Nutrition 29 (2013) 1152–1158

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Basic nutritional investigation

Long-term effect of green tea extract during lactation on AMPK expression in rat offspring exposed to fetal malnutrition Shin Sato Ph.D. a, *, Yuuka Mukai Ph.D. a, Mai Hamaya B.A. a, Yongkun Sun Ph.D. b, Masaaki Kurasaki Ph.D. b a b

Department of Nutrition, Aomori University of Health and Welfare, Aomori, Japan Environmental Adaptation Science, Graduate School of Environmental Science, Hokkaido University, Sapporo, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 December 2012 Accepted 22 March 2013

Objective: The fetal and neonatal environments are important determinants of disease risk in adult life. The aim of this study was to determine whether maternal green tea extract (GTE) intake during lactation affects the expression and activity of adenosine monophosphate-activated protein kinase (AMPK) in the kidneys of male offspring of protein-restricted dams during gestation. Methods: Pregnant Wistar rats were fed control (C) or low-protein diets (LP) during gestation. Following delivery, dams received a control or GTE-containing control diet during lactation as follows: C on control diet (CC), LP on control diet (LPC), LP on 0.12% GTE-containing control diet (LPL), or LP on 0.24% GTE-containing control diet (LPH). Some of the male pups from each dam were sacrificed at week 3, and the remaining male pups were fed a standard diet and sacrificed at week 30. Blood chemistry and expression levels of AMPKa, mammalian target of rapamycin (mTOR), and Akt in the kidneys of the male offspring were examined. Results: The level of phosphorylated AMPKa in the LPH group at week 3 was higher than that in the LPC group. At week 30, the protein levels of total and phosphorylated AMPK in the LPL and LPH groups were lower than those in the LPC group. The protein levels of mTOR and Akt at week 30 in the LPL and LPH groups were lower than those in the LPC group. Conclusion: GTE intake during lactation modulates AMPK, Akt, and mTOR expression in the kidneys of the adult male offspring of dams fed a protein-restricted diet and may induce long-term alterations in the expressions of these proteins in the kidneys. Ó 2013 Elsevier Inc. All rights reserved.

Keywords: Green tea extract Adenosine monophosphate-activated protein kinase Mammalian target of rapamycin Kidney Offspring Maternal protein restriction

Introduction Epidemiological and experimental studies demonstrate relationships between the periconceptional, fetal, and early infant stages of life and the subsequent development of diseases in the adult stage [1]. For instance, maternal low-protein diets induce glucose intolerance, renal disease, hypertension, and obesity early in the lives of offspring [2–4]. Several studies report that maternal undernutrition reduces the number of nephrons in the kidneys of offspring [5]. Maternal protein restriction during lactation affects the glomerular filtration rate and renal The authors had the following responsibilities in the preparation of this contribution: Shin Sato, study concept, writing, Western blotting, and discussion; Yuuka Mukai, study design, discussion, and manuscript submission; Mai Hamaya, animal treatment and part of the discussion; Yongkun Sun, blood biochemistry; and Masaaki Kurasaki, part of the discussion. * Corresponding author: Tel.: þ81-17-765-4184; fax: þ81-17-765-4184. E-mail address: [email protected] (S. Sato). 0899-9007/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nut.2013.03.021

Naþ transporters in adult offspring [6]. Additionally, animals with low-protein levels develop glomerular injury and progressive loss of renal function with aging [7,8]. Therefore, fetal and neonatal environments are important determinants of disease risk in adult life. Theys et al [9] report that although islets from the female and male offspring of rat dams that were fed a low-protein diet during gestation exhibited mitochondrial dysfunction, the consequences of protein restriction during early life are more evident in males. Moreover, protein level and the activity of endothelial nitric oxide (NO) synthase, which contributes to NO production, are reduced in the kidneys of male rats with advancing age and maintained during aging in female rats; these results suggest that renal NO deficiency may be a factor contributing to the gender difference in renal function [10]. Thus, the metabolism of male offspring seems to be affected more strongly. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that plays a central role in

S. Sato et al. / Nutrition 29 (2013) 1152–1158

regulating cellular metabolism and energy balance [11]. AMPK activation elicits various beneficial effects with a potential to ameliorate the defects associated with metabolic syndrome [12]. In the kidneys, AMPK is thought to regulate ionic concentration and mediate NO synthase activity [13]. Metformin, used in hypoglycemic therapy for type 2 diabetes, mediates its antidiabetic effects via the activation of AMPK in humans [14,15]. One of the major signaling pathways regulated by AMPK is the mammalian target of rapamycin (mTOR) pathway. When AMPK is active, it phosphorylates at least two proteinsdtumor suppressor tuberous sclerosis complex 2 (TSC2) and regulatory-associated protein of mTOR (raptor)dto suppress mTOR complex 1 (mTORC1) activation [16,17]. Akt is known to upregulate mTOR activity in two steps: 1) the phosphorylation and inhibition of TSC2, which suppresses the GTP-binding protein Rheb, leading to mTOR activation and 2) the stimulation of mTOR activity via PRAS40 phosphorylation [18]. Furthermore, mTOR downregulates the phosphorylation of proteins that play a role in the regulation of the initiation phase of mRNA translation [19,20]. Thus, the regulatory effects of mTOR are associated with renal diseases such as acute kidney injury and diabetic nephropathy [17]. Green tea, which is a popular beverage in East Asia, contains several polyphenolic compounds such as catechins, particularly ()-epigallocatechin gallate (EGCG), and ()-epigallocatechin. Green tea polyphenols are involved in the activation of AMPK [21]. EGCG, one of the bioactive components of green tea extract (GTE), activates AMPK in cultured cells [22,23] and suppresses hepatic gluconeogenesis via AMPK activation [24]. Moreover, it suppresses the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway by decreasing Akt phosphorylation in mouse mesangial cells [25]. These findings suggest that GTE has beneficial effects on metabolism in the kidneys. These reports prompted us to examine the effect of GTE intake on the kidneys of offspring of dams fed a low-protein diet. Maternal protein restriction causes early life programming via the remodeling of specific tissues such as the kidneys [5,8]. However, little is known about the effects of GTE intake during

lactation on the activation of AMPK and the AMPK signaling pathway in the kidneys of adult offspring programmed by maternal protein restriction. Therefore, we believe it is important to examine the effects of GTE intake on the expressions of key proteins of the metabolic signaling cascade, such as AMPK, Akt, and mTOR, in the kidneys of offspring of protein-restricted dams. This study evaluated whether maternal GTE intake during lactation affects the expression and activity of AMPK in the kidneys of the adult offspring of dams exposed to protein restriction during gestation and whether GTE intake modulates the expression and activity of the Akt/mTOR signaling pathway in renal metabolic responses in adult offspring. Methods and materials Animals All procedures were performed in accordance with the Guidelines for Animal Experimentation of the Aomori University of Health and Welfare (Permission number: 10009). Seven wk-old virgin female Wistar rats weighing 204–238 g were obtained from Charles River Laboratories Japan, Inc. (Yokohama, Japan). They were maintained at 23  1 C under a 12-h light/dark cycle and had access to food and tap water ad libitum. To determine whether the female rats were in the appropriate stage of the estrus cycle for mating, a vaginal impedance reader (Model MK-10C; Muromachi Kikai Co. Ltd., Osaka, Japan) was used as described previously [26]; this assessment was routinely performed in the afternoon. A reading of >3 kU indicated that the female was in proestrus and presumably in estrus. One appropriate female was mated with one male overnight. The next morning, the presence of a vaginal plug indicated successful mating; this day was taken as day 0 of gestation. Pregnant rats were randomly allocated to feed ad libitum on a diet containing either 20% (control group: C, n ¼ 4) or 8% (low-protein group: LP, n ¼ 12) casein during gestation (Fig. 1). Following delivery, each dam received a control or a GTE-containing control diet during lactation as follows: C on control diet (CC, n ¼ 4), LP on control diet (LPC, n ¼ 4), LP on 0.12% GTE-containing control diet (LPL, n ¼ 4), or LP on 0.24% GTEcontaining control diet (LPH, n ¼ 4). The diets were isocaloric (Table 1). The GTE, Polyphenon E, contained 80% to 98% total catechins by weight (the main component was EGCG, composing approximately 65% of the material) and was supplied by Mitsui-Norin Co. Ltd. (Shizuoka, Japan). In this study, we administered diets containing 0.12% and 0.24% GTE (Polyphenon E) because the no-observed adverse-effect level in a two-generation reproductive toxicity study in rats was equivalent to 200 mg/kg daily EGCG preparation [27], which is greater

Sacrifice at week 3 Mating

Birthing (Gestation)

CC

Control diet

LPL

LPH

Low-protein diet

Sacrifice at week 30

Weaning (Lactation)

Control diet

Control diet

LPC

1153

Standard laboratory diet

0.12% GTE +control diet

0.24% GTE +control diet

Fig. 1. Experimental design. Pregnant Wistar rats were fed control (C, 20% casein) or low-protein (LP, 8% casein) diets during gestation. During lactation, each dam received a control or green tea extract (GTE)-containing control diet: CC, control on control diet; LPC, low-protein on control diet; LPL, low-protein on 0.12% GTE-containing control diet; LPH, low-protein on 0.24% GTE-containing control diet.

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S. Sato et al. / Nutrition 29 (2013) 1152–1158 20 for 8 h and incubated overnight at 4 C with rabbit AMPKa, phospho-AMPKaThr172, mTOR, phospho-mTOR-Ser2448, Akt, and phospho-Akt-Ser473 polyclonal antibodies (1:1000; Cell Signaling Technology, Danvers, MA, USA). After washing, the membranes were incubated with the appropriate secondary horseradish peroxidase-conjugated antibodies. Protein bands were visualized with ECL Western Blotting Detection Reagents (GE Healthcare UK Ltd.) on Hyperfilm (GE Healthcare UK Ltd.). Quantitative analysis of specific band density was performed using ATTO densitometry software (ATTO Corp., Tokyo, Japan). Protein levels were normalized to those of b-actin from the same sample.

Table 1 Composition of the diets Ingredient

Control Low-protein 0.12% GTE0.24% GTEdiet diet containing diet containing diet

Casein l-Cystine Cornstarch a-Corn starch Sucrose Soybean oil Cellulose Mineral mixture* Vitamin mixturey Choline chlorhydrate tert-Butylhydroquinone GTE

20.000 0.300 39.749 13.200 10.000 7.000 5.000 3.500 1.000 0.250 0.001 –

g/100 g of diet 20.000 20.000 0.300 0.300 39.749 39.749 13.200 13.200 10.000 10.000 7.000 7.000 4.880 4.760 3.500 3.500 1.000 1.000 0.250 0.250 0.001 0.001 0.120 0.240

8.000 0.123 48.826 16.300 10.000 7.000 5.000 3.500 1.000 0.250 0.001 –

Statistical analysis Statistical analyses were performed using one-way analysis of variance followed by the Tukey test. All values are expressed as mean  SE.

Results Body and kidney weights

GTE, green tea extract (Polyphenon E) * AIN-93G mineral mixture. y AIN-93G vitamin mixture (Oriental Yeast, Tokyo, Japan).

The body weights of the C and LP groups did not differ significantly during pregnancy (C, 435  19 g, n ¼ 4 vs. LP, 434  6 g, n ¼ 12 at day 20 of gestation). There were no significant differences in body weight between the four groups at postnatal week 3 or 30 (data not shown). The daily food intake levels at postnatal week 10 were 30  0.6 (n ¼ 9), 29.9  0.8 (n ¼ 9), 27.7  1.1 (n ¼ 10), and 28.5  1 g per rat per day (n ¼ 10) in the CC, LPC, LPL, and LPH groups, respectively; at week 20, the levels were 28.1  1.4 (n ¼ 9), 26.0  0.6 (n ¼ 9), 25.4  0.5 (n ¼ 10), and 25.9  0.6 g per rat per day (n ¼ 10), respectively. There were no significant differences in kidney weight between the four groups at week 3 or 30 (data not shown).

than 160 mg/kg daily EGCG in the 0.24% GTE-containing diet used in our experiment. Additionally, it is reported that malignant stroke-prone spontaneously hypertensive rats fed 0.5% Polyphenon E exhibit delayed stroke onset [28]. The pups were weighed on postnatal day 4, and four male and four female pups were housed together to ensure adequate nutrition during lactation. At weaning, the male pups were separated from the CC (n ¼ 6), LPC (n ¼ 6), LPL (n ¼ 7), and LPH (n ¼ 6) groups and sacrificed. The animals were weighed, and blood samples were collected under anesthesia. The kidneys were immediately removed and weighed. The remaining pups (CC, n ¼ 9; LPC, n ¼ 9; LPL, n ¼ 10; LPH, n ¼ 10) continued to receive a standard commercial laboratory diet (MF diet; Oriental Yeast, Tokyo, Japan) and were weighed. Before sacrifice at week 30, the animals were fasted overnight and weighed, and blood samples were collected. Under ether anesthesia, the kidneys were immediately removed and weighed. The kidneys of all offspring were stored at 80 C before evaluation.

Plasma parameters of offspring The plasma BUN levels at week 30 were significantly higher in the LPL and LPH groups than the LPC group (Table 2). At week 30, plasma CRE levels were significantly lower in the LPH group than in the CC and LPC groups.

Blood chemistry Plasma samples were separated by centrifugation at 800g for 10 min at 4 C and tested for glucose, blood urea nitrogen (BUN), and creatinine (CRE) using a commercially available kit (Wako Pure Chemical Industries Ltd., Osaka, Japan). Plasma levels of insulin were measured using a Rat Insulin enzyme-linked immunosorbent assay kit (TMB; AKRIN-010T, Shibayagi, Gunma, Japan).

Effect of the GTE diet on total and phosphorylated AMPK levels Although the level of total AMPK at week 3 was lower in the LPH group than in the LPC group, the level of phosphorylated AMPK in the LPH group was significantly higher than those in the CC and LPC groups (Fig. 2A), indicating that GTE intake during lactation activated AMPK in the kidneys of the young offspring of dams fed a protein-restricted diet. Conversely, at week 30, the protein levels of total and phosphorylated AMPK in LPL and LPH offspring were lower than those in LPC offspring (Fig. 2B). However, the ratios of phosphorylated to total AMPK did not differ significantly between

Western blot analysis The kidneys were homogenized in homogenizing buffer (50 mM HEPES, 150 mM NaCl, 1 mM DTT, and 0.5% (v/v) Tween-20; pH 7.4) containing protease inhibitor cocktail tablets (Roche Applied Science, Indianapolis, IN, USA). The homogenates were centrifuged at 5000g for 45 min at 4 C. Supernatants were collected, and the protein concentration was determined using the BCAÔ Protein Assay Kit (Pierce, Rockford, IL, USA). For Western blot analysis, the proteins were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel and subsequently electrotransferred onto polyvinylidene difluoride membranes (GE Healthcare UK Ltd., Buckinghamshire, UK). The membranes were then blocked in 5% nonfat dry milk in Tris-buffered saline containing 0.1% v/v Tween

Table 2 Plasma parameters of offspring at weeks 3 and 30 Group

Week 3 CC

Glc (mg/dL) Insulin (ng/mL) BUN (mg/dL) CRE (mg/dL)

69.6 0.27 9.84 0.68

Week 30 LPC

   

5.4 0.09 1.24 0.05

79.9 0.45 8.80 0.73

LPL    

3.6 0.17 0.91 0.05

64.7 0.51 9.56 0.75

LPH    

7.7 0.13 0.69 0.02

70.4 0.42 9.77 0.77

CC    

6.2 0.12 1.11 0.04

151.3 6.72 10.69 1.13

LPC    

8.8 0.81 1.11 0.04

136.5 4.44 8.23 1.08

LPL    

7.2 0.44*,z 0.47 0.02

149.1 5.80 14.38 1.13

LPH    

5.2 0.43 0.43*,y 0.04

135.3 3.43 14.77 0.95

   

4.8 0.44*,z 0.66*,y 0.03*,y,z

BUN, blood urea nitrogen; CC, control on control diet; CRE, creatinine; Glc, glucose; GTE, green tea extract; LPC, low-protein on control diet; LPL, low-protein on 0.12% GTE-containing control diet; LPH, low-protein on 0.24% GTE-containing control diet During lactation, each dam received a control or GTE-containing control diet. Values are means  SE (week 3, n ¼ 6–7; week 30, n ¼ 9–10) * P < 0.05 vs. CC. y P < 0.05 vs. LPC. z P < 0.05 vs. LPL.

S. Sato et al. / Nutrition 29 (2013) 1152–1158

A

1155

B

β-actin

β-actin

p-AMPK

p-AMPK

β-actin

β-actin

a

CC

LPC

LPL

a,b

a,b a,b

p-AMPK/β-actin

p-AMPK/β-actin

b

t-AMPK/β-actin

t-AMPK

t-AMPK/β-actin

t-AMPK

b b

CC

LPH

LPC

LPL

LPH

Fig. 2. Total and phosphorylated AMP-activated protein kinase (AMPK) levels in the kidneys of offspring at postnatal weeks 3 (A) and 30 (B). Values are mean  SE (week 3, n ¼ 6–7; week 30, n ¼ 8–10). a P < 0.05 versus CC. b P  0.05 versus LPC. CC, control on control diet; LPC, low protein on control diet; LPL, low protein on 0.12% GTE-containing control diet; LPH, low protein on 0.24% GTE-containing control diet.

groups at week 30: 1.034  0.100 (n ¼ 9), 1.512  0.106 (n ¼ 9), 1.291  0.155 (n ¼ 10), and 1.172  0.187 (n ¼ 10) in the CC, LPC, LPL, and LPH groups, respectively.

expression in the kidneys of the adult offspring of dams fed a protein-restricted diet. The ratio of phosphorylated to total mTOR did not differ significantly between groups.

Effect of GTE diet on total and phosphorylated mTOR levels

Effect of GTE diet on total and phosphorylated Akt levels

Because AMPK suppresses mTORC1 expression via the phosphorylation of TSC2 and raptor [16], we examined whether the level of mTOR phosphorylation was altered in the kidneys of offspring. At week 3, the protein levels of total and phosphorylated mTOR were significantly lower in LPH offspring than in LPC offspring (Fig. 3A). Likewise, the protein levels of total and phosphorylated mTOR at week 30 in LPL and LPH offspring were lower than those in LPC offspring (Fig. 3B). These results indicate that GTE intake during lactation reduced mTOR

Because EGCG, a major component of green tea, inhibits Akt phosphorylation [29], we examined Akt protein expression in the kidneys. The levels of total and phosphorylated Akt protein did not change at postnatal week 3 in any of the LP groups. However, at week 30, the level of total Akt protein was significantly lower in LPL and LPH offspring than LPC offspring (Fig. 4). These results indicate that GTE intake during lactation is associated with Akt protein expression and phosphorylation in the kidneys.

A t-mTOR

B

t-mTOR

p-mTOR

p-mTOR

β-actin

β-actin

b

CC

LPC

LPL

LPH

a a,b a,b

p-mTOR/β-actin

p-mTOR/β-actin

b

t-mTOR/β-actin

β-actin

t-mTOR/β-actin

β-actin

a,b a,b

CC

LPC

LPL

LPH

Fig. 3. Total and phosphorylated mammalian target of rapamycin (mTOR) levels in the kidneys of offspring at postnatal weeks 3 (A) and 30 (B). Values are mean  SE (week 3, n ¼ 6; week 30, n ¼ 8–10). a P < 0.05 versus CC. b P < 0.05 versus LPC. CC, control on control diet; LPC, low protein on control diet; LPL, low protein on 0.12% GTE-containing control diet; LPH, low protein on 0.24% GTE-containing control diet.

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A t-Akt

B t-Akt

β-actin

β-actin

t-Akt/β-actin

p-Akt

p-Akt/β-actin

p-Akt

t-Akt/β-actin

β-actin

p-Akt/β-actin

β-actin

CC

LPC

LPL

LPH

a,b a,b

CC

LPC

a

a

LPL

LPH

Fig. 4. Total and phosphorylated Akt levels in the kidneys of offspring at postnatal weeks 3 (A) and 30 (B). Values are mean  SE (week 3, n ¼ 6–7; week 30, n ¼ 7–9). a P < 0.05 versus CC. b P < 0.05 versus LPC. CC, control on control diet; LPC, low protein on control diet; LPL, low protein on 0.12% GTE-containing control diet; LPH, low protein on 0.24% GTE-containing control diet.

Discussion Maternal undernutrition and dietary protein restriction during pregnancy are thought to be associated with long-term metabolic consequences in offspring, such as obesity, diabetes, and hypertension. In the kidneys, maternal dietary protein restriction is involved in physiological and morphological changes in the kidneys, leading to renal dysfunction. For instance, a deficiency of zinc, which is an essential trace element for several metabolic processes, during fetal life and lactation induces an early decrease in renal function, which is associated with a decrease in NO activity and an increase in oxidative stress [30]. Thus, a nutritional imbalance during gestation and lactation periods is important determinants of disease risk in adult life. The beneficial effects of green tea, a popular beverage in East Asia, in the treatment and prevention of human disease have been widely studied [31]. However, there is limited information about the long-term effects of GTE intake on renal metabolic pathways in adult offspring programmed by maternal protein restriction. Therefore, we hypothesized that GTE intake during lactation may contribute to the prevention of the development of early and irreversible renal alterations in offspring programmed by maternal protein restriction. The major findings of the present study are as follows: 1. GTE intake during lactation downregulated AMPK protein expression and upregulated its phosphorylation in LPH offspring at postnatal week 3, whereas GTE intake downregulated both AMPK protein expression and phosphorylation at postnatal week 30. 2. The expression and phosphorylation of mTOR and Akt protein decreased significantly at week 30 with GTE intake during lactation. In this study, plasma BUN levels at week 30 were significantly higher in the LPL and LPH groups than the LPC group (Table 2). At week 3, the BUN levels in the LPL and LPH groups tended to be higher than those in LPC group. Additionally, at week 30, plasma

CRE levels were significantly lower in the LPH group than in the CC and LPC groups but not in a dose-dependent manner. The precise reasons for the increased BUN levels in the LPL and LPH groups and decreased CRE level in the LPH group remain unclear. Green tea beverages given to healthy male smokers are reported to increase plasma NO oxide levels [32]. Therefore, GTE intake during lactation might affect nitrogen metabolism in the offspring of dams fed a protein-restricted diet. Plasma insulin levels at week 30 were significantly lower in the LPH group than the CC and LPL groups, although there was no difference between these levels in the LPC and LPH groups. Several studies report that the islet cells of adult rat offspring from proteinrestricted dams have increased vulnerability [33,34]. The lower levels of plasma insulin in the LPC and LPH offspring may be associated with the impairment of pancreatic islet function. The present results revealed elevated phosphorylated AMPK protein levels in the kidneys of the offspring of protein-restricted dams at week 3 when GTE was given during lactation. EGCG, which is present in GTE, is involved in AMPK activation [24,35]. For instance, oral administration of EGCG to mice induces an increase in AMPK activity in the liver concomitant with increases in AMPK and acetyl-CoA carboxylase phosphorylation [23]. Furthermore, EGCG significantly increases the protein levels of phosphorylated AMPK in the livers of experimental mice with obesity-related liver tumorigenesis [36]. Therefore, the present results suggest that GTE intake during lactation activates AMPK in the kidneys of young offspring programmed by maternal protein restriction. Conversely, the protein levels of total and phosphorylated AMPK at week 30 in LPL and LPH offspring were significantly lower than those in LPC offspring in the present study. However, the ratio of phosphorylated to total AMPK did not differ significantly between groups. These results suggest that although GTE intake during lactation does not affect AMPK activity, GTE suppresses AMPK protein expression in the kidneys of adult offspring. Interestingly, at week 3, the levels of total and phosphorylated mTOR protein in the kidneys of LPH offspring were lower than those in LPC offspring. The mTOR signaling pathway

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regulates gene transcription and protein translation in response to nutrients and growth factors [37,38]. AMPK activation suppresses downstream substrates such as mTOR and eukaryotic initiation factor 4E-binding protein-1 [20,39]. Activated AMPK is known to phosphorylate and suppress mTOR [40]. Therefore, we hypothesized that increased phosphorylated AMPK in LPH offspring at week 3 may downregulate the expression and phosphorylation of mTOR, leading to the reduction in the translation and synthesis of proteins including AMPK protein, at week 30. Additionally, the EGCG present in GTE may affect epigenetic processes via DNA methylation status, because EGCG is reported to decrease global DNA methylation levels and inhibit DNA methyltransferases (DNMTs) in vitro [40,41]. Although DNMT activity was not measured in this study, we assumed that at week 30, the protein levels of total and phosphorylated AMPK in the LPL and LPH groups were reduced by modifications of the epigenetic processes due to EGCG action. Furthermore, EGCG has been shown to inhibit the PI3K/Akt/mTOR signaling pathway in animal and cell experiments [21,25]. Van Aller et al [29] report that in physiologically relevant concentrations, EGCG is an ATP-competitive inhibitor of PI3K and mTOR in in vitro systems. Therefore, we assumed that greater GTE intake during lactation plays a role in downregulating total and phosphorylated mTOR protein levels in the kidneys of young offspring. However, it is more interesting that total and phosphorylated mTOR protein levels decreased significantly in the kidneys of LPL and LPH offspring even at week 30, although there were no significant differences in the kidneys of LPL or LPH offspring with respect to the ratio of phosphorylated to total mTOR. Although the precise mechanism(s) underlying the persistence of mTOR protein expression into adulthood are not currently understood, our findings suggest that GTE intake during lactation downregulates mTOR in protein expression but not activity in the kidneys with increasing age. Surprisingly, the protein expressions of AMPK, Akt, and mTOR were downregulated in the kidneys of the adult offspring of protein-restricted dams, although no GTE was administered after weaning. Similar to AMPK and mTOR, total Akt protein levels were significantly lower in the kidneys of LPL and LPH offspring than LPC offspring at week 30. Akt is the main target of PI3K, and the PI3K/Akt signaling pathway plays an important role in the metabolic effects of insulin and glucose [42,43]. In an animal experiment, the consumption of green tea polyphenols attenuated the effects of high-fructose diets on insulin signaling, including PI3K and Akt as well as lipid metabolism [44]. On the other hand, EGCG inhibits the PI3K/Akt/mTOR pathway independent of AMPK by decreasing Akt phosphorylation in mouse mesangial cells [25]. Thus, green tea polyphenols appear to modulate the insulin signaling pathway. However, the mechanism underlying decreased Akt protein expression in the kidneys at week 30 remains unclear. This phenomenon may be interpreted as follows. A decrease in phosphorylated Akt may downregulate total and phosphorylated mTOR protein expression. Akt is reported to upregulate mTOR activity via TSC2 and PRAS40 phosphorylation [17,40]. Therefore, downregulation of total and phosphorylated mTOR protein may lead to the reduction of the translation and synthesis of proteins including Akt. Another possibility is that maternal nutrition during gestation and lactation has long-term effects on gene and protein expression in adult offspring. For instance, maternal consumption of red raspberry leaf and its constituents leads to long-term alterations to cytochrome P450 activity in adult rat offspring [45]. Insulin resistance was observed in adult male offspring of lactating rats fed a diet containing flaxseed [46]. We previously

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demonstrated that the intake of quercetin, a naturally occurring flavonoid and AMPK activator during lactation, upregulates AMPK activation in the liver of the adult offspring of dams fed a low-protein diet during pregnancy [47]. Therefore, we hypothesize that GTE intake during lactation at least induces long-term alterations in the expression and phosphorylation of AMPK, Akt, and mTOR protein in the kidneys of the adult male offspring of dams fed a protein-restricted diet. Several studies report that the downregulation of the mTOR signaling pathway through pharmacologic manipulation (e.g., rapamycin), calorie restriction, nutrients, and growth-factor restriction positively affects longevity and age-related diseases such as diabetes and obesity [48–50]. For instance, treatment with grape polyphenols such as resveratrol, quercetin, and catechin inhibits Akt/mTOR signaling in breast cancer cells [51]. Although future studies are required to elucidate the precise underlying mechanism(s), the current results are useful for understanding the effects of maternal GTE intake during lactation on the development of age-related diseases and extended longevity in adult offspring. Conclusions The present study demonstrates that GTE intake during lactation upregulates AMPK activation in young offspring and modulates the expressions of AMPK, Akt, and mTOR, which are key proteins of the metabolic signaling cascade, in the kidneys of the adult male offspring of protein-restricted dams. Acknowledgments The authors thank Keiko Tamakuma for her technical assistance. This study was supported by a Grant-in-Aid for Scientific Research (C) (No. 23500960) from the Japan Society for the Promotion of Science. References [1] Barker DJ, Bagby SP, Hanson MA. Mechanisms of disease: in utero programming in the pathogenesis of hypertension. Nat Clin Pract Nephrol 2006;2:700–7. [2] Curhan GC, Willett WC, Rimm EB, Spiegelman D, Ascherio AL, Stampfer MJ. Birth weight and adult hypertension, diabetes mellitus, and obesity in US men. Circulation 1996;94:3246–50. [3] McMullen S, Langley-Evans SC. Maternal low-protein diet in rat pregnancy programs blood pressure through sex-specific mechanisms. Am J Physiol Regul Integr Comp Physiol 2005;288:R85–90. [4] Warner MJ, Ozanne SE. Mechanisms involved in the developmental programming of adulthood disease. Biochem J 2010;427:333–47. [5] Langley-Evans SC, Welham SJ, Jackson AA. Fetal exposure to a maternal low protein diet impairs nephrogenesis and promotes hypertension in the rat. Life Sci 1999;64:965–74. [6] Luzardo R, Silva PA, Einicker-Lamas M, Ortiz-Costa S, do Carmo Mda G, Vieira-Filho LD, et al. Metabolic programming during lactation stimulates renal Naþ transport in the adult offspring due to an early impact on local angiotensin II pathways. PLoS One 2011;6. e21232. [7] Nwagwu MO, Cook A, Langley-Evans SC. Evidence of progressive deterioration of renal function in rats exposed to a maternal low-protein diet in utero. Br J Nutr 2000;83:79–85. [8] Woods LL, Weeks DA, Rasch R. Programming of adult blood pressure by maternal protein restriction: role of nephrogenesis. Kidney Int 2004;65:1339–48. [9] Theys N, Bouckenooghe T, Ahn MT, Remacle C, Reusens B. Maternal low-protein diet alters pancreatic islet mitochondrial function in a sexspecific manner in the adult rat. Am J Physiol Regul Integr Comp Physiol 2009;297:R1516–25. [10] Erdely A, Greenfeld Z, Wagner L, Baylis C. Sexual dimorphism in the aging kidney: effects on injury and nitric oxide system. Kidney Int 2003;63:1021–6. [11] Hardie DG. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev 2011;25:1895–908.

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