Cinnamon Extract And Polyphenols Affect The Expression Of Tristetraprolin, Insulin Receptor, And Glucose Transporter 4 In Mouse 3T3-L1

Adipocytes

Advisor : Tzou Chi Huang

Speaker :

Adelya Desi K. (M10118043)

December, 27th 2012

DEPARTMENT BIOLOGICAL SCIENCE AND TECHNOLOGY NATIONAL PINGTUNG UNIVERSITY SCIENCE AND TECHNOLOGY

Introduction
Objectives Methods Results and Disscussion Conclusion

North America 2011 = 37.7 2030 = 51.2

Europe 2011 = 52.6 2030 = 64.0

World 2011 = 366.2 to 2030 = 551.8 Increase 51%

Africa 2011 = 14.7 2030 = 28.0

South-East Asia 2011 = 71.4 2030 = 120.9

International Diabetes Federation, IDF Regions and Global Projection of the Number of People with Diabetes (20-79 years), 2011 and Prediction 2030 (in millions)

Chronic disease that occurs when the pancreas does not produce enough insulin, or when the body cannot effectively use the insulin

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.

Fasting Glucose

Glucose Tolerance

peptide hormone, produced by beta cells of the human pancreas

Source : The National Institutes of Health resource for stem cell research, 2001.

it is response by presence of glucose in bloodstream

Hyperglycemia

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

Insulin
Glucose

Cells

Insulin resistance combined with reduced insulin secretion

Glucose uptake to muscle and fat cells is dependent upon activation of GLUT4 by insulin

1988

The 1st evidence for this glucose transport protein was provided by Davis James 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 phosphorylates the 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

Source : Pearson Education Inc., 2012

PIP3 binds a protein called Akt, which is activated by other protein kinases
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
Source : Pearson Education Inc., 2012

Number of death attribute to diabetes in 2011

Total healthcare expenditures due to diabetes in USD (billions) USD 1,274 is spent on not feasible and diabetes care per person too expensive with diabetes in 2011

4.6 million deaths 60% due to > diabetes in 2011

Source : The IDF Diabetes Atlas 5th Edition

Extracted Cinnamomum the cinnamon burmannii samples have insulin-mimetic with acetic acid and function ethanol and isolated fractions with insulin-enhancing activity using HPLC Heping Cao, 2001 Anderson et. al, 1988

Identified Isolation and polyphenolic characterization polymers of polyphenol that increase type-A glucose metabolism polymers from roughly cinnamon 20-fold with in vitro insulin-like in the epididymal biological fat cell activity assay Polansky Anderson et. al, 2004 et. al, 1998

Kingdom:

Plantae

Cinnamomum burmannii , also Subkingdom: Tracheobionta known as Indonesian Cinnamon, Superdivision: Padang Cassia, or Spermatophyta Korintje, is one of several plants in the genus Division: Magnoliophyta Cinnamomum whose bark is sold as Class: Magnoliopsida the spice cinnamon. The most common and cheapest type of Subclass: Magnoliidae cinnamon in the US is made from Order: Laurales powdered Cinnamomum burmannii Family: Lauraceae
Genus:
Species:

Cinnamomum
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

Resulting in reduced insulin action (insulin resistance)

Increased phosphorylation of IR or IRS proteins on discrete serine decreases the extent of insulin-stimulated tyrosine phosphorylation

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.

Cinnamon extract (CE), like insulin, stimulates TTP gene expression in mouse adipocytes.

TTP binding AUrich element (ARE) removal of the poly(A) tails

degradation of the RNA bodies
mRNA stable cytokine expression

No transcription

 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 (IR), glucose transporter 4 (GLUT4) and Tristetraprolin (TTP) in mouse 3T3-L1 adipocytes.

Ground cinnamon extracted with absolute ethanol

Cinnamon Extract (CE)

Purified by HPLC 8 fractions of Cinnamon Polyphenols (CP) CP1A, CP1B, CP2, CP3, CP4, CP5, CP6, and CP7.

100mg/ml DMSO

reconstituted at DMSO before being added to the culture medium

10mg/ml DMSO

CE treatment 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

CP treatment CP prepared on 2 different concentrations (g/ml DMSO) 1 and 10 Treatment held on 3 hours

mouse cells 3T3-L1 fibroblas cell culture Cell extraction supernatant DMEM (+) medium added with the CE or CP

PCR primers and TaqMan probes

Protein concentration determination

RNA isolation cDNA synthesis

Western blotting RT PCR analysis

Effect of Cinnamon Extract on The Protein Levels of Insulin Receptor

A low concentration of CE (0.01mg/ml) did not significantly affect the amount of IR

high concentration of CE (1mg/ml) resulted in significant reductions of IR 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

0.1% DMSO is control

IR levels in the mouse 3T3L1 adipocytes were generally increased by most of the CP treatments for 3h

CP is strongly support the insulin signaling pathway by

increasing the amount of IR

Cinnamon Extract (CE) Crude extract

Cinnamon Polyphenols (CP) Purified by HPLC from CE

The reduction of IR level in high concentrations of CE treatment may suggest that the crude extract still contains

minor inhibitory compounds

Effect Of Cinnamon Polyphenols On The Protein Levels Of Insulin Receptor

0.1% DMSO is control

IR levels in the mouse 3T3L1 adipocytes were generally increased by most of the CP treatments for 3h

But, no significantly different activities between one fraction and another

CP is strongly support the insulin signaling pathway by

increasing the amount of IR

Effect of cinnamon extract and polyphenols on the protein levels of glucose transporter 4 (GLUT-4)
Immunoblotting showed that CP3 also increased GLUT4 accumulation in 3T3-L1 adipocytes after 3h treatment

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 protein levels of tristetraprolin (TTP)
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 Lane 1, DMSO control; lane 2, CE (10 g/ml); lane 3, CE (100 g/ml); lane 4, extract from the amount of TTP 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 68, CP3 (1, 10, and 100 g/ml, respectively). Lanes 13 (100 g of protein); lane 4 (40 g of protein); and lanes 58 (80 g of protein).

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 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 an anti-diabetes

in part due to increases in the amounts of TTP, that CP may have additional roles as anti-inflammatory

CP treatment showed increased the level of IR and GLUT4 in adipocytes

CP may increase the activity of TTP by decreasing its phosphorylation through inhibition of GSK3 activity cytokines

the beneficial effects of cinnamon in improving the conditions of diabetic people by down-regulating the synthesis of pro-inflammatory cytokines