Hydroxylamine acutely activates glucose uptake in L929 fibroblast cells

Abstract

Nitroxyl (HNO) has a unique, but varied, set of biological properties including beneficial effects on cardiac contractility and stimulation of glucose uptake by GLUT1. These biological effects are largely initiated by HNO’s reaction with cysteine residues of key proteins. The intracellular production of HNO has not yet been demonstrated, but the small molecule, hydroxylamine (HA), has been suggested as possible intracellular source. We examined the effects of this molecule on glucose uptake in L929 fibroblast cells. HA activates glucose uptake from 2 to 5-fold within two minutes. Prior treatment with thiol active compounds, such as iodoacetamide (IA), cinnamaldehyde (CA), or phenylarsine oxide (PAO) blocks HA activation of glucose uptake. Incubation of HA with the peroxidase inhibitor, sodium azide, also blocks the stimulatory effects of HA. This suggests that HA is oxidized to HNO by L929 fibroblast cells, which then reacts with cysteine residues to exert its stimulatory effects. The data suggest that GLUT1 is acutely activated in L929 cells by modification of cysteine residues, possibly the formation of a disulfide bond within GLUT1 itself.

Curcumin directly inhibits the transport activity of GLUT1

“…This treatment tended to reduce basal uptake, but not significantly (P = 0.19) and curcumin’s inhibitory effects were not curtailed, but enhanced. As a positive control, we show that IA treatment was able block the activation of hydroxylamine (HA), a compound whose activation has been previously shown to be dependent upon the availability of cysteine side chains [36]. In the second experiment, 75 μM curcumin was incubated with excess cysteine (2.0 mM) for an hour, to potentially react with curcumin, before its incubation with cells.…”

“…We investigated two acute activators that require a 20-min preincubation to maximize activation; azide treatment and glucose deprivation [34,35], where the activator plus or minus curcumin was present only during a 20-min pretreatment. We also measured 2DG uptake in the presence of two activators that act immediately; PAO and HA [28,36], where the activator with or without curcumin was present only during the measurement of 2DG uptake. The results, shown on Fig.…”

“…Thiol reactive compounds, such as cinnamaldehyde, phenylarsine oxide, hydroxylamine, and Angeli’s salt, have previously been shown to alter GLUT1 activity, suggesting a potential mode of action for curcumin [26–28,36]. To determine if curcumin requires free thiols to exert its inhibitory effects, we performed two experiments with results shown in Fig.…”

Alkaline pH activates the transport activity of GLUT1 in L929 fibroblast cells

“…However, there is accumulating evidence that GLUT1 can be quickly activated by a wide variety of reagents without an increase in either GLUT1 expression or total GLUT1 membrane concentration. Stimulants include cell stressors such as osmotic stress [8, 9], azide [10, 11], berberine [12, 13], methylene blue [14], and glucose deprivation [15, 16], as well as treatment with thiol reactive compounds such as phenylarsine oxide [17], cinnamaldehyde [18], nitroxyl [19], and hydroxylamine [20] all significantly activate GLUT1 within minutes. In addition, peptide C has shown to activate GLUT1 in erthryocytes [21].…”

“…This potential model for the acute activation of GLUT1 has not been directly demonstrated, though, we have generated significant data that suggests that thiols may be critical to the activation of GLUT1 in L929 fibroblast cells. This cell line expresses GLUT1 as the exclusive glucose transporter [28] and glucose uptake can be maximally activated within two minutes by thiol reactive compounds such as phenylarsine oxide, nitroxyl, and hydroxylamine, which is believed to be converted to nitroxyl [17, 19, 20]. Pretreatment of cells with iodoacetamide to react with free thiols, blocks the activation of these compounds.…”