Characterization of immortalized human brown and white pre-adipocyte cell models from a single donor

Abstract

Brown adipose tissue with its constituent brown adipocytes is a promising therapeutic target in metabolic disorders due to its ability to dissipate energy and improve systemic insulin sensitivity and glucose homeostasis. The molecular control of brown adipocyte differentiation and function has been extensively studied in mice, but relatively little is known about such regulatory mechanisms in humans, which in part is due to lack of human brown adipose tissue derived cell models. Here, we used retrovirus-mediated overexpression to stably integrate human telomerase reverse transcriptase (TERT) into stromal-vascular cell fractions from deep and superficial human neck adipose tissue biopsies from the same donor. The brown and white pre-adipocyte cell models (TERT-hBA and TERT-hWA, respectively) displayed a stable proliferation rate and differentiation until at least passage 20. Mature TERT-hBA adipocytes expressed higher levels of thermogenic marker genes and displayed a higher maximal respiratory capacity than mature TERT-hWA adipocytes. TERT-hBA adipocytes were UCP1-positive and responded to β-adrenergic stimulation by activating the PKA-MKK3/6-p38 MAPK signaling module and increasing thermogenic gene expression and oxygen consumption. Mature TERT-hWA adipocytes underwent efficient rosiglitazone-induced ‘browning’, as demonstrated by strongly increased expression of UCP1 and other brown adipocyte-enriched genes. In summary, the TERT-hBA and TERT-hWA cell models represent useful tools to obtain a better understanding of the molecular control of human brown and white adipocyte differentiation and function as well as of browning of human white adipocytes.

Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro

“…Next, we studied cellular respiration after prolonged exposure (24 hours) to 0.1 and 0.5 mM metformin in two additional human thermogenic cell models (TERT‐hWA and hMADS). Both cell‐lines were “browned” by treatment with rosiglitazone from days 12 to 15, as described previously . Oxygen consumption in the hMADS cell model was reduced by 9% during incubation with 0.1 mM metformin ( P < .01) and by 42% during incubation with 0.5 mM metformin ( P = .03; Figure A).…”

“…Immortalized brown adipocytes of human origin (TERT‐hBA), passage 10‐12, were cultured as previously described . On day 9 of differentiation, cells were replated into gelatin‐covered 96‐well XF Cell Culture Microplates (20 000 cells/well).…”

“…Cells were kept in growth medium (Advanced DMEM/F12) supplemented with 2% FBS, penicillin (62.5 μg/mL), streptomycin (100 μg/mL), L‐glutamine (2 mM) and T3 (1 nM) for 2 days. Immortalized white adipocytes of human origin (TERT‐hWA), passage 12‐13, and hMADS cells were cultured as described . On day 9 of differentiation, cells were replated as described above.…”

“…Cells were kept in growth medium without T3. On day 12, cells were treated with rosiglitazone (1 μM) to induce browning . Analyses were conducted on day 15.…”

Studying Brown Adipose Tissue in a Human in vitro Context

“…Similarly, subcloned immortalized BA-like cell lines have been generated from adult supraclavicular AT stromal vascular fraction (SVF) ( 43 ) and neck AT SVF ( 44 ). In addition, a more heterogeneous BA-like cell model was created from adult deep neck AT SVF without subcloning ( 45 ). These BA cell models demonstrated expression of UCP1 , lipolytic response to cAMP ( 43 ), and increased oxygen consumption in response to β-adrenergic stimulation ( 44 , 45 ), and two of the lines were maintained for at least 20 passages ( 44 , 45 ).…”