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V Poitout

Publications and source records attributed to V Poitout.

33 records · Page 2Linked to original sources
PubMed1996

An integrated view of beta-cell dysfunction in type-II diabetes.

Type-II (non-insulin-dependent) diabetes mellitus (NIDDM) is a heterogeneous disease resulting from insulin resistance and beta-cell dysfunction. beta-Cell dysfunction in Type-II diabetes is characterized by a specific lack of first-phase glucose-induced insulin secretion. This defect is readily reversible upon normalization of blood glucose levels. Chronic hyperglycemia itself is harmful to the beta-cell and affects both insulin biosynthesis and exocytosis. No unique intracellular defect has been demonstrated to be responsible for all common forms of the disease. However, mutations of the glucokinase gene have been identified in maturity onset diabetes in the young, a particular form of NIDDM.

Adult↗
PubMed1996

Insulin-secreting cell lines: classification, characteristics and potential applications.

The use of primary beta-cells in biochemical and molecular research is limited by the availability of pancreatic endocrine tissue. Numerous investigators have attempted to establish an insulin-secreting cell line that retains normal regulation of insulin secretion. Different approaches have been used, including induction of pancreatic tumors by irradiation or viral infection, immortalization of beta-cells in vitro, and development of transgenic mice with targeted expression of a recombinant oncogene in the beta-cell. Few of these attempts have proven successful, because cell differentiation and proliferation capacities are mutually exclusive. The most widely used insulin-secreting cell lines are RIN, HIT, beta TC, MIN6 and INS-1 cells. These cells contain mainly insulin and small amounts of glucagon and somatostatin. RIN cells, except for the subclone RIN-38, are not glucose-responsive. HIT cells and beta TC cells secrete insulin in response to glucose, but their dose-response curve is markedly shifted to the left MIN6, INS-1 and a newly available subclone of beta TC cells (beta TC-6 F7) are reported to retain normal regulation of glucose-induced insulin secretion. Although the behaviour of none of these cell lines perfectly mimics primary beta-cell physiology, they are extremely valuable tools for the study of molecular events underlying beta-cell function and dysfunction. In addition, insulin-secreting cell lines represent a potential source of transplantable tissue to overcome the limited availability of primary islets for this procedure.

Animals↗
PubMed1995

Somatostatin coordinately regulates glucagon gene expression and exocytosis in HIT-T15 cells.

Somatostatin (SRIF) regulates secretion from several endocrine cell types. SRIF inhibits both insulin and glucagon secretion and reduces insulin gene expression. However, whether SRIF inhibition of glucagon secretion from the pancreatic alpha cell is mediated via pertussis toxin-sensitive G-proteins is not presently known, nor has it been determined whether SRIF can regulate glucagon gene expression. Consequently, we performed studies in the transformed islet cell line HIT-T15 to determine whether the inhibitory effect of SRIF on glucagon exocytosis is preserved in this cell line, whether this effect is mediated through a pertussis toxin-sensitive mechanism, and whether SRIF has an inhibitory effect on glucagon gene expression. Confocal microscopy with immunostaining revealed that 15-25% of HIT-T15 cells contained glucagon. In static incubations forskolin (FSK, 1 microM) increased glucagon secretion 3.6 +/- 0.9-fold (P < 0.01) and mixed amino acids (15 mM) increased glucagon secretion 2.8 +/- 0.4-fold (P < 0.01). Addition of SRIF significantly inhibited both forskolin- and amino acid-stimulated secretion. Maximal inhibition of both FSK- and amino acid-stimulated secretion occurred at SRIF concentrations > or = 10(-8) M and these inhibitory effects were completely prevented by pertussis toxin pretreatment. In addition to inhibiting glucagon secretion, SRIF significantly reduced both basal and FSK-stimulated glucagon mRNA levels and this reduction in glucagon mRNA was completely prevented by the addition of cyclic AMP analogue. Glucagon gene promoter activity, as assessed by transient transfection experiments, was stimulated 2.1 +/- 0.25-fold by forskolin (P < 0.01). This effect was significantly inhibited by SRIF (71 +/- 4% reduction from FSK alone, P < 0.04) suggesting that SRIF inhibition of the glucagon promoter may, at least in part, account for the observed decrease in glucagon mRNA levels. These studies uniquely demonstrate that glucagon secretion from the HIT-T15 cell line is inhibited by SRIF through a pertussis toxin-sensitive mechanism and that SRIF also inhibits glucagon gene expression in part by reducing glucagon promoter activity. These findings indicate that SRIF can coordinately regulate glucagon delivery by the alpha cell both at the level of gene expression and hormone exocytosis.

Animals↗
PubMed1995

Morphological and functional characterization of beta TC-6 cells--an insulin-secreting cell line derived from transgenic mice.

Morphological analysis of hormone content and functional assessment of hormone secretion were conducted in beta TC-6 cells, an insulin-secreting cell line derived from transgenic mice expressing the large T-antigen of simian virus 40 (SV40) in pancreatic beta-cells. We observed by immunohistochemistry and confocal microscopy that beta TC-6 cells contain abundant insulin and small amounts of glucagon and somatostatin (SRIF). Glucagon usually co-localized with insulin, whereas cells containing SRIF did not contain insulin or glucagon. Static incubation and perifusion experiments demonstrated that beta TC-6 cells at passage 30-45 secrete insulin in response to glucose. In static incubations, maximal stimulation was achieved for glucose concentrations > 2.8 mmol/l glucose, and the half-maximal effect was observed at 0.5 mmol/l. Maximal stimulation was four times greater than HIT-T15 cells at passage 72-81, although HIT cells had a greater response over their basal levels. The magnitude of the insulin response to glucose in perifusion was 1,734 +/- 384 pmol.l-1. min and was 4.6-fold greater in the presence of 3-isobutyl-1-methylxanthine. Low amounts of glucagon were released in response to amino acids. Epinephrine (EPI), and to a lesser extent SRIF, inhibited phasic glucose-induced insulin secretion. A major portion of these inhibitory effects was mediated by pertussis toxin-sensitive substrates. Immunoblots detected the presence of the G-proteins Gi alpha 2, Gi alpha 3, and Go alpha 2. These results indicate that beta TC-6 cells are a glucose-responsive cell line in which insulin exocytosis is physiologically regulated by EPI and SRIF through Gi/Go-mediated mechanisms.

1-Methyl-3-isobutylxanthine↗
PubMed1995

A method for obtaining monodispersed cells from isolated porcine islets of Langerhans.

Micro or macroencapsulation of islets of Langerhans have been proposed as a bioartificial pancreas. Encapsulation of dispersed single cells instead of porcine islets should improve the oxygenation of encapsulated tissue. The aim of this work was, therefore, to develop techniques for dissociating porcine islets and test cell viability and function. After islet isolation and purification, islets were dispersed into single cells with collagenase and DNAse in either an extracellular type ionic solution or a UW solution. After culture, islets or cells were perfused with Krebs buffer. Two consecutive stimulations from 2.8 mM to 20 mM glucose were performed. Viability of cells (trypan blue) was higher than 85% after dispersion in ES or UW solutions. Islets or dispersed cells responded similarly to both stimulations with a return to basal rate between stimulations. No difference was found between cell function cultured during 18 hours or 6 days. However, islet function was improved by a long period of culture. In conclusion, this study demonstrates that dissociated cells performed as well as native islets up to six days culture.

Analysis of Variance↗
PubMed1994

Elimination of the acetaminophen interference in an implantable glucose sensor.

Acetaminophen has been one of the most serious electrochemical interferences to oxidase-based amperometric biosensors that measure H2O2. A study was carried out to investigate various polymer materials for their selectivity as the sensor inner membrane. A composite membrane of cellulose acetate and Nafion was found to eliminate acetaminophen and other electrochemical interferences effectively while at the same time maintaining reasonable diffusivity for hydrogen peroxide. The excellent in vivo performance of the sensor was attributed not only to significantly reduced steady-state sensitivity to acetaminophen but also to very slow acetaminophen response. These features, combined with rapid acetaminophen clearance pharmacokinetics, led to the decreased response as demonstrated in the rat.

Acetaminophen↗
PubMed1994

Reduction of acetaminophen interference in glucose sensors by a composite Nafion membrane: demonstration in rats and man.

Amperometric glucose sensors typically monitor the production of hydrogen peroxide generated in the course of the enzymatic oxidation of glucose. At the applied potential necessary to oxidize the peroxide produced, other species are also electroactive and contribute to the signal. Interference of ascorbate or urate has been effectively eliminated, but that resulting from the widely used analgesic acetaminophen is not. The aim of this work was to reduce this interference, which was found to be possible by introducing a membrane constructed of Nafion. We compared the in vitro sensitivity to acetaminophen of five Nafion sensors with that of five non-Nafion sensors with identical glucose sensitivity (2.0 +/- 0.4 vs 1.9 +/- 0.1 nA.mmol-1.l-1, NS): sensitivity to acetaminophen was 12.2 +/- 2.7 vs 30.8 +/- 6.3 nA.mmol-1.l-1, respectively (p < 0.05). These sensors were tested in rats by implanting in each animal one Nafion and one non-Nafion sensors. The in vivo sensitivity to glucose was similar (0.33 +/- 0.09 vs 0.30 +/- 0.05 nA.mmol-1.l-1, NS). The current generated by an acetaminophen infusion (plasma acetaminophen plateau = 140 +/- 10 mumol/l) was much decreased in the case of the Nafion sensor: 0.5 +/- 0.3 vs 2.0 +/- 0.7 nA, p < 0.05). Five Nafion sensors were implanted in the subcutaneous tissue of normal human volunteers who were given on oral dose of 500 mg acetaminophen.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaminophen↗
PubMed1994

Development of a glucose sensor for glucose monitoring in man: the disposable implant concept.

This paper describes the issues related to glucose sensing in the framework of the concept of disposable implants. The possibility of providing a diabetic patient with a continuous access to his blood glucose concentration and of detecting nocturnal hypoglycaemia would be a major breakthrough in diabetes therapy. To this end, our laboratories have developed a miniaturized, subcutaneous glucose sensor for glucose monitoring. It is based on the enzymatic, amperometric detection of glucose. This glucose sensor has been extensively evaluated in rats, dogs and more recently in human volunteers. Under experimental conditions, a controlled increase in blood glucose concentration is followed by an increase in the current delivered by the sensor. It is then possible to transform this current into an estimation of the glucose concentration. Experiments in rats have shown that the glucose sensor functions for up to 10 days when implanted in the subcutaneous tissue. Experiments in conscious dogs have shown that it works in a subcutaneous tissue closer to the human one. More recently, the glucose sensor was investigated in non-diabetic volunteers. These studies have demonstrated that this subcutaneous glucose sensor is able to provide a reliable estimation of blood glucose concentration in man, making it suitable for blood glucose monitoring. Biocompatibility of the glucose sensor, and particularly long-term tolerance, remains to be demonstrated.

Animals↗
PubMed1994

A one-step, operator-independent method for isolating islets of Langerhans from the porcine pancreas.

Large-scale isolation of islets of Langerhans is one of the major obstacles in islet transplantation. Until now, isolation methods relied on enzymatic digestion, the duration of which relies on a decision dictated by the operator's experience. This approach has always hindered development of an automated method. The aim of this study was to develop a one-step method based on complete digestion of the pancreas. The original aspect of the technique (derived from the Ricordi method) is use of the University of Wisconsin (UW) solution in the digestion medium and a continuous flow collagenase processing circuit with local cooling and rewarming to allow tissue digestion to proceed at 37 degrees C while settling of the cell suspension takes place at 4 degrees C. A stopcock system permits the alternate use of two settling chambers so that while one is in the circuit, the other can be removed for centrifugation, resuspension of the crude islet preparation in collagenase in free UW solution, and further purification in a density gradient system. Ten experiments were performed, and 545,750 +/- 48,670 purified pig islets were obtained per totally digested pancreas. Histological studies showed cell integrity. Insulin secretion in response to double glucose stimulation under perfusion conditions demonstrated the functional viability of the isolated islets. In conclusion, this one-step method makes it possible to obtain a high number of viable islets of Langerhans in the absence of any decision by an operator, and it should therefore provide basis for an automated method.

Adenosine↗
PubMed1993

A glucose monitoring system for on line estimation in man of blood glucose concentration using a miniaturized glucose sensor implanted in the subcutaneous tissue and a wearable control unit.

We have developed a miniaturized glucose sensor which has been shown previously to function adequately when implanted in the subcutaneous tissue of rats and dogs. Following a glucose load, the sensor output increases, making it possible to calculate a sensitivity coefficient to glucose in vivo, and an extrapolated background current in the absence of glucose. These parameters are used for estimating at any time the apparent subcutaneous glucose concentration from the current. In the previous studies, this calibration was performed a posteriori, on the basis of the retrospective analysis of the changes in blood glucose and in the current generated by the sensor. However, for clinical application of the system, an on line estimation of glucose concentration would be necessary. Thus, this study was undertaken in order to assess the possibility of calibrating the sensor in real time, using a novel calibration procedure and a monitoring unit which was specifically designed for this purpose. This electronic device is able to measure, to filter and to store the current. During an oral glucose challenge, when a stable current is reached, it is possible to feed the unit with two different values of blood glucose and their corresponding times. The unit calculates the in vivo parameters, transforms every single value of current into an estimation of the glucose concentration, and then displays this estimation. In this study, 11 sensors were investigated of which two did not respond to glucose. In the other nine trials, the volunteers were asked to record every 30 s what appeared on the display during the secondary decrease in blood glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Towards continuous glucose monitoring: in vivo evaluation of a miniaturized glucose sensor implanted for several days in rat subcutaneous tissue.

A miniaturized amperometric, enzymatic, glucose sensor (outer diameter 0.45 mm) was evaluated after implantation in the subcutaneous tissue of normal rats. A simple experimental procedure was designed for the long-term assessment of the sensor's function which was performed by recording the current during an intraperitoneal glucose load. The sensor was calibrated by accounting for the increase in the current during the concomitant increase in plasma glucose concentration, determined in blood sampled at the tail vein. This made it possible to estimate the glucose concentration in subcutaneous tissue. During the glucose load, the change in subcutaneous glucose concentration followed that in blood with a lag time consistently shorter than 5 min. The estimations of subcutaneous glucose concentration during these tests were compared to the concomitant plasma glucose concentrations by using a grid analysis. Three days after implantation (n = 6 experiments), 79 estimations were considered accurate, except for five which were in the acceptable zone. Ten days after implantation (n = 5 experiments), 101 estimations were accurate, except for one value, which was still acceptable. The sensitivity was around 0.5 nA.mmol-1.l-1 on day 3 and day 10. A longitudinal study on seven sensors tested on different days demonstrated a relative stability of the sensor's sensitivity. Finally, histological examination of the zone around the implantation site revealed a fibrotic reaction containing neocapillaries, which could explain the fast response of the sensor to glucose observed in vivo, even on day 10. We conclude that this miniaturized glucose sensor, whose size makes it easily implanted, works for at least ten days after implantation into rat subcutaneous tissue.

Animals↗
PubMed1992

Calibration in dogs of a subcutaneous miniaturized glucose sensor using a glucose meter for blood glucose determination.

The feasibility of calibrating a glucose sensor by using a wearable glucose meter for blood glucose determination and moderate variations of blood glucose concentration was assessed. Six miniaturized glucose sensors were implanted in the subcutaneous tissue of conscious dogs, and the parameters used for the in vivo calibration of the sensor (sensitivity coefficient and extrapolated current in the absence of glucose) were determined from values of blood glucose and sensor response obtained during glucose infusion. (1) Venous plasma glucose level and venous total blood glucose level were measured simultaneously on the same sample, using a Beckman analyser and a Glucometer II, respectively. The regression between plasma glucose (x) and whole blood glucose (y) was y = 1.12x-0.08 mM (n = 114 values, r = 0.96, p = 0.0001). The error grid analysis indicated that the use of a Glucometer II for blood glucose determination was appropriate in dogs. (2) The in vivo sensitivity coefficients were 0.57 +/- 0.11 nA mM-1 when determined from plasma glucose, and 0.51 +/- 0.07 nA mM-1 when determined from whole blood glucose (t = 1.53, p = 0.18, n.s.). The background currents were 0.88 +/- 0.57 nA when determined from plasma glucose, and 0.63 +/- 0.77 nA when determined from whole blood glucose (t = 0.82, p = 0.45, n.s.). (3) The regression equation of the estimation of the subcutaneous glucose level obtained from the two methods was y = 1.04x + 0.56 mM (n = 171 values, r = 0.98, p = 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Progress toward the development of an implantable sensor for glucose.

The development of an electrochemically based implantable sensor for glucose is described. The sensor is needle-shaped, about the size of a 28-gauge needle. It is flexible and must be implanted subcutaneously by using a 21-gauge catheter, which is then removed. When combined with a monitoring unit, this device, based on the glucose oxidase-catalyzed oxidation of glucose, reliably monitors glucose concentrations for as long as 10 days in rats. Various design considerations, including the decision to monitor the hydrogen peroxide produced in the enzymatic reaction, are discussed. Glucose constitutes the most important future target analyte for continuous monitoring, but the basic methodology developed for glucose could be applied to several other analytes such as lactate or ascorbate. The success in implementation of such a device depends on a reaction of the tissue surrounding the implant so as not to interfere with the proper functioning of the sensor. Histochemical evidence indicates that the tissue response leads to enhanced sensor performance.

Biocompatible Materials↗
PubMedDate not supplied

In vitro and in vivo evaluation in dogs of a miniaturized glucose sensor.

A miniaturized glucose sensor was developed, consisting of a platinum wire entirely coated with teflon, except for a 1 mm section near its extremity where glucose oxidase is immobilized. The in vitro sensitivity to glucose of the sensors was 2.3 +/- 0.4 nA/mM, mean +/- SEM (n = 23). These sensors were implanted in the subcutaneous tissue of normal beagles. Two consecutive glucose infusions (15-30 mg/kg/min) were performed. The current generated by the sensor was used for calculation of the sensitivity coefficient (SC) (nA/mM), and the background current in the absence of glucose (lo) (nA). These parameters were used for determination of the apparent subcutaneous glucose concentration. The in vivo sensitivity was less than the in vitro sensitivity (0.5 +/- 0.1 vs. 2.2 +/- 0.2; n = 12 comparisons; p less than 0.01). Stability of sensor function was demonstrated by the absence in variation of SC and lo, calculated from the different plateaus obtained during the glucose infusions. This study provides a simple method for evaluating in vivo the function of a miniaturized sensor implanted in subcutaneous tissue.

Animals↗