Subramanian Senthivinayagam1, Vlad Serbulea1, Clint M Upchurch1, Renata Polanowska-Grabowska1, Suresh K Mendu1, Srabani Sahu1, Prathiba Jayaguru2, Kevin W Aylor3, Mahendra D Chordia4, Limor Steinberg1, Nathaniel Oberholtzer1, Seichii Uchiyama5, Noriko Inada6, Ulrike M Lorenz7, Thurl E Harris1, Susanna R Keller3, Akshaya K Meher1, Alexandra Kadl2, Bimal N Desai1, Bijoy K Kundu8, Norbert Leitinger9. 1. Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA. 2. Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Virginia, Charlottesville, VA, 22908, USA. 3. Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia, Charlottesville, VA, 22908, USA. 4. Department of Radiology and Medical Imaging, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA. 5. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan. 6. Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan. 7. Department of Microbiology, Immunology and Cancer Biology, Center for Cell Clearance, the Beirne B. Carter Center for Immunology Research, USA. 8. Department of Radiology and Medical Imaging, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA; Department of Biomedical Engineering, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA. 9. Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA; Robert M Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA, 22908, USA. Electronic address: nl2q@virginia.edu.
Abstract
OBJECTIVE: Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of β-adrenergic receptor (βAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis. METHODS: In an immortalized brown pre-adipocytes cell line, Panx1 currents were measured using patch-clamp electrophysiology. Flow cytometry was used for assessment of dye uptake and luminescence assays for adenosine triphosphate (ATP) release, and cellular temperature measurement was performed using a ratiometric fluorescence thermometer. We used RNA interference and expression plasmids to manipulate expression of wild-type and mutant Panx1. We used previously described adipocyte-specific Panx1 knockout mice (Panx1Adip-/-) and generated brown adipocyte-specific Panx1 knockout mice (Panx1BAT-/-) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of 18F-fluorodeoxyglucose (18F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe. RESULTS: In brown adipocytes, Panx1 channel activity was induced either by apoptosis-dependent caspase activation or by β3AR stimulation via a novel mechanism that involves Gβγ subunit binding to Panx1. Inactivation of Panx1 channels in cultured brown adipocytes resulted in inhibition of β3AR-induced lipolysis, UCP-1 expression, and cellular thermogenesis. In mice, adiponectin-Cre-dependent genetic deletion of Panx1 in all adipose tissue depots resulted in defective β3AR agonist- or cold-induced thermogenesis in BAT and suppressed beigeing of white adipose tissue. UCP1-Cre-dependent Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis without affecting beigeing of white adipose tissue and aggravated diet-induced obesity and insulin resistance. CONCLUSIONS: These data demonstrate that Gβγ-dependent Panx1 channel activation is involved in β3AR-induced thermogenic regulation in brown adipocytes. Identification of Panx1 channels in BAT as novel thermo-regulatory elements downstream of β3AR activation may have therapeutic implications.
OBJECTIVE: Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of β-adrenergic receptor (βAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis. METHODS: In an immortalized brown pre-adipocytes cell line, Panx1 currents were measured using patch-clamp electrophysiology. Flow cytometry was used for assessment of dye uptake and luminescence assays for adenosine triphosphate (ATP) release, and cellular temperature measurement was performed using a ratiometric fluorescence thermometer. We used RNA interference and expression plasmids to manipulate expression of wild-type and mutant Panx1. We used previously described adipocyte-specific Panx1 knockout mice (Panx1Adip-/-) and generated brown adipocyte-specific Panx1 knockout mice (Panx1BAT-/-) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of 18F-fluorodeoxyglucose (18F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe. RESULTS: In brown adipocytes, Panx1 channel activity was induced either by apoptosis-dependent caspase activation or by β3AR stimulation via a novel mechanism that involves Gβγ subunit binding to Panx1. Inactivation of Panx1 channels in cultured brown adipocytes resulted in inhibition of β3AR-induced lipolysis, UCP-1 expression, and cellular thermogenesis. In mice, adiponectin-Cre-dependent genetic deletion of Panx1 in all adipose tissue depots resulted in defective β3AR agonist- or cold-induced thermogenesis in BAT and suppressed beigeing of white adipose tissue. UCP1-Cre-dependent Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis without affecting beigeing of white adipose tissue and aggravated diet-induced obesity and insulin resistance. CONCLUSIONS: These data demonstrate that Gβγ-dependent Panx1 channel activation is involved in β3AR-induced thermogenic regulation in brown adipocytes. Identification of Panx1 channels in BAT as novel thermo-regulatory elements downstream of β3AR activation may have therapeutic implications.
Authors: C Brent Wakefield; Vanessa R Lee; Danielle Johnston; Parastoo Boroumand; Nicolas J Pillon; Samar Sayedyahossein; Brooke L O'Donnell; Justin Tang; Rafael E Sanchez-Pupo; Kevin J Barr; Robert Gros; Lauren Flynn; Nica M Borradaile; Amira Klip; Frank Beier; Silvia Penuela Journal: Int J Obes (Lond) Date: 2021-12-13 Impact factor: 5.551