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)

Introduction

Objectives

Methods

Results and Disscussion

Conclusion

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

2011 = 366.2 to 2030 = 551.8Increase 51%

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.

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

Hyperglycemia

Hypoglycemia

Source URL: http://www.scienceinschool.org/2006/issue1/diabetes

Glucose

Insulin

Glucose uptake to muscle and fat cells is dependent upon

activation of GLUT4 by insulin

Insulin resistance combined

with reduced insulin secretion

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.

When the IR binds insulin,

the activated phosphorylatesthe IRS-1 protein

Source : Pearson Education Inc., 2012

IRS-1 activates PI-3-kinase, which

catalyzes the addition of phosphate

group to the membrane lipid PIP2,

thereby converting it to PIP3

PIP3 binds a protein calledAkt, which is activated by

other protein kinases

Akt catalyzes phosphorylation of key

proteins, leading to an increase in

glycogen synthase activity and

recruitment of the GLUT4 to membrane

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

not feasible and

too expensive

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

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

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

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

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)

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.

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.

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.

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

10mg/ml

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

CE treatment

CP prepared on 2

different concentrations

(µg/ml DMSO)

1 and 10

Treatment held on 3 hours

CP treatment

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

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

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

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)

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

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

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

Effect of cinnamon extract on the mRNA levels of TTP in

3T3-L1 adipocytes

TTP mRNA levels were

rapidly increased by CE

treatment

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

Effect of cinnamon extract on the mRNA levels of GLUT4 in

3T3-L1 adipocytes

GLUT4 mRNA levels were

not signiWcantly affected

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

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

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