目的 探讨低聚半乳糖（galacto-oligosaccharides,Gos）干预对慢性冷暴露小鼠棕色脂肪组织（brown adipose tissue,BAT）产热功能的影响及其可能的分子机制。方法 将8 w龄雄性C57BL/6J小鼠按照体重随机分为常温对照组（RT_Con）、低温对照组（Co_Con）和低温Gos组（Co_Gos）,每组11只;RT_Con组在常温条件下饲养,Co_Con组和Co_Gos组置于4℃寒冷模拟舱中持续暴露14 d。Co_Gos组每日灌胃1500 mg/kg体重Gos（灌胃体积10ml/kg,灌胃浓度150mg/ml）,RT_Con和Co_Con组每日灌胃等体积纯水;实验期间监测小鼠的摄食量、饮水量、体重、直肠温度及四肢抓力;实验结束后分离肩胛区BAT,称重后进行HE染色观察脂肪细胞脂滴形态变化,采用酶联免疫吸附测定(ELISA) BAT中腺苷酸环化酶（adenyl cyclase,AC）、蛋白激酶A（protein kinase A,PKA)、激素敏感性脂肪酶（hormone-sensitive lipase,HSL）、脂蛋白脂肪酶（lipoprotein lipase,LPL）、脂肪甘油三酯脂肪酶（adipose triglyceride lipase,ATGL）及产热关键蛋白过氧化物酶体增殖物激活受体γ共激活因子1α（peroxisome proliferator-activated receptor gamma coactivator 1-alpha,PGC-1α）和解偶联蛋白1（uncoupling protein 1,UCP1）的蛋白含量及环磷酸腺苷（cyclic adenosine monophosphate,cAMP）的浓度,采用荧光定量PCR（RT-qPCR）检测BAT中Adcy3、Adcy5、Adcy6、Hsl、Lpl、Atgl、Pgc-1α、Ucp1、Prdm16和Pparg的mRNA表达水平,采用蛋白印迹（Western blot）和免疫组化（IHC）检测BAT中UCP1的蛋白表达水平。结果 与RT_Con组相比,冷暴露导致小鼠体重下降、直肠温度降低、四肢抓力减弱（P<0.05）;BAT脂滴面积明显减少,AC/cAMP/PKA信号通路相关分子及UCP1、PGC-1α蛋白表达均上调（P<0.05）,与Co_Con组相比,Gos干预后小鼠直肠温度回升,四肢抓力增强,BAT重量和相对重量增加,脂滴面积进一步减小（P<0.05）,AC、PKA、HSL、LPL、UCP1、PGC-1α蛋白含量及cAMP浓度显著上调（P<0.05）;Adcy3、Adcy6、Lpl、Ucp1、Pgc-1α、Pparg mRNA水平亦显著提升（P<0.05）。结论 Gos干预可能通过激活BAT中AC/cAMP/PKA信号通路,上调产热关键蛋白UCP1和PGC-1α的表达,增强脂解作用,从而促进慢性冷暴露小鼠的BAT产热功能,改善其寒冷适应能力。
Abstract
Objective To investigate the effect of galacto-oligosaccharides on the thermogenic function of brown adipose tissue in chronic cold-exposed mice and its possible molecular mechanism. Methods Eight-week-old male C57BL/6J mice were randomly divided, based on body weight, into a room-temperature control group (RT_Con), a cold-control group (Co_Con), and a cold-Gos group (Co_Gos), with 11 mice per group. The RT_Con group was housed under normal room-temperature conditions, while the Co_Con and Co_Gos groups were placed in a cold simulation chamber at 4℃ for continuous exposure for 14 days. The Co_Gos group received a daily intragastric administration of Gos at 1500 mg/kg body weight (gavage volume 10 ml/kg, concentration 150 mg/ml), whereas the RT_Con and Co_Con groups received an equal volume of distilled water by gavage. During the experiment, food and water intakes, body weight, rectal temperature, and four-limb grip strength were monitored. After the experiment, the interscapular BAT was dissected, weighed, and processed for hematoxylin-eosin (HE) staining to observe morphological changes in adipocyte lipid droplets. The protein contents of adenylyl cyclase (AC), protein kinase A (PKA), hormone-sensitive lipase (HSL), lipoprotein lipase (LPL), adipose triglyceride lipase (ATGL), and the key thermogenic proteins peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and uncoupling protein 1 (UCP1), as well as the concentration of cyclic adenosine monophosphate (cAMP), in BAT were determined by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of Adcy3, Adcy5, Adcy6, Hsl, Lpl, Atgl, Pgc-1α, Ucp1, Prdm16, and Pparg in BAT were detected by real-time quantitative PCR (RT-qPCR). Protein expression levels of UCP1 in BAT were measured by Western blot and immunohistochemistry (IHC). Results Compared with the RT_Con group, cold exposure resulted in decreased body weight, reduced rectal temperature, and weakened four-limb grip strength in mice (P < 0.05). BAT lipid droplet areas were significantly reduced, and the expressions of the AC/cAMP/PKA signaling pathway molecules, as well as UCP1 and PGC-1α, were upregulated (P<0.05). Compared with the Co_Con group, Gos intervention led to a recovery in rectal temperature, enhanced four-limb grip strength, increased BAT weight and relative BAT weight, and further reduction in lipid droplet area (P<0.05) . The protein expressions of AC, PKA, HSL, LPL, UCP1 and PGC-1α, as well as the cAMP concentration were significantly upregulated (P < 0.05), and the mRNA levels of Adcy3, Adcy6, Lpl, Ucp1, Pgc-1α, and Pparg were also significantly elevated (P<0.05). Conclusion Galacto-oligosaccharide intervention may promote BAT thermogenic function and improve cold adaptation capacity in chronic cold-exposed mice, possibly by activating the AC/cAMP/PKA signaling pathway in BAT, upregulating the expression of the key thermogenic proteins UCP1 and PGC-1α, and enhancing lipolysis.
关键词
冷暴露 /
小鼠 /
低聚半乳糖 /
棕色脂肪组织 /
产热 /
解偶联蛋白1
Key words
cold exposure /
mice /
galacto-oligosaccharides /
brown adipose tissue /
thermogenesis /
uncoupling protein 1
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