Real-time RT-PCR and proteomic analysis of adipose tissue from ob/ob mice treated with leptin

Abstract

Leptin, a hormone secreted by adipose tissue, plays a critical role in regulating body weight, energy expenditure, lipid metabolism, and adipocyte apoptosis. In order to elucidate the underlying signaling pathways of leptin on adipocytes, advanced real-time RT-PCR and proteomic technologies were performed to compare mRNA and protein levels from white adipose tissue between control and leptin-treated ob/ob mice. ob/ob mice were treated with either PBS (control), or leptin (2.5 µg/d or 10 µg/d) for 14 days via sc osmotic mini pumps, and three fat depots were collected at the end of the experiment. In the first study, eighteen genes were found to be the potential targets of leptin in inguinal fat pad of ob/ob mice by real-time RT-PCR and TaqMan low-density arrays (24 genes format). They are classified as genes related to energy expenditure (UCP2, ADRB3, MFN2, SIRT3), genes involved in lipid metabolism (HSL, SCD1, FAS, RBP4), transcription factor SREBF1, genes related to apoptosis (Bcl-2, Bax, Caspase 3), cytokines (TNF alpha., adiponectin, complement 3), as well as genes taking part in inhibition of angiogenesis (Angiopoietin 2). In the second study, different detergent combinations were tested, and a modified protocol was described as the basis of extraction buffer, including 8.4M urea, 2.4M thiourea, 2% CHAPS (v/v), 2% ASB14 (v/v), 1% IPG buffer, 15mM Tris and 50 mM DTT. In the third study, comparative proteomic analysis was performed between control and leptin-treated ob/ob mice in parametrial adipose tissue. Twelve protein entries were differentially expressed, functionally classified as molecular chaperones (CRT, PDIA3, PHB, and Prx-VI), cytoskeleton proteins (beta-actin, alpha-tubulin and desmin) and other proteins (PC, GDEase, UQCRC, PTRF, and fibrinogen). The mRNA levels of CRT, PDIA3 and PHB were consistent with the fold changein protein expression level by real-time RT-PCR. In general, our results demonstrated that proteomic analysis offers a promising tool to study the global analysis of proteins, and real-time RT-PCR is a reliable, sensitive and medium-throughput platform to measure mRNA quantitatively in a complex biological system (ob/ob mice) under certain external stimuli (leptin treatment).