Accelerated fat cell aging

links oxidative stress and

insulin resistance in

adipocytesFinny Monickaraj, Sankaramoorthy Aravind, Pichamoorthy

Nandhini,

Paramasivam Prabu, Chandrakumar Sathishkumar, Viswanathan

Mohan and Muthuswamy Balasubramanyam

Natalia Perilla Hernández

Salomé Rave DiosaThird semester of medicine

Molecular 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. Storeslipids 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 mRNART-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 MÉTODOS

WESTERN

BLOT

RESULTADOS

RESULTADOS

RESULTADOS

Morley.2008

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

Monickaraj agrees

Tchkonia et al. 2010

“Despite the fact that senescence in adipocytescould have profound clinical consequences becauseof the large size of the fat organ and its centralmetabolic role, there are only very few studies thathave looked at the senescence mechanisms inadipocytes”.

Monickaraj agrees

Beliveau and Yaswen.2007

“In fact, ectopic expression of hTERT was shown toreduce the p53-dependent cellular stressresponses”.

Monickaraj agrees

Campisi.2005

Minamino and Komuro.2007

“Senescent cells are known to secrete moleculesthat can alter the local microenvironment, such aspro – 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 thatwoul be reflected in insulin resistance.

•Type 2 diabetes and obesity induce oxidative stress

which causes a shortened telomeres, aging and

functional impairment

CONCEPTUAL MAP

NATALIAObesity and

type 2 diabetes

Insulineresistance

• Shortenedtelomeres

• Functionalimpairment

AgingOxidative

stress

Apoptosis

IncreaseTNFa, IL6

ROS

Hyperglycemia

Dcreasedadiponectin

Damageof:

DNAPROTEIN

LIPIDS

Increasep53

Increasep21

Pro-inflamatorycytokines

Induced:H202, HG-LG,

ADMA,GONATALIA

CONCEPTUAL MAP

SALOME

GRACIAS

THANKS