Soy protein–based compared with casein-based diets fed during pregnancy and lactation increase food intake and characteristics of metabolic syndrome less in female than male rat offspring

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Nutrition Research 31 (2011) 644 – 651 www.nrjournal.com

Soy protein–based compared with casein-based diets fed during pregnancy and lactation increase food intake and characteristics of metabolic syndrome less in female than male rat offspring☆ Alireza Jahan-mihan, Christopher E. Smith, Atyeh Hamedani, G. Harvey Anderson ⁎ Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 Received 26 May 2011; revised 30 July 2011; accepted 2 August 2011

Abstract We hypothesized that soy protein (S)–based diets fed during pregnancy and lactation increase food intake and the presence of characteristics of the metabolic syndrome to a lesser extent in female than in male rats. Soy protein– and casein (C)-based American Institute of Nutrition–93G diets were fed to 2 groups (n = 12 per group) of pregnant Wistar rats from day 3 of gestation and throughout lactation. Their effects on characteristics of metabolic syndrome and food intake regulation in female pups maintained for 15 weeks on the C diet were compared. Body weight (BW) and food intake (FI) were measured weekly. Fat pad mass was measured at birth, at weaning, and at week 15. Glucose and insulin tolerance tests were conducted at weeks 8 and 12; and systolic and diastolic blood pressures were measured at weeks 4, 8, and 12. Plasma was collected at weaning and at the end of the studies for glucose, insulin, glucagon-like peptide 1, peptide YY, and ghrelin. Food intake in response to protein preloads was measured at week 7. Feeding the S diet throughout gestation and lactation resulted in higher systolic blood pressure (P b .005), FI (P b .05), and glucagon-like peptide 1 and lower peptide YY at weaning and higher BW during weeks 11 to 15 and fat pad mass at week 15 (all Ps b .05). However, no sign of insulin resistance was found; nor was short-term FI in response to protein preloads affected. In conclusion, S- compared with C-based American Institute of Nutrition–93 G diets consumed throughout gestation and lactation increased BW and FI later and resulted in fewer characteristics of metabolic syndrome in female than in male offspring. © 2011 Elsevier Inc. All rights reserved. Keywords: Abbreviations:

Protein; Programming; Metabolic syndrome; Food intake; Female offspring AIN 93G, American Institute of Nutrition (1993); AUC, area under the curve; BW, body weight; C, casein; Cat, catalogue; DBP, diastolic blood pressure; DEXA, dual-energy X-ray absorptiometry; FI, food intake; GLP-1, glucagon-like peptide 1; HOMA-IR, homeostatic model assessment of insulin resistance; PYY, peptide YY; S, soy protein; SBP, systolic blood pressure.

1. Introduction Numerous epidemiological and clinical studies plus investigations in animal models provide substantial

☆ Supported by the Natural Sciences and Engineering Research Council of Canada. ⁎ Corresponding author. Tel.: +1 416 978 1832; fax: +1 416 978 5882. E-mail address: [email protected] (G.H. Anderson).

0271-5317/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2011.08.003

evidence that fetal and early postnatal nutrition alters development of somatic structures, endocrine systems, and homeostatic mechanisms in the fetus and infant [1-3]. These effects influence the risk of obesity, hypertension, diabetes, and other components of metabolic syndrome in later life. The expression of later-life responses to nutrition in utero are markedly affected by postweaning and chronic diets, leading to the suggestion that the adverse consequences of maternal malnutrition arise from a mismatch

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between the in utero and the external environment as conceptualized as the predictive adaptive response hypothesis [4-6]. Both low- and high-protein diets fed during gestation and lactation have detrimental effects on the physiologic and metabolic phenotype of offspring in animals [7-9]. In addition, we have shown that source of protein in nutritionally complete diets fed to pregnant rats influences phenotype of the male offspring [10,11]. Because casein (C) and soy protein (S) are 2 significant sources of proteins in human diets and in infant formulas [12] and are the proteins most often used in rodent test diets for metabolic studies [13,14], we compared the effect of Sand C-based diets fed during gestation alone or gestation and lactation on the development of food intake (FI) regulation and characteristics of metabolic syndrome in offspring of Wistar rats. Male offspring of dams fed the S diet had higher FI, consistent with alterations of intake regulatory systems [10], and higher body fat, blood pressure, and insulin resistance [11]. However, the effect of these diets on the phenotype of the female offspring has not been reported. There is substantial evidence indicating that the dams' diet affects phenotypes of the offspring in a sex-dependent manner [15-24], including offspring of dams fed high- and low-protein diets [17,20,23]. Female offspring from dams fed a high-multivitamin diet during pregnancy, unlike their male littermates, do not exhibit increased body weight (BW) and characteristics of metabolic syndrome if fed the American Institute of Nutrition (AIN) 93G diet at weaning [24]. However, when fed an obesogenic diet, they become more obese compared with the male rats and demonstrate earlier characteristics of the metabolic syndrome [24]. Protein content of maternal diets also affects male and female offspring differently. When rat dams were fed a high-protein (40% rather than 20% of total calories) diet during gestation and lactation, blood pressure and glomerulosclerosis were elevated in male offspring, whereas increased food efficiency, BW, and fat pads were observed in female offspring [17]. Male compared with female Wistar rats born to dams fed low-protein (8% wt/wt) diets were more hyperinsulinemic and insulin resistant at 20 weeks of age [20]. The present study is based on the 2 observations that the effects of maternal diets on rat offspring are sex dependent [17] and that AIN 93G diets based on S increase FI and presence of characteristics of metabolic syndrome in male rats [10,11]. Because of the relatively short duration of the present study, we hypothesized that S-based diets fed during pregnancy and lactation increase FI and the presence of characteristics of the metabolic syndrome to a lesser extent in female than in male rats. Therefore, the objective of this report is to describe effects of these maternal diets on FI and characteristics of metabolic syndrome and FI regulation in female offspring of Wistar rats.

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2. Methods and materials 2.1. Experimental design Two groups of pregnant rats (n = 18 per group) were fed either the C or the S diets during gestation and lactation. Six dams from each group were euthanized at day 20 of gestation, and the fetus was obtained. At birth, the pups of each litter of the remaining 12 dams were weighed and culled to 10 pups. At weaning, one female from each mother in each diet group was assigned to the C diet for 15 weeks (n = 12 per group). For the remaining pups, BW was measured at birth (day 1) and days 7, 14, and 21 and then weekly for 15 weeks. At birth, weaning (day 21 of age), and week 15 postweaning, one rat from each dam (n = 12 per dam diet group) was euthanized. Body fat composition was determined at birth, weaning, and week 15. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse rate were measured at weeks 4, 8, and 12 in the pups. Glucose and insulin tolerance tests were conducted at weeks 8 and 12. To obtain sufficient blood volume for hormone assays, blood samples from the offspring of the 6 dams in each diet group were obtained by pooling trunk blood of unsexed fetuses (n = 3-4 per dam) at day 20 of gestation and at birth. Blood samples were also obtained from pups (n = 5-6 per group) at weaning. Pups (n = 12 per group) were euthanized by decapitation after a 12-hour overnight food deprivation at weaning and at the end of the experiment (week 15) for blood collection. At week 15, pups in each diet group were allocated to 2 groups of 6 and received either glucose or water preloads and were euthanized 30 minutes later. Plasma concentrations of glucose, insulin, glucagon-like peptide 1 (GLP-1), ghrelin, peptide YY (PYY), homocysteine, and corticosterone were measured at weaning and at week 15. The protocol was approved by the University of Toronto Animal Care Committee, and care and maintenance of the animals conformed to the guidelines of the Canadian Council on Animal Care. 2.2. Animals and diets First-time pregnant Wistar rats were received at day 3 of gestation (Charles River, Quebec, Canada) and were housed individually in ventilated plastic transparent cages with bedding at 22°C ± 1°C and 12-hour light-dark cycle (lights off at 10:00 PM to 10:00 AM). At weaning, female offspring were housed individually in ventilated plastic transparent cages with bedding. The powdered diets were provided ad libitum in stainless steel cups with a mesh disk insert to reduce spillage. All rats had free access to water throughout the experiments. The composition (in grams per kilogram) of the test diets was as follows. The C diet contained C (200.0), cornstarch (529.4), sucrose (100.1), soybean oil (70.0), cellulose (50.0), vitamin mixture (10.0), mineral mixture (35.0), cystine (3.0), choline bitartrate (2.5), and tert-butylhydroquinone (0.014). The composition of the S diet was identical to the C diet,

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Table 1 Amino acid composition of C and S AIN 93G diets (per kilogram diet)

Alanine Arginine Aspartic acid + Asn Cystine Glutamic acid + Gln Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

Micellar C

Isolated S

(g) 4.7 5.7 10.6 3.5 34.8 2.8 4.4 8.7 13.9 12.2 4 7.7 16 8.4 6.6 1.7 8 10.4

(g) 6.7 11.9 18 4.5 29.9 6.7 4.1 7.7 12.8 9.9 4.5 8.1 7.9 8.1 5.9 2.2 5.9 7.9

The amino acid content of the diets is calculated based on purity of the protein sources (87% and 90% for C and S, respectively).Micellar C: Harlan Teklad; isolated S: Dyets Inc. Cystine was added to both C (3 g/kg diet) and S (2.54 g/kg diet) diets. Methionine was added to S diet (2.54 g/kg diet). The amounts shown in the table are the totals of the addition plus the bound amino acids.

except that S replaced C, and methionine (2.54) and cystine (2.54) were added as recommended for the AIN 93G S diet [25]. Contributions of protein, fat, and carbohydrate to total energy content were 19%, 17%, and 64% in the C diet and 20%, 17%, and 63% in the S diet, respectively. Amino acid content of the C and S diets as fed is shown in Table 1. Cornstarch, high-protein C (87%), and cellulose were purchased from Harlan Teklad (Madison, WI). The vitamin and mineral mixtures, cystine, methionine, choline bitartrate, and tert-butylhydroquinone were purchased from Dyets Inc (Bethlehem, PA); sucrose was from Allied Food Service (Toronto, Ontario, Canada); and soybean oil was from Loblaws (Toronto, Ontario, Canada). The genistein, daidzein, and glycitein content (micrograms per gram) of the S diet was 36.1, 31.3, and 4.4, respectively. 2.3. Glucose tolerance test Rats were fasted overnight for 10 hours. A blood sample was withdrawn from the tail vein before and at 15, 30, and 60 minutes after a glucose gavage (0.375 g glucose per milliliter, 5 g glucose per kilogram BW). 2.4. Insulin tolerance test Rats were fasted overnight for 10 hours. Insulin (Humulin-R; Eli Lilly and Company, Indianapolis, IN) injections were given intraperitoneally (0.5 U/mL, 0.75 U insulin per kilogram BW); and blood was obtained before and at 15, 30, and 60 minutes after an insulin injection.

2.5. Blood pressure Systolic and diastolic blood pressures were measured by the noninvasive tail-cuff method with optical plethysmography using a tail manometer-tachometer system (BP-2000; Visitech Systems, Apex, NC). Rats were restrained in holders on a platform with constant temperature of 30°C. They were adapted daily to the device for 5 days. On the day of measurement, 5 mock measurements preceded a series of 10 measurements; and only the latter were used in calculating the average as previously reported [26]. 2.6. Blood glucose Tail vein glucose concentration was assayed using a handheld commercial glucose meter (MediSense Precision Xtra; Abbott Laboratories, Alameda, CA) using test strips [26]. Glucose in plasma from trunk blood obtained upon decapitation was measured using a glucose oxidase kit (Ascensia Elite XL, Bayer AG, Leverkusen, Germany). 2.7. Short-term FI Short-term FI (1 hour) was measured 30 minutes after water and protein preloads at week 7 when rats were sexually mature. Before testing, rats were adapted to the experimental procedures. They were gavaged with water over 7 days before the adaptation test, performed as follows. Food was deprived for 12 hours before measurement. On day 1, one half of the rats were gavaged with the water preload, whereas the rest were untreated. On the next day, this testing order was reversed. The experiment began when it was determined that the process of gavage did not affect FI. At week 7 after weaning, a randomized design was applied to compare the effect of C and S preloads on shortterm FI. At week 7, on the first day of the experiment and after 12-hour fasting, rats received at 9:30 PM C, S (3 g/kg BW per 6 mL distilled water), or water control (6 mL distilled water) as preloads in random order. Food was introduced at 10:00 PM and was measured for 1 hour in both experiments. After a washout day, the testing order was reversed. Ultimately, all rats received all treatments in random order. Food intake was measured to the nearest 0.1 g under red light. 2.8. Long-term FI Weaned rats were housed individually in ventilated plastic transparent cages with bedding. Diets were provided ad libitum. The powdered diet was provided in jars with a mesh disk insert to minimize spillage. Food intake was measured weekly for 14 weeks after weaning. Food intake over 24 hours was not measured during weeks 7 to 8 when preload experiments were conducted. 2.9. Blood collection Trunk blood was collected in chilled Vacutainer tubes (BD, Franklin Lakes, NJ) containing EDTA + Trasylol (Bayer AG, Leverkusen, Germany) solution (10% blood volume,

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500 kIU/mL). A dipeptidyl peptidase IV inhibitor (Cat # DPP4; Linco Research Inc, St Charles, MO) was added at 1% ratio for the determination of active GLP-1 in plasma. Blood samples were centrifuged for 20 minutes at 3000g and 4°C for 10 minutes. Plasma was separated and immediately stored at −80°C. 2.10. Hormone assays Plasma insulin was measured using insulin enzyme immunoassay (Cat # 80-INSRT-E01; Alpco Diagnostics, Salem, NH) with assay sensitivity of 0.124 ng/mL. Plasma homocysteine was measured by enzyme immunoassay (Cat # 1945361; Bio-Rad Laboratories, Inc, Hercules, CA) with assay sensitivity of 1.0 μmol/L. Plasma corticosterone was measured using enzyme immunoassay (Cat # DSL-10-81100; Beckman Coulter, Webster, TX) with assay sensitivity of 1.6 ng/mL. Total plasma PYY concentrations were measured using radioimmunoassay method (Cat # RMPYY-68HK; Millipore Research Inc, St Charles, MO) with assay sensitivity of 15.6 pg/mL. Total plasma ghrelin concentrations were measured using radioimmunoassay method (Cat # GHRT89HK; Linco Research Inc) with assay sensitivity of 93 pg/mL. Plasma GLP-1 concentrations were measured using the enzyme-linked immunosorbent assay method (Cat # EGLP35K; Linco Research Inc) with assay sensitivity of 2 pmol/L. 2.11. Body composition Fat mass and lean mass were measured at birth by dual-energy x-ray absorptiometry (DEXA) (pSabre; Orthometrix, Naples, Fla, USA) applying a specialized software program (Host Software version 3.9.4; Scanner Software version 1.2.0) [27]. After euthanization by carbon dioxide, carcasses were scanned by DEXA at a speed of 10 mm/s and a resolution of 0.5 × 1.0 mm. At weaning and at the end of experiments, fat pad mass was measured by dissection of extracted abdominal and perirenal fat. 2.12. Isoflavones measurement Homogenized S samples were analyzed for isoflavones (genistein, daidzein, and glycitein) by gas chromatography– mass spectrometry, as previously described [28]. The isoflavones analysis involved extraction of samples twice with 5 mL 70% methanol, passing a portion of extraction through a C18 solid-phase extraction column (Octadecyl C18/14%, 200 mg/3 mL; Applied Separations, Allentown, PA), hydrolysis with β-glucuronidase (Helix Pomatia; Sigma Aldrich, St Louis, MO), and passage through another C18 solid-phase extraction column. An internal standard (5α-androstane-3β,17β-diol; Steraloids Inc, Wilton, NH) was added to the column eluent, and the sample was then derivatized with Tri-Sil Reagent (Pierce Co, Rockford, IL) before injection to the gas chromatography–mass spectrometry (Agilent 6890 series GC system interfaced with an Agilent 5973 network mass selective detector; Agilent Technologies, Wilmington, DE).

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2.13. Statistical analyses All data are expressed as means ± SEM. The effect of dams' diet on BW, FI, glucose response, SBP, and DBP was analyzed by PROC MIXED MODEL procedure with diet and time as main factors. A 1-way repeated-measure analysis of variance followed by post hoc Tukey test was conducted when treatment effects or interactions were statistically significant. Student unpaired t test was applied to compare the dependent measures (eg, hormones) at individual time points. Blood glucose response was calculated as the total incremental area under the curve (tAUC) of the blood glucose concentration over 1 hour after receiving glucose by gavage for the glucose tolerance test and after receiving insulin injection for the insulin tolerance test. For the former, the reported tAUC is positive above baseline, whereas for the latter, the tAUC is a negative representing the area below baseline. The homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated as fasting glucose multiplied by fasting insulin divided by 22.5 [29]. Food intake in response to protein preloads was analyzed by using PROC MIXED MODEL procedure with diet and time as main factors. To examine the suppressant effect of protein preload on FI, the Δ of FI (FI after water control − FI after protein preload) was calculated. The effect of the diets and preloads on plasma hormone concentrations was also analyzed by using PROC MIXED MODEL procedure with diet and preload as main factors. All analyses were conducted using SAS (version 9e; SAS Institute, Cary, NC). Statistical significance was defined at P b .05. 3. Results There were no differences due to the dams' diets (C vs S diets, respectively) on litter size (13.2 ± 0.7 and 13.2 ± 0.5), Table 2 Effect of dams' diet on BW and fat mass in female offspring (n = 11-12 per group) Dams' diet C Birth BW (g) TF3(g) TF/BW ratio (%BW) Weaning BW (g) FPM (g) FPM/BW ratio (%BW) Wk 15 postweaning BW (g) FPM (g) FPM/BW ratio (%BW)

S 6.2 ± 0.2 0.3 ± 0.0 6.2 ± 0.8

6.3 ± 0.1 0.4 ± 0.1 7.2 ± 0.8

59.7 ± 2.5 0.5 ± 0.0 0.8 ± 0.0

55.2 ± 2.0 0.4 ± 0.0 0.7 ± 0.0

322.0 ± 8.9a 20.2 ± 1.2a 6.2 ± 0.3

360.0 ± 12.0b 26.3 ± 2.5b 7.2 ± 0.5

Data are means ± SEM. Unpaired t test. Values in a row with different superscript letters are significantly different; P b .05. TF indicates total fat (measured by DEXA at birth); FPM, fat pad mass (abdominal + perirenal).

Weight (g)

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A. Jahan-mihan et al. / Nutrition Research 31 (2011) 644–651 S C

400 350 300 250 200 150 100 50

*

*

*

*

Dams' diet

C

S

Water preload

(g) 6.5 ± 0.4

6.4 ± 0.5

C preload S preload

4.4 ± 0.4 3.8 ± 0.5

5.2 ± 0.7 4.3 ± 0.5

C preload S preload

2.1 ± 0.6 a 2.7 ± 0.8 a

1.2 ± 0.7 2.2 ± 0.8 a

Protein preload 1

2

3

4

5

6

7 8 Week

9

10 11 12 13 14

Water − protein

Fig. 1. Effect of dams' diet on postweaning BW of female offspring (n = 12 per group). Data are means ± SEM; BW was analyzed by MIXED model with maternal and time as main factors. Time: P b .0001; maternal diet: P = .07; ⁎P b .05.

male to female ratio (0.53 ± 0.05 and 0.52 ± 0.03), or the pups' body fat or BW at birth (Table 2). In the PROC MIXED analysis, there was an overall trend over time for a higher BW (P = .07) in offspring born to dams fed the S diet. At 14 weeks, BW was higher by 12% in female offspring born to dams fed the S diet (P b .05) (Fig. 1), with the difference becoming statistically significant at week 11 (P b .05). Dams' diet had no effect on BW and fat pad mass at weaning (Table 2); but by 15 weeks postweaning, fat pad mass was larger in offspring born to dams on the S diet (P b .05) (Table 2). Food intake was influenced by time (P b .0001) and dams' diet (P b .05). It was higher in offspring born to the dams fed the S diet throughout weeks 13 to 14 (Fig. 2). At week 7, FI suppression of protein preloads (Δ of FI after water preload and after protein preload) was not influenced by the dams' diet at 1 hour (Table 3). A trend (P = .08) to higher plasma insulin concentrations (nanograms per milliliter) due to the S diet compared with the C diet was found both in the fetus (n = 5-6 per group) at day 20 of gestation (0.41 ± 0.060 vs 28 ± 0.03) and in the pups at birth (0.49 ± 0.08 vs 0.27 ± 0.07). When the data

S C

250

*

200 Food Intake (g)

Table 3 Effect of dams' diet on FI (1 hour) in response to protein preloads in female offspring at week 7 (n = 11-12 per group)

*

150 100 50

1

2

3

4

5

6

7

8

9

10 11 12

13 14

Week

Fig. 2. Effect of dams' diet on postweaning FI of female offspring (n = 12 per group). Data are means ± SEM. Food intake was analyzed by MIXED model with maternal diet and time as main factors. Time: P b .0001; diet: P b .05; ⁎P b .05. Food intake was not measured during weeks 7 to 8 when preload measures were conducted.

P Total intake D: NS P: b.0001 P × D: NS Water − protein D: NS P: NS P × D: NS

Data are means ± SEM. Food intake was analyzed by MIXED model with maternal diet and preload as main factors. Protein preload was given by gavage at 3 g/kg BW per 6 mL. Water preload was given in the same volume as protein preload. Water-protein: FI after water preload − FI after protein preload. D indicates diet; P, preload; NS, not significant. a Significant FI suppression: P b .05.

were analyzed by 2-way analysis of variance, day was not a factor; but maternal diet was (P b .01). Insulin was higher at 0.45 ± 0.05 ng/mL compared with 0.28 ± 0.03 ng/mL in the samples from offspring of the S-fed dams. At weaning, fasting plasma GLP-1 was higher and PYY was lower in rats born to dams fed the S diet compared with those born dams fed the C diet (P b .05) (Table 4). Dams' diet had no effect on fasting plasma concentrations of ghrelin, homocysteine, and corticosterone at weaning or at week 15 (Table 4). However, plasma GLP-1 was affected by preload at week 15 and was higher in pups from both dam groups after glucose preload (9.0 ± 1.2 pmol/L) than after

Table 4 Effect of dams' diet on fasting plasma measures in female offspring (n = 5-6 per group) Dams' diet Weaning Glucose, mmol/L Insulin, ng/mL HOMA-IR Corticosterone, ng/mL Homocysteine, μmol/L Ghrelin, ng/mL GLP-1, pmol/L PYY, pmol/L Wk 15 postweaning Glucose, mmol/L Insulin, ng/mL HOMA-IR Corticosterone, ng/mL Homocysteine, μmol/L Ghrelin, ng/mL GLP-1, pmol/L PYY, pmol/L

C

S

5.7 ± 0.24 0.2 ± 0.01 0.04 ± 0.01 250.8 ± 58.9 6.0 ± 0.95 2.2 ± 0.13 2.5 ± 0.23a 59.1 ± 3.14a

5.5 ± 0.20 0.2 ± 0.03 0.05 ± 0.01 288.2 ± 48.7 7.6 ± 1.73 2.4 ± 0.31 3.4 ± 0.32b 43.4 ± 2.28b

5.2 ± 0.11 3.9 ± 0.48 0.91 ± 0.10 295.2 ± 17.9 7.1 ± 0.87 3.6 ± 0.34 2.5 ± 0.23 53.1 ± 5.80

4.9 ± 0.24 3.8 ± 0.54 0.86 ± 0.13 314.2 ± 28.7 8.3 ± 1.27 3.9 ± 1.27 2.7 ± 0.44 42.9 ± 2.39

Data are means ± SEM. Unpaired t test. Values in a row with different superscript letters are significantly different; P b .05. The HOMA-IR index was calculated as fasting glucose (millimoles per liter) multiplied by fasting insulin (nanograms per milliliter) divided by 22.5.

A. Jahan-mihan et al. / Nutrition Research 31 (2011) 644–651 Table 5 Effect of dams' diet on SBP, DBP, and pulse rate in female offspring (n = 11-12 rat per group) Dams' diet SBP (mm Hg) Wk 4 8 12 DBP (mm Hg) 4 8 12 Pulse (BPM) 4 8 12

C

S

P

118 ± 3.9 122 ± 4.5 119 ± 5.7

121 ± 7.8 133 ± 3.5 141 ± 6.3

D: .05 T: NS D × T: NS

93 ± 8.9 93 ± 9.1 91 ± 8.8

99 ± 9.7 100 ± 7.5 115 ± 5.4

D: NS T: NS D × T: NS

381 ± 8.1 361 ± 18.1 419 ± 16.0b

D: NS T: NS D × T: .005

396 ± 14.8 393 ± 7.3 375 ± 7.9a

Data are means ± SEM. MIXED model with dams' diet and time as main factors followed by Tukey post hoc test when interaction was significant. Values in a row at each week with different superscript letters are significantly different; P b .05. T indicates time; BPM, beat per minute.

water control (2.6 ± 0.34 pmol/L) (P b .0005). Plasma glucose was also affected by preload at week 15 and was higher in pups from both dam groups after the glucose preload (5.7 ± 0.36 mmol/L) than after water control (5.1 ± 0.16 mmol/L) (P b .01). Dams' diet had no effect on HOMA-IR index and fasting plasma concentrations of either glucose or insulin at weaning and week 15 (Table 4) or on glucose response (tAUC) to glucose preloads or to insulin injections at weeks 8 and 12 (data are not shown). Systolic blood pressure and pulse rate were higher (P b .05) in rats born to dams fed the S diet (Table 5). With time to week 12, the effect of the S diet on pulse rate increased as shown by an interaction between diet and time (P b .005). 4. Discussion The results of the present study support the hypothesis that the AIN 93G diet with S compared with C in maternal diets increases FI and BW in female offspring but has less impact on characteristics of metabolic syndrome to 15 weeks postweaning than in male offspring. In female offspring, the S-based diet, when compared with the C-based diet, resulted in higher FI, BW, and SBP, similar to its effect in male offspring [11]. However, female offspring were more resistant to the effect of protein source in the dams' diet on several measures reported to be affected in the male offspring [11]. In the previously reported study, the increase in BW of male offspring born to dams fed the S diet started at week 4 [11]; but this effect was delayed to week 11 in the females. Similarly, an increase in FI was found beginning at week 4 in males born to dams fed the S diet [10] but not until week 13 in females. Moreover, the S diet resulted in higher SBP, DBP, and pulse rate in male offspring [11];

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but only SBP was affected in female offspring. Furthermore, FI (1 hour) in response to protein preloads was not influenced by the dams' diet in females but was in males [10]. At weaning, in female offspring born to dams fed the C diet, plasma PYY, an anorexigenic compound [30], was higher and consistent with their lower FI. However, a causative relationship is in doubt because there was no effect of the dams' diet on PYY at week 15. Similarly, plasma PYY concentrations were not influenced by the dams' diet in male offspring, whereas FI was higher in those born to dams fed the S diet [10]. Unlike its effects in males [10,11], the dams' diet had no effect in females on either plasma glucose response to glucose preloads or the HOMA-IR index, which are indicators of insulin resistance, and also had no effect on plasma concentrations of glucose, insulin, or GLP-1 at week 15. This may suggest that the observed higher insulin concentrations in the pooled unsexed offspring immediately and prior to birth was primarily due to the effects of the S diet on the males, which showed higher plasma insulin concentrations at weaning and early postweaning insulin resistance and more characteristics of metabolic syndrome than their female littermates [10,11]. It should be noted however that, although it is clear that the effects of the maternal diets were modest and much less in the female than in the male rats for the 15-week duration of the study, this may be both a time and diet effect. Female rats develop insulin resistance later in life (eg, 21 months) than males [19]. It may be suggested that the present study of 15-week duration postweaning was too short to show an effect in females. At 20 weeks of age, male but not female Wistar rats born to low protein–fed dams (8% wt/wt) were relatively hyperinsulinemic and insulin resistant [20]. Furthermore, there are 2 aspects of the weaning diet to consider. All of the female offspring were weaned to the C diet, which leaves uncertain whether those from the dams fed the S diet would be less affected if fed an S diet at weaning as suggested by the predictive adaptive hypothesis [6]. However, the effect of the maternal S diet on male offspring was not prevented by feeding them the S diet at weaning [10,11]. Weaning the female rats to an obesogenic diet may be more informative and may show a much greater consequence of feeding the S diet to the dams. Female rats born to dams fed high-multivitamin diets during pregnancy rapidly increased BW and FI and demonstrated characteristics of metabolic syndrome more than male rats when fed an obesogenic diet [24] but, unlike the male, did not respond when fed the AIN 93G diet [24]. The mechanisms by which the source of protein in nutritionally complete maternal diets influenced phenotype of their offspring are uncertain and remain to be explored. However, there are many differences in characteristics of C and S and in the diets as fed. These include amino acid composition, bioactive peptides, and digestion kinetics [31]. The additions of free amino acids also differed between the 2 diets. Furthermore, arginine, which is almost 2-fold higher

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in S than C, is one of the most potent insulinotropic amino acids [32-34] and may have contributed to higher in utero insulin exposure of offspring born to dams fed the S diet [11]. Furthermore, many bioactive peptides have been identified in both C and S. For example, C is rich in casomorphins capable of activating opioid receptors in the enteric nervous system and on the vagus, resulting in lower blood pressure through vasodilation [35-37]. Moreover, C and S differ in digestion kinetics. Casein is a slow protein, whereas S is classified as a fast protein [38]. The digestion kinetics of proteins influence their metabolic activities [39], brain amino acid concentrations, and neural activity [40]. For example, lower protein synthesis after ingestion of S compared with animal proteins [41] could be explained by the fact that the pattern of amino acids reaching the liver is not only more rapid but also more unbalanced in composition than milk protein [42,43]. The genistein content of the S diet was only 36 μg/g of the diets, far less than that reported (250 μg/g diet) in maternal diets affecting epigenetic and phenotypic changes in mice [44]. However, because the required physiological dose for isoflavones during gestation and lactation is not clear, their possible role cannot be dismissed. Various isoflavones in S with a wide range of pharmacokinetic properties, especially during gestation and lactation, have been identified [45-47]. Phytoestrogenic effects of isoflavones may also explain, in part, the differential effects of S on female vs male offspring observed in these studies. Moreover, these results cannot be explained by stress responses of the dams or pups because there were no differences due to diet in litter size or birth weight of the offspring, or in BW of dams at arrival, day 14, and day 20 of gestation and no difference was found in fasting corticosterone level in pups at weaning and week 15. Although this is primarily a descriptive study and mechanisms have not been described, the results of this and previous reports [10,11] show that nutritionally complete and macronutrient-balanced maternal diets may differ in their effects on fetal programming. Metabolic studies are often conducted with diets that differ in protein source or amino acid additions, or are composed of only free amino acids but are assumed to be similar based on their support of weight gain during pregnancy and lactation and weight gain in the offspring [25]. However, it is clear that these are not the only outcomes by which the composition of maternal diets should be judged. The relevance of the results of this study to humans is uncertain because human diets during gestation contain many protein sources. Infants are more vulnerable, however, because they are weaned to formulas that contain a constant source of protein (eg, whey/C mixtures, S) or their hydrolysates, with or without amino acid additions, during early life and may be fed the same formula for several months. Thus, it may be of interest to compare the presence of characteristics of metabolic syndrome in later life among infants sustained on formulas of different composition.

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