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. 2016 Jul 1;311(1):R115-23.
doi: 10.1152/ajpregu.00383.2015. Epub 2016 Apr 20.

Preproglucagon neurons in the hindbrain have IL-6 receptor-α and show Ca2+ influx in response to IL-6

Fredrik Anesten  1 , Marie K Holt  2 , Erik Schéle  1 , Vilborg Pálsdóttir  1 , Frank Reimann  3 , Fiona M Gribble  3 , Cecilia Safari  1 , Karolina P Skibicka  1 , Stefan Trapp  2 , John-Olov Jansson  4
Affiliations

Preproglucagon neurons in the hindbrain have IL-6 receptor-α and show Ca2+ influx in response to IL-6

Fredrik Anesten et al. Am J Physiol Regul Integr Comp Physiol. .

Abstract

Neuronal circuits in the hypothalamus and hindbrain are of importance for control of food intake, energy expenditure, and fat mass. We have recently shown that treatment with exendin-4 (Ex-4), an analog of the proglucagon-derived molecule glucagon-like peptide 1 (GLP-1), markedly increases mRNA expression of the cytokine interleukin-6 (IL-6) in the hypothalamus and hindbrain and that this increase partly mediates the suppression of food intake and body weight by Ex-4. Endogenous GLP-1 in the central nervous system (CNS) is produced by preproglucagon (PPG) neurons of the nucleus of the solitary tract (NTS) in the hindbrain. These neurons project to various parts of the brain, including the hypothalamus. Outside the brain, IL-6 stimulates GLP-1 secretion from the gut and pancreas. In this study, we aim to investigate whether IL-6 can affect GLP-1-producing PPG neurons in the nucleus of the solitary tract (NTS) in mouse hindbrain via the ligand binding part of the IL-6 receptor, IL-6 receptor-α (IL-6Rα). Using immunohistochemistry, we found that IL-6Rα was localized on PPG neurons of the NTS. Recordings of these neurons in GCaMP3/GLP-1 reporter mice showed that IL-6 enhances cytosolic Ca(2+) concentration in neurons capable of expressing PPG. We also show that the Ca(2+) increase originates from the extracellular space. Furthermore, we found that IL-6Rα was localized on cells in the caudal hindbrain expressing immunoreactive NeuN (a neuronal marker) or CNP:ase (an oligodendrocyte marker). In summary, IL-6Rα is present on PPG neurons in the NTS, and IL-6 can stimulate these cells by increasing influx of Ca(2+) to the cytosol from the extracellular space.

Keywords: IL-6; IL-6Rα; glucagon-like peptide-1; hindbrain; preproglucagon.

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Figures

Fig. 1.
Fig. 1.
A: PPG promoter (Glu)-Cre mice were mated with Rosa26-promoter-STOP-GCaMP3 mice. The Rosa26 promoter is active in most cell types. In cells with active PPG promoter, the cAMP response element (CRE) protein cleaved out the STOP sequence upstream of the GCaMP3 gene. This resulted in expression of the GCaMP3 protein and emission of green fluorescence after stimulation of Ca2+ influx into the cytosol, e.g., after stimulation by IL-6. B: immunofluorescence showing anti-glucagon-like peptide-1 (GLP-1) immunoreactivity in red in cells in the nucleus of the solitary tract (NTS) of Glu-Cre-GCaMP3 mice. C: immunofluorescence showing anti-GFP immunoreactivity in green in cells in the NTS of Glu-Cre-GCaMP3 mice. D: cells in the NTS of mice with anti-GLP-1 immunoreactivity in red, anti-GFP immunoreactivity in green, demonstrating colocalization of the two markers (yellow).
Fig. 2.
Fig. 2.
Interleukin-6 receptor α (IL-6Rα) is colocalized with glucagon-like peptide-1 (GLP-1) in the nucleus of the solitary tract (NTS) of the hindbrain. Immunohistochemistry showing IL-6Rα-immunoreactivity in red, GLP-1-immunoreactivity in green, and nuclear staining (DAPI) in blue in a coronal section of the mouse hindbrain. A and C: overviews of the NTS. B and D: magnifications of the areas indicated in A and C, respectively. One cell each in B, E, and F is subject to orthogonal analysis using z-stacks. D was made by focal stacking. Orange arrows indicate examples of colocalization between IL-6Rα and GLP-1. White and green arrows indicate examples of cells with only IL-6Rα (E) and only GLP-1 (F), respectively (D). Results of cell counting concerning the interrelation between GLP-1 and IL-6Rα are shown in 2G. Roughly 38% of all GLP-1-positive cells also stain positively for IL-6Rα and roughly 16% of all IL-6Rα-positive cells stain positively for GLP-1. Images were obtained using the confocal microscope system described in materials and methods. CC, central canal; NTS, nucleus of the solitary tract; GR, gracile nucleus. Scale bars = 100 μm (overview), 10 μm (magnification).
Fig. 3.
Fig. 3.
Preproglucagon neurons in the NTS respond to IL-6 with a rise in intracellular Ca2+. A: representative image of GCaMP3 fluorescence in PPG neurons in the NTS. Scale bar = 20 μm. B: traces showing the increase in intracellular Ca2+ in response to 2 nM IL-6. Gray trace shows individual cells, and the black trace shows the average. n = 6. C: rise in intracellular Ca2+ in response to IL-6 is dependent on influx of extracellular Ca2+. Top: representative trace showing the response to IL-6 (green bars) before, during, and after exposure to Ca2+-free solution. Note that removal of extracellular Ca2+ (0 Ca2+) also reduces the basal intracellular Ca2+ concentration. Bottom: mean peak change in intracellular Ca2+ in the presence (gray bars: n = 19 and 18, respectively) and absence (white bar, n = 28) of extracellular Ca2+. ***P < 0.001.
Fig. 4.
Fig. 4.
Interleukin-6 receptor α (IL-6Rα) is localized on neurons and oligodendrocytes. Immunohistochemistry showing IL-6Rα-immunoreactivity in green, NeuN immunoreactivity in red (A and B), CNP:ase immunoreactivity in red (C and D), and nuclear staining (DAPI) in blue in a coronal section of the mouse hindbrain. A and C: overview of the NTS. B and D: magnification of the areas indicated in A and C. Examples of cells with both IL-6Rα and NeuN (B, orange arrows), cells with both IL-6Rα and CNP:ase (D, orange arrows), as well as a cell only positive for NeuN (B, red arrows) or IL-6Rα (D, white arrows) are shown. Images were obtained using the confocal microscope system described in materials and methods. Scale bars = 100 μm (overview), 10 μm (magnification).
Fig. 5.
Fig. 5.
IL-6Rα is not localized on astrocytes or microglia. Immunohistochemistry showing IL-6Rα immunoreactivity in green and nuclear staining (DAPI) in blue (A–D) in a coronal section of the hindbrain. Iba-1-immunoreactivity (A and B) and GFAP-immunoreactivity (C and D) are shown in red. A and C: overviews of the mouse NTS, and a magnification (D) of the area indicated in C. B show examples of IL-6-Rα-positive and -negative neurons. White arrows indicate cells containing IL-6-Rα. There was no colocalization between glial cell markers, GFAP, and Iba-1 on one hand, and IL-6Rα on the other. Images were obtained using the confocal microscope system described in materials and methods. Scale bars = 100 μm (overview), 10 μm (magnification).
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