Medical student delivery of alcohol education to high school pupils: the MEDALC programme.

There is increasing concern about the inappropriate use of alcohol by teenagers and young adults. The objective of this study was to assess the practicality of developing an alcohol education programme for school pupils delivered by medical students. The study design was of a prospective, observational, interventional cohort study. The primary outcomes were the number of schools, school pupils and medical students participating in the programme. Secondary outcomes were quantitative and qualitative measures derived from the feedback received from the participants. Over a three-year period, 60 medical students provided alcohol education sessions to 1,780 high school pupils (aged 13-15 years) within seven schools in Wales. Strongly positive feedback was obtained from all stakeholders and all schools asked to be included in future programmes. In conclusion, medical student-delivered teaching of alcohol education programmes to school pupils appears to be feasible and welcome by schools, teachers, pupils and medical students.

Diabetes due to exocrine pancreatic disease--a review of patients attending a hospital-based diabetes clinic.

BACKGROUND: Diabetes secondary to underlying exocrine pancreatic disease is a specific, but heterogeneous, type of diabetes mellitus. Studies such as UKPDS and DCTT excluded patients with pancreatic diabetes, so there is a paucity of evidence regarding best clinical practice in this group. AIM: To characterize the clinical features of patients with diabetes secondary to underlying pancreatic disease attending general diabetes clinics in a single hospital. DESIGN AND METHODS: A cross-sectional observational cohort study, identifying patients with pancreatic diabetes from clinic letters and medical notes at the University Hospital of Wales, Cardiff, UK. RESULTS: The notes of 38 patients with pancreatic diabetes were reviewed. Of these, six had pancreatic malignancy and the remainder had a range of benign disorders. The majority (29/38) had diabetes diagnosed at or shortly after the pancreatic diagnosis was made. There was a lack of consistency regarding initial hypoglycaemic therapy, with metformin alone being the most common initial therapy, but with 30/38 taking insulin within 12 months of diagnosis. Similarly, a broad range of insulin regimens were employed with twice daily pre-mixed insulin being most prevalent. Sixty-three percent of patients were prescribed lipid lowering therapy and 42% were taking anti-hypertensives. Glycaemic control, as estimated by the latest HbA1C, was no different in patients with pancreatic diabetes compared to the general clinic population and there were no reports of severe hypoglycaemia. CONCLUSION: There is great variability in how patients with pancreatic diabetes are currently managed. Future clinical trials should specifically address this group.

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.

Type 2 diabetes mellitus--genes or intrauterine environment? An embryo transfer paradigm in rats.

AIMS/HYPOTHESIS: The familial predisposition to Type 2 diabetes mellitus is mediated by both genetic and intrauterine environmental factors. In the normal course of events, maternal genes always develop in the same uterus, thus restricting studies aimed at investigating the relative contribution of these factors. We have developed an embryo transfer paradigm in rats to overcome this difficulty. METHODS: Euglycaemic female Wistar rats were superovulated and mated with male Wistar rats. The following day, fertilised eggs were transferred into pseudo-pregnant female Wistar rats or hyperglycaemic Goto Kakizaki (GK) rats. Pregnancies were allowed to go to term. Offspring were weighed at 6 weeks, 3 months and 6 months of age and an intravenous glucose tolerance test was carried out at 6 months of age. RESULTS: Offspring from Wistar into Wistar embryo transfers (n=20) were not significantly hyperglycaemic compared to the non-manipulated Wistar stock colony (n=26). However, offspring from Wistar gametes reared in hyperglycaemic GK mothers (n=51) were significantly lighter at 6 weeks of age (156+/-4.1 g vs 180+/-6.1 g [mean +/- SEM], p<0.01) and significantly more hyperglycaemic at 6 months of age (fasting glucose 6.6+/-0.18 mmol/l vs 4.8+/-0.21 mmol/l, mean blood glucose during glucose tolerance test 14.3+/-0.31 mmol/l vs 11.1+/-0.28 mmol/l, p<0.01) than Wistar gametes transferred back into euglycaemic Wistar mothers. When GK rats were superovulated and mated together, transfer of 1-day-old embryos into pseudo-pregnant Wistar dams did not alleviate hyperglycaemia in adult offspring. CONCLUSIONS/INTERPRETATION: In GK rats, a euglycaemic intrauterine environment cannot overcome the strong genetic predisposition to diabetes. However, in Wistar rats with a low genetic risk of diabetes, exposure to hyperglycaemia in utero significantly increases the risk of diabetes in adult life.

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. METHODS: Goto Kakizaki (GK) rats (a model of Type 2 diabetes) were mated with non-diabetic Wistar rats. Offspring were produced from 20 GK female vs. Wistar male and 20 Wistar female vs. GK male crosses. Fasting blood glucose was measured at 6 weeks and 3 months of age and an intravenous glucose tolerance test (0.8 g/kg) performed at 6 months of age. RESULTS: Wistar mothers produced litters with almost twice as many viable offspring as GK mothers (14.1 vs. 7.4, P < 0.001). Despite the larger litter size, offspring in the two groups were of comparable weight at 6 weeks and 6 months of age. At 3 months of age, male offspring of Wistar mothers were heavier than offspring of GK mothers (415.7 g vs. 379.5 g, P = 0.016) but this difference was not sustained at 6 months of age. Fasting blood glucose at all ages and average blood glucose during the glucose tolerance test were similar in both groups. CONCLUSIONS: We therefore conclude that there is no evidence for maternal transmission of diabetes in the GK rat. Mothers were able to adjust their supply of milk so that offspring attained similar weights independent of litter size. The weight of the offspring remained independent of litter size into adult life.

Examination of the hands: an insight into the health of a Welsh population.

When asked to examine a patient in a clinical examination, undergraduates and postgraduates characteristically begin with a painstaking observation of the hands. It was of concern that this is done mainly for the benefit of the examiners and that, in routine clinical practice, examination of the hands receives scant attention. The case notes of 70 general medical and surgical inpatients were reviewed and it was found that in only seven was there any mention of physical examination of the hands; in three of these "no clubbing" was the only entry. However, when the authors carefully examined the hands of 197 patients, at least one clinical sign was found in 87 (44%) cases. Heberden's nodes, cigarette tar staining, and the spectrum of Dupuytren's contractures were the most common findings. In this population in the South Wales valleys, coal deposits were also commonly seen in men.

Maternal transmission of diabetes.

Type 2 diabetes mellitus represents a heterogeneous group of conditions characterized by impaired glucose homeostasis. The disorder runs in families but the mechanism underlying this is unknown. Many, but not all, studies have suggested that mothers are excessively implicated in the transmission of the disorder. A number of possible genetic phenomena could explain this observation, including the exclusively maternal transmission of mitochondrial DNA (mtDNA). It is now apparent that mutations in mtDNA can indeed result in maternally inherited diabetes. Although several mutations have been implicated, the strongest evidence relates to a point substitution at nucleotide position 3243 (A to G) in the mitochondrial tRNA(leu(UUR)) gene. Mitochondrial diabetes is commonly associated with nerve deafness and often presents with progressive non-autoimmune beta-cell failure. Specific treatment with Coenzyme Q10 or L-carnitine may be beneficial. Several rodent models of mitochondrial diabetes have been developed, including one in which mtDNA is specifically depleted in the pancreatic islets. Apart from severe, pathogenic mtDNA mutations, common polymorphisms in mtDNA may contribute to variations of insulin secretory capacity in normal individuals. Mitochondrial diabetes accounts for less than 1% of all diabetes and other mechanisms must underlie the maternal transmission of Type 2 diabetes. Possibilities include the role of maternally controlled environments, imprinted genes and epigenetic phenomena.

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. METHODS: The region around 16189 nt was investigated by polymerase chain reaction-restriction fragment length polymorphism analysis and automated sequencing. RESULTS: We find that the T to C substitution at 16189 nt is associated with heteroplasmic length variation only when the resultant polycytosine tract is not interrupted by a second mutation. There are no significant differences in the prevalence of the 16189 nt variant or heteroplasmic length variation between patients with the 3243 nt mutation, patients with Type 2 diabetes or race-matched normal controls. CONCLUSIONS: We conclude that these variants are likely to represent normal polymorphisms and that previously reported associations should be treated with caution unless they can be replicated in other populations.

Identification of mtDNA mutation in a pedigree with gestational diabetes, deafness, Wolff-Parkinson-White syndrome and placenta accreta.

Mitochondrial DNA (mtDNA) defects are associated with a number of human disorders. Although many occur sporadically, maternal transmission is the hallmark of diseases due to mtDNA point mutations. The same mutation may manifest strikingly different phenotypes; for example, the A to G substitution at np 3243 was first reported in patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (the MELAS syndrome), but is also found in patients with diabetes and deafness. Here we present a case of gestational diabetes, deafness, premature greying, placenta accreta and Wolff-Parkinson-White (WPW) syndrome associated with a mtDNA mutation. Although this is the first report of such an association, study of 27 other patients with WPW syndrome failed to confirm that this mtDNA mutation is a common cause of such pre-excitation disorders.

Differential expression of mRNA in human thyroid cells depleted of mitochondrial DNA by ethidium bromide treatment.

A wide variety of human diseases have been associated with defects in mitochondrial DNA (mtDNA). The exact mechanism by which specific mtDNA mutations cause disease is unknown and, although the disparate phenotypes might be explained on the basis of impaired mitochondrial gene function alone, the role of altered nuclear gene expression must also be considered. In recent years, the experimental technique of depleting cells of mtDNA by culturing them with ethidium bromide has become a popular method of studying mitochondrial disorders. However, apart from depleting mtDNA, ethidium bromide may have many other intracellular and nuclear effects. The aim of the present study was to investigate the effects of ethidium bromide treatment on nuclear gene expression. A simian-virus-40-transformed human thyroid cell line was depleted of mtDNA by culture in ethidium bromide, and differential display reverse transcriptase-PCR (DDRT-PCR) was then employed to compare mRNA expression between wild-type, mtDNA-replete (rho(+)) and ethidium bromide-treated, mtDNA-depleted (rho(0)) cells. Expression of the majority of nuclear-encoded genes, including those for subunits involved in oxidative phosphorylation, remained unaffected by the treatment. Seven clones were found to be underexpressed; three of the clones showed significant similarity with sequences of the human genes encoding RNase L inhibitor, human tissue factor and ARCN1 (archain vesicle transport protein 1), a highly conserved species which is related to vesicle structure and trafficking proteins. We conclude that the effects of ethidium bromide treatment on nuclear gene expression are not simply limited to changes in pathways directly associated with known mitochondrial function. Further studies will be required to elucidate which of these changes are due to mtDNA depletion, ATP deficiency or other disparate effects of ethidium bromide exposure. Given that most genes appear unaffected, the results suggest that depleting cells of mtDNA by ethidium bromide treatment is a valuable approach for the study of mitochondrial mutations by cybrid techniques.

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.

Phylogenetic analysis of mitochondrial DNA in type 2 diabetes: maternal history and ancient population expansion.

Several studies have suggested a maternal excess in the transmission of type 2 (non-insulin-dependent) diabetes. However, the majority of these reports rely on patients recalling parental disease status and hence are open to criticism. An alternative approach is to study mitochondrial DNA (mtDNA) lineages. The hypervariable region 1 of the rapidly evolving noncoding section of mtDNA is suitable for investigating maternal ancestry and has been used extensively to study the origins of human racial groups. We have sequenced this 347-bp section of mtDNA from leukocytes of subjects with type 2 diabetes (n = 63) and age- and race-matched nondiabetic control subjects (n = 57). Consensus sequences for the two study groups were identical. Pairwise sequence analysis showed unimodal distribution of pairwise differences for both groups, suggesting that both populations had undergone expansion in ancient times. The distributions were significantly different (chi2 = 180, df = 11, P < 0.001); mean pairwise differences were 4.7 and 3.8 for the diabetic and control subjects, respectively. These data suggest that the diabetic subjects belong to an ancient maternal lineage that expanded before the major expansion observed in the nondiabetic population. Phylogenetic trees constructed using maximum parsimony, neighbor-joining, Fitch-Margolish, or maximum likelihood methods failed to show the clustering of all (or a subset) of the diabetic subjects into one or more distinct lineages.

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.

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 defects: a widening clinical spectrum of disorders.

1. Mitochondrial DNA has a number of interesting properties including maternal transmission, the ability to replicate in post-mitotic cells, a high mutation rate and an extremely compact molecular architecture with no introns and no large non-coding sequences. 2. Point mutations, deletions and duplications of mitochondrial DNA may occur. Mitochondrial DNA defects may co-exist with wild-type sequence within a cell (heteroplasmy). The level of heteroplasmy may vary in different tissues within the same individual (segregative replication). 3. A number of neurological disorders are characterized by morphological and biochemical mitochondrial defects. It is now clear that mitochondrial DNA mutations underlie these conditions although there is not always a clear correlation between a particular mutation and clinical presentation. 4. Mitochondrial DNA defects, particularly deletions, accumulate in senescent tissue and studies have been performed with the aim of linking such somatic mutations with degenerative disorders. 5. Recently mitochondrial DNA mutations have been implicated in a wider range of clinical disorders including diabetes and nerve deafness. 6. Nuclear gene defects may result in mitochondrial disorders by predisposing to multiple mitochondrial DNA deletions or quantitative depletions of mitochondrial DNA content.

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.

Molecular scanning of candidate mitochondrial tRNA genes in type 2 (non-insulin dependent) diabetes mellitus.

Mitochondrial DNA (mtDNA) gene defects may play a role in the development of non-insulin dependent diabetes mellitus (NIDDM). In order to search for potentially diabetogenic mtDNA defects we have applied the technique of single stranded conformational polymorphism (SSCP) analysis to 124 patients with a history of NIDDM and 40 non-diabetic controls. No new heteroplasmic mutations were detected. However, a variety of homoplasmic variants were found in patients with NIDDM; some of these merit further investigation.