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Open Access 21-03-2024 | Insulins | Article

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity

Authors: Vanessa Yu, Fiona Yong, Angellica Marta, Sanjay Khadayate, Adrien Osakwe, Supriyo Bhattacharya, Sneha S. Varghese, Pauline Chabosseau, Sayed M. Tabibi, Keran Chen, Eleni Georgiadou, Nazia Parveen, Mara Suleiman, Zoe Stamoulis, Lorella Marselli, Carmela De Luca, Marta Tesi, Giada Ostinelli, Luis Delgadillo-Silva, Xiwei Wu, Yuki Hatanaka, Alex Montoya, James Elliott, Bhavik Patel, Nikita Demchenko, Chad Whilding, Petra Hajkova, Pavel Shliaha, Holger Kramer, Yusuf Ali, Piero Marchetti, Robert Sladek, Sangeeta Dhawan, Dominic J. Withers, Guy A. Rutter, Steven J. Millership

Published in: Diabetologia | Issue 6/2024

Abstract

Aims/hypothesis

Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected ‘hub’ cells, important for the propagation of intercellular Ca²⁺ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion.

Methods

Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca²⁺ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging.

Results

Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT⁺ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. ‘Hub’ cells were less abundant in the NNAT⁺ population, consistent with epigenetic control of this functional specialisation.

Conclusions/interpretation

These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

Graphical Abstract

Available only for authorised users

Rutter GA, Pullen TJ, Hodson DJ, Martinez-Sanchez A (2015) Pancreatic β-cell identity, glucose sensing and the control of insulin secretion. Biochem J 466(2):203–218. https://doi.org/10.1042/BJ20141384CrossRefPubMed

Salinno C, Cota P, Bastidas-Ponce A, Tarquis-Medina M, Lickert H, Bakhti M (2019) β-cell maturation and identity in health and disease. Int J Mol Sci 20(21):5417. https://doi.org/10.3390/ijms20215417CrossRefPubMedPubMedCentral

Hoffman BG, Robertson G, Zavaglia B et al (2010) Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver. Genome Res 20(8):1037–1051. https://doi.org/10.1101/gr.104356.109CrossRefPubMedPubMedCentral

Dhawan S, Tschen SI, Zeng C et al (2015) DNA methylation directs functional maturation of pancreatic β cells. J Clin Invest 125(7):2851–2860. https://doi.org/10.1172/JCI79956CrossRefPubMedPubMedCentral

Lu TT, Heyne S, Dror E et al (2018) The polycomb-dependent epigenome controls β cell dysfunction, dedifferentiation, and diabetes. Cell Metab 27(6):1294-1308 e1297. https://doi.org/10.1016/j.cmet.2018.04.013CrossRefPubMedPubMedCentral

Parveen N, Wang JK, Bhattacharya S et al (2023) DNA methylation dependent restriction of tyrosine hydroxylase contributes to pancreatic β-cell heterogeneity. Diabetes 72(5):575–589. https://doi.org/10.2337/db22-0506CrossRefPubMedPubMedCentral

Dror E, Fagnocchi L, Wegert V et al (2023) Epigenetic dosage identifies two major and functionally distinct β cell subtypes. Cell Metab 35(5):821-836 e827. https://doi.org/10.1016/j.cmet.2023.03.008CrossRefPubMed

Salomon D, Meda P (1986) Heterogeneity and contact-dependent regulation of hormone secretion by individual B cells. Exp Cell Res 162(2):507–520. https://doi.org/10.1016/0014-4827(86)90354-xCrossRefPubMed

Bosco D, Meda P (1991) Actively synthesizing β-cells secrete preferentially after glucose stimulation. Endocrinology 129(6):3157–3166. https://doi.org/10.1210/endo-129-6-3157CrossRefPubMed

10.

Dorrell C, Schug J, Canaday PS et al (2016) Human islets contain four distinct subtypes of β cells. Nat Commun 7:11756. https://doi.org/10.1038/ncomms11756CrossRefPubMedPubMedCentral

11.

Wang YJ, Schug J, Won KJ et al (2016) Single-cell transcriptomics of the human endocrine pancreas. Diabetes 65(10):3028–3038. https://doi.org/10.2337/db16-0405CrossRefPubMedPubMedCentral

12.

Segerstolpe A, Palasantza A, Eliasson P et al (2016) Single-cell transcriptome profiling of human pancreatic islets in health and type 2 diabetes. Cell Metab 24(4):593–607. https://doi.org/10.1016/j.cmet.2016.08.020CrossRefPubMedPubMedCentral

13.

Camunas-Soler J, Dai XQ, Hang Y et al (2020) Patch-seq links single-cell transcriptomes to human islet dysfunction in diabetes. Cell Metab 31(5):1017-1031 e1014. https://doi.org/10.1016/j.cmet.2020.04.005CrossRefPubMedPubMedCentral

14.

Johnston NR, Mitchell RK, Haythorne E et al (2016) Beta cell hubs dictate pancreatic islet responses to glucose. Cell Metab 24(3):389–401. https://doi.org/10.1016/j.cmet.2016.06.020CrossRefPubMedPubMedCentral

15.

Salem V, Silva LD, Suba K et al (2019) Leader β-cells coordinate Ca²⁺ dynamics across pancreatic islets in vivo. Nat Metab 1(6):615–629. https://doi.org/10.1038/s42255-019-0075-2CrossRefPubMed

16.

Bader E, Migliorini A, Gegg M et al (2016) Identification of proliferative and mature β-cells in the islets of Langerhans. Nature 535(7612):430–434. https://doi.org/10.1038/nature18624CrossRefPubMed

17.

Westacott MJ, Ludin NWF, Benninger RKP (2017) Spatially organized β-cell subpopulations control electrical dynamics across islets of Langerhans. Biophys J 113(5):1093–1108. https://doi.org/10.1016/j.bpj.2017.07.021CrossRefPubMedPubMedCentral

18.

Chabosseau P, Rutter GA, Millership SJ (2021) Importance of both imprinted genes and functional heterogeneity in pancreatic beta cells: is there a link? Int J Mol Sci 22(3):1000. https://doi.org/10.3390/ijms22031000CrossRefPubMedPubMedCentral

19.

Karaca M, Castel J, Tourrel-Cuzin C et al (2009) Exploring functional β-cell heterogeneity in vivo using PSA-NCAM as a specific marker. PLoS One 4(5):e5555. https://doi.org/10.1371/journal.pone.0005555CrossRefPubMedPubMedCentral

20.

Salinno C, Buttner M, Cota P et al (2021) CD81 marks immature and dedifferentiated pancreatic β-cells. Mol Metab 49:101188. https://doi.org/10.1016/j.molmet.2021.101188CrossRefPubMedPubMedCentral

21.

Muraro MJ, Dharmadhikari G, Grun D et al (2016) A single-cell transcriptome atlas of the human pancreas. Cell Syst 3(4):385-394 e383. https://doi.org/10.1016/j.cels.2016.09.002CrossRefPubMedPubMedCentral

22.

Rodnoi P, Rajkumar M, Moin ASM, Georgia SK, Butler AE, Dhawan S (2017) Neuropeptide Y expression marks partially differentiated β cells in mice and humans. JCI Insight 2(12):e94005. https://doi.org/10.1172/jci.insight.94005CrossRefPubMedPubMedCentral

23.

Rubio-Navarro A, Gomez-Banoy N, Stoll L et al (2023) A beta cell subset with enhanced insulin secretion and glucose metabolism is reduced in type 2 diabetes. Nat Cell Biol 25(4):565–578. https://doi.org/10.1038/s41556-023-01103-1CrossRefPubMedPubMedCentral

24.

van der Meulen T, Mawla AM, DiGruccio MR et al (2017) Virgin beta cells persist throughout life at a neogenic niche within pancreatic islets. Cell Metab 25(4):911-926 e916. https://doi.org/10.1016/j.cmet.2017.03.017CrossRefPubMedPubMedCentral

25.

Chabosseau P, Yong F, Delgadillo-Silva LF et al (2023) Molecular phenotyping of single pancreatic islet leader beta cells by “Flash-Seq.” Life Sci 316:121436. https://doi.org/10.1016/j.lfs.2023.121436CrossRefPubMed

26.

Kravets V, Dwulet JM, Schleicher WE et al (2022) Functional architecture of pancreatic islets identifies a population of first responder cells that drive the first-phase calcium response. PLoS Biol 20(9):e3001761. https://doi.org/10.1371/journal.pbio.3001761CrossRefPubMedPubMedCentral

27.

Nasteska D, Fine NHF, Ashford FB et al (2021) PDX1^LOW MAFA^LOW β-cells contribute to islet function and insulin release. Nat Commun 12(1):674. https://doi.org/10.1038/s41467-020-20632-zCrossRefPubMedPubMedCentral

28.

Wang G, Chiou J, Zeng C et al (2023) Integrating genetics with single-cell multiomic measurements across disease states identifies mechanisms of beta cell dysfunction in type 2 diabetes. Nat Genet 55(6):984–994. https://doi.org/10.1038/s41588-023-01397-9CrossRefPubMedPubMedCentral

29.

Katsuta H, Aguayo-Mazzucato C, Katsuta R et al (2012) Subpopulations of GFP-marked mouse pancreatic β-cells differ in size, granularity, and insulin secretion. Endocrinology 153(11):5180–5187. https://doi.org/10.1210/en.2012-1257CrossRefPubMedPubMedCentral

30.

Xin Y, Dominguez Gutierrez G, Okamoto H et al (2018) Pseudotime ordering of single human β-cells reveals states of insulin production and unfolded protein response. Diabetes 67(9):1783–1794. https://doi.org/10.2337/db18-0365CrossRefPubMed

31.

Millership SJ, Van de Pette M, Withers DJ (2019) Genomic imprinting and its effects on postnatal growth and adult metabolism. Cell Mol Life Sci 76(20):4009–4021. https://doi.org/10.1007/s00018-019-03197-zCrossRefPubMedPubMedCentral

32.

Villanueva-Hayes C, Millership SJ (2021) Imprinted genes impact upon beta cell function in the current (and potentially next) generation. Front Endocrinol (Lausanne) 12:660532. https://doi.org/10.3389/fendo.2021.660532CrossRefPubMed

33.

Ferguson-Smith AC (2011) Genomic imprinting: the emergence of an epigenetic paradigm. Nat Rev Genet 12(8):565–575. https://doi.org/10.1038/nrg3032CrossRefPubMed

34.

John RM, Lefebvre L (2011) Developmental regulation of somatic imprints. Differentiation 81(5):270–280. https://doi.org/10.1016/j.diff.2011.01.007CrossRefPubMed

35.

Millership SJ, Da Silva Xavier G, Choudhury AI et al (2018) Neuronatin regulates pancreatic beta cell insulin content and secretion. J Clin Invest 128(8):3369–3381. https://doi.org/10.1172/JCI120115CrossRefPubMedPubMedCentral

36.

Vrang N, Meyre D, Froguel P et al (2010) The imprinted gene neuronatin is regulated by metabolic status and associated with obesity. Obesity (Silver Spring) 18(7):1289–1296. https://doi.org/10.1038/oby.2009.361CrossRefPubMed

37.

Dalgaard K, Landgraf K, Heyne S et al (2016) Trim28 haploinsufficiency triggers bi-stable epigenetic obesity. Cell 164(3):353–364. https://doi.org/10.1016/j.cell.2015.12.025CrossRefPubMedPubMedCentral

38.

Millership SJ, Tunster SJ, Van de Pette M et al (2018) Neuronatin deletion causes postnatal growth restriction and adult obesity in 129S2/Sv mice. Mol Metab 18:97–106. https://doi.org/10.1016/j.molmet.2018.09.001CrossRefPubMedPubMedCentral

39.

Byrnes LE, Wong DM, Subramaniam M et al (2018) Lineage dynamics of murine pancreatic development at single-cell resolution. Nat Commun 9(1):3922. https://doi.org/10.1038/s41467-018-06176-3CrossRefPubMedPubMedCentral

40.

Sasaki S, Lee MYY, Wakabayashi Y et al (2022) Spatial and transcriptional heterogeneity of pancreatic beta cell neogenesis revealed by a time-resolved reporter system. Diabetologia 65(5):811–828. https://doi.org/10.1007/s00125-022-05662-0CrossRefPubMed

41.

Sachs S, Bastidas-Ponce A, Tritschler S et al (2020) Targeted pharmacological therapy restores β-cell function for diabetes remission. Nat Metab 2(2):192–209. https://doi.org/10.1038/s42255-020-0171-3CrossRefPubMed

42.

Tritschler S, Thomas M, Bottcher A et al (2022) A transcriptional cross species map of pancreatic islet cells. Mol Metab 66:101595. https://doi.org/10.1016/j.molmet.2022.101595CrossRefPubMedPubMedCentral

43.

Herrera PL (2000) Adult insulin- and glucagon-producing cells differentiate from two independent cell lineages. Development 127(11):2317–2322. https://doi.org/10.1242/dev.127.11.2317CrossRefPubMed

44.

Luche H, Weber O, Nageswara Rao T, Blum C, Fehling HJ (2007) Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies. Eur J Immunol 37(1):43–53. https://doi.org/10.1002/eji.200636745CrossRefPubMed

45.

Parton LE, McMillen PJ, Shen Y et al (2006) Limited role for SREBP-1c in defective glucose-induced insulin secretion from Zucker diabetic fatty rat islets: a functional and gene profiling analysis. Am J Physiol Endocrinol Metab 291(5):E982-994. https://doi.org/10.1152/ajpendo.00067.2006CrossRefPubMed

46.

Herman JS, Sagar, Grun D (2018) FateID infers cell fate bias in multipotent progenitors from single-cell RNA-seq data. Nat Methods 15(5):379–386. https://doi.org/10.1038/nmeth.4662CrossRefPubMed

47.

Jima DD, Skaar DA, Planchart A et al (2022) Genomic map of candidate human imprint control regions: the imprintome. Epigenetics 17(13):1920–1943. https://doi.org/10.1080/15592294.2022.2091815CrossRefPubMedPubMedCentral

48.

Pullen TJ, Huising MO, Rutter GA (2017) Analysis of purified pancreatic islet beta and alpha cell transcriptomes reveals 11β-hydroxysteroid dehydrogenase (Hsd11b1) as a novel disallowed gene. Front Genet 8:41. https://doi.org/10.3389/fgene.2017.00041CrossRefPubMedPubMedCentral

49.

Georgiadou E, Muralidharan C, Martinez M et al (2022) Mitofusins Mfn1 and Mfn2 are required to preserve glucose- but not incretin-stimulated β-cell connectivity and insulin secretion. Diabetes 71(7):1472–1489. https://doi.org/10.2337/db21-0800CrossRefPubMedPubMedCentral

50.

Ivanova E, Chen JH, Segonds-Pichon A, Ozanne SE, Kelsey G (2012) DNA methylation at differentially methylated regions of imprinted genes is resistant to developmental programming by maternal nutrition. Epigenetics 7(10):1200–1210. https://doi.org/10.4161/epi.22141CrossRefPubMedPubMedCentral

51.

Van de Pette M, Abbas A, Feytout A et al (2017) Visualizing changes in Cdkn1c expression links early-life adversity to imprint mis-regulation in adults. Cell Rep 18(5):1090–1099. https://doi.org/10.1016/j.celrep.2017.01.010CrossRefPubMedPubMedCentral

52.

Ebrahimi AG, Hollister-Lock J, Sullivan BA, Tsuchida R, Bonner-Weir S, Weir GC (2020) Beta cell identity changes with mild hyperglycemia: implications for function, growth, and vulnerability. Mol Metab 35:100959. https://doi.org/10.1016/j.molmet.2020.02.002CrossRefPubMedPubMedCentral

53.

Ou K, Yu M, Moss NG et al (2019) Targeted demethylation at the CDKN1C/p57 locus induces human β cell replication. J Clin Invest 129(1):209–214. https://doi.org/10.1172/JCI99170CrossRefPubMed

Title: Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity
Authors: Vanessa Yu
Fiona Yong
Angellica Marta
Sanjay Khadayate
Adrien Osakwe
Supriyo Bhattacharya
Sneha S. Varghese
Pauline Chabosseau
Sayed M. Tabibi
Keran Chen
Eleni Georgiadou
Nazia Parveen
Mara Suleiman
Zoe Stamoulis
Lorella Marselli
Carmela De Luca
Marta Tesi
Giada Ostinelli
Luis Delgadillo-Silva
Xiwei Wu
Yuki Hatanaka
Alex Montoya
James Elliott
Bhavik Patel
Nikita Demchenko
Chad Whilding
Petra Hajkova
Pavel Shliaha
Holger Kramer
Yusuf Ali
Piero Marchetti
Robert Sladek
Sangeeta Dhawan
Dominic J. Withers
Guy A. Rutter
Steven J. Millership
Publication date: 21-03-2024
Publisher: Springer Berlin Heidelberg
Keywords: Insulins
Insulins
Type 2 Diabetes
Published in: Diabetologia / Issue 6/2024
Print ISSN: 0012-186X
Electronic ISSN: 1432-0428
DOI: https://doi.org/10.1007/s00125-024-06123-6

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