cytochalasin-b and Pancreatic-Neoplasms

Mammalian cells take in D-glucose as an essential fuel as well as a carbon source. In contrast, L-glucose, the mirror image isomer of D-glucose, has been considered merely as a non-transportable/non-metabolizable control for D-glucose. We have shown that 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), a D-glucose analogue combining a fluorophore NBD at the C-2 position, is useful as a tracer for monitoring D-glucose uptake through glucose transporters (GLUTs) into mammalian cells. To more precisely evaluate the stereoselectivity of 2-NBDG uptake, we developed an L-glucose analogue 2-NBDLG, the mirror-image isomer of 2-NBDG. Interestingly, 2-NBDLG was taken up into mouse insulinoma MIN6 cells showing nuclear heterogeneity, a cytological feature of malignancy, while remaining MIN6 cells only exhibited a trace amount of 2-NBDLG uptake. The 2-NBDLG uptake into MIN6 cells was abolished by phloretin, but persisted under blockade of major mammalian glucose transporters. Unfortunately, however, no such uptake could be detected in other tumor cell lines. Here we demonstrate that human osteosarcoma U2OS cells take in 2-NBDLG in a phloretin-inhibitable manner. The uptake of 2-NBDG, and not that of 2-NBDLG, into U2OS cells was significantly inhibited by cytochalasin B, a potent GLUT inhibitor. Phloretin, but neither phlorizin, an inhibitor of sodium-glucose cotransporter (SGLT), nor a large amount of D/L-glucose, blocked the 2-NBDLG uptake. These results suggest that a phloretin-inhibitable, non-GLUT/non-SGLT, possibly non-transporter-mediated yet unidentified mechanism participates in the uptake of the fluorescent L-glucose analogue in two very different tumor cells, the mouse insulinoma and the human osteosarcoma cells.

Topics: 4-Chloro-7-nitrobenzofurazan; Animals; Bone Neoplasms; Cytochalasin B; Deoxyglucose; Depression, Chemical; Glucose; Glucose Transport Proteins, Facilitative; Humans; Insulinoma; Isomerism; Mice; Osteosarcoma; Pancreatic Neoplasms; Phloretin; Sodium-Glucose Transporter 2 Inhibitors; Tumor Cells, Cultured

D-mannoheptulose was recently proposed as a possible tool to label preferentially insulin-producing cells in the pancreatic gland. In the present study, D-[3H]-mannoheptulose uptake by rat pancreatic islets or dispersed islet cells was found to represent a time-related and temperature-sensitive process inhibited by cytochalasin B. This mould metabolite also inhibited the efflux of D-[3H]-mannoheptulose from prelabelled islets. After 60 min incubation at 37 degrees C, the apparent intracellular distribution space of the tritiated heptose was close to or somewhat higher than that of D-[5-3H]glucose and close to 50% of the intracellular 3HOH space. It was further enhanced by D-glucose and a high concentration of 10 mM of D-mannoheptulose. The uptake of D-[3H]mannoheptulose was much lower however than that of D-[3H]mannoheptulose hexaacetate. As judged from the fate of D-mannoheptulose hexa[2-14C]acetate, the latter ester was efficiently hydrolyzed in the islet cells. The internalization of D-[3H]mannoheptulose (or its ester) coincided with the generation of tritiated acidic metabolites, reflecting phosphorylation of the heptose. The situation found in normal islet cells sharply differed from that found in tumoral islet cells of either the RINm5F or INS-1 line, in which the apparent distribution space of D-[3H]mannoheptulose represented only about 3 and 9%, respectively, of the intracellular 3HOH space. These results indicate that the entry of D-mannoheptulose into islet cells represents a carrier-mediated process, possibly mediated at the intervention of GLUT2 and, hence, provide further support to the possible use of a suitable D-mannoheptulose analog as a tool for the preferential labelling of insulin-producing cells in the pancreatic gland.

Topics: Animals; Cells, Cultured; Cytochalasin B; DNA; Glucose; Glucose Transporter Type 2; Humans; Insulin; Islets of Langerhans; Mannoheptulose; Monosaccharide Transport Proteins; Pancreatic Neoplasms; Rats; Temperature; Time Factors; Tumor Cells, Cultured

Previous studies have shown that lactogenic hormones stimulate beta-cell proliferation and insulin production in pancreatic islets. However, all such studies have been conducted in cells incubated in medium containing glucose. Since glucose independently stimulates beta-cell replication and insulin production, it is unclear whether the effects of prolactin (PRL) on insulin gene expression are exerted directly or through the uptake and/or metabolism of glucose. We examined the interactions between glucose and PRL in the regulation of insulin gene transcription and the expression of glucose transporter-2 (glut-2) and glucokinase mRNAs in rat insulinoma (INS-1) cells. In the presence of 5.5 mM glucose, the levels of preproinsulin and glut-2 mRNAs in PRL-treated cells exceeded the levels in control cells (1.7-fold, P<0.05 and 2-fold, P<0.05 respectively). The maximal effects of PRL were noted at 24-48 h of incubation. PRL had no effect on the levels of glucokinase mRNA. The higher levels of glut-2 mRNA were accompanied by an increase in the number of cellular glucose transporters, as demonstrated by a 1. 4- to 2.4-fold increase in the uptake of 2-deoxy-d-[(3)H]glucose in PRL-treated INS-1 cells (P<0.001). These findings suggested that the insulinotropic effect of PRL is mediated, in part, by induction of glucose transport and/or glucose metabolism. Nevertheless, even in the absence of glucose, PRL stimulated increases in the levels of preproinsulin mRNA (3.4-fold higher than controls, P<0.0001) and glut-2 mRNA (2-fold higher than controls, P<0.01). These observations suggested that PRL exerts glucose-independent as well as glucose-dependent effects on insulin gene expression. Support for this hypothesis was provided by studies of insulin gene transcription using INS-1 cells transfected with a plasmid containing the rat insulin 1 promoter linked to a luciferase reporter gene. Glucose and PRL, alone and in combination, stimulated increases in cellular luciferase activity. The relative potencies of glucose (5.5 mM) alone, PRL alone, and glucose plus PRL in combination were 2.2 (P<0.001), 3.4 (P<0.01), and 7.9 (P<0.0001) respectively. Our findings suggest that glucose and PRL act synergistically to induce insulin gene transcription.

Topics: Animals; Blotting, Northern; Culture Media; Cytochalasin B; Drug Synergism; Gene Expression Regulation; Glucokinase; Glucose; Glucose Transporter Type 2; Insulin; Insulinoma; Luciferases; Monosaccharide Transport Proteins; Pancreatic Neoplasms; Proinsulin; Prolactin; Protein Precursors; Rats; RNA, Messenger; Tumor Cells, Cultured

Patients with cancer cachexia exhibit increased glucose flux and lactate production in skeletal muscle. The aim of this study was to examine the direct effect of cancer cell-conditioned media on glucose metabolism in L6 myoblasts. Media from PANC-1 and Colo 320 cells caused a marked time-dependent and concentration-dependent increase of 2-deoxyglucose uptake in GLUT-4 transfected L6 myoblasts. This effect was greater than maximal acute stimulation by insulin and the effect of insulin was additive. Glucose utilization and lactate production increased in parallel to glucose uptake. The effect was inhibited by the protein synthesis inhibitor, cycloheximide and the glucose transport inhibitor, cytochalasin B. The bioactive factor had a molecular weight of approximately 5,000 and the biological activity was destroyed by proteinase K digestion. Radioimmunoassay and immunoneutralization studies indicated the major factor involved is not TNFalpha, IL-1beta, insulin, IGF-I or IGF-II. Further purification and characterization are needed to reveal the identity of this novel factor or factors which may have other metabolic effects that contribute to the cancer cachexia and insulin resistance.

Topics: Animals; Biological Factors; Cell Line; Colonic Neoplasms; Culture Media, Conditioned; Cycloheximide; Cytochalasin B; Deoxyglucose; Dose-Response Relationship, Drug; Endopeptidase K; Glucose; Glucose Transporter Type 4; Humans; Insulin; Lactic Acid; Molecular Weight; Monokines; Monosaccharide Transport Proteins; Muscle Proteins; Muscles; Pancreatic Neoplasms; Rats; Somatomedins; Tumor Cells, Cultured

Tumoral pancreatic islet cells of the RINm5F line are equipped with two classes of [3H]cytochalasin B binding sites with respective Kd of 0.4 and 7 microM. The binding of the fungal metabolite and its dissociation from the binding sites display rapid time courses. The binding is inhibited by D-glucose, more than by L-glucose, by phlorizin and by cytochalasin E. These findings are considered in the light of the dual action of cytochalasin B upon hexose transport and motile activity in islet cells.

Topics: Adenoma, Islet Cell; Binding Sites; Cytochalasin B; Cytochalasins; Glucose; Islets of Langerhans; Kinetics; Pancreatic Neoplasms; Phlorhizin; Tumor Cells, Cultured

The rat insulinoma-derived RINm5F cell line retains many differentiated functions of islet beta-cells. However, it fails to recognize glucose as an insulin secretagogue in the physiological concentration range. With this cell line, glucose-transport kinetics were investigated, by using a double-label technique with the non-metabolizable glucose analogue 3-O-methylglucose (OMG). RINm5F cells possess a passive glucose-transport system with high capacity and low affinity. Equilibration across the plasma membrane of extracellular OMG concentrations up to at least 20 mM is achieved within 2 min at 37 degrees C. The half-saturation of OMG uptake occurs at 32 mM. At lower temperatures OMG uptake is markedly retarded, with a temperature coefficient (Q10) of 2.9. As indicated by efflux measurements, transport is symmetrical. Cytochalasin B at micromolar concentrations and phlorrhizin in millimolar concentrations are potent inhibitors of OMG uptake. Neutralization of the secreted insulin with antibodies does not alter OMG uptake kinetics. The glucose metabolism of RINm5F cells is much exaggerated compared with that of islet beta-cells. Nonetheless, when measured in parallel to uptake, transport exceeds by far the rate of metabolism at glucose concentrations above 3 mM. Measurements of intracellular D-glucose reveal a lower intracellular glucose concentration relative to the extracellular in RINm5F cells. This seems to be due to abnormalities in the subsequent steps of glucose metabolism, rather than to abnormalities in hexose uptake. The loss of glucose-induced insulin release in RINm5F cells cannot be explained by alterations in hexose transport.

Topics: 3-O-Methylglucose; Adenoma, Islet Cell; Animals; Biological Transport; Cell Line; Cytochalasin B; Glucose; Insulin; Insulin Antibodies; Insulinoma; Methylglucosides; Pancreatic Neoplasms; Phlorhizin; Rats

We studied the release of insulin, glucagon, and somatostatin in response to glucose, glyceraldehyde (GA), and alpha-ketoisocaproate (KIC) from rat kidneys containing transplanted insulinomas. Kidneys were perfused about 11 wk after transplantation when the plasma glucose concentration of the fed animals had decreased from 180 +/- 7 to 95.1 +/- 9.9 mg/dl and plasma insulin concentrations had increased from 2.6 +/- 0.5 to 14.2 +/- 2.0 ng/ml. The insulin content of the tumor-containing kidney ranged from 40 to 679 micrograms; the glucagon and somatostatin concentrations ranged from undetectable levels to 3.7 micrograms and 248 ng, respectively. The average response to 30 mM glucose and 10 mM GA was a four- to fivefold increase in insulin secretion, whereas 30 mM KIC caused a 16- to 28-fold increase. In vitro stimulation of the insulinoma with 30 mM glucose primed the beta-cell response to a second stimulus following a short rest period. Cytochalasin B did not enhance this primed glucose response. Diazoxide inhibited glucose, GA, and KIC-stimulated insulin release. Glucose, GA, and KIC stimulated glucagon release in 2 of 17 insulinomas studied here. Somatostatin release was not seen in any of the experiments. These findings show that this islet cell tumor transplanted under the kidney capsule releases insulin in response to physiologic and model fuel substances. Thus, this particular transplantable tumor offers an opportunity to study the biochemistry and biophysics that underlie fuel-stimulated insulin release.

Topics: Adenoma, Islet Cell; Animals; Cytochalasin B; Diazoxide; Glucagon; Glucose; Glyceraldehyde; Humans; In Vitro Techniques; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Keto Acids; Kidney; Male; Neoplasm Transplantation; Pancreatic Neoplasms; Radioimmunoassay; Rats; Somatostatin