Abstract
Insulin receptor-specific polyclonal antipeptide serum was generated against a synthetic pentadecapeptide (residues 657-670) of the deduced amino acid sequence of human insulin proreceptor cDNA for use in the analysis of insulin receptors in the retina. The affinity-purified antibodies recognized peptide antigen but not keyhole limpet hemocyanin as determined by dot blot analysis and solid phase radioimmunoassay. Addition of either synthetic peptide or the affinity-purified serum had no effect on 125I-insulin binding to placental membranes or to cells in culture. alpha-Subunits of approximately 125 kDa from human placental membranes and liver membranes were labeled by immunoblot analysis with this antiserum. In membranes isolated from human retina and brain, two classes of alpha-subunits of approximately 125 and 115 kDa were detectable. The 115-kDa subunit was neuraminidase resistant whereas the 125-kDa subunit was digested to a band of 115 kDa, indicating that these bands represent peripheral and neuronal receptors, respectively. Analysis of human retinas obtained from type I diabetic donors revealed an increased level of neuronal receptor as compared with normal retinas. These data indicate that human retina expresses neuronal insulin receptor subtypes that are up-regulated in diabetes.
Highlights
Antipeptide pentadecapeptide of the deduced amino acid sequence of human insulin proreceptor cDNA for use in the analysis of insulin receptors in the retina
These data indicate that human retina expresses neuronal insulin receptor subtypes that are up-regulated in diabetes
Analysis of neuronal insulin of type I diabetic receptors in the human retinas revealed an increase in the level of neuronal insulin receptor protein compared with normal controls. These results indicate that retinal insulin receptors are up-regulated in diabetes as described previously for hepatic receptors
Summary
Materials-CH-Sepharose was obtained from Pharmacia LKB Biotechnology Inc. Keyhole limpet hemocyanin, Pansorbin, and endoglycosidase H were purchased from Calbiochem. iz51-Labeled human insulin, human IGF-I, and the enhanced chemiluminescence (ECL). O.l-ml final volume) at a 1:lOO dilution was added and incubated in the tubes for 30 min at room temperature after which the solution was aspirated and the tubes washed three times with wash buffer. The filters were incubated with alkaline phosphatase-conjugated or peroxidase-conjugated goat anti-rabbit antibody for 1 h at room temperature. Membranes were prepared from human retinas by homogenization in 0.25 M sucrose containing 5 mM EDTA, 1 mM PMSF, 1 mM bacitracin, 1 pg/ml aprotinin, and 5 pM leupeptin using six strokes of a motor-driven Teflon pestle at 2,000 rpm. Membranes were pelleted in a microcentrifuge, washed, repelleted, and cross-linked with disuccinimidyl suberate for 5 min at 0 “C in a total volume of 100 ~1 of 25 mM HEPES, pH 7.4,104 mM.
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