Background

Objective: To examine the molecular mechanism of cellular reprogramming of primary human adipocyte cells into Brown Adipose Tissue (BAT)–like cells.

Methods

Previous results from our lab have demonstrated lipid-accumulation, lack of cellular proliferation, stimulation of genes involved in the BAT pathway and increased number of mitochondria when heparin binding EGF-like growth factor (HB-EGF) and a soluble form of a disintegrin and metalloproteinase 12 (ADAM 12S) were co-transfected in cells. HB-EGF and ADAM 12S adenoviral expression vectors were engineered in order to recapitulate these results with the goal of using these vectors in vivo and in vitro. Human primary adipocytes were infected with either mock or ADAM 12S high titer adenovirus, monitored for fluorescence, lipid accumulation, and RNA was extracted after three weeks of infection. Gene expression patterns were examined using qRT-PCR for the canonical BAT genes, PRDM16, PGC-1α, and UCP-1. Metabolic analysis for functionality of BAT-like cells was determined using a Seahorse XF24 analyzer.

Results

Infection of the primary human adipocyte cells was confirmed by the presence of enhanced green fluorescent protein. Both the mock infected and the ADAM 12S adenovirus infected cells exhibited fluorescence. The ADAM 12S infected cells demonstrated noticeable lipid droplet accumulation in comparison to mock infected cells. This was confirmed by significant and specific Oil Red O staining in Ad-ADAM 12S infected cells, indicating the accumulation of oil droplets. RT-PCR results demonstrated the presence of endogenous hHB-EGF. The BAT-like gene PGC-1α was found to be statistically significantly upregulated in the cells that were infected with ADAM 12S (p = 0.05), while the mock infected cells did not exhibit this pattern of gene expression. The BAT-like gene UCP-1 was upregulated but not statistically significant (p = 0.05). PRDM16, a BAT-like gene, was found to be statistically significantly downregulated in ADAM 12S infected cells. The Seahorse metabolic assay demonstrated an increase rate of glycolysis for ADAM 12S cells after exposure to a stressor mix, composed of FCCP and Oligomycin, when compared to their basal rate. ECAR was significantly increased in ADAM 12S cells compared to MOCK cells after exposure to catecholamines and the stressor mix (FCCP and Oligomycin).

Conclusions

These results further support previous findings that co-expression of HB-EGF and ADAM 12S stimulates cellular reprogramming into brown adipose tissue (BAT)-like cells. We believe that ADAM 12S stimulates cellular reprogramming into BAT-like cells utilizing endogenous hHB-EGF. These novel insights may provide the first evidence demonstrating BAT-like cellular reprogramming occurs in vivo in humans. This research has possible therapeutic applications to combat obesity and type II diabetes.