Method for measuring a comprehensive energy budget in a proliferating cell system over multiple cell cycles

Guppy, Michael; Kong, Sung‐Eun; Niu, Xianwa; Busfield, Samantha J.; Klinken, S. Peter

doi:10.1002/(sici)1097-4652(199701)170:1<1::aid-jcp1>3.0.co;2-s

Cited by 19 publications

(14 citation statements)

References 27 publications

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“…Thus, we queried the uptake of nutrients in the basal media. We had calculated that the media contained sufficient amino acids and glucose to not be depleted within two doublings; these calculations were based on published data for differentiated cells [8, 9]. These calculations were borne out by direct measurement in the case of amino acids (Additional file 2: Figure S2); the only amino acid to undergo significant loss within 3 days was glutamine, and this was due to hydrolysis (as also seen by the increase in glutamic acid, and synthesis of glycine and proline).…”

Section: Resultsmentioning

confidence: 99%

Mesenchymal stem cells/multipotent stromal cells (MSCs) are glycolytic and thus glucose is a limiting factor of in vitro models of MSC starvation

Nuschke

Rodrigues

Wells³

et al. 2016

Stem Cell Res Ther

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BackgroundMesenchymal stem/multipotent stromal cells (MSCs) contribute to tissue repair but are challenged during wound healing when the blood supply is disrupted, thereby limiting nutrient delivery. Survival mechanisms against ‘starvation’ include autophagy, which we previously found to enhance differentiation efficiency. MSC response to models of in vitro nutrient deprivation are of great interest for improving MSC survival and therapeutic efficacy; however, the rate-limiting nutrients are unknown.MethodsMSC responses to culture nutrient and/or serum deprivations were assessed through light microscopy, cell survival, and measurements of metabolic levels. Glucose uptake was determined through conditioned media analyses over 3 days of culture. The Seahorse XF24 Flux analysis system was used to determine oxygen consumption and extracellular acidification for glycolytic metabolism. MSC autophagic response to these conditions was assessed via immunoblots for LC3-I and LC3-II, markers of autophagosome turnover.ResultsWe more closely examined limiting nutritional factors to MSC survival in vitro, finding that glucose is rapidly utilized/depleted whereas amino acids and other required nutrients were used sparingly. This finding concurred with metabolic analyses that showed a primarily glycolytic character to the MSCs at steady state. MSC autophagy, previously linked to MSC function through a unique accumulated autophagosome phenotype, also responded quickly to changes in glucose concentration, with drastic LC3-II changes within 24 h of glucose concentration shifts.ConclusionsOur results demonstrated a rapid uptake of glucose in MSC cultures that was due to a highly glycolytic phenotype for the cells; MSC starvation with serum or other nutrients appears to have a less notable effect on the cells. These findings highlight the importance of glucose and glucose metabolism on MSC function. The conditions and cellular responses outlined here may be essential in modeling MSC nutrient deprivation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0436-7) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Mesenchymal stem cells/multipotent stromal cells (MSCs) are glycolytic and thus glucose is a limiting factor of in vitro models of MSC starvation

Nuschke

Rodrigues

Wells³

et al. 2016

Stem Cell Res Ther

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“…Aerobic glycolysis was originally thought to be a unique characteristic of cancer cells (Wang et al, 1976), and certainly does manifest in these cell types (Neermann and Wagner, 1996;Fitzpatrick et al, 1993). However, numerous studies show that it is a common phenomenon in a variety of cells (see Guppy et al, 1996), rather than an artifact of in vitro measurement systems, unphysiological media or cell lines. This study shows that it is a significant energy-producing pathway that occurs under aerobic conditions in the presence of all the other fuels in plasma.…”

Section: Discussionmentioning

confidence: 99%

Fuel Choices by Human Platelets in Human Plasma

Guppy

Abas

Neylon

et al. 1997

European Journal of Biochemistry

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Despite the fact that homogeneous preparations of isolated cells are now being used very effectively to study a range of important biochemical questions, it is still not known what combination of fuels and energy-producing pathways is used by cells when offered the complex mixture characteristic of plasma or extracellular fluid. We have developed an in vitro system whereby highly purified and functional human platelets are incubated in human plasma that has been minimally modified from its native state. The concentration of platelets and fuels, and the complexity of fuels in the incubation are similar to those in vivo. The preparation thus represents a reasonable approximation of the physiological condition, considering the complex nature of the system being studied. Measurements carried out simultaneously during the incubation are rates of oxygen consumption, lactate production and fuel oxidation. The data allow the calculation of total ATP turnover, and contributions to this turnover by lactate production and the oxidation of individual fuels. Lactate production accounts for 24% of the ATP turnover. The oxidation of glucose and 3-hydroxybutyrate each account for under S%, palmitate for 21 %, oleate for 7% and acetate for 9%, leaving 32% of the ATP turnover as yet unaccounted for. The results confirm some previous measurements in the literature, but show that data collected under non-physiological experimental conditions can be misleading.Keywords: platelet; oxygen consumption ; plasma; glycolysis ; oxidation.The preparations used for the study of cellular metabolism have progressed over the last 40 years from tissue homogenates and slices to homogeneous preparations of isolated cells. The latter systems are now being used very effectively to study a range of important biochemical questions. However, there is still a question that has never really been adequately addressed, which could have important implications for the relevance of these preparations, and which is fundamental to our understanding of cell biology. This question concerns the combination of fuels and energy-producing pathways used by a cell when it is offered the complex mixture characteristic of plasma or extracellular fluid.Where energy budgets have been presented previously, it is often the case that total ATP turnover has been underestimated due to the omission of any measurement of oxygen consumption. 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. Similarly it is now obvious that aerobic glycolysis contributes significantly (20 -50%) to ATP production under aerobic conditions...

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“…The oxygen tension was constantly recorded before and after glucose stimulation. Oxygen consumption was calculated as previously described (15).…”

Section: Methodsmentioning

confidence: 99%

Cellular Hypoxia of Pancreatic β-Cells Due to High Levels of Oxygen Consumption for Insulin Secretion in Vitro

Sato

Endo

Okuyama

et al. 2011

Journal of Biological Chemistry

143

138

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Cellular oxygen consumption is a determinant of intracellular oxygen levels. Because of the high demand of mitochondrial respiration during insulin secretion, pancreatic ␤-cells consume large amounts of oxygen in a short time period. We examined the effect of insulin secretion on cellular oxygen tension in vitro. We confirmed that Western blotting of pimonidazole adduct was more sensitive than immunostaining for detection of cellular hypoxia in vitro and in vivo. The islets of the diabetic mice but not those of normal mice were hypoxic, especially when a high dose of glucose was loaded. In MIN6 cells, a pancreatic ␤-cell line, pimonidazole adduct formation and stabilization of hypoxia-inducible factor-1␣ (HIF-1␣) were detected under mildly hypoxic conditions. Inhibition of respiration rescued the cells from becoming hypoxic. Glucose stimulation decreased cellular oxygen levels in parallel with increased insulin secretion and mitochondrial respiration. The cellular hypoxia by glucose stimulation was also observed in the isolated islets from mice. The MIN6 cells overexpressing HIF-1␣ were resistant to becoming hypoxic after glucose stimulation. Thus, glucose-stimulated ␤-cells can become hypoxic by oxygen consumption, especially when the oxygen supply is impaired.

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Method for measuring a comprehensive energy budget in a proliferating cell system over multiple cell cycles

Cited by 19 publications

References 27 publications

Mesenchymal stem cells/multipotent stromal cells (MSCs) are glycolytic and thus glucose is a limiting factor of in vitro models of MSC starvation

Mesenchymal stem cells/multipotent stromal cells (MSCs) are glycolytic and thus glucose is a limiting factor of in vitro models of MSC starvation

Fuel Choices by Human Platelets in Human Plasma

Cellular Hypoxia of Pancreatic β-Cells Due to High Levels of Oxygen Consumption for Insulin Secretion in Vitro

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