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ABSTRACT
Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo “browning.” In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.
Abbreviations
| BAT | = | brown adipose tissue |
| Camk2a | = | calcium/calmodulin-dependent protein kinase IIα |
| DMEM | = | Dulbecco's Modified Eagle's Medium |
| Fabp4 | = | fatty acid-binding protein 4 |
| FBS | = | fetal bovine serum |
| FCCP | = | carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone |
| GAPDH | = | glyceraldehyde-3-phosphate |
| Glut4 | = | glucose transporter 4 |
| hMADS | = | human multipotent adipose-derived stem cells |
| ISO | = | isoproterenol |
| iWAT | = | inguinal white adipose tissue |
| OCR | = | oxygen consumption rate |
| Pkm | = | pyruvate kinase, muscle |
| RNAi | = | RNA interference |
| RT-qPCR | = | reverse transcription-quantitative polymerase chain reaction |
| SEM | = | standard error of the mean |
| shRNA | = | short-hairpin RNA |
| siRNA | = | small interfering RNA |
| SV | = | stromal vascular |
| T3 | = | thyroid hormone |
| Tbp | = | TATA-binding protein |
| Ucp1 | = | uncoupling protein 1 |
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Funding
This work was supported by the EU FP7 project DIABAT (HEALTH-F2-2011-278373). MSI and MCHP were funded in part by The Danish Diabetes Academy supported by The Novo Nordisk Foundation.