BMP7 drives human adipogenic stem cells into metabolically active beige adipocytes

Abstract

Adult humans have a substantial amount of inducible-brown (or beige) fat, which is associated with increased energy expenditure and reduced weight gain via thermogenesis. Despite the identification of key regulators of beige adipogenesis, impacts of dietary factors on adaptive thermogenesis are largely unknown, partly due to a lack of validated human cell models. Bone morphogenetic protein 7 (BMP7) is known to promote brown adipogenesis in rodent and human progenitor cells. However, controversy still surrounds the cellular identity in BMP7-mediated transition of white to brown adipocytes. The aim of this study was to confirm BMP7-derived human adipocytes as a relevant in vitro model of human beige adipocyte by verifying the cellular lineage and metabolic activity. In this study, we hypothesized that pre-exposure of the stromal vascular (SV) fraction of primary human adipogenic precursor cells (hASC) to BMP7 would convert metabolically active brown adipocytes. Our results showed that exposure of hASC to human BMP7 was associated with significant escalation of (1) UCP1 gene expression, a signature gene of brown adipocytes, (2) beige specific marker gene expression (i.e., CD137 and TMEM26), (3) glucose and fatty acid uptake, and (4) basal and cAMP-stimulated oxygen consumption rate compared to white adipocyte control. Taken together, we demonstrated that BMP7 mediates conversion of hASC into metabolically active beige adipocytes. By confirming the cellular identity and metabolic activity, this BMP7-induced human beige adipocytes from hASC should aid in the discovery and assessment of bioactive molecules to promote adaptive thermogenesis.

Figure 1. Preparation of BMP7 conditioned medium using Ad-BMP7

A. BMP7 mRNA expression at 72 h after Ad-BMP7-GFP infection (0–5 MOI), B. Florescent image of hASC infected with GFP-tagged Ad-BMP7BMP7, C. Secretion to the media at 72 h after Ad-BMP7 infection (0–5 MOI) by ELISA. D. Western blot analysis of 1μl of BMP7-containing condition medium (from MOI 5 infection). Values not sharing a common letter differ significantly by one-way ANOVA.

Figure 2. BMP7 promotes brown adipocyte-like gene profiles in human adipocytes

A. hASC treated with or without BMP7 conditioned medium were differentiated into adipocyte. Phase contrast images were taken 10 d after differentiation. Arrows indicate timeline for BMP7 addition and initiation of adipogenic differentiation by adding differentiation medium (DM) and adipocyte maintenance medium (AM), B. BMP7-mediated changes in white- and brown-adipocyte gene expression measured after 10 d of differentiation by qPCR. C. Kinetics of white- and brown-adipocyte gene expression with or without BMP7 incubation to hASC during 10 d of adipogenic differentiation. Changes of UCP1 (D) and Leptin E) gene expression in response to( continuous exposure BRL (1 and 5 μmol/L) in differentiated adipocytes with or without BMP7. BRL treatment after first 3 days of differentiation has shown for experiment in D and E. Classical BAT from monkey (mkBAT) was used as a classical brown fat control for UCP1 and leptin expression. In B (n=4), D and E (n=3), ^*p<0.05 and ^**p<0.01, ***p<0.001 and ****p<0.0001 by student t-tests, ns= not significant. In C, *p<0.05, ***p<0.001, ****p<0.0001 by two-way ANOVA with multiple comparisons at each time point (n=3).

Figure 3. Expression of beige vs. classical brown adipocyte selective markers in BMP7 treated adipocytes

mRNA expression of beige adipocyte-selective markers CD137 and TMEM26 in supraclavicular BAT collected from nonhuman primates (A), cultured adipocytes with or without BMP7 (B), and in the presence and absence of Bt₂-cAMP stimulation (C). D. Classical BAT marker LHX8 in BAT of nonhuman primates and cultured adipocytes with or without BMP7 infection. ns = not significant, ^*p<0.05, ^**p<0.01 and ^***p<0.001 by student t-tests (n=4–5 for each experiments).

Figure 4. Measurement of metabolic capacity in BMP7-derived beige adipocytes

A. [³H]-2-deoxy glucose uptake (2-DOG) in the presence and absence of insulin (100 nmol/L) in triplicate samples (n=3), B. Fatty acid uptake using [¹⁴C]-oleic acid over 4 h, n=6 per each time point C. Lipolysis was determined by measuring [¹⁴C]-oleate release to the medium for 2 h (n=3). Data expressed as relative [¹⁴C] radioactivity to the cellular radioactivity, ^*p<0.05, ^**p<0.01, and ^***p<0.001 by student t-tests.

Figure 5. Measurement of oxygen consumption rate in BMP7-derived beige adipocytes

Oxygen consumption was measured using a fluorescent oxygen probe. A. Effectiveness of oxygen quenching was validated in the presence (+) and absence (−) of Antimycin A (AA) for 4 h. B. Oxygen consumption in adipocytes grown with or without BMP7. C. Cultured adipocytes grown with or without BMP7 then stimulated with or without of Bt₂-cAMP for last 12 hr before measurement of oxygen consumption rate (OCR). Slope of OCR was obtained by linear regression for all data points (n=8–10 per group). RPU; relative phosphorescent unit, OCR; oxygen consumption rate, Values not sharing a common letter differ significantly by one-way ANOVA.