Neuropeptide Y expression marks partially differentiated β cells in mice and humans
Pope Rodnoi, Mohan Rajkumar, Abu Saleh Md Moin, Senta K. Georgia, Alexandra E. Butler, Sangeeta Dhawan
Pope Rodnoi, Mohan Rajkumar, Abu Saleh Md Moin, Senta K. Georgia, Alexandra E. Butler, Sangeeta Dhawan
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Research Article Development Endocrinology

Neuropeptide Y expression marks partially differentiated β cells in mice and humans

Abstract

β Cells are formed in embryonic life by differentiation of endocrine progenitors and expand by replication during neonatal life, followed by transition into functional maturity. In this study, we addressed the potential contribution of neuropeptide Y (NPY) in pancreatic β cell development and maturation. We show that NPY expression is restricted from the progenitor populations during pancreatic development and marks functionally immature β cells in fetal and neonatal mice and humans. NPY expression is epigenetically downregulated in β cells upon maturation. Neonatal β cells that express NPY are more replicative, and knockdown of NPY expression in neonatal mouse islets reduces replication and enhances insulin secretion in response to high glucose. These data show that NPY expression likely promotes replication and contributes to impaired glucose responsiveness in neonatal β cells. We show that NPY expression reemerges in β cells in mice fed with high-fat diet as well as in diabetes in mice and humans, establishing a potential new mechanism to explain impaired β cell maturity in diabetes. Together, these studies highlight the contribution of NPY in the regulation of β cell differentiation and have potential applications for β cell supplementation for diabetes therapy.

Authors

Pope Rodnoi, Mohan Rajkumar, Abu Saleh Md Moin, Senta K. Georgia, Alexandra E. Butler, Sangeeta Dhawan

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Figure 4

NPY expression is epigenetically regulated and modulates β cell maturation.

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NPY expression is epigenetically regulated and modulates β cell maturati...
(A–C) ChIP analysis showing the enrichment of histone modifications (A) H3K9me3 (histone H3-lysine 9 trimethylation; repressive) and H3K9Ac (histone H3 lysine 9 acetylation; activating), (B) histone acetyl transferase CBP (CREB-binding protein) and histone deacteylase HDAC2, (C) along with control rabbit and mouse IgGs, to the Npy promoter region in β cells sorted from MIP-GFP mice at P5 and P30. These data show the epigenetic repression of the Npy promoter as a function of maturation. (D) Npy mRNA expression levels, normalized to the housekeeping gene CyclophilinA in β cells sorted from MIP-GFP mice, in which GFP expression is driven by insulin promoter at P5 and P30, showing reduced expression as a function of maturation. (E) A representative pancreatic section from wild-type, neonatal (P5) mice showing immunostaining for NPY (green), insulin (Ins; red), and Ki67 (cyan). Arrows mark replicating NPY+ β cells. (F) Quantification of replication marker Ki67 in the NPY+ and NPY– subpopulations of β cells in wild-type neonatal mice at P5 and P14. (G) Immunohistochemistry and (H) quantification for replication of β cells in islets isolated from neonatal (P5) mice treated with an siRNA targeting Npy or a control, scrambled siRNA. Immunostaining for replication marker Ki67 (red) and β cell marker Pdx1 (green) was used to measure β cell replication. Arrows mark Ki67 Pdx1 double-positive cells. (I–K) Npy mRNA levels and static incubation glucose-stimulated insulin secretion (GSIS) assay in islets from (I) wild-type, (J) P5 pups, or (K) adult (2.5-month-old) mice, treated either with an siRNA targeting Npy or a scrambled (Scr) siRNA. Insulin secretion was measured at 2.8 mM glucose (basal) and 16.7 mM (stimulated) glucose and reported as a percentage of insulin content. Scale bar: 50 μm. For A–D, average of n = 3 independent sorts; each sort had 6–8 pups at P5 and 4 mice at P30. For E and F, n = 4 animals. For G–K, n = 3 independent experiments, with each replicate representing a pool of islets from 6–8 pups. The error bars represent SEM of the mean. *P < 0.05, **P < 0.01, ***P < 0.005. A 2-tailed Student’s t test was used for A–D, H, and I to determine statistical significance. For F, J, and K (which involved repeat measures), statistical significance was determined by 1-way ANOVA followed by Bonferroni’s post-hoc test.