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PubMed1993

Subcellular localization of Bcr, Abl, and Bcr-Abl proteins in normal and leukemic cells and correlation of expression with myeloid differentiation.

We used specific antisera and immunohistochemical methods to investigate the subcellular localization and expression of Bcr, Abl, and Bcr-Abl proteins in leukemic cell lines and in fresh human leukemic and normal samples at various stages of myeloid differentiation. Earlier studies of the subcellular localization of transfected murine type IV c-Abl protein in fibroblasts have shown that this molecule resides largely in the nucleus, whereas transforming deletion variants are localized exclusively in the cytoplasm. Here, we demonstrate that the murine type IV c-Abl protein is also found in the nucleus when overexpressed in a mouse hematopoietic cell line. However, in both normal and leukemic human hematopoietic cells, c-Abl is discerned predominantly in the cytoplasm, with nuclear staining present, albeit at a lower level. In contrast, normal endogenous Bcr protein, as well as the aberrant p210BCR-ABL and p190BCR-ABL proteins consistently localize to the cytoplasm in both cell lines and fresh cells. The results with p210BCR-ABL were confirmed in a unique Ph1-positive chronic myelogenous leukemia (CML) cell line, KBM5, which lacks the normal chromosome 9 and hence the normal c-Abl product. Because the p210BCR-ABL protein appears cytoplasmic in both chronic phase and blast crisis CML cells, as does the p190BCR-ABL in Ph1-positive acute leukemia, a change in subcellular location of Bcr-Abl proteins between cytoplasm and nucleus cannot explain the different spectrum of leukemias associated with p210 and p190, nor the transition from the chronic to the acute leukemia phenotype seen in CML. Further analysis of fresh CML and normal hematopoietic bone marrow cells reveals that p210BCR-ABL, as well as the normal Bcr and Abl proteins, are expressed primarily in the early stages of myeloid maturation, and that levels of expression are reduced significantly as the cells mature to polymorphonuclear leukocytes. Similarly, a decrease in Bcr and Abl levels occurs in HL-60 cells induced by DMSO to undergo granulocytic differentiation. The action of p210BCR-ABL and its normal counterparts may, therefore, take place during the earlier stages of myeloid development.

Animals↗

PubMed2005

Protein subcellular localization prediction for Gram-negative bacteria using amino acid subalphabets and a combination of multiple support vector machines.

BACKGROUND: Predicting the subcellular localization of proteins is important for determining the function of proteins. Previous works focused on predicting protein localization in Gram-negative bacteria obtained good results. However, these methods had relatively low accuracies for the localization of extracellular proteins. This paper studies ways to improve the accuracy for predicting extracellular localization in Gram-negative bacteria. RESULTS: We have developed a system for predicting the subcellular localization of proteins for Gram-negative bacteria based on amino acid subalphabets and a combination of multiple support vector machines. The recall of the extracellular site and overall recall of our predictor reach 86.0% and 89.8%, respectively, in 5-fold cross-validation. To the best of our knowledge, these are the most accurate results for predicting subcellular localization in Gram-negative bacteria. CONCLUSION: Clustering 20 amino acids into a few groups by the proposed greedy algorithm provides a new way to extract features from protein sequences to cover more adjacent amino acids and hence reduce the dimensionality of the input vector of protein features. It was observed that a good amino acid grouping leads to an increase in prediction performance. Furthermore, a proper choice of a subset of complementary support vector machines constructed by different features of proteins maximizes the prediction accuracy.

Algorithms↗

PubMed2006

A graphical model approach to automated classification of protein subcellular location patterns in multi-cell images.

BACKGROUND: Knowledge of the subcellular location of a protein is critical to understanding how that protein works in a cell. This location is frequently determined by the interpretation of fluorescence microscope images. In recent years, automated systems have been developed for consistent and objective interpretation of such images so that the protein pattern in a single cell can be assigned to a known location category. While these systems perform with nearly perfect accuracy for single cell images of all major subcellular structures, their ability to distinguish subpatterns of an organelle (such as two Golgi proteins) is not perfect. Our goal in the work described here was to improve the ability of an automated system to decide which of two similar patterns is present in a field of cells by considering more than one cell at a time. Since cells displaying the same location pattern are often clustered together, considering multiple cells may be expected to improve discrimination between similar patterns. RESULTS: We describe how to take advantage of information on experimental conditions to construct a graphical representation for multiple cells in a field. Assuming that a field is composed of a small number of classes, the classification accuracy can be improved by allowing the computed probability of each pattern for each cell to be influenced by the probabilities of its neighboring cells in the model. We describe a novel way to allow this influence to occur, in which we adjust the prior probabilities of each class to reflect the patterns that are present. When this graphical model approach is used on synthetic multi-cell images in which the true class of each cell is known, we observe that the ability to distinguish similar classes is improved without suffering any degradation in ability to distinguish dissimilar classes. The computational complexity of the method is sufficiently low that improved assignments of classes can be obtained for fields of twelve cells in under 0.04 second on a 1600 megahertz processor. CONCLUSION: We demonstrate that graphical models can be used to improve the accuracy of classification of subcellular patterns in multi-cell fluorescence microscope images. We also describe a novel algorithm for inferring classes from a graphical model. The performance and speed suggest that the method will be particularly valuable for analysis of images from high-throughput microscopy. We also anticipate that it will be useful for analyzing the mixtures of cell types typically present in images of tissues. Lastly, we anticipate that the method can be generalized to other problems.

Algorithms↗

PubMed2004

Endotoxin leads to rapid subcellular re-localization of hepatic RXRalpha: A novel mechanism for reduced hepatic gene expression in inflammation.

BACKGROUND: Lipopolysaccharide (LPS) treatment of animals down-regulates the expression of hepatic genes involved in a broad variety of physiological processes, collectively known as the negative hepatic acute phase response (APR). Retinoid X receptor alpha (RXRalpha), the most highly expressed RXR isoform in liver, plays a central role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism and homeostasis. Many of the genes regulated by RXRalpha are repressed during the negative hepatic APR, although the underlying mechanism is not known. We hypothesized that inflammation-induced alteration of the subcellular location of RXRalpha was a common mechanism underlying the negative hepatic APR. RESULTS: Nuclear RXRalpha protein levels were significantly reduced (~50%) within 1-2 hours after low-dose LPS treatment and remained so for at least 16 hours. RXRalpha was never detected in cytosolic extracts from saline-treated mice, yet was rapidly and profoundly detectable in the cytosol from 1 hour, to at least 4 hours, after LPS administration. These effects were specific, since the subcellular localization of the RXRalpha partner, the retinoic acid receptor (RARalpha), was unaffected by LPS. A potential cell-signaling modulator of RXRalpha activity, c-Jun-N-terminal kinase (JNK) was maximally activated at 1-2 hours, coincident with maximal levels of cytoplasmic RXRalpha. RNA levels of RXRalpha were unchanged, while expression of 6 sentinel hepatic genes regulated by RXRalpha were all markedly repressed after LPS treatment. This is likely due to reduced nuclear binding activities of regulatory RXRalpha-containing heterodimer pairs. CONCLUSION: The subcellular localization of native RXRalpha rapidly changes in response to LPS administration, correlating with induction of cell signaling pathways. This provides a novel and broad-ranging molecular mechanism for the suppression of RXRalpha-regulated genes in inflammation.

Journal Article↗

PubMed2004

Automated interpretation of protein subcellular location patterns: implications for early cancer detection and assessment.

Fluorescence microscopy is a powerful tool for analyzing the subcellular distributions of proteins, but that power has not been fully utilized because most analysis of those distributions has been done by visual examination. This limitation can be overcome using automated pattern recognition methods widely used in other fields. This article summarizes work demonstrating that automated systems can recognize the patterns of major organelles in both two- and three-dimensional images of cultured cells, and that these systems can distinguish similar patterns better than visual examination. The basis for these systems are sets of Subcellular Location Features that capture the essence of subcellular patterns without being sensitive to the extensive variation that occurs in the size, shape, and orientation of cells in microscope images. These features can also be used to make sensitive, statistical comparisons of the distribution of a protein between two conditions, such as in the presence and absence of a drug. The possible use of automated pattern analysis methods for improving detection of abnormal cells in cancerous or precancerous tissues is also discussed.

Automation↗

PubMed1990

Aberrant subcellular localization of peroxisomal 3-ketoacyl-CoA thiolase in the Zellweger syndrome and rhizomelic chondrodysplasia punctata.

Fibroblasts from patients with the inherited disorder Zellweger syndrome have few or no peroxisomes; multiple biochemical processes that normally occur in this organelle are defective. Rhizomelic chondrodysplasia punctata (RCDP) is another inherited disorder in which two unrelated peroxisomal metabolic processes, plasmalogen synthesis and phytanic acid oxidation, are impaired despite the normal appearance of peroxisomal structure. It was previously reported that one of the enzymes of peroxisomal fatty acid beta-oxidation, 3-ketoacyl-CoA thiolase (beta-keto-thiolase), was present in precursor rather than mature form in both of these diseases. Immunofluorescent staining for peroxisomal beta-ketothiolase showed the immunoreactivity to be localized in subcellular particles in fibroblasts from both Zellweger syndrome and RCDP patients, even though the former lack normal peroxisomes. Immunoblot studies were performed to determine the subcellular location of the thiolase precursor in fractionated fibroblasts from Zellweger and RCDP patients. In both disorders, thiolase immunoreactivity was detected in subcellular fractions having a lower density than normal peroxisomes and mitochondria, and was resistant to digestion by proteinase K. The density of the thiolase precursor-containing fractions was similar to that of peroxisomal membrane "ghost" fractions recently described by Santos et al. (J Biol Chem 263:10502-10509, 1988). Our results suggest that these are not empty membrane vesicles but contain at least one peroxisomal matrix protein. Furthermore, they exist not only in cells in which normal peroxisomes fail to form (Zellweger syndrome), but also in some cells which have catalase-containing peroxisomes (RCDP).

Acetyl-CoA C-Acyltransferase↗

PubMed1975

Subcellular localization of LH releasing activity in the rat hypothalamus.

Separation of particulate fractions associated with LH releasing activity has been effected by differential centrifugation followed by sucrose density gradient centrifugation. The fractions were monitored for LH releasing activity by an in vitro incubation with rat pituitaries and for subcellular organelles by electron microscopy. LH released into the incubation medium and contained in the pituitaries was measured by radioimmunoassay (RIA). After differential centrifugation only the "crude mitochondrial fraction" caused an increase of LH release accompanied by a depletion of pituitary LH and a rise in total LH. Further fractionation of this pellet on a discontinuous sucrose gradient resulted in three opaque bands and three clear areas which were removed as sic fractions. Only one fraction, not associated with myelin, synaptosomes, or mitochondria, caused an increase in LH release and total LH while pituitary LH remained unchanged. This fraction appears to be predominantly composed of electron dense vesicles of varying sizes. Also of interest is another fraction which decreased LH release and was again not associated with myelin, synaptosomes, or mitochondria. The decreased release was associated with an increased pituitary LH while total LH remained unchanged. Of the six subcellular fractions obtained from the "crude mitochondrial pellet" only these two caused significant changes in LH release. Two other neural tissues, the cerebellum and cerebral cortex, were similarly processed. None of these subcellular fractions significantly altered LH release and/or synthesis.

Animals↗

PubMed2005

Role of the subcellular localization of ALK tyrosine kinase domain in neuronal differentiation of PC12 cells.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed in specific areas of the developing central and peripheral nervous systems. We previously demonstrated that a membrane-bound and constitutively active form of the ALK protein tyrosine kinase (PTK) domain induced the neuron-like differentiation of PC12 cells through specific activation of the mitogen-activated protein kinase (MAP kinase) pathway. Its PTK domain had been originally identified in a nucleo-cytosolic and constitutively active transforming protein, NPM-ALK. Downstream targets involved in oncogenic proliferation and survival processes have been proposed to include phospholipase Cgamma (PLCgamma), phosphoinositide 3-kinase (PI 3-kinase)/AKT, STAT 3/5 and Src. We therefore postulated that activation of specific signaling pathways leading to differentiation or proliferation can be differently controlled depending on the subcellular localization of ALK PTK domain. To increase knowledge of its physiological role in the nervous system, we focused in the present study on the influence of its subcellular localization on neuronal differentiation. To achieve this goal, we characterized biological responses and transduction pathways in PC12 cells elicited by various constructs encoding membrane-bound (through transmembrane or myristyl sequences) or cytosolic ALK-derived proteins. In order to control the activation of their PTK domain, we used an inducible dimerization system. Here, we demonstrate that membrane attachment of the ALK PTK domain, in PC12 cells, is crucial for initiation of neurite outgrowth and proliferation arrest through a decrease of DNA synthesis. Furthermore, we show that this differentiation process relies on specific and sustained activation of ERK 1/2 proteins. By contrast, activation of the cytosolic form of this domain fails to induce MAP kinase activation and cell differentiation but promotes a PI 3-kinase/AKT-dependent PC12 cell proliferation. These data indicate that subcellular localization of the ALK PTK domain was a determinant for the control and specificity of downstream transduction cascades and was crucial for deciding the fate to which the neuronal cell will be committed.

Anaplastic Lymphoma Kinase↗

PubMed2000

Subcellular localization of Bic-D::GFP is linked to an asymmetric oocyte nucleus.

Bicaudal-D (Bic-D) is essential for the establishment of oocyte fate and subsequently for polarity formation within the developing Drosophila oocyte. To find out where in the germ cells Bic-D performs its various functions we made transgenic flies expressing a chimeric Bic-D::GFP fusion protein. Once Bic-D::GFP preferentially accumulates in the oocyte, it shows an initial anterior localization in germarial region 2. In the subsequent egg chamber stages 1-6 Bic-D::GFP preferentially accumulates between the oocyte nucleus and the posterior cortex in a focus that is consistently aligned with a crater-like indentation in the oocyte nucleus. After stage 6 Bic-D::GFP fluorescent signal is predominantly found between the oocyte nucleus and the dorso-anterior cortex. During the different phases several genes have been found to be required for the establishment of the new Bic-D::GFP distribution patterns. Dynein heavy chain (Dhc), spindle (spn) genes and maelstrom (mael) are required for the re-localization of the Bic-D::GFP focus from its anterior to its posterior oocyte position. Genes predicted to encode proteins that interact with RNA (egalitarian and orb) are required for the normal subcellular distribution of Bic-D::GFP in the germarium, and another potential RNA binding protein, spn-E, is required for proper transport of Bic-D::GFP from the nurse cells to the oocyte in later oogenesis stages. The results indicate that Bic-D requires the activity of mRNA binding proteins and a negative-end directed microtubule motor to localize to the appropriate cellular domains. Asymmetric subcellular accumulation of Bic-D and the polarization of the oocyte nucleus may reflect the function of this localization machinery in vectorial mRNA localization and in tethering of the oocyte nucleus. The subcellular polarity defined by the Bic-D focus and the nuclear polarity marks some of the first steps in antero-posterior and subsequently in dorso-ventral polarity formation.

Animals↗

PubMed1990

Ultrastructural localization of the regulatory (RII) subunit of cyclic AMP-dependent protein kinase to subcellular compartments active in endocytosis and recycling of membrane receptors.

The subcellular distribution of the regulatory (RII) subunit of cyclic AMP-dependent protein kinase (PK-A) was analyzed at the electron-microscopical level using thawed cryo-sections of Madin Darby Bovine Kidney (MDBK) cells. The highest density of labelling for RII was found on membranes of the prelysosomal compartment (PLC; marked with the cation-independent mannose 6-phosphate receptor, MPR) and the trans-Golgi network, TGN (at 20 degrees C, marked with the G protein of vesicular stomatitis virus, VSV), as well as in coated buds on the latter. Significant labelling was also localized to the cytoplasmic surface of the plasma membrane, including clathrin-coated pits and microvilli, and to early endosomes (identified using internalized HRP). In contrast, no significant label was seen over the Golgi compartments proximal to the TGN, the endoplasmic reticulum (ER) or over lysosomes. From these results we conclude that PK-A type II is associated with the membranes of precisely those subcellular compartments that are active in endocytosis and recycling of cell surface receptors. We believe these findings to be related to the well-established role of cyclic AMP in signal transduction. In particular, we propose that activation of PK-A in endocytic compartments may contribute to regulation (via phosphorylation) of the subcellular distribution of internalized surface receptors or their functional coupling to effector systems involved in signal propagation.

Animals↗

PubMed2004

Subcellular localization of frizzled receptors, mediated by their cytoplasmic tails, regulates signaling pathway specificity.

The Frizzled (Fz; called here Fz1) and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/beta-catenin pathway or Fz/planar cell polarity (PCP) signaling in Drosophila. The regulation of signaling specificity remains largely obscure. We show that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/beta-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, we propose that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways.

Animals↗

PubMed2025

Subcellular proteomics of the protist Paradiplonema papillatum reveals the digestive capacity of the cell membrane and the plasticity of peroxisomes across euglenozoans.

Diplonemids are among the most diverse and abundant protists in the deep ocean, have extremely complex and ancient cellular systems, and exhibit unique metabolic capacities. Despite this, we know very little about this major group of eukaryotes. To establish a model organism for comprehensive investigation, we performed subcellular proteomics on Paradiplonema papillatum and localized 4,870 proteins to 22 cellular compartments. We additionally confirmed the predicted location of several proteins by epitope tagging and fluorescence microscopy. To probe the metabolic capacities of P. papillatum, we explored the proteins predicted to the cell membrane compartment in our subcellular proteomics dataset. Our data revealed an accumulation of many carbohydrate-degrading enzymes (CDZymes). Our predictions suggest that these CDZymes are exposed to the extracellular space, supporting proposals that diplonemids may specialize in breaking down carbohydrates in plant and algal cell walls. Further exploration of carbohydrate metabolism revealed an evolutionary divergence in the function of glycosomes (modified peroxisomes) in diplonemids versus kinetoplastids. Our subcellular proteome provides a resource for future investigations into the unique cell biology of diplonemids.

Peroxisomes↗

PubMed1997

Subcellular localization of dihydropyrimidine dehydrogenase.

Although dihydropyrimidine dehydrogenase (DPD) has been purified and characterized from liver tissues of various mammals conflicting data exist on its subcellular localization. To determine the localization of DPD we prepared crude subcellular fractions of a rat liver homogenate by means of differential centrifugation. In the fractions obtained (heavy mitochondrial, light mitochondrial, microsomal and cytosolic) the activities of different marker enzymes were measured as well as the activity of DPD. These results showed that almost all of the activity of DPD was located in the cytosolic fraction. To exclude any particulate-associated DPD, a light mitochondrial fraction was subsequently subjected to equilibrium density gradient centrifugation. The distribution profile of the activity of DPD and the various marker enzymes indicated that DPD from rat liver was exclusively located in the cytosol since no significant activity of DPD could be detected in any subcellular organelle.

Animals↗

PubMed1989

On the subcellular localization of the polyamines.

Putrescine, spermidine and spermine were determined in the nuclear fraction of rat liver which was obtained by density gradient centrifugation in non-aqueous media, i.e. under conditions which avoid migration of water-soluble compounds. Calculations of the distribution of the polyamines between nuclear and extranuclear compartments were based on the assumption that the DNA is concentrated in the nuclei. No significant losses of the polyamines occurred during fractionation. From the polyamine determination in tissue and nuclear fraction it appeared that 16-17% of the liver spermidine and spermine, and about 8% of the putrescine content was localized in the nuclei. The spermidine/spermine-ratios in nuclei and whole tissue were not significantly different. Pretreatment of the animals with inhibitors of ornithine decarboxylase caused a decrease of putrescine exclusively in the extranuclear compartments, in agreement with a higher proportion of the inhibitors in the cytoplasm. Since the nuclear volume of rat liver corresponds to about 5% of total liver volume, the concentration of spermidine and spermine is higher in the nucleus than in extranuclear compartments. Published histochemical localizations of the polyamines suggested very low polyamine concentrations in the nuclei of non-dividing liver and HeLa cells, but dramatic polyamine accumulations in metaphase and anaphase nuclei. These results are in disagreement with previously reported autoradiographic data, subcellular localizations based on density gradient centrifugations, and with our present results. Since subcellular localization is a key issue in all attempts to clarify cellular functions of the polyamines the careful revision of the techniques involved in subcellular polyamine localizations seems imperative.

Adenosylmethionine Decarboxylase↗

PubMed2005

Heterogeneity in expression and subcellular localization of tight junction proteins, claudin-10 and -15, examined by RT-PCR and immunofluorescence microscopy.

Tight junctions regulate paracellular permeability, create the luminal fluid microenvironment of blood vessels and the digestive tract, and also form the protective barrier in the stratified epithelium including the epidermis. Claudins are the integral membrane proteins at tight junctions and form a multigene family composed of at least 24 members, but knowledge of the subcellular localization of each claudin is still fragmentary. We performed RT-PCR for fifteen claudin species to examine the mRNA expression in various mouse tissues, and focused on investigating the subcellular localization of claudin-10 and -15 by immunofluorescence microscopy in various rat tissues. Neither claudin-10 nor -15 was detected in vascular endothelial cells in most tissues, and these claudins were restricted to the vasa recta in the kidney medulla. Both claudins were also detected at apical tight junctions in the epithelium of the jejunum with no intensity gradients along the crypt-to-villus axis. However, both claudins were expressed only in the basal half of the crypt epithelium in the colon, showing obvious gradients along crypt-to-surface axis. Moreover, claudin-10 showed the ectopic subcellular localization where tight junction strands do not exist. Claudin-10 was detected along the entire lateral membranes of acinar cells in addition to the apical tight junctions in exocrine glands, and in the cytoplasm of basal cells in the stratified epithelium including the dorsal skin and cutaneous stomach. These heterogeneous distributions of claudin-10 and -15 in tissues may be related to the differences in paracellular permeability among tissues.

Animals↗

PubMed2004

Subcellular accumulation of polychlorinated biphenyls in the green alga Chlamydomonas reinhardtii.

In this study, the subcellular accumulation of 13 polychlorinated biphenyl (PCB) congeners was investigated in the green alga Chlamydomonas reinhardtii. Two main arguments pertaining to the mechanism of bioaccumulation of persistent bioaccumulative toxic chemicals (PBTs) in phytoplankton were evaluated, including the controversy of whether the limitation of uptake of ultra-hydrophobic pollutants is kinetic or due to a physical barrier presented by the membrane that prevents transfer into the internal parts of the cell, and second, the role of surface adsorption for the bioaccumulation process. The first argument was addressed by studying the time-dependent subcellular uptake of PCBs into thylakoids (photosynthetic membranes) as representative internal membranes. The second issue was addressed by investigating the role of the algal cell wall as a potential extracellular sorbate. Accumulation of PCBs in thylakoids was found to be limited kinetically and slow compared to total accumulation in the alga. Super-hydrophobic PCBs with Kow > 6 were not restricted from entering the cell. Sorption to the cell wall was found to be less than 10% and insignificant compared to total bioaccumulation in C. reinhardtii. To our knowledge, this is the first study in which the subcellular uptake of a class of PBTs was investigated directly. The results offer a mechanistic framework for improving kinetic modeling of PBT bioaccumulation in phytoplankton.

Adsorption↗

PubMed1993

Multiple defects in the adipocyte glucose transport system cause cellular insulin resistance in gestational diabetes. Heterogeneity in the number and a novel abnormality in subcellular localization of GLUT4 glucose transporters.

Mechanisms causing cellular insulin resistance in gestational diabetes mellitus are not known. We, therefore, studied isolated omental adipocytes obtained during elective cesarean sections in nondiabetic (control) and GDM gravidas. Cellular insulin resistance was attributed to impaired stimulation of glucose transport; compared with control subjects, basal and maximally insulin-stimulated transport rates (per surface area) were reduced 38 and 60% in GDM patients, respectively. To determine underlying mechanisms, we assessed the number, subcellular distribution, and translocation of GLUT4, the predominant insulin-responsive glucose transporter isoform. The cellular content of GLUT4 was decreased by 44% in GDM patients as assessed by immunoblot analysis of total postnuclear membranes. However, GDM patients segregated into two subgroups; half expected profound (76%) cellular depletion of GLUT4 and half had GLUT4 levels in the normal range. Cellular GLUT4 was negatively correlated with adipocyte size in the control subjects and GDM patients with normal GLUT4 (r = 0.60), but fell way below this continuum in GDM patients with low GLUT4, indicating that heterogeneity was not caused by differences in obesity. All GDM. distribution. In basal cells, increased amounts of GLUT4 were detected in membranes fractionating with (such that the plasma membrane GLUT4 level in GDM (such that the plasma membrane GLUT4 level in GDM patients was equal to that observed in insulin-stimulated cells from control subjects). Furthermore, insulin stimulation induced translocation of GLUT4 from low-density microsomes to plasma membranes in control subjects but did not alter subcellular distribution in GDM patients. In other experiments, cellular content of GLUT1 was normal in GDM patients, and GLUT1 did not undergo insulin-mediated recruitment to plasma membranes in either control subjects or GDM patients. A faint signal was detected for GLUT3 only in low-density microsomes and only with one of two different antibodies. In GDM, we conclude that insulin resistance in adipocytes involves impaired stimulation of glucose transport and arises from a heterogeneity of defects intrinsic to the glucose transport effector system. GLUT4 content in adipocytes is profoundly depleted in approximately 50% of GDM patients, whereas all patients are found to exhibit a novel abnormality in GLUT4 subcellular distribution. This latter defect is characterized by accumulation of GLUT4 in membranes cofractionating with plasma membranes and high-density microsomes in basal cells and absence of translocation in response to insulin. The data suggest that abnormalities in cellular traffic or targeting relegate GLUT4 to a membrane compartment from which insulin cannot recruit transporters to the cell surface and have important implications regarding skeletal muscle insulin resistance in GDM and NIDDM.

Adipocytes↗

PubMed1999

Human diabetes is associated with hyperreactivity of vascular smooth muscle cells due to altered subcellular Ca2+ distribution.

Alterations of vascular smooth muscle function have been implicated in the development of vascular complications and circulatory dysfunction in diabetes. However, little is known about changes in smooth muscle contractility and the intracellular mechanisms contributing to altered responsiveness of blood vessels of diabetic patients. Therefore, smooth muscle and endothelial cell function were assessed in 20 patients with diabetes and compared with 41 age-matched control subjects. In rings from uterine arteries, smooth muscle sensitivity to K+, norepinephrine (NE), and phenylephrine (PE) was enhanced by 1.4-, 2.3-, and 9.7-fold, respectively, and endothelium-dependent relaxation was reduced by 64% in diabetic patients, as compared with control subjects. In addition, in freshly isolated smooth muscle cells from diabetic patients, an increased perinuclear Ca2+ signaling to K+ (30 mmol/l >73%; 60 mmol/l >68%) and NE (300 nmol/l >86%; 10 micromol/l >67%) was found. In contrast, subplasmalemmal Ca2+ response, which favors smooth muscle relaxation caused by activation of Ca2+-activated K+ channels, was reduced by 38% in diabetic patients as compared with control subjects, indicating a significant change in the subcellular Ca2+ distribution in vascular smooth muscle cells in diabetic patients. In contrast to the altered Ca2+ signaling found in freshly isolated cells from diabetic patients, in cultured smooth muscle cells isolated from control subjects and diabetic patients, no difference in the intracellular Ca2+ signaling to stimulation with either K+ or NE was found. Furthermore, production of superoxide anion (*O2-) in intact and endothelium-denuded arteries from diabetic patients was increased by 150 and 136%, respectively. Incubation of freshly isolated smooth muscle cells from control subjects with the *O2- -generating system xanthine oxidase/hypoxanthine mimicked the effect of diabetic patients on subcellular Ca2+ distribution in a superoxide dismutase-sensitive manner. We conclude that in diabetic subjects, smooth muscle reactivity is increased because of changes in subcellular Ca2+ distribution on cell activation. Increased *O2- production may play a crucial role in the alteration of smooth muscle function.

Arteries↗