Distinct cellular and molecular mechanisms for β3 adrenergic receptor-induced beige adipocyte formation

Abstract

Beige/brite adipocytes are induced within white adipose tissues (WAT) and, when activated, consume glucose and fatty acids to produce heat. Classically, two stimuli have been used to trigger a beiging response: cold temperatures and β3-adrenergic receptor (Adrb3) agonists. These two beiging triggers have been used interchangeably but whether these two stimuli may induce beiging differently at cellular and molecular levels remains unclear. Here, we found that cold-induced beige adipocyte formation requires Adrb1, not Adrb3, activation. Adrb1 activation stimulates WAT resident perivascular (Acta2+) cells to form cold-induced beige adipocytes. In contrast, Adrb3 activation stimulates mature white adipocytes to convert into beige adipocytes. Necessity tests, using mature adipocyte-specific Prdm16 deletion strategies, demonstrated that adipocytes are required and are predominant source to generate Adrb3-induced, but not cold-induced, beige adipocytes. Collectively, we identify that cold temperatures and Adrb3 agonists activate distinct cellular populations that express different β-adrenergic receptors to induce beige adipogenesis.

Keywords: Brite; SMA; adipose; beige adipocytes; beta adrenergic receptors; cell biology; cold exposure; developmental biology; mouse; stem cells.

Figure 1.. Differences between cold exposure and Adrb3 agonists-induced beiging.

(A) Representative H&E-stained images of sections from IGW depots from two-month-old male mice were cold exposed (6.5°C) or treated with CL (1 mg/Kg/mouse/day) for one, three or seven days (n= 6/group). (B) Rectal temperatures from six-month-old male mice maintained at room temperature, cold exposed or treated with CL for seven days (n= 10/group). (C) Sera glucose from mice described in (B). (D) Representative H&E-stained images of IGW depot sections from mice described in (B). (E) Representative Ucp1 immunohistochemistry (IHC)-stained images of IGW depot sections from mice described in (B). (F) mRNA expression of beige and thermogenic genes from mice described in (B) maintained at room temperature or cold exposed for seven days. (G) mRNA expression of beige and thermogenic genes from mice described in (B) maintained at room temperature or treated with CL for seven days. *p<0.05 unpaired t-test, two tailed: Cold exposed compared to room temperature mice. #p<0.05 unpaired t-test, two tailed: CL-treated compared to vehicle room temperature-treated mice. Scale bar = 100 μm.

Figure 1—figure supplement 1.. Differences between cold exposure and Adrb3 agonists induced beiging.

(A) Representative H&E staining of IGW depot sections from two-month-old male mice cold exposed (6.5°C) or treated with CL (1 mg/Kg/mouse/day) for one, three or seven days (n= 6/group) (lower magnification images). Scale bar = 100 µm. (B) Representative Ucp1 IHC staining of IGW depot sections from mice described in (A) (n= 6/group). Scale bar = 100 µm. (C) mRNA expression of denoted genes from IGW depots from mice described in (A) cold exposed (right) or treated with CL (left) for zero, one, three, or seven days (n= 6/group in triplicate). #p<0.002 unpaired t-test, two tailed: days of CL treatment compared to Day 0 (no treatment). *p<0.001 unpaired t-test, two tailed: days of cold exposure compared to Day 0 (no cold).

Figure 2.. Smooth muscle cells are not a source of Adrb3-induced beiging.

(A) Illustration of mice and experimental regime: two-month-old Acta2^Cre-ERT2; RosaR26^RFP male mice were administered one dose of TM for two consecutive days. Two weeks post TM, mice were randomized to vehicle, CL or mirabegron for seven days. (B) Representative images of fate mapping analysis of Acta2^Cre-ERT2-RFP+ cells into Ucp1 beige adipocytes within IGW depots from mice described in (A). (C) Illustration of mice and experimental regime: two-month-old Acta2^rtTA; TRE-Cre; RosaR26^RFP male mice were administered doxycycline (Dox) for four consecutive days. Two weeks post Dox, mice were randomized to vehicle, CL or mirabegron for seven days. (D) Representative images of fate mapping analysis of Acta2^rtTA-RFP+ cells into Ucp1 beige adipocytes within IGW depots from mice described in (C). Scale bar = 200 µm.

Figure 2—figure supplement 1.. Induction of beige adipocytes by CL316,243 and mirabegron.

(A) Representative H&E staining of IGW and PGW depot sections from two-month-old male mice were treated with vehicle, CL or mirabegron for seven days (n= 6/group). Scale bar = 100 µm.

Figure 2—figure supplement 2.. Acta2+ smooth muscle cells are not a source of Adrb3 induced beiging.

(A) Representative RFP and Ucp1 IHC staining of IGW depot sections from Acta2^Cre-ERT2; RosaR26^RFP treated with CL for seven days (n= 6/group). Scale bar = 100 µm.

Figure 2—figure supplement 3.. Acta2+ smooth muscle cells are a source for cold induced beiging but not for Adrb3.

(A) Experimental procedure to TM induce two-month-old Acta2^Cre-ERT2; RosaR26^RFP; (Acta2-RFP) male mice which were subsequently cold exposed or CL treated for 2 weeks (n = 5/group). (B) Graphical representation of Acta2-RFP fate mapping studies in response to cold exposure and CL treatments. Representative IHC images from mice described in (A) were quantified for Acta2-RFP and Ucp1 co-localization (n= 4 mice/group). (C) Representative IHC staining of IGW depot sections from cold mice described in (A). (D) Representative IHC staining of IGW depot sections from CL mice described in (A). Two representative IHC staining of IGW depots of CL-treated mice were shown to demonstrate variability throughout the tissue. All data are means ±SEM. Scale bar = 200 µm.

Figure 2—figure supplement 4.. Acta2+ smooth muscle cells are not a source of Adrb3 induced beiging at different ages.

(A) Experimental procedure and respresentative IHC staining of TM induce one-month-old Acta2^Cre-ERT2; RosaR26^RFP; (Acta2-RFP) male mice treated with CL seven days (n = 5/group). (B) Experimental procedure and respresentative IHC staining of TM induce three-month-old Acta2^Cre-ERT2; RosaR26^RFP; (Acta2-RFP) male mice treated with CL seven days (n = 5/group). (C) Experimental procedure and respresentative IHC staining of TM induce six-month-old Acta2^Cre-ERT2; RosaR26^RFP; (Acta2-RFP) male mice treated with CL seven days (n = 5/group). Scale Bar = 200 µm.

Figure 2—figure supplement 5.. Acta2 genetic tools do not mark or track Adrb3 induced beiging.

(A) I Representative RFP and Ucp1 IHC staining of IGW depot sections from Acta2^rtTA; TRE-Cre; RosaR26^RFP treated with CL for seven days (n= 6/group). Scale bar = 100 µm.

Figure 2—figure supplement 6.. Acta2 Adrb3 necessity tests.

(A) Genetic schema to generate two-month-old Acta2^Cre-ERT2; Pparg^fl/fl or DTA male mice. (B) Experimental procedure for mice described in (A) (n = 10/group). (C) Rectal temperature from mice in (A). (D) Sera glucose from mice in (A). (E) Inguinal adipose tissue (IGW) weights from mice in (A). (F) Representative H&E staining of IGW depot sections from mice described in (A). Scale bar = 100 µm. (G) Representative Ucp1 IHC staining of IGW depot sections from mice described in (A). Scale bar = 100 µm. (H) mRNA expression of beige and thermogenic gene expression from mice described in (A) (n= 6 in triplicate). ♯p<0.001 unpaired t-test, two tailed: CL compared to vehicle-treated mice (control and mutant). All data are means ±SEM.

Figure 2—figure supplement 7.. Myh11+ smooth muscle cells are not a source of Adrb3 induced beiging.

(A) Illustration of mice and experimental regime: two-month-old Myh11^Cre-ERT2; RosaR26^RFP male mice were administered one dose of TM for two consecutive days. Two weeks post TM, mice were randomized to vehicle, CL or mirabegron for seven days (n= 6/group). (B) Representative images of fate mapping analysis of Myh11^Cre-ERT2-RFP+ cells into Ucp1 [Ucp1 (green) and perilipin (blue)] beige adipocytes within IGW depots from mice described in (A) Scale bar = 100 µm. (C) Representative RFP and Ucp1 IHC of IGW depot sections from mice described in (A) treated with CL for seven days. Scale bar = 100 µm.

Figure 2—figure supplement 8.. Myh11+ smooth muscle cells are not a source of Adrb3 induced beiging.

(A) Illustration of mice and experimental regime: two-month-old Myh11^Cre-ERT2; RosaR26^RFP male mice were administered one dose of TM for two consecutive days. 30 days post TM, mice were randomized to vehicle or CL for seven days (n= 6/group). (B) Representative images of fate mapping analysis of Myh11^Cre-ERT2-RFP+ cells into Ucp1 [Ucp1 (green) and Perilipin (blue)] beige adipocytes within IGW depots from mice described in (A) Scale bar = 100 µm.

Figure 3.. Pdgfra+ cells are not required for Adrb3-induced beiging.

(A) Representative images of Pdgfra-RFP fate mapping immunostaining of IGW depots from two-month-old male Pdgfrα^Cre-ERT2; RosaR26^RFP mice. Mice were administered one dose of tamoxifen (TM) for two consecutive days and after a two-week TM washout period mouse were treated with vehicle, CL or mirabegron for seven days. White arrowheads indicate RFP+ and Ucp1 +beige adipocytes. (B) TM-pulsed Pdgfra-RFP+ cells were FACS isolated from the WAT SV compartment and antibody stained for Pdgfra and analyzed by flow cytometry (n= 6/group). (C) SV cells were isolated from TM-pulsed Pdgfra-RFP+ mice. Cells were immunostained for Pdgfra and examined for Pdgfra-RFP positivity by flow cytometry (n= 6/group). (D) Cells described in (B) were FACS isolated and examined for mRNA expression of Pdgfra. ★p<0.001 RFP+ compared to RFP- cells (n= 6/group). (E) Cells described in (B) were FACS isolated and mRNA expression of denoted genes were examined. ★p<0.001 RFP+ compared to RFP- cells (n= 6/group). (F) Illustration (left) and experimental procedure (right) used to generate TM-induced two-month-old Pdgfra^Cre-ERT2; Pparg^fl/fl (PRa-Pparg) male mice (n= 8/group). (G) Representative H&E-staining images of IGW depot sections from mice described in (F). (H) Representative Ucp1 IHC staining of IGW depot sections from mice described in (F). (I) mRNA expression of beige and thermogenic markers from IGW depots from mice described in (F) (n= 6/group in triplicate). (J) mRNA expression of beige and thermogenic markers from PGW depots from mice described in (F). †p<0.05 unpaired t-test, two tailed: PRa-Pparg CL treated compared to control CL treated. Scale bar = 200 µm.

Figure 3—figure supplement 1.. PDGFRa+ cells are not a major source of Adrb3 induced beiging.

(A) Illustration of mice and experimental regime: two-month-old Pdgfra^Cre-ERT2; RosaR26^RFP male mice were administered one dose of TM for two consecutive days. Two weeks post TM, mice were randomized to vehicle, CL or mirabegron for seven days (n= 6/group). (B) Representative lower magnification images of IGW adipose sections from mice described in (A) treated with vehicle or CL. Scale bar = 100 µm. (C) Representative images of fate mapping analysis of Pdgfra^Cre-ERT2-RFP+ cells into Ucp1 [Ucp1 (green) and Perilipin (blue)] beige adipocytes within PGW depots from mice described in (A) treated with CL. (D) Representative images immunostaining of Pdgfra-RFP (red), Acta2 (green) and DAPI (blue) from IGW depot section from TM-pulsed Pdgfra^Cre-ERT2; RosaR26^RFP male mice. Scale bar = 100 µm.

Figure 3—figure supplement 2.. PDGFRa+ necessity tests for Adrb3 induced beiging.

(A) Rectal temperatures from Pdgfra^Cre-ERT2; Pparg^fl/fl (PRa-Pparg) male mice treated with vehicle or CL for seven days (n= 10/group). (B) Sera glucose from mice in (A). (C) IGW weights from mice in (A). (D) PGW weights from mice in (A). (E) Representative H&E staining of PGW depot sections from mice described in (A). Scale bar = 100 µm. (F) Representative Ucp1 IHC staining of PGW depot sections from mice described in (A). Scale bar = 100 µm. All data are means ±SEM.

Figure 3—figure supplement 3.. Adrb3 induced beiging is not generated by an adipose progenitor cell.

(A) Genetic illustration to generate AdipoTrak mice. (B) Illustration of experimental paradigm: AdipoTrak mice were Dox suppressed from E0 until P60. Two weeks off Dox, mice were administered vehicle or CL for seven days (n= 6/group). (C) Representative immunostaining of GFP, Perilipin, and DAPI from IGW depot sections from mice described in (B). Scale bar = 200 µm.

Figure 4.. Pre-existing white adipocytes are the major source of Adrb3-induced beiging.

(A) Genetic schema of mice used to generate two-month-old Adiponectin^Cre-ERT2; RFP (Adpn-RFP) male mice (n = 6/group). (B) Experimental paradigm of TM induction and subsequent treatment with vehicle, CL or mirabegron of Adpn-RFP mice. (C) Representative images of Adpn-RFP fate mapping and Ucp1 immunostaining of IGW depot sections from mice described in (B). Scale bar = 200 µm. (D) Representative images of RFP and Ucp1 IHC of IGW depot sections from mice described in (B). Scale bar = 100 µm. (E) Quantitation of co-localized Adpn-RFP+/Ucp1+ beige adipocytes in response to CL (n= 1000 beige adipocytes). (F) Experimental procedure to stimulate beiging in vitro. (G) Representative images of Ucp1-RFP fluorescence co-localization with lipid staining (LipidTox^TM) after CL treatment of cultured white adipocytes.

Figure 4—figure supplement 1.. Pre-existing white adipocytes are the major source of Adrb3 induced beiging.

(A) FACS analysis from SV cells isolated from TM-induced Adiponectin^Cre-ERT2; RosaR26^RFP (Adpn-RFP) mice (n = 6/group). (B) Representative micrograph and RFP images from SV cells isolated from Adpn-RFP mice and TM induced in culture. (C) Experimental procedure for TM induction and CL treatment of Adpn-RFP pulse-chase experiment. (D) Representative IHC staining of Adpn-RFP fate mapping after 7 days CL treatment. (E) Cell culture experimental procedure. (F) mRNA expression of beige and thermogenic genes from cells described in (E) (n = 4/group in triplicate). ♯p<0.001 CL compared to vehicle-treated cells. ✗p<0.01 unpaired t-test, two tailed: Mirabegron compared to vehicle-treated cells. All data are means ± SEM. Scale bar 100 μm.

Figure 5.. Pre-existing white adipocytes are required for Adrb3-induced beiging.

(A) Illustration of genetic alleles used to generate two-month-old Adiponectin^Cre-ERT2; RosaR26^RFP; Prdm16^fl/fl (Adiponectin-Prdm16) male mice (n = 10/group). (B) Experimental procedure to TM induced and treated Adiponectin-Prdm16 mice. (C) IGW depot weight from mice described in (B). ★p<0.001 unpaired t-test, two tailed: Mutant CL-treated compared to control CL-treated mice. (D) Representative photographs of intact IGW depots from mice described in (B). (E) Representative H&E staining of IGW depot sections from mice described in (B). (F) Representative Ucp1 IHC staining of IGW depot sections from mice described in (B). (G) mRNA expression of beige markers from mice described in (B). ★p<0.001 unpaired t-test, two tailed: Mutant CL- treated compared to control CL-treated mice. (H) Experimental procedure for in vitro beiging. (I) Triglyceride levels in denoted cells after 24 hr vehicle or CL treatment (n = 4/group in triplicate). ♯p<0.001 unpaired t-test, two tailed: CL- treated compared to vehicle-treated cells. †p<0.05 unpaired t-test, two tailed: mutant CL-treated compared to vehicle-treated control cells. (J) mRNA expression of beige and thermogenic genes from cells described in (H) (n = 6/group in triplicate). ♯p<0.001 unpaired t-test, two tailed: CL-treated compared to vehicle-treated cells. ★p<0.001 unpaired t-test, two tailed: Mutant CL-treated compared to control CL-treated cells. All data are means ±SEM. Scale bar = 200 µm.

Figure 5—figure supplement 1.. White adipocyte necessity tests for cold and Adrb3 induced beiging.

(A) Rectal temperature from two-month-old cold-exposed Adpn-control and Adpn-Prdm16 mutant male mice (n = 10/group). (B) mRNA expression of beige and thermogenic genes from cold-exposed mice described (C) (n = 6/group in triplicate). (C) Representative H&E staining of IGW depot sections from cold-exposed mice taken at 10x and 20x magnification. Scale bar = 100 µm. (D) Representative Ucp1 IHC staining of IGW depot sections from cold-exposed mice. Scale bar = 100 µm. (E) Rectal temperatures from two-month-old Adpn-control and Adpn-Prdm16 mutant male mice after CL treatment (n = 10/group). ♯p<0.001 unpaired t-test, two tailed: CL-treated mutant compared to CL-treated control mice. (F) Sera glucose from Adpn-control and Adpn-Prdm16 mutant mice described in (E). (G) mRNA expression of Prdm16 from isolated floated adipocytes and SV cells generated from Adpn-control and Adpn-Prdm16 mutant mice (n = 4/group). ★p<0.001 unpaired t-test, two tailed: control compared to mutant. All data are means ± SEM.

Figure 5—figure supplement 2.. Acta2+ smooth muscle cells are required for cold induced beiging.

(A) Illustration of experimental procedure for TM-induced two-month-old Acta2^Cre-ERT2; RosaR26^RFP (Acta2-control) male mice and Acta2^Cre-ERT2; Prdm16; RosaR26^RFP (Acta2-Prdm16) mice. (B) mRNA expression of Prdm16 from isolated adipocytes and SV cells from mice described in (A) after TM pulse (prior to cold exposure). (C) Rectal temperatures from Acta2-control and Acta2-Prdm16 mutant mice after cold treatment. (D) Sera glucose from Acta2-control and Acta2-Prdm16 mutant mice after cold treatment. (E) H&E staining from mice described in (G). (F) Fate mapping of Acta2-RFP: Immunostaining of Acta2-RFP (red), Ucp1 (green), and Perilipin (blue) from mice described in (G). (G) mRNA expression of beige and thermogenic genes from mice described in (G). ★p<0.001 cold exposed mutant compared to cold exposed control mice. All data are means ± SEM. Scale bar = 100 µm.

Figure 6.. Adrb3 is not required for cold-induced beiging.

(A) Illustration of experimental paradigm: Two-month-old male mice were administered vehicle or SR59230A (SR59) for 5 days and then maintained at room temperature, cold exposed or treated with CL for seven days (n= 10/group). (B) Rectal temperature from mice described in (A). ★p<0.001 unpaired t-test, two tailed: cold exposed compared to room temperature vehicle-treated mice. ★★p<0.05 unpaired t-test, two tailed: cold + SR59 compared to vehicle room temperature-treated mice. (C) Sera glucose from mice described in (A). (D) Representative H&E staining of IGW depot sections from mice described in (A). (E) Representative Ucp1 IHC staining of IGW depot sections from mice described in (A). (F) mRNA expression of beige and thermogenic genes from mice described in (A) treated with vehicle and SR59 and then maintained at room temperature or cold exposed for seven days (n= 6/group in triplicate). ★p<0.001 unpaired t-test, two tailed: cold exposed compared to room temperature vehicle-treated mice. ★★p<0.05 unpaired t-test, two tailed: cold + SR59 compared to vehicle room temperature-treated mice. (G) mRNA expression of beige and thermogenic genes from mice described in (A) treated with vehicle and SR59 and then administered vehicle or CL for seven days (n= 6/group in triplicate). ♯p<0.01 unpaired t-test, two tailed: CL-treated compared to vehicle-treated mice. ✗p<0.01 unpaired t-test, two tailed: CL316 + SR59-treated mice compared to CL-treated mice. All data are means ±SEM. Scale bar = 200 µm.

Figure 6—figure supplement 1.. Inhibiting Adrb3 does not alter brown adipose tissue.

(A) H&E staining of brown adipose tissue (BAT) from mice treated with vehicle or SR59 maintained at room temperature, cold exposed or CL treated for seven days. (B) Ucp1 IHC of BAT from mice described in (A). (C) mRNA expression of BAT genes from mice described in (A) cold exposed. ★p<0.01 cold exposed to room temperature vehicle-treated mice. ★★p<0.05 cold+SR59 compared to vehicle room temperature-treated mice. (D) mRNA expression of BAT genes from mice described in (A) treated with CL. ♯ p<0.01 CL-treated compared to vehicle-treated mice. §p<0.01 CL+SR59-treated compared to vehicle-treated mice. All data are means ± SEM. Scale bar = 100 µm.

Figure 7.. Adrb1 is required for cold-induced beiging.

(A) Experimental paradigm: Two-month-old male mice were first treated with vehicle or talinolol (1 mg/Kg/day) for five days. Mice were then maintained at room temperature, treated with CL or cold exposed for seven days (n= 10/group). (B) Rectal temperature from mice described in (A). §p<0.05 unpaired t-test, two tailed: cold + talinolol compared to cold + vehicle. (C) Sera glucose from mice described in (A). §p<0.05 unpaired t-test, two tailed: cold + talinolol compared to cold + vehicle. (D) Representative H&E staining of IGW depot sections from mice described in (A). (E) Representative Ucp1 IHC staining of IGW depot sections from mice described in (A). (F) mRNA expression of beige and thermogenic genes from mice described in (A) treated with vehicle and talinolol and then administered vehicle or CL for seven days. ♯ p<0.001 unpaired t-test, two tailed: CL-treated mice compared to vehicle-treated mice. ✗p<0.001 unpaired t-test, two tailed: CL + Talinolol compared to vehicle-treated mice. (G) mRNA expression of beige and thermogenic genes from mice described in (A) treated with vehicle and talinolol and then maintained at room temperature or cold exposed for seven days. ★p<0.001 unpaired t-test, two tailed: cold compared to room temperature vehicle-treated mice. §p<0.05 unpaired t-test, two tailed: cold + talinolol compared to cold + vehicle. All data are means ±SEM. Scale bar = 200 µm.

Figure 7—figure supplement 1.. Adrb expression.

(A) SV cells were isolated from two-month-old male mice and given white adipogenic media for 6 days. mRNA was harvested at denoted times and Adrb1, 2, and 3 expression was assessed throughout adipogenesis (n= 4/group in triplicate). (B) TM pulsed (Acta2^Cre-ERT2; RFP) Acta2-RFP+ cells were FACS isolated from the WAT SV compartment and mRNA expression of denoted genes were examined (n= 6/group in triplicate). ★p<0.001 unpaired t-test, two tailed: RFP+ compared to RFP- cells. (C) TM-pulsed (Pdgfra^Cre-ERT2; RFP) Pdgfra-RFP+ cells were FACS isolated from the WAT SV compartment and mRNA expression of denoted genes were examined (n= 6/group). ★p<0.001 unpaired t-test, two tailed: RFP+ compared to RFP- cells. All data are means ± SEM.

Figure 7—figure supplement 2.. Adrb1 is required for cold-induced beiging.

(A) Rectal temperatures from two-month-old male mice treated with vehicle or talinolol (1 mg/Kg/day) for five days. Mice were then maintained at room temperature or treated with CL (1 mg/Kg/day) for seven days (n = 10/group). (B) Adipose tissue weights from mice described in (A). ♯p<0.001 CL-treated mice compared to vehicle-treated mice. ✗p<0.001 unpaired t-test, two tailed: CL +talinolol- compared to vehicle-treated mice. (C) Rectal temperatures from two-month-old male mice treated with vehicle or talinolol (1 mg/Kg/day) for five days. Mice were then maintained at room temperature or cold exposed (6.5°C) for seven days. ★p<0.001 unpaired t-test, two tailed: cold-exposed compared to room temperature vehicle-treated mice. §p<0.001 unpaired t-test, two tailed: cold exposed + talinolol-treated compared to cold-exposed mice. (D) Adipose tissue weights from mice described in (C). ★p<0.001 unpaired t-test, two tailed: cold-exposed compared to room temperature vehicle-treated mice. §p<0.001 unpaired t-test, two tailed: cold-exposed + talinolol-treated compared to cold-exposed mice.