Comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines, insect and primary liver cells

Abstract

Glucose and glutamine metabolism in several cultured mammalian cell lines (BHK, CHO, and hybridoma cell lines) were investigated by correlating specific utilization and formation rates with specific maximum activities of regulatory enzymes involved in glycolysis and glutaminolysis. Results were compared with data from two insect cell lines and primary liver cells. Flux distribution was measured in a representative mammalian (BHK) and an insect (Spodoptera frugiperda) cell line using radioactive substrates. A high degree of similarity in many aspects of glucose and glutamine metabolism was observed among the cultured mammalian cell lines examined. Specific glucose utilization rates were always close to specific hexokinase activities, indicating that formation of glucose-6-phosphate from glucose (catalyzed by hexokinase) is the rate limiting step of glycolysis. No activity of the key enzymes connecting glycolysis with the tricarboxylic acid cycle, such as pyruvate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase, could be detected. Flux distribution in BHK cells showed glycolytic rates very similar to lactate formation rates. No glucose- or pyruvate-derived carbon entered the tricarboxylic acid cycle, indicating that glucose is mainly metabolized via glycolysis and lactate formation. About 8% of utilized glucose was metabolized via the pentose phosphate shunt, while 20 to 30% of utilized glucose followed pathways other than glycolysis, the tricarboxylic acid cycle, or the pentose phosphate shunt. About 18% of utilized glutamine was oxidized, consistent with the notion that glutamine is the major energy source for mammalian cell lines. Mammalian cells cultured in serum-free low-protein medium showed higher utilization rates, flux rates, and enzyme activities than the same cells cultured in serum-supplemented medium. Insect cells oxidized glucose and pyruvate in addition to glutamine. Furthermore, insect cells produced little or no lactate and were able to channel glycolytic intermediates into the tricarboxylic acid cycle. Metabolic profiles of the type presented here for a variety of cell lines may eventually enable one to interfere with the metabolic patterns of cells relevant to biotechnology, with the hope of improving growth rate and/or productivity.

Carbonylation of glycolytic proteins is a key response to drug-induced oxidative stress and apoptosis

“…As glycolysis is the most common source for ATP in cultured cells, due to poor coupling between glycolysis and the TCA, 34 we explored whether a reduction of glycolysis, through glucose deprivation, had any effect on the levels of VP16-induced cell death. We also looked at whether growth in serum-free media had any effect on the susceptibility of these cells to VP16-induced apoptosis.…”

Effects of overexpression of the liver subunit of 6-phosphofructo-1-kinase on the metabolism of a cultured mammalian cell line

“…These rates are high compared with those measured in isolated hepatocytes [28]. However, this was expected, since cultured cells tend to be very glycolytic, oxidizing very little glucose carbon via the citric acid cycle [35,36]. For instance, in wild-type Chinese hamster fibroblasts, more than 97 % of the glucose consumed is converted into lactate [37,38].…”

Fuel Choices by Human Platelets in Human Plasma

“…This approach results in overestimates of the contributions of particular fuels and pathways to total ATP turnover as the 'total ATP turnover' figure is too low. For example Petch and Butler (1994) and Neermann and Wagner (1996) conclude that the various pathways of glucose and glutamine utilization account for 100% of ATP turnover in various cell lines in the presence of glucose, glutamine and fetal calf serum. Oxygen consumption was not measured and would almost certainly demonstrate the oxidation of other fuels.…”

“…Oxygen consumption was not measured and would almost certainly demonstrate the oxidation of other fuels. Similarly it is now obvious that aerobic glycolysis contributes significantly (20 -50%) to ATP production under aerobic conditions in a variety of cell types (reviewed in Neermann and Wagner, 1996;Guppy et al, 1993) so lactate production must be measured if one is to get a meaningful measure of total ATP turnover. Studies in which comprehensive energy budgets were the primary aim, and in which there was a measurement of total ATP turnover (oxygen consumption and lactate production) are not common, but do exist for human platelets (Guppy et al, 1990), rat thymocytes (Guppy et al, 1993), pig enterocytes (Posho et al, 1994), mouse erythroid cells (Kim et al, 1992), human and rat lymphocytes (Roos and Loos, 1973;Ardawi and Newsholme, 1985), fish erythrocytes (Sephton et al, 1991) and rat coronary endothelial cells (Noll et al, 1990).…”

“…Oxygen consumption was not measured and would almost certainly demonstrate the oxidation of other fuels. Similarly it is now obvious that aerobic glycolysis contributes significantly (20 -50%) to ATP production under aerobic conditions in a variety of cell types (reviewed in Neermann and Wagner, 1996;Guppy et al, 1993) so lactate production must be measured if one is to get a meaningful measure of total ATP turnover.…”