Supplemental arginine above the requirement during suckling causes obesity and insulin resistance in rats

    Supplemental arginine above the requirement during suckling causes obesity and insulin resistance in rats Lila Otani, Tomomi Mori, Ayaka Koyama, Shin-Ichiro Takahashi, Hisanori Kato PII: DOI: Reference:

S0271-5317(16)00016-6 doi: 10.1016/j.nutres.2016.01.007 NTR 7597

To appear in:

Nutrition Research

Received date: Revised date: Accepted date:

5 October 2015 18 January 2016 25 January 2016

Please cite this article as: Otani Lila, Mori Tomomi, Koyama Ayaka, Takahashi Shin-Ichiro, Kato Hisanori, Supplemental arginine above the requirement during suckling causes obesity and insulin resistance in rats, Nutrition Research (2016), doi: 10.1016/j.nutres.2016.01.007

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ACCEPTED MANUSCRIPT Supplemental arginine above the requirement during suckling causes obesity and insulin resistance in rats

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Lila Otani1, Tomomi Mori1, Ayaka Koyama1, Shin-Ichiro Takahashi2, and Hisanori Kato1 1

Animal Sciences, Graduate School of Agricultural and Life Sciences, The University of

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Food for Life, Organization for Interdisciplinary Research Projects, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

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Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

*Corresponding author: Prof. Hisanori Kato, Food for Life, Organization for Interdisciplinary Research Projects, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel&Fax: +81-3-5841-1607 E-mail: [email protected]

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Running title: Arginine supplementation and obesity

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Word count (not including title page, Abbreviations list and References): 5761

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ACCEPTED MANUSCRIPT Abbreviations

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5,10-CH3 THF: 5,10-methylenetetrahydrofolate, AUC: area under the curve, CT: computed

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tomography, GH: growth hormone, IGF-1: insulin like growth factor-1, NO: nitric oxide,

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OGTT: oral glucose tolerance test, SAH: S-adenosylhomocysteine, SAM:

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S-adenosylmethionine, TG: triglyceride

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ACCEPTED MANUSCRIPT Abstract

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Nutrition in early life is important in determining susceptibility to adult obesity, and arginine

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may promote growth acceleration in infants. We hypothesized that maternal arginine

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supplementation may promote growth in their pups and contribute to obesity and alteration of the metabolic system in later life. Dams and pups of Wistar rats were given a normal diet

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(15% protein) as a control (CN) or a normal diet with 2% arginine (ARG). Altered profiles of free amino acids in breast milk were observed in that the concentrations of threonine and glycine were lower in the ARG dams compared to the CN dams. The offspring of the CN and

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ARG dams were further subdivided into normal diet (CN-CN and ARG-CN) groups and a

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high-fat diet groups (CN-HF and ARG-HF). In response to the high-fat diet feeding, the

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visceral fat deposits were significantly increased in the ARG-HF group (although not

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compared to the CN-HF group); no difference was observed between the CN-CN and ARG-CN groups. The blood glucose and insulin levels after glucose loading were significantly higher in the ARG-HF group compared to the CN-HF group. The results suggest that the offspring of dams supplemented with arginine during lactation acquired increased susceptibility to a high-fat diet, resulting in visceral obesity and insulin resistance. The lower supply of threonine and glycine to pups may be one of the contributing causes to the programming of lifelong obesity risk in offspring. Our findings also indicated that maternal arginine supplementation during suckling causes obesity and insulin resistance in rats. 3

ACCEPTED MANUSCRIPT Key words: Postnatal nutrition, arginine, obesity, insulin sensitivity, breast milk, obesity,

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metabolic syndrome

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ACCEPTED MANUSCRIPT 1. Introduction

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Metabolic syndrome — which includes central obesity, insulin resistance, and

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hypertension — has a multifactorial etiology that involves a series of complex interactions

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between individual dietary habits and the genetic background. The current prevalence of metabolic syndrome is a consequence of pervasive obesity. Increasing evidence indicates that

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the risk of obesity can be developmentally induced during the prenatal or postnatal period by unbalanced maternal nutrition. As such, alterations of nutrient delivery during early periods can significantly impact an individual’s susceptibility to obesity, diabetes and cardiovascular

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diseases later in life.

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The nursing period is an important critical window in the determination of the future metabolic system. Babies born small who experience rapid catch-up growth have been shown to tend to develop obesity in later life [1]. In addition, rapid weight gain during the first 3 months of life is associated with central adiposity and reduced insulin sensitivity in early adulthood, compared to slower weight gain [2]. Rapid early growth may thus be linked to adult obesity.

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ACCEPTED MANUSCRIPT Singahal et al. suggested that low birth weight with rapid catch-up growth in infants is

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associated with the feeding of formula milk [3, 4]. The disposition to obesity during the

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suckling period has also been observed in animal models. In rats, the adult offspring of

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mothers that were over-nourished while suckling are susceptible to obesity despite a normal post-weaning diet [5, 6]. Overnutrition during pre-weaning also induced adiposity in

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young-adult baboons [7]. It was demonstrated that dietary arginine supplementation

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stimulated protein synthesis and accretion in the skeletal muscle of neonatal pigs [8, 9].

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Arginine is necessary for the optimal growth of young mammals including rats, pigs, and

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dogs [10–12]. It is one of the major sources of tissue proteins and the precursor of nitric oxide

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(NO), urea, polyamines, proline, glutamate, and agmatine [13]. Arginine is a well-known growth hormone (GH) stimulator, and GH is an important modulator of linear growth. However, little is known about the influence of arginine supplementation in early life on metabolic systems in later life. We hypothesized that excessive maternal arginine intake during the suckling period may promote early growth and cause metabolic changes in rat offspring. In the present study, we thus examined the effect of arginine supplementation on susceptibility to obesity and insulin resistance in rat offspring, as well as its effect on the composition of breast milk. Our findings demonstrated that excessive arginine 6

ACCEPTED MANUSCRIPT supplementation during the suckling period resulted in visceral obesity with insulin resistance

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in offspring. This result suggests that additional care may be required regarding the arginine

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intake level in lactating women.

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2. Methods and materials 2.1. Animals

Eight pregnant (14 days) Wistar rats were purchased from Charles River Laboratories

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(Kanagawa, Japan). Rats were fed a commercial diet (MF-2, Oriental BioService) until

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delivery. After delivery, we randomly divided the dams and pups into two groups; the first

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was given a normal diet (15% protein diet) as a control group (CN, 4 dams in the group), and

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the second group was given a normal diet supplemented with 2% arginine, as the arginine-treated group (ARG, 4 dams per). All litters were culled to 8 pups on postnatal day 4. The mean body weight of the pups kept alive was similar in both litters. The dams and their pups were maintained under these conditions until weaning on day 21. After weaning, only the male offspring were studied and were placed in individual wire mesh cages (KN-615, Natsume Seisakusho. Tokyo). The size of cage was 750 mm wide × 210 mm long × 170 mm high.

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From 3 weeks of age, all animals were given a normal diet (15% protein diet). At 6 weeks of

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age, the offspring in the CN and ARG groups were further divided into a normal diet group

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(15% protein, 12.5% calories from fat) and a high-fat diet group (15% protein, 35% calories from fat). The composition of each purified diet is described in Table 1.The number of rats in

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each group was 9–10. The offspring were weighed once a week. Their food intake was

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measured every day.

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An oral glucose tolerance test (OGTT) was performed when the pups reached 11 weeks of

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age, as follows. After a 16-h overnight fast, the rats were administered glucose (2 g kg–1)

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orally. Blood was collected from the tail vein in heparinized tubes chilled on ice. Blood glucose levels were measured using a commercial kit (Wako Pure Chemical Industries, Osaka, Japan), and insulin was measured using an insulin measurement kit (Morinaga Institute of Biological Science, Yokohama, Japan) following the manufacturer’s instructions. All samples were run in duplicate.

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At 12 weeks of age, the rats were anesthetized with pentobarbital (i.p., 30 mg·kg–1, Abbott,

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North Chicago, IL) after a 1-h fast, and blood was taken from the carotid artery. All rats were

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euthanized by exsanguination under pentobarbital anesthesia immediately after the blood sampling. The liver, longissimus muscles and mesentery fats were excised, snap-frozen in

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liquid nitrogen and stored at −80°C until the analyses. All animal experiments were carried out in accord with the guidelines of the Animal Usage Committee of the Faculty of Agriculture, University of Tokyo, and were approved by the committee (Permission No.

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P09-375).

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2.2. Computed tomography scan analysis (body composition) For the computed tomography (CT) analysis of body fat mass, 11-week-old rats were anesthetized with isoflurane (3%–4%, 5 L/min, Dainippon Sumitomo Pharma, Osaka, Japan) and then placed in the chamber of a CT scanner for small animals (Latheta LCT-100, Hitachi Aloka Medical, Tokyo), according to the manufacturer's instructions. CT scans were performed at 2-mm intervals. Body fat composition images between thoracic vertebra T11 and S1 (the tip of the ischium) were used for the quantitative assessment with the Latheta software (ver. 3.00). The visceral adipose tissue was analyzed by assessing the fat content 9

ACCEPTED MANUSCRIPT within the visceral adipose tissue, and the subcutaneous adipose tissue and muscle were

2.3. Measurement of triglyceride (TG) in tissue

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distinguished and evaluated quantitatively.

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Lipids were extracted from the liver and muscle using the method described by Folch et al [14]. A piece of tissue was briefly homogenized in 4 ml of chloroform:methanol (2:1, v/v), after which 2% potassium chloride solution was added to the mixture and vortexed

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vigorously. The organic phase was transferred and dried in a vacuum. It was then dissolved in

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isopropanol, and samples were processed for analysis using a TG measurement kit (Wako),

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following the manufacturer’s instructions. The measurements of TG were run in duplicate.

2.4. Amino acids and hormone concentrations in the breast milk and plasma of lactating dams or pups Breast milk and dam’s plasma were collected on day 14 of lactation from additional groups of dams fed the control diet (CN dams, n=5) or the 2% arginine-supplemented diet (ARG dams, n=5). After separation from the pups for 30 min, the dams were anesthetized with isoflurane (3%–4%, 5 L/min). Oxytocin (i.p., 0.1 ml, 2IU, Sigma-Aldrich Japan, Tokyo) was injected 10

ACCEPTED MANUSCRIPT into the dams to facilitate the collection of milk, and 0.5–1 ml milk was manually obtained

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from all teats. Dam’s blood was also collected from the tail vein in heparinized tubes chilled

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on ice. Pups were decapitated for the collection of trunk blood on day 14 or 21 of lactation.

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Samples were frozen at −80°C for further analysis. Whole milk samples were analyzed after sonication (Microson™ Ultrasonic cell disruptor, Misonix Inc., Farmingdale, NY) with 10

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bursts of 10-sec duration. Skimmed milk was prepared by centrifuging 500 μL of whole milk at 11,400×g, 4°C for 2 min. The fat layer was removed, and the aqueous phase was assayed

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as described [15].

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The plasma and milk samples were each separately mixed with an equal volume of 3% (w/w)

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trichloroacetic acid. The mixture was centrifuged, and the supernatant was filtered through a 0.45-μm membrane prior to vialing as described [16]. The amino acid concentrations were measured by an automatic amino acid analyzer (L-8900; Hitachi, Tokyo). Single amino acid measurements were performed. The levels of the following components in plasma and milk were determined by commercial kits: leptin (R&D Systems, Minneapolis, MN), adiponectin (Otsuka Pharmaceuticals, Tokyo), insulin like growth factor-1 (IGF-1; R&D Systems), insulin (Morinaga Institute of Biological Science), and corticosterone (Assaypro, St. Charles, MO) according to the manufacturers' instructions. All hormone measurements were run in 11

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duplicate.

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2.5. Statistical analyses

The data are presented as the means ± standard error of the mean (SE). We used a statistical

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software package (Ekuseru-Toukei 2010, Social Survey Research Information Co.) for all data analyses. Statistical significance was calculated using Student’s t-test or a two-way analysis of variance (ANOVA) with Bonferroni and Tukey post hoc testing as indicated. A

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probability value of P < 0.05 was considered significant.

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We analyzed the values of body weight, CT-based body compositions, blood biochemical parameters, and liver and muscle TG in the pups fed the control diet or high-fat diet by performing a two-way ANOVA, followed by Bonferroni and Tukey post hoc tests to assess the differences between groups. The sample sizes were n=8 for the CN-CN group, n=9 for the ARG-CN group, n=9 for the CN-HF group, and n=10 for the ARG-HF group. The statistical differences in the body weight changes of the dams and pups at 6 weeks of age were assessed using Student’s t-test. The sample sizes were n=17 for the CN group and n=19 for the ARG group. The differences in the amino acid concentrations of the dams and pups were assessed 12

ACCEPTED MANUSCRIPT by Student’s t-test. The sample sizes of the dams were n=4 for the CN group and n=5 for the

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ARG group. Two pups per litter were subjected to the analyses (CN group, n=8; ARG group,

3. Results 3.1. Growth and body composition

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n=10).

At the end of the supplementation, the body weights were slightly lower in the pups of the

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arginine-supplemented group compared to the control group, although the difference was not

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significant (CN, 65 ± 1; ARG, 60 ± 2 g). After the further subdivision into the normal diet

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and high-fat diet groups at 6 weeks of age, no difference was observed in body weight

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between the CN-CN and ARG-CN groups under the normal diet feeding conditions (Fig. 1A). In contrast, in response to the high-fat diet, the body weights of the ARG-HF group were modestly increased (Fig. 1B), but the difference was not significant (p=0.07).

We also estimated the body fat mass of the rats’ abdominal and subcutaneous adipose tissue using a CT scan analysis on the abdominal area (Fig. 2). The subcutaneous fat volume was also increased in the ARG-HF group, although the difference was not significant (Fig. 2A,B). 13

ACCEPTED MANUSCRIPT The adipose volume was significantly increased in the intra-abdominal tissue of the rats in the

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ARG-HF group compared to the CN-CN and ARG-CN groups (Fig. 2A,C). The visceral fat

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weights (including mesenteric, epididymal, and retroperitoneal depots) were also

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significantly increased in the ARG-HF group (38.8 ± 1.6 g) compared to the CN-HF group (33.9 ± 1.2 g), as shown in Table 1. Under the normal diet feeding condition, the visceral fat

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weight was 30.9 ± 0.7 g in the CN-CN group, and 32.2 ± 1.6 g in the ARG-CN group (Table

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3.2 Glucose tolerance test

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1).

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Figure 3 shows the results of the oral glucose tolerance test (OGTT) in the 11-week-old rats.

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With the normal dietary intake, no difference was observed in glucose levels between the CN and ARG rats (Fig. 3A). The insulin levels after glucose loading were slightly higher in the ARG-CN group than in the CN-CN group (Fig. 3C). The data of the CN-HF group did not differ from those of the control animals (CN-CN) with respect to plasma glucose and insulin concentrations. On the other hand, in the high-fat diet groups, the rats that were supplemented with arginine during lactation had significantly higher levels of glucose and insulin levels after glucose loading (Fig. 3B,D). The total area under curve (AUC) for glucose and insulin is presented in Figure 3E,F. The insulin AUC was significantly increased in the ARG-HF group 14

ACCEPTED MANUSCRIPT compared to the CN-HF group. The glucose AUC was increased in the ARG-HF group, but

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the difference was not significant.

3.3. TG concentration in the plasma, liver and longissimus muscle

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Under the normal dietary conditions, the TG content in the longissimus muscle was significantly increased in the ARG-CN rats compared to the CN-CN rats (Fig. 4C). There was no significant difference between the ARG-CN and CN-CN groups or between the ARG-HF

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and CN-HF groups with respect to the liver TG (Fig. 4B). Figure 4C shows the significant

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difference between the ARG-HF and CN-HF groups with respect to plasma TG.

3.4. Amino acid concentrations in the breast milk and plasma of lactating dams or pups We measured the leptin, corticosterone, adiponectin, and insulin levels in the dams’ breast milk and plasma at 14 days of lactation. There were no significant differences in the profiles of these hormones (IGF-1, leptin, corticosterone, adiponectin, and insulin) between the CN and ARG groups, in the breast milk or in the plasma of the dams and pups (data not shown). Figure 5 shows the results of the measurement of free amino acid levels in the dams’ plasma and breast milk at 14 days of lactation. The arginine supplementation during lactation 15

ACCEPTED MANUSCRIPT produced an incremental increase of amino acids that are closely related to the urea cycle

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(arginine, proline, ornithine) in dam’s plasma. The plasma levels of valine, methionine,

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isoleucine, and leucine were also higher in the arginine-supplemented group.

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Isoleucine was significantly increased in the breast milk from the dams fed the arginine-supplemented diet. Surprisingly, the threonine levels in the dams’ plasma were dramatically decreased by the maternal arginine supplementation. Moreover, the threonine

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concentrations in the breast milk were also decreased in the arginine-supplemented dams. The

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glycine concentration was also decreased in the breast milk of the ARG dams. Breast milk

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samples could not collected at 21 days of lactation. In the pups’ plasma, the threonine and

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lysine concentrations at 14 days of lactation were significantly decreased in the pups from dams fed the arginine-supplemented diet. However, the decreases in these amino acids were not found at 21 days of lactation.

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ACCEPTED MANUSCRIPT 4. Discussion

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We hypothesized that early postnatal arginine supplementation in rats induces rapid growth in

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early life and affects the abdominal fat accumulation and insulin resistance in later life. In

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fact, our present findings indicate that maternal arginine supplementation during the suckling period did cause obesity and insulin resistance in the offspring, although it did not induce

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rapid growth.

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When the rats in our study were fed a normal diet, there was no apparent alteration in their

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metabolic status including their body weights and visceral fat weight, regardless of arginine

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supplementation. Since a high-calorie diet is often used to amplify metabolic abnormalities

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induced by pre- or postnatal dietary manipulations, we gave rats a high-fat diet (35% calories from fat) from 6 to12 weeks of age in the present study. A reduction of food intake, commonly observed in rats fed high-fat diets containing more than and is an important amino, is accompanied by a lower protein intake. It has been reported that a low-protein diet induces visceral fat accumulation in rats [17]. In our present findings, the interference of the food and protein consumption in rats fed the high-fat diet (35% calories from fat) can be excluded. Here, when rats were fed a high-fat diet, intra-abdominal fat was markedly accumulated in the offspring of dams fed the arginine-supplemented diet during the suckling period, but not 17

ACCEPTED MANUSCRIPT in the control offspring. In contrast, there was little difference in the subcutaneous fat

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volumes among all groups. Accordingly, arginine supplementation during the suckling period

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may be said to induce visceral obesity but not subcutaneous obesity in the offspring. Our

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findings demonstrated that abdominal fat accumulation was brought about as an alteration of the susceptibility to high-fat intake by arginine supplementation to dams and pups during the

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suckling period.

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There is growing evidence that the suckling period plays an important role in determining the

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later metabolic system. In humans, babies born small who undergo rapid catch-up growth

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have been shown to be more obese in later life [1]. In animal models, pre-weaning

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overnutrition induced adiposity in young-adult baboons [7] and rats [6]. It was also demonstrated that dietary arginine supplementation stimulates protein synthesis and accretion in skeletal muscle of neonatal pigs [8, 9, 18]. In the present study, arginine supplementation to dams did not induce rapid body weight increase in their pups. Moreover, the maternal arginine supplementation during the suckling period did not influence the levels of IGF-1, leptin corticosterone, adiponectin and insulin in the breast milk or plasma of the dams and pups. However, intra-abdominal fat was accumulated in the pups from dams fed an arginine-supplemented diet during the suckling period. Our results thus imply that adiposity 18

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and susceptibility to obesity may be determined by nutrient conditions in early life.

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Visceral obesity has been strongly associated with the development of major risk factors for insulin resistance [19–21]. In the present study, obese rats on a high-fat diet showed elevated

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plasma glucose levels after glucose administration compared to the control rats fed a normal-fat diet. Moreover, the high-fat feeding brought about high insulin secretion in the ARG-HF rats, but not in CN-HF rats. Therefore, the early arginine supplementation induced

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more severe insulin resistance under high-fat diet feeding. These results are in line with the

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established concept that adipocytes in the visceral adipose tissue are a stronger determinant of

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insulin sensitivity than subcutaneous adipose tissue [22–24].

Regarding the rats on a normal diet, the insulin secretion of the ARG-CN rats was slightly higher after glucose loading, but no change in glucose levels was observed. In addition, ectopic fat accumulation was observed in the muscles of the ARG-CN rats. It has been reported that ectopic fat accumulation in the liver and skeletal muscle in humans is a critical determinant of insulin resistance, and may also predispose to the development of Type 2 diabetes [25, 26]. Arginine supplementation to young pigs increased the lipid content in the 19

ACCEPTED MANUSCRIPT longissimus muscle [18]. Taken together, these findings suggest that insulin resistance

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potentially existed in the present arginine-supplemented group and was manifested by the

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high-fat diet. As a result, high insulin secretion and high plasma glucose were found in the

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ARG-HF group.

We also evaluated the amino acid concentrations in the dams’ breast milk and plasma. Arginine supplementation increased the arginine concentration in the dams’ plasma but not in

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their breast milk. Mateo et al. demonstrated that in sows’ breast milk, arginine

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supplementation did not increase the arginine concentration but did increase the plasma

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arginine concentration [27]. Arginine and lysine use the same membrane transporter system.

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In our study, however, arginine supplementation to suckling dams did not affect the dams’ plasma lysine concentration, and it did not affect the early growth pattern of their pups. Tan et al. reported that 1% arginine supplementation to pigs’ diet did not change the plasma lysine concentrations [28]. In a rat model, the oral arginine administration at 250 mg·kg−1 was reported to cause no abnormal histopathological changes [29]. It may be that arginine fed to the dams in our study was not directly transferred to their pups via maternal breast milk.

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ACCEPTED MANUSCRIPT An unexpected finding of our investigation was that the Thr levels in the maternal plasma and

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breast milk were greatly decreased by maternal arginine supplementation. The threonine

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concentration was also decreased in the pups’ plasma. In a study of swine, the arginine

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supplementation of lactating sows significantly decreased the breast-milk threonine level at 7 days of lactation (Mateo et al. 2008). Threonine is one of the essential amino acids, and can

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be cleaved by threonine dehydrogenase to yield glycine and 5,10-methylenetetrahydrofolate (5,10-CH3 THF) [30, 31]. Threonine is involved in folate and the methionine cycle, and it is an important amino acid in controlling epigenetic modifications. Threonine and its catabolites

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control the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), which

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is a key factor for histone modifications [31]. Lower supplementation of threonine and lysine

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to pups may program higher susceptibility to a high-fat diet in later life by modifying the

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methylation status.

In the present study, no decrease in threonine concentration was found in the pups’ plasma at 21 days of lactation. It is likely that the pups obtained large proportions of nutrients from the purified diet at 21 days after birth, since the dams’ breast milk production was low and samples could not be collected at 21 days of lactation. We could not address how arginine supplementation changes the composition of breast milk or how such changes influence the 21

ACCEPTED MANUSCRIPT pups’ metabolism. The demand for arginine increases in specific conditions such as burns,

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sepsis, hypertension, and angina pectoris [32]. Arginine seems to be safe and effective

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therapy for many health conditions. A pair of studies demonstrated beneficial effects of

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arginine: an improvement of insulin resistance [33] and an anti-obesity effect [34]. However, we observed that the arginine supplementation during the suckling period led to higher

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susceptibility to a high-fat diet and obesity in offspring. Our data indicate that we need to be cautious in the application of arginine supplementation to pregnant and lactating women, and

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that this matter should be investigated further.

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In conclusion, our present findings demonstrated that the offspring of dams supplemented

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with arginine during lactation acquired higher susceptibility to obesity and insulin resistance when fed a high-fat diet later in life. Interactions among these factors could lead to the alteration of susceptibility to diet-induced metabolic disorders. Growth acceleration was not observed in the pups from dams supplemented with arginine during suckling. In addition, the arginine supplementation to dams lowered the supply of threonine and glycine to their pups, and this may be one of the causes contributing to the programming of a lifelong risk of obesity in the offspring. Our study did not determine how arginine supplementation results in the changes that we observed. 22

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5. Acknowledgment

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This work was supported by the Program for the Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (to S.-I. Takahashi and H. Kato). The authors declare

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no conflict of interest.

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ACCEPTED MANUSCRIPT 6. References

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[9] Yao K, Yin YL, Chu WY, Li ZQ, Deng D, Li TJ, et al. Dietary arginine supplementation

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[14] Folch J, Lees M, Stanley GHS. A Simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226:497–509. [15] Yamawaki N, Yamada M, Kan-no T, Kojima T, Kaneko T, Yonekubo A. Macronutrient, mineral and trace element composition of breast milk from Japanese women. J Trace Elem Med Bio. 2005;19:171–81. [16] Hsu JM, Anthony WL, Rider AA. Free amino acids in plasma and tissue of rats born to underfed dams. Proc Soc Exp Biol Med. 1975;148:1087–9. 25

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ACCEPTED MANUSCRIPT 7. Figure legends

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Fig. 1. Body weights of the rats supplemented with arginine during the lactation period fed a

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normal diet (A) or high-fat diet (B) from 6 to 12 wks of age. Dams and their pups were given

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a normal diet (15% protein) as the control group (CN; open circles) or a normal diet with 2% arginine as the arginine-treated group (ARG; closed rectangles) from birth to weaning (3 wks

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of age). After weaning, both groups of rats were fed a normal diet. The offspring of the CN and ARG groups were further divided into a normal diet (15% protein, 12.5% calories from fat, CN-CN: n=8, ARG-CN: n=9) or a high-fat diet (15% protein, 35% calories from fat,

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CN-HF: n=9, ARG-HF: n=10) at 6 wks of age. Only male pups were used. Values are means

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± SE. Data were significantly different by a two-way ANOVA with Bonferroni and Tukey

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post-hoc tests.

Fig. 2. CT-based body composition analysis of rats supplemented with arginine during lactation and fed a normal diet or a high-fat diet from 6 to 12 wks of age. Body fat composition images between T11 and S1 (the tip of the ischium) were used for the quantitative assessment. The representative CT images shown were taken at the L3 slice area (A). Pink and yellow areas represent the visceral and subcutaneous fat, respectively. The mean areas of subcutaneous (B) and visceral (C) fat are shown. The offspring of the CN and 29

ACCEPTED MANUSCRIPT ARG rats were further divided into a normal diet (15% protein, 12.5% calories from fat,

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CN-CN: n=8, ARG-CN: n=9) or high-fat diet (15% protein, 35% calories from fat, CN-HF:

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n=9, ARG-HF: n=10) at 6 wks of age. Only male pups were studied. Values are means ± SE.

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Data were significantly different using a two-way ANOVA with Bonferroni and Tukey

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post-hoc tests. *p<0.05, **p<0.01.

Fig. 3. The glucose and insulin levels after the glucose loading of rats supplemented with

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arginine during lactation fed a normal or high-fat diet from 6 to 12 wks of age. The OGTT in

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the CN (open circles) and ARG (closed rectangles) offspring was performed at 11 wks of age.

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After a 16-h overnight fast, 2 g kg–1 body weight of glucose was orally administered. A small

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amount of blood from the tail vein was collected at 0, 15, 30, 60 and 120 min after the glucose administration. Plasma glucose (A,B), insulin concentrations (C,D) and area under the curve (E,F) are shown. The offspring of the CN and ARG dams were further divided into a normal diet (15% protein, 12.5% calories from fat, CN-CN: n=8, ARG-CN: n=9) or high-fat diet (15% protein, 35% calories from fat, CN-HF: n=9, ARG-HF: n=10) at 6 wks of age. Only male pups were studied. Values are means ± SE. Data were significantly different by a two-way ANOVA with Bonferroni and Tukey post-hoc tests. *p<0.05, **p<0.01.

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Fig. 4. The longissimus muscle, liver and plasma TG content of the rats supplemented with

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arginine during lactation fed a normal diet or high-fat diet from 6 to 12 wks of age. Blood

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was collected from the carotid artery after a 1-h fast at 12 wks of age. At the same time, the longissimus muscle and liver were obtained. Plasma TG (C), extracted lipid from the

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longissimus muscle (A) and liver (B) were measured using a TG measurement kit. The offspring of the CN and ARG groups were further divided into a normal diet (15% protein, 12.5% calories from fat, CN-CN: n=8, ARG-CN: n=9) or high-fat diet (15% protein, 35%

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calories from fat, CN-HF: n=9, ARG-HF: n=10) at 6 wks of age. Only male pups were

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studied. Values are means ± SE. Data were significantly different by a two-way ANOVA with

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Bonferroni and Tukey post-hoc test. *p<0.05, **p<0.01.

Fig. 5. Free amino acid levels in the dams’ plasma and breast milk on day 14 of lactation. Dams’ plasma (A) and breast milk (B) were collected from dams fed the control diet (CN dams, n=5) or 2% arginine-supplemented diet (ARG dams, n=5). Values are means ± SE. Data were significantly different by Student’s t-test. *p<0.05.

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ACCEPTED MANUSCRIPT Fig. 6. Free amino acid levels in the pups’ plasma on day 14 or 21 of lactation. Plasma was

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collected from suckling pups from the dams fed the control diet (CN dams, n=4) or 2%

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arginine-supplemented diet (ARG dams, n=5) on day 14 (A) or 21 (B) of lactation. Two pups

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per litter were subjected to the analysis (CN group, n=8; ARG group, n=10). Pups were decapitated for the collection of trunk blood for the determination of plasma free amino acids.

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Values are means ± SE. Data were significantly different by Student’s t-test. *p<0.05.

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ACCEPTED MANUSCRIPT Table 1 Semi-purified ingredient composition of the diets fed to the rats 15% protein with 2% arginine

15% protein

High fat diet

12.5% 620.5 -

Casein1 DL-methionine Soy bean oil Vitamin mixture2 Mineral mixture2

175.0 2.5 20.0 10.0 40.0

175.0 2.5 20.0 10.0 40.0

175.0 2.5 20.0 10.0 40.0

Cellulose powder Choline chloride

100.0 2.0

100.0 2.0

100.0 2.0

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Calories from fat Corn starch L-arginine Lard

g/kg 12.5% 600.5 20.0 -

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Crude protein 85.71%

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AIN-76 prescription (Oriental Bio Service, Inc., Tokyo, Japan)

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1

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35.0% 506.7 113.8

ACCEPTED MANUSCRIPT Table 2 Body weight gain and adipose tissue weight of the rats supplemented with arginine during lactation, fed a normal diet or high-fat diet from 6 to 12 wks of age

n=8 28 ± 1 a 225 ± 4 475 ± 6

n=9 24 ± 1b 226 ± 5 478 ± 12

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n=9 27 ± 1 a 226 ± 3 475 ± 9

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High fat diet CN-HF ARG-HF

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Food intake (g/day) Initial body weight (g) Final body weight (g)

Normal diet CN-CN ARG-CN

n=10 26 ± 1 ab 226 ±3 497 ± 9

Mesenteric depots fat (g) Visceral depots fat (g)

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Body weight gain (g) 250 ± 4 2491 ± 9 253 ± 8 272 ± 7.9 Liver weight (g) 17.2 ± 0.5 17.8 ± 0.6 16.4 ± 0.8 17.7 ± 0.4 Gastrocnemius muscle (g) 5.0 ± 0.1 4.8 ± 0.1 5.1 ± 0.2 4.9 ± 0.1 a a ab Epididymal depots fat (g) 12.3 ± 0.5 11.9 ± 0.6 13.0 ± 0.5 15.3 ±0.9 b Retroperitoneal depots fat (g) 11.8 ± 0.8 a 13.0 ± 0.8 a 13.7 ± 0.5 ab 15.6 ±0.5 b 6.8 ± 0.3 30.9 ± 0.7a

7.4 ± 0.5 32.2 ± 1.6a

7.2 ± 0.5 33.9 ± 1.2ab

8.0 ±0.5 38.8 ±1.6b

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Values are means ± SE. Data were significantly different by two-way ANOVA with

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Bonferroni and Tukey post-hoc test. Different superscript letters denote significant differences.

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