To the Editor: The LLC-PK1 pig renal cell line has been widely used as a model system to study renal proximal tubular functions in vitro, because it retains many properties of proximal tubular cells. LLC-PK1 cells express some functions in vitamin D metabolism (9), possess a Na+-dependent phlorizin-sensitive glucose transport sys-tem (1, 17, 21, 23, 25) and Na+-dependent transport of amino acids (26, 27, 35) and phosphate (6, 24), and exhibit high activities of proximal tubule brush border membrane enzymes (11, 22, 28, 34). In addition to these characteristics dissimilarities with respect to proximal nephron properties have also been reported (10). Al-though the proximal tubule does not respond to antidi-uretic hormone or serum calcitonin, LLC-PK1 cells do respond by increasing intracellular adenosine 3’, 5’-cyclic monophosphate content, whereas no response to parathyroid hormone, known to act at proximal nephron portions (19), could be found. In mammals, aside from the liver, the proximal tubule is the main tissue capable of gluconeogenesis (7, 13, 31, 33). In this context Mullin and co-workers (20) studied the ability of LLC-PK1 cells to synthesize glucose from added lactate. After feeding labeled L-lactate to confluent monolayers of LLC-PK1 cells, Mullin et al. failed to detect appreciable amounts of labeled glucose. They found that [U-14C] L-lactate is metabolized to [14C]~-alanine,[14C] L-glutamate, and unlabeled urea. This lack of gluconeogenesis led us to examine the enzymes for glucose biosynthesis in LLC-PK1 cell cul-tures. Activities of the gluconeogenic enzymes phos-phoenolpyruvate carboxykinase(PEPCK) and glucose-6-phosphatase in LLC-PK, cells have been reported (18, ZO), but no activity values for fructose-bisphosphatase (FBPase) have been available so far. Therefore we looked for the activity of FBPase in LLC-PK1 cells but could find no activity. At the same time enzymes of alternative pathways of lactate metabolism yielding alanine, aspartate, and glutamate have been determined. LLC-PK1 ceils (Flow Laboratories, Glasgow) were used from passages 200 to 220. Serial cultures were maintained in plastic culture flasks in a 50: 50 mixture of Dulbecco’s modified Eagle’s Medium and Ham’s F-12 Medium, supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 pg/ml streptomycin (8). This medium contained-7 mM glucose. Confluent monolayers were subcultured by treatment with 0.03% ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS), pH 7.4. Cells were plated at 1: 6 dilution in 250-ml plastic culture flasks and incubated in a humidified 5% C02-95% air mixture at 37OC. Cultures were fed with fresh medium twice a week, and reached confluence on day 3 to 4 after subculturing. For enzyme assays confluent monolayers of LLC-PK1 cells were used on day 6 to 8 after seeding, at which time monolayers started to form domes. Confluent monolayers were rinsed twice with PBS and harvested by EDTA treatment. The cell suspension was then centrifuged at 1000 rpm for 5 min and resuspended in PBS. Crude cell homogenates were obtained by freezing cell suspensions three times in liquid nitrogen with subsequent rewarming and final homoge-nization at 0 C with 40 strokes by a glass teflon homogenizer.

Enzyme activities were measured immediately after homogenization, using the crude cell homogenate. FBPase (EC 3.1. 3.11) was assayed as described by Latzko and Gibbs (15). Cuvettes containing 1.0 ml of the assay mixture were placed in thermostated cuvette holders in a Shimadzu Scientific Instruments double-beam spectrophotometer. Enzyme reaction was initiated by addition of …