Roberto Pizzuto

Mitochondria and l-lactate metabolism

We review here the novel insights arisen from investigations on l‐lactate metabolism in mammalian, plant and yeast mitochondria. The presence of l‐lactate dehydrogenases inside mitochondria, where l‐lactate enters in a carrier‐mediated fashion, suggests that mitochondria play an important role in l‐lactate metabolism. Functional studies have demonstrated the occurrence of several l‐lactate carriers. Moreover, immunological investigations have proven the existence of monocarboxylate translocator isoforms in mitochondria.

Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings.

Abiotic stresses, such as high salinity or drought, can cause proline accumulation in plants. Such an accumulation involves proline transport into mitochondria where proline catabolism occurs. By using durum wheat seedlings as a plant model system, we investigated how proline enters isolated coupled mitochondria. The occurrence of two separate translocators for proline, namely a carrier solely for proline and a proline/glutamate antiporter, is shown in a functional study in which we found the following: (1) Mitochondria undergo passive swelling in isotonic proline solutions in a stereospecific manner. (2) Externally added l-proline (Pro) generates a mitochondrial membrane potential (ΔΨ) with a rate depending on the transport of Pro across the mitochondrial inner membrane. (3) The dependence of the rate of generation of ΔΨ on increasing Pro concentrations exhibits hyperbolic kinetics. Proline transport is inhibited in a competitive manner by the non-penetrant thiol reagent mersalyl, but it is insensitive to the penetrant thiol reagent N-ethylmaleimide (NEM). (4) No accumulation of proline occurs inside the mitochondria as a result of the addition of proline externally, whereas the content of glutamate increases both in mitochondria and in the extramitochondrial phase. (5) Glutamate efflux from mitochondria occurs at a rate which depends on the mitochondrial transport, and it is inhibited in a non-competitive manner by NEM. The dependence of the rate of glutamate efflux on increasing proline concentration shows saturation kinetics. The physiological role of carrier-mediated transport in the regulation of proline catabolism, as well as the possible occurrence of a proline/glutamate shuttle in durum wheat seedlings mitochondria, are discussed.

Genistein and daidzein prevent low potassium-dependent apoptosis of cerebellar granule cells

We have investigated the ability of certain dietary flavonoids, known to exert beneficial effects on the central nervous system, to affect neuronal apoptosis. We used cerebellar granule cells undergoing apoptosis due to potassium deprivation in a serum-free medium in either the absence or presence of the flavonoids genistein and daidzein, which are present in soy, and of catechin and epicatechin, which are present in cocoa. These compounds were used in a blood dietary concentration range. We found that genistein and daidzein, but not catechin and epicatechin, prevented apoptosis, with cell survival measured 24h after the induction of apoptosis being higher than that of the same cells incubated in flavonoid free medium (80% and 40%, respectively); there was no effect in control cells. A detailed investigation of the effect of these compounds on certain mitochondrial events that occur in cells en route to apoptosis showed that genistein and daidzein prevented the impairment of glucose oxidation and mitochondrial coupling, reduced cytochrome c release, and prevented both impairment of the adenine nucleotide translocator and opening of the mitochondrial permeability transition pore. Interestingly, genistein and daidzein were found to reduce the levels of reactive oxygen species, which are elevated in cerebellar granule cell apoptosis. These findings strongly suggest that the prevention of apoptosis depends mainly on the antioxidant properties of genistein and daidzein. This could lead to the development of a flavonoid-based therapy in neuropathies.

l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria.

As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.

The mitochondrial L-lactate dehydrogenase affair

The existence of a mitochondrial L-lactate dehydrogenase (m-L-LDH) suggested by Dianzani (1951), was shown by Baba and Sharma (1971) with the enzyme located in the mitochondrial matrix; later Brooks et al. (1999) proposed the intracellular lactate shuttle and in the third millennium the existence of m-L-LDH was definitively been confirmed in mammalian, plant and yeast mitochondria as reviewed by Schurr (2006), Passarella et al. (2008), and Brooks (2009), being its existence finally recognized by inclusion of m-L-LDH in the Mitocarta (http://www.broadinstitute.org/pubs/MitoCarta/index.html). The experimental strategy to be used to show whether and how L-lactate can enter mitochondria to be metabolized is well-established and has been applied to a variety of mitochondria including heart (Brooks et al., 1999; Valenti et al., 2002), liver (Brooks et al., 1999; de Bari et al., 2004), skeletal muscle (Dubouchaud et al., 2000; de Bari et al., 2008; Passarella et al., 2008) plant (Paventi et al., 2007), brain (Schurr, 2006; Atlante et al., 2007; Schurr and Payne, 2007; Hashimoto et al., 2008), and cancer cells (de Bari et al., 2010a; Pizzuto et al., 2012). Thus, it is a matter for considerable surprise that the overwhelming evidence for an m-L-LDH located inside mitochondria is not by now universally accepted (Rasmussen et al., 2002; Sahlin et al., 2002; Ponsot et al., 2005; Gladden, 2007; Yoshida et al., 2007; Elustondo et al., 2013).

The post-thaw irradiation of avian spermatozoa with He-Ne laser differently affects chicken, pheasant and turkey sperm quality

The effects of post-thaw Helium-Neon (He-Ne) laser irradiation on mobility and functional integrity of frozen/thawed chicken, pheasant and turkey spermatozoa were investigated. Cytochrome C oxidase (COX) activity was also determined as a measure of the effect of irradiation on mitochondrial bioenergetics. Semen samples from each species were collected, processed and frozen according to the pellet procedure. After thawing, each semen sample was divided into two subsamples: the first one was the control; the second one was irradiated with a single mode continuous He-Ne laser wave (wavelength 632.8 nm; 6 mW; 3.96 J/cm(2)). Then the samples were assessed for sperm mobility (Accudenz(®) swim-down test), viability (SYBR-14/PI staining), osmotic-resistance (HOS test) and COX activity. The irradiation was effective P<0.05 increasing sperm motility in the turkey semen (0.228 ± 0.01 compared with 0.294 ± 0.02). The irradiation also caused an increase (P<0.05) of the COX activity in pheasant (+135 ± 4%) and turkey (+116 ± 4%) sperm, without affecting viability and osmotic-resistance. The COX was positively correlated (P<0.05) with the viability of chicken sperm, however no significant interactions were found between mobility and COX activity in the three avian species. Due to the difference in energetic metabolism among avian species used in this study, the He-Ne laser irradiation has a differential action on bio-stimulation of turkey, chicken and pheasant spermatozoa. The present results are the first to elucidate the possibility for restoration of motility of cryopreserved avian spermatozoa by bio-stimulation provided via He-Ne laser irradiation.

L‐Lactate metabolism in potato tuber mitochondria

We investigated the metabolism of l‐lactate in mitochondria isolated from potato tubers grown and saved after harvest in the absence of any chemical agents. Immunologic analysis by western blot using goat polyclonal anti‐lactate dehydrogenase showed the existence of a mitochondrial lactate dehydrogenase, the activity of which could be measured photometrically only in mitochondria solubilized with Triton X‐100. The addition of l‐lactate to potato tuber mitochondria caused: (a) a minor reduction of intramitochondrial pyridine nucleotides, whose measured rate of change increased in the presence of the inhibitor of the alternative oxidase salicyl hydroxamic acid; (b) oxygen consumption not stimulated by ADP, but inhibited by salicyl hydroxamic acid; and (c) activation of the alternative oxidase as polarographically monitored in a manner prevented by oxamate, an l‐lactate dehydrogenase inhibitor. Potato tuber mitochondria were shown to swell in isosmotic solutions of ammonium l‐lactate in a stereospecific manner, thus showing that l‐lactate enters mitochondria by a proton‐compensated process. Externally added l‐lactate caused the appearance of pyruvate outside mitochondria, thus contributing to the oxidation of extramitochondrial NADH. The rate of pyruvate efflux showed a sigmoidal dependence on l‐lactate concentration and was inhibited by phenylsuccinate. Hence, potato tuber mitochondria possess a non‐energy‐competent l‐lactate/pyruvate shuttle. We maintain, therefore, that mitochondrial metabolism of l‐lactate plays a previously unsuspected role in the response of potato to hypoxic stress.

Pyruvate kinase in pig liver mitochondria.

The existence of the pyruvate kinase (PK) in pig liver mitochondria was shown by monitoring photometrically the PK reaction in solubilised mitochondria with either phosphoenolpyruvate (PEP) or ADP used as a substrate. In distinction with the cytosolic isoenzyme, the mitochondrial PK showed a sigmoidal dependence on either PEP or ADP concentrations. The occurrence of the mitochondrial PK was confirmed by immunological analysis. Titration with digitonin showed that mPK is restricted to the matrix. PEP addition to mitochondria resulted in reduction of the intramitochondrial NAD(P)+ inhibited by either the non-penetrant thiol reagent mersalyl or by arsenite, an inhibitor of the pyruvate dehydrogenase complex. Citrate/oxaloacetate appearance outside mitochondria also occurred as result of PEP addition to PLM. Taken together these findings support a role for PEP itself in triggering fatty acid synthesis via its mitochondrial metabolism.

The occurrence of l-lactate dehydrogenase in the inner mitochondrial compartment of pig liver

Although pig represents a model species in biomedical research including studies dealing with liver patho-physiology, some aspects of liver metabolism need to be addressed. In particular, whether and how pig mitochondria can metabolize l-lactate remains to be established. We show here that pig liver mitochondria (PLM) possess their own l-lactate dehydrogenase (mL-LDH). This was shown both via immunological analysis and by assaying photometrically the L-LDH reaction in solubilised PLM. The mL-LDH reaction shows hyperbolic dependence on the substrate concentration, it is inhibited by oxamate and proves to differ from the cytosolic activity (cL-LDH), as revealed by the difference found in both pH profiles and temperature dependence of m- and cL-LDH. Titration experiments with digitonin show that mL-LDH is restricted in mitochondrial inner compartment. In agreement with the above findings, three genes in Sus scrofa genome encoded for L-LDH subunits which are predicted to have mitochondrial localization, as investigated by Target P 1.1 and PredSL analysis.

The He-Ne laser irradiation improves the quality of stored rabbit semen

Abstract The aim of the study was to investigate the effect of different energy doses of Helium-Neon (He-Ne) laser irradiation on rabbit semen quality during 48 h of storage. From 30 commercial hybrid bucks semen was collected by artificial vagina. The ejaculates of bucks were pooled (3-4 ejaculates/pool) and in total 8 semen pools were used. Each pool was divided into four aliquots. One sample was not irradiated (control), the other ones were irradiated with a single mode continuous He-Ne laser wave (wavelenght 632,8) and exposed to the following energy doses: 3.96, 6.12 and 9 J/cm2 respectively. Samples were kept at 15° C during laser irradiation. They were subsequently stored again at the same temperature for a further 48 h. Samples were evaluated for sperm motility (Accudenz procedure), sperm viability (SyBr-PI procedure) and acrosome integrity (PSA-FITC procedure) at 3,24 and 48h of storage. Laser irradiation at all energy doses utilized significantly improved motility (P>0.01), viability (P>0.01) and acrosome integrity (P>0.05) than in control either at 24 and 48 h of storage. Moreover, the energy dose of 6.12J/cm2 recorded a better semen quality during storage compared to the 3,96 and 9J/cm2 ones respectively. No significant differences for measured seminal quality parameters were found at 3h; this might be due to the fact that irradiation treatment requires a longer period of time to determine modification on semen quality. The present study shows that laser irradiation, as shown in a previous research for turkey semen, seems to improve the quality of rabbit semen stored for up 48h even if higher energy doses were utilized. Therefore, the irradiation laser might be a useful technique to enhance quality of stored rabbit semen, although further studies are need.