Effect of Incubation Temperature on the Proliferation and Differentiation of Pig Preadipocytes in Primary Culture

Abstract Text: Better understanding molecular mechanisms governing the proliferation and differentiation of pig preadipocytes may provide insight into the regulation of adipose tissue development in vivo. Primary cultures of pig preadipocytes have served as a useful tool for investigating these mechanisms. However, to date, such cultures have generally been maintained at 37°C while normal body temperature in pigs is 39.2°C.  This raises questions concerning the physiological relevance of culturing primary pig preadipocytes at 37°C. The objective of this study was to investigate the effect of culture temperature, 37 vs 39°C, on the proliferation and differentiation of pig preadipocytes in primary culture. The effect of temperature on preadipocyte proliferation was determined using the MTT, resazurin, and cell count assays as markers for proliferation. Preadipocyte number was increased 30-50% when cultures were incubated at 39 vs 37°C based upon cleavage of the tetrazolium salt, MTT (P < 0.001), reduction of resazurin (P < 0.001), and daily cell counts (P < 0.001). Differentiation was monitored on d 8 after induction morphologically, enzymatically, and by measuring the mRNA abundance of key adipogenic transcription factors via real-time PCR. Glycerol-3-phosphate dehydrogenase (GPDH) activity was higher in differentiating cultures maintained at 39°C than in cultures differentiated at 37°C (P < 0.01) in agreement with increased lipid accumulation observed in these cultures as measured by Oil Red O staining suggesting that cultures maintained at 39°C differentiated to a greater degree than those maintained at 37°C. Finally, the effect of temperature on gene expression was investigated. Expression of peroxisome proliferator-activated receptor gamma (PPARγ; P < 0.01), CCAAT/enhancer binding protein alpha (C/EBPα; P < 0.01), bone morphogenic protein 2 (BMP2; P < 0.05), bone morphogenic protein 4 (BMP4; P < 0.05), and adiponectin (P < 0.001) were higher while mRNA expression of CCAAT/enhancer binding protein beta (C/EBPβ; P < 0.01) was lower in cultures differentiated at 39°C versus those differentiated at 37°C. Collectively these data indicate that culturing primary preadipocytes at 37 rather than 39°C decreases their proliferation rates and suppresses their adipogenic potential. Furthermore the observation that incubation temperature influences gene expression in primary cultures of pig preadipocytes raises the possibility that studying porcine adipocyte hyperplasia at temperatures below physiological body temperatures may confound the ability to extrapolate observations concerning the underlying mechanisms regulating proliferation and differentiation of primary cultures of pig preadipocytes to the live animal.

Keywords: Adipocyte, Primary culture, Pig