John C. Alcolado

Animal models of diabetes mellitus

Animal models have been used extensively in diabetes research. Early studies used pancreatectomised dogs to confirm the central role of the pancreas in glucose homeostasis, culminating in the discovery and purification of insulin. Today, animal experimentation is contentious and subject to legal and ethical restrictions that vary throughout the world. Most experiments are carried out on rodents, although some studies are still performed on larger animals. Several toxins, including streptozotocin and alloxan, induce hyperglycaemia in rats and mice. Selective inbreeding has produced several strains of animal that are considered reasonable models of Type 1 diabetes, Type 2 diabetes and related phenotypes such as obesity and insulin resistance. Apart from their use in studying the pathogenesis of the disease and its complications, all new treatments for diabetes, including islet cell transplantation and preventative strategies, are initially investigated in animals. In recent years, molecular biological techniques have produced a large number of new animal models for the study of diabetes, including knock‐in, generalized knock‐out and tissue‐specific knockout mice.

The detection of mitochondrial DNA mutations using single stranded conformation polymorphism (SSCP) analysis and heteroduplex analysis.

A number of mitochondrial (mt) point mutations have been associated with inherited disorders. These pathogenic mutations are usually heteroplasmic. Here we describe the identification of three heteroplasmic mtDNA point mutations using the techniques of single stranded conformation polymorphism (SSCP) and heteroduplex analysis.

Is human type 2 diabetes maternally inherited? : insights from an animal model

Aims  Patients with Type 2 diabetes mellitus more often report a history of an affected mother than father. However, in the few studies where both parents and offspring have been directly tested, this apparent maternal excess has not been confirmed. Rodent models of diabetes have the advantage that all parents and offspring can undergo glucose tolerance testing at a specific age in adult life. The aim of this study was to gain insights into the inheritance of human Type 2 diabetes by using a rat model.

Analysis of a polycytosine tract and heteroplasmic length variation in the mitochondrial DNA D-loop of patients with diabetes, MELAS syndrome and race-matched controls

Aim The T to C substitution at position 16189 nt of the human mitochondrial genome has been associated with the development of heteroplasmic length variation in the control region of mtDNA. Previous reports have suggested that this defect may be associated with the development of other pathogenic mtDNA mutations, including the diabetogenic A to G mutation in the tRNALEU(UUR). Recently the 16189 nt variant has also been associated with insulin resistance in British adult men. In order to investigate these associations further we studied 23 patients with the 3243 nt mutation, 150 patients with Type 2 diabetes and 149 non‐diabetic controls.

Current methods of transfer of young people with Type 1 diabetes to adult services

We read with interest the recent paper [1] on transfer of people with diabetes from paediatric (PDS) to adult (ADS) services. We have undertaken a similar study in a single centre, to evaluate the outcome of transfer and to identify factors associated with success or failure of the process. Individuals transferred aged 17 years from an evening paediatric clinic staffed by paediatric and adult physicians to an adult clinic within the same teaching hospital or to one of two neighbouring district general hospitals (DGHs), or at the subject’s request, to their GP. Patients transferred from the PDS in the preceding 5 years were identified. An ADS hospital clinic attendance rate of 75% (known to be associated with lower serum HbA 1c concentrations [2]) was used to define ‘attenders’ (gross attendance rates > 75%) and ‘defaulters’ (attendance rates < 75%). Hospital case records were reviewed for demographic, diabetes-related and clinic-related data. Patients were invited by letter to a structured interview, either in person or by telephone, based on two questionnaires: 1 The Experience of Diabetes Care Questionnaire (EDCQ)— a measure developed for this study, to address individual’s experience of the diabetes clinics and of living with diabetes. 2 The Personal Model of Diabetes Interview (PMDI)—an abbreviated version of Personal Model of Diabetes Questionnaire [3] which addresses individual’s beliefs about the seriousness of the condition and treatment effectiveness. Of 92 patients transferred, records were available for 84 (91%) and 43 were interviewed. Half were transferred to the ADS in the same hospital, 28% to their local DGH ADS and 22% to their GP. There were no significant differences between attendance groups in gender, age of diagnosis or transfer, family history of diabetes, presence of retinopathy, injection regimen, diabetes-related admissions, attendance in the PDS in the last year or HbA 1c at transfer. As previously reported [1], administrative arrangements for transfer were generally good, with transfer letters and appointments given for the ADS in all but three cases. There were more missed appointments during the first year in the ADS than the last year in the PDS (median (range) 2 (0–5) and 0 (0–5), respectively; P < 0.001). Although the prior definition of the groups would predict more ‘attenders’ than ‘defaulters’ would attend the first appointment in the ADS, the difference was large, with 96% of ‘attenders’ keeping that first appointment compared with 33% of ‘defaulters’. ‘Defaulters’ lived further ( P < 0.001) from the clinic than ‘attenders’. There were no significant differences between ‘attender’ and ‘defaulter’ groups in their perceptions of the adequacy or effectiveness of self-care. Both rated the importance of managing diabetes similarly. However, ‘attenders’ were more confident ( P < 0.05) in their ability to manage their diabetes well. Although not statistically significant, defaulters tended to be more troubled by having diabetes and regarded treatment as less effective. In terms of changes to the ADS, 62% said they would prefer a clinic for under 30-year-olds, 40% would prefer different clinic times, in particular an evening clinic, and 27% wanted more frequent appointments. The main findings of this study were the importance of geographical distance between the patient’s home and clinic, the possible longer-term impact of failure to attend the first ADS appointment, and the ‘attenders’ confidence in their ability to manage diabetes well. Whereas this confidence may be engendered by the ADS, this finding also suggests that it is confidence which assists in the transition. Factors such as distance to clinic, familiarity with staff and hospital setting may impact on this confidence. Given these findings, we have developed a ‘handover’ clinic physically located in the ADS clinic. This ‘handover’ clinic is staffed by nurse specialists and doctors from both services, but responsibility for ensuring attendance is delegated to the paediatric team. Individuals with diabetes are seen once in this clinic before follow-up in a new ADS clinic designed specifically for those who have previously attended the ‘handover’ clinic, with the focus of effort being to help the young adult develop confidence in the management of their diabetes.

Genetic linkage study of a major susceptibility locus (D2S125) in a British population of non-insulin dependent diabetic sib-pairs using a simple non-isotopic screening method

Abstract A polymorphic microsatellite marker (D2S125) was recently reported to show significant linkage to non-insulin dependent diabetes mellitus (NIDDM) in a population of Mexican-American affected sib-pairs. We have used a simple non-isotopic screening technique employing the polymerase chain reaction (PCR) with a biotinylated primer to study the genetic linkage and allele frequency distribution of the D2S125 marker in a population of 109 British NIDDMs (62 possible affected sib-pairs). The analysis provided no evidence for linkage of the D2S125 marker in the British subjects (MLS = 0.029, P > 0.05). The PCR screening method used proved to be a convenient and reliable alternative to the radiolabelling of PCR products.

Mitochondrial DNA variations in patients with Type 2 (non-insulin dependent) diabetes mellitus and a Welsh control population

Type 2 (non‐insulin dependent) diabetes mellitus may be inherited along the maternal line and a variety of mitochondrial DNA (mtDNA) variants have been implicated in the pathogenesis. We have previously reported mutations in five regions of the mitochondrial genome which encompass 11 of the 22 tRNA genes. Now we employ the technique of single stranded conformational polymorphism (SSCP) analysis to investigate a further 6 regions of the mitochondrial genome, covering the remaining 11 tRNA genes in 40 patients with Type 2 diabetes and 30 racially‐matched normal controls. A variety of homoplasmic mutations were detected in patients with diabetes and these will be of value in further population association studies. Hum Mutat 13:412–413, 1999.

Nonradioactive characterization of low-level heteroplasmic mitochondrial DNA mutations by SSCP-PCR enrichment.

Mitochondrial DNA (mtDNA) mutations have been implicated in an increasing number of human diseases. Many of these mutations are heteroplasmic and are only present at low levels in readily accessible human tissue such as blood. The technique of single-stranded conformational polymorphism (SSCP) allows the detection of mtDNA variants from peripheral blood, but characterization of these variants by automated sequencing is hampered by the low level of heteroplasmy. We have therefore developed a technique for the enrichment of mtDNA mutations that allows reliable sequence data to be obtained even if the variant mtDNA represents only 1% of the total mtDNA. The procedure involves the excision, purification and subsequent PCR amplification of selected DNA fragments from SSCP gels. The techniques can be applied to other heterogeneous mutations such as mosaic mutations in skin biopsies or somatic oncogene mutations in tumor tissue.

Mitochondrial DNA variations in patients with Type 2 (non-insulin dependent) diabetes mellitus and a Welsh control population. Mutation in brief no. 239. Online.

Type 2 (non‐insulin dependent) diabetes mellitus may be inherited along the maternal line and a variety of mitochondrial DNA (mtDNA) variants have been implicated in the pathogenesis. We have previously reported mutations in five regions of the mitochondrial genome which encompass 11 of the 22 tRNA genes. Now we employ the technique of single stranded conformational polymorphism (SSCP) analysis to investigate a further 6 regions of the mitochondrial genome, covering the remaining 11 tRNA genes in 40 patients with Type 2 diabetes and 30 racially‐matched normal controls. A variety of homoplasmic mutations were detected in patients with diabetes and these will be of value in further population association studies. Hum Mutat 13:412–413, 1999.