Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25ºC in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40% dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).

Association between the -174 G/C promoter polymorphism of the interleukin-6 gene and cardiovascular disease risk factors in Brazilian older women

In worldwide studies, interleukin-6 (IL-6) is implicated in age-related disturbances. The aim of the present report was to determine the possible association of IL-6 -174 C/G promoter polymorphism with the cytokine profile as well as with the presence of selected cardiovascular risk features. This was a cross-sectional study on Brazilian women aged 60 years or older. A sample of 193 subjects was investigated for impaired glucose regulation, diabetes, hypertension, and dyslipidemia. Genotyping was done by direct sequencing of PCR products. IL-6 and C-reactive protein were quantified by high-sensitivity assays. General linear regression models or the Student t-test were used to compare continuous variables among genotypes, followed by adjustments for confounding variables. The chi-square test was used to compare categorical variables. The genotypes were consistent with Hardy-Weinberg equilibrium proportions. In a recessive model, mean waist-to-hip ratio, serum glycated hemoglobin and serum glucose were markedly lower in C homozygotes (P = 0.001, 0.028, and 0.047, respectively). In a dominant hypothesis, G homozygotes displayed a trend towards higher levels of circulating IL-6 (P = 0.092). Non-parametric analysis revealed that impaired fasting glucose and hypertension were findings approximately 2-fold more frequent among G homozygous subjects (P = 0.042 and 0.043, respectively). Taken together, our results show that the IL-6 -174 G-allele is implicated in a greater cardiovascular risk. To our knowledge, this is the first investigation of IL-6 promoter variants and age-related disturbances in the Brazilian elderly population.

Evaluation of a 1-h 75-g oral glucose tolerance test in the diagnosis of gestational diabetes

In order to evaluate the performance of a 1-h 75-g oral glucose tolerance test (OGTT) for the diagnosis of gestational diabetes mellitus (GDM), a cohort of 4998 women, 20 years or older, without previous diabetes being treated in prenatal care clinics in Brazil answered a questionnaire and performed a 75-g OGTT including fasting, 1-h and 2-h glucose measurements between their 24th and 28th gestational weeks. Pregnancy outcomes were transcribed from medical registries. GDM was defined according to WHO criteria (fasting: ≥126 mg/dL; 2-h value: ≥140 mg/dL) and macrosomia as a birth weight equal to or higher than 4000 g. Areas under the receiver operator characteristic curve (AUC) were compared and diagnostic properties of various cut-off points were evaluated. The AUCs for the prediction of macrosomia were 0.606 (0.572-0.637) for the 1-h and 0.589 (0.557-0.622) for the 2-h plasma glucose test. Similar predictability was demonstrable regarding combined adverse outcomes: 0.582 (0.559-0.604) for the 1-h test and 0.572 (0.549-0.595) for the 2-h test. When the 1-h glucose test was evaluated against a diagnosis of GDM defined by the 2-h glucose test, the AUC was 0.903 (0.886-0.919). The cut-off point that maximized sensitivity (83%) and specificity (83%) was 141 mg/dL, identifying 21% of the women as positive. A cut-off point of 160 mg/dL, with lower sensitivity (62%), had higher specificity (94%), labeling 8.6% as positive. Detection of GDM can be done with a 1-h 75-g OGTT: the value of 160 mg/dL has the same diagnostic performance as the conventional 2-h value (140 mg/dL). The simplification of the test may improve coverage and timing of the diagnosis of GDM.

A link between sleep loss, glucose metabolism and adipokines

The present review evaluates the role of sleep and its alteration in triggering problems of glucose metabolism and the possible involvement of adipokines in this process. A reduction in the amount of time spent sleeping has become an endemic condition in modern society, and a search of the current literature has found important associations between sleep loss and alterations of nutritional and metabolic contexts. Studies suggest that sleep loss is associated with problems in glucose metabolism and a higher risk for the development of insulin resistance and type 2 diabetes mellitus. The mechanism involved may be associated with the decreased efficacy of regulation of the hypothalamus-pituitary-adrenal axis by negative feedback mechanisms in sleep-deprivation conditions. In addition, changes in the circadian pattern of growth hormone (GH) secretion might also contribute to the alterations in glucose regulation observed during sleep loss. On the other hand, sleep deprivation stress affects adipokines - increasing tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and decreasing leptin and adiponectin -, thus establishing a possible association between sleep-debt, adipokines and glucose metabolism. Thus, a modified release of adipokines resulting from sleep deprivation could lead to a chronic sub-inflammatory state that could play a central role in the development of insulin resistance and type 2 diabetes mellitus. Further studies are necessary to investigate the role of sleep loss in adipokine release and its relationship with glucose metabolism.

Serum uric acid and disorders of glucose metabolism: the role of glycosuria

Hyperuricemia has been associated with hypertension, diabetes mellitus, and metabolic syndrome. We studied the association between hyperuricemia and glycemic status in a nonrandomized sample of primary care patients. This was a cross-sectional study of adults ≥20 years old who were members of a community-based health care program. Hyperuricemia was defined as a value >7.0 mg/dL for men and >6.0 mg/dL for women. The sample comprised 720 participants including controls (n=257) and patients who were hypertensive and euglycemic (n=118), prediabetic (n=222), or diabetic (n=123). The mean age was 42.4±12.5 years, 45% were male, and 30% were white. The prevalence of hyperuricemia increased from controls (3.9%) to euglycemic hypertension (7.6%) and prediabetic state (14.0%), with values in prediabetic patients being statistically different from controls. Overall, diabetic patients had an 11.4% prevalence of hyperuricemia, which was also statistically different from controls. Of note, diabetic subjects with glycosuria, who represented 24% of the diabetic participants, had a null prevalence of hyperuricemia, and statistically higher values for fractional excretion of uric acid, Na excretion index, and prevalence of microalbuminuria than those without glycosuria. Participants who were prediabetic or diabetic but without glycosuria had a similarly elevated prevalence of hyperuricemia. In contrast, diabetic patients with glycosuria had a null prevalence of hyperuricemia and excreted more uric acid and Na than diabetic subjects without glycosuria. The findings can be explained by enhanced proximal tubule reabsorption early in the course of dysglycemia that decreases with the ensuing glycosuria at the late stage of the disorder.

GUAR GUM EFFECTS ON BLOOD SERUM LIPIDS AND GLUCOSE CONCENTRATIONS OF WISTAR DIABETIC RATS1

The effects of guar gum derived from the endosperm of Cyamopsis tetragonoloba (75% soluble fiber, 7.6% insoluble fiber, 2.16% crude protein, 0.78% total lipids, 0.54% ash and 9.55% moisture) on diabetic rats were studied concerning food intake, body weight gain, blood serum cholesterol, triacylglycerols, glucose, LDL-, and HDL-cholesterol concentrations. The effect of gum on indexes of protein absorption and utilization was also investigated. Diets containing 0%, 10% and 20% (w/w) guar gum were fed to diabetic rats for 28 days. In spite of the fact that diabetes elevated blood lipids in all animals, guar gum diet significantly decreased (p <0.05) serum concentrations of cholesterol and triacylglycerols. Furthermore, a concomitant increase in HDL-cholesterol with a substancial elevation of the HDL/LDL cholesterol ratio was found. The most significant result in this assay was the drastic reduction of blood glucose in diabetic rats treated with guar gum diet. The gum promoted a general improvement in the condition of the diabetic rats, in body weight gain and indexes of protein absorption and utilization. The results of this research suggest that guar gum, at concentrations equal to or higher than 10%, should be effective in the treatment of hypercholesterolemia and diabetes, in humans.

The effect of a diet education with six iso-caloric meals on the body weight and blood glucose of diabetes type 2 patients

The treatment of Diabetes should not only be sought through drug administration; diet is also a part of its treatment. The aim of this study was to determine the effect of a diet with six meals having equal calories on the body weight and blood glucose on diabetes type 2 patients. This research is an Experimental study conducted in 2009 on 181 patients with diabetes. The patients visited the IDSF (Iranian Diabetes Society of Fars) weekly and the patients to be studied were randomly divided into two groups of 85 and 96 patients, respectively. The participants were repeatedly requested to consume their calculated calorie in six equal parts. The average age in the Experimental and Control groups were 51.2 ± 13.3 and 53.1 ± 9.4, respectively. The mean body weight and fasting blood glucose at the beginning of the study in Experimental and Control groups were 66.3 ± 9.4 and 69.1 ± 11.1 kg, 198.9 ± 35.1, and 199.8 ± 39.1 mg.dL-1, respectively. At the end of the study, however, the values were 63.5 ± 7.5 and 66.98 ± 9 kg, 139.5 ± 34.6 and 164.2 ± 22.1 mg.dL-1, respectively. Only the mean fasting blood glucose at the end of the study revealed a significant difference (p-value = 0.001). The results show that educating those afflicted with Diabetes Type 2 aiming at changing their diet can greatly help them manage their blood glucose.

Sesame and flaxseed oil: nutritional quality and effects on serum lipids and glucose in rats

This study evaluated the nutritional value of sesame and flaxseed oils and their effects on the lipid and glucose profile of rats fed diets containing different fat combinations. Fatty acid composition, refractive index, and iodine and saponification values were analyzed to characterize the oils. In the biological assay, Wistar rats were fed different diets, whose fat composition consisted of varying combinations of flaxseed oil, sesame oil, and animal fat. The primary constituents of the sesame oil were oleic (28.6%), linoleic (28.4%), and lauric acid (14.6%); for the flaxseed oil they were alpha-linolenic (39.90%), oleic (17.97%) and linoleic acid (12.25%). The iodine and saponification values of the oils were within the reference range. Rats fed flaxseed oil-based diets had the lowest serum cholesterol values, whereas rats fed diets with flaxseed oil + sesame oil + animal fat had the highest glucose levels. HDL levels decreased significantly with flaxseed oil. Sesame and flaxseed oils are sources of polyunsaturated fatty acids (PUFA), and the flaxseed oil-based diet had a hypocholesterolemic effect, whereas sesame oil showed oxidative stability since it contains high levels of monounsaturated and saturated fatty acids.

Effect of preparation practices and the cowpea cultivar Vigna unguiculata L.Walp on the quality and content of myo-inositol phosphate in akara (fried bean paste)

Akara is one of Brazil's national treasures prepared from cowpea (Vigna unguiculata L.Walp), grated onions and salt and deep-fried in crude palm oil. The results of this study on akara preparation methods showed that, in general, cowpeas were soaked for up 3 hours at room temperature, and the seed coats were then removed. The akara makers preferred the olho de pombo cultivar, because of its cream hue, or the macassar cultivar because it produces a crispier paste. The seeds purchased from street markets had lower ranges of InsP6, InsP5, and InsP4 (1.03-7.62 ∝mol.g- 1; 0.14-1.31 ∝mol.g- 1; and 0.0-0.10 ∝mol.g- 1, respectively) than both the paste and akara (6.72-19.24 ∝mol.g- 1; 1.29-4.57 ∝mol.g- 1; 0.0-0.76 ∝mol.g- 1; 3.31-13.71 ∝mol.g- 1; 0.0-4.48 ∝mol.g- 1; and 0.0-1.32 ∝mol.g- 1). These results suggest that other beans or cowpea varieties have been used in the preparation of akara and that the phytate levels do not affect its nutritional quality.

Inhibition of renal glucose reabsorption as a novel treatment for diabetes patients

The importance of the kidney in glucose homeostasis has been recognized for many years. Recent observations indicating a greater role of renal glucose metabolism in various physiologic and pathologic conditions have rekindled the interest in renal glucose handling as a potential target for the treatment of diabetes. The enormous capacity of the proximal tubular cells to reabsorb the filtered glucose load entirely, utilizing the sodium-glucose co-transporter system (primarily SGLT-2), became the focus of attention. Original studies conducted in experimental animals with the nonspecific SGLT inhibitor phlorizin showed that hyperglycemia after pancreatectomy decreased as a result of forced glycosuria. Subsequently, several compounds with more selective SGLT-2 inhibition properties (“second-generation”) were developed. Some agents made it into pre-clinical and clinical trials and a few have already been approved for commercial use in the treatment of type 2 diabetes. In general, a 6-month period of therapy with SGLT-2 inhibitors is followed by a mean urinary glucose excretion rate of ~80 g/day accompanied by a decline in fasting and postprandial glucose with average decreases in HgA1C ~1.0%. Concomitant body weight loss and a mild but consistent drop in blood pressure also have been reported. In contrast, transient polyuria, thirst with dehydration and occasional hypotension have been described early in the treatment. In addition, a significant increase in the occurrence of uro-genital infections, particularly in women has been documented with the use of SGLT-2 inhibitors. Conclusion: Although long-term cardiovascular, renal and bone/mineral effects are unknown SGLT-2 inhibitors, if used with caution and in the proper patient provide a unique insulin-independent therapeutic option in the management of obese type 2 diabetes patients.

Glucose dynamics in rat skeletal muscle : recovery from osmotic stress

The purpose of this study was to examine cell glucose kinetics in rat skeletal muscle during iso-osmotic recovery from hyper- and hypo-osmotic stress. Rat EDL muscles were incubated for sixty minutes in either HYPO (190 mmol/kg), ISO (290 mmol/kg), or HYPER (400 mmol/kg) media (Sigma medium-199, 8 mM glucose) according to an established in vitro whole muscle model. In addition to sixty minute baseline measures in aniso-osmotic conditions, (HYPO-0 n=8; ISO- 0, n=S; HYPER-0, n=8), muscles were subjected to either one minute (HYPO-1 n=8; ISO-1, n=8; HYPER-1, n=8) or five minutes (HYPO-5 n=8; ISO-5, n=8; HYPER-5, n=8) of iso-osmotic recovery media and analyzed for metabolite content and glycogen synthase percent activation. To determine glucose uptake during iso-osmotic recovery, muscles (n=6 per group) were incubated for sixty minutes in either hypo-, iso-, or hyper-osmotic media immediately followed by five minutes of iso-osmotic media containing 3H-glucose and 14 C-mannitol. Increased relative water content/decreased [glucose] (observed in HYPO-0) and decreased water content/increased [glucose] (observed in HYPER-0) returned to ISO levels within 5 minutes of recovery. Glycogen synthase percent activation increased significantly in HYPO-5 over iso-osmotic controls. Glucose uptake measurements revealed no significant differences between groups. It was determined that [glucose] and muscle water content rapidly recovered from osmotic stress demonstrating skeletal muscle's resilience to osmotic stress.

Structure d'une tagatose-1,6-bisphosphate aldolase de classe I : étude d'une apparente perte de stéréospécificité

La tagatose-1,6-biphosphate aldolase de Streptococcus pyogenes est une aldolase de classe I qui fait montre d'un remarquable manque de spécificité vis à vis de ses substrats. En effet, elle catalyse le clivage réversible du tagatose-1,6-biphosphate (TBP), mais également du fructose-1,6-biphosphate (FBP), du sorbose-1,6-biphosphate et du psicose-1,6-biphosphate, quatre stéréoisomères, en dihydroxyacétone phosphate (DHAP) et en glycéraldéhyde-3-phosphate (G3P). Afin de mettre à jour les caractéristiques du mécanisme enzymatique, une étude structurale de la TBP aldolase de S. pyogenes, un pathogène humain extrêmement versatile, a été entreprise. Elle a permis la résolution de la structure native et en complexe avec le DHAP, a respectivement 1.87 et 1.92 Å de résolution. Ces mêmes structures ont permis de se représenter plus clairement le site actif de l'enzyme en général, et les résidus catalytiques en particulier. Le trempage des cristaux de TBP aldolase dans une solution saturante de DHAP a en outre permis de piéger un authentique intermédiaire iminium, ainsi que sa géométrie particulière en atteste. Des expériences d'échange de proton, entreprises afin d'évaluer le stéréoisomérisme du transfert de proton catalytique, ont également permis de faire une intéressante découverte : la TBP aldolase ne peut déprotoner le coté pro-R du C3 du DHAP, mais peut le protonner. Ce résultat, ainsi que la comparaison de la structure du complexe TBP aldolase-DHAP avec la structure du complexe FBP aldolase de muscle de lapin- DHAP, pointe vers un isomérisme cis-trans autour du lien C2-C3 de la base de Schiff formée avec le DHAP. De plus, la résolution de ces deux structures a permis de mettre en évidence trois régions très mobiles de la protéine, ce qui pourrait être relié au rôle postulé de son isozyme chez S. pyogenes dans la régulation de l’expression génétique et de la virulence de la bactérie. La cristallographie par rayons X et la cinétique enzymatique ont ainsi permis d'avancer dans l'élucidation du mécanisme et des propriétés structurales de cette enzyme aux caractéristiques particulières.

Étude structure-fonction des fructose-1,6-bisphosphate aldolases métallo-dépendantes : mécanisme catalytique et développement d’antimicrobiens

Les fructose-1,6-bisphosphate aldolases (FBPA) sont des enzymes glycolytiques (EC 4.1.2.13) qui catalysent la transformation réversible du fructose-1,6-bisphosphate (FBP) en deux trioses-phosphates, le glycéraldéhyde-3-phosphate (G3P) et le dihydroxyacétone phosphate (DHAP). Il existe deux classes de FBPA qui diffèrent au niveau de leur mécanisme catalytique. Les classes I passent par la formation d’un intermédiaire covalent de type iminium alors que les classes II, métallodépendantes, utilisent généralement un zinc catalytique. Contrairement au mécanisme des classes I qui a été très étudié, de nombreuses interrogations subsistent au sujet de celui des classes II. Nous avons donc entrepris une analyse détaillée de leur mécanisme réactionnel en nous basant principalement sur la résolution de structures cristallographiques. De nombreux complexes à haute résolution furent obtenus et ont permis de détailler le rôle de plusieurs résidus du site actif de l’enzyme. Nous avons ainsi corrigé l’identification du résidu responsable de l’abstraction du proton de l’O4 du FBP, une étape cruciale du mécanisme. Ce rôle, faussement attribué à l’Asp82 (chez Helicobacter pylori), est en fait rempli par l’His180, un des résidus coordonant le zinc. L’Asp82 n’en demeure pas moins essentiel car il oriente, active et stabilise les substrats. Enfin, notre étude met en évidence le caractère dynamique de notre enzyme dont la catalyse nécessite la relocalisation du zinc et de nombreux résidus. La dynamique de la protéine ne permet pas d’étudier tous les aspects du mécanisme uniquement par l’approche cristallographique. En particulier, le résidu effectuant le transfert stéréospécifique du proton pro(S) sur le carbone 3 (C3) du DHAP est situé sur une boucle qui n’est visible dans aucune de nos structures. Nous avons donc développé un protocole de dynamique moléculaire afin d’étudier sa dynamique. Validé par l’étude d’inhibiteurs de la classe I, l’application de notre protocole aux FBPA de classe II a confirmé l’identification du résidu responsable de cette abstraction chez Escherichia coli (Glu182) mais pointe vers un résidu diffèrent chez H. pylori (Glu149 au lieu de Glu142). Nos validations expérimentales confirment ces observations et seront consolidées dans le futur. Les FBPA de classe II sont absentes du protéome humain mais sont retrouvées chez de nombreux pathogènes, pouvant même s'y révéler essentielles. Elles apparaissent donc comme étant une cible idéale pour le développement de nouveaux agents anti-microbiens. L’obtention de nouveaux analogues des substrats pour ces enzymes a donc un double intérêt, obtenir de nouveaux outils d’étude du mécanisme mais aussi développer des molécules à visée pharmacologique. En collaboration avec un groupe de chimistes, nous avons optimisé le seul inhibiteur connu des FBPA de classe II. Les composés obtenus, à la fois plus spécifiques et plus puissants, permettent d’envisager une utilisation pharmacologique. En somme, c’est par l’utilisation de techniques complémentaires que de nouveaux détails moléculaires de la catalyse des FBPA de classe II ont pu être étudiés. Ces techniques permettront d’approfondir la compréhension fine du mécanisme catalytique de l’enzyme et offrent aussi de nouvelles perspectives thérapeutiques.

Rôle du stress oxydant en période néonatale dans l'hypertension artérielle et la dysfonction vasculaire et métabolique de l'adulte

Thèse réalisée dans le cadre d'une cotutelle entre l'Université de Montréal et l'Université d'Auvergne en France

Évaluation d’un prototype de détecteur de glucose dans le tissu interstitiel sans aiguille, le PGS (Photonic Glucose Sensor)

Objectif : Déterminer la fiabilité et la précision d’un prototype d’appareil non invasif de mesure de glucose dans le tissu interstitiel, le PGS (Photonic Glucose Sensor), en utilisant des clamps glycémiques multi-étagés. Méthodes : Le PGS a été évalué chez 13 sujets avec diabète de type 1. Deux PGS étaient testés par sujet, un sur chacun des triceps, pour évaluer la sensibilité, la spécificité, la reproductibilité et la précision comparativement à la technique de référence (le Beckman®). Chaque sujet était soumis à un clamp de glucose multi-étagé de 8 heures aux concentrations de 3, 5, 8 et 12 mmol/L, de 2 heures chacun. Résultats : La corrélation entre le PGS et le Beckman® était de 0,70. Pour la détection des hypoglycémies, la sensibilité était de 63,4%, la spécificité de 91,6%, la valeur prédictive positive (VPP) 71,8% et la valeur prédictive négative (VPN) 88,2%. Pour la détection de l’hyperglycémie, la sensibilité était de 64,7% et la spécificité de 92%, la VPP 70,8% et la VPN : 89,7%. La courbe ROC (Receiver Operating Characteristics) démontrait une précision de 0,86 pour l’hypoglycémie et de 0,87 pour l’hyperglycémie. La reproductibilité selon la « Clark Error Grid » était de 88% (A+B). Conclusion : La performance du PGS était comparable, sinon meilleure que les autres appareils sur le marché(Freestyle® Navigator, Medtronic Guardian® RT, Dexcom® STS-7) avec l’avantage qu’il n’y a pas d’aiguille. Il s’agit donc d’un appareil avec beaucoup de potentiel comme outil pour faciliter le monitoring au cours du traitement intensif du diabète. Mot clés : Diabète, diabète de type 1, PGS (Photonic Glucose Sensor), mesure continue de glucose, courbe ROC, « Clark Error Grid».