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

<div><p>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.</p></div

Conversion of mature TERT-hWA adipocytes into brown-like adipocytes upon exposure to rosiglitazone.

<p><b>(A)</b> The rosiglitazone-induced browning protocol. <b>(B)</b> Relative mRNA levels of <i>UCP1</i> in mature TERT-hWA adipocytes (day 15, passage 7–14), SGBS and hMADS adipocytes exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. <b>(C)</b> Protein levels of UCP1 in TERT-hWA adipocytes (day 15, passage 13) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. TFIIB was used as a loading control. <b>(D)</b> Relative mRNA levels of the thermogenesis-related genes <i>EBF2</i>, <i>DIO2</i> and <i>PDK4</i> in mature TERT-hWA adipocytes (day 15, passage 12) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. <b>(E)</b> Relative mRNA levels of the β-adrenoceptors <i>ADRB1-3</i> in mature TERT-hWA adipocytes (day 15, passage 12) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. <b>(F)</b> Relative mRNA levels of the mitochondrial markers <i>CPT1B</i>, <i>CS</i> and <i>COXII</i> in mature TERT-hWA adipocytes (day 15, passage 12) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. In (B) and (D-F), expression levels were normalized to <i>TBP</i> levels. The normalized expression in vehicle-treated cells was set to 1. Data are presented as mean +SEM of one representative experiment done in technical triplicate. Statistical significance was determined by unpaired two-tailed Student’s t-test. *, p < 0.05 versus vehicle-treated cells.</p

Subject and biopsy characterization.

<p><b>(A)</b> Patient information. <b>(B)</b> Relative mRNA levels of the thermogenic genes <i>UCP1</i>, <i>PPARGC1A</i> and <i>EBF2</i> in hBAT and hWAT. <b>(C)</b> Relative mRNA levels of the mitochondrial genes <i>CPT1B</i>, <i>CS</i> and <i>COXII</i> in hBAT and hWAT. In (B) and (C), expression levels were normalized to <i>TBP</i> levels. The normalized expression in hWAT was set to 1, except for <i>UCP1</i> in which hBAT was set to 1. Data represent the mean of a technical duplicate without error bars, since only one patient was included. Statistical analyses were not applied.</p

Immortalized human neck pre-adipocytes differentiate into mature adipocytes.

<p><b>(A)</b> Population doubling time for TERT-hWA and TERT-hBA pre-adipocytes in passage 8, 13 and 17. <b>(B)</b> The general differentiation protocol for TERT-hBA and TERT-hWA. <b>(C)</b> Representative overview photos of Oil red O-stained TERT-hBA and TERT-hWA adipocytes at day 12 at passage 10 (P10), 15 (P15) and 20 (P20). <b>(D)</b> Relative mRNA levels of the differentiation markers <i>GLUT4</i>, <i>CEBPA</i> and <i>FABP4</i> in pre-adipocytes (day 0) and mature adipocytes (day 12) at P10, P15 and P20. Expression levels were normalized to <i>TBP</i> levels. RT-qPCR data are represented as mean of means +SEM from 4 independent experiments (two experiments in P10 and one experiment in P15 and P20). Statistical significance was determined by paired two-tailed Student’s t-test. *, p < 0.05 versus pre-adipocytes (day 0). <b>(E)</b> Protein levels of FABP4 in TERT-WA and TERT-hBA pre-adipocytes (day 0) and adipocytes (day 12) at P9. AKT was used as a loading control.</p

TERT-hBA display features of thermogenic adipocytes.

<p><b>(A)</b> Relative mRNA levels of the thermogenic marker genes <i>UCP1</i>, <i>EBF2</i> and <i>DIO2</i> in mature TERT-hBA and TERT-hWA adipocytes (day 12) at passage 10, 15 and 20. Expression levels were normalized to <i>TBP</i> levels. The normalized expression in TERT-hWA adipocytes was set to 1. RT-qPCR data are presented as mean of means +SEM from 5 independent experiments (two experiments in passage 10 and passage 15 and one experiment in passage 20). Statistical significance was determined by paired two-tailed Student’s t-test. *, p < 0.05 versus TERT-hWA. <b>(B)</b> Immunocytochemistry for UCP1 in mature TERT-hBA and TERT-hWA adipocytes (day 12, passage 5). <b>(C)</b> Protein levels of CYC1 and UCP1 in mature TERT-hBA and TERT-hWA adipocytes [day 12, passage 6 (P6) and 7 (P7)]. TFIIB was used as a loading control. <b>(D)</b> Time course of oxygen consumption rates (OCR) of mature TERT-hBA and TERT-hWA adipocytes (day 11, passage 7), after sequential injection of oligomycin (5 μM), FCCP (1 μM), rotenone/antimycin A (1 μM of each). Cell culture media contained 2% BSA. Seahorse data are presented as mean +/- SEM of one representative experiment done with 9–12 wells per condition.</p

Response to β-adrenergic stimulation.

<p><b>(A)</b> Relative mRNA levels of the β-adrenoceptors <i>ADRB1-3</i> in mature TERT-hBA and TERT-hWA adipocytes at passage 10, 15 and 20. Expression levels were normalized to <i>TBP</i> levels. The normalized expression in TERT-hWA adipocytes was set to 1. RT-qPCR data are presented as mean of means +SEM from 5 independent experiments (two experiments in passage 10 and passage 15 and one experiment in passage 20). Statistical significance was determined by paired two-tailed Student’s t-test. *, p < 0.05 versus TERT-hWA. <b>(B)</b> Relative mRNA levels of thermogenic genes in mature TERT-hBA adipocytes (day 12, passage 12) stimulated with 0.1 μM ISO for 6 h. <b>(C)</b> Relative mRNA levels of thermogenic genes in mature TERT-hBA adipocytes (day 12, passage 13) stimulated with 10 μM FSK for 6 h. In (B) and (C), expression levels were normalized to <i>TBP</i> levels. The normalized expression in vehicle-treated cells was set to 1. Data are presented as mean +SEM of one representative experiment done in technical triplicate. Statistical significance was determined by unpaired two-tailed Student’s t-test. *, p < 0.05 versus vehicle-treated cells. <b>(D)</b> UCP1 protein levels in mature TERT-hBA [day 12, passage 10 (P10) and 15 (P15)] stimulated with 0.1 μM ISO for 24 h. TFIIB was used as a loading control. <b>(E)</b> Western blot analysis for phosphorylated adipocyte mediators in mature TERT-hBA adipocytes (day 12, P9) pretreated with 10 μM propranolol or vehicle for 1 h before being stimulated with 10 μM FSK or 0.1 μM ISO for an additional 1 h. <b>(F-G)</b> Representative time course of oxygen consumption and extracellular acidification rates (OCR and ECAR, respectively) in mature TERT-hBA adipocytes (day 12, passage 9) before and after injection of 10 μM ISO or 10 μM FSK. Data are presented as mean +/- SEM of one representative experiment with 9–12 wells per condition. (H) Western blot analysis for phosphorylated HSL in mature TERT-hWA adipocytes (day 12, passage 9) pretreated with 10 μM propranolol or vehicle for 1 h before being stimulated with 10 μM FSK or 0.1 μM ISO for an additional 1 h.</p

GO analysis of regulated proteins by LPS.

<p>(A) Upregulated proteins by LPS treatment belonged to the GO classes: respiratory electron transport chain and generation of precursor metabolites and energy processes. (B) Downregulated proteins especially belonged to the GO class protein glycosylation and to a smaller degree lipid metabolic processes. (C and D) Schematic overview of the distribution of upregulated (C) and downregulated (D) proteins in different GO classes represented here by gene name. Abbreviations: please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159747#pone.0159747.s001" target="_blank">S1 Table</a>.</p

STAT1 expression measured by qPCR and SILAC-MS.

<p>(A) STAT1 gene expression measured by qPCR in 3T3-L1 cells incubated with LPS and resveratrol. (B) IFIT1 protein expression measured by SILAC-MS in 3T3-L1 cells incubated with LPS and resveratrol.</p

Thirty most downregulated proteins.

<p>Thirty most downregulated proteins.</p

IFIT1 expression in whole adipose tissue measured by qPCR and in 3T3-L1 cells measured by and SILAC-MS.

<p>(A) Mice treated with LPS for 28 days showed increased gene expression of IFIT1, which was ameliorated by resveratrol delivered through the diet. (B) IFIT1 protein expression measured by SILAC-MS in 3T3-L1 cells incubated with LPS and resveratrol.</p