Targeted deletion of the Vgf gene indicates that the encoded secretory peptide precursor plays a novel role in the regulation of energy balance.

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Multiple low-dose streptozotocin-induced diabetes in the mouse. Evidence for stimulation of a cytotoxic cellular immune response against an insulin-producing beta cell line.

Mice were examined for the presence of splenocytes specifically cytotoxic for a rat insulinoma cell line (RIN) during the induction of diabetes by streptozotocin (SZ) in multiple low doses (Multi-Strep). Cytotoxicity was quantitated by the release of 51Cr from damaged cells. A low but statistically significant level of cytolysis (5%) by splenocytes was first detectable on day 8 after the first dose of SZ. The cytotoxicity reached a maximum of approximately 9% on day 10 and slowly decreased thereafter, becoming undetectable 42 d after SZ was first given. The time course of the in vitro cytotoxic response correlated with the degree of insulitis demonstrable in the pancreata of the Multi-Strep mice. The degree of cytotoxicity after Multi-Strep was related to the number of effector splenocytes to which the target RIN cells were exposed and was comparable to that detectable after immunization by intraperitoneal injection of RIN cells in normal mice. The cytotoxicity was specific for insulin-producing cells; syngeneic, allogeneic, and xenogeneic lymphocytes and lymphoblasts, 3T3 cells, and a human keratinocyte cell line were not specifically lysed by the splenocytes of the Multi-Strep mice. This phenomenon was limited to the Multi-Strep mice. Splenocytes from mice made diabetic by a single, high dose of SZ exhibited a very low level of cytotoxicity against the RIN cells. The cytotoxic response was also quantitated in splenocytes from control and Multi-Strep mice (10 d after the first dose of SZ) before and after culture with mitomycin-treated RIN cells in the presence of T cell growth factor (TCGF). The cytotoxicity of the Multi-Strep splenocytes was enhanced more than fivefold after such culture, suggesting the proliferation of an effector cell that could be stimulated and supported in vitro by TCGF. These results support the hypothesis that cell-mediated anti-beta cell autoimmunity may play a role in the destruction of the beta cells in this animal model. The stimulation of this response by TCGF may provide a tool by which enough cytotoxic effector cells could be obtained to establish their possible direct pathogenetic role in the induction of insulin-dependent diabetes. In addition, such cells will be a valuable tool to define the specific beta-cell antigens that may direct the highly selective cell-mediated destruction of these cells in experimental models and, perhaps, in human insulin-dependent diabetes mellitus.

Virus-induced alterations in insulin release in hamster islets of Langerhans.

After the inoculation of Golden Syrian hamsters with the TC-83 vaccine strain of Venezuelan encephalitis (VE) virus, a sustained diminution in glucose-stimulated insulin release and glucose intolerance of shorter duration develops. To understand better the mechanism of this defect in insulin release, we examined insulin secretion in response to several test agents in isolated perifused islets from control and 24-d post-VE virus-infected hamsters. 50 islets were used in all perifusion experiments, and data were expressed as total insulin released as well as peak response for each test agent during a 30-min perifusion period from control and VE-infected islets. After perifusion with 20 mM glucose, a 45% diminution of insulin release was noted in VE-infected islets in comparison with control islets, which in turn was similar to in vivo findings. However, following 1-mM tolbutamide stimulation, insulin release was similar in control and VE-infected islets. In separate studies, 1 mM tolbutamide, 10 mM theophilline, 1 mM dibutyryl cyclic (c)AMP, and 1 mM 8-bromo-cAMP resulted in statistically similar insulin-release curves in control and VE-infected islets. Additional experiments assessing [5-3H]glucose use in control and infected islets after 20 min of perifusion with 20 mM glucose revealed virtually identical values (239 +/- 30-control; and 222 +/- 27-VE-infected islets). Morphological and morphometric evaluation of VE-infected islets (21 d following virus inoculation) showed no changes in islet volume density, beta cell density, and beta cell granulation. Thus, VE virus induces a defect in glucose-stimulated insulin release from hamster beta cells that can be corrected by cAMP analogues and does not alter islet glucose use.

Changes in the volumes of the A-, B-, and D-cell populations in the pancreatic islets during the postnatal development of the rat.

The changes in the volumes of three of the principal islet cell types in the developing rat pancreas were quantitated from day 10 to day 210 of life. The insulin-, glucagon-, and somatostatin-positive cell populations of the islets were identified by immunocytochemical staining, and their volumes were determined by linear scanning. The insulin and glucagon content of the pancreata, and the concentrations of these hormones in plasma, were also determined at each age by radioimmunoassays. The volumes of the A- and D-cells reached their maximum values by day 50 and day 35, respectively. The content of glucagon within the pancreas reached adult levels by day 50. In contrast, the volume of B-cells and the insulin content were highest on day 210. The concentrations of both insulin and glucagon in the plasma reached adult levels by approximately day 50. There were no differences in the pancreatic parameters between male and female rats until day 25, when the wet weight of the male gland became significantly greater. The concentrations of the islet hormones and the percentages of the islet cell types within the pancreas did not differ between the sexes at any age. However, the greater weight of the pancreas resulted in a greater total content of islet cells and hormones in the male gland after day 25. The data suggest that, in the rat, the B-cell volume may continue to increase with age, while the A- and D-cells apparently do not. The physiologic consequences of these changes remain to be determined.

Syngeneic transplantation of fetal rat pancreas. III. Effect of insulin treatment on the growth and differentiation of the pancreatic implants after reversal of diabetes.

Eight 18-316 fetal pancreases were transplanted to syngeneic alloxan diabetic male rats. Some of the recipients were treated with insulin for a 7-day period immediately after transplant. By previously published clinical criteria, three groups of recipients could be identified after reversal of diabetes by the transplanted tissue: insulin-treated rapid reversal; insulin-treated slow reversal; and control (not treated with insulin). Five animals in each group were sacrificed after glucose tolerance testing for morphologic and hormonal analysis of the transplanted tissue. The insulin-,glucagon-, and somatostatin-positive islet cell masses of the fetal pancreatic implants were quantitated. There was a correlation between the beta cell mass of the implants and the glucose tolerance exhibited by the host animals. The rapid response insulin-treated recipients had significantly greater implant beta cell mass and insulin content compared with the other groups. There was no difference in implant alpha cell mass among the groups, but the insulin-treated implants had a significantly greater glucagon content. The delta cell mass of insulin-treated rapid response was less than that of the other two groups. The results are discussed in relation to previously reported morphometric analysis 15 days after transplantation. The relationships of transplanted beta cell mass, beta cell differentiation, transplant site, and cell-to-cell interactions within the transplanted islet to the control of glucose homeostasis are also discussed.

Syngeneic transplantation of fetal rat pancreas. II. Effect of insulin treatment on the growth and differentiation of pancreatic implants fifteen days after transplantation.

Eight 18-days-postcoitum fetal pancreases were transplanted to isogenic alloxan-diabetic male rats. Some recipients were treated with insulin for seven days immediately after transplantation. Eight animals in both the insulin-treated group and control group were killed 15days after transplantation for morphologic and hormonal studies of the transplanted tissue. Using the morphometric technique of linear scanning, the insulin, glucagon, and somatostatin immunocytochemically positive, cell masses of the fetal pancreatic implants were quantitated. The beta cell mass of the implants from the control animals increased roughly eightfold from the time of transplant; insulin treatment resulted in a further two- to threefold increase. The insulin content of the implants increased more than did the beta cell mass, resulting in the fivefold increase in insulin per beta cell. The alpha cell and delta cell masses did not change during the transplant site, the mass of functional beta cells, and the cell-to-cell content of the implanted tissue. These results are discussed in relation to previous quantitative studies of pancreatic islet cell growth. The relationships of the transplant site, the mass of functional beta cell, and the cell-to-cell interaction within the islet to the maintenance of glucose homeostasis are also discussed.

Morphometric quantitation of the pancreatic insulin-, glucagon-, and somatostatin-positive cell populations in normal and alloxan-diabetic rats.

The pancreatic insulin-, glucagon-, and somatostatin-positive cell populations were quantitated in normal and alloxan-diabetic rats. The method of quantitation (linear scanning) allowed an estimation of absolute changes in these cell populations through 14 months of diabetes. The changes in cell masses were correlated with changes in plasma and pancreatic immunoreactive insulin and glucagon. A marked reduction in the insulin-positive beta cells was demonstrated within seven days after alloxan treatment. No significant change in the glucagon-positive alpha cell population was noted in the diabetic rats when compared with normoglycemic controls. A statistically significant increase in the pancreatic somatostatin-positive delta cell population was demonstrable only after 14 months of alloxan diabetes. The results would suggest that the hyperglucagonemia of insulin-deficient diabetes is not a consequence of an increased pancreatic alpha cell population. In addition, since the increase in the pancreatic delta cell mass was found only late in the course of alloxan diabetes in the rat, the increase in delta cells is probably not of significance in the pathophysiology of diabetes in this experimental model.

Transplantation of fetal rat islets into the cerebral ventricles of alloxan-diabetic rats. Amelioration of diabetes by syngeneic but not allogeneic islets.

Syngeneic fetal rat islets were isolated and transplanted into alloxan-diabetic inbred male Lewis rats. The effect of transplantation of islets into the cerebral ventricles on the diabetic state of the recipients was compared with that of the conventional transplantation of islets homeotypically into the liver via the portal vein. Fourteen of fourteen rats surviving after stereotaxic implantation of islets into the ventricles returned to normoglycemia; normoglycemia has been maintained for up to 34 wk. Glucose tolerance tests revealed an improved, although not completely normalized, pattern. Histologic examination of the brains of these recipients revealed clusters of intact islets in the ventricle. These data provided a physiologic basis for further investigation of the immunologically privileged nature of the intraventricular space as a site for implantation of allogeneic pancreatic islets. Islets from Wistar-Furth rats (major histocompatibility difference) or Fischer 344 rats (minor histocompatibility difference) were transplanted into the ventricles of alloxan-diabetic Lewis rats. There were only small and unsustained changes in body weight and blood and urine glucose of any of the rats receiving the allogeneic islets. Histologic examination of the ventricles of these rats 3 wk after transplantation revealed only glial scar tissue. These data suggest that the cerebral ventricles cannot serve as a privileged site for islet transplantation, at least using the type of islet preparation employed in these experiments.

Anti-insulin antibodies in children with type I diabetes mellitus. Genetic regulation of production and presence at diagnosis before insulin replacement.

We evaluated the production of antibodies against insulin in a genetically well-defined population. In the first study, 124 young patients with type I diabetes for longer than 6 mo were included. Anti-insulin antibodies were detected by polyethyleneglycol (PEG) precipitation after incubation of acidified, charcoal-stripped sera with 125I-labeled pork insulin and were expressed as microunits insulin bound per milliliter whole serum. For comparison, the patients were divided into six groups based on HLA DR antigens: 3/3, 3/-, 4/4, 4/-, 3/4, and -/-(-is non-DR3 or -DR4). The mean age of the patients was 14.7 +/- 0.5 yr; the duration of diabetes was 5.8 +/- 0.4 yr; and the glucose control, as measured by hemoglobin A1c was average (7.6 +/- 0.2%). There were no significant differences in any of these parameters among the patients in any of the HLA DR groups. Patients expressing DR3/3 had significantly lower insulin binding than the rest of the groups (2.5 +/- 0.4 vs. 13.6 +/- 1.4 microU/ml, P less than 0.0001). Patients with DR3/ - did not differ in insulin-binding capacity from the other groups. The type of insulin used for replacement was not correlated with the serum insulin-binding capacity. In a second study, sera from 48 children, newly diagnosed with type I diabetes, were examined for the presence of insulin binding before treatment with exogenous insulin and compared with sera from 80 children without diabetes or a family history of diabetes and from 103 unaffected HLA-identical or haploidentical siblings of a child with type I diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of MtTW15 mammosomatotropic tumors on pancreatic islet hormones.

The effects of hypersecretion of growth hormone and prolactin on islet endocrine cells have been studied by radioimmunoassays, immunocytochemistry, and morphometry in randomized samples of pancreata from MtTW15 mammosomatotropic tumor-bearing and control rats. The randomized sampling procedure, validated by immunoassays, allowed evaluation of both hormone content (immunoassay) and endocrine cell population (immunocytochemistry) on samples derived from the same origin. Hyperinsulinemia (2x) and non-fasting hypoglycemia in 10-wk-tumor rats were normalized 3 wk after tumor removal. Pancreatic weight was doubled, but proportional to body weight increases. Islet/pancreas ratio was constant (1.29 +/- 0.05%) and the same in tumor, tumor-removed, and control animals, but average islet dimensions were increased by 30% and average area doubled in tumor animals. Frequency analysis showed fewer small (less than 70 micrometers) and more large (greater than 140 micrometers) islets in tumor animals, but no change in average islet shape shown by average axis ratios of 1.4 in all groups. Pancreatic content of insulin and glucagon was doubled, while that of somatostatin was constant. These changes were not completely reversed in tumor-removed animals. Similarly, a significant doubling in islet-derived mass was mainly due to a doubling of the B-cell mass as the average proportion of endocrine cells per islet shifted from 66%, 26%, and 18% to 81%, 18%, and 3% for B-, A-, and D-cells of control and tumor-bearing rats, respectively. Immunocytochemically detectable insulin was found in duct cells of tumor animals, but not controls. Whether such cells represent a functional reserve remains to be determined.

Syngeneic transplantation of fetal rat pancreas. I. Effect of insulin treatment of the reversal of alloxan diabetes.

Sixty-nine alloxan-diabetic male Fischer rats received syngeneic transplants of eight 18-days-postcoitum fetal pancreases at the renal subcapsular site. One half of the recipients were given 2 to 4 U. protamine-zinc insulin for seven days immediately after transplantation. This insulin-treatment regimen effectively normalized blood glucose rapidly. Forty-seven transplant recipients survived, and diabetes was reversed in all. Insulin treatment had no effect on recovery time or glucose tolerance. Those animals requiring longer periods to reach normoglycemia had impaired glucose tolerance. Some insulin-treated recipients returned to normoglycemia rapidly while others required an extended period. Those animals that showed rapid reversal exhibited elevated concentrations of plasma insulin both in the fasting state and during glucose tolerance tests. No pretransplant parameters could be identified as predictors of rapid reversal.

Strong association between diabetes and displacement of mouse anti-rat insulinoma cell monoclonal antibody by human serum in vitro.

In an attempt to identify novel pancreatic beta-cell surface antigens, mouse monoclonal antibodies (MoAbs) were raised against rat insulinoma (RIN5F) cells with standard techniques. Several clones were identified whose antibodies bound specifically to RIN5F cells but not to other rat, mouse, and human target cells. Each of these MoAbs was radiolabeled, and the specificity of binding of each MoAb was determined by the ability of excess cold homologous MoAb to displace the labeled MoAb. Six RIN5F cell-specific MoAbs of different epitopic specificities were identified. The relevance of these beta-cell epitopes to human insulin-dependent diabetes (IDDM) was demonstrated by the differential ability of human serums from control and diabetic children to displace the radiolabeled MoAbs from the RIN5F cells. Serums from 333 children without diabetes or a family history of diabetes and from 156 newly diagnosed IDDM patients were tested. Only one IgM MoAb was specifically displaced by the IDDM serums, i.e., 146 of 156, compared to serums from control children, i.e., 10 of 333. With immunofluorescence, the serum component responsible for the displacement of the mouse MoAb was identified as IgG. Most of the positive control serums were from children with active autoimmune thyroiditis. Serums from children with other forms of glucose intolerance did not displace MoAb 1A2. There was no correlation between age and the degree of displacement of 1A2. Thus, the displacement of 1A2 is a specific and sensitive marker of diabetes susceptibility easily applicable to mass screening.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic control and quality-of-life self-assessment in adolescents with IDDM.

OBJECTIVE: To examine the relation between metabolic control and self-assessed quality of life in adolescents with IDDM. RESEARCH DESIGN AND METHODS: The Diabetes Quality of Life (DQOL) questionnaire for youths was given to 69 subjects with IDDM aged 10-20 years at the time of their outpatient visit. Subjects with IDDM of < 1 year's duration or with documented psychotic disorder or mental retardation were excluded. Metabolic control was assessed by the mean HbA1c during the preceding year (long-term), by a single HbA1c at the time of the visit (short-term), and by the number of acute events related to IDDM in the preceding year. RESULTS: The DQOL score correlated with mean HbA1c (beta = 6.13, R2 = 0.22, P = 0.0122) and single HbA1c (beta = 3.94, R2 = 0.18, P = 0.05). Self-health assessment was the best predictor of DQOL score (beta = -44.42, R2 = 0.45, P < 0.0001). The Worries subscale score on DQOL correlated with the occurrence of acute events (beta = 6.97, R2 = 0.2, P = 0.006), but did not correlate with either HbA1c level. Correlations of mean HbA1c with the predictors were stronger than the correlations of single HbA1c with the same predictors. CONCLUSIONS: Metabolic control and quality of life are two important outcomes of IDDM care. In our study, adolescents in better metabolic control report better quality of life. Both components need to be addressed in developing successful diabetes treatment strategies for adolescents with IDDM.

Tissue culture of fetal rat islets: corticosterone promotes D cell maintenance and function.

Fetal pancreatic islets were cultured using a recently described technique (1). After 1 day in culture, half of the plates were continued in control medium and half were grown in identical medium supplemented with corticosterone (0.1 microgram/ml). Media were renewed daily, and culture was continued for a total of 8 days. Insulin, glucagon, and somatostatin contents in the media were determined daily. These hormones were also estimated in the tissue at the time of plating, and after 1 and 8 days in culture. Islets were fixed on day 8, and the cells containing each of these hormones were identified by immunocytochemical staining. Corticosterone supplementation of the medium resulted in a 3-fold increase in the somatostatin concentration of the medium. The insulin and glucagon contents of the supplemented medium were slightly reduced. On day 8, there was no difference in the insulin content of the cultured tissue regardless of medium. The glucagon and somatostatin contents of the tissue grown in the steroid-supplemented medium were greater (1.8- and 3.1-fold, respectively) than those of the tissue grown in control medium. D cells were rare in the islets grown in control medium volume density, 0.4%, but were more numerous in the islets maintained in supplemented medium (2.2%). Islets grown in corticosterone-supplemented medium lacked an insulin secretory response to 22 mM glucose. These findings indicate that the volume densities of the cells within the islets can be altered during an 8-day period in culture, suggesting that nutritional and other requirements of the individual subpopulations of islet cells may be different. In addition, corticosterone may prevent the maturation of the secretory responsiveness of cultured B cells to glucose.

Insulin-like growth factor I receptor expression and function in fibroblasts from two patients with deletion of the distal long arm of chromosome 15.

Most patients with deletion of the distal long arm of chromosome 15 have intrauterine growth retardation and postnatal growth deficiency in addition to developmental abnormalities. It has been proposed that the absence of one copy of the insulin-like growth factor I (IGF-I) receptor gene may play a role in the growth deficiency seen in this syndrome. To address this question we examined IGF-I receptor expression and function in fibroblasts from two patients with deletion of the distal long arm of chromosome 15 (15q26.1-->qter). Quantitative Southern blot analysis of the IGF-I receptor gene was performed on HindIII digests of fibroblast DNA. Radioactivity in the 1.7-kilobase receptor fragment in the two patients was 55% and 51% of the values in controls, consistent with the absence of one copy of the IGF-I receptor gene. IGF-I receptor messenger ribonucleic acid levels were quantitated by a solution hybridization/nuclease protection assay. Receptor messenger ribonucleic acid levels in the two patients were 45% and 52% of the values in controls. Northern blotting demonstrated normal size IGF-I receptor transcripts and affinity crosslinking of [125I]IGF-I to Triton X-100-solubilized fibroblasts demonstrated a normal size receptor in the patients. Analysis of placental membranes prepared from one patient revealed no difference in [125I]IGF-I binding. In the patients' fibroblasts, however, binding of [125I]long [R3]-IGF-I to the IGF-I receptor was significantly reduced, as assessed by the amount of radioactivity competed by the monoclonal antibody alpha IR-3 or insulin and Scatchard analysis of binding data. To assess IGF-I receptor function, stimulation of [alpha-1-14C]-methylaminoisobutyric acid transport and stimulation of [methyl-3H]thymidine incorporation into DNA by a full range of IGF-I concentrations was examined in patient and control fibroblasts. There was a significant decrease in the maximal response to IGF-I in both assays for one of the two patients when data were expressed as fold response over the basal value. However, there was no evidence for impairment of response to IGF-I in either patient's fibroblasts when data were expressed as net stimulation (maximal response minus basal). In conclusion, although IGF-I receptor expression was decreased in fibroblasts from two patients with deletion of the distal long arm of chromosome 15, we were unable to provide conclusive evidence for impairment of the biological response to IGF-I.

Escape from self-tolerance leads to neonatal insulin-dependent diabetes mellitus.

Double transgenic (dTg) mice expressing the hemagglutinin (HA) of influenza virus under the insulin promoter and the TCR specific for the immunodominant CD4 T cell epitope of HA (HA110-120) develop insulin-dependent diabetes mellitus (IDDM). In order to gain information on the breaking down of neonatal self-tolerance we studied the occurrence of IDDM after birth. Our results showed that newborn mice develop fulminant IDDM characterized by occurrence of insulitis as early as 3 days after birth, followed by hyperglycemia by 7 days, and significant hypoinsulinemia by 28 days. The neonatal breakdown of self-tolerance of T cells positively selected in the thymus is supported by the facts that: (i) peripheral HA110-120 specific T cells from neonates are fully functional and proliferated upon stimulation with the nominal peptide, and (ii) peptide-specific T cells were accumulated in the pancreas of dTg mice as early as 3 days after birth. Our results demonstrate that diabetes occurring in young dTg mice is due to early activation of self-reactive T cells immediately after birth. Accumulation of specific T cells in the target organ leads to destruction of pancreatic beta-cells and IDDM. These mice may provide a useful model to evaluate new strategies for the prevention of diabetes.

Syngeneic transplantation of the fetal rat pancreas IV. Dissociated versus whole organ implantation.

Reversal of insulinopenia, hyperglycemia, glycosuria, and polyuria associated with severe alloxan diabetes in the rat was accomplished by syngeneic transplantation of whole late-gestation fetal rat pancreata. Intravenous glucose tolerance test (GTT) revealed an improved yet still abnormal glucose and insulin response in reversed recipients reconstituted with as few as two pancreata from fetal donors. Eight fetal donors were sufficient to return glucose and insulin response following GTT to normal. Seventy to eighty percent fewer donors were required when the pancreata were transplanted in their entirely as opposed to transplantation of pancreata subjected to prior enzymatic and mechanical dissociation. The facility and simplicity of the whole fetal pancreas implantation technique makes it an appealing model for further study of islet growth and differentiation at the transplant site and of its effect on the metabolic state of the recipient.

Evidence for the presence of serotonergic perikarya in the fetal rat pancreas as demonstrated by the high affinity uptake of [3H] 5-HT.

Evidence has been obtained for the presence of serotonergic fibers in the adult rat pancreas; however, the location of the serotonergic cell bodies remains unknown. Fetal rat pancreata (18, 20 and 22 days) were demonstrated to possess a high-affinity uptake of serotonin. Radioautography revealed the uptake sites to be similar to those seen in the adult, indicating the presence of serotonergic fibers in early development. In addition, cell bodies, possibly representing primitive neurons, were demonstrated to be heavily labelled. This suggests a possible intra-pancreatic location for the cell bodies of the pancreatic nerves. The preservation of the specific serotonin uptake in 18-day fetal tissue after 4 days in organ culture substantiates this view.

Human insulin B chain but not A chain decreases the rate of diabetes in BB rats.

The autoimmune response seen in insulin-dependent diabetes mellitus (IDDM) includes a humoral immune response against human insulin. Early insulin treatment has been used to prevent IDDM in the rodent models of IDDM, and a prevention trial is underway in humans. The metabolic effects of insulin may not be involved in this prevention since, in NOD mice, the use of metabolically inert human insulin B chain was effective. We wished to ascertain whether immunization of diabetes-prone BB/W rats with insulin B chain, A chain, or both could alter the incidence of diabetes. Immunizations began by 30 days of age and the rats were followed until 120 days of age. Only immunization with insulin B chain plus adjuvant was effective in reducing the rate of diabetes. All immunization frequencies were effective, but a significantly lower rate of diabetes was achieved with injections every week. All of the doses tested resulted in significantly lower rates of diabetes. These data confirm in the BB rat model that immunization with insulin B chain in the presence of adjuvant can reduce diabetes incidence. The absence of any metabolic effect of B chain and the requirement for adjuvant suggest that this effect is mediated via modulation of the autoimmune response.