Information on the thesis

The dissertation is dedicated to the expansion and validation of the methodology of
assessing the adaptive capacity of rat pancreatic acinar cells mitochondria.
New parameters of adaptive capacity of rat pancreatic acinar cells mitochondria were
characterized: maximal uncoupled respiration, optimal protonophore concentration, respi-
ration acceleration and deceleration. These parameters depended on the oxidative substrate
and functional states of pancreatic acini. In most cases (except for glutamine oxidation) the
acceleration was much higher after 1.5 μM FCCP and maximal peak respiration – after
0.5 μM FCCP. Only when malate or isocitrate was present, deceleration was significantly
higher. It has been found that, the increase in the respiration rate was proportional to the
decrease in membrane potential. The coefficient of elasticity and the area under the curves
of the dependence of respiratory rate on the concentration of FCCP were used for formalize
the assessment of adaptive capacity of mitochondria. Elasticity coefficient was the highest
when the combination of glucose, glutamine and pyruvate was present and the lowest when
monomethyl-succinate was oxidized. The combination of three substrates (glucose, py-
ruvate and glutamine) maintains the highest respiration rate in response to FCCP load.
Upon acetylcholine or cholecystokinin stimulation, maximal uncoupled respiration
rate increased only upon the oxidation of pyruvate with glucose; insulin stimulated only
when glucose-fueled uncoupled respiration, but abolished the stimulative effect of chole-
cystokinin on pyruvate-fueled uncoupled respiration.
Ethanol in vitro suppressed rate of cholecystokinin-stimulated uncoupled respiration
of pancreatic acini, while combination of ethanol with cholecystokinin caused an increase
in number of necrotic cells and cells with plasma membrane blebs. These effects were de-
fendant on the oxidative substrates in the medium. Combination of ethanol and CCK in vitro
caused the decrease of uncoupled respiration of pancreatic acini only upon the oxidation of
glutamine independently of other substrates. Moreover, mitochondrial membrane depolari-
zation and NADH autofluorescence increase caused by CCK and ethanol were not observed
upon glutamine supplementation. Combination of ethanol and CCK caused increase necro-
sis only in case of glucose presence, but not when either pyruvate or glutamine were added.
Substantial increase in plasma membrane blebbing was observed only after incubation with
combination of glucose, glutamine, pyruvate and combination of ethanol and CCK.
After a single in vivo administration of ethanol (2 h), CCK (1 h) or combination
thereof, the fraction of necrotic cells and plasma amylase level did not change indicating the
lack of pancreatic damage. However, the number of cells with membrane blebbing substan-
tially increased in animal group treated with both ethanol and CCK. Basal respiration rate
of isolated pancreatic acini was lower in animals after ethanol administration irrespectively
of the oxidative substrate. Neither EtOH nor CCK on their own affected the uncoupled res-
piration, but their combination caused a significant decrease of the maximal uncoupled res-
piration.
Chronic alcohol administration (14 days) caused an increase of FCCP-stimulated res-
piration upon glucose oxidation, but not when pyruvate was co-administered for 7 last days.
Pyruvate also ameliorated the negative effects of ethanol administration on other mitochon-
drial parameters: oligomycin-insensitive respiration and IC 50 of FCCP on mitochondrial
membrane potential. Number of necrotic cells mildly increased in the group of animals after
ethanol administration, but no such effect was observed in the group of animals that were
also injected with pyruvate. Plasma amylase level in animals after ethanol administration
did not increase indicating no full-blown pancreatitis.
Thus, the parameters of adaptive mitochondrial capacity are useful tools to study both
different aspects of normal pancreatic acinar cells functioning and mechanisms of patho-
genesis of pancreatic diseases.
Key words: pancreatic acini, adaptive capacity of mitochondria, uncoupled respira-
tion, FCCP, ethanol, oxidation substrate.