Control of Glycogen Content in Retina: Allosteric Regulation of Glycogen Synthase

Retinal tissue is exceptional because it shows a high level of energy metabolism. Glycogen content represents the only energy reserve in retina, but its levels are limited. Therefore, elucidation of the mechanisms controlling glycogen content in retina will allow us to understand retina response under local energy demands that can occur under normal and pathological conditions. Thus, we studied retina glycogen levels under different experimental conditions and correlated them with glucose-6-phosphate (G-6-P) content and glycogen synthase (GS) activity

In the Early Stages of Diabetes, Rat Retinal Mitochondria Undergo Mild Uncoupling due to UCP2 Activity

<div><p>In order to maintain high transmembrane ionic gradients, retinal tissues require a large amount of energy probably provided by a high rate of both, glycolysis and oxidative phosphorylation. However, little information exists on retinal mitochondrial efficiency. We analyzed the retinal mitochondrial activity in <i>ex vivo</i> retinas and in isolated mitochondria from normal rat retina and from short-term streptozotocin-diabetic rats. In normal <i>ex vivo</i> retinas, increasing glucose concentrations from 5.6mM to 30mM caused a four-fold increase in glucose accumulation and CO₂ production. Retina from diabetic rats accumulated similar amounts of glucose. However, CO₂ production was not as high. Isolated mitochondria from normal rat retina exhibited a resting rate of oxygen consumption of 14.6 ± 1.1 natgO (min.mg prot)^-1 and a respiratory control of 4.0. Mitochondria from 7, 20 and 45 days diabetic rats increased the resting rate of oxygen consumption and the activity of the electron transport complexes; under these conditions the mitochondrial transmembrane potential decreased. In spite of this, the ATP synthesis was not modified. GDP, an UCP2 inhibitor, increased mitochondrial membrane potential and superoxide production in controls and at 45 days of diabetes. The role of UCP2 is discussed. The results suggest that at the early stage of diabetes we studied, retinal mitochondria undergo adaptations leading to maintain energetic requirements and prevent oxidative stress.</p></div

Superoxide production was determined by the reduction of NBT by incubation of isolated mitochondria at 30° in IM at state 4 conditions.

<p>(A) Superoxide production from normal and diabetic rat retina. (B) Mitochondria were incubated in the absence (white columns) or in the presence of 1mM GDP (black columns). Each value represents the average ± SEM of three to six independent experiments * p< 0.5.</p

Mitochondrial ATP synthesis.

<p>Mitochondrial fraction was incubated at 30°C in IM, using glutamate/malate (10mM) as oxidative substrate and 100μM ADP. The ATP production was determined by NADPH reduction by a coupled reaction with HK and G6P-DH enzymes as described in Methods. Incubation was carried out in the absence and presence of oligomycine (10μg/mg prot.) to obtain the specific ATP produced by ATP synthase. Each value is the mean ± SEM of three to five independent experiments.</p

Effect of GDP on mitochondrial transmembrane potential.

<p>∆ψ was determined in the absence (white bars) and presence of 1mM GDP (black bars). Each value corresponds to the average ± SEM of at least three independent experiments.</p

Activity of glycogen synthase and glycogen phosphorylase in rat retina.

<p>Retina from normal, diabetic, and insulin-treated diabetic animals (diabetic+insulin) were processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030822#s2" target="_blank">Methods</a>. Activity of glycogen synthase was measured in the presence of 6.2 mM glucose-6-phosphate. Activity of glycogen phosphorylase was measured in the presence of 1 mM AMP. Activities are expressed as nmol/mg protein/min. Values are the means ± SEM from 5 to 8 rats.</p>*<p>p<0.01 with respect to the normal rats.</p

Glucose-6-phosphate and glycogen content in the retina of rats under different conditions.

<p>Normal and 20 day diabetic rats were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030822#s2" target="_blank">Methods</a>. Normal (N), normal-fasted rats (F) and normal-fasted rats plus glucose administration (F+glucose) were sacrificed 2 h after saline or glucose administration. Normal (N) or diabetic (D) rats were killed after 3 h insulin or saline injection. Glycogen, nmol glycosyl residues/mg protein; glucose-6-P, pmol/mg protein; glucose, mg/dl.</p>*<p>p<0.01;</p>**<p>p<0.001 with respect to normal rats.</p

Total and phosphorylated GS in retina.

<p>Western blot analysis of representative experiments, top panels. (<b>A–D</b>) Graphic representation of the relative levels of total GS (<b>A, C</b>) and phospho (Ser 641)-GS (<b>B, D</b>). N, normal rat; F, fasted rat; F+G, fasted rat with glucose administration; N+I, normal rat plus insulin administration; D, 20 day streptozotocin-diabetic rat; D+I, diabetic rat plus insulin injection. Values are the mean ± SEM of four experiments. * p<0.05; ** p<0.02.</p

Mitochondrial membrane potential.

<p>(A) Representative mitochondrial transmembrane potential trace, (M) mitochondria (100μg protein) from normal rat retina were incubated at 30°C, in IM, monitoring the Safranine O fluorescence. The ∆ψ was dissipated by the addition of 5μM CCCP, as described in Methods. (B) ∆ψ from normal and diabetic (7, 20, 45 days) rat retinas. Values are the mean ± SEM from at least four separated experiments.* p< 0.5, ** p< 0.05.</p

Uncoupling UCP2 and COX expression levels in retina.

<p>Western blot of (A) COX-IV and (B) UCP2 in normal, 7, 20 and 45 days after diabetes induction. Upper part, a representative western blot experiment; lower part, densitometric analysis of three to five independent experiments.</p