Evidence for the Involvement of Sulfhydryl Oxidation in the Regulation of Fat Cell Hexose Transport by Insulin

Abstract

Previous studies have shown that the oxidants Cu++, H202, and diamide mimic the stimulatory effect of insulin on 3-0-methylglucose transport in isolated fat cells. The present experiments were designed to determine whether sulfhydryl oxidation plays a key role in the activation of the glucose transport system. It was found that reductants such as dithiothreitol inhibited 3-0-methylglucose transport rates and that this effect was reversible when cells were washed free of reducing agent. Treatment of cells with 1 mM N-ethylmaleimide for 5 min completely blocked the actions of insulin and oxidants on hexose transport without affecting control transport system activity. Under these conditions, binding of 1251_ labeled insulin to fat cell surface receptors was inhibited by only about 50%. Addition of insulin or oxidants to fat cells for 10 min before addition of N-ethylmaleimide completely prevented the inhibitory effect of N-ethylmaleimide on the activated transport system. This protective effect on transport rates appears to reside at a site that is altered by insulin subsequent to hormone-receptor interaction, since prior treatment of fat cells with insulin did not prevent the partial inhibitory effect of N-ethylmaleimide on insulin receptors. Furthermore, treatment of cells with N-ethylmaleimide after incubation with insulin prevented the elevated transport rates from returning to control levels when either the cells were washed free of hormone or insulin binding to its receptors was disrupted by trypsin digestion. However, transport rates in these cells treated with N-ethylmaleimide remained sensitive to cytochalasin B, phlorizin, and reductants. These data suggest that a component of the glucose transport system in isolated fat cells must be maintained in its disulfide state for expression of transport activity. Further, the results are consistent with the concept that the binding of insulin to cell surface receptors triggers sulfhydryl oxidation in this component, which prevents its reaction with N-ethylmaleimide.The well-known stimulatory effects of insulin on transport processes are thought to result from events triggered by the interaction of insulin with specific cell surface receptors. Although considerable information about the hormonereceptor interaction has become available in the last few years, nothing is known about the subsequent steps that lead to the physiological response. Studies on the actions of chemically specific agents, such as certain oxidants (1,2) which mimic effects of insulin on fat cell glucose transport, may be useful in this regard since they appear to act at steps subsequent to the hormone-receptor interaction (3). A major Abbreviation: MalNEt, N-ethylmaleimide. 3-0-Methyl-D-glucose transport into these cells is readily monitored, occurs by facilitated diffusion, and is inhibited by D-glucose, phlorizin, and cytochalasin 13 (4).Using this rationale we found that agents such as H202, Cu++, and diamide, which readily oxidize sulfhydryls to disulfides, markedly enhanced 3-0-m...

The Major Target of the Endogenously Generated Reactive Oxygen Species in Response to Insulin Stimulation Is Phosphatase and Tensin Homolog and Not Phosphoinositide-3 Kinase (PI-3 Kinase) in the PI-3 Kinase/Akt Pathway

“…A role for ROS in insulin-mediated signaling has been suggested by the observation that insulin stimulation induces the rapid production of ROS and that H 2 O 2 can mimic some of the action of insulin (Czech et al, 1974;KriegerBrauer et al, 1997). Recent reports (Mahadev et al, 2001a;Mahadev et al, 2001b) have demonstrated that insulin-stimulated ROS generation modulated the steady-state tyrosine phosphorylation of the insulin receptor and its cellular substrates by reversible inhibition of PTPases.…”

“…Our data show that the insulin-mediated increase of PI-3 kinase activity is independent of ROS production and suggest that PI-3 kinase is not the major target of ROS. Moreover, these results suggest that the binding motifs of the p85 regulatory subunit of PI-3 kinase located in the insulin receptor or IRS are more resistant to the member of PTP family, the known target of the reversible inactivation by ROS, than other tyrosine phosphorylation sites.A role for ROS in insulin-mediated signaling has been suggested by the observation that insulin stimulation induces the rapid production of ROS and that H 2 O 2 can mimic some of the action of insulin (Czech et al, 1974; KriegerBrauer et al, 1997). Recent reports (Mahadev et al, 2001a;Mahadev et al, 2001b) have demonstrated that insulin-stimulated ROS generation modulated the steady-state tyrosine phosphorylation of the insulin receptor and its cellular substrates by reversible inhibition of PTPases.…”

Protein cysteine sulfinic acid reductase (sulfiredoxin) as a regulator of cell proliferation and drug response

“…This can be oxidized readily by cellular activities associated with redox cycling (Nakamura et al, 1997;Denu and Dixon, 1998). More than 30 years ago, Czech et al (1974), documented the involvement of sulfhydryl oxidation in insulin signaling, which more recent studies have shown to be mediated through one of the cysteine residues of PTP1B. It is interesting to note that insulin positively regulates Srx transcription and protein expression (Figure 4), which in turn can promote a negative regulatory activity on insulin signaling.…”

Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells

“…These include anti-(insulin receptor) antibodies (Kahn et spermine (Lockwood & East, 1974), vitamin K5 (Livingston et al, 1977, and H202 (Czech et al, 1974;May & de Haen, 1979). Among these, only orthovanadate has been reported to increase the tyrosine phosphorylation of the fJ subunit of insulin receptors in an intact cell system (Tamura et al, 1984).…”

“…One example of these agents is H202. Although this is a simple molecule it is able to stimulate 3-O-methylglucose uptake (Czech et al, 1974), lipogenesis (May & de Haen, 1979), and to activate pyruvate dehydrogenase (May & de Haen, 1979). Using the rat hepatoma cell line H-35, in this study we have examined the possibility that H202 elicitates its insulin-like effects by stimulating tyrosine phosphorylation of the insulin receptor and activating its receptor kinase.…”