Evidence of delayed β-cell destruction in Type 1 (insulin-dependent) diabetic patients with persisting complement-fixing cytoplasmic islet cell antibodies

Summary Forty-four children with Type 1 (insulin-dependent) diabetes (aged 0.7–16.7 years) were observed from diagnosis for cytoplasmic islet cell antibodies and serum C-peptide concentrations. Islet cell antibodies were analysed by indirect immunofluorescence for both conventional IgG and complement-fixing antibodies. Thirty-seven children (84%) were found to be positive for conventional islet cell antibodies at diagnosis, and 21 (48%) remained positive over the observation period. Twenty-six patients (59%) were positive for complement-fixing antibodies at diagnosis and eight remained so during the follow-up period. The serum C-peptide concentrations increased significantly during the first 3 months after diagnosis, after which there was a gradual decrease in the levels. Those children who remained positive for complement-fixing antibodies over the observation period had significantly higher serum C-peptide concentrations on several occasions during the second year and had also a higher integrated serum C-peptide concentration over the initial 2 years than those who became negative for complement-fixing antibodies. These observations suggest that the continuous production of complement-fixing islet cell antibodies in those patients who are positive for these antibodies at diagnosis presupposes the preservation of a sufficient amount of functioning cells for antigenic stimulation. These results support the view that the complement-fixing islet cell antibodies reflect ongoing destructive processes in the cells.     

Plasma 6-keto-PGF1α, thromboxane B2 and PGE2 in Type 1 (insulin-dependent) diabetic patients during exercise

Summary The capacity of prostacyclin production determined as plasma 6-keto-PGF₁ was investigated in 12 Type 1 (insulin-dependent) diabetic patients with a median duration of diabetes of 14 years during ordinary metabolic control. Using high pressure liquid chromatography preceding radioimmunoassay, the plasma concentration of 6-keto-PGF₁, the stable metabolite of prostacyclin, was determined at rest and after a standardised bicycle exercise test. The plasma 6-keto-PGF₁ in diabetic patients at rest did not differ from that of 25 healthy volunteers; 2.9 pg/ml (range<0.2–15.3) versus 1.7 pg/ml (range<0.2–16.6). During the exercise test plasma 6-keto-PGF₁ increased significantly in the diabetic patients as well as in the control group (p< 0.05). The increment of 6-keto-PGF₁ in the diabetic patients was neither related to the metabolic regulation, duration of diabetes nor to changes in platelet volume, platelet number or the production of thromboxane B₂ and prostaglandin E₂. Our results do not support the hypothesis that Type 1 diabetic patients have a decreased capacity of prostanoid production, as suggested from in vitro studies.     

Absence of otoacoustic emissions in insulin-dependent diabetic patients: is there evidence for diabetic cochleopathy?

In order to evaluate cochlear function in Type 1 diabetes mellitus, this study analyses otoacoustic emissions (OAEs) on normal hearing subjects with diabetes and on controls. Patients with Type 1 diabetes (n=60), with a mean age of 31+/-6.23 years, mean disease duration of 17.5+/-8.9 years, and mean HbA1c of 8.1+/-1.8%, of whom 43% had signs of retinopathy and 28% had clinical signs of neuropathy, were studied. All patients underwent an OAE analysis and brainstem-evoked potentials. Fifty-eight normal volunteers were used as controls for the OAE analysis. Seventeen patients (28.3%) had no OAEs in at least one ear and 10% in both ears. The mean intensity of the response was lower in diabetic subjects [7.1+/-4.4 vs. 10.9+/-9.3 dB SPL (sound pressure level)] than in controls. The cochlear impairment was over 5 dB for the 1-kHz frequency, which is the critical level for speech understanding. These findings suggest that cochleopathy can be detected in a relatively high proportion of subjects with Type 1 diabetes in spite of a normal audiometric hearing threshold.     

TNF-α gene polymorphisms in Type 1 (insulin-dependent) diabetes mellitus

Summary Type 1 (insulin-dependent) diabetes mellitus, like some other autoimmune diseases, is linked to certain alleles coded by genes in the HLA-D region. Sequence analysis of DQ chains indicates that aspartic acid at codon 57 confers resistance to the development of Type 1 diabetes. However, a full explanation for the HLA-association of Type 1 diabetes, particularly the increased susceptibility of DR3/4 heterozygotes is still awaited.The localisation of tumour necrosis factor genes on the short arm of chromosome 6 between HLA-B and the complement genes (Class III) prompted us to investigate a possible polymorphism of TNF- at the genomic level in relation to Type 1 diabetes susceptibility. A dialleleic TNF- restriction fragment length polymorphism was found with Ncol and its segregation with HLA-haplotypes analysed in diabetic families. We describe here a strong linkage of TNF- alleles with certain DR haplotypes. For example, the common extended haplotype HLA A1-B8-DR3 was almost exclusively associated with the 5.5 kb TNF- allele whereas Bw62-DR4 with the 10.5 kb allele. Thus both alleles segregate to diabetic patients.DR matched haplotypes of affected family members differed significantly from those of the non-affected at the TNF alpha locus. All affected sibling pairs in 11 multiplex affected families were identical for TNF- alleles, even if they were only haploidentical for HLA-B-DR haplotypes. In addition, heterozygosity for the TNF- alleles was significantly more frequent in the patients.This tight linkage of TNF- alleles with some extended haplotypes could help to explain the HLA-association of Type 1 diabetes as well as some other autoimmune diseases.     

The diabetic syndrome of the ‘BB’ Wistar rat: Possible relevance to Type 1 (insulin-dependent) diabetes in man

Summary The diabetes which occurs spontaneously in the BB Wistar rat has many affinities with human Type 1 (insulin-dependent) diabetes. It occurs in a non-obese, standard, laboratory rat derived from a non-inbred Wistar line. Both sexes are affected, with onset corresponding approximately to the time of sexual maturation. Both genetic and immune factors are involved in the aetiology, but their precise nature remains to be defined. Evolution of the overt clinical syndrome occurs over a period of hours to a few days. An intense insulitis is found, accompanied by selective destruction of B cells. Although insulitis may precede diabetes by many weeks, within 7–21 days after glycosuria the B cells are completely destroyed and have disappeared and the islets are few, small and with little residual inflammation. If untreated, marked wasting of body tissues, including fat and muscle protein, dehydration, and ketosis supervene. Careful study of littermates reveals glucose intolerance in 10%–25%, accompanied always by insulitis and these rats may subsequently develop insulin-dependent diabetes. Marked lymphopenia, mainly of thymus-derived (T) lymphocytes, both precedes and is sustained during glucose intolerance and overt diabetes. This lymphopenia appears to be associated reliably with insulitis, and may be a simple marker of susceptibility thereto. Abnormalities of nerves, testicles, and a tendency towards increased frequency of lymphomas have been found. Further research in this animal could lead to insights into aetiology, pathophysiology and complications potentially applicable to man.     

Genetic variation in insulin receptor β-chain exons among members of familial Type 2 (non-insulin-dependent) diabetic pedigrees

Summary Insulin resistance appears to be an essential component of Type 2 (non-insulin-dependent) diabetes mellitus. Both hyperinsulinaemia and insulin resistance are inherited and may precede the onset of Type 2 diabetes. To determine whether insulin receptor gene mutations, and specifically whether mutations of the -chain could account for the observed insulin resistance, we studied members of 16 pedigrees ascertained for two or more Type 2 diabetic siblings and members of four additional pedigrees ascertained for a mixture of Type 1 and Type 2 diabetes. We previously demonstrated insulin resistance among unaffected members of these pedigrees. Each pedigree was initially examined with insulin receptor restriction fragment length polymorphisms to determine whether any allele segregated with Type 2 diabetes in these pedigrees. Of the 16 pedigrees ascertained for Type 2 diabetes, at least one recombinant event between diabetes and the insulin receptor locus was present in seven pedigrees. An additional two pedigrees showed no linkage if individuals with impaired glucose tolerance were also considered affected. In all but one of the remaining pedigrees, apparent sharing of haplotypes may have resulted from insufficient polymorphism to distinguish all parental alleles. Subsequently, exons 13–21 of each allele which appeared in a Type 2 diabetic individual were examined by single strand conformation polymorphisms to detect any mutations in this region. A total of five mutations were detected, but DNA sequence analysis showed each mutation to be silent and thus not likely to result in defective insulin receptor function. No mutation detected in this fashion was present on an allele which appeared to segregate with Type 2 diabetes. We conclude that although some insulin receptor exon mutations are very common, the insulin receptor gene and particularly the -chain region, including the tyrosine kinase domain, is unlikely to be a significant cause of Type 2 diabetes and insulin resistance among White familial Type 2 diabetic pedigrees.     

β-Adrenergic blockade is more effective in suppressing adrenaline-induced glucose production in Type 1 (insulin-dependent) diabetes

Summary To examine whether diabetes affects the ability of -blockade to suppress adrenaline-stimulated hepatic glucose production, we infused adrenaline with and without propranolol into normal subjects and diabetic patients receiving a constant insulin infusion in basal amounts. In normal subjects, propranolol did not block the transient 50%–60% rise in glucose production during adrenaline infusion. In contrast, propranolol virtually abolished adrenaline-induced hyperglycaemia and glucose production was virtually abolished by propranolol in the diabetic patients, even though they demonstrated an exaggerated response to adrenaline alone (persistent increase in glucose production of 50%–90% above baseline). When insulin was infused together with adrenaline and propranolol in normal subjects in doses exceeding those given to the diabetics (plasma insulin rose threefold), the rise in glucose production was still threefold greater than in the diabetic patients (p<0.02). We conclude that -blockade is more effective in suppressing the hepatic response to adrenaline in diabetics than in normal subjects. Our data may explain why diabetic subjects are more vulnerable to hypoglycaemia during treatment with propranolol.     

Analysis of the HNF4α gene in Caucasian Type II diabetic nephropathic patients

Aims/hypothesis. Linkage and association studies in Caucasian patients with Type II (non-insulin-dependent) diabetes mellitus suggest that one or more diabetes susceptibility gene(s) reside within human chromosome 20q12–13.1. This region of chromosome 20 contains the maturity-onset diabetes of the young type 1 gene, HNF4 α. The purpose of this study was to assess the possible involvement of HNF4 α in Type II diabetes.¶Methods. Mutation analysis was done on the 12 exons and promoter regions of the HNF4 α gene in 182 Caucasian diabetic nephropathic patients and 100 Caucasian control subjects. The functional consequences of a novel promoter mutation were examined using a reporter system in the HepG2 liver cell line and electrophoretic mobility shift assays.¶Results. We identified two novel mutations in the HNF4α, an R323H missense mutation in exon 8, and a 7 bp deletion (Δ7) in the proximal promoter region resulting in deletion of a single putative Sp1 binding site. Using a reporter assay system, the Δ7 sequence was found to exhibit a 51.2 % (standard error ± 4.2 %) reduction in promoter activity relative to the normal sequence. In electrophoretic mobility shift assays using specific and non-specific competitors, the Δ7 sequence had a 45.5 % (range 40.4–46.6) reduction in binding compared with the normal sequence. The Δ7 allele occurs in a family with multiple cases of Type II diabetes in a pattern consistent with coinheritance of the Δ7 allele and diabetes.¶Conclusion/interpretation. Analysis of the HNF4 α gene revealed two possible mutations in 182 diabetic patients which suggests that the HNF4 α gene does not make a large contribution to diabetes susceptibility in the general population of Caucasian diabetic nephropathic patients. Functional analysis of the Δ7 promoter deletion suggests, however, that promoter mutations in otherwise normal genes could contribute to diabetes susceptibility. [Diabetologia (2000) 43: 364–372]     

The 5′ insulin gene polymorphism and the genetics of vascular complications in Type 1 (insulin-dependent) diabetes mellitus

Summary Recent data suggest genetic contributions to the microvascular complications of Type 1 (insulin-dependent) diabetes mellitus. Most research has focused on the HLA region, and the potential role of other genetic loci has not been adequately explored. We examined the possible relationship between DNA polymorphisms in the region 5 to the insulin gene on chromosome 11 and diabetic nephropathy. This was done by comparison of those diabetic patients homozygous for class 1 alleles at the 5 insulin gene polymorphism locus to 1/3 heterozygotes in a well-characterized series of 324 insulin-requiring diabetic patients from the Wisconsin Epidemiologic Study of Diabetic Retinopathy. Proteinuria (defined as 0.3 g protein/l urine), was used as suggestive evidence for diabetic nephropathy. Hypertension, a frequent associated finding in diabetic patients with nephropathy, was defined as a blood pressure greater than 140/90 or a history of previous treatment of hypertension. The two genotypically defined groups did not differ from each other in regard to sex ratio, age at diagnosis, age at examination, duration of diabetes, body mass, HbA_lc or C-peptide. The 1/1 group had a higher prevalence of proteinuria, 29% as compared to 16.2 % in other genotypes (p<0.05). There was no significant difference in the frequency of hypertension between the two genotypic groups. This finding suggests that the 5 insulin gene polymorphism may be associated with risk for nephropathy, but the pathophysiologic mechanism remains unclear.     

Insulin resistance in Type 1 (insulin-dependent) diabetes following hypoglycaemia — evidence for the importance of β-adrenergic stimulation

Summary The insulin effect, evaluated with the euglycaemic clamp technique, was studied before and after hypoglycaemia in 7 patients with Type 1 (insulin-dependent) diabetes. Following an initial 2 h clamp (clamp I) hypoglycaemia was induced and 2 h later a second clamp (clamp II), identical to the former, was performed. Each subject was studied twice; during infusion with saline (placebo) or propranolol. Glucose production and disposal were studied with the 3(³H)glucose technique. During placebo infusion, hypoglycaemia elicited an insulin resistance leading to approx. 50% reduction in the steady state glucose infusion rate during clamp II as compared to clamp I (clamp I 2.58±0.32, clamp II 1.26±0.08 mg·kg^–1·min^–1, p<0.02). The insulin resistance was prevented by infusing propranolol (clamp I 2.29±0.29, clamp II 2.85±0.56 mg·kg^–1·min^–1). The posthypoglycaemic insulin resistance was due to a less pronounced insulin effect on both glucose production (clamp I 0.29±0.21, clamp II 0.86±0.19 mg·kg^–1·min^–1, p<0.05) and glucose utilisation (clamp I 2.84±0.26, clamp II 2.13±0.23 mg·kg^–1·min^–1, p<0.05). The insulin resistance on both glucose production and utilisation was prevented by propranolol. Thus, the present study demonstrates that hypoglycaemia elicits a prolonged insulin resistance which is due to a less pronounced effect of insulin to both inhibit splanchnic glucose production and to stimulate peripheral glucose utilisation. The insulin resistance is due to -adrenergic stimulation and can be prevented by propranolol.     

Urinary N-acetyl-β-D-glucosaminidase (NAG) activity in the early detection of diabetic nephropathy

One of the serious outcomes of diabetes mellitus is nephropathy. Measurement of microalbuminuria is a routine clinical practice for screening the diabetic nephropathy, but the injury to the kidney may be happening even without microalbuminuria. The association between urinary enzyme activities of N-acetyl-β-D-glucosaminidase (NAG) and angiotensin converting enzyme (ACE) and urine microalbumin was assessed in this study to define the possible biofactor for detection of early diabetic nephropathy. Urinary enzyme activities of NAG and ACE and urine microalbumin of 24 h, serum ACE, and some other clinical features are investigated in 35 type 2 diabetic patients. Glycated hemoglobin (HbA1c), triglycerides, serum ACE, and urine NAG were significantly elevated in diabetic groups compared to the healthy controls. There was no relation between urine NAG and microalbuminuria except in the group of diabetic patients which had urine NAG activity upper than 25 IU/ml; there was a correlation between urine NAG and serum ACE. We may conclude that before the finding of microalbumin in urine, the elevated urine NAG, as an early indicator of renal damage, is associated with serum ACE which is related to kidney vascular microangiopathies.     

The increased angiotensin II (type 1) receptor density in myocardium of type 2 diabetic patients is prevented by blockade of the renin–angiotensin system

Aims/hypothesis The angiotensin II (type 1) (AT1) receptor mediates many biological effects of the renin–angiotensin system (RAS), leading to remodelling of cardiac tissue. The present study was designed to analyse changes in the function and expression of the AT1 receptor as principal effector of the RAS in myocardium from type 2 diabetic patients compared with non-diabetic myocardium as control. In addition, we determined the effect of treatment with ACE inhibitors or AT1 receptor blockers on expression levels of the receptor in diabetic patients.Methods Gene expression of the AT1 receptor was analysed by quantitative RT-PCR and protein expression was determined by immunoblot analysis in human right atrial myocardium. We investigated functional coupling of the receptors by measuring contractility in isolated trabeculae stimulated with increasing concentrations of angiotensin II.Results Diabetic myocardium showed a significant increase in protein expression (170 ± 16% of control) and median mRNA expression (186% of control) of the AT1 receptor. The additional receptors were functionally coupled, resulting in a stronger inotropic response upon stimulation with angiotensin II (89 ± 5.5% vs 29 ± 1.6% in controls), whereas receptor affinity was similar in both groups. However, myocardium from diabetic patients treated with ACE inhibitors or AT1 receptor blockers showed no increase in AT1 receptor expression.Conclusions/interpretation AT1 receptor expression in myocardium of type 2 diabetic patients is dynamic, depending on the level of glycaemic control and the activity of the RAS. These findings could at least in part explain the strong therapeutic benefit of RAS inhibition in diabetic patients.     

The Swedish childhood diabetes study III: IgM against coxsackie B viruses in newly diagnosed Type 1 (insulin-dependent) diabetic children — no evidence of increased antibody frequency

Summary Sera from essentially all Swedish children aged 0–14 years with Type 1 (insulin-dependent) diabetes mellitus with onset during an autumn period (October–December 1985) and a late spring period (May–June 1986) were selected. In all, 98 patients were analysed for IgM antibodies against coxsackie B virus serotypes 1 through 5 by a -antibody capture radio immunoassay technique. Sera from 94 referent children matched for age, sex and residential area, collected during the same period, were also analysed. During the autumn period, 10 out of 67 (15%) diabetic children were IgM positive while 14 out of 75 (19%) of the healthy referent children demonstrated positivity. During the late spring period only one out of 31 (3%) children with diabetes and two out of 19 (10%) referent children were IgM positive. In the diabetic patients, five were coxsackie B2 positive while coxsackie B1, 3, 4 and 5 were represented by one to three patients each. Eight referent children were coxsackie B4 positive, six were B3 positive and two B2 positive, while no referent children were positive against coxsackie B1 and 5. During these two periods in late 1985 and early 1986 these data demonstrate no evidence of increased antibody frequency against coxsackie B virus 1 through 5 at the onset of childhood diabetes in Sweden.     

Susceptibility to Type 1 (insulin-dependent) diabetes mellitus in Spanish patients correlates quantitatively with expression of HLA-DQ α Arg 52 and HLA-DQ β non-Asp 57 alleles

Summary HLA-DQ and alleles were chosen as the most sensitive Type 1 (insulin-dependent) diabetes mellitus susceptibility markers for evaluating the disease associations and Type 1 diabetes risk in a population-based registry from Madrid. The absence of aspartic acid in position 57 of the DQ chain (non-Asp 57), and the presence of arginine in position 52 of the DQ a chain (Arg 52) were found to be reliable markers of Type 1 diabetes susceptibility among the Spanish population, with significantly higher frequencies among the cases of Type 1 diabetes compared to randomly selected non-diabetic control subjects from the general Madrid population. While non-Asp 57 homozygosity conferred an absolute risk of 32.3 per 100,000 per year and Arg 52 of 31.5 per 100,000 per year, the risk for double homozygotes for both non-Asp 57 and Arg 52 was estimated as 101.7 per 100,000 per year. Individuals homozygous for only one of these alleles, and heterozygous at the other locus, had a markedly lower Type 1 diabetes risk (12.8 per 100,000 per year), approximating the general population incidence for Madrid. Thus, susceptibility to Type 1 diabetes in Spanish patients is associated, quantitatively, with non-Asp 57 DQ and Arg 52 DQ alleles.     

What is hypertension in diabetes? Ambulatory blood pressure in 137 normotensive and normoalbuminuric Type 1 diabetic patients

AIMS: To establish reference data for ambulatory blood pressure (AMBP) in normotensive, normoalbuminuric Type 1 diabetic patients and characterize the relation to clinic blood pressure (BP). To evaluate the statement of the third working party of the British Hypertension Society (BHS) that a target clinic BP in diabetes < 140/80 corresponds to a target day-time AMBP < 130/75 mmHg. PATIENTS AND METHODS: AMBP were performed in 172 normoalbuminuric, adult Type 1 diabetic patients, who had never received anti-hypertensive drugs. Clinic BP was determined as the mean of at least three auscultatory (Hawskley random zero manometer) and as the mean of at least three oscillometric (Spacelabs) BP values obtained just prior to ambulatory monitoring. Five patients with more than three missing hours/24 h were excluded. RESULTS: For 30 patients auscultatory clinic BP exceeded 140 mmHg systolic and/or 90 mmHg diastolic. For the remaining 137 normotensive patients day-time AMBP was 125.7/77.2 mmHg and oscillometric clinic BP was 125.3/76.5 mmHg (mean difference 0.3/0.7 mmHg; 95% confidence interval (CI) -0.9 to 1.5/-0.3 to 1.7 mmHg, P = 0.6/P = 0.2). Sixty-five percent of the patients had a diastolic day-time AMBP > 75 mmHg. CONCLUSIONS: Clinic BP and day-time AMBP measured by the same method were indistinguishable. The target for day-time diastolic AMBP (< 75 mmHg) proposed by the BHS is too low and is based on the misconception that in normotensive subjects day-time AMBP is lower than clinic BP. If the BHS guidelines are strictly adhered to, the consequence may be overtreatment in patients with normoalbuminuria and no end organ damage.     

Effects of short-term high dose intake of evening primrose oil on plasma and cellular fatty acid compositions, α-tocopherol levels,and erythropoiesis in normal and Type 1 (insulin-dependent) diabetic men

Summary In addition to their usual diet, nine Type 1 (insulin-dependent) diabetic men and ten male control subjects took 20 g d,ga-tocopheryl acetate enriched evening primrose oil (14.45 g 182c,6, 1.73g 183c,6, 400 mg d,-tocopheryl acetate) daily for one week. At start, diabetic patients had more 140, 150 and 18 2c,6, and less 160, 161c,7, 181c,7, 183c,6, 203c,9, 203c,6, 204c,6 and 226c,3 in plasma, erythrocytes and/or platelets. Furthermore, they had lower 161c,7/160, 181c,7/160, and 204c,6/203c,6 ratios and a higher 203c,6/183c,6 ratio. In diabetic patients, -tocopherol levels in erythrocytes were lower, whereas those in plasma were normal. In both groups, oil intake changed fatty acid profiles. Most markedly, 203c,6 increased, whereas the ratios 203c,6/ 183c,6 and 204c,6/203c,6 decreased. 204c,6 increased in control subjects, but not in diabetic patients. Erythrocytes and platelets responded differently in their fatty acid profiles, -tocopherol rose in plasma and, although less for diabetic patients, in erythrocytes. In diabetic patients as well as in control subjects, erythrocyte count, haemoglobin level, mean corpuscular haemoglobin content and concentration increased and glycosylated haemoglobin percentage decreased without an apparent decline in blood glucose levels. Plasma -thromboglobulin and platelet factor 4 decreased, especially in diabetic patients. In conclusion, diabetic patients had abnormal fatty acid patterns, suggesting an impaired 9, 6 and 5 desaturation and an enhanced chainelongation, and had lower erythrocyte a-tocopherol levels; and short-term high dose intake of evening primrose oil increased 203c,6 in both groups, but 204c,6 only in control subjects, gave fatty acid responses which were different for erythrocytes and platelets, enhanced erythropoiesis, and lowered indices of in vivo platelet activation.     

Aspartic acid at position 57 of DQβ chain does not protect against Type 1 (insulin-dependent) diabetes mellitus in Japanese subjects

Summary HLA DQ chain, in particular amino acid at position 57, has been reported to contribute to susceptibility and resistance to Type 1 (insulin-dependent) diabetes mellitus in Caucasians. Resistance has been proposed to be conferred by aspartic acid at this position. To ascertain the association of HLA DQ and DR genes with Type 1 diabetes in Japanese subjects, ten Japanese Type 1 diabetic patients were investigated at DNA level. Genomic DNA was amplified by polymerase chain reaction, and dot blot analysis was carried out using the amplified DNA with allele specific oligonucleotide probes. All patients had aspartic acid at position 57 of at least one of their two DQ chains, and there was no significant difference of amino acids at the same position of DR chain in patients compared to control subjects. These data indicate that the protective role of aspartic acid at position 57 of DQ chain is less significant in Japanese compared with Caucasian subjects.