A Novel Gluconeogenic Strain of OK Cells with Metabolic Properties Different from Gluconeogenic LLC-PK₁ Cells

Abstract

By selective adaptation to glucose-free tissue culture conditions, a gluconeogenic substrain of the opossum kidney (OK) cell line, designated OK_gNg+, was established, analogous to the method recently employed for the isolation of gluconeogenic LLC-PK₁-FBPase+ cells [Gstraunthaler and Handler, Am J Physiol 1987;252:C232-C238]. OK_Gng+ cells express considerable amounts of fructose-1, 6-bisphosphatase activity, and metabolic flux through phosphoenolpyruvate carboxykinase (PEPCK), which is indicative for their gluconeogenic capacity, in contrast to OK wild-type cells. OK_gng+ cells also seem to exhibit a higher degree of differentiation indicated by an increased expression of γ-glutamyltranspeptidase activity. Karyotype analysis revealed that OK_gng+ cells are descendants of OK wild-type cells, exhibiting slight increases in tetraploidy. Remarkable differences between OK_gng+ and LLC-PK₁^-FBPase+ cells have been found in terms of substrate metabolism of the two gluconeogenic renal sub-strains. Cultures were incubated in culture medium containing either Na-L-lactate, Na-pyruvate or lactate/pyruvate mixtures (10:1, 5:5, 1:10) as main gluconeogenic substrates. OKg_gng+ cultures grew better in lactate than in pyruvate, whereas the opposite was observed with LLC-PK₁^-FBPase+ cells. This was further confirmed by the consumption rates of lactate and pyruvate by the substrains. When lactate or lactate/pyruvate were supplied, OKq_gng+ cells consumed lactate at much higher rates as compared to LLC-PK₁^-FBPase+ cultures. When pyruvate was added as the sole gluconeogenic substrate, pyruvate consumption rates by LLC-PK₁^-FBPase+ cells were linear over time, without considerable lactate production. However, pyruvate consumption by OK_gng+ cells was almost completed after 24-48 h, and resulted in an equimolar production and accumulation of lactate in the culture medium, respectively, which was consumed by the cells thereafter. In summary, a novel gluconeogenic strain of OK cells has been established (OK_gng+), as previously shown for gluconeogenic LLC-PK₁^-FBPase+ cells. Both cell strains, however, differ markedly in the metabolism of lactate and pyruvate, and thus obviously in the subcellular localization of PEPCK, in their regulation of gluconeogenesis by the intracellular NADH/NAD ratio, and/or in the rate of mitochondrial substrate fluxes and shuttle activities, respectively.

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

“…Cell Culture LLC-PK 1 cells (ATCC CRL 1392) and opossum kidney (OK) cells were grown in DMEM with 5.5 mM D-glucose, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 Ìg/ml streptomycin as described elsewhere [8,27,[38][39][40]. Serial cultures were grown in monolayer culture technique in 10-cm tissue culture dishes containing 7.0 ml of culture medium.…”

“…After incubation in different media volumes for 24-72 h, culture media aliquots were taken, and metabolites (glucose, lactate, glutamine, and ammonia) were analyzed enzymatically by established methods [7,8,27,37,39,40].…”

“…Cell homogenates were prepared using hypo-osmotic shock by resuspending cell pellets in diluted PBS (1:10) and freezing 3 times in liquid nitrogen followed by subsequent rewarming in an ultrasonic water bath. Glycolytic enzymes and phosphate-dependent glutaminase (schematically shown in figure 2) were assayed as described elsewhere [8,26,38,39,46]. Protein was determined by the method of Lowry with bovine serum albumin as a standard.…”

pH-responsive, gluconeogenic renal epithelial LLC-PK1-FBPase+cells: a versatile in vitro model to study renal proximal tubule metabolism and function

“…Furthermore, glucose-free selection was successfully used to isolate a gluconeogenic strain of opossum kidney (OK) cells (29,40). The OK cell line (ATCC CRL-1840) was initiated in 1975 from a female American opossum (Didelphys virginiana) (51).…”

Nephrotoxicity Testing in Vitro: What We Know and What We Need to Know

“…As mentioned, omission or drastic reduction of glucose and replacement against pyruvate in the media used for cultivation of LLC-PK1 cells enables reexpression of gluconeogenesis (45,46). Fusion of cells of continuous cell lines (120) stemming from the same in vivo ancestor cell of different species and with different retainment of cellular functions, i.e., metabolic pathways, transport systems, or hormone receptors, may be used to establish new continuous lines more closely resembling the characteristics of the cell type of origin.…”