Application of a Novel Pancreas Perfusion Technique to Characterize Exocrine Pancreas Metabolism.

The pancreas functions as both an endocrine and exocrine gland. The endocrine component consists of the islets of Langerhans, which contain several pancreatic cell types that produce hormones. In the islets, α-cells produce glucagon and β-cells produce insulin, both of which serve to regulate glucose metabolism. The exocrine tissue produces digestive enzymes such as chymotrypsin and is connected through the ductal system. Type I Diabetes (T1D) is an autoimmune disease in which immune cells attack the pancreatic β-cells, resulting in the pancreas producing little to no insulin. Although the vast majority of research has focused on the islets, the exocrine tissue may be implicated in T1D as well. This research introduces a novel method to investigate exocrine pancreas metabolism specifically using a pancreas perfusion system through the common bile duct. I hypothesized that the exocrine pancreas would demonstrate metabolic activity through uptake and utilization of [ H ]glucose during a perfusion. Using C57BL/6 male mice, the surgical procedure consisted of clamping the ampulla of Vater with a John Hopkins bulldog clamp and cannulation of the common bile duct. The pancreas was then perfused for 20 minutes with perfusate containing Krebs electrolytes, free fatty acids, and 16.7mM of either unlabeled or [ H ]glucose. The tissue was flash frozen in liquid nitrogen and subjected to an acetonitrile isopropanol water extraction. The extracts were derivatized using methoxyamine and MTBSTFA before gas chromatography-mass spectrometry (GC-MS) analysis. GC-MS measurements confirmed the pancreas exocrine tissue metabolized [ H ]glucose, as shown by an increased enrichment in lactate mass isotopologues in the tissue perfused with isotopically labeled glucose compared to the unlabeled control. Additional central carbon metabolites also demonstrated increased enrichment, namely malate and fumarate. The observed isotopologue labeling patterns are indicative of anaplerosis. This research demonstrates metabolism specific to the exocrine pancreas can be measured using a pancreas perfusion system and isotopic labeling. Future work will examine real-time central carbon metabolic flux using dissolution dynamic nuclear polarization (dDNP) combined with the pancreas perfusion system. This research will use the T1D mouse model NOD. Rag1-/-.AI4α/β and compare the results to the NOD.Rag1-/- control mouse model to examine how exocrine pancreas central carbon metabolism is altered in T1D models in a more physiologically relevant manner than previously possible.