Generation of mega brown adipose tissue in adults by controlling brown adipocyte differentiation in vivo

Significance

Brown adipose tissue (BAT) as a highly specialized tissue in its invariable size and location significantly participates in global energy metabolism by producing heat. In this study, we show that the adult BAT tissue mass is tightly controlled by a platelet-derived growth factor receptor α (PDGFRα) signaling mechanism. Surprisingly, suppression of PDGFRα alone in adult animals produces an enlarged BAT mass (megaBAT) owing to increasing adipocyte numbers. Mechanistic insights into megaBAT demonstrate that PDGFRα inhibition instigates the differentiation of progenitor cells into mature and functional BAT adipocytes. On the basis of these findings, we reasonably propose a therapeutic paradigm of generation of megaBAT for treating metabolic diseases such as obesity, liver steatosis, and diabetes by manipulating the BAT mass.

Abstract

Brown adipose tissue (BAT) is a highly specialized adipose tissue in its immobile location and size during the entire adulthood. In response to cold exposure and other β3-adrenoreceptor stimuli, BAT commits energy consumption by nonshivering thermogenesis (NST). However, the molecular machinery in controlling the BAT mass in adults is unknown. Here, we show our surprising findings that the BAT mass and functions can be manipulated in adult animals by controlling BAT adipocyte differentiation in vivo. Platelet-derived growth factor receptor α (PDGFα) expressed in BAT progenitor cells served a signaling function to avert adipose progenitor differentiation. Genetic and pharmacological loss-of-function of PDGFRα eliminated the differentiation barrier and permitted progenitor cell differentiation to mature and functional BAT adipocytes. Consequently, an enlarged BAT mass (megaBAT) was created by PDGFRα inhibition owing to increases of brown adipocyte numbers. Under cold exposure, a microRNA-485 (miR-485) was identified as a master suppressor of the PDGFRα signaling, and delivery of miR-485 also produced megaBAT in adult animals. Noticeably, megaBAT markedly improved global metabolism, insulin sensitivity, high-fat-diet (HFD)-induced obesity, and diabetes by enhancing NST. Together, our findings demonstrate that the adult BAT mass can be increased by blocking the previously unprecedented inhibitory signaling for BAT progenitor cell differentiation. Thus, blocking the PDGFRα for the generation of megaBAT provides an attractive strategy for treating obesity and type 2 diabetes mellitus (T2DM).

Brown adipose tissue (BAT) located in interscapular, subscapular, axillary, perirenal, and periaortic regions in rodents and interscapular, supraclavicular, and perivertebral regions in humans bestows energy consumption by nonshivering thermogenesis (NST) (1–6). Although relatively small in size, BAT provides about 60% NST in small rodents to protect their survival in response to low ambient temperature (7–10). Recent human studies using the 18-fluorodexyglucose (¹⁸FDG) imaging analysis show that the total volume of BAT is substantially larger than the typically estimated 50 to 150 mL, suggesting considerable contribution of BAT in total energy expenditure (11). The existence of a substantial amount of BAT in adult humans and its reverse correlation with obesity and type 2 diabetes mellitus (T2DM) has generated exciting opportunities for potentially treating metabolic disorders by augmenting BAT activation. Unlike white adipose tissue (WAT) that constantly undergoes expansion and shrinkage during the entire adulthood, BAT is homeostatically stable in volume and energy dissipation (11). BAT-NST is induced by norepinephrine released from binding to β3-adrenoreceptors in sympathetic nerves (12).

Morphologically, BAT adipocytes exhibit abundant multivacuolar lipid depots and dense mitochondrial structures (13, 14), whereas WAT adipocytes contain a single lipid droplet that occupies nearly the entire intracellular compartment (15). Activation of β3-adrenoreceptors by cold exposure and other stimuli, WAT adipocytes, especially adipocytes in subcutaneous WAT (sWAT), exhibit a phenotypical transformation toward brown-like adipocytes, i.e., browning or beige adipocytes (16–18). The hallmark of BAT activation is the increased expression of uncoupling protein 1 (UCP1) in the inner membrane of mitochondria, which uncouples respiration from ATP synthesis (6, 11, 19). UCP1 stipulates NST through dissipating the electrochemical proton gradient across the mitochondrial inner membrane and abolishing ATP synthesis (20). Although BAT activation accompanies high amounts of glucose uptake, glucose metabolism may not necessarily correlate with the levels of heat production (11, 21–24).

BAT constitutes a myriad of cell types, including mature BAT adipocytes, preadipocytes, vascular cells, stromal fibroblasts, inflammatory cells, and various immune cells (25). Genetic lineage tracking demonstrates that brown adipocytes, skeletal muscle cells, and dorsal dermal cells are derived from the Engrailed 1⁺/Paired box transcription factor 7 (Pax7)⁺ multipotential progenitor cells, which are originated from the central dermomyotome (26). Interestingly, brown adipocytes and skeletal muscle progenitor cells expressed myogenic factor 5 (MYF5), which is one of the muscle-specific lineage-determining genes (17, 27–29). These findings suggest that brown adipocytes and skeletal muscle cells originated from the same progenitor cells. By contrast, WAT and beige progenitor cells lacked Myf5 expression, and a recent study suggests the mosaic origins and plasticity of adipocyte lineages (30).

Platelet-derived growth factor receptor α (PDGFRα) is a cell surface tyrosine kinase receptor expressed in perivascular cells, stromal fibroblasts, and adipose progenitor cells (31, 32). PDGFRα is a commonly used marker for defining adipose progenitor cells, and it plays a crucial role in adipocyte differentiation during adipogenesis (33–36). Genetic lineage tracking experiments show that PDGFRα⁺ cells can differentiate into WAT adipocytes (37). Moreover, in response to β3-adrenergic activation resident PDGFRα⁺ cells in WAT or BAT can differentiate into browning adipocytes and mature brown adipocytes (37). Activation of the PDGFRα signaling by its specific ligands including PDGF-AA, PDGF-AB, PDGF-BB, and PDGF-CC triggers multiple signals in targeted cells to control proliferation, differentiation, migration, and survival (32, 38). However, the net balance of the PDGFRα signaling–transduced functional outcomes could be dependent on spatiotemporal and cell type contexts. In particular, the availability of ligands and levels of receptor activation may play crucial roles in determining the balance between cell proliferation and differentiation. In this regard, the roles of PDGFRα in BAT homeostasis, tissue mass, progenitor cell differentiation, and regulation of NST remain uncharacterized.

In this study, we aimed to address the crucial issues of the PDGFRα signaling in controlling BAT progenitor cell differentiation and metabolic functions in adult BAT tissues. We employed multidisciplinary approaches, including in vitro and in vivo gain- and loss-of-function experiments to investigate the PDGFRα signaling in modulating BAT functions under thermoneutrality and cold acclimation. Inhibition of the PDGFRα signaling permitted differentiation of progenitor cells into mature BAT adipocytes, leading to marked enlargement of the BAT tissue mass by increasing the number of BAT adipocytes. On the basis of these findings, we propose that the PDGFRα signaling serves as a gatekeeper in adults for controlling the BAT stem cell differentiation and tissue mass. Moreover, the differentiated mature adipocytes were metabolically active and augmented NST. In high-fat-diet (HFD)-induced obese mice, the PDGFRα inhibition–induced megaBAT improved insulin sensitivity and obesity by enhancing NST. Thus, targeting the PDGFRα signaling provides exciting opportunities for treating T2DM and other metabolic complications.

Results

Blocking PDGFRα Promotes BAT Progenitor Cell Differentiation.

PDGFRα is known to be expressed in adipose progenitor cells and has been used as a reliable marker to define adipose stem cells (33–36). However, biological functions of the PDGFRα-mediated signaling in modulating BAT progenitor cell differentiation and metabolic functions are largely unknown. To study the role of the PDGFRα signaling in BAT adipocyte differentiation, mouse BAT progenitor (BAT1) cells were isolated from interscapular BAT (17) and subjected to differentiation using a BAT-progenitor differentiation medium. BAT1 cells are immortalized primary brown preadipocytes that exhibit a typical brown adipocyte phenotype and function with a robust UCP1 expression after differentiation (17). Along differentiation, BAT1 cells accumulated lipid droplets and progressively diminished PDGFRα expression (Fig. 1 A–D). On day 8 after differentiation, PDGFRα expression levels were barely detectable (Fig. 1 A and B). Interestingly, the expression level of PDGFRα was transiently increased on day 2 after stimulation with the differentiation medium (Fig. 1 A and B). Consistent with immunohistochemical staining, the messenger RNA (mRNA) levels of PDGFRα decreased after prolonged differentiation period, except the day 2 spike (Fig. 1 A–C). Concordantly, PDGFRα protein levels exhibited the same pattern of fluctuation (Fig. 1D). Accumulation of lipid droplets in differentiated BAT cells became apparent along differentiation (Fig. 1E).

Fig. 1.

Inhibition of PDGFRα promotes brown preadipocyte differentiation. (A) Immunostaining of BAT1 progenitor cells with PDGFRα⁺ (PRα, red), BODIPY⁺ (green), and DAPI (blue) in the presence of and absence of differentiation medium (DM). Yellow arrows point to PDGFRα⁺ cells. (B) Quantification of PDGFRα⁺ BAT1 cells stained by a specific anti-PDGFRα antibody (n = 6 to 8 random fields/group). (C) qPCR analysis of Pdgfra mRNA levels in differentiated and undifferentiation BAT1 cells (n = 9 samples/group). (D) Immunoblot analysis of PDGFRα protein levels in differentiated and nondifferentiation BAT1 cells (n = 5 samples/group). (E) Quantification of percentages of BODIPY⁺ BAT1 cells under differentiated and undifferentiated conditions (n = 6 random fields/group). (F) Staining and quantification of differentiated and undifferentiated BAT1 cells with Oil Red O and hematoxylin. Pdgfra-siRNA- and scramble-siRNA-transfected BAT1 cells under the differentiation condition were also analyzed (n = 6 random fields/group). Arrows point to differentiated mature brown adipocytes. (G) qPCR analysis of Pdgfra mRNA level in Pdgfra-siRNA- and scramble-siRNA-transfected BAT1 cells. (n = 4 samples/group). (H) qPCR analysis of mRNA levels of browning makers, including Cidea, Cox4, Cox7a, Ppargc1a, and Ucp1 in BAT1 cells treated with DM⁻, DM⁺-Non-Treatment (NT), DM⁺-Scramble, and DM⁺-si-Pdgfra (n = 4 samples/group). (I) Staining and quantification of differentiated and undifferentiated BAT1 cells with Oil Red O and hematoxylin. Anti-PDGFRα (αPRα)- and NIIgG-treated BAT1 cells under the differentiation condition were analyzed (n = 6 random fields/group). Arrows point to differentiated mature brown adipocytes. (J) qPCR quantification of mRNA levels of browning makers, including Cidea, Cox4, Cox7a, Ppargc1a, and Ucp1 in DM⁻-, DM⁺-NT-, DM⁺-Vehicle-, and DM⁺ αPRα-treated BAT1 cells (n = 4 samples/group). *P < 0.05; **P < 0.01; ***P < 0.001; ns = not significant. Data are presented as means ± SEM. Scale bars in A, F, and I: 50 μm.

We next employed PDGFRα blockades to BAT1 progenitor differentiation experimental settings. Inhibition of PDGFRα by Pdgfra-small interfering RNA (siRNA) markedly increased the number of the differentiated cell population under the differentiation condition (Fig. 1F). The siRNA knockdown effect was confirmed by qPCR (Fig. 1G). Reconciling with the phenotypic alteration of BAT1 cells, mRNA levels of browning markers, including Cidea, Cox4, Cox7a, Ppargc1a, and Ucp1 were also significantly increased by knocking down the Pdgfra (Fig. 1H). Similar to the Pdgfra-siRNA knockdown approach, blocking PDGFRα by an anti-mouse PDGFRα neutralizing antibody (PDGFRα blockade) (39–41) also markedly enhanced BAT1 cell differentiation (Fig. 1 I and J). Additionally, PDGFRβ functions were also inhibited by the siRNA and neutralizing antibody approaches, and similar enhancements of BAT1 cell differentiation were observed, albeit a slightly weaker effect was observed (SI Appendix, Fig. S1). These data indicate that anti-PDGFRα markedly increases the potency of BAT1 cell differentiation toward a brown adipocyte lineage.

Increase of BAT Mass and Activation by Anti-PDGFRα Treatment.

To study the impact of PDGFRα blockade on BAT tissue mass and activation, adult C57BL/6 mice received PDGFRα blockade treatment under thermoneutrality and cold exposure according to the experimental design (Fig. 2A). The experimental animals were adapted at 18 °C for 1 week, followed by randomized division into 30 °C and 4 °C groups for exposure of consecutive 4 week. After the 4-week exposure, markedly increased BAT masses were detected in both 30 °C- and 4 °C-exposed groups (Fig. 2B). Under thermoneutrality, the average BAT mass of the anti-PDGFRα-treated group was substantially larger than that of the nonimmune IgG (NIIgG)-treated group (Fig. 2B). Approximately, 25% and 37.5% increases of the BAT mass by PDGFRα blockade were detected under thermoneutrality and cold exposure, respectively (Fig. 2C). These data demonstrate that anti-PDGFRα treatment substantially increases the BAT mass in adult mice.

Fig. 2.

Anti-PDGFRα treatment increases BAT mass and thermogenic activation. (A) Anti-PDGFRα treatment schedule under cold and thermoneutral conditions. (B) Morphology and quantification of BAT. Representative BAT in anti-PDGFRα (αPRα)- and NIIgG-treated adult animals under cold and thermoneutral acclimation. Bar: 1 cm. Arrows point to the αPRα-treated BAT in the cold and thermoneutral groups. BAT weight was quantified in each group (n = 4 to 5 samples/group). (C) Percentage increases of BAT mass were quantified in the αPRα-treated groups versus the NIIgG-treated control groups (n = 4 to 5 samples/group). (D) Histological and immunohistochemical analyses of BAT adipocyte morphology (H&E), adipocyte boundaries (MCT1⁺), PERILIPIN (PERI⁺), and microvessel density (CD31⁺) (n = 4 to 5 samples/group). Double-headed arrows mark adipocyte diameters. Yellow arrows point to CD31⁺ structures. (E) Quantification of BAT adipocyte (AC) numbers, average adipocyte (Ave. AC) diameters, and CD31⁺ areas (n = 5 to 10 random fields/group). (F) Immunohistochemical staining of PDGFRα (PRα), MYF5, SCA1, COX4, and UCP1 in the αPRα- and NIIgG-treated adult animals under cold and thermoneutral acclimation. Arrows point to positive signals. PERI (green) and DAPI (blue) were used for counterstaining. (G) Quantification of MYF5⁺PRα⁺, PRα⁺, MYF5⁺, SCA1⁺, COX4⁺, and UCP1⁺ signals (n = 4 to 10 random fields/group, 4 to 5 animals/group). (H) Quantification of total amounts of UCP1 (n = 3 to 4 samples/group). (I) qPCR quantification of mRNA levels of browning makers of Cidea, Cox4, Cox7a, Ppargc1a, and Ucp1 (n = 4 samples/group). (J) qPCR quantification of mRNA levels of BAT Pdgfra, Ly6a, and Pecam1 (n = 4 samples/group). *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as means ± SEM. Scale bars in B: 1 cm; scale bars in D and F: 50 μm.

Because both increases of adipocyte sizes and numbers might significantly contribute to the megaBAT mass, we next performed histological and immunohistochemical staining. Hematoxylin and eosin (H&E) staining showed that the total number of BAT adipocytes, but not sizes, were significantly increased under thermoneutrality and cold in the anti-PDGFRα-treated group relative to their respective controls (Fig. 2D). Under 30 °C, the anti-PDGFRα-treated BAT contained multivacuolar structures and the average diameter of adipocytes decreased. Cold exposure augmented a high density of intracellular structures (Fig. 2D). In order to define adipocyte sizes and numbers, monocarboxylate transporter 1 (MCT1) as a plasma membrane protein expressed in BAT adipocytes was used for immunohistochemical staining. MCT1 was previously reported to successfully define adipocyte boundaries in mouse BAT (42). Interestingly, under thermoneutrality, the average adipocyte sizes in anti-PDGFRα-treated BATs were significantly smaller than the NIIgG-treated controls (Fig. 2 D and E). Similarly, average BAT adipocyte sizes under 4 °C in the anti-PDGFRα-treated group were also smaller compared with the relevant controls (Fig. 2 D and E). Increases of the total BAT mass and decreases of the average BAT adipocyte size suggested the increase of adipocyte numbers as a mechanism for generating megaBAT. Indeed, MCT1⁺ adipocyte numbers were markedly increased in anti-PDGFRα-treated groups relative to their controls (Fig. 2 D and E). Additionally, CD31⁺ microvessels were increased under cold exposure, although PDGFRα blockade treatments had no impact on microvessel density (Fig. 2 D and E).

Consistent with the in vitro differentiation assay, anti-PDGFRα treatment significantly mitigated the expression PDGFRα levels under thermoneutrality and cold conditions (Fig. 2 F and G), supporting the fact that PDGFRα blockade induced progenitor cell differentiation. Indeed, MYF5 as a BAT progenitor marker (27) was markedly decreased in PDGFRα blockade–treated BATs under both thermoneutrality and cold exposure (Fig. 2 F and G). Independent data using the stem cell marker SCA1 validated the decrease of BAT progenitor cell populations in anti-PDGFRα-treated groups under 4 °C and 30 °C (Fig. 2 F and G). These findings demonstrate that inhibition of PDGFRα induces progenitor cell differentiation into mature BAT adipocytes.

Notably, anti-PDGFRα-treated 4 °C BAT showed significant increases of mitochondrial contents and UCP1 expression relative to controls (Fig. 2 F and G). In order to accurately define the total amounts of UCP1 in the entire BAT tissue, a previous published quantitative and standard method was employed using immunoblot-based methodology (43). Quantification of total amounts of UCP1 showed that anti-PDGFRα treatment under 4 °C markedly increased the total UCP1 protein in megaBAT (Fig. 2H). It should be emphasized that the anti-PDGFRα-treated BAT under 4 °C showed the highest levels of COX4⁺ mitochondria and UCP1. In concordance with protein levels, quantitative measurements of browning markers, including Cidea, Cox4, Cox7a, Ppargc1a, and Ucp1 validated the PDGFRα blockade–augmented BAT activation under cold exposure (Fig. 2I). Similarly, mitigation of Pdgfra and Ly6a expression levels and increase of Pecam1 under 4 °C were also corroborated in the PDGFRα blockade–treated group (Fig. 2J). However, tissue expression levels of PDGFRβ⁺ and NG2⁺ fibrotic cells, F4/80⁺ macrophages, CD80⁺ M1-macrophages, and CD206⁺ M2-macrophages remained unchanged (SI Appendix, Fig. S2). Together, these findings demonstrate that blocking PDGFRα induces a megaBAT phenotype in adult animals.

Defining miR-485 as an Endogenous Pdgfra Regulator for BAT Progenitor Differentiation.

The discovery of inhibiting PDGFRα in instigating BAT progenitor differentiation and increase of BAT mass promoted us to define endogenous in vivo pathways that antagonize the PDGFRα blockade signaling. For this purpose, we performed a microRNA and total mRNA array using the stromal-vascular fraction (SVF) isolated from the β3-adrenoreceptor agonist CL-316,243-treated WAT in C57BL/6 mice. Female 8-week-old mice received an intraperitoneal daily injection of CL-316,243 at a dose of 1 mg/kg for consecutive 3 d. At the end of the day, CL-316,243-treated mice were euthanized and WATs were dissected. Fresh WATs were immediately minced and digested to isolate SVF. Isolated miRNAs and total RNAs from SVF were subjected to microRNA array analysis and mRNA microarray. Among all microRNAs that targeted growth factor signaling pathways, microRNA-485 (miR-485) was identified as a unique PDGFRα regulating microRNAs in humans and mice (Fig. 3 A and B). Expression levels of miR-485 in browning WAT were markedly decreased (Fig. 3A), which is similar to most other miRNAs that target the 3′ untranslated region (3′ UTR) of target mRNAs to induce mRNA degradation and translational repression. miR-485 binds to the 3′ UTR region of Pdgfra to form a double-stranded RNA, which is more susceptible for degradation (44, 45). Consistent with reduction of miR-485, a genome-wide expression profiling analysis showed that Pdgfra was markedly increased in browning WAT (Fig. 3C). A 1.65-fold increase of Pdgfra existed in the browning WAT relative to the control.

Fig. 3.

miR-485 as a bona fide inhibitor for PDGFRα. (A) Heatmap analysis of miRNAs targeting growth factors in browning WAT (n = 5 mice/sample; n = 2 samples/group). The expression levels less than twofold miRNAs are listed, and miR-485 was identified as a Pdgfra-specific miRNA. CL = CL 316,243. GF = growth factor. (B) Detailed alignment of miR-485 against the Pdgfra 3′ UTR mouse and human origins. × marks nucleotides that were mutated to generate Mut-mPdgfra and Mut-hPdgfra. (C) Genome-wide microarray analysis in fold changes of PDGF ligands and receptors of 3-d CL 316,243-treated WAT-SVF (n = 4 mice/sample; n = 3 samples/group). (D) Quantification of Renilla luciferase activity normalized to Firefly luciferase activity in the miR-485 mimic-treated HEK293T cells that were transfected with psiCHECK-2 vector, psiCHECK-2-mPdgfra, psiCHECK-2-hPdgfra, psiCHECK-2-Mut-mPdgfra, or psiCHECKTM-2- Mut-hPdgfra (n = 3 samples/group). (E) qPCR quantification of miR-485 levels in CL 316,243-treated and control BAT-SVFs (n = 3 samples/group). Sno-202 was used as an internal control. (F) qPCR quantification of Pdgfra levels in CL 316,243-treated and control BAT-SVFs (n = 3 samples/group). Actb was used as an internal control. (G) qPCR quantification of Pdgfra in miR-485 mimic (miR-485)-transfected and miR-scrambled negative control (miR-NC)-transfected BAT1 cells (n = 4 samples/group). Immunoblotting and quantification of PDGFRα in miR-485- and miR-NC-transfected BAT1 cells (n = 3 samples/group). (H) qPCR quantification of Pdgfra in the miR-485 inhibitor (Inh-miR-485)-transfected and miR-scrambled NC (Inh-miR-NC)-transfected BAT1 cells (n = 6 samples/group). Immunoblotting and quantification of PDGFRα in Inh-miR-485- and Inh-miR-NC-transfected BAT1 cells (n = 3 samples/group). (I) Staining and quantification of differentiated and undifferentiated BAT1 cells with Oil Red O and hematoxylin. miR-485- and miR-NC-transfected BAT1 cells under the differentiation condition were analyzed (n = 6 random fields/group). Arrows point to differentiated mature brown adipocytes. (J) Staining and quantification of differentiated and undifferentiated BAT1 cells with Oil Red O and hematoxylin. Inh-miR-485- and Inh-miR-NC-transfected BAT1 cells under the differentiation condition were analyzed (n = 6 random fields/group). Arrows point to differentiated mature brown adipocytes. (K) qPCR quantification of mRNA levels of browning makers in miR-485- and miR-NC-transfected BAT1 cells under the differentiation condition, including Cidea, Cox4, Cox7a, Ppargc1a, and Ucp1 in DM⁻-, DM⁺-NT-, DM⁺-miR-NC-, and DM⁺ miR-485-treated BAT1 cells (n = 4 samples/group). (L) qPCR quantification of mRNA levels of browning makers in Inh-miR-485- and Inh-miR-NC-transfected BAT1 cells under the differentiation condition, including Cidea, Cox4, Cox7a, Ppargc1a, and Ucp1 in DM⁻-, DM⁺-NT-, DM⁺-Inh-miR-NC-, and DM⁺ Inh-miR-485-treated BAT1 cells (n = 4 samples/group). *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as means ± SEM. Scale bars in I and J: 50 μm.

To further validate Pdgfra as a target of miR-485, we constructed a luciferase reporter system to dictate the luciferase signals in the presence and absence of miR-485. In HEK293T cells, cotransfection of a miR-485 mimic with a human or mouse Pdgfra 3′ UTR luciferase reporter markedly inhibited the luciferase activity (Fig. 3D). Mutation of the critical miR-485-binding domain of human and mouse Pdgfra 3′ UTR completely abolished the suppressive activity of miR-485 (Fig. 3 B and D). Thus, miR-485 specifically targets Pdgfra and inhibits its transcription.

Next, SVF was isolated from CL-316,243-stimulated and nonstimulated BAT and quantitatively measured the expression levels of miR-485 and Pdgfra by qPCR. The level of miR-485 was immediately decreased to a barely detectable level after 1 d of stimulation with CL-316,243 (Fig. 3E). However, prolonged activation of BAT by CL-316,243 resulted in full recovery of miR-485 on day 7 (Fig. 3E). Inversely, a correspondingly reverse pattern was observed with Pdgfra expression, a transient up-regulation followed by normalization (Fig. 3F). Additionally, treatment of BAT1 progenitor cells with miR-485 markedly inhibited expression levels of PDGFRα mRNA and protein, whereas a miR-485 inhibitor further enhanced PDGFRα mRNA and protein levels (Fig. 3 G and H).

A miR-485 mimic significantly promoted BAT1 cell differentiation as seen with other PDGFRα inhibitors (Figs. 1 and 3I). A robustly opposing effect was observed with the miR-485 inhibitor, which potently blocked BAT1 cell differentiation (Fig. 3J). Similarly, the miR-485 mimic promoted activation of differentiated brown adipocytes, whereas the miR-485 inhibitor suppressed the activation (Fig. 3 K and L). Together, these results demonstrate that miR-485 is an endogenous inhibitor of PDGFRα and effectively regulates BAT progenitor differentiation through PDGFRα.

Delivery of the Pdgfra-Targeting miR-485 Induces a megaBAT Phenotype.

To further investigate the functional role of Pdgfra-targeting miR-485 in vivo, miR-485 was constructed into an adenoviral local delivery system (Adv-miR-485) for in vivo expression. Adv-miR-485 was directly injected into BAT, and the high transduction efficiency was validated by the detection of GFP expression (SI Appendix, Fig. S3). After a 2-week treatment according to the schedule shown in SI Appendix, Fig. S4A, Adv-miR-485-treated BAT exhibited significantly enlarged tissue masses relative to controls under both thermoneutrality and cold exposure (SI Appendix, Fig. S4B). Under thermoneutrality, a ∼35% increase of BAT mass by weight under thermoneutrality and 40% increase of BAT in the cold were observed (SI Appendix, Fig. S4C). These independent in vivo experimental data validated the findings using the PDGFRα blockade.

Similar to PDGFRα blockade, the Adv-miR-485-treated BATs under thermoneutrality and cold exposure contained higher numbers of smaller adipocytes compared with their relative controls as defined by MCT1 staining (SI Appendix, Fig. S4 D and E). Additionally, higher densities of microvessels were present in the 4 °C-exposed BAT (SI Appendix, Fig. S4 D and E), corroborating the phenotype of BAT activation. Consistent with BAT activation, Adv-miR-485-treated BATs showed markedly decreased PDGFRα⁺ signals, confirming preadipocyte differentiation toward a mature phenotype (SI Appendix, Fig. S4 F and G). Concordantly, the stem cell marker SCA1 and BAT progenitor marker MYF5 were significantly decreased in Adv-miR-485-treated BATs under both conditions (SI Appendix, Fig. S4 F and G). Noticeably, the Adv-miR-485-treated BAT under cold showed a significantly increased browning phenotype compared with the Adv-scrambled miR-treated BAT by expressing higher levels of the COX4⁺ mitochondrial content and UCP1 (SI Appendix, Fig. S4 F and G). The immunohistochemical findings of activation of the Adv-miR-485-treated BAT under 4 °C were further validated by quantification of total UCP1 proteins levels in the entire BAT tissues (SI Appendix, Fig. S4H). qPCR quantification using a set of browning markers, including Cidea, Cox4, Cox7a, Ppargc1a, and Ucp1 further corroborated these results (SI Appendix, Fig. S4I). Similarly, adipocyte progenitor markers Pdgfra and Ly6a were significantly mitigated in the Adv-miR-485-treated BATs (SI Appendix, Fig. S4J). Together, these findings obtained from in vivo delivery of Adv-miR-485 further strengthen our conclusions that targeting PDGFRα augments a megaBAT phenotype and activation.

Improvement of Global Metabolism and Insulin Sensitivity by Inhibition of PDGFRα.

Next, we performed functional experiments to study the impact of inhibition of PDGFRα on global metabolism under thermoneutrality and cold conditions. Despite the mass increase of BAT under the thermoneutral condition, down-regulation of PDGFRα levels produced virtually no effects on NST metabolism, blood glucose, glucose tolerance test (GTT), insulin tolerance test (ITT), and blood lipid profiles (SI Appendix, Fig. S5). These results showed that megaBAT had no impacts on global metabolic changes under thermoneutrality and browning activation is required for metabolic activity.

By contrast, under cold exposure, PDGFRα inhibition-induced megaBAT markedly improved global metabolism by enhancing NST (SI Appendix, Fig. S5 A and B). In concordance with NST improvement, down-regulation of Pdgfra by Adv-miR-485 substantially improved fasting blood glucose levels and GTT (SI Appendix, Fig. S5 C and E). Importantly, PDGFRα inhibition also markedly increased insulin sensitivity by decreasing insulin levels and ITT values (SI Appendix, Fig. S5 D and F). Additionally. PDGFRα inhibition also improved blood lipid profiles, including decreasing levels of cholesterol, triglyceride, and free fatty acid (FFA) (SI Appendix, Fig. S5G). These findings demonstrate that PDGFRα inhibition markedly improves insulin sensitivity and mitigates blood lipids under cold acclimation.

PDGFRα Inhibition–Augmented megaBAT Improves Global Metabolism and Liver Steatosis in HFD-Induced Obese Animals.

To study if inhibition of PDGFRα would also produce a megaBAT phenotype and activation in obese animals, mice were fed with HFD and doubled body weight (SI Appendix, Fig. S3B). In HFD-fed obese mice, PDGFRα inhibition produced a similar phenotype of megaBAT and robust activation under cold as seen in lean mice (Fig. 4 and SI Appendix, Fig. S4). In particular, megaBATs in obese animals exhibited elevated levels of mitochondrial content and total UCP1 expression (Fig. 4). These data indicate that the suppression of the PDGFRα signaling also creates a megaBAT that is functionally active.

Fig. 4.

miR-485 increases BAT mass and thermogenic activation in obese mice. (A) miR-485 treatment schedule under cold and thermoneutral conditions. (B) Morphology and quantification of BAT. Representative BAT in Adv-miR-485- and Adv-miR-scrambled NC (Adv-miR-NC)-treated BAT under cold and thermoneutral acclimation. Bar: 1 cm. Arrows point to the Adv-miR-485-treated BAT in the cold and thermoneutral groups. BAT weight was quantified in each group (n = 4 to 5 samples/group). (C) Percentage increases of BAT mass were quantified in Adv-miR-485- and Adv-miR-NC-treated BAT under cold and thermoneutral acclimation (n = 4 to 5 samples/group). (D) Histological and immunohistochemical analyses of BAT adipocyte morphology (H&E), adipocyte boundaries (MCT1⁺), PERILIPIN (PERI⁺), microvessel density (CD31⁺), and nuclear (DAPI) (n = 4 to 5 samples/group). Double-headed arrows mark adipocyte diameters. Yellow arrows point to CD31⁺ structures. (E) Quantification of BAT adipocyte (AC) numbers, average AC diameters, and CD31⁺ areas (n = 5 to 10 random fields/group). (F) Immunohistochemical staining PDGFRα (PRα), MYF5, SCA1, COX4, and UCP1 in Adv-miR-485-and Adv-miR-NC-treated adult animals under cold and thermoneutral acclimation. Arrows point to positive signals. PERI (green) and DAPI (blue) were used for counterstaining. (G) Quantification of MYF5⁺PRα⁺, PRα⁺, MYF5⁺, SCA1⁺, COX4⁺, and UCP1⁺ signals (n = 7 to 10 random fields/group, 4 to 5 animals/group). (H) Quantification of the total amount of UCP1 (n = 3 samples/group). (I) qPCR quantification of mRNA levels of browning makers of Cidea, Cox4, Cox7a, Ppargc1a, and Ucp1 (n = 4 samples/group). (J) qPCR quantification of mRNA levels of BAT Pdgfra, Ly6a, and Pecam1 (n = 4 samples/group). *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as means ± SEM. Scale bars in B: 1 cm; scale bars in D and F: 50 μm.

Reconciling with morphological changes, down-regulation of PDGFRα by Adv-miR-485 in cold significantly increased the NST metabolic rates (Fig. 5 A and B). Consequently, cold acclimation of the Adv-miR-485-treated animals normalized the values of hyperglycemia, hyperinsulinemia, hypercholesterolemia, and hyperlipidemia in these HFD-obese mice (Fig. 5). It should be emphasized that inhibition of PDGFRα by miR-485 reverted hepatic steatosis in obese mice (Fig. 5 H and I). In the scramble-miR-treated control mice, cold exposure alone had a significant anti-steatotic effect. However, Adv-miR-485 plus cold produced a remarkably enhanced effect against liver steatosis (Fig. 5I). Thus, suppression of PDGFRα improves both insulin sensitivity and liver steatosis in obese animals.

Fig. 5.

Improvement of metabolic dysfunction in obese mice by miRNA 485-induced PDGFRα inhibition. (A) Measurement of volume of O₂ consumption of NST-metabolism under thermoneutrality and cold in Adv-miR-485- and Adv-miR-NC-treated BAT (n = 5 mice/group). AUC = area under curve. (B) Measurement of volume of CO₂ production of NST-metabolism under thermoneutrality and cold in Adv-miR-485- and Adv-miR-NC-treated BAT (n = 5 mice/group). (C) GTT under thermoneutrality and cold in Adv-miR-485- and Adv-miR-NC-treated BAT (n = 3 to 5 mice/group). (D) ITT under thermoneutrality and cold in Adv-miR-485- and Adv-miR-NC-treated BAT (n = 4 to 5 mice/group). (E) Measurement of fasting blood glucose levels under thermoneutrality and cold in Adv-miR-485- and Adv-miR-NC-treated BAT (n = 3 to 5 mice/group). (F) Measurements of fasting blood insulin levels under thermoneutrality and cold in Adv-miR-485- and Adv-miR-NC-treated BAT (n = 4 mice/group). (G) Quantitative measurements of blood lipid profiles of cholesterol, triglyceride, FFA, and glycerol under thermoneutrality and cold in Adv-miR-485- and Adv-miR-NC-treated BAT (n = 4 to 5 mice/group). (H) Liver weight of animals under thermoneutrality and cold in Adv-miR-485- and Adv-miR-NC-treated BAT (n = 4 to 5 mice/group). (I) Liver histology and the content of Oil Red O⁺ lipid droplets under thermoneutrality and cold in Adv-miR-485- and Adv-miR-NC-treated BAT (n = 5 mice/group). Arrows point to Oil-Red-O⁺ signals. Quantification of Oil Red O⁺ signals (n = 6 to 8 random fields/group). *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as means ± SEM. Scale bars in I: 50 μm.

Genetic Deletion of Pdgfrα-Targeting miR-485 Aggravates Metabolic Dysfunction by Mitigating BAT Activation in HFD-Induced Obese Mice.

To further study the impact of suppression of PDGFRα on global metabolism, miR-485 was genetically deleted from mice. Genetic removal of miR-485 significantly reduced the number of BAT adipocytes in adult mice. The average BAT adipocyte size of miR-485 knock-out (KO) mice was significantly larger relative to that in wild-type mice (SI Appendix, Fig. S6A). Expression of several NST-related gene products, including Cox4, Ucp1, and Cox7a was significantly reduced (SI Appendix, Fig. S6B). Similarly, the total amount of UCP1 protein in miR-485 KO mice was significantly decreased (SI Appendix, Fig. S6B). By contrast, expression levels of stem cell markers such as Pdgfra and Ly6a were elevated in BAT of miR-485 KO mice (SI Appendix, Fig. S6C). Immunohistochemical analysis of BAT corroborated the mRNA alterations, showing up-regulation of PDGFRα and SCA1 and mitigation of UCP1 and COX4 in miR-485 KO mice (SI Appendix, Fig. S6 D–F). Similarly, the MYF5⁺ population was significantly increased in miR-485 KO mice. Importantly, in response to cold exposure, the blood glucose level of GTT in miR-485 KO mice were significantly higher relative to control animals (SI Appendix, Fig. S6 G and H). miR-485 KO animals also showed insulin resistance by elevation of ITT values under cold acclimation (SI Appendix, Fig. S6 I and J). Together, these results demonstrate that genetic deletion of Pdgfra-targeting miR-485 produces global metabolic dysfunction by mitigating BAT activation.

Feeding the miR-485 KO mice with HFD also resulted in increases in average BAT adipocyte sizes and cell number reduction (Fig. 6A). The total amount of UCP1 protein in miR-485 KO mice was significantly decreased (Fig. 6B). The BAT browning phenotype was markedly alleviated by expressing reduced levels of Cidea, Cox4, Cox7a, Ppargc1a, and Ucp1 (Fig. 6B). In miR-485 KO mice, BAT Pdgfra and Ly6a stem cell markers were markedly increased (Fig. 6C). In concordance with mRNA alterations, levels of UCP1 and COX4 proteins in miR-485 KO BAT were markedly mitigated, whereas MYF5, PDGFRα, and SCA1 were elevated (Fig. 6 D–F). The metabolic rate of oxygen consumption was significantly reduced in miR-485 KO mice relative to control animals (Fig. 6G). Strikingly, the blood glucose and GTT were markedly higher in miR-485 KO mice compared with control mice (Fig. 6 H and I). Consequently, miR-485 KO mice exhibited insulin resistance (Fig. 6 J and K). These genetic studies provide convincing evidence to support the concept of suppression of PDGFRα for improving global metabolic dysfunctions in HFD-induced obese mice.

Fig. 6.

Genetic deletion of miR-485 exacerbates global metabolic dysfunction in obese animals. (A) Histological and immunohistochemical analyses of BAT adipocyte morphology (H&E) and adipocyte boundaries (MCT1⁺). Double-headed arrows mark adipocyte. Quantification of BAT adipocyte numbers and sizes in adult wild type (WT) and miR-485 KO HFD-obese mice (n = 5 to 7 random fields/group). (B) Quantification of the total amount of UCP1 (n = 3 samples/group). qPCR quantification of mRNA levels of browning makers of Cidea, Cox4, Cox7a, Ppargc1a, and Ucp1 (n = 4 samples/group). (C) qPCR quantification of mRNA levels of BAT Pdgfra and Ly6a (n = 4 samples/group). (D) Immunohistochemical staining of PDGFRα (PRα), MYF5, SCA1, COX4, and UCP1 in BAT of WT and miR-485 KO HFD-obese mice. Arrows point to positive signals. PERILIPIN (green) and DAPI (blue) were used for counterstaining. (E) Quantification of MYF5⁺PRα⁺, PRα⁺, MYF5⁺, and SCA1⁺ signals (n = 6 to 10 random fields/group, 4 animals/group). (F) Quantification of COX4⁺ and UCP1⁺ signals (n = 8 random fields/group, 4 animals/group). (G) Measurement of volume of O₂ consumption of NST metabolism under cold in obese WT and miR-485 KO (n = 3 mice/group). (H) Measurement of fasting blood glucose levels during GTT under cold in obese WT and miR-485 KO (n = 8 mice/group). (I) Quantification of GTT AUC under cold in obese WT and miR-485 KO (n = 8 mice/group). (J) Percentages of basal glucose levels during ITT under cold in obese WT and miR-485 KO (n = 5 mice/group). (K) Quantification of ITT AUC under cold in obese WT and miR-485 KO (n = 5 mice/group). *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as means ± SEM. Scale bars in A and D: 50 μm.

Discussion

BAT is probably the most effective organ for dissipating energy. It has been estimated that if the 60 g of supraclavicular BAT depot was fully activated in an adult human, it would dissipate a relatively large amount of energy equivalent to approximate 4.0 kg of adipose tissue per year (3, 46). If the BAT mass can be expanded to 150 g, slightly over the double amount, the fully activated BAT would burn 10 kg of adipose tissue each year. Thus, expansion of the BAT mass is one of the most effective approaches for energy expenditure and losing body weight. Moreover, activation of a large BAT depot would also markedly reduce blood glucose. In a healthy adult, a rate of 12.2 μmol of glucose uptake per 100 g per min is estimated, corresponding to a total amount of 7.7 μmol of a 63-g depot (47). On the basis of this calculation, activation of BAT provides an attractive approach for treating hyperglycemia-associated metabolic syndromes such as T2DM. Unfavorably, obese and diabetic humans have been reported to possess less amounts of BAT (5, 48, 49).

Unlike WAT that constantly experiences expansion and shrinkage during the entire adulthood, the BAT mass remains nearly unchanged in adults. At this time of writing, the mechanism underlying BAT homeostasis and signaling pathways sustaining the perpetual tissue mass are unknown. Increasing evidence demonstrates the presence of specialized progenitor cells in BAT, and these BAT progenitor cells have potentials to differentiate into mature and functional BAT adipocytes (26–28, 34). What are the signals to control BAT progenitor differentiation and to prevent their differentiation by maintaining stemness features? Our present findings provide compelling evidence of the existence of inhibitory signals for preventing BAT progenitor cell differentiation, which we name “stemness gatekeeper” signaling. In this case, PDGFRα is an example of the gatekeeper signaling that determines the fate of BAT progenitors.

Amplification of the BAT stem cell reservoir prior to differentiation makes seamless sense for ensuring the homeostatic level of adipose progenitors and a sufficiently increased number of differentiated mature BAT adipocytes (Fig. 7). Thus, amplifying the stem cell number is a prerequisite for subsequent differentiation into mature BAT adipocytes (Fig. 7). According to this concept, the PDGFRα gatekeeper signaling that determines progenitor cell proliferation and differentiation should be tightly balanced by optimally regulated stimulatory and inhibitory signals. For uncovering this balancing mechanism, we show that miR-485 targets PDGFRα and accurately controls its expression in a temporospatial manner. miR-485 serves as an additionally regulatory machinery or safeguard to ensure the optimal expression and the gatekeeper role of PDGFRα in controlling BAT progenitor proliferation and differentiation. While a decreased level of miR-485 promotes BAT progenitor proliferation, high miR-485 levels drive progenitor cell differentiation. Again, the miR-485 machinery is controlled by physiopathological demands of energy deposition or expenditure of BAT.

Fig. 7.

Mechanistic diagram of the stemness gatekeeper of PDGFRα in controlling BAT mass and thermogenic metabolism. Under physiological conditions in adults, the optimal activation of the PDGFRα signaling by the basal levels of PDGF ligands sufficiently maintains the homeostatic numbers of BAT stem cells, which sustain the immobilized BAT mass. Inhibition of PDGFRα permits the differentiation of BAT progenitors to become mature BAT adipocytes and thus increases the total number of mature adipocytes. Expansion of mature adipocytes produces a megaBAT. Activation of megaBAT is dependent on secondary BAT activation signals such as cold exposure and sympathetic activation by various drugs and agents. Without BAT activation signals, megaBAT remains in a metabolically inert state. BAPC = BAT adipocyte progenitor cell.

Along with the differentiation of BAT progenitors, expansion of BAT in adult animals by inhibiting the PDGFRα signaling is achieved by increasing the number of mature BAT adipocytes but not by enlarging adipocyte sizes. In fact, the enlarged megaBAT by PDGFRα inhibition consists of increased numbers of smaller adipocytes relative to controls. Independent evidence against the enlarged BAT adipocyte size is that lipid droplets in megaBAT are mitigated compared with controls. These findings provocatively demonstrate that the adult tissue mass of BAT can be manipulated by targeting the PDGFRα signaling–regulated stem cell differentiation. If so, a concept of in vivo stem cell differentiation in adults is proposed to augment metabolic functions of BAT. This concept is provocative because it is believed that the tissue mass of adult BAT remains homeostatically unchanged, except with a slight fluctuation under cold exposure owing to the content of lipid droplets. Using both genetic and pharmacological approaches, we provide convincing evidence that the expansion of BAT mass can be achieved without cold exposure. It appears that the PDGFRα gatekeeper controls differentiative signals of BAT progenitor cells. It does not provide signals for BAT activation, and additional signals are required to activate thermogenic metabolism. For example, under cold exposure, the differentiated BAT cells accelerate metabolic rates in megaBAT. Thus, these newly differentiated BAT cells are fully functional, which are indistinguishable from the preexisting BAT adipocytes.

Although PDGFRα⁺ progenitor cells actively participate in BAT and beige adipogenesis as shown by our present data and other published work (50, 51), we cannot exclude the possibility of PDGFRα⁻ preadipocytes and smooth muscle–derived preadipocyte populations in BAT adipogenesis in our experimental system. In particular, ∼10 to 20% of adipocytes arising from a PDGFRα-negative lineage have been reported (52). It is highly possible that PDGFRα loss of function or miR-485 gain of function increase the contribution of PDGFRα-negative, vascular smooth muscle–derived preadipocytes to the pool of mature, thermogenic fat cells. This possibility warrants further investigation in future studies. Also, BAT1 cells are a mixture of multiple cell types and alterations in PDGFRα signaling could result in changes in the proliferation of multiple cell types that may indirectly influence adipocyte progenitor proliferation and differentiation. Again, we cannot exclude the indirect effect of PDGFRα inhibition on other progenitor cells in differentiation.

In the HFD-induced obese and diabetic mouse model, we show that the inhibition of PDGFRα-induced megaBAT markedly improves global metabolic dysfunction relative to regular BAT. Insulin sensitivity and hyperglycemia are substantially normalized in megaBAT mice. On the basis of these data, we reasonably speculate that the expansion of BAT mass by targeting the PDGFRα signaling provides an effective paradigm for treating T2DM, metabolic complications, and other metabolic disorders. This therapeutic approach is harmless because BAT is physiologically present in our body and remains thermogenically inert, except by activation of the sympathetic system by cold exposure and other agents. Expansion of BAT mass is particularly important for obese and diabetic individuals because of smaller BAT masses in their bodies. An extended speculation is that creating megaBAT by targeting PDGFRα in combination with other existing therapeutic modalities would be likely to produce additive or even synergistical effects for treating T2DM. This possibility warrants future validation. Together, our work provides a concept of expanding the BAT mass in adults by progenitor cell differentiation and a possible therapeutic paradigm for effective treatment of metabolic diseases. Although we provide the PDGFRα signaling as an example of stemness gatekeeper, we believe that other signaling molecules independent from the PDGF system may also act as a gatekeeper since intricate regulatory systems exist in controlling adipose progenitor differentiation.

Materials and Methods

Animals.

Six-week-old female C57BL/6 mice, NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice, male C57BL/6 mice at the age of 4 to 5 months were fed with HFD or normal diet and miR-485 KO mice were used in this study. See SI Appendix for antibodies and adenovirus treatment, glucose and ITTs, indirect calorimetry, and blood lipid profile.

In Vitro Experiment.

Mouse BAT1 preadipocytes were maintained in Dulbecco’s modified Eagle medium/F-12 supplemented with 10% fetal bovine serum (FBS). See SI Appendix, Materials and Methods for cell differentiation assay, RNA transfection, RNA isolation, qPCR, immunoblotting, total UCP1 protein quantification, H&E staining, immunohistochemistry, cell isolation from adipose tissues, flow cytometry cell counting, dual luciferase reporter assay, and microarray analysis.

Statistical Analysis.

Quantitative values were presented as mean determinants (±SEM. For two-group comparisons, a two-sided unpaired Student’s t-test was performed. For multiple comparisons, the one-way ANOVA statistical analysis was employed. More details are provided in SI Appendix, Materials and Methods.

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix.