Phosphorylation of PLIN5 is dynamically regulated in mice, required for IRS2 expression in the male mice liver, but dispensable for PGC1a target genes previously associated with PLIN5

Perilipin 5 (PLIN5) is a lipid droplet (LD) protein highly expressed in tissues that are active in fatty acid oxidation such as the heart, oxidative muscle, the liver, and brown adipocytes. PLIN5 shares homology with PLIN1: it prevents lipolysis in basal state but promotes lipolysis upon the activation of protein kinase A (PKA). In vitro studies expressing exogenous PLIN5 have shown that PLIN5 is phosphorylated at serine 155 (S155) by PKA. In addition to the regulation of lipolysis, phosphorylation of PLIN5 at S155 has been shown to regulate expression of genes in lipid metabolism, mitochondria, inflammation, and autophagy by traveling to the nucleus and activating peroxisome proliferater-activated-receptor-gamma-coactivator-1- (PGC1a). However, a role of PLIN5 phosphorylation at S155 has been primarily studied in vitro with exception of a few studies expressing exogenous wild type (WT) and phosphorylation resistant PLIN5 (S155A) in the heart or the liver of mice. Here, we aim to determine the extent of endogenous PLIN5 phosphorylation in vivo and its role in the regulation of lipid metabolism and gene expression in the liver using PLIN5 S155A knock in mice (S155A). First, we enriched LDs from the liver of fed and fasted C57BL6 WT mice to measure non-phosphorylated and S155 phosphorylated PLIN5 by LC-MS/MS. While peak area of non-phosphorylated PLIN5 was similar, S155 phosphorylation was increased in the liver from fasted mice compared with fed mice confirming that endogenous PLIN5 is phosphorylated at S155 when PKA is activated during fasting (3.0-fold increase, p<0.05). Then, we utilized Phos-tag gel as a simple method to assess the extent of PLIN5 phosphorylation. When the heart and the liver from WT and S155A mice were run on Phos-tag gel and immunoblotted for PLIN5, a phosphorylated band was significantly reduced in S155A tissues confirming that S155 is the dominant phosphorylation site in vivo. Next, we assessed phenotypes of S155A mice to gauge the necessity of S155 phosphorylation for metabolism and gene expression in the fasted liver. For the glucose tolerance test, 4-month-old male and female S155A mice showed no differences, but one year-old S115A males had impaired glucose tolerance. There was not a significant difference in weight or blood glucose in fasted young S155A females compared to wild type, but aged male S155A mice had a higher fasted blood glucose (p<0.05) without difference in weight. There was not a significant difference in serum insulin, triglycerides (TG), beta-hydroxybutyrate, or non-esterified fatty acids levels between WT and S155A mice in either males or females. The liver TG contents did not differ between WT and S155A mice of both genders. When qPCR tested the liver of fasted WT and S155A mice, Pgc1a target genes upregulated by fasting and proposed to be regulated by PLIN5 phosphorylation did not show striking difference in expression. Pgc1a, Ppara, Acot1, Pdk4, and Ascl1 expression was similar between WT and S155A liver of fasted mice. Cpt1a and G6pc showed trend of reduction in S155A liver but did not reach statistical significance (n=5 to 7). Inflammation markers such as IL1b and Ccl2 did not differ between WT and S155A liver of fasted mice either. We performed unbiased RNA sequencing of WT and S155A liver from fasted female mice to identify genes differentially regulated by phosphorylation of PLIN5 at S155. There were limited number of genes differentially expressed in S155A female livers with 3 genes < 0.05 for adjusted p value. Interestingly, RNA seq identified insulin receptor substrate 1 (Irs1) as a gene with significantly decreased expression in the liver of female S155A mice (Log2 fold change -0.74, adjusted p value 0.003). The difference was confirmed in qPCR of fasted liver for the female mice (p<0.05), but not for males. Interestingly, insulin receptor substrate 2 (Irs2) measured by qPCR was not different in the liver of female S155A mice but was reduced in male S155A mice (p<0.05). The reduction of IRS2 protein in the liver of S155A male mice was confirmed by Western blot. In conclusion, endogenous PLIN5 in the liver increases phosphorylation at S155 upon fasting in mice. However, the loss of S155 phosphorylation does not affect serum lipids or liver TG. Also, changes in the expression of PGC1a target genes were subtle in the liver of fasted S155A mice compared with WT mice indicating that the loss of PLIN5 S155 phosphorylation can be readily compensated by other mechanisms. Unexpectedly, IRS2 showed decreased expression in the liver of S155A male mice that may explain glucose intolerance in aged male mice. Thus, PLIN5 phosphorylation may impact the insulin signaling in the liver by supporting IRS2 expression in male mice. To investigate the role of PLIN5 S155 phosphorylation in vivo, C57BL/6J were obtained and Crispr Cas9 was used to introduce S155A mutation to these mice. RNAseq was done to compare gene expression in the liver of the WT C57BL/6J and the phosphorylation deficient mice.周脂素5（Perilipin 5, PLIN5）是一种脂滴（lipid droplet, LD）蛋白，在脂肪酸氧化活跃的组织中高表达，包括心脏、氧化型骨骼肌、肝脏以及棕色脂肪细胞。PLIN5与PLIN1具有同源性：其在基础状态下可抑制脂解，但在蛋白激酶A（protein kinase A, PKA）激活后会促进脂解。体外过表达外源性PLIN5的研究表明，PLIN5可被PKA磷酸化于丝氨酸155（serine 155, S155）位点。除调控脂解外，PLIN5在S155位点的磷酸化还可通过转位入核并激活过氧化物酶体增殖物激活受体γ辅激活因子1-α（peroxisome proliferator-activated-receptor-gamma-coactivator-1-α, PGC1α），进而调控脂质代谢、线粒体功能、炎症反应及自噬相关基因的表达。 然而，目前针对PLIN5 S155位点磷酸化的功能研究主要集中于体外实验，仅有少数研究在小鼠心脏或肝脏中外源性过表达野生型（wild type, WT）及磷酸化抗性PLIN5（S155A）。本研究旨在通过构建PLIN5 S155A敲入小鼠（S155A），探究内源性PLIN5在体内的磷酸化水平，及其在肝脏脂质代谢与基因表达调控中的作用。 首先，我们从禁食与正常进食的C57BL/6野生型小鼠肝脏中富集脂滴，通过液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）检测非磷酸化及S155磷酸化的PLIN5水平。结果显示，非磷酸化PLIN5的峰面积无显著差异，但禁食小鼠肝脏中S155磷酸化PLIN5的水平较进食小鼠升高3.0倍（p<0.05），证实了当禁食期间PKA激活时，内源性PLIN5会在S155位点发生磷酸化。 随后，我们利用Phos-tag凝胶作为简便方法评估PLIN5的磷酸化程度。将野生型与S155A小鼠的心脏及肝脏组织进行Phos-tag凝胶电泳并针对PLIN5进行免疫印迹，结果显示S155A组织中的磷酸化条带显著减少，证实S155是体内PLIN5的主要磷酸化位点。 接下来，我们评估了S155A小鼠的表型，以探究S155磷酸化对于禁食状态下肝脏代谢与基因表达的必要性。葡萄糖耐受实验结果显示，4月龄的雌雄S155A小鼠未表现出葡萄糖耐受异常，但1岁龄的S155A雄性小鼠出现了葡萄糖耐受受损。与野生型小鼠相比，年轻雌性S155A小鼠在禁食状态下的体重与空腹血糖均无显著差异，但老龄雄性S155A小鼠的空腹血糖更高（p<0.05），体重无明显变化。无论雌雄，野生型与S155A小鼠的血清胰岛素、甘油三酯（triglyceride, TG）、β-羟丁酸以及非酯化脂肪酸水平均无显著差异。两组小鼠的肝脏甘油三酯含量也无显著差异。 通过实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qPCR）检测禁食状态下野生型与S155A小鼠的肝脏组织，发现受禁食上调且被认为受PLIN5磷酸化调控的PGC1α靶基因，其表达并无显著差异：Pgc1a、Ppara、Acot1、Pdk4及Ascl1的表达在两组禁食小鼠肝脏中无明显差异；Cpt1a与G6pc在S155A小鼠肝脏中呈现下调趋势，但未达到统计学显著性（样本量n=5~7）。炎症标志物如IL1b与Ccl2的表达在两组禁食小鼠肝脏中也无显著差异。 我们对禁食雌性小鼠的野生型与S155A肝脏组织进行了无偏倚RNA测序（RNA sequencing, RNA-seq），以筛选受PLIN5 S155磷酸化调控的差异表达基因。结果显示，S155A雌性小鼠肝脏中差异表达基因数量有限，校正后p值<0.05的基因仅有3个。有趣的是，RNA-seq结果显示胰岛素受体底物1（insulin receptor substrate 1, Irs1）在雌性S155A小鼠肝脏中的表达显著下调（对数2倍变化值为-0.74，校正p值为0.003）。该差异在雌性小鼠的禁食肝脏qPCR验证中得到了证实（p<0.05），但在雄性小鼠中未观察到该现象。实时定量PCR检测显示，胰岛素受体底物2（insulin receptor substrate 2, Irs2）的表达在雌性S155A小鼠肝脏中无显著差异，但在雄性S155A小鼠肝脏中出现下调（p<0.05），该结果通过蛋白质免疫印迹实验得到了证实。 综上，小鼠肝脏中的内源性PLIN5在禁食状态下会增加S155位点的磷酸化水平。然而，丧失S155位点的磷酸化并不会影响血清脂质水平或肝脏甘油三酯含量。此外，与野生型小鼠相比，禁食S155A小鼠肝脏中PGC1α靶基因的表达变化较为微弱，这表明PLIN5 S155磷酸化的缺失可通过其他机制得到代偿。出乎意料的是，雄性S155A小鼠肝脏中的IRS2表达出现下调，这或许可以解释老龄雄性小鼠的葡萄糖耐受受损现象。因此，PLIN5磷酸化可能通过维持雄性小鼠肝脏中的IRS2表达，从而影响肝脏胰岛素信号通路。 为探究PLIN5 S155磷酸化在体内的功能，我们获取了C57BL/6J小鼠，并利用CRISPR-Cas9技术向其基因组中引入S155A突变，随后通过RNA测序比较野生型C57BL/6J小鼠与磷酸化缺陷型小鼠的肝脏基因表达差异。