6.1.1. Role of calcium in the control of mitochondrial respiration during fatty acid oxidation

Mitochondrial respiration involves energy-producing machinery including the oxygen-consuming respiratory chain, which produces an electrochemical gradient over the inner mitochondrial membrane, and F₁F_o-ATPase, which uses this gradient in ATP synthesis (Mitchell 1976). The fuel for this ATP-producing machinery is produced in reactions at the substrate level, which must also be activated during times of increased energy requirement.

Calcium has been suggested to regulate mitochondrial respiration both at the substrate level and at the level of oxidative phosphorylation. Calcium has been convincingly shown to regulate carbohydrate metabolism by affecting PDH activity. Calcium has also been suggested to regulate enzymes of the TCA cycle and β -oxidation (McCormack et al. 1990). In oxidative phosphorylation, calcium has been proposed to stimulate F₁F_o-ATPase (Harris & Das 1991).

The current results show that flavin oxidation occurs upon a calcium-induced increase in energy consumption. This is observed especially during a high workload when energy is gained from glucose oxidation. Flavin oxidation is less clear upon smaller workload changes during fatty acid oxidation. These results support the view that mitochondrial respiration is primarily regulated at the level of the respiratory chain, but stimulation at the substrate level rapidly follows, keeping the cellular redox-state nearly unchanged especially during slighter workload alterations. As has been convincingly shown, calcium stimulates PDH and the oxidation of glucose. In spite of this, a calcium-induced increase in energy consumption during glucose oxidation caused flavin oxidation, which subsequently returned near to the original level. This means that calcium-stimulated glucose oxidation cannot match the increased activity of the respiratory chain at the moment of the transition of energy consumption, thus causing oxidation of the cellular redox state.

It was confirmed that when fatty acids were added as exogenous substrates, they totally overrode glucose in substrate oxidation, as they concomitantly caused a permanent reduction in the cellular redox state. During fatty acid oxidation, the increase in energy consumption caused hardly any noticeable flavin oxidation, suggesting that when reducing power is gained from fatty acid oxidation, it is better matched to the increased respiratory chain activity. This could mean that calcium is a more effective substrate level stimulator during fatty acid oxidation than during glucose oxidation, and it is known that calcium stimulates the TCA cycle enzymes. This alone, however, is not sufficient, as the production of acetyl-CoA must also be increased, which means increased β -oxidation. The results in isolated rat liver mitochondria show the activation of β -oxidation in high Ca²⁺-concentrations (Lopaschuk et al. 1994, Ontko & Otto 1975), but the evidence for calcium as a β -oxidation activator in a more physiological situation in myocardium is vague, especially compared to PDH (McCormack et al. 1990). So, the theory of calcium as an effective stimulator of β -oxidation in the heart does not receive much support from previous studies.

The other explanation for an effective substrate level response upon workload alterations could be the energy-linked regulation of β -oxidation. Earlier results suggest that the myocardial substrate supply is the primary cause for alterations in fatty acid oxidation rates (Grynberg & Demaison 1996, van der Vusse et al. 1992). As Hassinen et al. have previously shown, increased fatty acid oxidation causes NAD reduction, i.e. increased thermodynamic driving force, concomitantly with an increased cellular energy state (Hassinen et al. 1990). The present results support this view, since the addition of fatty acids caused flavin reduction with an initial overshoot, as well as a slight increase in the cellular energy state. After the redox overshoot, a new steady state is formed with an increased NADH/NAD ratio and increased cellular energy state, which fit to the thermodynamic model. Also, the acetyl-CoA/CoA ratio increases upon fatty acid oxidation. The increased NADH/NAD and acetyl-CoA/CoA ratios cause product inhibition of enzymes of β -oxidation (Lopaschuk et al. 1994). The increased energy demand by calcium tends to decrease this product inhibition, which results in increased β -oxidation, stabilizing the redox-changes better than during glucose oxidation.

If the primary site of regulation lies above the substrate level, what is the primary regulator connecting the needs of increased energy consumption to increased energy production? Calcium has been suggested to stimulate substrate level reactions and F₁F_o-ATPase activity, but our results on the cellular redox state suggest that substrate level stimulation is an auxiliary mechanism that responds to increased energy consumption. The observation that the cellular energy state is mainly decreased upon a calcium-induced increase in energy consumption argues against a primary role of ATP synthase stimulation by calcium. The current results best fit to the thermodynamic model of regulation, where a decrease in the cellular energy state stimulates respiratory chain activity, leading to an increased rate of phosphorylation and the delivery of reducing equivalents to the respiratory chain (Erecinska & Wilson 1982). The results do not argue against the kinetic theories either, but considering previous data, where ANT shows no control strength in the myocardium, the thermodynamic model prevails (Brown 1992, Doussiere et al. 1984, Hassinen 1986a). The thermodynamic model includes the principles of shared control as it anticipates substrate level reactions to be regulated in coordination with the machinery of oxidative phosphorylation. It seems that during fatty acid oxidation, when the thermodynamic driving force is increased because of NAD reduction, the substrate level reactions better support the cellular energy state. During glucose oxidation, the cellular energy state is more affected by workload increase. It appears that the function of the substrate level reactions is not to control ATP turnover, but rather to prevent a large fall in the cellular energy state. This is achieved at least partly by stimulating calcium sensitive dehydrogenases, which seems to be the main role of calcium in cellular energetics.

6.1.2. Role of energy consumption in substrate selection and anaplerosis

The present results support the view that fatty acids are the preferable substrate for the heart. This is observed even with a minor (50 µM) octanoate concentration, when virtually all of the acetyl-CoA input (97 ± 2%) into the TCA-cycle, i.e. substrate oxidation, originated from octanoate, as the competing exogenous substrate was 5.0 mM glucose in the presence of 12 IU/L insulin. This is in good agreement with a previous study (Yu et al. 1996), but usually the fatty acid contribution has been evaluated to be smaller depending on the use of competing substrates and methods (Jeffrey et al. 1995a, Saddik & Lopaschuk 1991, Sherry et al. 1992).

During fatty acid oxidation, any significant increase in the oxidation of glucose or endogenous substrates upon workload increase was not observed, but the fatty acid dominance in substrate oxidation continued. This is in disagreement with many studies, which suggest that the contribution of carbohydrates to energy production increases as the energy needs are increased (Collins-Nakai et al. 1994, Drake et al. 1980, Goodwin et al. 1998, Keul et al. 1966). In these studies, methods have been used that measure the extraction of substrates rather than their oxidation, and therefore their real use in energy production (Drake et al. 1980, Keul et al. 1966). Additionally, in some experiments, cardiac stimulation by adrenalin has been used, which favours the use of carbohydrates, as adrenalin has been documented to stimulate PDH complex (Hiraoka et al. 1980). In the present experiments, calcium was used to increase cardiac workload, which is also a well-known activator of PDH. In spite of that, octanoate superseded glucose in substrate oxidation. This is possibly due to an increased thermodynamic driving force and β -oxidation raising the NADH/NAD-, ATP/ADP-, and acetyl-CoA/CoA-ratios, which all inhibit PDH activity. An increased acetyl-CoA concentration also stimulates pyruvate carboxylase, resulting in increased anaplerosis and citrate concentration, which inhibits phosphofructokinase, and thereby glycolysis (Depre et al. 1998). Apparently, increased Ca²⁺-stimulation of PDH is not sufficient to overrun the inhibitory signals originating from fatty acid oxidation.

In studies where the methods used have been comparable to the current ones, the results are more in agreement with the present results (Goodwin & Taegtmeyer 2000, Jeffrey et al. 1995b, Saddik & Lopaschuk 1991). The difference from other studies is at least partially explainable by the observation that fatty acids inhibit more cardiac glucose oxidation than its uptake (Henning et al. 1996). Therefore, glucose metabolism in the myocardium is conveyed to alternative routes rather than glycolysis.

The anaplerotic rate during octanoate oxidation was 0.13 ± 0.03 (mol/mol of acetyl-CoA input into the TCA cycle) in the high workload group, which is in good agreement with previous results (Lewandowski 1992a, Malloy et al. 1988, Peuhkurinen et al. 1982). During the low work output, the anaplerotic rate increased to 0.29 ± 0.05, which means that the absolute anaplerotic flux increased from 1.63 ± 0.48 to 2.42 ± 0.55 µmol/min⋅g dry wt. The work output of the low workload group was near to zero, which is the value in the potassium-arrested heart where relative anaplerosis has been found to be 0.32–0.47 (Lewandowski 1992b, Peuhkurinen et al. 1982). So, the present finding is in good agreement with previous observations. A possible reason for the increased anaplerotic rate during low energy expenditure is the increased acetyl-CoA/CoA ratio, which has been found upon arrest of the heart (Hiltunen & Hassinen 1976). Similarly, when workload is reduced near to zero, an increase in the acetyl-CoA/CoA ratio may occur, which is a known activator of pyruvate carboxylase. This would lead to increased anaplerotic flux from pyruvate to oxaloacetate. Also, an increased ATP/ADP ratio is known to stimulate pyruvate carboxylase (Hiltunen & Hassinen 1976), but the observed ATP/ADP increase upon workload decrease during fatty acid oxidation was so small that this probably does not have any effect on anaplerosis.