HB-EGF and ADAM 12S directed cellular reprogramming results in metabolically active brown adipose tissue-like cells

Abstract

Brown adipose tissue (BAT) is considered a potential tool for the treatment of obesity and type 2 diabetes due to its ability to uncouple oxidative phosphorylation and stimulate non-shivering thermogenesis that utilizes glucose and lipids as its source of energy. Previous results from our lab demonstrated that co-expression of HB-EGF and ADAM 12S resulted in lipid accumulation and a BAT-like phenotype, including up-regulation of BAT genes, down-regulation of genes involved in formation of white adipose tissues, and increased mitochondrial staining in three cell lines including mouse fibroblasts, human epidermoid carcinoma cells, and human preadipocytes. Furthermore, qRT-PCR results demonstrated up-regulation of cellular reprogramming factors such as KLF4, KLF3, and FGF-2 and down-regulation of LMNA, a marker gene involved in differentiation, in the BAT-like reprogrammed cells. This study substantiates these finding using immunohistochemical analysis of reprogrammed BAT-like cells that demonstrate increased immunofluorescent detection of FGF-2, KLF3, and PGC-1α and decreased immunofluorescence for C/EBPα. Supportive evidence of cellular reprogramming involves the use of a stem-cell transcription factor RT-profiler array that results in enhanced expression of HOXA10 (3.04-fold) and HOXC5 (6.46-fold). In order to demonstrate that HB-EGF/ADAM 12S reprogrammed BAT-like cells function as BAT, oxygen consumption and extracellular acidification rates were measured using a Seahorse XFe24 Analyzer with and without catecholamine exposure followed by FCCP + Oligomycin exposure. HB-EGF/ADAM 12S reprogrammed BAT-like cells demonstrate a significant metabolic increase compared to MLC, HB-EGF, ADAM 12S. HB-EGF/ADAM12S reprogrammed BAT-like cells exhibit a metabolic profile similar to 3T3-L1 induced BAT cells. Collectively, these results demonstrate that HB-EGF/ADAM 12S co-expression stimulates cellular reprogramming into metabolically active BAT and may be a putative therapeutic tool to combat obesity and type 2 diabetes.