Assessment of Alimentación Normal con Ajuste de Insulina (ANAIS), a Spanish version of the DAFNE programme, in people with Type 1 diabetes: a randomized controlled parallel trial.

AIM: To evaluate the effects of Alimentación Normal con Ajuste de Insulina (ANAIS), a group-based, therapeutic education programme for Type 1 diabetes based on a flexible insulin regimen adjusted to the individual's food intake. METHODS: Participants with Type 1 diabetes and HbA1c levels of 53-86 mmol/mol (7-10%) were recruited from outpatient clinics at a tertiary care centre. They were randomized (using opaque, sealed envelopes, with a 2:1 treatment allocation ratio) to attend the training course immediately (immediate ANAIS; intervention group) or a year later (delayed ANAIS; control group). The main outcome was HbA1c level at 1 year. Secondary outcomes included lipid levels, weight, hypoglycaemic events, insulin dose, treatment satisfaction, self-perceived dietary freedom, quality of life and participant-defined goals. RESULTS: A total of 48 participants were assigned to the intervention group and 32 to the control group. Twelve months after completing the training, adjusted HbA1c was not significantly different in the intervention group [64 ± 1.3 vs 68 ± 1.6 mmol/mol (8.0 ±0.1% vs 8.4 ±0.1%); P=0.081]. Treatment satisfaction was significantly higher in the intervention group, but no differences were found in hypoglycaemic events, weight, insulin dose or changes in dietary freedom. At 1 year after the intervention, 72% of the participants (vs 33% in the control group; P=0.046) reported exceeding their expectations regarding achievement of their main personal goal. CONCLUSION: Promoting dietary freedom and empowering people with Type 1 diabetes through structured education programmes, such as ANAIS, improves treatment satisfaction and self-defined goals. No significant improvement in HbA1c level was achieved.

Genetics: Is LADA just late onset type 1 diabetes?

Background: There is a controversy regarding Latent Autoimmune Diabetes in Adults (LADA) classification and whether it should be considered a slowly progressing form of type 1 (T1) diabetes (DM) or a distinct type of DM altogether. Methods: This cross-sectional study assessed major genes associated with T1DM (class II HLA, PTPN22 [rs2476601] and INS [rs689]) in patients with LADA, as compared with participants with T1DM (stratified according to age of diagnosis before or after 30) and T2DM. HLA genotyping of the DRB1, DQA1 and DQB1 loci was performed by reverse PCR sequence-specific oligonucleotides. HLA haplotypes were assigned according to those most frequently described in the European population. INS and PTPN22 SNPs were genotyped by real-time PCR. Results: A total of 578 participants were included: 248 with T1DM (70 diagnosed after the age of 30), 256 with T2DM and 74 with LADA. High risk HLA alleles were significantly more frequent in LADA than in T2DM, whereas the opposite was true for protective alleles. We found a lower frequency of the high-risk DRB1*04-DQB1*03:02-DQA1*03:01 haplotype in LADA (21.1%) than in the overall T1DM (34.7%) (p<0.05), whereas no differences were found between these groups for DRB1*03-DQB1*02:01-DQA1*05:01 or for protective alleles. Only 12% the overall T1DM group had no risk alleles vs 30% of LADA (p<0.0005). However, HLA allele distribution was similar in LADA and T1DM diagnosed after the age of 30. A total of 506 individuals (195 with T1DM [21 diagnosed after age 30] 253 with T2DM and 58 with LADA) were genotyped for the PTPN22 and INS SNPs. The G/A genotype of the PTPN22 rs2476601 was more frequent and the T/T genotype of the INS SNP rs689 was less frequent in T1DM compared to LADA. We did not find any significant differences in the frequency of the mentioned SNPs between LADA and T2DM, or between LADA and T1DM diagnosed after the age of 30. Conclusion: In this relatively small cross-sectional study, the genetic profile of subjects with LADA showed a similar T1DM-related risk allele distribution as in participants with T1DM diagnosed after the age of 30, but fewer risk alleles than those diagnosed before 30. Differences were present for HLA, as well as PTPN22 and INS genes.

Insulin resistance explains the relationship between novel cardiovascular risk factors and hypertension. The Telde Study.

BACKGROUND: Most studies describing an association between hypertension and an inflammatory/pro-thrombotic state do not assess insulin resistance. AIM: To examine the association between hypertension and new cardiovascular risk factors when considering both classical risk factors and insulin resistance. METHODS: In a population-based sample of 1030 subjects, clinical information and blood samples were obtained. Subjects were classified according to the presence or absence of hypertension, and insulin resistance was estimated using the homeostasis model of assessment (HOMA). To identify variables independently associated with hypertension, a four-model multiple logistic regression was performed: model 1 included novel risk factors (Plasminogen Activator Inhibitor- 1 [PAI-1], fibrinogen, von Willebrand Factor [vWF], lipoprotein(a), homocysteine and C-reactive Protein [CRP]); model 2, novel risk factors plus HOMA; model 3 included both classical (smoking, triglycerides, HDL cholesterol, total cholesterol, waist circumference and diabetes) and novel risk factors and model 4, model 3 plus HOMA. All were adjusted for age, BMI and gender and compared using Akaike's Information Criterion (AIC). RESULTS: In model 1, only PAI-1, age and BMI showed association with hypertension.When HOMA and classical risk factors were also included, PAI-1 was replaced by triglyceride, smoking and diabetes. The lowest AIC value (best adjustment) was displayed by model 4, comprising all of the variables. Only age, BMI, HOMA and smoking remained significantly associated with hypertension. CONCLUSIONS: The novel cardiovascular risk factors assessed do not add information as markers of hypertension when classical risk factors or insulin resistance are included in the evaluation.

Kidney function and glucose metabolism in overweight and obese cats.

Background: In people, obesity and prediabetes mellitus might predispose to chronic kidney disease (CKD).Aims: To assess the association of overweight [Body condition score (BCS) >5] and glucose metabolism alterations, with established or potential markers of CKD. In addition, fructosamine and fasted blood glucose were compared as predictors of early abnormal glucose metabolism.Methods: 54 clinically healthy cats were included in a cross-sectional study comprising 25 neutered males and 29 (28 neutered) females aged 7.2 (5.5-9.4) years. Two potential markers of CKD, namely urinary free active transforming growth factor-ß1-creatinine ratio and urinary retinol binding protein-creatinine ratio were measured along with other parameters to assess CKD. A receiver operating curve was used to identify the best sensitivity and specificity of fructosamine to identify cats with fasting glucose >6.5 mmol/L.Results: No association was found between BCS and markers of CKD. Fructosamine was greater in cats with fasting glucose >6.5 mmol/L compared to those with fasting glucose ≤6.5 mmol/L. A fructosamine concentration ≥250 µmol/L was able to detect cats with hyperglycemia with a sensitivity of 77% and a specificity of 65%. Furthermore, fructosamine was more strongly correlated with fasting glucose than albumin-corrected fructosamine (r = 0.43, p = 0.002 vs r = 0.32, p = 0.026). Cats with higher fructosamine had lower serum symmetric dimethylarginine concentrations.Conclusion: The present study does not suggest an effect of obesity on renal function in domestic cats.Clinical relevance: Fructosamine might be of value for the diagnosis of prediabetes mellitus in cats.

Differences in traditional and emerging cardiovascular risk factors of subjects discordantly classified by metabolic syndrome definitions of the International Diabetes Federation and the National Cholesterol Education Program.

BACKGROUND AND AIM: Several working definitions of metabolic syndrome have been proposed for clinical use. However, individuals can be discordantly classified as having or not having metabolic syndrome depending on the choice of one or another definition. This study compared the cardiovascular risk profile of subjects concordantly and discordantly diagnosed by the criteria of the National Cholesterol Education Program (NCEP) and the criteria of the International Diabetes Federation (IDF). METHODS AND RESULTS: Nine hundred and twenty-nine non-diabetic adult subjects belonging to a cross-sectional population-based study in Gran Canaria island (Spain) were assessed. Participants completed a questionnaire and underwent physical examination, fasting blood analyses, and a standardized oral glucose tolerance test. Two hundred and four subjects (22%) had metabolic syndrome according to both definitions, 31 (3.3%) only by the IDF criteria, and 5 (0.5%) only by the NCEP criteria. Participants fulfilling both proposals showed more adverse age and sex-adjusted measures of BMI, waist, HDL cholesterol, triglycerides, post-load glucose, HOMA-IR and plasminogen inhibitor activator-1 (PAI-1) than individuals exclusively satisfying the IDF criteria. Moreover, in contrast to subjects meeting both criteria, those that fulfilled only the IDF criteria had levels of BMI, waist, total and HDL cholesterol, post-load glucose, glycated HbA1c, C-reactive protein, PAI-1 and fibrinogen not significantly different from those observed in subjects without metabolic syndrome. CONCLUSION: The IDF definition identifies a surplus of individuals whose cardiovascular risk profile, particularly regarding to some non-traditional cardiovascular risk factors, is less adverse than that observed in subjects also diagnosed by the NCEP definition.

Effect of liraglutide on physical performance in type 2 diabetes: Results of a randomized, double-blind, controlled trial (LIPER2).

AIMS: To assess the effect of the GLP-1 analogue liraglutide on measures of cardiac function and physical performance in patients with type 2 diabetes (T2D). METHODS: In this phase-IV randomized double-blind placebo-controlled parallel-group clinical trial at a tertiary hospital, T2D patients with HbA1c levels of 7-10% with oral agents and/or intermediate-/long-acting insulin were allocated (computer-generated randomization, ratio 1:1) to either liraglutide 1.8 mg/day or a placebo for 6 months. The primary endpoint was maximum oxygen consumption (VO2max) during cycle ergometry, while other procedures included a 6-min walk test, echocardiography, anthropometry and blood tests. Safety endpoints were also monitored, and an intention-to-treat analysis was performed. RESULTS: A total of 24 patients (15 women) aged 52 (11.7) years, with diabetes duration of 8.7 (5.8) years, BMI 34.98 (6.2) kg/m2 and HbA1c 8.2% (0.68%), were randomized to liraglutide 1.8 mg daily or placebo. There were no differences in VO2max [17.98 (4.8) vs. 15.90 (4.96) mL/kg/min; P > 0.10], VE/VCO2 slope [30.18 (4.8) vs. 32 (4.49)], left ventricular ejection fraction or 6-min walk test [530.7 (86) vs. 503.9 (84) m] at 6 months. HbA1c was lower (6.7% vs. 7.7%; P = 0.005), with a trend towards lower maximum systolic blood pressure during ergometry [171.7 (24.4) vs. 192.5 (25.6); P = 0.052] in the liraglutide group at the end of the study. There were no severe adverse events. CONCLUSION: In this trial, liraglutide improved glycaemic control in T2D, but had no significant effects on either physical performance or myocardial function.

Measurements of in Vivo Ubiquinone Reduction Levels in Plant Cells.

A method is described for the determination of in vivo ubiquinone (UQ) reduction levels in nongreen tissues by extraction and subsequent detection of ubiquinone-10 and ubiquinol-10 with high-performance liquid chromatography. In Petunia hybrida cell suspensions UQ reduction remained at a stable level of about 60%, despite the changing conditions during the batch culture (from excess sugar to starvation) and the concomitant variations in respiration. Also, in the presence of uncoupler, which causes a large increase in respiration via both the cytochrome pathway and the alternative pathway, UQ reduction levels stayed at 60%. In mitochondria isolated from these cells, activity of the alternative pathway was only observed at UQ reduction levels higher than 80%. It is proposed that in vivo the relationship between UQ reduction and the activity of the alternative oxidase is modulated by mechanisms such as thiol modifications and accumulation of organic acids. Accordingly, pyruvate concentration in P. hybrida cells increased in the presence of uncoupler.

Changes in Mitochondrial Respiratory Chain Components of Petunia Cells during Culture in the Presence of Antimycin A.

When petunia (Petunia hybrida Vilm, cv Rosy Morn) cells are cultured in the presence of 2 [mu]M antimycin A (AA), respiration proceeds mainly via the cyanide-resistant pathway. Cyanide-resistant respiratory rates were higher in mitochondria from AA cells than in control mitochondria. Compared with control cells, an increase in alternative oxidase protein was observed in AA cells, as well as an increase in ubiquinone (UQ) content. A change in the kinetics of succinate dehydrogenase was observed: there was a much higher activity at high UQ reduction in mitochondria from AA cells compared with control mitochondria. No changes were found for external NADH dehydrogenase kinetics. In AA cells in vivo, UQ reduction was only slightly higher than in control cells, indicating that increased electron transport via the alternative pathway can prevent high UQ reduction levels. Moreover, O2 consumption continues at a similar rate as in control cells, preventing O2 danger. These adaptations to stress conditions, in which the cytochrome pathway is restricted, apparently require, in addition to an increase in alternative oxidase protein, a new setup of the relative amounts and/or kinetic parameters of all of the separate components of the respiratory network.

Stimulation of the Alternative Pathway by Succinate and Malate.

Stimulation of the cyanide-resistant oxidation of exogenous NADH in potato (Solanum tuberosum L. cv Bintje) tuber callus mitochondria was obtained with succinate, malate, and pyruvate. Half-maximal stimulation was observed at a succinate or malate concentration of 3 to 4 mM, which is considerably higher than that found for pyruvate (0.128 mM). No effect of succinate or malate addition was found when duroquinone was the electron acceptor. Duroquinol oxidation via the alternative pathway was poor and not stimulated by organic acids. Under stimulating conditions, no swelling or contraction of the mitochondria could be observed. Conversely, variation of the osmolarity did not affect the extent of stimulation. However, the assay temperature had a significant effect: no stimulation occurred at temperatures below 16 to 20[deg]C. Membrane fluidity measurements showed a phase transition at about 17[deg]C. Ubiquinone reduction levels were not significantly higher in the presence of succinate and malate, but the kinetics of the alternative oxidase were changed in a way comparable to that found for stimulation by pyruvate. At low temperatures the alternative oxidase displayed "activated" kinetics, and a role for membrane fluidity in the stimulation of the alternative pathway by carboxylic acids is suggested.

Cytoplasmic Acidification and Secondary Metabolite Production in Different Plant Cell Suspensions (A Comparative Study).

In this study, a correlation is described between low cytoplasmic pH, measured with the fluorescent probes 2[prime],7[prime]-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (acetoxymethyl ester) and bis- [3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol, and the production of secondary metabolites for several plant cell-suspension systems. Anthraquinone production in Morinda citrifolia suspensions is negligible in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), whereas with naphthalene acetic acid (NAA) a significant accumulation is realized. NAA-grown cells showed a lower cytoplasmic pH than did 2,4-D-grown cells. Addition of 2,4-D or parachlorophenoxy acetic acid to NAA-grown cells resulted in an inhibition of anthraquinone production and an increase of the cytoplasmic pH, whereas addition of parachlorophenyl acetic acid had no effect on either parameter. Lignin production in Petunia hybrida cells could be induced by subculturing them in a medium without iron. These cells showed a lower cytoplasmic pH than control cells. Addition of Fe3+ led to a decreased lignin content and an increased cytoplasmic pH. Two cell lines of Linum flavum showed a different level of coniferin and lignin concentration in their cells. Cells that accumulated coniferin and lignin had a lower cytoplasmic pH than cells that did not accumulate these secondary metabolites. Apparently, in different species and after different kinds of treatment there is a correlation between acidification of the cytoplasm and the production of different secondary metabolites. The possible role of this acidification in secondary metabolite production is discussed.

Quantitative effect of glycaemic improvement on the components of diabetic dyslipidaemia: a longitudinal study.

In order to assess the effect of glycaemic improvement on lipoprotein concentrations, we studied 73 type 2 diabetic subjects before (HbA1c 10.1 (6.2-16)%) and after (HbA1c 6.6 (3.8-8.0)%) glycaemic improvement. Total triglyceride and cholesterol (c), LDLc, HDLc, non-HDLc and apolipoproteins AI (apoAI) and B (apoB) were measured. Bivariate correlations and step-wise, multivariate analysis were performed to find predictors of change in the different components of diabetic dyslipidaemia. Changes in HDLc (r = -0.358, P = 0.001), apoAI (r = -0.355, P = 0.003), apoAI/apoB ratio (r = -0.333, P = 0.005), weight (r = -0.245, P = 0.046) and BMI (r = -0.253, P = 0.039) correlated with that of HbA1c, but, in multivariate analysis, only changes in HDLc, apoAI and apoAI/apoB ratio were predicted by the decrease in HbA1c. For the median observed change in HbA1c (-3.3 percentage-points), the estimated changes were +0.14 mmol/l, +0.12 g/l and +0.20 for HDLc, apoAI and apoAI/apoB ratio, respectively, accounting for 81, 92 and 80% of the observed changes. In conclusion, for the component of diabetic dyslipidaemia for which less therapeutic tools are available, glycaemic improvement is most effective.

Prevalence and phenotypic distribution of dyslipidemia in type 1 diabetes mellitus: effect of glycemic control.

BACKGROUND: Data on the prevalence of dyslipidemia in type 1 diabetes mellitus are scarce and are based on total triglyceride and total cholesterol concentrations alone. OBJECTIVE: To assess the effect of glycemic optimization on the prevalence of dyslipidemia and low-density lipoprotein cholesterol (LDL-C) concentrations requiring intervention in patients with type 1 diabetes. PATIENTS: A total of 334 adults with type 1 diabetes and 803 nondiabetic control subjects. METHODS: Levels of glycosylated hemoglobin, total cholesterol, total triglyceride, high-density lipoprotein cholesterol (HDL-C), and LDL-C were assessed at baseline and after 3 to 6 months of intensive therapy with multiple insulin doses. RESULTS: Levels of LDL-C greater than 4.13 mmol/L (>160 mg/dL) and total triglyceride greater than 2.25 mmol/L (>200 mg/dL) and low HDL-C levels (<0.9 mmol/L [<35 mg/dL] in men or <1.1 mmol/L [<45 mg/dL] in women) were found in 16%, 5%, and 20% of patients and 13%, 6%, and 9% of controls, respectively (P<.001 for HDL-C). Diabetic women showed more hypercholesterolemia than nondiabetic women (15.6% vs 8.5%; P =.04). After glycemic optimization (mean +/- SD glycosylated hemoglobin decrease, 2.2 +/- 1.96 percentage points), the prevalence of LDL-C levels greater than 4.13 mmol/L (>160 mg/dL) became lower in diabetic men than in nondiabetic men (9.7% vs 17.5%; P =.04), but women showed frequencies of dyslipidemia similar to their nondiabetic counterparts. The proportion of patients with LDL-C concentrations requiring lifestyle (>2.6 mmol/L [>100 mg/dL]) or drug (>3.4 mmol/L [>130 mg/dL]) intervention decreased from 78% and 42% to 66% and 26%, respectively. CONCLUSIONS: Low HDL-C is the most frequent dyslipidemic disorder in patients with poorly controlled insulin-treated type 1 diabetes, and a high proportion show LDL-C levels requiring intervention. Less favorable lipid profiles could explain the absence of sex protection in diabetic women. The improvement caused by glycemic optimization puts forward intensive therapy as the initial treatment of choice for dyslipidemia in poorly controlled type 1 diabetes.

Apolipoprotein(B) identifies dyslipidemic phenotypes associated with cardiovascular risk in normocholesterolemic type 2 diabetic patients.

OBJECTIVE: Apolipoprotein(B) [apo(B)] reflects the total mass of atherogenic particles (VLDL, IDL, and LDL), and its increase is associated with cardiovascular disease independently of LDL cholesterol (LDLc) levels. Apo(B) determination has been recently standardized, but attention to regional reference limits is advisable. Our aim was to analyze the frequency of dyslipidemic phenotypes, including those dependent on increased apo(B) in normocholesterolemic type 2 diabetic patients. RESEARCH DESIGN AND METHODS: A total of 100 consecutively seen type 2 diabetic patients (63 men, 37 women; aged 59 +/- 11 years) were included, after excluding those on lipid-lowering therapy. Apo(B) cutoff (1.1 g/l) was obtained from a group of normolipidemic (47 men, 21 women) control subjects, and LDLc, triglycerides, and HDL cholesterol (HDLc) cutoff points were those from the National Cholesterol Education Program guidelines. LDLc levels were obtained by ultracentrifugation if triglyceride levels were > 3.45 mmol/l; otherwise, they were calculated (Friedwald). Apo(B) levels were measured by immunoturbidimetry. RESULTS: Normocholesterolemia (LDLc < 4.13 mmol/l) appeared in 75 of the 100 patients, of whom 55 were normo- and 20 hypertriglyceridemic. Hyperapolipoprotein(B) [hyperapo(B)] was the most frequent lipid disorder, present in 34 (45%) of the normocholesterolemic patients (22 normo- and 12 hypertriglyceridemic). Low HDLc levels were more prevalent (53%) in patients with hyperapo(B) than in the rest (24%). CONCLUSIONS: Hyperapo(B) was found in almost half of the normocholesterolemic type 2 diabetic patients and was frequently associated with low HDLc levels and hypertriglyceridemia. Thus, given its independent association with cardiovascular disease and that it identifies high-risk phenotypes in normocholesterolemic diabetic patients apo(B) should be used to evaluate the lipidic pattern of these patients.

A role for active oxygen species as second messengers in the induction of alternative oxidase gene expression in Petunia hybrida cells.

Incubation of Petunia hybrida cells with H2O2 leads to an increase in alternative oxidase activity measured after 24 h. This increased activity is accompanied by an increase in alternative oxidase protein. A model is presented for the regulation of alternative oxidase protein synthesis in which active oxygen species and especially H2O2 play a crucial role as second messengers in the signal transducing pathway from the mitochondria to the nucleus. It is proposed that also the induction of the alternative oxidase by salicylic acid is mediated via H2O2.

Immunological identification of the alternative oxidase of Acanthamoeba castellanii mitochondria.

Mitochondria of the protozoa Acanthamoeba castellanii possess a cyanide-insensitive oxidase cross-reacting with monoclonal antibodies raised against the plant alternative oxidase. Immunoblotting revealed three monomeric forms (38, 35, and 32 kDa) and very low amounts of a single 65 kDa dimeric form. Cross-linking studies suggest that while in plants the alternative oxidase occurs as a dimer, in amoeba it functions as a monomer. Immunologically detectable protein levels change with the age of amoeba cell culture. Increased amounts of the 35 kDa protein are accompanied by an increase in the activity of cyanide-resistant respiration.

Structure and function of the plant alternative oxidase: its putative role in the oxygen defence mechanism.

Current understanding of the structure and function of the plant alternative oxidase is reviewed. In particular, the role of the oxidase in the protection of tissues against oxidative stress is developed.

Stress-induced changes in ubiquinone concentration and alternative oxidase in plant mitochondria.

We have investigated the influence of stress conditions such as incubation at 4 degrees C and incubation in hyperoxygen atmosphere, on plant tissues. The ubiquinone (Q) content and respiratory activity of purified mitochondria was studied. The rate of respiration of mitochondria isolated from cold-treated green bell peppers (Capsicum annuum L) exceeds that of controls, but this is not so for mitochondria isolated from cold-treated cauliflower (Brassica oleracea L). Treatment with high oxygen does not alter respiration rates of cauliflower mitochondria. Analysis of kinetic data relating oxygen uptake with Q reduction in mitochondria isolated from tissue incubated at 4 degrees C (bell peppers and cauliflowers) and at high oxygen levels (cauliflowers) reveals an increase in the total amount of Q and in the percentage of inoxidizable QH2. The effects are not invariably accompanied by an induction of the alternative oxidase (AOX). In those mitochondria where the AOX is induced (cold-treated bell pepper and cauliflower treated with high oxygen) superoxide production is lower than in the control. The role of reduced Q accumulation and AOX induction in the defense against oxidative damage is discussed.