Alfredo Rigalli

Proteomic analysis of gastrocnemius muscle in rats with streptozotocin-induced diabetes and chronically exposed to fluoride.

Administration of high doses of fluoride (F) can alter glucose homeostasis and lead to insulin resistance (IR). This study determined the profile of protein expression in the gastrocnemius muscle of rats with streptozotocin-induced diabetes that were chronically exposed to F. Male Wistar rats (60 days old) were randomly distributed into two groups of 18 animals. In one group, diabetes was induced through the administration of streptozotocin. Each group (D-diabetic and ND-non-diabetic) was further divided into 3 subgroups each of which was exposed to a different F concentration via drinking water (0 ppm, 10 ppm or 50 ppm F, as NaF). After 22 days of treatment, the gastrocnemius muscle was collected and submitted to proteomic analysis (2D-PAGE followed by LC-MS/MS). Protein functions were classified by the GO biological process (ClueGO v2.0.7+Clupedia v1.0.8) and protein-protein interaction networks were constructed (PSICQUIC, Cytoscape). Quantitative intensity analysis of the proteomic data revealed differential expression of 75 spots for ND0 vs. D0, 76 for ND10 vs.D10, 58 spots for ND50 vs. D50, 52 spots for D0 vs. D10 and 38 spots for D0 vs. D50. The GO annotations with the most significant terms in the comparisons of ND0 vs. D0, ND10 vs. D10, ND50 vs. D50, D0 vs. D10 and D0 vs. D50, were muscle contraction, carbohydrate catabolic processes, generation of precursor metabolites and energy, NAD metabolic processes and gluconeogenesis, respectively. Analysis of subnetworks revealed that, in all comparisons, proteins with fold changes interacted with GLUT4. GLUT4 interacting proteins, such as MDH and the stress proteins HSPB8 and GRP78, exhibited decreased expression when D animals were exposed to F. The presence of the two stress proteins indicates an increase in IR, which might worsen diabetes. Future studies should evaluate whether diabetic animals treated with F have increased IR, as well as which molecular mechanisms are involved.

Luminal calcium concentration controls intestinal calcium absorption by modification of intestinal alkaline phosphatase activity.

Intestinal alkaline phosphatase (IAP) is a brush-border phosphomonoesterase. Its location suggests an involvement in the uptake of nutrients, but its role has not yet been defined. IAP expression parallels that of other proteins involved in Ca absorption under vitamin D stimulation. Experiments carried out in vitro with purified IAP have demonstrated an interaction between Ca and IAP. The gut is prepared to face different levels of Ca intake over time, but high Ca intake in a situation of a low-Ca diet over time would cause excessive entry of Ca into the enterocytes. The presence of a mechanism to block Ca entry and to avoid possible adverse effects is thus predictable. Thus, in the present study, Sprague-Dawley rats were fed with different amounts of Ca in the diet (0.2, 1 and 2 g%), and the percentage of Ca absorption (%Ca) in the presence and absence of L-phenylalanine (Phe) was calculated. The presence of Phe caused a significant increase in %Ca (52.3 (SEM 6.5) % in the presence of Phe v. 31.1 (sem 8.9) % in the absence of Phe, regardless of the amount of Ca intake; paired t test, P = 0.02). When data were analysed with respect to Ca intake, a significant difference was found only in the group with low Ca intake (paired t test, P = 0.03). Additionally, IAP activity increased significantly (ANOVA, P < 0.05) as Ca concentrations increased in the duodenal lumen. The present study provides in vivo evidence that luminal Ca concentration increases the activity of IAP and simultaneously decreases %Ca, acting as a minute-to-minute regulatory mechanism of Ca entry.

Physical exercise ameliorates the toxic effect of fluoride on the insulin–glucose system

Daily intake of water with fluoride concentrations >1.5 mg/l produces insulin resistance (IR). On the other hand, physical activity increases insulin sensitivity in the muscle. Therefore, the aim of this study was to evaluate the effect of physical activity on IR in rats treated with sodium fluoride (NaF) in drinking water. Sprague-Dawley rats were divided into three groups (n=10/group): Control (drinking water without NaF), NaF (drinking water with NaF 15 mg/l for 30 days), and Exercise (daily running on a treadmill for 60 min at 2.25 m/min and drinking water with NaF 15 mg/l for 30 days). IR was evaluated with the homeostasis model assessment-IR (HOMA-IR) index using fasting plasma levels of glucose and insulin. IR increased in rats treated with 15 mg/l NaF in drinking water. A decrease in IR was observed in rats that performed physical activity and drank water with 15 mg/l NaF; the Exercise group also showed an increase in the amounts of bone fluoride. The variation in the HOMA-IR values could be the consequence of variation in the sensitivity of tissues to insulin or decrease in plasma fluoride levels due to bone fluoride intake. These findings indicate that the performance of daily physical activity could reduce the negative effects of the chronic ingestion of NaF on glucose homeostasis.

Low-Level Fluoride Exposure Increases Insulin Sensitivity in Experimental Diabetes

The effect of chronic fluoride (F) exposure from the drinking water on parameters related to glucose homeostasis was investigated. Wistar rats were randomly distributed into 2 groups (diabetic [D] and nondiabetic [ND]; n = 54 each). In D, diabetes was induced with streptozotocin. Each group was further divided into 3 subgroups (0, 10, or 50 mgF/L in drinking water). After 22 days of treatment, plasma and liver samples were collected. No alterations in glycemia, insulinemia, KITT, and HOMA2-IR (homeostasis model assessment 2 of insulin resistance) were seen for ND. F-exposure of D rats led to significantly lower insulinemia, without alterations in glycemia (increased %S). Proteomic analysis detected 19, 39, and 16 proteins differentially expressed for the comparisons D0 vs. D10, D0 vs. D50, and D10 vs. D50, respectively. Gene Ontology with the most significant terms in the comparisons D0 vs. D10, D0 vs. D50, and D50 vs. D10 were organic acid metabolic process and carboxylic acid metabolic process, organic acid metabolic process, and cellular ketone metabolic process. Analysis of subnetworks revealed that proteins with fold changes interacted with GLUT4 in comparison D0 vs. D10. Among these proteins, ERj3p was present in D10. Upregulation of this protein in the presence of F might help to explain the higher %S found in these animals. These data suggest that fluoride might enhance glucose homeostasis in diabetes and identify specific biological mechanisms that merit future studies.

Fluoride increases superoxide production and impairs the respiratory chain in ROS 17/2.8 osteoblastic cells.

It is known that fluoride produces oxidative stress. Inflammation in bone tissue and an impairment of the respiratory chain of liver have been described in treatments with fluoride. Whether the impairment of the respiratory chain and oxidative stress are related is not known. The aim of this work was to study the effects of fluoride on the production of superoxide radical, the function of the respiratory chain and the increase in oxidative stress in ROS 17/2.8 osteoblastic cells. We measured the effect of fluoride (100 µM) on superoxide production, oxygen consumption, lipid peroxidation and antioxidant enzymes activities of cultured cells following the treatment with fluoride. Fluoride decreased oxygen consumption and increased superoxide production immediately after its addition. Furthermore, chronic treatment with fluoride increased oxidative stress status in osteoblastic cells. These results indicate that fluoride could damage bone tissue by inhibiting the respiratory chain, increasing the production of superoxide radicals and thus of the others reactive oxygen species.

Extended adaptive predictive controller with robust filter to enhance blood glucose regulation in type I diabetic subjects

Abstract In this paper, an improved adaptive predictive control with robust filter is developed to be applied in an artificial pancreas. Several problems inherent to endocrine systems for diabetic persons have to be tackled such as nonlinearities, long time delays or daily variations of parameters. Three Finite Impulse Response models for insulin input and the same for meal intake (perturbations) corresponding to normal, hyper-hypoglycaemia levels to implement three zones control are taken into account. The glycaemia reference trajectory is shaped from a healthy person response. A variable weighting factor in the cost function is included to prevent dangerous glycaemia excursions out of the allowed limits. Additionally, a noisy blood glucose subcutaneous sensor model is used. This control strategy is tested on 30 virtual subjects from the UVa – Padova Simulator. Simultaneous meals and physiological disturbances are taken into account and the main conclusions are drawn from Control Variability Grid Analysis.

Effect of disodium monofluorphosphate on plasma and blood viscosity in the rat.

Alpha macroglobulins (AM) are plasma proteins whose main function is to inactivate proteinases, protecting the tissues from the action of these enzymes. AM have influence on plasma viscosity (PV) and binds monofluorophosphate (MFP), which disturbs its homeostasis. The aim of this work was to evaluate whether the administration with MFP could modify blood viscosity. AM levels (micromol/l), PV (mPa.s), viscosity of red blood cells suspensions in NaCl 9 g/l (VES) and in autologue plasma (VEP) were measured in fifty-day old rats after a single dose of 80 micromol MFP or after 30 days of treatment with 80 micromol of MFP. Relative viscosity (RV) was calculated as the ratio VEP/PV. AM and PV increased significantly after 30 min of an oral dose of MFP. Controls (n=6), AM: 19.65+/-0.85, PV: 1.39+/-0.01, treated (n=6), AM: 22.88+/-0.75 (p<0.05), PV: 1.76+/-0.14 (p<0.05). After 30 days of treatment with MFP, AM and PV increased significantly. Controls (n=6), AM: 10.76+/-1.33, PV: 1.19+/-0.04, treated (n=6), AM: 17.66+/-1.27 (p<0.05), PV: 1.38+/-0.03 (p<0.05). The treatment with MFP modifies neither the VEP nor the RV. These results would indicate that AM and/or MFP did not interact with erythrocyte membrane and did not modify erythrocyte deformability.

Mathematical model of glucose-insulin homeostasis in healthy rats

According to the World Health Organization there are over 220 million people in the world with diabetes and 3.4 million people died in 2004 as a consequence of this pathology. Development of an artificial pancreas would allow to restore control of blood glucose by coupling an infusion pump to a continuous glucose sensor in the blood. The design of such a device requires the development and application of mathematical models which represent the gluco-regulatory system. Models developed by other research groups describe very well the gluco-regulatory system but have a large number of mathematical equations and require complex methodologies for the estimation of its parameters. In this work we propose a mathematical model to study the homeostasis of glucose and insulin in healthy rats. The proposed model consists of three differential equations and 8 parameters that describe the variation of: blood glucose concentration, blood insulin concentration and amount of glucose in the intestine. All parameters were obtained by setting functions to the values of glucose and insulin in blood obtained after oral glucose administration. In vivo and in silico validations were performed. Additionally, a qualitative analysis has been done to verify the aforementioned model. We have shown that this model has a single, biologically consistent equilibrium point. This model is a first step in the development of a mathematical model for the type I diabetic rat.

Testing PFC Controller On A Well Validated In Silico Model of a Type I Diabetic Patient

Abstract Diabetes technology has been focused since three decades ago on developing the artificial pancreas through several closed-loop control algorithms linking glucose measurements and insulin delivery. This work is focused on rigorously analyzing the Predictive Functional Control (PFC) algorithm capabilities for deciding about the correct insulin dosage under everyday circumstances. The study is done by applying the PFC in a recently developed model of the endocrine system, approved by the FDA in 2008, as a substitute to animal trial. The platform used here consists only of a limited number of patients: 10 children, 10 adolescents, and 10 adults. To realistically represent the full closed loop system, a model of a subcutaneous glucose sensor was added and the constraints related to the insulin pump was taken into account by the predictive controller. The performance of the controller, with and without the sensor model, was evaluated by means of the Control Variability Grid Analysis (CVGA) technique and the results were satisfactory in all patients.

Regulation of intestinal calcium absorption by luminal calcium content: Role of intestinal alkaline phosphatase

SCOPE Intestinal alkaline phosphatase is a brush border enzyme that is stimulated by calcium. Inhibition of intestinal alkaline phosphatase increases intestinal calcium absorption. We hypothesized that intestinal alkaline phosphatase acts as a minute-to-minute regulatory mechanism of calcium entry. The aim of this study was to evaluate the mechanism by which intestinal luminal calcium controls intestinal calcium absorption. METHODS AND RESULTS We performed kinetic studies with purified intestinal alkaline phosphatase and everted duodenal sacs and showed that intestinal alkaline phosphatase modifies the luminal pH as a function of enzyme concentration and calcium luminal content. A decrease in pH occurred simultaneously with a decrease in calcium absorption. The inhibition of intestinal alkaline phosphatase by l-phenylalanine caused an increase in calcium absorption. This effect was also confirmed in calcium uptake experiments with isolated duodenal cells. CONCLUSION Changes in luminal pH arising from intestinal alkaline phosphatase activity induced by luminal calcium concentration modulate intestinal calcium absorption.