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.

Insulin secretion by pancreas of athymic mice injected with peripheral mononuclear cells from insulin-dependent diabetic patients.

We studied the effect of peripheral blood mononuclear cells (PBMNC) from insulin-dependent diabetic (IDDM) children on the insulin secretion pattern of the pancreas from recipient athymic mice. PBMNC from healthy controls or IDDM patients in different stages of disease were injected into athymic mice. PBMNC from newly diagnosed IDDM children elicited basal nonfasting hyperglycemia and in vitro inhibition of the first and second phases of glucose-stimulated insulin secretion in recipient mice. Animals injected with cells from chronically IDDM children showed normoglycemia, abnormal tolerance to glucose, and inhibition of first-phase insulin secretion. Mitomycin C treatment of MNC from IDDM patients abolished insulin secretion inhibition in recipient mice. PBMNC from newly diagnosed and chronically IDDM patients showed positive anti-beta-cell cellular immune aggression. Mice injected with cells from patients during the remission period showed normoglycemia and no alteration of insulin secretion patterns. When relapsed to their former clinical stage, injection of the cells significantly inhibited first-phase glucose-induced insulin secretion in recipients. PBMNC from newly diagnosed IDDM patients were found to migrate to the pancreas of recipient mice preferably as compared with cells from controls. Cells from chronically IDDM patients cultured with concanavalin A (Con A) increased insulin secretion inhibition; despite this, cells from children during the remission period cultured with Con A failed to modify insulin secretion in recipients. These results show that injection of PBMNC from diabetic patients leads to insulin secretion impairment in recipient mice pancreas, and provide a basis for the study of mechanisms involved in the onset and modulation of anti-beta-cell cellular immune aggression induced by human PBMNC.

Beta-cell function in mice injected with mononuclear splenocytes from multiple-dose streptozotocin diabetic mice.

Multiple low doses of streptozotocin (mid sz 40 mg/kg/day, five consecutive days) induce autoimmune diabetes in mice. The aim of the present work was to study beta-cell function in mice injected with splenocytes from mid-sz diabetic mice. Mononuclear splenocytes (MS) from control or diabetic donors were injected into syngeneic C57BL/6J healthy mice (5 x 10(7) MS, ip). MS from diabetic donors did not produce basal hyperglycemia, but they induced abnormal ip glucose tolerance in recipient mice. These "diabetic" MS were also preferentially trapped by the recipient's pancreas. Perifused pancreas from mice injected with MS from mid sz-diabetic donors showed a diminished first and second phase of glucose-induced insulin secretion after 15 days of the cell injection. At this time, pancreatic insulin content among MS recipients did not differ from that found in controls or diabetic donors. Diabetic MS treated with Mitomycin C prior to transfer did not inhibit insulin secretion in recipient mice. Injection of MS from mice made diabetic by a single high sz dose (200 mg/kg) did not induce any alterations of the insulin secretion in recipients. There is enough evidence when using athymic and euthymic (BALB/c nu/nu and +/nu) mice to suggest that proliferation of the injected splenocytes enhanced the progression to the diabetic state, and that both donor and recipient T lymphocytes played an important part in this progression. The results suggest that injection of MS from mid sz-diabetic mice interfere with glucose-stimulated insulin secretion in recipient mice and provide a basis for the study of the mechanisms involved in the onset and modulation of autoimmune pancreatic aggression.

Cellular and humoural autoimmunity markers in type 2 (non-insulin-dependent) diabetic patients with secondary drug failure.

In some cases patients with Type 2 (non-insulin-dependent) diabetes mellitus fail to respond to treatment with oral hypoglycaemic agents. These patients may respond in the same way as Type 1 (insulin-dependent) diabetic patients. Cellular immune aggression (defined as the capacity of peripheral mononuclear cells to inhibit stimulated insulin secretion by dispersed rat islet cells), insulin autoantibodies, C-peptide response and HLA antigens were determined in 31 Type 2 diabetic patients with secondary failure to oral hypoglycaemic agents and in 22 control subjects. Nine (29.03%) of the 31 Type 2 diabetic patients showed positive cellular immune aggression (2 SD below control group) and 22 (70.97%) presented no cellular immune aggression. There was a relationship between positive cellular immune aggression and each of the following parameters: age, body mass index and microangiopathy. No correlation was found between positive cellular immune aggression and glycaemia, HbA1, blood lipids or atherosclerosis. Patients with positive cellular immune aggression showed a significantly lower glucagon-stimulated C-peptide response vs those with no cellular immune aggression. Within a sub-group of patients who had never been treated with insulin, insulin autoantibodies were present in four of six patients with positive cellular immune aggression. DR2 antigen was found with decreased frequency in patients whereas no DR3/DR4 heterozygotes were observed. Our data support the hypothesis that a group of Type 2 diabetic patients with secondary failure to oral hypoglycaemic agents presented autoimmunity towards pancreatic Beta cells.

Insulin secretion stimulated by allogeneic lymphocytes in an inbred strain of mice.

Effects of intraperitoneal injection of allogeneic lymphocytes on insulin secretion were studied in incubated pancreas slices from BALB/c mice. Injection of allogeneic lymphocytes from C57BL/6J (H2b) mice increased insulin secretion, both in basal and 11-mM glucose-stimulated conditions. This effect was only present when at least 5 X 10(6) or 1 X 10(6) cells were injected (in basal and stimulated conditions, respectively). Glucose-induced insulin secretion (3.3-27.5 mM) was significantly increased in pancreata from mice injected with allogeneic lymphocytes. No effect was observed when glucose was not included in the incubation medium. Intraperitoneal injection of Dextran 70 produced no change in glucose-elicited insulin secretion. There were no differences in glucagon and somatostatin (SRIF) secretion obtained from pancreas of mice injected with allogeneic or syngeneic lymphocytes. Injection of allogeneic cells increases insulin secretion (basal and both phases of 11 mM glucose-stimulated secretion). Puromycin significantly inhibited the second phase of insulin secretion. These results suggest that: Injection of allogeneic lymphocytes raises both basal and glucose-stimulated insulin secretion. This effect seems to be connected with the major histocompatibility complex, and to be related to the number of allogeneic cells injected. Injection of allogeneic lymphocytes seems to sensitize the beta cell response to glucose stimulus. Neither glucagon nor SRIF secretion are altered by alloantigen injection. The stimulatory effect of allogeneic lymphocytes is related, at least in part, to insulin synthesis.

A comparative study of two insulin secretion inhibitors: somatostatin and diazoxide.

We studied the effect of the ionophere A 23187 and of phosphodiesterase inhibitors and activators (Theophylline, Pentoxiphylline and Imidazol) on insulin secretion and on the pool of free tubulin in rat pancreas in the presence of somatostatin and diazoxide. The results suggest that: 1. The inhibitory effect of somatostatin on insulin secretion does not seem to be related mainly to an inhibition of cAMP production. The decrease in calcium translocation induced by somatostatin could inhibit the cAMP participation in the mechanism of hormonal secretion. 2. Somatostatin seems to inhibit the movement of calcium towards the cytoplasm from outside and from within the cell. Diazoxide seems to inhibit only the entrance of calcium from outside the cell but does not seem to inhibit the entrance determined by theophylline and pentoxiphylline from intracellular compartments. 3. Arginine glucose stimulation in the presence of A 23187-induced calcium translocation is able to determine insulin secretion although cAMP degradation is increased by imidazol. 4. Somatostatin and diazoxide inhibit pancreatic tubulin polimerization; however, the effect seem to be indirect and related to the inhibition of calcium translocation determined by both substances.