Abstract
The effects of the microtubule inhibitor, colchicine, on insulin or glucagon stimulation of alpha-amino[1-14C]-isobutyric acid (AIB) transport were investigated in isolated hepatocytes from normal fed rats. Under all conditions tested, AIB uptake appeared to occur through two components of transport: a low affinity (Km approximately 50 mM) component and a high affinity (Km approximately 1 mM) component. Within 2 h of incubation, insulin and glucagon, at maximal concentrations, increase AIB (0.1 mM) uptake by 2- to 3-fold and 4- to 6-fold, respectively. Colchicine, at the low concentration of 5 X 10(-7) M, slightly reduces basal AIB transport, decreases by 80% the simulatory effect of insulin, and diminishes by 40% the stimulatory effect of either glucagon or dibutyryl cAMP. Kinetic analysis of AIB influx indicates that the drug inhibits the increase in Vmax of a high affinity (Km approximately 1 mM) component of transport stimulated by insulin or glucagon, without affecting the kinetic parameters of a low affinity component of transport (Km approximately 50 mM). Various short term hormonal effects of insulin and glucagon (changes in glucose, urea, and lactate production) were found not to be modified by the drug. Vinblastine elicits similar changes as colchicine on AIB uptake. Lumicolchicine, a colchicine analogue that does not bind to tubulin, has no effect. The concentration of colchicine (10(-7) M) required for half-maximal inhibition of hormone-stimulated AIB transport is in the appropriate range for specific microtubule disruption. These data suggest that microtubules are involved in the regulation of the insulin or glucagon stimulation of AIB transport in isolated rat hepatocytes.
Highlights
- - through two components of transport: a low affinity ( K, 50 m ~ c)omponent and a high affinity ( K, 1 m ~ co)mponent
Dose responses of insulin,glucagon, and dibutyryl cAMP stimulation of AIB transport indicate that, at submaximal insulin concentration (1-7 nM), colchicine totally suppresses the insdin effect while at higher concentrations of the hormone a 60-85% inhibitory effect by the drug is observed.In contrast, colchicinedecreases but fails to completely abolish the glucagon stimulation of AIB uptake at submaximal concentraions of the hormone (0.5-5 nM).The inhibition at maximal glucagonconcentrations (30-50% inhibition) is less than that observed with insulin
This study indicates that colchicine markedly inhibits insulin- or glucagon-stimulated AIB transport by isolated hepatocytes fromfed rats
Summary
Effect of Colchicine on Insulin- or Glucagon-stimulated AIB Transport-Time course experiments (data not shown) have indicated that basal or insulin- and glucagon-stimulated AIB uptake, both in the presence or absence of colchicine, increased linearly with time for at least 8 min and with AIB concentrations ranging from 0.1 to 40 mM. Dose responses of insulin,glucagon, and dibutyryl cAMP stimulation of AIB transport indicate (data not shown) that, at submaximal insulin concentration (1-7 nM), colchicine totally suppresses the insdin effect while at higher concentrations of the hormone a 60-85% inhibitory effect by the drug is observed.In contrast, colchicinedecreases but fails to completely abolish the glucagon stimulation of AIB uptake at submaximal concentraions of the hormone (0.5-5 nM).The inhibition at maximal glucagonconcentrations (30-50% inhibition) is less than that observed with insulin. Kinetic Analysis of AIB Influx-The initial rate of AIB uptake (influx)was measured at various substrate concentrations, in the presence or absence of colchicine, insulin, and glucagon (Fig. 2).
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