铁过负荷对小鼠血脂代谢及相关细胞因子作用研究
摘要
研究背景
铁是人体含量最多的一种必需微量元素。长期以来，对铁代谢的研究主要集中在铁缺乏/缺铁性贫血的防治。随着人们对铁代谢认识的发展，如今已经从铁缺乏扩展到机内铁过负荷的原因以及铁代谢紊乱引发的一系列相关疾病的机制探讨，尤其是铁过量与代谢性疾病之间的关系。
近年来，诸多流行病学调查已经明确糖尿病，非酒精性脂肪肝等代谢性疾病患者存在铁过负荷。前瞻性研究进一步发现体内铁蛋白、转铁蛋白的升高与代谢综合征的发病率成正相关。多项META分析得出结论：机体铁含量过高是引起糖脂代谢紊乱、心血管疾病等发病的诱因之一，但具体机制尚未明确。血脂代谢的紊乱是代谢综合征的主要症状之一，血脂参数的持续异常显著增加心血管疾病的发病风险。流行病学调查显示铁过负荷人群常常伴有高脂血症，LDL-C、HDL-C等脂蛋白水平改变；动物给予单纯的高铁膳食也可见到血脂参数的异常。由于肝脏是调节脂代谢的重要器官，铁沉积于肝脏引起的氧化应激损伤是导致脂代谢紊乱的一个原因。事实上，机体铁沉积不仅仅发生在肝脏，脂肪、脑、胰腺等均有铁沉积的存在。
与肝脏同样，脂肪组织是维持脂代谢平衡的重要器官。近年来，有研究证实脂肪组织是一个敏感的铁感应器官，它可以表达所有与铁代谢相关的调控、储存及转运的因子如铁蛋白、铁调节蛋白、转铁蛋白受体、HEF、hepcidin、ferroportin等。目前，脂肪组织不再是一个单纯的储脂产能组织，其分泌的细胞因子如瘦素、脂联素等对于维持机体能量和糖脂代谢的平衡具有重要的调节作用。是否脂肪组织像肝脏一样，会因为铁过负荷影响其内分泌功能，成为铁过量引起脂代谢紊乱的机制之一，这方面研究一直没见报道是个值得探索的问题。
因此，本实验通过给小鼠腹腔注射右旋糖酐铁，观察血脂代谢指标的变化，并且用高通量细胞因子芯片观察血液中与脂代谢相关细胞因子的变化特点；进一步细胞实验证明不同浓度的结合型和离子型的铁干预能否直接影响脂肪细胞因子的分泌；最后在铁过负荷动物造模的同时，补充与血脂代谢最为密切的细胞因子脂联素至正常水平，检测脂联素作用的酶类活性及血脂参数的恢复情况，明确脂联素异常是否为铁过载引起的血脂异常的机制之一。
研究目的
观察铁过负荷小鼠血脂代谢变化以及与脂代谢密切相关的多个细胞因子的变化特点；观察铁过负荷对脂肪细胞细胞因子分泌的直接影响并证实其分泌的异常是否为铁过负荷引起血脂代谢异常的机制之一。为阐明铁过负荷引起的代谢紊乱的原因机制提供证据支持，同时也为铁过负荷引起的血脂代谢紊乱的预防和治疗方法提供一定新的思路。
研究方法
1.铁过负荷对小鼠血脂代谢的影响
1.1实验动物小鼠铁过负荷模型的建立
雄性C57小鼠（购自上海斯莱克公司），体重(20士2)g。按体重随机分为空白对照组、铁过负荷实验组，每组10只。实验组右旋糖酐铁腹腔注射2mg/d，对照组注射生理盐水，右旋糖酐铁注射总量为12mg。于第7天检测血清铁、铁蛋白、转铁蛋白饱和度、总铁结合力等血清铁相关参数；原子吸收法检测肝脏、脂肪铁含量；脂肪组织普鲁士蓝染色。
1.2铁过负荷小鼠血脂参数及其他生化指标测定
检测血糖、TG、TC、LDL-C、HDL-C、ALT、AST等血清生化指标。血糖仪检测小鼠血糖水平。放射免疫法检测小鼠血液胰岛素水平。
1.3肝脏甘油三酯合成酶类活性测定
实时定量荧光PCR法检测小鼠肝脏FAS、ACC等甘油三酯合成酶mRNA水平；Western blot检测ACC酶磷酸化水平。
2.铁过负荷小鼠血清各类细胞因子含量及脂肪组织细胞因子mRNA的变化
2.1高通量细胞因子芯片检测血清细胞因子的改变
选取IL-1β，IL-2，IL-6，IL-10，IL-15，IL-12(p70)，TNF-α，IFNγ，GLP-1，GIP、Leptin，Resistin，Ghrelin，PAI-1，MCP-1，RANTES，VEGF-basic、FGF-bb、PDGF等19个细胞因子定制细胞因子芯片，检测血清含量变化。
2.2Elisa重复验证芯片结果
2.3检测脂肪组织中细胞因子的mRNA表达水平
采用实时定量荧光PCR法测定小鼠血清中显著改变的细胞因子脂肪组织中的mRNA表达变化。
3.不同浓度的结合型和游离型铁对脂肪细胞瘦素、脂联素分泌的影响
3.13T3-L1细胞培养分化
选用3T3-L1前脂肪细胞，DMEM培养液中加入10%小牛血清、双抗，培养箱生长。细胞接触抑制至融合后用DMEM+10%胎牛血清培养，培养液中加入胰岛素（10μg/ml）、地塞米松(1μM)、IBMX(0.5mM)分化液分化，3天后换液，完全培养液并加入胰岛素（10μg/ml）干预，每隔两天换液一次，直至8-12天分化为成熟脂肪细胞，油红O染色鉴定。
3.2不同浓度的结合型铁和游离铁干预后细胞活性检测
用MTT法检测不同浓度的结合型铁和游离铁干预后细胞活性情况。
3.3不同饱和度的转铁蛋白结合型铁对脂肪细胞瘦素、脂联素分泌影响
用不同比例的apo-transferrin/holo-transferrin配成转铁蛋白饱和度为0、25%、50%、75%、100%5个浓度梯度的混合液，干预成熟脂肪细胞24h，转铁蛋白总浓度为30μM，Elisa法检测细胞培养液中瘦素、脂联素含量，实时定量荧光PCR法测定mRNA的表达水平。
3.4不同浓度的细胞外游离FeSO_4对脂肪细胞瘦素、脂联素分泌的影响
用0μM，10μM，100μM，1000μM游离FeSO_4干预脂肪细胞24h，检测瘦素、脂联素mRNA水平及细胞培养液中的含量。
4.外源性脂联素补充对铁过负荷小鼠血脂代谢及相关酶活性的影响
4.1血清脂联素含量恢复后观察血液脂代谢变化
小鼠右旋糖酐铁注射同时，腹腔注射重组脂联素，浓度为3μg/g，使其血液中含量恢复正常，检测小鼠血清中TG、LDL-C、HDL-C等血脂代谢指标变化。放射免疫法检测胰岛素水平变化。
4.2甘油三酯分解酶活性的变化
血清中脂联素水平恢复至正常后，比色法检测各组小鼠血清脂蛋白酯酶、肝酯酶活性变化。
5.数据的统计与处理
采用quantity one图像分析系统对western条带进行灰度分析，实验数据用(X±S)表示。应用SPSS11.5统计软件进行实验数据分析，两组间差异采用成组设计资料的t检验，多组间差异采用单因素方差分析。各组方差齐时，对照组与各实验组间比较采用Dunnett法，各组间两两比较采用LSD-t检验和SNK-q检验；方差不齐时采用Dunnett’s C检验。p<0.05为差别有统计学意义，p <0.01为非常显著性水平。
结果
1.铁过负荷对小鼠血脂的影响
1.1铁过负荷小鼠机体铁状况
与对照组相比，右旋糖酐铁注射小鼠血清铁、转铁蛋白饱和度（TS%）、总铁结合力（TIBC）均显著升高（p<0.01）；模型组小鼠肝脏铁水平较对照组升高4倍，脂肪组织铁含量升高5倍（p<0.01），通过普鲁士蓝染色进一步确定脂肪组织存在明显的铁沉积。
1.2铁过负荷小鼠血清生化指标变化
与对照组相比，铁过负荷模型小鼠血清甘油三酯水平（TG）显著升高（p<0.05）；低密度脂蛋白胆固醇酯（LDL-C）显著升高（p<0.01），高密度脂蛋白胆固醇酯（HDL-C）显著降低（p<0.01）；血糖、胆固醇（TC）、谷丙转氨酶（ALT）、天冬氨酸转氨酶（AST）没有明显改变，实验组、对照血清胰岛素水平差别没有统计学意义（p>0.05）。
1.3铁过负荷小鼠肝脏甘油三酯合成相关酶ACC、FAS变化
实验组肝脏ACC、FAS mRNA水平与对照组没有统计学差异（p>0.05），ACC的磷酸化水平没有明显变化。
2.铁过负荷小鼠血清各类细胞因子含量及脂肪组织细胞因子mRNA变化
2.1细胞因子芯片结果
从芯片结果来看，IL-1β，IL-2，IL-6，IL-10，IL-15，IL-12(p70)，TNF-α，IFN-γ等炎性细胞因子含量与对照组没有统计学差异；GIP、GLP-1两个促胰岛素分泌细胞因子没有显著改变；生长因子和趋化因子中，实验组趋化因子RANTES显著升高（p<0.01），VEGF， FGF、PDGF、MCP-1等与对照组差别均没有统计学意义；由脂肪细胞分泌的细胞因子瘦素、抵抗素、脂联素出现显著的降低（p<0.01），PAI-1有降低趋势，但没有统计意义（p=0.27），胃底细胞分泌的Ghrelin与对照组相比显著升高（p<0.01）。
2.2Elisa重复验证结果
实验组小鼠血清中，由脂肪细胞分泌的脂联素、瘦素出现了显著地降低（p<0.01），与芯片结果一致。
2.3脂肪组织细胞因子的mRNA水平变化
实验组小鼠脂肪组织中，脂联素、瘦素的mRNA水平降低显著（p<0.01）。
3.不同浓度的结合型铁和离子型铁干预对脂肪细胞细胞因子分泌的影响
3.13T3-L1前脂肪细胞分化结果
用完全培养液加入分化液分化三天，再用完全培养液加胰岛素干预约十天后，细胞由梭形变圆，核周围脂滴聚集，呈“戒环样”，经油红O染色鉴定前脂肪细胞分化为成熟脂肪细胞，成熟脂滴形成。
3.2不同浓度的离子型和结合型铁干预后细胞活性情况
用MTT法检测后发现，最高浓度30μM的holo-transferrin和1000μM的硫酸亚铁干预后，细胞活性与对照组没有明显差异（p<0.05）。
3.3不同饱和度的转铁蛋白结合型铁对脂肪细胞因子分泌的影响
转铁蛋白饱和度在大于等于50%，脂肪细胞TfR-1mRNA水平出现降低（p<0.05），提示细胞内的铁含量升高；细胞中的瘦素、脂联素mRNA水平随转铁蛋白饱和度升高，逐渐降低（p<0.05）；转铁蛋白饱和度达到75%时，细胞培养液中瘦素、脂联素含量显著降低（p<0.05）。
3.4不同浓度的细胞外游离FeSO_4干预影响脂肪细胞因子分泌
FeSO_4干预成熟脂肪细胞后，浓度在10μM时，细胞TfR-1mRNA水平就出现了显著降低（p<0.05），细胞中瘦素、脂联素的mRNA水平随着游离铁浓度的升高，逐渐降低（p<0.05）。100μM浓度的FeSO_4干预后，细胞培养液中瘦素、脂联素含量降低（p<0.05）。
4．外源性补充重组脂联素对铁过负荷小鼠血脂代谢的影响
4.1铁过负荷小鼠补充重组脂联素至正常后血脂代谢参数变化
外源性补充重组脂联素至正常水平后，可见血清TG的升高得到有效缓解，差别有统计学意义（p<0.05），HDL-C有升高趋势，LDL-C也有一定程度的恢复，但与对照组相比仍有统计学差异。
4.2铁过负荷小鼠补充重组脂联素后血清LPL、HL酶活性变化
铁过负荷小鼠血清中分解甘油三酯的脂蛋白酯酶和肝酯酶活性降低（p<0.05）；重组脂联素水平恢复正常后，脂蛋白酯酶活性较模型组显著升高（p<0.05），肝酯酶活性与实验组差别没有统计学意义。
结论
1.铁过负荷可引起血清甘油三酯、低密度脂蛋白升高，高密度脂蛋白降低；但对肝脏中与甘油三酯合成相关酶类没有受到影响；
2.铁过负荷小鼠血液中与脂代谢密切相关的脂肪细胞因子瘦素、脂联素的含量降低，而炎性因子、趋化因子、生长因子等其他细胞因子没有变化，提示细胞因子含量的异常有可能为血脂代谢紊乱原因之一；
3.细胞内铁含量升高后影响脂肪细胞瘦素、脂联素的分泌，结合整体动物脂肪组织细胞因子mRNA的结果，认为铁过负荷在转录水平影响了脂联素、瘦素的表达；
4.外源性补充重组脂联素使血液中含量恢复至正常后，铁过负荷小鼠血清TG水平得到一定程度缓解，说明脂联素异常为铁过负荷导致的机体血脂代谢紊乱的机制之一；与此同时，调控血清甘油三酯分解代谢的脂蛋白酯酶活性得到一定程度的恢复；推测脂联素可能通过影响脂蛋白酯酶的活性参与了铁过载引起的血液中甘油三酯的升高。
Background
Iron is an essential trace element and an important structural or functionalcomponent of many physiological systems. Iron deficiency and iron overload canresult in deviation from optimal health. The most common disorder associated withiron depletion is iron deficiency anemia, which affects more than30%of the world’spopulation. At the other end of the spectrum, Increased iron stores are associated withincreased risk of type2diabetes, prediabetes, metabolic syndrome (MetS), centraladiposity, and cardiovascular disease. Epidemiological studies have demostrated astatistically significant association between ferritin levels and lipid metabolism.Meanwhile, increasing evidence now suggested a potential role for iron in theetiopathogenesis of dyslipidemia. Recently, a large number of primary studiesregarding ferritin levels and T2D have been published, The meta-analysis andsystematic review suggest that increased ferritin levels and heme-iron intake are bothassociated with higher risk of T2D. Elevated plasma triglyceride levels, as often seenin these subjects, are independently associated with an increased risk ofcardiovascular diseases. The mechanisms underlying these associations are poorlyunderstood
The liver is the major recipient of the excess of iron and is important in theregulation of iron metabolism. Several researches have reported a link betweenchronic iron overload and hepatic lipid peroxidation. For example, markedperturbations in plasma lipid transport and hepatobiliary sterol metabolism occur indietary carbonyl iron overload. Iron-induced lipid peroxidation in hepaticmitochondria and microsomes results in defective electron transport and reducedconcentrations in cytochromes P-450and b5, respectively.
Recent research has shown that adipose tissue is not simply a storage depot forlipids but is also an important endocrine organ which played a key role in the controlof energy homeostasis. White adipose tissue has been found to produce more than50cytokines and other molecules. These adipokines are associated with obesity,metabolic syndrome, insulin resistance and other pathological or physiopathologicalconditions and processes through endocrine, paracrine, autocrine mechanisms ofaction. Circulating adipokines can fundamentally influence lipid metabolism inseveral target tissues. Adiponectin, an insulin-sensitizing adipokine, plays a important role in lipid metabolism. Adiponectin has been demonstrated to play an important rolein the modulation of glucose and lipid metabolism in both humans and animals.Several clinical reports have pointed to an association between plasma adiponectinand dyslipidaemia.Plasma adiponectin concentrations were not only inversely linkedto triglyceride levels, LDL cholesterol, and apolipoproteins (apos) B and E, but alsopositively correlated to serum HDL cholesterol. Hypoadiponectinemia observed indyslipidemia may accelerate the atherosclerotic changes seen in the metabolicsyndrome. It has also been reported that adiponectin has direct actions on vascularendothelium that could protect against cardiovascular disease in part by suppressinglipid accumulation in macrophages. Recent studies have also found a negativecorrelation between serum ferritin and the insulin-sensitizing adipokine, adiponectin.Whether the hypoadiponectinemia was caused by increased body iron have not beenverified.
Adipocytes are well suited for their iron-sensing role. They express not onlycommon regulators of iron homeostasis, such as ferritin and iron regulatory proteins,but also iron-related proeins with restricted tissue expression, including transferrinrecepor2, HFE, hepcidin, and, as shown herein, ferroportin. These findings prompteda causal role for iron as a risk factor for metabolic syndrome and a role for adipocytesin modulating metabolism through adiponectin in response to iron stores.
Taken together, it appears that excess iron may increase the adipose tissue ironlevel, which can bring changes to the expression of adipokines such as adiponectin、leptin etc. It is important to interpret the mechanism of iron accumulation induceddyslipidemia by reducing adipokines secretion, and these found may help to furtherexplore the mechanism and prevention of lipid metabolism caused by iron loading andprovide experimental basis for new therapy method.
Objective
This study is based on the latest research progress of relationship betweendyslipidemia and iron overload. We used dextran iron supplement prepared ironaccumulating model. It aimed to find out the effect and underlying mechanism of ironoverload on TG metabolism, and taking adipokine intervention to explain the role ofadipokines in the occurrence and development of dyslipidemia related to iron stores.Cell experiment used to determine whether iron stored in adipocyte could directlydisturbed the tissue endocrine function.
Method
1. Effects of iron overload on plasma lipids and adipokines
1.1To divide experimental animals into groups
All experimental procedures involving animals received the approval from theAnimal Care and Use Committee of the Second Military Medicine University.Guidelines and Policy on using and caring of the laboratory animals were followed atall time. Male c57mouse (20±2g body weight) fed with a standard diet werepurchased from the Shanghai-BK Ltd. Co, and were housed individually in a cage in atemperature-controlled room (24±1℃,55±5%humidity) with a12-hour light and12-hour dark cycle. After adaptation for7days, the mouse were divided into the ironoverload group (IO), the control group (control).IO group received2mg/d dextran ironby intraperitoneal injection. A total of12mg of iron was administered to mouse inmodel groups.). Control group was similarly injected with0.2mL of sterile salineduring the same period. The time of continuous disposal is6days. Body weight andfeeds consumption of mouse were weighted by electronic balance. After treatment,anesthetized rats immediately mouse received heart perfusion, and take serum, liver,and adipose tissues. Atomic absorption spectrophotometer was used to detect ironconcentration of mouse serum, liver and adipose.Perls’ Prussian blue staining wasperformed for showing adipocyte irons.
1.2Testing serum biochemical parameters
The fasting levels of glucose，TG，TC，HDL-C，LDL-C were measured byautomatic biochemical analyzer. The insulin concentration was determined byradioimmunoassay. And elisa repeated the result.
1.3Determination of leptin and adiponectin mRNA levels in adipose tissue
Real time Q-RT-PCR was performed using IQ5Real-Time PCR DetectionSystem. Two step RT-PCR method was performed using Real Time PCR Master Mix.Primers used to analyze all the transcripts have been reported else where. TheQ-RT-PCR data were analyzed by2-ΔΔCT method as described.
1.4Western blotting analysis of ACC/p-ACC expression
Dissected tissues from liver were homogenized separately by a dounce homogenizer in lysis buffer. Proteins were incubated overnight at4°C with aprimary antibody against ACC (rabbit polyclonal l,1:1000, Abcam), p-ACC (rabbitpolyclonal,1:500, Abcam), or GAPDH (rabbit polyclonal,1:10000, Sigma). Theblots were developed by incubation in ECL chemiluminescence reagent andsubsequently exposed to BioMax Light Film.
2. Effects of iron overload on plasma adipokines
2.1To divide experimental animals into groups
Same as in part1.
2.2Testing serum biochemical parameters
Same as in part1
2.3Perls’ Prussian blue staining
For Perls’ Prussian blue staining, sections were processed through a series ofgraded alcohols, into xylene, and rehydrated back to water. Sections werecounterstained with Neutral Red, dehydrated in increasing concentrations of ethanol,cleared in xylene, and mounted on slides.
2.4Measurement of serum cytokines levels
Choose19cytokines including IL-1β，IL-2，IL-6，IL-10，IL-15，IL-12(p70)，TNF-α，IFNγ， GLP-1，GIP、Leptin，Resistin，Ghrelin，PAI-1，MCP-1，RANTES，VEGF-basic、FGF-bb、PDGF, made an cytokine assay chip. The serum concentrationof these cytokines were be measured. Using a commercially available ELISA kitscheck the chip result.
2.5Determination of adiponectin,leptin mRNA levels
Real time PCR was performed using IQ5Real-Time PCR Detection System.Two step RT-PCR method was performed using Real Time PCR Master Mix.Primers used to analyze all the transcripts have been reported elsewhere.
3. Effects of adipocyte iron on adipokines secretion
3.13T3-L1adipocyte culture and differentiation.
3T3-L1adipocytes (ATCC) were maintained in high-glucose DMEM(HG-DMEM) supplemented with10%bovine serum and penicillin/streptomycin.For differentiation, cells were incubated in HG-DMEM with10%FBS for48hoursafter confluence. Cells were then cultured in differentiation media I (HG-DMEM,10%FBS,1μg/ml insulin,0.25μg/ml dexamethasone,0.5mM IBMX) for3days,followed by differentiation media II (HG-DMEM,10%FBS,1μg/ml insulin) for48hours. Prior to experiments, cells were cultured overnight in low-glucose DMEMwith10%FBS. All experiments were performed in LG-DMEM.
3.2Effect of different TS%transferrin bounding iron on adipokines secretion
Mixtures of apo-transferrin and holo-transferrin were added to3T3-L1adipocytes cultures at a total transferrin concentration of30μM. All cells weretreated for24hours before collection in Trizol. Determined the secretion andexpression of leptin and adiponectin by enzyme linked immunosorbent assay(ELISA) and real-time RT-PCR
3.3Effect of non-transferrin-bound iron on adipokines secretion
3T3-L1adipocytes cultures were treated with iron sulfate, concentrationranging from0to1000μM (0,10,100,1000μM).All cells were treated for24hoursbefore collection in Trizol. Determined the secretion and expression of leptin andadiponectin by enzyme linked immunosorbent assay (ELISA) and real-time RT-PCR
4. Effect of recombinant adiponectin supplement on serum lipid metabolismin iron overload mouse
4.1Testing serum lipid parameters
The purified protein was administered at3μg/g i.p. b.i.d.to mouse whenbuilding iron overload model. Adiponectin in mouse plasma was measured byELISA. The fasting levels of TG， HDL-C，LDL-C were measured by automaticbiochemical analyzer.
4.2Measurement of LPL,HTGL activity
Postheparin and plasma LPL and HTGL activity was measured using a kit andprotocol.
Result
1. Iron overload caused plasma dyslipidemia and abnormal adipokines level.
1.1Determination of iron status
We found that the iron levels in mouse serum were significantly higher in ironoverload group than in the control group (P<0.05);concentrations of liver non-hemeiron were4times higher than control; and adipose tissue non-heme iron were4timesincreased.
1.2Effect of iron overload on plasma lipid profiles
The concentrations of serum lipids differ significantly between the two groups.Treatment with iron dextran increased serum triacylglycerols and LDL-C level.HDL-C concentration decreased significantly in iron overload mouse（p<0.05）.
1.3Determination of ACC FAS levels.
The ACC, FAS mRNA levels showed no difference in iron overload groupcompared with control group (P>0.05). Both of hepatic ACC and phosphoryl-ACCprotein levels were no significantly change in iron overload model group comparedwith normal control group.
2. Iron deposition decreased plasma adipokines level in iron overload model.
2.1Iron status and plasma lipid profiles
The result was same as part1
2.2Iron stored in adipose tissue
Perls’ Prussian blue staining of adipose tissue revealed the presence ofiron-containing vesicles or endosomes in the cytoplasm。
2.3Iron overload decreased adipokines protein and mRNA expression in serum andtissue.
The serum level of adiponectin decreased apparently in iron-loaded mouse,leptin also occured this change（p<0.05）. However, other kind of cytokine such asinflammatory cytokine, chemotactic factor and growth factor have no differencesbetween two group.Real time-PCR analysis showed that iron overload mouse havedecreased adiponectin and leptin mRNA levels（p<0.05）.
3. Cultured adipocytes treated with iron exhibited decreased adiponectinmRNA and protein
3.1Different TS%transferrin bounding iron decreased adipokine secretion in cellculture model
Increasing transferrin saturations resulted in progressive decrease in leptinmRNA in3T3-L1adipocytes（p<0.05）. Adiponectin mRNA concentration alsoshowed a steady decrease when treated with increasingly iron-saturated transferrin（p<0.05）. In the presence of75%iron-saturated holotransferrin, Protein levels wasdecreased apparently in comparison to iron-free transferrin treatment (30μMapotransferrin alone)（p<0.05）.
3.2Different concentration of non-transferrin-binding iron decreased adipokinesecretion in cell culture model.
Treatment of3T3-L1adipocytes with iron sulfate decreased adiponectin andleptin mRNA levels in a dose-dependent manner（p<0.05）; The two cytokinesprotein in medium also decreased in presence of100μM FeSO_4（p<0.05）.
4. Recombinant adiponectin lower plasma TG in iron overload mouse
4.1Effect of recombinant adiponectin supplement on serum lipid index in ironoverload mouse
Injection of purified recombinant adiponectin to iron overload mouse led to amildly elevation in circulating adiponectin, which triggered a decrease in triglyceridelevel（p<0.05）. However, recombinant adiponectin supplement have no effect onserum LDL-C,HDL-C in iron overload mouse.
4.2Effect of recombinant adiponectin supplement on lipase activity.
Adiponectin could increase LPL activity in model group（p<0.05）,have nochange in activity of HTGL or degree of ACC phosphorylation.
5. Statistical analysis
Descriptive statistics in the text and figures are represented as average±SEM.An unpaired.2-tailed Students t-test was used to determine significance betweencontrols and individual experimental groups, One-way ANOVA was used to compareseries of data. P <0.05was considered significant for all tests. All statistical analyses were performed with SPSS11.5.
Conclusions
1. Body iron stores enhanced serum triacylglycerols, LDL-C, and decreasedserum HDL-C levels, but did not affect serum cholesterol concentration,
2. Circulating adipokines such as adiponectin、leptin levels also dropedapparently. The findings suggest that iron excess in the mouse probably causeddyslipidemia and abnormal adipokines level.
3. We have verified that adipocyte iron loading might suppress adiponectin andleptin mRNA and protein express. The decreased results could be found in3T3-L1cell after either tranferrin bounding iron or non-tranferrin bounding iron intervention.These findings demonstrate a causal role for iron as a factor affect adipocyteendocrine function.
4. Adiponectin appeared to reduces plasma triglyceride by increasing postheparinplasma lipoprotein lipase (LPL) activity in iron overload model. That meansadiponectin supplement might be benefit to improve dyslipidemia caused by ironaccumulating.
铁是人体含量最多的一种必需微量元素。长期以来，对铁代谢的研究主要集中在铁缺乏/缺铁性贫血的防治。随着人们对铁代谢认识的发展，如今已经从铁缺乏扩展到机内铁过负荷的原因以及铁代谢紊乱引发的一系列相关疾病的机制探讨，尤其是铁过量与代谢性疾病之间的关系。
近年来，诸多流行病学调查已经明确糖尿病，非酒精性脂肪肝等代谢性疾病患者存在铁过负荷。前瞻性研究进一步发现体内铁蛋白、转铁蛋白的升高与代谢综合征的发病率成正相关。多项META分析得出结论：机体铁含量过高是引起糖脂代谢紊乱、心血管疾病等发病的诱因之一，但具体机制尚未明确。血脂代谢的紊乱是代谢综合征的主要症状之一，血脂参数的持续异常显著增加心血管疾病的发病风险。流行病学调查显示铁过负荷人群常常伴有高脂血症，LDL-C、HDL-C等脂蛋白水平改变；动物给予单纯的高铁膳食也可见到血脂参数的异常。由于肝脏是调节脂代谢的重要器官，铁沉积于肝脏引起的氧化应激损伤是导致脂代谢紊乱的一个原因。事实上，机体铁沉积不仅仅发生在肝脏，脂肪、脑、胰腺等均有铁沉积的存在。
与肝脏同样，脂肪组织是维持脂代谢平衡的重要器官。近年来，有研究证实脂肪组织是一个敏感的铁感应器官，它可以表达所有与铁代谢相关的调控、储存及转运的因子如铁蛋白、铁调节蛋白、转铁蛋白受体、HEF、hepcidin、ferroportin等。目前，脂肪组织不再是一个单纯的储脂产能组织，其分泌的细胞因子如瘦素、脂联素等对于维持机体能量和糖脂代谢的平衡具有重要的调节作用。是否脂肪组织像肝脏一样，会因为铁过负荷影响其内分泌功能，成为铁过量引起脂代谢紊乱的机制之一，这方面研究一直没见报道是个值得探索的问题。
因此，本实验通过给小鼠腹腔注射右旋糖酐铁，观察血脂代谢指标的变化，并且用高通量细胞因子芯片观察血液中与脂代谢相关细胞因子的变化特点；进一步细胞实验证明不同浓度的结合型和离子型的铁干预能否直接影响脂肪细胞因子的分泌；最后在铁过负荷动物造模的同时，补充与血脂代谢最为密切的细胞因子脂联素至正常水平，检测脂联素作用的酶类活性及血脂参数的恢复情况，明确脂联素异常是否为铁过载引起的血脂异常的机制之一。
研究目的
观察铁过负荷小鼠血脂代谢变化以及与脂代谢密切相关的多个细胞因子的变化特点；观察铁过负荷对脂肪细胞细胞因子分泌的直接影响并证实其分泌的异常是否为铁过负荷引起血脂代谢异常的机制之一。为阐明铁过负荷引起的代谢紊乱的原因机制提供证据支持，同时也为铁过负荷引起的血脂代谢紊乱的预防和治疗方法提供一定新的思路。
研究方法
1.铁过负荷对小鼠血脂代谢的影响
1.1实验动物小鼠铁过负荷模型的建立
雄性C57小鼠（购自上海斯莱克公司），体重(20士2)g。按体重随机分为空白对照组、铁过负荷实验组，每组10只。实验组右旋糖酐铁腹腔注射2mg/d，对照组注射生理盐水，右旋糖酐铁注射总量为12mg。于第7天检测血清铁、铁蛋白、转铁蛋白饱和度、总铁结合力等血清铁相关参数；原子吸收法检测肝脏、脂肪铁含量；脂肪组织普鲁士蓝染色。
1.2铁过负荷小鼠血脂参数及其他生化指标测定
检测血糖、TG、TC、LDL-C、HDL-C、ALT、AST等血清生化指标。血糖仪检测小鼠血糖水平。放射免疫法检测小鼠血液胰岛素水平。
1.3肝脏甘油三酯合成酶类活性测定
实时定量荧光PCR法检测小鼠肝脏FAS、ACC等甘油三酯合成酶mRNA水平；Western blot检测ACC酶磷酸化水平。
2.铁过负荷小鼠血清各类细胞因子含量及脂肪组织细胞因子mRNA的变化
2.1高通量细胞因子芯片检测血清细胞因子的改变
选取IL-1β，IL-2，IL-6，IL-10，IL-15，IL-12(p70)，TNF-α，IFNγ，GLP-1，GIP、Leptin，Resistin，Ghrelin，PAI-1，MCP-1，RANTES，VEGF-basic、FGF-bb、PDGF等19个细胞因子定制细胞因子芯片，检测血清含量变化。
2.2Elisa重复验证芯片结果
2.3检测脂肪组织中细胞因子的mRNA表达水平
采用实时定量荧光PCR法测定小鼠血清中显著改变的细胞因子脂肪组织中的mRNA表达变化。
3.不同浓度的结合型和游离型铁对脂肪细胞瘦素、脂联素分泌的影响
3.13T3-L1细胞培养分化
选用3T3-L1前脂肪细胞，DMEM培养液中加入10%小牛血清、双抗，培养箱生长。细胞接触抑制至融合后用DMEM+10%胎牛血清培养，培养液中加入胰岛素（10μg/ml）、地塞米松(1μM)、IBMX(0.5mM)分化液分化，3天后换液，完全培养液并加入胰岛素（10μg/ml）干预，每隔两天换液一次，直至8-12天分化为成熟脂肪细胞，油红O染色鉴定。
3.2不同浓度的结合型铁和游离铁干预后细胞活性检测
用MTT法检测不同浓度的结合型铁和游离铁干预后细胞活性情况。
3.3不同饱和度的转铁蛋白结合型铁对脂肪细胞瘦素、脂联素分泌影响
用不同比例的apo-transferrin/holo-transferrin配成转铁蛋白饱和度为0、25%、50%、75%、100%5个浓度梯度的混合液，干预成熟脂肪细胞24h，转铁蛋白总浓度为30μM，Elisa法检测细胞培养液中瘦素、脂联素含量，实时定量荧光PCR法测定mRNA的表达水平。
3.4不同浓度的细胞外游离FeSO_4对脂肪细胞瘦素、脂联素分泌的影响
用0μM，10μM，100μM，1000μM游离FeSO_4干预脂肪细胞24h，检测瘦素、脂联素mRNA水平及细胞培养液中的含量。
4.外源性脂联素补充对铁过负荷小鼠血脂代谢及相关酶活性的影响
4.1血清脂联素含量恢复后观察血液脂代谢变化
小鼠右旋糖酐铁注射同时，腹腔注射重组脂联素，浓度为3μg/g，使其血液中含量恢复正常，检测小鼠血清中TG、LDL-C、HDL-C等血脂代谢指标变化。放射免疫法检测胰岛素水平变化。
4.2甘油三酯分解酶活性的变化
血清中脂联素水平恢复至正常后，比色法检测各组小鼠血清脂蛋白酯酶、肝酯酶活性变化。
5.数据的统计与处理
采用quantity one图像分析系统对western条带进行灰度分析，实验数据用(X±S)表示。应用SPSS11.5统计软件进行实验数据分析，两组间差异采用成组设计资料的t检验，多组间差异采用单因素方差分析。各组方差齐时，对照组与各实验组间比较采用Dunnett法，各组间两两比较采用LSD-t检验和SNK-q检验；方差不齐时采用Dunnett’s C检验。p<0.05为差别有统计学意义，p <0.01为非常显著性水平。
结果
1.铁过负荷对小鼠血脂的影响
1.1铁过负荷小鼠机体铁状况
与对照组相比，右旋糖酐铁注射小鼠血清铁、转铁蛋白饱和度（TS%）、总铁结合力（TIBC）均显著升高（p<0.01）；模型组小鼠肝脏铁水平较对照组升高4倍，脂肪组织铁含量升高5倍（p<0.01），通过普鲁士蓝染色进一步确定脂肪组织存在明显的铁沉积。
1.2铁过负荷小鼠血清生化指标变化
与对照组相比，铁过负荷模型小鼠血清甘油三酯水平（TG）显著升高（p<0.05）；低密度脂蛋白胆固醇酯（LDL-C）显著升高（p<0.01），高密度脂蛋白胆固醇酯（HDL-C）显著降低（p<0.01）；血糖、胆固醇（TC）、谷丙转氨酶（ALT）、天冬氨酸转氨酶（AST）没有明显改变，实验组、对照血清胰岛素水平差别没有统计学意义（p>0.05）。
1.3铁过负荷小鼠肝脏甘油三酯合成相关酶ACC、FAS变化
实验组肝脏ACC、FAS mRNA水平与对照组没有统计学差异（p>0.05），ACC的磷酸化水平没有明显变化。
2.铁过负荷小鼠血清各类细胞因子含量及脂肪组织细胞因子mRNA变化
2.1细胞因子芯片结果
从芯片结果来看，IL-1β，IL-2，IL-6，IL-10，IL-15，IL-12(p70)，TNF-α，IFN-γ等炎性细胞因子含量与对照组没有统计学差异；GIP、GLP-1两个促胰岛素分泌细胞因子没有显著改变；生长因子和趋化因子中，实验组趋化因子RANTES显著升高（p<0.01），VEGF， FGF、PDGF、MCP-1等与对照组差别均没有统计学意义；由脂肪细胞分泌的细胞因子瘦素、抵抗素、脂联素出现显著的降低（p<0.01），PAI-1有降低趋势，但没有统计意义（p=0.27），胃底细胞分泌的Ghrelin与对照组相比显著升高（p<0.01）。
2.2Elisa重复验证结果
实验组小鼠血清中，由脂肪细胞分泌的脂联素、瘦素出现了显著地降低（p<0.01），与芯片结果一致。
2.3脂肪组织细胞因子的mRNA水平变化
实验组小鼠脂肪组织中，脂联素、瘦素的mRNA水平降低显著（p<0.01）。
3.不同浓度的结合型铁和离子型铁干预对脂肪细胞细胞因子分泌的影响
3.13T3-L1前脂肪细胞分化结果
用完全培养液加入分化液分化三天，再用完全培养液加胰岛素干预约十天后，细胞由梭形变圆，核周围脂滴聚集，呈“戒环样”，经油红O染色鉴定前脂肪细胞分化为成熟脂肪细胞，成熟脂滴形成。
3.2不同浓度的离子型和结合型铁干预后细胞活性情况
用MTT法检测后发现，最高浓度30μM的holo-transferrin和1000μM的硫酸亚铁干预后，细胞活性与对照组没有明显差异（p<0.05）。
3.3不同饱和度的转铁蛋白结合型铁对脂肪细胞因子分泌的影响
转铁蛋白饱和度在大于等于50%，脂肪细胞TfR-1mRNA水平出现降低（p<0.05），提示细胞内的铁含量升高；细胞中的瘦素、脂联素mRNA水平随转铁蛋白饱和度升高，逐渐降低（p<0.05）；转铁蛋白饱和度达到75%时，细胞培养液中瘦素、脂联素含量显著降低（p<0.05）。
3.4不同浓度的细胞外游离FeSO_4干预影响脂肪细胞因子分泌
FeSO_4干预成熟脂肪细胞后，浓度在10μM时，细胞TfR-1mRNA水平就出现了显著降低（p<0.05），细胞中瘦素、脂联素的mRNA水平随着游离铁浓度的升高，逐渐降低（p<0.05）。100μM浓度的FeSO_4干预后，细胞培养液中瘦素、脂联素含量降低（p<0.05）。
4．外源性补充重组脂联素对铁过负荷小鼠血脂代谢的影响
4.1铁过负荷小鼠补充重组脂联素至正常后血脂代谢参数变化
外源性补充重组脂联素至正常水平后，可见血清TG的升高得到有效缓解，差别有统计学意义（p<0.05），HDL-C有升高趋势，LDL-C也有一定程度的恢复，但与对照组相比仍有统计学差异。
4.2铁过负荷小鼠补充重组脂联素后血清LPL、HL酶活性变化
铁过负荷小鼠血清中分解甘油三酯的脂蛋白酯酶和肝酯酶活性降低（p<0.05）；重组脂联素水平恢复正常后，脂蛋白酯酶活性较模型组显著升高（p<0.05），肝酯酶活性与实验组差别没有统计学意义。
结论
1.铁过负荷可引起血清甘油三酯、低密度脂蛋白升高，高密度脂蛋白降低；但对肝脏中与甘油三酯合成相关酶类没有受到影响；
2.铁过负荷小鼠血液中与脂代谢密切相关的脂肪细胞因子瘦素、脂联素的含量降低，而炎性因子、趋化因子、生长因子等其他细胞因子没有变化，提示细胞因子含量的异常有可能为血脂代谢紊乱原因之一；
3.细胞内铁含量升高后影响脂肪细胞瘦素、脂联素的分泌，结合整体动物脂肪组织细胞因子mRNA的结果，认为铁过负荷在转录水平影响了脂联素、瘦素的表达；
4.外源性补充重组脂联素使血液中含量恢复至正常后，铁过负荷小鼠血清TG水平得到一定程度缓解，说明脂联素异常为铁过负荷导致的机体血脂代谢紊乱的机制之一；与此同时，调控血清甘油三酯分解代谢的脂蛋白酯酶活性得到一定程度的恢复；推测脂联素可能通过影响脂蛋白酯酶的活性参与了铁过载引起的血液中甘油三酯的升高。
Background
Iron is an essential trace element and an important structural or functionalcomponent of many physiological systems. Iron deficiency and iron overload canresult in deviation from optimal health. The most common disorder associated withiron depletion is iron deficiency anemia, which affects more than30%of the world’spopulation. At the other end of the spectrum, Increased iron stores are associated withincreased risk of type2diabetes, prediabetes, metabolic syndrome (MetS), centraladiposity, and cardiovascular disease. Epidemiological studies have demostrated astatistically significant association between ferritin levels and lipid metabolism.Meanwhile, increasing evidence now suggested a potential role for iron in theetiopathogenesis of dyslipidemia. Recently, a large number of primary studiesregarding ferritin levels and T2D have been published, The meta-analysis andsystematic review suggest that increased ferritin levels and heme-iron intake are bothassociated with higher risk of T2D. Elevated plasma triglyceride levels, as often seenin these subjects, are independently associated with an increased risk ofcardiovascular diseases. The mechanisms underlying these associations are poorlyunderstood
The liver is the major recipient of the excess of iron and is important in theregulation of iron metabolism. Several researches have reported a link betweenchronic iron overload and hepatic lipid peroxidation. For example, markedperturbations in plasma lipid transport and hepatobiliary sterol metabolism occur indietary carbonyl iron overload. Iron-induced lipid peroxidation in hepaticmitochondria and microsomes results in defective electron transport and reducedconcentrations in cytochromes P-450and b5, respectively.
Recent research has shown that adipose tissue is not simply a storage depot forlipids but is also an important endocrine organ which played a key role in the controlof energy homeostasis. White adipose tissue has been found to produce more than50cytokines and other molecules. These adipokines are associated with obesity,metabolic syndrome, insulin resistance and other pathological or physiopathologicalconditions and processes through endocrine, paracrine, autocrine mechanisms ofaction. Circulating adipokines can fundamentally influence lipid metabolism inseveral target tissues. Adiponectin, an insulin-sensitizing adipokine, plays a important role in lipid metabolism. Adiponectin has been demonstrated to play an important rolein the modulation of glucose and lipid metabolism in both humans and animals.Several clinical reports have pointed to an association between plasma adiponectinand dyslipidaemia.Plasma adiponectin concentrations were not only inversely linkedto triglyceride levels, LDL cholesterol, and apolipoproteins (apos) B and E, but alsopositively correlated to serum HDL cholesterol. Hypoadiponectinemia observed indyslipidemia may accelerate the atherosclerotic changes seen in the metabolicsyndrome. It has also been reported that adiponectin has direct actions on vascularendothelium that could protect against cardiovascular disease in part by suppressinglipid accumulation in macrophages. Recent studies have also found a negativecorrelation between serum ferritin and the insulin-sensitizing adipokine, adiponectin.Whether the hypoadiponectinemia was caused by increased body iron have not beenverified.
Adipocytes are well suited for their iron-sensing role. They express not onlycommon regulators of iron homeostasis, such as ferritin and iron regulatory proteins,but also iron-related proeins with restricted tissue expression, including transferrinrecepor2, HFE, hepcidin, and, as shown herein, ferroportin. These findings prompteda causal role for iron as a risk factor for metabolic syndrome and a role for adipocytesin modulating metabolism through adiponectin in response to iron stores.
Taken together, it appears that excess iron may increase the adipose tissue ironlevel, which can bring changes to the expression of adipokines such as adiponectin、leptin etc. It is important to interpret the mechanism of iron accumulation induceddyslipidemia by reducing adipokines secretion, and these found may help to furtherexplore the mechanism and prevention of lipid metabolism caused by iron loading andprovide experimental basis for new therapy method.
Objective
This study is based on the latest research progress of relationship betweendyslipidemia and iron overload. We used dextran iron supplement prepared ironaccumulating model. It aimed to find out the effect and underlying mechanism of ironoverload on TG metabolism, and taking adipokine intervention to explain the role ofadipokines in the occurrence and development of dyslipidemia related to iron stores.Cell experiment used to determine whether iron stored in adipocyte could directlydisturbed the tissue endocrine function.
Method
1. Effects of iron overload on plasma lipids and adipokines
1.1To divide experimental animals into groups
All experimental procedures involving animals received the approval from theAnimal Care and Use Committee of the Second Military Medicine University.Guidelines and Policy on using and caring of the laboratory animals were followed atall time. Male c57mouse (20±2g body weight) fed with a standard diet werepurchased from the Shanghai-BK Ltd. Co, and were housed individually in a cage in atemperature-controlled room (24±1℃,55±5%humidity) with a12-hour light and12-hour dark cycle. After adaptation for7days, the mouse were divided into the ironoverload group (IO), the control group (control).IO group received2mg/d dextran ironby intraperitoneal injection. A total of12mg of iron was administered to mouse inmodel groups.). Control group was similarly injected with0.2mL of sterile salineduring the same period. The time of continuous disposal is6days. Body weight andfeeds consumption of mouse were weighted by electronic balance. After treatment,anesthetized rats immediately mouse received heart perfusion, and take serum, liver,and adipose tissues. Atomic absorption spectrophotometer was used to detect ironconcentration of mouse serum, liver and adipose.Perls’ Prussian blue staining wasperformed for showing adipocyte irons.
1.2Testing serum biochemical parameters
The fasting levels of glucose，TG，TC，HDL-C，LDL-C were measured byautomatic biochemical analyzer. The insulin concentration was determined byradioimmunoassay. And elisa repeated the result.
1.3Determination of leptin and adiponectin mRNA levels in adipose tissue
Real time Q-RT-PCR was performed using IQ5Real-Time PCR DetectionSystem. Two step RT-PCR method was performed using Real Time PCR Master Mix.Primers used to analyze all the transcripts have been reported else where. TheQ-RT-PCR data were analyzed by2-ΔΔCT method as described.
1.4Western blotting analysis of ACC/p-ACC expression
Dissected tissues from liver were homogenized separately by a dounce homogenizer in lysis buffer. Proteins were incubated overnight at4°C with aprimary antibody against ACC (rabbit polyclonal l,1:1000, Abcam), p-ACC (rabbitpolyclonal,1:500, Abcam), or GAPDH (rabbit polyclonal,1:10000, Sigma). Theblots were developed by incubation in ECL chemiluminescence reagent andsubsequently exposed to BioMax Light Film.
2. Effects of iron overload on plasma adipokines
2.1To divide experimental animals into groups
Same as in part1.
2.2Testing serum biochemical parameters
Same as in part1
2.3Perls’ Prussian blue staining
For Perls’ Prussian blue staining, sections were processed through a series ofgraded alcohols, into xylene, and rehydrated back to water. Sections werecounterstained with Neutral Red, dehydrated in increasing concentrations of ethanol,cleared in xylene, and mounted on slides.
2.4Measurement of serum cytokines levels
Choose19cytokines including IL-1β，IL-2，IL-6，IL-10，IL-15，IL-12(p70)，TNF-α，IFNγ， GLP-1，GIP、Leptin，Resistin，Ghrelin，PAI-1，MCP-1，RANTES，VEGF-basic、FGF-bb、PDGF, made an cytokine assay chip. The serum concentrationof these cytokines were be measured. Using a commercially available ELISA kitscheck the chip result.
2.5Determination of adiponectin,leptin mRNA levels
Real time PCR was performed using IQ5Real-Time PCR Detection System.Two step RT-PCR method was performed using Real Time PCR Master Mix.Primers used to analyze all the transcripts have been reported elsewhere.
3. Effects of adipocyte iron on adipokines secretion
3.13T3-L1adipocyte culture and differentiation.
3T3-L1adipocytes (ATCC) were maintained in high-glucose DMEM(HG-DMEM) supplemented with10%bovine serum and penicillin/streptomycin.For differentiation, cells were incubated in HG-DMEM with10%FBS for48hoursafter confluence. Cells were then cultured in differentiation media I (HG-DMEM,10%FBS,1μg/ml insulin,0.25μg/ml dexamethasone,0.5mM IBMX) for3days,followed by differentiation media II (HG-DMEM,10%FBS,1μg/ml insulin) for48hours. Prior to experiments, cells were cultured overnight in low-glucose DMEMwith10%FBS. All experiments were performed in LG-DMEM.
3.2Effect of different TS%transferrin bounding iron on adipokines secretion
Mixtures of apo-transferrin and holo-transferrin were added to3T3-L1adipocytes cultures at a total transferrin concentration of30μM. All cells weretreated for24hours before collection in Trizol. Determined the secretion andexpression of leptin and adiponectin by enzyme linked immunosorbent assay(ELISA) and real-time RT-PCR
3.3Effect of non-transferrin-bound iron on adipokines secretion
3T3-L1adipocytes cultures were treated with iron sulfate, concentrationranging from0to1000μM (0,10,100,1000μM).All cells were treated for24hoursbefore collection in Trizol. Determined the secretion and expression of leptin andadiponectin by enzyme linked immunosorbent assay (ELISA) and real-time RT-PCR
4. Effect of recombinant adiponectin supplement on serum lipid metabolismin iron overload mouse
4.1Testing serum lipid parameters
The purified protein was administered at3μg/g i.p. b.i.d.to mouse whenbuilding iron overload model. Adiponectin in mouse plasma was measured byELISA. The fasting levels of TG， HDL-C，LDL-C were measured by automaticbiochemical analyzer.
4.2Measurement of LPL,HTGL activity
Postheparin and plasma LPL and HTGL activity was measured using a kit andprotocol.
Result
1. Iron overload caused plasma dyslipidemia and abnormal adipokines level.
1.1Determination of iron status
We found that the iron levels in mouse serum were significantly higher in ironoverload group than in the control group (P<0.05);concentrations of liver non-hemeiron were4times higher than control; and adipose tissue non-heme iron were4timesincreased.
1.2Effect of iron overload on plasma lipid profiles
The concentrations of serum lipids differ significantly between the two groups.Treatment with iron dextran increased serum triacylglycerols and LDL-C level.HDL-C concentration decreased significantly in iron overload mouse（p<0.05）.
1.3Determination of ACC FAS levels.
The ACC, FAS mRNA levels showed no difference in iron overload groupcompared with control group (P>0.05). Both of hepatic ACC and phosphoryl-ACCprotein levels were no significantly change in iron overload model group comparedwith normal control group.
2. Iron deposition decreased plasma adipokines level in iron overload model.
2.1Iron status and plasma lipid profiles
The result was same as part1
2.2Iron stored in adipose tissue
Perls’ Prussian blue staining of adipose tissue revealed the presence ofiron-containing vesicles or endosomes in the cytoplasm。
2.3Iron overload decreased adipokines protein and mRNA expression in serum andtissue.
The serum level of adiponectin decreased apparently in iron-loaded mouse,leptin also occured this change（p<0.05）. However, other kind of cytokine such asinflammatory cytokine, chemotactic factor and growth factor have no differencesbetween two group.Real time-PCR analysis showed that iron overload mouse havedecreased adiponectin and leptin mRNA levels（p<0.05）.
3. Cultured adipocytes treated with iron exhibited decreased adiponectinmRNA and protein
3.1Different TS%transferrin bounding iron decreased adipokine secretion in cellculture model
Increasing transferrin saturations resulted in progressive decrease in leptinmRNA in3T3-L1adipocytes（p<0.05）. Adiponectin mRNA concentration alsoshowed a steady decrease when treated with increasingly iron-saturated transferrin（p<0.05）. In the presence of75%iron-saturated holotransferrin, Protein levels wasdecreased apparently in comparison to iron-free transferrin treatment (30μMapotransferrin alone)（p<0.05）.
3.2Different concentration of non-transferrin-binding iron decreased adipokinesecretion in cell culture model.
Treatment of3T3-L1adipocytes with iron sulfate decreased adiponectin andleptin mRNA levels in a dose-dependent manner（p<0.05）; The two cytokinesprotein in medium also decreased in presence of100μM FeSO_4（p<0.05）.
4. Recombinant adiponectin lower plasma TG in iron overload mouse
4.1Effect of recombinant adiponectin supplement on serum lipid index in ironoverload mouse
Injection of purified recombinant adiponectin to iron overload mouse led to amildly elevation in circulating adiponectin, which triggered a decrease in triglyceridelevel（p<0.05）. However, recombinant adiponectin supplement have no effect onserum LDL-C,HDL-C in iron overload mouse.
4.2Effect of recombinant adiponectin supplement on lipase activity.
Adiponectin could increase LPL activity in model group（p<0.05）,have nochange in activity of HTGL or degree of ACC phosphorylation.
5. Statistical analysis
Descriptive statistics in the text and figures are represented as average±SEM.An unpaired.2-tailed Students t-test was used to determine significance betweencontrols and individual experimental groups, One-way ANOVA was used to compareseries of data. P <0.05was considered significant for all tests. All statistical analyses were performed with SPSS11.5.
Conclusions
1. Body iron stores enhanced serum triacylglycerols, LDL-C, and decreasedserum HDL-C levels, but did not affect serum cholesterol concentration,
2. Circulating adipokines such as adiponectin、leptin levels also dropedapparently. The findings suggest that iron excess in the mouse probably causeddyslipidemia and abnormal adipokines level.
3. We have verified that adipocyte iron loading might suppress adiponectin andleptin mRNA and protein express. The decreased results could be found in3T3-L1cell after either tranferrin bounding iron or non-tranferrin bounding iron intervention.These findings demonstrate a causal role for iron as a factor affect adipocyteendocrine function.
4. Adiponectin appeared to reduces plasma triglyceride by increasing postheparinplasma lipoprotein lipase (LPL) activity in iron overload model. That meansadiponectin supplement might be benefit to improve dyslipidemia caused by ironaccumulating.
引文
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