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H F Lodish

Publications and source records attributed to H F Lodish.

At least 163 records · Page 9Linked to original sources
PubMed1991

Decreased in vivo glucose uptake but normal expression of GLUT1 and GLUT4 in skeletal muscle of diabetic rats.

This study was designed to determine whether altered glucose transporter expression is essential for the in vivo insulin-resistant glucose uptake characteristic of streptozocin-induced diabetes. Immunofluorescence in rat skeletal muscle colocalizes GLUT4 with dystrophin, both intrinsic to muscle fibers. In contrast, GLUT1 is extrinsic to muscle fibers, probably in perineurial sheath. Immunoblotting shows that levels of GLUT1 and GLUT4 protein per DNA in hindlimb muscle are unaltered from control levels at 7 d of diabetes but decrease to approximately 20% of control at 14 d of diabetes. This decrease is prevented by insulin treatment. In adipose cells of 7 d diabetic rats, GLUT4 levels are depressed. Thus, GLUT4 undergoes tissue-specific regulation in response to diabetes. GLUT4 and GLUT1 mRNA levels in muscle are decreased 62-70% at both 7 and 14 d of diabetes and are restored by insulin treatment. At 7 d of diabetes, when GLUT4 protein levels in muscle are unaltered, in vivo insulin-stimulated glucose uptake measured by euglycemic clamp is 54% of control. This reflects impairment in both glycogen synthesis and glycolysis and the substrate common to these two pathways, glucose-6-phosphate, is decreased approximately 30% in muscle of diabetic rats. These findings suggest a defect early in the pathway of glucose utilization, probably at the step of glucose transport. Because GLUT1 and GLUT4 levels are unaltered at 7 d of diabetes, reduced glucose uptake in muscle probably reflects impaired glucose transporter translocation or intrinsic activity. Later, at 14 d of diabetes, GLUT1 and GLUT4 protein levels are reduced, suggesting that sequential defects may contribute to the insulin-resistant glucose transport characteristic of diabetes.

Amino Acid Sequence↗
PubMed1991

Expression of the low Km GLUT-1 glucose transporter is turned on in perivenous hepatocytes of insulin-deficient diabetic rats.

In normal fed rats the low Km glucose transporter GLUT-1 is expressed only in one row of hepatocytes immediately surrounding a terminal hepatic venule, while the high Km GLUT-2 is expressed in every hepatocyte. Previously, we showed that additional perivenous hepatocytes express GLUT-1 in fasting animals. In diabetes, as in starvation, the liver functions to release glucose into the circulation, but unlike starvation, circulating extracellular glucose is high in diabetes. By immunofluorescence and Western blotting we studied whether glucose or insulin is the primary extracellular signal for inducing GLUT-1 expression in hepatocytes. We observed that streptozocin-induced diabetes causes induction of GLUT-1 expression in the plasma membrane of hepatocytes within four cell rows of a terminal hepatic venule; GLUT-2 expression is unaltered. Chronic insulin treatment of diabetic rats reduces the number of rows of hepatocytes expressing GLUT-1 from approximately four to approximately two. In contrast, chronic insulin infusion into nondiabetic rats does not affect the number of hepatocytes expressing GLUT-1. Thus, both fasting and diabetes induce GLUT-1 expression in specific hepatocytes that normally do not express this gene. This induction correlates with low insulin levels in the blood, and not with circulating glucose levels.

Animals↗
PubMed1990

Point mutation in the exoplasmic domain of the erythropoietin receptor resulting in hormone-independent activation and tumorigenicity.

The receptors for erythropoietin and other cytokines constitute a new superfamily. They have no tyrosine-kinase or other enzyme motif and their signal-transducing mechanism is unclear. Here we describe two classes of activating mutations in the erythropoietin receptor (EPOR). A single point mutation in the exoplasmic domain enables it to induce hormone-independent cell growth and tumorigenesis after expression in nontumorigenic, interleukin-3-dependent haematopoietic cells. A C-terminal truncation in the cytoplasmic domain of the EPOR renders the receptor hyperresponsive to erythropoietin, but is insufficient to induce hormone-independent growth or tumorigenicity. The activating point mutation retards intracellular transport and turnover of the receptor. These alterations in metabolism and tumorigenicity caused by the EPOR with activating point mutations are similar to those observed in erythropoietin-independent activation of the wild type EPOR by association with gp55, the Friend spleen focus-forming virus glycoprotein.

Amino Acid Sequence↗
PubMed1990

Isolation, characterization, and expression of cDNA encoding a rat liver endoplasmic reticulum alpha-mannosidase.

We have isolated a cDNA encoding an endoplasmic reticulum alpha-mannosidase, an asparagine-linked oligosaccharide processing enzyme, from a rat liver lambda gt11 library. Two degenerate oligonucleotides, based on amino acid sequence data from the purified enzyme, were used as primers in the polymerase chain reaction with liver cDNA as a template to generate an unambiguous cDNA probe. The cDNA fragment (524 base pair) obtained was then used to isolate cDNA clones by hybridization. We isolated two overlapping clones which were used to construct a full-length cDNA of 3392 base pairs. A single open reading frame of 1040 amino acids encodes a protein with a molecular mass of 116 kilodaltons containing the six known peptide sequences. The deduced amino acid sequence revealed no classical signal sequence or membrane-spanning domain. The alpha-mannosidase encoding cDNA can be expressed transiently in COS cells using the mammalian expression vector pXM, causing a 400-fold increase in alpha-mannosidase activity as well as a dramatic increase in immunoreactive polypeptide. The rat liver endoplasmic reticulum alpha-mannosidase bears striking homology to the vacuolar alpha-mannosidase from Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1990

Perturbation of cellular calcium blocks exit of secretory proteins from the rough endoplasmic reticulum.

In the cultured human hepatoma HepG2, Ca2+ ionophores block secretion of different secretary proteins to different extents, alpha 1-antitrypsin secretion being more sensitive to A23187 and ionomycin than is alpha 1-antichymotrypsin, and albumin secretion the least of the three proteins studied. As judged by subcellular fractionation experiments and by treatment of pulse chase labeled protein with endoglycosidase H, A23187 and ionomycin cause newly made secretory proteins to remain within the rough endoplasmic reticulum (ER). Experiments in which A23187 is added at different times during a pulse or chase show that secretion of newly made alpha 1-antitrypsin becomes resistant to the ionophore, on average, 15 min after synthesis; this is about 20 min before it reaches the trans-Golgi, and while it is still within the rough ER. We speculate that a high concentration of Ca2+ within the ER may be essential for certain secretory proteins to fold properly, that folding is inhibited when ER Ca2+ levels are lowered by ionophore treatment, and that unfolded proteins, particularly alpha 1-antitrypsin, cannot exit the rough ER. Treatment of murine 3T3 fibroblasts or human hepatoma HepG2 cells with the Ca2+ ionophores A23187 or ionomycin also induces a severalfold accumulation of the ER lumenal protein Bip (Grp78). These findings disagree with a recent report that Ca2+ ionophores cause secretion of Bip and other resident ER proteins, but is consistent with other reports that A23187 causes accumulation of mRNAs for Bip and other ER lumenal proteins.

Acetylglucosaminidase↗
PubMed1990

The high Km glucose transporter of islets of Langerhans is functionally similar to the low affinity transporter of liver and has an identical primary sequence.

The liver has been shown to contain a facilitated diffusion glucose transporter with high Km for glucose that is structurally distinct from the low Km glucose transporters found in most other tissues. We find that 3-O-methyl glucose is greater than 90% equilibrated across dispersed islet cells within 60 s, consistent with a facilitated diffusion transport mechanism. L-Glucose uptake was minimal throughout the time course, indicating stereospecificity. Measurement of glucose transport over a range of 3-O-methyl glucose concentrations from 0.05 to 60 mM revealed the presence of a component of glucose transport with an apparent Km of 17 mM, a value essentially identical to that previously reported for liver. Interestingly, a second component of glucose transport was also observed with an apparent Km of 1.4 mM, as has been reported for other tissues such as erythrocytes that are known to contain the "HepG2" or "erythroid/brain" type glucose transporter. Further evidence for the existence of two transport components is provided by the observation that a low concentration of cytochalasin B (0.4 microM) completely inhibits the low Km transport activity but has no effect on the high Km transporter. The kinetic similarity of high Km glucose transport in liver and islets is readily understood in light of our structural analysis. Sequence analysis of cDNA clones indicates that the liver and islet glucose transporters have identical sequences and, thus, are the products of the same gene.

3-O-Methylglucose↗
PubMed1990

Activation of cell growth by binding of Friend spleen focus-forming virus gp55 glycoprotein to the erythropoietin receptor.

Friend spleen focus-forming virus (SFFV) is a defective murine C-type retrovirus which causes a multi-stage erythroleukaemia in mice and erythroblastosis in bone marrow cultures. The SFFV env gene encodes a membrane glycoprotein, gp55, which is located on the cell surface and in the rough endoplasmic reticulum and is essential both for the induction of leukaemia in vivo and erythroblast proliferation in vitro. The mechanism by which gp55 causes increased erythroblastosis and ultimately leukaemia is unknown, but a reasonable suggestion is that gp55 can mimic the action of erythropoietin by binding to its receptor (Epo-R), thereby triggering prolonged proliferation of erythroid cells. To test this possibility, we have co-expressed gp55 and the murine Epo-R in a fibroblast cell line. We show here that in such cells, the SFFV glycoprotein binds directly to Epo-R. Furthermore, when an interleukin-3 (IL-3)-dependent lymphoid cell line was co-infected by SFFV and a virus that carries the Epo-R gene, it could grow without IL-3. We suggest that through direct binding to Epo-R, gp55 can stimulate the receptor and by-pass the normal requirement for Epo, causing prolonged proliferation of infected erythroid cells. This could be the first step of leukaemogenesis induced by Friend virus.

Animals↗
PubMed1990

Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies.

We have generated four high affinity monoclonal antibodies (MoAbs) to recombinant human erythropoietin (EPO). All four MoAbs immunoprecipitate radioiodinated native EPO, and the concentrations of MoAbs required for maximum binding range from 10 nmol/L to 100 nmol/L. Two MoAbs, designated Group I MoAbs, bind to an epitope within the N-terminal 20 amino acids of EPO and also immunoprecipitate sodium dodecyl sulfate (SDS)-denatured EPO. Two other MoAbs (Group II MoAbs) do not immunoprecipitate SDS-denatured EPO and do not bind to any of the eight endo C fragments of EPO. We first used murine erythroleukemia (MEL) cells to test the MoAbs for inhibition of EPO-receptor binding. MEL cells, although unresponsive to EPO, express 760 high affinity receptors for EPO per cell (Kd = 0.24 nmol/L). To assay our MoAbs, MEL cells were grown as monolayers on fibronectin-coated Petri dishes and incubated at 4 degrees C with radioiodinated EPO. Group I MoAbs do not inhibit binding of radioiodinated EPO to the MEL EPO-receptor, but Group II MoAbs do inhibit binding in a dose-dependent manner. We next examined the neutralization of EPO bioactivity by our MoAbs, using EPO-dependent cell line. Only Group II MoAbs inhibit a newly developed EPO-dependent cell growth, demonstrating that inhibition of EPO-receptor binding correlates with neutralization of EPO bioactivity.

Animals↗
PubMed1990

Multiple episodes of induced secretion of human growth hormone from recombinant AtT-20 cells.

Recombinant AtT-20 cells expressing human growth hormone (hGH) secreted the hormone at a constant, basal rate of 0.3-0.5 ng/10(5) cells-hour when exposed to medium without secretagogues. When triggered with 8 bromo-cyclic AMP, cells secreted hGH at an initial rate of 1.7 ng/10(5) cells-hour while intracellular hGH declined sharply. Upon extended exposure to secretagogue, secretion decreased gradually to the basal rate and intracellular hGH stabilized at a value 40% the initial. In cells switched from secretion to growth medium, the total rate of hGH accumulation intracellularly and in medium was 2.2 times that observed with cells never exposed to secretagogue; however, only a fraction of the hormone was stored intracellularly and the rest was secreted. When cells were exposed alternately to growth and secretion medium, induced cells secreted at rates at least two times higher than uninduced controls during the first five cycles. The induced response deteriorated with time, however, in parallel with outgrowth of attached cells by foci of round cells, and by the eighth cycle induced secretion did not occur. Operational modifications that may improve the performance of cycling schemes are discussed.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Friend spleen focus-forming virus glycoprotein gp55 interacts with the erythropoietin receptor in the endoplasmic reticulum and affects receptor metabolism.

The Friend spleen focus-forming virus envelope glycoprotein, gp55, binds to the murine erythropoietin receptor (EPO-R) and triggers growth activation in the absence of EPO. Interleukin 3-dependent lymphoid cell lines that have been stably transfected with the EPO-R cDNA grow in the presence of EPO or interleukin 3. In these cells, the EPO-R is synthesized as a minor 62-kDa unglycosylated form and a major 64-kDa form carrying one high-mannose N-linked oligosaccharide. A fraction of the 64-kDa form is processed to a 66-kDa species with complex-type sugars. Very little of the EPO-R is expressed on the cell surface and all three forms of EPO-R are degraded rapidly. Cells transfected with both EPO-R and gp55 cDNAs grow in the absence of EPO. Most of the EPO-R associated with gp55 is endoglycosidase H-sensitive, suggesting that the interactions between these proteins occur in the endoplasmic reticulum. Furthermore, the endoglycosidase H-sensitive EPO-R is more stable than in the absence of gp55, a result suggesting that interaction of gp55 with the EPO-R causes it to remain within the rough endoplasmic reticulum. It is possible that gp55 EPO-R complexes within this compartment send a growth-promoting signal to the cell.

Animals↗
PubMed1990

Reduced expression of the liver/beta-cell glucose transporter isoform in glucose-insensitive pancreatic beta cells of diabetic rats.

Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.

Animals↗
PubMed1990

Oligomeric structure of the human asialoglycoprotein receptor: nature and stoichiometry of mutual complexes containing H1 and H2 polypeptides assessed by fluorescence photobleaching recovery.

The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein receptor (ASGP-R), were investigated by immunofluorescence and lateral mobility measurements combined with antibody-mediated cross-linking and immobilization. Immunofluorescence microscopy revealed two ASGP-R populations on the cell surface, one homogeneously distributed and the other in micropatches. This was observed both in stably transfected NIH 3T3 lines expressing H1 and/or H2, and in the human hepatoma cell line HepG2. In transfected cells expressing both polypeptides (the 1-7-1 line), H1 and H2 were colocalized in the same micro aggregates. Moreover, enhancement of the patching of, e.g., H1 by IgG-mediated crosslinking was accompanied by copatching of H2. To quantify H1-H2 complex formation, the lateral diffusion of H1 and H2 was measured at 12 degrees C (to avoid internalization) by fluorescence photobleaching recovery. H1 (or H2) was immobilized by crosslinking with specific IgG molecules; the other chain was labeled with fluorescent monovalent Fab' fragments, and is lateral mobility was measured. In HepG2 cells, immobilization of either H1 or H2 led to an equal immobilization of the other, indicating that all the mobile H1 and H2 are in stable heterooligomers. In 1-7-1 cells, immobilization of H2 immobilized H1 to the same degree, but immobilization of H1 reduced the mobile fraction of H2 only by 2/3. Thus, in 1-7-1 cells all surface H1 molecules are associated with H2, but 1/3 of the H2 population is independent of H1. From these data and from measurements of the relative surface densities of H1 and H2, conclusions are drawn regarding the oligomeric structure and stoichiometry of the ASGP-R.

Animals↗
PubMed1990

Structure and transcription of the mouse erythropoietin receptor gene.

The complete gene encoding the mouse erythropoietin receptor was isolated by using a cDNA probe derived from a mouse erythroleukemia (MEL) cell library. The gene spans approximately 5 kilobases and is present in a single copy per haploid genome. It contains eight exons, and the nucleotide sequence of the coding region from the genomic DNA is identical to the sequence of one of the MEL cDNA clones except for a single amino acid substitution (Leu----Val) at codon 163. There is a cluster of three major transcriptional start sites approximately 150 nucleotides upstream of the initiator ATG codon which is conserved in erythropoietin-dependent and -independent erythroleukemic cells, in MEL cells at different stages of differentiation, and in normal bone marrow cells. The promoter region contains a potential binding site for Sp1, erythroid-specific transcription factor GF-1, and several CACCC boxes, but not typical TATA or CAAT sequences. A fusion gene containing 452 nucleotides of 5' noncoding sequence linked to a promoterless human growth hormone gene directed the transcription of the latter in MEL cells but not in mouse fibroblasts, T cells, B cells, or macrophagelike cells, suggesting that this promoter functions in an erythroid-specific manner.

Animals↗
PubMed1990

Liver glucose transporter: a basolateral protein in hepatocytes and intestine and kidney cells.

The "liver" isoform of the facilitated diffusion glucose transporter is expressed predominantly in liver, intestine, kidney, and pancreatic islet beta-cells. The apparent molecular mass of the transporter in liver, kidney, and intestine is different, as detected by Western blot analysis of membrane proteins using antipeptide antibodies. However, as assessed by Northern blot analysis and molecular cloning, the same mRNA is expressed in these tissues, indicating that there are tissue-specific posttranslational modifications of the same transporter polypeptide. As determined by immunofluorescence analysis on frozen tissue sections, the liver glucose transporter is present on the sinusoidal membrane of hepatocytes, on the basolateral membrane of fully differentiated absorptive intestine epithelial cells, and on the basolateral membrane of proximal tubule cells of the kidney nephron. This localization is consistent with the involvement of the liver glucose transporter in several key steps of glucose metabolism: glucose uptake and release by the liver and absorption or reabsorption by epithelial cells of the intestine and kidney, respectively.

Animals↗
PubMed1990

Differential localization of two glucose transporter isoforms in rat kidney.

The localization of two glucose transporter isoforms was mapped in the rat kidney: the high-Michaelis constant (Km; 15-20 mM) low-affinity "liver" transporter and the low-Km (1-2 mM) high-affinity "erythroid/brain" transporter. Both are basolateral membrane proteins, but the liver transporter was present exclusively in the S1 part of the proximal tubule, whereas the erythroid/brain transporter was expressed at variable levels in different nephron segments. Staining intensity was low in the straight proximal tubule (S3), intermediate in the medullary thin and thick ascending limbs, and highest in connecting segments and collecting ducts. In the collecting duct, the erythroid/brain glucose transporter was expressed at the highest level in intercalated cells; less was present in principal cells. In the papilla, only intercalated cells expressed this transporter isoform. These results suggest specific involvements of each transporter isoform in transepithelial glucose reabsorption by different segments of the proximal tubule. They also indicate that while the liver glucose transporter is present in gluconeogenic cells, there is a good correlation between the level of expression of the erythroid/brain glucose transporter and the glycolytic activity of the different nephron segments.

Animals↗