Effects of fluorescein isothiocyanate on insulin actions in rat adipocytes

Goto, Yuichi; Sumida, Yasuhiro; Flanagan, J E; Robinson, F W; Simpson, Ian A.; Cushman, Samuel W.; Kôno, Tetsuro

doi:10.1016/0003-9861(92)90389-e

Cited by 14 publications

(5 citation statements)

References 32 publications

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“…The membrane is washed twice by gentle shaking in 30 mL of PBS-T for 5 min and then incubated for 1 h at room temperature with the primary antibody added in the same buffer containing 10 mg/mL bovine serum albumin. The antibodies used were as follows: GLUT1 monoclonal antibody 7F7.5 at a 1:5 dilution of tissue culture supernatant (Tai & Carter-Su, 1988), a gift from Dr. Christin Carter-Su, and affinity-purified GLUT4 polyclonal antibody at a 1:5000 dilution (Goto et al, 1992). Following incubation with the primary antibody, the membrane was washed once with 30 mL of PBS-T for 15 min and then four additional times for 5 min each in the same volume of PBS-T. Antibody 7F7.5 was detected with horseradish peroxidase conjugated to goat anti-mouse antibody (1:1000 dilution in PBS-T with 10 mg/mL of bovine serum albumin).…”

Section: Methodsmentioning

confidence: 99%

Role of the C-terminal tail of the GLUT1 glucose transporter in its expression and function in Xenopus laevis oocytes

Due

Qu²,

Thomas³

et al. 1995

Biochemistry

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Structural determinants for the glucose transport kinetics of the erythrocyte glucose transporter have not been established. In this work the role of the cytosolic carboxy-terminal tail in the expression and function of the human GLUT1 isoform in Xenopus oocytes was investigated. Oocyte plasma membrane expression of GLUT1 was a saturable function of the amount of mRNA injected. Transport activity increased as a linear function of the amount of immunoreactive transporter in the plasma membrane. Transport kinetics of human GLUT1 expressed in oocytes resembled those of human erythrocyte GLUT1. Addition of up to 31 extra amino acids to the carboxy-terminal tail of GLUT1 was without effect on its function in oocytes. Removal of the carboxy-terminal 21 amino acids also did not affect GLUT1 expression or transport kinetics in oocytes. Removal of the entire carboxy-terminal tail to Phe-450 resulted in a transporter that had moderately decreased plasma membrane expression compared to that of GLUT1. However, transport activity of this construct was less than 5% of that of GLUT1, and was associated with loss of its outward-facing inhibitor binding site. When the carboxy-terminal 29 amino acids of GLUT1 were replaced with the corresponding region of GLUT4, transporter expression in the plasma membrane and the transport Vmax fell to low levels, similar to those of native GLUT4. When the carboxy-terminal 29 or 73 amino acids of GLUT1 were swapped into the corresponding region of GLUT4, the transport Vmax markedly increased to about one-third to one-half that of GLUT1, although the affinity for substrate was halved. These results show that the carboxy-terminal tail of the GLUT1 is not critical for targeting of the protein to the plasma membrane, but that this region is an important determinant of transport function.

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Section: Methodsmentioning

confidence: 99%

Role of the C-terminal tail of the GLUT1 glucose transporter in its expression and function in Xenopus laevis oocytes

Due

Qu²,

Thomas³

et al. 1995

Biochemistry

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“…Adipocytes prestimulated with 0.3 nM insulin were exposed to KCN and further incubated with 1 mM chromate or 20 nM insulin before measuring 3-0-MG uptake. As described previously (11,16,17), with this KCN intervention method, we could test effects of agents under the condition in which glucose transporters recruited by a submaximal concentration of insulin were fixed on the cell surface because their dynamic cycling was stopped with ATP depletion. As shown in Table 2, no stimulatory effect of 1 mM chromate or 20 nM insulin was seen under such a condition.…”

Section: Atp-dependentmentioning

confidence: 99%

Insulin-like Action of Chromate on Glucose Transport in Isolated Rat Adipocytes

Goto

Kida

1995

Japanese Journal of Pharmacology

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ABSTRACT-The effects of chromium compounds on 3-O-methylglucose (3-0-MG) transport were studied in isolated rat adipocytes. Sodium chromate significantly stimulated 3-0-MG uptake into adipocytes in a dose-dependent manner without altering the equilibrium space of 3-0-MG in adipocytes. The stimulatory effect reached the maximum at 300 pM, and the effect was 60 -70070 of the maximal insulin effect that was obtained with 20 nM insulin. The chromate concentration achieving a half-maximal effect was estimated at 50 pM. The effect of the combination of 1 mM chromate and 20 nM insulin was equipotent to that of 20 nM insulin alone, which showed that these two effects were not additive. The stimulatory effects of 1 mM chromate and 20 nM insulin were entirely abolished in adipocytes deprived of ATP, which indicated that these effects were completely ATP-dependent.Judging from experiments using various chromium compounds, Cr042-was responsible for the insulinomimetic action. These results indicate that the action of Cr042-is exerted through a mechanism analogous to that of insulin action, and that Cr042-is a novel and useful tool for studying issues involved in insulin actions.

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“…This is difficult to explain if insulin actually caused the insertion into the membrane of fully functional transporters that were not altered by the drug treatment [8]. Although this work was challenged in experiments incubating cells for an extensive time (45 minutes) [9] the data under our careful conditions are clear.…”

Section: Am J Biomed Sci and Resmentioning

confidence: 92%