Increases in mRNA levels of glucose transporters types 1 and 3 in Ehrlich ascites tumor cells during tumor development

A common feature of many tumors is an increase in glucose catabolism during tumor growth. We studied the mechanism of this phenomenon by using Ehrlich ascites tumor bearing mice as the animal model. We found that Ehrlich ascites tumor cells possess only glucose transporter 1 (GLUT1) and GLUT3 but no GLUT2, GLUT4, or GLUT5. The mRNA levels of GLUT1 and GLUT3 increased progressively in the tumour during development; however, there were no changes observable in mRNA levels of glucose transporters of all types in brain, liver, and heart of the host mice. These findings suggest that Ehrlich ascites tumor augments its glucose transport mechanism relative to other tissues in response to its unique growth needs. J. Cell. Biochem. 67:131–135, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of fibroblast proliferation in three-dimensional collagen gel matrix

Summary Fibroblastsin vivo reside in a three-dimensional (3-D) matrix. The 3-D culture method using collagen gels provides valuable information, but is also has some practical difficulties. In particular, the changes caused by the contraction of gels and the occasional abrupt detachment from the underlying surface have made extended culture difficult. In this study, the 3-D culture method was modified in order to observe the cells with minimal change of substrata for longer periods. The proliferation characteristics of fibroblasts cultured in gels in response to fetal calf serum (FCS), to two defined growth factors, insulin and platelet-derived growth factor (PDGF), and to a growth inhibitory factor, prostaglandin E₂ (PGE₂), were evaluated with this system in comparison with monolayer cultured fibroblasts. The DNA content of fibroblasts cultured both in gels and on dishes increased in response to FCS in a concentration-dependent manner. The proliferation of gel-cultured fibroblasts, however, was lower than that of dish-cultured cells, and higher concentrations of serum were necessary for proliferation. The response of gel-cultured cells to PDGF was also less than that of dish-cultured cells. In addition, fibroblasts cultured in gel culture did not respond to insulin, while the fibroblasts on dishes responded to insulin in a concentration-dependent manner. In contrast to the reduced response to growth stimulators, PGE₂ inhibited proliferation in gel culture and in monolayer culture similarly. The reduced responsiveness to growth stimulation but equivalent response to growth inhibition may account for reduced proliferation of fibroblasts in 3-D culture.     

Changes in muscle mRNAs for hexokinase, phosphofructokinase-1 and glycogen synthase in acute and persistent hypoglycemia induced by tolbutamide in chickens

To elucidate the specificity of glucose metabolism in chicken skeletal muscle, changes in mRNA levels of hexokinase I (HKI), hexokinase II (HKII), phosphofructokinase-1 (PFK-1) and glycogen synthase (GS) were characterized in acute and persistent hypoglycemia induced by tolbutamide administration. In acute hypoglycemia, induced by a single dose of tolbutamide (100 mg/kg body mass), HKII, PFK-1 and GS mRNA levels remained unchanged; however, levels of HKI mRNA and glucose transporter 1 (GLUT1) were significantly increased 4 h after administration. In persistent hypoglycemia, induced by sequential administration of tolbutamide (100 mg/kg body mass) 3 times a day for 5 days, GS mRNA was significantly increased at day 5, while HKI, HKII and PFK-1 mRNA levels remained unchanged. These results suggest that HKI is responsible for glucose transport into skeletal muscle in acute hypoglycemia and that glucose preferentially enters the glycogenic pathway before the glycolytic pathway in persistently hypoglycemic chickens.     

Release of glycopeptides and mucopolysaccharides from ascites hepatoma cells by tryptic treatment.

Two types of ascites hepatoma cells, AH 66 and AH 130 FN, were treated with trypsin to observe the release of complex carbohydrates constituting the plasma membranes. From AH 66 cells, mucopolysaccharide (heparan sulfate) was preferentially released. From AH 130 FN cells, N-glycosidic glycopeptides were preferentially released whereas no mucopolysaccharide (chondroitin sulfate A) was released.     

Development of an intracellular pool of glucose transporters in 3T3-L1 cells.

The membrane-impermeant bis-mannose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) has been used to study the development of an intracellular pool of glucose transporters in 3T3-L1 cells. The subcellular distributions of the transporter isoforms GLUT1 and GLUT4 were determined by comparing the labeling obtained in cells in which the impermeant reagent only had access to the cell surface and the labeling obtained in digitonin-permeabilized cells. ATB-BMPA labeling showed that only GLUT1 was present in preconfluent fibroblasts and that most of the transporters were distributed to the cell surface. In preconfluent fibroblasts, the 2-deoxy-D-glucose transport activity was approximately 5 times higher than in confluent fibroblasts. ATB-BMPA labeling showed that the decrease in transport as cells reached confluence was associated with a decrease in the proportion of GLUT1 distributed to the cell surface. The sequestration of these transporters was associated with the development of an insulin-responsive transport activity which increased by approximately 2.5-fold compared with unstimulated confluent cells. ATB-BMPA labeling showed that insulin stimulation resulted in an approximately 2-fold increase in surface GLUT1 so that about one-half of the available transporters became recruited to the cell surface. Measurements of the changes in the distribution of both GLUT1 and GLUT4 throughout the differentiation of confluent fibroblasts into adipocytes showed that both transporters were sequestered in parallel. Basal levels of transport and photolabeling remained low throughout the differentiation period when the total pool of transporters (GLUT1 plus GLUT4) was increased by approximately 5-fold. These results suggest that the sequestration process was present before new transporters were synthesized. Thus, the sequestration mechanism develops in confluent growth-arrested fibroblasts although the capacity to sequester additional transporters may increase as differentiation proceeds.     

Kinetics of transport and phosphorylation of glucose in cancer cells

Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS‐30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D ‐[2‐³H‐glucose] as physiological substrate. Kinetic analysis revealed two components at low‐ and high‐glucose concentrations in both tumor cells. At low glucose and 37°C, the V_max was 55 ± 20 and 17.2 ± 6 nmol (min × mg protein)^?1, whereas the K_m was 0.52 ± 0.7 and 9.3 ± 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC₅₀ = 0.44 ± 0.1; K_i = 0.3 ± 0.1 µM) and phloretin (IC₅₀ = 8.7 µM) in AS‐30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS‐30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS‐30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS‐30D carcinoma and HeLa cells. The much lower GLUT V_max and catalytic efficiency (V_max/K_m) values in comparison to those of G6P‐sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target. J. Cell. Physiol. 221: 552–559, 2009. © 2009 Wiley‐Liss, Inc.     

Protein synthesis inhibitors activate glucose transport without increasing plasma membrane glucose transporters in 3T3-L1 adipocytes

In this study, we tested the hypothesis that hexose transport regulation may involve proteins with relatively rapid turnover rates. 3T3-L1 adipocytes, which exhibit 10-fold increases in hexose transport rates within 30 min of the addition of 100 nM insulin, were utilized. Exposure of these cells to 300 microM anisomycin or 500 microM cycloheximide caused a maximal, 7-fold increase in 2-deoxyglucose transport rate after 4-8 h. The effects due to either insulin (0.5 h) or anisomycin (5 h) on the kinetics of zero-trans 3-O-methyl[14C]glucose transport were similar, resulting in 2.5-3-fold increases in apparent Vmax values (control Vmax = 1.6 +/- 0.3 x 10(-7) mmol/s/10(6) cells) coupled with approximately 2-fold decreases in apparent Km values (control Km = 23 +/- 3.3 mM). Insulin elicited the expected increases in plasma membrane levels of HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) transporters (1.6- and 2.8-fold, respectively) as determined by protein immunoblotting. In contrast, neither total cellular contents nor plasma membrane levels of these two transporter isoforms were increased when 3T3-L1 adipocytes were treated with either anisomycin or cycloheximide. 3-[125I]Iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n labeling of glucose transporters in plasma membrane fractions of similarly treated cells was also unaffected by these agents. Thus, a striking discrepancy was observed between the marked increase in cellular hexose transport rates due to these protein synthesis inhibitors and the unaltered amounts of glucose transporter proteins in the plasma membrane fraction. These data indicate that short-term protein synthesis inhibition in 3T3-L1 adipocytes leads to large increases in the intrinsic catalytic activity of one or both of the GLUT1 and GLUT4 transporter isoforms.     

PC12 cells grown on cellulosic filters differentiate in response to NGF and exhibit a polarity not seen when they are grown on solid substrata

Summary Studies were performed with cellulosic filters and standard culture plates to compare methods of cell culture and differentiation of the cell line PC12, a clone originating from a rat pheochromocytoma. PC12 cells respond to nerve growth factor (NGF) by flattening of the cell body and subsequent extension of neurite-like processes. When PC12 cells are cultured in dishes without NGF, they have a diameter of approximately 3 to 7 μm and exhibit short processes of no longer than 3 to 5 μm. If PC12 cells are grown on a cellulosic filter they have the same average soma diameter and similar short processes extending laterally, but in addition have branching processes which extend as far as 10 to 15 μm into the filter substrate. When dish-cultured and filter-cultured cells are incubated with 50 ng/ml NGF they both exhibit differentiation-specific ultrastructural changes by 3 d of treatment. In the case of dish-cultured cells, large cytoplasmic processes exhibit an increase in the number of chromaffin cell-like secretory granules by 3 d of treatment. This characteristic is also demonstrated by filter-cultured cells, but the processes containing these granules are found concentrated within the cellulosic meshwork. Thus the timing of the NGF-elicited differentiation program is similar to both filter-cultured and dish-cultured cells, but the ultrastructural consequences are different. The filter-cultured PC12 cells exhibit a polarity not demonstrated by dish-cultured cells. Growing PC12 cells on cellulosic filters is a technique useful for “anchoring” neurons without the complication of the addition of extracellular matrix components. Filter-culture may represent a more in vivo-like method for studying neuronal growth and differentiation. This work was supported by grants S07RR07149-13 to R.V.B. and CA40929 to J.A.W. from the National Institutes of Health, Bethesda, MD.     

Protein synthesis, ribosomal protein S6 phosphorylation in vitro and the effects of amiloride: SDS gel electrophoresis studies in the Yoshida ascites tumor (AH 130) grown in vivo

Cell-free cytosolic extracts from the Yoshida (AH 130) rat ascites hepatoma cell line, grown in vivo, showed high ribosomal protein S6 kinase activity in vitro, as measured by transfer of 32P to exogenous 40S rat liver ribosomal subunits, in both exponential growing and stationary phase cells. A significant decrease of protein synthesis (3H-leucine incorporation into total cell protein) was found to occur in cells reaching the stationary phase of growth, suggesting that S6 phosphorylation was not tightly coupled to the rate of the intraperitoneal cell growth and of protein synthesis in these tumor cells. When the cell-free cytosolic extracts were prepared from cells exposed to amiloride, at concentrations that inhibit the Na+/H+ exchange, a decrease of S6 kinase activity was observed only in exponential growing cells, suggesting the possibility of coupling of the Na+/H+ exchange with phosphorylation of intracellular proteins in these tumor cells. Actually, stationary phase cells showed unchanged S6 kinase activity under the same conditions, possibly due to the extremely low Na+/H+ exchange activity, previously demonstrated (Cell Biol. Int. Rep., 1985, 9, 1017-1025). The present experiments support the hypothesis that the regulation of protein synthesis is not tightly coupled to phosphorylation-dephosphorylation cycles, at least of ribosomal protein S6, in cells characterized by a rather uncontrolled growth such as the Yoshida (AH 130) rat ascites hepatoma. In this connection, an elevated degree of protein phosphorylation, such as that of the ribosomal protein S6, could be a general phenomenon of neoplastic transformation.     

Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester

A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. This compound is 2-N-4(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4- yloxy)-2- propylamine. We have used this reagent in combination with immunoprecipitation by specific antibodies against the GLUT4 and GLUT1 glucose transporter isoforms to estimate the relative abundance of these two transporters on the surface of the intact adipose cell following stimulation by insulin and phorbol 12-myristate 13-acetate (PMA). In the basal state, GLUT4 and GLUT1 are both present at the cell surface but GLUT4 is more abundant than GLUT1. In response to insulin, GLUT4 increases 15-20-fold and GLUT1 increases approximately 5-fold while 3-O-methyl-D-glucose transport is stimulated 20-30-fold. By contrast, PMA only induces a approximately 4-fold increase in GLUT4 while GLUT1 increases approximately 5-fold to the same level as seen with insulin. In addition, PMA stimulates 3-O-methyl-D-glucose transport approximately 3-fold to only 13% of the insulin-stimulated state. Thus GLUT4 is the major glucose transporter isoform under all conditions, and it is selectively and markedly enriched in response to insulin but not PMA which increases GLUT1 and GLUT4 equally. Furthermore, stimulation of glucose transport activity correlates closely with the appearance of GLUT4 on the cell surface in response to both insulin and PMA but does not correlate with the sum of GLUT1 and GLUT4 appearance. These results suggest that GLUT4 may be inherently more active than GLUT1 due to a higher TK (turnover/Km).     

Chemical denervation using botulinum toxin increases Akt expression and reduces submaximal insulin-stimulated glucose transport in mouse muscle

Botulinum toxin A (botox) is a toxin used for spasticity treatment and cosmetic purposes. Botox blocks the excitation of skeletal muscle fibers by preventing the release of acetylcholine from motor nerves, a process termed chemical denervation. Surgical denervation is associated with increased expression of the canonical insulin-activated kinase Akt, lower expression of glucose handling proteins GLUT4 and hexokinase II (HKII) and insulin resistant glucose uptake, but it is not known if botox has a similar effect. To test this, we performed a time-course study using supra-maximal insulin-stimulation in mouse soleus ex vivo. No effect was observed in the glucose transport responsiveness at day 1, 7 and 21 after intramuscular botox injection, despite lower expression of GLUT4, HKII and expression and phosphorylation of TBC1D4. Akt protein expression and phosphorylation of the upstream kinase Akt were increased by botox treatment at day 21. In a follow-up study, botox decreased submaximal insulin-stimulated glucose transport. The marked alterations of insulin signaling, GLUT4 and HKII and submaximal insulin-stimulated glucose transport are a potential concern with botox treatment which merit further investigation in human muscle. Furthermore, the botox-induced chemical denervation model may be a less invasive alternative to surgical denervation.     

Chinese hamster ovary cells resistant to beta-aspartylhydroxamate contain increased levels of asparagine synthetase

The growth of Chinese hamster ovary cells in a complete medium lacking asparagine is inhibited by beta-aspartylhydroxamate. The inhibition is overcome by the presence of asparagine in the growth medium. beta-Aspartylhydroxamate inhibits the activity of both asparagine synthetase and asparaginyl-tRNA synthetase in vitro. beta-Aspartylhydroxamate-resistant clones of Chinese hamster ovary cells have been isolated and three of these have been characterized. One clone, AH12, is 3-fold more resistant to beta-aspartylhydroxamate than the parental line and has 2 times higher levels of asparagine synthetase activity. Strains AH2 and AH5 are 6- to 7-fold more resistant to beta-aspartylhydroxamate and have 5 times higher levels of asparagine synthetase. The regulation of the expression of asparagine synthetase is altered in all three resistant cell lines. Whereas asparagine synthetase activity varies 2- to 3-fold in response to the asparagine content of the medium or to the extent of aminoacylation of tRNALeu in the parental cells, the activity of asparagine synthetase in the resistant cells is elevated under all growth conditions. No significant changes in the Km for substrates, Ki for beta-aspartylhydroxamate, or thermal stability were found for the asparagine synthetase of the resistant cells. These variants should prove useful in understanding the mechanisms involved in regulating the levels of asparagine synthetase in mammalian cells.     

Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3-L1 adipocytes.

Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.