3.1. Mitochondrial Ca2+ Transport in Diabetes
Ca
2+ is a universal regulator of many intracellular processes. The appearance of Ca
2+ in the cytosol of pancreatic β-cells is one of the important steps in the mechanism of insulin secretion and regulation of glucose metabolism in humans and animals [
147]. On the other hand, higher intracellular Ca
2+ level has been found in primary adipocytes, hepatocytes, and cardiomyocytes isolated from obese human subjects with insulin resistance as well as diabetic animals [
148,
149,
150]. In this regard, maintaining a low Ca
2+ concentration inside cells is an important function of a number of structures, including mitochondria. Activation of mitochondrial Ca
2+ uptake occurs at a sufficiently high ion concentration in the cytosol. Mitochondria have the ability to rapidly transport and store high concentrations of Ca
2+ in the matrix, which is extremely important for the regulation of calcium homeostasis under stressful conditions. Mitochondrial matrix Ca
2+ regulates a fairly wide range of proteins of the tricarboxylic acid cycle (in particular, pyruvate dehydrogenase, citrate dehydrogenase, and α-ketoglutarate dehydrogenase), respiratory chain complexes, contributing to the maintenance of cell energy metabolism, and ATP generation. It should also be noted that excessive accumulation of Ca
2+ in mitochondria leads to the opening of the MPT pore in the inner membrane and cell death initiation [
84,
151].
Ca
2+ enters the mitochondria through the voltage-dependent anion channel (VDAC) located on the outer mitochondrial membrane. VDAC is one of the components of the MAM (mitochondria-associated membrane) contacts, which allows Ca
2+ released from the endoplasmic reticulum to be immediately redirected via IP
3 receptors into mitochondria. The main component responsible for mitochondrial Ca
2+ handling is the Ca
2+ uniporter of the inner mitochondrial membrane. The uniporter has a remarkably high affinity for Ca
2+, since other divalent cations are transported inside the mitochondria much more slowly and, moreover, inhibit Ca
2+ uptake. It is inhibited by the polycationic dye ruthenium red and its analogues. Nowadays it is recognized that the mitochondrial calcium uniporter is a multicomponent system—the pore channel is formed by MCU integral membrane proteins (there is also an inactive MCU paralogue—MCUb), Ca
2+ uptake is regulated by MICU (MICU1–3) gate proteins, as well as regulatory proteins EMRE and MCUR1. It is important to note that the level of these proteins varies in different tissues, and the ratio of regulatory and channel subunits determines the ability of the mitochondria of a particular tissue to absorb calcium ions. Along with the mitochondrial uniporter, other structures are considered as possible Ca
2+ uptake mechanisms: the rapid mode of uptake or RaM, the mitochondrial ryanodine receptor, and the Ca
2+/H
+ exchanger Letm1. However, their contribution to mitochondrial Ca
2+ uptake is not as significant compared to the Ca
2+ uniporter. The structures responsible for the release of Ca
2+ from mitochondria include Na
+/Ca
2+ and H
+/Ca
2+ exchanges. These systems are supposed to function in different tissues: Na
+/Ca
2+ exchange takes place in excitable tissues, while H
+/Ca
2+ exchange occurs in non-excitable tissues. It has been shown that these are systems of slow release of Ca
2+ from mitochondria, the rate of ion transport through them is much lower than the rate of Ca
2+ entry through the Ca
2+ uniporter. The carrier responsible for the Na
+/Ca
2+ exchange is an antiporter of the inner mitochondrial membrane, capable of releasing Ca
2+ in exchange for Na
+ or Li
+ (NCLX— Na
+/Li
+/Ca
2+ exchanger). It is assumed that Letm1 functions as a Ca
2+/2H
+ exchanger. In addition to these systems of slow release of Ca
2+ from mitochondria, a sharp rapid discharge of mitochondria from Ca
2+ occurs by the opening of non-specific Ca
2+-dependent mitochondrial pores. The balance between mitochondrial calcium entry and release is responsible for the maintenance of intracellular Ca
2+ homeostasis under normal and pathological conditions. More details about mitochondrial Ca
2+ transport systems are described in reviews [
84,
133,
151,
152].
Intramitochondrial Ca
2+ is shown to be involved in the regulation of insulin secretion in pancreatic β-cells under normal conditions [
147]. The intake of glucose in β-cells leads to the accumulation of Ca
2+ in the mitochondria, an increase in the concentration of ATP in the cells, and insulin secretion. Indeed, MCU knockout mice show inhibition of the first phase of insulin secretion [
153]. It should also be noted that glucolipotoxicity is associated with suppression of Ca
2+ transport in the mitochondria of β-cells and an increase in the level of ATP in the cytosol. However, the expression of MCU and NCLX did not change. The authors suggested that the dysregulation of Ca
2+ transport in the mitochondria of β-cells under glucolipotoxicity is due to a change in the ultrastructure of organelles [
154]. At the same time, palmitic acid upregulated MCU protein expression in mouse clonal β-cell MIN6 under normal glucose, but not high glucose medium. The authors suggested that high glucose attenuates the compensatory mechanism involving MCU in palmitate-induced cytotoxicity and causes further serious consequences related to Ca
2+ overload in β-cell lipotoxicity [
155].
One of the diabetes-related pathological changes in β-cells is the development of ER stress [
156]. Along with the suppression of Ca
2+ transport in mitochondria, this causes an increase in free Ca
2+ in the cytoplasm, which, in turn, can lead to an imbalance of diverse Ca
2+-dependent signaling pathways. In addition, the induction of mitochondrial dysfunction and ER stress in the pancreas may eventually result in the death of β-cells and an increase in diabetic complication rates.
Data on the functioning of the calcium uniporter in diabetic tissues are quite contradictory (
Figure 2). A decrease in the rate of Ca
2+ transport was observed in heart mitochondria in streptozotocin- induced T1DM rats and in T2DM
ob/ob mice, as well as in pancreatic cells [
154,
157,
158,
159]. Heart mitochondria also showed a decrease in MCU expression in a murine model of streptozotocin-induced T1DM, which was accompanied by a suppression of mitochondrial Ca
2+ uptake [
160]. An elevated level of the dominant negative MCUb subunit of the uniporter is also expected to contribute to this picture. Indeed, normalization of the MCU level in hearts restored mitochondrial Ca
2+ handling, increased pyruvate dehydrogenase activity, and reprogrammed a metabolism toward normal glucose oxidation [
160,
161]. In addition, the heart of
db/db mice showed reduced expression of the peripheral membrane MiCU1 protein acting as a gatekeeper. The reconstitution of MiCU1 in diabetic hearts significantly inhibited the development of diabetic cardiomyopathy by increasing mitochondrial Ca
2+ uptake and subsequently activating the antioxidant system [
162].
On the other hand, there is evidence that the development of diabetes is accompanied by activation of the calcium uniporter. Indeed, as far back as the 70s, it was shown that the induction of alloxan diabetes stimulates the entry of Ca
2+ into liver mitochondria [
163]. Recently, we also demonstrated that two weeks after streptozotocin administration to Sprague-Dawley rats, the rate of Ca
2+ uptake by liver mitochondria significantly increased. The analysis showed that an increase in the Ca
2+ transport rate was due to a decrease in the expression of the dominant-negative MCUb subunit of the uniporter [
41]. Adipose tissue mitochondria also show an increase in Ca
2+ handling. It was shown that mitochondrial Ca
2+ uptake increased and MCU components (MCU and MICU1) were upregulated in insulin-resistant adipocytes. Similar results were observed in mouse (
db/db and
ob/ob) and human visceral adipose tissue during the progression of obesity and diabetes [
164].
The development of diabetes mellitus changes not only the activity and expression of Ca
2+ uniporter, but also NCLX. Indeed, the endothelia of streptozotocin-induced T1DM rats demonstrated an increase in NCLX expression. In this case, silencing of NCLX expression increased ROS generation and NLRP3 inflammasome activation [
165].
Insulin resistance and T2DM cause a disruption in the structure of MAM contacts [
157,
166,
167,
168]. The antidiabetic drugs metformin and rosiglitazone restore the structure of MAM contacts in diabetic animals [
168]. It should be noted that diabetes mellitus is associated with overexpression of VDAC1 in certain tissues (pancreatic β-cells, vascular endothelial cells) [
169,
170,
171]. In parallel, an increased amount of Ca
2+ accumulates in mitochondria, which ultimately leads to the activation of apoptosis. Inhibition of VDAC1 overexpression leads to the suppression of apoptosis in endothelial cells and improves insulin secretion in islets [
170,
171].
The contradictions observed in studies of mitochondrial Ca2+ transport in diabetes mellitus are difficult to explain. It is possible that the development of diabetes shows tissue specificity. As mentioned above, liver cells react differently to diabetes. In particular, this organ shows PGC1-1α overexpression and stimulation of biogenesis. It is worth noting that similar adaptive changes may possibly occur in other tissues in the early stages of diabetes. Several studies on the induction of diabetes have shown an increase in the concentration of Ca2+ in the cytosol and mitochondria. In this regard, it can be speculated that under these conditions, the observed activation of Ca2+ uptake and release systems from mitochondria will lead to Ca2+ recyclization through the mitochondrial membrane (futile cycle) and ΔΨ decrease. Like UCP expression, this adaptive reaction will suppress oxidative stress, at least in the initial stages of the development of the disease. Such a futile cycle, causing a slight depolarization, is expected to stimulate mitophagy. Along with increased biogenesis, this will trigger the renewal of the mitochondrial population in the cell. Meanwhile, excessive accumulation of Ca2+ in mitochondria will undoubtedly cause the opening of the MPT pore and cell death initiation.
3.2. Mitochondrial Permeability Transition Pore
Excessive accumulation of Ca
2+ in the mitochondrial matrix is known to lead to an abrupt increase in nonspecific permeability of the inner mitochondrial membrane (referred to as the mitochondrial permeability transition (MPT) pore) for various ions and hydrophilic compounds with a molecular weight of up to 1.5 kDa. This leads to swelling of the mitochondria, equilibration of ionic gradients across the inner membrane, a decrease in the mitochondrial membrane potential, and impaired ATP synthesis. The final consequence of the opening of the MPT pore is cell death. Ca
2+-dependent permeabilization of the inner mitochondrial membrane is one of the key elements in the process of cell death during hypoxia and subsequent reoxygenation. Moreover, convincing evidence has accumulated over the years supporting an essential role of the MPT pore opening in the development of cardiovascular diseases, neurodegenerative processes, viral diseases, muscular dystrophies, etc. [
84,
172,
173,
174,
175].
By the mid-90s of the last century, most of the modulators of the MPT pore had been elucidated. This is described in detail in a review by Zoratti and Szabo. Ca
2+ is perhaps the main pore activator. In addition to Ca
2+, inorganic phosphate (and polyphosphates), SH-oxidizing agents, oxidative stress, uncouplers, a decrease of the mitochondrial adenine nucleotide content, and other factors stimulate MPT pore opening. Inhibitors of the mitochondrial pore are the cyclosporins (cyclosporin A is most effective), adenine nucleotides, SH-reducing agents, reduced pyridine nucleotides, etc. [
175].
Despite significant progress in the study of MPT pore induction and regulation, its molecular structure and protein composition are still under discussion (
Figure 3). An analysis of the literature data suggests that the MPT pore is a nonselective, high-conductance megachannel consisting of proteins of the inner and outer mitochondrial membranes. However, to date, cyclophilin D is the only, precisely established component of this structure; it is the pharmacological target of cyclosporin A and its analogues, which can specifically block pore opening [
176,
177]. Cyclophilin D is considered as a regulatory protein, which in the presence of Ca
2+ stimulates rearrangement in the proteins responsible for the formation of the MPT pore channel. Knockout of cyclophilin D or its binding to an inhibitor leads to a significant increase in the threshold concentration of Ca
2+ necessary for the pore opening. In 2015, using RNAi-based screening, it was suggested that, along with cyclophilin D, spastic paraplegia 7 (SPG7) is an important regulatory component of MPT [
176]. However, it has recently been shown that SPG7 does not constitute a core component of MPT, but instead regulates activity by lowering the basal mitochondrial Ca
2+ levels via regulation of MCUR1 and Ca
2+ uniporter assembly [
178].
Several MPT models have been proposed over the past 40 years. Indeed, only cyclophilin D is an integral component of the pore. In this case, proteins of the outer membrane and intermembrane space (VDAC, TSPO, HK, and CrK) are auxiliary in the assembly of the pore complex in intact mitochondria [
179]. However, the question of which protein is the main pore component in the inner mitochondrial membrane has not yet been resolved. Until the mid-2000s the prevailing hypothesis was that such a channel-forming pore protein of the inner mitochondrial is adenine nucleotide translocator. This assumption was because the adenine nucleotide translocator inhibitors, atractyloside and carboxyatractyloside, stimulated pore opening, and bongkrekic acid showed an inhibitory effect. Adenine nucleotides carried by the translocator under normal conditions also suppressed the pore opening [
175]. In addition, it was shown that ANT is able to bind to VDAC and hexokinase, as well as to cyclophilin D, forming channels in liposomes whose properties resemble MPT [
180,
181,
182]. However, the discovery that the opening of the MPT pore also occurred in the mitochondria from ANT1 and ANT2 null mice led to the abandonment of this model [
183]. After this, the idea of a phosphate carrier as a channel component of the MPT pore was considered [
184].
According to the data of the last decade, mitochondrial ATP synthase is considered the main candidate for the role of the channel-forming component of the MPT pore, whose subunits and, in particular, OSCP, are able to combine with cyclophilin D, which, as expected, leads to pore opening [
84,
172,
173,
174]. In addition, it was shown that the OSCP subunit of ATP synthase contains a unique pH-sensitive histidine (H112), which has a significant modulating effect on the opening of the MPT pore [
185]. Several suggestions have been made as to how ATP synthase can form an MPT pore channel. Two main hypotheses can be distinguished: (1) «dimer» and (2) «c-ring» [
174]. According to the first one, ATP synthase dimers, but not monomers, conduct currents when inserted into planar lipid bilayers which are activated by Ca
2+ and oxidizing agents and closed by ADP/Mg
2+ (established MPT pore desensitizers) [
186]. Further, it was shown that currents were strongly attenuated in yeast mutants that lacked ATP synthase subunits e and g necessary for the formation of dimers [
187]. Indeed, channel formation by ATP synthase dimers was shown on mitochondria of evolutionarily distant species (
S. cerevisiae, and
D. Melanogaster), which makes this hypothesis quite convincing [
187,
188,
189]. At the same time, it was found that the ATP synthase monomer is sufficient, and dimer formation is not required, for MPT pore activity [
190]. According to an alternative «c-ring» hypothesis, the c-subunit of ATP synthase localized in the inner membrane of organelles may act as a channel component of the MPT pore [
191,
192]. In this case, it is assumed that Ca
2+ and (or) ROS-induced dissociation of the F1 sector of ATP synthase leads to conformational changes in the c-ring of the Fo sector, which allows the formation of an MPT pore channel. However, this hypothesis runs into a series of contradictions. Indeed, in this case, the process of dissociation must be fast and reversible. First, there must be a quick detachment of the F1 sector, and second, the c-ring channel must be emptied and hydrated, which makes this hypothesis unlikely [
174,
193]. Moreover, the data of model experiments suggest that the hypothetical MPT pore based on the c-ring will have a significantly lower conductivity than that shown for MPT [
194]. These data are supported by the results of patch-clamp measurements. Finally, it has recently been shown that mitochondria of mutant cells with disrupted c-subunits of ATP synthase still display a cyclosporin A-sensitive Ca
2+-induced MPT pore opening and, moreover, in some cases demonstrate increased sensitivity to the induction of this process [
195].
Recent data again bring us back to the question of ANT as a structural element of the MPT pore. Indeed, it has recently been shown that the MPT pore in c-subunit-deficient mitochondria is sensitive to cyclosporin A, ADP, and bongkrekic acid [
195]. Finally, knockout of the genes of three ANT isoforms (not two, as in 2005) significantly increased mitochondrial resistance to MPT induction and the calcium capacity of organelles, nevertheless, did not prevent it [
196]. These contradictions led to the emergence of a model for the joint participation of ANT and ATP-synthase in the MPT pore opening. It is assumed that, depending on the threshold concentration of Ca
2+, these proteins will form channels of various currents that provide different modes of MPT functioning [
197] and contribute to both the rapid release of Ca
2+ and metabolites and the maintenance of the functioning of the futile cycles mentioned above.
It should also be mentioned that the search for MPT modulators made it possible to establish conditions when the mitochondrial Ca
2+-dependent swelling was insensitive to cyclosporin A. This type of permeabilization includes the lipid pore induced by saturated fatty acids and Ca
2+. In our previous papers, we described this type of permeabilization of the mitochondrial membrane in sufficient detail [
84,
198,
199]. We found that the opening of this pore occurs by the mechanism of a chemotropic phase transition in a lipid bilayer. Indeed, palmitic acid in the presence of Ca
2+ was able to permeabilize both natural and artificial membranes. The physiological significance of this pore has also been described [
200]. The features of the formation and physiological significance of cyclosporin A-insensitive lipid pore are presented in more detail in our previous review [
84].
3.4. MPT Pore as a Target for Diabetes Management
The issue of MPT pore as a target in the treatment of diabetes mellitus is rather complicated. According to a wide range of studies, cyclosporin A and other MPT pore inhibitors contribute to the suppression of mitochondrial dysfunction and improve the quality of life of animals [
217,
219,
232]. Administration of high-dose cyclosporin A has been demonstrated to induce remission of type 1 diabetes mellitus [
240]. On the other hand, the same cyclosporin causes suppression of mitochondrial biogenesis in liver cells [
220]. Mice with the deletion of cyclophilin D show the development of hyperglycemia, insulin resistance, and glucose intolerance, albeit resistant to diet-induced obesity [
241]. However, it should be noted that the in vivo interpretation of the effects of cyclosporin A as an MPT inhibitor may not always be correct. Cyclosporin A is a well-known immune suppressor, and its effect can be associated not only with the suppression of MPT, but also with the effect on various signaling pathways in humans and animals.
Synthetic and natural antidiabetic compounds exhibit a bi-directional effect on MPT pore opening. Notably, metformin has been shown to inhibit MPT pore opening in mitochondria, enhances biogenesis, and prevents cell death [
61,
242,
243]. However, there is an evidence that metformin stimulates MPT in rat liver mitochondria [
244]. The thiazolidinedione class of antidiabetic agents also shows a similar stimulating effect on MPT pore [
245]. Moreover, troglitazone enhanced MPT pore induction in the liver of diabetic Zucker (
fa/fa) rats [
246]. The natural polyphenolic compound luteolin, reduces mortality from coronary artery diseases, including diabetes [
107]. It has been shown to inhibit MPT pore opening [
247]. On the other hand, the plant alkaloid berberine, used in traditional Chinese medicine and possessing antidiabetic properties [
248], causes an inhibition of mitochondrial respiration and a decrease on Ca
2+ loading capacity through induction of the mitochondrial permeability transition [
249]. All this suggests that it is necessary to carefully approach the issue of diabetes mellitus therapy through the modulation of MPT pore activity.