ABSTRACT
Acylation-stimulating protein (ASP), a human plasma protein, is a potent stimulator of triglyceride synthesis and glucose transport in both human adipocytes and fibroblasts. The purpose of the present in vitro study was to examine the effect of ASP on glucose transport in muscle cells. ASP stimulated 2-deoxy-glucose transport (2-DG) in differentiated rat L6 myotubes in a time (30 min to 24 h) and concentration dependent manner (97% increase). The magnitude of the ASP effect on glucose transport was comparable to the time- and concentration-dependent effects seen with insulin (125% increase), but was additive to insulin, pointing to involvement of differential signalling pathways. ASP stimulation was dependent on cell differentiation in that glucose transport increased by only 12% in myoblasts, comparable to the effect of insulin in myoblasts (15% increase) demonstrating selective responsiveness of the differentiated myotubes to ASP and insulin. The mechanism for the ASP induced increase in glucose transport was also examined. ASP increased the Vmax for 2-DG transport by 183% (4.02 vs. 1.42 nmol/mg cell protein/30 s; ASP vs. Control, respectively). This could be explained by an increased translocation of glucose transporters (GLUT 1, GLUT 4 and GLUT 3) to the plasma membrane surface as demonstrated by Western analysis (+43% P < 0.05, +30% P < 0.05, and +49% P < 0.05, respectively). The effects of ASP were equal to those of insulin (+47%, +26% and +53% for GLUT 1, GLUT 4 and GLUT 3, respectively) and in all cases were paralleled by comparable glucose transport increases under the same incubation conditions. After long-term stimulation (24 h), Western analysis indicated that ASP had a permissive effect on insulin stimulated increases in total GLUT3 and GLUT4 cellular transporter content. These results suggest that muscle is also responsive to ASP and that ASP may play a role in glucose metabolism in both muscle and adipose tissue.
Subject(s)
Blood Proteins/pharmacology , Complement C3a/analogs & derivatives , Glucose/metabolism , Muscle Proteins , Muscles/metabolism , Nerve Tissue Proteins , Animals , Biological Transport , Cell Differentiation , Cell Line , Cell Membrane/metabolism , Deoxyglucose/metabolism , Glucose Transporter Type 1 , Glucose Transporter Type 3 , Glucose Transporter Type 4 , Insulin/pharmacology , Kinetics , Monosaccharide Transport Proteins/metabolism , Muscles/cytology , RatsABSTRACT
In this study, sugar transport and the cellular content of the human Glut 1 and 3 glucose transporters were ascertained in uninfected and chronically HIV-infected Jurkat and H9 cell lines (T-cell lines) and U937 cells (a promonocytic cell line). Sugar transport was determined by monitoring 2-deoxy glucose uptake (2DG) and glut transporter content was determined by Western analysis. Although 'acute' HIV infection of H9 cells led to increased cellular transport activity and Glut 3 transporter content, chronic HIV infection exhibited no significant differences in sugar transport in any of the cell types investigated whether log or stationary phase cultures were employed. When uninfected and chronically HIV-infected cell lines were compared, all cell lines expressed the Glut 1 transporter, however, significant differences in Glut 1 transporter content were not observed. The Glut 3 transporter which could only be detected in the H9 cell line exhibited no differences in Glut 3 content in uninfected or chronically HIV-infected cells (2.1 +/- 0.6 versus 3.8 +/- 2.1 x 10(-3) arbitrary units/microgram protein). A trend towards lower amino acid uptake was seen in the chronically HIV-infected cells but this was not significantly different from uninfected cell cultures. The data indicate that: (1) glucose transport and the Glut 1 and 3 transporters are not increased in cells chronically infected with HIV-1 and (2) the expression of the Glut 3 sugar transporters is not the same in all target cells.
Subject(s)
Carbohydrate Metabolism , HIV Infections/metabolism , HIV-1 , Monosaccharide Transport Proteins/metabolism , Nerve Tissue Proteins , Biological Transport, Active , Cell Line , Deoxyglucose/metabolism , Glucose Transporter Type 1 , Glucose Transporter Type 3 , Humans , Jurkat Cells , U937 CellsABSTRACT
Insulin-like growth factor 1 and insulin, considered primarily as metabolic and growth modulatory hormones, were found to inhibit the replication of HIV-1 in cultured cord blood mononuclear cells and chronically HIV-infected U937 cells. The effect of IGF-1 was seen at physiological concentrations or lower (1.7 x 10(-10) M) while that of insulin was observed at supraphysiological concentrations (8 x 10(-7) M). The EC50 for IGF-1 was found to be in the physiological range (2.5-4.5 x 10(-9) M) while that for insulin was considerably higher (1.1-3.3 x 10(-6) M). Insulin-like growth factor 1 and insulin at the concentrations employed exhibited no toxicity on the cells used in these studies. Furthermore, neither IGF-1 nor insulin exhibited any inhibitory activity on purified reverse transcriptase in vitro. Epidermal growth factor from 1.6 x 10(-10) to 1.6 x 10(-8) M demonstrated no inhibition of HIV-1 replication, while IGF-1 inhibited p24 antigen production 49 and 42% at 1.3 x 10(-9) and 1.3 x 10(-8) M IGF-1, respectively. These results suggest that IGF-1 under certain conditions has significant inhibitory effects on HIV-1 replication at physiological concentrations. This may prove to be of therapeutic value in patients infected with HIV-1.
Subject(s)
HIV-1/drug effects , Insulin-Like Growth Factor I/pharmacology , Insulin/pharmacology , Virus Replication/drug effects , Cell Division/drug effects , Cell Survival/drug effects , Cells, Cultured , Fetal Blood , HIV-1/physiology , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/virologyABSTRACT
The objective of this study was to investigate the long-term effects of anti-retroviral protease inhibitors (PIs) on 2-deoxy-d -glucose (2-DG) transport in L6 cells in vitro. Exposure of L6 cells to saquinavir, ritonavir, indinavir and amprenavir resulted in significant increases in 2-DG transport using PI concentrations of 1-10 microM with continual exposure to PI. After removal of the PI for up to 48 h, 2-DG transport increases did not change and remained at pre-reversal levels. These changes in 2-DG transport were not related to stress-induced sugar transport or to apoptosis. The examination of glucose transporter (GLUT) 1, 3 or 4 translocation with subcellular fractionation indicated that insulin (i.e. 67 nM) could induce the translocation of all the GLUTs to the plasma membrane. Also, ritonavir (10 microM), which leads to a 2-fold increase in 2-DG transport, demonstrated increased GLUT (i.e. 1, 3 or 4) presence in the plasma membrane fraction, in the presence or absence of insulin. This increased 2-DG transport involved transporter presence in plasma membrane preparations and did not affect the ability of insulin to stimulate 2-DG transport with continual PI exposure. The mechanism(s) involved indicates ready reversibility of PI effects on transporters. The mechanism(s) why reversibility of PI-induced 2-DG transport was similar plus or minus PI was not apparent.
Subject(s)
Deoxyglucose/metabolism , HIV Protease Inhibitors/pharmacology , Muscle Proteins , Myoblasts/metabolism , Nerve Tissue Proteins , Biological Transport/drug effects , Blotting, Western/methods , Carbamates , Cell Membrane/metabolism , Cells, Cultured , Furans , Glucose Transporter Type 1 , Glucose Transporter Type 3 , Glucose Transporter Type 4 , Humans , Indinavir/pharmacology , Insulin/pharmacology , Monosaccharide Transport Proteins/metabolism , Myoblasts/drug effects , Ritonavir/pharmacology , Saquinavir/pharmacology , Sulfonamides/pharmacologyABSTRACT
In this study, we have investigated the ability of insulin-like growth factor I (IGF-I) to inhibit HIV long terminal repeat (LTR)-driven gene expression. Using COS 7 cells cotransfected with tat and an HIV LTR linked to a chloramphenicol acetyltransferase (CAT) reporter, we observed that physiological levels of IGF-I (10(-9) M) significantly inhibited CAT expression in a concentration- and time-dependent manner. IGF-I did not inhibit CAT expression in COS 7 cells transfected with pSVCAT, and did not affect CAT expression in the absence of cotransfection with tat. Transfection of HIV-1 proviral DNA into COS 7 cells +/- IGF-I resulted in a significant decrease (p < 0.05) in infectious virion production. Both IGF-I and Ro24-7429 inhibited LTR-driven CAT expression, while TNF-alpha-enhanced CAT expression was not affected by IGF-I. On the other hand, a plasmid encoding parathyroid hormone-related peptide exhibited dramatic additivity of inhibition of CAT expression in COS 7 cells. Finally, we show that in Jurkat or U937 cells cotransfected with HIVLTRCAT/tat, IGF-I significantly inhibited CAT expression. Further, interleukin 4 showed in U937 cells inhibition of CAT expression that was not additive to IGF-I induced inhibition. Our data demonstrate that IGF-I can specifically inhibit HIVLTRCAT expression. This inhibition may occur at the level of the tat/TAR interaction. Finally, this IGF-I effect is seen in target cell lines and similar paths of inhibition may be involved in the various cell types employed.
Subject(s)
Gene Expression Regulation, Viral , HIV Long Terminal Repeat/genetics , HIV-1/genetics , Insulin-Like Growth Factor I/metabolism , Pyrroles , Animals , Anti-HIV Agents/pharmacology , Benzodiazepines/pharmacology , COS Cells , Chloramphenicol O-Acetyltransferase/genetics , Gene Expression Regulation, Viral/drug effects , Gene Products, tat/genetics , Gene Products, tat/metabolism , Genes, Reporter , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , Humans , Insulin-Like Growth Factor I/pharmacology , Interleukin-4/metabolism , Interleukin-4/pharmacology , Jurkat Cells , Parathyroid Hormone-Related Protein , Proteins/metabolism , Proteins/pharmacology , U937 Cells , tat Gene Products, Human Immunodeficiency VirusABSTRACT
Using a standard radio-receptor assay, we have demonstrated that [125I]insulin can bind specifically to each of two types of purified enveloped viruses, influenza A virus and Rous sarcoma virus. A non-enveloped icosahedral virus (echovirus 11) and herpes simplex virus type 2, which acquires its envelope from the nuclear membrane of the cell, did not possess insulin receptor activity. Displacement of specifically bound radiolabelled insulin from the viral surface was achieved by addition of an excess of unlabelled insulin but not by addition of another unrelated protein, cytochrome C. We conclude that certain types of enveloped viruses may acquire insulin binding sites from the plasma membrane of their host cell.
Subject(s)
Avian Sarcoma Viruses/metabolism , Influenza A virus/metabolism , Receptor, Insulin/metabolism , Animals , Autoimmune Diseases/etiology , Binding Sites , Cell Membrane/metabolism , Cells, Cultured , Diabetes Mellitus, Type 1/etiology , Enterovirus B, Human/metabolism , Humans , Insulin/metabolism , Receptor, Insulin/immunology , Simplexvirus/metabolismABSTRACT
Acylation-stimulating protein (ASP) is the most potent recognized stimulant of triacylglycerol synthesis in human adipocytes. However, its mechanism(s) of action have not yet been elucidated in detail. The present study examines the effects of ASP on membrane transport of glucose and fatty acids in cultured human skin fibroblasts. The data demonstrate that ASP stimulated carrier-mediated glucose transport in a time- and concentration-dependent manner, an effect that was greater than and independent of that observed with insulin. ASP increased the Vmax for glucose transport with no change in the transport Km, suggesting that ASP might result in increased sugar transporters in the plasma membrane. This finding was supported by quantitative Western blot analyses using a monoclonal antibody (G3) that demonstrated an increase in the plasma membrane content of the glucose transporter (Glut 1) with a concomitant decrease in the glucose transporter content of an intracellular membrane fraction. By contrast, ASP had no effect on specific membrane transport of fatty acids. Maximal effects of ASP on triacylglycerol synthesis were demonstrated at saturating levels of both glucose and oleate. Comparable stimulation by ASP in the absence of glucose (with or without pyruvate) was also demonstrated, although the absolute rates of triacylglycerol synthesis were substantially lower. Finally, it was shown that ASP increased the apparent Vmax for triglyceride synthesis without changing its Km. Since ASP will act in the absence of extracellular glucose, ASP must have additional actions, independent of its effect on specific membrane transport of glucose by which it accelerates intracellular triglyceride synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Blood Proteins/pharmacology , Complement C3a/analogs & derivatives , Glucose/metabolism , Triglycerides/biosynthesis , Biological Transport/drug effects , Cells, Cultured , Deoxyglucose/metabolism , Fatty Acids/metabolism , Fibroblasts/metabolism , Glucose/pharmacology , Humans , Insulin/pharmacology , Oleic Acid , Oleic Acids/pharmacologyABSTRACT
We investigated the mechanism by which glucose refeeding can reverse the enhancement of glycolysis caused by glucose starvation. Human fibroblasts were deprived of glucose for 18 hr and then refed for 1 hr with either (a) medium from sister glucose-starved cultures (controls), (b) fresh, glucose-containing medium (fresh medium), or (c) medium conditioned for 18 hr by glucose-fed cells (conditioned medium). Despite a lower glucose content, conditioned medium was significantly more effective at inhibiting the accumulation of radio-labeled glucose than fresh medium (74 vs. 49% inhibition). The uptake of 2-deoxyglucose was not affected by either medium, indicating that the site of control of glycolysis was distal to glucose transport and phosphorylation. The active principle was heat labile, dialyzable (Mr less than 12,000) and unrelated to the lactate content of conditioned medium. Medium conditioned by cells exposed to 3-O-methylglucose did not inhibit glycolysis in glucose-starved cells even though long-term exposure to this hexose, like glucose, results in the repression of transport.
Subject(s)
Glycolysis , Culture Media , Deoxyglucose/metabolism , Dialysis , Fibroblasts/metabolism , Glucose/metabolism , Hexoses/metabolism , Hot Temperature , HumansABSTRACT
Under conditions employed in our laboratory, tumors which are induced by avian sarcoma virus (ASV) usually grow progressively for several weeks and then regress. In order to further understand the basis for tumor regression in this model, we compared avian sarcoma cells which were cultured from tumors at different stages of development in terms of various phenotypic properties. The results indicate that tumor cells which are derived from progressively-growing sarcomas are rapidly growing, produce large quantities of the enzyme plasminogen activator, and have much in common generally with chicken embryo fibroblast (CEF) cells that have been transformed by ASV. In contrast, tumor cells that are obtained from regressors have elevated levels of hexose transport, grow very slowly, are greatly enlarged and display properties that are characteristic of senescent cells in culture.
Subject(s)
Sarcoma, Avian/pathology , Animals , Avian Sarcoma Viruses , Cell Division , Cell Transformation, Neoplastic , Cell Transformation, Viral , Chick Embryo , Chickens , DNA, Neoplasm/metabolism , Deoxyglucose/metabolism , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Male , Neoplasm Proteins/metabolism , Neoplasm Regression, Spontaneous , Phenotype , Plasminogen Activators/metabolism , Sarcoma, Avian/metabolismABSTRACT
The replicative ability of variously 'aged' cell cultures, their insulin binding and biological responsiveness under control and glucocorticoid (i.e. hydrocortisone) amplified conditions have been studied in human fibroblast cultures. Insulin stimulation of DNA synthesis in early and late passage cultures and in cultures from young and old donors showed no age-related difference in insulin responsiveness. Hydrocortisone amplification of insulin-stimulated DNA synthesis in early and late passage cells expressed no age-related differences. Hydrocortisone affected basal DNA synthesis in cultures from in vivo young and old donors differently. Additionally, hydrocortisone amplified insulin-stimulated DNA synthesis in young donor cell cultures was observed to be higher than in old donor cell cultures. Specific 125I-insulin binding was increased by hydrocortisone in both early and late passage cultures and in cultures from young and old donors but no age-related differences in 125I-insulin binding were observed in the presence or absence of hydrocortisone. The data suggest that an age-related loss of an insulin postreceptor interaction during hydrocortisone amplification of insulin-stimulated DNA synthesis is being expressed in the cultures from old donors.
Subject(s)
Aging , DNA/biosynthesis , Hydrocortisone/pharmacology , Insulin/pharmacology , Skin/metabolism , Adult , Aged , Cell Division , Cell Survival , Cells, Cultured , Child , Child, Preschool , Female , Fibroblasts/metabolism , Humans , Insulin/metabolism , Male , Middle AgedABSTRACT
We have constructed an insulin-diphtheria hormono-toxin which migrates as a single 29 kd band on 10% SDS polyacrylamide gel electrophoresis. This corresponds to a one to one molar ratio of the diphtheria A-chain (23 kDa) and insulin (6 kDa) molecules. The diphtheria A-chain: insulin (DTaI) hormono-toxin demonstrates cytotoxicity in V-79 Chinese hamster cells exhibiting an LD50 of 1.1 x 10(-8) M, which is 22 x more potent than whole diphtheria toxin. Also, DTaI can competitively displace [125I]-insulin with an ED50 of 1.1 x 10(-8) M, which is identical to the ED50 of insulin (1.1 x 10(-8) M) and showed limited cross-reactivity with the IGF-1 receptor (12% displacement of [125I]-IGF-1 with a DTaI concentration of 1.1 x 10(-8) M). We have used DTaI to select conjugate-resistant clones from the V-79 Chinese hamster fibroblast parental cell line. Conjugate-resistant variants expressed insulin binding levels ranging from 8.0 +/- 2.0 fmoles/mg protein down to 3.6 +/- 0.5 fmoles/mg protein while insulin binding in the V-79 parental cell line was 11.2 +/- 0.2 fmoles/mg protein. Additionally, a number of conjugate resistant clones expressed variable ability to grow in medium containing 5% serum. The altered ability of these clones to grow in a serum-containing medium did not correlate directly with the changes observed for insulin binding. One mutant, IV-A1-j, did not grow in a serum-free defined medium containing insulin as the predominant mitogen. This IV-A1-j mutant had a lower number of insulin receptors, no change in insulin binding affinity, no change in the rate of internalization of [125I]-insulin and no apparent difference in [125I]-IGF-1 binding. Further, insulin-stimulated sugar transport was similar to that observed in the parental cell line. Based on these observations we suggest that 1) DTaI elicits its cytotoxicological effects through the insulin receptor trafficking pathway, 2) DTaI can be used to isolate cells altered at the level of insulin binding and/or action, and 3) signal transduction mechanisms responsible for mediating insulin-dependent cell growth can be pursued using mutants such as IV-A1-j.
Subject(s)
Diphtheria Toxin/toxicity , Insulin , Peptide Fragments/toxicity , Animals , Blood , Cattle , Cell Division/drug effects , Cells, Cultured , Cricetinae , Cricetulus , Cross-Linking Reagents , Culture Media, Serum-Free , Deoxyglucose/metabolism , Fibroblasts/drug effects , Insulin/metabolism , Insulin/pharmacology , Insulin-Like Growth Factor I/metabolism , Receptor, Insulin/metabolism , Signal Transduction , SuccinimidesABSTRACT
The effects of various sulfhydryl modifying reagents on hexose transport in cultured human skin fibroblasts were studied. H2O2 was observed to have no effect on 2-deoxy-D-glucose transport in serum-starved glucose-fed cells. The elevation of hexose transport rates in cells by glucose deprivation, insulin, or serum stimulation rendered them sensitive to H2O2. Hexose transport in glucose-deprived cells was inhibited 51-55% by 1-2 mM H2O2, while hexose transport in insulin or serum-stimulated glucose-fed cells was inhibited 45% and 46%, respectively. H2O2 inhibition was blocked or reversed by 8 mM dithiothreitol. N-ethyl-maleimide (NEM), a permeant, sulfhydryl reagent, elicited effects on hexose transport similar to those effected by H2O2 (i.e., in glucose-deprived and insulin-stimulated cells, inhibition of hexose transport was 44% and 23%, respectively). Impermeant sulfhydryl reagents such as dithio(bis)nitrobenzoic acid (DTNB) and N-iodoacetyl-N'-(5-sulfo-1-naphthly-ethylenediame (1,5,-I-AEDANS) had no inhibitory effect on hexose transport under any conditions (i.e., glucose-fed, glucose-deprived, and insulin-stimulated cells). DTNB and 1,5-I-AEDANS afforded no protection from the action of H2O2 on hexose transport. The data suggest that the sensitive sites are thiol in nature and are located at an intramembrane or intracellular site and probably not exofacial.
Subject(s)
Deoxy Sugars/metabolism , Deoxyglucose/metabolism , Insulin/pharmacology , Monosaccharide Transport Proteins/metabolism , Skin/metabolism , Sulfhydryl Reagents/pharmacology , Cells, Cultured , Dithionitrobenzoic Acid/pharmacology , Dithiothreitol/pharmacology , Ethylmaleimide/pharmacology , Fibroblasts/metabolism , Humans , Hydrogen Peroxide/pharmacology , KineticsABSTRACT
Hexose transport in glucose-starved human fibroblasts was readily reversed by glucose refeeding. This hexose transport reversal was not inhibited by tunicamycin (1.5 microgram/ml) but was blocked by cycloheximide (20 micrograms/ml). The ability of insulin (100 mU/ml) to stimulate hexose transport was returned by glucose refeeding and this was not affected by tunicamycin. Cycloheximide which blocked the glucose refeeding effect on hexose transport, decreased the ability of insulin to stimulate hexose transport. Specific 125I-insulin binding was increased by glucose refeeding of glucose-starved cells and this change in binding was inhibited by tunicamycin and cycloheximide. Thus, it appears that under the conditions employed in human fibroblasts, the ability of insulin to stimulate hexose transport is differentially regulated more by factors affecting basal hexose transport than by those affecting changes in insulin binding.
Subject(s)
Cycloheximide/pharmacology , Glucose/physiology , Hexoses/metabolism , Insulin/pharmacology , Tunicamycin/pharmacology , Biological Transport/drug effects , Cells, Cultured , Fibroblasts/drug effects , Humans , Insulin/metabolism , Receptor, Insulin/drug effects , Receptor, Insulin/physiologyABSTRACT
NS-1 mouse plasmacytoma cells were examined for their insulin and insulinlike growth factor-1 (IGF-1) binding characteristics and ability to produce peptide-dependent cellular effects. At concentrations of labelled insulin (i.e., 1.7 x 10(-10) M) or IGF-1 (i.e., 1.5 x 10(-10) M), NS-1 cells specifically bind 0.2 +/- 0.06 fmol insulin per 10(6) cells (n = 7), where little, if any, IGF-1 specific binding was observed (0.02 +/- 0.01 fmol/10(6) cells) (n = 3). Additionally, the data indicate that the total number of insulin binding sites per cell was 3200 +/- 390 (n = 3). Insulin was employed at various concentrations (6.7-667 nM) and failed to stimulate either sugar or amino acid transport. Insulin at low concentrations (i.e., 6.7 or 67 nM) did not stimulate DNA synthesis, yet a small but significant increase was observed at a concentration of 667 nM insulin. IGF-1 did not stimulate DNA synthesis at all concentrations employed (1.4-143 nM). In summary, there exists a small but significant number of insulin receptors, little insulin-stimulated DNA synthesis, and no apparent insulin stimulation of sugar or amino acid transport. Also, since there is no significant IGF-1 binding and no IGF-1 stimulation of DNA synthesis, these findings indicate that this cell line might be a good candidate for the study of insulin receptor function as a transfection recipient of insulin receptor genes.
Subject(s)
Amino Acids/metabolism , Carbohydrate Metabolism , DNA/biosynthesis , Insulin-Like Growth Factor I/pharmacology , Insulin/pharmacology , Plasmacytoma/metabolism , Aminoisobutyric Acids/metabolism , Animals , Binding Sites , Biological Transport/drug effects , Deoxyglucose/metabolism , Insulin/metabolism , Insulin-Like Growth Factor I/metabolism , Mice , Receptor, Insulin/metabolism , Receptors, Cell Surface/metabolism , Receptors, Somatomedin , Thymidine/metabolism , Tumor Cells, CulturedABSTRACT
A procedure for the direct solubilization of fibroblast monolayers allowing precise determination of DNA and protein content has been developed. This simple procedure is applicable to the analysis of these macromolecules in fibroblast cultures containing small numbers of cells (less than 1 X 10(5].
Subject(s)
DNA/analysis , Fibroblasts/analysis , Proteins/analysis , Buffers , Cells, Cultured , Humans , Indoles , Polyethylene Glycols , Sodium Hydroxide , Solubility , Spectrometry, Fluorescence , SpectrophotometryABSTRACT
Non-proliferating cells of Lactobacillus plantarum accumulated about 1.3 mug of pantothenic acid (PA) per milligram dry cells when placed in a phosphate-buffered solution containing glucose, NaCl, and PA for 21 h. Under the conditions applied for PA uptake, pH 7.5 and 38 degrees C, the accumulated PA existed almost exclusively in a bound form. Free PA was observed only occasionally and then at questionably significant levels. All bound PA was released quantitatively by enzyme hydrolysis with alkaline (intestinal) phosphatase. Chromatographic analysis coupled with differential microbiological analysis of cell extracts using Saccharomyces cerevisiae, Lactobacillus helveticus, and L. plantarum identified the accumulated PA as phosphopantothenic acid (P-PA). Assay of P-PA loaded cell extracts (devoid of free PA) by L. plantarum before and after alkaline phosphatase hydrolysis showed that the growth of this primary assay organism was stimulated by 8.3%.
Subject(s)
Lactobacillus/metabolism , Pantothenic Acid/analogs & derivatives , Pantothenic Acid/metabolism , Alkaline Phosphatase/metabolism , Cell-Free System , Hydrolysis , Lactobacillus/drug effects , Lactobacillus/growth & development , Pantetheine/pharmacology , Pantothenic Acid/pharmacology , Saccharomyces/drug effects , Saccharomyces/growth & developmentABSTRACT
Sugar transport regulation was characterized in terms of the expression and subcellular distribution of the glucose transporter (GT) in the V79 hamster fibroblast cell line and in a respiration deficient mutant (G14) of the V79 cell line. Comparison of GT content in V79 and G14 cells and cell fractions revealed that the 3-fold elevation in basal sugar transport observed in the G14 cell line did not coincide with any significant difference in either the whole cell or plasma membrane GT content when compared to the V79 parental cell line. Determination of delta-antibody binding to intact cell monolayers supported the finding that the two cell lines demonstrate equivalent plasma membrane GT content. Further, D-glucose inhibitable cytochalasin B binding to total cell membranes indicates that additional, unrecognized GT isoforms do not occur in either cell line. A higher average molecular weight GT was detected in the G14 cell line, and treatment of GT enriched preparations with endoglycosidase F established that the G14 cell line exhibits a hyperglycosylated form of the same core GT protein expressed in V79. These results suggest that an enhancement of the intrinsic activity of the GT expressed in G14 is responsible for its increased sugar transport capabilities and this may be related to differences in GT post-translational processing.
Subject(s)
Deoxyglucose/metabolism , Monosaccharide Transport Proteins/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Cricetinae , Cricetulus , Cytochalasin B/metabolism , Glycosylation , In Vitro Techniques , Insulin-Like Growth Factor I/metabolism , Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase/pharmacology , Oxygen Consumption , Protein Processing, Post-TranslationalABSTRACT
Studies were carried out on cultures of human skin fibroblasts to explore the effects of culture medium glucose levels on insulin binding and action. Cell cultures in 5.55 mM glucose-containing medium depleted their medium glucose within 3 days, and at that time exhibited elevated deoxy-D-glucose (2-DG) transport (84% greater than control cultures fed 22.2 mM glucose) and failure of insulin to stimulate 2-DG transport (an insulin:control transport ratio of 1.02). There was also a significant negative correlation between basal 2-DG transport and insulin binding (r = -0.621; n = 29; P less than 0.01), while insulin binding exhibited a significant positive correlation with insulin action (r = 0.816; n = 12; P less than 0.01). Glucose starvation of cultures for 18 h resulted in several changes: a 49% decrease in specific 125I-insulin binding due to a reduction in binding capacity; elevated basal 2-DG transport; and an absence of insulin stimulation of 2-DG transport. Exposure to increasing concentrations of glucose for 18 h led to a glucose concentration-dependent increase in specific insulin binding. Additionally, the various changes in the glucose-starved group were reversed after as little as 6 h of glucose refeeding. The results indicate that basal sugar transport, and insulin binding and action can be regulated by the amount of glucose in the medium.
Subject(s)
Deoxy Sugars/metabolism , Deoxyglucose/metabolism , Fibroblasts/metabolism , Glucose/pharmacology , Insulin/metabolism , Biological Transport/drug effects , Cells, Cultured , Culture Media , Fibroblasts/drug effects , Humans , Insulin/pharmacology , Time FactorsABSTRACT
The regulation of hexose transport was studied in a human diploid fibroblast respiration-deficient cell strain (WG750). Transport of 2-deoxy-D-glucose (2-DG) was greater than sixfold higher compared with an in vivo age-matched normal cell strain (MCH55). In addition, 3-O-methylglucose transport and 14CO2 production were elevated in the mutant cell strain. Kinetic analysis revealed that the increased sugar transport in mutant cells was due to an average 5.7-fold increase in the 2-DG maximal transport rate, with no observed differences in the transport Michaelis constant for both normal and mutant cells. Also, the inhibitor constants for D-glucose inhibition of 2-DG transport were nearly identical for both cell types. Glucose deprivation led to a similar time-dependent increase in hexose transport in both cell strains. Serum refeeding of glucose-fed serum-deprived cultures led to a progressive increase in 2-DG transport in normal cells, whereas mutant cells displayed a time-delayed increase in 2-DG transport. Exposure to 67 and 670 nM insulin stimulated 2-DG transport on average 1.99 +/- 0.25- and 2.33 +/- 0.26-fold, respectively, over basal transport in the normal cells, whereas the mutant cells were significantly less sensitive to the stimulatory effects of the hormone. Insulin binding and amino acid transport (i.e., alpha-aminoisobutyric acid uptake) in the normal and mutant cells were not different. Data obtained using Western blot analysis showed that WG750 (mutant) cells expressed an increase (approximately 4-fold) in total cellular HepG2 (erythroid-brain) transporter protein compared with normal cells, thus reflecting the changes seen in hexose transport.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Cell Line/metabolism , Fibroblasts/metabolism , Hexoses/metabolism , Oxygen/metabolism , Biological Transport , Blotting, Western , Carbon Dioxide/metabolism , Deoxyglucose/metabolism , Diploidy , Fibroblasts/cytology , Humans , Insulin/metabolism , Kinetics , Lactates/metabolism , Mutation , Up-RegulationABSTRACT
Recently, we have isolated a Chinese hamster cell variant (IV-A1-j) resistant to an insulin-diphtheria-A chain toxic conjugate (Leckett and Germinario: Cytotechnology [in press]. This cell line exhibited a decreased level of insulin binding, but normal growth in serum-containing medium when compared to the parental cell line (V-79). In this paper we further demonstrate that while IV-A1-j cells are capable of growing in serum-containing medium, they are insensitive to the mitogenic actions of either insulin or IGF-1. In contrast, epidermal growth factor (EGF) and/or alpha-thrombin (THR) generate a mitogenic effect in IV-A1-j cells comparable to that observed in the parental V-79 cells. The combination of EGF and/or THR with either insulin or IGF-1 results in an increase in V-79 cell growth above EGF and/or THR alone. On the other hand, insulin or IGF-1 in the presence of other mitogens did not stimulate further growth in IV-A1-j cells. While insulin binding was lower in IV-A1-j cells, internalization of 125I-insulin was not different in the two cell types. Additionally, insulin-stimulated glycogen synthesis and protein synthesis were not different in the two cell types. These observations are consistent with insulin and IGF-1 sharing a mitogenic signalling pathway in Chinese hamster fibroblasts and that this pathway is distinct from other growth factor signalling pathways. The fact that this pathway is defective in the IV-A1-j cell line indicates the potential usefulness of these cells in identifying a key step(s) in the insulin (IGF-1) mitogenic pathway.