Neuronatin regulates pancreatic β cell insulin content and secretion
Steven J. Millership, Gabriela Da Silva Xavier, Agharul I. Choudhury, Sergio Bertazzo, Pauline Chabosseau, Silvia M.A. Pedroni, Elaine E. Irvine, Alex Montoya, Peter Faull, William R. Taylor, Julie Kerr-Conte, Francois Pattou, Jorge Ferrer, Mark Christian, Rosalind M. John, Mathieu Latreille, Ming Liu, Guy A. Rutter, James Scott, Dominic J. Withers
Steven J. Millership, Gabriela Da Silva Xavier, Agharul I. Choudhury, Sergio Bertazzo, Pauline Chabosseau, Silvia M.A. Pedroni, Elaine E. Irvine, Alex Montoya, Peter Faull, William R. Taylor, Julie Kerr-Conte, Francois Pattou, Jorge Ferrer, Mark Christian, Rosalind M. John, Mathieu Latreille, Ming Liu, Guy A. Rutter, James Scott, Dominic J. Withers
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Research Article Cell biology Genetics

Neuronatin regulates pancreatic β cell insulin content and secretion

Abstract

Neuronatin (Nnat) is an imprinted gene implicated in human obesity and widely expressed in neuroendocrine and metabolic tissues in a hormone- and nutrient-sensitive manner. However, its molecular and cellular functions and precise role in organismal physiology remain only partly defined. Here we demonstrate that mice lacking Nnat globally or specifically in β cells display impaired glucose-stimulated insulin secretion leading to defective glucose handling under conditions of nutrient excess. In contrast, we report no evidence for any feeding or body weight phenotypes in global Nnat-null mice. At the molecular level neuronatin augments insulin signal peptide cleavage by binding to the signal peptidase complex and facilitates translocation of the nascent preprohormone. Loss of neuronatin expression in β cells therefore reduces insulin content and blunts glucose-stimulated insulin secretion. Nnat expression, in turn, is glucose-regulated. This mechanism therefore represents a novel site of nutrient-sensitive control of β cell function and whole-animal glucose homeostasis. These data also suggest a potential wider role for Nnat in the regulation of metabolism through the modulation of peptide processing events.

Authors

Steven J. Millership, Gabriela Da Silva Xavier, Agharul I. Choudhury, Sergio Bertazzo, Pauline Chabosseau, Silvia M.A. Pedroni, Elaine E. Irvine, Alex Montoya, Peter Faull, William R. Taylor, Julie Kerr-Conte, Francois Pattou, Jorge Ferrer, Mark Christian, Rosalind M. John, Mathieu Latreille, Ming Liu, Guy A. Rutter, James Scott, Dominic J. Withers

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Figure 5

ER membrane topology of NNAT and its direct effect on SPC processing.

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ER membrane topology of NNAT and its direct effect on SPC processing. (A...
(A) Representative Western blotting analysis of in vitro–translated preproinsulin converted to proinsulin in the presence (+) of pancreatic microsomes with and without coexpression of NNAT, expressed as percentage processing of preproinsulin. Coexpression of GFP was used as a control (n = 5 reactions per group, *P < 0.05, Mann-Whitney U test). (B) INS1E cells with Nnat siRNA knockdown versus scramble siRNA control were permeabilized with digitonin or Triton X-100, immunostained using an antibody that detects all insulin species (Insulins, green) and also NNAT (red), and visualized by confocal microscopy. The luminal ER protein PDI (green) was used to assess membrane permeabilization, and nuclei were visualized with DAPI. Scale bar: 10 μm. Fields of view were quantified for total fluorescence using ImageJ (NIH) from insulin-stained cells permeabilized with digitonin and normalized to cell number (Student’s t test, ***P < 0.001). (C) Representative Western blotting analysis of C-terminal c-Myc–tagged SPCS3 and SEC11A, and FLAG-tagged NNAT translated in vitro in the presence (+) or absence (–) of pancreatic microsomes and treated with proteinase K (Prot. K) (n = 3 reactions per group, mean vs. absence of microsomes, **P < 0.01, Student’s t test vs. presence of microsomes). (D) Immunofluorescent staining of INS1E cells permeabilized with digitonin or Triton X-100 with use of antibodies against NNAT (red) and PDI (green) visualized by confocal microscopy. PDI was used to assess membrane permeabilization, and nuclei were visualized with DAPI. Scale bar: 10 μm. (E) Topology of NNAT (green) and subunits of the SPC (black) on the ER membrane. The catalytic site for signal peptidase cleavage in SEC11A/C is shown in blue (N and C, amino- and carboxy terminal, respectively).