Intrinsic differences in BRITE adipogenesis of primary adipocytes from two different mouse strains

Highlights

• Two inbred mouse strains differ in BRITE adipogenesis (129Sv > C57BL/6J).

• This difference is sustained in cultured primary adipocytes (129Sv > C57BL/6J).

• However, serum from C57BL/6J is more potent in stimulating BRITE adipogenesis.

• Strain differences in co-culture are independent of paracrine or autocrine signals.

• Cell autonomous differences in BRITE adipogenesis depend on trans-acting factors.

Abstract

BRITE (brown-in-white) cells are brown adipocyte-like cells found in white adipose tissue (WAT) of rodents and/or humans. The recruitment of BRITE adipocytes, referred to as the browning of WAT, is hallmarked by the expression of UCP1 and exerts beneficial metabolic effects. Here we address whether beyond systemic cues depot- and strain-specific variation in BRITE recruitment is determined by a cellular program intrinsic to progenitors. Therefore we compared the browning capacity of serum and investigated brown and BRITE adipogenesis in primary cultures of stromal-vascular cells isolated from interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) in two inbred mouse strains C57BL/6J (B6, a strain with low browning propensity) and 129/S6SvEv (129, a strain with high browning propensity). Paradoxically, serum collected from B6 mice was more potent in the promotion of browning than serum collected from 129 mice. Nevertheless, we demonstrate that depot- and strain-specific differences observed in vivo are pheno-copied in primary cultures in vitro, as judged by UCP1 expression and by functional analysis. Notably, primary adipocytes from 129 mice had a higher capacity for isoproterenol-induced uncoupled respiration than B6. We conclude that cues intrinsic to the progenitor cells contribute to differential BRITE adipogenesis. Further analyses demonstrate that these cues are independent of autocrine/paracrine mechanisms, BRITE progenitor abundance and genetic variation in the gene regulatory region of Ucp1 but rather depend on trans-acting factors. These results provide new insights on the molecular basis of strain and depot-specific differences in BRITE adipogenesis.

Introduction

In mammals, two types of adipose tissues are present, white (WAT) and brown (BAT). The former stores energy as triglycerides, whereas the latter catabolizes lipids to produce heat through uncoupling protein 1 (UCP1)-mediated thermogenesis. BAT was originally identified in infants and rodents, but recent studies have also found functional BAT in adult humans [1]. Interestingly, UCP1-expressing brown adipocyte-like cells (BRITE, brown-in-white, also termed ‘beige’) can also develop in typical WAT depots in response to cold exposure, beta3-adrenergic receptor stimulation and PPARγ agonist treatment, a process termed “browning” of WAT [2], [3], [4], [5], [6], [7]. During post-natal development these BRITE cells spontaneously emerge around weaning [6], [8]. Although the browning phenomenon has been first described in 1984, the developmental origin, transcriptional control and physiological function of BRITEs are largely unknown. It has been suggested that BRITE cells represent a newly identified type of adipocytes distinct from white and brown adipocytes in respect to their development, molecular outfit and hormonal-sensitivity [9], [10], [11]. Notably, the abundance of BRITEs in WAT has been associated with obesity-resistance, enhanced fat oxidation and energy expenditure as well as improved systemic insulin sensitivity [2], [12]. Even more, current evidence points to the presence of both classical brown and BRITE adipocytes in humans, both as babies and adults [10], [13], [14], [15], [16]. Thus, increasing the numbers of BRITE adipocytes within WAT depots may represent an innovative approach to treat obesity and associated metabolic conditions [17], [18], [19]. Understanding the molecular mechanisms underlying BRITE adipocyte recruitment is therefore of great scientific and medical interest [1].

In mice a large accumulation of BRITE cells can be found most readily in the subcutaneous inguinal adipose tissue, but is rather scarce in epididymal/perigonadal adipose tissue [12]. The propensity to accumulate BRITE cells, however, differs not only between WAT depots but also among inbred mouse strains [8], [12], [20]. Mice of some strains, such as C57BL/6J, upon adrenergic stimulation exhibit only minor induction of BRITE adipocytes, whereas others, such as A/J and 129, are very responsive to this stimulus [21], [22]. The density of sympathetic fibers in the parenchyma of adipose tissues increases during cold acclimation and positively correlates with the number of BRITE adipocytes in mice of the B6 and 129 strains, thus suggesting that strain differences in the sympathetic tone may be the cause for different browning propensity between strains [23]. It is not clear, however, whether different browning propensities are caused by cell-extrinsic cues only (such as innervation, angiogenesis, hormones, cytokines, etc.) or also by cell-intrinsic properties (such as progenitor abundance, cis-elements and trans-acting factors, etc.). Elucidating the underlying mechanisms will not only help us to understand the development of BRITE cells but also may help us to enrich these cells in the attempt to treat obesity.

Primary cultures represent invaluable tools to characterize cell autonomous or non-autonomous traits. Previous studies have shown that white and brown fat precursor cells in culture proliferate and develop into adipocytes which on the molecular level have distinct inherent characteristics resembling white and brown adipocytes differentiated in vivo [9]. Moreover, an induction of Ucp1 gene expression has been observed in cultured primary adipocytes upon chronic treatment with peroxisome proliferator-activated receptor- (PPAR) ligands [9]. In fact, several recent studies have reported that exposure of white adipocytes in culture or in vivo to potent PPARγ agonists induces browning [7], [9], [24]. The molecular basis of browning induced by PPARγ agonists has not been finally resolved. Nevertheless, mechanisms such as induction of peroxisome proliferator activated receptor gamma coactivator 1-alpha (PGC-1α) expression, stabilization of PRD1-BF-1-RIZ1 homologous domain containing-16 protein (PRDM16), increment of fibroblast growth factor 21 (FGF21) secretion, and posttranslational modifications of PPARγ (deacetylation, desumoylation) activity seem to be involved [7], [25], [26].

Pertaining to the transcriptional control of brown and BRITE adipogenesis, in addition to PPARγ, other core transcriptional regulators of brown and BRITE adipogenesis such as PGC-1α and PRDM16 have been suggested. Among those factors, PRMD16 is considered the first transcriptional regulator that is absolutely required to promote the differentiation of brown/BRITE adipocytes. A recent study describes that PRDM16 is selectively expressed in subcutaneous white adipocytes in comparison to other white fat depots in mice, suggesting that this transcription factor could act as a determinant of browning propensity among fat depots [7], [27]. Nevertheless, whether PRDM16 determines the differential browning capacity of white fat between inbred mouse strains is unknown.

By taking advantage of the highly variable trait of induction of BRITE cells in WAT between mouse strains (C57BL/6J and 129S6/SvEv) and using the primary culture method combined with co-culture strategies and cell transfection, we demonstrate that differences in Ucp1 expression both between depots and strains are maintained in primary cultures, a condition definitely excluding extrinsic cues such as innervation, angiogenesis and blood-borne hormones. In support of this point, we demonstrate that the B6 serum is paradoxically more potent in Ucp1 induction compared to 129, thus questioning a major hormonal contribution to the strain difference in Ucp1 expression. Furthermore, gene profiling and co-culture experiments verify that this cell-autonomous trait is contributed neither by differences in progenitor abundance nor by autocrine or paracrine factors. Importantly, cell transfection experiments confirm that the strain specific differences in Ucp1 expression are caused by cell intrinsic trans-acting factors. Taken together, these data demonstrate that there are intrinsic differences between progenitors from different fat depots and mouse strains which contribute to the differential browning propensity of WAT.