Korean J Physiol Pharmacol.  2021 Jul;25(4):355-363. 10.4196/kjpp.2021.25.4.355.

Deletion of adipose triglyceride lipase abolishes blood flow increase after β3-adrenergic stimulation in visceral adipose tissue of mice

• Lee HJ
• Jin BY
• Park MR
• Seo KS
• Jeong YT
• Choi SH
• Kim DH

• KMID: 2516874
• DOI: http://doi.org/10.4196/kjpp.2021.25.4.355

Abstract

Dynamic changes in adipose tissue blood flow (ATBF) with nutritional status play a role in the regulation of metabolic and endocrine functions. Activation of the sympathetic nervous system via β-adrenergic receptors (β-AR) contributes to the control of postprandial enhancement of ATBF. Herein, we sought to identify the role of each β-AR subtype in the regulation of ATBF in mice. We monitored the changes in visceral epididymal ATBF (VAT BF), induced by local infusion of dobutamine, salbutamol, and CL316,243 (a selective β1-, β2-, and β3-AR agonist, respectively) into VAT of lean CD-1 mice and global adipose triglyceride lipase (ATGL) knockout (KO) mice, using laser Doppler flowmetry. Administration of CL316,243, known to promote lipolysis in adipocytes, significantly increased VAT BF of CD-1 mice to a greater extent compared to that of the vehicle, whereas administration of dobutamine or salbutamol did not produce significant differences in VAT BF. The increase in VAT BF induced by β3-AR stimulation disappeared in ATGL KO mice as opposed to their wild-type (WT) littermates, implying a role of ATGL-mediated lipolysis in the regulation of VAT BF. Different vascular reactivities occurred despite no significant differences in vessel density and adiposity between the groups. Additionally, the expression levels of the angiogenesis-related genes were significantly higher in VAT of ATGL KO mice than in that of WT, implicating an association of ATBF responsiveness with angiogenic activity in VAT. Our findings suggest a potential role of β3-AR signaling in the regulation of VAT BF via ATGL-mediated lipolysis in mice.

Keyword

Adipose tissue blood flow; Adipose triglyceride lipase; Beta adrenergic receptors; Lipolysis; Vasculature

Figure

• 

  Fig. 1 Effects of β-adrenergic receptor subtypes on the regulation of visceral adipose tissue blood flow (VAT BF) in CD-1 mice. (A) Experimental timeline diagram. After stabilizing VAT BF for 5 min, vehicle and agonists (10–4 and 10–2 M) were infused into VAT a rate of 0.5 µl min–1 for 10 min, respectively. Arrows represent the time point of the switch in the agonist delivery. (B) Changes in VAT BF induced by stimulation of dobutamine, a selective β1-adrenergic receptor agonist. (C) Changes in VAT BF induced by stimulation of salbutamol, a selective β2-adrenergic receptor agonist. (D) Changes in VAT BF induced by stimulation of CL316,243, a selective β3-adrenergic receptor agonist. *p < 0.05.

• 

  Fig. 2 Distribution and colocalization of β-adrenergic receptor subtypes in visceral epididymal fat (VAT) of mice. (A) Comparison of the expression levels of Adrb1, Adrb2, and Adrb3 in VAT of CD-1 male mice fed a normal chow diet at 8 weeks of age. The ΔCt method (2–ΔCt) was used to calculate the relative expression level of each gene. (B–D) Comparison of the expression levels of Adrb1 (B), Adrb2 (C), and Adrb3 (D) between fractionated adipocytes and stromal vascular fraction (SVF) in VAT of C57BL/6N male mice fed a normal chow diet at 9 weeks of age. The relative mRNA expression was calculated using the ΔΔCt method (2–ΔΔCt). (E, F) Representative images of double immunofluoresnce staining of whole-mount VAT using ADRB1, ADRB2, or ADRB3 antibodies with PECAM1 antibodies (E) or BODIPY (F) dye in C57BL/6N male mouse fed a normal chow diet at 8 weeks of age. White arrowheads indicate localization of ADRB2 in PECAM1+ vessels. Images are shown at a magnification of ×500. Scale bars, 20 µm. *p < 0.05.

• 

  Fig. 3 Changes in visceral adipose tissue blood flow (VAT BF) to β3-adrenergic receptor stimulation in wild-type (WT) and adipose triglyceride lipase (ATGL) knockout (KO) mice. (A–D) Phenotype comparison of body weight (A), total fat mass (B), fat pad weights (C), and total lean mass (D) between WT and ATGL KO mice at 10 weeks of age. (E) Timeline diagram for the experiment on changes in VAT BF by stimulation of β3-adrenergic receptor agonist CL316,243. Arrows represent the time point of the switch in the agonist delivery. (F) Comparison of changes in VAT between the vehicle (n = 8) and CL316,243 (n = 8) infusion in WT mice at time point 30 min. (G) Comparison of changes in VAT between the vehicle (n = 4) and CL316,243 (n = 7) infusion in ATGL KO mice at time point 30 min. *p < 0.05.

• 

  Fig. 4 Expression of β3-adrenergic receptor-mediated lipolysis associated mRNA and protein in visceral adipose tissue (VAT) of wild-type (WT) and adipose triglyceride lipase (ATGL) knockout (KO) mice. (A) Comparison of the gene expression of Adrb3, Pnpla2, and Lipe in VAT of WT and ATGL KO mice. The relative mRNA expression was calculated using the ΔΔCt method (2–ΔΔCt). (B) Western blot analysis of lipolysis associated proteins, including ADRB3, ATGL, HSL, and p-HSL Ser563. (C–E) The relative intensity of ADRB3 (C), ATGL (D), and phosphorylation of HSL on serine 563 (E). Adrb3, beta-3 adrenergic receptor; Pnpla2, Patatin Like Phospholipase Domain Containing 2 known as ATGL; Lipe, Lipase E, Hormone Sensitive Type known as hormone-sensitive lipase (HSL). *p < 0.05: WT (n = 7) vs. ATGL KO (n = 5) mice.

• 

  Fig. 5 Comparison of adipose tissue vessel morphology and angiogenesis-related mRNA expression in visceral adipose tissue (VAT) of wild-type (WT) and adipose triglyceride lipase (ATGL) knockout (KO) mice. (A) Representative images of vasculature and adipocytes of whole-mount VAT in WT and ATGL KO mice. Images are shown at a magnification of ×100. Scale bars, 100 µm. (B, C) Quantitative total vessel area (B) and vessel density normalized to average adipocyte size (C) between WT and ATGL KO mice. (D, E) Quantification of mean area of adipocyte (D) and distribution of adipocyte size (E) between WT and ATGL KO mice. (F) Comparison of the expression levels of genes related to angiogenesis in VAT of WT and ATGL KO mice. The relative mRNA expression was calculated using the ΔΔCt method (2–ΔΔCt). Hif1a, hypoxia-inducible factor 1, alpha subunit; Vegfa, vascular endothelial growth factor A; Kdr, kinase insert domain protein receptor; Fgf1, fibroblast growth factor 1; Fgf2, fibroblast growth factor 2; Pecam1, platelet endothelial cell adhesion molecule 1. *p < 0.05.

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  Fig. 6 Schematic diagram of the role of β3-adrenergic receptor-mediated lipolysis in the regulation of visceral adipose tissue blood flow. β3-adrenergic receptor (AR) signaling contributes to β-AR-mediated enhancement of visceral adipose tissue blood flow (VAT BF) in mice. Adipose triglyceride lipase (ATGL) deficiency negates the enhancement of VAT BF induced by β3-AR stimulation, suggesting the role of ATGL-mediated lipolysis in the regulation of VAT BF. This process may contribute to VAT remodeling in the development of obesity. TAG, triacylglycerol; HSL, hormone-sensitive lipase; MGL, monoglyceride lipase.

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