Accelerated fat cell aging links oxidative

stress and insulin resistance in adipocytes

Finny Monickaraj, Sankaramoorthy Aravind, Pichamoorthy Nandhini,

Paramasivam Prabu, Chandrakumar Sathishkumar, Viswanathan Mohan and Muthuswamy Balasubramanyam

Natalia Perilla HernándezSalomé Rave Diosa

Third semester of medicineMolecular Biology

INTRODUCTION

Recent studies have shown the importance of fat tissue in telomere shortening and its relationship with obessity and type 2 diabetes. This study was made inducing oxidative stress in 3T3-L1 adipocytes to test shortened telomeres, senescence and functional impairment. This adipocytes showed ROS, DNA damage, mRNA and protein expression of senescence and pro-inflamatory markers, telomere length and glucose uptake.The researchers wanted to study adipocytes rather than white blood cells due to its reflection on target tissues.

Become from fibroblasts, stores lipids such as triglyceride and cholesteryl ester, as an energetic reserve.

There are two types:

•White: one fat vesicle. Stores lipids as a long term energetic reserve.

•Brown: multiple vesicles. Produces heat.

ADIPOCYTES

OXIDATIVE STRESS

Appears when there are unpaired reactive oxygen species (ROS) that generates free radicals and hydrogen peroxide which damages the cellular components including proteins, lipids and DNA.This damage lowers the ATP levels leading to an uncontrolled apoptosys that releases cytotoxic components.

INSULIN RESISTANCE

Its a genetic or acquired cell inability to uptake insulin-dependent glucose in tissues such as liver, muscle and fat. It leads to hyperglycemia and disminished glucose uptake if is not treated. Some risk factors are obesity and low physical activity.

ADIPOCYTE

OBJECTIVE

•Determine how oxidative stress and accelerated senescence impacts fat tissue function and how this, in turn, leads to age-related diseases.

MATERIALES Y MÉTODOS

•DMEM• IBMX, insulina, dexametasona y FBS Cultivo

•H2O2 - medio sin suero •H2O2 - glucosa oxidasa•ADMA•HG-LG

Tratamiento de células e inducción de estrés oxidativo

•PBSTinción β galactosidasa asociada a senectud

(SA – β-gal)

•Tinción DCF-DA•HEPES•PMA ROSMedición ROS

•PBS – agarosa – extendido – lisis – electroforesis – neutralización – tinción – microscopio.

Ensayo cometa

MATERIALES Y MÉTODOS

• DNA genómico – amplificación PCR – relación T/S.

Medición telómeros RT - PCR

• cDNA – amplificación PCR – β actina.Cuantificación mRNA RT-PCR

• PBS – RIPA – Nano drop – SDS PAGE – Western blot – Ac – β actina.

Extracción de proteínas y Western

blot

• KRH – insulina – KRH – DOG – PBS – SDS – conteo. Captación 2 – DOG

MATERIALES Y MÉTODOS

MATERIALES Y MÉTODOS

ABI 7000

MATERIALES Y MÉTODOSW

E S T E R N

B L O T

RESULTADOS

RESULTADOS

RESULTADOS

Morley. 2008

“In fact, diabetes mellitus has recently been considered as a cause of accelerated aging”.

Monickaraj agrees

Tchkonia et al. 2010

“Despite the fact that senescence in adipocytes could have profound clinical consequences because of the large size of the fat organ and its central metabolic role, there are only very few studies that have looked at the senescence mechanisms in adipocytes”.

Monickaraj agrees

Beliveau and Yaswen. 2007

“In fact, ectopic expression of hTERT was shown to reduce the p53-dependent cellular stress responses”.

Monickaraj agrees

Campisi.2005

Minamino and Komuro.2007

“Senescent cells are known to secrete molecules that can alter the local microenvironment, such as pro – inflammatory cytokines”.

Monickaraj agrees

DISCUSSION

CONCLUSIONS

•Adipocytes subjected to glucose levels oscillation causes a hyperactivation of p53 which is associated with insulin resistance, pro-inflamatory effects and premature cell-aging.

•Adipocytes exposed to oxidative stress showed increased cellular aging due to the following:

• Telomere shortening• Increased expression of mRNA of p53 and p21• Decreased adiponectin expression.

CONCLUSIONS

• The oxidative stress cause a increased secretion of IL6, TNFa and decreased secretion of adiponectin and that woul be reflected in insulin resistance.

• Type 2 diabetes and obesity induce oxidative stress which causes a shortened telomeres, aging and functional impairment

CONCEPTUAL MAP NATALIA

Obesity and type 2 diabetes

Insuline resistance

• Shortened telomeres

• Functional impairment

AgingOxidative stress

ApoptosisIncrease TNFa, IL6

ROS

Hyperglycemia

Dcreasedadiponectin

Damage of:

DNAPROTEIN

LIPIDS

Increase p53

Increase p21

Pro-inflamatory cytokines

Induced:H202, HG-LG,

ADMA,GONATALIA

CONCEPTUAL MAP

SALOME

GRACIASTHANKS