Modulation of glycolysis induction in primary cultures of rabbit kidney proximal tubule cells: the role of shaking, glucose and insulin.

Monteil, Christelle; Leclere, Caroline; Dantzer, Françoise; Elkaz, Veronika; Fillastre, J P; Morin, Jean–Paul

doi:10.1006/cbir.1993.1019

Cited by 8 publications

(12 citation statements)

References 17 publications

(31 reference statements)

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“…As seen with LLC-PK 1 and OK cultures in the present study, glucose availability markedly affects glycolytic metabolism, i.e. the expression of glycolytic enzyme activities as well as the rates of lactate production [18,19,35]. It is thus likely that these alterations in cell metabolism generally observed in cultured proximal tubular cells represent adaptive responses of the cells to the in vitro tissue culture environment resulting in a combination of aerobic glycolysis and oxidate metabolism for the supply of cellular energy.…”

Section: Glucose and Glutamine Metabolismmentioning

confidence: 54%

“…Recently, the effect of glucose and insulin on carbohydrate metabolism was studied in rabbit kidney proximal tubule primary cultures [18,19,35]. Gluconeogenic capacity as well as activities of phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase fell markedly in primary cultures over time, regardless of the presence or absence of glucose.…”

Section: Glucose and Glutamine Metabolismmentioning

confidence: 99%

“…Several attempts were undertaken in order to improve culture conditions for renal proximal primary cultures, with main emphasis on improving the oxygenation of cultured epithelial monolayers [15,21,23,35,36]. In these studies the method of choice was rocking the cultures by placing the culture vessels on a rocker platform.…”

Section: Oxygen Supply In Monolayer Culturesmentioning

confidence: 99%

“…As a result, the increase in lactate dehydrogenase activity was delayed in shake cultures [15], which was paralleled by decreased rates of lactate production, which under glucose-free culture conditions even changed to net lactate consumption [21]. However, despite those improvements gluconeogenesis completely dropped, and the activities of gluconeogenic key enzymes declined to zero after 1 week in culture [20,23,35]. Rocking also promoted cellular metabolism and differentiated functions of LLC-PK 1 cells, which was evidenced by enhanced ammonia production, a decrease in lactate dehydrogenase activity, lower rates of lactate formation, increased cellular ATP levels, and increased dome formation [36].…”

Section: Roller Culturesmentioning

confidence: 99%

“…The maneuvers applied in these studies included glucose limitation in culture media [22,[30][31][32], the use of hormonally defined, serum-free media supplemented with insulin [18,19,33], and/or improved oxygenation of primary cultures by shaking cell monolayers on a rocker platform [15,21,23,[34][35][36].…”

Section: Introductionmentioning

confidence: 99%

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Impact of Culture Conditions, Culture Media Volumes, and Glucose Content on Metabolic Properties of Renal Epithelial Cell Cultures

Gstraunthaler¹,

Seppi²,

Pfaller³

1999

Cell Physiol Biochem

100

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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-PK₁ (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-PK₁ 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-PK₁ 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-PK₁ 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-PK₁ 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-PK₁ 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-PK₁ 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 o...

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Section: Glucose and Glutamine Metabolismmentioning

confidence: 54%

Section: Glucose and Glutamine Metabolismmentioning

confidence: 99%

Section: Oxygen Supply In Monolayer Culturesmentioning

confidence: 99%

Section: Roller Culturesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Impact of Culture Conditions, Culture Media Volumes, and Glucose Content on Metabolic Properties of Renal Epithelial Cell Cultures

Gstraunthaler¹,

Seppi²,

Pfaller³

1999

Cell Physiol Biochem

100

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Comparative impact of cephaloridine on glutathione and related enzymes in LLC-PK1, LLC-RK1, and primary cultures of rat and rabbit proximal tubule cells

et al. 1996

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Among kidney tubular epithelial cell types, proximal tubule cells are one of the major renal targets for xenobiotics. Several in vitro culture models have been proposed for use of proximal tubule cells for in vitro pharmacotoxicology studies. This paper reports a comparative study of the response to cephaloridine exposure of two established cell lines from pig (LLC-PK1) and rabbit (LLC-RK1) kidneys and primary cultures of rat and rabbit proximal tubule cells. These cultured cells were first compared for their levels of activity of alpha-methylglucopyranoside transport, alkaline phosphatase, succinate dehydrogenase, and NADPH cytochrome c reductase, their glutathione-dependent activity levels, and their adenylate cyclase response pattern to stimulation by PTH and AVP. The results presented show major phenotypic differences between these four cellular models. The differences observed in glutathione-dependent mechanism activities and regulation may in part be responsible for the variability of the responses of these four cellular models when exposed to cephaloridine.

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Morphological and biochemical characterization of primary culture of rabbit proximal kidney tubule cells grown on collagen-IV coated millicell-cm

Genestie¹,

Morin

Guery³

et al. 1997

In Vitro Cell.Dev.Biol.-Animal

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The aim of this study was to better characterize rabbit proximal kidney tubule cells cultured on collagen IV-coated porous inserts, as compared to the same cells seeded in standard plastic wells. Total protein contents in confluent monolayers on permeable membranes were about twofold higher than those measured in confluent cultures in plastic wells. Microscopy examinations suggested that such a difference was probably due to a higher cell density and to an impressive development of the apical brush-border membrane. Moreover, measurement of unidirectional transport of p-aminohippuric acid and tetraethylammonium bromide confirmed the high polarization level of cultures on porous inserts. Results of methyl(alpha-D-[U-14C]glyco)pyranoside uptake suggested that cell phenotype was probably influenced by culture conditions. Analysis of different markers as a function of time in culture showed decreases of alkaline phosphatase (AP), gamma-glutamyltranspeptidase (GGT), and Na(+)-K(+)-ATPase activities as well as increases in LDH, ATP, and glutathione levels, similar to those formerly reported for cells cultured in standard plastic plates. However, comparative data from 6-d-old monolayers have shown that AP, GGT, Na(+)-K(+)-ATPase, glutathione reductase (GRED), and selenium-dependent glutathione peroxidase (Se-GPX) activities were 2.8-, 2.6-, 1.6-, 1.2-, and 2.1-fold, respectively, better preserved on precoated permeable membranes. On the other hand, this paper reports for the first time in the literature that GRED and SE-GPX, two phase II detoxification enzymes, were well maintained in cultures of rabbit proximal kidney tubule cells. Our results show that culturing rabbit proximal kidney tubule cells on collagen IV-coated porous membranes was accompanied by an improvement of both morphological and biochemical properties of the cells.

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Modulation of glycolysis induction in primary cultures of rabbit kidney proximal tubule cells: the role of shaking, glucose and insulin.

Cited by 8 publications

References 17 publications

Impact of Culture Conditions, Culture Media Volumes, and Glucose Content on Metabolic Properties of Renal Epithelial Cell Cultures

Impact of Culture Conditions, Culture Media Volumes, and Glucose Content on Metabolic Properties of Renal Epithelial Cell Cultures

Comparative impact of cephaloridine on glutathione and related enzymes in LLC-PK1, LLC-RK1, and primary cultures of rat and rabbit proximal tubule cells

Morphological and biochemical characterization of primary culture of rabbit proximal kidney tubule cells grown on collagen-IV coated millicell-cm

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