Nitric oxide‐sensitive protein ADP‐ribosylation is altered in rat diabetic neuropathy

Abstract

Endogenous ADP-ribosylation of proteins was studied in retina crude extract, membrane and cytosolic fractions of control and diabetic rats. ADP-ribosyltransferase activity is present in all cellular fractions, but protein ADP-ribosylation is reduced in diabetic rat retina. At least 6 proteins are labelled in the crude extract fraction and a similar number in the membrane preparation of control animals. In these preparations from diabetic retina, only two bands were labelled, the 85 K and 36 K for the crude extract, and the 97 K and 39 K for membranes. Labelling of 36 K and 39 K proteins was much less than in controls. In the cytosolic preparations of controls, two proteins of 85 K and 39 K are ADP-ribosylated, while in diabetic rat retina cytosol, only the 85 K is labelled. Treatment of diabetic rats with insulin normalized plasma glucose levels and prevented the alterations of the extent of ADP-ribosylation for the 38 K cytosolic, 39 K membrane and 36 K crude extracts proteins, but it failed to affect the other bands. These results suggest a hyperactivity of endogenous ADP-ribosylases in diabetic rat retina, so that the protein sites for ADP-ribosylation are no longer available. Since insulin treatment prevents the onset of neuropathy and of retinal G protein impairment (Abbracchio et al., J Neurosci Res 29:196-220, 1991) in diabetic rats and, in this study, normalizes ADP-ribosylation of 39 K, 38 K and 36 K proteins, we suggest that the abnormal endogenous ADP-ribosylation of these proteins might play a role in the onset of diabetic neuropathy.

Inhibition of high glucose-induced protein mono-ADP-ribosylation restores neuritogenesis and sodium-pump activity in SY5Y neuroblastoma cells

“…mono-ADP-ribosylation. The present results confirm a previous hypotheses that alterations of protein mono-ADPribosylation is triggered in experimental diabetes and that such protein posttranslational modification may be involved in the mechanisms leading to the onset of diabetic neuropathy Gorio et al, 1995Gorio et al, , 1996.…”

“…The supplementation to SY5Y neuroblastoma cell culture medium of an inhibitor of protein-mono-ADP-ribosylation, silybin, selectively prevented the neuritogenic and Na ϩ -pump changes induced by 30 mM glucose, whereas the alterations produced by fructose and galactose were unaffected. The restorative effect of silybin was accompanied by the prevention of glucoseinduced excessive protein mono-ADP-ribosylation in SY5Y neuroblastoma cells, thus confirming previous observations linking this protein posttraslational modification to hyperglycemia and to neuronal dysfunction occurring in diabetes mellitus Gorio et al, 1995Gorio et al, , 1996. Our data also suggest that high fructose and high galactose may perturb cells in a similar manner by means of mechanisms different from those affected by high glucose and certainly not via protein 0.03 Ϯ 0.01** † Cells were incubated for 2 weeks in control medium, medium supplemented with 30 mM glucose (Hgluc), with 30 mM glucose ϩ 10 nM silybin (Hgluc ϩ sil), or with 5 mM sodium nitroprussiate (SNP).…”

“…This report shows that 30 mM glucose affect the extent of mono-ADP-ribosylation of proteins in SY5Y neuroblastoma cells; the effect is particularly evident in the bands larger than 100, 71, 63, and 36.5 K. The latter protein band may correspond to the 39-K retina membrane-bound protein described by Pozdnyakov et al (1993) as the ␣-subunit of G-proteins. This subunit in diabetic retina was shown to be less labeled by ADPribose in experimental diabetes and to have its labeling restored by treatment with insulin or with silybin Gorio et al, 1995Gorio et al, , 1996. Also, in the present study, the excessive mono-ADP-ribosylation of this band was prevented by silibyn treatment with the restoration of neurite formation even in presence of high glucose, an effect that is reminiscent of the restoration of axonal transport by silybin treatment of diabetic rats (Gorio et al, 1996).…”

“…This hypothesis would explain the higher susceptibility of sympathetic and sensory nervous system to diabetes. By preventing the excessive protein mono-ADP-ribosylation with silybin, we had also prevented the drop of substance P axonal transport, showing that mono-ADP-ribosylation of protein may be involved in the onset of diabetic neuropathy Gorio et al, 1995;1996).…”

Progressive and selective changes in neurotrophic factor expression and substance p axonal transport induced by perinatal diabetes: Protective action of antioxidant treatment

“…The function of sensory neurons was monitored evaluating substance P axonal transport (Tomlison and Mayer, 1984). A group of pregnant diabetic rats was treated with silybin, a flavonoid with antioxidant activity (Pietrangelo et al, 1995); this compound inhibits the excessive neuronal protein mono-ADP-ribosylation and prevents the reduction of substance P axonal transport in diabetic rats Gorio et al, 1995Gorio et al, , 1996a.…”