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Nutrient Metabolism

Fish Oil Supplementation of Rats during Pregnancy Reduces Adult Disease Risks in Their Offspring^1,2

Sadhana Joshi, Shobha Rao³, Ajit Golwilkar^*, Manisha Patwardhan^* and Ramesh Bhonde

Department of Biometry and Nutrition Unit, Agharkar Research Institute, Pune 411 004, India; ^* NPIL-Dr, Golwilkar Laboratories, Pune 411 004, India; and National Centre for Cell Science, Pune 411 007, India

³To whom correspondence should be addressed. E-mail: raoari{at}yahoo.com.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Metabolic programming in utero due to maternal undernutrition is considered to increase the risk of adult diseases in offspring. It is therefore of relevance to investigate how dietary supplementation of specific nutrients can ameliorate the negative effects of maternal malnutrition. We examined the effects of supplementing fish oil or folic acid, both of which are conventional supplements in maternal intervention, on risk factors in the offspring as adults. Pregnant female rats from 4 groups (n = 6/group) were fed casein diets with 18 g/100 g protein (control diet), 12 g/100 g protein supplemented with 8 mg folic acid/kg diet (0.08 mg/kg diet) (FAS), 12 g/100 g protein without folic acid (FAD) or 12 g/100 g protein supplemented with 7 g/100 g fish oil (FOIL). Pups were weaned to a standard laboratory diet with 18 g/100 g protein. Serum glucose, insulin and cholesterol and plasma homocysteine levels were measured in the offspring at 6 and 11 mo of age. Serum glucose in 11-mo-old male and female pups was greater (P < 0.05) in both the FAS (males 2.46 ± 0.51, females 2.49 ± 0.29 mmol/L) and FAD groups (2.48 ± 0.28 and 2.67 ± 0.41 mmol/L) than in controls (2.03 ± 0.15 and 2.02 ± 0.18 mmol/L). Serum insulin concentrations were higher (P < 0.05) in the FAD group (males 1476 ± 317, females 1441 ± 220 pmol/L) but were lower in males from the FAS group (483 ± 165 pmol/L) compared with controls (males 917 ± 373, females 981 ± 264 pmol/L). Glucose and insulin concentrations did not differ between the control and FOIL groups. Plasma homocysteine levels were lower (P < 0.05) only in 11-mo-old folate-deficient males; none of the other groups differed from the controls. Maternal supplementation of fish oil to a diet containing marginal protein was beneficial in maintaining circulating glucose, insulin, cholesterol and homocysteine levels in the offspring as adults.

KEY WORDS: • fish oil • folate • glucose • insulin • homocysteine

The prevalence of low birth weight continues to be a public health problem in many developing countries. Perinatal malnutrition resulting in low birth weight and early growth retardation may be important risk factors for syndrome X in later life (1–3). A large amount of data was reported by Barker and his group (4) in support of fetal programming "in utero" leading to increased risks of adult diseases. However, the nutrients involved in fetal "metabolic programming" are not yet known. Therefore, identifying possible involvement of specific nutrients is an important step in planning effective intervention to mothers in countries such as India.

Recent studies revealed that increased intake of long-chain PUFA (LC-PUFA)³ during pregnancy increases the length of gestation and birth size (5), suggesting that maternal LC-PUFA status during pregnancy is critical in the development of the fetus. It was shown in mice that inadequacy of LC-PUFA during fetal development and infancy may lead to a defect in the expression and/or function of insulin receptors in the brain (3). In fact, subtle deficiencies in the accretion of essential fatty acids into cell membranes may contribute to metabolic derangements in children such as insulin resistance (6).

Iron and folic acid are the other important nutrients most commonly supplemented in pregnancy in many developing countries including India. Their short-term benefits in terms of improved fetal growth are well documented (7,8). However, it is not known whether these micronutrients have any role in "metabolic programming." In particular, it would be interesting to examine the role of maternal folic acid supplementation in maintaining adult glucose levels in offspring. Folic acid and LC-PUFA have independent effects on fetal growth. However, folic acid can also modify LC-PUFA metabolism. Folic acid may increase the concentration of (n-3) PUFA and decrease vitamin K-dependent coagulation factors, which may eventually lower the risk of thrombosis (9,10). Thus, folic acid and (n-3) fatty acids must be investigated in the context of fetal adaptation to maternal undernutrition.

A number of animal studies (11,12) reported that maternal undernutrition modifies glucose and cholesterol metabolism in the offspring as adults. However, it is not known whether maternal supplementation of specific micronutrients has beneficial effects that carry into adulthood. Because maternal diets in many poor communities are inadequate in protein, identifying the micronutrients involved in the metabolic programming of fetal adaptation will require studies of maternal supplementation of micronutrients at low or marginal protein levels. Therefore, we examined the effects of supplementation of folic acid and fish oil, rich in docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), at a marginal protein level during pregnancy, on various blood variables in offspring at 6 and 11 mo of age.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
All experimental procedures were in accordance with the guidelines of Institutional Animal Ethics Committee (IAEC). Animal maintenance and handling were in accordance with the National Institute of Nutrition, India guidelines (13).

Diets.

The composition of the control diet followed the AIN 93 purified diets for laboratory rodents (14) and contained 18 g/100 g protein. Three other isoenergetic treatment diets were formulated. One was folic acid deficient (FAD), one was supplemented with folic acid (FAS) and the third was supplemented with fish oil (FOIL). In the experimental diets, the protein level was reduced to 12 g/100 g to satisfy the optimum requirement during pregnancy and the amount of cornstarch was 45.75 g/100 g diet. Vitamin-free casein was used for the experimental groups.

Control rats received 2 mg folic acid/kg diet (0.02 mg/kg diet). Folate deficiency was obtained exclusively through dietary means rather than by using folate antagonists or antibiotics. This more closely resembles the overall effects of folate deprivation upon pregnancy as it might occur in humans, rather than using a drug to produce deficiency. The FAS diet was enriched with 8 mg folic acid/kg diet (0.08 mg/kg diet). This is 4 times the requirement of normal rats and agrees with the fact that the folate requirement for Indian women during pregnancy is 400 µg/d, which is 4 times the requirement of women who are not pregnant. Cod liver oil (Seven Seas, Mumbai, India), which is a rich source of DHA and EPA (Table 1), was supplemented (7 g/100 g) to the 12 g/100 g protein diet of the FOIL group.

Virgin female Wistar rats weighing 200–250 g were obtained from the animal house of Agharkar Research Institute, Pune, India, and kept for breeding in the ratio 3 females:1 male. Pups born were separated according to sex on d 21 and 24 female pups were randomly selected. They were maintained at 22°C on a controlled 12-h light:dark cycle with appropriate ventilation. They were fed a control diet for 3 mo and kept for breeding. On confirmation of pregnancy (through sperm positive vaginal smears), i.e., d 0 of pregnancy, they were randomly allocated to one of the four groups (n = 6/group).

Rats in the FAD group were kept in cages with plastic wire bottoms to reduce coprophagy. A day before the expected delivery, dams were placed in plastic solid bottom cages with rice husk as the bedding material. After delivery, the litters in each group were culled to eight pups each. The experimental dams received the control (18 g/100 g protein) diet after delivery and the pups were also weaned onto the same diet. The offspring from each group were killed by cardiac puncture at 6 and 11 mo of age, to study the effects of dietary treatments "in utero" on long-term health outcomes of the progeny.

Blood samples.

The offspring were food deprived overnight before dissection; blood samples were taken the next morning via heart puncture under anesthesia with diethyl ether. Serum and plasma were separated and stored at -20°C until analysis.

Biochemical assays.

Serum glucose was estimated by the GOD-POD method (Randox Laboratories, Crumlin, UK) (15). Cholesterol was estimated by the CHOD-PAP method (Randox) (16). HDL cholesterol was measured by the new clearance method using diagnostic kits (Randox) (17). LDL cholesterol was calculated using the Friedewald formula (18). Serum triglycerides (TG) were estimated by the method of Buccolo et al. (19) using a diagnostic kit from Randox. Homocysteine estimation was done by the microparticle enzyme immunoassay method (Abbott Diagnostics, Abbott Park, IL) (20). Folate and vitamin B-12 were estimated by the fluorescence polarization immunoassay (Abbott Diagnostics) (21). Insulin resistance was calculated as fasting insulin x fasting glucose, whereas the quick insulin sensitivity index was calculated as 1/(log glucose) + (log insulin).

Statistical methods.

Values are expressed as means ± SD. The data were analyzed using the SPSS/PC+ package (Version 11.0, Chicago, IL). The experimental groups were compared with the control group by ANOVA and the post-hoc least significant difference test. These comparisons were done separately for the data at 6 and 11 mo and separately for sexes. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Body weight.

Weight gain during gestation was significantly lower for dams in the FAD group than those in the control group. Although the FAD, FAS and FOIL diets did not affect litter weight or litter size, weight gain during the preweaning period was lower in the FAD group than in controls (Table 2). In the postweaning period up to 3 mo, the weight of pups in all four groups did not differ (Table 2). At 6 and 11 mo, male and female rats in the FOIL group and males in the FAD group weighed less than controls (P < 0.05).

At 6 mo of age, male and female offspring in the FAD group and males in the FAS group had higher serum glucose concentrations than controls (P < 0.05). In contrast, the offspring in the FOIL group had glucose levels that did not differ from those of the control group. At 11 mo, both male and female offspring from the FAD and FAS groups had higher serum glucose levels, whereas rats from the FOIL group did not differ from the controls (Table 3).

At 11 mo of age, male and female offspring from the FAD group had higher (P < 0.05) serum insulin levels compared with control rats (Table 3). In contrast, males in the FAS group had lower insulin concentrations than the control rats (P < 0.05). Rats in the FOIL group did not differ from controls. The estimated values of insulin resistance were higher (P < 0.05), whereas those for insulin sensitivity were lower (P < 0.05) in the FAD group compared with control rats (data not shown).

HDL, LDL and total cholesterol.

Males in the FAD group had higher (P < 0.05) serum total, HDL and LDL cholesterol at 6 mo but not at 11 mo than control males (Table 3). In other experimental groups, cholesterol levels didnot differ from the controls in either sex at 6 mo of age. At 11 mo, females in the FAS group had higher (P < 0.05) LDL cholesterol than the controls (Table 3).

Triglycerides (TG).

Serum TG levels in males in the FAD and FOIL groups were lower (P < 0.05), whereas those in the FAS group did not differ from the controls at 6 mo of age. However, at 11 mo, only rats from the FAD group had lower (P < 0.05) serum TG levels. The females were not affected by the treatments.

Folate and vitamin B-12 status.

Serum folate and vitamin B-12 concentrations were not affected by the treatments at any time tested (data not shown).

Homocysteine.

Plasma homocysteine concentrations did not differ from the controls in any experimental group at 6 mo (Table 3). However, homocysteine concentrations in males in the FAD group at 11 mo of age were lower than in control rats (P < 0.05). Homocysteine levels in females in all three experimental groups did not differ from female controls.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The recent literature (22,23) suggests that fetal undernutrition during critical periods of growth and development has lasting effects on the structure and function of the tissues and body systems. Because maternal diets in developing countries are often inadequate in protein, it is important to investigate how dietary manipulations ameliorate the negative effects of maternal malnutrition. Therefore, we examined the effect of maternal supplementation of folic acid or fish oil at marginal levels of protein, in reducing risks associated with adult noncommunicable diseases in offspring with a rat model (11,12) commonly used for examining the long-term consequences of undernutrition "in utero." Our study highlights the fact that unlike folate supplementation, fish oil supplementation was able to maintain glucose and cholesterol concentrations in the offspring as adults.

Most studies examined the effects of folate deficiency before breeding, for periods ranging from 2 wk to 6 mo, and showed adverse effects on birth outcomes (24–26). We observed that folate deficiency during gestation did not affect mean litter weight, but significantly affected growth of offspring during weaning, even though the dams consumed the control diet from the day of delivery. There are reports of beneficial effects of maternal folate supplementation on birth outcome at varying time points such as before and during pregnancy and lactation (25,27,28) or with varying levels (25) except at an excess level of folate, i.e., 40 mg/kg (28). Negative effects were also reported in well-nourished South African women (29). Our observation that folate supplementation supported fetal growth as well as postnatal growth suggests, therefore, that its effect can be better assessed at marginal levels of protein than at adequate or higher levels.

Glucose concentrations were studied in offspring at adult age (12,30–31) in relation to maternal protein restriction (8 or 9 g/100 g) but not with respect to maternal folate deficiency. Higher serum glucose levels in the FAS group in our study may be a result of folate supplementation leading to increased iron stores because it was reported recently that increased iron stores may indicate the development of diabetes (32). In contrast, fish oil supplementation during pregnancy was beneficial in maintaining glucose concentrations in the offspring as adults. Purified EPA ethyl ester had beneficial effects on glucose metabolism (33,34). In fact, fish oil also was shown to improve glucose tolerance at higher levels of dietary fat by enhancing insulin secretion (35).

Malnutrition during the critical period of growth was shown to reduce insulin secretion in response to the glucose load (31,12). Maternal protein deficiency was reported to impair insulin secretion but increase the sensitivity to insulin in weaned rats (12). In our study, rats from the FAD group had significantly higher serum glucose levels, higher insulin resistance and lower insulin sensitivity than controls, suggesting adverse effects on glucose metabolism. Serum glucose levels were higher than controls even in the folate-supplemented group. In contrast, these adverse effects did not occur in the FOIL group. A maternal low protein diet was shown to decrease the activity of -6 desaturase and -5 desaturase in offspring, resulting in low amounts of various LC-PUFA in liver and muscle (36). Maternal supplementation of cod liver oil may therefore have positively influenced carbohydrate metabolism and improved glucose homeostasis in the offspring as adults.

Cholesterol metabolism was affected at 6 mo of age in male offspring from the FAD group, whereas LDL cholesterol increased in 11-mo-old females in the FAS group. Low protein levels during gestation were reported to alter cholesterol metabolism in offspring (11). It was shown that fish oil lowers circulating TG (37). The generally low serum TG levels in the FOIL group suggest that maternal supplementation with fish oil may yield similar benefits to the offspring. The significantly lower TG concentrations in male rats from the FAD group are difficult to explain.

Whether homocysteine is an independent risk factor for vascular disease is controversial (38). The association of vitamin B-12 and folate deficiency with increased serum homocysteine levels (39,40) is well documented, but studies relating folate deficiency in utero and homocysteine levels in the offspring in adulthood are scarce. In the present study, we found an inverse relationship between plasma homocysteine concentrations and serum insulin levels in male rats in the FAD group (r = -0.596, P = 0.032). It was reported that hyperinsulinemia affects the catabolism of homocysteine (41). An inverse relationship was also found in a type 1 diabetic animal model (42) as well as in healthy subjects (43). Interference of hyperinsulinemia with homocysteine metabolism leads to increased remethylation and/or transsulfuration and subsequently reduces homocysteine concentrations (42). The inverse relationship may have been found only in males and not in females in the FAD group because of the faster rate of tissue growth in the former (11). Another possible explanation is that sex steroid hormones may function differently in the homocysteine regulating pathway (43). Our findings thus highlight the need for further investigations to examine the role of insulin in homocysteine metabolism.

In our earlier study of rural Indian mothers, most of whom were undernourished, we found a significant relationship between consumption of micronutrient rich foods and birth size (44). Although this study underscored the importance of micronutrients and their combinations in improving fetal growth, supplementation trials are required for identifying the roles of specific micronutrients. The present study shows for the first time that at marginal levels of maternal protein, supplementation of fish oil, which is rich source of (n-3) fatty acids (DHA and EPA), maintained glucose and cholesterol metabolism in the offspring as adults. Thus, the findings suggest that (n-3) fatty acids may play an important role in the "metabolic programming" of fetal adaptation to maternal undernutrition. In vegetarian diets, sources of (n-3) fatty acids are scarce; therefore, it would be worthwhile to examine the role of their precursors such as -linolenic acid in fetal growth as well as in reducing risks related to adult diseases. Similarly, in communities in which maternal nutrition intervention programs with iron and folate have been in operation for last two decades or more, evaluation of their long-term effects is essential. The wider implication of our findings is that improving maternal nutrition qualitatively (micronutrient rich) rather than quantitatively, will yield both short- and long-term benefits, especially in undernourished populations.

	ACKNOWLEDGMENTS

 
The authors thank V. S. Rao, Director, Agharkar Research Institute, Pune for providing the necessary facilities to carry out this research work. Thanks are also due to S. Girigosavi for preparing the experimental diets.

	FOOTNOTES

 
¹ Presented in part at the IX Asian Congress of Nutrition Feb 23–27, 2003, New Delhi, India [Rao, S., Kanade, A. & Joshi, S. (2003) Micronutrients, fetal growth and risk for increased glucose levels, Symposium no. S12–03 (abs.)].

² Supported by the Indian Council of Medical Research.

⁴ Abbreviations used: DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FAD, folic acid deficient; FAS, folic acid supplemented; FOIL, fish oil supplemented; LC-PUFA, long-chain PUFA; TG, triglycerides.

Manuscript received 10 April 2003. Initial review completed 15 May 2003. Revision accepted 9 July 2003.

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