In most mammals, glucokinase (GK) acts as a hepatic "glucose sensor" that permits hepatic metabolism to respond appropriately to changes in plasma glucose concentrations. GK activity is potently regulated by the glucokinase regulatory protein (GKRP), which is encoded by the GCKR gene. GKRP binds GK in the nucleus and inhibits its activity. GK becomes active when it is released from GKRP and translocates to the cytosol. Low glucokinase (GK) activity is reported to be a principal feature of feline hepatic carbohydrate metabolism but the molecular pathways that regulate GK activity are not known. This study examined the hypothesis that species-specific differences in GKRP expression parallel the low GK activity observed in feline liver. Hepatic GKRP expression was examined using RT-PCR, immunoblot, and confocal immunomicroscopy. The results show that the GCKR gene is present in the feline genome but GCKR mRNA and the GKRP protein were absent in feline liver. The lack of GKRP expression in feline liver indicates that the low GK activity cannot be the result of GKRP-mediated inhibition of the GK enzyme. However, the absence of the permissive effects of GCKR expression on GK expression and activity may contribute to reduced GK enzyme activity in feline liver. The study results show that the cat is a natural model for GCKR knockout and may be useful to study regulation of GCKR expression and its role in hepatic glucose-sensing and carbohydrate metabolism.

Background: Pancreatic beta cells express ATP-sensitive potassium (K ATP ) channels that are needed for normal insulin secretion and are targets for drugs that modulate insulin secretion. The K ATP channel is composed of two subunits: a sulfonylurea receptor (SUR 1) and an inward rectifying potassium channel (Kir 6.2 ). K ATP channel activity is influenced by the metabolic state of the cell and initiates the ionic events that precede insulin exocytosis. Although drugs that target the K ATP channel have the expected effects on insulin secretion in dogs, little is known about molecular aspects of this potassium channel. To learn more about canine beta cell K ATP channels, we studied K ATP channel expression by the normal canine pancreas and by insulin-secreting tumors of dogs.

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.