Proportional activities of glycerol kinase and glycerol 3-phosphate dehydrogenase in rat hepatomas

Harding, Joseph W.; Pyeritz, E A; Morris, H. P.; White, Harold B.

doi:10.1042/bj1480545

Cited by 26 publications

(12 citation statements)

References 42 publications

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“…Negligible α‐glycerophosphate dehydrogenase activity was found in both tumor cell types. A similar observation has been described for the Morris hepatomas 3924A, 5123D, 7793 and 44 [26], which are fast or moderate‐growth tumor lines [27].…”

Section: Resultssupporting

confidence: 80%

Determining and understanding the control of glycolysis in fast‐growth tumor cells

et al. 2006

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It is well documented that fast-growth tumor cells have higher rates of lactate formation even under aerobic conditions than nontumorigenic cells. For instance, different types of hepatoma (Reuber, Morris, Dunings LC18) and fibrosarcoma 1929 exhibit rates of 0.2-2.7 lmol lactateAEh )1 AE(mg protein))1, whereas normal liver and kidney cells have rates of 0.05 lmol lactateAE hThe increase in tumor glycolysis has been associated with the activation of several oncogenes (c-myc, ras and src) or with the expression of the hypoxia-inducible factor (HIF-1a) in transformed human lymphoblastoid Control analysis of the glycolytic flux was carried out in two fast-growth tumor cell types of human and rodent origin (HeLa and AS-30D, respectively). Determination of the maximal velocity (V max ) of the 10 glycolytic enzymes from hexokinase to lactate dehydrogenase revealed that hexokinase (153-306 times) and phosphfructokinase-1 (PFK-1) (22-56 times) had higher over-expression in rat AS-30D hepatoma cells than in normal freshly isolated rat hepatocytes. Moreover, the steady-state concentrations of the glycolytic metabolites, particularly those of the products of hexokinase and PFK-1, were increased compared with hepatocytes. In HeLa cells, V max values and metabolite concentrations for the 10 glycolytic enzyme were also significantly increased, but to a much lesser extent (6-9 times for both hexokinase and PFK-1). Elasticity-based analysis of the glycolytic flux in AS-30D cells showed that the block of enzymes producing Fru(1,6)P 2 (i.e. glucose transporter, hexokinase, hexosephosphate isomerase, PFK-1, and the Glc6P branches) exerted most of the flux control (70-75%), whereas the consuming block (from aldolase to lactate dehydrogenase) exhibited the remaining control. The Glc6P-producing block (glucose transporter and hexokinase) also showed high flux control (70%), which indicated low flux control by PFK-1. Kinetic analysis of PFK-1 showed low sensitivity towards its allosteric inhibitors citrate and ATP, at physiological concentrations of the activator Fru(2,6)P 2 . On the other hand, hexokinase activity was strongly inhibited by high, but physiological, concentrations of Glc6P. Therefore, the enhanced glycolytic flux in fast-growth tumor cells was still controlled by an over-produced, but Glc6P-inhibited hexokinase.Abbreviations DHAP, dihydroxyacetone phosphate; G6PDH, glucose-6-phosphate dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GluT, glucose transporter; LDH, lactate dehydrogenase; PFK-1, phosphofructokinase type 1.

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Section: Resultssupporting

confidence: 80%

Determining and understanding the control of glycolysis in fast‐growth tumor cells

et al. 2006

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“…In agreement with data reported for Sarcoma 180 [14], other carbohydrates such as galactose and glycerol did not affect oxidative phosphorylation in AS‐30D cells. Moreover, it has been described that the activity of glycerol 1‐kinase is negligible in some tumor lines [35]. However, the activity of this enzyme has not yet been evaluated in AS‐30D cells.…”

Section: Discussionmentioning

confidence: 99%

Multisite control of the Crabtree effect in ascites hepatoma cells

Rodrı́guez-Enrı́quez¹,

Juárez²,

Rodríguez‐Zavala³

et al. 2001

European Journal of Biochemistry

130

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AS-30D hepatoma cells, a highly oxidative and fastgrowing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mm glucose or 10 mm fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular P i (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca 21 and Mg 21 were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low P i (0.6 mm) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.Keywords: Crabtree effect; fast-growth tumor cells; multisite control; oxidative phosphorylation.Fast-growing tumor cells are characterized by a high glycolytic activity, even in the presence of saturating oxygen. In these cells it is also known that cellular oxidative phosphorylation is strongly inhibited by exogenous glucose [1]. A diminished or absent Pasteur effect would explain the former phenomenon. However, for the glucose-induced inhibition of oxidative phosphorylation, known as the Crabtree effect [2], there is no satisfactory explanation. The Crabtree effect is not restricted to malignant cells as it is also observed in normal proliferative cells [3]. In fact, in several nontumoral tissues (pig platelets, hamster embryos, proliferating thymocytes, retina, intestinal mucosa) [4±7] and in bacteria and yeast [8,9], it has been reported that oxidative phosphorylation is inhibited by exogenous hexoses.Eigenbrodt et al. proposed that this process could be a selective advantage for tumor cells, as the consequence of a glucose-dependent accumulation of essential metabolic intermediaries, such as serine, phosphoribosyl-pyrophosphate, fructose 1,6-bisphosphate, and glycerol 3-phosphate, which can trigger the mitogenic events In this work, we have evaluated some of these hypotheses, using the oxidative ascites tumor AS-30D cell line as the experimental model. We show that several factors are involved in the onset of the Crabtree effect. M A T E R I A L S A N D M E T H O D SCell preparation AS-30D ascites tumor cells were propagated by inoculation of 2 Â 10 8 cells into the peritoneal cavity of female Wistar rats of 200±250 g weight. The cells were harvested, and washed as described previously [17]. They were stored in ice at a density of 2±3 Â 10 8 cells´mL 21 until use. Isolation of mitochondriaAS-30D mitochondria were isolated by the digitonin permeabilization procedure...

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“…This might explain the limited flow of glucose carbons to G3P and hence to TG glycerol we observed in some muscle groups. Indeed, the activity of cytosolic glycerophosphate dehydrogenase in skeletal muscle is at least 2-3 times lower than that of liver in rats (23,24). In addition, due to oxidative shift of redox status as a result of fasting (25), a high NAD ϩ /NADH ratio in fasted rats may have inhibited glucose incorporation into muscle TG glycerol because it favors the equilibrium from G3P to DHAP.…”

Section: Discussionmentioning

confidence: 99%

Blood Glycerol Is an Important Precursor for Intramuscular Triacylglycerol Synthesis

Guo

Jensen²

1999

Journal of Biological Chemistry

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The utilization of blood glycerol and glucose as precursors for intramuscular triglyceride synthesis was examined in rats using an intravenous infusion of [2-14 C]glycerol and [6-3 H]glucose or [6-14 C]glucose. In 24-h fasted rats, more glycerol than glucose was incorporated into intramuscular triglyceride glycerol in soleus (69 ؎ 23 versus 4 ؎ 1 nmol/mol triglyceride/h, respectively, p ‫؍‬ 0.02 glycerol versus glucose) and in gastrocnemius (25 ؎ 5 versus 9 ؎ 2 nmol/mol triglyceride/h, respectively, p ‫؍‬ 0.02). Blood glucose was utilized more than blood glycerol for triglyceride glycerol synthesis in quadriceps. In fed rats, the blood glycerol incorporation rates (4 ؎ 2, 8 ؎ 3, and 9 ؎ 3 nmol/mol triglyceride/h) were similar (p > 0.3) to those of glucose (5 ؎ 2, 8 ؎ 2, and 5 ؎ 2 nmol/mol triglyceride/h for quadriceps, gastrocnemius, and soleus muscle, respectively). Glucose incorporation into intramuscular triglycerides was less with [6- 13 C]glycerol) was complete in quadriceps and gastrocnemius, but not soleus, within 2 h after beginning the tracer infusion. We conclude that blood glycerol is a direct and important precursor for muscle triglyceride synthesis in rats, confirming the presence of functionally important amounts of glycerol kinase in skeletal muscle.It is a biochemistry precept that the glycerol moiety of triacylglycerols and phospholipids in non-hepatic mammalian tissues is primarily derived from glucose via glycolysis (1). Dihydroxyacetone phosphate (DHAP), 1 originating from glucose, is reduced to glycerol 3-phosphate (G3P) by the action of glycerophosphate dehydrogenase. G3P then undergoes sequential acylation steps to incorporate three fatty acids to form a triacylglycerol (TG) (2). DHAP can also take a different path (DHAP pathway) via 1-acyldihydroxyacetone phosphate and then 1-acylglycerol 3-phosphate, but this appears to be a quantitatively minor reaction (3, 4). Glycerol can be converted directly to G3P by glycerol kinase, but this is believed to occur primarily, if not solely, in the liver and kidney because the glycerol kinase activity in these tissues is sufficient to permit large quantities of blood glycerol to be used for gluconeogenesis and TG synthesis. The direct conversion of free glycerol to G3P is thought to be negligible in skeletal muscle and adipose tissue because of their low activities of glycerol kinase (5-8). A corollary to this supposition is that glycerol generated by the hydrolysis of TG in skeletal muscle quantitatively enters the circulation. These assumptions form the basis for using systemic glycerol appearance rate, measured by isotope dilution techniques, as a quantitative measure of whole body lipolysis (9, 10).The presence of small but measurable glycerol kinase activity in skeletal muscle of various animal species, including rat (5) and humans (6), raises concerns as to the validity of these assumptions, however. For example, although low in specific activity, skeletal muscle glycerol kinase could be important in the metabolism of circulating glycerol consid...

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Proportional activities of glycerol kinase and glycerol 3-phosphate dehydrogenase in rat hepatomas

Cited by 26 publications

References 42 publications

Determining and understanding the control of glycolysis in fast‐growth tumor cells

Determining and understanding the control of glycolysis in fast‐growth tumor cells

Multisite control of the Crabtree effect in ascites hepatoma cells

Blood Glycerol Is an Important Precursor for Intramuscular Triacylglycerol Synthesis

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