Preclinical data demonstrate that the gut microbiota can promote pancreatic ductal adenocarcinoma (PDAC), but mechanisms remain unclear. We hypothesized that intestinal microbiota alters anti-tumor innate immunity response to facilitate PDAC progression. Human PDAC L3.6pl cells were heterotopically implanted into Rag1-/- mice after microbiota depletion with antibiotics, while syngeneic murine PDAC Pan02 cells were implanted intrapancreatic into germ-free (GF) C57BL/6 J mice. Natural killer (NK) cells and their IFNγ expression were quantitated by flow cytometry. NK cells were depleted in vivo using anti-Asialo GM1 antibody to confirm the role of NK cells. Bacteria-free supernatant from SPF and GF mice feces was used to test its effect on NK-92MI cell anti-tumor response in vitro. SPF and ex-GF mice (reconstituted with SPF microbiota) developed larger PDAC tumors with decreased NK cell tumor infiltration and IFNγ expression versus GF-Rag1-/-. Microbiota-induced PDAC tumorigenesis was attenuated by antibiotic exposure, a process reversed following NK cell depletion in both Rag1-/- and C57BL/6 J mice. Compared to GF, SPF-Rag1-/- abiotic stool culture supernatant inhibited NK-92MI cytotoxicity, migration, and anti-cancer related gene expression. Gut microbiota promotes PDAC tumor progression through modulation of the intratumoral infiltration and activity of NK cells.

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related death in the United States. Our group and others have demonstrated that the intestinal microbiota accelerates pancreatic carcinogenesis. The relationship of intestinal bacteria, immune response, and PDAC development is unclear. To that end, we investigated the role of intestinal bacterial soluble factors in modulating the immune environment of PDAC and its progression. Methods: Mice intestinal microbiota was depleted with wide-spectrum antibiotics. The human PDAC cell line, L3.6pl, was heterotopically implanted into Rag1-/- mice, while the syngeneic murine PDAC cell line, Pan02, was orthotopically implanted into the pancreas of C57Bl/6 mice. Tumor and pancreas infiltrated natural killer (NK) cells were quantitated by flow cytometry. In vivo NK cell depletion was attained by intraperitoneal injection of anti-Asialo-GM1 antibody twice weekly. Germ-free (GF) and conventionally housed Rag1-/- mice stool was cultured and bacteria-free supernatant extracted. The ability of these bacteria-free supernatants to regulate NK-92mi cell cytotoxicity and migration was tested in vitro by flow cytometry and a Transwell assay, respectively. Results: Compared to microbiota-intact Rag1-/- mice, microbiota depletion yielded 74% smaller tumors (p<0.05) with a 1.5-fold increase in PDAC infiltrating NK cells (p<0.001). Confirmation by immunohistochemistry demonstrated a 6.5-fold increase in NK cell tumor infiltration in microbiota-depleted mice (p<0.0001). Notably, a 3-fold increase of intrapancreatic NK cells in GF Rag1-/- mice versus conventionally housed mice was noted, suggesting that bacteria regulate immune cell trafficking. Antibody-mediated NK cell depletion with concomitant microbiota depletion increased PDAC tumor growth, negating the antitumor phenotype of microbiota depletion alone in both immunodeficient Rag1-/- (2.8-fold increase; p<0.05) and immunocompetent C57Bl/6 mice (2.4-fold increase; p<0.05). Quantitative PCR revealed 9-fold higher IFN-gamma gene expression in PDAC xenografts of microbiota-depleted mice versus microbiota-intact (p<0.05). Cell-free stool bacteria culture supernatant from conventionally housed Rag1-/- mice, but not from GF mice, inhibited NK-92mi cell migration by 26% (p<0.05) and cytotoxicity against L3.6pl cells by 51% (p<0.01). Compared to NK-92mi cells exposed to cultured stool supernatant from GF Rag1-/- mice, SPF cultured stool supernatant resulted in decreased gene expression associated with activation/recruitment of NK cells including FASLG, CCL18, IL-13, CXCR2, CCL4, and increased expression associated with inhibition of NK cell activity including CCR1 and IL-6. Conclusion: These findings suggest that intestinal bacteria modulate PDAC development through suppression of NK cell recruitment and activation, a phenomenon potentially mediated by yet to be identified small molecules. Citation Format: Qin Yu, Mark Beveridge, Ryan Thomas, Christian Jobin. Intestinal microbiota mediates pancreatic carcinogenesis through modulation of tumor infiltrating natural killer cells [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B05.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States yet data are scant regarding host factors influencing pancreatic carcinogenesis. Increasing evidence support the role of the host microbiota in carcinogenesis but its role in PDAC is not well established. Herein, we report that antibiotic-mediated microbial depletion of KrasG12D/PTENlox/+ mice showed a decreased proportion of poorly differentiated tumors compared to microbiota-intact KrasG12D/PTENlox/+ mice. Subsequent 16S rRNA PCR showed that ~50% of KrasG12D/PTENlox/+ mice with PDAC harbored intrapancreatic bacteria. To determine if a similar observation in humans correlates with presence of PDAC, benign and malignant human pancreatic surgical specimens demonstrated a microbiota by 16S bacterial sequencing and culture confirmation. However, the microbial composition did not differentiate PDAC from non-PDAC tissue. Furthermore, murine pancreas did not naturally acquire a pancreatic microbiota, as germ-free mice transferred to specific pathogen-free housing failed to acquire intrapancreatic bacteria over time, which was not augmented by a murine model of colitis. Finally, antibiotic-mediated microbial depletion of Nod-SCID mice, compared to microbiota-intact, showed increased time to PDAC xenograft formation, smaller tumors, and attenuated growth. Interestingly, both xenograft cohorts were devoid of intratumoral bacteria by 16S rRNA PCR, suggesting that intrapancreatic/intratumoral microbiota is not the sole driver of PDAC acceleration. Xenografts from microbiota-intact mice demonstrated innate immune suppression by immunohistochemistry and differential regulation of oncogenic pathways as determined by RNA sequencing. Our work supports a long-distance role of the intestinal microbiota on PDAC progression and opens new research avenues regarding pancreatic carcinogenesis.

Abstract We previously reported that within the pancreatic ductal adenocarcinoma (PDAC) microenvironment, miR-145 and miR-199a are exclusively expressed in tumor-associated stroma (TAS) cells, but these miRNAs are present in PDAC cells following co-culture with TAS cells. We hypothesized that miRNAs function as paracrine signals via exosomal exchange between TAS cells and adjacent PDAC cells. Primary cultures of human TAS and PDAC cells were employed. Membrane-bound microparticles were isolated from TAS conditioned, serum-free culture media by sequential ultracentrifugation followed by ultrafiltration. Exosomes and microvesicles were then assayed for particle size distribution using nanoparticle tracking analysis and electronic microscopy. miRNA expression levels were determined using quantitative PCR. miRNA transfection was performed with RNAiMax reagents. Cell viability was measured by Alamar Blue. Statistics were performed using Prism 6 software. Following transfection of human TAS cells with cel-miR-39, a nonhuman miRNA, we demonstrated that miRNA exchanges occurred between TAS cells and neighboring PDAC cells via a process that is not dependent upon cell-cell contact. We next confirmed the presence and enrichment of miR-145-5p in TAS-cell-derived exosomes (8-fold higher concentrations in exosomes than parental cells, p<0.05). Feeding of TAS-derived exosomes or transfection of miR-145-5p mimics into PDAC cells led to dose-dependent decreases in PDAC cell viability (p<0.05). Taken together, our data suggest that stroma derived exosomes deliver miRNAs to adjacent PDAC cells and may function as tumor-suppressing paracrine signals in the case of miR-145. This finding provides a potential explanation for the observation that stroma depletion paradoxically accelerates PDAC progression in murine models. Citation Format: Song Han, Sayali Belsare, DongYu Zhang, Mark Beveridge, Carlos Rinaldi, Jose G. Trevino, Thomas D. Schmittgen, Steven J. Hughes. Exosomal delivery of stroma-derived miR-145 inhibits pancreatic cancer cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4322. doi:10.1158/1538-7445.AM2017-4322

The pancreatic adenocarcinoma (PDAC) microenvironment is largely comprised of fibrotic tumor associated stroma (TAS) that contributes to the lethal biology of PDAC. microRNA (miRNA) are small non-coding RNAs that regulate gene expression. We hypothesized that interactions between PDAC cells and TAS cells within the microenvironment modulate miRNA expression and thus, tumor biology. We observed that miR-205 and members of the miR-200 family (miR-200a, -200b, -200c, -141 and miR-429) were exclusively expressed in PDAC cells, consistent with an epithelial miRNA signature, while miR-145 and miR-199 family members (miR-199a and -199b) were solely expressed in TAS cells, consistent with a stromal miRNA signature. This finding was confirmed by qRT-PCR of RNA obtained by laser-capture microdissection of surgical specimens. Using an in vitro co-culture model, we further demonstrated regulation of miRNA expression by cell-cell contact. Forced expression in TAS cells of miR-200b/-200c and miR-205 to mimic these observed changes in miRNA concentrations induced secretion of GM-CSF and IP10, and notably inhibited migration. These data suggest interactions within the tumor microenvironment alter miRNA expression, which in turn have a functional impact on TAS.

pancreatitis.Methods.Stool and salivary samples were collected from 14 control subjects (7 males and 7 females) and 8 patients (6 males and 2 females) with autoimmune pancreatitis from the same geographical region.The average age was 50 years for control subjects, 56 years for patients.All patients were naïve and enrolled at diagnosis.Biological samples were obtained before medical treatment.Total DNA was extracted from each sample and purified, the V3-V5 region of the 16s rRNA gene was amplified and massive ultra-deep pyrosequencing was performed by 454-GS Junior.Sequences with high quality score and length >250bp were analyzed with QIIME (v1.6.0).Chimeric sequences were removed by ChimeraSlayer tool and good coverage index was evaluated for every sample.Results.Comprehensive comparison of the salivary and fecal microbiota between patients with treatment naive autoimmune pancreatitis and healthy control subjects revealed a significant variation of salivary/fecal microflora.NGS analysis showed an increase of Proteobacteria phylum (p= 0,008) in fecal samples of patients with autoimmune pancreatitis compared to control subjects: in particular, we observed an increase in Enterobacteriaceae family (p=0,005).Moreover in patients with autoimmune pancreatitis we found at genera level an increase of Lactobacillus (p=0,05) and Oscillospira (p=0,05) and a decrease of Faecalibacterium (p=0,01) compared to control subjects.In salivary samples, we observed a decrease of Haemophilus parainfluenzae (p=0,02) in patients with autoimmune pancreatitis but no significant differences at any other taxonomic level of analyses.Conclusions.We observed a significant association between variations of patients' salivary/fecal microbiome with autoimmune pancreatitis when compared with normal controls.Interestingly Proteobacteria that are known to increase in local and systemic inflammatory diseases or can be causative agents of immune proliferative diseases such as MALT lymphomas are also significantly increased in autoimmune pancreatitis patients.These results open the way to larger trials and to novel therapeutic approaches with the aim of re-establish a beneficial intestinal environment and microbiome composition in patients with autoimmune pancreatitis.

ObjectiveAdipose tissue derived stem cells (ADSCs) transplantation has recently gained widespread enthusiasm, particularly in the perspective to use them as potential alternative cell sources for hepatocytes in cell based therapy, mainly because of their capability of hepatogenic differentiation in vitro and in vivo. But some challenges remain to be addressed, including whether ADSCs can be provided effectively to the target organ and whether subsequent proliferation of transplanted cells can be achieved. To date, intrasplenic injection is the conventional method to deliver ADSCs into the liver; however, a number of donor cells retained in the spleen has been reported. In this study, our objective is to evaluate a novel route to transplant ADSCs specifically to the liver. We aimed to test the feasibility of in situ transplantation of ADSCs by injecting bioencapsulated ADSCs into the liver in mouse model.MethodsThe ADSCs isolated from human alpha 1 antitrypsin (M-hAAT) transgenic mice were used to allow delivered ADSCs be readily identified in the liver of recipient mice, and alginate was selected as a cell carrier. We first evaluated whether alginate microspheres are implantable into the liver tissue by injection and whether ADSCs could migrate from alginate microspheres (study one). Once proven, we then examined the in vivo fate of ADSCs loaded microspheres in the liver. Specifically, we evaluated whether transplanted, undifferentiated ASDCs could be induced by the local microenvironment toward hepatogenic differentiation and the distribution of surviving ADSCs in major tissue organs (study two).ResultsOur results indicated ADSCs loaded alginate microspheres were implantable into the liver. Both degraded and residual alginate microspheres were observed in the liver up to three weeks. The viable ADSCs were detectable surrounding degraded and residual alginate microspheres in the liver and other major organs such as bone marrow and the lungs. Importantly, transplanted ADSCs underwent hepatogenic differentiation to become cells expressing albumin in the liver. These findings improve our understanding of the interplay between ADSCs (donor cells), alginate (biomaterial), and local microenvironment in a hepatectomized mouse model, and might improve the strategy of in situ transplantation of ADSCs in treating liver diseases.

Background. Gestational protein restriction (GPR) can program a fetal phenotype prone to develop metabolic syndrome (MetS) in successive generations. Objectives. To understand the placental-fetal adaptations underpinning metabolic syndrome (MetS) prone phenotype in successive generations. Material and Methods. Rats (F0) were pair-fed either a 19% normal protein diet (NPD) or an 8% low protein diet (LPD) through pregnancy and lactation. Male and female offspring (F1) were bred to control animals, and the growth of F2 animals monitored for 15 months. [(14)C]-2-(methylamino) isobutyric acid) (MeAIB) was used to monitor the activity of placental amino acid transport system A (SysA). Results. Maternal weight gain (g) in F0 pregnancies of the LPD group was less than the NPD group (105 +/- 15 vs. 120 +/- 25, p <= 0.005). Fetal, 3.8 +/- 0.9 g (LPD) vs. 3.7 +/- 0.7 g (NPD); p <= 0.2, and placental weights, 0.56 +/- 0.01 g (LPD) vs. 0.6 +/- 0.02 g (NPD); p <= 0.6 were comparable. MeAIB transfer expressed as (DPM) of gram fetus/mL maternal serum (0.08 +/- 0.010 vs. 0.14 +/- 0.02; p <= 0.003) and (DPM) gram fetus/DPM gram placenta were lower (0.10 +/- 0.01 vs. 0.14 +/- 0.01; p <= 0.02) in LPD than NPD group. Transport in apical membrane vesicles from LPD group was decreased (15 +/- 2 vs. 23 +/- 4, pmol.mg(-1) protein 10sec(-1); p = 0.05). Maternal-fetal MeAIB transfers, fetal and placental weights, and maternal weight gains in F1 pregnancies were comparable between animals descended from NPD and LPD groups. However, F2 generation postnatal weight gains were impacted by F1 gestational nutrition (LPD vs. NPD; p <= 0.0001). Conclusions. Moderate GPR impacted placental nutrient transfer in F0 pregnancies; F2 descended from LPD exposed F1 generation tended to be larger than their NPD derived counterparts through 15 months of age samples (Adv Clin Exp Med 2010, 19, 3, 301-312).

Amino acid transport System A (SysA) activity is present within the rodent and human placentas. Inhibition of this transport system is associated with fetal growth retardation. Several cDNAs encoding SysA transport proteins have been discovered, and their presence documented within the human placenta. We have demonstrated the presence of mRNA encoding three of these transporters, SNAT1, 2, and 4 within the rat placenta over the final third of gestation. Abundance of these mRNA species increases from day 14 to day 20 of gestation. Immunohistochemistry demonstrates the presence of SNAT1 and 2 within the placental labyrinth at both days 14 and 20. Transport proteins are also present within marginal giant cells and, for SNAT1, within fetal endothelium. In conclusion, several proteins capable of SysA transport activity are present within the rodent placenta. mRNA expression increases over the final third of gestation, coincident with the period of greatest need for fetal amino acid delivery.

The yolk sac plays an important role in fetal nutrition. Transport of amino acids by the rodent visceral yolk sac has been shown previously. We have demonstrated the presence of several amino acid transport proteins capable of the Na(+)-dependent transport of anionic amino acids within late gestation mouse visceral yolk sac and uterine epithelium. We speculate that these proteins may be involved in the efflux of glutamate from the fetal to the maternal circulations.

The role of growth hormone (GH), insulin-like growth factor (IGF)-II and the IGF-I receptor (IGF-Ir) in the regulation of the in vivo expression of Na+-coupled anionic [System X−AG; GLAST1 (EAAT1), GLT1 (EAAT2), EAAC1 (EAAT3), EAAT4; where the human homologues of amino acid transport proteins first cloned in the rat are given in parentheses] and Na+-independent cationic (System y+;CAT1) amino acid transport proteins was evaluated by comparing transporter expression in day 17 placentae of mice that overexpressed bovine GH (GH+) or that carried null gene mutations for IGF-II or IGF-Ir. Northern analysis revealed no apparent difference in the mRNA content of GLAST1 (EAAT1), EAAC1 (EAAT3), or EAAT4, in homogenates of GH+ placentae, but levels of GLT1 (EAAT2) and CAT1 mRNA were increased. Immunoblot analysis revealed that whole-placental steady-state GLAST1 (EAAT1), EAAC1 (EAAT3), and EAAT4 protein levels were not affected by GH+, whereas GLT1 (EAAT2) levels were increased. Immunohistochemical analysis showed that the cell-specific expression of the anionic and CAT1 transporters was not affected by overexpression of GH. Similar analyses of null IGF-II placentae demonstrated increases in GLAST1 (EAAT1), EAAT4 and CAT1 mRNAs. Parallel immunoblot analysis demonstrated decreased expression of GLT1 (EAAT2), GLAST1 (EAAT1) and EAAC1 (EAAT3) protein, but an increased expression of EAAT4. In null IGF-II and IGF-Ir placentae, however, GLT1 (EAAT2) and EAAC1 (EAAT3) protein content was decreased in junctional zone cells, whereas CAT1 content was increased in junctional and labyrinth zone cells. These data indicate that an excess level of GH stimulates GLT1 (EAAT2) expression and that a normal level of IGF-II is required for typical expression of GLT1 (EAAT2), GLAST1 (EAAT1) and EAAC1 (EAAT3), but that IGF-II downregulates the expression of EAAT4 and CAT1.

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Concentrative absorption of glutamate by the developing placenta is critical for proper fetal development. The expression of GLAST1, GLT1, EAAC1, and EAAT4, known to be capable of D-aspartate-inhibitable and Na(+)-coupled glutamate transport (system X-AG), was evaluated in day 14 vs. day 20 rat chorioallantoic placenta. Steady-state mRNA levels were greater at day 20 for all transporters. Immunohistochemistry determined that the expression of GLAST1, GLT1, and EAAC1 was greater throughout the day 20 placenta and was asymmetric with respect to cellular localization. EAAT4 protein was not detected. System X-AG activity was responsible for most of the Na(+)-dependent glutamate uptake and was greater in day 20 than in day 14 apical and basal membrane subdomains of the labyrinth syncytiotrophoblast. Greater quantities of EAAC1 and GLAST1 protein were identified on day 20, and quantities were greater in basal than in apical membranes. GLT1 expression, unchanged in apical membranes, was decreased in basal membranes. These data correlate transporter mRNA and protein content with transport activity and demonstrate an increasing capacity for glutamate absorption by the developing placenta.

Glutamine plays an important role in fetal nutrition. This study explored the transport of [3H]glutamine into apical and basal predominant membrane vesicles derived from rat and human placenta. Na+-dependent glutamine transport was present in both apical and basal predominant vesicles derived from 20- and, to a lesser degree, 14-day gestation rat placenta. Amino-acid transport systems A, ASC-like, B(o,+) (in apical membrane vesicles) and, perhaps, y+L were involved in Na+-dependent glutamine transport. Na+-dependent glutamine uptake into human placental microvillus and basolateral membrane vesicles also occurred via several distinct transport activities. Glutamine transport via system N was not detected in either rat or human placental preparations. Na+-dependent glutamine transport in the rat was more pronounced in basal as compared to apical membrane vesicles. Conversely, in the human preparations, activity was significantly higher in microvillus as compared to basolateral membrane vesicles. It is concluded that Na+-dependent glutamine transport occurs through a variety of transport agencies in both the rat and human placenta. Transport varies with ontogeny and between species.

Na+-independent cationic amino acid transport in the rat placenta occurs by leucine-sensitive and leucine-insensitive pathways. The ontogeny of these transport mechanisms within the rat placenta has been described recently. To assign the leucine-inhibitable portion of uptake definitively the uptake of [3H]arginine was studied in the presence of both BCH (to inhibit system B0,+) and varied concentrations of leucine. Uptake of arginine into basal-enriched membrane vesicles derived from rat placenta was, in the presence of sodium, inhibited by micromolar concentrations of leucine, consistent with assignment of this activity to system y+L. In contrast, the majority of arginine uptake into apical-enriched membrane vesicles was leucine insensitive. Messenger RNA derived from rat placenta at days 14, 16, 18 and 20 of gestation was hybridized with full-length rat cDNA probes against NBAT and 4F2HC (thought to encode proteins associated with system b0,+ and y+L activities, respectively). No NBAT mRNA was detected, whereas 4F2HC mRNA was present at all gestational stages, increasing 12-fold over the last third of gestation. It is concluded that system y+L is present in the basal plasma membrane of the rat placenta syncytium and is subject to developmental regulation by a mechanism that alters the steady content of 4F2HC mRNA.

Conference Article| August 01 1996 Rat placental amino acid transport after protein-deprivation-induced intrauterine growth retardation M. S. Malandro; M. S. Malandro *Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Box 100245, Gainesville, FL 32610-0245, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar M. J. Beveridge; M. J. Beveridge †Department of Pediatrics, University of Florida College of Medicine, Box 100245, Gainesville, FL 32610-0245, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar D. A. Novak; D. A. Novak †Department of Pediatrics, University of Florida College of Medicine, Box 100245, Gainesville, FL 32610-0245, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar M. S. Kilberg M. S. Kilberg ‡ *Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Box 100245, Gainesville, FL 32610-0245, U.S.A. †To whom correspondence should be addressed. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1996) 24 (3): 839–843. https://doi.org/10.1042/bst0240839 Article history Received: March 29 1996 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation M. S. Malandro, M. J. Beveridge, D. A. Novak, M. S. Kilberg; Rat placental amino acid transport after protein-deprivation-induced intrauterine growth retardation. Biochem Soc Trans 1 August 1996; 24 (3): 839–843. doi: https://doi.org/10.1042/bst0240839 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: IUGR, intrauterine growth retardation, AIB, 2-aminoisobutyric acid This content is only available as a PDF. © 1996 Biochemical Society1996 Article PDF first page preview Close Modal You do not currently have access to this content.

Given the central role of the placenta in nutrient transport to the fetus, one might propose that maternal nutrition would have a regulatory effect on this nutrient delivery. We have examined the effect of a low-protein adequate-calorie diet on specific amino acid transport processes by the rat placenta. Maternal weight, fetal weight, and placental weight were all significantly reduced in dams fed a low-protein (5% casein), isocaloric diet when compared with dams pair-fed a control (20% casein) diet. Even though maternal serum amino acid levels were maintained in the low-protein animals, fetomaternal serum amino acid ratios were significantly reduced, suggesting a reduction in nutrient transfer to the fetus. Apical and basal membrane vesicles were isolated from the placental trophoblast and were used to examine the amino acid transport capacity of both maternal-facing and fetal-facing membranes, respectively. Na+-dependent neutral amino acid transport mediated by system A was decreased in both membrane preparations, while transport mediated by system ASC was unaffected. The Na+-dependent anionic amino acid uptake by system X(-)AG (EAAC1) was reduced on the basal membrane, while the Na+-independent component was similar between the low-protein and control diet-fed dams. Cationic amino acid uptake was also reduced on both membrane surfaces. A decreased steady-state mRNA content for EAAC1 and CAT1 (system y+) suggests that reduced synthesis of the transporter proteins is responsible for the decrease in transport activity. Taken together, these data support the hypothesis that maternal protein malnutrition affects nutrient delivery to the fetus by downregulation of specific amino acid transport proteins.