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Gene regulation
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Insulin
• Insulin affects the expression of many more
genes (>200)
• Insulin affects transcription, mRNA stability, and
translation (from gene to protein)
• Focusing on insulin-regulated gene transcription
– Positive and negative effects on the transcription of
specific genes even within the same cell.
– Insulin-regulated genes  coding proteins involved in
a variety of biologic phenomena (F25.1)
– liver, muscle, adipose tissue >> other tissue
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F 25.1 Insulin regulated genes
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Cis/trans model for insulin
regulated gene transcription
• Transcription is mediated by the
interaction of cis-acting DNA elements with
trans-acting factors.
• Specific sequence of the cis-acting
element determines which trans-acting
factor will bind.
=> These cis-acting elements are referred to
as insulin response sequences or
elements (IRSs/IREs)
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• Impaired insulin-regulated gene expression
decreased
: peripheral glucose utilization
: rate of insulin secretion
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Specific defects in gene expression
in the etiology of in MODY
•
•
•
•
MODY-1: Hepatocyte nuclear factor (HNF)-4
MODY-3: HNF-1α
MODY-4: IPF-1
MODY-5: HNF-1β
=> Complex : these genes encodes a
transcription factor that could regulate the
expression of multiple islet genes.
• MODY-2: glucokinase
=> a key element in glucose sensing by the Betacell
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How a signal passes from the insulin receptor in
the plasma membrane to a specific trans-acting
factor binding to an IRS ?
* Insulin actions on gene transcription by two ways
A) through plasma membrane-initiated changes in
nuclear protein phosphorylation
/dephosphorylation
B) more directly via intracellular insulin receptors.
• Recent reports suggest
– cell surface receptors translocate to the nucleus.
– Insulin receptor substrate-1 and 3 in the nucleus.
– PI3-kinase/PKB signaling system in the nucleus.
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Identification of positive insulin
response sequences
1.
2.
3.
4.
5.
The serum response element
The Ets motif
The Activator protein-1 motif
The Sp1 Motif
The Sterol response element and the E-Box
motif
6. The Thyroid Transcription Factor-2 Motif
7. Miscellaneous
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The serum response element
• C-fos SRE
– C-fos gene is cellular homologue of the transforming
gene of FBJ murine osteosarcoma virus
– Insulin stimulates c-fos gene transcription in cell
system
– -356~+109 of c-fos promoter sequence mediates the
insulin response
• SRES in the β-actin and GLUT-1
– Mediate positive effects of insulin
=> SRE mediates effects of several hormones on
gene transcription rather than insulin specific
effect
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Signal transmission from insulin receptor in
plasma membrane to c-fos IRS in nucleus
• Insulin  Insulin Rc. in plasma
membrane  Shc (thyrosinephosphorylated IRS family
member) binds GRB-2
(adaptor molecule)  mSOS
(guanine nucleotide exchange
factor)  p21ras 형성을 촉진 
raf-1 (serine /threonine kinase)
활성화  MEK1/2 활성화 
Erk1/2  p90RSK  cFOS
(p211 참조)
RSK
cFos
• Thus, ERK1/2 kinase pathway involves in insulin-stimulated c-fos
gene transcription
– MEK1/2 inhibitor (PD98059) blocks insulin-stimulated c-fos
expression
=> However, target of insulin signaling at the c-fos remains unclear
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SRE binding proteins
•
•
p67SRF, C/EBP-β, Phox 1, DBF/MAPF1
ERK1/2 kinase pathway 는 p62TCF 를 target으로 하며, 인슐린에 의해 활성화
된 다른 경로는 p67SRF을 Target 으로 한다.
=> insulin can regulate c-fos gene transcription either directly through the SRE
or through the 5’ Ets motif.
5’
Ets motif
p62TCF
Erk1/2
C-fos SRE
p67SRF
Other pathway
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The Ets Motif
• 인슐린에 의한 gene expression 이 Ets motif를 통해서 일
어남은 prolactin gene 에서 처음 보고되었다 (in the GH3
rat pituitary cell line)
– Prolactin IRS was mapped to a region of the rat promoter
between -212~+73
– Primary insulin response sequence: -97~-87
– For the full insulin response: -76~-67 also required
• Prolactin IRS is composed of the dyad repeat sequence
CGGAA(A/G)(T/A)(A/G)CAT with a 10-bp intervening
sequence.
– CGGAA is critical sequence
• Ets motif is important for insulin-induced somatostatin
and thymidine kinase gene transcription
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• Prolactin IRS에 binding 하는 Ets family :
GABP-α, GABP-β1, Ets-1, Elk-1, CREbinding protein (CREB)
• Pit-1 (pituitary specific factor)
– when Pit-1 binding sites are mutated, the
stimulatory effect of insulin on prolactin gene
transcription is reduced
– Deletion of Pit-1 binding sites dramatically
reduces basal prolactin gene transcription
– accessory factor vs. a direct target of insulin
signaling
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PI 3-kinase pathway: most important
for metabolic action of insulin
• PI 3-kinase pathway가 Gene transcription 을 포함한
insulin action에 있어서 가장 중요한 것으로 알려져 있다.
• PI-3kinase는 110kd catalytic subunit와 85-kd의
regulatory subunit로 구성되어 있으며, p85 subunit의 두
개의 SH2 domain 이 IRS-1과 상호 작용을 하고, 결과적
으로 p110 을 활성화 시킴.
• Wortmannin, a specific inhibitor of PI 3-kinase, blocks
many of insulin’s actions in various cell types.
=> PI 3-kinase is required for the insulin mediated prolactin
gene transcription.
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The Activator Protein-1 Motif
• AP-1 motifs are important for the gene
expression by insulin;
1. Matrix metalloproteinases (MMPs).
– First documented
– Accessory factor가 필요: Ets motif
2. malic enzyme
3. hepatitis B virus X gene
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Action of insulin through the AP-1
motif at two levels (F25.2)
=> Insulin has both short- and long-term effects
on gene expression through the same element. 18
• AP-1을 매개로 한 인슐린의 효과는
ERK1/2 kinase pathway를 필요로 한다
• But it depends on cell type
– In CHO. T cell: ERK1/2 kinase activation is
sufficient
– In adipocytes: FRAP/mTOR pathway
activation is also required
• 최근에 Maf 와 activating transcription
factor (ATF) 도 AP-1 motif에 결합하여 작
용을 나타냄을 보고.
– target gene은 아직 밝혀지지 않음
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The Sp1 Motif
• Insulin-regulated calmodulin gene transcription
을 통해 처음 밝혀짐
• Delta1-crystallin, PAI-1, apolipoprotein A1 are
stimulated by insulin through Sp1
• Interesting issues regarding Sp1
1. Sp1 binds the promoters of many genes, (not all)
which are regulated by insulin.
2. Sp1 may oppose the action of insulin
3. Sp1 can also mediate effects of glucose on gene
expression.
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• Sp1 belong to highly related family members:
Sp2, Sp3, Sp4 (may be much larger)
• Mechanism of Sp1 in insulin action: complex
– Sp1 is phosphorylated by multiple protein kinases,
known to be insulin regulated.
– Insulin can also regulate Sp1 gene expression.
• Insulin induces Egr-1 gene expression
– Sp1과 Egr-1은 비슷한 DNA sequence를 인지 함으로
DNA binding 에 있어서 경쟁을 할 수 있고 이러한 점
때문에 인슐린이 복잡한 효과를 나타냄.
– 예) malic enzyme promoter에서 Sp1은 인슐린
signaling 을 촉진 하지만, Egr-1은 이를 억제함
• 또한 Sp1 은 인슐린에 의해 조절되는 GAPDH
gene의 발현에 관여하는 것으로 보고됨
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The Sterol Response Element and
the E-box Motif
• Unusual transcription factor
– SREBPs can bind two motifs: sterol response element and the
E-box motif.
• Released from the Endoplasmic reticulum
• SREBP-1a, SREBP-1c, SREBP-2
– SREBP-1 activates genes required for fatty acid synthesis
– SREBP-2 activates genes required for cholesterol synthesis
• Insulin preferentially induces SREBP-1c, but , through
the stimulation of the ERK1/2 kinase pathway, might
activate both SREBP-1 and SREBP-2
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• Insulin은 SREBP-1c 를 통하여 직접적으로 fatty acid
biosynthesis 에 관여하는 multiple hepatic genes 의 발현
에 관여한다고 알려져 있지만, 실제적으로 glucose 와
insulin 이 둘 다 높을 때 이러한 gene들이 발현함.
=> Glucose rather than insulin was the key signaling
molecule in hepatic gene expression
• Kahn demonstrated that glucose was sufficient for the
induction of pyruvate kinase gene expression in primary
hepatocytes.
=> insulin mainly play a permissive role in the regulation of
hepatic gene expression through
– stimulating glucokinase gene transcription
– promoting glucose phosphorylation.
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fatty acid synthase (FAS)
• FAS: central role in de novo lipogenesis in
mammal.
• Insulin increases FAS gene transcription in
the livers of diabetic mice.
• IRS of FAS is located in a region between
-332 and +67 in the FAS promoter.
• E-box motif mediates the effect of insulin
through Fas.
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Relative roles of glucose and insulin in the
regulation of FAS gene expression
1. Insulin/glucose → through the proximal region of the
FAS promoter → 2 to 3-fold stimulation of FAS gene
expression
• fasting/refeeding → 20-30 fold induction of the
endogenous FAS gene
=> unidentified upstream FAS promoter region must be
important for the fasting/refeeding response.
2. SREBP-1c is not a direct target of glucose signaling itself
and other transcription factors mediate the action of
glucose on FAS gene expression.
• Full induction of FAS gene expression requires
1) glucose action, mediated through a carbohydrate-responsive
factor
2) insulin action, mediated through SREBP-1c
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Upstream stimulatory factor (USF)
• Insulin-regulated FAS gene transcription
involves another transcription factor, USF
• Sul demonstrated that E-box motif (-68 to -52
FAS promoter region) mediates the effect of
insulin (in tissue culture cells)
– Selective mutations in the SRE (E-box intact) had no
effect on the insulin response
– Instead, the binding of USF1/2 to E-box correlated
with the effect of insulin on FAS gene transcription (in
3T3-L1 adipocytes)
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• In contrast, Osborne demonstrated that
mutation of the SRE (intact E-box motif)
blunted insulin-stimulated FAS gene
expression
=> Conflicting data !
• Possible solution
=> SREBPs have a dual binding specificity
for both SREs and E-box motifs in their
DNA binding domains .
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The Thyroid Transcription Factor-2
Motif (TTF-2)
• Thyroglobulin (Tg), the macromolecular precursor of
thyroid hormone, is the major protein synthesized by
thyroid cells.
• IGF-1 과 insulin 은 이러한 Tg gene transcription 을 자극
하며, 이러한 효과는 Tg promoter 의 -168~+39 region에
의해 매개
• 이 region 내에 3개의 transcription factor binding sites가
존재: TTF-1, TTF-2, ubiquitous factor (UFA)
• Insulin & IGF-1: dramatically stimulate binding of TTF-2
• TTF-2 gene transcription 은 thyroperoxidase expression
(enzyme involved in the synthesis of thyroid hormone) 에
도 필요 (TTF-2만으로는 부족하고, TTF-1과 ubiquitous
factor (UFB) 도 있어야 한다).
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UFB (nuclear factor-1)
1. Insulin also stimulates NF-1 expression in thyroid cells
=> indicating that insulin regulates thyroperoxidase gene
expression through a combination of two transcription
factors, TTF-2 and NF-1.
2. Insulin also stimulates expression of an NF-1 in liver
cells.
3. 그런데, NF-1 이 지방세포에서 인슐린의 GLUT-4 의 발현
에 대한 negative effect를 매개
* NF-1 의 역할은 아직 명확하지 않아 IRS로 구분하지는 않
음
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Miscellaneous
Amy-2.1 and Amy-2.2
• IRS in the amylase promoter
• Pancreas-specific
• Amy-2.2 is stimulated by insulin and
carbohydrate
• Amy-2.1 is not responsive to either
• 당뇨 쥐에서 amylase promoter의 30-bp
fragment (from -167~-138) 의 activity 가 정
상 쥐보다 훨씬 떨어짐을 발견=> 이 부위가
IRS
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Identification of Negative insulin
response sequences
• The Phosphoenolpyruvate Carboxykinase-like Motif
–
–
–
–
Phosphoenolpyurvate Carboxykinase
IGF binding protein-1
Tyrosine Aminotransferase
Glucose-6-phosphatase catalytic subunit
• Apolipoprotein CIII
• Candidates for the insulin response factor
– Models of insulin response Factor Action
– Mechanisms of insulin signaling through the insulin response
Factor
• Miscellaneous
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