THE EFFECT OF GLUCOSE AND GALACTOSE TOXICITY ON MYOINOSITOL TRANSPORTAND METABOLISM IN HUMAN SKIN FIBROBLASTS IN CULTURE

Myo-inositol transport and metabolism were studied in cultured human s kin fibroblasts exposed to potentially toxic levels of glucose or gala ctose. Although variable among 11 different cell lines, the myo-inosit ol level in confluent cells, ranging from 10-50 nmol/mg protein, was c onstant with passage. A high-affinity transport system for myo-inosito l had an apparent K-t of 55 mu M and V-max of 16 pmol/min/mg protein. No obvious relationship existed between cellular levels and transport capacity. Dependency on sodium was complex. When medium sodium was low ered to 23 mM, myo-inositol uptake ceased after about 1 h. However, th e initial rate of myo-inositol uptake only showed a sodium dependence at low myo-inositol concentrations. Both phloretin and phloridzin inhi bited myo-inositol uptake. Phloridzin had a K-i of 60 mu M and phloret in was either a noncompetitive or uncompetitive inhibitor. Glucose and galactose were only weak competitive inhibitors, with a K-i of 30 mM and 65 mM, respectively. After 24 h of incubation with myo-[2-H-3]inos itol, only 10% of the total cell label was incorporated into phospholi pid. Compared with control media with 5 mM glucose, the incubation of confluent cells in media with 20 mM glucose had little effect on intra cellular glucose and sorbitol, whereas cells incubated in control medi a supplemented with 5 mM galactose showed a large increase in galactos e and polyol levels, In media with more than 200 mu M of myo-inositol, neither treatment had an effect on myo-inositol levels after 24 h. Th e uptake and incorporation of 11 mu M myo-[2-H-3]inositol and incorpor ation into phospholipid were studied after cells had been previously e xposed for 24 to 48 h to media supplemented with 15 mM glucose or gala ctose. Compared with controls, fibroblasts with a 24-h exposure to 20 mM glucose showed a 10% decrease is myo-inositol uptake. When the expo sure was extended to 48 h, preconditioning with galactose as well as g lucose elicited the same 10% reduction in uptake. Phosphoinositide lab eling in fibroblasts exposed to 20 mM glucose was reduced in parallel. These cells offer a unique opportunity for the study of sugar toxicit y in human tissue: they can be exposed to high levels of glucose witho ut significant glucose or polyol accumulation or can be made to accumu late polyol by exposure to moderate levels of galactose. The expressio n of a hexose-induced reduction in myo-inositol transport required 24 to 48 h of exposure of the fibroblasts to elevated concentrations of g lucose or galactose and may not be related to a competitive inhibitory effect of these sugars on transport.