Preliminary studies on CD36 in type 2 diabetic patients from north India

Sunaina Gautam, C. G. Agrawal, Hemant Kumar Bid & Monisha

Banerjee

July 2011

María Isabel Henao MontañoLuis Carlos Fuentes Acosta

III Semestre MedicinaUPB

Agosto 26 de 2011

• Diabetes mellitus often referred as simply  diabetes, it is a  group of metabolic diseases  which affects persons with a high blood sugar levels.

• The body does either because not produce enough insulin, or cells don`t respond the insulin receptor. 

• High blood sugar levels produces the classical symptoms of polyuria (frequent urination), polydipsia (increased thirst) and polyphagia (incrased hunger).

INTRODUCTION

Diabetes

• Comprises an array of dysfunctions resulting from the combination of resistance to insulin action and inadequate insulin secretion.

• Is characterized by hyperglycemia and associated with microvascular (retinal, renal, possibly neuropathic), macrovascular (coronary, peripheral vascular), and neuropathic (autonomic, peripheral) complications.

Diabetes Type 2

• Unlike patients with type 1 diabetes mellitus, patients with type 2 are not absolutely dependent upon insulin for life. This distinction was the basis for the older terms for types 1 and 2, insulin dependent and non–insulin dependent diabetes. However, many patients with type 2 diabetes are ultimately treated with insulin.

• Because they retain the ability to secrete some endogenous insulin, they are considered to require insulin but not to depend on insulin.

CD36

• Protein: is an integral membrane protein found on the surface of many cell types in vertebrate animals.

• Is a member of the class B scavenger receptor family of cell surface proteins.

• Binds many ligands including collagen, thrombospondin, erythrocytes parasitized with Plasmodium falciparum, oxidized low density lipoprotein, native lipoproteins, oxidized phospholipids, and long-chain fatty acids.

• Gen: The gene is Located on the Long arm of chromosome 7 at band 11.2 (7q11.2) and is encoded by exons 15 more than that extend over 32 kilobases. 

• Both the 5‘ and the 3‘ untranslated regions contain introns: the 5 'with two and the 3' one.

• Exons 1, 2 and first nucleotides of exon 89 as well as 3 and exon 15 arenon-coding. 

• Exon 3 contains the N-terminal encoder cytoplasmic and transmembrane domains. The C-terminal cytoplasmic and transmembraneregions is encoded by exon 14. 

• The extracellular domain is encoded by the 11central exons. Alternative splicing of the untranslated regions at least Gives Rise to two mRNA species.

Relationship between CD36 and DM2

The CD36 is a protein responsible for the endocytosis of LDL and if it’s faulty will not be able to enter the LDL. Therefore quickly the macrophages would transform them into foam cells which would generate an increase of free fatty acids. This increase will generate an insulin resistance and then it would generate a mellitus diabetes type 2.     

GENERAL OBJECTIVE

We undertook this study to investigate CD36 gene status in north Indian subjects by screening for the deletion of exons 3, 4 and 5 and certain polymorphisms.

Recolección de la muestra

• Fundamento: Método por el cual se va a realizar la extracción de la muestra.

• ¿Para que?: Para poder determinar las diferencias entre pacientes sanos y pacientes con DM2

MATERIALES Y METODOS

Parámetros Bioquímicos

• Fundamento: Se emplean químicos para medir concentraciones plasmáticas de sustancias.

• ¿Para que?: Determinación de glucosa y perfil lipidico en plasma.

Extracción de DNA

• Fundamento: Aislamiento del DNA a partir de los glóbulos blancos.

• ¿Para que?: Para observar los exones 3, 4 y 5 mediante la espectofometria.

Reacción en cadena de la Polimerasa (PCR)

• Fundamento: Aumentar un segmento especifico de ADN usando polimerasa

• ¿Para que?: Para verificar la funcionalidad del gen.

Recoleccion de la Muestra

• Marzo-noviembre de 2009 • Muestras de sangre al azar en 300 pacientes con DM-2

• Se registraron DM y enfermedades asociadas

• Muestra control: 100 pacientes

• Detección y tratamiento de los pacientes se realizaron de acuerdo con la American Diabetes Association

RESULTADOS

• Criterios para muestra problema: Prueba de Tolerancia a la Glucosa

- glicemia en ayunas > 126 mg/dL - glicemia pospandrial > 200 mg/dL

• Criterios muestra control:

- glicemia en ayunas: 70-109 mg/dl

Paràmetro Bioquìmico

• Muestra de sangre + 0.5 mL EDTA + estimaciones bioquìmicas

• Muestra de sangre en vial plano para extraccion de DNA + estimaciones bioquìmicas

Centrifugación 10 minutos

Espectrofotometría

550 nm: TGL510 nm: creatinina500 nm TC560 nm HDL. 

• Se mide altura, peso y circunferencia de la cintura para calcular el índice de masa corporal (IMC) y la relación cintura cadera (WHR)

• PA: paciente sentado, con material apropiado, luego de 5 minutos de reposo

Extracción de ADN y análisis de supresión

• DNA de pacientes con DM-2

• Lisis, Buffer y SDS

• Añadir NaCl frio

• Extracción de fenol cloroformo

• Verificación de DNA en gel agarosa

• Amplificación de exones 3, 4 y 5 y gen CD36 por PCR y respectivo primer.

Análisis del Genotipo

• Dos polimorfismos en el gen CD36 por supresión de los exones 4 y 5

• Analizados en pacientes DM-2

• Controles por PCR y los fragmentos de restricción

•Enzimas de restricción: Cfr 131 y BoxI.

• Los productos amplificados y digeridos en el gel, se tiñeron conbromuro de etidio y se documenta en Gel Doc

Análisis Estadístico

• Se aplicó a los datos bioquímicos utilizando el software SPSS 

• Desviación estándar de todos los parámetros fue calculado en cada grupo de edad ((≤ 40, 41-59 y ≥ 60 años)

• IMC: <18,5; 18,5 - 25 y> 25 kg/m2

• Promedio de edad: 48.61 años• Glucosa en ayuno: 165.40 – 69.65 mg/dL• Glucosa pospandrial: 266.40 – 97.04 mg/dL• Presion Sistolica media: 84.27 + 10.77 mmHg• Colesterol total: aumentado• LDL: aumentado• HDL: bajo• IMC, WHR, trigliceridos y niveles de creatinina no presentaron cambios significativos

Figura 1

Figura 2

DISCUSSION

AUTOR COMMENTSAGREE /

DESAGREE

(32) Febbraio M, Hajjar DP, Silverstein RL.

(33) Furuhashi M, Ura N, Nakata T, Shimamoto K.

“Studies have shown its involvement in diverse disorders such as insuline resistance, dyslipidaemia, hyperlipidaemia, atherosclerosis”

(15) Handberg A, Levin K, Hojlund K, et al.

“CD36 expression in monocytes is upregulated by oxidized low density lipoprotein (Ox-LDL), whose levels increase in case of T2DM, hyperglicaemia and related atherosclerosis”

AUTOR COMMENTSAGREE /

DESAGREE(13) Miyaoca K Kuwasako T, et al.

(35) Imai M, Tanaka T, et al.

(36) Kintaka T, Tanaka T, et al.

“CD36 genotype was identified as a fundamental determinant of myocardial long chain fatty-acid uptake”

(37) Banerjee M, Gautam S, Saxena M, Bid HK, Agrawal CG.

“We have recently reported that one of the several SNPs in the CD36 gene (rs1761667, G>A) shows a significant association with T2DM in the north Indian population.”

The conclusions of these studies lead to a guide of genotypic of CD36 and its associations with T2DM. These studies also have shown that the results are backed up in different multi-ethnical and genotipical analysis focused on the specific mutation related to CD36 gen.

CONCLUSSIONS

CD36, associated with metabolic and hypertense syndromes, is encoded by different gens that despite being on the same chromosome, do not have the nucleotidal identical sequences, that means that there is no sustitution or replace due damaged or deficiency of one of them. Therefore, from the begin of the protein codification exists a damaged sequence, provoking its inactivity, leading to the appearance of diferent diseases.

Most of the studied cases in north india shown that a possible relations between the high rating of people affected by T2DM and the deletion and deficiency of CD36 gen can exist. deletion of exons 3, 4 and 5 and certain polymorphisms lead to investigators to further studies about genetical factors and deletion related to CD36 gen.

This study confirmed that the ages are not relevant because they can relate to young adults from 10 to 50 years so we conclude that CD36 is a problem of deficiency in any human in this case the population of northern India.

MAPA CONCEPTUALMaria Isabel Henao

MAPA CONCEPTUALLuis Carlos Fuentes