Changes in intracellular metabolite pools during growth of adherent MDCK cells in two different media.

In bioprocess engineering, the growth of continuous cell lines is mainly studied with respect to the changes in cell concentration, the resulting demand for substrates, and the accumulation of extracellular metabolites. The underlying metabolic process rests upon intracellular metabolite pools and their interaction with enzymes in the form of substrates, products, or allosteric effectors. Here, we quantitatively analyze time courses of 29 intracellular metabolites of adherent Madin-Darby canine kidney cells during cultivation in a serum-containing medium and a serum-free medium. The cells, which originated from the same pre-culture, showed similar overall growth behavior and only slight differences in their demand for the substrates glucose (GLC), glutamine (GLN), and glutamate (GLU). Analysis of intracellular metabolites, which mainly cover the glycolytic pathway, the citric acid cycle, and the nucleotide pools, revealed surprisingly similar dynamics for both cultivation conditions. Instead of a strong influence of the medium, we rather observed a growth phase-specific behavior in glycolysis and in the lower citric acid cycle. Furthermore, analysis of the lower part of glycolysis suggests the well-known regulation of pyruvate kinase by fructose 1,6-bisphosphate. The upper citric acid cycle (citrate, cis-aconitate, and isocitrate) is apparently uncoupled from the lower part (α-ketoglutarate, succinate, fumarate, and malate), which is in line with the characteristics of a truncated cycle. Decreased adenosine triphosphate and guanosine triphosphate pools, as well as a relatively low energy charge soon after inoculation of cells, indicate a high demand for cellular energy and the consumption of nucleotides for biosynthesis. We finally conclude that, with sufficient availability of substrates, the dynamics of GLC and GLN/GLU metabolism is influenced mainly by the cellular growth regime and regulatory function of key enzymes.

The relation between growth phases, cell volume changes and metabolism of adherent cells during cultivation.

In biotechnology, mathematical models often consider changes in cell numbers as well as in metabolite conversion to describe different cell growth phases. It has been frequently observed that the cell number is only a delayed indicator of cell growth compared to the biomass, which challenges the principle structure of corresponding models. Here, we evaluate adherent cell growth phases in terms of cell number and biomass increase on the basis of detailed experimental data of three independent cultivations for Madin Darby canine kidney cells. We develop a model linking cell numbers and mean cell diameters to estimate cell volume changes during growth without the need for diameter distribution measurements. It simultaneously describes the delay between cell number and cell volume increase, cell-specific volume changes and the transition from growth to maintenance metabolism while taking different pre-culture conditions, which affect the cell diameter, into account. In addition, inspection of metabolite uptake and release rates reveals that glucose is mainly used for generation of cellular energy and glutamine is not required for cellular maintenance. Finally, we conclude that changes in cell number, cell diameter and metabolite uptake during cultivation contribute to the understanding of the time course of intracellular metabolites during the cultivation process.

2-Deoxy-D-glucose and mercaptoacetate induce different patterns of macronutrient ingestion.

2-Deoxy-D-glucose (2DG) and mercaptoacetate (MA) are antimetabolic agents that reduce the metabolism of glucose and fatty acids, respectively, and stimulate feeding. The present study compared the effects of MA and 2DG on macronutrient self-selection. Because 2DG and MA have different metabolic actions and appear to activate different neural pathways, our hypothesis was that 2DG and MA would elicit different patterns of macronutrient selection. The first experiment examined macronutrient selection in response to 2DG, MA, and 0.9% saline in rats maintained on a three-macronutrient self-selection diet consisting of cornstarch, casein, and vegetable oil. Subsequently, one macronutrient source was replaced in each of three similar experiments with Polycose, albumin, or solid vegetable shortening. Finally, 2DG and MA tests were conducted in which only one macronutrient (cornstarch, casein, or oil) was available during the test. Results show that MA and 2DG elicit different macronutrient preferences. 2DG elicits intake of all three macronutrients in the same relative proportion consumed during spontaneous feeding across a number of dietary conditions, suggesting that glucoprivation activates interoceptive signals and neural pathways similar to those involved in normal hunger. MA elicits a selective intake of protein. Conditions in which carbohydrate palatability is enhanced or protein palatability is diminished lead to a relative increase in carbohydrate intake in response to MA. However, MA did not increase the intake of fat. Results suggest that intake of each macronutrient is subject to separate neural or endocrine control, and that these controls are linked to metabolic cues.

The health information manager as lawmaker.

The authors argue that the economic well-being of the healthcare industry depends on the work of its health information managers. These managers will play a critical role in guiding the transformation to electronic records and in shaping the laws that will govern these records.