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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...

We have studied the inhibitory action of long-and short-chain fatty acids on hepatic glucose utilization in hepatocytes isolated from fasted rats. The rates of hepatic glucose phosphorylation and glycolysis were determined from the tritiated products of [2-'H] and [6-3H]glucose metabolism, respectively. The difference between these was taken as an estimate of the 'cycling' between glucose and glucose-6-phosphate. In the presence of 40 mM glucose this cycling was estimated at 0.68 pmol/min/g wet wt. Glucose phosphorylation was unaffected during palmitate and hexanoate oxidation to ketone bodies but glycolysis was inhibited. The rate of glucose cycling was increased during this phase to 1.25 pmol/min/g. Following the complete metabolism of the fatty acids, glycolysis was reinstated and cycling rates returned to control levels. Hepatic glucose cycling appears to be an important component of the glucose/fatty acid cycle.

Morphologically intact muscle cells were prepared by perfusing adult rat hearts with a balanced salt medium containing 0.1% collagenase and 0.2% hyaluronidase. Yields of intact cells representing up to 25% of the weight of the heart were obtained. The cells separated along the line of the intercalated discs, widi cleavage of desmosomes but with tearing of the plasma membrane in the region of the gap junction, so that when two contiguous cells were parted, the gap junction was retained intact attached to one of the cells. The fine structure of undamaged cells was indistinguishable from that of normal myocardial cells in situ, whereas damaged cells characteristically revealed numerous cytoplasmic vacuoles, clumping of myofilaments, and blebbing of the cell membrane, but morphologically normal mitochondria. The earliest lesion detected was a dilatation of the T (transverse tubular) system.Respiration in the intact cells was linear for 30 to 60 minutes and approximately two to three times the rate observed with heart muscle slices or the arrested isolated perfused heart. Oxygen uptake was stimulated by pyruvate but not by lactate. These observations demonstrate the feasibility of preparing intact isolated cells from adult rat heart and their potential value in histologic, pharmacologic, and metabolic studies.

Suspensions of isolated liver cells were prepared by the method of Branster and Morton (3). Livers of mice anesthetized with ether were perfused via the on

Fluoromalate (an inhibitor of malate dehydrogenase and the malate carrier) or difluorooxaloacetate (an inhibitor of aspartate aminotransferase) inhibited gluconeogenesis from both pyruvate and lactate, but had no effect on glucose formation from fructose or endogenous substrates. When cells were incubated with both fluorocarboxylic acids simultaneously, an additive effect on the inhibition of glucose formation from lactate was observed, but the inhibition was not additive when pyruvate was the glucogenic precursor. Pyruvate removal was reduced I5 O/ , , , by difluorooxaloacetate and 44O/, by fluoromalate. Lactate production from pyruvate was inhbited 36O/, by fluoromalate but was almost unaffected by ditluorooxaloacetate.The results suggest that both malate-oxaloacetate and glutamate-aspartate shuttles participate, but to different extents, in glucose formation from either pyruvate or lactate. The malateoxaloacetate shuttle predominates during gluconeogenesis from pyurvate whereas the glutamateaspartate shuttle is of greater importance when lactate is the gluconeogenic precursor. However, the inhibition by fluoromalate of glucose formation from lactate indicates that not all the reducing equivalents arising during lactate oxidation are directly available for the reductive step of gluconeogenesis.Neither of the two shuttles seems to compensate for inhibition of the alternative system. This lack of compensation implies that each shuttle is working close to or a t its maximal capacity. Hence, transfer processes between mitochondria and cytoplasm must be considered as potential rate-limiting and regulatory sites for gluconeogenesis. The failure of fluoromalate to inhibit ethanol oxidation suggests that alternatives to the malate-oxaloacetate shuttle exist for the transfer to the mitochondria of reducing equivalents generated in the cytoplasmic compartment.

Rates of cycling between glucose and glucose 6-phosphate and between glucose and pyruvate, and the effects of these cycles on glucose metabolism, were compared in hepatocytes isolated from fasted normal or streptozotocin-induced diabetic rats. In diabetic hepatocytes the rate of glucose phosphorylation was 30% lower than that in normal hepatocytes, and there was a doubling of the rate of glucose/glucose 6-phosphate cycling. In addition, the rate of glycolysis was 60% lower in diabetic hepatocytes. This inhibition of glycolysis and stimulation of glucose/glucose 6-phosphate cycling appeared to be a consequence of the elevated rates of endogenous fatty acid oxidation observed in diabetic hepatocytes. The proportion of glycolytically derived pyruvate that was recycled to glucose was more than doubled in hepatocytes from diabetic rats compared with normal animals. This increase also appeared to be linked to the high rates of endogenous fatty acid oxidation in diabetic cells. As a consequence of the increased rates of both these cycles, 85% of all glucose molecules taken up by diabetic hepatocytes were recycled to glucose, compared with only 50% in normal hepatocytes. Glucose cycling is therefore likely to make a substantial contribution to the hyperglycemia of diabetes.

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