**6.2. Mitochondrial phosphate carrier**

The role of mitochondrial phosphate carrier (PiC, SLC25A3) is importing inorganic phosphate into the mitochondrial matrix. A part of ATP synthasome enables efficient energy production, since Pi is essential for F1 F0 -ATP synthase to catalyze formation of ATP from ADP. For PiC, two isoforms differing in alternative splicing of mutually exclusive exon are documented in tissue-specific expression pattern. PiC-A is expressed in heart and skeletal muscle, while PiC-B is expressed in liver, kidney, and other tissues [128]. The PiC gene is located on chromosome 12q23.1. In 2007, the presence of a homozygous mutation in PiC-A was found in two siblings of nonconsanguineous Turkish parents. Given the role of PiC in energy production, the clinical manifestation of PiC deficiency is associated with multisystemic disorder characterized by muscle hypotonia, lactic acidosis, severe hypertrophic cardiomyopathy, and shortened lifespan [129, 130]. Besides that, PiC has been suggested to impact mtPTP opening [131].

#### **6.3. Mitochondrial pyruvate carrier**

increased glycolysis, maintaining mitochondrial membrane potential, and finally prevention of apoptosis [115]. The effect of hexokinase has been described in Section 4.1. Similarly, the ATP synthasome, a complex of ANT, F0F1-ATPase, and phosphate carrier (PiC), facilitates a mechanism for adenine nucleotide and pyrophosphate release. Under pathological conditions, the imported ATP may also be hydrolyzed by F0F1-ATPase to maintain mitochondrial membrane potential [115]. This type of hydrolysis has also been reported in order to prevent neuro-

Roussel et al. [119] found isoforms ANT1 and 2 to mediate uncoupling by fatty acids and to lower mitochondrial membrane potential in heart and skeletal muscle in rats. ANT is inhibited by competitive displacement of the nucleotide by long-chain acyl-carnitines (LCAC) [120]. LCAC may accumulate under pathological conditions with excess lipid supply, obesity, and mitochondrial β-oxidation defects [121]. ANT inhibition is thought to contribute to

The ANT function in subcellular compartment energy supply is important and therefore it is not surprising that the altered structure and subsequently properties of this solute carrier protein associated with DNA mutations are also associated with serious clinical conditions. Mutations linked to mitochondrial disorders with autosomal recessive inheritance cause mitochondrial myopathy and cardiomyopathy presented in childhood or early adulthood. It is characterized by lactic acidosis, fatigue, proximal muscle weakness, and exercise intolerance [123, 124]. Several different autosomal-dominant mutations in ANT1 gene have been associated with an adulthood-onset disorder, autosomal-dominant progressive external ophthalmoplegia, characterized by ptosis, restriction of eye movement, and accumulation of clonally expanded mtDNA deletions in postmitotic tissues [125, 126]. Thompson et al. [127] have recently described recurrent *de novo*–dominant mutation with severe early-onset of mitochondrial disease. Mutations in the gene encoding ANT1 are associated with the presence of mtDNA deletions. The most likely mechanism of how the defective carrier affects the appearance of mtDNA mutations is the unsufficient adenine nucleotide availability for dATP

The role of mitochondrial phosphate carrier (PiC, SLC25A3) is importing inorganic phosphate into the mitochondrial matrix. A part of ATP synthasome enables efficient energy

ADP. For PiC, two isoforms differing in alternative splicing of mutually exclusive exon are documented in tissue-specific expression pattern. PiC-A is expressed in heart and skeletal muscle, while PiC-B is expressed in liver, kidney, and other tissues [128]. The PiC gene is located on chromosome 12q23.1. In 2007, the presence of a homozygous mutation in PiC-A was found in two siblings of nonconsanguineous Turkish parents. Given the role of PiC in energy production, the clinical manifestation of PiC deficiency is associated with multisystemic disorder characterized by muscle hypotonia, lactic acidosis, severe hypertrophic cardiomyopathy, and shortened lifespan [129, 130]. Besides that, PiC has been suggested to


F0

degeneration [117] and in activated macrophages [118].

262 Mitochondrial Diseases

mitochondrial defects in metabolic syndrome [122].

synthesis and consequent imbalance in dNTP pools [127].

**6.2. Mitochondrial phosphate carrier**

production, since Pi is essential for F1

impact mtPTP opening [131].

Important one is another of the mentioned carriers. Pyruvate is the end product of glycolysis in the cytosol. In mitochondria, pyruvate entering the tricarboxylic acid cycle supports the ATP generation but also serves as a link to anabolic pathways for lipid, amino acid biosynthesis, and gluconeogenesis. The main sources of pyruvate in the cytoplasm are reactions catalyzed by pyruvate kinase. Two more sources are lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), which are important to mention in terms of linking metabolic pathways between tissues. Reversible transamination of pyruvate and glutamate to alanine and α-ketoglutarate catalyzed by ALT converts the pyruvate from muscles into a transport form, alanine, which is reused in the liver for gluconeogenesis. LDH reversibly catalyzes reduction of pyruvate to lactate concurrently with oxidation of NADH to NAD+ . Enzyme gains importance especially in muscle tissue in conditions requiring excessive energy production and in cancer cells. During increased need for ATP for muscle activity, the energy requirements to support continued muscle activity exceed mitochondrial capacity for ATP production. As glycolysis requires NAD+ , ATP production is limited when NAD+ depletes and NADH accumulates. In this case, LDH ensures ATP production in glycolysis by regenerating NAD+ . With a steady supply of NAD<sup>+</sup> , and until acidosis becomes limiting, glycolysis can produce ATP to support work rates exceeding those that could be supported by oxidative phosphorylation alone [132]. The lactate is transported to the circulatory system from where it is taken up by the liver and converted back into pyruvate.

The cross-connection of pyruvate with catabolic and anabolic pathway in mitochondria depends on its passage through mitochondrial membranes. Pyruvates cross MOM through VDAC; however, transport through MIM requires specific carrier. Although the existence of carrier was known earlier, the existence of genes on chromosome 6q27 encoding of mitochondrial pyruvate carrier (MPC) formed by hetero-oligomeric complex of two proteins, MPC1 and MPC2, has been revealed recently. Both proteins are needed for sufficient activity [133, 134]. Proteins do not contain any sequence homology to other mitochondrial carriers. Instead, they have been proposed to belong to the PQ-loop/MtN3/MPC superfamily [135]. Members of PQ-loop family are located in a variety of organelles performing diverse functions. They combine common characteristic features that are seven transmembrane domains and two conserved glutamine motifs. Subunits MPC1 and 2 contain three of seven transmembrane domains and are only half size of other PQ-loop family members [136]. Pyruvate uptake has been proposed to be coupled with the electrochemical gradient, occurring with the symport of one proton, or exchange with one hydroxide ion [137].

MPC activity increases in response to glucagon and decreases in response to insulin. Adrenaline and cortisol also have been found to increase pyruvate carboxylation by increasing mitochondrial pyruvate import [138]. The MPC2 transcript levels have been found to increase up to 1.5-fold under fasting conditions. In a physiological response to fasting (e.g., excessive exercise or prolonged food deprivation), hepatic mitochondrial ketone export and pyruvate import through MPC allows to enhance hepatic gluconeogenesis and maintains membrane potential [132]. Conversely, unregulated rate of gluconeogenesis contributes to chronic hyperglycemia in diabetes. Except for some substances (e.g., α-cyano-4-hydroxy cinnamate, UK-5099, and several thiazolidinediones) acting as specific MPC inhibitors, inhibitory effects of α-ketoacids and phenylpyruvate were found [139, 140]. The accumulation of phenylpyruvate in phenylketonuria prevents pyruvate transfer to mitochondria. Malate was shown to significantly increase mitochondrial pyruvate uptake while not affecting affinity. A genetic background of diminished pyruvate utilization due to pyruvate transport deficiency accompanied by lactic acidosis resulting also from the described metabolic possibilities of pyruvate are the mutations in MPC1 and/or MPC2 genomic loci [133, 141].

to periods of increased levels of glutamate. High levels of glutamate cause complex I inhibition through receptor-stimulated Ca2+ overload, which is an attribute of acute neuropathologies [151]. MPC1 and MPC2 are highly expressed in brown adipose tissue compared with other tissues [152]. Brown adipocytes use predominantly fatty acids as an energy source for uncoupled respiration and thermogenesis, which requires replenishment of oxaloacetate through pyruvate carboxylation to enter citric acid cycle. MPC is supposed to be important in shifting between

Nuclear Encoded Mitochondrial Proteins in Metabolite Transport and Oxidation Pathway…

http://dx.doi.org/10.5772/intechopen.72937

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Alteration in pyruvate metabolism plays a conspicuous role in heart disease. Heart muscle predominantly metabolizes fatty acids, ketone bodies, lactate, and glucose depending on their availability and neurohormonal signaling. Up to 95% of the heart's ATP generation comes from mitochondrial oxidation, and typically approximately 60–90% of this mitochondrial ATP production comes from fatty acids, whereas 10–40% is from pyruvate oxidation. The myocardium is a significant consumer of lactate even at the maximum load, because of specific expression of LDH-B isoform preferring reaction catalysis toward pyruvate [153]. It was found that acute stress (such as ischemia) and chronic stress (hypertrophy and heart failure) change substrate availability and metabolism [143]. Reduction in pyruvate oxidation leads to increased anaerobic glycolysis and lactate formation. However, age-related decrease in MPC activity was observed [154]. Shift in substrate utilization in order to maintain citric acid cycle can lead to serious states of energy deficiency called "starved heart." The effect was observed in cancer treatment with doxorubicin reducing carnitine transport followed by reduced fatty acid oxidation [155].

The tricarboxylate carrier (SLC25A1) catalyzes an electroneutral exchange of the dibasic form of a tricarboxylic acid (citrate, isocitrate, and cis-aconitate) with proton for another tricarbox-

of the citrate carrier (CiC) results from formation of a link between carbohydrate catabolism and lipogenesis. The CiC overlaps with oxoglutarate carrier ((OGC) SLC25A11) by transporting the 2-oxoglutarate in exchange for malate and malonate [157]. CiC facilitates transport of citrate across MIM, followed by passive diffusion through VDAC in MOM into the cytosol. In the cytosol, fatty acids and cholesterol are synthesized from citrate. Citrate also acts as inhibitor of phosphofructokinase 1, thus affecting the rate of glycolysis, positive allosteric modulator of acetyl-CoA carboxylase in fatty acid synthesis pathway, and serves as a substrate for the formation of malate, the conversion of which into pyruvate facilitates NADPH production necessary for lipogenesis [158]. High CiC mRNA levels in liver, kidney, and pancreas; lower levels in heart, skeletal muscle, and placenta; and no detectable mRNA in brain and lung were detected [128]. High liver and kidney CiC mRNA levels are supposed to be due to gluconeogenesis and lipogenesis. Moreover, CiC plays a role in gluconeogenesis from lactate where phosphoenolpyruvate carboxykinase is located in mitochondria. Similarly as in other animals, mitochondrial phosphoenolpyruvate isoforms are present mainly in the liver, kidney, and adipose tissue [159]. The relatively high CiC mRNA level in pancreas could be explained with the role of CiC in regulation of insulin secretion. On the other hand, the low CiC mRNA level in skeletal muscle correlates to the very low activity of gluconeogenesis and fatty acid synthesis [160].

, dicarboxylate (malate and succinate), or phosphoenolpyruvate [156]. An importance

formation and oxidation of fatty acids in fat cell metabolism.

**7.1. Mitochondrial di- and tricarboxylic acid transport**

*7.1.1. Tricarboxylate carrier*

ylate-H+

A 13C metabolic flux analysis of cells after transcriptional or pharmacological inhibition of MPC, published by Vacanti et al. [142], revealed that inhibition of MPC activity leads to shift from glucose to amino acid and fatty acid oxidation. Citric acid cycle and fatty acid synthesis were maintained due to malic enzyme flux, glutaminolysis, fatty acid, and branched chain amino acid oxidation. Alternatively, pyruvate interconversion into gluconeogenic substrates (e.g., alanine) that can enter mitochondria independently of the MPC could compensate for loss of the MPC [143].
