Quantitative Subcellular Imaging of Glucose Metabolism within Intact Pancreatic Islets

Mark A. Magnuson,Thomas L. Jetton,David W. Piston,Guangtao Ying,Brian D. Bennett

doi:10.1074/jbc.271.7.3647

Abstract

Studies of dispersed beta cells have been used to infer their behavior in the intact pancreatic islet. When dispersed, beta cells exhibit multiple metabolic glucose-response populations with different insulin secretion properties. This has led to a model for glucose-dependent insulin secretion from the islet based on a step-wise recruitment of individual beta cells. However, previously reported synchronous and uniform Ca2+ activity and electrical responses indicate that beta cell behavior within intact islets is more uniform. Therefore, uncertainty remains whether beta cell metabolic heterogeneity is functionally important in intact islets. We have used two-photon excitation microscopy to measure and compare the glucose-induced NAD(P)H autofluorescence response in dispersed beta cells and within intact islets. Over 90% of beta cells in intact islets responded to glucose with significantly elevated NAD(P)H levels, compared with less than 70% of dispersed beta cells. In addition, all responding beta cells within intact islets exhibited a sigmoidal glucose dose response behavior with inflection points of approximately 8 mm glucose. These results suggest that beta cell heterogeneity may be functionally less important in the intact islet than has been predicted from studies of dispersed beta cells and support the role of glucokinase as the rate-limiting enzyme in the beta cell glucose response.

Highlights

Metabolic behavior of cells within the intact islet, has not been performed
To validate the results obtained from intact islets, we examined the NAD(P)H response of isolated ␤ cells
We have used two-photon excitation microscopy to measure and compare the metabolic responses to glucose of individual cells within intact pancreatic islets

Summary

EXPERIMENTAL PROCEDURES

Islet Isolation and Culture—Rat islets were isolated by pancreatic distention followed by collagenase digestion [15]. Islets were attached to a coverslip with Cell-Tak (Collaborative Biomedical Products, Bedford, MA), and the coverslip was placed in a temperature-controlled perifusion micro incubator (TLC-MI, Adam and List Associates, Westbury, NY). This micro incubator held the temperature of the sample at 37 °C (measured by thermocouple adjacent to the islet) by heating both the coverslip chamber and incoming perifusate. Cells were titurated briefly and plated onto marked coverslips (Bellco) coated with a monolayer of Cell-Tak. Quantitative NAD(P)H Imaging in Living Islets and Isolated ␤ Cells—Two-photon excitation microscopy was performed using a previously described instrument [14].

Quantitative Imaging of Glucose Metabolism

RESULTS

DISCUSSION