Information on the thesis

Thesis for PhD degree in Biology, specialty 03.00.13 – Human and animal
physiology. – Ivan Franko National University of Lviv, Lviv, 2016.
Mechanisms of regulation of rat hepatocytes respiration by Ca ²⁺ were investigated.
Suspension of isolated liver cells was obtained using collagenase by Seglen method. Rate
of oxygen consumption was registered at 37°C using Clark electrode.
A model of permeabilized hepatocytes for mitochondria in situ respiration studying
was developed. It was revealed that hepatocytes permeabilization depends on digitonin
concentration in medium and on cell number in suspension: the higher is suspension
density, the higher digitonin concentration is required. Optimal ratio of digitonin amount
to cell number equaled 20–22 μg/million.
The dependence of permeabilized hepatocytes oxygen consumption on Ca ²⁺
concentration within the medium (0.01 – 10 μM) was investigated. The highest respiration
rate upon either succinate along or malate, glutamate and pyruvate mixture oxidation was
in the solution with 1 μM Ca ²⁺ . ADP-stimulated oxygen consumption was the most
intensive within the medium containing 0.1 μM Ca ²⁺ . Concentration of 10 μM Ca ²⁺ has
been revealed to be toxic for hepatocytes mitochondria as ADP and dinitrophenol did not
stimulate oxidative processes upon such conditions.
The dependence of permeabilized hepatocytes respiration rate on oxidative
substrates concentration was studied at different [Ca ²⁺ ] – 0.1 and 1 μM in the medium.
Parameters of Hill equation were calculated applying modified Eadie-Hofstee coordinates –
{v; v/[S] h }. The kinetic dependence of respiration upon either succinate or pyruvate
oxidation was well described by Hill equation in the mediums with concentrations of Са ²⁺
mentioned earlier. Hill coefficient h and semi activation constant K_0.5 for succinate, at
rotenone presence, were not changed after increase of Са ²⁺ concentration from 0.1 to
1 μM. And maximal velocity V _max slightly increased under such conditions. There was
more essential difference between kinetic parameters of pyruvate-stimulated respiration at
various Ca ²⁺ concentrations. Thus, V _max was 1.5 times lower, and K_0.5 – 10.7 times higher
at 0.1 μM Са ²⁺ in comparison with 1 μM Са ²⁺ . Hill coefficient became less than 1 at both
Са ²⁺ concentrations.
Prolonged influence of taurine on kinetic parameters of rat permeabilized
hepatocytes respiration upon various Са ²⁺ concentrations in medium was investigated. The
kinetic dependence of respiration upon succinate and pyruvate oxidation was well
described by Hill equation under taurine action, similar to control. Kinetic parameters of
respiration, upon succinate oxidation at both Са ²⁺ concentrations, were not changed
significantly under prolonged taurine injection. As a result of taurine injection, V _max upon
pyruvate oxidation was 1.6 times less at low Са ²⁺ and K_0.5 was higher, but only 6 times.
Hill coefficient of this process increased at 0.1 μM Са ²⁺ and was 1.12, upon taurine
impact. Substrate inhibition, inherent to the dependence of respiration rate on pyruvate
concentration within the medium with 1 μM Са ²⁺ , started developing, under taurine effect,
at higher concentration of this substrate in comparison with control (3mM comparatively
to 0.35 mM).
Insulin effect on energy supplying processes in isolated rat hepatocytes at different
duration of hormone action at succinate and a-ketoglutarate oxidation was investigated.
Short-term insulin action in vitro did not influence the examined processes. In vivo insulin
effect was characterized by time dependence with activation of ADP-stimulated succinate
and α-ketoglutarate oxidation in isolated hepatocytes in 4 hours after injection and absence
of influence in case of prolonged action (6, 12 days).
Са ²⁺ influence on respiration processes of permeabilized hepatocytes upon
streptozotocin-induced diabetes mellitus was studied. The greater intensification of
respiration by mixture of malate, glutamate and pyruvate in diabetic animals was observed
at 10 μM Са ²⁺ . ADP-stimulated respiration rate in diabetic rats was maximal at 1 μM Са ²⁺ .
Upon succinate oxidation the maximal mitochondrial functional ability in control animals
was observed at 0.1 μM Са ²⁺ within the medium and in experimental rats at 1 μM Са ²⁺ .
Hill coefficient h and V _max of succinate-stimulated mitochondrial respiration in diabetic
animals were decreased upon low Са ²⁺ concentration (0.1 μM). These parameters
remained similar to the control at 1 μM Са ²⁺ in the medium. So, at the early stages of
streptozotocin-induced diabetes mellitus development, the hepatocytes respiration was
intensified thanks to, in particular, the increase of resistance to toxic Са ²⁺ amounts within
mitochondrial matrix.
Key words: hepatocytes, respiration, Са ²⁺ , mitochondria in situ, kinetics, taurine,
insulin, diabetes mellitus.