Download PowerPoint - 埼玉医科大学総合医療センター 内分泌・糖尿病内科

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.
2011年9月15日 8:30-8:55
８階 医局
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
膵β細胞機能不全の病因
Age
Incretin
Effect
Amyloid
(IAPP)
Deposition
Glucose
Toxicity
Genetics
(TCF 7L2)
Beta Cell
Failure
Insulin
Resistance
Lipotoxicity
FFA
T2DM Candidate Polymorphisms
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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
CDKAL1 β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
TCF7L2 β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
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.
Diabetes Division, University of Texas
Health Science Center,
7703 Floyd Curl Drive,
San Antonio, TX 78229, USA
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.
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
DNAbinding patterns of TCF7L2 across the
whole genome.
Abbreviations
ChIP Chromatin-immunoprecipitation
ChIP-Seq ChIP 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
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.
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-Seq ChIP DNA 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 ng ChIP 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).
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, IgG ChIP. 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 Illumina ChIP-Seq
libraries were approximately 200 to
250 bp in length, as shown by the
Bioanalyzer gel image (Agilent
Technologies, Santa Clara, CA, USA)
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 TCF7L2silenced 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.
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.
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。
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.
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.
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.
Figure. Study flow diagram.
Figure. Adjusted odds ratios for new-onset diabetes, by number of lifestyle
factors in the low-risk category and stratified by sex.
Figure. Adjusted odds ratios for new-onset diabetes, by number of lifestyle
factors in the low-risk category and stratified by sex.
Table. Comparison of Included and Excluded Participants Without Prevalent Heart
Disease, Cancer, or Diabetes at Baseline
Table. Adjusted ORs for New-Onset Diabetes, by Number of Lifestyle Factors in the
Low-Risk Category, Sex, and Family History of Diabetes
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.
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 NIHAARP Diet and Health Study was
supported by the Intramural Research
Program of the NIH.
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。