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Placental efficiency is a predictor of fetal growth and development, which is also controlled by maternal gestational health and diet. The present study investigated the effects of a gestational low-protein diet on offspring growth capacity as well as the diet's contribution to altered expression of placental genes associated with the mammalian amino acid response (AAR) pathway. To assess these outcomes, timed-pregnant Sprague Dawley rats were fed a control (C) diet with 18% protein or a low-protein (LP) diet with 9% protein throughout gestation (Expt. 1) or throughout gestation and lactation (Expt. 2). Placentas were collected during natural delivery and quantitative RT-PCR and Western-blot analyses were performed to determine placental mRNA and protein levels. By the end of the lactation period, offspring of dams fed the LP diet had stunted growth in both experiments, mRNA expression of target genes in the AAR pathway, such as activating transcription factor-3 (Atf3), asparagine synthetase (Asns), and Sodium-dependent neutral amino acid transporter-2 (Snat2), was greater in placentas of rats fed the LP diet compared with controls, as were placental ATF4 and p-elF2[alpha] protein levels. The increase in mRNA expression of AAR pathway-associated genes was correlated with the stunting of offspring growth (Atf3: [R.sup.2] = 0.32, P = 0.086; Asns: [R.sup.2] = 0.44, P < 0.05; Snat2: [R.sup.2] = 0.33, P = 0.084). Our study showed that the mammalian AAR pathway in placenta is upregulated by a maternal low-protein diet and this activation may act as a cue for the fetus to develop an adaptive response suited to their predicted postnatal environment, i.e. a more favorable phenotype for their survival. J. Nutr. 140: 2116-2120, 2010. doi: 10.3945/jn.110.127803.

This study investigates the mechanism by which maternal protein restriction induces hepatic autophagy-related gene expression in the offspring of rats. Pregnant Sprague-Dawley rats were fed either a control diet (C, 18 % energy from protein) or a low-protein diet (LP, 8·5 % energy from protein) during gestation, followed by the control diet during lactation and post-weaning. Liver tissue was collected from the offspring at postnatal day 38 and divided into four groups according to sex and maternal diet (F-C, F-LP, M-C and M-LP) for further analysis. Autophagy-related mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. In addition, chromatin immunoprecipitation (ChIP) was performed to investigate the interactions between transcription factors and autophagy-related genes. Protein levels of p- eukaryotic translation initiation factor 2a and activating transcription factor 4 (ATF4) were increased only in the female offspring born to dams fed the LP diet. Correlatively, the mRNA expression of hepatic autophagy-related genes including Map1lc3b, P62/Sqstm1, Becn1, Atg3, Atg7 and Atg10 was significantly greater in the F-LP group than in the F-C group. Furthermore, ChIP results showed greater ATF4 and C/EBP homology protein (CHOP) binding at the regions of a set of autophagy-related genes in the F-LP group than in the F-C group. Our data demonstrated that a maternal LP diet transcriptionally programmed hepatic autophagy-related gene expression only in female rat offspring. This transcriptional programme involved the activation of the eIF2α/ATF4 pathway and intricate regulation by transcription factors ATF4 and CHOP. [ABSTRACT FROM AUTHOR]

To investigate the kinetics of peptide transport by the peptide transporter, PepT1, Chinese hamster ovary cells were transfected with an expression vector containing our cloned ovine PepT1 cDNA. Transport was assessed by uptake studies using the radiolabeled dipeptide, [[sup.3]H]-Gly-Sar. Expression of oPepT1 was detected at 8-24 h post-transfection with an optimal time of 16-24 h. Uptake of Gly-Sar by oPepT1 was pH-dependent with an optimal pH of 5.5-6.0, concentration-dependent and saturable with an apparent [K.sub.m] value of 1.0 [+ or -] 0.1 mmol/L and a maximum velocity of 14.3 [+ or -] 0.4 nmol/(mg protein . 40 min). Competition studies with nonradiolabeled peptides and [[sup.3]H]-Gly-Sar showed that all di- and tripeptides inhibited uptake of [[sup.3]H]-Gly-Sar. In addition, three tetrapeptides (Met-Gly-Met-Met, Pro-Phe-Gly-Lys, and Val-Gly-Ser-Glu) also inhibited [[sup.3]H]-Gly-Sar uptake. There was no inhibition of [[sup.3]H]-Gly-Sar uptake detected in the presence of nonradiolabeled free amino acids. Treatment of the cells with staurosporine, an inhibitor of protein kinase C (PKC) significantly increased the transport system. This increase was specific and could be blocked if treatment was done in the presence of phorbol 12-myristate-13-acetate (PMA), an activator of PKC. The staurosporine- and PMA-induced changes in peptide transport activity were not affected by cotreatment with cycloheximide. These data demonstrate that the transport of peptide substrates by oPepT1 in transfected mammalian cells is similar to that in microinjected Xenopus oocytes and that PKC phosphorylation plays a regulatory role in oPepT1 function. KEY WORDS: * cloning * protein kinase * sequence * inhibitor * activator

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