Effect Of Iron Overload On Plasma Lipid Metabolism And The Related Cytokines In Mouse

BackgroundIron 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 poorlyunderstoodThe 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.ObjectiveThis 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. Method1. Effects of iron overload on plasma lipids and adipokines1.1To divide experimental animals into groupsAll 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 parametersThe 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 tissueReal 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 expressionDissected 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 adipokines2.1To divide experimental animals into groupsSame as in part1.2.2Testing serum biochemical parametersSame as in part12.3Perls’ Prussian blue stainingFor 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 levelsChoose19cytokines 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 levelsReal 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 secretion3.13T3-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 secretionMixtures 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-PCR3.3Effect of non-transferrin-bound iron on adipokines secretion3T3-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-PCR4. Effect of recombinant adiponectin supplement on serum lipid metabolismin iron overload mouse4.1Testing serum lipid parametersThe 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 activityPostheparin and plasma LPL and HTGL activity was measured using a kit andprotocol. Result1. Iron overload caused plasma dyslipidemia and abnormal adipokines level.1.1Determination of iron statusWe 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 profilesThe 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 profilesThe result was same as part12.2Iron stored in adipose tissuePerls’ 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 protein3.1Different TS%transferrin bounding iron decreased adipokine secretion in cellculture modelIncreasing 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 FeSO₄（p<0.05）.4. Recombinant adiponectin lower plasma TG in iron overload mouse4.1Effect of recombinant adiponectin supplement on serum lipid index in ironoverload mouseInjection 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 analysisDescriptive 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.Conclusions1. 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.