Evidence that the gluconeogenic pathway is confined to an enriched Müller cell fraction derived from the amphibian retina

Abstract

An enriched Müller cell fraction was prepared from bullfrog retina, employing the endoprotease bromelain to digest the retina, and iso-osmotic Percoll gradient centrifugation to resolve the retinal digest. The Müller cell was the most resistant of all retinal cells to bromelain digestion, thus large numbers of morphologically intact cells were produced. The only other retinal cells that survived bromelain digestion were cones and some bipolar cells. The enriched Müller cells were incubated in 10 mm lactate Ringer containing U-[ 14C]lactate and the amount of [ 14C]glucose synthesized was used as the biochemical endpoint for assessing the rate of gluconeogenesis. There was no incorporation of [ 14C]glucose into Müller cell glycogen: however, 2·8 nmol glucose (mg Müller cell protein × hr) −1 was released to the medium. The synthesis and release of glucose to the medium was increased four- to sixfold by glucagon, vasoactive intestinal peptide or a mixture of forskolin/IBMX. In addition, glycolytic flux of isolated intact retina was determined with 3-[ 3H]glucose. In lactate-free and lactate-containing Ringer, glycolytic flux was 92·5 and 67·9 nmol (mg protein × hr) −1, respectively. In the presence of 100 n m vasoactive intestinal peptide, glycolytic flux in lactate-free and lactate-containing Ringer was reduced by 50 and 66%, respectively. These data strongly suggest that a Cori-like cycle exists in the amphibian retina and the Müller cell may be the predominant site for the gluconeogenic pathway. In addition, vasoactive intestinal peptide may serve both as a gluconeogenic and antiglycolytic neurohormone in the amphibian retina.