Energy metabolism in human retinal Müller cells.

PURPOSE: To measure selected parameters of energy metabolism and adenosine triphosphate (ATP) production in passaged monolayer cultures of human retinal glial (Müller) cells to assess the effects of varying substrate and oxygen availability on the biochemistry and histologic integrity of these cells. METHODS: Confluent Müller cell cultures were incubated for up to 4 hours at 37 degrees C in a modified minimal essential medium (no serum) under aerobic or mitochondrial-inhibited conditions in the presence and absence of 5 mM glucose or in the presence of lactate, pyruvate, glutamate, or glutamine. Cellular ATP levels, lactic acid production, and (14)CO(2) production from labeled glucose or glutamate were measured along with an examination of cellular morphology. Immunohistochemistry with antibodies to glial cell-specific proteins was also performed. Cells were positive for vimentin, but negative for glial fibrillary acidic protein and glutamine synthetase. RESULTS: Human Müller cells maintained ATP content aerobically at the same level for 4 hours in the presence and absence of glucose. ATP content was also maintained anaerobically at a value equal to that found aerobically, but only in the presence of glucose. ATP content in human Müller cells declined to a very low level when glycolysis was blocked by iodoacetate, and inclusion of lactate, pyruvate, glutamate, or glutamine did not restore the level of ATP. Aerobically, lactic acid production accounted for 99% of the total glucose used, whereas the oxidation of glucose by the mitochondria accounted for only 1%. When mitochondria were inhibited with antimycin A, there was only a modest (1.3-fold) increase in the rate of lactic acid production. No significant differences were found in the histologic appearance of the cells after mitochondrial blockade, but there was massive death of cells after inhibition of glycolysis with iodoacetate. CONCLUSIONS: These results suggest that, in the presence of glucose and oxygen, cultured Müller cells obtain their ATP principally from glycolysis and have a low rate of oxygen consumption. This metabolic pattern may spare oxygen for retinal neurons, particularly in the inner nuclear and ganglion cell layers under normal physiological conditions. Furthermore, retinal Müller cells in culture are resistant to anoxia or absence of glucose, which provides a basis for understanding why Müller cells are less susceptible than neurons to ischemia or hypoglycemia.

Glucose dependence of glycolysis, hexose monophosphate shunt activity, energy status, and the polyol pathway in retinas isolated from normal (nondiabetic) rats.

PURPOSE: To measure glucose-dependent metabolic activities and selected parameters of the polyol pathway in retinas isolated from normal rats to test the hypothesis recently proposed by Van den Enden et al that incubation of whole retinas for 2 hours with elevated concentrations of glucose results in activation of the polyol pathway, which is the cause of a redox imbalance, as measured by an increase in the retinal cytosolic lactate-pyruvate ratio and a diabetic-like state. METHODS: Retinas obtained from nondiabetic rats and separated from other ocular tissues were incubated for several hours in incubation medium containing glucose at concentrations ranging from 5 to 30 mM. Measurements were made under aerobic and anaerobic conditions of lactic acid production, retinal adenosine triphosphate (ATP), lactic acid content, the hexose monophosphate shunt pathway, aldose reductase activity, and levels of sorbitol and galactitol. Morphology was examined by light microscopy at the end of the incubations. RESULTS: Incubation of isolated rat retinas with 20 mM glucose increased lactic acid production by approximately 25% in comparison to the rate observed in 5mM glucose under aerobic and anaerobic conditions. The content of ATP and lactate in the retinas after a 2-hour incubation in the presence of oxygen and 20 mM glucose was equal to the amounts found in fresh tissues, whereas these metabolites declined, respectively, by 25% and 45% when 5 mM glucose was used. The activity of the hexose monophosphate shunt pathway in isolated rat retinas was not increased increased significantly when the concentration of glucose was raised from 5 to 30 mM. Aldose reductase activity and polyols were below our limits of detection, 0.5 nmol/minute.mg protein and 3.5 nmol/retina, respectively, under all conditions tested. The morphologic appearance of the retina was similar in the presence of normal and high concentrations of glucose. CONCLUSIONS: These results show that incubation of isolated rat retinas, obtained from nondiabetic rats, with elevated concentrations of glucose for 2 hours leads to increases in glycolysis and a higher tissue content of lactic acid and ATP in comparison to values obtained with 5 mM glucose. However, the magnitude of the glucose-dependent increase in the retinal level of lactate in the current study and in that of Van den Enden et al is six to seven times greater than the calculated flux of glucose through the polyol pathway. These results, therefore, do not support the hypothesis of Van den Enden et al. Rather, it is suggested that supranormal concentrations of glucose yield more lactate and ATP in a whole retina because they optimize the supply of this essential nutrient to cells throughout the tissue by overcoming diffusional limitations that result when the retina is separated from its normal choroidal and intraretinal blood supplies.