Regulatory Role of Reducing‐Equivalent Transfer from Substrate to Oxygen in the Hepatic Metabolism of Glycerol and Sorbitol

Abstract

Suspensions of morphologically intact isolated rat-liver cells were used in conjunction with specific inhibitors to identify and quantitate the hepatic hydrogen-trans'ocating systems involved in the transfer ofreducing-equivalents from sorbitol or glycerol to 0,. 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 a t saturating substrate concentrations, rates of sorbitol or glycerol 3-phosphate oxidation were closely similar (about 1.8 pmol x g wet weight-l x min-I). There was an inverse relationship between rates of sorbitol and glycerol uptake so that the rate of hydrogen flux to 0, 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 a t 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 0, 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 1501, of the flux of reducing equivalents to 0, involved antimycin-insensitive pathways, presumably microsomal. A further 40°/, passed to 0, by a rotenone-insensitive path, most likely involving flavinlinked mitochondrial glycerolphosphate dehydrogenase. The remainder of the flux was rotenonesensitive, but less than half of this utilized malate-oxaloacetate or malate-aspartate shuttles.The pathway for this residual rotenone-sensitive fraction (about 20-30 ,Ilo 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 7001, 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...

Stimulatory Effects of Thyroxine Administration on Reducing‐Equivalent Transfer from Substrate to Oxygen during Hepatic Metabolism of Sorbitol and Glycerol

“…After appropriate correction, the rates of substrate uptake are equivalent to the rates of hydrogen flux from these substrates to 0, [l]. As in previous studies [1,3,12] the dry weight of the cells was obtained after precipitation with trichloracetic acid and a conversion factor of 3.77 [12] …”

“…After perfusion for 15 to 25 min, the softened liver was dispersed in 60 ml of the same medium and the dispersion transferred to two 250-ml conical flasks, gassed with 95O/, O,, 50/, CO, and shaken a t 120 cycles/min for 12 min a t 37 "C to break up cell clumps and to digest debris. The cells were filtered through nylon mesh of 210 pm pore size (Tobler, Ernst and Traber Inc., New York, U.S.A.), washed three times in ice-cold enzyme-free medium, devoid of added bicarbonate, and use immediately for metabolic studies, as described previously [1,3,12]. The cells (100-160 mg wet weight) were incubated for 40min in a final volume of 4 m l a t 37 "C with a gas phase of air.…”

THE PRODUCTION OF |14C| OXALATE DURING THE METABOLISM OF |14C| CARBOHYDRATES IN ISOLATED RAT HEPATOCYTES

“…Secondly, glycerol and xylitol show a marked similarity in all areas of metabolism including oxalate production, yet sorbitol, which is also very similar to xylitol in the pattern of metabolites produced, produces little oxalate in comparison. Thirdly, sorbitol produces fructose and NADH in the first step of its hepatic breakdown yet fructose alone produces twice as much glucose, 3 times more lactate, 28 times more pyruvate and 10 times more oxalate than sorbitol, thus emphasizing the major regulatory effect which elevated cytosolic NADH levels play in sorbitol metabolism (Berry, Kun and Werner, 1973) and oxalate synthesis (Rofe ct al., 1976). A correlation exists between oxalate and glycerophosphate production with the polyols, but not with fructose, as substrates.…”

“…and those which do not. This distinction is important in that a reduced cellular redox state limits the further metabolism of polyols (Berry et al, 1973;Williamson. Jakob and Refmo, 1971) and also limits the oxidation of the immediate oxalate precursors, glycollate and glyoxylate (Rofe etal., 1916).…”

The Role of Hydrogen Translocating Shuttles during Ethanol Oxidation in Hepatocytes from Euthyroid and Hyperthyroid Rats

“…With dihydroxyacetone present, production of glucose (see below) utilizes ATP and makes ADP available for oxidation of mitochondrial reducing equivalents by oxygen. With glycerol present the total flux of reducing equivalents from cytosol to mitochondria can be calculated as the sum of ethanol uptake, lactate production and twice the amount of glucose produced [30], assuming that oxidation of acetaldehyde occurs in the mitochondria [31] and that the contribution of total ethanol consumption of ethanol oxidation not mediated by alcohol dehydrogenase is relatively small, as appears to be the case [l, 2,6,32]. Under these conditions total hydrogen flux to the mitochondria amounted to 492 pmol x (g dry weight of cells)-' x h-', which includes 28 f 3 pmol lactate formed (not shown in Table 1).…”