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Mitochondria play a central role in metabolic reprograming that occurs in numerous disease conditions. A precise evaluation of the extent of mitochondrial involvement in the metabolic alterations is essential for a better definition of metabolically based therapeutic strategies. In this chapter, some simple protocols are presented, using carbon 13 tracers and nuclear magnetic resonance isotopomer analysis, for the evaluation of mitochondrial contributions to intermediary metabolism and the metabolic effects of the implementation of some mitochondrial regulatory mechanisms.

The effect of duration of chronic treatment with fluoride (F, 50 mg/L as NaF) on the lipid profile, lipid droplets and triglycerides (TG) in liver was evaluated in mice with nonalcoholic fatty liver disease (NAFLD) previously induced by hyperlipidic diet and in animals fed normocaloric diet. In addition, the effect of F administered for a short period (20 days) was evaluated on de novo lipogenesis, by nuclear magnetic resonance. GRP78, Apo-E, and sterol regulatory element-binding protein (SREBP) were quantified by Western blotting. Our data indicate that F interferes in lipid metabolism and lipid droplets, having a different action depending on the exposure time and type of diet administered. F improved lipid parameters and reduced steatosis only when administered for a short period of time (up to 20 days) to animals fed normocaloric diet. However, when NAFLD was already installed, lipid parameters were only slightly improved at 20 days of treatment, but no effect was observed on the degree of steatosis. In addition, lipid profile was in general impaired when the animals were treated with F for 30 days, regardless of the diet. Moreover, F did not alter de novo lipogenesis in animals with installed NAFLD. Furthermore, hyperlipidic diet increased F accumulation in the body. GRP78 increased, while Apo-E and SREBP decreased in the F-treated groups. Our results provide new insights on how F affects lipid metabolism depending on the available energy source.

Metabolic reprogramming has been associated to a plethora of diseases, and there has been increased demand for methodologies able to determine the metabolic alterations that characterize the pathological states and help developing metabolically centered therapies. In this chapter, methodologies for monitoring TCA cycle turnover and its interaction with pyruvate cycling and anaplerotic reactions will be presented. These methodologies are based in the application of stable 13 C isotope "tracers"/substrates and 13 C-NMR isotopomer analysis of metabolic intermediates. These methodologies can be applied at several organizational levels, ranging from isolated organelles and organs to whole organisms/humans. For the sake of simplicity, only very simple and well-defined models will be presented, including isolated heart mitochondria and isolated perfused hearts and livers.

Nuclear magnetic resonance (NMR)-based metabolomics is widely used in the research of metabolic conditions of complex biological systems under various conditions, and its use has been found in the field of male fertility. Here we describe the implementation of total and targeted NMR-based metabolomics in the research on Sertoli cell metabolism. Main principles and techniques of cell medium, cellular extracts, and intact cells are explained, as well as some classical experiments that can give complementary information on the Sertoli cell metabolism.

Nuclear magnetic resonance (NMR) spectroscopy is a technique with an increasing importance in the study of metabolic diseases. Its initial important role in the determination of chemical structures (1, 2) has been considerably overcome by its potential for the in vivo study of metabolism (3-5). The main characteristic that makes this technique so attractive is its noninvasiveness. Only nuclei capable of transitioning between energy states, in the presence of an intense and constant magnetic field, are studied. This includes abundant nuclei such as proton ((1)H) and phosphorous ((31)P), as well as stable isotopes such as deuterium ((2)H) and carbon 13 ((13)C). This allows a wide range of applications that vary from the determination of water distribution in tissues (as obtained in a magnetic resonance imaging scan) to the calculation of metabolic fluxes under ex vivo and in vivo conditions without the need to use radioactive tracers or tissue biopsies (as in a magnetic resonance spectroscopy (MRS) scan). In this chapter, some technical aspects of the methodology of an NMR/MRS experiment as well as how it can be used to study mitochondrial bioenergetics are overviewed. Advantages and disadvantages of in vivo MRS versus high-resolution NMR using proton high rotation magic angle spinning (HRMAS) of tissue biopsies and tissue extracts are also discussed.

H9c2 cells, derived from the ventricular part of an E13 BDIX rat heart, possess a proliferative and relatively undifferentiated phenotype but can be readily directed to differentiate under reduced serum conditions originating cells presenting muscle features. Skeletal or cardiac phenotypes can be originated depending on whether or not serum reduction is accompanied by a daily treatment with all-trans-retinoic acid. In the present study, we aimed to characterize and compare the metabolic profile of H9c2 cells at various differentiation states, correlating the differences between different populations with muscle-specific development. We determined that H9c2 myoblasts remodel their metabolism upon differentiation, with undifferentiated cells more reliant on glycolysis, as demonstrated by higher lactate production rates. Differentiated cells adopted a more oxidative metabolism with better coupling between the glycolytic and oxidative pathways, which is indicative of a metabolic evolvement toward a higher energetic efficiency state. Our findings emphasize the metabolic differences between differentiated and undifferentiated H9c2 cells and raise caution on how to adequately select the H9c2 differentiation state that will act as the better model for the design of experimental studies.

Myocardial ischemia is the main cause of death in the Western societies. Therapeutic strategies aimed to protect the ischemic myocardium have been extensively studied. Reperfusion is the definitive treatment for acute coronary syndromes, especially acute myocardial infarction; however, reperfusion has the potential to exacerbate tissue injury, a process termed reperfusion injury. Ischemia/reperfusion (I/R) injury may lead to cardiac arrhythmias and contractile dysfunction that involve apoptosis and necrosis in the heart. The present review describes the mitochondrial role on cardiomyocyte death and some potential pharmacological strategies aimed at preventing the opening of the box, i.e., mitochondrial dysfunction and membrane permeabilization that result into cell death. Data in the literature suggest that mitochondrial disruption during I/R can be avoided, although uncertainties still exist, including the fact that the optimal windows of treatment are still fairly unknown. Despite this, the protection of cardiac mitochondrial function should be critical for the patient survival, and new strategies to avoid mitochondrial alterations should be designed to avoid cardiomyocyte loss.

Various stressful conditions such as ischemia in cold cardioplegic solutions and reperfusion occur during heart transplantation. Since ATP production is essential for the maintenance of contractile activity, mitochondrial function may be a mediator of ischemia and ischemia/reperfusion (I/R) injury. We aimed at testing the ability of two distinct cardioplegic solutions, Celsior (Cs) and Histidine Buffer (HBS), to protect rat heart mitochondria (HM) function during ischemia alone or ischemia followed by reperfusion. A standard Krebs-Henseleit solution (KH) was used as "negative" control. Male and Female Wistar rats were divided into control (Ctrl), reperfusion control (Ctrl_R), ischemia and I/R groups. Ischemia and I/R were divided into three subgroups depending on the cardioplegic solution used (Cs, HBS or KH) and subjected to 4-or 6-h ischemia alone or followed by reperfusion. HM respiration and transmembrane electric potential (Deltapsi) were measured with oxygen and TPP(+)-selective electrodes, respectively. Mitochondrial electron microscopy and detection of protein carbonyl groups content were also performed. After ischemic heart preservation, mitochondrial respiration and Deltapsi were not significantly affected except for the respiratory control ratio (RCR). After I/R, state 3 respiration, RCR and Deltapsi were decreased, especially in HM from male and for complex I substrates (CxI). HM preserved in HBS had less membrane disruption, segregation or disintegration. We conclude that (a) female HM were less sensitive to I/R, (b) CxI was particularly affected by I/R, (c) two cardioplegic solutions tested act in different mitochondrial targets preventing mitochondrial collapse.