Acute nutritional stress during pregnancy affects placental efficiency, fetal growth and adult glucose homeostasis.

Exposure to maternal malnutrition impairs postnatal health. Acute nutritional stress is less clearly implicated in intrauterine programming. We studied the effects of stressing pregnant mothers on perinatal growth and adult glucose homeostasis. We compared one group ("stressed", mothers fasted for 16 hours) with controls ("unstressed"). We found that fasting stress had adverse effects on the weight of the fetuses conceived (p<0.005) and the placental efficiency (p<0.001) in stressed compared to unstressed offspring. Placental weight was increased (p<0.001) presumably in compensation. Stress affected the glucose homeostasis of the offspring when they became adults (p<0.005) when analysed as individuals. We previously linked nutritional stress throughout pregnancy with a mitochondrial stress response. We modelled placenta with cultured human trophoblast cells (BeWos) and fetal tissues with mouse embryonic fibroblasts (MEFs). High throughput imaging showed that the mitochondria of both cell types underwent a similar sequence of changes in morphology, induced by nutritional stresses. The contrasting stress responses on fetal and placental weight were not captured by the cellular models. The stress of maternal fasting may be an important determinant of perinatal outcome in the mouse and might be relevant to nutritional stress in human pregnancy.

The functional and molecular entities underlying amino acid and peptide transport by the mammary gland under different physiological and pathological conditions.

This review describes the properties and regulation of the membrane transport proteins which supply the mammary gland with aminonitrogen to support metabolism under different physiological conditions (i.e. pregnancy, lactation and involution). Early studies focussed on characterising amino acid and peptide transport pathways with respect to substrate specificity, kinetics and hormonal regulation to allow a broad picture of the systems within the gland to be established. Recent investigations have concentrated on identifying the individual transporters at the molecular level (i.e. mRNA and protein). Many of the latter studies have identified the molecular correlates of the transport systems uncovered in the earlier functional investigations but in turn have also highlighted the need for more amino acid transport studies to be performed. The transporters function as either cotransporters and exchangers (or both) and act in a coordinated and regulated fashion to support the metabolic needs of the gland. However, it is apparent that a physiological role for a number of the transport proteins has yet to be elucidated. This article highlights the many gaps in our knowledge regarding the precise cellular location of a number of amino acid transporters within the gland. We also describe the role of amino acid transport in mammary cell volume regulation. Finally, the important role that individual mammary transport proteins may have in the growth and proliferation of mammary tumours is discussed.

Review: Epithelial aspects of human placental trophoblast.

The placenta must act as a surrogate lung, gastrointestinal tract and kidney for the fetus as well as acting as an endocrine gland necessary for the maintenance of a successful pregnancy: to achieve this, to what extent does the trophoblast necessarily share a similar epithelial phenotype? Here I review from a historical and a contemporary perspective some relevant studies with an emphasis on the similarities and differences between small intestinal and trophoblast biology. Certain physiological, structural and cell biological similarities are striking.

IDO induces expression of a novel tryptophan transporter in mouse and human tumor cells.

IDO is the rate-limiting enzyme in the kynurenine pathway, catabolizing tryptophan to kynurenine. Tryptophan depletion by IDO-expressing tumors is a common mechanism of immune evasion inducing regulatory T cells and inhibiting effector T cells. Because mammalian cells cannot synthesize tryptophan, it remains unclear how IDO(+) tumor cells overcome the detrimental effects of local tryptophan depletion. We demonstrate that IDO(+) tumor cells express a novel amino acid transporter, which accounts for ∼50% of the tryptophan uptake. The induced transporter is biochemically distinguished from the constitutively expressed tryptophan transporter System L by increased resistance to inhibitors of System L, resistance to inhibition by high concentrations of most amino acids tested, and high substrate specificity for tryptophan. Under conditions of low extracellular tryptophan, expression of this novel transporter significantly increases tryptophan entry into IDO(+) tumors relative to tryptophan uptake through the low-affinity System L alone, and further decreases tryptophan levels in the microenvironment. Targeting this additional tryptophan transporter could be a way of pharmacological inhibition of IDO-mediated tumor escape. These findings highlight the ability of IDO-expressing tumor cells to thrive in a tryptophan-depleted microenvironment by expressing a novel, highly tryptophan-specific transporter, which is resistant to inhibition by most other amino acids. The additional transporter allows tumor cells to strike the ideal balance between supply of tryptophan essential for their own proliferation and survival, and depleting the extracellular milieu of tryptophan to inhibit T cell proliferation.

The apical (hPepT1) and basolateral peptide transport systems of Caco-2 cells are regulated by AMP-activated protein kinase.

The effect of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) activation of the AMP-activated protein kinase (AMPK) on the transport of the model radiolabeled dipeptide [(3)H]-D-Phe-L-Gln was investigated in the human epithelial colon cancer cell line Caco-2. Uptake and transepithelial fluxes of [(3)H]-D-Phe-L-Gln were carried out in differentiated Caco-2 cell monolayers, and hPepT1 and glucose transporter 2 (GLUT2) protein levels were quantified by immunogold electron microscopy. AICAR treatment of Caco-2 cells significantly inhibited apical [(3)H]-D-Phe-L-Gln uptake, matched by a decrease in brush-border membrane hPepT1 protein but with a concomitant increase in the facilitated glucose transporter GLUT2. A restructuring of the apical brush-border membrane was seen by electron microscopy. The hPepT1-mediated transepithelial (A-to-B) peptide flux across the Caco-2 monolayers showed no significant alteration in AICAR-treated cells. The electrical resistance in the AICAR-treated monolayers was significantly higher compared with control cells. Inhibition of the sodium/hydrogen exchanger 3 (NHE3) had an additive effect to AICAR, suggesting that the AMPK effect is not via NHE3. Fluorescence measurement of intracellular pH showed no reduction in the proton gradient driving PepT1-mediated apical uptake. The reduction in apical hPepT1 protein and dipeptide uptake after AICAR treatment in Caco-2 cells demonstrates a regulatory effect of AMPK on hPepT1, along with an influence on both the microvilli and tight junction structures. The absence of an associated reduction in transepithelial peptide movement implies an additional stimulatory effect of AICAR on the basolateral peptide transport system in these cells. These results provide a link between the hPepT1 transporter and the metabolic state of this model enterocyte.

Proton-assisted amino-acid transporters are conserved regulators of proliferation and amino-acid-dependent mTORC1 activation.

The phosphoinositide3-kinase (PI3K)/Akt and downstream mammalian target of rapamycin complex 1 (mTORC1) signalling cascades promote normal growth and are frequently hyperactivated in tumour cells. mTORC1 is also regulated by local nutrients, particularly amino acids, but the mechanisms involved are poorly understood. Unexpectedly, members of the proton-assisted amino-acid transporter (PAT or SLC36) family emerged from in vivo genetic screens in Drosophila as transporters with uniquely potent effects on mTORC1-mediated growth. In this study, we show the two human PATs that are widely expressed in normal tissues and cancer cell lines, namely PAT1 and PAT4, behave similarly to fly PATs when expressed in Drosophila. Small interfering RNA knockdown shows that these molecules are required for the activation of mTORC1 targets and for proliferation in human MCF-7 breast cancer and HEK-293 embryonic kidney cell lines. Furthermore, activation of mTORC1 in starved HEK-293 cells stimulated by amino acids requires PAT1 and PAT4, and is elevated in PAT1-overexpressing cells. Importantly, in HEK-293 cells, PAT1 is highly concentrated in intracellular compartments, including endosomes, wherein mTOR shuttles upon amino-acid stimulation. Therefore our data are consistent with a model in which PATs modulate the activity of mTORC1 not by transporting amino acids into the cell but by modulating the intracellular response to amino acids.

Cerebellar agenesis revisited.

New clinical and employment information, together with over-looked previously published information, on a patient (H.C.) is reviewed. H.C., who died at the age of 76 in 1939, was found, by chance during anatomical dissection, to lack a cerebellum. This synthesis challenges an unusual and interesting account of cerebellar agenesis published in Brain in 1994 by Glickstein (see also Glickstein, 2006), in which the allegedly 'bogus' oral history of this individual's motor skills was held to have led to 'medical myth making'. Part of the burden of the 1994 paper was to show that 'cerebellar agenesis is always associated with profound motor deficits'. Glickstein therefore focussed on an apparent 'exception' to this conclusion, concerning the brain of a single case, H.C., who died 70 years ago, who 'had given rise to an oral tradition alleging that normal movement is possible despite total cerebellar agenesis'. Glickstein (1994) concludes 'despite an oral tradition to the contrary there is absolutely no evidence about the motor capacities of this man during his life'. Rather remarkably, an extensive history of this individual has become available, its significance becoming noted only this year; this complements and adds to a previous brief history published on H.C. (and not mentioned in the 1994 paper; see below). The new evidence includes the death certificate stating the man's occupation to have been 'manual labourer' with all the implications relevant to his supposed incapacity. The written historical record thus confronts the alleged 'myth'. It is interesting to note how medical records on an undoubtedly very ordinary citizen were recorded in London in the 1930s (before the NHS was set up in 1949) and how they could be made accessible to clinical colleagues in east London in the middle of World War II blitz bombing of the capital.

Sir Joseph Barcroft, Cambridge placental and fetal research (1933-1966) and inter-generational Science.

The nature of Cambridge (UK) placental and fetal research in the middle third of the twentieth century is reviewed on the basis of published literature and personal recollection. Joseph Barcroft is a central figure who came to fetal research late in an extremely productive career which is briefly sketched. Contemporaneous Cambridge academics in the field included J.D. Boyd (the authors father), J. Hammond, F.H.A. Marshall, R.A. McCance, J. Needham, A.S. Parkes and Elsie Widdowson. The then current Cambridge academic geography is explained and features of its scientific life such as funding, institutional structure and ethos, teaching and clinical duties, domestic and gender roles, and political context, including war and empire, are briefly considered. The testing of research findings against general principles and use of quantitative thinking are identified as important features. Intergenerational connections, often within individual families, are identified as a striking feature. The long-term impact of Cambridge work of this period; locally, in current trophoblast and feto-placental genetic research, in Oxford in probably influencing G.S. Dawes research leadership, and internationally, especially through D.H. Barron, and through him to the Denver School, is considered. That human placental and embryological specimens collected by J.D. Boyd have received a new lease of life as the "Boyd Collection", including use by Allen Enders is noted. Mechanisms for the maintenance of scientific quality and productivity during the period, mainly through the scientist himself relying on an internalised sense of "obligation", are contrasted with those current in the UK and more widely; formal peer-review at frequent intervals, with subsequent allocation of short-term funding. The strengths and weaknesses of each are considered.

Placental expression of 2,3 bisphosphoglycerate mutase in IGF-II knock out mouse: correlation of circulating maternal 2,3 bisphosphoglycerate and fetal growth.

Bisphosphoglycerate mutase (BPGM) catalyses the formation of 2,3 bisphosphoglycerate (BPG) a ligand of haemoglobin. BPG facilitates liberation of oxygen from haemoglobin at low oxygen tension enabling efficient delivery of oxygen to tissues. We describe expression of BPGM in mouse labyrinthine trophoblasts, located at the maternal-placental interface. Expression is lower in placentae of igf2(+/-) knockout mice, a widely used model of growth restriction, compared to wild type placentae. Circulating maternal BPG increased throughout gestation but this increase was less in wt mothers carrying igf2(+/-) pups than in those carrying exclusively wt pups. This reduction was observed well before term and may contribute to the low birth weight of igf2(+/-) pups. Strikingly, we also measured reductions of fetal and placental weight in wt littermates of igf2(+/-) pups compared to pups developing in an exclusively wt environment. These data suggest that placental expression of BPGM can influence maternal BPG concentrations and supports a hypothesis under which BPG synthesized in the placenta may act on maternal haemoglobin to enhance delivery of oxygen to the developing fetus.

Drosophila expresses a CD98 transporter with an evolutionarily conserved structure and amino acid-transport properties.

Mammalian CD98 heterodimeric amino acid transporters consist of a promiscuous single-pass transmembrane glycoprotein, CD98hc (CD98 heavy chain), and one of six multipass transmembrane proteins or 'light chains'. The heterodimeric complexes of CD98hc and the light chains LAT1 (L-type amino acid transporter 1) or LAT2 specifically promote sodium-independent System L exchange of neutral amino acids, including leucine. CD98hc is also implicated in other processes, including cell fusion, cell adhesion and activation of TOR (target of rapamycin) signalling. Surprisingly, recent reports suggested that insects lack a membrane-bound CD98hc, but in the present study we show that Drosophila CG2791 encodes a functional CD98hc orthologue with conservation in intracellular, transmembrane and extracellular domains. We demonstrate by RNA-interference knockdown in Drosophila Schneider cells that CG2791 and two Drosophila homologues of the mammalian CD98 light chains, Mnd (Minidiscs) and JhI-21, are required for normal levels of System L transport. Furthermore, we show that System L activity is increased by methoprene, an analogue of the developmentally regulated endocrine hormone juvenile hormone, an effect that is potentially mediated by elevated Mnd expression. Co-expression of CG2791 and JhI-21, but not CG2791 and Mnd, in Xenopus oocytes mediates System L transport. Finally, mapping of conserved sequences on to the recently determined crystal structure of the human CD98hc extracellular domain highlights two conserved exposed hydrophobic patches at either end of the domain that are potential protein-protein-interaction surfaces. Therefore our results not only show that there is functional conservation of CD98hc System L transporters in flies, but also provide new insights into the structure, functions and regulation of heterodimeric amino acid transporters.

mTOR: dissecting regulation and mechanism of action to understand human disease.

mTOR (mammalian target of rapamycin) is a highly conserved serine/threonine protein kinase that has roles in cell metabolism, cell growth and cell survival. Although it has been known for some years that mTOR acts as a hub for inputs from growth factors (in particular insulin and insulin-like growth factors), nutrients and cellular stresses, some of the mechanisms involved are still poorly understood. Recent work has implicated mTOR in a variety of important human pathologies, including cancer, Type 2 diabetes and neurodegenerative disorders, heightening interest and accelerating progress in dissecting out the control and functions of mTOR.

Amino acid sensing and mTOR regulation: inside or out?

mTOR (mammalian target of rapamycin) plays a key role in determining how growth factor, nutrient and oxygen levels modulate intracellular events critical for the viability and growth of the cell. This is reflected in the impact of aberrant mTOR signalling on a number of major human diseases and has helped to drive research to understand how TOR (target of rapamycin) is itself regulated. While it is clear that amino acids can affect TOR signalling, how these molecules are sensed by TOR remains controversial, perhaps because cells use different mechanisms as environmental conditions change. Even the question of whether they have an effect inside the cell or at its surface remains unresolved. The present review summarizes current ideas and suggests ways in which some of the models proposed might be unified to produce an amino acid detection system that can adapt to environmental change.

Facts, fantasies and fun in epithelial physiology.

The hallmark of epithelial cells is their functional polarization. It is those membrane proteins that are distributed differentially, either to the apical or to the basal surface, that determine epithelial physiology. Such proteins will include 'pumps', 'channels' and 'carriers', and it is the functional interplay between the actions of these molecules that allows the specific properties of the epithelium to emerge. Epithelial properties will additionally depend on: (a) the extent to which there may be a route between adjacent cells (the 'paracellular' route); and (b) the folding of the epithelium (as, for example, in the loop of Henle). As for other transporters, there is polarized distribution of amino-acid carriers; the molecular basis of these is of considerable current interest with regard to function, including 'inborn errors' (amino-acidurias); some of these transporters have additional functions, such as in the regulation of cell fusion, in modulating cell adherence and in activating intracellular signalling pathways. Collaboration of physiologists with fly geneticists has generated new insights into epithelial function. One example is the finding that certain amino-acid transporters may act as 'transceptors' and play a role as sensors of the extracellular environment that then regulate intracellular pathways controlling cell growth.

The Abeta1-42M35C mutated amyloid peptide Abeta1-42 and the 25-35 fragment fail to mimic the subtype-specificity of actions on recombinant human nicotinic acetylcholine receptors (alpha7, alpha4beta2, alpha3beta4).

Alzheimer's disease (AD) is a neurodegenerative condition involving accumulation of the beta-amyloid peptide, Abeta1-42. Previously we have shown that amyloid peptides (Abeta1-42, Abeta1-40) have different actions on the three major brain nicotinic acetylcholine receptor (nAChR) subtypes (alpha7, alpha4beta2 and alpha3beta4). The methionine in position 35 of Abeta (M35) has been shown to be important in the toxicity of Abeta and the 25-35 fragment can mimic some of the actions of the Abeta1-42 peptide. However, the extent to which this mutant and the fragment mimic subtype selectivity is unknown. Two-electrode voltage-clamp electrophysiology has been used to study the actions on alpha7, alpha4beta2 and alpha3beta4 recombinant nAChRs expressed in Xenopus laevis oocytes of full length Abeta1-42, and Abeta peptide fragments, scrambled peptides, and the Abeta1-42 peptide containing mutations of the methionine in position 35. The Abeta25-35 fragment did not display subunit specificity. Abeta1-42 with an M35C mutation showed similar subtype-specificity to wild-type Abeta1-42. However, Abeta1-42 with an M35V substitution reduced the peak amplitude of ACh-induced currents recorded from alpha4beta2 nAChRs, but did not affect those recorded from alpha7 or alpha3beta4. These results indicate that the amino acid in position 35 of Abeta1-42 is an important determinant of the subtype-specificity of this peptide on human recombinant alpha7, alpha4beta2 and alpha3beta4 nAChRs and that the 25-35 fragment fails to mimic all of the actions of the full-length peptide.

Amino acid transporters and nutrient-sensing mechanisms: new targets for treating insulin-linked disorders?

The IIS (insulin/IGF (insulin-like growth factor) signalling) cascade has an important role in regulating normal development and physiology, as evidenced by its effects in a host of major human diseases including cancer, Type 2 diabetes and neurodegeneration. Recently, it has become clear that multiple types of local nutrient-sensing mechanisms have an impact on cellular insulin-sensitivity through the downstream kinase TOR (target of rapamycin). In vivo analysis in flies has surprisingly highlighted PATs (proton-assisted amino acid transporters) as having a uniquely potent role in regulating IIS/TOR activity and growth, potentially via a novel signalling mechanism. Other molecules such as the heterodimeric amino acid transporter, CD98, which provides the principal route for cellular uptake of leucine, an amino acid implicated in regulating TOR, also appear to have important effects. As our understanding of how nutrient sensing has an impact on IIS/TOR increases, novel targets to modulate aberrant IIS in disease are likely to emerge, which could complement current strategies designed to block kinases in this pathway.

Membrane trafficking of CD98 and its ligand galectin 3 in BeWo cells--implication for placental cell fusion.

CD98 heavy chain (CD98hc), expressed at high levels in developing human trophoblasts, is an integral membrane protein with multiple N-linked glycosylation sites and known to be important for cell fusion, adhesion, and amino acid transport. Western blotting and flow cytometry were used to study the effect of brefeldin A, an inhibitor of protein translocation through the Golgi, on CD98hc in the human placental trophoblast cell line BeWo. Although brefeldin A treatment caused increased cell surface expression of CD98hc, a novel partially glycosylated form of the protein was found and, concomitantly, cell fusion was reduced. Western blotting showed that CD98 and galectin 3, a proposed ligand for the glycosylated extracellular domain of CD98hc, co-immunoprecipitated, and double-label immuno-electron microscopy confirmed that CD98hc associated with galectin 3. Furthermore, cell fusion was reduced (specifically) by the disaccharide lactose, a known ligand for the carbohydrate recognition domain of galectin 3, suggesting that the association was functional. Taken together, the data suggest that N-glycosylation of CD98 and subsequent interaction with galectin 3 is critical for aspects of placental cell biology, and provides a rationale for the observation that, in the mouse, truncation of the CD98hc extracellular domain leads to early embryonic lethality [Tsumura H, Suzuki N, Saito H, Kawano M, Otake S, Kozuka Y, Komada H, Tsurudome M & Ito Y (2003) Biochem Biophys Res Commun 308, 847-851].

Extracellular and subcellular regulation of the PI3K/Akt cassette: new mechanisms for controlling insulin and growth factor signalling.

The PI3K (phosphoinositide 3-kinase)/Akt (also called protein kinase B) signalling cassette plays a central role in the response to growth factors, particularly insulin-like molecules, and its misregulation is a characteristic feature of diabetes and many forms of human cancer. Recent molecular genetic studies initiated in the fruitfly, Drosophila melanogaster, have highlighted two new cell-type-specific mechanisms regulating PI3K/Akt signalling and its downstream effects. First, the cellular response to this cassette is modulated by several classes of cell-surface transporters and sensors, suggesting an important role for extracellular nutrients in insulin-sensitivity. Secondly, various cell types show a markedly different subcellular distribution of the activated kinase Akt, influencing the cellular functions of this molecule. These findings reveal new mechanisms by which processes such as growth, lipogenesis and insulin resistance can be differentially regulated and may suggest novel strategies for treating insulin-linked diseases.

Differential effect of cross-linking the CD98 heavy chain on fusion and amino acid transport in the human placental trophoblast (BeWo) cell line.

CD98 (otherwise known as 4F2) is an integral membrane protein with multiple functions including amino acid transport, integrin activation, cell fusion and cell activation. The molecular mechanisms coordinating these multiple functions remain unclear. We have studied CD98 heavy chain (hc) function in a human placental trophoblast cell line (BeWo). We show that cross-linking of CD98hc by incubation of cells in the presence of functional monoclonal antibodies causes cellular re-distribution of the protein from the cytoplasm to the plasma membrane as measured by flow cytometry, western blotting and quantitative immuno-electron microscopy. The latter technique also indicated that CD98hc is trafficked between cell surface and cytoplasmic pools in vesicles. Increased cell surface CD98 correlates with increased cellular fusion in BeWo cells. In addition, we show reduced LAT 1 surface expression and neutral amino acid transport in the presence of the CD98 mabs. The results thus suggest that the function of CD98 in cell fusion is distinct from its role in cellular nutrient delivery.