Dynamics of blood electrolytes in repeated hyper- and/or hypoglycaemic events in patients with type 1 diabetes.

AIMS/HYPOTHESIS: Electrolyte disturbances are well-known consequences of the diabetic pathology. However, less is known about the cumulative effects of repeated changes in glycaemia, a characteristic of diabetes, on the electrolyte balance. We therefore investigated the ionic profiles of patients with type 1 diabetes during consecutive hyper- and/or hypoglycaemic events using the glucose clamp. METHODS: In protocol 1, two successive hyperglycaemic excursions to 18 mmol/l were induced; in protocol 2, a hypoglycaemic excursion (2.5 mmol/l) was followed by a hyperglycaemic excursion (12 mmol/l) and another hypoglycaemic episode (3.0 mmol/l). RESULTS: Blood osmolarity increased during hyperglycaemia and was unaffected by hypoglycaemia. Hyperglycaemia induced decreases in plasma Na(+) Cl(-) and Ca(2+) concentrations and increases in K(+) concentrations. These changes were faithfully reproduced during a second hyperglycaemia. Hypoglycaemia provoked rapid and rapidly reversible increases in Na(+), Cl(-) and Ca(2+). In sharp contrast, K(+) levels displayed a rapid and substantial fall from which they did not fully recover even 2 h after the re-establishment of euglycaemia. A second hypoglycaemia caused an additional fall. CONCLUSIONS/INTERPRETATION: Repeated hyperglycaemia events do not lead to any cumulative effects on blood electrolytes. However, repeated hypoglycaemias are cumulative with respect to K(+) levels due to a very slow recovery following hypoglycaemia. These results suggest that recurring hypoglycaemic events may lead to progressively lower K(+) levels despite rapid re-establishment of euglycaemia. This warrants close monitoring of plasma K(+) levels combined with continuous glucose monitoring particularly in patients under intensive insulin therapy who are subject to repeated hypoglycaemic episodes. TRIAL REGISTRATION: Clinicaltrial.gov NCT01060917.

Suppression of Pdx-1 perturbs proinsulin processing, insulin secretion and GLP-1 signalling in INS-1 cells.

AIMS/HYPOTHESIS: Mutations in genes encoding HNF-4alpha, HNF-1alpha and IPF-1/Pdx-1 are associated with, respectively, MODY subtypes-1, -3 and -4. Impaired glucose-stimulated insulin secretion is the common primary defect of these monogenic forms of diabetes. A regulatory circuit between these three transcription factors has also been suggested. We aimed to explore how Pdx-1 regulates beta cell function and gene expression patterns. METHODS: We studied two previously established INS-1 stable cell lines permitting inducible expression of, respectively, Pdx-1 and its dominant-negative mutant. We used HPLC for insulin processing, adenovirally encoded aequorin for cytosolic [Ca2+], and transient transfection of human growth hormone or patch-clamp capacitance recordings to monitor exocytosis. RESULTS: Induction of DN-Pdx-1 resulted in defective glucose-stimulated and K+-depolarisation-induced insulin secretion in INS-1 cells, while overexpression of Pdx-1 had no effect. We found that DN-Pdx-1 caused down-regulation of fibroblast growth factor receptor 1 (FGFR1), and consequently prohormone convertases (PC-1/3 and -2). As a result, DN-Pdx-1 severely impaired proinsulin processing. In addition, induction of Pdx-1 suppressed the expression of glucagon-like peptide 1 receptor (GLP-1R), which resulted in marked reduction of both basal and GLP-1 agonist exendin-4-stimulated cellular cAMP levels. Induction of DN-Pdx-1 did not affect glucokinase activity, glycolysis, mitochondrial metabolism or ATP generation. The K+-induced cytosolic [Ca2+] rise and Ca2+-evoked exocytosis (membrane capacitance) were not abrogated. CONCLUSIONS/INTERPRETATION: The severely impaired proinsulin processing combined with decreased GLP-1R expression and cellular cAMP content, rather than metabolic defects or altered exocytosis, may contribute to the beta cell dysfunction induced by Pdx-1 deficiency.

The K-Cl cotransporter in the lobster stretch receptor neurone--a kinetic analysis.

Experiments were performed to define quantitatively the substrate (K(+) and Cl(-)) dependence of the transport function (production of equally large and oppositely directed K(+)and Cl(-) flows/currents) of an earlier (Theander et al., 1999) identified electroneutral K-Cl cotransporter in the slowly adapting stretch receptor neurone of the European lobster. The experiments were based on microelectrode techniques. This allowed us to perform steady-state measurements of the so-called "instantaneous" current-voltage relationships (around a holding voltage of -65 mV after a blockage of the cell's action potential and hyperpolarization-activated currents) and intracellular ion concentrations at various settings of the extracellular K(+) and Cl(-) concentrations. From the results, we could then define steady-state values of all of the cell's non-KCl cotransporter K(+) and Cl(-) currents. Finally, the negative sums of the inferred non-KCl cotransporter K(+) and Cl(-) currents could be taken as equivalents of the K-Cl cotransporter's K(+) and Cl(-) currents for the reason that, in steady state, all membrane currents add up to zero. For the cotransporter currents, thus inferred for a range from 2.5/410.5 to 40.0/448.0 mM external K(+)/Cl(-), we found that their absolute values increased in a nonlinear fashion from about 5 nA cell(-1) at the lowest, to about 20 nA cell(-1) at the highest external K(+)/Cl(-) concentrations. Formally, this relationship could be reproduced by a Hill function-based enzyme kinetic expression simulating inward and outward transmembrane electroneutral ion transports. Following insertion of this expression into a comprehensive model of electrical membrane functions and intracellular solute and solvent control in the lobster stretch receptor neurone, the model predictions suggested that the K-Cl cotransporter does play an important role in (a) keeping intracellular Cl(-) low for a proper function of the cell's inhibitory system, and (b) enabling rapid transmembrane K(+) shifts that provide for a stabilization of the cell's membrane voltage and membrane excitability in cases of varying extracellular K(+) concentrations. The model predictions gave, however, no clear evidence that the K-Cl cotransporter is critically involved in the cell's volume regulation in conditions of varying extracellular osmolalities.

Cl- transport in the lobster stretch receptor neurone.

Experiments were performed to identify mechanisms underlying non-leakage and non-H+/HCO3--linked transmembrane Cl- transports in the slowly adapting stretch receptor neurone of the European lobster, using intracellular microelectrode and pharmacological techniques. In methodological tests, it was established that direct estimates of intracellular Cl- with ion-sensitive microelectrodes are statistically identical with indirect estimates by means of a GABA method, where 1-2 mM GABA is transforming the cell's membrane voltage into its Cl- equilibrium voltage from which the Cl- concentration is inferred by the Nernst equation. From experiments using sodium orthovanadate and ethacrynic acid, supposed to block primary Cl- pumps, and bumetanide, supposed to block Na-K-Cl co-transporters, it appeared that neither of the two Cl- transport systems exists in the stretch receptor neurone. It could be shown, however, that the cell is equipped with an electroneutral K-Cl co-transporter that (a) is blockable by furosemide in high (Km approximately 350 microM), by 4-acetamido-4'-isothiocyanato-stilbene-2,2-disulphonic acid (SITS) in medium-high (Km approximately 35 microM), and by 4, 4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) in low (Km approximately 15 microM) doses, (b) is (transiently) activatable by (1 mM) n-ethylmaleimide, (c) is not suppressed by extracellular Rb+ or NH4+, and (d) is not directly coupled to any transmembrane transports of Na+, H+ or HCO3-. From functional tests, with varying transmembrane K+ and Cl- gradients, evidence obtained that the K-Cl co-transporter is able to reverse its transport direction and to adjust its transport rate in a considerable range. As a whole, the results speak in favour of the K-Cl co-transporter being responsible (a) for normally keeping the intracellular Cl- concentration at low levels, for an optimization of the cell's inhibitory system, and (b) for achieving fast transmembrane shifts of K+ (and Cl-), as a means of stabilizing the cell's membrane excitability in conditions of varying extracellular K+ concentrations.

Standardized mortality in eating disorders--a quantitative summary of previously published and new evidence.

Ten eating disorder (ED) populations were reviewed using the standardized mortality ratio (SMR) presenting new evidence for several studies. In eight of the ten samples, strong evidence (in one sample weak evidence and in one sample no evidence) supports an hypothesis of elevated SMR. We found strong evidence for an increase in SMR for anorexia nervosa (AN), whereas no firm conclusions could be drawn for bulimia nervosa (BN). Bias caused by loss to follow-up was quantified and found non-negligable in some samples (possible increase in SMR from 25% to 240%). We did not find a significant effect of gender or time period on SMR. Survival analysis showed a significant difference among the life-tables for males and females; female risk of death averaged 0.59% per year, whereas all male deaths occurred within the first 2 years after presentation. Weight at presentation had a highly significant effect on SMR, and lower weight at presentation was associated with higher SMR. Age at presentation exerted a significant unimodal effect on SMR; aggregate overall SMR was 3.6 for the youngest age group (< 20 years), 9.9 for those aged 20-29 years, and 5.7 for those aged > or = 30 years at presentation. Length of follow-up had a highly significant inverse effect on SMR; maximal SMR was 30 for female AN patients in the first year after presentation. A statistically significant increase in SMR was documented for at least up to 15 years after presentation. One study indicated a treatment effect on SMR. New evidence on causes of death suggests there are more deaths from suicide and other and unknown causes and fewer deaths related to ED than previously reported. Our findings have both research and clinical implications, with the most important clinical implication being the need for vigorous and well-directed treatment efforts from the initial presentation for treatment. An important research implication is that no single measure of mortality is sufficient; that is, only a combination of different statistics will maximize the available information.

Changes in ultrasound appearance of the internal female genital organs during treatment for eating disorders.

OBJECTIVE: To confirm that changes of the internal female genital organs in patients with eating disorders can be detected with ultrasound and that successive normalization can be followed during treatment. STUDY DESIGN: Thirty-five women with the diagnoses of eating disorders were examined with ultrasound while undergoing psychiatric treatment. The endometrial thickness and ovarian volume were measured. The sonographic picture of the ovaries was classified in four classes. RESULTS: Bulimics had changes of their ovaries in spite being of normal weight. After psychiatric treatment and a normal diet, the ovaries and the bleeding pattern normalized without a change in body weight. In anorectics, undetectable ovaries or ovaries without follicles were associated with low body mass index (BMI), but multifollicular ovaries or presence of a dominant follicle and ovarian volume had no clear relation to BMI. The endometrial thickness correlated with BMI. CONCLUSION: Ovarian morphology appeared more important than ovarian size. Changes of the ovaries appeared more related to eating patterns than to BMI. Eating disorders should be considered in women with bleeding disorders. Ultrasound examination can contribute to the differential diagnosis.

Analysis of leak current properties in the lobster stretch receptor neurone.

Experiments were performed to characterize the so-called leak current of the slowly adapting stretch receptor neurone of the European lobster with respect to its ionic basis, its kinetics and its pharmacology. Estimates of the leak current were obtained by subtraction of a Na-K pump current and of an unspecific impalement current from a non-dynamic ('instantaneous') current, recorded in a voltage range from approximately -120 to approximately -30 mV, after blockage of spike-generating currents and a hyperpolarization-activated inwardly rectifying current (Q-current). The leak current, estimated in this way, was seen to reverse direction at the cell's K+ equilibrium voltage, thus indicating that it is carried by K+ passing through channels which, also, proved to be permeable to Rb+ and NH4+, but not permeable to Na+ or Cl- to any significant extent. Kinetically, the leak current was found to be characterized by being enhanced by increases in extracellular K+ and by being subject to outward rectification, most distinctly at elevated extracellular [K+]. In quantitative terms, these kinetic properties could be accounted for by a mathematical model comprising (1) a one-site two-barrier Eyring formulation describing ion permeation through membrane channels and (2) an ordinary dose-response relationship describing the channel-opening effect of K+ at an extracellular regulatory site. Pharmacologically, the leak current proved to be distinguished by being reversibly blockable, in a non-voltage dependent manner, by CO2+ (Kd = 0.9 mM, Hill coefficient 1.1) and procaine, but not by Ba2+, Gd3+, bupivacaine (a local anesthetic), or other K+ channel blockers such as TEA, 4-AP and Cs+. It is concluded that, in native unimpaled cells, the K+ carried leak current (1) is setting the resting voltage together with the (mainly) Na(+)-carried Q-current and the Na-K pump current, (2) is determining the cell's firing threshold, together with the spike generating currents, and (3) is also stabilizing the cell's membrane excitability in conditions of varying extracellular [K+], by virtue of its K+ sensitivity.

Buffer capacity of rat cortical tissue as well as of cultured neurons and astrocytes.

The primary objective of this work was to assess the intrinsic nonbicarbonate buffer capacity (beta i) of cultured neurons and astrocytes and to compare the beta i values obtained to those of neocortical tissue. A second objective was to determine the pH dependence of beta i. Titration of homogenates of whole-brain cortical tissue and cultured neurons with NaOH and HCl gave beta i values of 25-30 mmol.l-1 x pH-1. The buffer capacity was essentially constant in the pH range of 6-7. Astrocytes showed a higher buffer capacity and a clear relationship between beta i and pH. However, beta i decreased when pH was reduced from 7 to 6. The beta i values derived from microspectrofluorometric studies on neurons and astrocytes were surprisingly variable, ranging from 10 to 50 mmol.l-1 x pH-1. The ammonia "step method" suggested that beta i increased dramatically when pH was lowered from 7 to 6 but the propionic "step method" failed to reveal such a pH dependence. Some techniques obviously give erroneous values for beta i, presumably because changes in buffer base concentration (due to transmembrane fluxes of H+, HCO3-, NH4+ or anions of weak acids) violate the principles upon which the calculations are based. From the results obtained by direct titration and with the propionate technique, we tentatively conclude that beta i in neurons and astrocytes are approximately 20 and 30 mmol.l-1 x pH-1, respectively. We further suggest that the term "intrinsic buffer capacity", as commonly used, is redefined.

Functional effects of a hyperpolarization-activated membrane current in the lobster stretch receptor neurone.

The functional effects of a hyperpolarization-activated membrane current (IQ) in the slowly adapting lobster stretch receptor neurone were investigated. From comparisons between changes in membrane excitability due to blockage of IQ by Cs+, in normally impaled and native unimpaled (Edman et al. 1987 b) cells, it could be concluded that the resting voltage of native cells is distinctly more negative than -65 mV (average membrane voltage of impaled cells) and, therefore, under the control of an activated IQ. Starting from this conclusion, impaled cells were polarized to holding (resting) voltages around -75 mV and their polarization and excitability properties studied after tetanic impulse activity and variation of various external influences (K+, pH, temperature), both in control conditions and after blockage of IQ by 2 mM Cs+. It was found that an unblocked IQ (a) greatly accelerates the initial (90%) decay of post-tetanic hyperpolarization, and (b) depresses distinctly any polarization and excitability alterations due to increases in extracellular K+ concentration (from 2.5 to 10 mM), variations in extracellular pH (between 6.4 and 8.6), and changes in temperature (between 14 and 24 degrees C). It was inferred that in well polarized cells, IQ plays a role as a stabilizer of membrane polarization and excitability in conditions of varying external influences. From a model study of IQ it could be concluded that, with its slow dynamic responses, the current is well adapted to its functional purposes and to the rather slow homeostatic effects of the cell's Na-K pump.

The clinical relevance of salivary amylase monitoring in bulimia nervosa.

The aim of this study was to evaluate the clinical relevance of amylase level monitoring as an objective measure in diagnosis and assessment of treatment response in bulimia nervosa. Thirty-three subjects who fulfilled DSM-111-R criteria for bulimia nervosa had serum levels of total and salivary amylase monitored during an 8-week treatment trial. At the beginning of treatment, the average total amylase level was within the upper limits of normal, whereas average salivary amylase levels were abnormally high. During the course of treatment, there was a significant reduction in the average salivary isoenzyme to within the normal range. Significant reductions in amylase levels were recorded in patients with good treatment outcome, but not in those with poor outcome. Amylase levels were not significantly correlated with severity of bulimic symptoms. These results do not justify the use of amylase assays as a routine diagnostic or monitoring test, but isoenzyme monitoring may provide useful clinical information in selected cases.

The effect of microwave radiation on the stability and formation of gramicidin-A channels in lipid bilayer membranes.

The effects of microwaves on the single-channel kinetics of gramicidin-A channels in lipid bilayer membranes were examined. Attempts were made to separate thermal and athermal effects by accurate measurements of temperature at the site of the membrane and by relating the measured parameters to their previously characterized temperature dependence. It was found that microwave radiation does not affect single-channel conductance or channel life time to a degree that is significantly different from that expected of a purely thermal effect. On the other hand, the rate of channel formation is decreased during exposure, which is opposite to that expected of a purely thermal effect. The mechanism of this effect is discussed in terms of the dimerization process of channel formation.

Acid-base changes during complete brain ischemia.

We examined the proposal that preischemic hyperglycemia causes exaggerated brain damage by decreasing intracellular or extracellular pH to below a specified threshold value. We also provide a critical appraisal of two related hypotheses. The first is that hyperglycemia enhances brain damage by causing excessive intraglial acidosis; the second, that the critical degree of acidosis is reached not during the ischemia but when recirculation is instituted. The following conclusions are drawn. First, the evidence is inconclusive in favor of marked compartmentation of H+ during ischemia, based on a discontinuous delta lactate/delta PCO2 relation and on direct intracellular pH measurements. In fact, results obtained with identical techniques in normoglycemic animals suggest that the acid compartment assumed to be glia is very small and may be of another origin. Second, although recirculation may give rise to a further increase in either extracellular or intracellular acidosis under certain conditions, this acidosis is not a prerequisite for increased tissue damage or infarction. Third, a critical appraisal of reports supports the contention that enhanced damage is triggered below a specified threshold pH value. In complete or near-complete ischemia, this value corresponds to a tissue lactate content of 17-20 mM.kg-1 wet wt. No correlation exists between subthreshold values for delta lactate and the severity of tissue damage. Furthermore, hyperglycemia cannot be expected to enhance damage if conditions prevent lactate from reaching threshold values or if they uncouple changes in lactate and pH.

Preservation of brain temperature during ischemia in rats.

Our objectives were to study the loss of heat from ischemic brain and to devise a method of maintaining brain temperature. Reversible forebrain ischemia was induced by carotid clamping and exsanguination in 30 anesthetized and artificially ventilated rats. Rectal, skull, and brain temperatures were measured, confirming previous findings that brain temperature falls by 4-5 degrees C during 15 minutes of ischemia unless measures are taken to maintain head temperature by external heating. Temperature gradients developed within the ischemic brain, superficial tissues being cooler than deep ones. These temperature gradients were reversed when skull temperature was maintained at core body (rectal) temperature by external heating. With rectal and skull temperatures maintained at 38 degrees, 37 degrees, 35 degrees, or 33 degrees C, brain temperatures nonetheless decreased by approximately 1 degree C during ischemia. This decrease in brain temperature could be prevented by placing the rat in a Plexiglas box with circulating air at temperatures close to that of the body core and a relative humidity of approximately 100%. We also found that, unless special precautions are taken, a temperature gradient develops between the brain and body core during recirculation.

Microcalorimetric study of muscle and platelet thermogenesis in anorexia nervosa and bulimia.

Muscle and platelet thermogenesis was measured by direct microcalorimetry in patients with anorexia nervosa. The median heat production, as calculated per unit of tissue mass, was 0.35 mW/g in vastus lateralis muscle, which is significantly lower (p less than 0.001), by approximately 50% than that in matched healthy subjects, 0.74 mW/g. The average deficit in body weight was 35%. Platelet heat production was decreased to a lesser degree with median values being 51.3 fW per cell and 60.9 fW per cell, respectively (p less than 0.02). Patients with bulimia without concomitant anorexia nervosa had normal heat production values. In the control group a positive correlation appeared (rs = 0.75, p less than 0.02) between resting muscle heat production and body surface area whereas no such relationship was found in the anorexic group.

A diffusion limited reaction theory for a microtiter plate assay.

Calculations are presented describing the diffusion limited kinetics of a solid-phase immunoassay in which reactants are immobilized at the inner surface of a cylindrical well. The calculations refer to an unstirred situation and simplified expressions are presented which can be used for calibration and optimization of the assay.