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1.
M R Sly D G Walker 《Comparative biochemistry and physiology. B, Comparative biochemistry》1978,61(3):471-477
1. A method for the preparation of hepatocytes from livers of 11-15-day old rats is described. These cells in general behave similarly to hepatocytes made from adult rats with respect to stimulation of gluconeogenesis by glucagon and adrenaline and the effects of added oleate. 2. Significant differences in the behaviour of hepatocytes from neonatal and adult rats were nevertheless seen in certain situations, e.g. with alanine as gluconeogenic substrate, and appeared to be related to the redox state of the cells. 3. The importance of redox state upon gluconeogenesis was examined in more detail by determining the effects of oleate, ethanol and DL-3-hydroxybutyrate alone and in combinations. Major differences between neonatal and adult hepatocytes were again observed with alanine as substrate. 4. A discussion concludes that, while some relevant differences in the enzyme complements of neonatal and adult rat livers are known, it is the high capacity of the neonatal liver to generate reducing power by oxidation of fatty acid that can explain the observed differences. 相似文献
2.
Stimulation by glucose of gluconeogenesis in hepatocytes isolated from starved rats. 总被引:2,自引:1,他引:1
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Control properties of the gluconeogenic pathway in hepatocytes isolated from starved rats were studied in the presence of glucose. The following observations were made. (1) Glucose stimulated the rate of glucose production from 20 mM-glycerol, from a mixture of 20 mM-lactate and 2 mM-pyruvate, or from pyruvate alone; no stimulation was observed with 20 mM-alanine or 20 mM-dihydroxyacetone. Maximal stimulation was obtained between 2 and 5 mM-glucose, depending on the conditions. At concentrations above 6 mM, gluconeogenesis declined again, so that at 10 mM-glucose the glucose production rate became equal to that in its absence. (2) With glycerol, stimulation of gluconeogenesis by glucose was accompanied by oxidation of cytosolic NADH and reduction of mitochondrial NAD+ and was insensitive to the transaminase inhibitor amino-oxyacetate; this indicated that glucose accelerated the rate of transport of cytosolic reducing equivalents to the mitochondria via the glycerol 1-phosphate shuttle. (3) With lactate plus pyruvate (10:1) as substrates, stimulation of gluconeogenesis by glucose was almost additive to that obtained with glucagon. From an analysis of the effect of glucose on the curves relating gluconeogenic flux and the steady-state intracellular concentrations of gluconeogenic intermediates under various conditions, in the absence and presence of glucagon, it was concluded that addition of glucose stimulated both phosphoenolpyruvate carboxykinase and pyruvate carboxylase activity. 相似文献
3.
1. The fatty acid synthesis in isolated liver cells from fed rats was studied with tritiated water as the radioactive precursor. The cells incorporated 3H20 at a rate of 1.26 mumol per min per g packed cells. 2. Addition of ethanol caused a 20% decrease in the incorporation of tritium into fatty acids. The decrease was correlated to the increase in the NAD-redox level. Probably, the decreased tritium incorporation into fatty acids during ethanol metabolism is due to a decrease in the specific activity of the NADPH used for the synthesis of fatty acids, rather than to a real inhibition of the fatty acid synthesis. 3. Ethanol oxidation via NADPH-consuming pathways and ethanol per se at a concentration of 80 mM had no effect upon the incorporation of tritium into fatty acids. 4. Fructose in a concentration of 15 mM inhibited the fatty acid synthesis by 75%, and this inhibition was further augmented by ethanol. 5. The ioslated rat liver cells oxidized ethanol at a rate of 2.72, 2.93 and 3.48 mumol per min per g packed cells at 5, 20 and 80 mM ethanol, respectively. Fructose had no effect upon ethanol oxidation neither at low nor at high concentrations of ethanol. 6. Ethanol oxidation via the non alcohol dehydrogenase pathway(s) may involve a transfer of reducing equivalents from mitochondrial NADH to cyctosolic NADP+ as judged from measurements of metabolite levels. This conclusion is supported by determinations of 14C yield in glucose from [1-14C] ethanol, and the results are taken as evidence for the presence of hydrogen shuttle activity during metabolism of ethanol, catalyzed by the NAD-dependent alcohol dehydrogenase. A metabolic scheme is proposed to account for the observed changes at low and high concentrations of ethanol. 相似文献
4.
We examined the possibility of quantitative differences in lactate entry into periportal and perivenous hepatocytes under different nutritional states. The rate of14C-L(+)-lactate uptake was determined after 15-second incubations with freshly isolated zonally separated hepatocytes using a centrifuge stop technique at 37 °C and 4 °C, in the presence or absence of either differing amounts of unlabelled lactate or of a hepatocyte lactate transport inhibitor,-cyano-3-hydroxycinnamate. Total entry as well as carrier mediated entry of14C-L(+)-lactate into the isolated cell populations was found to be similar in periportal and perivenous hepatocytes, irrespective of the nutritional state of the animal. Periportal and perivenous hepatocytes showed a greater tendency to transport lactate when isolated from starved animals, in agreement with previously reported data from non-zonally separated isolated hepatocytes. The activity of the hepatocyte plasma-membrane lactate transporter was diminished between fourfold and eightfold in transport studies conducted at 4 °C; similar results were obtained in unseparated and zonally separated suspensions. Temperature dependence of the hepatocyte transporter is markedly less than that reported for the erythrocyte transporter. 相似文献
5.
Control of glycogen metabolism by gluconeogenic and ketogenic substrates in isolated hepatocytes from fed rats. 总被引:1,自引:0,他引:1
1. This study was conducted to examine the effects of gluconeogenic and ketogenic substrates on the activities of the glycogen-metabolizing enzymes and on glycogenolysis in isolated hepatocytes from fed rats. 2. Gluconeogenic substrates like fructose, dihydroxyacetone or lactate turned out to stimulate the glucose-induced activation of glycogen synthase and this effect may be linked, to some extent, to the increase of the cellular glucose 6-phosphate concentration. 3. The effect of fructose was accompanied by the onset of glycogen synthesis. 4. Energetic substrates like fatty acids were also potent activators of glycogen synthase, especially in the presence of glucose. 5. When fatty acids were added alone or together with a physiological concentration of glucose, they induced or potentiated the inhibition of glycogen phosphorylase-a. 6. This inhibitory effect was mediated by a decrease of lactate release. 7. The stimulatory effect of amino acids on glycogen synthase seemed to be direct, non mediated by an inhibition of the phosphorylase-a activity although hepatic glycogenolysis markedly decreased. 8. Moreover, the amino acid action could be linked to their capacities to induce cell swelling and/or to limit proteolysis. 相似文献
6.
7.
M E Tischler D Friedrichs K Coll J R Williamson 《Archives of biochemistry and biophysics》1977,184(1):222-236
Hepatocytes isolated from fed or starved rats were rapidly lysed using the recently described technique of turbulent flow (M. E. Tischler, P. Hecht, and J. R. Williamson, 1977, Arch. Biochem. Biophys., 181, 278–292). Pyridine nucleotide and metabolite contents were measured in the particulate fraction of both whole and disrupted cells after centrifugation through silicone oil. Lactate/pyruvate, β-hydroxybutyrate/acetoacetate, isocitrate/α-ketoglutarate, and malate/pyruvate ratios were determined for calculation of the free and ratios in the cytosol and mitochondria. Lactate/pyruvate ratios measured in the extracellular and cytosolic compartments were in good agreement. Ratios of β-hydroxybutyrate/acetoacetate measured in the extracellular, cytosolic, and mitochondrial compartments also agreed well. Addition of ammonia to fed or starved rat liver cells incubated with lactate, pyruvate, β-hydroxybutyrate, and acetoacetate caused an oxidation of both the NAD and NADP redox states in the mitochondria and cytosol, although the NADP system was oxidized to a greater extent. Calculation of the free NADH and NAD concentrations in the cytosol provided values of about 1 and 400 to 500 μm, respectively, under control conditions. The concentrations of free NADH and NAD in the mitochondria were considerably higher, being 300 to 400 μm and 4 to 6 mm, respectively. The free andm bound NAD systems in both the cytosol and mitochondria were more oxidized in the presence of ammonia. NAD and NADP redox potential differences across the mitochondrial membrane (ΔEh) were not significantly affected by ammonia addition and were generally similar in cells from both fed and starved rats: ?52 and ?56 mV for the NAD system and ?19 to ?29 mV for the NADP system. For the NAD system the cytosolic potential was ?260 mV in the absence of ammonia and ?250 mV in its presence, the mitochondrial values being ?315 and ?303 mV, respectively. The average cytosolic NADP potential, on the other hand, was ?400 mV in the absence and ?384 mV in the presence of ammonia. The mitochondrial fractions yielded NADP potentials of ?420 mV in the absence of ammonia with both fed and starved rats. Ammonia decreased the mitochondrial NADP potential to ?404 mV in fed rats and to ?415 mV in starved rats. The calculated free and ratios as well as metabolite concentrations were used to evaluate the mass action ratios of both cytosolic and mitochondrial enzymes. Cytosolic alanine aminotransferase remained near equilibrium in the absence and presence of ammonia, while cytosolic and mitochondrial aspartate aminotransferase reactions deviated up to fivefold. The glutamate dehydrogenase reaction was in near equilibrium with the NAD system, but deviated by three to four orders of magnitude from equilibrium with the NADP system in the direction favoring glutamate synthesis rather than deaminatión. Cytosolic malate dehydrogenase deviated from equilibrium by about one order of magnitude, while mitochondrial malate dehydrogenase and citrate synthase deviated by two to six orders of magnitude. These data emphasize the importance of regulation of the citric acid cycle at the citrate synthase step. 相似文献
8.
Interactions between vasopressin and glucagon on ketogenesis and oleate metabolism in isolated hepatocytes from fed rats. 总被引:8,自引:8,他引:0
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Vasopressin (10nM) inhibited ketogenesis (56%) in hepatocytes from fed rats when oleate (1 mM) was the substrate, but had no effect with butyrate (10mM). The hormone increased the accumulation of lactate and stimulated the esterification of [1(-14)C]oleate (70%). These effects of vasopressin were reversed by glucagon (10 nM). The physiological implications of these findings are discussed. 相似文献
9.
Alterations in energy status by menadione metabolism in hepatocytes isolated from fasted and fed rats 总被引:2,自引:0,他引:2
F A Redegeld R M Moison A S Koster J Noordhoek 《Archives of biochemistry and biophysics》1989,273(1):215-222
The biochemical mechanism of cytotoxicity, induced by the quinoid compound 2-methyl 1,4-naphthoquinone (menadione), was investigated in hepatocytes freshly isolated from fasted and fed rats. Hepatocytes from fasted rats were significantly more vulnerable to the toxicity of menadione than hepatocytes from fed rats. Menadione (150 microM) induced a 50% loss of viability of cells (LT50) from fasted rats after 55 min of incubation, whereas a LT50 of 80 min was observed after exposure of hepatocytes from fed rats to menadione. Glutathione and NADPH levels were rapidly depleted by menadione metabolism. This depletion was sustained during the incubation period. No significant differences were found in the time course and extent of the menadione-induced glutathione and NADPH depletion in hepatocytes of both nutritional states. Menadione also affected the energy status of the hepatocytes. The ATP content of cells from fasted rats decreased to 50% (AT50) within 18 min of exposure to menadione, whereas a 50% loss of ATP content of hepatocytes from fed rats was reached at 65 min. In contrast to depletion of glutathione and NADPH, the time course and extent of menadione-induced ATP depletion correlated well with the time of onset and rate of cell killing. Our results suggest that menadione metabolism may interfere with both mitochondrial and glycolytic ATP production. Depletion of ATP might be a critical step in menadione-induced cytotoxicity. 相似文献
10.
M J Geelen 《Life sciences》1977,20(6):1027-1034
Hepatocytes isolated from the liver of rats starved for two days synthesized glycogen only when incubated in the presence of both glucose and glucogenic precursors (combinations of alanine, glycerol, pyruvate, lactate or fructose). Unlabeled glucogenic precursors facilitated the incorporation of [U-14C]glucose into glycogen. Unlabeled glucose likewise greatly enhanced glycogen synthesis from isotopically labeled lactate and other glucogenic precursors.In those systems which contained no added endocrines glucose dampened glycogen phosphorylase activity in a cAMP-independent fashion. Fructose is unable to mimic the effects of glucose on glycogen deposition and on glycogen phosphorylase activity. 相似文献
11.
The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose.
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The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction. 相似文献
12.
H K Metcalfe J P Monson R D Cohen C Padgham 《The Journal of biological chemistry》1988,263(36):19505-19509
Hepatic plasma membrane lactate transport was studied in isolated hepatocytes prepared from fed, starved, and streptozotocin diabetic rats. Carrier-mediated lactate entry was determined using the lactate transport inhibitors alpha-cyano-3-hydroxycinnamate and D-3-hydroxybutyrate and was significantly greater in hepatocytes from starved compared to fed rats and in hepatocytes from diabetic fed compared to fed rats. The saturable component of lactate entry which corresponds to carrier-mediated transport was higher in the starved than in the fed state with results from diabetic fed being intermediate between the two. Insulin treatment prevented the increment in carrier-mediated lactate transport observed in hepatocytes from diabetic fed rats. The data indicate that hepatic plasma membrane lactate transport is increased under conditions of starvation and diabetes mellitus. This may partly explain the increased gluconeogenic flux under these conditions. 相似文献
13.
Isolated parenchymal cells from fed rat liver rapidly lose glycogen when incubated with glucose. The addition of glycerol or fructose but not insulin prevents much of the breakdown. When cells are incubated with glycerol and glucose, more glycogen is retained with the further addition of xylitol than of fructose or pyruvate. Oleate stimulates glycogen breakdown. The results indicate that glycerol may play an important physiological role in the control of glycogen synthesis in the liver, possibly by esterifying fatty acids. Furthermore, the results support the concept that the main effect of insulin on liver glycogen levels may be the results of diminished flow of free fatty acids to the liver. 相似文献
14.
E Chico J S Olavarría I N de Castro 《Biochemical and biophysical research communications》1978,83(4):1422-1429
In hepatocytes isolated from fed rats, the addition of fructose caused an inhibition of respiration. In hepatocytes isolated from starved rats the Crabtree effect was not observed. No difference in oxygen uptake was found by addition of glucose to hepatocytes from fed or starved animals. The inhibition of respiration was parallel with a rise in the glycolytic flux and the oxidation of the mitochondrial respiratory carriers. The metabolic conditions in which the Crabtree effect can be operative in liver cells are discussed. 相似文献
15.
Sybil Golden Gad Riesenfeld Joseph Katz 《Archives of biochemistry and biophysics》1982,213(1):118-126
Hepatocytes were isolated from livers of mature male and female starved Japanese quail (Coturnix coturnix japonica). The hepatocytes take up lactate and dihydroxyacetone extensively, and have a very high rate of glucose synthesis from these substrates. Fructose uptake and incorporation into glucose is much less. Pyruvate and alanine are taken up extensively, but form little glucose. There is negligible lipogenesis in cells of starved quail. Alanine increases up to 10-fold incorporation of 3HOH and 14C from several substrates into fatty acids, but it remains insignificant as compared to lipogenesis by cells of fed quail. There is little utilization of glucose, even in the presence of alanine, in marked contrast to hepatocytes from fed quail. However, glucose is phosphorylated at high rates, but most of the glucose 6-phosphate is recycled to glucose. There is a marked difference in the metabolism of polyols between the sexes. Glycerol, xylitol, and sorbitol are converted nearly quantitatively into glucose by hepatocytes of starved female quail. In cells of starved males, the uptake of polyols is higher, but conversion to glucose less efficient. In cells of starved male quail, alanine markedly stimulates the uptake of glycerol and xylitol and their conversion to glucose, but has no effect on sorbitol metabolism. In cells of female quail, alanine is without a significant effect on polyol metabolism. 相似文献
16.
Loranne Agius 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,800(3):306-308
Parenchymal hepatocytes isolated from lactating rats had similar rates of amino acid incorporation into protein, but increased rates of urea formation compared to hepatocytes from non-lactating rats. The increased urea formation may be due to increased amino acid transport and degradation. The liver contributes to the increased utilization of amino acids during lactation. 相似文献
17.
1. The influence of ethanol on the metabolism of livers from fed and starved rats has been studied in liver-perfusion experiments. Results have been obtained on oxygen consumption and carbon dioxide production, on glucose release and uptake by the liver and on changes in the concentrations of lactate and pyruvate and of β-hydroxybutyrate and acetoacetate in the perfusion medium. 2. Oxygen consumption and carbon dioxide production were lower in livers from starved rats than in livers from fed rats. Ethanol had no effect on the oxygen consumption of either type of liver. After the addition of ethanol to the perfusion medium carbon dioxide production ceased almost completely, the change being faster in livers from starved rats. 3. With livers from fed rats glucose was released from the liver into the perfusion medium. This release was slightly greater when ethanol was present. With livers from starved rats no release of glucose was observed, and when ethanol was added a marked uptake of glucose from the medium was found. A simultaneous release of glycolytic end products, lactate and pyruvate, into the medium occurred. 4. Acetate was the main metabolite accumulating in the perfusion medium when ethanol was oxidized. With livers from starved rats a slightly increased formation of ketone bodies was found when ethanol was present. 5. The lactate/pyruvate concentration ratio in the perfusion medium increased from 10 to 87 with livers from fed rats and from 20 to 171 with livers from starved rats when the livers were perfused with ethanol in the medium. The β-hydroxybutyrate/acetoacetate concentration ratio increased from 0·8 to 7·6 with livers from fed rats and from 1·0 to 9·5 with livers from starved rats when ethanol was added to the medium. 6. The effects of ethanol are discussed and related to changes in the redox state of the liver that produce new conditions for some metabolic pathways. 相似文献
18.
19.
Ammonia overloading was investigated during glucose and fructose metabolism in isolated hepatocytes under a variety of metabolic conditions. In all assay conditions, the glycolytic flux and oxygen uptake was not modified by 10 mM ammonia. In hepatocytes isolated from rats fed as libitum, the presence of ammonia caused a decrease in the production of lactate (pyruvate); this effect was not observed in anaerobic incubations, in hepatocytes isolated from starved animals, or in fetal hepatocytes. In spite of an overproduction of urea, ammonia detoxification also takes place by the synthesis of alanine, glutamate and aspartate. Addition of 1 mM aminooxyacetate, an inhibitor of aminotransferases, to the incubation medium prevents the formation of these amino acids, and also prevents the decrease of lactate in hepatocytes isolated from fed animals. 相似文献
20.