Development of infrared imaging to measure thermogenesis in cell culture: thermogenic effects of uncoupling protein-2, troglitazone, and beta-adrenoceptor agonists.
OBJECTIVE
Although the effects of thermogenic agents in cell culture can be measured by direct microcalorimetry, only a few samples can be analyzed over several hours. In this report, we describe a robust non-invasive technique to measure real-time thermogenesis of cells cultured in microtiter plates using infrared thermography.


METHODS
Yeast were transformed with uncoupling protein-2 (UCP2) or exposed to carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or rotenone. Adipocytes were exposed to rotenone, FCCP, cycloheximide. troglitazone, or CL316243. Thermogenesis was measured using infrared thermography.


RESULTS
Thermogenesis increased after exposing yeast to the mitochondrial uncoupler, FCCP, or transforming the cells with UCP2. Further, thermogenesis in adipocytes was stimulated by CL316243, a beta3-adrenoceptor agonist being developed to treat obesity. The protein synthesis inhibitor, cycloheximide, did not inhibit CL316243-mediated thermogenesis. In contrast, the mitochondrial proton transport inhibitor, rotenone, inhibited thermogenesis in yeast and adipocytes. Similarly, the antidiabetic agent, troglitazone, suppressed thermogenesis in adipocytes. Although increased UCP synthesis resulted in increased thermogenesis in yeast, UCP expression did not correlate with thermogenesis in adipocytes.


CONCLUSIONS
The results, taken together with the high resolution (0.002 degrees C) and robustness (384-well format) of the approach, indicate infrared-imaging is a rapid and effective method for measuring thermogenesis in vitro.
Paulik
M A
MA
Department of Metabolic Diseases, GlaxoWellcome Inc., Research Triangle Park, North Carolina 27709, USA. map16209@glaxowellcome.com
Buckholz
R G
RG
Lancaster
M E
ME
Dallas
W S
WS
Hull-Ryde
E A
EA
Weiel
J E
JE
Lenhard
J M
JM
D017667:Adipocytes / Q000187:drug effects* / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D001483:Base Sequence; D002478:Cells, Cultured; D002839:Chromans / Q000494:pharmacology*; D017931:DNA Primers; D004734:Energy Metabolism; D006801:Humans; D007473:Ion Channels; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D011506:Proteins / Q000235:genetics / Q000494:pharmacology*; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D013055:Spectrophotometry, Infrared / Q000379:methods*; D013844:Thiazoles / Q000494:pharmacology*; D045162:Thiazolidinediones; D014157:Transcription Factors; D000077288:Troglitazone; D000071246:Uncoupling Protein 2
D000318:Adrenergic beta-Agonists; D002839:Chromans; D017931:DNA Primers; D007473:Ion Channels; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D013844:Thiazoles; D045162:Thiazolidinediones; D014157:Transcription Factors; C000604389:UCP2 protein, human; D000071246:Uncoupling Protein 2; D000077288:Troglitazone