Genome-wide analysis of DNA methylation in subjects with type 1 diabetes identifies epigenetic modifications associated with proliferative diabetic retinopathy.

BACKGROUND: Epigenetic variation has been linked to several human diseases. Proliferative diabetic retinopathy (PDR) is a major cause of vision loss in subjects with diabetes. However, studies examining the association between PDR and the genome-wide DNA methylation pattern are lacking. Our aim was to identify epigenetic modifications that associate with and predict PDR in subjects with type 1 diabetes (T1D). METHODS: DNA methylation was analyzed genome-wide in 485,577 sites in blood from cases with PDR (n = 28), controls (n = 30), and in a prospective cohort (n = 7). False discovery rate analysis was used to correct the data for multiple testing. Study participants with T1D diagnosed before 30 years of age and insulin treatment within 1 year from diagnosis were selected based on 1) subjects classified as having PDR (cases) and 2) subjects with T1D who had had diabetes for at least 10 years when blood DNA was sampled and classified as having no/mild diabetic retinopathy also after an 8.7-year follow-up (controls). DNA methylation was also analyzed in a prospective cohort including seven subjects with T1D who had no/mild diabetic retinopathy when blood samples were taken, but who developed PDR within 6.3 years (converters). The retinopathy level was classified by fundus photography. RESULTS: We identified differential DNA methylation of 349 CpG sites representing 233 unique genes including TNF, CHI3L1 (also known as YKL-40), CHN2, GIPR, GLRA1, GPX1, AHRR, and BCOR in cases with PDR compared with controls. The majority of these sites (79 %) showed decreased DNA methylation in cases with PDR. The Natural Killer cell-mediated cytotoxicity pathway was found to be significantly (P = 0.006) enriched among differentially methylated genes in cases with PDR. We also identified differential DNA methylation of 28 CpG sites representing 17 genes (e.g. AHRR, GIPR, GLRA1, and BCOR) with P <0.05 in the prospective cohort, which is more than expected by chance (P = 0.0096). CONCLUSIONS: Subjects with T1D and PDR exhibit altered DNA methylation patterns in blood. Some of these epigenetic changes may predict the development of PDR, suggesting that DNA methylation may be used as a prospective marker of PDR.

Cardiovascular disease in relation to diabetes status in immigrants from the Middle East compared to native Swedes: a cross-sectional study.

BACKGROUND: Type 2 diabetes is highly prevalent in immigrants to Sweden from Iraq, but the prevalence of cardiovascular disease (CVD) and its risk factors are not known. In this survey we aimed to compare the prevalence of CVD and CVD-associated risk factors between a population born in Iraq and individuals born in Sweden. METHODS: This population-based, cross-sectional study comprised 1,365 Iraqi immigrants and 739 Swedes (age 30-75 years) residing in the same socioeconomic area in Malmö, Sweden. Blood tests were performed and socio-demography and lifestyles were characterized. To investigate potential differences in CVD, odds ratios (ORs) with 95% confidence intervals (CIs) were estimated by multivariate logistic regression analysis with adjustment for metabolic, lifestyle and psychosocial risk factors for CVD. Outcome measures were odds of CVD. RESULTS: There were no differences in self-reported prevalence of CVD between Iraqi- and Swedish-born individuals (4.0 vs. 5.5%, OR 0.9, 95% CI 0.4-1.8). However, the prevalence of type 2 diabetes was higher in Iraqi compared to Swedish participants (8.4 vs. 3.3%, OR = 4.2, 95% CI 2.6-6.7). Moreover, among individuals with type 2 diabetes, Iraqis had a higher prevalence of CVD (22.8 vs. 8.0%, OR = 4.2, 95% CI 0.9-20.0), after adjustment for age and sex. By contrast, among those without diabetes, immigrants from Iraq had a lower prevalence of CVD than Swedes (2.2 vs. 5.5%, OR = 0.6, 95% CI 0.3-0.9).Type 2 diabetes was an independent risk factor for CVD in Iraqis only (OR = 6.8, 95% CI 2.8-16.2). This was confirmed by an interaction between country of birth and diabetes (p = 0.010). In addition, in Iraqis, type 2 diabetes contributed to CVD risk to a higher extent than history of hypertension (standardized OR 1.5 vs. 1.4). CONCLUSIONS: This survey indicates that the odds of CVD in immigrants from Iraq are highly dependent on the presence or absence of type 2 diabetes and that type 2 diabetes contributes with higher odds of CVD in Iraqi immigrants compared to native Swedes. Our study suggests that CVD prevention in immigrants from the Middle East would benefit from prevention of type 2 diabetes.

Nuclear factor of activated T cells regulates osteopontin expression in arterial smooth muscle in response to diabetes-induced hyperglycemia.

OBJECTIVE: Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. METHODS AND RESULTS: An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3(-/-) mice. CONCLUSIONS: These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.

High prevalence of type 2 diabetes in Iraqi and Swedish residents in a deprived Swedish neighbourhood--a population based study.

BACKGROUND: Immigrants from the Middle-East are at high risk of developing type 2 diabetes (T2D). The aim of the present survey was to measure, in a single deprived neighbourhood, the prevalence rates of impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and T2D in residents originating from Iraq and to compare them to those in residents born in Sweden. An additional aim was to identify metabolic, lifestyle and socioeconomic risk factors associated with IFG/IGT and T2D in these residents. METHODS: The study was conducted February 1'st to March 31'st 2010. Men and women aged 45 to 65 years of Swedish or Iraqi origin, living in the neighbourhood of Rosengård, Malmö, Sweden, were randomly selected from the census register. Each participant signed a written informed consent form, underwent a physical examination and an oral glucose tolerance test (OGTT), provided blood samples and filled in a questionnaire. A total of 175 subjects participated (Swedish origin n = 79, Iraqi origin n = 96), reflecting an overall response rate of almost 60%. RESULTS: In total, 21.9% and 19.0% of the Iraqi and Swedish participants, respectively, suffered from T2D, while 24.0% of the Iraqi participants and 25.3% of the Swedish participants had IFG/IGT. There were no significant differences in prevalence rates relating to country of origin.Obesity (BMI ≥30 kg/m2) and sedentary leisure time physical activity were highly prevalent in both groups, while a family history of diabetes was more prevalent in participants from Iraq (49.2%) than in those from Sweden (22.8%) (p = 0.001).Being obese or having a sedentary leisure time were, independently associated with T2D (OR 5.43 (95% CI 2.10-14.02) and 2.89 (95% CI 1.03-8.10) respectively), while economic difficulties were independently associated with IFG/IGT (OR 2.55 (95% CI 1.06-6.15)) after adjustment for the confounding effects of other common risk factors for T2D. CONCLUSIONS: This study reveals a high prevalence of T2D, independently of country of origin (Iraq or Sweden), in a socially vulnerable area and additionally presents a risk factor profile that is markedly different from that of Sweden in general.

Tumor necrosis factor-alpha does not mediate diabetes-induced vascular inflammation in mice.

OBJECTIVE: Vascular inflammation is a key feature of both micro- and macrovascular complications in diabetes. Several lines of evidence have implicated the cytokine tumor necrosis factor (TNF) alpha as an important mediator of inflammation in diabetes. In the present study we evaluated the role of TNF alpha in streptozotocin (STZ)-induced diabetes on vascular inflammation in C57BL/6 wild-type and apoE-/- mice. METHODS AND RESULTS: Diabetes increased the expression of vascular cell adhesion molecule (VCAM)-1 in cerebral arteries 150 m in diameter as well as the macrophage accumulation in aortic root atherosclerotic plaques in apoE-/- mice. A more pronounced vascular inflammatory response was observed in diabetic TNF alpha-deficient apoE-/- mice. These mice were also characterized by increased accumulation of IgG and IgM autoantibodies in atherosclerotic lesions. Diabetes also increased VCAM-1 expression and plaque formation in apoE-competent TNF alpha -/- mice, whereas no such effects were observed in C57BL/6 wild-type mice. CONCLUSIONS: The present findings suggest that TNF alpha does not mediate diabetic-induced vascular inflammation in mice and reveal an unexpected protective role for TNF alpha. These effects are partly attributable to a direct antiinflammatory role of TNF alpha, but may also reflect a defective development of the immune system in these mice.

Coeliac disease-specific tissue transglutaminase autoantibodies are associated with osteoporosis and related fractures in middle-aged women.

OBJECTIVE: To investigate whether the serological marker for coeliac disease, tissue transglutaminase autoantibody (tTGAb), is associated with decreased bone mass density (BMD) and increased frequency of fractures in middle-aged women screened for osteoporosis. MATERIAL AND METHODS: The study comprised 6480 women (mean age 56 years, range 50-64) who answered a number of questionnaires and who underwent dual X-ray absorptiometry of the wrist bone. Serum samples were analysed for tTGAb using radioligand binding assays. A tTGAb level of >4 U/ml was used to determine a positive value and a level of >17 U/ml was used as an alternative discrimination of high levels. RESULTS: A tTGAb level >4 U/ml was found among 90/6480 (1.4%) women and correlated with lower BMD (multiple linear regression coefficient -382.1; 95% CI = - 673.6-90.7, p=0.011) and with fracture frequency (r=0.18, p=0.023). The 59 women with tTGAb levels >or=17 U/ml had a lower BMD (0.41+/-0.08 g/cm(2) versus 0.44+/-0.08 g/cm(2), p=0.001) and a lower T-score (-1.40+/-1.28 versus -0.90+/-1.40, p=0.003) as well as a higher prevalence of osteoporosis (13.4% versus 6.5%, p=0.008) compared with the remaining 6421 women with tTGAb levels <17 U/ml. Furthermore, fracture frequency was more pronounced in women with tTGAb levels >or=17 U/ml, among whom 19/59 (32.2%) had fractures during the study period compared with 1204/6421 (18.8%) among women with tTGAb levels <17 U/ml (p=0.009). CONCLUSIONS: High levels of tTGAb indicating coeliac disease are associated with lower BMD and higher fracture frequency in women between 50 and 64 years of age. Osteometry is therefore warranted in middle-aged women detected with tTGAb.

Inflammatory activity increases with haemoglobin A1c in patients with acute coronary syndrome.

OBJECTIVE: To study the relationship between inflammation, diabetes and HbA(1)c levels in patients with acute coronary syndrome (ACS). DESIGN: Single-centre cross-sectional study comprising 688 consecutive patients with ACS (108 with diabetes) and 341 with stable coronary artery disease (SCAD) (51 with diabetes). High-sensitive C-reactive protein (hsCRP), albumin and fibrinogen concentrations, erythrocyte sedimentation rates (ESR) and leukocyte counts were measured. RESULTS: hsCRP, fibrinogen and ESR levels were higher and albumin lower in ACS patients. ESR was higher, albumin lower and hsCRP borderline significantly higher (p=0.053) in ACS patient with diabetes compared to those without. All inflammatory markers were associated with HbA(1)c in all 688 ACS patients as well as in 540 non-diabetic ACS patients with normal HbA(1)c. In multivariate analyses, all inflammatory markers were independently associated with HbA(1)c in the entire ACS group, regardless of diabetes being present or not. When non-diabetic ACS patients were analyzed separately, only ESR and leukocyte counts were independently correlated with HbA(1)c. CONCLUSIONS: Patients with ACS had increased inflammatory activity, which increased with HbA(1)c levels in patients who neither had a history of diabetes nor HbA(1)c above normal, and was further exaggerated in the presence of diabetes.

The aldose reductase inhibitor fidarestat suppresses ischemia-reperfusion-induced inflammatory response in rat retina.

Recent studies suggest that increased aldose reductase (AR) activity plays an important role in ischemia-reperfusion injury in the retina. The mechanisms are not completely understood, but may be linked to inflammation. In the present study, we investigated whether the AR inhibitor fidarestat suppressed the retinal inflammatory response induced by ischemia-reperfusion in a rat model. The inflammatory response was manifested by increased gene expression of tumor necrosis factor-alpha and intercellular adhesion molecule-1 (ICAM-1) as well as elevated protein levels of soluble ICAM-1. This response was partially suppressed by the AR inhibitor fidarestat. The findings may reveal beneficial effects of AR inhibition on retinal inflammation associated with ischemia-reperfusion and are in agreement with recent developments in pharmacological research suggesting that pathological conditions other than diabetes may benefit from AR inhibitors.

Blood pressure in middle-aged women: are androgens involved? A population-based study of Swedish women: the Women's Health in the Lund Area study.

OBJECTIVE: To assess the prevalence of hypertension and use of antihypertensive drug therapy in relation to menopausal status and to delineate perceived associations between androgens and blood pressure in perimenopausal women. METHODS: A population-based sample of women aged 50-59 (n = 6893). Women were divided into three groups according to their hormonal status: premenopausal, postmenopausal without hormone therapy, and postmenopausal with hormone therapy. RESULT: In the premenopausal, postmenopausal without hormone therapy, and postmenopausal with hormone therapy groups, the prevalence of high blood pressure (>/= 140 mmHg systolic or >/= 90 mmHg diastolic) was 43.9, 49.9 and 45.8%, respectively. In women with normal blood pressure, adjusting for age, body mass index and smoking, there were negative associations between serum testosterone and systolic blood pressure in the total sample (P < 0.01) and the postmenopausal without hormone therapy group (P < 0.05). In women using antihypertensive drug therapy with a blood pressure of at least 140/90 mmHg, positive associations were found between serum testosterone and systolic blood pressure in the total series (P < 0.05) and in the postmenopausal without hormone therapy group (P < 0.05). CONCLUSION: Abnormal blood pressure is common in middle-aged women regardless of hormonal status. Our findings suggest that testosterone could have a dual influence on blood pressure in perimenopausal women.

Insulin resistance and self-rated symptoms of depression in Swedish women with risk factors for diabetes: the Women's Health in the Lund Area study.

Previous studies have suggested that depression increases the risk for diabetes and that this may be mediated through insulin resistance. The study aimed to analyze if self-rated symptoms of depression are related to insulin resistance among middle-aged and older Swedish women with features of the metabolic syndrome and being at risk for type 2 diabetes mellitus. We analyzed data from 1047 Swedish women aged 50 to 64 years without a history of diabetes and living in the southern part of Sweden. A variable self-rated symptoms of depression (SRSD) was defined by using the Gothenburg Quality of Life instrument. We estimated odds ratios (ORs) to determine whether or not SRSD was associated with the homeostasis model assessment of insulin resistance. The variable SRSD was not associated with insulin resistance. However, it was positively associated with waist-hip ratio (OR, 1.95; 95% confidence interval, 1.28-3.00) and negatively associated with physical exercise (OR, 1.29; 95% confidence interval, 0.99-1.68) after multivariate adjustment. In conclusion, lifestyle factors such as physical inactivity and abdominal obesity, but not insulin resistance, seem to be related to self-rated symptoms of depression in women with risk factors for diabetes mellitus. The relationship between insulin resistance and major depression needs to be further examined.

Health hazards in middle-aged women with cardiovascular disease: a case-control study of swedish women. the women's health in the Lund area (WHILA) study.

OBJECTIVE: To delineate the health profile in middle-aged women with cardiovascular disease (CVD). METHODS: The Women's Health in the Lund Area (WHILA) project covered all women born 1935-1945 (n = 10,766) living in the Lund area; 6917 (64.2%) women completed a generic questionnaire and underwent physical and laboratory assessments. Of the 6917 women, 6416 were postmenopausal women, of whom 104 had CVD. For each woman with CVD, two controls were selected and matched for age, smoking habits, body mass index (BMI), waist/hip ratio (WHR), low-density lipoprotein cholesterol (LDL-C), high-density liproprotein cholesterol (HDL-C), diastolic blood pressure and hormonal status. RESULTS: One hundred four women (1.6%) reported CVD. Forty-nine had a myocardial infarction (MI), 49 had a stroke, and 6 women had both events; 71.4% were postmenopausal, with never use of hormone therapy (HT) (PM0), and 28.6% were postmenopausal with ever use of HT (PMT). Compared with the control group, serum androstendione was lower (p = 0.004) in the case group, and menopausal estradiol (E(2)) values were less frequent (p = 0.037) in cases from the PM0 group. Among psychological and somatic symptoms, nervousness (p < 0.05), difficulty relaxing, crying easily, visual disturbance (p

Obese women and the relation between cardiovascular risk profile and hormone therapy, glucose tolerance, and psychosocial conditions.

OBJECTIVE: To evaluate the relation between cardiovascular disease (CVD) risk factors and hormone therapy, serum hormone levels, glucose tolerance, and psychosocial and psychological conditions in subjectively healthy obese female subjects. RESEARCH DESIGN AND METHODS: The study included 606 women, aged 50-64 years, with BMI 30-40 kg/m(2) and no history of cardiovascular or other severe diseases. One group with a CVD risk profile (n = 473) (i.e., cholesterol >7.0 mmol/l, HDL cholesterol <1.2 mmol/l, triglycerides >2.0 mmol/l, systolic or diastolic blood pressure >140/90 mmHg, or waist-to-hip ratio >0.85) was compared with women without such risk (n = 133). Steroid hormones, leptin, insulin, and oral glucose tolerance tests (OGTTs) were analyzed. A subgroup of women with baseline impaired glucose tolerance (IGT) completed a 2.5-year follow-up OGTT. RESULTS: Fewer obese postmenopausal women with CVD risk had ever used hormone therapy (odds ratio 0.24 [95% CI 0.07-0.75]), after multivariate adjustments. Furthermore, women with CVD risk had a higher testosterone index (1.07 [1.01-1.13]) and more had insulin resistance (1.04 [1.00-1.08]) and IGT (2.92 [1.50-5.69]), while OGTT was similar at follow-up. No differences were observed regarding family history or lifestyle, except that fewer women with CVD risk consumed fruits, boiled vegetables, or whole-grain cereals. More women with CVD risk lived alone (3.26 [1.28-8.31]) and had more mental problems (1.16 [1.05-1.28]). CONCLUSIONS: Previously healthy obese women with a CVD risk profile seemed to have a high risk of diabetes, as well as psychosocial or psychological problems. Hormone therapy was associated with reduced CVD risk. Obesity's growing burden on society makes it more important to further target individuals that are at greatest risk of future health hazards.

Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

Expression of antioxidant enzymes in rat retinal ischemia followed by reperfusion.

To evaluate the expression and protein levels of antioxidant enzymes in the rat retina exposed to oxidative stress induced by ischemia-reperfusion injury. Retinal ischemia was induced in female Wistar rats by ligation of the optic nerve and vessels behind the left eye bulb, and was followed by reperfusion for 0, 3, 6, or 24 hours. The right eye served as control. RNA and protein were extracted simultaneously from each retina. Expressions of the endogenous antioxidant enzymes glutathione peroxidase (GPx1), catalase (CAT), copper/zinc superoxide dismutase, manganese superoxide dismutase, and the catalytic subunit of glutamylcysteine ligase (GCLc) were analyzed with real-time reverse transcription polymerase chain reaction and related to the endogenous control cyclophilin B. Protein levels were measured with Western blot analysis. During the early phase (0 or 3 hours) of reperfusion, no changes were seen in enzyme expression. After 6 hours, GCLc expression increased by a factor of 1.14 (P = .034), followed by a decline of 0.80 after 24 hours (P = .00004), according to the comparative Ct method. After 24 hours of reperfusion, GPx1 expression increased by a factor of 1.14 (P = .028), and CAT had decreased by 0.82 (P = .022). Expressions of copper/zinc superoxide dismutase and manganese superoxide dismutase showed a tendency toward a decrease by factors of 0.86 (P = .055) and 0.88 (P = .053), respectively, after 24 hours. Protein levels did not differ for any of the antioxidants, regardless of reperfusion time. The slightly increased messenger RNA expression of GPx1 after 24 hours of reperfusion with a concomitant very modest decrease in CAT and GCLc expression and no change in protein levels indicate a very modest, if any, response to oxidative stress generated by ischemia followed by reperfusion in rat retina.

Increased serum levels of the specific advanced glycation end product methylglyoxal-derived hydroimidazolone are associated with retinopathy in patients with type 2 diabetes mellitus.

Advanced glycation end products (AGEs) are thought to play a major pathogenic role in diabetic retinopathy. The most important AGE is unknown, but as increased serum methylglyoxal-derived hydroimidazolone has been demonstrated in patients with type 2 diabetes mellitus, the aim of the present study was to elucidate possible associations between serum levels of hydroimidazolone and retinopathy in patients with type 2 diabetes mellitus. We recruited 227 patients with type 2 diabetes mellitus and retinopathy ranging from none to proliferative. Level of retinopathy was determined from 7 standard field stereo photographs per eye according to the Early Treatment Diabetic Retinopathy Study. The patients were 66 +/- 11 years old, with a known diabetes duration of 14 +/- 9 years. Serum levels of hydroimidazolone were determined with a competitive immunoassay. Serum levels of hydroimidazolone were increased in nonproliferative (median, 4.50 U/mL; interquartile range, 3.69-5.77 U/mL) and proliferative retinopathy (median, 4.88 U/mL; interquartile range, 3.70-6.52 U/mL) compared with patients without retinopathy (median, 4.02 U/mL; interquartile range, 3.47-4.88 U/mL) (P = .008 and .002, respectively). There was no association between hydroimidazolone and hemoglobin A1c (r = 0.04, P = .57). In addition, patients with proliferative retinopathy and a relatively short known duration of diabetes, that is, less than the median of 14 years, had increased serum levels of hydroimidazolone (median, 6.91 U/mL; interquartile range, 4.70-8.91 U/mL) compared with those with nonproliferative retinopathy (median, 4.34; interquartile range, 3.86-5.53U/mL, P = .015). Serum levels of hydroimidazolone are increased in type 2 diabetic patients with retinopathy. This association is independent of hitherto known associated factors, such as hemoglobin A1c.

Women living alone have an increased risk to develop diabetes, which is explained mainly by lifestyle factors.

OBJECTIVE: The purpose of this study was to assess the role of household conditions for the progression to diabetes in women with impaired glucose tolerance (IGT). RESEARCH DESIGN AND METHODS: A total of 461 women, aged 50-64 years, with IGT defined by an oral glucose tolerance test, had baseline advice on physical exercise, diet, smoking, and alcohol habits. Physical examination, blood tests, and questionnaires were completed at baseline and after 2.5 years. Household status was categorized into living alone or with a partner, other adults, or children. RESULTS: Women living alone had a 2.68-fold increased risk (95% CI 1.02-7.05) of developing diabetes after adjustments for biological risk factors. Further stepwise adjustments for education, occupation, subjective mental health, exercise, diet, and alcohol showed remaining significant odds ratios (ORs), decreasing from 3.26 (1.19-8.96) to 3.03 (1.02-8.99). However, when smoking status was added, the OR became nonsignificant, 2.07 (0.62-6.88). More women who lived alone smoked and did not reduce their daily cigarette consumption compared with women in other household conditions. At follow-up, women living alone had reduced their alcohol consumption and were more often abstainers and fewer had healthy dietary habits or had improved their diet. Physical exercise did not differ among the groups. Separate analyses of any other household status did not show any excess risk for development of diabetes. CONCLUSIONS: Women living alone had a higher risk to progress from IGT to diabetes, mostly explained by smoking, alcohol, and dietary habits. Household conditions should be accounted for when assessing future risk for diabetes.

Bovine retinal pericytes are resistant to glucose-induced oxidative stress in vitro.

Diabetic retinopathy is a sight-threatening complication of diabetes, and loss of pericytes represents early signs of its development. We tested the hypothesis that high glucose levels may induce signs of oxidative stress in cultured bovine retinal pericytes. Pericytes were exposed to either normal (5.5 mM) or high (22 mM) glucose levels for 1, 3, and 5 days. Signs of oxidative stress were measured by expression of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, and glutathione peroxidase using real-time RTPCR. To elucidate the role of oxidative stress, we also measured glutathione (GSH) concentration in the cells and investigated the impact of thiol-reactive metal ions and hydrogen peroxide (H(2)O(2)) on intracellular GSH. Despite the stimulation with high glucose, thiol-reactive metal ions, or H(2)O(2), there was no clear increased expression of antioxidant enzymes or influence of GSH levels. Lipid peroxidation (malondialdehyde level) was increased in bovine aortic smooth muscle cells, but not in bovine retinal pericytes. The data indicate that pericytes do not develop oxidative stress in response to hyperglycemia. However, it is not definitively excluded that oxidative stress may occur after longer time periods of glucose stimulation.

Albuminuria and hypertension are independently associated with circulating antipericyte autoantibodies in type 2 diabetic patients.

AIMS: To determine whether albuminuria, hypertension, or HbA 1c are independently associated with antipericyte autoantibodies (APAAs) in type 2 diabetes mellitus. METHODS: Two hundred ninety-nine subjects with different degrees of retinopathy according to the Early Treatment Diabetic Retinopathy Study Scale participated in this study. Albuminuria was defined as an albumin/creatinine ratio above the normal cutoff limit, that is, 2.0 g/mol for men and 2.8 g/mol for women. Hypertension was defined as a diastolic blood pressure more than 90 mm Hg, a systolic blood pressure more than 140 mm Hg, or pharmacological antihypertensive treatment. Serum APAAs were detected by immunofluorescence on tissue-cultured bovine retinal pericytes. Association analysis was performed using univariate and multivariate statistical tools. RESULTS: In type 2 diabetes, APAAs were independently associated with albuminuria (OR = 0.56; P < .04), hypertension (OR = 2.21; P < .01), as well as with proliferative retinopathy (OR = 0.39; P < .01). CONCLUSIONS: The increased prevalence of APAA in patients with hypertension may suggest that these antibodies are related to tissue damage and repair and that the decline in frequency with albuminuria may serve as a marker for more advanced angiopathy. Future longitudinal studies are needed to determine whether the frequency of APAA is associated with the progression of angiopathy, and to determine the biological activity and antigens recognized by the antibody.

Clinical evidence for the safety of GAD65 immunomodulation in adult-onset autoimmune diabetes.

The purpose of this Phase II study was to evaluate if alum-formulated human recombinant GAD65 is safe and does not compromise beta cell function. The study was conducted as a randomized, double blind, placebo-controlled, dose-escalation clinical trial in a total of 47 Latent Autoimmune Diabetes in Adults (LADA) patients who received either placebo or 4, 20, 100, or 500 microg Diamyd subcutaneously at Weeks 1 and 4. Safety evaluations, including neurology, beta cell function tests, diabetes status assessment, hematology, biochemistry, and cellular and humoral immunological markers, were repeatedly assessed over 24 weeks. None of the patients had significant study-related adverse events (AE). Fasting c-peptide levels at 24 weeks were increased compared with placebo (P=.0015) in the 20 microg but not in the other dose groups. In addition, both fasting (P=.0081) and stimulated (P=.0236) c-peptide levels increased from baseline to 24 weeks in the 20 microg dose group. GADA log levels clearly increased (P=.0002) in response to 500 microg Diamyd. The (CD4+)(CD25+)/(CD4+)(CD25-) cell ratio increased (P=.0128) at 24 weeks in the 20 microg group. No sudden increase in HbA1c or plasma glucose or decrease in beta cell function was observed in any of the dose groups. These positive findings for clinical safety further support the clinical development of Diamyd as a therapeutic to prevent autoimmune diabetes.