Amiloride derivatives enhance insulin release in pancreatic islets from diabetic mice

BACKGROUND: Amiloride derivatives, commonly used for their diuretic and antihypertensive properties, can also cause a sustained but reversible decrease of intracellular pH (pH(i)). Using dimethyl amiloride (DMA) on normal rodent pancreatic islets, we previously demonstrated the critical influence of islet pH(i )on insulin secretion. Nutrient-stimulated insulin secretion (NSIS) requires a specific pH(i)-range, and is dramatically enhanced by forced intracellular acidification with DMA. Furthermore, DMA can enable certain non-secretagogues to stimulate insulin secretion, and induce time-dependent potentiation (TDP) of insulin release in mouse islets where this function is normally absent. The present study was performed to determine whether pH(i)-manipulation could correct the secretory defect in islets isolated from mice with type 2 diabetes. METHODS: Using two mouse models of type 2 diabetes, we compared a) pHi-regulation, and b) NSIS with and without treatment with amiloride derivatives, in islets isolated from diabetic mice and wild type mice. RESULTS: A majority of the islets from the diabetic mice showed a slightly elevated basal pH(i )and/or poor recovery from acid/base load. DMA treatment produced a significant increase of NSIS in islets from the diabetic models. DMA also enabled glucose to induce TDP in the islets from diabetic mice, albeit to a lesser degree than in normal islets. CONCLUSION: Islets from diabetic mice show some mis-regulation of intracellular pH, and their secretory capacity is consistently enhanced by DMA/amiloride. Thus, amiloride derivatives show promise as potential therapeutic agents for type 2 diabetes

Long-Term Safety of Roflumilast in Patients with Chronic Obstructive Pulmonary Disease, a Multinational Observational Database Cohort Study

Edeltraut Garbe,1 Fabian Hoti,2 Tania Schink,1 Kristian Svendsen,3 Haydar Al-Eid,4 Per Arkhammar,5 Marie Carlholm,6 Harald Fjällbrant,6 Stefan Franzén,7,8 Cecilia Hedlund,9 Bianca Kollhorst,10 Atul Kumar,11 Muriel Lobier,2 Vasili Mushnikov,2 Tore Persson,9 Xu Qiao,2 Aaro Salosensaari,12 Wiebke Schäfer,1 Nicholas M Sicignano,13 Gunnar Johansson,14 Eileen O Dareng15 1Department Clinical Epidemiology, Leibniz Institute for Prevention Research and Epidemiology – BIPS, Bremen, Germany; 2Biostatistics, Scientific Services, Real World Solutions, IQVIA, Espoo, Finland; 3Department of Pharmacy, UiT The Arctic University of Norway, Tromsø, Norway; 4United States Navy, Naval Medical Center, Portsmouth, Virginia, USA; 5Global Patient Safety Biopharma, AstraZeneca, Gothenburg, Sweden; 6Late-Stage Respiratory and Immunology, BioPharmaceuticals Research and Development, AstraZeneca, Gothenburg, Sweden; 7BPM Evidence Statistics, Medical Evidence, BioPharmaceuticals Medical, Gothenburg, Sweden; 8School of Public Health and Community Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 9Biometrics, Late-stage Respiratory and Immunology, BioPharmaceuticals Research and Development, AstraZeneca, Gothenburg, Sweden; 10Department Biometry and Data Management, Leibniz Institute for Prevention Research and Epidemiology – BIPS, Bremen, Germany; 11Global Patient Safety Biopharma, AstraZeneca, Bangalore, India; 12Biostatistics, Scientific Services, Real World Solutions, IQVIA, Turku, Finland; 13Health ResearchTx LLC, Trevose, Pennsylvania, USA; 14Department of Public Health and Caring Sciences, Uppsala University, Uppsala, Sweden; 15Safety Epidemiology and Risk Management, AstraZeneca, Cambridge, UKCorrespondence: Eileen O Dareng, AstraZeneca, City House, 126-130 Hills Road, Cambridge, CB2 1RY, United Kingdom, Tel +44 7789447801, Email [email protected]: This study evaluated the long-term safety of roflumilast in patients with chronic obstructive pulmonary disease or chronic bronchitis using electronic healthcare databases from Germany, Norway, Sweden, and the United States (US).Patients and Methods: The study population consisted of patients aged ≥ 40 years who had been exposed to roflumilast and a matched cohort unexposed to roflumilast. The matching was based on sex, age, calendar year of cohort entry date (2010– 2011, 2012, or 2013), and a propensity score that included variables such as demographics, markers of chronic obstructive pulmonary disease (COPD) severity and morbidity, and comorbidities. In comparison to the unexposed matched cohort (never use), three exposure definitions were used for the exposed matched cohort: ever use, use status (current, recent, past use), and cumulative duration of use. The main outcome was 5-year all-cause mortality. Cox regression models were used to estimate crude and adjusted hazard ratios (HRs) and 95% confidence intervals (CI).Results: 112,541 unexposed and 23,239 exposed patients across countries were included. Some variables remained unbalanced after matching, indicating higher COPD disease severity among the exposed patients. Adjusted HRs of 5-year all-cause mortality for “ever use” of roflumilast, compared to “never use”, were 1.12 (95% CI, 1.08– 1.17) in Germany, 1.00 (95% CI, 0.92– 1.08) in Norway, 0.98 (95% CI, 0.92– 1.04) in Sweden, and 1.16 (95% CI, 1.12– 1.20) in the US. Compared to never users, there was a decrease in 5-year mortality risk observed among “current users” in Germany (HR: 0.93, 95% CI: 0.88– 0.98), Norway (HR: 0.77, 95% CI: 0.67– 0.87), and Sweden (HR: 0.80, 95% CI: 0.73– 0.88).Conclusion: There was no observed increase in 5-year mortality risk with the use of roflumilast in Sweden or Norway. A small increase in 5-year mortality risk was observed in Germany and the US in the ever versus never comparison, likely due to residual confounding by indication.Keywords: roflumilast, 5-year all-cause mortality, COPD, electronic healthcare database, propensity scor

Direct evidence for opposite effects of D-glucose and D-glyceraldehyde on cytoplasmic pH of mouse pancreatic beta-cells.

The effects of D-glucose, D-glyceraldehyde, glibenclamide, D-600, NH4+ and high concentrations of K+ on cytoplasmic pH (pHi) were investigated in dispersed and cultured pancreatic beta-cells from ob/ob mice. The cytoplasmic pH was measured with the fluorescent H+-indicator quene 1. The average pHi value in resting beta-cells was 6.71. Addition of 20 mM of the physiological stimulus D-glucose increased pHi with 0.05 units. Both glibenclamide and high concentrations of K+ decreased pHi. The latter effects were completely reversed by D-600, supporting the notion that free cytoplasmic Ca2+ can be involved in the regulation of pHi. In contrast to D-glucose, 10 mM of D-glyceraldehyde decreased pHi by 0.09 units, an effect persisting even in the presence of D-600. From the present study it is evident that D-glyceraldehyde and D-glucose have opposite effects on pHi in pancreatic beta-cells

Voltage-activated Na+ currents and their suppression by phorbol ester in clonal insulin-producing RINm5F cells.

The whole-cell configuration of the patch-clamp technique was applied on the clonal insulin-producing cell line RINm5). Thus attempts were made to characterize voltage-activated inward and outward membrane currents and to examine to what extent these were affected by both long-term and acute exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Current responses to voltage-clamp steps up to -40 mV were small. A pulse to -28 mV evoked an inward current, and slowly activating outward currents developed at potentials above -20 mV. The inward current had a V-shaped current-voltage relationship, reaching a peak between -10 and 0 mV, whereas the outward current increased linearly at potentials beyond -20 mV. It was demonstrated that the inward currents are carried primarily by Ca2+ and Na+ and the outward current by K+. After long-term exposure to TPA, there was a suppression of Na+ currents in one-third of the cells, whereas the Ca2+ and K+ currents were unaffected. Acute exposure to the phorbol ester increased the Ca2+ currents with little effect on the Na+ currents. The extent to which the differences in effects on membrane currents initiated by respective acute and long-term exposure to TPA may reflect two separate mechanisms of protein kinase C activation, the latter related to regulation of differentiation of the RINm5F cells, merits further investigation

Inhibition of ATP-regulated K+ channels precedes depolarization-induced increase in cytoplasmic free Ca2+ concentration in pancreatic beta-cells.

The effects of glucose, diazoxide, K+, and tolbutamide on the activity of K+ channels, membrane potential, and cytoplasmic free Ca2+ concentration were investigated in beta-cells from the Uppsala colony of obese hyperglycemic mice. With [K+]e = [K+]i = 146 mM, it was demonstrated that the dominating channel at the resting potential is a K+ channel with a single-channel conductance of about 65 picosiemens and a reversal potential of about +70 mV (pipette potential). This channel is characterized by complex kinetics with openings grouped in bursts. The channel was completely inhibited by 20 mM glucose in intact cells or by intracellularly applied Mg-ATP (1 mM). The number of active channels was markedly reduced already by 5 mM glucose. However, the single channel current of the channels remaining active was unaffected, indicating no major depolarization. To evoke a substantial depolarization of the membrane and thereby action potentials, a total block in channel activity was necessary. This could be achieved either by increasing the concentration of glucose to 20 mM or by combining 5 mM glucose with 100 microM tolbutamide. In both cases, the effect was counteracted by the hyperglycemic sulfonamide diazoxide. The effects on single channel activity were paralleled by changes in membrane potential and cytoplasmic free Ca2+ concentration, also when the latter measurements were performed at room temperature. The transient increase in the number of active channels and the resulting hyperpolarization observed after raising the glucose concentration to 20 mM probably reflected a drop in cytoplasmic ATP concentration. It is suggested that ATP works as a key regulator of the beta-cell membrane potential and thereby the opening of voltage-activated Ca2+ channels