Ulla F. Rasmussen

Experimental evidence against the mitochondrial theory of aging A study of isolated human skeletal muscle mitochondria

The mitochondrial theory of aging was tested with optimised preparation techniques. Mitochondria were isolated from approximately 90 mg quadriceps muscle from healthy humans at age 70+ and 20+. The content of mitochondrial protein was approximately 10 mg g(-1) muscle and the yields were approximately 40%. The mitochondrial integrity was high as judged from the respiratory control and P/O ratios. No general membrane alterations or changes in the cytochrome contents were observed. BSA decreased the non-phosphorylating rates of respiration equally in both age groups. Thirteen different enzyme activities were assayed and normalised to protein content and citrate synthase activity. Most of the critical levels for detection of declines were <10%. In the 70+ group, the activity for fatty acid oxidation was decreased by approximately 20%. Two inherently low activities associated with oxidation of sarcoplasmic NADH were also decreased, probably related to the age change of fibre types. The remaining activities measured, e.g. those of pyruvate dehydrogenase, tricarboxylic acid cycle, respiratory chain, and ATP synthesis, were not observed to be lowered. Thus, the central bioenergetic systems appeared unaltered with age. The obvious discord with reported age declines of human skeletal muscle mitochondrial function is discussed. It is concluded that the present results are incompatible with the mitochondrial theory of aging.

Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle.

The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit ∼0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore, showed high specific rates of state 3 respiration. This excluded artificial loss from the mitochondria of all activity of a possible LDH. It was concluded that skeletal muscle mitochondria are devoid of LDH and unable to metabolize lactate.

Human quadricepts muscle mitochondria: A functional characterization

Human quadriceps mitochondria were isolated from ca. 80 mg tissue in ca. 45% yield. The preparation is described with respect to content of mitochondrial markers and nine different respiratory activities. The specific state 3 activities were high in comparison with literature data, indicating high integrity and purity of the preparation. Examples of state 3 rates, in µmol O min-1 g protein-1 (25°C): pyruvate + malate, 400; succinate, 514; malate + glutamate, 444. The notion of high integrity was also supported by the reproducibility of the preparation and the magnitude of the respiratory control ratios and the P/O ratios. The mitochondria most likely had lost ca. 30% of their cytochrome c upon isolation, but it was substantiated that this loss had not influenced the state 3 rates. Functional assays of single reactions or groups of reactions could be based on respiration experiments. The respiratory chain activity, for instance, was measured as respiration of NADH in freeze-permeabilized mitochondria (1263 μmol O min-1 g protein-1). Comparison of uncoupled rates of respiration and state 3 rates indicated that the ATP synthesis exerted major flux control over respiration of succinate + glutamate, malate + glutamate and pyruvate + malate. These reactions, showing very similar rates of ATP synthesis, could be used as a functional assay of ATP synthesis (1200 μmol ATP min-1 g protein-1). Respiration of succinate, palmitoyl-carnitine + malate, or glutamate could not support the maximal rate of ATP synthesis and the upstream reactions probably exerted major flux control in these cases. The specific activities appeared very constant in this group of young men, only the respiratory activity with glutamate might show biological variation.

SMALL SCALE PREPARATION OF SKELETAL MUSCLE MITOCHONDRIA, CRITERIA OF INTEGRITY, AND ASSAYS WITH REFERENCE TO TISSUE FUNCTION

Mitochondria prepared in small scale from skeletal muscle were studied with respiration measurements and low temperature spectroscopy. The method of preparation was developed for 25–100 mg tissue with pigeon breast muscle as model organ. The yield was 40%. Data collected during the developmental work were used to evaluate criteria of mitochondrial quality. The cytochrome c conservation, i.e. cytochrome c per mitochondrial quantity in the preparation relative to that in the tissue, is a most useful test parameter. It is bounded between 0-100%. Proportionality between the state 3 rate and the cytochrome c conservation was not rejected by statistical tests. The respiratory control ratio (RCR) was also highly correlated to the cytochrome c conservation. These correlations might be extrapolated to 100% conservation to give hypothetical tissue values. The cause for the correlations is discussed. The P/O ratio showed only weak dependence on the cytochrome c conservation and the state 4 rate showed no dependence. Other, rather insensitive test parameters are also discussed. The pigeon breast muscle mitochondria isolated by the final method showed cytochrome c conservation of 73 ± 9% (n = 16). They are compared with pig biceps femoris mitochondria prepared by the same method. The two types of mitochondria show many similarities. Some differences may be explained by a different amount of inner mitochondrial membrane relative to mitochondrial protein. The pig tissue contains ten times less mitochondrial protein than the pigeon tissue does. (Mol Cell Biochem 174: 55–60, 1997)

The oxidation of added NADH by intact heart mitochondria.

The exchange of reducing equivalents between the cytoplasm and the mitochondria involves a very important metabolic control mechanism (e.g. refs. [ 1,2]). The liver gluconeogeneses require a high rate of NADH formation in the cytoplasm, while in heart cytoplasmic NADH must be reoxidized in order to maintain pyruvate formation from glucose. It is generally accepted that the mitochondrial membrane is virtually impermeable to NADH [3] and this has resulted in the proposal of several shuttle mechanisms suggesting substrate transfer instead of coenzyme transfer across this membrane (e.g. refs. [ 1,2]). NADH oxidation catalyzed by mitochondria but stimulated by added cyt. c and not completely inhibited by amytal and antimycin A has often been reported in the literature (e.g. refs. [4,5]). This paper describes, however, a NADH-oxidase which was destroyed upon ageing of the mitochondria and which was highly sensitive to the normal respiratory chain inhibitors. This oxidative system present in pigeon heart mitochondria presumably oxidized added NADH on the mitochondrial surface. It was furthermore characterized by a Km for NADH below 2 /.IM and a turnover equal to the turnover of succinate (in the presence of glutamate) in the same mitochondria. The enzyme therefore possesses some of the characteristics which might be expected from a cytoplasmicmitochondrial NADH-oxidase working in Go. Some preliminary results have been published elsewhere [6].

Regulation of succinate oxidation by endogenous reduced nicotinamide adenine dinucleotides in intact heart mitochondria

It is well established that succinate may cause complete reduction of the endogenous pool of nicotinamide adenine dinucleotides (NAD(P))* in intact mitochondria [2,3]. The reaction is usually called the energy-linked reduction of NAD by succinate, and a very labile mechanism is indeed involved. The reduction rate decreased 50% in the pigeon heart mitochondria used in these studies when they were kept for only 3 min at 35” before dilution. The response of the NAD(P)H to ADP was half maximal after 710 min at 35” [4] while the respiratory activities were not affected by this treatment. This paper provides evidence that the level of NAD(P)H serves as a direct controlling factor in the metabolism of succinate in intact mitochondria. Pigeon heart mitochondria were very suitable for these studies for the following reasons: (i) endogenous substrates able to reduce NAD(P) in the presence of rotenone were absent. (ii) Experimental conditions could be obtained without use of inhibitors under which the NAD(P) reduction by succinate was com-

The energy requirement for activation of succinate metabolism in intact heart mitochondria

The so-called energy-linked reduction of NAD(P)* by succinate in mitochondria and subm~tochond~al particles has been extensively studied (e.g. [l-7] , for review see [8] ). The activation mechanism of succinate dehydrogenase (SDH) has been studied from a very different point of view [9-l l] . It seems possible, however, to correlate these phenomena because the rate of NAD(P) reduction must somehow be determined by the SDH activity which in turn is controlled by the extent of NAD(P) reduction [ 121. This control was explained by a transformation of SDH from a deactivated to an activated form as a result of NAD(P)H binding to the enzyme. The activation was a reversible function of the redox level under very different conditions [ 121 , It is generally accepted that NAD(P) reduction by succinate is energy-linked and that it occurs by a reversal of the respiratory chain through site 1 of oxidative phosphorylation [8] . This view has, however, been questioned by Krebs and coworkers [ 131, because uncouplers did not prevent the reduction of NAD(P) by succinate in the presence of amytal. The

Propavane an inhibitor of oxidative phosphorylation connected with mitochondrial glutamate metabolism

Abstract A description is given of an inhibitor, propavane, which: 1. 1) is specific for glutamate oxidation in low concentrations, 2. 2) gives half maximal state 3 (i.e. ADP supplemented) respiration for about 12 uM, i.e. for 25 mu moles/mg protein, 3. 3) inhibits state 4 (i.e. ADP limited) respiration only very slightly, 4. 4) is not influenced by the presence of inorganic phosphate, 5. 5) cannot be reversed by uncouplers (2,4-dinitrophenol), 6. 6) maintains a completely oxidized steady state of the pyridine nucleotides in the presence of glutamate even when preincubated with amytal (2.7 mM).