Hexose transport in L6 rat myoblasts. I. Rate‐limiting step, kinetic properties, and evidence for two systems

D’Amore, Tony; Lo, Theodore C. Y.

doi:10.1002/jcp.1041270113

Cited by 43 publications

(52 citation statements)

References 39 publications

(71 reference statements)

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“…Furthermore, virtually all of the 2-DOG taken up by the mutants was present in the phosphorylated form; free 2-DOG could not be detected. This is similar to the results obtained with the parental L6 strain (D'Amore and Lo, 1986a). Table 2 shows that the rate of 2-DOG phosphorylation is reduced to the same extent as the rate of 2-DOG uptake.…”

Section: Mutants Are Impaired In Hexose Transportsupporting

confidence: 91%

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Isolation and characterization of hexose transport mutants in L6 rat myoblasts

D’Amore

Duronio

Cheung

et al. 1986

Journal Cellular Physiology

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A method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2-deoxy-D-glucose (2-DOG)-and 2-deoxy-2-fluoro-D-glucose (2FG)-resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2-DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2-DOG, 2FG, or 3-O-methyl-D-glucose (3-OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four- to five-fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for alpha-aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2-DOG and 3-OMG transport in mutant F72 demonstrated that the Vmax for 2-DOG uptake was significantly reduced, whereas the Vmax for 3-OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) involved in the high-affinity hexose transport system.

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Section: Mutants Are Impaired In Hexose Transportsupporting

confidence: 91%

“…We have established that transport is the rate-limiting step of hexose uptake (Lo and Duronio, 1984a;D'Amore and Lo, 1986a). Furthermore, our results indicate that two hexose transport systems may be operating in these cells.…”

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confidence: 74%

Isolation and characterization of hexose transport mutants in L6 rat myoblasts

D’Amore

Duronio

Cheung

et al. 1986

Journal Cellular Physiology

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“…Therefore, the inhibition of fructose uptake by glucose and its analogs is noncompetitive which provides an additional evidence that glucose and fructose did not share a common binding site and the absolute value of the slope represents the inhibitor constant Ki. As demonstrated previously, Ki is an accurate measurement of the affinity of inhibitors in sugar transport studies (5). The values found with glucose, 3-OMG, and dGlc were each close to the Km values of their high affinity transport system.…”

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confidence: 79%

Characterization of the Hexose Transport System in Maize Root Tips

Xia

Saglio²

1988

Plant Physiol.

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Sugar-depleted excised maize (Zea mays L.) root tips were used to study the kinetics and the specificity of hexose uptake. It was found that difficulties induced by bulk diffusion and penetration barriers did not exist with root tips. Several lines of evidence indicate the existence of a complex set of uptake systems for hexoses showing an overall biphasic dependence on external sugar concentrations. The results suggest that the high and the low affinity components might be located on the same carrier. One uptake system was specific for fructose, but the high affinity component was repressed by high concentrations of external glucose. A second system was specific for glucose and its analogs (2-deoxy-D-glucose and 3-0-methyl-D-glucose), and a third one, more complex, had a high affinity for glucose and its analogs but could transport fructose when glucose was not present in the external solution. A simple method is proposed to determine the inhibitor constants in competition experiments.The seminal maize root tip has few carbohydrate reserves but is a strong sink for sugars. Therefore, its energy metabolism, either in normoxia through oxidative phosphorylation (18), or in anoxia through fermentation (19) competitors were added together. After appropriate times, the uptake was terminated by washing the tips five times for 1 min each with 20 ml of ice-cold MOPS solution. The ice-cold tips were counted by groups of 10 in 2 ml of aqueous scintillation fluid (ACS II, Amersham). All the determinations were done in duplicate.Phosphorylation of 2-deoxy-glucose. Root tips were placed in groups of 40 in 2 ml of medium containing 1 mM ['4C]dGlc (65 MBq/mmol) as described above. After appropriate times they were rapidly rinsed three times within 20 s with ice-cold MOPS solution and immediately frozen in liquid N2. The soluble sugars were then extracted with 0.6 M TCA as for adenine nucleotides (18). The extract was dried under vacuum and resuspended in 100 ,L H20. One aliquot (10-20 uL) was used for the determination ofthe total radioactivity and a second (5-20 1L depending on radioactivity) was spotted and thin layer chromatographed as described previously (16). After a 7-d exposure period on a Kodak X-ray safety film ARD, two radioactive spots, corresponding to dGlc and dGlc-P, were located on the chromatogram and recovered for counting.Determination of Fructose Carrier Inhibitor Constants. The experimental results obtained in the course of this work suggest that two different sets of fructose carriers were operating with similar affinities (Kin) for the substrate (Fig. 4, A and B). One set was not affected by the presence of competitors, whereas the other one could be completely inhibited. Dixon's representation of inhibition (6) which applies to a homogeneous population of enzyme, could not be used for the calculation of inhibitor constants of such a complex system. We have therefore, developed a simple mathematical and graphical method to describe this

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“…As noted above, evidence has been accumulating to support the existence of several tissuespecific GTs (9,10,12). Our findings do not exclude the possibility that multiple GTs can be expressed by hepatocytes, much as it has been suggested for undifferentiated L6 myocytes (11). However, our data provide circumstantial evidence that the GT transcript induced by starvation is identical to that induced in isolated hepatocytes: the two transcripts are identical in size (2.5 kb) and hybridize to the GT probes under similar conditions of stringency (data not shown).…”

Section: Discussioncontrasting

confidence: 40%

“…Chang (10) has presented immunological data distinguishing insulin-sensitive GT(s) from that found in brain. On the basis of kinetic evidence, D'Amore and Lo (11) have suggested that undifferentiated 3T3-L6 adipocytes possess two independent GTs. Moreover, detection in Southern blots of multiple bands hybridizing to GT probes has led Lodish et al (12) to postulate a family of GTs.…”

mentioning

confidence: 99%

Evidence for expression of the facilitated glucose transporter in rat hepatocytes.

Rhoads

Takano

Gattoni‐Celli

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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The eukaryotic facilitated glucose transporter (GT) is expressed by many cell types, with the notable exception of hepatocytes; however, GT is expressed by several hepatoma cell lines, including the well-differentiated lines Fao, Hep3B, and HepG2. We report on studies carried out to determine the aspect(s) of the transformed phenotype that might be responsible for activating GT expression. Using RNA blot analysis with probes derived from rat GT cDNA, we found that GT was expressed by rat hepatocytes under two conditions (i) in vitro, when isolated hepatocytes were placed in cell culture, and (a) in vivo, when rats were subjected to starvation for .2 days. However, GT expression was not an obligatory feature of hepatomas, since two primary hepatocellular carcinomas did not express any GT mRNA. GT expression in hepatocytes was reduced by addition of dimethyl sulfoxide or sodium butyrate to the culture medium. Since these reagents are known to promote differentiation in some cell culture systems, their effect on hepatocytes may be to maintain the GT repression normally observed in vivo. Inclusion or exclusion in the culture medium of several other agents that enhance hepatocyte viability (serum, insulin, corticosteroids, epidermal growth factor, or triiodothyronine) did not affect GT expression. It is unclear whether the two conditions that led to GT expression in hepatocytes are related by a common signaling mechanism. Possibly, both cases involve a "stress" response: in vivo, a normal physiological response to starvation; in vitro, a response to a major alteration in the cellular environment.

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Hexose transport in L6 rat myoblasts. I. Rate‐limiting step, kinetic properties, and evidence for two systems

Cited by 43 publications

References 39 publications

Isolation and characterization of hexose transport mutants in L6 rat myoblasts

Isolation and characterization of hexose transport mutants in L6 rat myoblasts

Characterization of the Hexose Transport System in Maize Root Tips

Evidence for expression of the facilitated glucose transporter in rat hepatocytes.

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