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...

kDa pleiotropic protein that participates in multiple biological pathways, including DNA damage repair, transcription, cell growth, and apoptosis [11, 12]. Evidence in support of a tumor suppressor role for BRCA1 was indirectly provided by studies showing somatic allelic loss of 17q21 in breast and ovarian tumors [13, 14]. Direct evidence for its tumor suppressor activity was provided by experiments showing that BRCA1 arrested growth of breast and ovarian cancer cells whereas inactivation of the endogenous BRCA1 gene accelerated the growth of normal and malignant cells and induced cellular transformation [15]. While clearly inactivating, BRCA1 germline mutations substantially increase breast and / or ovarian cancer risk, little information is available regarding the cellular and secreted factors involved in regulation of BRCA1 gene expression [11, 16]. Given the intimate involvement of both BRCA1 and the IGF system in normal cell cycle progression as well as in breast cancer cell proliferation, we hypothesized that the regulatory mechanisms controlling BRCA1 and IGF-IR gene expression

I n order to identify rate-limiting processes of hepatic sorbitol and glycerol metabolism in the hyperthyroid state, suspensions of isolated liver cells were prepared from thyroxine-treated rats. Rates of utilization of sorbitol and glycerol by these cells were compared with the data obtained in previous studies with euthyroid animals. At saturating substrate concentrations hydrogen flux from sorbitol or glycerol to 0, was increased 50-60°/, over control values to 2.7-2.8 pmol x g-1 x min-l following thyroxine treatment. When cells were exposed to two sources of reducing-equivalents by incubation with sorbitol and glycerol in combination, the rate of hydrogen flux to 0, was increased about 160°/, above control levels to 4.7 p r n o l~g -~x m i n -~. However, this value was only 85O/, of the sum of the flux rates in cells incubated with sorbitol and glycerol separately, since each member of the substrate mixture partially inhibited the uptake of the other. Pyruvate, added as a cytoplasmic hydrogen acceptor, did not enhance sorbitol and glycerol uptake by liver cells from thyroxine-treated rats except when the cells were incubated with these substrates in combination. In this circumstance pyruvate addition overcame the inhibition of sorbitol uptake by glycerol. It is inferred from these findings that in the hyperthyroid state the transfer of reducing-equivalents from substrate to 0, is not rate-limiting for the hepatic metabolism of sorbitol or glycerol individually, as it is in control animals, but it remains the rate-limiting process when liver cells from thyroxine-treated rats are incubated with mixtures of these substrates.Glucose was the major end-product of sorbitol and glycerol metabolism in cells from both euthyroid and hyperthyroid rats. As anticipated, therefore, rates of gluconeogenesis in cells from thyroxine-treated rats were increased in a manner corresponding to the enhanced capacity for hydrogen translocation. Maximal rates of glucose formation (3.39 pmol x g-l x min-l) were observed when cells were incubated with a mixture of sorbitol, glycerol and pyruvate. It was found that up to 7501, of the hydrogen flux to 0, during sorbitol and glycerol metabolism could utilize an antimycin-sensitive, rotenone-insensitive pathway, most likely involving mitochrondrial glycerolphosphate dehydrogenase. Less than 10 of the reducing-equivalent flux in these experiments was mediated by NAD-linked shuttles involving malate dehydrogenase. Significant stimulation of hydrogen translocation was observed a t 12 h after thyroxine administration and reached a plateau about 4 days after the initial thyroxine injection. This timecourse is consistent with the hypothesis that the effects of thyroxine are mediated through new protein synthesis and may reflect the time-course for the induction of mitochondria1 glycerolphosphate dehydrogenase. It is suggested that induction of this enzyme may represent an example of a general action of thyroxine to increase the capacity of the carrier systems which mediate intercompartmental hyd...

Normal female rats were given 15mug of ethynyloestradiol/kg body wt. for 14 days and were killed on day 15 after starvation for 12-14h. The livers were isolated and were perfused with a medium containing washed bovine erythrocytes, bovine serum albumin, glucose and [1-(14)C]oleic acid; 414mumol of oleate were infused/h during a 3h experimental period. The output of bile and the flow of perfusate/g of liver were decreased in livers from animals pretreated with ethynyloestradiol, whereas the liver weight was increased slightly. The rates of uptake and of utilization of [1-(14)C]oleate were measured when the concentration of unesterified fatty acid in the perfusate plasma was constant. The uptake of unesterified fatty acid was unaffected by pretreatment of the animal with oestrogen; however, the rate of incorporation of [1-(14)C]oleate into hepatic and perfusate triacylglycerol was stimulated, whereas the rate of conversion into ketone bodies was impaired by treatment of the rat with ethynyloestradiol. Pretreatment of the rat with ethynyloestradiol increased the output of very-low-density lipoprotein triacylglycerol, cholesterol, phospholipid and protein. The production of (14)CO(2) and the incorporation of radioactivity into phospholipid, cholesteryl ester and diacylglycerol was unaffected by treatment with the steroid. The net output of glucose by livers from oestrogen-treated rats was impaired despite the apparent increased quantities of glycogen in the liver. The overall effect of pretreatment with oestrogen on hepatic metabolism of fatty acids is the channeling of [1-(14)C]oleate into synthesis and increased output of triacylglycerol as a moiety of the very-low-density lipoprotein, whereas ketogenesis is decreased. The effect of ethynyloestradiol on the liver is apparently independent of the nutritional state of the animal from which the liver was obtained. It is pertinent that hepatocytes prepared from livers of fed rats that had been treated with ethynyloestradiol produced fewer ketone bodies and secreted more triacylglycerol than did hepatocytes prepared from control animals. In these respects, the effects of the steroid were similar in livers from fed or starved (12-14h) rats. Oestrogens may possibly inhibit hepatic oxidation of fatty acid, making more fatty acid available for the synthesis of triacylglycerol, or may stimulate the biosynthesis of triacylglycerol, or may be active on both metabolic pathways.

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