Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 2, pp. 299-312 © Pleiades Publishing, Ltd., 2024.

299

Age-Dependent Changes in the Production

of Mitochondrial Reactive Oxygen Species

in Human Skeletal Muscle

Mikhail Yu. Vyssokikh

1,2,3,a

*, Maksim A. Vigovskiy

, Vladislav V. Philippov

Yakov R. Boroday

, Mariya V. Marey

, Olga A. Grigorieva

, Tatiana F. Vepkhvadze

Nadezhda S. Kurochkina

, Ludmila A. Manukhova

, Anastasiya Yu. Efimenko

Daniil V. Popov

, and Vladimir P. Skulachev

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia

National Medical Research Center for Obstetrics, Gynecology and Perinatology

Named after Academician V.I. Kulakov, 117997 Moscow, Russia

Institute of Biomedical Problems, Russian Academy of Sciences, 123007 Moscow, Russia

Medical Research and Education Center, Lomonosov Moscow State University, 119192 Moscow, Russia

e-mail: mikhail.vyssokikh@gmail.com; mike@genebee.msu.ru

Received December 12, 2023

Revised January 30, 2024

Accepted February 1, 2024

Abstract— A decrease in muscle mass and its functionality (strength, endurance, and insulin sensitivity) is one of

the integral signs of aging. One of the triggers of aging is an increase in the production of mitochondrial reactive

oxygen species. Our study was the first to examine age-dependent changes in the production of mitochondrial

reactive oxygen species related to a decrease in the proportion of mitochondria-associated hexokinase-2 in human

skeletal muscle. For this purpose, a biopsy was taken from m. vastus lateralis in 10 young healthy volunteers

and 70 patients (26-85 years old) with long-term primary arthrosis of the knee/hip joint. It turned out that aging

(comparing different groups of patients), in contrast to inactivity/chronic inflammation (comparing young healthy

people and young patients), causes a pronounced increase in peroxide production by isolated mitochondria. This

correlated with the age-dependent distribution of hexokinase-2 between mitochondrial and cytosolic fractions,

a decrease in the rate of coupled respiration of isolated mitochondria and respiration when stimulated with glu-

cose (a hexokinase substrate). It is discussed that these changes may be caused by an age-dependent decrease in

the content of cardiolipin, a potential regulator of the mitochondrial microcompartment containing hexokinase.

The results obtained contribute to a deeper understanding of age-related pathogenetic processes in skeletal mus-

cles and open prospects for the search for pharmacological/physiological approaches to the correction of these

pathologies.

DOI: 10.1134/S0006297924020093

Keywords: aging, skeletal muscle, mitochondria, mitochondrial reactive oxygen species, hexokinase

Abbreviations: mROS, mitochondrial reactive oxygen species.

* To whom correspondence should be addressed.

# Deceased.

INTRODUCTION

Aging is a complex process inherent in all organ-

isms that includes a number of common features, in

particular, a progressive decrease in the functional

and regenerative capabilities of the organism, devel-

oping against the background of impaired adaptation

to external and internal stressors, and ultimately lead-

ing to death. One of the most developed theories of ag-

ing is the mitochondrial theory, first proposed by Har-

man in 1956, according to which the trigger of aging

is considered to be mitochondrial dysfunction associ-

ated with their production of reactive oxygen species

and oxidative damage to biological macromolecules [1].

VYSSOKIKH et al.300

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

One of the integral signs of aging is a decrease in the

mass and strength of skeletal muscles, leading to a pro-

nounced decrease in physical performance and quali-

ty of life. The last of the above is due to the fact that

skeletal muscles not only provide movement and main-

tain posture, but also play an important role in regu-

lating the metabolism of the organism. Thus, normally,

skeletal muscles, constituting up to 40% of body mass,

consume from 20 to 30% of the energy produced [2]

and are one of the main insulin-dependent consumers

of glucose [3]. Unlike other tissues, decline in skeletal

muscle mitochondrial function is thought to be a ma-

jor mediator of declines in muscle mass and strength

in older adults [4].

The most important role in the regulation of me-

tabolism and consumption of glucose from the blood [5]

is played by the enzyme hexokinase (EC 2.7.1.1), which

catalyzes the rate-limiting reaction of glycolysis and

transfers the phosphoryl from ATP to glucose with the

formation of glucose-6-phosphate, an intermediate of

glycolysis and the pentose phosphate pathway. In mus-

cles, as in other insulin-sensitive tissues, I and II of the

five known isozymes of hexokinase are predominantly

expressed; they have high similarity in amino acid

sequences, however, they differ quite significantly in

kinetic parameters and metabolic functions (for re-

view, see [6]). In skeletal muscle, most hexokinase is

localized on the surface of mitochondria, forming a

metabolic compartment that uses ATP synthesized in

mitochondria to phosphorylate hexoses, which stimu-

lates mitochondrial respiration and functionally cou-

ples glycolysis with oxidative phosphorylation [7]. The

binding of hexokinase to mitochondria is mediated by

protein–protein interactions with VDAC1, the main pro-

tein of the outer membrane of mitochondria, which

ensures the transport of various compounds between

mitochondria and the rest of the cell [8]. The forma-

tion of this protein complex leads to the convergence

of mitochondrial membranes and stabilization of the

so-called contact sites, which are the structural basis

of the metabolic microcompartment of mitochondria,

responsible for the shuttle movement of ATP/ADP be-

tween the active centers of ATP synthase and hexoki-

nase [9, 10].

Approximately 1-2% of molecular oxygen in the cell,

with the participation of complexes  I and  III of the

respiratory chain, is converted into superoxide, spon-

taneously or with the help of enzyme systems that

regulate the production of hydrogen peroxide with

the subsequent formation of mitochondrial reactive

oxygen species (mROS), which can damage biological

macromolecules [11]. It has been established that an

imbalance in the production and utilization of ROS(re-

active oxygen species) underlies oxidative stress and

the induction of programmed cell death, which is one

of the key reasons for the development of cardiovas-

cular and neurodegenerative pathologies [12]. It is im-

portant to note that a slight decrease in mitochondrial

membrane potential almost completely stops the gen-

eration of mROS [13]. In particular, in isolated mito-

chondria, activation of oxidative phosphorylation by

the addition of ADP led to a decrease in transmem-

brane potential by a small amount – approximately

20% (mild depolarization), preventing the formation

of H

. Investigating this effect, A. Galina with col-

leagues described a new antioxidant mechanism con-

sisting in the cyclic movement of ADP, formed during

phosphorylation of glucose at the expense of ATP, pro-

duced during oxidative phosphorylation and reaching

the active center of hexokinase through the VDAC1

channel [14].

Previously, using models of short- and long-lived

rodents, we showed that mild depolarization of mito-

chondria, associated with activation of bound hexoki-

nase, leads to a decrease in the production of mROS

in various tissues (including skeletal muscles), and dis-

ruption of this mechanism correlates with the age of

short-lived animals [15]. The purpose of this study was

to investigate the age-related distribution of hexoki-

nase, as well as the dependence of the rate of hydro-

gen peroxide production by mitochondria on the acti-

vation of mitochondrial hexokinase by its substrates in

human skeletal muscle.

MATERIALS AND METHODS

Study design. The study was approved by the Bio-

medical Ethics Committee of the State Scientific Cen-

ter of the Russian Federation– Institute of Biomedical

Problems of the Russian Academy of Sciences and the

Local Ethics Committee of the Lomonosov Moscow State

University (protocol no. 2/20 dated March 16, 2020).

All volunteers signed a voluntary consent to partici-

pate in the study.

The study involved 10 healthy volunteers (age from

25 to 43 years) and 62 patients with long-term prima-

ry arthrosis of the knee/hip joint (age from 26 to 85

years), divided into groups of young (n = 8, 39 (26-45)

years), middle (n = 20, 59 (58-62) years) and elderly (n=

= 42, 72 (66-83) years) age (Table 1). This pathology was

previously used as a model to study the effects of de-

creased physical activity and chronic inflammation on

the skeletal muscles of the thigh, including during ag-

ing [16-19]. The criteria for non-inclusion of volunteers

into the study were: a history of cancer or systemic

diseases; mental, physical, and other reasons that do

not allow you to adequately assess behavior and cor-

rectly comply with the conditions of the research pro-

tocol; a history of any significant, in the opinion of the

research physician, condition/disease or circumstance

that prevents inclusion in the study; contraindications

MITOCHONDRIAL HEXOKINASE AND ROS PRODUCTION 301

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

for surgical treatment at the time of inclusion; preg-

nancy and lactation. All participants had an anamnesis

collected and the subjective level of physical capabil-

ities (questionnaire SF12 [20]) and fasting blood glu-

cose and insulin levels were assessed. A biopsy of the

lateral head of the quadriceps muscle (~500 mg) was

taken from patients at the start of elective hip/knee

replacement surgery. In healthy volunteers, samples

from the vastus lateralis muscle (~200 mg) were collect-

ed by needle biopsy with aspiration under local anesthe-

sia (1 ml of 2% lidocaine) as previously described [21].

The muscle tissue sample was immediately placed into

a tube containing a cardioplegic solution (Custodiol,

USA); after 10min, part of the sample was taken for

histological and biochemical studies (see below), and

the remaining part was transferred to a fresh aliquot

of Custodiol solution to isolate mitochondria.

Histological analysis. Skeletal muscle samples

were fixed in 10% buffered neutral formalin for 24-48h.

Histological processing was carried out according to

standard methods using 8 changes of isopropyl alcohol

(total duration – 5.5 h at 37°C) and 3 changes of paraf-

fin (total duration – 5 h at 62°C). Then the preparations

were embedded in paraffin blocks, sections 1 µm thick

were prepared and mounted on glass slides (Menzel

GmbH & Co KG, Germany). Staining was performed ac-

cording to standard techniques using Mayer’s hema-

toxylin and eosin (PanReac AppliChem, Spain). Mi-

croscopic examination was carried out on a Leica

DM600Β microscope with a Leica DFC 420X camera

(Leica Microsystems GmbH, Germany), using repre-

sentative fields of view to obtain microphotographs.

Image processing and analysis were performed using

LasX software (Leica Microsystems GmbH) and FiJi.

Isolation of mitochondria. Mitochondrial iso-

lation was performed as described previously [15].

Biopsy samples of muscle tissue were rinsed with an

ice-cold solution of 0.9% NaCl, dried with filter paper,

weighed, and fragmented into pieces of 0.5-1 mm in

size with cooled and washed with the isolation medi-

um (see below) using scissors with curved ends. There-

sulting fragments were homogenized using a Potter

microhomogenizer (glass/Teflon) with a clearance of

200 microns for 2 min at 4°C, in a ratio of 10/1 (vol-

ume/mass) in an isolation medium of the following

composition: 300 mM mannitol, 0.5 mM EGTA, 20 mM

HEPES/ NaOH, pH 7.6 and 0.1% BSA. The homogenate

was centrifuged at 1000g for 10 min at 4°C on a 5410

centrifuge (Eppendorf, Germany). The supernatant was

collected and centrifuged at 9000g under the same

conditions. The pellet was suspended in the same vol-

ume of isolation medium without BSA (by a micro-

homogenizer) and centrifuged at 10,500g for 10 min

and4°C. The resulting pellet was suspended in a min-

imal volume, the typical protein concentration of the

resulting mitochondrial preparation was 90-100 mg/ml.

Theprotein content in mitochondrial preparations was

determined with bicinchoninic acid and 1 mg/ml BSA

solution as a standard according to manufacturer’s

instructions (Pierce, USA). All procedures for the iso-

lation and storage of mitochondria during the experi-

ments were carried out at 4°C.

Respiratory rate measurement. The rate of ox-

ygen consumption by mitochondria was measured at

30°C using a closed-type Clark electrode on an oxy-

graph Hansatech (UK), as previously described [15, 22].

Mitochondria (0.05-0.1 mg protein) were incubated

in an oxygraph cell containing 0.5 ml of MIR05 respi-

ratory medium [23] (0.5 mM EGTA, 3 mM MgCl

, 60 mM

potassium lactobionate, 20 mM taurine, 10 mM KH

20 mM HEPES, 110 mM sucrose, 1 g/liter bovine serum

albumin (free from fatty acids), 100 µM potassium di-

adenosine pentaphosphate) and assessed respiration

efficiency in the presence of 5.5 mM pyruvate/malate

or 10 mM succinate / 2 µM rotenone, 1 µM oligomycin,

0.1 mM ADP, 5 mM glucose, 10 nM carbonyl cyanide- 4-

(trifluoromethoxy) phenylhydrazone (FCCP) (all Sigma-

Aldrich, USA).

Measuring the rate of peroxide production. The

rate of hydrogen peroxide production by mitochondria

was estimated using the method of Chow et al. [24]

with the modifications we described earlier [25, 26].

For registration of H

production Amplex reagent

Red (10-acetyl-3,7-dihydroxyphenoxazine; Invitrogen,

USA) was used. Resorufin, a product of H

-induced

oxidation

of Amplex Red, was measured by moni-

toring its fluorescence (excitation/emission maxima

~550/595 nm) on a spectrophotometer Cary Eclipse

(Agilent, USA) for 10-15min. Mitochondria (0.15mg/ml

mitochondrial protein) were incubated at 37°C with

stirring with a built-in magnetic stirrer in MIR05 me-

dium containing 5 μM Amplex Red, horseradish per-

oxidase (12  units/ml, Sigma, USA) and superoxide dis-

mutase (45 units/ml, Sigma, USA). The reaction was ini-

tiated by adding 10 mM succinate, after reaching the

maximum rate of peroxide production, ADP (up to

0.1 mM final concentration) and glucose (final concen-

tration 5 mM) were added. The rate of H

production

was calculated from the change in fluorescence inten-

sity, as described previously [27]. Calibration curves

were obtained by adding freshly diluted H

to the

analytical medium

(the concentration of the hydrogen

peroxide stock solution was checked at 240 nm using a

molar extinction coefficient of 43.6).

Enzyme activity measurement. Determination of

the enzymatic activity of hexokinase was carried out

in unfractionated muscle tissue homogenate, cytosolic

and mitochondrial fractions according to the method

of Scheer et al. [28] with minor modifications. The as-

say buffer contained 50 mM Tris-HCl, 5 mM mercap-

toethanol, 5 mM ATP, 10 mM MgCl

, 0.5 mM glucose,

0.8 mM NAD

and 1 U/ml glucose-6-phosphate dehy-

VYSSOKIKH et al.302

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

drogenase from Leuconostoc mesenteroides (Roche)

at pH 7.5. All assays were performed at 25°C in a total

volume of 1.0 ml, activity was determined using a spec-

trophotometer Cary Varian 300 (Agilent, USA). Enzyme

activity was determined by measuring the increase

in optical density at a wavelength of 340 nm, where

1 unit of enzyme was defined as the amount that cat-

alyzes the conversion of 1 µmol of substrate to product

within 1 min. Calculations were carried out consider-

ing the dilution factor, which was different for different

fractions. Three technical replicates were performed

for each measurement. Reagents from Sigma-Aldrich

were used.

PAGE and Western blot analysis of hexokinase

content in mitochondrial preparations. Electropho-

resis in polyacrylamide gel was carried out according

to the Laemmli method. [29]. Mitochondria were lysed

in buffer: 150 mM sodium chloride, 50 mM Tris-HCl,

pH  8.0, 0.5% Nonidet P-40, 1% sodium deoxycholate,

0.5% sodium dodecyl sulfate with a protease inhibitor

cocktail (ThermoFisher Scientific, USA). After separa-

tion (30 μg of protein per lane), proteins from a 12%

polyacrylamide gel were transferred to a nitrocellu-

lose membrane using a combined electrophoresis/

immunoblotting system (Bio-Rad, USA) and immuno-

blotting was performed as previously described [30].

Primary mouse monoclonal antibodies against human

isozymes of hexokinase  I and  II (ab150013, ab227198,

Abcam, USA) and against the mitochondrial outer mem-

brane protein VDAC1 (ab186321, Abcam) were used, as

well as secondary antibodies conjugated to horserad-

ish peroxidase (ab97023, Abcam) in accordance with

the manufacturer’s recommendations. To visualize the

signal, a Novex ECL Kit (Invitrogen) and a ChemiDoc

scanner (Bio-Rad) were used.

Lipid extraction and cardiolipin analysis

by high-performance thin-layer chromatography

(HPTLC). Lipid extraction was carried out accord-

ing to the Bligh and Dyer method in a nitrogen flow

with oxygen-free solutions bubbled overnight with

99.9%N

[31]. Extracted lipids were dissolved in chlo-

roform/methanol 2 : 1 (v/v) and stored under nitrogen

at –80°C in a silica gel desiccator. Thin layer chroma-

tography was carried out according to the method

[32], using organic solvents of analytical grade and

HPTLC chromatographic plates with silica gel on an

aluminum substrate (Merck, Germany). Before appli-

cation of the sample or standard on the day of use,

10 by 10 cm HPTLC plates were prepared as follows: im-

mersed once in 2.3% boric acid in ethanol, dried for

2 h in a fume hood, and activated at 110°C for 20min

in a sand bath. Samples were applied using a home-

made glass capillary applicator with a ball valve (ac-

tuated by a stream of nitrogen) in the form of strips

10 mm long at a distance of 15 mm from the edge of

the plate with a constant application rate of about

200 nl/s while drying continuously with a stream of

nitrogen at a pressure of 1.5-2.0 bar. For phospholipid

standards (Avanti Polar Lipids, France) prepared a

stock solution (1 mg/ml) in a mixture of chloroform/

methanol (2 : 1, v/v). Elution was carried out in a glass

chamber equilibrated with eluent vapor for at least

1 h before chromatography. The eluent consisted of a

mixture of chloroform/ethanol/triethylamine /water

(3/3.5/3.5/0.7, v/v). Chromatography was stopped when

the front reached 10 mm from the upper edge of the

plate, after two hours of drying in a fume hood the

plate was stained by immersing for 2 min in 0.5% cop-

per sulfate (w/v) in 1.16 M phosphoric acid, drying in

a fume hood for 2 h at room temperature in a stream

of nitrogen and developed in a sand bath for 15 min

at 155°C. The plates were photographed in a ChemiDoc

scanner (Bio-Rad). The measured phospholipid band

intensities were integrated and the peak surface area

values were expressed in arbitrary units using the

ImageJ tool, and the concentrations of cardiolipin and

monolysocardiolipin (nmol/mg mitochondrial protein)

were then calculated using calibration curves for the

corresponding standards.

Statistical methods. Group comparisons were

made using analysis of variance and Tukey’s multiple

comparison test. Data are presented as median with in-

terquartile range; p < 0.05 were considered significant.

Correlation analysis was performed using Spearman’s

rank correlation coefficient. Statistical processing was

carried out using the Prism 7.0 program (Graph Soft-

ware Inc., USA).

RESULTS

Characteristics of volunteer groups. Clinical

and anamnestic characteristics of study participants

presented in Table1; it was shown that the subjective

assessment of physical capabilities in patients is sig-

nificantly lower than in young healthy people, and the

body mass index and insulin resistance index are in-

creased only in middle-aged and elderly patients.

Histological analysis revealed progressive impair-

ment of muscle fibers in patients with age, namely:

muscle fiber atrophy, displacement of nuclei from the

periphery of the fiber, infiltration by immune cells and

an increase in the distance between fibers, presum-

ably associated with an increase in the proportion of

the connective tissue component (Fig.1).

Distribution of hexokinase between cellular

fractions. No differences in hexokinase activity were

found between healthy volunteers and young patients,

either in the homogenate or in the isolated fractions

(Fig. 2). Elderly patients showed a decrease in total

hexokinase activity compared to other groups (p < 0.01

for HV, p < 0.05 for YP and EP, Fig.2a) and a significant

MITOCHONDRIAL HEXOKINASE AND ROS PRODUCTION 303

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Table 1. Characteristics of individuals in various groups

Individuals n Age, years BMI, kg/m

HOMA-IR, c.u. Physical capabilities SF-12, c.u.

Norm —— 18.5-25.0 <2.7 40-60

Healthy volunteers (HV) 10 25-43 (34.5) 18.9-29.4 (22.5) 0.14-3.3 (1.3) 52.0-59.8 (56.3)

Young patients (YP) 8 26-44 (38) 20.8-30.9 (25.7) 1.43-4.65 (2.7) 16.1-31.7 (26.8)

Middle-aged patients (MP) 20 52-64 (59) 21.3-49.3 (34.5) 0.81-12.5 (4.6) 19.1-29.8 (25.2)

Elderly patients (EP) 42 65-85 (72) 20.8-45 (30.4) 1.12-10.96 (3.4) 15.5-37.7 23.0

Note. Medians, minimum and maximum values are presented. BMI,body mass index; HOMA-IR,insulin resistance index.

Fig. 1. Representative microphotographs of longitudinal sections of skeletal muscle stained with hematoxylin-eosin. Lens20×.

a)Sample of a 35-year-old healthy individuals (HV group, n=10); b)sample of a 37-year-old patient (YP group, n=8); c)sample

of a 56-year-old patient (MP group, n=20); d)sample of a 72-year-old patient (EP group, n=42).

increase in activity for the cytosolic fraction compared

to the group of young patients (p<0.05, Fig.2b). How-

ever, in middle-aged and elderly patients, hexokinase

activity in the mitochondrial fraction was lower com-

pared to both healthy volunteers (p < 0.01 and 0.001,

respectively) and young patients (p < 0.05 and 0.01, re-

spectively); moreover, differences were found between

middle-aged and elderly patients (p < 0.05) (Fig.2c).

It is known that in human skeletal muscles the ratio

of I and II hexokinase’s isozymes varies and, according

to different authors, ranges from 1/10 to 1/3 [5, 33, 34].

At the same time, more than 90% isozymeI associated

VYSSOKIKH et al.304

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 2. Hexokinase activity in skeletal muscle of young healthy volunteers (HV, n = 10), young (YP, n =8), middle-aged (MP,

n=20) and elderly (EP, n=42) patients. a)Total hexokinase activity in the homogenate; b)hexokinase activity cytosolic frac-

tion; c)activity of hexokinase-2 associated with mitochondria; d)activity of hexokinase-1 associated with mitochondria. Medi-

ans and interquartile ranges are presented. *p<0.05.

with mitochondria [35] and the level of expression of

this gene, as well as the protein representation in skel-

etal muscle, do not depend on metabolic load, physical

activity, or the concentration of glucose or insulin in the

blood [36]. On the contrary, for isozymeII, this depen-

dence is clearly expressed– mRNA expression and pro-

tein concentration for hexokinaseII in skeletal muscle

can change significantly with changes in the concentra-

tion of glucose or insulin in the blood, as well as with

changes in the level of physical activity [2, 5, 8, 37, 38].

Taking into account the different levels of bind-

ing of these isozymes to the mitochondrial membrane

and the different regulation of their expression, activ-

ity measurements were carried out at 42°C, when the

overwhelming majority of hexokinaseII is inactivated,

and hexokinase activity I changes slightly [5]. It was

shown that there were no differences in hexokinase I

activity in the mitochondrial fraction, regardless of

age and clinical status (Fig.2d).

Data from the analysis of isozyme distribution in

groups of different ages were confirmed using mito-

chondrial protein electrophoresis followed by immu-

noblotting. Representative blots are shown in Fig.3.

The data obtained suggested that, firstly, the ob-

served differences in the distribution of hexokinase

activity between mitochondria and the cytosol are

largely determined by age rather than by a decrease

in motor activity/chronic inflammation, and secondly,

the identified differences are associated with changes

in hexokinase contentII, not I.

Fig. 3. Contents I and II isozymes hexokinase (GK-1 and 2, respectively) in skeletal muscle mitochondria of patients of different

ages. a)Representative electrophoregrams: 1 and 2)young patients (YP) 37 and 39 years old, 3 and 4)middle-aged patients (MP)

59 and 60 years old, 5 and 6)elderly patients (EP) 77 and 81 years old. Membrane protein VDAC1 is used as a control for mito-

chondrial protein loading; b)age-related changes in the content of GK-1 (left) and GK-2 (right), for all groups of patients n=8,

*p<0.001.

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BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 4. Respiration of skeletal muscle mitochondria in young healthy volunteers (HV, n=10), young (YP, n=8), middle-aged (MP,

n=20), and elderly (EP, n=42) patients. a)Representative polarograms: P,pyruvate+ malate; S,succinate+ rotenone; A,ADP;

G,glucose; F,FCCP; K,KCN; b)respiration rate on pyruvate with malate; c)respiration rate on succinate in the presence of ro-

tenone; d)rate of phosphorylating respiration on succinate with rotenone in the presence of ADP (state3); e)respiration rate

on succinate with rotenone in state4; f)rate of phosphorylating respiration on succinate with rotenone in the presence of ADP

and glucose; g)rate of uncoupled breathing in the presence of FCCP; h and i)respiratory control by ADP and FCCP, respectively.

Medians and interquartile ranges are presented; *p<0.05.

Respiration of isolated mitochondria. The respi-

ration rate of isolated mitochondria was determined us-

ing a polarograph, assessing the change in oxygen con-

centration over time. Representative polarograms are

presented in Fig. 4a. The obtained values of the respi-

ration rate of skeletal muscle mitochondria in different

states depending on the age of the study participants are

presented in Fig.4,b-g. Endogenous respiration (state1)

was initiated by adding the respiratory substrate pyru-

vate with the addition of malate, as described above,

and after adding the complexI inhibitor– rotenone, res-

piration was again activated by succinate, which some-

what stimulated respiration; it was now limited by the

absence of ADP (energy acceptor state 2). Addition of

0.1 mM ADP increased respiration rate to a maximum

level (state3); however, within minutes, O

consumption

decreased

to levels prior to the addition of ADP as ADP

was depleted upon phosphorylation to ATP (state 4).

The maximum rate of O

consumption was

achieved

after the addition of the uncoupler of respiration and

phosphorylation (FCCP). Blocking of respiration was

achieved by adding 1 mM potassium cyanide (KCN), the

rate of respiration in its presence was subtracted from

all previously obtained values [22].

The data shown in Fig.4,a, f, andg, demonstrate

a change in the respiration rate when 5 mM glucose

VYSSOKIKH et al.306

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 5. Hydrogen peroxide production by skeletal muscle mitochondria of young healthy volunteers (HV, n=10), young (YP,

n=8), middle-aged (MP, n=20), and elderly (EP, n=42) patients. a)Products in the presence of succinate; b)production in the

presence of succinate upon activation of mitochondrial hexokinase by substrates of the enzymatic reaction. Medians and in-

terquartile ranges are presented; *p<0.05.

is added to mitochondria in state4 in the presence of

succinate after the depletion of ADP. At the same time,

in fact, state 3 continues and respiration occurs at al-

most maximum speed, as if the added ADP had not

been exhausted, which indicates its constant level in

the microcompartment due to the hexokinase reaction

at a saturating glucose concentration and access to mi-

tochondrial ATP, which fully confirms the previously

obtained S. Bessman and D. Wilson data [7,39]. Thus, it

can be argued that hexokinase bound on the surface of

mitochondria retained its functional conjugacy within

the microcompartment during mitochondrial fraction-

ation, i.e., after isolation mitochondria were a highly

coupled (Fig.4,g-i).

It was shown that substrate respiration in state2

significantly decreases, both with activation of com-

plex  I and complex  II (Fig.  4, b and c) for groups of

middle-aged and elderly patients in comparison not

only with healthy volunteers, but also with young pa-

tients. At the same time, stimulated respiration (ADP

or FCCP) did not differ between healthy volunteers and

young patients, but showed a pronounced (multiple)

age-related decrease: for the “MP” and “EP” groups

compared with healthy volunteers and for “EP” com-

pared with young patients (Fig.  4, d and g). The effect

of continued state 3 (see above) when measuring the

respiratory rate in the presence of glucose disappeared

with age, and for the “EP” group the differences were

significant in relation to all other groups, including

middle-aged patients (Fig.4f).

Peroxide production by mitochondria. It has

been shown that the generation of peroxide by mito-

chondria, energized by succinate, tends to increase

even in young patients compared to healthy volun-

teers of the same age (Fig. 5a), however, a significant

increase in this indicator was observed with increas-

ing age of patients, which correlated with the degree

of dissociation of hexokinase with the mitochondrial

membrane (Fig.2, Table2) and a decrease in the rate

of phosphorylating respiration (Fig. 3, Fig. S1c and

Table2). Confirming the data previously obtained by us

and other researchers [15, 40-46], we found a signifi-

cant decrease in the generation of hydrogen peroxide

by mitochondria upon stimulation of respiration in the

presence of the substrate of the hexokinase reaction

(p < 0.05 for “HV” and “YP” in Fig.5, a and b, Fig.S1e)

and revealed the absence of significant changes for the

“MP” and “EP” groups. In addition, it turned out that

for young people, regardless of clinical status, the rate

of peroxide production significantly decreases in the

presence of glucose, i.e., upon activation of the ATP/

ADP shuttle in the microcompartment hexokinase and

the outer membrane of mitochondria, while in middle

and old age, peroxide production with the addition of

a hexokinase reaction substrate remains high (as in

the presence of succinate) (Fig.5, a and b, Fig.S2).

Cardiolipin content in mitochondria. We have

previously shown the importance of maintaining the

intact structure and total content of the mitochondrial

lipid cardiolipin, and also demonstrated that cardiolipin

is present in the mitochondrial proteolipid complexes,

including hexokinase associated with VDAC1 [10, 47].

In this study, significant differences were identified

in the content of cardiolipin in the mitochondria of

skeletal muscles of elderly patients compared with a

group of healthy volunteers (p < 0.01) and a group of

young patients (p < 0.05) (Fig. 6b), while for monoly-

socardiolipin, significant differences were observed

between the following groups: middle-aged patients

in relation to healthy volunteers (p < 0.01) and young

patients (p < 0.05); elderly patients and healthy volun-

teers (p < 0.001); elderly patients and young patients

(p < 0.01). Thus, a decrease in the content of mitochon-

drial cardiolipin and accumulation of monolysocardi-

olipin does not occur in young patients in relatively

healthy people, but is observed with increasing age of

patients (Fig. 6), and this correlates with the produc-

tion of mROS (Table2).

MITOCHONDRIAL HEXOKINASE AND ROS PRODUCTION 307

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Table 2. Correlation (Spearman correlation coefficient) of indicators characterizing mitochondrial functions in

human skeletal muscles in various groups

Groups VH (n=10) YP (n=8) MP (n=20) EP (n=42)

HK bound/age no

r = –0.809

p = 0.015

r = –0.953

p = 0.0012

r = –0.866

p = 0.0002

Respiration (succ.+ADP)/age

r = –0.814

p = 0.004

r = –0.88

p = 3.2e-7

r = –0.514

p = 3.6e-4

Respiration (succ.+ ADP+gluc.)/age

r = –0.843

p = 0.002

r = –0.8

p = 0.017

r = –0.917

p = 0.001

r = –0.746

p = 0.002

Respiration (succ.+ADP+gluc.)/HK bound.

r = 0.793

p = 0.006

r = 0.921

p = 0.001

r = 0.836

p = 4.5e-6

r = 0.824

p = 6.37e-12

production (succ.)/age

r = 0.87

p = 0.01

r = 0.77

p = 0.025

r = 0.872

p = 5.5e-7

r = 0.555

p = 9.57e-5

production (succ.)/HK bound

r = –0.761

p = 0.037

r = –0.887

p = 1.85e-7

r = –0.556

p = 8.92e-5

production (succ.)/respiration (succ.+ADP+gluc.)

r = –0.703

p = 0.023

r = –0.762

p = 9.34e-5

r = –0.477

p = 0.001

production (succ.+ADP+gluc.)/respiration

(succ.+ADP+gluc.)

r = –0.765

p = 0.01

r = –0.791

p = 3.25e-5

r = –0.766

p = 1.35e-9

production (succ.+ADP+gluc.)/HK bound no no

r = –0.932

p = 2.32e-9

r = –0.813

p = 2.07e-11

production (succ.+ADP+gluc.)/age

r = 0.94

p = 5.21e-5

r = 0.882

p = 0.004

r = 0.925

p = 5.67e-9

r = 0.674

p = 5.22e-7

production (succ.+ADP+gluc.)/respiration (succ.+ADP)

r = –0.754

p = 0.012

r = –0.854

p = 1.7e-6

HK total/age no no no

r = –0.361

p = 0.016

Cardiolipin content/respiration (succ.+ADP+gluc.) no no no

r = 0.622

p =6.7e-06

Cardiolipin content /HK bounded no no no

r = 0.841

p =9.53e-13

Cardiolipin content/H

production (succ.) no no no

r = –0.521

p = 0.00029

Cardiolipin content/H

production (succ.+ADP+gluc.)

r = –0.802

p = 0.005

no no

r = –0.612

p =1.03e-05

MLCL content/ respiration (succ.+ADP+gluc.) no

r = –0.71

p = 0.048

r = –0.594

p = 2.13e-05

MLCL content/HK bound no

r = –0.805

p = 0.016

r = –0.718

p = 3.99e-08

MLCL content/H

production (succ.) no no no

r = 0.432

p = 0.003

VYSSOKIKH et al.308

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Table 2 (cont.)

Groups VH (n=10) YP (n=8) MP (n=20) EP (n=42)

MLCL content/H

production (succ.+ADP+gluc.) no no no

r = 0.544

p = 0.00014

Respiratory control (FCCP)/age no no no

r = –0.874

p = 0.0002

Respiration (succ.+ADP)/BMI no

r = 0.719

p = 0.044

no no

HOMA-IR/HK bound no no no no

HOMA-IR/respiration (succ.+ADP) no no no no

HOMA-IR/age

r = 0.94

p = 5e-5

r = 0.882

p = 0.004

r = 0.925

p = 0.0001

r = 0.674

p = 2e-5

HOMA-IR/BMI no no

r = 0.453

p = 0.045

r = 0.472

p = 0.001

HOMA-IR/respiration (succ.+ADP+gluc.) no no

r = 0.448

p = 0.047

Fig. 6. Cardiolipin content in skeletal muscle mitochondria of young healthy volunteers (HV), young (YP), middle-aged (MP), and

elderly (EP) patients, for all groups n=8. a)Representative thin-layer chromatogram: 1-3)young patients, 4-6)elderly patients.

FFA,free fatty acids; CL and MLCL,cardiolipin and monolysocardiolipin; FA,phosphatidic acid; PE,phosphatidylethanolamine;

PI,phosphatidylinositol; PS, phosphatidylserine; PC and LPC, phosphatidylcholine and lysophosphatidylcholine. b and c)Aver-

aged values of the content of cardiolipin and monolysocardiolipin, normalized to the content of phosphatidylcholine.

DISCUSSION

The study examined the influence of physical inac-

tivity/chronic inflammation in young people, as well as

age in patients with arthrosis of the knee/hip joint on

the regulation of mROS production in human skeletal

muscle. For the first time, an association of an age-de-

pendent increase in the production of hydrogen per-

oxide by mitochondria with a decrease in the propor-

tion of hexokinase-2 associated with mitochondria was

shown for human muscle tissue. This is confirmed by

data on an age-dependent change in the distribution of

hexokinase-2 between the mitochondrial and cytosolic

fractions and a decrease in the rate of coupled respi-

ration of isolated mitochondria and respiration upon

stimulation with glucose– the substrate of hexokinase.

These observations are in good agreement and com-

plement our previous data on the production of mROS

by tissues of long- and short-lived rodents[15].

It can be assumed that the age-dependent de-

crease in the efficiency of coupled respiration of skel-

etal muscle mitochondria that we described (Fig.4d) is

associated with an increase in energy deficiency in ag-

ing muscle, which was previously shown in studies on

rodents and primary human myoblasts (for a review,

see [48, 49]). A pronounced age-related decrease in the

sensitivity of isolated mitochondria to ADP (Fig. 4d)

confirmed the effect of a decrease in the ADP-induced

decrease in peroxide production in permeabilized

muscle fibers of elderly people compared to young

MITOCHONDRIAL HEXOKINASE AND ROS PRODUCTION 309

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

people shown by G. Holloway and colleagues [50].

At the same time, the lack of effect of the addition of

hexokinase on respiration and peroxide generation

in the cited work can be explained by the fact that,

firstly, to maintain the microcompartment in an intact

state during mitochondrial fractionation, the presence

of Mg

ions in the medium is necessary; secondly,

when isolating mitochondria, reassociation hexoki-

nase with the outer membrane does not undergo mir-

rored dissociation and requires special conditions;

thirdly, the added enzyme is approximately ten times

less effective, in comparison with the adsorbed one, in

stimulating the respiration of ADP, formed during the

phosphorylation of glucose [39, 51].

Pathological conditions of skeletal muscles caused

by oxidative stress are especially pronounced during

aging, leading to apoptosis, atrophy, and muscle dys-

function [52-54]. Increasing evidence in the literature

points to a link between oxidative stress caused by ex-

cess mROS production by hyperpolarized mitochondria,

and hyperglycemia [13, 15, 44, 55]. Our data confirm

this observation and, in part, explain the mechanisms

responsible for these changes [52]. Hexokinase  II, un-

like other isozymes of this enzyme, has a second (non-

catalytic) glucose binding site. Occupation of this site

caused by hyperglycemia blocks chaperone binding

to it HSP7C (Heat shock cognate 71, encoded by the

HSPA8 gene), inducing proteolytic degradation of hex-

okinase [44]. This leads to an increase in the intracel-

lular content of hexokinase  II and the accumulation of

glucose-6-phosphate, which has a solubilizing effect

and causes desorption of hexokinase from the surface

of mitochondria. Apparently, these events underlie

the age-dependent destruction of the outer membrane

microcompartment, including hexokinase  II, observed

by us and other authors [14, 15], which leads not only

to an increase in membrane potential, increased pro-

duction of mROS and oxidative stress, but also may

induce apoptosis by binding the proapoptotic protein

Bax to VDAC1 [56].

We found an age-correlated decrease in cardio-

lipin content in skeletal muscle mitochondria (Fig.  5,

Table  2). It is known that ADP/ATP translocase1 (ANT),

which is part of the microcompartment and local-

ized in the inner membrane, is part of the proteolipid

complex of mitochondrial contact sites [57], which in-

cludes 3 molecules of tightly bound cardiolipin; in this

case, the oxidation of cardiolipin leads to disruption of

the structure of this complex [58,  59]. It was previous-

ly shown that disruption of ANT conformations leads

to the destruction of its complex with VDAC1 and mi-

tochondrial contact sites [60]. It can be assumed that

the age-dependent decrease in cardiolipin content in

human skeletal muscle mitochondria that we discov-

ered, which correlates with the amount of bound hex-

okinase, the production of mROS and phosphorylating

respiration, is part of the general mechanism of im-

paired soft depolarization of skeletal muscle mitochon-

dria during aging. This assumption opens up prospects

for further research into the mechanisms underlying

age-dependent disruption of the structure of mitochon-

drial contact sites, which are associated with remod-

eling cardiolipin during oxidative stress. The study

of these mechanisms seems promising for the search

for pharmacological/physiological approaches to the

correction of age-related skeletal muscle pathologies

caused by disturbances in glucose oxidation and mROS

production.

Supplementary information. The online version

contains supplementary material available at https://

doi.org/10.1134/S0006297924020093.

Contributions. M.Yu.V., A.Yu.E., D.V.P., and V.P.S.

concept and management of the work; M.Yu.V., M.A.V.,

V.V.Ph., Ya.R.B., M.V.M., O.A.G., T.F.V., N.S.K., and L.A.M.

conducting experiments; M.Yu.V., M.A.V., A.Yu.E., D.V.P.,

and V.P.S. discussion of the research results; M.Yu.V.,

A.Yu.E., and D.V.P. text writing; M.Yu.V., A.Yu.E., and

D.V.P. editing the text of the article.

Funding. The work was supported by the Russian

Science Foundation (grant no.21-15-00405, study with

healthy volunteers, experiments with mitochondrial

respiration) and by the State Assignment of Lomonosov

Moscow State University (study with patients, clinical

and histological studies).

Ethics declarations. All studies were conducted

in accordance with the principles of biomedical eth-

ics as outlined in the 1964 Declaration of Helsinki and

its later amendments. Each participant in the study

provided a voluntary written informed consent after

receiving an explanation of the potential risks and

benefits, as well as the nature of the upcoming study.

The authors of this work declare that they have no

conflicts of interest.

REFERENCES

1. Harman, D. (1972) The biologic clock: the mitochon-

dria?, J. Am. Geriatr. Soc., 20, 145-147, doi: 10.1111/

j.1532-5415.1972.tb00787.x.

2. Anderson, C.M., Hu,J., Barnes, R.M., Heidt, A.B., Cor-

nelissen,I., and Black, B.L. (2015) Myocyte enhancer

factor 2C function in skeletal muscle is required for

normal growth and glucose metabolism in mice, Skel-

et. Muscle, 5, 7, doi:10.1186/s13395-015-0031-0.

3. Hargreaves,M. (2004) Muscle glycogen and metabolic

regulation, Proc. Nutr. Soc., 63, 217-220, doi: 10.1079/

PNS2004344.

4. Fealy, C. E., Grevendonk, L., Hoeks,J., and Hesselink,

M.K.C. (2021) Skeletal muscle mitochondrial network

dynamics in metabolic disorders and aging, Trends

VYSSOKIKH et al.310

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Mol. Med., 27, 1033-1044, doi: 10.1016/j.molmed.

2021.07.013.

5. Vogt, C., Yki-Jarvinen, H., Iozzo, P., Pipek, R., Pender-

grass,M., Koval,J., Ardehali,H., Printz,R., Granner,D.,

Defronzo,R., and Mandarino,L. (1998) Effects of insu-

lin on subcellular localization of hexokinase II in hu-

man skeletal muscle in vivo, J.Clin. Endocrinol. Metab.,

83, 230-234, doi:10.1210/jcem.83.1.4476.

6. Wilson, J. E. (2003) Isozymes of mammalian hexoki-

nase: structure, subcellular localization and metabol-

ic function, J.Exp. Biol., 206, 2049-2057, doi: 10.1242/

jeb.00241.

7. Gots, R.E., Gorin, F. A., and Bessman, S. P. (1972) Ki-

netic enhancement of bound hexokinase activity by

mitochondrial respiration, Biochem. Biophys. Res.

Commun., 49, 1249-1255, doi: 10.1016/0006-291x(72)

90602-x.

8. Sui, D., and Wilson, J. E. (1997) Structural determi-

nants for the intracellular localization of the isozymes

of mammalian hexokinase: intracellular localization

of fusion constructs incorporating structural elements

from the hexokinase isozymes and the green fluores-

cent protein, Arch. Biochem. Biophys., 345, 111-125,

doi:10.1006/abbi.1997.0241.

9. Brdiczka,D., Knoll,G., Riesinger,I., Weiler,U., Klug,G.,

Benz, R., and Krause, J. (1986) Microcompartmen-

tation at the mitochondrial surface: its function in

metabolic regulation, Adv. Exp. Med. Biol., 194, 55-69,

doi:10.1007/978-1-4684-5107-8_5.

10. Vyssokikh, M., and Brdiczka, D. (2004) VDAC and pe-

ripheral channelling complexes in health and disease,

Mol. Cell Biochem., 256-257, 117-126, doi: 10.1023/

b:mcbi.0000009863.69249.d9.

11. Cadenas, E., Boveris, A., Ragan, C. I., and Stoppani,

A.O. (1977) Production of superoxide radicals and hy-

drogen peroxide by NADH-ubiquinone reductase and

ubiquinol-cytochrome c reductase from beef-heart

mitochondria, Arch. Biochem. Biophys., 180, 248-257,

doi:10.1016/0003-9861(77)90035-2.

12. Morse, P.T., Wan,J., Bell,J., Lee,I., Goebel, D.J., Malek,

M. H., Sanderson, T. H., and Huttemann, M. (2022)

Sometimes less is more: inhibitory infrared light

during early reperfusion calms hyperactive mitochon-

dria and suppresses reperfusion injury, Biochem. Soc.

Trans., 50, 1377-1388, doi:10.1042/BST20220446.

13. Korshunov, S. S., Skulachev, V. P., and Starkov, A. A.

(1997) High protonic potential actuates a mecha-

nism of production of reactive oxygen species in

mitochondria, FEBS Lett., 416, 15-18, doi: 10.1016/

s0014-5793(97)01159-9.

14. Da-Silva, W. S., Gomez-Puyou, A., de Gomez-Puyou,

M.T., Moreno-Sanchez,R., De Felice, F.G., de Meis,L.,

Oliveira, M. F., and Galina, A. (2004) Mitochondrial

bound hexokinase activity as a preventive antioxidant

defense: steady-state ADP formation as a regulatory

mechanism of membrane potential and reactive oxy-

gen species generation in mitochondria, J.Biol. Chem.,

279, 39846-39855, doi:10.1074/jbc.M403835200.

15. Vyssokikh, M.Y., Holtze,S., Averina, O.A., Lyamzaev,

K.G., Panteleeva, A. A., Marey, M.V., Zinovkin, R. A.,

Severin, F.F., Skulachev, M.V., Fasel,N., Hildebrandt,

T. B., and Skulachev, V. P. (2020) Mild depolarization

of the inner mitochondrial membrane is a crucial

component of an anti-aging program, Proc. Natl.

Acad. Sci. USA, 117, 6491-6501, doi: 10.1073/pnas.

1916414117.

16. Callahan, D.M., Miller, M.S., Sweeny, A.P., Tourville,

T. W., Slauterbeck, J. R., Savage, P.D., Maugan, D.W.,

Ades, P.A., Beynnon, B.D., and Toth, M.J. (2014) Mus-

cle disuse alters skeletal muscle contractile function

at the molecular and cellular levels in older adult

humans in a sex-specific manner, J. Physiol., 592,

4555-4573, doi:10.1113/jphysiol.2014.279034.

17. Callahan, D.M., Tourville, T.W., Miller, M.S., Hackett,

S.B., Sharma,H., Cruickshank, N.C., Slauterbeck, J.R.,

Savage, P. D., Ades, P. A., Maughan, D. W., Beynnon,

B.D., and Toth, M.J. (2015) Chronic disuse and skeletal

muscle structure in older adults: sex-specific differ-

ences and relationships to contractile function, Am.J.

Physiol. Cell Physiol., 308, C932-C943, doi: 10.1152/

ajpcell.00014.2015.

18. Miller, M.S., Callahan, D.M., Tourville, T.W., Slauter-

beck, J.R., Kaplan,A., Fiske, B.R., Savage, P.D., Ades,

P.A., Beynnon, B.D., and Toth, M.J. (2017) Moderate-in-

tensity resistance exercise alters skeletal muscle mo-

lecular and cellular structure and function in inactive

older adults with knee osteoarthritis, J.Appl. Physiol.,

122, 775-787, doi:10.1152/japplphysiol.00830.2016.

19. Suetta, C., Aagaard, P., Magnusson, S. P., Andersen,

L. L., Sipila, S., Rosted, A., Jakobsen, A. K., Duus, B.,

and Kjaer,M. (2007) Muscle size, neuromuscular acti-

vation, and rapid force characteristics in elderly men

and women: effects of unilateral long-term disuse due

to hip-osteoarthritis, J. Appl. Physiol., 102, 942-948,

doi:10.1152/japplphysiol.00067.2006.

20. Ware, J., Jr., Kosinski, M., and Keller, S. D. (1996)

A 12-Item Short-Form Health Survey: construction

of scales and preliminary tests of reliability and va-

lidity, Med. Care, 34, 220-233, doi: 10.1097/00005650-

199603000-00003.

21. Popov, D. V., Makhnovskii, P. A., Shagimardanova,

E.I., Gazizova, G.R., Lysenko, E.A., Gusev, O.A., and

Vinogradova, O. L. (2019) Contractile activity-specific

transcriptome response to acute endurance exercise

and training in human skeletal muscle, Am. J. Physi-

ol. Endocrinol. Metab., 316, E605-E614, doi: 10.1152/

ajpendo.00449.2018.

22. Skulachev, V.P., Bogachev, A.V., and Kasparinsky, F.O.

(2013) Principles of Bioenergetics, Springer-Verlag

Berlin Heidelberg, doi:10.1007/978-3-642-33430-6.

23. Gnaiger, E., Kuznetsov, A. V., Schneeberger, S., Seiler,R.,

Brandacher, G., Steurer, W., and Margreiter, R.

MITOCHONDRIAL HEXOKINASE AND ROS PRODUCTION 311

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

(2000) Mitochondria in the Cold, in Life in the Cold

(Heldmaier, G., Klingenspor, M., Eds.), Springer, Ber-

lin, Heidelberg, pp. 431-442, doi: 10.1007/978-3-662-

04162-8_45.

24. Zhou,M., Diwu,Z., Panchuk-Voloshina,N., and Haug-

land, R. P. (1997) A stable nonfluorescent derivative

of resorufin for the fluorometric determination of

trace hydrogen peroxide: applications in detecting

the activity of phagocyte NADPH oxidase and other

oxidases, Anal. Biochem., 253, 162-168, doi: 10.1006/

abio.1997.2391.

25. Shabalina, I. G., Vyssokikh, M. Y., Gibanova, N.,

Csikasz, R. I., Edgar, D., Hallden-Waldemarson, A.,

Rozhdestvenskaya,Z., Bakeeva, L.E., Vays, V. B., Pus-

tovidko, A.V., Skulachev, M.V., Cannon,B., Skulachev,

V.P., and Nedergaard,J. (2017) Improved health-span

and lifespan in mtDNA mutator mice treated with the

mitochondrially targeted antioxidant SkQ1, Aging

(Albany NY), 9, 315-339, doi:10.18632/aging.101174.

26. Antonenko, Y. N., Avetisyan, A. V., Bakeeva, L. E.,

Chernyak, B.V., Chertkov, V.A., Domnina, L.V., Ivano-

va, O.Y., Izyumov, D.S., Khailova, L.S., Klishin, S.S.,

Korshunova, G. A., Lyamzaev, K. G., Muntyan, M. S.,

Nepryakhina, O. K., Pashkovskaya, A. A., Pletjushki-

na, O.Y., Pustovidko, A.V., Roginsky, V.A., Rokitskaya,

T. I., Ruuge, E. K., Saprunova, V. B., Severina, I. I., Si-

monyan, R.A., Skulachev, I.V., Skulachev, M.V., Sum-

batyan, N.V., Sviryaeva, I.V., Tashlitsky, V.N., Vassiliev,

J.M., Vyssokikh, M.Yu., Yaguzhinsky, L.S., Zamyatnin,

A.A.,Jr., and Skulachev, V.P. (2008) Mitochondria-tar-

geted plastoquinone derivatives as tools to interrupt

execution of the aging program. 1. Cationic plasto-

quinone derivatives: synthesis and in vitro studies,

Biochemistry (Moscow), 73, 1273-1287, doi: 10.1134/

s0006297908120018.

27. Shabalina, I.G., Vrbacky,M., Pecinova,A., Kalinovich,

A. V., Drahota,Z., Houstek, J., Mracek,T., Cannon, B.,

and Nedergaard, J. (2014) ROS production in brown

adipose tissue mitochondria: the question of UCP1-

dependence, Biochim. Biophys. Acta, 1837, 2017-2030,

doi:10.1016/j.bbabio.2014.04.005.

28. Scheer, W.D., Lehmann, H.P., and Beeler, M.F. (1978)

An improved assay for hexokinase activity in hu-

man tissue homogenates, Anal. Biochem., 91, 451-463,

doi:10.1016/0003-2697(78)90531-6.

29. Laemmli, U.K. (1970) Cleavage of structural proteins

during the assembly of the head of bacteriophage T4,

Nature, 227, 680-685, doi:10.1038/227680a0.

30. Buscher, T., Luh, E., and Pette, D. (1964) Simple and

Compound Optical Tests with Pyridine Nucleotides,

in Hoppe-Seyler/Thierfelder, Handbook of Physiological

and Pathological Chemical Analysis [in German], VI/A,

292-339, doi:10.1007/978-3-662-11689-0_6.

31. Bligh, E. G., and Dyer, W. J. (1959) A rapid method of

total lipid extraction and purification, Can.J. Biochem.

Physiol., 37, 911-917, doi:10.1139/o59-099.

32. Pinault,M., Guimaraes,C., Dumas, J.-F., Servais,S., Che-

valier,S., Besson,P., and Goupille,C. (2020) A 1D high

performance thin layer chromatography method val-

idated to quantify phospholipids including cardiolip-

in and monolysocardiolipin from biological samples,

Eur. J. Lipid Sci. Technol., 122, 1900240, doi: 10.1002/

ejlt.201900240.

33. Kruszynska, Y. T., Mulford, M. I., Baloga, J., Yu, J. G.,

and Olefsky, J. M. (1998) Regulation of skeletal mus-

cle hexokinase II by insulin in nondiabetic and

NIDDM subjects, Diabetes, 47, 1107-1113, doi:10.2337/

diabetes.47.7.1107.

34. Vestergaard,H., Bjorbaek,C., Hansen,T., Larsen, F.S.,

Granner, D. K., and Pedersen, O. (1995) Impaired ac-

tivity and gene expression of hexokinase II in mus-

cle from non-insulin-dependent diabetes mellitus

patients, J. Clin. Invest., 96, 2639-2645, doi: 10.1172/

JCI118329.

35. Ritov, V. B., and Kelley, D. E. (2001) Hexokinase iso-

zyme distribution in human skeletal muscle, Diabetes,

50, 1253-1262, doi:10.2337/diabetes.50.6.1253.

36. Mandarino, L. J., Printz, R. L., Cusi, K. A., Kinching-

ton, P., O’Doherty, R. M., Osawa, H., Sewell, C., Con-

soli, A., Granner, D. K., and DeFronzo, R. A. (1995)

Regulation of hexokinase II and glycogen synthase

mRNA, protein, and activity in human muscle, Am.J.

Physiol., 269, E701-E708, doi: 10.1152/ajpendo.1995.

269.4.E701.

37. Wilson, J.E. (1995) Hexokinases, Rev. Physiol. Biochem.

Pharmacol., 126, 65-198, doi:10.1007/bfb0049776.

38. Cunha, T. F., Vieira, J. S., Santos, J. B., Coelho, M. A.,

Brum, P.C., and Gabriel-Costa,D. (2022) Lactate mod-

ulates cardiac gene expression in mice during acute

physical exercise, Braz. J. Med. Biol. Res., 55, e11820,

doi:10.1590/1414-431X2022e11820.

39. BeltrandelRio, H., and Wilson, J. E. (1991) Hexoki-

nase of rat brain mitochondria: relative importance

of adenylate kinase and oxidative phosphorylation

as sources of substrate ATP, and interaction with in-

tramitochondrial compartments of ATP and ADP,

Arch. Biochem. Biophys., 286, 183-194, doi: 10.1016/

0003-9861(91)90026-f.

40. Saraiva, L.M., Seixas da Silva, G.S., Galina,A., da-Sil-

va, W. S., Klein, W. L., Ferreira, S. T., and De Felice,

F.G. (2010) Amyloid-beta triggers the release of neu-

ronal hexokinase 1 from mitochondria, PLoS One, 5,

e15230, doi:10.1371/journal.pone.0015230.

41. De-Souza-Ferreira, E., Rios-Neto, I. M., Martins, E. L.,

and Galina, A. (2019) Mitochondria-coupled glucose

phosphorylation develops after birth to modulate

release and calcium handling in rat brain, J.Neu-

rochem., 149, 624-640, doi:10.1111/jnc.14705.

42. Silva-Rodrigues, T., de-Souza-Ferreira, E., Machado,

C. M., Cabral-Braga, B., Rodrigues-Ferreira, C., and

Galina,A. (2020) Hyperglycemia in a type 1 Diabetes

Mellitus model causes a shift in mitochondria cou-

VYSSOKIKH et al.312

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

pled-glucose phosphorylation and redox metabolism

in rat brain, Free Radic. Biol. Med., 160, 796-806,

doi:10.1016/j.freeradbiomed.2020.09.017.

43. Tan, V.P., Smith, J.M., Tu,M., Yu, J.D., Ding, E.Y., and

Miyamoto, S. (2019) Dissociation of mitochondrial

HK-II elicits mitophagy and confers cardioprotection

against ischemia, Cell Death Dis, 10, 730, doi:10.1038/

s41419-019-1965-7.

44. Rabbani,N., Xue,M., and Thornalley, P.J. (2022) Hex-

okinase-2-linked glycolytic overload and unscheduled

glycolysis-driver of insulin resistance and develop-

ment of vascular complications of diabetes, Int.J. Mol.

Sci., 23, 2165, doi:10.3390/ijms23042165.

45. Bryson, J.M., Coy, P.E., Gottlob,K., Hay,N., and Robey,

R. B. (2002) Increased hexokinase activity, of either

ectopic or endogenous origin, protects renal epithe-

lial cells against acute oxidant-induced cell death,

J. Biol. Chem., 277, 11392-11400, doi: 10.1074/jbc.

M110927200.

46. Ahmad, A., Ahmad, S., Schneider, B. K., Allen, C. B.,

Chang, L. Y., and White, C. W. (2002) Elevated ex-

pression of hexokinase II protects human lung epi-

thelial-like A549 cells against oxidative injury, Am. J.

Physiol. Lung Cell. Mol. Physiol., 283, L573-L584,

doi:10.1152/ajplung.00410.2001.

47. Shilovsky, G. A., Putyatina, T. S., Ashapkin, V. V.,

Yamskova, O. V., Lyubetsky, V. A., Sorokina, E. V.,

Shram, S.I., Markov, A.V., and Vyssokikh, M.Y. (2019)

Biological diversity and remodeling of cardiolip-

in in oxidative stress and age-related pathologies,

Biochemistry (Moscow), 84, 1469-1483, doi: 10.1134/

S000629791912006X.

48. Bratic,I., and Trifunovic,A. (2010) Mitochondrial en-

ergy metabolism and ageing, Biochim. Biophys. Acta,

1797, 961-967, doi:10.1016/j.bbabio.2010.01.004.

49. Pedersen, Z. O., Pedersen, B. S., Larsen, S., and Dys-

gaard, T. (2023) A scoping review investigating the

“gene-dosage theory” of mitochondrial DNA in the

healthy skeletal muscle, Int. J. Mol. Sci., 24, 8154,

doi:10.3390/ijms24098154.

50. Holloway, G. P., Holwerda, A. M., Miotto, P.M., Dirks,

M.L., Verdijk, L.B., and van Loon, L.J.C. (2018) Age-as-

sociated impairments in mitochondrial ADP sensi-

tivity contribute to redox stress in senescent human

skeletal muscle, Cell Rep., 22, 2837-2848, doi:10.1016/

j.celrep.2018.02.069.

51. BeltrandelRio, H., and Wilson, J. E. (1992) Coordinat-

ed regulation of cerebral glycolytic and oxidative

metabolism, mediated by mitochondrially bound

hexokinase dependent on intramitochondrially gen-

erated ATP, Arch. Biochem. Biophys., 296, 667-677,

doi:10.1016/0003-9861(92)90625-7.

52. Vincent, A. M., Olzmann, J. A., Brownlee, M., Sivitz,

W. I., and Russell, J. W. (2004) Uncoupling proteins

prevent glucose-induced neuronal oxidative stress

and programmed cell death, Diabetes, 53, 726-734,

doi:10.2337/diabetes.53.3.726.

53. Bellanti, F., Lo Buglio, A., and Vendemiale, G. (2022)

Muscle delivery of mitochondria-targeted drugs

for the treatment of sarcopenia: rationale and per-

spectives, Pharmaceutics, 14, 2588, doi: 10.3390/

pharmaceutics14122588.

54. Russell, J.W., Golovoy,D., Vincent, A.M., Mahendru,P.,

Olzmann, J.A., Mentzer,A., and Feldman, E.L. (2002)

High glucose-induced oxidative stress and mitochon-

drial dysfunction in neurons, FASEBJ., 16, 1738-1748,

doi:10.1096/fj.01-1027com.

55. Green,K., Brand, M.D., and Murphy, M.P. (2004) Pre-

vention of mitochondrial oxidative damage as a ther-

apeutic strategy in diabetes, Diabetes, 53, S110-S118,

doi:10.2337/diabetes.53.2007.s110.

56. Vyssokikh, M. Y., Zorova, L., Zorov, D., Heimlich, G.,

Jurgensmeier, J.J., and Brdiczka,D. (2002) Bax releases

cytochromec preferentially from a complex between

porin and adenine nucleotide translocator. Hexoki-

nase activity suppresses this effect, Mol. Biol. Rep.,

29, 93-96, doi:10.1023/a:1020383108620.

57. Beutner,G., Ruck,A., Riede,B., and Brdiczka,D. (1997)

Complexes between hexokinase, mitochondrial porin

and adenylate translocator in brain: regulation of hex-

okinase, oxidative phosphorylation and permeabil-

ity transition pore, Biochem. Soc. Trans., 25, 151-157,

doi:10.1042/bst0250151.

58. Kunji, E. R., Aleksandrova, A., King, M. S., Majd, H.,

Ashton, V.L., Cerson,E., Springett,R., Kibalchenko,M.,

Tavoulari,S., Crichton, P.G., and Ruprecht, J.J. (2016)

The transport mechanism of the mitochondrial ADP/

ATP carrier, Biochim. Biophys. Acta, 1863, 2379-2393,

doi:10.1016/j.bbamcr.2016.03.015.

59. Duncan, A. L., Ruprecht, J. J., Kunji, E.R.S., and Rob-

inson, A. J. (2018) Cardiolipin dynamics and bind-

ing to conserved residues in the mitochondrial ADP/

ATP carrier, Biochim. Biophys. Acta Biomembr., 1860,

1035-1045, doi:10.1016/j.bbamem.2018.01.017.

60. Allouche, M., Pertuiset, C., Robert, J. L., Martel, C.,

Veneziano, R., Henry, C., Sharaf el dein, O., Saint,N.,

Brenner, C., and Chopineau, J. (2012) ANT-VDAC1 in-

teraction is direct and depends on ANT isoform con-

formation in vitro, Biochem. Biophys. Res. Commun.,

429, 12-17, doi:10.1016/j.bbrc.2012.10.108.

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