Hypoxia and proliferation are primarily responsible for induction of lactate dehydrogenase activity in cultured cells.

Abstract

In vitro to in vivo extrapolations require cellular models that closely mimic cells in vivo. Typically, cultured cells revert to glycolysis, have increased lactate dehydrogenase (LDH) activity, have decreased oxidative metabolism, and dedifferentiate. This study examined the role of hypoxia and proliferation in the regulation of LDH activity, and the temporal relationships among induction of glycolysis, LDH activity, and proliferation in primary cultures of rabbit renal proximal tubular cells (RPTC). LDH activity in RPTC grown under standard conditions (STILL) did not increase during the first 3 d of culture, but increased 18-fold by d 7. LDH activity in RPTC grown under conditions of increased oxygen supply (SHAKE) did not increase during the first 3 d of culture, but increased 5.5-fold by d 7. Hypoxia and proliferation were responsible for 73% and 27%, respectively, of the increase in LDH activity in STILL RPTC. Hypoxia had no effect on RPTC proliferation. Neither medium glucose nor insulin concentrations had any effect on LDH activity in SHAKE RPTC. Supplementation of the culture medium with ribose 5-phosphate or ribose diminished the increase in LDH activity in SHAKE RPTC to 62% and 52% of controls but had no effect on monolayer DNA content. Two-day treatment of confluent SHAKE RPTC with epidermal growth factor (EGF) resulted in 1.6-, 1.4-, and 1.9-fold increases in LDH and G6PDH activities and monolayer DNA content, respectively. The stimulatory effect of EGF on LDH and G6PDH activities, but not monolayer DNA content, was abolished by ribose or ribose 5-phosphate. In contrast, transforming growth factor-beta 1 (TGF-beta 1) treatment stimulated lactate production but had no effect on LDH and G6PDH activities, or proliferation of SHAKE RPTC. These results show that (1) both hypoxia and proliferation are primarily responsible for the induction of LDH activity in cultured cells, (2) LDH activity is not an accurate indicator of glycolysis, (3) induction of LDH activity is secondary and due, in part, to the induction of the pentose phosphate pathway, (4) EGF stimulates glycolysis, LDH activity, and proliferation, and (5) TGF-beta 1 stimulates glycolysis but has no effect on LDH activity or proliferation.