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7/30/2019 Presentation Seminar Terbaru
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Cinnamon Extract And Polyphenols
Affect The Expression Of
Tristetraprolin
,
Insulin Receptor, And Glucose Transporter 4
In Mouse 3T3
-
L1 Adipocytes
DEPARTMENT BIOLOGICAL SCIENCE AND TECHNOLOGY
NATIONAL PINGTUNG UNIVERSITY SCIENCE AND TECHNOLOGY
December, 27th 2012
Advisor : Tzou Chi Huang
Speaker :艾蝶
Adelya Desi K. (M10118043)
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Introduction
Objectives
Methods
Results and Disscussion
Conclusion
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International Diabetes Federation, IDF Regions and Global Projection of the Number
of People with Diabetes (20-79 years), 2011 and Prediction 2030 (in millions)
North America
2011 = 37.7
2030 = 51.2
Europe
2011 = 52.62030 = 64.0
Africa2011 = 14.7
2030 = 28.0South-East Asia
2011 = 71.4
2030 = 120.9
World
2011 = 366.2 to 2030 = 551.8Increase 51%
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Chronic disease that occurs when the pancreasdoes not produce enough insulin, or when the
body cannot effectively use the insulin
Fasting Glucose Glucose Tolerance
a person must not to eat for
12 to 14 hours before test
a standard dose of glucose
(75-gram glucose drink) is
ingested by mouth and
blood levels are checked 2
hours later.
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it is response by
presence of glucose in
bloodstreamSource : The National Institutes of Health resource for
stem cell research, 2001.
peptide hormone,
produced by beta cells
of the human pancreas
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Hyperglycemia
Hypoglycemia
Source URL: http://www.scienceinschool.org/2006/issue1/diabetes
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Glucose
Insulin
Cells
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Glucose uptake to muscle and fat cells is dependent upon
activation of GLUT4 by insulin
Insulin resistance combined
with reduced insulin secretion
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The 1st evidence for this glucose transport
protein was provided by Davis James1988
GLUT4 is the insulin regulated glucose
transporter found in adipose tissue, that is
responsible for insulin glucose transport into
the cell
Defects in GLUT4 activity have been
implicated in some forms of insulin resistance
or pre-type 2 diabetes.
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When the IR binds insulin,
the activated phosphorylatesthe IRS-1 protein
Source : Pearson Education Inc., 2012
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IRS-1 activates PI-3-kinase, which
catalyzes the addition of phosphate
group to the membrane lipid PIP2,
thereby converting it to PIP3
Source : Pearson Education Inc., 2012
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Source : Pearson Education Inc., 2012
PIP3 binds a protein calledAkt, which is activated by
other protein kinases
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Source : Pearson Education Inc., 2012
Akt catalyzes phosphorylation of key
proteins, leading to an increase in
glycogen synthase activity and
recruitment of the GLUT4 to membrane
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Total healthcare expenditures due todiabetes in USD (billions)
Number of death attribute todiabetes in 2011
USD 1,274 is spent on
diabetes care per person
with diabetes in 2011
Source : The IDF Diabetes Atlas 5th
Edition
> 60%4.6 million deaths
due to diabetes in
2011
not feasible and
too expensive
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Extracted the cinnamon samples with acetic acid and
ethanol and isolated fractions with insulin-enhancing
activity using HPLC
Anderson et. al, 1988
Isolation and characterization of polyphenol type-A
polymers from cinnamon with insulin-like biologicalactivity
Polansky et. al, 2004
Cinnamomum burmannii have insulin-mimetic function
Heping Cao, 2001
Identified polyphenolic polymers that increase glucose
metabolism roughly 20-fold in vitro in the epididymal fatcell assay
Anderson et. al, 1998
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Cinnamomum burmannii , also
known as Indonesian Cinnamon ,
Padang Cassia, or Korintje, is one of
several plants in the genusCinnamomum whose bark is sold as
the spice cinnamon. The most
common and cheapest type of
cinnamon in the US is made from
powdered Cinnam omum bur mannii
Kingdom: Plantae
Subkingdom: Tracheobionta
Superdivision: Spermatophyta
Division: Magnoliophyta
Class: Magnoliopsida
Subclass: Magnoliidae
Order: Laurales
Family: Lauraceae
Genus: Cinnamomum
Species: C. burmannii
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polymers that increase glucose metabolism roughly 20-fold
in vitro from an aqueous extract of commercial cinnamon
Spectral analysis indicated the presence of type A
procyanidin
these A-type procyanidin were shown to have insulin-enhancing
biological activity in an in vitro assay
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This compound have been shown to inhibit serine phosphorylation in the insulin-receptor domain and to activate
insulin receptor kinase, and to function as a mimetic for
insulin
catechin (1) ; epicatechin (2) ; procyanidins B2 (3) and B3 (4) ;
Procyanidins A1 (5) and A2 (6) ; Procyanidin C
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Increased phosphorylation of IR or IRS proteins
on discrete serine decreases the extent of
insulin-stimulated tyrosine phosphorylation
Resulting in reduced insulin
action (insulin resistance)
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Tristetraprolin is an anti-inflammatory protein that binds to the
unstable elements of mRNAs coding for inflammation-related
factors such as TNF-a, COX2, and some interleukins (ILs)
mRNAs, and decreases their stability.
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TTP binding AU-
rich element (ARE)
removal of the
poly(A) tails
degradation of the
RNA bodies
No transcription
mRNA stable
cytokine
expression
Cinnamon extract (CE), like insulin,
stimulates TTP gene expression in
mouse adipocytes.
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To understand the molecular basis of insulin-like
activity
To explore additional benefits of cinnamon
To investigate the effects of Cinnamon Extract (CE)
and Cinnamon Polyphenols (CP) on the regulation
of Insulin Receptor (IRβ), glucose transporter 4(GLUT4) and Tristetraprolin (TTP) in mouse 3T3-L1
adipocytes.
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Ground cinnamon
extracted withabsolute ethanol
Cinnamon
Extract (CE)
Purified by HPLC
8 fractions of Cinnamon
Polyphenols (CP)
CP1A, CP1B, CP2, CP3,
CP4, CP5, CP6, and CP7.
reconstituted at DMSO before being
added to the culture medium
100mg/ml
DMSO
10mg/ml
DMSO
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CE prepared on 3
different concentrations
(mg/ml DMSO)
0.01 ; 0.1 ; and 1
Treatment held on 5
different times (hours)0.5 ; 1 ; 1.5 ; 2 ; and
2.5
CE treatment
CP prepared on 2
different concentrations
(µg/ml DMSO)
1 and 10
Treatment held on 3 hours
CP treatment
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mouse cells 3T3-L1 fibroblas
cell cultureDMEM (+) medium
added with the CE
or CP Cell extraction
supernatant
Protein concentration
determination
Western blotting
RNA isolation
cDNA synthesis
PCR primers and
TaqMan probes
RT PCR analysis
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Effect of Cinnamon Extract on The Protein Levels
of Insulin Receptor β
A low concentration of CE (0.01mg/ml) did
not significantly affect the amount of IRβ
high concentration of CE (1mg/ml) resulted in significant
reductions of IRβ levels at all of the time points
analyzed, ranging from 30 min to 3h
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Effect Of Cinnamon Polyphenols On The Protein Levels Of
Insulin Receptor β
IR β levels in the mouse 3T3-L1 adipocytes were generally
increased by most of the CP
treatments for 3h
0.1% DMSO is
control
CP is strongly support the insulin signaling pathway by
increasing the amount of IR β
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The reduction of IR β level in high concentrations of CE
treatment may suggest that thecrude extract still containsminor inhibitory compounds
CinnamonExtract (CE)
Crude extract Purified by HPLC
from CE
CinnamonPolyphenols (CP)
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Effect Of Cinnamon Polyphenols On The Protein Levels Of
Insulin Receptor β
IR β levels in the mouse 3T3-L1 adipocytes were generally
increased by most of the CP
treatments for 3h
0.1% DMSO is
control
CP is strongly support the insulin signaling pathway by
increasing the amount of IR β
But, no significantly different activities between
one fraction and another
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increase
Lane 1, protein size standard, lane 2, DMSO control (1%); lane 3,
CE (10 g/ml); lane 4, CP3 (1 g/ml).
Effect of cinnamon extract and polyphenols on the proteinlevels of glucose transporter 4 (GLUT-4)
Immunoblotting showed that CP3
also increased GLUT4 accumulation
in 3T3-L1 adipocytes after 3htreatment
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Effect of cinnamon extract and polyphenols on the protein
levels of tristetraprolin (TTP)
Lane 1, DMSO control; lane 2, CE (10 g/ml); lane 3, CE (100 g/ml); lane 4, extract from
RAW264.7 cells treated with LPS (0.1 g/ml) for 2 h as a positive TTP control; lane 5,
DMSO control (1%); and lane 6 – 8, CP3 (1, 10, and 100 g/ml, respectively). Lanes 1 – 3 (100
g of protein); lane 4 (40 g of protein); and lanes 5 – 8 (80 g of protein).
The size of TTP induced by
CP in the adipocytes was
similar to that induced by
0.1 g/ml LPS in mouse
RAW264.7 cells
The purified CP3 at higher concentration also increased
the amount of TTP
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Effect of cinnamon extract on the mRNA levels of TTP in
3T3-L1 adipocytes
TTP mRNA levels were
rapidly increased by CE
treatment
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Effect of cinnamon extract on the mRNA levels of Insulin Receptor in 3T3-L1 adipocytes
IR β mRNA levels were
rapidly decreased by a higher
concentration of CE
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Effect of cinnamon extract on the mRNA levels of GLUT4 in
3T3-L1 adipocytes
GLUT4 mRNA levels were
not signiWcantly affected
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CP treatment showed
better result than CE as ananti-diabetes
CP treatment showed
increased the level of IR β and GLUT4 in adipocytes
in part due to increases in
the amounts of TTP, that
CP may have additional
roles as anti-inflammatory
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the beneficial effects of cinnamon in
improving the conditions of diabetic people by down-regulating the synthesis
of pro-inflammatory cytokines
CP may increase the activity
of TTP by decreasing its
phosphorylation throughinhibition of GSK3 activity
cytokines