Meta-analysis and cross-validation of a hyperglycemic status predictor model based on salivary glucose

Poster presented at the First International Congress of CiiEM - From Basic Sciences To Clinical Research. Egas Moniz, Caparica, Portugal, 27-28 November 2015.

Impaired Incretin Secretion And Pancreatic Dysfunction With Older Age And Diabetes.

To estimate the impact of aging and diabetes on insulin sensitivity, beta-cell function, adipocytokines, and incretin production. Hyperglycemic clamps, arginine tests and meal tolerance tests were performed in 50 non-obese subjects to measure insulin sensitivity (IS) and insulin secretion as well as plasma levels of glucagon, GLP-1 and GIP. Patients with diabetes and healthy control subjects were divided into the following groups: middle-aged type 2 diabetes (MA-DM), aged Type 2 diabetes (A-DM) and middle-aged or aged subjects with normal glucose tolerance (MA-NGT or A-NGT). IS, as determined by the homeostasis model assessment, glucose infusion rate, and oral glucose insulin sensitivity, was reduced in the aged and DM groups compared with MA-NGT, but it was similar in the MA-DM and A-DM groups. Insulinogenic index, first and second phase insulin secretion and the disposition indices, but not insulin response to arginine, were reduced in the aged and DM groups. Postprandial glucagon production was higher in MA-DM compared to MA-NGT. Whereas the GLP-1 production was reduced in A-DM, no differences between groups were observed in GIP production. In non-obese subjects, diabetes and aging impair insulin sensitivity. Insulin production is reduced by aging, and diabetes exacerbates this condition. Aging associated defects superimposed diabetic physiopathology, particularly regarding GLP-1 production. On the other hand, the glucose-independent secretion of insulin was preserved. Knowledge of the complex relationship between aging and diabetes could support the development of physiopathological and pharmacological based therapies.

Changes In Liver Cell Dna Methylation Status In Diabetic Mice Affect Its Ft-ir Characteristics.

Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas -CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of -CH3 groups. Other spectral differences were found at 1700-1500 cm(-1) and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

Enhanced Glucose-induced Intracellular Signaling Promotes Insulin Hypersecretion: Pancreatic Beta-cell Functional Adaptations In A Model Of Genetic Obesity And Prediabetes.

Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca(2+) mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca(2+) signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca(2+) signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.

Epicardial And Pericardial Fat In Type 2 Diabetes: Favourable Effects Of Biliopancreatic Diversion.

Ectopic fat is often identified in obese subjects who are susceptible to the development of type 2 diabetes mellitus (T2DM). The ectopic fat favours the decrease in insulin sensitivity (IS) and adiponectin levels. We aimed to evaluate the effect of biliopancreatic diversion (BPD) on the accumulation of ectopic fat, adiponectin levels and IS in obese with T2DM. A nonrandomised controlled study was performed on sixty-eight women: 19 lean-control (23.0 ± 2.2 kg/m(2)) and 18 obese-control (35.0 ± 4.8 kg/m(2)) with normal glucose tolerance and 31 obese with T2DM (36.3 ± 3.7 kg/m(2)). Of the 31 diabetic women, 20 underwent BPD and were reassessed 1 month and 12 months after surgery. The subcutaneous adipose tissue, visceral adipose tissue, epicardial adipose tissue and pericardial adipose tissue were evaluated by ultrasonography. The IS was assessed by a hyperglycaemic clamp, applying the minimal model of glucose. One month after surgery, there was a reduction in visceral and subcutaneous adipose tissues, whereas epicardial and pericardial adipose tissues exhibited significant reduction at the 12-month assessment (p < 0.01). Adiponectin levels and IS were normalised 1 month after surgery, resembling lean-control values and elevated above the obese-control values (p < 0.01). After 12 months, the improvement in IS and adiponectin was maintained, and 17 of the 20 operated patients exhibited fasting glucose and glycated haemoglobin within the normal range. After BPD, positive physiological adaptations occurred in grade I and II obese patients with T2DM. These adaptations relate to the restoration of IS and decreased adiposopathy and explain the acute (1 month) and chronic (12 months) improvements in the glycaemic control.

Jnk And Ikkβ Phosphorylation Is Reduced By Glucocorticoids In Adipose Tissue From Insulin-resistant Rats.

Peripheral insulin resistance (IR) is one of the main side effects caused by glucocorticoid (GC)-based therapies, and the molecular mechanisms of GC-induced IR are not yet fully elucidated. Thus, we aimed to investigate the effects of dexamethasone treatment on the main components of insulin and inflammatory signaling in the adipose tissue of rats. Male Wistar rats received daily injections of dexamethasone (1mg/kg body weight (b.w.), intraperitoneally (i.p.)) for 5 days (DEX), whereas control rats received saline (CTL). The metabolic status was investigated, and the epididymal fat fragments were collected for lipolysis and western blot analyses. The DEX rats became hyperglycemic, hyperinsulinemic, insulin resistant and glucose intolerant, compared with the CTL rats (P<0.05). The basal glycerol release in the fat fragments was 1.5-fold higher in the DEX rats (P<0.05). The phosphorylation of protein kinase B (PKB) at ser(473) decreased by 44%, whereas, the phosphorylation of insulin receptor substrate (IRS)-1 at ser(307) increased by 93% in the adipose tissue of the DEX rats after an oral bolus of glucose (P<0.05). The basal phosphorylation of c-jun-N-terminal kinase (JNK) and inhibitor of nuclear factor kappa-B (IKKβ) proteins was reduced by 46% and 58%, respectively, in the adipose tissue of the DEX rats (P<0.05). This was paralleled with a significant reduction (47%) in the glucocorticoid receptor (GR) protein content in the adipose tissue of the DEX rats (P<0.05). The insulin-resistant status of rats induced by dexamethasone administration have PKB and IRS-1 activity attenuated in epididymal fat without increases in the phosphorylation of the proinflammatory signals JNK and IKKβ.

Biópsia hepática por laparotomia paracostal em bovinos e búfalos

A técnica de biópsia hepática em ruminantes tem importante valor no diagnóstico clínico de doenças tóxicas e metabólicas, em especial nos desequilíbrios minerais. As técnicas mais comumente utilizadas restringem análises devido ao limitado volume de tecido obtido. No presente trabalho, avaliou-se o uso de uma técnica de biópsia hepática por laparotomia paracostal em bovinos e búfalos. Foram utilizados 10 bovinos e 10 búfalos hígidos. Os animais foram mantidos em estação, sedados com xilazina e infiltrados localmente com lidocaína e epinefrina. O acesso à cavidade abdominal foi realizado por meio de uma incisão dorso-ventral de 15cm no flanco direito, iniciada ventralmente (cerca de 4-5cm) ao processo transverso da 2a ou 3a vértebra lombar e situada caudalmente (cerca de 4cm) e paralelamente à 13a costela, obtendo-se visualização do fígado. Foi então realizado pinçamento do bordo caudal do órgão com pinça Doyen para remoção de fragmento hepático (2 a 4g). Procedeu-se o fechamento da cavidade abdominal como de rotina. Foram analisados os parâmetros bioquímicos e hematológicos antes do procedimento (tempo zero) e após 24 horas, 48 horas, 5 dias e 10 dias após a biópsia. Todas as variáveis bioquímicas estudadas retornaram aos valores basais 5 e 10 dias após o procedimento nos bovinos e búfalos, respectivamente. O tempo médio de cirurgia por animal foi de 25 minutos. A biópsia hepática por laparotomia paracostal demonstrou ser uma técnica eficaz e de baixo risco à saúde dos animais, permitindo a coleta de suficiente quantidade de tecido hepática para realização de múltiplas análises.

Intrahepatic Glissonian Approach for Pure Laparoscopic Left Hemihepatectomy

Background: Recent advances in laparoscopic devices and experience with advanced techniques have increased the indications for laparoscopic liver. Aim: The aim of this work was to present a video with technical aspects of a pure laparoscopic left hemi-hepatectomy (segments 2, 3, and 4) by using the intrahepatic Glissonian approach and control of venous outflow without hilar dissection or the Pringle maneuver. Patient and Method: A 63-year-old woman with a 5-cm solitary liver metastasis was referred for treatment. Four trocars were used. The left lobe was pulled upward and the lesser omentum was divided, exposing Arantius' ligament. This ligament is a useful landmark for the identification of the main left Glissonian pedicle. A small anterior incision was made in front of the hilum, and a large clamp was introduced behind the Arantius' ligament toward the anterior incision, allowing control of the left main sheath. Ischemic discoloration of the left liver was achieved and marked with cautery. The vascular clamp was replaced by a stapler. If ischemic delineation was coincident with a previously marked area, the stapler was fired. The left hepatic vein was dissected and encircled. Parenchymal transection and vascular control of the hepatic veins were accomplished with a Harmonic scalpel and an endoscopic stapling device, as appropriate. All these steps were performed without the Pringle maneuver and without hand assistance. Results: Operative time was 220 minutes with minimum blood loss. Hospital stay was 4 days. Pathology showed free surgical margins. The patient is alive with no signs of recurrence 18 months after the operation. Conclusion: Totally laparoscopic left hemihepatectomy is safe and feasible in selected patients and should be considered for patients with benign or malignant liver neoplasms. The described technique, with the use of the intrahepatic Glissonian approach and control of venous outflow, may facilitate laparoscopic left hemihepatectomy by reducing the technical difficulties in pedicle control and may decrease bleeding during liver transection.

Fretting fatigue in overhead conductors: Rig design and failure analysis of a Grosbeak aluminium cable steel reinforced conductor

The performance optimisation of overhead conductors depends on the systematic investigation of the fretting fatigue mechanisms in the conductor/clamping system. As a consequence, a fretting fatigue rig was designed and a limited range of fatigue tests was carried out at the middle high cycle fatigue regime in order to access an exploratory S-N curve for a Grosbeak conductor, which was mounted on a mono-articulated aluminium clamping system. Subsequent to these preliminary fatigue tests, the components of the conductor/clamping system, such as ACSR conductor, upper and lower clamps, bolt and nuts, were subjected to a failure analysis procedure in order to investigate the metallurgical free variables interfering on the fatigue test results, aiming at the optimisation of the testing reproducibility. The results indicated that the rupture of the planar fracture surfaces observed in the external At strands of the conductor tested under lower bending amplitude (0.9 mm) occurred by fatigue cracking (I mm deep), followed by shear overload. The V-type fracture surfaces observed in some At strands of the conductor tested under higher bending amplitude (1.3 mm) were also produced by fatigue cracking (approximately 400 mu m deep), followed by shear overload. Shear overload fracture (45 degrees fracture surface) was also observed on the remaining At wires of the conductor tested under higher bending amplitude (1.3 mm). Additionally, the upper and lower Al-cast clamps presented microstructure-sensitive cracking, which was folowed by particle detachment and formation of abrasive debris on the clamp/conductor tribo-interface, promoting even further the fretting mechanism. The detrimental formation of abrasive debris might be inhibited by the selection of a more suitable class of as-cast At alloy for the production of clamps. Finally, the bolt/nut system showed intense degradation of the carbon steel nut (fabricated in ferritic-pearlitic carbon steel, featuring machined threads with 190 HV), with intense plastic deformation and loss of material. Proper selection of both the bolt and nut materials and the finishing processing might prevent the loss in the clamping pressure during the fretting testing. It is important to control the specification of these components (clamps, bolt and nuts) prior to the start of large scale fretting fatigue testing of the overhead conductors in order to increase the reproducibility of this assessment. (c) 2008 Elsevier Ltd. All rights reserved.

Functionality of bioactive compounds in Brazilian strawberry (Fragaria x ananassa Duch.) cultivars: Evaluation of hyperglycemia and hypertension potential using in vitro models

Fruits of seven fully ripened strawberry cultivars grown in Brazil (Dover, Camp Dover, Camarosa, Sweet Charlie, Toyonoka, Oso Grande, and Piedade) were evaluated for total phenolics, antioxidant activity based on DPPH radical scavenging assay, and functionality such as inhibition of alpha-amylase, alpha-glucosidase, and angiotensin I-converting enzyme (ACE) relevant for potentially managing hyperglycemia and hypertension. The total phenolics content ranged from 966 to 1571 mu g of gallic acid/g of fruit fresh weight for Toyonoka and Dover, respectively. No correlation was found between total phenolics and antioxidant activity. The major phenolic compounds in aqueous extracts of strawberries were ellagic acid, quercetin, and chlorogenic acid. Strawberries had high alpha-glucosidase inhibitory activity. However, alpha-amylase inhibitory activity was very low in all cultivars. This suggested that strawberries could be considered as a potential dietary source with anti-hyperglycemic potential. The evaluated cultivars had no significant ACE inhibitory activity, reflecting low anti-hypertensive potential.

Purification and characterization of Ts15, the first member of a new alpha-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus

Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: alpha-, beta-, gamma- and kappa-KTx. These peptides display varying selectivity and affinity for K(v) channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Nay channels (Na(V)1.4; Na(V)1.5; Na(V)1.6; Na(V)1.8 and DmNa(V)1) and 12 different subtypes of Kv channels (K(V)1.1 - K(V)1.6; K(V)2.1; K(V)3.1; K(V)4.2; K(V)4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is crosslinked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K(V)1.2 and K(V)1.3 channels with an IC(50) value of 196 +/- 25 and 508 +/- 67 nM, respectively. No effect on Na(V) channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new alpha-Ktx21 subfamily and therefore was classified as alpha-Ktx21.1. (C) 2011 Elsevier Ltd. All rights reserved.

Characterization of calcium-activated chloride channels in patches excised from the dendritic knob of mammalian olfactory receptor neurons

We investigated the properties of calcium-activated chloride channels in inside-out membrane patches from the dendritic knobs of acutely dissociated rat olfactory receptor neurons. Patches typically contained large calcium-activated currents, with total conductances in the range 30-75 nS. The dose response curve for calcium exhibited an EC50 of about 26 mu M. In symmetrical NaCl solutions, the current-voltage relationship reversed at 0 mV and was linear between -80 and +70 mV. When the intracellular NaCl concentration was progressively reduced from 150 to 25 mM, the reversal potential changed in a manner consistent with a chloride-selective conductance. Indeed, modeling these data with the Goldman-Hodgkin-Katz equation revealed a P-Na/P-Cl of 0.034. The halide permeability sequence was P-Cl > P-F > P-I > P-Br indicating that permeation through the channel was dominated by ion binding sites with a high field strength. The channels were also permeable to the large organic anions, SCN-, acetate(-), and gluconate(-), with the permeability sequence P-Cl > P-SCN > gluconaie. Significant permeation to gluconate ions suggested that the channel pore had a minimum diameter of at least 5.8 Angstrom.

Are there functional P2X receptors on cell bodies in intact dorsal root ganglia of rats?

P2X purinoceptors have been suggested to participate in transduction of painful stimuli in nociceptive neurons. In the current experiments, ATP (1-10 mM), alpha,beta-methylene-ATP (10-30 mu M) and capsaicin (10 nM-1 mu M) were applied to neurons impaled with high resistance microelectrodes in rat dorsal root ganglia (L4 and L5) isolated in vitro together with the sciatic nerve and dorsal roots. The agonists were either bath applied or focally applied using a picospritzer. GABA (100 mu M) and 40-80 mM K+ solutions gave brisk responses when applied by either technique. Only three of 22 neurons with slowly conducting axons (C cells) showed evidence of P2X-purinoceptor-mediated responses. Only two of 13 cells which responded to capsaicin (putative nociceptors), and none of 29 cells with rapidly conducting axons (A cells), responded to the purinergic agonists. When acutely dissociated dorsal root ganglion cells were studied using patch-clamp techniques, all but four of 30 cells of all sizes responded with an inward current to either ATP or alpha,beta-methylene-ATP (both 100 mu M). Our data suggest that few sensory cell bodies in intact dorsal root ganglia express functional purinoceptors. (C) 1998 IBRO. Published by Elsevier Science Ltd.

Zinc potentiation of the glycine receptor chloride channel is mediated by allosteric pathways

Molecular mechanisms of zinc potentiation were investigated in recombinant human alpha 1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [H-3]strychnine binding assays. In the wild-type (WT) GlyR, 1 mu M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1-M2 and M2-M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2-M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.

Excitatory synaptic inputs to pyramidal neurons of the lateral amygdala

Whole-cell patch clamp recordings were made from pyramidal neurons in the rat lateral amygdala (LA). Synaptic currents were evoked by stimulating in either the external capsule (ec), internal capsule (ic) or basolateral nucleus (BLA). Stimulation of either the ic, ec or BLA evoked a glutamatergic excitatory synaptic current (EPSC) which was mediated by both non-NMDA and NMDA (N-methyl-D-aspartic acid) receptors, The ratio of the amplitude of the NMDA receptor-mediated component measured at +40 mV to the amplitude of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) component measured at -60 mV was similar regardless of whether EPSCs were evoked in the ec, ic or BLA. At resting membrane potentials, excitatory synaptic potentials evoked from either the ec or putative thalamic inputs were unaffected by application of the NMDA receptor antagonist APV. Spontaneous glutamatergic currents had two components to their decay phase. The slow component was selectively blocked by the NMDA receptor antagonist D-APV, indicating that AMPA and NMDA receptors are colocalized in spiny neurons. We conclude that pyramidal cells of the LA receive convergent inputs from the cortex, thalamus and basal nuclei. At all inputs, both AMPA/kainate and NMDA-type receptors are active and colocalized in the postsynaptic density.