biomolecules

Article Comparison of Effects of Metformin, Phenformin, and Inhibitors of Mitochondrial Complex I on Mitochondrial Permeability Transition and Ischemic Brain Injury

Kristina Skemiene, Evelina Rekuviene, Aiste Jekabsone , Paulius Cizas, Ramune Morkuniene and Vilmante Borutaite *

Neuroscience Institute, Lithuanian University of Health Sciences, LT-50161 Kaunas, Lithuania; [email protected] (K.S.); [email protected] (E.R.); [email protected] (A.J.); [email protected] (P.C.); [email protected] (R.M.) * Correspondence: [email protected]



Received: 18 August 2020; Accepted: 29 September 2020; Published: 1 October 2020


Abstract: Damage to cerebral mitochondria, particularly opening of mitochondrial permeability transition pore (MPTP), is a key mechanism of ischemic brain injury, therefore, modulation of MPTP may be a potential target for a neuroprotective strategy in ischemic brain pathologies. The aim of this study was to investigate whether biguanides—metformin and phenformin as well as other inhibitors of Complex I of the mitochondrial electron transfer system may protect against ischemia-induced cell death in brain slice cultures by suppressing MPTP, and whether the effects of these inhibitors depend on the age of animals. Experiments were performed on brain slice cultures prepared from 5–7-day (premature) and 2–3-month old (adult) rat brains. In premature brain slice cultures, simulated ischemia (hypoxia plus deoxyglucose) induced necrosis whereas in adult rat brain slice cultures necrosis was induced by hypoxia alone and was suppressed by deoxyglucose. Phenformin prevented necrosis induced by simulated ischemia in premature and hypoxia-induced—in adult brain slices, whereas metformin was protective in adult brain slices cultures. In premature brain slices, necrosis was also prevented by Complex I inhibitors rotenone and amobarbital and by MPTP inhibitor cyclosporine A. The latter two inhibitors were protective in adult brain slices as well. Short-term exposure of cultured neurons to phenformin, metformin and rotenone prevented ionomycin-induced MPTP opening in intact cells. The data suggest that, depending on the age, phenformin and metformin may protect the brain against ischemic damage possibly by suppressing MPTP via inhibition of mitochondrial Complex I.

Keywords: metformin; phenformin; brain ischemia; hypoxia; mitochondrial complex I; permeability transition

1. Introduction Ischemia-induced brain injury is serious, life-threatening pathology which may cause death or severe complications such as physical disability and cognitive impairment. The incidence of stroke is increasing and seeks about 15 million new strokes worldwide each year [1]. Even when patients survive acute stroke episodes, neurological consequences may cause disability which negatively affect the quality of patients life, and that becomes an important issue as well. On the other hand, hypoxia at birth may affect developing brains and this can also have a devastating effect on the growth and maturation of the central nervous system. Therefore, it is important to explore the possibilities not only to reduce stroke mortality, but also to ease the consequences of hypoxic/ischemic episodes for the

Biomolecules 2020, 10, 1400; doi:10.3390/biom10101400 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 1400 2 of 17 patients’ health and disability. Consequently, new therapies are constantly being sought and one of the research directions is a search for effective neuroprotective drugs. For this purpose, understanding of the molecular and cellular mechanisms of ischemia-induced injuries is crucial. Mitochondrial injury and opening of the mitochondrial permeability transition pore (MPTP) has been suggested as a key mechanism of irreversible damage during ischemia (for review see [2–5]. Oxygen and substrate deficiency acutely impairs oxidative phosphorylation, while prolonged ischemia results in irreversible uncoupling of oxidative phosphorylation, energy depletion, opening of MPTP and necrotic cell death [6,7]. In several studies a decade ago, it has been observed that reversible inhibition of Complex I of mitochondrial electron transfer system may exert cardioprotective effect against ischemic damage possibly via suppression of reverse electron transfer and inhibition of production of reactive oxygen species (ROS) [8,9]. There were also studies suggesting that Complex I activity may modulate MPTP opening [10]. Though the structure and regulation of MPTP is still the hot issue of the research [11–13], it has been demonstrated that MPTP suppression by cyclosporine A and inhibitors of mitochondrial Complex I decrease cell death in cardiomyocytes, U937 and KB cells [14,15]. Opposing data showed different sensitivity of MPTP to cyclosporine A and inhibitors of Complex I in various tissues [16]. It is still unclear whether MPTP in the brain can be modulated by the activity of Complex I and whether this exerts any neuroprotective effect during ischemia/reperfusion. Metformin is one of the well-known biguanide derivatives and is widely used as anti-diabetic drug decreasing hepatic glucose output by inhibiting gluconeogenesis [17]. Depletion of energy reserves in the liver activates AMP-dependent protein kinase (AMPK) [18,19] and inhibits adenylate cyclase [20], resulting in a decrease in blood glucose and an increased sensitivity to insulin. Besides this action, metformin has been suggested to activate AMPK signaling pathway [10] via mild inhibition of mitochondrial Complex I and to reduce tumorigenesis due to effects on AMPK activity and reactive oxygen species (ROS) formation [21]. It has also received attention as a possible neuroprotective agent. In clinical studies on patients with diabetes, the treatment with metformin has been shown to reduce the risk of stroke [22]. Also, there are data that therapy of metformin applied to diabetic patients improved prognosis and severity of neurological damages of acute ischemic stroke [23]. The molecular mechanisms of metformin action during stroke are unclear but may include inhibition of endogenous ROS production and oxidative stress [24,25], up-regulation of AMPK and down-regulation of expression of apoptotic proteins such as Bax and caspase 3 [26]. On the other hand, recent studies suggest that metformin can exert protective effects against cardiac ischemia/reperfusion-induced damages independently of AMPK activation but inhibiting Complex I and MPTP [10]. Whether similar mechanisms may operate in the brain and exert neuroprotective effects during brain ischemia is not investigated. Another derivative of biguanides, phenformin, is no longer used as anti-diabetic drug in clinical practice due to its toxicity during long-term treatment. It has been demonstrated that phenformin has a stronger inhibitory effect on mitochondrial Complex I than metformin possibly due to its hydrophobic properties and easier transport into mitochondria [19]. Phenformin has been also shown to inhibit complexes II and IV of the mitochondrial electron transfer system [27]. There is some evidence suggesting that sensitivity to ischemic damage may be different in developing and aging individuals. Studies with aged animals showed reduced cardiac mitochondrial Ca2+ handling [28], increased sensitivity to calcium overload [29] and increased susceptibility to Ca2+-induced MPTP opening, associated with an elevated release of cytochrome c and other mitochondrial proteins, compared with young animals [30,31]. An enhanced susceptibility to calcium-induced MPTP opening and cell death was also shown in aging rodent brains [32–34], and this may change the response of cerebral cells to hypoxia/ischemia. Consequently, effects of mitochondria-targeted compounds may also vary with the age of individuals. In this study, we sought to get deeper insight into the mechanism of neuroprotective action of biguanide derivatives and aimed to investigate whether metformin, phenformin, and inhibitors of Biomolecules 2020, 10, 1400 3 of 17 mitochondrial Complex I can prevent cell death by inhibiting opening of MPTP in ischemia-affected brain slice cultures from premature and adult rats.

2. Materials and Methods

2.1. Preparation and Cultivation of Organotypic Brain Slices All experimental procedures were reviewed and approved by the National Ethical Committee for Animal Care (Licenses No 0217 and No 0228) according to Directive 2010/63/EU of the European Parliament. The rats were maintained and handled at Lithuanian University of Health Sciences animal house in agreement with the Guide for the Care and Use of Laboratory Rats. Brain slices were prepared from the cerebral hemispheres of 5–7-days and 2–3-months old Wistar rats. The animals were anesthetized with inhalation of CO2, after cervical dislocation sculls were dissected, brains removed and placed in 4 ◦C PBS with 10 mM glucose. The cerebral hemispheres were separated from cerebella and meninges were removed. The brains were immobilized caudal end down and sectioned with the vibratome (Vibratome 1000, Technical Products International Inc., Saint Louis, MO, United States) in PBS and glucose-filled cooling bath at 1 ◦C. The brains were coronally sliced with low profile microtome blade (Leica Biosystems) into 300 µm thick slices with an amplitude of 2 mm and minimal advancing speed. The slices selected for culturing were collected from vibratome bath and placed on porous tissue culture inserts (BD-Falcon, 1 µm pores) in 6-well insert-adapted cell culture plates (BD-Falcon) at the air–liquid interface with medium. Medium for cultivating organotypic brain slices from 5–7 days old rats was Dubelco’s Modified Eagle Medium (DMEM) with Glutamax (Gibco) supplemented with 12% horse serum, 12% fetal bovine serum, Antibiotic-Antimycotic (10 units/mL-10 µg/mL) and HEPES (25 mM). Medium for cultivating organotypic brain slices from 2–3 months old rats was DMEM with Glutamax (Gibco) supplemented with 2.5% horse serum, 2.5% fetal bovine serum, Antibiotic-Antimycotic (10 units/mL-10 µg/mL) and HEPES (25 mM). The slices from 5–7 days old rats were cultivated for 1 week and slices from 2–3 months old rats for 3 days in the incubator (37 ◦C, humidified atmosphere, 5% CO2/95% air) before the start of treatments.

2.2. Preparation of Cultures of Cerebellar Granule Cells Cultures of cerebellar granule cells (CGC) were prepared from 7 to 8 days old Wistar rats as described in [35]. Cells were grown in vitro for 6–7 days at density 0.25 106 cells/cm2 in 24-well × plates in cell culture growth medium (DMEM Glutamax supplemented with 5% fetal bovine serum, 5% horse serum, 13 mM glucose, 20 mM KCl, and 1% penicillin/streptomycin) coated with 0.001% poly-l-Lysine, (Sigma-Aldrich). Cells were maintained at 37 ◦C in a humidified atmosphere containing of 5% CO2/95% air. To inhibit proliferation of glial cells CGC cultures were treated with 10 µM of cytosine β- D–arabinofuranoside (Ara-C) at 1 DIV. These cultures (called pure neuronal cultures) contained 97.2 0.4% neurons, 0.3 0.1% microglia, and 2.4 0.4% astrocytes. ± ± ± 2.3. Simulated Ischaemia Model After one week (slice cultures from 5–7 days old rats) or after 3 days (slice cultures from 2–3 months old rats) ex vivo brain slice cultures were subjected to 24 h simulated ischemia by placing them in a humidified chamber perfused by gas mixture of 2% O2/5% CO2/93% N2 in the presence/absence of 10 mM D-deoxyglucose (DOG).

2.4. Determination of Lactate Dehydrogenase Activity in the Incubation Medium For the evaluation of necrosis, activity of lactate dehydrogenase (LDH) released into the brain incubation medium was measured spectrophotometrically recording NADH oxidation rate at 340 nm wavelength. The incubation medium of the slices (control group and ischemic groups) were collected and centrifuged at 10,000 g for 20 min. Aliquots of the medium were added to the buffer containing × 0.1 M TRIS-HCl (pH 7.5), 1 mM potassium salt of pyruvic acid, 0.1 mM NADH. LDH release was Biomolecules 2020, 10, 1400 4 of 17 calculated from the rate of the decrease of absorbance and expressed as nmol NADH/min/mg brain slice tissue [36].

2.5. Evaluation of Cell Viability by Fluorescence Microscopy The viability of cells in organotypic brain slice cultures was assessed by propidium iodide (PI, 7 µM) staining using an inverted fluorescence microscope OLYMPUS IX71S1F-3 (Olympus Corporation, Tokyo, Japan), x20 objective. Pictures of at least 5 randomly selected fields per slice (2 slices per group of individual experiment) were taken. Pictures were analyzed by ImageJ freeware, version 1.49, and raw data expressed as the mean level of red fluorescence intensity. Neuronal viability in CGC cultures was assessed by staining with Hoechst-33342 (4 µg/mL) and PI (7 µM). Neurons were recognized according to characteristic shape and morphology by phase contrast microscopy. Cells with homogeneously Hoechst-33342 stained nuclei were classified as viable, with condensed/fragmented chromatin as apoptotic and PI-positive cells as necrotic. Cells were counted in at least 5 randomly selected microscopic fields in each well, 2 wells per treatment.

2.6. Isolation of Brain Mitochondria Brain mitochondria were isolated by differential centrifugation [37]. The brains were cut into small pieces and homogenized with a Teflon-glass homogenizer in the isolation buffer containing 222 mM manitol, 75 mM sucrose, 5 mM HEPES, 1mM EGTA (pH 7.4, 4 ◦C). Cytosolic and mitochondrial fractions were separated by differential centrifugation (5 min at 1000 g, then 10 min at 10,000 g). × × Total cytosolic and mitochondrial protein was measured by the Biuret method.

2.7. Measurement of Mitochondrial Respiration Mitochondrial respiration was measured with high-resolution respirometry OROBOROS Oxygraph-2k (Oroboros Instruments, Insbruck, Austria) at 37 ◦C in 2 mL respiration buffer containing 110 mM KCl, 10 mM Tris-HCl, 5 mM KH2PO4, 2.24 mM MgCl2, pH 7.2 and 0.25 mg/mL mitochondrial protein. Respiration assay started with addition of pyruvate/malate (1/1 mM) and mitochondria (0.25 mg/mL), this respiration represented proton leak-driven respiration (LEAK) [38]. Then the respiration buffer was supplemented with 2 mM ADP and ADP-stimulated phosphorylating respiration (OXPHOS) was registered with pyruvate/malate. Rates of oxygen consumption were expressed in pmol O2/s/mg mitochondrial protein.

2.8. Measurement of Mitochondrial Calcium Retention Capacity Calcium retention capacity (CRC) of brain mitochondria was determined fluorometrically (Fluorescence Spectrometer Perkin Elmer LS55) using 100 nM Calcium Green 5N (excitation at 507 nm, emission at 536 nm) in medium containing 200 mM sucrose, 10 mM Tris-HCl, 1 mM KH2PO4, 10 µM EGTA and 5 mM succinate, pH 7.4 at 37 ◦C as described in [39]. Isolated mitochondria (0.2 mg protein/mL) were incubated with rotenone (0.05 and 1 µM), amobarbital (2.5 mM), cyclosporine A (0.5 µM) for 2 min or phenformin (Phen, 0.1 and 1 mM), metformin (1 and 10 mM) for 5 min and then pulses of 1.67 µM CaCl2 were applied in 2 min intervals. Calcium ions were continually loaded until large increase of Calcium Green 5N fluorescence was observed which indicated the release of stored in mitochondria calcium due to opening of MPTP.

2.9. Assesment of MPTP Opening in Neuronal Cell Cultures. For the experiments, pure neuronal cell cultures (see Section 2.2) were pre-incubated 1 h with 50–100 µM phenformin, 2–3 mM metformin, 0.05–1 µM rotenone, or 3–5 µM cyclosporine A. After incubation, cells were washed and resuspended in modified Hank’s Balanced Salt Solution (HBSS) containing 10 mM HEPES, 92 mM L-glutamine and 100 µM succinate. Cells were loaded with 1 µM Calcein-AM (Invitrogen) for 30 min at 37 ◦C in the dark. Then 1 mM CoCl2 (ChemCruz) was added and Biomolecules 2020, 10, 1400 5 of 17 cells were incubated for another 15 min. Then cells were twice washed in warm HBSS buffer to remove residual dye and imaged with a fluorescence microscope. To induce MPTP opening, 1 µM ionomycin was added and changes in fluorescence were measured at 5 min intervals. Cells were imaged in at least five microscopic fields per well (2 wells per condition). Image analyses were performed using ImageJ software and results were expressed as fluorescence arbitrary units per cell.

2.10. Measurement of the Mitochondrial Electron Transfer System Complex I Activity The activity of mitochondrial electron transfer system Complex I was determined in brain mitochondria isolated from 5–7 days and 2–3 months aged rats spectrophotometrically at a wavelength of 340 nm following the oxidation of 100 µM NADH in the presence of 2 µg/mL antimycin, 2 mM sodium azide, 3 mg/mL bovine serum albumin (BSA), 60 µM coenzyme Q1 (CoQ1) and investigated biguanides—phenformin or metformin—in a medium containing 25 mM KH2PO4 (pH 7.4 at 37 ◦C) and 0.125 mg/mL freeze–thawed mitochondria. Complex I activity was calculated by substracting nonspecific NADH oxidation rate (obtained after rotenone supplement) from total NADH oxidation rate and expressed as nmol NADH/min/mg protein [40].

2.11. Statistical Analysis All data are expressed as means standard errors of at least three experiments on separate ± preparations of brain slice or CGC cultures. The graphs were created and statistical significance was evaluated by SigmaPlot v13 software. Data were tested for normality using Shapiro–Wilk test and statistically compared between experimental groups by One Way Anova. Data presented in Figure 2a and Figure 7 did not pass Shapiro–Wilk normality test, and were tested by One Way Anova using Fisher LSD test. A value of p < 0.05 was considered statistically significant.

3. Results

3.1. Effect of Phenformin and Metformin on Neuronal Viability in CGC Cultures Phenformin and metformin are known to inhibit mitochondrial Complex I, which if complete and prolonged may lead to neuronal death. To find the optimal concentrations that are not toxic to neurons over 24 h incubation, we investigated effects of phenformin (0.005–0.2 mM) and metformin (0.1–3 mM) on viability of neurons in CGC cultures. The numbers of viable, apoptotic and necrotic cells were counted and expressed as percentage of all neurons counted (see Figure1 in result section). As shown in (Figure1A) phenformin at 0.05–0.25 mM concentrations had no e ffect on neuronal viability: There was only negligible percentage of necrotic cells in the cultures 2.5–9.5%, which was − not different from percentage in the control cultures 3.7%. Higher concentrations of phenformin − (0.5, 1 and 2 mM) caused an increase in neuronal necrosis in CGC cultures compared to the control group by 41%, 62%, and 63%, respectively. At any concentration, phenformin did not induce neuronal apoptosis in CGC cultures. The data presented in Figure1b show that metformin concentrations up to 0.5 mM have no e ffect on neuronal viability in CGC cultures: The level of necrosis at these concentrations did not exceed 6.6%. At higher concentrations of metformin, 1, 2, and 3 mM, an increase of necrotic neuronal death was observed by 33%, 61% and 58%, respectively (Figure1b). Metformin did not cause neuronal apoptosis at any concentration investigated. Based on these findings 0.025 mM phenformin and 0.5 mM metformin were chosen as the highest non-toxic concentrations to be used in further experiments on brain slice cultures. Biomolecules 2020, 10, x 5 of 17

ionomycin was added and changes in fluorescence were measured at 5 min intervals. Cells were imaged in at least five microscopic fields per well (2 wells per condition). Image analyses were performed using ImageJ software and results were expressed as fluorescence arbitrary units per cell.

2.10. Measurement of the Mitochondrial Electron Transfer System Complex I Activity The activity of mitochondrial electron transfer system Complex I was determined in brain mitochondria isolated from 5–7 days and 2–3 months aged rats spectrophotometrically at a wavelength of 340 nm following the oxidation of 100 µM NADH in the presence of 2 µg/mL antimycin, 2 mM sodium azide, 3 mg/mL bovine serum albumin (BSA), 60 µM coenzyme Q1 (CoQ1) and investigated biguanides—phenformin or metformin—in a medium containing 25 mM KH2PO4 (pH 7.4 at 37 °C) and 0.125 mg/mL freeze–thawed mitochondria. Complex I activity was calculated by substracting nonspecific NADH oxidation rate (obtained after rotenone supplement) from total NADH oxidation rate and expressed as nmol NADH/min/mg protein [40].

2.11. Statistical Analysis All data are expressed as means ± standard errors of at least three experiments on separate preparations of brain slice or CGC cultures. The graphs were created and statistical significance was evaluated by SigmaPlot v13 software. Data were tested for normality using Shapiro–Wilk test and statistically compared between experimental groups by One Way Anova. Data presented in Figure 2a and Figure 7 did not pass Shapiro–Wilk normality test, and were tested by One Way Anova using Fisher LSD test. A value of p < 0.05 was considered statistically significant.

3. Results

3.1. Effect of Phenformin and Metformin on Neuronal Viability in CGC Cultures Phenformin and metformin are known to inhibit mitochondrial Complex I, which if complete and prolonged may lead to neuronal death. To find the optimal concentrations that are not toxic to neurons over 24 h incubation, we investigated effects of phenformin (0.005–0.2 mM) and metformin (0.1–3 mM) on viability of neurons in CGC cultures. The numbers of viable, apoptotic and necrotic cells were counted and expressed as percentage of all neurons counted (see Figure 1 in result section). As shown in (Figure 1A) phenformin at 0.05–0.25 mM concentrations had no effect on neuronal viability: There was only negligible percentage of necrotic cells in the cultures −2.5–9.5%, which was not different from percentage in the control cultures −3.7%. Higher concentrations of phenformin (0.5, 1 and 2 mM) caused an increase in neuronal necrosis in CGC cultures compared to the control group by 41%, 62%, and 63%, respectively. At any concentration, phenformin did not induce neuronal Biomoleculesapoptosis2020 in, CGC10, 1400 cultures. 6 of 17

Biomolecules 2020, 10, x 6 of 17

Figure 1. Effect of phenformin (a) and metformin (b) on neuronal viability in cerebellar granule cells (CGC) cultures. CGC were treated with phenformin and metformin at indicated concentrations. White column—viable neurons (%), black—necrosis (%), grey—apoptosis (%). The number of all neuronal cells (live, apoptotic and necrotic) in the vision field was equal to 100%. The data are presented as means ± standard errors of 3 experiments on separate CGC cultures.

The data presented in Figure 1b show that metformin concentrations up to 0.5 mM have no effect

on neuronal viability in CGC cultures: The level of necrosis at these concentrations did not exceed 6.6%. At higher concentrations(a) of metformin, 1, 2, and 3 mM, an increase of(b )necrotic neuronal death was Figureobserved 1. Ebyffect 33%, of phenformin61% and 58%, (a) andrespectively metformin (Figure (b) on neuronal1b). Metformin viability did in cerebellarnot cause granule neuronal apoptosiscells (CGC) at any cultures. concentration CGC were investigated. treated with phenformin and metformin at indicated concentrations. WhiteBased column—viableon these findings neurons 0.025 (%), mM black—necrosis phenformin (%),and grey—apoptosis0.5 mM metformin (%). Thewere number chosen of as all the highestneuronal non-toxic cells (live,concentrations apoptotic and to necrotic)be used inin the further vision experiments field was equal on to brain 100%. slice The datacultures. are presented as means standard errors of 3 experiments on separate CGC cultures. 3.2. Effects of Phenformin± and Metformin on Simulated Ischemia- and Hypoxia-Induced Necrosis in Brain 3.2.Slice E Culturesffects of Phenformin and Metformin on Simulated Ischemia- and Hypoxia-Induced Necrosis in Brain Slice Cultures Brain slice cultures were used in further experiments as they are considered to preserve complexityBrain slice of brain cultures tissue were architecture used in further and experimentsmaintain native as they synaptic are considered circuitry to which preserve is important complexity ofinvestigating brain tissue effects architecture on pharmacological and maintain compounds native synaptic [41]. circuitryFirst, we which investigated is important the effects investigating of 24 h ehypoxiaffects on and pharmacological 24 h simulated compounds ischemia (hypoxia [41]. First, plus we investigatedDOG to inhibit the glycolysis) effects of 24 on h hypoxiacell viability and 24in h simulatedbrain slice ischemiacultures prepared (hypoxia from plus DOGpremature to inhibit (5–7 glycolysis)days old) and on celladult viability (2–3 months in brain old) slice rats. cultures The preparedlevel of necrosis from premature in these experiments (5–7 days old) was and determi adultned (2–3 by months measuring old) rats.LDH The release level into of necrosisthe incubation in these experimentsmedia of the was slice determined cultures. As by can measuring be seen LDHin Figure release 2a, into simulated the incubation ischemia media (indicated of the sliceas hypoxia cultures. Aswith can DOG) be seen caused in Figure about2a, 5-fold simulated increase ischemia in LDH (indicated release into as hypoxia 5–7 days with rat DOG)brain slice caused culture about media 5-fold increasecompared in to LDH the releasenormoxia into with 5–7 DOG days group rat brain (5.5 slice ± 0.8 culture and 0.9 media ± 1.1 mU/L×mg, compared respectively) to the normoxia (Figure with DOG group (5.5 0.8 and 0.9 1.1 mU/L mg, respectively) (Figure2a). Twenty-four hour hypoxia 2a). Twenty-four± hour hypoxia± without DOG× had no effect on LDH release in brain slices cultures of without5–7 days DOG old rats had compared no effect onwith LDH the releasenormoxia in brain group slices (1.4 ± cultures 0.3 and of2.3 5–7 ± 2.2 days mU/L×mg, old rats comparedrespectively). with theSimilar normoxia results group were obtained (1.4 0.3 measuring and 2.3 necrosis2.2 mU/ Laccordmg,ing respectively). to PI staining Similar of cellresults nuclei: were24 h hypoxia obtained ± ± × measuringwith DOG caused necrosis 5-fold according increase to of PI PI staining fluorescence of cell intensity nuclei: 24in 5–7 h hypoxia days old with rat brain DOG slice caused cultures 5-fold increasecompared of to PI the fluorescence normoxia intensity with DOG in 5–7group days (16.8 old ± rat 1.8 brain and slice2.6 ± cultures0.6 FU, respectively) compared to the(Figure normoxia 2b). withTwenty-four DOG group hour (16.8hypoxia1.8 without and 2.6 DOG0.6 had FU, no respectively) effect on brain (Figure slices2b). cultures Twenty-four from premature hour hypoxia rats ± ± withoutcompared DOG with had the no normoxic effect on group brain (3.9 slices ± 0.6 cultures and 2.6 from ± 0.8 premature FU, respectively). rats compared with the normoxic group (3.9 0.6 and 2.6 0.8 FU, respectively). ± ±

(a) (b)

Figure 2. Cont. Biomolecules 2020, 10, x 7 of 17 Biomolecules 2020, 10, 1400 7 of 17

(c)

Figure 2. TheThe effect effect of of hypoxia hypoxia and and simula simulatedted ischemia ischemia on oncell cell death death in brain in brain slice slice cultures cultures from from 5–7 days5–7 days and and2–3 months 2–3 months old rats. old rats. Afte Afterr 24 h 24hypoxia h hypoxia or hypoxia or hypoxia with with 10 mM 10 mMD-deoxyglucose D-deoxyglucose (DOG), (DOG), the levelthe level of necrosis of necrosis was was evaluated evaluated by bymeasuring measuring increa increasese in in lactate lactate dehydrogenas dehydrogenasee (LDH) (LDH) activity activity in in the slice culture medium ( a) and and PI PI fluorescence fluorescence in in the the slice slice cultures cultures ( (bb).). FU—arbitrary FU—arbitrary fluorescence fluorescence units. units. Representative fluorescencefluorescence microscopy microscopy images images of brain of slicebrain cultures slice stainedcultures with stained PI (red with fluorescence) PI (red fluorescence)and Hoechst-33342 and Hoechst-33342 (blue fluorescence) (blue are fluorescence) presented in are (c). presented The data are in presented(c). The data as means are presentedstandard as ± meanserrors of± standard 7–18 experiments errors of on7–18 separate experiment brains slices on separate cultures. brain— slicesp < 0.01; cultures.—p **—< 0.001p < 0.01; compared ***—p to< ∗∗ ∗∗∗ 0.001normoxic compared control to with normoxic DOG, control ###—p with< 0.001 DOG, compared ###—p < to 0.001 normoxic compared control to withoutnormoxic DOG. control without DOG. Unlike 5–7 days age group, the level of LDH in media from 2–3 months old rat brain slice cultures afterUnlike simulated 5–7 ischemiadays age was group, not statisticallythe level of significantlyLDH in media diff erentfrom from2–3 months the corresponding old rat brain control slice culturesgroup (1.4 after0.2 simulated and 1.7 ischemia0.3 mU/ Lwasmg, not respectively). statistically significantly Meanwhile, 24different h hypoxia from without the corresponding DOG caused ± ± × control2-fold increase group (1.4 in ± LDH 0.2 and release 1.7 ± as0.3 compared mU/L×mg, to respectively). control group Meanwhile, (3.9 0.2 24 and h hypoxia 1.7 0.19 without mU/L DOGmg, ± ± × causedrespectively) 2-fold increase (Figure2 ina), LDH suggesting release thatas compared adult brain to control slices group cultures (3.9 exhibit ± 0.2 and extreme 1.7 ± 0.19 sensitivity mU/L×mg, to respectively)hypoxia alone. (Figure Interestingly, 2a), suggesting hypoxia-induced that adult brain damageslices cultures at this exhibit age was extreme prevented sensitivity by DOG to hypoxiatreatment alone. (Figure Interestingly,2a). Measurement hypoxia-induced of PI fluorescence brain intensitydamage at in brainthis age slice was cultures prevented of 2–3 by months DOG treatmentrats revealed (Figure that there2a). Measurement was a similar of increase PI fluorescence in hypoxia-induced intensity in necrosisbrain slice compared cultures withof 2–3 normoxia months ratsas it revealed was observed that there in the was measurements a similar increase of LDH in hypoxia-induced release (Figure2 a,b):necrosis In slices compared exposed with to normoxia hypoxia asalone it was the observed level of fluorescence in the measurements was increased of LDH to 9.7 release1.4 (Figure FU compared 2a,b): In to s 3.9lices exposed0.6 FU in to normoxia, hypoxia ± ± whereasalone the fluorescencelevel of fluorescence in slices exposedwas increased to hypoxia to 9.7 in± 1.4 the FU presence compared of DOG to 3.9 was ± 0.6 4.8 FU 0.5in normoxia, FU which ± whereasis at the samefluorescence level as in in slices normoxia exposed plus to DOGhypoxia4.0 in the0.7 presence FU (Figure of 2DOGb,c). was These 4.8 data ± 0.5 suggest FU which that is − ± atslice the cultures same level from as adult in normoxia brains are plus more DOG sensitive −4.0 ± 0.7 to hypoxiaFU (Figure alone 2b,c). than Th prematureese data suggest brains that and slice that cultureshypoxia-induced from adult necrosis brains in adultare more brains sensitive can be preventedto hypoxia by alone DOG. than As both premature methods—determination brains and that hypoxia-inducedof necrosis by LDH necrosis release in and adult PI fluorescence, brains can gavebe theprevented same results, by DOG. in further As both experiments methods— we determinationevaluated necrosis of necrosis according by to LDH LDH release release and into PI slice fluorescence, culture media. gave the same results, in further experimentsWhen 5–7 we daysevaluated brain necrosis slices cultures according were to preincubatedLDH release into with slice 0.025 culture mM phenforminmedia. and then exposedWhen to 5–7 24 hdays simulated brain slices ischemia cultures (hypoxia were pr pluseincubated DOG), the with level 0.025 of necrosismM phenformin was substantially and then exposeddecreased, to as24 measured h simulated by LDH ischemia release (hypoxia (106 10%) plus comparedDOG), the to level hypoxia of necrosis+DOG group was substantially (434 115%) ± ± decreased,(Figure3). Preincubation as measured by of slicesLDH withrelease 0.5 (106 mM ± metformin 10%) compared had no to statistically hypoxia+DOG significant group e (434ffect ± and 115%) the (Figurelevel of LDH3). Preincubation release remained of slices similar with as 0.5 in mM the simulatedmetformin ischemia had no statistically group (316 significant134%) (Figure effect3). and ± the level of LDH release remained similar as in the simulated ischemia group (316 ± 134%) (Figure 3). Biomolecules 2020, 10, x 8 of 17 Biomolecules 2020, 10, 1400 8 of 17 Biomolecules 2020, 10, x 8 of 17

Figure 3. Effect of phenformin and metformin on simulated ischemia-induced cell death in brain slice

cultures from 5–7 days old rats. Where indicated, 0.025 mM phenformin (Phen) or 0.5 mM metformin Figure 3. Effect of phenformin and metformin on simulated ischemia-induced cell death in brain slice (Metf)Figure was 3. Effect added of phenforminto slice culture and metforminincubation onmedi simulateum befored ischemia-induced the start of simulated cell death ischemia in brain sliceas cultures from 5–7 days old rats. Where indicated, 0.025 mM phenformin (Phen) or 0.5 mM metformin describedcultures fromin Methods. 5–7 days The old level rats. ofWhere necrosis indicated, after simulated 0.025 mM ischemia, phenform indicain (Phen)ted as or treatment 0.5 mM metformin with 10 (Metf) was added to slice culture incubation medium before the start of simulated ischemia as described mM(Metf) DOG was and added 24 h hypoxia,to slice culturewas evaluated incubation by measurmediuming before increase the in start LDH of activity simulated in slice ischemia culture as in Methods. The level of necrosis after simulated ischemia, indicated as treatment with 10 mM DOG described in Methods. The level of necrosis after simulated ischemia, indicated as treatment with 10 medium.and 24 h The hypoxia, data were was normalized evaluated byagainst measuring normoxic increase controls in LDHof each activity group inassuming slice culture that normoxic medium. mM DOG and 24 h hypoxia, was evaluated by measuring increase in LDH activity in slice culture levelThe datais 100% were and normalized presented against as means normoxic ± standa controlsrd errors of each of group 8 separate assuming experiments. that normoxic *—p level< 0.05 is medium. The data were normalized against normoxic controls of each group assuming that normoxic compared100% and presentedwith normoxic as means controlstandard with DOG, errors #— ofp < 8 0.05 separate compared experiments. to hypoxia *— pwith< 0.05 DOG. compared with level is 100% and presented± as means ± standard errors of 8 separate experiments. *—p < 0.05 normoxic control with DOG, #—p < 0.05 compared to hypoxia with DOG. Incompared 2–3 month with old normoxic rats brain control slice with cultures, DOG, preincubation #—p < 0.05 compared with 0.025 to hypoxia mM phenformin with DOG. and 0.5 mM metforminIn 2–3 monthbefore oldhypoxia rats brain caused slice 2-fold cultures, decre preincubationase in LDH with release 0.025 mM(121 phenformin± 16 and 126 and ± 0.5 29%, mM respectively)metforminIn 2–3 before month compared hypoxia old rats with caused brain the slicehypoxia 2-fold cultures, decrease group preincubation in(250 LDH ± 12%) release (Figure with (121 0.025 4a).16 mM andUnder phenformin 126 simulated29%, respectively) and ischemia 0.5 mM ± ± conditions,comparedmetformin withmetformin before the hypoxiahypoxia and phenformin groupcaused (250 2-fold had12%) no decre effect (Figurease on in 4LDH a).LDH Under release release simulated which (121 was ± ischemia at16 theand same conditions,126 level ± 29%, as ± atmetforminrespectively) normoxia and and compared phenformin hypoxia withplus had DOGthe no hypoxia e(Figureffect on group 4b). LDH release(250 ± 12%) which (Figure was at 4a). the Under same level simulated as at normoxia ischemia andconditions, hypoxia metformin plus DOG (Figureand phenformin4b). had no effect on LDH release which was at the same level as at normoxia and hypoxia plus DOG (Figure 4b).

(a) (b)

FigureFigure 4. EEffectffect ofof phenformin phenformin(a) and and metformin metformin on hypoxia-inducedon hypoxia-induced (a) and (a) simulated and(b simulated) ischemia-induced ischemia- induced(b) necrosis (b) innecrosis brain slicein brain cultures slice from cultures 2–3 monthsfrom 2–3 old mo rats.nths Where old rats. indicated, Where 0.025indicated, mM of 0.025 phenformin mM of Figure 4. Effect of phenformin and metformin on hypoxia-induced (a) and simulated ischemia- phenformin(Phen) or 0.5 (Phen) mM of or metformin 0.5 mM of (Metf) metformin was added (Metf) to was slice added culture to incubation slice culture medium incubation before medium the start induced (b) necrosis in brain slice cultures from 2–3 months old rats. Where indicated, 0.025 mM of beforeof hypoxia the start (a) orof simulatedhypoxia (a ischemia) or simulated (b) as ischemia described (b in) as Methods. described The in Methods. level of necrosis The level after of necrosis hypoxia afterorphenformin simulated-ischemia hypoxia (Phen)or simulated-ischemia or was 0.5 evaluatedmM of metformin was by measuring evaluated (Metf) increaseby was measuring added in LDH to increase slice activity culture in in LDH slice incubation cultureactivity medium. inmedium slice cultureThebefore data medium.the were start normalized of The hypoxia data were against(a) or normalized simulated normoxic ischemia against controls normoxic (b of) as each described groupcontrolsassuming in ofMethods. each thatgroup The normoxic levelassuming of necrosis level that is normoxic100%after andhypoxia level presented isor 100% simulated-ischemia as meansand presentedstandard aswas means errors evaluated ± of standard 7–9 by separate measuring errors experiments. of 7–9 increase separate ***— in LDHexperiments.p < 0.001 activity compared ***—in slicep ±

Altogether, the data suggest that metformin and phenformin reduce hypoxia-induced necrotic cell death in brain slice cultures of adult rats, while in brain slices of premature animals only phenformin has the protective effect. Biomolecules 2020, 10, 1400 9 of 17 To test whether biguanides may act via inhibition of Complex I, we evaluated the effect of phenformin and metformin on the activity of Complex I in isolated brain mitochondria of both age groups.To test Data whether presented biguanides in Figure may5 show act that via 0.025 inhibition mM phenformin of Complex and I, we 0.5 evaluatedmM metformin the e ffdirectlyect of phenforminsuppress the and rate metformin of NADH onoxidation the activity by Complex of Complex I of premature I in isolated (by brain 34% mitochondria and by 27%, respectively) of both age groups.and adult Data (by presented 54% and by in Figure58%, respectively)5 show that 0.025rat br mMain mitochondria phenformin and compared 0.5 mM with metformin the respective directly suppresscontrol group. the rate Using of NADH another oxidation model of by global Complex brain I ofischemia premature in vitro, (by 34%we have and found by 27%, that respectively) andmitochondrial adult (by 54% Complex and by I activity 58%, respectively) was inhibited rat by brain 49% mitochondria in prematures compared and by 40% with in theadult respective brains controlafter 2 group.h ischemia, Using and another that direct model addition of global of brain phenformin ischemia andin vitro metformin, we have to found isolated that ischemia- mitochondrial Complexaffected mitochondria I activity was inhibiteddid not cause by 49% additional in prematures inhibitory and effect by 40% on inComplex adult brains I (unpublished after 2 h ischemia, data; anddata that not direct shown). addition of phenformin and metformin to isolated ischemia-affected mitochondria did not cause additional inhibitory effect on Complex I (unpublished data; data not shown).

Control 400 +Phen +Metf

300

* 200 ** * **

100

Complex I activity, nmol NADH/min/mg protein NADH/min/mg nmol activity, I Complex 0 5 - 7 days 2 - 3 months FigureFigure 5. 5.E Effectffect of of phenformin phenformin and and metformin metformin on Complexon Complex I activity I activity of brain of brain mitochondria mitochondria isolated isolated from 5–7from days 5–7 and days 2–3 and months 2–3 agemonths rats. age Complex rats. Complex I activity I was activity evaluated was evaluated by following by NADHfollowing oxidation NADH rateoxidation in isolated rate freeze–thawedin isolated freeze–thawed brain mitochondria brain mi withouttochondria (control) without or with(contr biguanides—phenforminol) or with biguanides— (Phen,phenformin 0.025 mM)(Phen, or 0.025 metformin mM) or (Metf, metformin 0.5 mM) (Metf, as described0.5 mM) as in described Methods. in Statistically Methods. significantStatistically difference compared with control group: *—p 0.05, **—p 0.01 (n = 3). significant difference compared with control ≤group: *—p ≤≤ 0.05, **—p ≤ 0.01 (n = 3). 3.3. Modulation of Simulated Ischaemia-Induced Necrosis by Inhibitors of Complex I and MPTP in Brain 3.3. Modulation of Simulated Ischaemia-Induced Necrosis by Inhibitors of Complex I and MPTP in Brain Slice Cultures Slice Cultures To test the involvement of mitochondrial respiratory Complex I and MPTP in simulated To test the involvement of mitochondrial respiratory Complex I and MPTP in simulated ischemia-induced damage in brains from different age groups, classical Complex I inhibitors rotenone ischemia-induced damage in brains from different age groups, classical Complex I inhibitors (0.05 µM) and amobarbital (2.5 mM), and an inhibitor of MPTP 0.5 µM CsA were added to brain rotenone (0.05 µM) and amobarbital (2.5 mM), and an inhibitor of MPTP 0.5 µM CsA were added to slice incubation media before simulated ischemia. As can be seen in Figure6a, rotenone, amobarbital, brain slice incubation media before simulated ischemia. As can be seen in Figure 6a, rotenone, and CsA completely prevented simulated ischemia-induced LDH release in brain slices from 5–7 days: amobarbital, and CsA completely prevented simulated ischemia-induced LDH release in brain slices The LDH release to the medium was the same as in the normoxic control (Figure6a). from 5–7 days: The LDH release to the medium was the same as in the normoxic control (Figure 6a). The effects of Complex I and MPTP inhibitors were also tested on hypoxia-induced LDH release in the samples from 2–3 months old animals. As presented above, hypoxia alone resulted in pronounced necrosis which was fully blocked by DOG. Rotenone used at 0.05 µM (the concentration effective in other age group during simulated ischemia) and at 1 µM, did not prevent hypoxia-induced necrosis measured as LDH release, though some tendency of protection was seen at lower rotenone concentration (Figure6b). Also, 1 µM rotenone itself was slightly toxic to normoxic samples of 2–3 months old rats inducing 1.5-fold increase in LDH release compared to control normoxic cultures (data not shown). However, another Complex I inhibitor amobarbital and MPTP inhibitor CsA were effective and significantly prevented hypoxia-induced increase in LDH release (Figure6b). These compounds had no effect on the level of necrosis under normoxic conditions (data not shown). The data show that effects of rotenone, amobarbital and CsA on simulated ischaemia- or hypoxia-triggered cell death in brain slice cultures is different among age groups investigated. All inhibitors efficiently prevented ischemia-induced necrosis in 5–7 days brains, and amobarbital and CsA (but not rotenone)—hypoxia-induced necrosis in adult rat brains. BiomoleculesBiomolecules 20202020, ,1010, ,x 1400 1010 of of 17 17

(a) (b)

FigureFigure 6. 6. TheThe effect effect of of mitochondrial mitochondrial respiratory respiratory Comple Complexx I Iand and mitochondrial mitochondrial permeability permeability transition transition porepore (MPTP) inhibitors onon thethe level level of of simulated simulated ischemia-induced ischemia-induced necrosis necrosis in brainin brain slice slice cultures cultures from from5–7 days 5–7 days (a) and (a) 2–3 and months 2–3 months old (b old) rats. (b) Where rats. Where indicated, indicated, 0.05 µ M0.05 or µM 1 µM or rotenone 1 µM rotenone (Ro), and (Ro), 2.5 and mM 2.5amobarbital mM amobarbital (Amo), (Amo), and 0.5 and mM 0.5 cyclosporin mM cyclosporin A (CsA) A were (CsA) added were to added slice cultureto slice incubationculture incubation medium mediumbefore the before start ofthe simulated start of simulated ischemia asischemia described as inde Methods.scribed in TheMethods. level of The necrosis level inof slicenecrosis cultures in slice was culturesevaluated was by evaluated measuring by increase measuring in LDH increase activity in LD inH slice activity culture in slice medium. culture The medium. data were The normalized data were normalizedagainst normoxic against controls normoxic of controls each age of group each age assuming group thatassuming normoxic that normoxic level is 100% level (white is 100% bars) (white and bars)presented and aspresented mean+standard as mean+standard error. N = 3–7;error. ***—statistically N = 3–7; ***—statistically significant diff erencesignificant compared difference with comparednormoxic controlwith normoxic of the corresponding control of the group, correspondingp 0.001. ###, group, ## and p #—statistical≤ 0.001. ###, significance## and #—statistical compared ≤ significanceto hypoxia +comparedDOG or hypoxia to hypoxia treatment, +DOGp or0.001, hypoxiap 0.01 treatment, and 0.05, respectively.p ≤ 0.001, p ≤ 0.01 and 0.05, ≤ ≤ respectively. 3.4. Acute Effects of Inhibitors of Complex I and MPTP on Ca2+ Retention Capacity of Brain Cortex Mitochondria TheIn the effects next seriesof Complex of experiments I and MPTP we investigatedinhibitors we whetherre also tested age of on rats hypoxia-induced may cause differences LDH onrelease Ca2+ - ininduced the samples MPTP regulationfrom 2–3 inmonths isolated old brain animals. mitochondria. As presented The respiratory above, hypoxia rates (with alone pyruvate resulted plus in pronouncedmalate as substrates) necrosis ofwhich mitochondria was fully isolated blocked from by DO prematureG. Rotenone rat brains used were: at 0.05 LEAK µM (the respiration concentration (State 2) effective98 15 in pmolO other /ages/mg group protein during and OXPHOS simulated (ADP-stimulated ischemia) and at respiration) 1 µM, did not383 prevent33 pmolO hypoxia-/s/mg − ± 2 − ± 2 inducedprotein; necrosis for mitochondria measured isolatedas LDH release, from adult though rat some brains—LEAK tendency of175 protection8 pmolO was seen/s/mg at protein lower − ± 2 rotenoneand OXPHOS concentration788 90(Figure pmolO 6b)./ sAlso,/mg protein.1µM roteno Thesene itself data indicatewas slightly high toxic quality to normoxic of mitochondrial samples − ± 2 ofpreparations 2–3 months of old both rats age inducing groups. 1.5-fold increase in LDH release compared to control normoxic culturesMPTP (data in not the shown). experiments However, was evaluatedanother Complex as mitochondrial I inhibitor calciumamobarbital retention and MPTP capacity inhibitor (CRC). CsAThere were was effective no significant and significantly difference prevented in CRC of isolatedhypoxia-induced brain mitochondria increase in LDH comparing release 5–7 (Figure days 6b). and These2–3 month compounds age groups had no (Figure effect7 ona). the CRC level of 5–7of necr daysosis old under rat brain normoxic was insensitive conditions to(data cyclosporine not shown). A, thoughThe it data was significantlyshow that effects higher inof therotenone, presence amobarbital of Complex Iand inhibitors: CsA on CRC simulated was increased ischaemia- by about or hypoxia-triggered150% with rotenone, cell by death 70% within br amobarbitalain slice cultures and by is aboutdifferent 50% among with metformin, age groups compared investigated. to control All inhibitors(Figure6a). efficiently Similarly, prevented in 2–3 months ischemia-induced age group, amobarbital necrosis in increased5–7 days brains, CRC by and 23% amobarbital and metformin and CsAby 23–29%, (but not however rotenone)—hypoxia-induced only higher 1 µM rotenone necrosis concentrationin adult rat brains. increased CRC by 43% (Figure7b). − Adult rat brain mitochondria were sensitive to cyclosporine A which increased CRC by 38%. However, 3.4.phenformin Acute Effects had of no Inhibitors effect on of brain Complex mitochondrial I and MPTP CRC on Ca in2+ both, Retention 5–7days Capacity and of 2–3 Brain months, Cortex age groups. MitochondriaAltogether, the data suggest that MPTP in premature rat brains is more sensitive to acute effects of ComplexIn the I inhibitorsnext series than of experiments mitochondria we from investigat adult brainsed whether whereas age cyclosporine of rats may A cause acutely differences desensitized on CaMPTP2+- induced to calcium MPTP in adultregulation brain in mitochondria. isolated brain mitochondria. The respiratory rates (with pyruvate plus malate as substrates) of mitochondria isolated from premature rat brains were: LEAK respiration (State 2) −98 ± 15 pmolO2/s/mg protein and OXPHOS (ADP-stimulated respiration) −383 ± 33 pmolO2/s/mg protein; for mitochondria isolated from adult rat brains—LEAK −175 ± 8 pmolO2/s/mg protein and OXPHOS −788 ± 90 pmolO2/s/mg protein. These data indicate high quality of mitochondrial preparations of both age groups. Biomolecules 2020, 10, x 11 of 17

MPTP in the experiments was evaluated as mitochondrial calcium retention capacity (CRC). There was no significant difference in CRC of isolated brain mitochondria comparing 5–7 days and 2–3 month age groups (Figure 7a). CRC of 5–7 days old rat brain was insensitive to cyclosporine A, though it was significantly higher in the presence of Complex I inhibitors: CRC was increased by about 150% with rotenone, by 70% with amobarbital and by about 50% with metformin, compared to control (Figure 6a). Similarly, in 2–3 months age group, amobarbital increased CRC by 23% and metformin by 23–29%, however only higher −1 µM rotenone concentration increased CRC by 43% (Figure 7b). Adult rat brain mitochondria were sensitive to cyclosporine A which increased CRC by 38%. However, phenformin had no effect on brain mitochondrial CRC in both, 5–7 days and 2–3 months, age groups. Altogether, the data suggest that MPTP in premature rat brains is more sensitive to acute effects of Complex I inhibitors than mitochondria from adult brains whereas cyclosporine A acutelyBiomolecules desensitized2020, 10, 1400 MPTP to calcium in adult brain mitochondria. 11 of 17

(a) (b)

FigureFigure 7. 7. CaCa2+ retention2+ retention capacity capacity of brain of brain cortex cortex mitochondria mitochondria isolated isolated from from5–7 days 5–7 days(a) and (a) 2–3 and months2–3 months (b) old (b) rats. old rats. Mitochondrial Mitochondrial calcium calcium retention retention capacity capacity (CRC) (CRC) was was measured measured fluorometrically fluorometrically usingusing Calcium Calcium Green-5N Green-5N as as described described in in Methods. Methods. Where Where indicated, indicated, rotenone rotenone (Rot, (Rot, 0.05 0.05 and and 1 1µM),µM), amobarbitalamobarbital (Amo, (Amo, 2.5 2.5 mM), mM), cyclopsorine cyclopsorine A A (CsA (CsA,, 0.5 0.5 µM),µM), metformin metformin (Metf, (Metf, 10 10 and and 20 20 mM) mM) or or phenforminphenformin (Phen, (Phen, 0.1 0.1 and and 1 1mM) mM) were were added added to to is isolatedolated mitochondria mitochondria 5 5min min before before calcium calcium pulses. pulses. TheThe data data are are presented asas meansmeans ±standard standard errors errors of of 3–10 3–10 separate separate experiments. experiments.— p*—

3.5. Effects of Metformin, Phenformin, and Inhibitors of Complex I on MPTP Opening in Intact Cultured 3.5. Effects of Metformin, Phenformin, and Inhibitors of Complex I on MPTP Opening in Intact Cultured CGC Neurons CGC Neurons We tested whether metformin and phenformin exert an effect on MPTP in intact neuronal We tested whether metformin and phenformin exert an effect on MPTP in intact neuronal cells. cells. In these experiments, pure neuronal cultures were pre-incubated 30 min with phenformin or In these experiments, pure neuronal cultures were pre-incubated 30 min with phenformin or metformin and then cells were loaded with fluorescent agent Calcein-AM which enters into cells metformin and then cells were loaded with fluorescent agent Calcein-AM which enters into cells and and accumulates in mitochondria. Calcein fluorescence in the cytosol is then quenched with CoCl2. accumulates in mitochondria. Calcein fluorescence in the cytosol is then quenched with CoCl2. MPTP MPTP opening was induced in these cells by adding selective calcium ionofore—ionomycin. As shown opening was induced in these cells by adding selective calcium ionofore—ionomycin. As shown in in Figure8, after addition of ionomycin there was a rapid decrease in fluorescence which is an indicator Figure 8, after addition of ionomycin there was a rapid decrease in fluorescence which is an indicator of MPTP opening allowing efflux of fluorescent calcein from mitochondria into cytosol. However, of MPTP opening allowing efflux of fluorescent calcein from mitochondria into cytosol. However, in in neurons pre-incubated with 100 µM phenformin the decrease in fluorescence was substantially neurons pre-incubated with 100 µM phenformin the decrease in fluorescence was substantially lower lower over 10 min incubation period (Figure8a). Lower concentration of phenformin was less e ffective. over 10 min incubation period (Figure 8a). Lower concentration of phenformin was less effective. Similarly, metformin at 2 mM and 3 mM concentrations substantially reduced ionomycin-induced Similarly, metformin at 2 mM and 3 mM concentrations substantially reduced ionomycin-induced decrease in fluorescence suggesting prevention of MPTP opening (Figure8b). Selective inhibitor of decrease in fluorescence suggesting prevention of MPTP opening (Figure 8b). Selective inhibitor of Complex I—rotenone effectively reduced ionomycin-induced MPTP opening even at lowest 0.05 µM Complex I—rotenone effectively reduced ionomycin-induced MPTP opening even at lowest 0.05 µM concentration (Figure8c). Ionomycin-induced decrease in fluorescence in neurons was also suppressed concentration (Figure 8c). Ionomycin-induced decrease in fluorescence in neurons was also by cyclosporine A—a specific inhibitor of MPTP (Figure8d). Altogether, these data suggest that suppressed by cyclosporine A—a specific inhibitor of MPTP (Figure 8d). Altogether, these data phenformin and metformin can suppress MPTP in neurons possibly acting via Complex I inhibition. Biomolecules 2020, 10, x 12 of 17

suggest that phenformin and metformin can suppress MPTP in neurons possibly acting via Complex BiomoleculesI inhibition.2020 , 10, 1400 12 of 17

(a) (b)

(c) (d)

FigureFigure 8. 8.Eff Effectsects of phenformin,of phenformin, metformin, metformin, rotenone rotenone and and cyclosporin cyclosporin A on A MPTP on MPTP opening opening in neuronal in cells.neuronal Pure cells. neuronal Pure culturesneuronal preparedcultures prepared treating tr CGCeating culturesCGC cultures with with Ara-C Ara-C (see (see Section 2.2 section) 2.2) were pre-incubatedwere pre-incubated for 1 h with:for 1 h (a with:) 50 µ M(a) and50 µM 100 andµM 100 phenformin µM phenformin (Phen), (Phen), (b) 2 mM (b) and 2 mM 3 mM and metformin 3 mM (Met);metformin (c) 0.05 (Met);µM and (c) 10.05µM µM rotenone and 1 µM (Rot); rotenone and (d (Rot);) 3 µM and and (d 5) µ3M µM cyclosporine and 5 µM cyclosporine A (CsA). Mitochondrial A (CsA). permeabilityMitochondrial transition permeability pore (MPTP) transition opening pore was (MPTP) quantified opening by measuring was quantified fluorescence by alterationmeasuring after stainingfluorescence with Calcein-AM alteration after as described staining inwith Methods. Calcein-AM The dataas described were normalized in Methods. against The controls data were of each normalized against controls of each group assuming that control level is 100% and presented as mean group assuming that control level is 100% and presented as mean standard errors of 4–6 experiments ± standard errors of 4–6 experiments on separate cell cultures. *—p± < 0.01. on separate cell cultures. —p < 0.01. ∗ 4.4. Discussion Discussion TheThe study study was was designed designed toto comparecompare the effects effects of of hypoxia hypoxia and and simulated simulated ischemia ischemia (hypoxia (hypoxia plus plus DOG)DOG) on on the the brain brain slices slices culturescultures prepared from from premature premature (5–7 (5–7 days) days) and and adult adult (2–3 (2–3 months months old) old) animals and to test whether two biguanide derivatives—metformin and phenformin—exert animals and to test whether two biguanide derivatives—metformin and phenformin—exert protective protective effects against hypoxic/ischemic damage and by which mechanism. We found that effects against hypoxic/ischemic damage and by which mechanism. We found that premature brain premature brain slice cultures were resistant to 24 h hypoxia alone; however, hypoxia in the presence slice cultures were resistant to 24 h hypoxia alone; however, hypoxia in the presence of DOG (simulated of DOG (simulated ischemia) induced necrosis in these brain slice cultures. Treatment of premature ischemia)brain slice induced cultures necrosis with phenformin in these brain before slice cultures.simulated Treatment ischemia protected of premature from brain necrotic slice cell cultures death with phenforminwhereas pretreatment before simulated with ischemiametformin protected was prac fromtically necrotic not effective cell death against whereas ischemia-induced pretreatment with metforminnecrosis in was these practically brain slice not cultures. effective against ischemia-induced necrosis in these brain slice cultures. BothBoth compounds, compounds, metformin and and phenformin, phenformin, at co atncentrations concentrations that were that protective were protective against againstcell celldeath, death, caused caused direct direct partial partial inhibition inhibition of Comple of Complexx I activity I activity in isolated in isolated brain mitochondria. brain mitochondria. The Themolecular molecular structure structure of ofphenformin phenformin slightly slightly differs diff ersfrom from metformin: metformin: Compared Compared to metformin, to metformin, phenforminphenformin has has additional additional phenylphenyl ring and a a two-carbon two-carbon linker linker between between the the phenyl phenyl ring ring and and the the biguanide moiety [42]. According to the literature, these structural features may lead to a significantly stronger inhibition of Complex I due to hydrophobic properties and easier transport to the mitochondrial matrix [19]. The data presented in this study are in agreement with this as we showed that about 10–20 times lower concentration of phenformin was needed to inhibit Complex I activity to the similar level as metformin. Biomolecules 2020, 10, 1400 13 of 17

Interesting and unexpected finding of this study was that adult rat brain slices (2–3 months old) were sensitive to hypoxia alone: In this case 24 h hypoxia induced substantial necrosis which was prevented in the presence of DOG. The results with cultures of 5–7 days old rat brain slices were opposite—DOG supplement caused greater ischemic necrotic damage, whereas in the absence of DOG there were no differences between normoxic and hypoxic levels of necrosis. These data correlate with findings by other researchers showing that 5–7 days old rats brain slice cultures were more sensitive to decreased glucose than slices from 2–3 months and older rats [43]. The exact mechanism of how DOG reduces hypoxia-induced cell death in adult rat brains is not clear, but inhibition of glycolysis by DOG may involve activation of some pro-survival signaling pathways, like hypoxia-inducible factor-1α [44], signal transducer and activator of transcription 3 and transcription factor 4 [45,46]. Hypoxia-induced necrosis in slice cultures from adult brains was found to be prevented by pre-treatment with both phenformin and metformin. These findings are in agreement with previously reported studies by other investigators: Zeng and colleges performed study in vivo with 2–3 month old mice and showed that metformin administered intraperitoneally immediately after induction of cerebral ischemia increased neuronal survival [46]. In another study with 2–3 month old rats administration of metformin by 1, 3, or 7 days before stroke resulted in the reduction of cerebral infarction volume [47]. Absence of the effect of metformin in 5–7 days age group observed in our study is not entirely clear, however it can be explained by the difference of energy metabolism that occur with development and aging. For example, in the study of healthy human population with age between 5 and 106 years, there was observed a shift in the glucose metabolism from aerobic to anaerobic, influencing the oxygen consumption not completely coupled with the ATP synthesis and increase in ROS production [48]. Importantly, in this study we demonstrated that necrosis induced by simulated ischemia in premature brain slice cultures as well as hypoxia-induced in adult brain slice cultures was prevented by a specific inhibitor of MPTP—cyclosporine A, suggesting that this cell death was mediated by MPTP. Necrosis in premature brain slices was also prevented by rotenone—a selective inhibitor of mitochondrial Complex I as well as by amobarbital which also inhibits Complex I. Amobarbital also prevented hypoxia-induced necrosis in adult brain slice cultures though rotenone was less effective in this case. We showed that preincubation of neuronal cell cultures with rotenone, phenformin and metformin substantially prevented cyclosporine-A-sensitive ionomycin-induced MPTP opening in intact neuronal cells suggesting that MPTP in neurons may be regulated by Complex I activity and that phenformin and metformin can inhibit MPTP. This allows to suggest that protective effects of phenformin in premature and adult brain slice cultures as well as effect of metformin in adult brain slice cultures against ischemia/hypoxia-induced cell death may be related to inhibition of MPTP due to suppression of Complex I activity. This is in line with our previous study, where we showed that infusion of rotenone to adult rats protected from brain ischemia-induced cell death by inhibiting MPTP [49], as well as data of other researchers who demonstrated that inhibitors of Complex I amobarbital [9,50,51] and rotenone [8] decreased dysfunction of myocardial mitochondria and ischemic heart injury. Further support for our conclusion was provided by assessment of calcium retention capacity of isolated brain mitochondria which was found to be increased by rotenone, amobarbital and metformin. The results of these experiments also revealed age-dependent differences in modulation of MPTP by Complex I inhibitors and cyclosporine A which inhibits MPTP by binding to cyclophilin D. MPTP in brain mitochondria isolated from premature (5–7 days old) rats were more sensitive to Complex I inhibitors—rotenone, amobarbital and metformin than in adult rat brain mitochondria where higher concentrations of rotenone were required to delay MPTP opening, as well as lower inhibition of MPTP by amobarbital and metformin was observed. Meanwhile MPTP in mitochondria from adult rat brains were more sensitive to cyclosporine A whereas MPTP in premature brain mitochondria were not acutely affected by the same concentration of cyclosporine A. Note that in neurons isolated from 7-day-old rats, cyclosporine A suppressed MPTP opening but at higher concentrations than used for isolated mitochondria indicating that even at that age neuronal MPTP can be modulated by cyclophilin D inhibitor. Biomolecules 2020, 10, 1400 14 of 17

It is important to mention that concentrations of metformin that inhibited MPTP in isolated brain mitochondria were higher than used in cultured neurons and exhibited protective effects in brain slice cultures. Phenformin even at the highest 1 mM concentration had no direct acute effect on CRC of isolated brain mitochondria though it inhibited ionomycin-induced MPTP opening in intact neurons at much lower concentration. One of the possible explanations for such findings may be related to different affinity of biguanides to Complex I conformational states [19,52]. Complex I can exist in active or deactive conformation, and ischemia has been shown to promote deactivation of this complex [52]. In the heart, metformin has been shown to inhibit Complex I in ischemia-damaged but not control mitochondria [10]. Our data show that Complex I is inhibited by metformin and phenformin in control mitochondria disrupted by freezing–thawing—a procedure which may affect conformational state of Complex I. Therefore it is possible to speculate that in isolated intact brain mitochondria Complex I is in active form which is less sensitive to biguanides. In intact cells (cultured neurons or brain slice cultures), Complex I may be in deactive conformation, particularly under hypoxic conditions, and this may promote phenformin and metformin binding and inhibiting Complex I. Recently, Matsuzaki and Humphries demonstrated supporting data that biguanides selectively inhibit the deactivated form of complex I [51]. Thus lower concentrations of biguanides would be needed to suppress MPTP in intact cells, particularly exposed to hypoxia. However, such hypothesis needs further experimental investigation. We cannot completely exclude other possible mechanisms of protective action of metformin and phenformin. It has been previously suggested that metformin suppresses oxidative stress in rat brain after ischemia and ischemia/reoxygenation [53]. Other investigators have proposed that treatment with metformin increases cerebral AMPK activation following ischemia [54], suggesting that metformin may confer neuroprotection by activating AMPK [55,56]. Other authors obtained contradictory data indicating that AMPK is highly expressed in neurons and is rapidly activated in the brain during energy-deprived states such as ischemia, though metformin ameliorated stroke-induced activation of AMPK [56]. However, recent study by Lesnefsky and colleges (2019) with isolated cardiac mitochondria and cultured H9c2 cells affected by ischemia suggested that protective effect of metformin is independent of AMPK activation [10]. Their data support our findings in this study that treatment of metformin before ischemia protects cells from ischemia-induced injury by inhibiting activity of Complex I and improving mitochondrial calcium retention capacity and delaying MPTP opening. The exact mechanism of regulation of MPTP opening by Complex I activity is unclear but may involve suppression of production of ROS (which are activators of MPTP) due to prevention of reverse electron transfer through the respiratory chain (a condition which may arise during ischemia due to accumulation of succinate) [10]. Another possible explanation is that Complex I activity may affect ubiquinone redox state, which in turn may modulate MPTP [15]. The third possibility is that Complex I may be involved in formation of MPTP, thus inhibitors binding to the complex may affect its structure depending on the redox state and subsequently modulate MPTP [16].

5. Conclusions The current study suggests that mitochondrial Complex I is one of the important targets in ischemia-induced injury. Pharmacological compounds such as metformin and phenformin that act on its activity may regulate opening of MPTP and lead to survival of brain cells under ischemic conditions.

Author Contributions: Conceptualization, V.B.; methodology A.J., R.M.; investigation, K.S., E.R., A.J., and P.C.; analysis K.S., E.R., A.J., P.C., and R.M.; data curation, K.S., A.J., R.M., and V.B.; writing—original draft preparation, K.S., E.R., A.J., and V.B., writing—review and editing—K.S., E.R., A.J., P.C., R.M., and V.B.; funding acquisition, V.B. All authors have read and agreed to the published version of the manuscript. Funding: This research has received funding from European Social Fund (project No 09.3.3-LMT-K-712-01-0131) under grant agreement with the Research Council of Lithuania. A.J. was supported by Global grant No VP1-3.1-SMM-07-K-01-130 from Research Council of Lithuania. Acknowledgments: We thank students Gerda Baranovaite and Paulius Balciunas for technical assistance performing experiments and Odeta Arandarcikaite for help with measurements of mitochondrial respiration. Biomolecules 2020, 10, 1400 15 of 17

Conflicts of Interest: The authors declare no conflict of interest.

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