Glucolipotoxicity Alters Insulin Secretion Via Epigenetic Changes in Human Islets
Total Page：16
File Type：pdf, Size：1020Kb
Page 1 of 4545 Diabetes Glucolipotoxicity alters insulin secretion via epigenetic changes in human islets Elin Hall1*, Josefine Jönsson1*, Jones K Ofori2, Petr Volkov1, Alexander Perfilyev1, Marloes Dekker-Nitert1,3, Lena Eliasson2, Charlotte Ling1, Karl Bacos1 1Epigenetics and Diabetes Unit, Department of Clinical Sciences, Lund University Diabetes Centre, Lund University, Malmö, Sweden 2Islet Cell Exocytosis Unit, Department of Clinical Sciences, Lund University Diabetes Centre, Lund University, Malmö, Sweden 3School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Australia (current affiliation) *Equal contribution Corresponding authors: Charlotte Ling and Karl Bacos, Lund University Epigenetics and Diabetes, Clinical Research Centre 91:12, Box 50332, 20213 Malmö, Sweden. Phone: +46-40391213; E-mail: [email protected] and [email protected] Running title: Glucolipotoxicity in human pancreatic islets Suggested Tweet: Treatment of human pancreatic islets with high levels of glucose and the fatty acid palmitate, i.e. mimicking a diabetic milieu, alters insulin secretion via epigenomic and transcriptomic changes #exodiab #LUDC @DiabetesLUDC @miRNA375 1 Diabetes Publish Ahead of Print, published online August 16, 2019 Diabetes Page 2 of 4545 Abstract Type 2 diabetes (T2D) is characterized by insufficient insulin secretion and elevated glucose levels, often in combination with high levels of circulating fatty acids. Long-term exposure to glucose or fatty acids impair insulin secretion in pancreatic islets and this may partly be due to epigenetic alterations. We aimed to study effects of combined high concentrations of glucose and palmitate for 48h (glucolipotoxicity) on the transcriptome, epigenome and cell function in human islets. Glucolipotoxic exposure impaired insulin secretion, increased apoptosis and significantly (FDR<5%) altered expression of 1,855 genes. These include 35 genes previously implicated in T2D by GWAS e.g. TCF7L2 and CDKN2B. Additionally, metabolic pathways were enriched for down-regulated genes. Of the differentially expressed genes, 1,469 also exhibited altered DNA methylation, e.g. CDK1, FICD, TPX2 and TYMS. Luciferase assay showed that increased methylation of CDK1 directly reduces its transcription in pancreatic beta-cells, supporting that DNA methylation underlies altered expression after glucolipotoxicity. Follow-up experiments in clonal beta-cells showed that knockdown of FICD and TPX2 alters insulin secretion. Together, our novel data demonstrate that glucolipotoxicity changes the epigenome in human islets thereby altering gene expression and possibly exacerbating the secretory defect in T2D. Keywords beta-cells, CDK1, DNA methylation, epigenetics, FICD, gene expression, glucolipotoxicity, pancreatic islets, TPX2, TYMS, Type 2 diabetes, CpG sites 2 Page 3 of 4545 Diabetes Introduction Type 2 diabetes (T2D) develops due to insufficient insulin release from the pancreatic beta- cells, often in combination with insulin resistance in liver, muscle and fat. This leads to elevated levels of glucose and fatty acids in the circulation. Extended exposure to high glucose (glucotoxicity) or fatty acid (lipotoxicity) levels, both alone and in combination (glucolipotoxicity), has detrimental effects on insulin secretion in vitro (1-3). Cell survival is also compromised under lipotoxic and glucolipotoxic conditions, depending on the length of treatment and type of fatty acid used (1; 3). The elevated levels of glucose and fatty acids seen in individuals with T2D may thus cause further impairment of insulin secretion and loss of functional beta-cell mass. In two recent studies, we have demonstrated that glucotoxicity (19mM glucose for 48h) or lipotoxicity (1mM palmitate for 48h) alone changes the DNA methylation pattern in human pancreatic islets and these changes may partly explain the impaired insulin secretion seen in the islets (4; 5). However, although most subjects with T2D experience both elevated glucose and lipid levels, the combined effects of glucolipotoxicity on DNA methylation, gene expression and function in human pancreatic islets has not been reported. DNA methylation in adult mammalian cells occurs mainly on cytosines followed by a guanine, so called CpG sites, and it regulates for example gene transcription and mRNA splicing (6). Over the last decade an increasing number of studies have identified altered DNA methylation levels in human islets which seem to contribute to impaired insulin secretion and T2D (7-15). For example, case-control studies have revealed widespread methylation changes in pancreatic islets from donors with T2D compared with non-diabetic donors (8; 13). Functional follow-up experiments in beta-cells showed that these alterations induce gene expression changes that impair insulin secretion. 3 Diabetes Page 4 of 4545 In this study, we investigated for the first time whether a 48h exposure to glucolipotoxic treatment (19mM glucose and 1mM palmitate) alters mRNA expression and DNA methylation patterns genome-wide in human pancreatic islets (Figure 1). We subsequently performed functional follow-up experiments to test whether the epigenetic changes underlie islet cell defects induced by glucolipotoxicity. 4 Page 5 of 4545 Diabetes Research Design and Methods Human pancreatic islets The cohort of human pancreatic islet donors (Table 1) has been described previously (4; 5). The islets were obtained from the Nordic Network for Islets Transplantation, Uppsala University, Sweden through the Human Tissue Laboratory at the Lund University Diabetes Centre. Informed consent was obtained from pancreatic donors or their relatives in accordance with the approval by the local ethics committee regarding organ donation for medical research. DNA methylation and mRNA expression was analyzed in a total of 13 islet donors; islets from eight donors were analyzed with both mRNA expression and DNA methylation arrays and islets from an additional ten donors were used for analysis of either mRNA expression or DNA methylation. The cohorts used for gene expression and DNA methylation analysis hence contain some individuals unique for each analysis. HbA1c was measured with the NGSP method. Glucolipotoxic treatment A 10mM solution of BSA-conjugated palmitate was prepared as described (5). Human islets were cultured for 24 or 48h in either control (5.6mM glucose) or glucolipotoxic (19mM glucose + 1mM palmitate) CMRL 1066 medium before analyses as indicated below (Figure 1A). 1mM palmitate is at the upper limit of circulating free fatty acid concentration in obese individuals with T2D (16). The molar ratio of palmitate/BSA in the culture medium was 6.6:1. The human beta-cell line EndoC-βH1 (17) was cultured as described (18). Glucose (total 19mM) and palmitate (1mM) were added to the culture media as indicated in the text. 5 Diabetes Page 6 of 4545 Insulin secretion Glucose-stimulated insulin secretion (GSIS) was analyzed in control and glucolipotox-treated islets from nine donors and EndoC-βH1 cells (passage 79-83). Ten replicates of ten islets per culture condition and donor were pre-incubated in HEPES-balanced salt solution (HBSS) containing (in mM) 114 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.16 MgSO4, 20 HEPES, 25.5 NaHCO3, 2.5 CaCl2 and 0.575 BSA, supplemented with 3.3mM glucose (1.7mM glucose for one sample), pH 7.2, for 1h at 37°C. Glucose was thereafter added to five of the replicates to a final concentration of 16.7mM (15mM for one sample) to study GSIS and the other five replicates were kept in 3.3mM glucose to study basal insulin secretion and the incubation was continued for one more hour. The supernatant was immediately removed and the insulin concentration in the medium was measured by radioimmunoassay (RIA) (Millipore, Uppsala, Sweden). GSIS from EndoC-βH1 cells was analysed as previously described (19). Secretion in islets and cells was normalized to number of islets and total insulin content, respectively. Assessment of apoptosis in human pancreatic islets and EndoC-βH1 Caspase-3 and -7 activity, as a measure for apoptosis, was analyzed as described (20). Full detail can be found in the supplementary information. mRNA expression and DNA methylation analysis DNA and RNA were extracted from islets with the AllPrep DNA/RNA kit (Qiagen, Hilden, Germany). The Affymetrix GeneChip® Human Gene 1.0 ST whole transcript-based array (Affymetrix, Santa Clara, CA, USA) and Infinium HumanMethylation450K BeadChip (Illumina, San Diego, CA, USA), was used to analyze mRNA expression and DNA methylation, respectively, in control and glucolipotox-treated 6 Page 7 of 4545 Diabetes pancreatic islets from 13 human donors (Figure 1, Table 1). Full detail can be found in the supplementary information. Gene set enrichment and pathway analysis Enrichment of KEGG pathways within the complete mRNA expression data set was analyzed with the gene set enrichment analysis (GSEA) tool (http://www.broad.mit.edu/gsea/, accessed September 2016) (21). Full detail can be found in the supplementary information. Pathway analysis was performed on subsets of the expression data set, as described in the text, by using the Reactome pathway database at www.webgestalt.org. The results were corrected for multiple testing with the Benjamini-Hochberg method. Luciferase assay Luciferase assay was performed as described previously (7). A brief description can be found in the supplementary information. siRNA transfection and functional experiments EndoC-βH1 cells (passage 72-78) were seeded on multi-well plates