A new substrain of the non-obese diabetic mouse which develops cataracts (NOD/Ba/Lop19)
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Animals↗
Biomedical subjects
P Pozzilli
Publications and source records attributed to P Pozzilli.
A common mutation of the insulin receptor substrate-1 gene is a risk factor for coronary artery disease.
Insulin resistance is associated with increased risk of atherosclerosis. Insulin receptor substrate-1 (IRS-1) plays a key role in tissue insulin sensitivity. A common mutation (G972R) of the IRS-1 gene has been shown to impair IRS-1 function, and it has been associated with reduced insulin sensitivity and lipid abnormalities. This led us to investigate the role of the G972R mutation in predisposing individuals to coronary artery disease (CAD). The DNA of 318 subjects with angiographically documented coronary atherosclerosis (>50% stenosis) and 208 population control subjects was analyzed for the presence of the G972R mutation. This mutation was detected by nested polymerase chain reaction and BstNI restriction enzyme digestion. The frequency of the G972R mutation was significantly higher among patients with CAD than controls (18. 9% versus 6.8%, respectively; P<0.001). After controlling for other coronary risk factors, the relative risk of CAD associated with the G972R mutation was 2.93 (95% CI 1.30 to 6.60; P<0.02) in the entire cohort. This risk was found to be even higher in the subgroups of obese subjects (odds ratio [OR] 6.97, 95% CI 2.24 to 21.4; P<0.001) and subjects with clinical features of insulin resistance syndrome (OR 27.3, 95% CI 7.19 to 104.0; P<0.001). The IRS-1 gene variant was associated with a higher frequency of diabetes mellitus (14.9% among carriers versus 6.5% among noncarriers; P<0.01) and with a 60% increase of plasma total triglycerides (P<0.001). Also, plasma concentrations of total cholesterol and the ratio of total cholesterol to HDL cholesterol were significantly (P<0.001) higher among carriers than noncarriers, although to lesser a extent. These effects were independent of CAD status. The G972R mutation in the IRS-1 gene was found to be a significant independent predictor of CAD. Moreover, this mutation greatly increased the risk of CAD in obese subjects and in patients with the cluster of abnormalities of insulin resistance syndrome. Besides the increased frequency of diabetes, carriers showed a more atherogenic lipid profile, suggesting a potential role of the IRS-1 gene in the pathogenesis of lipid abnormalities associated with CAD.
Adult↗
Beta-cell gene expression and functional characterisation of the human insulinoma cell line CM.
Animal insulinoma cell lines are widely used to study physiological and pathophysiological mechanisms involved in glucose metabolism and to establish in vitro models for studies on beta-cells. In contrast, human insulinoma cell lines are rarely used because of difficulties in obtaining and culturing them for long periods. The aim of our study was to investigate, under different experimental conditions, the capacity of the human insulinoma cell line CM to retain beta-cell function, particularly the expression of constitutive beta-cell genes (insulin, the glucose transporters GLUT1 and GLUT2, glucokinase), intracellular and secreted insulin, beta-cell granules, and cAMP content. Results showed that CM cells from an early-passage express specific beta-cell genes in response to glucose stimulation, in particular the insulin and GLUT genes. Such capacity is lost at later passages when cells are cultured at standard glucose concentrations. However, if cultured at lower glucose concentration (0.8 mM) for a longer time, CM cells re-acquire the capacity to respond to glucose stimulation, as shown by the increased expression of beta-cell genes (insulin, GLUT2, glucokinase). Nonetheless, insulin secretion could not be restored under such experimental conditions despite the presence of intracellular insulin, although cAMP response to a potent activator of adenylate cyclase, forskolin, was present indicating a viable system. In conclusion, these data show that the human insulinoma cell line CM, at both early-passage and late-passage, posseses a functional glucose-signalling pathway and insulin mRNA expression similar to normal beta-cells, representing, therefore, a good model for studies concerning the signalling and expression of beta-cells. Furthermore, we have previously shown that it is also a good model for immunological studies. In this respect it is important to note that the CM cell line is one of the very few existing human beta-cell lines in long-term culture.
Adenylyl Cyclases↗
Antibodies to GAD65 and a tyrosine phosphatase-like molecule IA-2ic in Filipino type 1 diabetic patients.
OBJECTIVE: Type 1 diabetes is more prevalent in Europeans than it is in Asians. The disease is associated with autoantibodies to GAD65 and a protein tyrosine phosphatase-like molecule (IA-2). The frequency of GAD antibodies in Asian patients with type 1 diabetes may be lower than that in Europeans. No data are available on IA-2 antibodies in Asians. We tested antibodies to GAD65 and IA-2ic (the intracellular fragment containing the antibody epitope) in Filipino diabetic patients because this population has mixed European and aboriginal racial origins. RESEARCH DESIGN AND METHODS: A cross-sectional study of antibodies to GAD65 and IA-2ic was performed on a consecutive series of 91 type 1 diabetic patients, 74 type 2 diabetic patients, and 100 control subjects attending a diabetes clinic in Manila, the Republic of the Philippines. All subjects were <40 years of age, with a mean age +/- SD of 24.8+/-9.8, 34.3+/-5.8, and 25.8+/-8.0 years, respectively. Diagnosis of type 1 diabetes was determined clinically and confirmed by baseline C-peptide. RESULTS: Of 91 type 1 diabetic patients, antibodies to GAD65 were detected in 25 (27.4%), but antibodies to IA-2ic were found in only 8 (8.8%) (P = 0.002); neither autoantibody was detected in either the type 2 diabetic or control subjects. Of the 25 recently diagnosed type 1 diabetic patients (disease duration <2.0 years), autoantibodies to GAD65 were detected in 14 (56%), but those to IA-2ic in only 4 (16%) (P = 0.007); GAD65 antibodies were detected in only 4 (6%) of 66 patients with a longer disease duration (P = 0.0004). Comparison with recently diagnosed European type 1 diabetic patients of age and disease duration similar to that of the Filipinos indicated that IA-2ic antibodies, unlike GAD antibodies, were significantly less prevalent in Filipino type 1 diabetic patients (P = 0.0007). CONCLUSIONS: This is the first study investigating the prevalence and pattern of humoral immune response in type 1 diabetic patients from the Philippines. Antibodies to IA-2ic, unlike GAD antibodies, were infrequent. Patterns of immune responses to type 1 diabetes-associated antigens may differ worldwide, with important implications for prediction of the disease and the potential for antigen-specific therapy.
Adult↗
T cell reactivity to human insulinoma cell line (CM) antigens in patients with type 1 diabetes.
Autoimmune (type 1) diabetes mellitus results from a progressive destruction of insulin secreting beta cells operated by T lymphocytes in pancreatic islets. Circulating autoreactive T cells to specific beta cell antigens are detected in patients with type 1 diabetes. To date, several beta cell autoantigens have been identified in this disease (GAD, IA-2, 38kD secretory protein, insulin, ICA69 etc.), however, it is possible that also other unidentified self molecules contribute to trigger beta cell autoimmunity. In this study we used the human insulinoma cell line CM as source of beta cell antigens to detect reactive T lymphocytes in patients with type 1 diabetes mellitus. This cell line has been previously shown to express a number of recognized beta cell antigens. Since the expression of several beta cell antigens is affected by glucose stimulation we tested two preparations of CM cells cultured under different conditions containing low (0.8 mM) and high glucose concentration (11 mM). T cell proliferation was measured using cells from 32 patients with type 1 diabetes (19 of recent onset and 13 at 3 to 22 months from diagnosis) and 27 age-matched control subjects. A significant increase in T cell proliferation to CM cells grown in high glucose conditions (11 mM) (p < 0.05) was found in type 1 diabetic patients compared to controls. No significant differences were observed when using CM cells cultured at the low glucose concentration. Furthermore, the response to both extracts of CM cells was independent of disease duration (p = 0.6 for both CM cells cultured at 0.8 and 11 mM glucose). These data indicate that T cell reactivity to homogenates of CM cells is detectable in patients with type 1 diabetes and suggest that this human insulinoma cell line is an interesting potential source of beta cell material for immunological studies of autoimmune diabetes.
Adolescent↗
Troglitazone prevents insulin dependent diabetes in the non-obese diabetic mouse.
Troglitazone has recently been introduced in the treatment of Type 2 diabetes. In addition to its anti-diabetic effects it acts as a perixosome proliferator activated receptor-gamma (PPAR-gamma) agonist and has anti-inflammatory properties by inhibiting macrophage tumour necrosis factor-alpha (TNF-alpha) secretion. It also inhibits the production of endothelial selectin (e-selectin). Troglitazone also reduces interleukin-1alpha induced nitric oxide production in pancreatic beta-cells, which may be relevant in preventing nitric oxide mediated damage to these cells in the Type 1 diabetes process. We tested troglitazone in the spontaneous model of autoimmune diabetes, the non-obese diabetic (NOD) mouse, to determine its effect on the disease process. When administered by gavage from weaning at a dose of 400 mg/kg body weight (n = 32), troglitazone reduced the incidence of diabetes by 16 weeks compared to controls (n = 32) in a pattern that was maintained up to the conclusion of the experiment at 31 weeks of age (p < 0.05). Insulitis was unaltered (index = 1.05 +/- 0.71 vs. 1.13 +/- 0.82, treated vs. controls, p = 0.78). The study was repeated using troglitazone in the diet of NOD mice (n = 24) to give a dose of approximately 200 mg/kg body weight in order to provide a more consistent level of troglitazone during the time course of the experiment. There was a reduction of diabetes incidence in this group but it did not reach significance. Insulin levels were reduced in gavage treated mice although such reduction did not reach significance (p < 0.07). We conclude that, in view of its effect on this model of autoimmune diabetes and because of its known function as an insulin sensitiser, troglitazone might be considered for potential use in those patients with Type 1 masquerading as Type 2 diabetes.
Age Factors↗
Sex steroids do not prevent amylin-induced apoptosis in human cells.
Formation of amylin-containing islet amyloid deposits may contribute to the progressive deterioration of beta cell function in non-insulin-dependent diabetes mellitus. As diabetes mellitus occurs in male, but rarely in female transgenic mice expressing human amylin in their pancreatic beta cells, it is of interest to study the influence of estradiol (E2) and testosterone (T) on amylin-induced cytotoxicity in human cells. The insulinoma cell line CM, thyroid epithelial cells (TEC) in primary culture, and nontransformed fibroblast lines were used. The occurrence of apoptotic cell death was assessed by nuclear labeling with propidium iodide. Amylin was cytotoxic on all cell types tested, but had the most pronounced effect on TEC and the weakest on the CM cell line. Although both E2 and T decreased the proportion of apoptotic cells in cultures kept in the absence of amylin, neither of the two hormones was able to counteract amylin-induced cytotoxicity. beta cell death and hyperglycemia can thus presumably not be prevented by the neutralization of amylin effects by sex steroids.
Amyloid↗
Prevention of insulin-dependent diabetes mellitus 1998.
Recent advances in the understanding of the pathogenesis of insulin-dependent diabetes mellitus (IDDM) have led to the first trials of disease prevention in susceptible individuals. Two main trials (nicotinamide and insulin) are now running but first results will not be available before the turn of the century. Pilot trials using different approaches, most of them based on the induction of immunotolerance, are also under way and should offer new insight for establishing larger multicentre studies including attempts aimed at primary prevention by removal of diabetogenic components in cow's milk. The field is moving fast and it is expected that intervention for IDDM prevention will be offered to an increasing number of individuals found at risk of developing the disease.
Clinical Trials as Topic↗
IDDM preventive trials: what's new?
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Clinical Trials as Topic↗
Prognostic relevance of pancreatic uptake of technetium-99m labelled human polyclonal immunoglobulins in patients with type 1 diabetes.
Insulin-dependent type 1 diabetes (IDDM) is caused by the autoimmune destruction of insulin-producing beta cells. Approximately 10%-20% of patients may benefit from adjuvant immunotherapy upon diagnosis of the disease in order to protect residual beta-cell function. It has been suggested that this subgroup of patients differs from others by virtue of the presence of residual pancreatic inflammation and beta-cell function. In this study we have investigated to what extent technetium-99m-labelled human polyclonal immunoglobulins (99mTc-HIG) accumulate in the pancreas of IDDM patients at the time of diagnosis and 1 year thereafter, with a view to ascertaining whether HIG scintigraphy is useful for the identification of IDDM patients with residual pancreatic inflammation. Patients with recent-onset IDDM (n=15) were investigated at the time of diagnosis and 1 year later, and ten age- and sex-matched normal subjects were also studied. Gamma camera imaging and target to background ratio, analysed blind by three independent readers, were used to quantify the radioactivity in the pancreatic region and findings were correlated with metabolic, immunological and clinical parameters. Seven out of 15 newly diagnosed IDDM patients showed a significant accumulation of radiolabelled HIG in the pancreas (pancreas/bone ratio higher than the mean +2SD of normal subjects). One year after diagnosis, pancreatic accumulation of HIG was still detectable in most IDDM patients who were positive at the time of diagnosis. Six out of seven patients with positive scintigraphy had a partial clinical remission. These results indicate that HIG scintigraphy at the time of onset of diabetes identifies a subset of patients with residual beta-cell function who may benefit from adjuvant immunotherapy.
Adolescent↗
High frequency of polymorphism but no mutations found in the GLUT1 glucose transporter gene in NIDDM and familial obesity by SSCP analysis.
To evaluate whether a structural defect in the human glucose transporter gene GLUT1 could be involved in the aetiology of insulin resistance, a key factor of non-insulin-dependent diabetes mellitus (NIDDM) and obesity, we performed single-strand conformation polymorphism (SSCP) analysis in 40 subjects (20 NIDDM patients and 20 subjects with familial obesity). The GLUT1 gene, which is involved in basal glucose transport in most tissues, consists of ten exons and encodes a 492 amino acid protein. Population studies have shown a strong association between the X1 allele of an XbaI restriction fragment length polymorphism of the GLUT1 gene and NIDDM. We therefore performed SSCP analysis in NIDDM subjects known to carry at least one X1 allele. Variant SSCP patterns were detected in exons 2, 4, 5 and 9. Sequence analysis of the SSCP variants revealed the presence, in all exons examined, of silent mutations consisting of single-nucleotide substitutions with no amino acid changes. Both NIDDM and obese patients showed a high frequency of polymorphism in the sequence (50% and 35%, respectively). We conclude that the GLUT1 gene is unlikely to play a role in the aetiology of NIDDM and obesity. However, the strong association between the GLUT1 gene and NIDDM, together with the recent family studies showing linkage between chromosome 1p and NIDDM warrant further studies on this chromosomal region.
Adult↗
Metabolic and immune parameters at clinical onset of insulin-dependent diabetes: a population-based study. IMDIAB Study Group. Immunotherapy Diabetes.
The age at diagnosis of insulin-dependent diabetes mellitus (type I DM) varies between childhood and adulthood. The aim of this study was to define the immunologic and metabolic characteristics of the disease according to the age at which it is diagnosed. We evaluated the residual beta-cell function (basal and stimulated C-peptide) and frequency of two major islet cell-related autoantibodies, glutamic acid decarboxylase (GAD) and tyrosine phosphatase-like molecule (IA-2ic), at the onset of type I DM. A population-based study was performed with 235 consecutive cases of recent-onset (<4 weeks) type I DM (ages 5 to 45 years) diagnosed in the Lazio region of central Italy. Five age groups were considered: patients diagnosed between ages 5 and 7 years (n = 10), 7 and 10 years (n = 38), 10 and 17 years (n = 94), 17 and 20 years (n = 17), and 20 and 45 years (n = 76). Patients diagnosed before puberty had significantly reduced C-peptide secretion compared with patients diagnosed at a later age (P < .02). Glycosylated hemoglobin (HbA1c) did not differ at diagnosis between the different age groups. Patients diagnosed at puberty or after required significantly less insulin compared with younger patients (P < .04). GAD antibodies were found in 65% and IA-2ic antibodies in 59% of patients. GAD antibodies tended to be more frequent in patients diagnosed after age 17 compared with younger patients (P = .05), while IA-2ic antibodies were not age-related. These data suggest that (1) the extent of beta-cell damage differs between patients diagnosed before and after puberty, the process being more destructive in children less than 7 years of age, when C-peptide levels are the lowest; and (2) residual beta-cell function at diagnosis is not influenced by the presence or absence of islet cell-related antibodies. These findings have implications for trials in type I DM diagnosis aimed at protecting beta cells from end-stage destruction and in attempts to prevent the disease in susceptible individuals.
Adult↗
Fine mapping of the diabetes-susceptibility locus, IDDM4, on chromosome 11q13.
Genomewide linkage studies of type 1 diabetes (or insulin-dependent diabetes mellitus [IDDM]) indicate that several unlinked susceptibility loci can explain the clustering of the disease in families. One such locus has been mapped to chromosome 11q13 (IDDM4). In the present report we have analyzed 707 affected sib pairs, obtaining a peak multipoint maximum LOD score (MLS) of 2.7 (lambda(s)=1.09) with linkage (MLS>=0.7) extending over a 15-cM region. The problem is, therefore, to fine map the locus to permit structural analysis of positional candidate genes. In a two-stage approach, we first scanned the 15-cM linked region for increased or decreased transmission, from heterozygous parents to affected siblings in 340 families, of the three most common alleles of each of 12 microsatellite loci. One of the 36 alleles showed decreased transmission (50% expected, 45.1% observed [P=.02, corrected P=.72]) at marker D11S1917. Analysis of an additional 1,702 families provided further support for negative transmission (48%) of D11S1917 allele 3 to affected offspring and positive transmission (55%) to unaffected siblings (test of heterogeneity P=3x10-4, corrected P=. 01]). A second polymorphic marker, H0570polyA, was isolated from a cosmid clone containing D11S1917, and genotyping of 2,042 families revealed strong linkage disequilibrium between the two markers (15 kb apart), with a specific haplotype, D11S1917*03-H0570polyA*02, showing decreased transmission (46.4%) to affected offspring and increased transmission (56.6%) to unaffected siblings (test of heterogeneity P=1.5x10-6, corrected P=4.3x10-4). These results not only provide sufficient justification for analysis of the gene content of the D11S1917 region for positional candidates but also show that, in the mapping of genes for common multifactorial diseases, analysis of both affected and unaffected siblings is of value and that both predisposing and nonpredisposing alleles should be anticipated.
Chromosome Mapping↗
Genetic analysis of chromosome 2 in type 1 diabetes: analysis of putative loci IDDM7, IDDM12, and IDDM13 and candidate genes NRAMP1 and IA-2 and the interleukin-1 gene cluster. IMDIAB Group.
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Chromosomes, Human, Pair 2↗
BCG vaccine in insulin-dependent diabetes mellitus. IMDIAB Group.
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Adolescent↗
Incidence of insulin-dependent diabetes mellitus among Sardinian-heritage children born in Lazio region, Italy.
BACKGROUND: The relative importance of genetic and environmental factors in causing insulin-dependent diabetes mellitus (IDDM) is unknown. We studied this question by assessing the incidence of the disease in children, born in a region with a low incidence of IDDM (Lazio), but whose parents came from a region with high incidence (Sardinia). METHODS: We identified all IDDM cases that occurred between 1989 and 1994. We used as the denominator the number of children aged 0-14 born in Lazio of Sardinian parents to calculate incidence. We compared this rate with the incidences of IDDM in the populations of Lazio and Sardinia. FINDINGS: The age-adjusted incidence of IDDM in Sardinian-heritage children born and living in Lazio was 33.8 per 100,000 per year (95% CI 7.0-99.0) for those with two Sardinian parents, and 15.9 (8.7-26.6) for those with only one parent from Sardinia. The former incidence was not different from that recorded in Sardinia (34.4, 31.3-37.9), but was fourtold that of Lazio-heritage children (7.9, 7.1-8.8). INTERPRETATION: Our results show that two different ethnic groups living in the same region have a fourfold difference in incidence of IDDM. Children of Sardinian-heritage born in Lazio have the same incidence as the population of origin, which is genetically prone to the disease. Moreover, children with one Sardinian parent had a rate half that of Sardinians and double that of the indigenous population. We conclude that in a given population genetic susceptibility determines the frequency of IDDM in response to the environmental challenge.
Adolescent↗
The role of insulin resistance in the natural history of type 1 diabetes.
It is well established that peripheral insulin sensitivity is a critical factor in the aetiology of non-insulin dependent (Type 2) diabetes mellitus (NIDDM). Insulin resistance may also play a role at various stages in the natural history of insulin dependent (Type 1) diabetes (IDDM) and this was the topic of a workshop held in London on Friday 14 July 1995. The mechanisms of insulin resistance in IDDM are ill-defined but probably include 'glucose toxicity'. In the pre-diabetic period, insulin resistance may affect rates of progression to frank hyperglycaemia. Following the clinical onset of IDDM, insulin resistance could influence the length of the 'honeymoon period', diabetic control and patterns of growth during puberty, insulin requirements and blood glucose control at any time, the birth weight of infants born to diabetic mothers, and, through an effect on lipid metabolism and hypertension, ultimately contribute to the excess mortality associated with IDDM. In NIDDM, insulin resistance could influence rates of progression to insulin dependence. Treatment using insulin enhancers in NIDDM patients with autoimmune changes might delay or arrest their usual high-risk of progression to insulin dependence. As it is likely that insulin resistance has a wide-ranging influence on the natural history of diabetes in IDDM patients we suggest that treatment with insulin enhancers may prove beneficial in selected patients.
Adolescent↗
Glutathione protects a human insulinoma cell line from tumor necrosis factor-alpha-mediated cytotoxicity.
It is postulated that glutathione acting as a free oxygen radical scavenger may protect beta-cells from cytokine-mediated cytotoxicity in insulin-dependent diabetes. In this study the effect of glutathione in preventing the cytotoxic damage mediated by tumor necrosis factor-a in vitro towards a human beta-cell line (CM insulinoma) was investigated. CM cells were exposed in vitro to tumor necrosis factor-alpha, tumor necrosis factor-alpha plus glutathione or glutathione alone at different concentrations. The resulting cytotoxicity was measured using a colorimetric assay. Glutathione significantly reduced the cytotoxicity mediated by tumor necrosis factor-alpha in a dose-dependent fashion (P < 0.001). These results suggest a protective effect of glutathione on beta-cell cytotoxicity induced by tumor necrosis factor-alpha and encourage the use of glutathione in trials aimed at reducing the beta-cell damage occurring in insulin-dependent diabetes.
Cell Survival↗