Effect of modified diabetic splenocytes on mice injected with splenocytes from multiple low-dose streptozotocin diabetic donors.

Etiological agents of autoimmune processes that have been made nonvirulent by several treatments, i.e., mitomycin C (Mit C), can be used as a vaccine to protect against disease. In this work we studied the effects of splenocytes from diabetic mice on animals that had been injected with modified splenocytes (Mit C-treated splenocytes from multiple low-dose streptozotocin [mld-sz] mice) 15 days before. Splenocytes from mld-sz diabetic donors altered i.p. glucose tolerance and the first peak of insulin secretion pattern when injected into normal singeneic recipients. These effects can be prevented partially (one injection in a vaccine form) or completely (two injections with a 15-day interval) by a previous injection of Mit C-treated mononuclear splenocytes (MS) from mld-sz mice. The fact that control splenocytes previously treated with Mit C were not able to achieve similar results indicates that donor splenocytes have to be diabetic to prevent the disease. On the other hand, Mit C-treated diabetic MS were not effective in preventing the alterations in glucose tolerance and in the pattern of insulin secretion when injected into athymic mice. This suggests that the preventive effect of Mit C-treated diabetic MS injection also implies an active role of the T cells from the recipient mice. Mit C-treated diabetic splenocytes are preferentially trapped by the pancreas and the lymph nodes from recipient mice. Our results show that the impairment in glucose tolerance and in the insulin secretion pattern produced by diabetic splenocyte transfer can be prevented by one or two previous injections of Mit C-modified diabetic splenocytes.

Branched-chain amino acid-enriched diet: effects on insulin secretion and cellular immune aggression.

Several reports have demonstrated that high-protein diets may have beneficial effects on experimental models of diabetes and have raised the possibility that branched-chain amino acids could play a role in these protective effects. We investigated the effect of a normoproteic, branched-chain amino acid-enriched diet (experimental diet) on insulin secretion from C57BL/6N mice transferred with splenocytes from diabetic syngeneic donors. Mice previously fed with the experimental or control diet received three intraperitoneal injections, every other day, of 5 x 107 viable mononuclear splenocytes obtained from control or diabetic donors. Results showed that mice fed with the experimental diet and transferred with "diabetic" splenocytes presented: i) normoglycemia, and (ii) significantly higher levels in both phases of glucose-induced insulin secretion and normal values of arginine-glucose-induced insulin secretion. To evaluate the in vitro cellular immune aggression, dispersed mouse islet cells were co-cultured with splenocytes from syngeneic diabetic mice. First, dispersed islet cells from mice on the experimental or control diet were co-cultured with splenocytes from control or diabetic mice on a commercial diet. In the presence of "diabetic splenocytes, dispersed islet cells from mice on the experimental diet presented a significantly lower in vitro cellular immune aggression. On the other hand, "diabetic" splenocytes from mice fed with the experimental diet produced a significantly reduced cellular immune aggression on dispersed islet cells. Our results showed that feeding branched-chain amino acids increased the capacity of beta cells to withstand a functional assault and diminished the extent of in vitro cellular immune aggression.

Inhibition of nitric oxide generation: normalization of in vitro insulin secretion in mice with multiple low-dose streptozotocin and in mice injected with mononuclear splenocytes from diabetic syngeneic donors.

We studied the effect on in vitro glucose-induced insulin secretion of in vivo administration of L-Ng-monomethyl-arginine (L-NMMA), a competitive inhibitor of nitric oxide (NO) synthase, to mice injected with multiple low-dose streptozotocin (mld-SZ). In addition, the effect of L-NMMA treatment on the capacity of mononuclear spleen cells (MS) from mld-SZ mice to transfer alterations in insulin secretion from normal syngeneic receptors was also investigated. We also studied the effect of in vivo treatment with L-NMMA on anti-beta-cell cellular immune aggression (CIA) by coculturing MS from mld-SZ mice with rat dispersed islet cells. Our results show that mld-SZ mice treated with 0.25 mg L-NMMA/g body weight had normoglycemia, first and second-phase glucose-stimulated insulin secretion similar to those obtained in nondiabetic mice-effects not observed with a lower dose of L-NMMA (0.17 mg/g body weight)-and a diminished anti-beta-cell CIA. We also demonstrate that mice injected with MS from syngeneic donors treated with mld-SZ plus 0.25 mg L-NMMA/g had normal levels for first-phase glucose-stimulated insulin secretion and an absence of CIA. Taken together, these findings seem to indicate that prevention of in vivo NO production may block the onset of diabetes in mld-SZ mice, and that L-NMMA administration to diabetic donor mice prevents inhibition of first-phase insulin secretion and CIA in the transferred recipient mice. Although a nonimmunological mechanism or mechanisms of diabetes prevention by L-NMMA cannot be excluded, these results suggest that L-NMMA treatment could also be acting on T-cell-dependent immune reactions.