Insulin expression in ß cells is reduced within islets before islet loss in diabetic cats.

OBJECTIVES: Diabetes mellitus is a common condition that requires intensive treatment and markedly impacts the welfare of affected cats. The aim of this study was to identify diabetes mellitus-associated perturbations in the feline pancreatic islet microenvironment. The utility of "clear, unobstructed brain/body imaging cocktails and computational analysis" (CUBIC) for three-dimensional pancreatic analysis was investigated. METHODS: Formalin-fixed paraffin-embedded tissues from cats with diabetes mellitus, or control cats without pancreatic pathology, were retrospectively identified. Immunohistochemistry for synaptophysin and ionised calcium binding adaptor molecule 1, and immunofluorescence for insulin and synaptophysin, were used to assess changes in islets. An image analysis pipeline was developed to analyse images acquired from two-dimensional immunofluorescence. CUBIC was used to optically clear selected pancreas samples before immunofluorescence and deep three-dimensional confocal microscopy. RESULTS: Diabetic cats have a significant reduction in synaptophysin-positive islet area. Whilst islets from diabetic patients have similar numbers of ß cells to islets from control cats, significantly lower intensity of insulin expression can be observed in the former. CUBIC facilitates clear visualisation of pancreatic islets in three dimensions. CLINICAL SIGNIFICANCE: The data presented support the theory that there is a decrease in function of ß cells before their destruction, suggesting a potentially significant step in the pathogenesis of feline diabetes mellitus. In parallel, we demonstrate CUBIC as a valuable new tool to visualise the shape of feline pancreatic islets and to interrogate pathology occurring in the islets of diabetic pets.

Dog leucocyte antigen (DLA) class II haplotypes and risk of canine diabetes mellitus in specific dog breeds.

BACKGROUND: Canine diabetes mellitus (DM) is a common endocrine disease in domestic dogs. A number of pathological mechanisms are thought to contribute to the aetiopathogenesis of relative or absolute insulin deficiency, including immune-mediated destruction of pancreatic beta cells. DM risk varies considerably between different dog breeds, suggesting that genetic factors are involved and contribute susceptibility or protection. Associations of particular dog leucocyte antigen (DLA) class II haplotypes with DM have been identified, but investigations to date have only considered all breeds pooled together. The aim of this study was to analyse an expanded data set so as to identify breed-specific diabetes-associated DLA haplotypes. METHODS: The 12 most highly represented breeds in the UK Canine Diabetes Register were selected for study. DLA-typing data from 646 diabetic dogs and 912 breed-matched non-diabetic controls were analysed to enable breed-specific analysis of the DLA. Dogs were genotyped for allelic variation at DLA-DRB1, -DQA1, -DQB1 loci using DNA sequence-based typing. Genotypes from all three loci were combined to reveal three-locus DLA class II haplotypes, which were evaluated for statistical associations with DM. This was performed for each breed individually and for all breeds pooled together. RESULTS: Five dog breeds were identified as having one or more DLA haplotype associated with DM susceptibility or protection. Four DM-associated haplotypes were identified in the Cocker Spaniel breed, of which one haplotype was shared with Border Terriers. In the three breeds known to be at highest risk of DM included in the study (Samoyed, Tibetan Terrier and Cairn Terrier), no DLA haplotypes were found to be associated with DM. CONCLUSIONS: Novel DLA associations with DM in specific dog breeds provide further evidence that immune response genes contribute susceptibility to this disease in some cases. It is also apparent that DLA may not be contributing obvious or strong risk for DM in some breeds, including the seven breeds analysed for which no associations were identified.

The Canine POMC Gene, Obesity in Labrador Retrievers and Susceptibility to Diabetes Mellitus.

BACKGROUND: Diabetes mellitus (DM) in dogs is a common endocrinopathy with a complex genetic architecture. Disease susceptibility in several breeds is associated with polymorphisms in immune response genes, but in the Labrador retriever breed, no genetic associations with DM have been identified. A deletion in the pro-opiomelanocortin (POMC) gene in Labrador retrievers is associated with increased appetite and risk of obesity. HYPOTHESIS/OBJECTIVES: To characterize the POMC deletion in Labrador retrievers, to develop a simple genetic test for this mutation, and to test the hypothesis that the POMC gene deletion is associated with an increased risk of DM in this breed. ANIMALS: Sixty-one non-diabetic Labrador retrievers aged >6 years and 57 Labrador retrievers with DM. METHODS: Case-control genotyping study to compare the frequency of the POMC deletion in dogs with and without DM. After polymerase chain reaction (PCR) and sequencing to characterize the mutation, a PCR-based test was developed and validated using 2 different restriction fragment length polymorphism assays. RESULTS: A 14-base-pair deletion was confirmed and localized to exon 3 of the canine POMC gene. A PCR-based test for the deletion was successfully developed. There was no association between the presence of the POMC deletion mutation and DM in this population of Labrador retriever dogs (P = .31). CONCLUSIONS AND CLINICAL IMPORTANCE: This study adds to the existing scientific literature indicating that there is little evidence for a direct link between obesity and DM in dogs.

Diabetes mellitus and pancreatitis--cause or effect?

Diabetes mellitus and pancreatitis are two distinct diseases encountered commonly in small animal practice. Whilst the clinical signs of diabetes mellitus are usually unmistakeable, a firm diagnosis of pancreatitis can prove more elusive, as clinical signs are often variable. Over the past 10 to 15 years, despite the fact that the clinical signs of diabetes mellitus are remarkably consistent, it has become more apparent that the underlying pathology of diabetes mellitus in dogs and cats is heterogeneous, with exocrine pancreatic inflammation accompanying diabetes mellitus in a number of cases. However, the question remains as to whether the diabetes mellitus causes the pancreatitis or whether, conversely, the pancreatitis leads to diabetes mellitus--as there is evidence to support both scenarios. The concurrence of diabetes mellitus and pancreatitis has clinical implications for case management as such cases may follow a more difficult clinical course, with their glycaemic control being "brittle" as a result of variation in the degree of pancreatic inflammation. Problems may also arise if abdominal pain or vomiting lead to anorexia. In addition, diabetic cases with pancreatitis are at risk of developing exocrine pancreatic insufficiency in the following months to years, which can complicate their management further.

Anti-insulin antibodies in diabetic dogs before and after treatment with different insulin preparations.

BACKGROUND: Anti-insulin antibodies (AIA) occur in diabetic dogs after insulin therapy, although their clinical significance is unclear. HYPOTHESIS: Treatment of diabetic dogs with heterologous insulin is more likely to stimulate production of AIA than is treatment with homologous insulin. ANIMALS: Diabetic dogs sampled before insulin therapy (n = 40), diabetic dogs sampled following treatment with porcine (homologous) insulin (n = 100), bovine (heterologous) lente insulin (n = 100), or bovine protamine zinc (PZI) insulin (n = 20), and nondiabetic control dogs (n = 120). METHODS: Prospective observational study. Sera were analyzed by ELISA for antibodies against porcine insulin, bovine insulin, insulin A, B, or C peptides, and control antigens; canine distemper virus (CDV) and canine thyroglobulin (TG). Canine isotype-specific antibodies were used to determine total and anti-insulin IgG1 : IgG2 ratios. RESULTS: There was no difference in CDV or TG reactivity among the groups. AIA were detected in 5 of 40 newly diagnosed (untreated) diabetic dogs. There was no significant difference in AIA (ELISA optical density reactivity) comparing control and porcine insulin-treated diabetic dogs (P > .05). Anti-insulin reactivity was most prevalent in bovine PZI insulin-treated dogs (90%; P < .01), and bovine lente insulin-treated dogs (56%; P < .01). AIA induced by treatment were enriched for the IgG1 isotype. CONCLUSIONS AND CLINICAL IMPORTANCE: This study indicates that bovine insulin is more immunogenic than porcine insulin when used for treatment of diabetic dogs.

Autoantibodies to GAD65 and IA-2 in canine diabetes mellitus.

Diabetes mellitus in dogs shares many characteristics with the human type 1 disease and virtually all diabetic dogs require insulin therapy to control hyperglycaemia. Insulin deficiency is suspected to result from immune-mediated destruction of pancreatic beta cells in some cases. Human patients suffering from Type 1A (immune-mediated) diabetes or latent autoimmune diabetes of the adult (LADA) demonstrate circulating autoantibodies against the 65kDa isoform of glutamic acid decarboxylase (GAD65) and/or insulinoma antigen-2 (IA-2). The aims of the current study were to develop radio-immunoassays to detect serum antibodies against recombinant canine GAD65 and IA-2 and to identify diabetic dogs showing serological evidence of autoreactivity to these pancreatic beta cell antigens. Canine GAD65 and the 3' end of IA-2 (coding for amino acids 771-979 of the intracellular domain) were amplified by PCR from cDNA prepared from canine insulinoma tissue and cloned into the pCRII vector. The canine sequences were later confirmed by identifying GAD2 and PTPRN genes from the dog genome assembly. Recombinant (35)S-methionine-radiolabelled canine GAD65 and IA-2 (771-979) proteins were used in radio-immunoprecipitation assays to screen sera from 30 newly diagnosed diabetic dogs and 30 control dogs. Four of 30 canine diabetic patients had significant GAD65 autoreactivity (p<0.01) compared to controls and 3 dogs were positive for autoantibodies to IA-2 (771-979). Two diabetic dogs showed dual autoantigen reactivity. These preliminary data indicate that serological reactivity to GAD65 and IA-2 is present in a proportion of diabetic dogs and suggests that, in some cases, canine diabetes is associated with an autoimmune response to these antigens.

Canine diabetes mellitus: from phenotype to genotype.

Breed differences in susceptibility to diabetes mellitus in dogs suggest an underlying genetic component to the pathogenesis of the disease. There is little evidence for an equivalent of human type 2 diabetes in dogs, and it has been proposed that canine diabetes is more comparable to the type 1 form of the disease. Certain immune response genes, particularly those encoding major histocompatibility complex molecules involved in antigen presentation, are important in determining susceptibility to human type 1 diabetes. We tested the hypothesis that canine major histocompatibility complex genes (known as the dog leucocyte antigen) are associated with diabetes in dogs. A total of 530 diabetic dogs and more than 1000 controls were typed for dog leucocyte antigen, and associations were found with three specific haplotypes. The DLA-DRB1*009/DQA1*001/DQB1*008 haplotype shows the strongest association with diabetes in the UK dog population. This haplotype is common in diabetes-prone breeds (Samoyed, cairn terrier and Tibetan terrier) but rare in diabetes-resistant breeds (boxer, German shepherd dog and golden retriever), which could explain differences in the prevalence of diabetes in these different breeds. There is evidence that the DLA-DQA1*001 allele is also associated with hypothyroidism, suggesting that this could represent a common susceptibility allele for canine immune-mediated endocrinopathies.

Identification of susceptibility and protective major histocompatibility complex haplotypes in canine diabetes mellitus.

Diabetes mellitus occurs spontaneously in dogs, which is believed to have an autoimmune component and to be a model of human latent autoimmune diabetes of adults (LADA). Some dog breeds (e.g. Samoyed) are particularly predisposed, whereas others (e.g. Boxer) are highly resistant. With the completion of the Dog Genome Assembly, comparative genomic studies of complex diseases in dogs, including diabetes, could provide an important investigative approach into such disorders. Type 1 diabetes in humans is strongly associated with major histocompatibility complex (MHC) class II polymorphisms. We have investigated whether canine dog leucocyte antigen (DLA) class II haplotypes are associated with diabetes. DNA from 460 cases and 1047 controls were genotyped for DLA-DRB1, DLA-DQA1 and DLA-DQB1 using sequence-based typing. Three DLA haplotypes, DRB1*009/DQA1*001/DQB1*008, DRB1*015/DQA1*0061/DQB1*023 and DRB1*002/DQA1*009/DQB1*001, were found at significantly increased frequency in cases with diabetes compared with controls. One DLA-DQ haplotype, DQA1*004/DQB1*013, was significantly reduced in cases with diabetes. Further analysis showed that DQA1 alleles carrying arginine at codon 55 of DQA1 were increased in dogs with diabetes. To our knowledge, this is the first report of a comparative study of MHC and diabetes in a non-rodent species. Since no laboratory model of LADA exists and dogs and humans share similar environments, further research into canine diabetes is warranted.

Management of 13 cases of canine respiratory disease using inhaled corticosteroids.

OBJECTIVES: To determine the value of inhaled corticosteroids in the management of chronic inflammatory airway disease in dogs. METHODS: Medical records of dogs that were presented for the investigation of respiratory disease were reviewed retrospectively. Criteria for inclusion were knowledge of previous medical treatment including side effects, diagnosis of the underlying disease, use of inhaled corticosteroids and at least two-months follow-up data. RESULTS: Thirteen dogs that fulfilled the criteria were identified. Ten dogs were diagnosed with chronic bronchitis and three with eosinophilic bronchopneumopathy. Four dogs had not previously received corticosteroid treatment for their respiratory disease, and all these showed a reduction or a resolution of clinical signs without obvious side effects after inhaled corticosteroid therapy. Nine dogs had previously received oral or parenteral corticosteroids for treatment of their respiratory disease, and all had exhibited side effects. Five of these dogs were treated with inhaled corticosteroids alone, and all exhibited an improvement in clinical signs without observable side effects. The remaining four dogs were treated with a combination of inhaled and oral corticosteroids, and all showed improvement in clinical signs and reduction in side effects. Inhaled medication was well tolerated in all dogs. CLINICAL SIGNIFICANCE: Inhaled corticosteroids were used for the management of chronic bronchitis and eosinophilic bronchopneumopathy in 13 dogs, and these may have the advantage of reducing side effects associated with oral corticosteroids.

Canine diabetes mellitus: can old dogs teach us new tricks?

BACKGROUND: Diabetes is common in dogs, with an estimated prevalence of 0.32% in the UK. Clinical signs, as in man, include polydipsia, polyuria and weight loss, associated with hyperglycaemia and glucosuria. Diabetes typically occurs in dogs between 5 and 12 years of age, and is uncommon under 3 years of age. Breeds predisposed to diabetes include the Samoyed, Tibetan Terrier and Cairn Terrier, while others such as the Boxer and German Shepherd Dog seem less susceptible. These breed differences suggest a genetic component, and at least one dog leucocyte antigen haplotype (DLA DRB1*009, DQA1*001, DQB1*008) appears to be associated with susceptibility to diabetes. METHODS: Canine diabetes can be classified into insulin deficiency diabetes (IDD), resulting from a congenital deficiency or acquired loss of pancreatic beta cells, or insulin resistance diabetes resulting mainly from hormonal antagonism of insulin function. RESULTS: There is no evidence for a canine equivalent of human type 2 diabetes. Adult-onset IDD, requiring insulin therapy, is the most common form, with pancreatitis and/or immune-mediated beta cell destruction considered to be the major underlying causes of the disease. DISCUSSION: Autoantibodies to insulin, recombinant canine GAD65 and/or canine islet antigen-2 have been identified in a proportion of newly diagnosed diabetic dogs, suggesting that autoimmunity is involved in the pathogenesis of disease in some patients. CONCLUSION: The late onset and slow progression of beta cell dysfunction in canine diabetes resembles latent autoimmune diabetes of the adult in man.

Study of 253 dogs in the United Kingdom with diabetes mellitus.

Clinical information and blood samples were collected from 253 dogs with naturally occurring diabetes mellitus. Over half of them were labrador retrievers, collies, Yorkshire terriers or crossbred dogs, and approximately 80 per cent of them were diagnosed between the ages of five and 12 years. The majority of the dogs were receiving insulin therapy once a day, but in the dogs receiving insulin injections twice a day there was a trend for lower serum fructosamine concentrations, suggesting better glycaemic control. The proportion of female dogs with diabetes was lower than in previous surveys. The disease was diagnosed more commonly in the winter months, a seasonal pattern also observed in human beings with diabetes, suggesting that similar environmental factors might be involved in the disease.

Evaluation of a continuous glucose monitoring system in diabetic dogs.

The generation of a blood glucose curve is important for assessing the response to insulin therapy in diabetic dogs. Disadvantages of this technique include patient discomfort and the potential for missing transient hypo- or hyperglycaemic episodes. The aim of the current study was to evaluate a continuous glucose monitoring system (CGMS) for use in diabetic dogs. Interstitial fluid glucose concentrations were recorded in 10 diabetic dogs, every five minutes for up to 48 hours, using a subcutaneous sensor attached to the CGMS device. Blood glucose concentrations were measured simultaneously using a glucometer. The correlation between interstitial fluid and blood glucose values was 0.81 (P < 0.01). The largest discrepancies between the two sets of data were seen during the one- to three-hour period following feeding, suggesting that postprandial hyperglycaemia might not be reflected in the interstitial fluid. The authors conclude that the CGMS is a potentially valuable tool in the management of canine diabetic patients.

Evaluation of two point-of-care analysers for measurement of fructosamine or haemoglobin A1c in dogs.

Measurement of glycosylated proteins such as fructosamine and haemoglobin A1c (HbA1c) can be used to assess glycaemic control in canine diabetic patients. Two point-of-care analysers, designed for human diabetics, were evaluated for use in dogs. Blood samples were collected from 50 normoglycaemic dogs, 100 diabetic patients and five dogs with insulinoma and tested using the In Charge fructosamine meter and the Haemaquant/Glycosal HbA1c meter. Readings were obtained in all cases except for 21 of 50 diabetics, which were above the upper limit of the In Charge meter. Diabetic dogs had higher fructosamine and HbA1c concentrations compared to controls. However, there was poor agreement between the In Charge meter readings and serum fructosamine concentrations, suggesting that there are problems associated with the use of this device in dogs. HbA1c concentrations showed a high degree of correlation with glycosylated haemoglobin measured at an external laboratory, suggesting that the Haemaquant/Glycosal meter warrants further evaluation for veterinary use.

Relative mutation rates at di-, tri-, and tetranucleotide microsatellite loci.

Using the generalized stepwise mutation model, we propose a method of estimating the relative mutation rates of microsatellite loci, grouped by the repeat motif. Applying ANOVA to the distributions of the allele sizes at microsatellite loci from a set of populations, grouped by repeat motif types, we estimated the effect of population size differences and mutation rate differences among loci. This provides an estimate of motif-type-specific mutation rates up to a multiplicative constant. Applications to four different sets of di-, tri-, and tetranucleotide loci from a number of human populations reveal that, on average, the non-disease-causing microsatellite loci have mutation rates inversely related to their motif sizes. The dinucleotides appear to have mutation rates 1.5-2 times higher than the tetranucleotides, and the non-disease-causing trinucleotides have mutation rates intermediate between the di- and tetranucleotides. In contrast, the disease-causing trinucleotides have mutation rates 3.9-6.9 times larger than the tetranucleotides. Comparison of these estimates with the direct observations of mutation rates at microsatellites indicates that the earlier suggestion of higher mutation rates of tetranucleotides in comparison with the dinucleotides may stem from a nonrandom sampling of tetranucleotide loci in direct mutation assays.