Targeting Apollo-NADP+ to Image NADPH Generation in Pancreatic Beta-Cell Organelles

Abstract

NADPH/NADP⁺ redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP⁺. To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP⁺ sensor by first selecting the most pH-stable version of the single-color sensor. We subsequently targeted mTurquoise2-Apollo-NADP⁺ to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome. Finally, we measured the glucose- and glutamine-stimulated NADPH responses by single- and dual-color imaging of the targeted sensors. Overall, we developed multiple organelle-targeted Apollo-NADP⁺ sensors to reveal the prominent role of beta-cell mitochondria in determining NADPH production in the cytoplasm, nucleus, and peroxisome.

Keywords: FRET; NADPH redox; fluorescence anisotropy; fluorescent protein; organelle targeting.

Figure 1

pH optimization of Apollo-NADP⁺. (a) Polarized excitation of fluorescent proteins results in polarized emission and high steady-state fluorescence anisotropy (Monomer). HomoFRET occurs when the proteins are within 10 nm causing depolarization of the emission and a drop in steady-state anisotropy (Dimer). A drop in pH below the pK_a results in “darkening” (darkened dimer) and an artifactual increase in anisotropy. (b) Tandem dimers of EGFP, mVenus, mCerulean (mCer1), mCerulean3 (mCer3), and mTurquoise2 (mTurq2) were expressed in AD293 cells and imaged under varying pH. (c) Cartoon representation of the Apollo-NADP⁺ sensor responding to NADP⁺ through allosteric dimerization. The sensor is composed of enzymatically inactivated human G6PD tagged with fluorescent protein. The crystal structures of human G6PD (PDB 6E07) and yellow fluorescent proteins (PDB 3V3D) as a surrogate for the GFP-derived fluorescent proteins were used to generate the cartoon. Structures shown are not to scale or an exact representation of the amino acid sequence. (d) Apollo-NADP⁺ and monomeric R198P-G6PD (R198I) tagged with mCer1, mCer3, and mTurq2 were imaged in AD293 cells in media. Apollo-NADP⁺ was subsequently imaged after treatment with diamide (5 min, 5 mM), n = 3. (e) Cells expressing the mTurq2 versions of Apollo-NADP⁺ and R198I were exposed to 5 mM diamide and imaged under varying pH, n = 3; * denotes significance <0.05.

Figure 2

Characterization of organelle-targeted Apollo-NADP⁺ constructs. (a) Representative wide-field intensity images of mTurq2-Apollo-NADP⁺ expressed in INS-1E cells to demonstrate the sensor shows correct organelle morphology and localizes to various subcellular compartments. (b) mTurq2-Apollo-NADP⁺ and R198I constructs targeted to each organelle were expressed in INS-1E beta-cells. Controls included the R198I monomeric control (198I, where expressed), Apollo-NADP⁺ in full media (media), and 5 mM diamide (diamide). The sensor was imaged using a two-photon microscope in sequential response to glucose and H₂O₂, n = 3. Not shown are the R198I values for the mitochondria (0.178 ± 0.002) and the peroxisome (0.097 ± 0.002) as the low intensity of the images and the variance in anisotropy suggested that these constructs did not properly refold when targeted to these organelles; * denotes significance <0.05.

Figure 3

Using organelle-specific Apollo-NADP⁺ to determine the role of mitochondrial metabolism on NADPH responses. (a) Representative wide-field images of INS1E cells coexpressing mVenus-Apollo-NADP⁺ (Cyto) and mTurq2-Apollo-NADP⁺ (Mito) with similar thresholding and a 1 pixel median filter. Images have a HiLo lookup table applied, where blue represents background intensity, grayscale represents increasing intensity, and red represents saturated pixels. (b) Temporal response to 15 mM glucose bolus was compared to cells pretreated for 1 h with 50 μM UK5099. Data are reported as mean change in anisotropy (Δanisotropy (×10^–3)) ± S.E.M., n = 5. (c) Temporal response to 10 mM BCH in cells pretreated with glutamine (5 mM, 1 h), n = 5. (d) Anisotropy response to glucose (1–15 mM, 5 min) in control cells and cells pretreated with UK5099 (50 μM, 1 h), FCCP (1 μM, 5 min), and rotenone (1 μM, 5 min), n = 3. (e) Anisotropy responses to a 10 (nuclear) or 20 (peroxisome) min glucose bolus in cells pretreated with UK5099, n = 5–8. ((f), left) Representative anisotropy images of dispersed mouse islet cells transduced with cytoplasmic Apollo-NADP⁺ with similar thresholding and a 1 pixel median filter. Both cells were treated with 15 mM glucose (5 min), while the bottom cell was also pretreated with UK5099 (50 μM, 1 h). The color map is Vik. ((f), right) Anisotropy responses to 15 mM glucose in controls or cells pretreated with UK5099, n = 3–5. Control replicates are shared with Figure 4f since the data were collected at the same time; * denotes significance <0.05.

Figure 4

Using organelle-specific Apollo-NADP⁺ to determine the roles of nonanaplerotic fatty acids and NNT activity on cytoplasmic and mitochondrial NADPH. (a) Acetyl-CoA produced by beta-oxidation of both fatty acids will inhibit pyruvate dehydrogenase (PDH) activity with dichloroacetate (DCA) reversing this effect. Longer-chain fatty acid palmitate is also a direct inhibitor of NNT. (b) Simultaneous wide-field imaging of cytoplasmic and mitochondrial anisotropy responses to 15 mM glucose (15 min) in cells pretreated with octanoate (400 μM, 30 min), octanoate +1 μM DCA (30 min), or no treatment (control). Data are reported as the mean change in anisotropy (Δanisotropy (×10^–3)) ± S.E.M., n = 6–9. (c) Same anisotropy changes from (b) shown in scatter plot. Dotted arrows denote the order they are described in the results. (d, e) Simultaneous wide-field imaging of cytoplasmic and mitochondrial anisotropy responses to 15 mM glucose (15 min) in cells pretreated with palmitate only (400 μM, 4 h), or palmitate (4 h) + 1 μM DCA (30 min) after a 15 mM glucose bolus, n = 5–9. ((f), left) Representative wide-field anisotropy images of dispersed mouse islets transduced with cytoplasmic Apollo-NADP⁺ with similar thresholding and a 1 pixel median filter. Cells were stimulated with 15 mM glucose (15 min) after pretreatment in 400 μM octanoate (30 min, top) or 400 μM octanoate + 1 μM DCA (30 min, bottom). ((f), right) Anisotropy responses of controls compared to cells pretreated with octanoate or octanoate + DCA, n = 4–5. Control replicates are shared with Figure 3f since the data were collected at the same time; * denotes significance <0.05.