PMID- 10494029
OWN - NLM
STAT- MEDLINE
DCOM- 20000508
LR  - 20190212
IS  - 1015-8987 (Print)
IS  - 1015-8987 (Linking)
VI  - 9
IP  - 3
DP  - 1999
TI  - Impact of culture conditions, culture media volumes, and glucose content on 
      metabolic properties of renal epithelial cell cultures. Are renal cells in tissue 
      culture hypoxic?
PG  - 150-72
AB  - When renal proximal tubular cells are brought into tissue culture, they revert 
      from oxidative metabolism and gluconeogenesis to high rates of glycolysis. Among 
      the factors possibly responsible for this metabolic conversion, limited oxygen 
      availability and/or substrate supply are discussed. In order to study the role of 
      these factors on long-term cultures, the impact of growth conditions, culture 
      media volume, and glucose content on carbohydrate metabolism of the continuous 
      renal cell lines LLC-PK(1) (porcine kidney) and OK (opossum kidney) was 
      investigated. The impact of culture media volumes and glucose content, 
      respectively, was determined by overlaying confluent monolayer cultures of 
      LLC-PK(1) and OK cells (i) with increasing volumes of culture medium and thus 
      increasing amounts of glucose, and (ii) with increasing culture medium volumes at 
      constant absolute amounts of glucose by adding glucose-free medium, in order to 
      increase volume at a constant glucose supply. Alternatively, and in order to 
      improve cell oxygenation, LLC-PK(1) cells were also cultured in roller bottles. 
      Cell carbohydrate metabolism was assessed by measuring rates of glucose 
      consumption and lactate production, respectively, and by determination of 
      specific activities of the key glycolytic enzymes hexokinase (HK), 
      phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH). 
      Mitochondrial phosphate-dependent glutaminase (PDG) was assayed as marker enzyme 
      of oxidative metabolism of glutamine. In LLC-PK(1) and OK cells, rates of glucose 
      consumption were independent of the initial glucose concentrations and/or the 
      culture media volumes used. Glucose was quantitatively converted to lactate, 
      which accumulated in a 1:2 molar ratio. Lactate in culture media reached a 
      maximum content after 24 h, and was reutilized by the cell lines thereafter. 
      Interestingly, the rates of lactate reuptake strictly depended on culture medium 
      volume, indicating a volume-induced stimulation of oxidative lactate metabolism. 
      Marked changes were found for the specific activities of glycolytic enzymes. In 
      LLC-PK(1) cells, increased glucose supply caused increases in HK, PFK, PK and LDH 
      activities, which were superimposed to the stimulatory effects of increased media 
      volumes. Enzyme activity showed a biphasic response, indicating that both glucose 
      supply and culture media volumes covering the cell monolayer are factors 
      determining glycolytic rates of LLC-PK(1) renal cells. Conversely, in OK cells 
      glycolytic enzyme activities decreased with increasing culture media volumes at 
      constant glucose levels. As expected, under conditions of enhanced oxygenation of 
      LLC-PK(1) cells in roller bottle culture, glycolytic enzyme activities decreased, 
      whereas PDG activity increased, which was paralleled by increased rates of 
      ammonia generation. Thus, changes in nutrient supply and oxygenation of renal 
      epithelial cell cultures by altered culture media volumes dramatically influence 
      metabolic rates and levels of enzyme activities, respectively.
FAU - Gstraunthaler, G
AU  - Gstraunthaler G
AD  - Institute of Physiology, University of Innsbruck, Austria. 
      gerhard.gstraunthaler@uibk.ac.at
FAU - Seppi, T
AU  - Seppi T
FAU - Pfaller, W
AU  - Pfaller W
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PL  - Germany
TA  - Cell Physiol Biochem
JT  - Cellular physiology and biochemistry : international journal of experimental 
      cellular physiology, biochemistry, and pharmacology
JID - 9113221
RN  - 0 (Culture Media)
RN  - 0 (Lactates)
RN  - 0RH81L854J (Glutamine)
RN  - EC 1.1.1.27 (L-Lactate Dehydrogenase)
RN  - EC 2.7.1.1 (Hexokinase)
RN  - EC 2.7.1.11 (Phosphofructokinase-1)
RN  - EC 2.7.1.40 (Pyruvate Kinase)
RN  - EC 3.5.1.2 (Glutaminase)
RN  - IY9XDZ35W2 (Glucose)
SB  - IM
MH  - Animals
MH  - Cell Culture Techniques/instrumentation/*methods
MH  - Cell Hypoxia/*physiology
MH  - Cell Line
MH  - Culture Media
MH  - Glucose/*metabolism
MH  - Glutaminase/metabolism
MH  - Glutamine/metabolism
MH  - Glycolysis
MH  - Hexokinase/metabolism
MH  - Kidney Tubules, Proximal/cytology/*physiology
MH  - L-Lactate Dehydrogenase/metabolism
MH  - Lactates/metabolism
MH  - Opossums
MH  - Phosphofructokinase-1/metabolism
MH  - Pyruvate Kinase/metabolism
MH  - Swine
MH  - Urothelium/cytology/*physiology
EDAT- 1999/09/24 09:00
MHDA- 2000/05/16 09:00
CRDT- 1999/09/24 09:00
PHST- 1999/09/24 09:00 [pubmed]
PHST- 2000/05/16 09:00 [medline]
PHST- 1999/09/24 09:00 [entrez]
AID - 16312 [pii]
AID - 10.1159/000016312 [doi]
PST - ppublish
SO  - Cell Physiol Biochem. 1999;9(3):150-72. doi: 10.1159/000016312.