Insulin-stimulated Sugar Transport Research Articles

Sugar Transport Regulation: Comparative Characterization of the Effect of NADH CoQ Reductase Deficiency in Two Cell Culture Systems

Abstract. In this report, we have characterized the upregulation of glucose transport in two different respiration-deficient fibroblast cell cultures. We have demonstrated that glucose transport increases in respiration-deficient cells as measured by 2 deoxy D-glucose transport and is readily observed in both the WG750 human and G14 Chinese hamster fibroblast respiration-deficient cell lines when compared with the MCH55 normal human and V79 parental Chinese hamster cell lines, respectively. Using subcellular fractionation techniques, the GLUT 1 glucose transporter was found located predominantly in the plasma membrane–enriched fraction of the human and hamster cell lines. In human cells, the expression of the GLUT 1 glucose transporter was elevated three-fold in the plasma membrane–enriched fraction of the WG750 respiration-deficient mutant cells. In the Chinese hamster cell lines, the respiration-deficient G14 cells exhibited no such GLUT 1 glucose transporter elevation in the plasma membrane–enriched fraction, yet expressed a >2-fold increase in glucose transport. Furthermore, the G14 cells had a similar content of GLUT 1 glucose transporter in the plasma membrane fraction when compared with the V79 parental cell line. Using Western blot analysis, the GLUT 1 glucose transporter in G14 cells exhibited a different mobility on a polyacrylamide gel when compared with the mobility of the GLUT 1 glucose transporter of the V79 cell line. This differential mobility of the glucose transporters in the hamster cells appeared to be related to glycosylation differences of the glucose transporters. Although normal human and hamster cell lines exhibited significant increases in insulin-stimulated sugar transport (P < 0.05), the two respective respiration-deficient cell lines exhibited no significant increases in insulin-stimulated sugar transport (P > 0.05). Additionally, the expression of the GLUT 1 mRNA in the human WG750 mutant cells was elevated when compared with GLUT 1 mRNA in normal cells. Insulin exposure significantly increased GLUT 1 mRNA in human cells (P < 0.05). No differences in the GLUT 1 mRNA were observed between both hamster cell lines. Thus, both respiration-deficient cell lines are insulin resistant (i.e., regarding their insulin-stimulated sugar transport). The respiration-deficient mutation results in an increased sugar transport in the human and hamster cells; however, the human cells adapt to the mutation by increasing their levels of GLUT 1 mRNA and eventually membrane-located glucose transporters. On the other hand, the hamster cells adapt by apparently modifying their glucose transporters' intrinsic activity via glycosylation. We feel that these cell systems can be effective models to study the multiple factors involved in sugar transport regulation in vertebrate cells.

Construction of a toxic insulin molecule: selection and partial characterization of cells resistant to its killing effects.

We have constructed an insulin-diphtheria hormono-toxin which migrates as a single 29 kd band on 10% SDS polyacrylamide gel electrophoresis. This corresponds to a one to one molar ratio of the diphtheria A-chain (23 kDa) and insulin (6 kDa) molecules. The diphtheria A-chain: insulin (DTaI) hormono-toxin demonstrates cytotoxicity in V-79 Chinese hamster cells exhibiting an LD50 of 1.1 x 10(-8) M, which is 22 x more potent than whole diphtheria toxin. Also, DTaI can competitively displace [125I]-insulin with an ED50 of 1.1 x 10(-8) M, which is identical to the ED50 of insulin (1.1 x 10(-8) M) and showed limited cross-reactivity with the IGF-1 receptor (12% displacement of [125I]-IGF-1 with a DTaI concentration of 1.1 x 10(-8) M). We have used DTaI to select conjugate-resistant clones from the V-79 Chinese hamster fibroblast parental cell line. Conjugate-resistant variants expressed insulin binding levels ranging from 8.0 +/- 2.0 fmoles/mg protein down to 3.6 +/- 0.5 fmoles/mg protein while insulin binding in the V-79 parental cell line was 11.2 +/- 0.2 fmoles/mg protein. Additionally, a number of conjugate resistant clones expressed variable ability to grow in medium containing 5% serum. The altered ability of these clones to grow in a serum-containing medium did not correlate directly with the changes observed for insulin binding. One mutant, IV-A1-j, did not grow in a serum-free defined medium containing insulin as the predominant mitogen. This IV-A1-j mutant had a lower number of insulin receptors, no change in insulin binding affinity, no change in the rate of internalization of [125I]-insulin and no apparent difference in [125I]-IGF-1 binding. Further, insulin-stimulated sugar transport was similar to that observed in the parental cell line. Based on these observations we suggest that 1) DTaI elicits its cytotoxicological effects through the insulin receptor trafficking pathway, 2) DTaI can be used to isolate cells altered at the level of insulin binding and/or action, and 3) signal transduction mechanisms responsible for mediating insulin-dependent cell growth can be pursued using mutants such as IV-A1-j.

Regulation of [formula omitted] uptake in isolated bovine adrenal chromaffin cells

We showed earlier that insulin stimulated sugar transport in adrenal chromaffin cells (Bigornia, L. and Bihler, I. Biochim. Biophys. Acta 885, 335–344). Transport regulation and its Ca 2+-dependence was further investigated in isolated bovine adrenal chromaffin cells, serving as a model of a homogeneous neuronal cell population. Uptake of the nonmetabolizable glucose analogue, 3-O- methyl- d-glucose was stimulated by hyperosmolar medium, and this effect was abolished in the absence of external Ca 2+, or depressed in the presence of La 3+ or the slow Ca 2+ channel blocker methoxyverapamil. Basal transport was also stimulated by factors (acetylcholine, carbamylcholine, low-Na + medium), which cause Ca 2+-dependent catecholamine release, and these effects were abolished in Ca 2+-free medium. In addition insulin, acetylcholine, hyperosmolar and low-Na + medium significantly increased 45Ca uptake. Thus, glucose transport in adrenal chromaffin cells was stimulated by insulin and hyperosmolarity in a Ca 2+-dependent manner, as in muscle. Sensitivity to secretory stimuli, a regulatory feature perhaps characteristic of this cell type, was also demonstrated. In contrast to muscle, sugar transport was not affected by Na +-pump inhibition, metabolic inhibitors or the Na + ionophore monensin, suggesting that Ca 2+ influx by Na + Ca 2+ exchange does not play a significant role in the activation of sugar transport in chromaffin cells.

Inhibitory effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport and On 125I-labelled insulin binding by rat soleus muscle

These experiments examined the effects of N-ethylmaleimide on insullin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661–668). Brief exposure (1 min) to N-ethylmaleimide (0.3−10 nM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U- 14C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H 2O 2 (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin was paralleled by the inhibition of 125I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and 125I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockae on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20–30%. Conversely, when muscles were first allowed to bind 125I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effet of sulphydryl blockade but no protective effect was observed with H 2O 2. None of the oxidants protected against the inhibitory effect of 3 nM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide.

Inhibition of insulin-stimulated xylose uptake in rat soleus muscle by cycloheximide.

Cycloheximide (20-200 mg/l) did not affect basal D-[U-14C]xylose uptake by rat soleus muscle (2.4 +/- 0.2 mumol . g-1 . h-1). However, the stimulatory effect of insulin on sugar transport was progressively reduced from 375% above basal in control muscles to 170% in muscles exposed to 200 mg cycloheximide/l but above this concentration cycloheximide inhibited basal xylose uptake without further effect on the incremental effect of insulin. Cycloheximide affected the insulin dose-response curve both by depressing insulin sensitivity and by reducing the maximum stimulatory effect of the hormone. In contrast to the inhibition of insulin action, which increased progressively over the range 20-200 mg cycloheximide/l, muscle protein synthesis was inhibited maximally at a concentration of 10 mg/l. Cycloheximide also inhibited the insulinomimetic effects of anoxia, 2:4-dinitrophenol, salicylate, cooling, hydrogen peroxide, diamide, vitamin K5, hyperosmolarity and EDTA, but did not affect concanavalin A-stimulated xylose uptake. It is concluded that cycloheximide inhibits insulin-stimulated sugar transport at some late post-receptor step, and that this effect of cycloheximide is not secondary to the inhibition of protein synthesis.

Enhanced insulin stimulation of sugar transport and DNA synthesis by glucocorticoids in cultured human skin fibroblasts

Glucocorticoids will enhance the growth of cultured human skin fibroblasts in serum-containing medium. In serum-free cultures hydrocortisone (5 × 10 −6 m) will enhance insulin stimulation of sugar transport and DNA synthesis (as measured by thymidine incorporation into trichloroacetic acid-precipitable material). The optimal concentration for the glucocorticoid effect on DNA synthesis was 5 × 10 −8 m for dexamethasone and 5 × 10 −7 m for hydrocortisone. In dexamethasone-treated cells, concentrations of insulin as low as 250 μU/ml (10 ng/ml) were effective in stimulating DNA synthesis. Further, hydrocortisone and dexamethasone (both at 5 × 10 −6 m) exhibited potentiating effects on insulin-stimulated sugar transport. These effects appeared to be mediated via inhibitory actions on the hexose transport system with the preservation of a functional insulin-receptor interaction resulting in insulin stimulation of deoxy- d-glucose transport at physiological insulin concentrations, 250 μU/ml (10 ng/ml). Hydrocortisone also enhanced specific [ 125I]insulin binding in these cells. The data indicate that the mechanism(s) of glucocorticoid enhancement of two actions of insulin may be different.

ATP depletion promotes deactivation of insulin-stimulated sugar transport in rat soleus muscle

It has been reported that deactivation of insulin-stimulated sugar transport in adipocytes is an energy-dependent process ( F. V. Vega, R. J. Key, J. E. Jordan, and T. Kono (1980) Arch. Biochem. Biophys. 203, 167–173 ). The stimulatory effect of insulin (0.1 U/ml) on the uptake of d-[U- 14C]xylose by rat soleus muscle was rapidly reversed when muscle ATP was depleted by exposure to 2,4-dinitrophenol (0.5 m m). Insulin action was not completely eliminated by ATP depletion; there was a small, residual stimulatory effect of the hormone which persisted for about 30 min after muscle ATP had been lowered to an unmeasurable level. The extent of deactivation was not altered when the rate of ATP depletion was accelerated, either by increasing the 2,4-dinitrophenol concentration, or by inducing leakiness by incubating muscles for 90 min at 37 °C prior to the addition of the uncoupler. 2,4-Dinitrophenol lowered steady-state 125I-insulin binding. These differences between the effect of ATP depletion on insulin-stimulated sugar transport in muscle and adipose tissue may be related to the action of the uncoupler in lowering steady-state insulin binding in muscle. Such a fall in bound insulin could be expected to promote deactivation during the period of ATP depletion. However, at present the possibility that these differences may represent some more fundamental difference in deactivation between muscle and adipose tissue cannot be excluded.

Inhibition of insulin-stimulated xylose uptake in denervated rat soleus muscle: a post-receptor effect

Insulin (100 U/l) stimulated xylose uptake in rat soleus muscle from a basal value of 2.3 +/- 0.5 to 11.6 +/- 2.1 mumol . g-1 . h-1. Denervation (section of the sciatic nerve) markedly reduced the stimulatory action of insulin (basal 1.3 +/- 0.4 mumol . g-1 . h-1; insulin-stimulated 4.5 +/- 0.6 mumol . g-1 . h-1). This effect appeared 3 days after denervation and was maximal after 5 days. Denervation also affected the insulin dose response curve; there was no effect of insulin in denervated muscle until the concentration exceeded 1 U/l. Denervation inhibited insulin-stimulated alpha-aminoisobutyrate uptake by 77% but did not affect 125I-insulin binding or glucose-independent activation of glycogen synthase by insulin. There was no effect of denervation on the insulinomimetic effects of concanavalin A, hydrogen peroxide, vitamin K5, anoxia, 2:4-dinitrophenol, cooling, hyperosmolarity or EDTA, but the effect of diamide was inhibited. It is concluded [1] that denervation inhibits insulin-stimulated sugar transport at some early post-receptor step, and [2] that the mechanism whereby insulin activates glycogen synthase is different from the activation of the membrane transport of sugars and amino acids.

Effect of ionophore A23187 on basal and insulin-stimulated sugar transport by rat soleus muscle.

The ionophore A23187 (10 micrograms/ml) did not affect the uptake of D-[U-14C]xylose by rat soleus muscle incubated under basal conditions. When muscles were incubated in a Ca2+/Mg2+-free (CMF) medium, A23187 promoted the efflux of intracellular Mg2+ and the efflux of 45Ca from preloaded muscles. Under these conditions, conditions, A23187 inhibited insulin-stimulated sugar transport without affecting 125I-insulin binding by the muscle. A23187 induced a slight fall in muscle ATP (16-18%); this does not appear to be responsible for the inhibitory effect of the ionophore on sugar transport. The inhibitory effect of A23187 was completely abolished when the CMF medium was supplemented with Mg2+ and partially reversed by Mn2+ or Zn2+; supplementation with Ca2+ did not reverse the inhibitory effect of the ionophore. These results suggest that insulin stimulates muscle sugar transport through a mechanism that involves intracellular Mg2+.

Effect of ionophore A23187 on basal and insulin-stimulated sugar transport by rat soleus muscle

Effect of ionophore A23187 on basal and insulin-stimulated sugar transport by rat soleus muscle

Effects of hyperosmolarity on basal and insulin-stimulated muscle sugar transport.

Xylose uptake was stimulated rapidly when rat soleus muscles were incubated in Krebs-bicarbonate medium made hyperosmolar by the addition of 200 mM mannitol. The stimulatory effect of hyperosmolarity increased as the concentration of mannitol was raised from 50 to 200 mM; above 200 mM, the effect tended to decline. Mannitol stimulated sugar transport submaximally compared with the effect of insulin. The stimulatory effect of hyperosmolarity persisted over a 60-min incubation period, but could be reversed by transferring the muscles to an isotonic incubation medium. Xylose uptake measured in the presence of insulin (0.1 U/ml) was depressed when muscles were exposed to concentrations of mannitol greater than 100 mM. This effect was not reversible; xylose uptake remained depressed when these muscles were transferred to an isotonic medium. Hyperosmolarity also depressed the binding of 125I-insulin irreversibly. These inhibitory effects of hyperosmolarity were associated with the lowering of muscle ATP. This effect on ATP provides an explanation for the inhibitory effects of hyperosmolarity on insulin-stimulated sugar transport and on insulin binding, in terms of the ATP-dependence of these two processes.

Permissive effect of ATP on insulin-stimulated sugar transport by rat soleus muscle.

The stimulatory effect of insulin (0.1 U/ml) on D-xylose uptake was progressively lost when rat soleus muscles were preincubated at 37 degrees C under anaerobic conditions for longer than 30 min; after 90 min these muscles were completely insensitive to insulin. This effect was associated with the loss of muscle ATP. When the breakdown of ATP was retarded either by lowering the preincubation temperature or by preincubation with 5 mM glucose, the effect of insulin in anaerobic muscle was correspondingly prolonged. Under certain conditions, externally added ATP promoted an effect of insulin in otherwise insulin-unresponsive muscles. This effect was small in magnitude and was complicated by the degradation of the added ATP in the incubation medium and by the fact that ATP also tended to inhibit insulin-stimulated xylose uptake. These results indicate that there is a relationship between insulin-stimulated sugar transport and muscle ATP levels. This supports the proposal that there may be some ATP-dependent reaction(s) involved in the mechanism whereby insulin promotes the process of muscle sugar transport.

Insulin-stimulated sugar transport and 125I-insulin binding by rat soleus muscle: Permissive effect of ATP

Summary The effect of insulin (0.1 U/ml) on the uptake of D-[U- 14 C] xylose by rat soleus muscle was abolished by preincubation for 90 min under anaerobic conditions. This loss of insulin sensitivity was associated with decreased binding of 125 I-insulin. Prolonged anoxia also depleted muscle ATP. These results are in accord with the proposal that the action of insulin on sugar transport involves some ATP-dependent step. Aerobic incubation (90 min) with cycloheximide (200 μg/ml) did not affect 125 I-insulin binding. This suggests that the effect of prolonged anoxia was not due simply to any effect on the synthesis of the insulin receptor, but rather to some more direct effect of ATP on the interaction of insulin with its receptor.