Journal Club

1. Journal Club Norton L, Fourcaudot M, Abdul-Ghani MA, Winnier D, Mehta FF, Jenkinson CP, Defronzo RA. Chromatin occupancy of transcription factor 7-like 2 (TCF7L2) and its role in hepatic glucose metabolism. Diabetologia. 2011 Sep 7. [Epub ahead of print] Reis JP, Loria CM, Sorlie PD, Park Y, Hollenbeck A, Schatzkin A. Lifestyle factors and risk for new-onset diabetes: a population-based cohort study. Ann Intern Med. 2011 Sep 6;155(5):292-9. 埼玉医科大学　総合医療センター　内分泌・糖尿病内科 Department of Endocrinology and Diabetes, Saitama Medical Center, Saitama Medical University 松田　昌文 Matsuda, Masafumi 2011年9月15日8:30-8:55 ８階　医局

2. Age Incretin Effect Beta CellFailure Lipotoxicity FFA 膵β細胞機能不全の病因 Genetics (TCF 7L2) Amyloid (IAPP) Deposition Insulin Resistance GlucoseToxicity

3. T2DMCandidate Polymorphisms • IGF2BP2　インスリンの作用を調整していると考えられているインスリン様成長因子2に関係する • CDKAL1β細胞に作用するタンパク質 • CDKN2AとCDKN2B β 細胞の成長に関与するタンパク質、ガンの成長でも研究されていた遺伝子 • TCF7L2β細胞の機能障害　インクレチンシグナル障害 • SCL30A8 β細胞だけで発現する亜鉛輸送体遺伝子 • KCNJ11　新生児糖尿病に関与 • HHEX • PPARα　脂肪酸化障害 • PPARγ • FTO　肥満 • GCKR中性脂肪を調節 • WFS1　インクレチンシグナル障害 • SLC30A8 • KCNQ1 • KCNJ15 • UBE2E2 • C2CD4A-C2CD4B

4. Transcription factor 7-like 2 (T-cell specific, HMG-box) also known as TCF7L2 or TCF4 is a protein acting as a transcription factor. In humans this protein is encoded by the TCF7L2 gene. A variant of the protein is linked to higher risk to develop type 2 diabetes. Function TCF7L2 is a transcription factor influencing the transcription of several genes thereby exerting a large variety of functions within the cell. It is a member of the Wnt signaling pathway. Stimulation of the pathway leads to the association of β-catenin with BCL9, translocation to the nucleus, and association with TCF7L2, which in turn results in the activation of Wnt target genes, specifically repressing proglucagon synthesis in enteroendocrine cells.

5. Diabetes Division, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA

6. Aims/hypothesis The mechanisms by which transcription factor 7-like 2 (TCF7L2) regulates the pathways that are important in the pathogenesis of type 2 diabetes are unknown. We therefore examined the role of TCF7L2 in hepatic glucose production (HGP) in vitro and characterised the whole genome chromatin occupancy of TCF7L2 in hepatocytes.

7. Methods We investigated the effect of TCF7L2 silencing and overexpression on HGP from gluconeogenic precursors and used chromatin-immunoprecipitation (ChIP) combined with massively parallel DNA sequencing (ChIP-Seq) to investigate the DNAbindingpatterns of TCF7L2 across the whole genome.

8. Abbreviations ChIP Chromatin-immunoprecipitation ChIP-SeqChIP combined with massively parallel DNA sequencing FDR False discovery rate GAPDH Glyceraldehyde-3-phosphate dehydrogenase HGP Hepatic glucose production HNF4α Hepatocyte nuclear factor 4α IC50 Half-maximal inhibitory concentration IPA Ingenuity Pathway Analysis pAKT Phosphorylated AKT PDC Pyruvate dehydrogenase complex PDK4 Pyruvate dehydrogenase kinase 4 siRNA Small interfering RNA TCF7L2 Transcription factor 7-like 2 TSS Transcriptional start site WNT Morphogenic wingless-type MMTV integration site family

9. Cell culture The rat hepatoma cell line, H4IIE, was used in the present study and has been described in detail elsewhere. H4IIE cells are highly differentiated, display a greater signal/noise ratio for HGP than other hepatic cell lines (i.e. HepG2, HuH6) and have been used extensively for the study of HGP in vitro. Low passage cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and routinely cultured in DMEM supplemented with 4.5 mmol/l glucose and 10% (vol./vol.) FBS. TCF7L2 silencing and glucose production assay We silenced TCF7L2 in H4IIE cells using the Neon Transfection System (Invitrogen, Carlsbad, CA, USA). Electroporated cells were allowed to grow for 24 h before the medium was replaced with serum-, Phenol Red- and glucose-free DMEM supplemented with 20 mmol/l D-lactate and 2 mmol/l sodium pyruvate. This medium was left on the cells for 10 h before being replaced with identical medium, with or without additives (insulin and metformin), and left overnight for the glucose production assay. The medium was then assayed for glucose using a kit (Amplex Red Glucose Assay; Invitrogen). Western blotting After treatment, H4IIE cells were washed in ice-cold PBS and cellular protein was collected by scraping the cells into 50 μl protein extraction buffer, after which western blotting was carried out as described.

10. Quantitative RT-PCR analysis Cells in 24-well plates were immediately washed with ice-cold PBS following treatment and lysed with 500 μl Trizol reagent (Invitrogen). Total RNA was extracted, cDNA prepared and real-time PCR carried out as previously described. ChIP-SeqChIPDNA was prepared using a kit (number 9003; Enzymatic SimpleChIP Magnetic Kit; Cell Signaling) and anti-TCF7L2 antibody (number 2569; Cell Signaling). This antibody was designed against a peptide sequence surrounding Leu330 of the TCF7L2 protein. This residue is at the 3′ end of exon 9 and represents a region of conservation in all Tcf7l2 mRNA transcripts [20, 21]. Sequencing libraries were prepared from 30 ngChIP DNA using a kit (ChIP-Seq Library Preparation; Illumina, San Diego, CA, USA). The TCF7L2 library was prepared from DNA obtained from replicate ChIP reactions (n=4). ChIP-Seq data analysis: identification of TCF7L2-enriched regions Analysis of ChIP-Seq data was performed with CLC GenomicsWorkbench version 4.5.1 (Cambridge, MA, USA). Raw 40 bp reads for the input control (29,226,566 reads) and TCF7L2 ChIP reaction (30,688,073 reads) were first mapped to the repeat-masked Rat Reference (RN4) Genome (UCSC Genome Browser, http://genome.ucsc.edu/, accessed 28 September 2010). ChIP-Seq peak detection was then carried out using a window size of 250 bp and a false discovery rate (FDR) of 1%. Motif analysis We searched for occurrence of the canonical (T/A–T/A–C–A–A–A–G) or evolutionarily conserved (A–C/G–T/A–T–C–A–A–A–G) TCF7L2 binding motif in each peak (±400 bp flanking DNA).

13. Fig. 3 ChIP of TCF7L2 in H4IIE cells and ChIP-Seq library preparation. a The WNT target gene Axin2 was used as a positive control region for the initial ChIP assay and was significantly bound by TCF7L2 in the 5′ promoter region. Glut4, also known as Slc2a4, is hepatic-silent and was used as a negative control region in the ChIP assay. Black bars, TCF7L2 ChIP; white bars, IgGChIP. b A figure from our CLC Genomics Workbench analysis shows a ChIP-Seq peak corresponding to the same site used as the positive control in the ChIP assay; it validates the strong binding of the Axin2 gene by TCF7L2 and confirms the specificity of our antibody in the ChIP-Seq experiments. The ChIP-Seq peak and flanking region are shown and the ChIP assay positive control PCR product is highlighted (bold and underlined). Red text in boxes indicates putative TCF7L2 binding sites. c Final IlluminaChIP-Seq libraries were approximately 200 to 250 bp in length, as shown by the Bioanalyzer gel image (Agilent Technologies, Santa Clara, CA, USA)

18. Results Silencing of TCF7L2 induced a marked increase in basal HGP, which was accompanied by significant increases in the expression of the gluconeogenic genes Fbp1, Pck1 and G6pc. Overexpression of Tcf7l2 reversed this phenotype and significantly reduced HGP. TCF7L2 silencing did not affect the half-maximal inhibitory concentration of insulin or metformin, but HGP remained elevated in TCF7L2-silenced cells due to the increased baseline HGP. Using ChIP-Seq, we detected 2,119 binding events across the genome. Pathway analysis demonstrated that diabetes genes were significantly over-represented in the dataset. Our results indicate that TCF7L2 binds directly to multiple genes that are important in regulation of glucose metabolism in the liver, including Pck1, Fbp1, Irs1, Irs2, Akt2, Adipor1, Pdk4 and Cpt1a.

19. Conclusions/interpretation TCF7L2 is an important regulator of HGP in vitro and binds directly to genes that are important in pathways of glucose metabolism in the liver. These data highlight the possibility that TCF7L2 may affect fasting and postprandial hyperglycaemia in carriers of at-risk TCF7L2 genetic polymorphisms.

20. Message/Comments TCF7L2は２型糖尿病の原因遺伝子としてチャンピオンのようなものだが機序は不明。 肝臓と膵β細胞に関連してインクレチンとも関連しているというが．．． この論文では肝臓に直接作用している可能性を示唆した。

22. the National Heart, Lung, and Blood Institute, Bethesda, and National Cancer Institute, Rockville, Maryland, and AARP, Washington, DC. Ann Intern Med. 2011;155:292-299.

23. Background: Epidemiologic data on the combined influence of several lifestyle factors on diabetes risk are rare, particularly among older adults. Objective: To examine how combinations of lifestyle risk factors relate to the 11-year risk for incident diabetes.

24. Design: Population-based prospective cohort study. Setting: National Institutes of Health (NIH)–AARP Diet and Health Study. Participants: 114 996 men and 92 483 women, aged 50 to 71 years in 1995 to 1996, without evidence of heart disease, cancer, or diabetes. Measurements: A comprehensive survey of demographic characteristics and lifestyle factors, including dietary intake, body weight and height, physical activity, smoking, and alcohol consumption at baseline (1995 to 1996). Low-risk groups were formed by dichotomizing each lifestyle factor. Incident self-reported, physician diagnosed diabetes was identified with a follow-up survey in 2004 to 2006.

25. Figure. Study flow diagram.

30. Figure. Adjusted odds ratios for new-onset diabetes, by number of lifestyle factors in the low-risk category and stratified by sex.

31. Figure. Adjusted odds ratios for new-onset diabetes, by number of lifestyle factors in the low-risk category and stratified by sex.

32. Table. Comparison of Included and Excluded Participants Without Prevalent Heart Disease, Cancer, or Diabetes at Baseline

33. Table. Adjusted ORs for New-Onset Diabetes, by Number of Lifestyle Factors in the Low-Risk Category, Sex, and Family History of Diabetes

34. Results: 11 031 men (9.6%) and 6969 women (7.5%) developed new-onset diabetes. For each additional lifestyle factor in the low risk group, the odds for diabetes were 31% lower (odds ratio [OR], 0.69 [95% CI, 0.68 to 0.71]) among men and 39% lower (OR, 0.61 [CI, 0.60 to 0.63]) among women. Men and women whose diet score, physical activity level, smoking status, and alcohol use were all in the low-risk group had ORs for diabetes of 0.61 (CI, 0.56 to 0.66) and 0.43 (CI, 0.34 to 0.55), respectively. When absence of overweight or obesity was added, the respective ORs were 0.28 (CI, 0.23 to 0.34) and 0.16 (CI, 0.10 to 0.24) for men and women. Results did not differ by family history of diabetes or level of adiposity.

35. Limitation: The study was observational, with potential for residual confounding. Conclusion: Lifestyle factors, when considered in combination, are associated with a substantial reduction in risk for diabetes. Primary Funding Source: The NIH-AARP Diet and Health Study was supported by the Intramural Research Program of the NIH.

36. Message/Comments 　癌や糖尿病などのない50-71歳の男性11万4996人・女性9万2483人を対象に、生活要因と糖尿病11年リスクの関連を集団ベースのコホート研究で調査。食生活、運動、喫煙、飲酒の面で低リスク群の糖尿病オッズ比は男女で0.61・0.43、さらに肥満や過体重がないと0.28・0.16で、生活要因の組み合わせと糖尿病リスク抑制が関連した。