One to two days after this separation the systolic blood pressure of all the pups was determined by an indirect tail-cuff method, as previously described (15). Arterial pulses were detected using a light sensor, and recorded by an IITC Blood Pressure Monitor (Linton Instruments, Diss, UK). A cuff was placed over the tail and inflated to 300 mm Hg to occlude blood flow. The cuff was allowed to deflate at 3 mm Hg per second, and systolic blood pressure taken as the pressure at which tail pulses reappeared. Care was taken to ensure that the cuff was located at the top of the tail. All pups were of similar size, so any artefact of tail size would be considered unlikely. The effects of low protein diet on the blood pressures of adult rats were determined in two separate studies. These animals included 18 females used in a breeding programme identical to that described above, and 12 males of a similar weight (200-225 g). All animals had been reared on CRMX chow throughout prenatal and postnatal life. Blood pressures were determined in males before and after 2 weeks of feeding on 18% casein or 9% casein. Similarly, the blood pressures of the females were assessed at the start of feeding, and then at weekly intervals, until day 14 of pregnancy. The pregnant females included the 14 rats used to generate the weanling offspring described above. These studies allowed the assessment of whether the low protein diet itself may directly raise blood pressure in adult rats, and, furthermore, whether the blood pressures observed in the offspring of rats fed the diets during pregnancy, might be accounted for by either an effect of the diets on maternal blood pressure during pregnancy, or genetic factors. Statistical analysis
Data are presented as means f SEM. Analyses were performed using one or two way analysis of variance (ANOVA) as appropriate. Where significant interactions were noted a Tukey test was performed as a secondary test. A probability of 5% or less was accepted as statistically significant.
Results The effects of the different dietary regimens upon systolic blood pressures of adult male and female rats
CLINICAL NUTRITION
Table 1 The effects of feeding low protein diet upon systolic blood pressure in adult male and female rats
Diet
n
18% casein 9% casein
15 1.5
Systolic blood pressure (mm Hg) Day 0 Day 14 133f5 136f4
138f5 143f6
Adult male and female rats (200-225 g) were fed the different diets for 14 days. Blood pressure was determined on day 0 and 14. Data are mean _+SEM for n observations. ANOVA indicated no effects of diet at either time point.
Table 2 The effects of feeding low protein diet upon systolic blood pressure in adult female rats during pregnancy
Diet
n
Systolic blood pressure (mm Hg) Day 0 Day 7 Day 14
18% casein 9% casein
11 6
139 * 10 141 f 8
130+ 11 126+6
124 f 5+ 120 + 7’
Adult female rats (200-225 g) were fed the different diets for 14 days, prior to mating and during pregnancy. Blood pressure was determined on days 0 and 14 of the pre-pregnancy period and on days 0,7 and 14 of pregnancy. Data are mean + SEM for n observations. ANOVA indicated an interaction of pregnancy and time (F = 5.41 p < 0.005). +indicates significantly different to pregnancy day 0 (P < 0.05).
are shown in Table 1. In males and females, feeding 18% casein or 9% casein over 2 weeks had no significant effects upon blood pressure. Pregnancy (Table 2) had no significant effects upon blood pressure, but during the first 2 weeks of gestation, significant falls in blood pressure were noted in both the 18% and 9% casein groups. Pregnant rat dams fed the 9% protein diet consumed significantly less protein than the 18% protein fed controls (2.52 f 0.25 g protein/day vs 4.89 + 0.10 g protein/day, F = 46.59, P < 0.0001). Food intake was similar in both groups. At mating, rats habituated to 9% casein over 14 days were significantly heavier than 18% casein fed controls (18% casein: 250 + 3 g; 9% casein: 274 f 3 g, P < 0.05). At the time of birth all rats were of similar weight (18% casein: 391 + 8 g; 9% casein 401 + 7 g). Weight gains during pregnancy were similar in both groups (Table 3), as were the resulting litter sizes. Immediately following delivery all litters were thinned to a maximum of 8 pups, and the body weights of the randomly removed pups recorded. Significant effects of maternal diet upon birthweight were observed (Table 3). Pups delivered by rats on the 9% casein diet were significantly smaller than those delivered by dams in the 18% protein control group. Survival in the perinatal period was similar in both groups.
321
Table 3 Effects of maternal diet on outcome of pregnancy and maternal growth Diet
18% casein n 9% casein n
Food intake g/day
Weight gain in pregnancy g
Litter size no pups
Birth weight g
27.0 f 1.5 5 28.0 f 1.O 5
141?sll 5 120+7 5
12.2+2.1 5 13.3 f 1.8 5
5.250.1 22 4.7 * 0.1+ 26
Virgin female rats were habituated to the dietary regimes over 14 days before mating. Food intake and weight gain were recorded through pregnancy. Litter size and birthweight of pups were assessed within an hour of birth. Data are means f SEM for n observations. “indicated significantly different to 18% casein group, P < 0.05. ANOVA indicated significant effects of diet on birthweight, (F = 34, 82. P < 0.0001).
Table 4 The effects of maternal diet upon systolic blood pressure of offspring at 4 weeks of age Systolic blood pressure (mm Hg) Diet 18% casein Male Female 9% casein Male Female
;o 16 21 20
91 f5 90+5 119fS 120 f 4’
Blood pressure was determined at 4 weeks of age. Data are mean & SEM for n observations. ANOVA indicated a significant effect of diet, (F = 8.48, P < 0.001) but no effect of sex. tindicates significantly different to 18% casein group (P < 0.01).
The systolic blood pressures of the pups were assessed at the age of 4 weeks (Table 4), when the animals were fully weaned. Maternal diet significantly influenced the blood pressure of the offspring (P < O.OOl),irrespective of sex. Blood pressures were significantly elevated in 9% casein exposed animals relative to the 18% casein group (18% casein; 91 + 5 mm Hg, 9% casein; 119 f 5 mm Hg, for combined males and females). All 77 pups surviving to 4 weeks of age were included in this study. Linear regression analyses revealed significant relationships between systolic blood pressures of the offspring at 4 weeks old, and aspects of maternal nutrition and weight gain. A striking inverse correlation between blood pressure and maternal protein intake was observed (r = -0.55, P < O.OOOl),with a positive relationship with maternal weight at the time of mating (P c 0.48, P < 0.0001). Mating weight and maternal protein intake were related variables (r = -0.67, P c 0.0001). Weight gain in pregnancy was inversely related to systolic blood pressure in the offspring (r = -0.30, P < 0.05).
322 MATERNALNUTRITIONANDHYPERTENSIONINWEANLINGRATS
Discussion Observations suggesting that the development of coronary heart disease and hypertension may be programmed in utero by maternal nutrition (1, 4-7), have provoked considerable interest and controversy in recent years. The lack of any defined molecular mechanism underlying the association between noncommunicable disease of adulthood and maternal nutrition, and the multiplicity of confounding factors involved in human studies, has prompted the development of an animal model. Preliminary studies in our laboratory (15, 18-20) have described such a model, with hypertension being induced in the offspring of rats fed low protein diets before, and during pregnancy. These preliminary findings have raised significant questions (25), which need to be resolved before any consideration of mechanistic issues may begin. The present study addresses a number of the limitations of the previous work, and serves to validate the rat model of maternal-diet-induced hypertension by including all surviving offspring of both sexes in the analysis. Hypertension, relative to the 18% casein exposed control group, was observed in the 9% casein exposed offspring at the age of 4 weeks, when the animals weighed between 70 and 100 g. The appearance of the hypertension has, thus, been defined at a much younger age than in our previous studies, in which blood pressures were first determined at the age of 9 weeks (15). The observed elevation of systolic blood pressure in the 9% casein exposed group was of a similar magnitude to that previously reported (20-30 mmHg). This would suggest that the mechanism(s) underlying the development of hypertension in the offspring operate in very early life, and possibly in utero. The full hypertensive syndrome would appear to be present at the age of weaning. The aetiology of hypertension is proposed to have two components (26). An initiating process raises the blood pressure, and a second component amplifies the effect throughout life. In humans it would appear that hypertension may be initiated in utero. Raised systolic blood pressure, associated with low birthweight is observable as early as 1 week old (7). The present data is strikingly similar, and our previous data show that hypertension in animals exposed to low protein diets in utero, persists into adulthood (15). No long term studies to examine a possible amplifier effect have yet been performed. It is proposed in the human studies that initiation of hypertension stems from changes in vascular structure, promoted by alterations to placental and
fetal blood flow (7). We have noted similarities between maternal-diet-induced hypertensive rats and spontaneously hypertensive rats following ACE inhibitor treatment (20-23), which, particularly given the early onset of the hypertension, may suggest alterations to vascular structure in utero. The basic principle suggested by the epidemiological studies of Barker et al (1, 3-7, 9) is that the risk of hypertension is increased in individuals with a low birthweight relative to placental weight. In the present study we have, importantly, demonstrated lower birthweight in the 9% casein exposed group, relative to 18% casein exposed controls, and the associated development of hypertension. Significantly larger placentas are observed in rats fed 9% protein diets, relative to 18% protein fed controls (12). The rat model is, therefore consistent with epidemiological evidence suggesting that lower birthweight, and a larger placenta increase the risk of developing hypertension (6). As in previous investigations (15) a strong relationship was noted between maternal protein intake and the systolic blood pressure of the offspring. A correlation between maternal protein intake and weight at mating was also observed. Those dams that were heavier at the time of mating tended to gain less weight during pregnancy (r = -0.47, P < 0.0001). Thus the strongest association between maternal protein intake and the blood pressure of the offspring was in those dams which were heavier at conception, and which gained least weight during pregnancy. Human studies show a similar relationship, with poor weight gain in the second trimester of pregnancy being associated with higher blood pressure in children aged 10-12 years (2). The present study suggests, as do human studies, that the plane of maternal nutrition prior to conception may be highly significant in determining the outcome for the fetus. The basic requirements of the rat for dietary protein alter with pregnancy. Non-pregnant animals have a minimum requirement for 9% protein in the diet, whilst pregnant rats require 12% protein (27). Animals fed the 9% casein diet were, therefore, protein deficient in pregnancy. Previous studies have shown this is not a prerequisite for the induction of hypertension in the offspring, with restriction to 12% protein being sufficient to exert the effect (15). In the present study no effect of low protein diets upon the systolic blood pressures of adult male and female rats were observed. It has been reported that increasing protein intake has no effect upon blood pressure in the pregnant rat (28), but there are no reports of the effects of low protein. The data would
CLINICAL NUTRITION
suggest that to exert an effect upon the blood pressure of the offspring, some fetal programming of control mechanisms is required, and that the blood pressure of the offspring does not simply track that of the mother. No effects of the diets upon blood pressure were observed in pregnancy, beyond a progressive fall of pressure in both groups of animals. This fall may be attributable to the high corn oil content of the diets. Diets containing 10% corn oil are rich in linoleic acid and have a hypotensive effect in rats (Langley-Evans et al, unpublished observations). Recent work has proposed that hypertension associated with poor maternal nutrition may be a consequence of increased fetal exposure to maternal glucocorticoids (29,30). Reduced activity of placental 11p hydroxysteroid dehydrogenase is associated with increased placental weight and lower birthweight. Lower activity of the enzyme exposes the fetus to maternal steroids. Preliminary data obtained in collaboration with Edwards et al suggests that rats fed low protein diets have lower placental activity of 1lp hydroxysteroid dehydrogenase (unpublished data). This finding may provide a molecular basis for alterations to the fetus in utero, induced by changes to maternal diet. The precise course of events linking fetal exposure to maternal glucocorticoids to the development of hypertension remains to be evaluated.
7.
8.
9.
10.
11. 12.
13.
14.
1.5.
16.
17.
Acknowledgement
18.
Work funded by Wessex Medical Trust.
19.
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20.
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Submission date: 8 April 1994; Accepted after revision: 9 June 1994
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