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In this investigation, the effects on proton leak of leptin administration to ob/ob mice was measured for liver mitochondria. We and others have shown that proton leak is approximately 3 times greater in liver mitochondria from ob/ob mice compared to lean controls at any given membrane potential. The results are consistent with obese mammals having higher lean mass-specific metabolic rates compared to lean controls. The increase in proton leak rate at any given membrane potential cannot be explained by the presence of free fatty acids associated with mitochondria isolated from the obese animals. The difference in proton leak must therefore represent a real difference in inner membrane permeability. Acute leptin (OB protein) administration restores the liver mitochondrial proton leak rate of ob/ob mice to that of lean controls. There was no effect on proton leak rate of liver mitochondria from sham-treated ob/ob mice. These novel results indicate a role for leptin, either directly or indirectly, in regulating the efficiency of oxidative phosphorylation.

We investigated why liver mitochondria from small mammals are leakier to protons than those from larger mammals. Sixty-nine percent (+/-23%) of the proton leak differences appeared to relate to membrane area (less inner membrane surface area in larger animals); any residual differences must reflect differences in membrane properties. There were differences in phospholipid fatty acid composition; unsaturation index, monounsaturates, palmitate (16:0), stearate (18:0), docosahexaenoate [22:6(n-3)], and the 22:6(n-3)/22:5(n-3) ratio all correlated with body mass. Proton flux per square centimeter did not correlate significantly with body mass or, in general, with phospholipid fatty acid composition, suggesting little role for fatty acid composition in determining proton leak in mammals of different body mass. However, unsaturation index and n-3 polyunsaturated fatty acid content correlated significantly with proton leak per milligram phospholipid when literature data from reptiles and rats in different thyroid states were included, giving some support to suggestions of a general role for phospholipid fatty acid composition in determining mitochondrial proton leak.

In this paper we examine the non-linearity of the relationship between the proton electrochemical gradient across the mitochondrial inner membrane (delta p) and oxygen consumption of non-phosphorylating mitochondria in situ in hepatocytes. Models proposing to explain the non-linear relationship were tested experimentally. It was shown that the mitochondrial proton conductance and the number of protons pumped to the cytosolic side of the mitochondrial inner membrane by the electron transport complexes per oxygen atom consumed (H+/O ratio) are independent of electron transport rate in mitochondria in isolated hepatocytes. The non-linearity of the plot of delta p against the non-phosphorylating oxygen consumption is due to either a potential-dependent slippage of the proton pumps of the mitochondrial inner membrane and/or a potential-dependent leakage of protons back across the mitochondrial inner membrane.

The degree of control exerted by the mitochondrial choline transporter over the oxidation pathway was measured in isolated rat liver mitochondria. Choline transporter activity was titrated with hemicholinium-3, a known competitive inhibitor of the transporter. It was shown that the rate of betaine efflux from mitochondria was an accurate measure of choline oxidation. The relative rate of choline oxidation was measured as a function of the relative degree of inhibition of the transporter. The resulting data gave a flux control coefficient over choline oxidation of 0.9 for the choline transporter. It is concluded that the choline transporter is the major site for control of choline oxidation in isolated rat liver mitochondria.

The standard metabolic rate of an animal is the rate of heat production under conditions that minimize known extra requirements for energy. In tissues and cells from aerobic organisms, energy expenditure can conveniently be measured as oxygen consumption. Measurements made using isolated rat hepatocytes have shown that a significant contribution to resting oxygen consumption (and hence heat production) is made by a futile cycle of proton pumping and proton leak across the mitochondrial inner membrane. Two important factors affecting standard metabolic rate, thyroid status and phylogeny, also affect the proton permeability. A third major factor affecting standard metabolic rate is body mass. Here we show that proton leak decreases with increasing body mass in mammals. We suggest that differences in proton leak may partly explain the differences in standard metabolic rate between mammals of different mass.

In order to investigate the control of endogenous betaine supply to the cytoplasmic enzyme betaine-homocysteine methyltransferase, it was necessary to understand how betaine synthesized within the mitochondrial matrix is transported across the mitochondrial inner membrane. Mitochondria were loaded with radiolabelled betaine and efflux was measured in a medium at physiological ionic strength. Efflux of radiolabelled betaine occurred continuously with time. The efflux rate was unaffected by the presence or absence of a source of energy except at high membrane potentials, where betaine efflux rate increased 2-3-fold. Titration of the membrane potential demonstrated a non-ohmic relationship between betaine efflux rate and membrane potential. The rate of betaine efflux was proportional to the matrix betaine concentration up to 9 mM. Efflux was unaffected by addition of analogues of betaine and known mitochondrial transport inhibitors. N-Ethylmaleimide did inhibit efflux by 50%, but evidence suggested that the effect was non-specific. The lack of saturability or other evidence for a transport system suggests that betaine escapes from mitochondria by simple diffusion. The relative diffusion rates of glycine, sarcosine, dimethylglycine and betaine suggest that increasing the degree of N-methylation lowers diffusion rate.

Rat liver mitochondria possess a specific choline transporter in the inner membrane. The transporter shows saturable kinetics at high membrane potential with a Km of 220 microM and a Vmax of 0.4 nmol/mg of protein/min at pH 7.0 and 25 degrees C. At physiological concentrations of choline, the rate of choline uptake by the transporter shows a linear dependence on membrane potential; uptake is distinct from the nonspecific cation diffusion process. Hemicholinium-3, hemicholinium-15, quinine, and quinidine, all analogues of choline, are high affinity competitive inhibitors of choline transport with Ki values of 17, 55, 15, and 127 microM, respectively. The choline transporter is distinct from other known mitochondrial transporters. Rat heart mitochondria do not appear to possess a choline transporter. Evidence suggests that the transporter is an electrophoretic uniporter. Analogue studies have shown that the hydroxyl and the quaternary ammonium groups of choline are necessary for binding to the transporter. A comparison of molecular models of choline and the high affinity inhibitors has provided evidence for the preferred conformation of choline for binding to the transporter. The presence of a choline transporter in the mitochondrial inner membrane provides a potential site for control of choline oxidation and hence supply of endogenous betaine.