Quantitative Subcellular Imaging of Glucose Metabolism within Intact Pancreatic Islets

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.

A subcellular model of glucose-stimulated pancreatic insulin secretion

“…Moreover, by imposing a glucose gradient across single islets, Rocheleau et al (2004) showed that active cells were not able to activate otherwise silent cells; in other words, a cell was active if and only if its expected single-cell threshold was reached. Similarly, Bennett et al (1996) observed that metabolism, as measured by NAD(P)H, is heterogeneous across an islet until glucose has equilibrated, such that cells that presumably are located at a low glucose concentration show a low NAD(P)H signal. Once glucose has equilibrated, and activated all cells, the NAD(P)H signal is synchronized across the islet.…”

Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet β cells

“…Because components other than NAD(P)H contribute to cell autofluorescence, for example, collagen, elastin, flavin adenine dinucleotide (FAD), or lipopigments (15), absolute NAD(P)H levels cannot be fluorescently determined. In fact, ␤ cell autofluorescence falls only Ϸ50% when glycolysis is inhibited by mannoheptulose, indicating a fluorescent component not related to glucose metabolism (8). However, the other major metabolically regulated autofluorescent component, FAD, does not contribute measurably to fluorescence generated by two-photon excitation at Ͻ780 nm (16,17).…”

“…By using two-photon excitation microscopy to image noninvasively islets of Langerhans (8), the fluorescence from a purified NADH standard curve was compared with subcellular NAD(P)H fluorescence to determine quantitatively the source and number of NAD(P)H molecules involved in glucose metabolic signaling in pancreatic ␤ cells. Isolated islets cultured on extracellular matrix provided ␤ cells that were sufficiently spread out to allow spatial separation of mitochondrial and cytoplasmic regions by fluorescence microscopy.…”

Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides

“…Elastase activity has been suggested to be involved in the pathogenesis of abdominal aortic aneurysm [4,16] as well as in remodeling of the vasculature occurring in aging [2]. In this study, we directly visualized the effect of minimally-oxidized LDL on the degradation of extracellular proteins in fresh rat aorta by means of two-photon near-infrared excitation microscopy [17][18][19]. This microscopy does not require sample treatment such as freezing-thawing, inclusion in matrices, fixation or staining [20 -22], which may produce artifacts, e.g., contraction produced by fixation, which dehydrates the sample.…”

“…Therefore, absorption in the ultraviolet region can be achieved by near-infrared excitation with the advantage of strongly reducing the sample photodamage and photobleaching, usually associated with UV excitation. In the absence of exogenous fluorophores, a variety of autofluorescence signals can be observed [18,23], arising from molecules absorbing in the UV range (onephoton absorption). We obtained for the first time highresolution images of the intense autofluorescence arising from extracellular matrix proteins of a fresh rat aorta.…”