Lack of Autoimmune Serological Reactions in Rodent Models of Insulin Dependent Diabetes Mellitus

Spontaneous insulitis with insulin-dependent diabetes mellitus (IDDM) in rodent models, the BB rat and NOD mouse, has clarified the pathogenesis of and guided decisions on interventional therapy for human IDDM. However, the occurrence in such models of a standard marker of human IDDM, autoantibodies to β islet cell constituents, has been controversial. Hence we assessed diabetes-prone rodents for the frequencies of raised levels of auto-antibodies to glutamic acid decarboxylase GAD (anti-GAD), insulin and heat shock protein 65 (HSP-65) in relation to levels in non-diabetes-prone animals and levels in human diabetic sera. Assays were performed sequentially at various ages of life. The immunoassays used for anti-GAD and anti-insulin were those validated for sensitivity and specificity for detection of the corresponding autoantibodies in human IDDM sera at international workshops. Positive controls included human IDDM sera with reactivity with GAD or insulin and, for mouse anti-GAD, the highly reactive monoclonal antibody, GAD-6. The results were that levels of autoantibodies in diabetes-prone BB rats or NOD mice to the ‘IDDM-relevant’ autoantigens in our panel did not exceed levels in control rats or mice, and were much lower than levels in humans with IDDM. We conclude that the BB rat and NOD mouse represent a model, but not a facsimile, of human IDDM and that therapeutic successes in such models should be interpreted with caution in relation to interventional therapy for human IDDM.     

Synaptic vesicle-associated glutamate decarboxylase: Identification and relationship to insulin-dependent diabetes mellitus

Glutamic acid decarboxylase (GAD) catalyzes the biosynthesis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). GAD has been suggested as an autoantigen in insulin-dependent diabetes mellitus and stiff-man syndrome. Recently, three forms of membrane-associated GAD (MGAD) have been characterized in porcine brain, but the subcellular localization and function of these proteins are unknown. We present evidence that GAD activity is associated with synaptic vesicles from porcine brain. These vesicles contain a 60 kDa protein recognized by serum from patients with insulin-dependent diabetes mellitus, probably MGADII, as shown by subcellular fractionation and immunoblotting. These results raise the possibility that the association of MGADII with synaptic vesicles may be crucial for its role as an autoantigen in insulin-dependent diabetes mellitus. © 1995 Wiley-Liss, Inc.     

Thrombin Generation and Factor VIII:C Levels in Patients with Type 1 Diabetes Complicated by Nephropathy

The association between plasma coagulant activity and the presence of diabetic nephropathy was investigated in 31 patients with Type 1 diabetes, 12 with and 19 without nephropathy, and in 11 healthy subjects. Thrombin generation was assessed by computer assisted chromogenic method and expressed as time (in seconds) to 50% maximal thrombin activity (T₅₀). Factor VIII:C levels related to thrombin activity, glycaemic control, and renal function. Median (IQ) FVIII:C concentration was increased in patients with nephropathy (1590 (1130–1900) IU l^?1) compared to those without renal disease and with controls (960 (750–1090); 1020 (810–1100) IU l^?1, p < 0.01, respectively). There were no significant differences in T₅₀ values between the groups. FVIII:C correlated with age in all subjects and in the diabetic group (r = 0.33, p = 0.036; r = 0.39, p = 0.031) and inversely with T₅₀ in all subjects and in controls (r = ?0.35, p = 0.02; r = ?0.62, p = 0.04). In all subjects and in patients, FVIII:C was related to urinary albumin excretion and creatinine clearance. FVIII:C and T₅₀ were not related to HbA_1c. The results show that FVIII:C levels are increased in Type 1 diabetes complicated by nephropathy and are related to degree of renal impairment but not levels of glycaemia. No associated enhancement of plasma procoagulant activity was detected.     

No evidence for linkage between an insulin-dependent diabetes mellitus-susceptibility locus and immunoglobulin loci KM or GM

We studied 52 families having more than one member affected with insulin-dependent diabetes mellitus (IDDM) for linkage of an IDDM-susceptibility locus to the immunoglobulin loci KM and GM. Linkage was analyzed by the LOD score method using single-locus recessive and dominant models of IDDM inheritance, with penetrances of the disease-susceptible genotypes being dependent on age and reaching a maximum of 20% by age 40. We found no evidence for linkage of IDDM to KM or GM. Close linkage (recombination fraction <5%) was rejected for KM under the recessive model and for GM under both models. These results suggest either that there are no IDDM-susceptibility loci closely linked to KM or GM or that use of a single-locus model of IDDM (which ignores the effects of the susceptibility locus in the HLA region) is inadequate for their detection.     

The immune response to islets in experimental diabetes and insulin-dependent diabetes mellitus

Over the past year, a number of important observations have been made in the nonobese diabetic mouse and in clinical insulin-dependent diabetes mellitus concerning the autoimmune response to islets. Assays have advanced to the point where individuals at risk for insulin-dependent diabetes mellitus can be readily identified prior to the onset of symptoms and a number of peptides of proteins expressed by the beta cell have been shown to protect nonobese diabetic mice from developing diabetes. The contributions of CD4⁺ and CD8⁺ T cells to beta cell destruction are beginning to be understood and this information will probably be of value in the design of intervention strategies for use in human subjects.     

Abnormal regulation of cell membrane fluidity in diabetic nephropathy

Summary An abnormality of the physical properties of the cell membrane may underlie the defect that unites the clinical and biochemical abnormalities found in subjects with diabetic nephropathy. The cell membrane is linked both structurally and functionally with the cytoskeleton. The fluorescence anisotropy, a measure of membrane fluidity, was studied at baseline and after modulation of cytoskeletal proteins by thiol group alkylation with N-ethylmaleimide (NEM). 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess anisotropy in the deep hydrophobic regions of the lipid bilayer and trimethylammonium-diphenylhexatriene (TMA-DPH) was used to assess the superficial, relatively hydrophilic regions. We compared 17 subjects with insulin-dependent diabetes mellitus (IDDM) and nephropathy with 17 control subjects with IDDM and 24 non-diabetic control subjects. Median TMA-DPH anisotropy (0.271 (0.239–0.332) vs 0.269 (0.258–0.281) vs 0.275 (0.246–0.287)) and DPH anisotropy (0.221 (0.193–0.261) vs 0.227 (0.197–0.253) vs 0.226 (0.193–0.245)) were similar in erythrocytes from the three groups. However after alkylation of protein thiol groups with NEM clear differences emerged. In the control subjects with and without IDDM there was a significant fall in TMA-DPH anisotropy compared to the subjects with diabetic nephropathy in whom the addition of NEM had no effect (ΔTMA-DPH anisotropy –0.005 (–0.020– + 0.006) vs –0.005 (–0.011– + 0.016) vs + 0.002 (–0.010 – + 0.008) p < 0.001). This finding was confirmed when the deep regions of the lipid bilayer were assessed using DPH (ΔDPH anisotropy –0.017 (–0.029 –– 0.007.) vs –0.015 (–0.029 – + 0.001) vs + 0.003 (–0.021 – + 0.018) p < 0.001). We conclude that cytoskeletal modulation of the physical properties of the cell membrane lipids by proteins is abnormal in subjects with diabetic nephropathy. Such an abnormality could explain some of the clinical and metabolic abnormalities found in this condition. [Diabetologia (1998) 41: 337–342] Received: 10 July 1997 and in revised form: 23 October 1997     

Incidence of Nocturnal Hypoglycaemia in Insulin-dependent Diabetic Patients on Intensive Therapy

ABSTRACT The frequency of nocturnal hypoglycaemia, i.e. blood glucose concentration (BG) <3.0 mmol/l, was evaluated in consecutively selected insulin-dependent patients on multiple insulin injections (MII), n =23, or continuous subcutaneous insulin infusions (CSII), n =25. Blood was sampled hourly from 23.00 to 07.00. Seven patients (30%) on MII had at least one BG <3.0 mmol/l during the night. Eleven patients (44%) on CSII had hypoglycaemia (NS). The total number of BGs <3.0 mmol/l was higher on CSII, 42 of 225, versus 16 of 207 on MII (p<0.025). The duration of hypoglycaemia was 2 hours (range 1–6) on MII and 4 hours (range 1–7) on CSII with a maximal prevalence at 4 hours and between 5 and 7 hours, respectively (p=<0.05). The frequency of nocturnal hypoglycaemia is high in patients on intensified insulin regimens. Nocturnal hypoglycaemia occurs later in the night and is of longer duration on CSII than on MIL HbA_1c, BG before bedtime and in the morning might be useful in the evaluation of nocturnal hypoglycaemia.     

Infection by Multiple Viruses and Lymphocyte Abnormalities at the Diagnosis of Diabetes

ABSTRACT. Evidence for an ongoing adenovirus infection and preceding EB and Coxsackie B virus infections was found in a 2-year-old boy at the diagnosis of insulin-dependent diabetes. An inverted T-helper/T-suppressor lymphocyte ratio and activation of T cells with a suppressor-cytotoxic phenotype were detected. A combination of multiple viral infections may be responsible for the rapid destruction of pancreatic beta cells in this case.     

Monocytic TNFα secretion patterns in IDDM patients with periodontal diseases*

Abstract The aim of the present study was to identify whether monocytic TNFα secretion patterns could serve as a potential phenotypic discriminator for periodontal disease susceptibility within insulin-dependent diabetes mellitus (IDDM) patients. In 32 IDDM individuals the lipopolysaccharide (LPS) stimulated monocytic TNFα secretion dose-response characteristics were analyzed and related to two different periodontal status categories. Diabetics were divided into group A (gingivitis or mild periodontal disease) and group B (moderate to severe periodontal disease). In addition, 17 non-diabetic individuals with various degrees of periodontal disease served as control patients. Diabetics as a group had a significantly higher monocytic TNFα production in response to increasing Porphyromonas gingivalis A 7436 lipopolysaccharide concentrations (0, 0.003, 0.03, 0.3 and 3.0 μg/ml) as compared to non-diabetic patients with gingivitis or adult periodontitis (p <0.05). A significant difference in the dose response was also noted in the level of TNFα secreted as a function of P. gingivalis LPS concentrations between group A and B diabetics, as determined by two-way repeated measurements ANOVA (p <0.05). Furthermore, there was no significant difference in the mean HbA_1C between the two diabetic groups, and the TNFα level was not significantly associated with the HbA_1C level within diabetic patients. These data suggest that the diabetic state results in an upregulated monocytic TNFα secretion phenotype (4.6-fold increase) which, in the presence of Gram-negative bacterial challenge, is associated with a more severe periodontal disease expression. In addition, approximately 40% (10 of 24) IDDM periodontitis patients in group B demonstrated a 62-fold elevation in TNFα secretion relative to non-diabetic gingivitis or periodontitis patients and a 13.5-fold increase relative to IDDM group A (gingivitis or mild periodontitis) patients.     

Studies of Insulin Resistance Following Hypoglycemia in Insulin-dependent Diabetes Mellitus

ABSTRACT Insulin resistance was assessed after a hypoglycemia induced by insulin (1.5 mU×kg^-1 ×min^-1) between 7 and 8 a.m. in 10 well-insulinized patients with insulin-dependent diabetes mellitus (IDDM). Blood glucose levels during a somatostatin (100 μg×h^-1)-insulin (0.4 mU×kg^-1×min^-1)-glucose (4.5 mg×kg^-1)-infusion test (SIGIT) performed between 11 a.m. and 3 p.m. served as an indicator of total body insulin resistance. Plasma epinephrine, growth hormone, and Cortisol increased in response to hypoglycemia, while blunted responses of glucagon were simultaneously registered. At the start of the subsequent SIGIT, blood glucose and plasma-free insulin concentrations were similar to those obtained in the control study without preceding hypoglycemia, and at this point all counter-regulatory hormones had returned to basal. During the SIGIT close to identical levels of plasma-free insulin and counter-regulatory hormones were registered, despite which a significant hyperglycemia was seen 2 hours after the start of the SIGIT when preceded by hypoglycemia. In a separate study, the SIGIT was shown to have a good reproducibility in IDDM patients. We conclude that hypoglycemia evokes a state of insulin resistance for several hours, as demonstrated by elevated blood glucose levels during a somatostatin-insulin-glucose-infusion test.