Altered intestinal and renal brush border amino-oligopeptidase structure in diabetes and metabolic acidosis: Normal and BioBreed (BB) rats

Abstract

Amino-oligopeptidase (AOP, aminopeptidase N), a major glycoprotein hydrolase in intestinal and kidney brush border membranes, plays a crucial role in digesting peptide nutrients and salvaging filtered peptides. The molecular structure of rat intestinal and kidney AOP was compared for normal Wistar and congenitally diabetic BB Wistar (BB d) rats. Brush border membranes were isolated, solubilized with Triton X-100, and the AOP specifically immunoprecipitated with polyvalent rabbit antiserum and analyzed on 7% sodium dodecyl sulfate (SDS)-acrylamide electrophoresis. While the specific hydrolytic activity was maintained, BB d rats displayed an altered migration of AOP on SDS gels. Intestinal AOP migrated as a smaller species (130 kd) in the BB d than in the normal Wistar (135 to 140 kd). In some BB d rats, additional intestinal AOP species were observed (a 130- to 135-kd doublet or a 125-, 130-, or 135-kd triplet). Kidney AOP migrated as a broader band (125 to 140 kd) than intestine for all rat groups, probably due to carbohydrate chain heterogeneity, and was approximately 5 kd smaller in the BB d rat than in the normal Wistar. In contrast, no mass change was found in diabetes induced by streptozotocin (STZ). The altered intestinal AOP in the BB d rat was present when first inserted into the brush border membrane (6 hours after intraperitoneal [ 35S]methionine labeling), and hence was not due to nonenzymatic glycosylation (NEG). Abnormal intestinal and kidney AOP structure appeared in early diabetes, irrespective of high plasma glucose levels or ketoacidosis, and was reversed following evolution of the diabetes under prolonged (21 to 120 days) insulin treatment. Removal of the glycan chains of intestinal AOP by trifluoromethanesulfonic acid (TFMS) yielded a residual protein product (110 kd) that was indistinguishable on SDS gels in nromal and BB d rats, indicating that the mass change in congenital diabetes is due to altered carbohydrate chains. When normal Wistar rats were made metabolically acidotic by ammonium chloride treatment, their intestinal AOP migrated normally, but a slight decrease in the apparent mass (−2 to −5 kd) was observed for the nondiabetic BB c littermate controls. For all groups of rats, ammonium chloride acidosis produced a smaller (−5 kd) species of kidney AOP on SDS gels. These reversible alterations in AOP glycan structure in congenital diabetes or produced by metabolic acidosis may reflect changes in carbohydrate processing enzymes and their substrates in these conditions. The BB rat may be more susceptible than the normal Wistar to these structural alterations.