β-Cell subcellular localization of glucose-stimulated Mn uptake by X-ray fluorescence microscopy: implications for pancreatic MRI

Abstract

Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β-cells and showed that, in the presence of MnCl(2), glucose-activated pancreatic islets yield significant signal enhancement in T(1)-weigheted MR images. In this study, we exploited for the first time the unique capabilities of X-ray fluorescence microscopy (XFM) to both visualize and quantify the metal in pancreatic β-cells at cellular and subcellular levels. MIN-6 insulinoma cells grown in standard tissue culture conditions had only a trace amount of Mn, 1.14 ± 0.03 × 10(-11)µg/µm(2), homogenously distributed across the cell. Exposure to 2 mM glucose and 50 µM MnCl(2) for 20 min resulted in nonglucose-dependent Mn uptake and the overall cell concentration increased to 8.99 ± 2.69 × 10(-11) µg/µm(2). When cells were activated by incubation in 16 mM glucose in the presence of 50 µM MnCl(2), a significant increase in cytoplasmic Mn was measured, reaching 2.57 ± 1.34 × 10(-10) µg/µm(2). A further rise in intracellular concentration was measured following KCl-induced depolarization, with concentrations totaling 1.25 ± 0.33 × 10(-9) and 4.02 ± 0.71 × 10(-10) µg/µm(2) in the cytoplasm and nuclei, respectively. In both activated conditions Mn was prevalent in the cytoplasm and localized primarily in a perinuclear region, possibly corresponding to the Golgi apparatus and involving the secretory pathway. These data are consistent with our previous MRI findings, confirming that Mn can be used as a functional imaging reporter of pancreatic β-cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast.

Figure 1. Quantification of intracellular manganese in MIN-6 cells

Manganese was quantified from XFM scans of fixed MIN-6 cells by selecting regions of interest corresponding to single cells. Samples were incubated for 20 minutes in KRB supplemented with a) 2mM glucose and 50 μM MnCl₂ (n=11) ; b) 16.7 mM glucose and 50 μM MnCl₂ (n=9); c) 16.7 mM glucose, 30mM KCl and 50 μM MnCl₂ (n=7). Cells grown in standard cell culture media were used as control (d, n=8). (mean ± SD, p<0.001)

Figure 2. Manganese distribution in nucleus and cytoplasm of MIN-6 cells

Manganese concentration, expressed as μg/μm², in nucleus and cytoplasm of MIN-6 cells. Manganese was quantified from XFM scans of fixed MIN-6 cells. ROIs corresponding to single cells were traced and phosphorous rich regions within each cell corresponding to the nucleus were traced. Samples were incubated for 20 minutes in KRB supplemented with a) 2mM glucose and 50 μM MnCl₂ (n=11) ; b) 16.7 mM glucose and 50 μM MnCl₂ (n=9); c) 16.7 mM glucose, 30mM KCl and 50 μM MnCl₂ (n=7). Cells grown in standard cell culture media were used as control (d, n=8). (mean ± SD, p<0.005)

Figure 3. Visualization of manganese in XFM scans of MIN-6 cells

Representative XFM scans of MIN-6 cells. Samples were incubated for 20 minutes in KRB supplemented with 50 μM MnCl₂ and top row) 2mM glucose; middle row) 16.7 mM glucose; bottom row) 16.7 mM glucose and 30mM KCl. Phosphorous rich regions corresponding to cell nuclei were pseudocolored in red (left column). Manganese rich regions are represented by green pseudocoloring (middle column). Cytoplasmic localization of manganese is shown in the image overlays (right column). Color intensity is dependent on the element concentration. XFM scans were acquired with 200×200 nm resolution.

Figure 4. Representative SEM and Fluorescent staining of MIN-6 cells

a)SEM micrograph of an ultra-thin section of MIN-6 cells. MIN-6 cells incubated with 50 μM MnCl₂ and 2mM glucose, 16 mM glucose, or 16mM glucose and 30mM KCl were fixed with osmium tetroxide and stained with uranyl acetate. A representative cell from the 50 μM MnCl₂ and 16mM glucose sample is shown. Among the cellular structures clearly visible are the nucleus (nu), mitochondria (m), and insulin granules (ig). b) Fluorescent staining of Golgi apparatus in MIN-6 cells. Golgi apparatus shown as intense green areas in MIN-6 cells was stained with green-fluorescent Alexa Fluor® 488 conjugate of HPA lectin. Nuclei were counterstained with DAPI.