*7.1.1. Tricarboxylate carrier*

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

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

Altered metabolism of pyruvate resulting from the inability to transfer pyruvate is present in cancer and other metabolic diseases. Pyruvate metabolism and carbon flux are altered in many cancer cells. Metabolic switch to enhanced glycolysis and decreased oxidative phosphorylation (Warburg effect) leads to elevated lactate production, which is advantageous for cancer

is proton-linked transport of lactate out of the cell, increasing the acidity of the extracellular space. Acidification of the extracellular environment provides protection from the immune system [144]. Furthermore, lactic acid appears to influence the activity of matrix metalloproteinases breaking down the extracellular matrix aiding in tumor proliferation and metastasis [145] and can be utilized as fuel source by cancer cells located at the surface of the tumor [146]. Schell et al. [147] found *MPC1* genomic locus as the most frequently deleted region across cancer cells, while *MPC2* locus does not appear to be frequently lost. MPC1 underexpression correlates with poor survival in almost all cancers examined, including colon, kidney, lung, bladder, and brain [147]. The correlation of survival with *MPC2* expression is more variable,

Increased pyruvate levels in cerebrospinal fluid reflecting an impaired metabolism of pyruvate have been detected in neurodegenerative disorders including Leigh's syndrome, Alzheimer's disease, and Parkinson's disease [148, 149]. Neuronal metabolism depends upon the uptake of lactate produced by astrocytes (astrocyte-neuron lactate shuttle), its conversion to pyruvate by LDH, and subsequent oxidation in mitochondria to form energy. Glucose is shifted into the pentose phosphate pathway for the NADPH generation to maintain reduced glutathione levels [150]. Due to the lack of pyruvate metabolism in neurodegenerative diseases, synthesis of acetylcholine is also insufficient because it requires acetyl-CoA [143]. To the present, there are not many findings available about MPC inhibition in neuronal cells except for α-cyano-4-hydroxy cinnamate or phenylpyruvate effects. Most likely, MPC efficacy and susceptibility to disease progression are also related to genetic predisposition. Mitochondrial pyruvate supply restriction can also display a neuroprotective effect by increase in glutamate oxidation. Maintaining the levels of synaptic glutamate during glutamatergic neurotransmission comes at energetic cost leading

for the continuation of glycolysis. Another

pyruvate are the mutations in MPC1 and/or MPC2 genomic loci [133, 141].

mitochondria independently of the MPC could compensate for loss of the MPC [143].

**7. Aberrant pyruvate transfer**

264 Mitochondrial Diseases

cells. The first advantage is regeneration of NAD<sup>+</sup>

but associated with poor prognosis in kidney and colon cancer [147].

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 tricarboxylate-H+ , dicarboxylate (malate and succinate), or phosphoenolpyruvate [156]. An importance 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]. Except for decisive interconnection between lipogenesis, gluconeogenesis, and glycolysis, CiC has been proposed to play a role in the maintenance of chromosome integrity and in the regulation of autophagy [161]. A particularly important role played by the CiC is in the regulation of insulin secretion by providing isocitrate for NADP-dependent isocitrate dehydrogenase [162].

oxoglutarate and aspartate. The oxoglutarate is then transported by OGC. Binding the succinate to the matrix side of the carrier increases the affinity for malate, while phenylsuccinate, pyridoxalphosphate, retinoic acid, or alcohol was found to inhibit OGC [171, 172]. The OGC inhibition causes a decrease in mitochondrial reduced glutathione levels by 40–50% [138]. The carrier has also been proposed as a porphyrin transporter, and its inhibition blocks porphyrin conversion to heme in mitochondria. The downregulation of OGC has been reported in horse muscle with

Another one from SLC25 family is a glutamate carrier. Two glutamate-transfer isoforms are known for the glutamate carrier: GC1 (SLC25A22) and GC2 (SLC25A18). They transport glutamate across the MIM in symport with a proton or in exchange for hydroxyl ions. In mitochondria, glutamate is converted by glutamate dehydrogenase into α-ketoglutarate while

respiratory chain. The mRNAs of GC1 have been found to be highly expressed than that of GC2 in liver, pancreas, and kidney but are similar in the brain. Moreover, the *K*m and *V*max values are higher than those of GC2 (5.2 vs. 0.26 mM; 12.2 vs. 3.9 μmol/min/g of proteins). Therefore, an acceptable explanation, for the expression pattern is that GC2 is responsible for

GC1 has been demonstrated to have an important physiological function in the control of glucose-stimulated insulin secretion in pancreatic β cells [96]. The signaling mechanism leads to adjustment of insulin release to levels greater than the sole contribution of Ca2+-induced triggering pathway in rats. Stimulation of β cells with high glucose might result in rapid saturation of the respiratory chain [175], favored by glucokinase properties (as mentioned in Section 4.1.) and low lactate release. Saturated electron transport chain would then promote export of metabolites (GTP, citrate, NADH, and glutamate) out of the mitochondria compensated by activity of anaplerotic pathways [176]. Energetic sufficiency favors the glutamate dehydrogenase reaction from α-ketoglutarate toward glutamate formation. Glutamate is taken up by secretory granules, which are consistent with the expression of vesicular glutamate transporters (VGLUT1 and 2) in insulin-secreting cells [177]. Inside the secretory granule, glutamate could induce pH changes and activate metabotropic receptors mGlu5, thereby mediating insulin release [178]. Alternative mechanisms include activation of acetyl-CoA carboxylase

GC1 is highly expressed in astrocytes from different structures (retina, spinal cord, and cortex) [180] and represents the principal gate for glutamate entry into the mitochondria of astrocytes. Restricting glutamate access to mitochondria results in reduced ATP and NAD(P)H formation. A defective glutamate carrier may lead to glutamate accumulation in the astrocytes cytosol and then to glutamate liberation in the synaptic cleft. The release could result in neuronal synchronicity, which may contribute to the generation of epileptic-like discharges in the brain [181]. Mutations in the human *GC1* gene (localized on chromosome 11p15.5) are responsible for the autosomal recessive form of early infantile epileptic encephalopathy

the basic function, whereas GC1 functions in tissues with increased demands [174].

and inhibition of phosphatase activities involved in insulin exocytosis [179].

. Ammonia is released and reduced coenzyme enters the complex I of

Nuclear Encoded Mitochondrial Proteins in Metabolite Transport and Oxidation Pathway…

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

267

recurrent exertional rhabdomyolysis [173].

**8.1. Mitochondrial glutamate carriers**

*8.1.1. Glutamate carrier*

reducing NAD (P)+

The studies have shown that the CiC activity and properties could be changed under specific conditions: starvation-induced decrease of CiC activity and considerable reduction of CiC mRNA in starved rats. The reduced CiC mRNA levels were ascribed to shortened half-life and accelerated degradation of CiC mRNA. Refeeding, however, leads to renewal of mRNA and increased activity of CiC [163]. The polyunsaturated fatty acid (PUFA) administration also dramatically affects CiC gene expression by transcriptional and posttranscriptional mechanisms. Rat liver mitochondria showed more decreased CiC activity and reduced transcriptional rate of CiC mRNA when affected by n-3 PUFA than by n-6 [164]. During the inflammation, CiC gene expression is activated by NF-κB, which causes an increased availability of cytosolic acetyl-CoA and NADPH + H+ for synthesis and activity of compounds and enzymes involved in inflammatory response (e.g., COX2, iNOS, and NADPH oxidase) [156].

The human SLC25A1 gene is localized on chromosome 22, within the region associated with allelic losses in DiGeorge/22q11 syndrome, velo-cardio-facial syndrome, and a subtype of schizofrenia [165]. To date, recessive mutations of CiC gene in 20 persons with combined D,L-hydroxyglutaric aciduria were described. The clinical phenotype of disorder is characterized by severe developmental delay, hypotonia, seizures, secondary microcephaly, hypoplasia or agenesis of the corpus callosum, optic nerve hypoplasia, dysmorphic feature, lactic acidosis, and recurrent apneic crises [166]. CIC has also been reported to be upregulated in ovarian and colon cancer [167].

#### **7.2. Dicarboxylate carrier**

Dicarboxylate carrier protein ((DIC) SLC25A10) transporting malate and succinate out of mitochondria in exchange for Pi is ubiquitously expressed in mammalian mitochondria. The carrier is inhibited by Pi and other phosphate and substrate analogues. Malate exchange for Pi provides a cytosolic source of malate for CiC and therefore plays a significant role in fatty acid synthesis [168]. DIC interacts with malate dehydrogenase by acting as an oxaloacetate shuttle, thus improving functional coupling of citric acid cycle with shuttle. In the cytosol, malate is converted into oxaloacetate following conversion into phosphoenolpyruvate by carboxykinase-catalyzed reaction. The reaction is rate-limiting for gluconeogenesis. No less important role of the carrier is the transport of reduced glutathione into the mitochondria shared with OGC. Therefore, limiting protein expression results in significantly reduced levels of glutathione in the mitochondria and subsequent altered redox conditions [169]. It was found that the activity of DIC is increased in type I diabetes, in contrast to decreased activities of CiC [170].
