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Neuronal nitric oxide synthase controls enzyme activity pattern of mitochondria and lipid metabolism

Lorenz Schild^*,1, Iveta Jaroscakova, Uwe Lendeckel, Gerald Wolf and Gerburg Keilhoff

^* Institut für Klinische Chemie und Pathologische Biochemie, Bereich Pathologische Biochemie;
Institut für Medizinische Neurobiologie; and
Institut für Experimentelle Innere Medizin, Medizinische Fakultät der Otto-von-Guericke-Universität Magdeburg, Magdeburg, Germany

¹Correspondence: Institut für Klinische Chemie und Pathologische Biochemie, Bereich Pathologische Biochemie, Medizinische Fakultät der Otto-von-Guericke Universität Magdeburg, Leipziger Str. 44, Magdeburg 39120, Germany. E-mail: lorenz.schild{at}medizin.uni-magdeburg.de

SPECIFIC AIMS

It is known that nitric oxide (NO) inhibits the electron flow in the mitochondrial respiratory chain, stimulates mitochondrial hydrogen peroxide generation, and promotes the biogenesis of mitochondria. This study aims to extend our knowledge about the effect of NO on mitochondria in the brain, muscle, heart, kidney, and liver. We hypothesize that endogenous NO which originates from the neuronal isoform of NO synthase (nNOS) affects the mitochondrial enzyme activity pattern of these tissues. Comparing mice lacking the nNOS (nNOS-KO) with wild-type animals (WT), we analyzed respiratory chain complex and citrate synthase in the corresponding organs. In analyzing the amount of fatty acid synthase and measuring the lipid content in brain, heart, and liver of nNOS-KO and WT animals, we considered the possibility that NO-dependent changes in citrate production are linked to changes in the activity of fatty acid synthesis.

PRINCIPAL FINDINGS

1. Endogenous NO derived from nNOS causes stimulation of mitochondrial biogenesis in tissue-specific cell types in the brain and kidney
Western blot analysis demonstrated that in WT mice, nNOS is expressed in the brain, muscle, heart, kidney, and liver (not shown). Electron microscopic analysis revealed that WT mice show higher densities of mitochondria expressed in the number of mitochondria per 40 µm² in neurons of the brain cortex (32.42±2.54 in WT vs. 25.08±2.04 in nNOS-KO) and in tubule cells of the kidney (59.17±3.52 in WT vs. 47.92±3.25 in nNOS-KO). In contrast, no significant differences in the number of mitochondria were found between WT and nNOS-KO mice in myocytes of the gastrocnemius muscle, cardiomyocytes, and hepatocytes. The mitochondria were similar in shape and size in the investigated tissues of WT and nNOS-KO mice and showed the organ-specific pattern of well-defined cristae structures. However, no significant difference was found comparing the ratio of mitochondrial and nuclear DNA and the tissue content of cytochrome c in homogenates of the brain, muscle, heart, kidney, and liver. These approaches for the evaluation of mitochondrial quantities take all cell types of a tissue into account.

2. WT animals differ in citrate synthase from nNOS-KO mice
The citric acid cycle enzyme citrate synthase is commonly used as a marker enzyme for the mitochondrial matrix. Between 2- and 3-fold as high activities of citrate synthase were determined in the brain, muscle, kidney, and liver for nNOS-KO in comparison to WT animals (Fig. 1 A). Western blot analysis revealed that the expression of this enzyme tended to be higher in brain and muscle and significantly higher in heart, kidney, and liver in nNOS-KO in comparison to WT mice (Fig. 1B ). Since similar ratios of mitochondrial and nuclear DNA as well as contents of cytochrome c were determined in the tissues of WT animals, this observation suggests a significantly higher citric acid generation, specifically by mitochondria of nNOS-KO mice.

View larger version (30K): [in this window] [in a new window]   Figure 1. Effect of nNOS-derived NO on the activities of the mitochondrial citrate synthase (A) and the amount of citrate synthase protein (B). The citrate synthase activities were determined in homogenates from the tissues of nNOS-KO and respective WT mice. Activities of citrate synthase are given in µmol/min/mg protein of homogenate. Data are presented as mean values ± SE of 10 preparations. The amount of citrate synthase protein was analyzed in tissue homogenates of nNOS-KO and respective WT mice by Western blot technique. The protein amount per lane was 20 µg. The ß-actin bands demonstrate equal sample loading. Band intensities were quantified by using BioDoc Analyze-System (Biometra) and given as mean ± SE in arbitrary units from 5 preparations. *Significant difference between the data of a tissue from WT and nNOS-KO mice, P < 0.05.

3. The livers of nNOS-KO contain more lipids compared with WT mice
Citrate is a key metabolite in cytosolic fatty acid synthesis. It is mitochondrially formed by the citrate synthase-catalyzed reaction and can be released from mitochondria via the tricarbonic acid carrier. In fact, higher citrate concentrations were found in the extramitochondrial space of liver mitochondria from nNOS-KO in comparison to WT mice. Higher amounts of fatty acid synthase protein were found in nNOS-KO mice. We tested the hypothesis that increases in citrate concentration and fatty acid synthase protein are paralleled by an elevation of the content of lipids in organs such as liver in which fatty acid synthesis takes place. Therefore, we compared the lipid content in brain and heart in which fatty acid synthesis is not supported with the liver of nNOS-KO and WT mice. The corresponding data are shown in Fig. 2 . Significantly higher content of lipids was only found in the livers of nNOS-KO mice. In contrast, in the brain and heart, equal contents of lipids were determined in nNOS-KO and WT mice.

View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of the expression of the nNOS isoenzyme on tissue lipids. The total lipid content determined for the brain, heart and liver of nNOS-KO and respective WT mice are presented as the ratio of lipid and tissue weight as mean ± SEM of at least 6 preparations of tissue homogenates. *Difference between the activities of tissue from WT and nNOS-KO mice was significant with P < 0.05.

CONCLUSIONS AND SIGNIFICANCE

We found the neuronal variant of nitric oxide synthase isoenzymes to be expressed in the brain, muscle, heart, and to a lesser extent in the kidney and liver. There it contributes, together with the endothelial (eNOS) and mitochondrial isoform (mtNOS), to the formation and distribution of NO. It is well known that mitochondria are specific targets of NO. Besides effects on the transport of electrons within the respiratory chain, endogenous NO has been shown to stimulate the biogenesis of mitochondria. The evidence for this suggestion was derived from the comparison of eNOS-KO and WT mice by quantifying mitochondrial DNA, subunit IV of the cytocrome oxidase complex, and the cytocrome c content in the brain, heart, and liver. Our electron microscopic analysis revealed the tendency of higher densities of mitochondria only in special cell types (neurons of the brain cortex and in tubule cells of the kidney) of WT compared with nNOS-KO mice. We found higher tissue activities of succinate cytochrome c oxidoreductase in brain, heart, kidney, and liver, and higher tissue activities of NADH cytochrome c oxidoreductase in the brain, muscle, and heart of WT in comparison to nNOS-KO mice. The higher activities of respiratory chain enzyme complexes and the higher quantity of mitochondria of special cell types in tissues of WT animals may compensate for the decrease in mitochondrial ATP production due to the inhibition of cytochrome c oxidase by NO as has been demonstrated many times in several laboratories.

An important result of our study is the finding that nNOS-KO mice exert up to 3-fold the levels of citrate synthase activities and protein in comparison to WT animals. An adequate rise in the citrate concentration in the mitochondrial matrix results in an increase in the cytosolic citrate concentration via transport through the mitochondrial membrane. This was really found in the extramitochondrial space of liver mitochondria from nNOS-KO mice. Subsequently, the ATP-dependent citrate lyase mediates the conversion to acetyl-CoA, ultimately stimulating the synthesis of fatty acids, particularly in liver. This is supported by the up-regulation of fatty acid synthase. Thus, a previously unrecognized effect of NO appears to be the regulation of fatty acid synthesis by suppressing the rate of citrate synthesis in mitochondria and expression of fatty acid synthase. Recently, another influence of NO on fatty acid synthesis was reported. It was shown that NO can nitrosylate acetyl CoA to the metabolically inactive S-nitrosoCoA, resulting in a direct and short-term control of fatty acid synthesis. Effects of NO on lipid metabolism should be reflected in the tissue content of lipids and in NO-dependent body weight changes. In fact, higher body weights of eNOS-KO mice compared to WT animals have been reported. It was concluded these differences were the result of the NO-dependent stimulation of mitochondrial biogenesis. In our study, we found no difference in the body weights of WT and nNOS-KO mice. In contrast to eNOS-KO mice, nNOS-KO are characterized by a higher locomotive activity compared with WT animals. This may explain our failure to detect differences in the body weight of WT and nNOS-KO animals. However, we found higher contents of lipids in the livers, but not in the brains and hearts, of nNOS-KO mice than in WT animals. Among the tissues investigated, only liver is equipped with enzymes of the fatty acid synthesis pathway. Therefore, our data supports the hypothesis that NO exerts a regulatory function in lipid metabolism at the level of citrate synthesis. This NO-dependent regulation of fatty acid synthesis may also contribute to the recently reported differences in body weight between WT and eNOS-KO mice. The relevant steps of the regulation of fatty acid synthesis by endogenous NO in the liver are summarized in Fig. 3 .

View larger version (19K): [in this window] [in a new window]   Figure 3. Hypothetical scheme of the regulatory effect of endogenous NO on fatty acid metabolism in the liver. Under normal conditions, endogenous tissue NO originates from different isoforms of NOS, such as endothelial (eNOS), neuronal (nNOS), and mitochondrial (mtNOS). Therefore, the tissue concentration of NO is somewhat higher in WT compared with nNOS-KO mice. This results in the higher activity of respiratory chain enzyme complexes in WT animals. Most striking is that the mitochondria from nNOS-KO mice exert up to 3-fold the activities of citrate synthase causing increased mitochondrial and cytosolic citrate concentrations. In turn, an increase in cytosolic citrate concentration stimulates fatty acid synthesis through several steps indicated by higher concentrations of fatty acids and lipids in the livers of nNOS-KO mice.

Here, we report for the first time that NO derived from constitutive nNOS plays a crucial role in the activity pattern of mitochondrial enzymes. In particular, the NO-mediated suppression of citrate synthase activity may be attributed to a regulatory function of NO in fatty acid synthesis. Inhibition of mitochondrial respiration by NO appears to be at least partially compensated for by a respective increase in the activity of respiratory chain complexes.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3898fje;

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