Regulatory Role of Reducing‐Equivalent Transfer from Substrate to Oxygen in the Hepatic Metabolism of Glycerol and Sorbitol
Authors: Berry Michael N. , Kun Ernest , Werner Harold V. 
Published: March 2005

Suspensions of morphologically intact isolated rat‐liver cells were used in conjunction with specific inhibitors to identify and quantitate the hepatic hy drogen‐trans ocating systems involved in the transfer of reducing‐equivalents from sorbitol or glycerol to O2. Rates of hydrogen translocation were derived either from measurement of the major products of substrate metabolism or from rates of substrate utilization. It was found that at saturating substrate concentrations, rates of sorbitol or glycerol 3‐phosphate oxidation were closely similar (about 1.8 μmol × g wet weight−1× min−1). There was an inverse relationship between rates of sorbitol and glycerol uptake so that the rate of hydrogen flux to O2 from substrate mixtures was no greater than that from either substrate added separately. Rates of sorbitol and glycerol consumption were increased by pyruvate acting as a cytoplasmic hydrogen acceptor.

It is concluded from these observations that at high substrate levels and in the absence of a cytoplasmic hydrogen acceptor, hydrogen translocation is the rate‐limiting process in the hepatic metabolism of sorbitol and glycerol and that the flux of reducing equivalents to O2 from these two substrates involves shared hydrogen‐translocating systems. At low levels of substrate, more likely to be encountered in vivo, the rate of sorbitol or glycerol metabolism is dependent also on substrate concentration, but even under these circumstances it was found that the capacity of the hydrogen‐translocating systems governs the over‐all rate of metabolism whenever substrate mixtures were present.

The nature of these systems was assessed by the use of specific inhibitors. About 15% of the flux of reducing equivalents to O2 involved antimycin‐insensitive pathways, presumably microsomal. A further 40% passed to O2 by a rotenone‐insensitive path, most likely involving flavin‐linked mitochondrial glycerolphosphate dehydrogenase. The remainder of the flux was rotenone‐sensitive, but less than half of this utilized malate‐oxaloacetate or malate‐aspartate shuttles. The pathway for this residual rotenone‐sensitive fraction (about 20–30% of the total flux) remains to be clarified. These data suggest that in parenchymal cells from normal rat liver the glycerol 3‐phosphate shuttle may be more important for the transfer of reducing equivalents from cytoplasm to mitochondria than has been previously recognized.

Sorbitol uptake by the cells was inhibited up to 70% by uncoupling agents. This inhibition could be overcome by addition of pyruvate as a cytoplasmic hydrogen acceptor or by artificial electron acceptors. This implies that uncoupling agents prevent the oxidation of cytoplasmic NADH by interfering with the operation of the normal hydrogen shuttles between cytoplasm and mitochondria and that these shuttles are energy‐dependent.

The rate‐limiting and energy‐dependent nature of the hydrogen translocating systems as revealed by these studies identify them as potential sites for metabolic regulation and as possible targets for hormonal action.