研究目的
高强度间歇训练（high-intensity interval training, HIIT）是一种高强度运动（大于等于最大摄氧量的90%）和低强度运动或休息交替进行的运动方式。HIIT具有强大的减脂作用，特别是对内脏脂肪组织和皮下脂肪组织的减脂效果尤为显著。但HIIT促进减脂作用的分子机制尚不清楚。乳酸是HIIT的标志性产物，多项研究发现乳酸对脂肪组织代谢具有调节作用，因此乳酸又被称为是糖代谢和脂代谢的重要连接点，但其是否参与了HIIT的减脂效应仍未见报道。最近研究发现乳酸可以诱导蛋白质发生乳酸化修饰，调节下游基因的表达和代谢酶的功能，参与多种病理生理进程。乳酸的浓度是影响乳酸化修饰水平的重要因素之一。HIIT过程中产生的大量乳酸是否会影响组织中蛋白质的泛乳酸化修饰从而改变蛋白的生物学功能，以及该修饰是否参与HIIT的减脂作用，目前均未见报道。 
在本研究中，我们建立了小鼠HIIT模型，采用乳酸抑制剂二氯乙酸钠（dichloroacetate，DCA）干预，观察乳酸在HIIT促进减脂中的作用，及其对多个组织中蛋白质泛乳酸化水平的影响。同时，采用4D非标记乳酸化修饰组学方法检测皮下脂肪组织的乳酸化修饰蛋白，通过对组学数据的进一步分析，筛选出脂肪酸合成酶（fatty acid synthase，FASN）为乳酸化修饰的目标蛋白。在此基础上进行离体实验验证乳酸对脂代谢、蛋白质乳酸化修饰的作用和乳酸化修饰对FASN功能的影响。本研究利用乳酸孵育3T3-L1脂肪细胞，检测FASN的乳酸化水平，确定乳酸化修饰对FASN功能的影响，观察乳酸对脂肪细胞脂质代谢的作用。根据上述研究结果，确定乳酸在HIIT促进减脂中的作用，探究HIIT对蛋白质乳酸化的影响及作用，为HIIT促进减脂的作用机制提供更多理论依据。
研究方法
1. 54只5周龄雄性C57/BL6小鼠，分成3组：对照（control，CON）组，HIIT组和DCA+HIIT组。HIIT组和DCA+HIIT组小鼠于跑台上进行8周的高强度间歇训练，DCA于每次训练前10 min通过腹腔注射（400mg/kg）。每周运动5次，自由饮水进食。运动方案为：以40%最大速度热身5 min后，85%最大速度高强度跑1.5 min和45%最大速度跑2 min，二者交替循环，共重复9次，结束时以40%最大速度恢复跑5 min。在运动后测量小鼠血乳酸的变化。在运动干预第8周末次干预结束24 h后，进行取材。对小鼠各个脂肪组织进行称重，通过苏木精伊红（hematoxylin-eosin，H&E）染色法观察脂肪组织细胞形态，采用超高效液相色谱法测量小鼠血浆脂肪酸含量。使用实时荧光定量PCR（Quantitative Real-time PCR, qPCR）检测小鼠皮下脂肪组织中脂质转运、脂质合成和脂质分解标志基因的表达。
2. 利用western-blot技术对三组小鼠的皮下脂肪组织、内脏脂肪组织、肝脏组织、心脏组织、比目鱼肌组织和腓肠肌组织进行泛乳酸化水平检测，选择变化最为明显的皮下脂肪组织进行4D非标记乳酸化修饰组学和生物信息学分析，对皮下脂肪组织样本进行质控检测，乳酸化修饰组学数据进行质控和重复性检测。对皮下脂肪组织中的差异乳酸化蛋白进行鉴定、定量和分析，确定蛋白富集的通路和目标蛋白。
3. 采用0 mM、3 mM和10 mM浓度的乳酸孵育分化的3T3-L1脂肪细胞24 h后，利用油红O染色观察细胞内脂滴，采用超高效液相色谱法测定3T3-L1细胞的脂肪酸水平，使用甘油三酯和甘油检测试剂盒测定甘油三酯和甘油含量。使用qPCR法检测脂质合成和脂质分解标志基因的表达，观察细胞内脂质代谢的情况。利用蛋白质免疫印迹技术检测细胞泛乳酸化水平和脂肪酸合成酶的含量。用免疫共沉淀法测定FASN的乳酸化水平。FASN活性测定试剂盒用于检测FASN的活性，观察乳酸化修饰对FASN活性的影响。
研究结果
1. 乳酸介导了HIIT对小鼠的减脂作用
与CON组相比，HIIT组小鼠体重显著降低（P<0.001），皮下脂肪组织和内脏脂肪组织重量与体重的比值显著降低（P<0.001），皮下脂肪细胞横截面积显著减小（P<0.001）；当使用乳酸生成抑制剂DCA后，DCA+HIIT组小鼠体重显著高于HIIT组（P<0.01），皮下脂肪组织和内脏脂肪组织重量与体重的比值显著增大（P<0.001），细胞横截面积显著增大（P<0.05），褐色脂肪无明显差异。脂质代谢基因表达也存在显著差异，与CON组相比，HIIT组中分化抗原簇36（cluster of differentiation 36，Cd36） mRNA表达显著上调（P<0.05），ATP-柠檬酸裂解酶（ATP citrate lyase，Acly） mRNA表达显著上调（P<0.05）；与HIIT组相比，DCA+HIIT组小鼠Cd36 mRNA显著下调（P<0.05）。
2. HIIT对小鼠脂肪组织乳酸化修饰的影响
在HIIT小鼠中皮下脂肪组织、内脏脂肪组织、肝脏组织和心脏组织中蛋白质泛乳酸化水平显著增加，其中皮下脂肪组织泛乳酸化上调程度最为显著。抑制乳酸生成后，小鼠皮下脂肪组织、肝脏和心脏泛乳酸化水平下调。根据多个组织泛乳酸化的结果，我们对皮下脂肪组织进行了蛋白质组学和乳酸化修饰组学分析，脂肪组织样本符合修饰组学检测要求，检测数据发现三组之间存在组间差异且重复性好。修饰组学的检测共鉴定到了15737个肽段，其中修饰肽段数为800个，同时鉴定到290个蛋白的812个修饰位点数，其中可定量的有217个蛋白所包含的602个乳酸化修饰位点。以乳酸化蛋白差异表达量超过1.3作为显著上调的变化阈值，小于0.7作为显著下调的变化阈值，且p <0.05作为差异乳酸化蛋白的判断标准，发现与CON组相比，HIIT组中存在25个蛋白的37个修饰位点发生上调，22个蛋白的27个修饰位点出现下调。与HIIT组相比，DCA+HIIT组鉴定到48个蛋白中75个修饰位点发生上调，17个蛋白中22个修饰位点出现下调。与CON组相比，DCA+HIIT组中鉴定到有66个蛋白中的94个修饰位点出现上调，35个蛋白中的55个修饰位点出现下调。对三组中两两之间均存在差异的蛋白进行统计后，发现存在22个蛋白有1个乳酸化位点，10个蛋白有2个乳酸化位点，有4个蛋白有3-4个修饰位点，3个蛋白有5-10个修饰位点，仅有一个蛋白存在14个修饰位点。其中在HIIT组显著上调且DCA+HIIT组显著下调的乳酸化蛋白共13个，包含10个蛋白存在1个修饰位点，2个蛋白存在2个位点，仅有1个蛋白存在4个修饰位点。这些蛋白主要富集在代谢通路，碳代谢、氧化磷酸化、丙酮酸代谢、三羧酸循环和支链氨基酸降解等通路。FASN有4个乳酸化修饰位点发生变化，因此确定FASN为接受乳酸化调控的主要靶蛋白。 
3. FASN的乳酸化对3T3-L1细胞脂代谢的影响
对分化10天的3T3-L1脂肪细胞进行24 h的乳酸孵育后，发现与0 mM组细胞相比，3 mM乳酸孵育组脂滴数量和体积明显减小，细胞上清中甘油含量显著上调（P<0.01），脂肪酸含量显著降低（P<0.05），甘油三酯脂肪酶（adipose triglyceride lipase，Atgl）、乙酰辅酶A羧化酶（acetyl-CoA carboxylase，Acc）和硬脂酰 COA 去饱和酶-1（stearoyl-CoA desaturase 1，Scd1）的表达显著增加（均P<0.05）；10 mM组细胞脂滴减小，细胞中甘油三脂的含量显著降低（P<0.01），细胞上清中甘油水平显著上调（P<0.001），细胞中脂肪酸含量显著降低（P<0.05），脂质分解标志基因激素敏感性甘油三酯脂肪酶（hormone-sensitive lipase，Hsl）、Atgl及脂质合成标志基因二脂酰甘油酰基转移酶1（diacylglycerol acyltransferase 1，Dgat1）、Acc 和Scd1 mRNA的表达显著增加（均P<0.05）。不同浓度的乳酸干预均显著上调了细胞泛乳酸化水平，Fasn基因有上升趋势但无显著性，FASN蛋白水平显著上调（P<0.05），FASN蛋白的乳酸化水平增加（P<0.01），FASN的活性显著降低（P<0.01）。
研究结论
乳酸参与高强度间歇训练促进减脂的作用。高强度间歇训练产生的乳酸显著上调多组织的泛乳酸化修饰水平，皮下脂肪组织是受乳酸化修饰调节的重要组织之一。皮下脂肪组织的修饰组学研究发现FASN存在4个乳酸化修饰位点。乳酸诱导细胞中FASN多个位点的乳酸化修饰增强，抑制了脂肪酸合成酶的活性，减少脂质从头合成，可能是HIIT促进减脂的机制之一。

Background and purpose: 
High-intensity interval training (HIIT) is an intense exercise which individuals train at an intensity equal to or above 90% of their maximum oxygen uptake in a short time, with intervals of low-intensity exercise or rest for recovery. HIIT is well-known for its fat-reducing effects and particularly effective in reducing both inguinal and visceral white adipose tissue. However, the mechanism of fat reduction induced by HIIT remains unclear. A hallmark product of HIIT is lactate. Many studies have found that lactate regulates adipose tissue metabolism. As an important link between glycolysis and lipid metabolism, whether lactate is involved in the fat-loss effect of HIIT is unclear. Recent researches have found that lactate can promote protein lactylation modification and regulate downstream gene expression or the function of metabolic enzymes, which involved in the pathophysiological processes of many diseases. The concentration of lactate is one of the important factors affecting the level of lactylation modification. The effects of lactate produced by HIIT on the protein lactylation, biological function and fat-loss induced by HIIT are still unknown.
In this study, a model of HIIT was established and the lactate inhibitor dichloroacetate (DCA) was used to investigate the role of lactate in promoting fat loss during HIIT and its effect on protein lactylation in multiple tissues. A 4D label-free lactylation modification proteomics was used to identify proteins lactylation in inguinal white adipose tissue and screen the target protein, fatty acid synthase（FASN）. On this basis, 3T3-L1 cells treated with lactate were utilized to measure the lactylation level of FASN, determine the effect of lactylation on the function of FASN, and observe the influence of lactate on lipid metabolism of adipocytes. According to all results, the role of lactate in fat reduction in HIIT was determined, and the influence of HIIT on protein lactation was explored, which might provide a theoretical basis for the mechanism of fat reduction in HIIT.
Methods:
1. Fifty-four 5-week-old male C57/BL6 mice were divided into 3 groups: sedentary control (CON) group, HIIT group, and DCA+HIIT group. The mice in the HIIT group and DCA+HIIT group underwent 8 weeks of high-intensity interval training on treadmill. 10 min before training session, DCA was injected intraperitoneally at a dose of 400mg/kg. The mice exercised 5 times a week and had free access to food and water. The exercise protocol consisted of a 5-min warm-up at 40% of the maximal speed, followed by 1.5 min of high-intensity running at 85% of the maximal speed and 2 min of low-intensity running at 45% of the maximal speed with 9 times. The exercise ended with a 5-min at 40% of the maximal speed. Changes in blood lactate were measured after exercise. At the end of the 8th week of exercise intervention, tissues was performed. The weight of the adipose tissues was measured, and the morphology of the adipose tissue was observed by H&E staining. The level of serum fatty acids was measured by ultra-high-performance liquid chromatography. qPCR were used to detect the expression of lipid transport, synthesis, and lipolysis marker genes in inguinal white adipose tissue. 
2. Western blotting was used to detect the level of pan-lactylation in multiple tissues. The inguinal white adipose tissue was selected to measure by 4D label-free lactylation modification proteomics and bioinformatics analysis to identify and analyze the lactylation proteins. Inguinal adipose tissue samples were tested for quality, and lactate modification omics data were tested for quality and repeatability. Differential lactylation proteins in inguinal white adipose tissue were identified, quantified and analyzed to determine the protein enrichment pathway and target proteins.
3. 3T3-L1 cells were treated by lactate at concentrations of 0mM, 3mM, and 10mM for 24 h. Intracellular lipid droplets were observed by Oil Red O staining, and the level of fatty acids in the cells was measured by ultra-high-performance liquid chromatography. The triglyceride and glycerol were determined by triglyceride and glycerol assay kits. qPCR was used to detect the expression of lipid transport, synthesis, and lipolysis marker genes. Western blotting was used to detect the level of pan-lactylation and fatty acid synthase in the cells. FASN lactylation was determined by immunoprecipitation, and FASN enzyme activity was measured using a FASN activity assay kit to observe the effect of lactylation modification on the activity of FASN.
Results
1.HIIT induced fat loss via lactate-mediated pathways
Compared with the CON group, the HIIT group showed significant decreases in body weight (P<0.001), inguinal white and visceral fat tissue weight-to-body weight ratio (P<0.001), and inguinal white fat cell area (P<0.001). Compared with the HIIT group, the DCA+HIIT group showed significant increases in body weight (P<0.01), inguinal white and visceral adipose tissue weight-to-body weight ratio (P<0.001), and cell size (P<0.05). There were also significant differences in lipid metabolism gene expression. Compared with the CON group, the HIIT group showed significant upregulation of Cd36 mRNA expression (P<0.05) and Acly mRNA expression (P<0.05), while Cd36 mRNA was significantly downregulated (P<0.05) in response to DCA intervention.
2. Effect of HIIT on protein lactylation in inguinal white adipose tissue
The pan-lactylation levels in inguinal white adipose tissue, visceral adipose tissue, liver, and heart of HIIT group were significantly increased, with the most significant upregulation observed in inguinal white adipose tissue. After lactate production was inhibited, the pan-lactylation level in inguinal white adipose tissue, liver, and heart was downregulated. Based on the results of pan-lactylation, we performed 4D label-free lactylation modification proteomics analysis on inguinal white adipose tissue, samples met the requirements of modification proteomics detection, and the detection data showed that there were differences between the three groups with well repeatability. We identified 15,737 peptide segments, 800 of which were modified peptide segments, and 290 proteins with 812 modified sites, of which 217 proteins containing 602 lactylation modification sites were quantifiable. Using a lactylation protein expression difference threshold more than 1.3 as the significant upregulation threshold and less than 0.7 as the significant downregulation threshold, and p <0.05 as the criteria for differential lactylation proteins. Compared with CON group, 37 modification sites of 25 proteins were up-regulated and 27 modification sites of 22 proteins were down-regulated in HIIT group. Compared with the HIIT group, 75 modification sites in 48 proteins were up-regulated and 22 modification sites in 17 proteins were down-regulated in the DCA+HIIT group. Compared with the CON group, 94 modification sites of 66 proteins were up-regulated in the DCA+HIIT group, and 55 modification sites of 35 proteins were down-regulated. After statistical analysis of the proteins with differences in pairs among the three groups, we found that there were 22 proteins with 1 lactate site, 10 proteins with 2 lactate sites, 4 proteins with 3-4 modification sites, 3 proteins with 5-10 modification sites, and only one protein with 14 modification sites. A total of 13 lactated proteins were significantly up-regulated in the HIIT group and significantly down-regulated in the DCA+HIIT group, including 10 proteins with 1 modification site, 2 proteins with 2 sites, and only 1 protein with 4 modification sites. These proteins enriched in metabolic pathways such as carbon metabolism, oxidative phosphorylation, pyruvate metabolism, tricarboxylic acid cycle, and branched-chain amino acid degradation. FASN had 4 modified sites, indicating that FASN is the main target protein for lactylation regulation.
3. The effect of FASN lactylation on lipid metabolism in 3T3-L1 cells
After 24 hours of lactate treatment on 3T3-L1 adipocytes, we found that compared with the 0mM group, the number and size of lipid droplets in the 3mM group were significantly reduced, and the glycerol in the cell supernatant was significantly upregulated (P<0.01). The content of fatty acids was significantly decreased (P<0.05), and the expressions of Atgl, Acc and Scd1 mRNA were significantly increased (both P<0.05). In the 10mM group, the lipid droplets were reduced, and the content of triglycerides in the cells was significantly reduced (P<0.01), while the glycerol in the cell supernatant was significantly upregulated (P<0.001). The content of fatty acids in the cells was significantly decreased (P<0.05), and the expressions of Hsl, Atgl, Dgat1, Acc and Scd1 mRNA were significantly increased (all P<0.05). Lactate intervention promoted a significant increase in cellular lactylation levels, upregulated the level of FASN (P<0.05) and lactylation levels (P<0.01), and significantly reduced FASN activity (P<0.01).
Conclusion
Lactate participated in promoting fat loss induced by high intensity interval training. Lactate produced by high-intensity interval training significantly up-regulated pan-lactylation modification in many tissues. Inguinal adipose tissue was one of the important tissues regulated by lactylation modification, in which FASN has four lactylation modification sites. Lactate induced the enhancement of multi-site lactylation modification of FASN in cells, inhibited the activity of FASN and reduced de novo lipid synthesis, which may be one of the mechanisms of HIIT promoting fat loss.