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Maternal protein restriction during pregnancy and lactation in rats imprints long-term reduction in hepatic lipid content selectively in the male offspring
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Nutrition Research 30 (2010) 410 – 417 www.nrjournal.com
Maternal protein restriction during pregnancy and lactation in rats imprints long-term reduction in hepatic lipid content selectively in the male offspring Rani J. Qasem a , Ganesh Cherala b , Anil P. D'mello a,⁎ a
b
Department of Pharmaceutical Sciences, University of the Sciences in Philadelphia, Philadelphia, PA 19104, USA Department of Pharmacy Practice, College of Pharmacy, Oregon State University & Oregon Health and Science University, Portland, OR 97239, USA Received 27 January 2010; revised 7 May 2010; accepted 26 May 2010
Abstract Maternal protein restriction during pregnancy and lactation reduces whole body lipid stores and alters lipid homeostasis in the adult offspring. Lipid homeostasis in the body is regulated, in part, by the liver via the metabolic processes of synthesis and utilization of lipids. The present study tested the hypothesis that maternal protein restriction will imprint changes in hepatic lipid metabolism and thereby alter the hepatic lipid content of the adult offspring. Pregnant rats were fed purified diets containing 19% protein (control group) or 8% protein (low-protein group) throughout pregnancy and lactation. On day 28, pups from both groups were weaned onto regular laboratory chow. On days 65 and 150, male and female pups from each litter in both groups were killed and blood and liver collected. Maternal protein restriction was found to reduce birth weight and produce long-term reduction in the body weight of the offspring. On day 65, liver triglyceride content was decreased by 40% in the male offspring that were fed a low-protein diet. The reduction in liver triglyceride content persisted until day 150, at which time it was accompanied by decreases in hepatic cholesterol content. No such changes were observed in the female offspring. To determine if the alterations in liver lipid content resulted in compensatory changes in liver carbohydrate stores, hepatic glycogen content was measured in male offspring. Hepatic glycogen content was similar between the 2 groups on days 65 and 150. In conclusion, the present study in rats showed that maternal protein restriction during pregnancy and lactation imprints long-term changes in hepatic lipid content selectively in the male offspring. © 2010 Elsevier Inc. All rights reserved. Keywords: Abbreviations:
Rats; Diet, protein restricted; Prenatal exposure delayed effects; Triglycerides; Lipids; Glycogen; Ketone bodies; Cholesterol ANOVA, analysis of variance; VLDL, very low density lipoprotein.
1. Introduction Epidemiologic studies have shown an association between low birth weight and increased susceptibility to developing one or more components of the metabolic syndrome during adulthood [1,2]. To explore this finding in more mechanistic detail, a variety of animal models of intrauterine growth ⁎ Corresponding author. Tel.: +1 215 596 8941. E-mail address: [email protected] (A.P. D'mello). 0271-5317/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2010.05.008
restriction have been developed. The most commonly used model involves exposure of rats to low-protein diets during pregnancy and lactation. Maternal protein restriction during pregnancy and lactation reduces birth weight of the pups and produces long-term reduction in the body weight of the offspring [3-5]. A review of the literature reveals that this model has been predominantly used to demonstrate the effect of a restricted maternal nutritional milieu and the resultant fetal growth retardation on altering glucose tolerance and insulin resistance in the offspring [6-8].
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Recent studies have examined the effects of maternal lowprotein diets on lipid metabolism in the offspring of rats. Maternal low-protein diets during pregnancy and lactation in rats alter whole body lipid homeostasis in the offspring as evidenced by lower plasma triglyceride and cholesterol levels [4,9] and decreased whole body lipid content [4] in the adult offspring. An important organ involved in the regulation of whole body lipid homeostasis is the liver [10]. The liver participates in lipid homeostasis by synthesizing lipids in the absorptive state and by using lipids via oxidation or export to peripheral tissues in the postabsorptive state. Disturbances in whole body lipid homeostasis in low-protein offspring could result, in part, from the imbalance between hepatic lipid synthesis and utilization. Insulin is the primary regulator of hepatic lipogenesis via its actions on sterol regulatory element binding protein 1c, a transcription factor that induces the expression of a number of lipogenic genes [11,12]. Low protein offspring exhibit lower plasma levels of insulin that can decrease hepatic lipid synthesis and account for the lower plasma triglyceride levels in these animals [4,13,14]. Interestingly, low-protein offspring exhibit an increased activity of carnitine palmitoyltransferase 1, a key ratelimiting enzyme in fatty acid oxidation [15]. The anticipated increase in hepatic fatty acid oxidation and consequent increased utilization of lipids could account, in part, for the decrease in the whole body lipid content in these rats. We hypothesized that decreased hepatic lipid synthesis coupled with increased hepatic lipid oxidation is partially responsible for the altered lipid homeostasis in adult lowprotein offspring and the imbalance will be reflected in reduced hepatic lipid content in these animals. Therefore, the primary objective of the present study was to determine the effect of maternal protein restriction during pregnancy and lactation on hepatic lipid content in the adult offspring. The next objective was to assess the status of lipid synthesis and utilization in low-protein offspring by measurement of plasma concentrations of select lipids, their biosynthetic precursors, and products of lipid oxidation. Furthermore, it is known that alterations in lipid metabolism in organs and tissues often provoke compensatory responses in carbohydrate metabolism [16,17]. These responses are reflected in perturbations in a number of biochemical parameters including organ/tissue glycogen content. Therefore, the final objective of the present study was to determine if putative changes in hepatic lipid content induced by maternal protein restriction are accompanied by changes in hepatic glycogen content in the adult offspring.
virgin female Sprague-Dawley rats were mated by housing 1 male with 2 females. Day 1 of pregnancy was confirmed by the appearance of sperm in the daily morning vaginal smear. Pregnant rats were randomly assigned to be fed a modified version of the AIN 76 A purified diet (control group, n = 7) containing 19% protein or its corresponding low-protein formulation AIN M76 A (low-protein group, n = 8) containing 8% protein. The diets are isoenergetic and their detailed compositions are described in Table 1. Pregnant rats were fed the diets throughout pregnancy and lactation. At birth, pups were weighed and sexed and at 72 hours postbirth, litters were randomly culled to 8 pups (4 males and 4 females) to ensure a standard litter size for each dam. On day 28 post-birth, offspring were weaned onto normal laboratory chow and subsequently kept on this diet for the entire duration of the study. It is important to note that the different dietary treatments were only confined to the gestation and lactation period. Body weight of the pups was periodically measured throughout the duration of the study. Morning fedstate blood samples (150 μL) were collected from the offspring by tail snip on days 65 and 150 post-birth, and plasma separated and stored at −20°C. On days 65 and 150 post-birth, one male and one female offspring from each litter in both groups were killed in the fed state by decapitation and livers dissected, blotted, weighed, and snap frozen in liquid nitrogen and stored at −80°C. Body weights of the remaining male and female offspring from each litter in the 2 groups were measured until day 180 when the study was terminated. 2.2. Measurement of plasma triglycerides, ketone bodies, and free fatty acids Plasma triglycerides, β-hydroxybutyrate, and free fatty acids were assayed using commercial kits available from Table 1 Composition (grams per kilogram) of the control and low-protein diets
Casein—vitamin-free Dextrinized cornstarch Sucrose Lard Corn oil Powdered cellulose RP vitamin mix no. 10 a RP mineral mix no. 10 b DL-methionine Choline chloride Gross energy (kJ/g) a
2. Methods and materials 2.1. Animals and experimental design The study was approved by the Institutional Animal Care and Use Committee of the University of the Sciences in Philadelphia. Ten- to twelve-week-old, approximately 275-g
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Control
Low protein
210 436 150 50 50 30 20 50 1.5 2 17.07
88 434 274 50 50 30 20 50 0.63 2 17.28
Provided per kilogram of diet: thiamin 20 mg, riboflavin 20 mg, pyridoxine 20 mg, nicotinic acid 90 mg, D-calcium pantothenate 60 mg, folic acid 4 mg, biotin 0.4 mg, cyanocobalamin 20 μg, retinyl acetate 22000IU, tocopheryl acetate 50 IU, cholecalciferol 2200 IU, menadione sodium bisulfite complex 20 mg. b Provided per kilogram of diet: calcium 6 g, phosphorus 4 g, sodium 2.1 g, potassium 4 g, magnesium 0.69 g, manganese 65 mg, iron 60 mg, copper 15 mg, zinc 20 mg, iodine 0.6 mg, selenium 0.2 mg, chromium 3 mg, chloride 2.4 g, sulfate 1.2 g, cobalt 3.2 g, fluoride 5 mg, molybdenum 0.8 g.
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Wako Chemicals (Richmond, VA). The interday coefficient of variation of assays for each analyte was less than 5%.
the sample size in each group did not exceed the number of treated dams in the group.
2.3. Extraction of liver triglycerides and cholesterol 3. Results Liver triglycerides and cholesterol were extracted using a minor modification of the Folch method [18]. Briefly, 50 mg of liver tissue was homogenized and lipids extracted in 5 mL of a chloroform/methanol (2:1) mixture. The extract was centrifuged at 2500g for 15 minutes and supernatant collected and evaporated to dryness under nitrogen. The residue was subsequently reconstituted in a solution of isopropyl alcohol containing 10% Triton X and centrifuged at 10 000g for 10 minutes. The supernatant was used for the determination of triglycerides and cholesterol content using commercial kits from Wako Chemicals (Richmond, VA). Preliminary studies were conducted to confirm that the 10% Triton X in isopropyl alcohol solvent used in reconstituting the residue did not affect the performance of the triglyceride and cholesterol assays.
3.1. Body weights and liver weights of the offspring Maternal low-protein diet during pregnancy reduced the birth weight of the male and female offspring (control males: 6.2 ± 0.1 g; low-protein males: 5.2 ± 0.2 g, P b .05, n = 7-8; control females: 5.9 ± 0.1 g; low-protein females: 5.0 ± 0.1 g, P b .05, n = 7-8). The body weights of the offspring over the entire duration of the study are shown in Fig. 1. The continued administration of a low-protein diet during lactation amplified the difference in body weight between
2.4. Measurement of liver glycogen levels Hepatic glycogen content was measured using a minor modification of the procedure described by Roehrig and Allred [19]. Briefly, 50 mg of liver tissue was weighed and homogenized on ice in 0.45 mL of cold sodium acetate buffer, pH 4.6. An aliquot of the homogenate was transferred to a screw capped glass tube, diluted with sodium acetate buffer and then incubated with 0.1 mL of aminoglycosidase (60U/mL) for 2 hours at 37°C. Upon complete digestion of glycogen to free glucose, samples were centrifuged and supernatant used for the measurement of glucose content using a kit supplied by Cayman Chemicals (Ann Arbor, Mich). 2.5. Statistical analyses All data are expressed as means ± SEM. Body weight measurements (conducted repeatedly in the same group of animals) were analyzed using a repeated measure 2-way analysis of variance (ANOVA) with maternal diet and age as the main factors. All other data were analyzed using a 2-way ANOVA with maternal diet and age as main factors. Wherever appropriate, multiple comparisons were conducted using the Student-Newman-Keuls post hoc test. All statistical tests were conducted at α level of 0.05. Birth weight and body weight were measured for all male and female pups within a litter and the litter mean computed. This procedure was repeated for all litters in the group. The mean of those values was reported as the group mean. Such a conservative method of data reporting and analyses is recommended for analyses of offspring data in multiparous species. It minimizes inflation of the .05 level of α and the spurious statistical significance that results as a consequence [20,21]. All other experiments included one male and one female offspring from each litter in the 2 groups. Therefore,
Fig. 1. Body weights of the male and female control and low-protein offspring up to 180 days post-birth. A, Male offspring. B, Female offspring. The inset shows the body weight of both groups of offspring during the lactation period. Pregnant rats were administered either control (19% protein) or low-protein (8% protein) diets during pregnancy and lactation. Body weights of the male and female offspring were periodically measured until day 180 post-birth as explained in the Methods and Materials section. All data are presented as mean ± SEM with an n = 7 – 8. Body weights were measured for all pups within a litter and the litter mean computed. This procedure was repeated for all litters in the group. The mean of those values was reported as the group mean. At most ages, error bars are within the symbol. Data were analyzed using 2-way repeated-measures ANOVA with maternal diet and age as the main factors. Body weight differences between control and low-protein offspring are statistically significant (P b .05) throughout the study.
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the control and low-protein offspring, and the differences were significant throughout lactation. On day 28 post-birth, both groups were weaned onto normal laboratory chow and maintained on it for the remainder of the study. Despite such nutritional rehabilitation, low-protein offspring of both sexes exhibited significantly lower body weight compared to their corresponding controls for the entire duration of the study (180 days). As shown in Table 2, maternal low-protein diet during pregnancy and lactation reduced absolute liver weights of 65- and 150- day-old male and female offspring. However, in both sexes, liver weights were not different when normalized to body weight. Male and female offspring in the control and low-protein groups exhibited lower body weight normalized liver weights on day 150 as compared to day 65.
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low-protein offspring. Liver cholesterol content was higher in the 150-day-old control male offspring compared to their 65-day-old littermates. No such age-related differences in the cholesterol levels of the liver were observed in the male low-protein offspring. Maternal low-protein diet did not affect liver cholesterol content in 65-day-old or 150-day-old female offspring (Fig. 3). 3.4. Liver glycogen content Because a reduction in hepatic triglyceride and cholesterol content was only observed in the male low-protein offspring, liver glycogen levels were measured in male offspring. As shown in Table 2, hepatic glycogen content was similar in the control and low-protein male offspring both on days 65 and 150. Hepatic glycogen content in the 150-day-old low-protein offspring was lower than in their 65-day-old littermates (P b .05). No such age-related differences were observed in the control offspring.
3.2. Plasma parameters As shown in Table 2, no significant differences in the plasma levels of β-hydroxybutyrate, triglycerides, and free fatty acids were observed between the low-protein offspring and their respective controls either on day 65 or on day 150. In the male low-protein offspring, plasma β-hydroxybutyrate concentrations were higher on day 150 compared to levels in their 65-day-old littermates.
4. Discussion A number of different laboratories have shown that maternal protein restriction during pregnancy and lactation reduces birth weight of the pups and produces long-term reduction in the body weight and absolute liver weight of the offspring [3-5,22]. We have reproduced this finding and, therefore, validated the model in our laboratory. We now demonstrate that the reduction in body weight is accompanied by marked and long-term reductions in hepatic triglyceride and cholesterol stores in the male offspring but not in the female offspring. These results substantiate our hypothesis and demonstrate that maternal protein restriction imprints long-term changes in hepatic lipid metabolism in the male offspring. Maternal protein restriction during pregnancy and lactation is known to reduce whole body lipid content [4]. However, the present study is the first to demonstrate the effect of this paradigm in specifically reducing liver
3.3. Liver triglyceride and cholesterol content As shown in Fig. 2, maternal low-protein diet during pregnancy and lactation caused a marked and significant reduction in liver triglyceride content in the 65-day-old male offspring. Interestingly, a similar magnitude of reduction was also observed on day 150, suggesting a long-term effect of maternal diet. Maternal low-protein diet did not affect liver triglyceride content in either 65- or 150-day-old female offspring. Fig. 3 shows that maternal protein restriction did not affect liver cholesterol content in 65-day-old male offspring. However, liver cholesterol content was significantly reduced in 150-day-old male
Table 2 Effect of maternal low-protein diet on liver weight, plasma lipid concentrations, and hepatic glycogen content of male and female offspring on day 65 and day 150 Parameter
Males Day 65
Liver weight (g) Liver weight (% of body weight) Plasma β-hydroxybutyrate (μmol/L) Plasma Triglycerides (mg/dL) Plasma free fatty acids (μmol/L) Liver glycogen (μg/g liver tissue)
Females Day 150
Day 65
Day 150
Control
Low protein
Control
Low protein
Control
Low protein
Control
Low protein
16.1 ± 0.8 3.8 ± 0.1 63 ± 15 96 ± 8 956 ± 77 46 ± 3
11.0 ± 0.5 ⁎ 3.9 ± 0.1 55 ± 12 90 ± 12 977 ± 56 50 ± 4
20.5 ± 0.9 # 3.2 ± 0.1 # 115 ± 24 112 ± 17 901 ± 77 39 ± 6
14.5 ± 0.5 ⁎, # 3.1 ± 0.1 # 128 ± 26 # 103 ± 25 832 ± 48 39 ± 4 #
9.1 ± 0.4 3.7 ± 0.1 33 ± 6 77 ± 15 921 ± 68 ND
7.2 ± 0.1 ⁎ 3.9 ± 0.1 69 ± 11 62 ± 8 931 ± 91 ND
8.9 ± 0.4 2.7 ± 0.1 # 69 ± 20 106 ± 18 865 ± 56 ND
7.8 ± 0.3 ⁎ 2.9 ± 0.1 # 64 ± 14 112 ± 26 752 ± 41 ND
Pregnant rats were administered either control (19% protein) or low-protein (8% protein) diets during pregnancy and lactation. One male and one female offspring from each litter in the 2 groups were sacrificed on day 65 and day 150, livers harvested, and plasma lipids and liver glycogen measured as described in the Methods and Materials section. All data are presented as mean ± SEM with an n = 5-7, and included 1 male and 1 female offspring from each litter. Data were analyzed using a 2-way ANOVA with maternal diet and age as the main factors. ND indicates not determined. ⁎ P b .05; statistically different from the control group of the same age. # P b .05; statistically different from the same group on day 65.
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imprints long-term alterations in one or more of these pathways in the offspring. We measured plasma lipid levels in a preliminary attempt to identify the affected pathways and to help guide more detailed future mechanistic studies. Plasma free fatty acids derived from diet or through hydrolysis of adipose tissue triglyceride stores are taken up by the liver to synthesize triglycerides. Decreased hepatic triglyceride content could result from decreased uptake of circulating plasma fatty acids that in turn would be expected to elevate plasma free fatty acid concentrations. In our study, maternal protein restriction did not affect plasma levels of free fatty acids suggesting that free fatty acid uptake is not affected in these rats. Decreased hepatic triglyceride content could also result from increased export of triglycerides from the liver in the form of triglyceride-rich VLDL particles [26]. An increased VLDL secretion rate increases plasma triglyceride levels [26]. However, in our studies, maternal
Fig. 2. Liver triglyceride content in male and female control and low-protein offspring on days 65 and 150 post-birth. A, Male offspring. B, Female offspring. ⁎Statistically different from control group of the same age; P b .05. Pregnant rats were administered either control (19% protein) or lowprotein (8% protein) diets during pregnancy and lactation. One male and one female offspring from each litter in the 2 groups were sacrificed on day 65 and day 150 and liver triglyceride measured as described in the Methods and Materials section. All data are presented as mean ± SEM with an n = 5-7, and included 1 male and 1 female offspring from each litter. Data were analyzed using a 2-way ANOVA with maternal diet and age as the main factors.
triglyceride content. Interestingly, and in contrast to these results, selective maternal protein restriction during pregnancy increases liver triglyceride content in the male offspring [23,24]. Collectively, these results suggest that the timing of protein restriction affects the final phenotype with restriction during pregnancy, increasing liver triglyceride content while continued restriction during lactation decreases liver triglyceride content. The molecular mechanisms mediating the effect of maternal protein restriction on liver triglyceride content are unclear. The extent of triglyceride accumulation in the liver is determined by the balance of 5 pathways: the degree of de novo hepatic synthesis of free fatty acids, the biosynthetic precursors of triglycerides; the rate of free fatty acid uptake by the hepatocytes that occurs by passive diffusion and through specialized transporters; the extent of hepatic mitochondrial free fatty acid oxidation; the magnitude of triglyceride synthesis via esterification of free fatty acids; and the rate of triglyceride assembly into very-lowdensity lipoproteins (VLDL) and their secretion from the liver [25]. It is conceivable that maternal protein restriction
Fig. 3. Liver cholesterol content in the male and female control and lowprotein offspring on days 65 and 150 post-birth. A, Male offspring. B, Female offspring. ⁎Statistically different from control group of the same age; P b .05. #Statistically different from the same group on day 65; P b .05. Pregnant rats were administered either control (19% protein) or low-protein (8% protein) diets during pregnancy and lactation. One male and one female offspring from each litter in the 2 groups were sacrificed on day 65 and day 150 and liver cholesterol measured as described in the Methods and Materials section. All data are presented as mean ± SEM with an n = 5-7, and included 1 male and 1 female offspring from each litter. Data were analyzed using a 2-way ANOVA with maternal diet and age as the main factors.
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protein restriction did not affect plasma triglyceride levels suggesting that VLDL secretion rate is not affected in these rats. Ketone bodies are end products of hepatic fatty acid oxidation and β-hydroxybutyrate is the predominant plasma ketone body [27]. Maternal protein restriction did not affect plasma levels of β-hydroxybutyrate in the offspring suggesting that the fatty acid oxidation pathway was not affected in these rats. However, plasma lipid levels are in a dynamic state and are affected both by their rate of production and their rate of utilization and, are therefore, of limited utility in understanding the mechanisms mediating the decreased hepatic triglyceride content of low-protein offspring. A review of the literature indicates that the effect of maternal protein restriction on plasma free fatty acid, triglyceride, and ketone body levels are inconsistent with reports of increases [13], decreases [9,13,23], and no change [4,28] in levels. The inconsistencies between laboratories can be accounted for by experimental design issues such as differences in timing of exposure (pregnancy vs lactation) to protein restriction [23], variations in composition of specific macro- and micronutrients between different low-protein diets, nutritional status (fed or fasted) of the animal [13], and the use of anesthesia during sacrifice and its effects on plasma lipid levels [29,30]. In view of these inconsistencies, future mechanistic studies will focus on the liver and will evaluate the status of each of the 5 aforementioned pathways that can affect liver triglyceride content. Status of each pathway will be assessed by measuring the activities of ratelimiting enzymes in each pathway, the amounts of key transcription factors governing activities of these rate limiting enzymes, and the amounts of relevant transporters. Maternal protein restriction also decreased levels of liver cholesterol in the male offspring, although this effect was only observed on day 150. This suggests that maternal protein restriction imprints the hepatic content of multiple lipids in the offspring and these effects are manifested through distinct ontogenic patterns. The mechanistic basis of this finding is not well understood at present. The activity of key enzymes mediating hepatic fatty acid, triglyceride, and cholesterol synthesis are partially controlled by common master regulators such as adenosine monophosphate–activated protein kinase [31]. It is conceivable that maternal protein restriction could affect the status of this or similar master regulators that, in turn, could decrease activity of rate-limiting enzymes responsible for the biosynthesis of each of these lipids and account for the decrease in their hepatic content. The final experiment determined if alterations produced by maternal protein restriction in hepatic lipid content would induce compensatory changes in hepatic carbohydrate metabolism. It has been hypothesized that the low-protein offspring are in a “glucose-sparing” mode of metabolism and peripheral tissues in these animals spare glucose utilization for use by critical organs such as the brain [8]. Implicit in this hypothesis is that low-protein offspring preferentially use lipids to meet their energy needs. Such
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preferential utilization of lipids and sparing of glucose would be expected to increase hepatic stores of glycogen. However, maternal protein restriction did not affect liver glycogen content in either 65- or 150-day-old offspring. Using a similar paradigm, Ozanne et al [32] also reported a lack of effect of maternal protein restriction on liver glycogen content in the adult offspring. In the present study, liver glycogen was measured in the fed state when hepatic glycogen stores were maximized. Therefore, the lack of change in liver glycogen content suggests that maternal protein restriction did not affect hepatic storage capacity of glycogen in the offspring. Hepatic glycogen stores are used during the postabsorptive state, and it is conceivable that maternal protein restriction could alter postabsorptive hepatic glycogen utilization. Maternal calorie restriction during pregnancy (another commonly used model to induce fetal growth restriction) is known to decrease postabsorptive glycogen utilization in the adult offspring as evidenced by smaller fasting-induced fall in liver glycogen content compared to control offspring [33]. Future studies will explore this possibility in the maternal protein restriction paradigm. Maternal protein restriction during pregnancy and lactation did not decrease liver triglyceride or cholesterol content in the female offspring. To the best of our knowledge, this is the first demonstration of a sex-specific effect of maternal protein restriction during pregnancy and lactation on liver lipid content. The mechanistic basis of this sex-specific effect is unclear. Few studies have examined the effects of maternal protein restriction on lipid metabolism in the female offspring. In support of our findings, 2 other studies have shown that male offspring are more susceptible to the imprinting effects of maternal protein restriction on plasma lipid levels [4,9]. Interestingly, previous studies have demonstrated that male offspring are also more susceptible to the imprinting effects of maternal protein restriction on glucose and insulin homeostasis [4,28,34]. As stated earlier, insulin is a major regulator of hepatic lipogenesis, and the selective alteration in hepatic lipid metabolism in the male low-protein offspring might be a consequence of the larger magnitude of perturbation in insulin homeostasis in these animals. Other investigators have attributed the increased susceptibility of male low-protein offspring to their faster growth and consequently more critical nutritional needs [9]. The reason for the lack of effect of maternal low-protein diet on liver lipid content in the female offspring is unclear. Female sex hormones affect the actions of sterol regulatory element binding protein 1c and peroxisome proliferator-activated receptor α, which are key transcription factors governing the expression of multiple genes in hepatic lipid synthesis and oxidation, respectively [35-37]. It is conceivable that sex hormones might prevent maternal low-protein diet– induced alterations in the actions of these transcription factors in the female offspring and thereby, protect against changes in lipid synthesis and utilization and liver lipid
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content. Alternatively, it is established that there are sex differences in lipid trafficking within the body with male rats directing dietary fat preferentially to the liver while female rats direct dietary fat preferentially to skeletal muscle [38,39]. It is therefore possible that in the female offspring the imprinting effects of maternal protein restriction on lipid metabolism will occur in the skeletal muscle rather than in the liver. Additional studies will be required to explore these possibilities in greater detail. In conclusion, the present study demonstrates that maternal protein restriction during pregnancy and lactation produces alterations in lipid metabolism with decreases in liver triglyceride and cholesterol stores in the male offspring but not in the female offspring. Future research will focus on the liver and explore the mechanisms underlying these changes. Acknowledgment This study was supported by a grant from Jenrin Discovery, with additional financial support from the Department of Pharmaceutical Sciences at the University of the Sciences in Philadelphia. References [1] Hales CN. Fetal and infant origins of adult disease. J Clin Pathol 1997; 50:359. [2] Barker DJ. The developmental origins of adult disease. Eur J Epidemiol 2003;18:733-6. [3] Langley SC, Jackson AA. Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets. Clin Sci (Lond) 1994;86:217-22 [discussion 121]. [4] Zambrano E, Bautista CJ, Deas M, Martinez-Samayoa PM, GonzalezZamorano M, Ledesma H, et al. A low maternal protein diet during pregnancy and lactation has sex- and window of exposure-specific effects on offspring growth and food intake, glucose metabolism and serum leptin in the rat. J Physiol 2006;571:221-30. [5] Desai M, Crowther NJ, Lucas A, Hales CN. Organ-selective growth in the offspring of protein-restricted mothers. Br J Nutr 1996;76: 591-603. [6] Langley SC, Browne RF, Jackson AA. Altered glucose tolerance in rats exposed to maternal low protein diets in utero. Comp Biochem Physiol Physiol 1994;109:223-9. [7] Hales CN, Desai M, Ozanne SE, Crowther NJ. Fishing in the stream of diabetes: from measuring insulin to the control of fetal organogenesis. Biochem Soc Trans 1996;24:341-50. [8] Petry CJ, Ozanne SE, Hales CN. Programming of intermediary metabolism. Mol Cell Endocrinol 2001;185:81-91. [9] Lucas A, Baker BA, Desai M, Hales CN. Nutrition in pregnant or lactating rats programs lipid metabolism in the offspring. Br J Nutr 1996;76:605-12. [10] Frayn KN, Arner P, Yki-Jarvinen H. Fatty acid metabolism in adipose tissue, muscle and liver in health and disease. Essays Biochem 2006; 42:89-103. [11] Kersten S. Mechanisms of nutritional and hormonal regulation of lipogenesis. EMBO Rep 2001;2:282-6. [12] Dentin R, Girard J, Postic C. Carbohydrate responsive element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c): two key regulators of glucose metabolism and lipid synthesis in liver. Biochimie 2005;87:81-6.
[13] Ozanne SE, Wang CL, Petry CJ, Smith JM, Hales CN. Ketosis resistance in the male offspring of protein-malnourished rat dams. Metabolism 1998;47:1450-4. [14] Petry CJ, Ozanne SE, Wang CL, Hales CN. Effects of early protein restriction and adult obesity on rat pancreatic hormone content and glucose tolerance. Horm Metab Res 2000;32:233-9. [15] Guzman M, Azzolin IR, Moulin CC, Perry ML. Pre- and postnatal protein undernutrition increases hepatic carnitine palmitoyltransferase I activity and decreases enzyme sensitivity to inhibitors in the suckling rat. Horm Metab Res 1992;24:471-3. [16] Frayn KN. The glucose–fatty acid cycle: a physiological perspective. Biochem Soc Trans 2003;31:1115-9. [17] Randle PJ, Garland PB, Hales CN, Newsholme EA. The glucose fattyacid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1963;1:785-9. [18] Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957; 226:497-509. [19] Roehrig KL, Allred JB. Direct enzymatic procedure for the determination of liver glycogen. Anal Biochem 1974;58:414-21. [20] Zorrilla EP. Multiparous species present problems (and possibilities) to developmentalists. Dev Psychobiol 1997;30:141-50. [21] Holson RR, Pearce B. Principles and pitfalls in the analysis of prenatal treatment effects in multiparous species. Neurotoxicol Teratol 1992; 14:221-8. [22] Woods LL, Ingelfinger JR, Nyengaard JR, Rasch R. Maternal protein restriction suppresses the newborn renin-angiotensin system and programs adult hypertension in rats. Pediatr Res 2001;49: 460-7. [23] Bieswal F, Ahn MT, Reusens B, Holvoet P, Raes M, Rees WD, et al. The importance of catch-up growth after early malnutrition for the programming of obesity in male rat. Obesity (Silver Spring) 2006;14: 1330-43. [24] Erhuma A, Salter AM, Sculley DV, Langley-Evans SC, Bennett AJ. Prenatal exposure to a low-protein diet programs disordered regulation of lipid metabolism in the aging rat. Am J Physiol Endocrinol Metab 2007;292:E1702-14. [25] Gibbons GF, Islam K, Pease RJ. Mobilisation of triacylglycerol stores. Biochim Biophys Acta 2000;1483:37-57. [26] Blasiole DA, Davis RA, Attie AD. The physiological and molecular regulation of lipoprotein assembly and secretion. Mol Biosyst 2007;3: 608-19. [27] Laffel L. Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes. Diabetes Metab Res Rev 1999; 15:412-26. [28] Desai M, Byrne CD, Meeran K, Martenz ND, Bloom SR, Hales CN. Regulation of hepatic enzymes and insulin levels in offspring of rat dams fed a reduced-protein diet. Am J Physiol 1997;273: G899-G904. [29] Saha JK, Xia J, Grondin JM, Engle SK, Jakubowski JA. Acute hyperglycemia induced by ketamine/xylazine anesthesia in rats: mechanisms and implications for preclinical models. Exp Biol Med (Maywood) 2005;230:777-84. [30] Bhathena SJ. Comparison of effects of decapitation and anesthesia on metabolic and hormonal parameters in Sprague-Dawley rats. Life Sci 1992;50:1649-55. [31] Viollet B, Foretz M, Guigas B, Horman S, Dentin R, Bertrand L, et al. Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders. J Physiol 2006; 574:41-53. [32] Ozanne SE, Smith GD, Tikerpae J, Hales CN. Altered regulation of hepatic glucose output in the male offspring of protein-malnourished rat dams. Am J Physiol 1996;270:E559-64. [33] Thompson NM, Norman AM, Donkin SS, Shankar RR, Vickers MH, Miles JL, et al. Prenatal and postnatal pathways to obesity: different underlying mechanisms, different metabolic outcomes. Endocrinology 2007;148:2345-54.
R.J. Qasem et al. / Nutrition Research 30 (2010) 410–417 [34] Sugden MC, Holness MJ. Gender-specific programming of insulin secretion and action. J Endocrinol 2002;175:757-67. [35] Yoon M. The role of PPARalpha in lipid metabolism and obesity: focusing on the effects of estrogen on PPARalpha actions. Pharmacol Res 2009;60:151-9. [36] D'Eon TM, Souza SC, Aronovitz M, Obin MS, Fried SK, Greenberg AS. Estrogen regulation of adiposity and fuel partitioning. Evidence of genomic and non-genomic regulation of lipogenic and oxidative pathways. J Biol Chem 2005;280:35983-91.
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[37] Paquette A, Wang D, Jankowski M, Gutkowska J, Lavoie JM. Effects of ovariectomy on PPAR alpha, SREBP-1c, and SCD-1 gene expression in the rat liver. Menopause 2008;15:1169-75. [38] Jackman MR, Kramer RE, MacLean PS, Bessesen DH. Trafficking of dietary fat in obesity-prone and obesity-resistant rats. Am J Physiol Endocrinol Metab 2006;291:E1083-91. [39] Priego T, Sánchez J, Picó C, Palou A. Sex-differential expression of metabolism-related genes in response to a high fat diet. Obesity 2008; 16:819-26.
Nutrition Research 30 (2010) 410 – 417 www.nrjournal.com
Maternal protein restriction during pregnancy and lactation in rats imprints long-term reduction in hepatic lipid content selectively in the male offspring Rani J. Qasem a , Ganesh Cherala b , Anil P. D'mello a,⁎ a
b
Department of Pharmaceutical Sciences, University of the Sciences in Philadelphia, Philadelphia, PA 19104, USA Department of Pharmacy Practice, College of Pharmacy, Oregon State University & Oregon Health and Science University, Portland, OR 97239, USA Received 27 January 2010; revised 7 May 2010; accepted 26 May 2010
Abstract Maternal protein restriction during pregnancy and lactation reduces whole body lipid stores and alters lipid homeostasis in the adult offspring. Lipid homeostasis in the body is regulated, in part, by the liver via the metabolic processes of synthesis and utilization of lipids. The present study tested the hypothesis that maternal protein restriction will imprint changes in hepatic lipid metabolism and thereby alter the hepatic lipid content of the adult offspring. Pregnant rats were fed purified diets containing 19% protein (control group) or 8% protein (low-protein group) throughout pregnancy and lactation. On day 28, pups from both groups were weaned onto regular laboratory chow. On days 65 and 150, male and female pups from each litter in both groups were killed and blood and liver collected. Maternal protein restriction was found to reduce birth weight and produce long-term reduction in the body weight of the offspring. On day 65, liver triglyceride content was decreased by 40% in the male offspring that were fed a low-protein diet. The reduction in liver triglyceride content persisted until day 150, at which time it was accompanied by decreases in hepatic cholesterol content. No such changes were observed in the female offspring. To determine if the alterations in liver lipid content resulted in compensatory changes in liver carbohydrate stores, hepatic glycogen content was measured in male offspring. Hepatic glycogen content was similar between the 2 groups on days 65 and 150. In conclusion, the present study in rats showed that maternal protein restriction during pregnancy and lactation imprints long-term changes in hepatic lipid content selectively in the male offspring. © 2010 Elsevier Inc. All rights reserved. Keywords: Abbreviations:
Rats; Diet, protein restricted; Prenatal exposure delayed effects; Triglycerides; Lipids; Glycogen; Ketone bodies; Cholesterol ANOVA, analysis of variance; VLDL, very low density lipoprotein.
1. Introduction Epidemiologic studies have shown an association between low birth weight and increased susceptibility to developing one or more components of the metabolic syndrome during adulthood [1,2]. To explore this finding in more mechanistic detail, a variety of animal models of intrauterine growth ⁎ Corresponding author. Tel.: +1 215 596 8941. E-mail address: [email protected] (A.P. D'mello). 0271-5317/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2010.05.008
restriction have been developed. The most commonly used model involves exposure of rats to low-protein diets during pregnancy and lactation. Maternal protein restriction during pregnancy and lactation reduces birth weight of the pups and produces long-term reduction in the body weight of the offspring [3-5]. A review of the literature reveals that this model has been predominantly used to demonstrate the effect of a restricted maternal nutritional milieu and the resultant fetal growth retardation on altering glucose tolerance and insulin resistance in the offspring [6-8].
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Recent studies have examined the effects of maternal lowprotein diets on lipid metabolism in the offspring of rats. Maternal low-protein diets during pregnancy and lactation in rats alter whole body lipid homeostasis in the offspring as evidenced by lower plasma triglyceride and cholesterol levels [4,9] and decreased whole body lipid content [4] in the adult offspring. An important organ involved in the regulation of whole body lipid homeostasis is the liver [10]. The liver participates in lipid homeostasis by synthesizing lipids in the absorptive state and by using lipids via oxidation or export to peripheral tissues in the postabsorptive state. Disturbances in whole body lipid homeostasis in low-protein offspring could result, in part, from the imbalance between hepatic lipid synthesis and utilization. Insulin is the primary regulator of hepatic lipogenesis via its actions on sterol regulatory element binding protein 1c, a transcription factor that induces the expression of a number of lipogenic genes [11,12]. Low protein offspring exhibit lower plasma levels of insulin that can decrease hepatic lipid synthesis and account for the lower plasma triglyceride levels in these animals [4,13,14]. Interestingly, low-protein offspring exhibit an increased activity of carnitine palmitoyltransferase 1, a key ratelimiting enzyme in fatty acid oxidation [15]. The anticipated increase in hepatic fatty acid oxidation and consequent increased utilization of lipids could account, in part, for the decrease in the whole body lipid content in these rats. We hypothesized that decreased hepatic lipid synthesis coupled with increased hepatic lipid oxidation is partially responsible for the altered lipid homeostasis in adult lowprotein offspring and the imbalance will be reflected in reduced hepatic lipid content in these animals. Therefore, the primary objective of the present study was to determine the effect of maternal protein restriction during pregnancy and lactation on hepatic lipid content in the adult offspring. The next objective was to assess the status of lipid synthesis and utilization in low-protein offspring by measurement of plasma concentrations of select lipids, their biosynthetic precursors, and products of lipid oxidation. Furthermore, it is known that alterations in lipid metabolism in organs and tissues often provoke compensatory responses in carbohydrate metabolism [16,17]. These responses are reflected in perturbations in a number of biochemical parameters including organ/tissue glycogen content. Therefore, the final objective of the present study was to determine if putative changes in hepatic lipid content induced by maternal protein restriction are accompanied by changes in hepatic glycogen content in the adult offspring.
virgin female Sprague-Dawley rats were mated by housing 1 male with 2 females. Day 1 of pregnancy was confirmed by the appearance of sperm in the daily morning vaginal smear. Pregnant rats were randomly assigned to be fed a modified version of the AIN 76 A purified diet (control group, n = 7) containing 19% protein or its corresponding low-protein formulation AIN M76 A (low-protein group, n = 8) containing 8% protein. The diets are isoenergetic and their detailed compositions are described in Table 1. Pregnant rats were fed the diets throughout pregnancy and lactation. At birth, pups were weighed and sexed and at 72 hours postbirth, litters were randomly culled to 8 pups (4 males and 4 females) to ensure a standard litter size for each dam. On day 28 post-birth, offspring were weaned onto normal laboratory chow and subsequently kept on this diet for the entire duration of the study. It is important to note that the different dietary treatments were only confined to the gestation and lactation period. Body weight of the pups was periodically measured throughout the duration of the study. Morning fedstate blood samples (150 μL) were collected from the offspring by tail snip on days 65 and 150 post-birth, and plasma separated and stored at −20°C. On days 65 and 150 post-birth, one male and one female offspring from each litter in both groups were killed in the fed state by decapitation and livers dissected, blotted, weighed, and snap frozen in liquid nitrogen and stored at −80°C. Body weights of the remaining male and female offspring from each litter in the 2 groups were measured until day 180 when the study was terminated. 2.2. Measurement of plasma triglycerides, ketone bodies, and free fatty acids Plasma triglycerides, β-hydroxybutyrate, and free fatty acids were assayed using commercial kits available from Table 1 Composition (grams per kilogram) of the control and low-protein diets
Casein—vitamin-free Dextrinized cornstarch Sucrose Lard Corn oil Powdered cellulose RP vitamin mix no. 10 a RP mineral mix no. 10 b DL-methionine Choline chloride Gross energy (kJ/g) a
2. Methods and materials 2.1. Animals and experimental design The study was approved by the Institutional Animal Care and Use Committee of the University of the Sciences in Philadelphia. Ten- to twelve-week-old, approximately 275-g
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Control
Low protein
210 436 150 50 50 30 20 50 1.5 2 17.07
88 434 274 50 50 30 20 50 0.63 2 17.28
Provided per kilogram of diet: thiamin 20 mg, riboflavin 20 mg, pyridoxine 20 mg, nicotinic acid 90 mg, D-calcium pantothenate 60 mg, folic acid 4 mg, biotin 0.4 mg, cyanocobalamin 20 μg, retinyl acetate 22000IU, tocopheryl acetate 50 IU, cholecalciferol 2200 IU, menadione sodium bisulfite complex 20 mg. b Provided per kilogram of diet: calcium 6 g, phosphorus 4 g, sodium 2.1 g, potassium 4 g, magnesium 0.69 g, manganese 65 mg, iron 60 mg, copper 15 mg, zinc 20 mg, iodine 0.6 mg, selenium 0.2 mg, chromium 3 mg, chloride 2.4 g, sulfate 1.2 g, cobalt 3.2 g, fluoride 5 mg, molybdenum 0.8 g.
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Wako Chemicals (Richmond, VA). The interday coefficient of variation of assays for each analyte was less than 5%.
the sample size in each group did not exceed the number of treated dams in the group.
2.3. Extraction of liver triglycerides and cholesterol 3. Results Liver triglycerides and cholesterol were extracted using a minor modification of the Folch method [18]. Briefly, 50 mg of liver tissue was homogenized and lipids extracted in 5 mL of a chloroform/methanol (2:1) mixture. The extract was centrifuged at 2500g for 15 minutes and supernatant collected and evaporated to dryness under nitrogen. The residue was subsequently reconstituted in a solution of isopropyl alcohol containing 10% Triton X and centrifuged at 10 000g for 10 minutes. The supernatant was used for the determination of triglycerides and cholesterol content using commercial kits from Wako Chemicals (Richmond, VA). Preliminary studies were conducted to confirm that the 10% Triton X in isopropyl alcohol solvent used in reconstituting the residue did not affect the performance of the triglyceride and cholesterol assays.
3.1. Body weights and liver weights of the offspring Maternal low-protein diet during pregnancy reduced the birth weight of the male and female offspring (control males: 6.2 ± 0.1 g; low-protein males: 5.2 ± 0.2 g, P b .05, n = 7-8; control females: 5.9 ± 0.1 g; low-protein females: 5.0 ± 0.1 g, P b .05, n = 7-8). The body weights of the offspring over the entire duration of the study are shown in Fig. 1. The continued administration of a low-protein diet during lactation amplified the difference in body weight between
2.4. Measurement of liver glycogen levels Hepatic glycogen content was measured using a minor modification of the procedure described by Roehrig and Allred [19]. Briefly, 50 mg of liver tissue was weighed and homogenized on ice in 0.45 mL of cold sodium acetate buffer, pH 4.6. An aliquot of the homogenate was transferred to a screw capped glass tube, diluted with sodium acetate buffer and then incubated with 0.1 mL of aminoglycosidase (60U/mL) for 2 hours at 37°C. Upon complete digestion of glycogen to free glucose, samples were centrifuged and supernatant used for the measurement of glucose content using a kit supplied by Cayman Chemicals (Ann Arbor, Mich). 2.5. Statistical analyses All data are expressed as means ± SEM. Body weight measurements (conducted repeatedly in the same group of animals) were analyzed using a repeated measure 2-way analysis of variance (ANOVA) with maternal diet and age as the main factors. All other data were analyzed using a 2-way ANOVA with maternal diet and age as main factors. Wherever appropriate, multiple comparisons were conducted using the Student-Newman-Keuls post hoc test. All statistical tests were conducted at α level of 0.05. Birth weight and body weight were measured for all male and female pups within a litter and the litter mean computed. This procedure was repeated for all litters in the group. The mean of those values was reported as the group mean. Such a conservative method of data reporting and analyses is recommended for analyses of offspring data in multiparous species. It minimizes inflation of the .05 level of α and the spurious statistical significance that results as a consequence [20,21]. All other experiments included one male and one female offspring from each litter in the 2 groups. Therefore,
Fig. 1. Body weights of the male and female control and low-protein offspring up to 180 days post-birth. A, Male offspring. B, Female offspring. The inset shows the body weight of both groups of offspring during the lactation period. Pregnant rats were administered either control (19% protein) or low-protein (8% protein) diets during pregnancy and lactation. Body weights of the male and female offspring were periodically measured until day 180 post-birth as explained in the Methods and Materials section. All data are presented as mean ± SEM with an n = 7 – 8. Body weights were measured for all pups within a litter and the litter mean computed. This procedure was repeated for all litters in the group. The mean of those values was reported as the group mean. At most ages, error bars are within the symbol. Data were analyzed using 2-way repeated-measures ANOVA with maternal diet and age as the main factors. Body weight differences between control and low-protein offspring are statistically significant (P b .05) throughout the study.
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the control and low-protein offspring, and the differences were significant throughout lactation. On day 28 post-birth, both groups were weaned onto normal laboratory chow and maintained on it for the remainder of the study. Despite such nutritional rehabilitation, low-protein offspring of both sexes exhibited significantly lower body weight compared to their corresponding controls for the entire duration of the study (180 days). As shown in Table 2, maternal low-protein diet during pregnancy and lactation reduced absolute liver weights of 65- and 150- day-old male and female offspring. However, in both sexes, liver weights were not different when normalized to body weight. Male and female offspring in the control and low-protein groups exhibited lower body weight normalized liver weights on day 150 as compared to day 65.
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low-protein offspring. Liver cholesterol content was higher in the 150-day-old control male offspring compared to their 65-day-old littermates. No such age-related differences in the cholesterol levels of the liver were observed in the male low-protein offspring. Maternal low-protein diet did not affect liver cholesterol content in 65-day-old or 150-day-old female offspring (Fig. 3). 3.4. Liver glycogen content Because a reduction in hepatic triglyceride and cholesterol content was only observed in the male low-protein offspring, liver glycogen levels were measured in male offspring. As shown in Table 2, hepatic glycogen content was similar in the control and low-protein male offspring both on days 65 and 150. Hepatic glycogen content in the 150-day-old low-protein offspring was lower than in their 65-day-old littermates (P b .05). No such age-related differences were observed in the control offspring.
3.2. Plasma parameters As shown in Table 2, no significant differences in the plasma levels of β-hydroxybutyrate, triglycerides, and free fatty acids were observed between the low-protein offspring and their respective controls either on day 65 or on day 150. In the male low-protein offspring, plasma β-hydroxybutyrate concentrations were higher on day 150 compared to levels in their 65-day-old littermates.
4. Discussion A number of different laboratories have shown that maternal protein restriction during pregnancy and lactation reduces birth weight of the pups and produces long-term reduction in the body weight and absolute liver weight of the offspring [3-5,22]. We have reproduced this finding and, therefore, validated the model in our laboratory. We now demonstrate that the reduction in body weight is accompanied by marked and long-term reductions in hepatic triglyceride and cholesterol stores in the male offspring but not in the female offspring. These results substantiate our hypothesis and demonstrate that maternal protein restriction imprints long-term changes in hepatic lipid metabolism in the male offspring. Maternal protein restriction during pregnancy and lactation is known to reduce whole body lipid content [4]. However, the present study is the first to demonstrate the effect of this paradigm in specifically reducing liver
3.3. Liver triglyceride and cholesterol content As shown in Fig. 2, maternal low-protein diet during pregnancy and lactation caused a marked and significant reduction in liver triglyceride content in the 65-day-old male offspring. Interestingly, a similar magnitude of reduction was also observed on day 150, suggesting a long-term effect of maternal diet. Maternal low-protein diet did not affect liver triglyceride content in either 65- or 150-day-old female offspring. Fig. 3 shows that maternal protein restriction did not affect liver cholesterol content in 65-day-old male offspring. However, liver cholesterol content was significantly reduced in 150-day-old male
Table 2 Effect of maternal low-protein diet on liver weight, plasma lipid concentrations, and hepatic glycogen content of male and female offspring on day 65 and day 150 Parameter
Males Day 65
Liver weight (g) Liver weight (% of body weight) Plasma β-hydroxybutyrate (μmol/L) Plasma Triglycerides (mg/dL) Plasma free fatty acids (μmol/L) Liver glycogen (μg/g liver tissue)
Females Day 150
Day 65
Day 150
Control
Low protein
Control
Low protein
Control
Low protein
Control
Low protein
16.1 ± 0.8 3.8 ± 0.1 63 ± 15 96 ± 8 956 ± 77 46 ± 3
11.0 ± 0.5 ⁎ 3.9 ± 0.1 55 ± 12 90 ± 12 977 ± 56 50 ± 4
20.5 ± 0.9 # 3.2 ± 0.1 # 115 ± 24 112 ± 17 901 ± 77 39 ± 6
14.5 ± 0.5 ⁎, # 3.1 ± 0.1 # 128 ± 26 # 103 ± 25 832 ± 48 39 ± 4 #
9.1 ± 0.4 3.7 ± 0.1 33 ± 6 77 ± 15 921 ± 68 ND
7.2 ± 0.1 ⁎ 3.9 ± 0.1 69 ± 11 62 ± 8 931 ± 91 ND
8.9 ± 0.4 2.7 ± 0.1 # 69 ± 20 106 ± 18 865 ± 56 ND
7.8 ± 0.3 ⁎ 2.9 ± 0.1 # 64 ± 14 112 ± 26 752 ± 41 ND
Pregnant rats were administered either control (19% protein) or low-protein (8% protein) diets during pregnancy and lactation. One male and one female offspring from each litter in the 2 groups were sacrificed on day 65 and day 150, livers harvested, and plasma lipids and liver glycogen measured as described in the Methods and Materials section. All data are presented as mean ± SEM with an n = 5-7, and included 1 male and 1 female offspring from each litter. Data were analyzed using a 2-way ANOVA with maternal diet and age as the main factors. ND indicates not determined. ⁎ P b .05; statistically different from the control group of the same age. # P b .05; statistically different from the same group on day 65.
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imprints long-term alterations in one or more of these pathways in the offspring. We measured plasma lipid levels in a preliminary attempt to identify the affected pathways and to help guide more detailed future mechanistic studies. Plasma free fatty acids derived from diet or through hydrolysis of adipose tissue triglyceride stores are taken up by the liver to synthesize triglycerides. Decreased hepatic triglyceride content could result from decreased uptake of circulating plasma fatty acids that in turn would be expected to elevate plasma free fatty acid concentrations. In our study, maternal protein restriction did not affect plasma levels of free fatty acids suggesting that free fatty acid uptake is not affected in these rats. Decreased hepatic triglyceride content could also result from increased export of triglycerides from the liver in the form of triglyceride-rich VLDL particles [26]. An increased VLDL secretion rate increases plasma triglyceride levels [26]. However, in our studies, maternal
Fig. 2. Liver triglyceride content in male and female control and low-protein offspring on days 65 and 150 post-birth. A, Male offspring. B, Female offspring. ⁎Statistically different from control group of the same age; P b .05. Pregnant rats were administered either control (19% protein) or lowprotein (8% protein) diets during pregnancy and lactation. One male and one female offspring from each litter in the 2 groups were sacrificed on day 65 and day 150 and liver triglyceride measured as described in the Methods and Materials section. All data are presented as mean ± SEM with an n = 5-7, and included 1 male and 1 female offspring from each litter. Data were analyzed using a 2-way ANOVA with maternal diet and age as the main factors.
triglyceride content. Interestingly, and in contrast to these results, selective maternal protein restriction during pregnancy increases liver triglyceride content in the male offspring [23,24]. Collectively, these results suggest that the timing of protein restriction affects the final phenotype with restriction during pregnancy, increasing liver triglyceride content while continued restriction during lactation decreases liver triglyceride content. The molecular mechanisms mediating the effect of maternal protein restriction on liver triglyceride content are unclear. The extent of triglyceride accumulation in the liver is determined by the balance of 5 pathways: the degree of de novo hepatic synthesis of free fatty acids, the biosynthetic precursors of triglycerides; the rate of free fatty acid uptake by the hepatocytes that occurs by passive diffusion and through specialized transporters; the extent of hepatic mitochondrial free fatty acid oxidation; the magnitude of triglyceride synthesis via esterification of free fatty acids; and the rate of triglyceride assembly into very-lowdensity lipoproteins (VLDL) and their secretion from the liver [25]. It is conceivable that maternal protein restriction
Fig. 3. Liver cholesterol content in the male and female control and lowprotein offspring on days 65 and 150 post-birth. A, Male offspring. B, Female offspring. ⁎Statistically different from control group of the same age; P b .05. #Statistically different from the same group on day 65; P b .05. Pregnant rats were administered either control (19% protein) or low-protein (8% protein) diets during pregnancy and lactation. One male and one female offspring from each litter in the 2 groups were sacrificed on day 65 and day 150 and liver cholesterol measured as described in the Methods and Materials section. All data are presented as mean ± SEM with an n = 5-7, and included 1 male and 1 female offspring from each litter. Data were analyzed using a 2-way ANOVA with maternal diet and age as the main factors.
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protein restriction did not affect plasma triglyceride levels suggesting that VLDL secretion rate is not affected in these rats. Ketone bodies are end products of hepatic fatty acid oxidation and β-hydroxybutyrate is the predominant plasma ketone body [27]. Maternal protein restriction did not affect plasma levels of β-hydroxybutyrate in the offspring suggesting that the fatty acid oxidation pathway was not affected in these rats. However, plasma lipid levels are in a dynamic state and are affected both by their rate of production and their rate of utilization and, are therefore, of limited utility in understanding the mechanisms mediating the decreased hepatic triglyceride content of low-protein offspring. A review of the literature indicates that the effect of maternal protein restriction on plasma free fatty acid, triglyceride, and ketone body levels are inconsistent with reports of increases [13], decreases [9,13,23], and no change [4,28] in levels. The inconsistencies between laboratories can be accounted for by experimental design issues such as differences in timing of exposure (pregnancy vs lactation) to protein restriction [23], variations in composition of specific macro- and micronutrients between different low-protein diets, nutritional status (fed or fasted) of the animal [13], and the use of anesthesia during sacrifice and its effects on plasma lipid levels [29,30]. In view of these inconsistencies, future mechanistic studies will focus on the liver and will evaluate the status of each of the 5 aforementioned pathways that can affect liver triglyceride content. Status of each pathway will be assessed by measuring the activities of ratelimiting enzymes in each pathway, the amounts of key transcription factors governing activities of these rate limiting enzymes, and the amounts of relevant transporters. Maternal protein restriction also decreased levels of liver cholesterol in the male offspring, although this effect was only observed on day 150. This suggests that maternal protein restriction imprints the hepatic content of multiple lipids in the offspring and these effects are manifested through distinct ontogenic patterns. The mechanistic basis of this finding is not well understood at present. The activity of key enzymes mediating hepatic fatty acid, triglyceride, and cholesterol synthesis are partially controlled by common master regulators such as adenosine monophosphate–activated protein kinase [31]. It is conceivable that maternal protein restriction could affect the status of this or similar master regulators that, in turn, could decrease activity of rate-limiting enzymes responsible for the biosynthesis of each of these lipids and account for the decrease in their hepatic content. The final experiment determined if alterations produced by maternal protein restriction in hepatic lipid content would induce compensatory changes in hepatic carbohydrate metabolism. It has been hypothesized that the low-protein offspring are in a “glucose-sparing” mode of metabolism and peripheral tissues in these animals spare glucose utilization for use by critical organs such as the brain [8]. Implicit in this hypothesis is that low-protein offspring preferentially use lipids to meet their energy needs. Such
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preferential utilization of lipids and sparing of glucose would be expected to increase hepatic stores of glycogen. However, maternal protein restriction did not affect liver glycogen content in either 65- or 150-day-old offspring. Using a similar paradigm, Ozanne et al [32] also reported a lack of effect of maternal protein restriction on liver glycogen content in the adult offspring. In the present study, liver glycogen was measured in the fed state when hepatic glycogen stores were maximized. Therefore, the lack of change in liver glycogen content suggests that maternal protein restriction did not affect hepatic storage capacity of glycogen in the offspring. Hepatic glycogen stores are used during the postabsorptive state, and it is conceivable that maternal protein restriction could alter postabsorptive hepatic glycogen utilization. Maternal calorie restriction during pregnancy (another commonly used model to induce fetal growth restriction) is known to decrease postabsorptive glycogen utilization in the adult offspring as evidenced by smaller fasting-induced fall in liver glycogen content compared to control offspring [33]. Future studies will explore this possibility in the maternal protein restriction paradigm. Maternal protein restriction during pregnancy and lactation did not decrease liver triglyceride or cholesterol content in the female offspring. To the best of our knowledge, this is the first demonstration of a sex-specific effect of maternal protein restriction during pregnancy and lactation on liver lipid content. The mechanistic basis of this sex-specific effect is unclear. Few studies have examined the effects of maternal protein restriction on lipid metabolism in the female offspring. In support of our findings, 2 other studies have shown that male offspring are more susceptible to the imprinting effects of maternal protein restriction on plasma lipid levels [4,9]. Interestingly, previous studies have demonstrated that male offspring are also more susceptible to the imprinting effects of maternal protein restriction on glucose and insulin homeostasis [4,28,34]. As stated earlier, insulin is a major regulator of hepatic lipogenesis, and the selective alteration in hepatic lipid metabolism in the male low-protein offspring might be a consequence of the larger magnitude of perturbation in insulin homeostasis in these animals. Other investigators have attributed the increased susceptibility of male low-protein offspring to their faster growth and consequently more critical nutritional needs [9]. The reason for the lack of effect of maternal low-protein diet on liver lipid content in the female offspring is unclear. Female sex hormones affect the actions of sterol regulatory element binding protein 1c and peroxisome proliferator-activated receptor α, which are key transcription factors governing the expression of multiple genes in hepatic lipid synthesis and oxidation, respectively [35-37]. It is conceivable that sex hormones might prevent maternal low-protein diet– induced alterations in the actions of these transcription factors in the female offspring and thereby, protect against changes in lipid synthesis and utilization and liver lipid
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content. Alternatively, it is established that there are sex differences in lipid trafficking within the body with male rats directing dietary fat preferentially to the liver while female rats direct dietary fat preferentially to skeletal muscle [38,39]. It is therefore possible that in the female offspring the imprinting effects of maternal protein restriction on lipid metabolism will occur in the skeletal muscle rather than in the liver. Additional studies will be required to explore these possibilities in greater detail. In conclusion, the present study demonstrates that maternal protein restriction during pregnancy and lactation produces alterations in lipid metabolism with decreases in liver triglyceride and cholesterol stores in the male offspring but not in the female offspring. Future research will focus on the liver and explore the mechanisms underlying these changes. Acknowledgment This study was supported by a grant from Jenrin Discovery, with additional financial support from the Department of Pharmaceutical Sciences at the University of the Sciences in Philadelphia. References [1] Hales CN. Fetal and infant origins of adult disease. J Clin Pathol 1997; 50:359. [2] Barker DJ. The developmental origins of adult disease. Eur J Epidemiol 2003;18:733-6. [3] Langley SC, Jackson AA. Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets. Clin Sci (Lond) 1994;86:217-22 [discussion 121]. [4] Zambrano E, Bautista CJ, Deas M, Martinez-Samayoa PM, GonzalezZamorano M, Ledesma H, et al. A low maternal protein diet during pregnancy and lactation has sex- and window of exposure-specific effects on offspring growth and food intake, glucose metabolism and serum leptin in the rat. J Physiol 2006;571:221-30. [5] Desai M, Crowther NJ, Lucas A, Hales CN. Organ-selective growth in the offspring of protein-restricted mothers. Br J Nutr 1996;76: 591-603. [6] Langley SC, Browne RF, Jackson AA. Altered glucose tolerance in rats exposed to maternal low protein diets in utero. Comp Biochem Physiol Physiol 1994;109:223-9. [7] Hales CN, Desai M, Ozanne SE, Crowther NJ. Fishing in the stream of diabetes: from measuring insulin to the control of fetal organogenesis. Biochem Soc Trans 1996;24:341-50. [8] Petry CJ, Ozanne SE, Hales CN. Programming of intermediary metabolism. Mol Cell Endocrinol 2001;185:81-91. [9] Lucas A, Baker BA, Desai M, Hales CN. Nutrition in pregnant or lactating rats programs lipid metabolism in the offspring. Br J Nutr 1996;76:605-12. [10] Frayn KN, Arner P, Yki-Jarvinen H. Fatty acid metabolism in adipose tissue, muscle and liver in health and disease. Essays Biochem 2006; 42:89-103. [11] Kersten S. Mechanisms of nutritional and hormonal regulation of lipogenesis. EMBO Rep 2001;2:282-6. [12] Dentin R, Girard J, Postic C. Carbohydrate responsive element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c): two key regulators of glucose metabolism and lipid synthesis in liver. Biochimie 2005;87:81-6.
[13] Ozanne SE, Wang CL, Petry CJ, Smith JM, Hales CN. Ketosis resistance in the male offspring of protein-malnourished rat dams. Metabolism 1998;47:1450-4. [14] Petry CJ, Ozanne SE, Wang CL, Hales CN. Effects of early protein restriction and adult obesity on rat pancreatic hormone content and glucose tolerance. Horm Metab Res 2000;32:233-9. [15] Guzman M, Azzolin IR, Moulin CC, Perry ML. Pre- and postnatal protein undernutrition increases hepatic carnitine palmitoyltransferase I activity and decreases enzyme sensitivity to inhibitors in the suckling rat. Horm Metab Res 1992;24:471-3. [16] Frayn KN. The glucose–fatty acid cycle: a physiological perspective. Biochem Soc Trans 2003;31:1115-9. [17] Randle PJ, Garland PB, Hales CN, Newsholme EA. The glucose fattyacid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1963;1:785-9. [18] Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957; 226:497-509. [19] Roehrig KL, Allred JB. Direct enzymatic procedure for the determination of liver glycogen. Anal Biochem 1974;58:414-21. [20] Zorrilla EP. Multiparous species present problems (and possibilities) to developmentalists. Dev Psychobiol 1997;30:141-50. [21] Holson RR, Pearce B. Principles and pitfalls in the analysis of prenatal treatment effects in multiparous species. Neurotoxicol Teratol 1992; 14:221-8. [22] Woods LL, Ingelfinger JR, Nyengaard JR, Rasch R. Maternal protein restriction suppresses the newborn renin-angiotensin system and programs adult hypertension in rats. Pediatr Res 2001;49: 460-7. [23] Bieswal F, Ahn MT, Reusens B, Holvoet P, Raes M, Rees WD, et al. The importance of catch-up growth after early malnutrition for the programming of obesity in male rat. Obesity (Silver Spring) 2006;14: 1330-43. [24] Erhuma A, Salter AM, Sculley DV, Langley-Evans SC, Bennett AJ. Prenatal exposure to a low-protein diet programs disordered regulation of lipid metabolism in the aging rat. Am J Physiol Endocrinol Metab 2007;292:E1702-14. [25] Gibbons GF, Islam K, Pease RJ. Mobilisation of triacylglycerol stores. Biochim Biophys Acta 2000;1483:37-57. [26] Blasiole DA, Davis RA, Attie AD. The physiological and molecular regulation of lipoprotein assembly and secretion. Mol Biosyst 2007;3: 608-19. [27] Laffel L. Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes. Diabetes Metab Res Rev 1999; 15:412-26. [28] Desai M, Byrne CD, Meeran K, Martenz ND, Bloom SR, Hales CN. Regulation of hepatic enzymes and insulin levels in offspring of rat dams fed a reduced-protein diet. Am J Physiol 1997;273: G899-G904. [29] Saha JK, Xia J, Grondin JM, Engle SK, Jakubowski JA. Acute hyperglycemia induced by ketamine/xylazine anesthesia in rats: mechanisms and implications for preclinical models. Exp Biol Med (Maywood) 2005;230:777-84. [30] Bhathena SJ. Comparison of effects of decapitation and anesthesia on metabolic and hormonal parameters in Sprague-Dawley rats. Life Sci 1992;50:1649-55. [31] Viollet B, Foretz M, Guigas B, Horman S, Dentin R, Bertrand L, et al. Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders. J Physiol 2006; 574:41-53. [32] Ozanne SE, Smith GD, Tikerpae J, Hales CN. Altered regulation of hepatic glucose output in the male offspring of protein-malnourished rat dams. Am J Physiol 1996;270:E559-64. [33] Thompson NM, Norman AM, Donkin SS, Shankar RR, Vickers MH, Miles JL, et al. Prenatal and postnatal pathways to obesity: different underlying mechanisms, different metabolic outcomes. Endocrinology 2007;148:2345-54.
R.J. Qasem et al. / Nutrition Research 30 (2010) 410–417 [34] Sugden MC, Holness MJ. Gender-specific programming of insulin secretion and action. J Endocrinol 2002;175:757-67. [35] Yoon M. The role of PPARalpha in lipid metabolism and obesity: focusing on the effects of estrogen on PPARalpha actions. Pharmacol Res 2009;60:151-9. [36] D'Eon TM, Souza SC, Aronovitz M, Obin MS, Fried SK, Greenberg AS. Estrogen regulation of adiposity and fuel partitioning. Evidence of genomic and non-genomic regulation of lipogenic and oxidative pathways. J Biol Chem 2005;280:35983-91.
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[37] Paquette A, Wang D, Jankowski M, Gutkowska J, Lavoie JM. Effects of ovariectomy on PPAR alpha, SREBP-1c, and SCD-1 gene expression in the rat liver. Menopause 2008;15:1169-75. [38] Jackman MR, Kramer RE, MacLean PS, Bessesen DH. Trafficking of dietary fat in obesity-prone and obesity-resistant rats. Am J Physiol Endocrinol Metab 2006;291:E1083-91. [39] Priego T, Sánchez J, Picó C, Palou A. Sex-differential expression of metabolism-related genes in response to a high fat diet. Obesity 2008; 16:819-26.