Biosynthetic regulation and intracellular transport of phosphatidylserine in mammalian cells

In mammalian cells, phosphatidylserine (PtdSer) is synthesized through the action of the endoplasmic reticulum enzymes, PtdSer synthase 1 and 2, and the decarboxylation of PtdSer accounts for the majority of phosphatidylethanolamine (PtdEtn) synthesis. PtdSer decarboxylation for PtdEtn formation occurs in the mitochondria. In addition, the transport of PtdSer from the endoplasmic reticulum to the mitochondria is probably a rate limiting step for PtdEtn synthesis through the decarboxylation pathway. Therefore, the regulation of PtdSer synthesis and its intracellular transport appear to be essential events for the maintenance of normal cellular PtdSer and PtdEtn levels. Here we describe the current understanding of the regulation of PtdSer biosynthesis and the transport of PtdSer from the ER to the mitochondria in mammalian cells.     

GLUT1-mediated glucose transport and its regulation by IGF-I in cultured bovine chromaffin cells

Regulation of glucose transport was studied in primary cultures of bovine chromaffin cells (BCC) using the glucose analogue 2-deoxyglucose (DOG) as a model substrate. The glucose transporter in freshly isolated and cultured BCC was identified as GLUT1 by Western immunoblots. The level of GLUT1 increased by time in culture and was followed by an enhancement in uptake of DOG. The DOG uptake was stimulated by insulin-like growth factor I (IGF-I) with an EC₅₀ of 1 nM and a maximal response (∼2-fold) was obtained at 10–100 nM IGF-I. Insulin was at least 100-fold less potent than IGF-I. Exposure to 10⁻⁸ M IGF-I also caused a redistribution of GLUT1 from an intracellular compartment to a plasma membrane-enriched fraction. Our results demonstrate a GLUT1-mediated glucose uptake in adrenomedullary cells. An enhanced glucose transport in response to IGF-I appears to be coupled to activation of IGF receptor type 1 and GLUT1 translocation. © 1996 Wiley-Liss, Inc.     

The molecular regulation of organelle transport in mammalian retinal pigment epithelial cells

Retinal pigment epithelial cells contain large numbers of melanosomes that can enter the apical processes extending between the outer segments of the overlying photoreceptors. Every day the distal portion of the photoreceptor outer segment is shed and phagocytosed by the retinal pigment epithelial cell. The phagosome is then transported into the cell body and the contents degraded by lysosomal enzymes. This review focuses on recent progress made in the identification of molecules that regulate the transport of melanosomes into the apical processes and the transport of phagosomes into the cell body. Myosin VIIa is a key player in both processes and, at least in the case of melanosome movement, myosin VIIa is recruited to the melanosome via the GTPase, Rab27a. The possible role played by defects in the transport of melanosomes and phagosomes in the development of retinal degenerative diseases is discussed.     

Insulin regulation of amino acid transport in mesenchymal cells from avian and mammalian tissues.

Insulin regulation of amino acid transport across the cell membrane was studied in a variety of mesenchymal cell directly isolated from avian and mammalian tissues or collected from confluent cultures. Transport activity of the principal systems of mediation in the presence and absence of insulin was evaluated by measuring the uptake of representative amino acids under conditions approaching initial entry rates. Insulin enhanced the transport rate of substrate amino acids from the A system(alpha-aminoisobutyric acid, L-proline, glycine, L-alanine and L-serine) in fibroblasts and osteoblasts from chick-embryo tissues, in mesenchymal cells (fibroblasts and smooth muscle cells) from immature rat uterus, in thymic lymphocytes from young rats and in chick-embryo fibroblasts from confluent secondary cultures. In these tissues, the uptake of amino acid substrates of transport systems L and Ly+ (L-leucine, L-phenylalanine, L-lysine) was not affected by the presence of the hormone. No insulin control of amino acid transport was detected in chick-embryo chondroblasts and rat peritoneal macrophages. These observations identify the occurrence of hormonal regulatory patterns of amino acid transport for different mesenchymal cells types and indicate that these properties emerge early during cell differentiation.     

Citrate transport and metabolism in mammalian cells

Citrate, an organic trivalent anion, is a major substrate for generation of energy in most cells. It is produced in mitochondria and used either in the Krebs' cycle or released into cytoplasm through a specific mitochondrial carriers. Citrate can also be taken up from blood through different plasma membrane transporters. In the cytoplasm, citrate can be used ultimately for fatty acid synthesis, which is increased in cancer cells. Here, we review the ways in which citrate can be transported and discuss the changes in transport and metabolism that occur in cancer cells. The primary focus is on the prostate gland, which is known to produce and release large amounts of citrate during its normal secretory function. The significant changes that occur in citrate‐related metabolism and transport in prostate cancer are the second focus. This review strives to relate these mechanisms to molecular biology on the one hand and to clinical applications on the other.     

Effect of plasma membrane cholesterol depletion on glucose transport regulation in leukemia cells

GLUT1 is the predominant glucose transporter in leukemia cells, and the modulation of glucose transport activity by cytokines, oncogenes or metabolic stresses is essential for their survival and proliferation. However, the molecular mechanisms allowing to control GLUT1 trafficking and degradation are still under debate. In this study we investigated whether plasma membrane cholesterol depletion plays a role in glucose transport activity in M07e cells, a human megakaryocytic leukemia line. To this purpose, the effect of cholesterol depletion by methyl-β-cyclodextrin (MBCD) on both GLUT1 activity and trafficking was compared to that of the cytokine Stem Cell Factor (SCF). Results show that, like SCF, MBCD led to an increased glucose transport rate and caused a subcellular redistribution of GLUT1, recruiting intracellular transporter molecules to the plasma membrane. Due to the role of caveolae/lipid rafts in GLUT1 stimulation in response to many stimuli, we have also investigated the GLUT1 distribution along the fractions obtained after non ionic detergent treatment and density gradient centrifugation, which was only slightly changed upon MBCD treatment. The data suggest that MBCD exerts its action via a cholesterol-dependent mechanism that ultimately results in augmented GLUT1 translocation. Moreover, cholesterol depletion triggers GLUT1 translocation without the involvement of c-kit signalling pathway, in fact MBCD effect does not involve Akt and PLCγ phosphorylation. These data, together with the observation that the combined MBCD/SCF cell treatment caused an additive effect on glucose uptake, suggest that the action of SCF and MBCD may proceed through two distinct mechanisms, the former following a signalling pathway, and the latter possibly involving a novel cholesterol dependent mechanism.     

Mathematical modeling and analysis of glucose and glutamine utilization and regulation in cultures of continuous mammalian cells

A number of factors have been shown to affect the metabolism of glucose and glutamine in mammalian cells and their mechanisms have been partially elucidated. Despite these efforts, a quantitative knowledge of the significance of these factors, the regulation of glucose and glutamine utilization, and particularly the interactions of these two nutrients is still lacking. Controversies exist in the literature. To clarify some of these controversies, mathematical models are proposed in this work which enable to separate and identify the effects of individual factors. Experimental data from five cell lines obtained in batch, fed-batch, and continuous cultures, both under steady-state and transient conditions, were used to verify the model formulations. The resulting kinetic models successfully describe all these cultures. According to the models, the specific consumption rate of glucose (Q(Glc)) of continuous animal cells under normal culture conditions can be expressed as a sum of three parts: a part owing to cell growth; a part owing to glucose excess; and a part owing to glutamine regulation. The specific consumption rate of glutamine (q(Glc)7) can be expressed as a sum of only two parts: a part owing to cell growth; and a part owing to glutamine excess. Using the kinetic models the interaction and regulation of glucose and glutamine utilizations are quantitatively analyzed. The results indicate that, whereas q(Glc) is affected by glutamine, q(Gln) appears to be not or less significantly affected by glucose. It is also shown that the relative utilizations of glucose and glutamine by anabolism and catabolism are mainly affected by the residual concentrations of the respective compounds and are less sensitive to growth rate and the nature of growth limitation.(c) 1995 John Wiley & Sons, Inc.     

Insulin signaling and the regulation of glucose transport

Gaps remain in our understanding of the precise molecular mechanisms by which insulin regulates glucose uptake in fat and muscle cells. Recent evidence suggests that insulin action involves multiple pathways, each compartmentalized in discrete domains. Upon activation, the receptor catalyzes the tyrosine phosphorylation of a number of substrates. One family of these, the insulin receptor substrate (IRS) proteins, initiates activation of the phosphatidylinositol 3-kinase pathway, resulting in stimulation of protein kinases such as Akt and atypical protein kinase C. The receptor also phosphorylates the adapter protein APS, resulting in the activation of the G protein TC10, which resides in lipid rafts. TC10 can influence a number of cellular processes, including changes in the actin cytoskeleton, recruitment of effectors such as the adapter protein CIP4, and assembly of the exocyst complex. These pathways converge to control the recycling of the facilitative glucose transporter Glut4.