Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA.
Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Virginia, Charlottesville, VA, 22908, USA.
Mol Metab. 2021 Feb;44:101130. doi: 10.1016/j.molmet.2020.101130. Epub 2020 Nov 25.
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.
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 (Panx1) and generated brown adipocyte-specific Panx1 knockout mice (Panx1) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of F-fluorodeoxyglucose (F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe.
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.
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.
棕色脂肪组织（BAT）专门进行产热。棕色脂肪细胞中能量向热量的转化是通过刺激β-肾上腺素能受体（βAR）依赖性信号传导和线粒体解偶联蛋白 1（UCP1）的激活来进行的。我们之前已经证明了 Pannexin-1（Panx1）通道在白色脂肪组织中的功能作用；然而，尚不清楚 Panx1 通道是否在调节棕色脂肪细胞功能中发挥作用。在这里，我们测试了 Panx1 通道参与棕色脂肪细胞激活和产热的假说。
在永生化的棕色前脂肪细胞系中，使用膜片钳电生理学测量 Panx1 电流。使用流式细胞术评估染料摄取，使用荧光光度计测定三磷酸腺苷（ATP）释放，使用比率荧光温度计测量细胞温度。我们使用 RNA 干扰和表达质粒来操纵野生型和突变型 Panx1 的表达。我们使用先前描述的脂肪细胞特异性 Panx1 敲除小鼠（Panx1）和生成棕色脂肪细胞特异性 Panx1 敲除小鼠（Panx1）来研究药理学或冷诱导产热。通过正电子发射断层扫描（PET）分析 F-氟脱氧葡萄糖（F-FDG）含量来量化棕色脂肪组织中的葡萄糖摄取。使用可植入遥测温度探头测量 BAT 温度。
在棕色脂肪细胞中，Panx1 通道活性通过凋亡依赖性半胱天冬酶激活或通过β3AR 刺激诱导，后者涉及 Gβγ 亚基与 Panx1 的结合的新机制。在培养的棕色脂肪细胞中失活 Panx1 通道导致β3AR 诱导的脂肪分解、UCP-1 表达和细胞产热受到抑制。在小鼠中，脂肪细胞特异性脂联素-Cre 依赖性 Panx1 敲除导致 BAT 中β3AR 激动剂或冷诱导产热受损，并抑制白色脂肪组织的米色形成。UCP1-Cre 依赖性棕色脂肪细胞中 Panx1 的特异性缺失会降低适应性产热能力，而不影响白色脂肪组织的米色形成，并加重饮食诱导的肥胖和胰岛素抵抗。
这些数据表明，Gβγ 依赖性 Panx1 通道激活参与了棕色脂肪细胞中β3AR 诱导的产热调节。鉴定 BAT 中的 Panx1 通道作为β3AR 激活的新型热调节元件可能具有治疗意义。
