Author Correction: NT5C2 methylation regulatory interplay between DNMT1 and insulin receptor in type 2 diabetes

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Article contained a reference to a circus plot, which was removed during revision of the manuscript prior to publication. In the corrected Results, in subsection 'DNA promoter hypermethylation and gene expression in T2D mice' the following sentence is now removed: "We also performed a gene expression array result circus plot of KK-Ay mice with T2D and control mice using RNA samples and GeneChip Mouse Exon 1.0 ST arrays. " Data supporting the statement that FUT8 mRNA levels are not significantly different between t2D and healthy controls, was not included. This is now added as Supplementary Figure S2 in the accompanying Supplementary information files, and included in the Results section, in subsection 'NT5C2 and FUT8 mRNA expression from peripheral blood of T2D patients' , where.
"However, FUT8 mRNA levels in T2D were showed no significant difference with healthy controls. " now reads: "However, FUT8 mRNA levels in T2D were showed no significant difference with healthy controls ( Figure S2). " The study uses a BMI of 24 as a normal weight/overweight threshold. The justification for this is now included in the Results, in subsection 'Effects of NT5C2 on BMI' where the following sentence was added in the first paragraph: "In Taiwan, BMI categories included underweight (BMI < 18.5 kg/m 2 ), normal weight (18.5 ≤ BMI < 24 kg/m 2 ), overweight (24 ≤ BMI < 27 kg/m 2 ), obesity (BMI ≥ 27 kg/m 2 ). " Data in the paper did not directly support the claim that NT5C2 is inhibited in mouse pancreatic β-cells specifically by DNMT1. Therefore, in the Results the title of section 'NT5C2 was inhibited in pancreatic β-cells in mice' .
In the Results, in subsection 'NT5C2 affected the insulin signaling pathway via DNMT1' the following sentence, which provides previously missing information, is now added, where "A plasmid containing a fragment of human DNMT1 or NT5C2 was constructed and transiently transfected into RIN-m5F cells. " now reads: "A plasmid containing human DNMT1 or NT5C2 was constructed and transiently transfected into RIN-m5F cells. " The information on how the sampling for methylation analysis was done, was missing. It is now included in the methods section together with previously missing reference to the article describing MAT-score calculation method, which is now cited as Ref 34. Therefore, in Methods and materials, subsection 'DNA methylation microarray analyses for human T2D and control' . "NT5C2 is expressed in skeletal muscle from tissue expression study 25,26,27 . NT5C2 in human myotubes, increases AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase phosphorylation, and promotes lipid oxidation and glucose transport. The NT5C2 and AMPK activity in T2D and obesity may play an important role in the regulation of insulin action and lipid metabolism in skeletal muscle 28 .
NT5C2 is present in the brain, heart, skeletal muscle, erythrocytes, spleen, testis, fibroblasts, and endothelial cells 29 . We checked the NT5C2 protein expression of the brain, heart, muscle, spleen, and testis by immunohistochemistry staining. Since the NT5C2 were low activity presentations in these tissues, there was no significant difference between KK and KK-Ay mice (data not showed). KK-Ay mice with the lethal yellow obese (A y ) mutation and develop diabetes of polygenic origin, showing severe obesity, hypertriglyceridemia, hyperglycemia, hyper-insulinemia, and insulin function loss by 42 weeks of age 18,21,30,31 . In our results, pancreatic β-cell mass was proliferation, and NT5C2 protein expression was inhibited in T2D mice's pancreatic β-cell. Our previously results showed DNMT1 was significantly overexpression in T2D mice's pancreatic β-cells 20,21 . Previously study have showed the importance of DNA methylation in pancreatic islet function 10 . In these studies, researchers identified the promoters of the insulin gene (INS) 32 ; pancreatic and duodenal homeobox 1 (PDX1) 33 , which encodes a transcription factor important for both pancreatic development 34 and the function of mature β-cells 35 ; PPARG coactivator 1 alpha (PPARGC1A) 36 ; and glucagon-like peptide 1 receptor (GLP1R) 37 , which stimulates insulin secretion and protects β-cell proliferation, as hypermethylated in islets from donors with T2D compared with those in islets from nondiabetic donors 38 . A hypermethylation was associated with reduced mRNA expression of the respective gene in pancreatic islets and with higher glycated HbA1c, suggesting the role in β-cell disturbs in T2D. High levels of glucose were also found to directly increase DNA methylation of Pdx1 and Ins in clonal β-cells 32,33 . " now reads: "NT5C2 is expressed in skeletal muscle from tissue expression study 23 . NT5C2 in human myotubes, increases AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase phosphorylation, and promotes lipid oxidation and glucose transport. The NT5C2 and AMPK activity in T2D and obesity may play an important role in the regulation of insulin action and lipid metabolism in skeletal muscle 23 . www.nature.com/scientificreports/ NT5C2 is present in the brain, heart, skeletal muscle, erythrocytes, spleen, testis, fibroblasts, and endothelial cells 23 . We checked the NT5C2 protein expression of the brain, heart, muscle, spleen, and testis by immunohistochemistry staining. Since the NT5C2 were low activity presentations in these tissues, there was no significant difference between KK and KK-Ay mice (data not showed). KK-Ay mice with the lethal yellow obese (A y ) mutation and develop diabetes of polygenic origin, showing severe obesity, hypertriglyceridemia, hyperglycemia, hyperinsulinemia, and insulin function loss by 42 weeks of age 18,21,25,26 . In our results, pancreatic β-cell mass was proliferation, and NT5C2 protein expression was inhibited in T2D mice's pancreatic β-cell. Our previously results showed DNMT1 was significantly overexpression in T2D mice's pancreatic β-cells 20,21 . Previously study have showed the importance of DNA methylation in pancreatic islet function 10 . In these studies, researchers identified the promoters of the insulin gene (INS) 27 ; pancreatic and duodenal homeobox 1 (PDX1) 28 , which encodes a transcription factor important for both pancreatic development 29 and the function of mature β-cells 30 ; PPARG coactivator 1 alpha (PPARGC1A) 31 ; and glucagon-like peptide 1 receptor (GLP1R) 32 , which stimulates insulin secretion and protects β-cell proliferation, as hypermethylated in islets from donors with T2D compared with those in islets from nondiabetic donors 33 . A hypermethylation was associated with reduced mRNA expression of the respective gene in pancreatic islets and with higher glycated HbA1c, suggesting the role in β-cell disturbs in T2D. High levels of glucose were also found to directly increase DNA methylation of Pdx1 and Ins in clonal β-cells 27,28 . " Additionally, there were referencing errors in the Methods and materials section, in subsection 'Subject participants' , where: "T2D subjects were enrolled from China Medical University Hospital (CMUH) 39 ". now reads: "T2D subjects were enrolled from China Medical University Hospital (CMUH) 35 ".
In subsection ' Animals' , "The experiment according to the criteria for the care and use of KK and KK-Ay mice laid out in the ''Guidebook for the Care and Use of Laboratory Animals'' 40 . " The experiment according to the criteria for the care and use of KK and KK-Ay mice laid out in the ''Guidebook for the Care and Use of Laboratory Animals'' 36 . " Lastly, in subsection 'Statistical analysis' , "Subgroup analyses were done by obesity level (BMI level) of subjects 41 . " now reads: "Subgroup analyses were done by obesity level (BMI level) of subjects 37 . " The numerical values shown in the Venn diagram in Figure 1 were incorrect. The original version of Figure 1 appears below as Figure 1.
The arrows in Figure 3 were not correctly aligned. The original version of Figure 3 appears below as Figure 2.
In the legend of the same Figure 3, the following description is added for clarity: "The brown color depicts NT5C2-positive cells in the pancreas. Representative images of anti-NT5C2 IHC on the pancreas, the NT5C2 expression was higher in KK mice than KK-Ay mice. The red arrowheads are pointing at NT5C2-positive cells. " The legend of Figure 5 was revised for accuracy, where "DNMT1 epigenetically regulate NT5C2 and insulin receptor. "   Finally, the Article did not include the list of 1091 differentially methylated genes identified in the study. These are listed in the new Supplementary Table 4, which now accompanies the Article.
All errors have now been corrected in the PDF and HTML versions of the Article, and in the Supplementary Information files that accompany the Article.
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