Proteins for glucose sensing found in canine insulinoma tumors

Abstract

Introduction: Canine insulinoma is a functional neoplasia of insulin‐producing endocrine cells that produces hypoglycemia as a result of unregulated secretion of insulin. Knowledge of the molecular defects responsible for the observed functional abnormalities might help understand the clinical behavior of these tumors, predict the response to therapy, and identify potential targets for therapeutic intervention. We examined canine insulinomas to determine expression of beta‐cell proteins known to be important for glucose recognition and signaling.Methods: Tissue samples were obtained from naturally‐occurring canine insulinomas at the time of surgery or necropsy. A pancreatic beta cell line was used as control in functional studies. Normal canine pancreas served as control tissue for expression studies. For functional studies, glucose‐induced insulin secretion by cultured canine insulinoma cells was determined using radioimmunoassay. For mRNA expression studies, total RNA was isolated and reverse transcription performed using commercial reagents. The expression of proteins of interest was determined using PCR. Identities of PCR products were confirmed by automated sequence analysis. For protein expression studies, proteins were solubilized in detergent‐containing buffer, resolved by SDS‐PAGE, and detected by immunoblot analysis using specific antibodies.Results: Canine insulinoma cells exhibited a half‐maximal glucose response of 1.3 mM (23 mg/dL). Insulinomas were found to express proteins important for glucose responsiveness. Proteins expressed include those involved in glucose recognition (glucokinase, hexokinase), glucose signal transduction (Kir6.2, SUR1), and insulin exocytosis (SNAP 25, syntaxin 1A).Conclusions: Canine insulinomas express proteins similar to those found in the normal canine pancreas but exhibit an increased sensitivity to glucose.