Glial cells transform glucose to alanine, which fuels the neurons in the honeybee retina

Abstract

The retina of honeybee drone is a nervous tissue with a crystal-like structure in which glial cells and photoreceptor neurons constitute two distinct metabolic compartments. The phosphorylation of glucose and its subsequent incorporation into glycogen occur in glia, whereas O2 consumption (QO2) occurs in the photoreceptors. Experimental evidence showed that glia phosphorylate glucose and supply the photoreceptors with metabolic substrates. We aimed to identify these transferred substrates. Using ion-exchange and reversed-phase HPLC and gas chromatography-mass spectrometry, we demonstrated that more than 50% of 14C(U)-glucose entering the glia is transformed to alanine by transamination of pyruvate with glutamate. In the absence of extracellular glucose, glycogen is used to make alanine; thus, its pool size in isolated retinas is maintained stable or even increased. Our model proposes that the formation of alanine occurs in the glia, thereby maintaining the redox potential of this cell and contributing to NH3 homeostasis. Alanine is released into the extracellular space and is then transported into photoreceptors using an Na(+)-dependent transport system. Purified suspensions of photoreceptors have similar alanine aminotransferase activity as glial cells and transform 14C- alanine to glutamate, aspartate, and CO2. Therefore, the alanine entering photoreceptors is transaminated to pyruvate, which in turn enters the Krebs cycle. Proline also supplies the Krebs cycle by making glutamate and, in turn, the intermediate alpha-ketoglutarate. Light stimulation caused a 200% increase of QO2 and a 50% decrease of proline and of glutamate. Also, the production of 14CO2 from 14C-proline was increased. The use of these amino acids would sustain about half of the light-induced delta QO2, the other half being sustained by glycogen via alanine formation. The use of proline meets a necessary anaplerotic function in the Krebs cycle, but implies high NH3 production. The results showed that alanine formation fixes NH3 at a rate exceeding glutamine formation. This is consistent with the rise of a glial pool of alanine upon photostimulation. In conclusion, the results strongly support a nutritive function for glia.

Lactate released by Muller glial cells is metabolized by photoreceptors from mammalian retina

“…This metabolic trafficking of substrate would serve to maintain glycolysis elevated in glial cells, and to fuel mitochondrial oxidative metabolism and glutamate resynthesis in photoreceptors, even in the presence of glucose. This trafficking is reminiscent of that of alanine recently demonstrated in insect retina (Tsacopoulos et al, 1994). Figure 2.…”

“…2: KH,PO,. 14C0, was collected on hyamine-impregnated filters for 60 min in darkness using the same microincubator as described by Tsacopoulos et al (1994). The amount of 14C-substrates was adjusted so that the specific activity of lactate was the same as that of glucose.…”

“…Since 14C-glucose was used at a concentration of about 5 mrvt, or 14-fold that used by Tsacopoulos et al (1994), the chromatographic peaks corresponding to 14C-a-ketoglutarate and r4C-pyruvate partially overlapped with that of 14C-glucose. Separation of 14C-pyruvate was improved by transforming enzymatically 14C-glucose to 14C-glucose-6-phosphate (G6P).…”

“…The method for the determination of carbohydrate intermediates (i.e., metabolites of glycolysis and of the Kreb's cycle) is that described by Tsacopoulos et al (1994). This is based on a refractive index (RI) detection system employing two Aminex HPX 87H+ ion exchange columns in series.…”

“…Amino acids. Analysis of amino acids with ultraviolet detection (UV) and precolumn derivatization using PITC was performed using the system described in Tsacopoulos et al (1994). This method was modified in the following way: the mobile phase consisted of 93 parts Na acetate (35 mM, pH = 5) and 7 parts acetonitrile, and the flow rate was 0.7 ml/min through a Nucleosil Cl8 (5 pm) (Macherey Nagel, Oensingen, Switzerland) reverse phase column at room temperature.…”