The dependence of uncoupled respiratory capacity of intact pancreatic acini on oxidative substrate supply and functional cell state has not yet been studied in detail. In this study, the respiratory responses of isolated pancreatic acini to FCCP were measured with Clark electrode and mitochondrial membrane potential was assessed with rhodamine123 fluorescence. The response of acini to FCCP was characteri zed with maximal uncoupled respiration rate, optimal FCCP concentration, respiration acceleration and deceleration. Maximal uncoupled respiration rate substantially increased upon the oxidation of glucose + glutamine (3.

Objectives:The objective of this study was to test whether pyruvate and glutamine affect the ethanol and cholecystokinin (CCK) effects on the mitochondrial function, viability, and morphology of rat pancreatic acini.Methods: Respiration was measured with Clark oxygen electrode. Mitochondrial membrane potential, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), cell morphology, and viability were studied with fluorescence microscopy. Results:In vitro, CCK (0.1 nM) caused pyruvate-dependent stimulation of basal and uncoupled respiration, and the effects were abolished by ethanol (20 mM). The combination of ethanol with CCK (2 hours) caused necrosis of approximately 40% acinar cells in medium with glucose, but not with pyruvate and/or glutamine. Cholecystokinin (10 nM) or ethanol with 0.1 nM CCK caused plasma membrane blebbing not related to apoptosis only when both glutamine and pyruvate were present. Glutamine, but not pyruvate, decreased NAD(P)H level and prevented the effects of ethanol with CCK on mitochondrial membrane potential and NAD(P)H, but, in combination with CCK and ethanol, decreased the uncoupled respiration. In vivo, the combination of ethanol (4 g/kg) and CCK (20 pmol/kg) suppressed basal and uncoupled respiration and caused acinar cell blebbing, but not necrosis. Conclusions:The lack of sufficient substrate supply in vitro makes pancreatic acinar cells susceptible to necrosis caused by ethanol and CCK in clinically relevant concentrations.

Insulin increases the basal and agonist-stimulated secretion of pancreatic acinar cells, which leads to increase of energy demand and requires sufficient oxidative substrates supply. Cholecystokinin substantially increases the respiration rate of pancreatic acinar cells upon pyruvate oxidation. However, it is not clear how insulin affects mitochondrial oxidative processes at rest and upon secretory stimulation. Experiments were carried out on male Wistar rats (250–300 g) kept on standard diet. Animals were fasted 12 h before the experiment. Pancreatic acini were isolated with collagenase. Basal and FCCP-stimulated respiration of rat pancreatic acini was measured with Clark electrode. Adaptive capacity of mitochondria was assessed by the maximal rate of uncoupled respiration. Statistical significance (P) of differenced between the means was assessed either with a paired t-test or with repeated measures two-way ANOVA and post-hoc Turkey test. Adaptive capacity of pan­creatic acinar mitochondria was significantly higher when pyruvate (2 mM) was used as oxidative substrate comparing with glucose (10 mM). Incubation with insulin (100 nM) for 20 minutes elevated the basal respiration and adaptive capacity of pancreatic acinar mitochondria upon glucose, but not pyruvate, oxidation. Cholecystokinin (0.1 nM, 30 min) stimulated the rate of basal and maximal uncoupled respiration of acinar cells upon pyruvate oxidation, but insulin completely negated this increase of mitochondrial adaptive capacity. Thus, insulin increases the glucose oxidation in pancreatic acinar cells at resting state, but suppresses pyruvate oxidation upon secretory stimulation with cholecystokinin. The mechanisms of insulin action of pyruvate metabolism in pancreatic acinar cells require further elucidation.

Вивчали базальне і FCCP-стимульоване дихання ацинарних клітин підшлункової залози щурів за допомогою електрода Кларка. Адаптаційну здатність мітохондрій оцінювали за максимальною швидкістю роз'єднаного дихання та оптимальною концентрацію FCCP. Під впливом ацетилхоліну та холецистокініну базальна швидкість дихання зростала за наявності у середовищі глюкози (на 16 і 25 %), а також суміші глюкози з піруватом (на 36 і 37 %) чи глутаміном (на 21 і 29 % відповідно), але не з монометилсукцинатом чи диметил-α-кетоглутаратом. Агоністстимульоване збільшення максимальної швидкості роз'єднаного дихання зареєстроване лише за окиснення суміші глюкози та пірувату: у разі дії ацетилхоліну від 2,32 у контролі до 3,62 відн.од., а холецистокініну-до 3,19 відн. од. Оптимальна концентрація FCCP збільшувалася лише у разі дії холецистокініну за наявності у середовищі інкубації суміші глюкози та пірувату-від 1,17 до 1,33 мкмоль/л. Отже, адаптаційна здатність мітохондрій ацинарних клітин підшлункової залози під впливом первинних агоністів збільшується і в основі лежить інтенсифікація окиснення пірувату, а не інших досліджуваних субстратів окиснення. Ключові слова: панкреатичні ацинуси; максимальна швидкість роз'єднаного дихання; оптимальна концентрація FCCP; ацетилхолін; холецистокінін; субстрати окиснення.