Quantitative NAD(P)H/Flavoprotein Autofluorescence Imaging Reveals Metabolic Mechanisms of Pancreatic Islet Pyruvate Response

Abstract

Glucose-stimulated insulin secretion is a multistep process dependent on ␤-cell metabolic flux. Our previous studies on intact pancreatic islets used two-photon NAD(P)H imaging as a quantitative measure of the combined redox signal from NADH and NADPH (referred to as NAD(P)H). These studies showed that pyruvate, a non-secretagogue, enters ␤-cells and causes a transient rise in NAD(P)H. To further characterize the metabolic fate of pyruvate, we have now developed one-photon flavoprotein microscopy as a simultaneous assay of lipoamide dehydrogenase (LipDH) autofluorescence. This flavoprotein is in direct equilibrium with mitochondrial NADH. Hence, a comparison of LipDH and NAD(P)H autofluorescence provides a method to distinguish the production of NADH, NADPH, or both. Using this method, the glucose dose response is consistent with an increase in both NADH and NADPH. In contrast, the transient rise in NAD(P)H observed with pyruvate stimulation is not accompanied by a significant change in LipDH, which indicates that pyruvate raises cellular NADPH without raising NADH. In comparison, methyl pyruvate stimulated a robust NADH and NADPH response. These data provide new evidence that exogenous pyruvate does not induce a significant rise in mitochondrial NADH. This inability likely results in its failure to produce the ATP necessary for stimulated secretion of insulin. Overall, these data are consistent with either a restricted pyruvate dehydrogenasedependent metabolism or a buffering of the NADH response by other metabolic mechanisms.

RETRACTED: Metabolic regulation of SIRT1 transcription via a HIC1:CtBP corepressor complex

“…Two-photon microscopy was performed to measure the nuclear autofluorescent NAD(P)H intensity as described (48). Cells were grown for 2 days on 35-mm glass dishes (MatTek, Ashland, MA) maintained at 37°C and 5% CO 2 in a humidified chamber on the microscope stage (Carl Zeiss, Thornwood, NY).…”

“…Cells were grown for 2 days on 35-mm glass dishes (MatTek, Ashland, MA) maintained at 37°C and 5% CO 2 in a humidified chamber on the microscope stage (Carl Zeiss, Thornwood, NY). NAD(P)H intensity was imaged by using a ϫ40, 1.3-NA oil immersion lens (Carl Zeiss), a 710-nm mode-locked Ti:Saph laser (Ϸ3.5 mW at the sample) (Coherent, Santa Clara, CA), and fluorescence collection through a nondescanned detector with a custom 380-to 550-nm filter (Chroma, Rockingham, VT) (48). Images were analyzed with Metamorph 5.0 (Molecular Devices, Sunnyvale, CA).…”

Direct Measurements of Oscillatory Glycolysis in Pancreatic Islet β-Cells Using Novel Fluorescence Resonance Energy Transfer (FRET) Biosensors for Pyruvate Kinase M2 Activity

“…Optical Measurements of Islet Redox Potential-To ascertain the kinetic relationship between PKM2 activity and mitochondrial metabolism in ␤-cells, we measured endogenous NAD(P)H (8, 21, 52-54) and flavin fluorescence, two methods previously used to assess metabolic activity of cells (54,55). The fluorescence from NAD(P)H is a combination of cellular NADH and NADPH fluorescence, produced in both the cytosolic and mitochondrial compartments.…”

Chronic Exposure to Excess Nutrients Left-shifts the Concentration Dependence of Glucose-stimulated Insulin Secretion in Pancreatic β-Cells

“…A deconvolution wide field epifluorescence microscope (Nikon) was used to measure NAD(P)H autofluorescence in INS-1 cells. This method cannot distinguish between NADH and NADPH, hence the designation of NAD(P)H (35). Prior to measurement, cells were preincubated as described for insulin secretion.…”