Modification of the Beckman-Coulter second-generation enzyme-linked immunosorbent assay protocol improves the reliability of serum antimüllerian hormone measurement.

OBJECTIVE: To determine whether the modified Beckman-Coulter 2nd-generation (Gen II) antimüllerian hormone (AMH) assay (Gen IIm) provides more consistent results following storage at room temperature and on dilution than the original Gen II assay, to compare AMH results from the modified assay with those obtained from the original assay, and to assess the relationship between new AMH values and the antral follicle count (AFC). DESIGN: Cohort. SETTING: Hospital fertility clinic. PATIENT(S): A total of 678 consecutive women (21-46 years old) investigated for subfertility. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): AMH was measured by means of the Gen IIm assay protocol in women with known AFC. AMH values were obtained on a subset of serum samples by means of both original and modified assays. RESULT(S): Specimens analyzed by Gen IIm exhibited a proportional AMH response on dilution, and AMH values decreased by an average of 12.1% after 7 days at room temperature, in contrast to the steady increase seen with the use of the original Gen II assay. Gen IIm assay values were, on average, 51.4% higher than Gen II values. Population analysis suggested a conversion factor of 1.35 (95% CI 1.23-1.47) between the Gen IIm and historical data obtained for the Diagnostic Systems Laboratories AMH assay. The relationship between the Gen IIm AMH measurement and AFC was adequately represented by a linear function. CONCLUSION(S): The Gen IIm assay gave more reliable AMH results on sample dilution and storage than the original Gen II protocol. Findings obtained with the use of the original Gen II ELISA method should be treated with caution.

The measurement of anti-Müllerian hormone: a critical appraisal.

CONTEXT: Measurement of anti-Müllerian hormone (AMH) is perceived as reliable, but the literature reveals discrepancies in reported within-subject variability and between-method conversion factors. Recent studies suggest that AMH may be prone to preanalytical instability. We therefore examined the published evidence on the performance of current and historic AMH assays in terms of the assessment of sample stability, within-patient variability, and comparability of the assay methods. EVIDENCE ACQUISITION: We reviewed studies (manuscripts or abstracts) measuring AMH, published in peer-reviewed journals between January 1, 1990, and August 1, 2013, using appropriate PubMed/Medline searches. EVIDENCE SYNTHESIS: AMH levels in specimens left at room temperature for varying periods increased by 20% in one study and by almost 60% in another, depending on duration and the AMH assay used. Even at -20°C, increased AMH concentrations were observed. An increase over expected values of 20-30% or 57%, respectively, was observed after 2-fold dilution in two linearity-of-dilution studies, but not in others. Several studies investigating within-cycle variability of AMH reported conflicting results, although most studies suggest that variability of AMH within the menstrual cycle appears to be small. However, between-sample variability without regard to menstrual cycle as well as within-sample variation appears to be higher using the GenII AMH assay than with previous assays, a fact now conceded by the kit manufacturer. Studies comparing first-generation AMH assays with each other and with the GenII assay reported widely varying differences. CONCLUSIONS: AMH may exhibit assay-specific preanalytical instability. Robust protocols for the development and validation of commercial AMH assays are required.

Suppression of inflammation reduces endothelial microparticles in active systemic lupus erythematosus.

BACKGROUND: In a prospective observational study, we investigated whether patients with active systemic lupus erythematosus (SLE) had higher indices of endothelial damage and dysfunction than healthy controls and whether improved disease control was associated with improvement in these indices. METHODS: Twenty-seven patients with active SLE (four or more American College of Rheumatology (ACR) criteria) and 22 age-matched controls were assessed. Endothelial microparticles (EMPs; CD31+/annexin V+/CD42b-) were quantified using flow cytometry. Brachial artery flow-mediated dilatation (FMD) was measured using automated edge-tracking software. Twenty-two patients had a second assessment at a median (IQR) of 20 (16, 22) weeks after initiating new immunosuppressive therapy. RESULTS: SLE patients had a median (IQR) baseline global British Isles Lupus Assessment Group Disease Activity Index (BILAG-2004) score of 14 (12, 22). CD31+/annexin V+/CD42b- EMPs were higher (157 548/ml (59 906, 272 643) vs 41 025(30 179, 98 082); p=0.003) and endothelial-dependent FMD was lower (1.63% (-1.22, 5.32) vs 5.40% (3.02, 8.57); p=0.05) in SLE patients than controls. CD31+/annexin V+/CD42b- EMPs correlated inversely with FMD (%) (r(2) -0.40; p=0.006). At follow-up, the median (IQR) change in global BILAG-2004 score was -11 (-18, -3). CD31+/annexin V+/CD42b- EMP levels were reduced (166 982/ml (59 906, 278 775 vs 55 655(29 475, 188 659; p=0.02) and FMD had improved (0.33% (-2.31, 4.1) vs 3.19% (0.98, 5.09); p=0.1) at the second visit. CONCLUSIONS: Active SLE is associated with evidence of increased endothelial damage and endothelial dysfunction, which improved with suppression of inflammation. Better control of active inflammatory disease may contribute to improved cardiovascular risk in patients with SLE.

Anti-Mullerian hormone: poor assay reproducibility in a large cohort of subjects suggests sample instability.

STUDY QUESTION: What is the variability of anti-Müllerian hormone (AMH) concentration in repeat samples from the same individual when using the Gen II assay and how do values compare to Gen I [Diagnostic Systems Ltd (DSL)] assay results? SUMMARY ANSWER: The Gen II AMH assay displayed appreciable variability, which can be explained by sample instability. WHAT IS KNOWN ALREADY: AMH is the primary predictor of ovarian performance and is used to tailor gonadatrophin dosage in cycles of IVF/ICSI and in other routine clinical settings. Thus, a robust, reproducible and sensitive method for AMH analysis is of paramount importance. The Beckman Coulter Gen II ELISA for AMH was introduced to replace earlier DSL and Immunotech assays. The performance of the Gen II assay has not previously been studied in a clinical setting. STUDY DESIGN, SIZE AND DURATION: We studied an unselected group of 5007 women referred for fertility problems between 1 September 2008 and 25 October 2011; AMH was measured initially using the DSL AMH ELISA and subsequently using the Gen II assay. AMH values in the two assays were compared using a regression model in log(AMH) with a quadratic adjustment for age. Additionally, women (n = 330) in whom AMH had been determined in different samples using both the DSL and Gen II assays (paired samples) identified and the difference in AMH levels between the DSL and Gen II assays was estimated using the age-adjusted regression analysis. A subset of 313 women had repeated AMH determinations (n = 646 samples) using the DSL assay and 87 women had repeated AMH determinations using the Gen II assay (n = 177 samples) were identified. A mixed effects model in log(AMH) was utilized to estimate the sample-to-sample (within-subject) coefficients of variation of AMH, adjusting for age. Laboratory experiments including sample stability at room temperature, linearity of dilution and storage conditions used anonymized samples. MAIN RESULTS AND THE ROLE OF CHANCE: In clinical practice, Gen II AMH values were ∼20% lower than those generated using the DSL assay instead of the 40% increase predicted by the kit manufacturer. Both assays displayed high within-subject variability (Gen II assay CV = 59%, DSL assay CV = 32%). In the laboratory, AMH levels in serum from 48 subjects incubated at RT for up to 7 days increased progressively in the majority of samples (58% increase overall). Pre-dilution of serum prior to assay, gave AMH levels up to twice that found in the corresponding neat sample. Pre-mixing of serum with assay buffer prior to addition to the microtitre plate gave higher readings (72% overall) compared with sequential addition. Storage at -20°C for 5 days increased AMH levels by 23% compared with fresh samples. The statistical significance of results was assessed where appropriate. LIMITATIONS, REASONS FOR CAUTION: The analysis of AMH levels is a retrospective study and therefore we cannot entirely rule out the existence of differences in referral practices or changes in the two populations. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggests that AMH may not be stable under some storage or assay conditions and this may be more pronounced with the Gen II assay. The published conversion factors between the Gen II and DSL assays appear to be inappropriate for routine clinical practice. Further studies are urgently required to confirm our observations and to determine the cause of the apparent instability. In the meantime, caution should be exercised in the interpretation of AMH levels in the clinical setting. CONFLICT OF INTEREST/STUDY FUNDING: S. Roberts is supported by the NIHR Manchester Biomedical Research Centre.

The reproducibility of serum anti-Müllerian hormone in subfertile women: within and between patient variability.

Serum anti-Müllerian hormone concentrations vary significantly over time and this should be taken into account when tailoring treatment protocols for patients undergoing controlled ovarian hyperstimulation (COH). Compared with FSH, serum anti-Müllerian hormone may have greater discriminatory power because of its modest intrapatient variation and the larger interpatient variation.

Electrophysiological effects of osmotic cell shrinkage in rat pancreatic ß-cells.

Electrical and secretory activity in the pancreatic ß-cell can be elicited by hypotonic cell swelling, due largely to activation of a volume-regulated anion channel (VRAC) leading to depolarisation and electrical activity. However, ß-cell responses to cell shrinkage are less well characterised. The present study has examined the effects of osmotic cell shrinkage on rat pancreatic ß-cells. Electrical activity and whole-cell current were studied in isolated ß-cells using the perforated patch and conventional whole-cell recording techniques. Insulin release was measured using intact islets by radioimmunoassay. Exposure to a 33% hypertonic bath solution resulted in an initial depolarisation and a period of electrical activity. In several cases, this depolarisation was transient and was followed by a hyperpolarisation. A similar pattern was observed with insulin release. In voltage-clamp experiments, osmotic shrinkage resulted in activation of a non-selective cation channel (NSCC) sensitive to inhibition by flufenamic acid and Gd3+. It is suggested that activation of this NSCC is responsible for the depolarisation evoked by hypertonic media. The secondary hyperpolarisation is likely to be the result of inhibition of VRAC activity. These opposing ionic effects could underlie the biphasic effect on insulin release following exposure to hypertonic media.

Explanations for the lower rates of diabetic neuropathy in Indian Asians versus Europeans.

OBJECTIVE: Risks of diabetes and cardiovascular disease are elevated worldwide in Indian Asians. However, risks of other diabetes-related complications, i.e., foot ulceration and amputation, also with a vascular basis, are substantially lower in Asians than in white Europeans in the U.K., possibly due to less neuropathy. We therefore compared signs, symptoms, and objective quantitative measures of diabetic neuropathy and their risk factors in Indian Asians and Europeans. RESEARCH DESIGN AND METHODS: This was a cross-sectional study of a population-based sample of age- and sex-matched adults with type 2 diabetes of European (95 male and 85 female) and Asian (96 male and 84 female) descent in the U.K. Patients were assessed for neuropathic symptoms, signs, nerve conduction, autonomic function, and quantitative sensory testing. Peripheral vascular function and other potential risk factors for neuropathy were measured. RESULTS Mean nerve conduction velocity Z scores were better in Asians (mean +/- SD 0.07 +/- 0.62) than in Europeans (-0.11 +/- 0.60; P = 0.007) and were explained by the shorter height, fewer pack-years smoked, and higher transcutaneous oxygen levels (TCpO(2)) in Indian Asians (P value for ethnic comparison attenuated to 0.2). Small fiber neuropathy was less prevalent in Indian Asians compared with Europeans (odds ratio 0.58 [95% CI 0.37-0.93]; P = 0.02) and was primarily accounted for by better TCpO(2) (0.70 [0.40-1.21]; P = 0.2). CONCLUSIONS: Asians with diabetes have substantially less large and small fiber neuropathy than Europeans, despite comparable traditional risk factors. Independent from smoking, the lower risk of neuropathy in Asians is due to better skin microvascularization and may help explain the substantially reduced Asian foot ulcer risk.

The relationships between AMH, androgens, insulin resistance and basal ovarian follicular status in non-obese subfertile women with and without polycystic ovary syndrome.

BACKGROUND: Hyperandrogenaemia and insulin resistance are prominent features of polycystic ovary syndrome (PCOS) and influence the process of folliculogenesis in women with the endocrinopathy. Anti-Müllerian hormone (AMH) levels are elevated in women with PCOS and studies including IVF subjects have shown that this is a reliable marker of ovarian performance. The aims of this prospective study were to assess the relationship between insulin resistance, androgens and AMH, and whether AMH contributes to altered folliculogenesis in non-obese women with PCOS. METHODS: A total of 232 IVF candidates, 49 of whom had PCOS according to the Rotterdam 2003 consensus criteria, were recruited. AMH levels and ovarian morphology were assessed. The relationships between AMH and insulin resistance and androgenaemia in patients with and without PCOS were studied. RESULTS: PCOS patients were slightly older than controls (median ages 34 and 30 years, respectively). AMH generally increased with antral follicle count (AFC), insulin, homeostatic model assessment of tissue insulin sensitivity (HOMA-IR), testosterone, free androgen index and luteinising hormone, and decreased with chronological age, homeostatic model assessment of steady state beta cell function (HOMA-B) and serum sex hormone binding globulin (SHBG). For these relationships there were no significant differences in the slopes between PCOS and non-PCOS patients. The ratio of AMH per antral follicle (AMH/AF) was higher in PCOS patients. Both PCOS and non-PCOS groups showed a very similar increase in AMH with increases in AFC, but the PCOS patients had consistently higher AMH across all AFC levels. CONCLUSIONS: These observations indicate that AMH is similarly related to insulin resistance and androgens in women with and without PCOS. This effect appears to be independent of age although an indirect causal effect due to ageing or some other mechanism cannot be ruled out. Excessive granulosa cell activity may be implicated in the abnormal follicular dynamic of the syndrome.

Biomarkers of oxidant stress, insulin sensitivity and endothelial activation in rheumatoid arthritis: a cross-sectional study of their association with accelerated atherosclerosis.

BACKGROUND: Women with rheumatoid arthritis (RA) have increased morbidity and mortality due to coronary heart disease. Chronic systemic inflammation is known to accelerate atherosclerosis and increase arterial stiffness in patients, but other mechanisms may also be involved. Biomarkers of oxidant stress, inflammation, insulinaemia and endothelial dysfunction were measured in blood and urine from 46 RA patients and 48 age-matched controls. Plaque formation and intima-medial thickness (IMT) were measured using B-mode carotid Doppler scan. FINDINGS: The prevalence of plaque was increased (p = 0.042) in RA patients between 50-59 years old compared to the same age group in controls. 8-isoprostane (p = 0.004), C-reactive protein (p < 0.001), interleukin-6 (p < 0.001), insulin (p = 0.035), adiponectin (p = 0.012), vascular cell adhesion molecule (VCAM) (p = 0.029) and E-selectin (p < 0.001) were all increased while selenium (p = 0.003) and LDL-cholesterol (p = 0.025) were both decreased in all RA patients. 8-isoprostane correlated with 10 year cardiac risk (r = 0.55, p < 0.001), VCAM with IMT (r = 0.37, p = 0.012) and E-selectin with rheumatoid factor titre (r = 0.43, p = 0.003) in RA patients. In the control group, age, carotid IMT, VCAM, systolic blood pressure and smoking status were all associated with plaque development whereas in RA patients only age was associated with plaque. CONCLUSION: The burden of atherosclerosis is particularly increased in middle-aged women with RA. Patients with RA have increased levels of oxidant stress, inflammation, insulin and soluble adhesion molecules. As the association between classical risk factors was much weaker in RA patients compared to controls, these additional factors may be more important in the accelerated development of atheroma in RA.

Contrasting effects of glycerol and urea transport on rat pancreatic beta-cell function.

BACKGROUND/AIMS: Pancreatic beta-cell function is influenced by changes in cell volume. Such volume changes depend on water permeability of the plasma membrane, conferred in part by aquaporins. Islet cells express aquaporin 7 (AQP7), which is permeable to urea and glycerol in addition to water. We therefore investigated the effects of glycerol and urea on rat pancreatic beta-cell function. METHODS: Electrical activity and whole-cell current were studied using the perforated patch technique. Cell volume was measured by video-imaging and insulin release by radioimmunoassay. Aquaporin 7 expression was studied by RT-PCR, Western blot and double fluorescent immunolabelling. RESULTS: The isosmotic addition of glycerol and urea resulted in depolarization of the plasma membrane and electrical activity, accompanied by beta-cell swelling, activation of the volume-regulated anion channel (VRAC) and insulin release. However, the effects of glycerol, in contrast to urea, persisted throughout exposure to the osmolyte. Glycerol also caused beta-cell activation when added hyperosmotically. A non-metabolizable glycerol analogue had comparable effects to urea on beta-cells. The expression of AQP7 was demonstrated in rat beta-cells. CONCLUSION: Glycerol and urea can activate beta-cells via their rapid uptake across the beta-cell plasma membrane, possibly via AQP7. This results in cell swelling, VRAC activation, electrical activity and insulin release. Glycerol appears to exert an additional effect, possibly related to its intracellular metabolism.

Expression of the electrogenic Na+-HCO3--cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells.

It was recently proposed that, in rat pancreatic islets, the production of bicarbonate accounts for the major fraction of the carbon dioxide generated by the oxidative catabolism of nutrient insulin secretagogues. In search of the mechanism(s) supporting the membrane transport of bicarbonate, the possible role of the electrogenic Na(+)-HCO(3) (-)-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells was investigated. Expression of NBCe1-A and NBCe1-B in rat pancreatic islet cells was documented by RT-PCR, western blotting, and immunocytochemistry. The latter procedure suggested a preferential localization of NBCe1-B in insulin-producing cells. Tenidap (3-100 microM), previously proposed as an inhibitor of NBCe1-A-mediated cotransport in proximal tubule kidney cells, caused a concentration-related inhibition of glucose-stimulated insulin secretion. It also inhibited 2-ketoisocaproate-induced insulin release and to a relatively lesser extent, the secretory response to L: -leucine. Tenidap (50-100 microM) also inhibited the metabolism of D: -glucose in isolated islets, increased (22)Na net uptake by dispersed islet cells, lowered intracellular pH and provoked hyperpolarization of plasma membrane in insulin-producing cells. This study thus reveals the expression of the electrogenic Na(+)-HCO(3) (-)-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells, and is consistent with the participation of such transporters in the process of nutrient-stimulated insulin secretion.

Local inflammation and hypoxia abolish the protective anticontractile properties of perivascular fat in obese patients.

BACKGROUND: Inflammation in adipose tissue has been implicated in vascular dysfunction, but the local mechanisms by which this occurs are unknown. METHODS AND RESULTS: Small arteries with and without perivascular adipose tissue were taken from subcutaneous gluteal fat biopsy samples and studied with wire myography and immunohistochemistry. We established that healthy adipose tissue around human small arteries secretes factors that influence vasodilation by increasing nitric oxide bioavailability. However, in perivascular fat from obese subjects with metabolic syndrome (waist circumference 111+/-2.8 versus 91.1+/-3.5 cm in control subjects, P<0.001; insulin sensitivity 41+/-5.9% versus 121+/-18.6% in control subjects, P<0.001), the loss of this dilator effect was accompanied by an increase in adipocyte area (1786+/-346 versus 673+/-60 mum(2), P<0.01) and immunohistochemical evidence of inflammation (tumor necrosis factor receptor 1 12.4+/-1.1% versus 6.7+/-1%, P<0.001). Application of the cytokines tumor necrosis factor receptor-alpha and interleukin-6 to perivascular fat around healthy blood vessels reduced dilator activity, resulting in the obese phenotype. These effects could be reversed with free radical scavengers or cytokine antagonists. Similarly, induction of hypoxia stimulated inflammation and resulted in loss of anticontractile capacity, which could be rescued by catalase and superoxide dismutase or cytokine antagonists. Incubation with a soluble fragment of adiponectin type 1 receptor or inhibition of nitric oxide synthase blocked the vasodilator effect of healthy perivascular adipose tissue. CONCLUSIONS: We conclude that adipocytes secrete adiponectin and provide the first functional evidence that it is a physiological modulator of local vascular tone by increasing nitric oxide bioavailability. This capacity is lost in obesity by the development of adipocyte hypertrophy, leading to hypoxia, inflammation, and oxidative stress.

Possible role of carbonic anhydrase in rat pancreatic islets: enzymatic, secretory, metabolic, ionic, and electrical aspects.

The presence of carbonic anhydrase (type V) was recently documented in rat and mouse pancreatic islet beta-cells by immunostaining and Western blotting. In the present study, the activity of carbonic anhydrase was measured in rat islet homogenates and shown to be about four times lower than in rat parotid cells. The pattern for the inhibitory action of acetazolamide on carbonic anhydrase activity also differed in islet and parotid cell homogenates, suggesting the presence of different isoenzymes. NaN3 inhibited carbonic anhydrase activity in islet homogenates and both D-[U-14C]glucose oxidation and glucose-stimulated insulin secretion. Acetazolamide (0.3-10.0 mM) also decreased glucose-induced insulin output but failed to affect adversely D-[U-14C]glucose oxidation, although it inhibited the conversion of D-[5-3H]glucose to [3H]OH and that of D-[U-14C]glucose to acidic metabolites. Hydrochlorothiazide (3.0-10.0 mM), which also caused a concentration-related inhibition of the secretory response, like acetazolamide (5.0-10.0 mM), decreased H(14)CO3- production from D-[U-14C]glucose (16.7 mM). Acetazolamide (5.0 mM) did not affect the activity of volume-sensitive anion channels in beta-cells but lowered intracellular pH and adversely affected both the bioelectrical response to d-glucose and its effect on the cytosolic concentration of Ca2+ in these cells. The lowering of cellular pH by acetazolamide, which could well be due to inhibition of carbonic anhydrase, might in turn account for inhibition of glycolysis. The perturbation of stimulus-secretion coupling in the beta-cells exposed to acetazolamide may thus involve impaired circulation in the pyruvate-malate shuttle, altered mitochondrial Ca2+ accumulation, and perturbation of Cl- fluxes, resulting in both decreased bioelectrical activity and insulin release.

Hyperinsulinemia, insulin resistance, and circulating oxidized low density lipoprotein in women with systemic lupus erythematosus.

OBJECTIVE: Women with systemic lupus erythematosus (SLE) have increased risk of coronary heart disease (CHD) that is not fully explained by the classic CHD risk factors. Insulin resistance is an established risk factor for CHD in the general population. We compared insulin secretion and sensitivity in patients with SLE and healthy controls, and assessed the prevalence of the metabolic syndrome in women with SLE and its relation to circulating oxidized low density lipoprotein (ox-LDL). METHODS: Fasting insulin, glucose, and lipid profiles were measured in nondiabetic women with SLE (>or= 4 revised 1997 criteria) not undergoing antimalarial therapy (n = 44), and in age matched controls recruited from the hospital staff and the local community (n = 45). Using the Homeostatic Model Assessment equations, insulin sensitivity (HOMA-S) and pancreatic beta cell function (HOMA-B) were calculated from fasting insulin and glucose. The metabolic syndrome, defined according to the Adult Treatment Panel (ATP III) criteria, was determined in a consecutive series of 61 women with SLE. RESULTS: Patients with SLE had significantly higher fasting insulin [median (range) 10 (2.8-38) vs 6.6 (3.1-26) mU/l; p < 0.01], higher pancreatic beta cell function (HOMA-B) [165 (54-1567) vs 111 (28-653); p < 0.01], and lower insulin sensitivity (HOMA-S) [0.46 (0.09-1.9) vs 0.73 (0.16-1.3); p < 0.01]. SLE patients also had significantly higher triglycerides (p < 0.01) and lower high density lipoprotein cholesterol (p < 0.01) than controls. HOMA-S did not correlate with disease activity or steroid therapy, but was associated with components of the insulin resistance syndrome. HOMA-S showed a significant negative correlation with levels of ox-LDL in patients, but not in controls. Eleven (18%) patients had the metabolic syndrome. Again, this was not related to current steroid therapy. SLE patients with the metabolic syndrome had no difference in LDL, but had significantly higher levels of ox-LDL. CONCLUSION: Nondiabetic patients with SLE have evidence of significant decrease in sensitivity to insulin, and overall this population has a high prevalence of the metabolic syndrome (18%). Insulin resistance in the context of SLE was not strongly related to current or recent steroid therapy; it was, however, associated with higher levels of ox-LDL. Insulin resistance may therefore represent an additional CHD risk factor in patients with SLE.

Stimulus-secretion coupling of hypotonicity-induced insulin release in BRIN-BD11 cells.

The stimulus-secretion coupling for hypotonicity-induced insulin release was investigated in BRIN-BD11 cells. A 50 mM decrease in extracellular NaCl caused a twofold increase in insulin release. The release of insulin evoked by hypotonicity progressively decreased in an exponential manner. The response to extracellular hypotonicity displayed a threshold value close to 20 mOsmol/L and a maximal response at about 70 mOsmol/ L. Hypotonicity also caused a rapid increase in cell volume followed by a regulatory volume decrease (RVD), cell membrane depolarization with induction of spike activity, and a rise in cytosolic Ca2+ concentration. 5-Nitro-2-(3-phenylpropylamino)benzoate inhibited the secretory response to hypoosmolarity, failed to affect the early increase in cell volume but prevented the RVD, and suppressed the hypotonicity-induced plasma membrane depolarization. Insulin release provoked by hypotonicity was inhibited by verapamil, absence of Ca2+, thapsigargin, furosemide, tributyltin, and diazoxide. On the contrary, tolbutamide augmented modestly insulin release recorded in the hypoosmolar medium. Last, a rise in extracellular K+ concentration, while augmenting basal insulin output, failed to affect insulin release in the hypoosmolar medium. Thus, the insulin secretory response to hypotonicity apparently represents a Ca2+-dependent process triggered by the gating of volume-sensitive anion channels with subsequent depolarization and gating of voltage-sensitive Ca2+ channels.

Actions of antipsychotic drugs on pancreatic beta-cell function: contrasting effects of clozapine and haloperidol.

The use of antipsychotic drugs is known to be associated with a number of adverse metabolic side effects, including diabetes mellitus. These side effects could be, at least in part, the result of impaired islet cell function, although the underlying mechanisms are unknown. We have studied the effects of the atypical antipsychotic clozapine and of the conventional drug haloperidol on electrical and secretory activity in rat pancreatic beta-cells. At a low glucose concentration (4mM), clozapine (5microM) had little or no effect on membrane potential. However, at a stimulatory glucose concentration (16mM), clozapine was found to hyperpolarize the cell membrane potential, resulting in a complete inhibition of electrical activity. In contrast, haloperidol (5microM) was found to cause a marked depolarization of the membrane potential in the presence of both low and high concentrations of glucose. Clozapine and haloperidol were found, respectively, to increase and decrease-cell input conductance, an index of K(+) permeability. Single channel recordings indicated that changes in K(ATP) channel activity contributed towards these effects. Neither clozapine nor haloperidol affected basal insulin release, although clozapine inhibited glucose-induced insulin release. It is concluded that clozapine and haloperidol exert contrasting actions on electrical activity in rat pancreatic -cells as a result of opposing effects on K(+) permeability. These findings may relate to the increased incidence of diabetes associated with clozapine treatment.

Progestogens of varying androgenicity and cardiovascular risk factors in postmenopausal women receiving oestrogen replacement therapy.

OBJECTIVE: Medroxyprogesterone (MP) was used as the progestogen in randomized clinical trials of postmenopausal hormone replacement on cardiovascular risk. To attempt to understand the lack of benefit in these trials, we have examined the effects of MP and two other progestogens, the less androgenic desogestrel (DG) and the more androgenic norethisterone (NE), on cardiovascular risk factors against a background of oestrogen therapy. DESIGN AND MEASUREMENTS: Thirty-four women were treated with conjugated equine oestrogens (CEE) 0.625 mg daily alone for 12 weeks, followed in random order by each of the three progestogens (DG 75 microg, MP 10 mg and NE 1 mg daily) given sequentially for three 12-week cycles while maintaining the same CEE treatment. We measured serum lipoproteins, paraoxonase activity, C-reactive protein (CRP), fibrinogen, fasting glucose and insulin levels at baseline, at the end of the oestrogen-only phase and at the end of each of the combined oestrogen and progestogen phases. RESULTS: The addition of progestogens to CEE maintained the oestrogen-induced reduction in apolipoprotein B (apo B) and lipoprotein (a) [Lp(a)], and further lowered total cholesterol (P < 0.01) and fibrinogen (P < 0.001). CEE raised serum triglyceride (P < 0.001) and CRP (P < 0.01) concentrations, which reverted towards pre-oestrogen levels with progestogens. Progestogens significantly reduced high density lipoprotein (HDL) cholesterol (P < 0.05). NE was associated with the greatest reduction in HDL cholesterol and apo A1, but was most effective in preserving paraoxonase activity and reducing the potentially unfavourable oestrogen-induced increases in triglycerides and CRP. CONCLUSION: Preconceptions that more androgenic progestogens necessarily have more unfavourable effects on cardiovascular risk factors may require revision.

Inhibition of glucose-induced electrical activity in rat pancreatic beta-cells by DCPIB, a selective inhibitor of volume-sensitive anion currents.

We have investigated the effects of the ethacrynic acid derivative 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB), an inhibitor of the volume-sensitive anion channel (VSAC), on electrical activity and insulin secretion in rat pancreatic beta-cells. DCPIB inhibited whole-cell VSAC currents in beta-cells with IC50 values of 2.2 and 1.7 microM for inhibition of outward and inward currents, respectively. DCPIB also inhibited the VSAC at the single channel level in cells activated by glucose. In intact cells, DCPIB caused a net increase in beta-cell input conductance and evoked an outward current that was sensitive to inhibition by tolbutamide, suggesting KATP channel activation. However, no KATP channel activation was evident under conventional whole-cell conditions, suggesting that the drug might activate the channel in intact cells via an indirect mechanism, possibly involving nutrient metabolism. DCPIB suppressed glucose-induced electrical activity in beta-cells, hyperpolarised the cell membrane potential at a substimulatory glucose concentration and prevented depolarisation when the glucose concentration was raised to stimulatory levels. The suppression of electrical activity by DCPIB was associated with a marked inhibition of glucose-stimulated insulin release from intact islets. It is concluded that DCPIB inhibits electrical and secretory activity in the beta-cell as a combined result of a reciprocal inhibition of VSAC and activation of KATP channel activities, thus producing a marked hyperpolarisation of the beta-cell membrane potential.