Arthur A. Like

Studies on Streptozotocin Diabetes

Both alloxan and streptozotocin produce β-cell necrosis in the rat. Previous studies have shown protection against alloxan toxicity by D-glucose, D-mannose, and the nonmetabolized analogue 3-0-methyl-D-glucose and removal of this protective effect by D-mannoheptulose. The effect of several agents (i.v. infusion) against the β-cell toxic effect of streptozotocin (60 mg./kg. i.v. in 24-hour-fasted 200-gm. male rats) was studied. Protection was determined by plasma glucose concentrations 24 and 48 hours later and, in certain experiments, by histologic examination of the islets. D-glucose and D-mannose provided no protection. Similarly, D-galactose, D-fructose, α-methyl-D-glucoside, D-L-glyceraldehyde, D-xylose, and D-glucosamine had no effect. However, 3-0-methyl-D-glucose administered immediately before streptozotocin resulted in progressive inhibition of β-cell toxicity with complete protection at 0.83 mMoles per rat. The protective effect of 3-0-methyl-D-glucose was not altered by mannoheptulose. 2-Deoxy-D-glucose, which has no effect against alloxan, provided nearly complete protection against streptozotocin at 2.2 mMoles per rat. The effects of 3-0-methyl-D-glucose and 2-deoxy-D-glucose were additive and were not altered by glucose. Furthermore, the 3-0-methyl-D-glucose as well as 2-deoxy-D-glucose protective effects were still present, albeit attenuated, when these agents were given following the administration of streptozotocin. This is in contrast to alloxan, against which 3-0-methyl-D-glucose provides protection only when given before alloxan. 3-0-Methyl-D-glucose is the only carbohydrate protective against both streptozotocin and alloxan in the rat. However, several silent differences seem to exist between the mechanisms of beta-cytotoxic effects of these two diabetogenic compounds.

Embryogenesis of the Human Pancreatic Islets: A Light and Electron Microscopic Study

The developing pancreases of twenty human embryos and fetuses, 8–23 weeks gestational age, were obtained under conditions optimum for cellular preservation and studied by light and electron microscopy. Primitive pancreatic tubules were the only epithelial structures present at 8 and 8.5 weeks. Alpha cells were first identified at 9 weeks, followed by delta and subsequently beta cells at 10.5 weeks. Endocrine cell progenitors, mixed endocrine cells, or mixed exocrine endocrine cells were not identified. Although potential sampling errors preclude a definitive recitation of the order of islet cell appearance, the alpha and delta cells are numerically the predominant pancreatic endocrine cells during early human embryogenesis. The observation of frequent, well-preserved delta cells and the identification of one undergoing mitotic division support their legitimacy as an independent cell type. Cytoplasmic glycogen was present in primitive tubular cells and differentiated exocrine cells but was excluded from endocrine cells. Other endocrine cells tentatively identified in the developing human pancreas are serotonin, gastrin, epinephrine and norepinephrine cells.

Genetic Influence of the Streptozotocin-induced Insulitis and Hyperglycemia

Multiple injections of subdiabetogenic doses of streptozotodn (SZ) to CD-I male mice produce a diabetic syndrome that includes a cell-mediated immune reaction against the pancreatic islet. The importance of the host genetic background in the pathogenesis of this model of diabetes was studied by comparing various inbred strains of mice. Of eight strains of mice studied, only C57BL/KsJ developed insulitis and hyperglycemie comparable to that observed in CD-I mice. In two mouse strains (DBA/2J and BALB/cJ) having an haplotype similar to the C57BL/KsJ, only mild insulitis and glucose intolerance were observed. These data suggest that major histocompatibility complex genes, as presently defined, cannot be the only determinant of the severity of hyperglycemia and insulitis in this model.

Influence of Environmental Viral Agents on Frequency and Tempo of Diabetes Mellitus in BB/Wor Rats

Elimination of environmental viruses by cesarean derivation of the University of Massachusetts colony of BB/Wor rats increased the frequency and accelerated the tempo of spontaneous diabetes among diabetes-prone (DP) rats. In contrast, the viral-antibody–free (VAF) environment did not alter the resistance of pre-VAF diabetes-resistant (DR) rats to spontaneous and RT6+ T-lymphocyte–depletion–induced diabetes. Pre-VAF and VAF rats have essentially the same lymphocyte subsets, and VAF-DP rats are susceptible to the adoptive transfer of diabetes and to the diabetes-accelerating effects of polyinosinic-polycytidylic acid injections. These results suggest that the presence of environmental viral pathogens may act to inhibit effector cell function in lymphopenic DP rats while enhancing effector cell activity in nonlymphopenic DR rats.

Spontaneous Autoimmune Diabetes Mellitus in the BB Rat

The BioBreeding Rat is a recently discovered model of spontaneous diabetes mellitus. Studies to date have revealed the following characteristics of the syndrome: genetic predisposition, equal frequency and severity among males and females, absence of obesity, life sustaining requirement for insulin therapy, lymphocytic insulitis with destruction of pancreatic beta-cells, lymphocytic thyroiditis and the presence of autoantibodies to smooth muscle, thyroid colloid and other cellular antigens. Animals raised in a germ-free environment evidence diabetes with equal frequency and severity. Support for a cell-mediated autoimmune pathogenesis of the diabetic syndrome is derived from the following experiments: administration of antiserum to rat lymphocytes prevents diabetes in susceptible animals and normaliies plasma glucose levels in 36% of diabetic rats; neonatal thymectomy almost completely prevents the occurrence of diabetes. Although the BB rat may not be an appropriate model for studying the vascular complications of diabetes, peripheral nerve functional and ultrastructural defects have been reported and renal glomerular immuneglobulin deposits have been observed in long-term diabetic animals.

Lymphocytic Thyroiditis and Diabetes in the BB/W Rat: A New Model of Autoimmune Endocrinopathy

The Bio Breeding/Worcester (BB/W) rat develops spontaneous insulin-dependent diabetes mellitus secondary to lymphocytic infiltration and destruction of the pancreatic beta-cells. This destructive process in the pancreas has been postulated to be based on a thymus-dependent cell-mediated autoimmune process. In view of the well recognized association in man of diabetes mellitus and another autoimmune endocrinopathy, chronic thyroiditis (Hashimotos thyroiditis), the present studies were carried out to determine whether lymphocytic thyroiditis occurred with increased frequency in the diabetic, insulin-treated BB/W rat. The incidence of lymphocytic thyroiditis was strikingly increased in 8–10-mo-old diabetic rats (59%) as compared with their nondiabetic cohorts (11%) (P < 0.001). Relative thyroid weight was significantly greater in diabetic as compared with nondiabetic rats (P < 0.01) and in diabetic rats with thyroiditis than in diabetic rats without thyroiditis (P < 0.025). Lymphocytic thyroiditis was not accompanied by any consistent changes in serum T4, T3, and TSH concentrations or in the serum TSH response to thyrotropin-releasing hormone (TRH) suggesting that the thyroiditis was not of sufficient severity or duration to induce primary thyroid gland failure. The BB/W rat represents the first animal model of multiple autoimmune endocrinopathies and provides a unique opportunity to study the pathogenesis of these disorders.

Susceptibility to diabetes is widely distributed in normal class IIu haplotype rats.

Aims/hypothesis. We did experiments to explore the pathways putatively leading to Type I (insulin-dependent) diabetes mellitus, and their association with the MHC locus, the major genetic determinant of disease susceptibility.¶Methods. Normal MHC congenic rat strains that do not spontaneously develop diabetes or any other autoimmune syndrome were injected with the interferon-alpha inducer polyinosinic-polycytidylic acid (Poly IC).¶Results. Insulitis and diabetes developed only in strains expressing Class IIu genes and was independent of the Class I haplotype. Poly IC induced islet cell Class I hyperexpression, up regulation of pancreatic endothelial intercellular adhesion molecule-1 and vascular adhesion molecule-1 and a T-cell and macrophage infiltration of the pancreatic interstitium in all rat strains studied, including diabetes-resistant strains. Poly IC also induced the generation of diabetes-transferring spleen cells in most Class IIu haplotype rats, including the diabetes-resistant WF rat.¶Conclusion/Interpretation. The minimum requirements for autoimmune diabetes development in the rat include: RT1 Class IIu genes, a T-cell repertoire containing beta-cell autoreactive T cells and a triggering event which breaks tolerance by the local up regulation of pancreatic endothelial adhesion receptors. Even when all of the minimum requirements have, however, been met, most Class IIu rats do not develop diabetes in response to autoimmune stimuli. It is clear, nonetheless, that susceptibility to diabetes is widely distributed in the RT1u rat. [Diabetologia (2000) 43: 890–898]

Kilham Rat Virus Triggers T-Cell–Dependent Autoimmune Diabetes in Multiple Strains of Rat

Kilham rat virus (KRV) infection of BB/Wor diabetes-resistant (DR) RT1u rats induces autoimmune diabetes without direct cytolytic infection of pancreatic β-cells and is a new model of virus-induced IDDM. To investigate genetic susceptibility to KRV-induced diabetes, major histocompatibility complex congenic and other inbred rats were infected with the virus and studied for the appearance of diabetes and insulitis. KRV infection alone induced insulitis, selective β-cell necrosis, and diabetes in BB/Wor DR and LEW1.WR1 (RT1 Au B/Du Ca) but not other rats. Thus, KRV, an environmentally ubiquitous rat parvovirus, can precipitate autoimmune diabetes in rats that are not susceptible to spontaneous diabetes. If rats are injected with poly(I·C) immediately before KRV infection, diabetes frequency increases to >90% in BB/Wor DR and LEW1.WR1 rats, and PVG.RT1u rats are converted from KRV-resistant to KRV-susceptible status. Susceptibility to KRV-induced diabetes thus requires the presence of class I Au and class II B/Du gene products, which are shared by DR, LEW1.WR1, and PVG.RT1u rats. The RT1u haplotype is not sufficient for susceptibility, however, because while WF rats are RT1u, they resist KRV-induced diabetes. If rats are depleted of RT6.1+ regulatory T-cells before KRV infection, the frequency of diabetes is dramatically increased in DR and LEW1.WR1, but not PVG.RT1u or other rats. These data confirm a regulatory role of RT6.1+ T-cells in diabetes induction, but indicate that they may not operate as such in all rat strains. KRV-induced diabetes is T-cell–mediated: DR and LEW1.WR1 rats are protected from diabetes by treatment with monoclonal antibodies directed against αβ T-cell receptor (TCR)+, CD5+, and CD8+ T-cells. Concanavalin A–activated spleen cells from KRV-infected DR rats adoptively transfer diabetes and insulitis into class IIu compatible rats, suggesting that KRV infection of susceptible rats leads to the activation of diabetogenic class IIu restricted T-cells. The ability of a common rat virus to initiate IDDM in multiple strains of rats strengthens the possibility that viruses may also initiate IDDM in human populations.

Lymphocyte transfusions prevent diabetes in the Bio-Breeding/Worcester rat.

The Bio-Breeding/Worcester (BB/W) rat develops spontaneous autoimmune diabetes similar to human insulin-dependent diabetes mellitus. Transfusions of whole blood from the nondiabetic W-line of BB/W rats prevent the syndrome in diabetes-prone recipients. We report three experiments designed to determine which blood component is protective. In all experiments, diabetes-prone BB/W rats 23 to 35 d of age were given four or six weekly intravenous injections. In the first experiment, animals received either saline or transfusions of erythrocytes, white blood cells, or plasma from W-line donors. Diabetes occurred in 7/22 (32%) erythrocyte, 2/27 (7%) white cell, 14/24 (58%) plasma, and 15/27 (56%) saline recipients (P less than 0.001). At 120 d of age, peripheral blood was obtained from nondiabetic rats. Fluorescence-activated cell sorter analysis of OX 19 tagged leucocytes revealed 35% T lymphocytes in white cell recipients (n = 13), compared with 9% in saline recipients (n = 7; P less than 0.001). Responsiveness to concanavalin A was also increased in the white cell group, whereas the frequency of both insulitis and thyroiditis was decreased. In the second experiment, 1/19 (5%) rats transfused with W-line spleen cells developed diabetes, as contrasted with 12/18 (67%) recipients of diabetes-prone spleen cells and 19/31 (61%) noninjected controls (P less than 0.001). In the third experiment, diabetes-prone rats received either W-line blood treated with a cytotoxic anti-T lymphocyte antibody plus complement, untreated blood, or saline. Diabetes occurred in 8/20 (40%), 1/20 (5%), and 13/19 (68%) rats in each group, respectively (P less than 0.001). We conclude that transfusions of W-line T lymphocytes prevent diabetes in the BB/W rat.

Autoantibodies in the BB/W Rat

The BioBreeding/Worcester (BB/W) rat is a model of spontaneous autoimmune diabetes mellitus and lymphocytic thyroiditis. Additional features supporting an immunologic pathogenesis of the BB/W syndrome include the protective action of antilymphocyte serum, neonatal bone marrow transfusions, and neonatal thymectomy. To evaluate other manifestations of immune dysregulation, the BB/W colony was surveyed for the presence of autoantibodies to a variety of tissue and cell constituents. Anti-smooth muscle and anti-thyroid colloid antibodies were present with great frequency in diabetic animals as well as in normoglycemic offspring of diabetic parents. Anti-parietal cell antibodies were less frequent and islet cell cytoplasmic and adrenal antibodies were not detected. These data suggest that the underlying defect in the BB/W rat is more likely to be an abnormal immune regulatory system than antigenically altered target tissues (“altered self”) under attack by a normal immune surveillance system.