目的:去甲肾上腺素通过β-肾上腺素受体(βAR)-环磷酸腺苷(cAMP)-蛋白激酶A(PKA)信号级联刺激脂肪组织产热程序。我们发现,脂肪组织褐变的βAR刺激需要PKA对雷帕霉素复合物1(mTORC1)的机械靶标进行非规范激活。然而,由PKA磷酸化mTORC1激活引发的驱动这种产热反应的下游事件尚不清楚.
方法:我们使用通过/用细胞培养物中的氨基酸(SILAC)标记稳定同位素的蛋白质组学方法来表征用βAR激动剂处理的棕色脂肪细胞中的整体蛋白质磷酸化谱。我们确定了盐诱导激酶3(SIK3)作为候选mTORC1底物,并进一步测试了SIK3缺乏或SIK抑制对棕色脂肪细胞和小鼠脂肪组织中产热基因表达程序的影响。
结果:SIK3与RAPTOR相互作用,mTORC1复合体的定义组件,并且在Ser884处以雷帕霉素敏感的方式磷酸化。pan-SIK抑制剂(HG-9-91-01)在棕色脂肪细胞中的药理SIK抑制作用会增加基础Ucp1基因的表达,并在阻断mTORC1或PKA后恢复其表达。Sik3的短发夹RNA(shRNA)敲低增加,而SIK3的过度表达被抑制,Ucp1基因在棕色脂肪细胞中的表达。SIK3的调节PKA磷酸化结构域对于其抑制是必需的。棕色脂肪细胞中CRISPR介导的Sik3缺失会增加IIa型组蛋白脱乙酰酶(HDAC)的活性,并增强与产热有关的基因的表达,例如Ucp1,Pgc1α,和线粒体OXPHOS复合蛋白。我们进一步表明,HDAC4在βAR刺激后与PGC1α相互作用,并减少了PGC1α中的赖氨酸乙酰化。最后,体内良好耐受的SIK抑制剂(YKL-05-099)可以刺激产热相关基因的表达和小鼠皮下脂肪组织的褐变。
结论:综合来看,我们的数据显示,SIK3,以及其他SIK的可能贡献,作为β-肾上腺素能激活的磷酸化开关,以驱动脂肪组织产热程序,并表明需要更多的工作来了解SIK的作用。我们的发现还表明,针对SIK的动作可能对肥胖和相关的心脏代谢疾病有益。
Norepinephrine stimulates the adipose tissue thermogenic program through a β-adrenergic receptor (βAR)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling cascade. We discovered that a noncanonical activation of the mechanistic target of rapamycin complex 1 (mTORC1) by PKA is required for the βAR-stimulation of adipose tissue browning. However, the downstream events triggered by PKA-phosphorylated mTORC1 activation that drive this thermogenic response are not well understood.
We used a proteomic approach of Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) to characterize the global protein phosphorylation profile in brown adipocytes treated with the βAR agonist. We identified salt-inducible kinase 3 (SIK3) as a candidate mTORC1 substrate and further tested the effect of
SIK3 deficiency or SIK inhibition on the thermogenic gene expression program in brown adipocytes and in mouse adipose tissue.
SIK3 interacts with RAPTOR, the defining component of the mTORC1 complex, and is phosphorylated at Ser884 in a rapamycin-sensitive manner. Pharmacological SIK inhibition by a pan-SIK inhibitor (HG-9-91-01) in brown adipocytes increases basal Ucp1 gene expression and restores its expression upon blockade of either mTORC1 or PKA. Short-hairpin RNA (shRNA) knockdown of
Sik3 augments, while overexpression of
SIK3 suppresses, Ucp1 gene expression in brown adipocytes. The regulatory PKA phosphorylation domain of
SIK3 is essential for its inhibition. CRISPR-mediated Sik3 deletion in brown adipocytes increases type IIa histone deacetylase (HDAC) activity and enhances the expression of genes involved in thermogenesis such as Ucp1, Pgc1α, and mitochondrial OXPHOS complex protein. We further show that HDAC4 interacts with PGC1α after βAR stimulation and reduces lysine acetylation in PGC1α. Finally, a SIK inhibitor well-tolerated in vivo (YKL-05-099) can stimulate the expression of thermogenesis-related genes and browning of mouse subcutaneous adipose tissue.
Taken together, our data reveal that
SIK3, with the possible contribution of other SIKs, functions as a phosphorylation switch for β-adrenergic activation to drive the adipose tissue thermogenic program and indicates that more work to understand the role of the SIKs is warranted. Our findings also suggest that maneuvers targeting SIKs could be beneficial for obesity and related cardiometabolic disease.