Diabetes mellitus (DM) is one of the most common metabolic diseases which is associated with either impairment of insulin secretion (type I diabetes) or tolerance of cells to insulin (type II diabetes). The common pathological syndrome in both cases is an increased level of blood glucose (hyperglycemia), which, in the long run, inflicts serious damage to many organs and systems of the organism. At the intracellular level, one of the main targets of diabetes are mitochondria. There are a lot of diabetes-related disorders, developing in the structure and function of mitochondria of various tissues and organs: Impairment of mitochondrial biogenesis, remodeling of the mitochondrial cell network, disorders in the system of oxidative phosphorylation, hyperproduction of reactive oxygen species, etc. [1
One of the manifestations of mitochondrial dysfunction in diabetes mellitus are disorders in the mitochondrial Ca2+
ion transport systems. As a major buffer system of this ion, mitochondria are involved in the regulation of intracellular Ca2+
homeostasis and, in addition, Ca2+
activates a number of mitochondrial respiratory enzymes and enzymes of the Krebs cycle. On the other hand, excessive accumulation of Ca2+
in mitochondria can lead to the opening of mitochondrial pores and initiation of cell death [4
enters mitochondria via Ca2+
uniporter, a protein complex consisting of the channel subunit MCU, its paralog (the dominant-negative channel subunit MCUb), the gate subunits MICU1-2, and the subunits EMRE and MCUR1 [4
]. It is generally accepted that the regulation of Ca2+
uniporter is mediated by the interaction of MCU with MCUb and MICU1 and by changes in the ratio of these proteins in the complex. For example, overexpression of MCUb subunit impairs the ion-transporting function of the Ca2+
]. A low level of MICU1 expression in the heart, as compared to the liver, explains why heart mitochondria have a low activation threshold for Ca2+
transport and a lower Ca2+
As mentioned above, excessive accumulation of Ca2+
in mitochondria leads to the opening of Ca2+
-dependent mitochondrial pores, which is a key step in the mechanism of the programmed cell death. The mitochondrial permeability transition pore (MPT pore) is considered to be a protein mega-channel, which includes proteins of the inner and outer membrane of mitochondria. It is not exactly clear which proteins form the pore—yet currently, they are believed to be either the mitochondrial ATP synthase or adenylate translocator. It is also established that MPT pore includes cyclophilin D, a regulatory protein targeted by the pore inhibitor cyclosporin A (CsA) [5
Earlier we showed that, in addition to the CsA-sensitive MPT pore, there was another variety of pores which could be opened in mitochondria in the presence of saturated fatty acids and Ca2+
. Those pores were not responsive to CsA and other modulators of the CsA-sensitive MPT pore. The studies performed on artificial lipid membranes (BLM and liposomes) showed that the mechanism of membrane permeabilization in that case was based on the ability of saturated fatty acid anions to form tight complexes with Ca2+
in the lipid bilayer followed by their segregation into solid-crystalline membrane domains. The process of phase separation was shown to be accompanied by the formation of hydrophilic lipid pores [5
The studies on the mitochondrial Ca2+
transport and MPT pores in the diabetic cells (at both types of diabetes) have been carried out for a long time, yet the data obtained are rather contradictory. In different studies of type I diabetes in animals, both the uptake of Ca2+
by cells and mitochondria and the opening of MPT pore have been reported to be either stimulated or repressed [10
]. Of particular interest are the data that type-I diabetic liver mitochondria are more resistant to the opening of MPT pores [15
]. The mechanisms of these effects are often not clear, although they should presumably depend on the species and age of the animal, as well as the type of tissue.
In this work, we have examined changes in the systems of mitochondrial Ca2+ transport (Ca2+ uniporter) and MPT pore opening in the liver of rats with streptozotocin-induced type-I diabetes. Taking into account the fact that diabetes also causes disorders in lipid metabolism, we have further investigated the formation of palmitate/Ca2+-induced pore in liver mitochondria of diabetic animals. The results have shown that: (1) streptozotocin-induced type-I diabetes increases the rate of Ca2+ uptake by liver mitochondria of Sprague-Dawley rats, which may be associated with a decreased expression of MCUb; (2) under streptozotocin-induced diabetes, the Ca2+ capacity of rat liver mitochondria is enhanced, and the amplitude of CsA-sensitive Ca2+/Pi-induced mitochondrial swelling is lowered; and (3) treatment of Sprague-Dawley rats with streptozotocin decreases the resistance of their liver mitochondria to palmitate/Ca2+-dependent CsA-insensitive permeability transition.
2. Materials and Methods
The laboratory animals (Sprague-Dawley male rats) were treated in accordance with the European Convention for the Protection of Vertebrates used for experimental and other purposes (Strasbourg, 1986) and the principles of the Helsinki Declaration (2000). All the protocols were approved by the Institute of Theoretical and Experimental Biophysics RAS Ethics Committee (Order No. 173/k of 03.10.2011, Protocol No. 04/2019 of 05.03.2019).
2.2. Induction of Diabetes
Experiments were performed on Sprague-Dawley male rats with induced diabetes mellitus (n
= 5) and without diabetes (n
= 5). Diabetes was induced by a single injection of streptozotocin (STZ; 70 mg/kg, IP). This is considered to be the optimal diabetogenic dose: At higher concentrations, STZ can have a toxic effect not only on the pancreatic β-cells but on the cells of liver and kidney as well [16
]. Control rats received an equal volume of vehicle (0.1 M citrate buffer, pH 4.5). Rats with blood glucose over 300 mg/dL were considered diabetic. Animals were sacrificed after two weeks, while blood glucose (BG) and body weight (BW) were regularly monitored.
2.3. Isolation of Rat Liver Mitochondria
Mitochondria were isolated from the liver of Sprague-Dawley rats (150–200 g) by differential centrifugation as described earlier [18
]. The homogenization buffer contained 210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM Hepes/KOH buffer, pH 7.4. Subsequent centrifugations were performed in the same buffer, except that, instead of EDTA, 100 μM EGTA was used. Final suspensions contained 70–80 mg of mitochondrial protein/mL, as determined by the Lowry method [19
2.4. Ca2+ Uptake by Mitochondria
The concentration of Ca2+
in the reaction medium (external [Ca2+
]) was measured with an ion-selective electrode [20
]. The reaction medium contained 150 mM sucrose, 50 mM KCl, 2 mM KH2
, 5 mM succinate, 1 μM rotenone, 5 μM EGTA, and 10 mM Hepes/KOH buffer, pH 7.4. The measurements were carried out in in a stirred cuvette at room temperature (~22 °C). The concentration of mitochondrial protein was 1–1.5 mg/mL. In the experiments, 25 μM Ca2+
was added to the reaction medium every 60 s. After several additions, external [Ca2+
] increased, indicating a massive release of the ion from the organelles due to the opening of MPT pore in the inner mitochondrial membrane. The amount of Ca2+
released upon permeability transition (defined as Ca2+
capacity) was used as a measure of the MPT pore opening probability.
2.5. Mitochondrial Respiration and Ca2+/O
The rate of oxygen consumption was measured polarographically with a Clark-type gold electrode Oxygraph-2k (O2k, OROBOROS Instruments, Innsbruck, Austria) at 25 °C under continuous stirring [20
]. The reaction medium contained 130 mM KCl, 5 mM KH2
, 5 mM succinate, 1 μM rotenone, 10 µM EGTA, and 10 mM Hepes/KOH, pH 7.4. The concentration of mitochondrial protein was ~0.5 mg/mL. The respiratory control of succinate-oxidizing mitochondria was in the range of four to five. The Ca2+
/O ratio was estimated under limited conditions of Ca2+
load, by measuring stimulated respiration after the addition of 200 nmoles of CaC12
to the suspension of respiring mitochondria [21
2.6. Mitochondrial Swelling
The swelling of mitochondria (0.4 mg/mL) was measured as a decrease in absorbance at 540 nm (A540
) in a stirred cuvette at room temperature (~22 °C) using a USB-2000 spectroscopy fiber-optic system (Ocean Optics, Dunedin, FL, USA) [20
]. The incubation medium contained 210 mM mannitol, 70 mM sucrose, 5 mM succinate, 10 μM EGTA, 1 μM rotenone, and 10 mM Hepes/KOH buffer, pH 7.4. The rate of swelling (Vmax
/min per mg protein) was calculated as a change in absorbance within the first 30 s from the beginning of the high-amplitude swelling. The amplitude of swelling (%) was determined 3 min after the beginning of high-amplitude swelling. The change in absorbance upon the addition of alamethicin (7.5 μg/mL) was taken as 100% swelling.
2.7. Fluidity of the Mitochondrial Membrane
Fluidity of the mitochondrial membrane was determined by measuring the fluorescence intensity of laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) in temperature-controlled (37 °C) 96-well plates [22
]. Freshly prepared liver mitochondria were suspended (0.5 mg/mL) in a medium containing 210 mM mannitol, 70 mM sucrose, 5 mM succinate, 10 μM EGTA, 1 μM rotenone, 1 mM KH2
, and 10 mM Hepes/KOH buffer, pH 7.4. After that, 1 μM laurdan was added, and the samples were incubated in dark for 30 min at 37 °C. Laurdan fluorescence was excited at 340 (20) nm, and the emission was measured at two wavelengths: 430 (20) and 495 (10) nm—using a Tecan Spark 10 M reader (Tecan, Männedorf, Switzerland). The generalized polarization (GP) values were calculated as follows: GP = (I430
), where I430
were the laurdan emission intensities at the respective wavelengths. GP can theoretically assume values from −1 (the least ordered state) to +1 (the most ordered state).
2.8. Lipid Peroxidation
Lipid peroxidation in the suspension of liver mitochondria was estimated spectrophotometrically by measuring the levels of thiobarbituric acid-reactive substances (TBARS). The TBARS assay quantifies the levels of malondialdehyde (MDA) and other minor aldehyde species through their reaction with thiobarbituric acid. The concentration of TBARS was calculated using the molar absorption coefficient of the colored TBA–MDA complex (E535
= 1.56 × 105
2.9. Electrophoresis and Immunoblotting of Mitochondrial Proteins
To prepare samples for quantifying the levels of mitochondrial proteins, aliquots of native mitochondria (2 mg/mL) were solubilized in Laemmli buffer in Eppendorf tubes and heated for 3 min at 95 °C. Aliquots of the mitochondrial samples equalized by the protein concentration (10 µg of mitochondrial protein) were applied to the gel lanes and subjected to electrophoresis followed by Western blot analysis. Mitochondrial samples were separated by 12.5% SDS-PAGE and transferred to a 0.45 µm nitrocellulose membrane (Amersham, Germany). The proteins of PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA) was used as markers. After overnight blocking, the membrane was incubated with the appropriate primary antibody. The monoclonal rabbit anti-MCU (#14997), anti-CBARA/MICU1 antibody (#12524) and anti-ANT2/SLC25A5 (#14671) antibodies were from Cell Signalling technology Inc (Danvers, MA, USA). The total OXPHOS Rodent WB Antibody Cocktail (ab110413), containing α-subunit of complex V (CV-ATP5A-55 kDa), anti-VDAC1 (ab154856) and the polyclonal rabbit antibodies Anti-CCDC109B (ab170715), anti-cyclophilin F (CypD) (ab64935) and anti-ANT1 (ab102032) were from Abcam (Cambridge, GB). The immunoreactivity was detected using the appropriate secondary antibody conjugated to horseradish peroxidase (#7074, Cell Signaling technology Inc., Danvers, MA, USA). Peroxidase activity was detected with ECL chemiluminescence reagents (Pierce, Rockford, IL, USA). The relative levels of the detected proteins were visualized using an LI-COR system (LI-COR, Lincoln, NE, USA). Optical density measurements were performed using LI-COR Image Studio software.
2.10. Fatty Acid Composition of Mitochondrial Phospholipids
The fatty acid composition of mitochondrial phospholipids was determined using the method of gas chromatography. Lipids were extracted from liver mitochondria using the modified method by Bligh and Dyer [24
]. Mitochondria (1 mg/mL) were placed in a centrifuge tube, then 200 μL of water and 900 μL of chloroform/methanol mixture (2:1, v
) were added. The mixture was kept at room temperature for 30 min and stirred periodically. Heptadecanoic acid (C17:0, 15 μg/mL) in hexane was added to each sample as an internal standard. Chloroform and water/methanol layers were separated by centrifugation at 10,000× g
for 10 min at 4 °C. The lower layer of the extract containing the lipid fraction was collected and evaporated to dryness in a stream of argon at 25 °C. The obtained fractions were methylated with one volume of 5% sulfuric acid/methanol (v
) at 100 °C for 3 h. The reaction was stopped by the addition of three volumes of 5% K2
). Methyl ethers were extracted by four volumes of hexane and then evaporated in a stream of nitrogen to a volume of 100 μL. Samples were analyzed using a Chromatek-Crystal 5000 gas chromatography system (Chromatek, Yoshkar-Ola, Russia). On the basis of the chromatograms obtained, the following indexes were calculated: TFA, the total amount of fatty acids; SFA, the total amount of saturated fatty acids; PUFA, the total amount of polyunsaturated fatty acids; and the unsaturation index (UI), which was calculated by multiplying the amount of a fatty acid by the number of double bonds in its structure.
2.11. Statistical Analysis
The data were analyzed using the GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) and Microsoft Excel 2016 (Microsoft, Redmond, WA, USA) software and were presented as means ± SEM of four to five experiments. Statistical differences between the means were determined by a two-tailed t-test; p < 0.05 was considered to be statistically significant.
It is generally accepted that mitochondria are one of the main intracellular targets of DM. In diabetic cells, the mitochondrial network is damaged, the process of ATP synthesis is impaired, and oxidative stress is developed. Diabetes also affects Ca2+ homeostasis of the cell, which is regulated with a direct involvement of mitochondria. Disorders in the system of mitochondrial Ca2+ transport not only affect the bioenergetic parameters of the cell but can also induce cell death via the opening of mitochondrial pores. The purpose of our work was to study in more detail how type-I DM affects the system of Ca2+ transport, as well as the probability of pore formation, in rat liver mitochondria.
Studies on the effects of type-I DM on mitochondrial Ca2+
transport systems have been conducted for a long time. The uptake of Ca2+
by the organelles is primarily achieved through the mitochondrial Ca2+
uniporter complex, the basic component of which is the pore-forming protein MCU. The MCU pore is a highly selective Ca2+
channel, which transfers Ca2+
across the inner mitochondrial membrane and is associated with other subunits, both structural and regulatory: MCUb, MICU1-2, EMRE, and MCUR1 [4
]. The literature data on the effect of type-I DM on mitochondrial Ca2+
transport is rather contradictory. It has been shown, for example, that the development of STZ-induced diabetes lowers the rate of Ca2+
uptake by mice heart mitochondria, and this effect has been revealed to be associated with the decreased expression of MCU [13
]. In this study, we have found that the rate of Ca2+
uptake by liver mitochondria increased significantly in diabetic rats (1). Similar results on liver mitochondria were obtained earlier on the model of alloxan diabetes [12
]. As shown here, the cause for the DM-related stimulation of Ca2+
uptake by rat liver mitochondria is not an elevated expression of MCU, but a decreased expression of the MCU paralogue, MCUb (Figure 2
It is believed that the MCU pore is formed by 4 transmembrane channel subunits, which can be either MCU or MCUb. MCUb is a dominant-negative MCU paralog; its overexpression and presence in the uniporter complex impairs the Ca2+
-transporting properties of the Ca2+
]. Owing to the reducing of the MCUb level, the ratio of MCU/MCUb in diabetic mitochondria grows more than 2-fold (Figure 2
B), and this can explain the enhanced rates of mitochondrial Ca2+
uptake. At the same time, the expression of the gate subunit MICU1 in the liver of STZ-treated rats almost does not change. A similar observation was reported earlier for heart mitochondria [28
]. It can, therefore, be assumed that the DM-related changes in the mitochondrial Ca2+
transport occurs primarily at the level of pore, rather than regulatory, subunits of the uniporter complex. Moreover, the structural-functional alterations in the Ca2+
uniporter complex seem to be tissue-dependent.
As mentioned above, excessive accumulation of Ca2+
in mitochondria leads to the opening of pores in the membrane of organelles: CsA-sensitive MPT pores and CsA-insensitive lipid palmitate/Ca2+
-induced pores [5
]. As a result, transmembrane ion gradients and membrane potential (Δψm
) collapse, and mitochondria swell, this leading to the rupture of their outer membrane and the release of proapoptotic proteins from the organelles. One of the factors facilitating the opening of MPT pores in mitochondria is oxidative stress, and our experiments show that the level of malondialdehyde (TBARS) in the liver mitochondria of STZ-treated rats grows (Figure 7
), indicating an increased lipid peroxidation. Yet, surprisingly, these mitochondria become more resistant to the opening of MPT pores (Figure 3
). Earlier, liver mitochondria of STZ-treated rats were shown to have an increased lag period before they start to swell due to the opening of MPT pores [15
]. The increased resistance of the organelles to MPT might be underlain by metabolic changes, such as an increase in the content of coenzyme Q or cardiolipin [29
]. In any case, this effect seems to depend on the type of tissue: in diabetic heart mitochondria, the probability of MPT pores to open increases [14
To probe into the molecular mechanism of resistance of diabetic liver mitochondria to MPT, we assayed for levels of MPT-associated mitochondrial proteins: Cyclophilin D, ATP synthase, and adenylate translocator isoforms, ANT1 and ANT2 (Figure 4
). It can be seen that in mitochondria of diabetic animals, the level of cyclophilin D remains almost unchanged. There is, however, a significant decrease in the level of ANT1 and ATP synthase—proteins that supposedly form the MPT pore channel. Thus, the increased resistance of diabetic mitochondrial to MPT may be underlain by a lowered expression of the MPT pore-forming proteins.
An important result of this work is the finding that liver mitochondria of diabetic rats become less resistant to the formation of CsA-insensitive palmitate/Ca2+
-induced lipid pores (Figure 5
). Diabetes is known to be accompanied by the impairment of lipid metabolism—and our results show that the content of total fatty acids in diabetic mitochondria grows substantially (Table 2
). This can facilitate the formation of lipid pores in the mitochondrial membrane.
Formation of lipid pores in a membrane depends on the physicochemical properties and structure of its lipid bilayer. It was demonstrated that changes in the lipid composition and microviscosity of the membrane (induced by unsaturated cardiolipin or unsaturated free fatty acids) led to the stimulation of lipid pore formation—both in liposomes and mitochondria [18
]. The results of this work show that the level of polyunsaturated fatty acids in the liver mitochondria of diabetic animals grows significantly (Table 2
). Correspondingly, peroxidation of lipids goes up (Figure 7
). The products of lipid peroxidation can alter membrane packing and increase its microviscosity [32
]—exactly what we observed when diabetic mitochondria were probed with laurdan (Figure 6
). Altogether, these data give an explanation for the increased lipid pore formation in the mitochondria of diabetic animals.
It should be noted that, as lipid pores, the palmitate/Ca2+
-induced pores tend to close spontaneously, which can lead to the restoration of Δψm
. The restoration of Δψm
was demonstrated in our previous work, in which we also suggested that these pores may be a nonspecific system for Ca2+
release from the organelles [33
]. The formation of lipid pores can be considered as an emergency option for quick release of Ca2+
from mitochondria which, in contrast to the CsA-sensitive MPT pore, may not lead to the collapse of Δψm
and damage of the organelles.