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

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 ^-11μg/μm ², homogenously distributed across the cell. Exposure to 2m m glucose and 50μ m MnCl ₂ for 20min resulted in nonglucose-dependent Mn uptake and the overall cell concentration increased to 8.99±2.69×10 ^-11μg/μm ². When cells were activated by incubation in 16m m glucose in the presence of 50μ m MnCl ₂, a significant increase in cytoplasmic Mn was measured, reaching 2.57±1.34×10 ^-10μg/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 ^-10μg/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.