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PubMed1991

Antiobesity effects of dehydroepiandrosterone are mediated by futile substrate cycling in hepatocytes of BHE/cdb rats.

This study investigated the hypothesis that dehydroepiandrosterone (DHEA) functions as an antiobesity agent by promoting energy wastage via hepatic substrate cycling in prediabetic male BHE/cdb rats. Weanling BHE/cdb rats fed a 65% glucose diet were injected intraperitoneally daily with either DHEA (0.35 mol/kg body wt) or vehicle (1 mL/kg body wt) for 7 wk. The DHEA treatment significantly (P less than 0.05) reduced body weight gain. The DHEA-treated rats had epididymal and retroperitoneal fat pads that were 40% and 66% lighter, respectively, than those of control rats. The residual carcasses (i.e., minus fat pads, liver and ingesta) of DHEA-treated rats contained a significantly lower percentage of fat than those of control rats. The DHEA treatment significantly reduced fasting serum glucose and triglycerides without affecting total or HDL cholesterol. Isolated hepatocytes from DHEA-treated rats converted 2.5 times as much [U-14C]glucose to 14CO2 and one-half as much alanine to glucose as did hepatocytes from control rats. The DHEA treatment increased the specific activities of malic enzyme and lactate dehydrogenase 4.0- and 1.8-fold, respectively. Hepatocytes from DHEA-treated rats tended (P less than 0.08) to have lower phosphoenolpyruvate carboxykinase activities than hepatocytes from control rats. These data suggest that DHEA treatment exerts some of its antiobesity and antidiabetic effects in prediabetic, lipemic BHE/cdb rats by promoting hepatic glucose oxidation and reducing gluconeogenesis.

Animals↗
PubMed1988

The influence of starvation and natural refeeding on the rate of triacylglycerol/fatty acid substrate cycling in brown adipose tissue and different white adipose sites of the rat in vivo. The role of insulin and the sympathetic nervous system.

Triacylglycerol/fatty acid substrate cycling was measured in vivo in brown adipose tissue (BAT) and white adipose tissue (WAT) of fed, starved and refed rats. Starvation (24h) significantly decreased the rate of cycling in BAT, and refeeding chow diet led to a rapid, 6-fold increase in cycling. Cycling rate in WAT was much lower than in BAT, and was not influenced by fasting or refeeding. Similar rates of cycling were found in epididymal, mesenteric, subcutaneous, and scapular WAT depots. Sympathetic denervation of interscapular BAT abolished the response of the tissue to refeeding, as did acute suppression of insulin secretion. Similarly, rats fasted for 3 days showed no acute increase in the activity of the cycle following refeeding.

Adipose Tissue↗
PubMed1983

The rate of substrate cycling between glucose and glucose 6-phosphate in muscle and fat-body of the hawk moth (Acherontia atropos) at rest and during flight.

1. The rate of substrate cycling between glucose and glucose 6-phosphate was measured in tissues of the hawk moth (Acherontia atropos). 2. The insect was injected with [2-3H,2-14C]glucose, and after periods of time at rest or flying the animal was freeze-clamped. Separation of glucose and hexose monophosphate from the tissues was performed by paper chromatography and t.l.c., and the 3H and 14C radioactivities in these compounds were measured. 3. On the basis of the 3H/14C ratios in these compounds and the measured rate of glycolysis, the rate of cycling was calculated. The rates of cycling were 0.03, 0.10, 0.06 and 3.9 mumol/min per g for fat-body at rest and during flight and for flight muscle at rest and during flight respectively. 4. The marked increase in the cycling rate between glucose and glucose 6-phosphate upon flight contrasts with the finding of Clark, Bloxham, Holland & Lardy [(1973) Biochem. J. 134, 589-597] in the bumble-bee, in which this condition inhibited cycling. It is suggested that the increased rate of cycling increases the sensitivity of glucose phosphorylation to changes in the concentrations of effectors of hexokinase should it be necessary to increase the rate of glycolysis in muscle, for example, to increase power output of the flight muscle for increased speed of flight.

Adipose Tissue↗
PubMed1987

Regulation of pyrimidine deoxyribonucleotide metabolism by substrate cycles in dCMP deaminase-deficient V79 hamster cells.

A mutant V79 hamster fibroblast cell line lacking the enzyme dCMP deaminase was used to study the regulation of deoxynucleoside triphosphate pools by substrate cycles between pyrimidine deoxyribosides and their 5'-phosphates. Such cycles were suggested earlier to set the rates of cellular import and export of deoxyribosides, thereby influencing pool sizes (V. Bianchi, E. Pontis, and P. Reichard, Proc. Natl. Acad. Sci. USA 83:986-990, 1986). While normal V79 cells derived more than 80% of their dTTP from CDP reduction via deamination of dCMP, the mutant cells had to rely completely on UDP reduction for de novo synthesis of dTTP, which became limiting for DNA synthesis. Because of the allosteric properties of ribonucleotide reductase, CDP reduction was not diminished, leading to a large expansion of the dCTP pool. The increase of this pool was kept in check by a shift in the balance of the deoxycytidine/dCMP cycle towards the deoxynucleoside, leading to massive excretion of deoxycytidine. In contrast, the balance of the deoxyuridine/dUMP cycle was shifted towards the nucleotide, facilitating import of extracellular deoxynucleosides.

Animals↗
PubMed1984

Measurement of the rate of substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate in skeletal muscle by using a single-isotope technique.

The effects of several agents on the rates of the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle were measured in incubated epitrochlearis muscles of the rat by monitoring the transfer of radiolabel from [6-14C]glucose to the 1-position of glucose residues in glycogen. The cycling rates observed were almost identical with those previously obtained by using the well-established dual-isotope technique. In particular, it was found that the beta-adrenoceptor agonist isoprenaline increased the cycling rate about 12-fold.

Animals↗
PubMed1987

Experimental evidence for a zero-order ultrasensitivity in a simple substrate cycle.

It was shown [Goldbeter & Koshland (1981), Proc. Natl. Acad. Sci. USA, 78, 6840-6844] that amplified sensitivity may arise in reversible covalent modification systems, when the converter enzymes operate in their zero-order region. We show that "zero-order ultrasensitivity" may also occur in simple substrate cycles. The experimental study deals with the Formate/Lactic dehydrogenases model cycle, interconverting the reduced and oxidized forms of NAD. For NAD(H) concentrations high enough (with respect to the enzyme KM's), abrupt changes in the steady-state substrate concentrations may result from small variations in the ratio of maximal enzyme activities. The amplification factors are measured. Implications in metabolic regulation are also taken up.

Aldehyde Oxidoreductases↗
PubMed2000

Kinetics of a self-amplifying substrate cycle: ADP-ATP cycling assay.

A kinetic study of an ATP-ADP amplification cyclic system involving the enzymes adenylate kinase, pyruvate kinase and L-lactate dehydrogenase has been made. The stoichiometry of the cycle is 2:1, because two molecules of ADP are synthesized from one each of ATP and AMP, and one molecule of ADP is converted back into one of ATP at each turn of the cycle. This results in a continuous exponential increase in the concentrations of ATP and ADP in the reaction medium, according to the equations obtained. This is therefore a substrate cycle that amplifies itself, the cycling rate increasing continuously with time. The background signal of the reagent was reduced by using apyrase to degrade ATP and ADP in the reagent, permitting detection limits as low as 16 pmol of ATP and/or ADP in a continuous spectrophotometric assay.

Adenosine Diphosphate↗
PubMed1994

Existence and role of substrate cycling between AMP and adenosine in isolated rabbit cardiomyocytes under control conditions and in ATP depletion.

BACKGROUND: Adenosine, a physiological coronary vasodilator, has been proposed to regulate coronary circulation according to myocardial oxygen demand. In the present study, we investigated the mechanisms of adenosine formation and utilization in isolated rabbit cardiomyocytes and, in particular, the existence and the role of substrate cycling between AMP and adenosine in the regulation of its concentration. METHODS AND RESULTS: Rabbit cardiomyocytes were isolated by collagenase perfusion and incubated in HEPES-buffered Krebs-Henseleit solution at 37 degrees C, pH 7.4, in control conditions and in ATP depletion achieved by inhibiting glycolysis with 5 mmol/L iodoacetate. Under control conditions, adenosine accumulated at a rate of 4 pmol.min-1.10(-6) cells. The 13-fold elevation of adenosine accumulation induced by iodotubercidin (ITu), an inhibitor of adenosine kinase, proves that adenosine is normally recycled into AMP. This recycling involves 95% of the adenosine formed. In ATP depletion, adenosine accumulated at the rate of 335 pmol.min-1.10(-6) cells and was no longer rephosphorylated after 20 minutes, as shown by the absence of effect of ITu after this time interval. Moreover, adenosine was deaminated, as indicated by the twofold increase of its accumulation induced by deoxycoformycin (dCF), an inhibitor of adenosine deaminase. Both in control conditions and in ATP depletion, adenosine-dialdehyde, an inhibitor of S-adenosylhomocysteine (SAH) hydrolase, had no significant effect on adenosine formation, indicating that the transmethylation pathway is not an important source of adenosine in rabbit cardiomyocytes. CONCLUSIONS: The results indicate that recycling of adenosine into AMP is essential for the maintenance of low, nonvasodilatory concentrations of the nucleoside under control conditions and that interruption of recycling plays an important role in elevating adenosine during ATP depletion.

Adenine Nucleotides↗
PubMed1986

Interrelations between substrate cycles and de novo synthesis of pyrimidine deoxyribonucleoside triphosphates in 3T6 cells.

Degradation of pyrimidine deoxyribonucleoside triphosphates plays a major role in the regulation of their pool sizes in 3T6 cells. During normal growth, these cells excrete deoxyribonucleosides (mostly deoxyuridine) into the medium. When DNA strand elongation is inhibited, de novo synthesis of dCTP and dTTP continues, followed by degradation of the deoxyribonucleotides. We now demonstrate that inhibition of de novo synthesis with hydroxyurea stops degradation of deoxyribonucleotides. We now demonstrate that inhibition of de novo synthesis with hydroxyurea stops degradation of deoxyribonucleotides and leads to an influx of deoxyuridine from the medium. This effect appears to be caused by a large drop in the size of the intracellular dUMP pool. We propose that substrate cycles, involving phosphorylation of deoxyribonucleosides by kinases and dephosphorylation of deoxyribonucleoside 5'-phosphates by a nucleotidase, participate in the regulation of the size of pyrimidine deoxyribonucleoside triphosphate pools by directing the flow of deoxyribonucleosides across the cell membrane. While kinases are regulated mainly by allosteric effects, the activity of the nucleotidase appears to be regulated by substrate concentration.

Animals↗
PubMed1991

An in vitro model showing different rates of substrate cycle for phosphofructokinases of Escherichia coli with different kinetic properties.

An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fructose-bisphosphatase. The model is applied to the study of three PFK of Escherichia coli: two isoenzymes, phosphofructokinase-1 (PFK-1) and phosphofructokinase-2 (PFK-2), and a mutant form of phosphofructokinase-2 (PFK-2*). Results show that for a variety of conditions the PFK-1/fructose-bisphosphatase pair gives the lowest and the PFK-2*/fructose-bisphosphatase pair the highest rates of substrate cycle, with the PFK-2/fructose-bisphosphatase pair in an intermediate position. The effects of variables such as maximum activity ratios and MgATP concentration were explored. The possible role of MgATP in decreasing the futile cycle of the PFK-2/fructose-bisphosphatase pair is described. The results are discussed in terms of possible metabolic consequences of PFK-2* and of predictions of the model to be tested in vivo.

Adenosine Triphosphate↗
PubMed1983

Development of rat liver and colon substrate cycle enzymes in offspring with and without prenatal exposure to a carcinogenic dose of 1,2-dimethylhydrazine.

Pregnant F-344 rats were exposed by intubation to a single dose (35 mg/kg) of 1,2-dimethylhydrazine dihydrochloride or to an acetate buffer on day 14 of gestation. A detailed examination of the effects of this dose of 1,2-dimethylhydrazine on brush border enzymes in the offspring was performed. Changes in the liver and colon levels of the alpha-glycerophosphate and malate/aspartate substrate cycle enzymes were measured during the development; at days 17 and 20 of gestation and at 2, 6, 13, 20, 27, 55, 110 days and 1 year after birth. It is concluded that metabolic energy enzymes, glutamate oxaloacetate transaminase and malate dehydrogenase, are more sensitive to 1,2-dimethylhydrazine treatment than are the brush border hydrolases or NAD- and Fp-linked alpha-glycerophosphate dehydrogenases.

1,2-Dimethylhydrazine↗
PubMed1986

Nuclear magnetic resonance studies of carbohydrate metabolism and substrate cycling in Fasciola hepatica.

We have been interested in clarifying unique features of glycolytic metabolism in parasitic trematodes and in developing improved methods for monitoring the effects of pharmacologic agents that may alter functions of the pathway. In the present study metabolism of [1-13C]glucose by the common liver fluke, Fasciola hepatica, was studied both directly with 13C NMR and indirectly by observation of 13C-induced multiplet splitting of the 1H NMR resonances from the glycolytic end-products propionate and acetate. The extent of 13C enrichment of the end-products demonstrated that exogenous glucose was the predominant source of glycolytic substrate under the incubation conditions used. Specific enrichments of propionate and acetate in 13C were similar and enrichments at the acetate C-1 carboxyl and C-2 methyl were identical, demonstrating that acetate is generated preferentially from pyruvate formed by the malic enzyme reaction. End-product synthesized in substrate-free medium following incorporation of a small fraction of [1-13C]glucose into endogenous glycogen demonstrates that glucose equivalents from the most recently synthesized polymeric chains, which have a specific activity in 13C equal to that of the exogenous glucose, are preferentially used for glycogenolysis. Stimulation of flukes with 0.1 mM serotonin results in a reduction of the propionate/acetate 13C enrichment ratio consistent with functional "compartmentation" of glycogen pools having different structures and/or specific enrichment in 13C. Glucose equivalents were incorporated into glycogen in intact flukes with label at both the C-1 and C-6 positions during perfusion with [1-13C]glucose as a consequence of "substrate cycling" at the phosphofructokinase/fructosebisphosphatase enzyme couple. The observed glycogen C-6/C-1 labeling ratio of 0.42 and the net glycolytic flux of 11 mumol/g wet weight/hr imply a total forward flux of about 29 mumol/g wet weight/hr through phosphofructokinase with a reverse flux of about 17 mumol/g wet weight/hr through fructosebisphosphatase. Net glycolytic flux is therefore a poor estimate of the true flux through phosphofructokinase in this preparation.

Acetates↗
PubMed1989

The rate of the AMP/adenosine substrate cycle in concanavalin-A-stimulated rat lymphocytes.

The effect of adenosine on the metabolism of prelabelled adenine nucleotides was investigated in concanavalin-A-stimulated rat lymphocytes. Adenosine in the presence of the adenosine deaminase inhibitor, deoxycoformycin, caused a 2-fold increase in the ATP concentration. This effect was, in part, countereacted by an increased rate of adenine nucleotide catabolism, which could be explained by a stimulation of AMP deaminase (EC 3.5.4.6). At the same time a continuous rate of labelled adenosine production was found, which was not affected by the increased ATP concentration and which could only be detected by the trapping effect of a high concentration of added unlabelled adenosine. It is concluded that the rate of the substrate cycle between AMP and adenosine is low (1.9 +/- 0.2 nmol/h per 10(7) cells) in comparison to the rate of AMP deamination.

5'-Nucleotidase↗
PubMed1994

The involvement of fructose 2,6-bisphosphate in substrate cycle control in the nonoxidative stage of the pentose phosphate pathway. A phosphorus magnetic resonance spectroscopy study.

The role of fructose 2,6-bisphosphate in the interconversion of sedoheptulose 7-phosphate and sedoheptulose 1,7-bisphosphate in rat liver cytosol fractions was studied by means of phosphorus magnetic resonance spectroscopy. When the activity of 6-phosphofructo-1-kinase was inhibited by a high concentration of ATP, the addition of fructose 2,6-bisphosphate led to a marked decrease in sedoheptulose 7-phosphate levels, accompanied by an increased concentration of ADP. Fructose 2,6-bisphosphate essentially inhibited both the decrease in sedoheptulose 1,7-bisphosphate concentration and the accumulation of Pi in the incubation mixture. The data provided evidence that fructose 2,6-bisphosphate can regulate the substrate cycle: sedoheptulose 7-phosphate<-->sedoheptulose 1,7-bisphosphate in the liver, and thus control the flux through the nonoxidative stage of the pentose phosphate pathway.

Animals↗
PubMed2005

Corticotropin-releasing hormone directly stimulates thermogenesis in skeletal muscle possibly through substrate cycling between de novo lipogenesis and lipid oxidation.

The mechanisms by which CRH and related peptides (i.e. the CRH/urocortin system) exert their control over thermogenesis and weight regulation have until now focused only upon their effects on brain centers controlling sympathetic outflow. Using a method that involves repeated oxygen uptake determinations in intact mouse skeletal muscle, we report here that CRH can act directly on skeletal muscle to stimulate thermogenesis, an effect that is more pronounced in oxidative than in glycolytic muscles and that can be inhibited by a selective CRH-R2 antagonist or blunted by a nonselective CRH receptor antagonist. This thermogenic effect of CRH can also be blocked by interference along pathways of de novo lipogenesis and lipid oxidation, as well as by inhibitors of phosphatidylinositol 3-kinase or AMP-activated protein kinase. Taken together, these studies demonstrate that CRH can directly stimulate thermogenesis in skeletal muscle, and in addition raise the possibility that this thermogenic effect, which requires both phosphatidylinositol 3-kinase and AMP-activated protein kinase signaling, might occur via substrate cycling between de novo lipogenesis and lipid oxidation. The effect of CRH in directly stimulating thermogenesis in skeletal muscle underscores a potentially important peripheral role for the CRH/urocortin system in the control of thermogenesis in this tissue, in its protection against excessive intramyocellular lipid storage, and hence against skeletal muscle lipotoxicity and insulin resistance.

Adenylate Kinase↗
PubMed1991

Inhibition of gluconeogenesis by tolbutamide in isolated rat hepatocytes: modulation of glucose-6-phosphate substrate cycle.

In hepatocytes isolated from 24-hour fasted rats, the oral hypoglycemic agent tolbutamide (1 mmol/L) inhibited glucose formation from different concentrations (1 to 20 mmol/L) of galactose, dihydroxyacetone, glycerol, and a mixture of L-lactate:pyruvate (molar ratio, 10:1). Parallel to the reduction of gluconeogenesis, tolbutamide stimulated L-lactate formation when cells were incubated with either galactose, dihydroxyacetone, or glycerol. All these tolbutamide effects occurred without significant modification of hepatocyte fructose-2,6-bisphosphate (F-2,6-P2) levels. Only when glucose was included in the incubation medium was the inhibition of gluconeogenesis caused by the sulfonylurea accompanied by a significant increment of the cellular F-2,6-P2 concentration. Under these conditions, tolbutamide potentiated the effect of glucose in promoting the increase of this regulatory metabolite, as well as the stimulation of glycolysis; in addition, tolbutamide increased the cellular pool of hexose-6-phosphates and the rate of tritium release from (2-3H)glucose. These results support the hypothesis that tolbutamide regulates hepatic glucose metabolism, at least, by modulating the glucose-6-phosphate substrate cycle.

Animals↗
PubMed1985

Evidence for the involvement of substrate cycles in the regulation of deoxyribonucleoside triphosphate pools in 3T6 cells.

Pool sizes of deoxyribonucleoside triphosphates (dNTPs) in cultured cells are tightly regulated by i.al., the allosteric control of ribonucleotide reductase. We now determine the in situ activity of this enzyme from the turnover of the deoxycytidine triphosphate (dCTP) pool in rapidly growing 3T6 mouse fibroblasts, as well as in cells whose DNA replication was inhibited by aphidicolin or amethopterin, by following under steady state conditions the path of isotope from [5-3H]cytidine into nucleotides, DNA, and deoxynucleosides excreted into the medium. In normal cells as much as 28% of the dCDP synthesized was excreted as deoxynucleoside (mostly deoxyuridine), leading to an accumulation of deoxyuridine in the medium. Inhibition with amethopterin slightly increased ribonucleotide reductase activity, while aphidicolin halved the activity of this enzyme (and thymidylate synthase). In both instances all dCDP synthesized was degraded and excreted as nucleosides. This continued synthesis and turnover in the absence of DNA synthesis is in contrast to the earlier found inhibition of dCTP (and dTTP) turnover when hydroxyurea, an inhibitor of ribonucleotide reductase, was used to block DNA synthesis. To explain our results, we propose that substrate cycles between deoxyribonucleosides and their monophosphates, involving the activities of kinases and phosphatases, participate in the regulation of pool sizes. Within the cycles, a block of the reductase activates net phosphorylation, while inhibition of DNA polymerase stimulates degradation.

Animals↗