| BackgroundAtherosclerosis(AS)is an epidemic disease in Western countries,and as people’s living standards have improved and dietary habits have changed,the disease has also become the leading cause of death in our population.According to the World Health Organization(WHO)health statistics report,AS is the most common potential trigger of coronary artery disease(CAD),causing related cardiovascular diseases seriously endanger life and health,and is currently the leading cause of death in the worldwide population.As a result,research into AS prevention and treatment is critical.A large number of studies have shown that AS is caused by multiple risk factors,with dyslipidemia being the most important.Dyslipidemia is defined as abnormal concentrations of circulating lipids or lipoproteins,and changes in the formation,breakdown,and clearance of circulating lipoproteins.Blood lipids mainly included total cholesterol(TC)and triglyceride(TG).Blood lipids are transported in the circulation in the form of lipoprotein,which are classified as chylomicron,very low density lipoprotein(VLDL),low density lipoprotein(LDL),intermediate density lipoprotein(IDL),and high density lipoprotein(HDL).Currently,TG-rich VLDL is believed to have no atherogenic effect,but LDL and lipoprotein(a)[Lp(a)],the end products of VLDL metabolism,can cause atherosclerosis.Changes in these components can be caused by environmental or genetic factors.As early as the 1930s,some scholars proposed that cold environment significantly increased the incidence of myocardial infarction,and there was a significant negative correlation between temperature and the occurrence of acute cardiovascular events such as myocardial infarction and stroke.A recent study found that for every 1℃ decrease in average temperature,the incidence of myocardial infarction increased by 2.2%.More and more epidemiological evidence shows that cold is closely related to cardiovascular events,and low density lipoprotein cholesterol(LDL-C),AS an important pathogenic factor of AS,also has significant seasonal differences.A large population study found that subjects had higher levels of LDL-C in winter compared with the summer.The incidence of LDL-C>130mg/dl increased by 8%.However,the exact mechanism of how cold stimulation or hypothermia induced cardiovascular events occur remains unclear.One of the key factors in LDL metabolism is low density lipoprotein receptor(LDLR),which can bind circulating LDL-C,internalize LDL-C and move it to lysosome for degradation,while LDLR itself circulates back to the cell membrane and binds LDL-C again.As the molecular companion of LDLR,proprotein convertase subtilisin kexin type 9(PCSK9)binds to LDLR-LDLC complex and internalizes together.PCSK9 can block the structural transformation of LDLR in endosomes,inhibit its recirculation,and due to its degradation in lysosomes.Therefore,PCSK9 plays an important role in lipid metabolism and has become a new target for clinical lipid-lowering therapy.Sterol regulatory element binding proteins(SREBPs)are transcription factors.Their subtype,SREBP2,is relatively specific to cholesterol synthesis and plays a central role in regulating cholesterol homeostasis.SREBP2 is regulated primarily at the level of precursor protein hydrolysis.When it is not activated,SREBP2 precursor is anchored in the endoplasmic reticulum(ER)membrane by tightly binding with SREBP cleavage activating protein(SCAP).When it is stimulated by several factors(such as low sterol concentration or insulin),the SREBP2 precursor underwent two cleavage to release the NH2-terminal active domain,which contains a nuclear localization signal that enables SREBP2 to transport into the nucleus and promote the expression of many genes involved in cholesterol production and adipogenesis.The expression of both LDLR and PCSK9 mRNA is regulated by SREBP2.For example,when cholesterol content is lower than 5%of total lipids in the ER,SREBP2 can activate the transcription of LDLR mRNA by binding to the cholesterol regulatory element of the LDLR gene promoter,accompanied by the expression of PCSK9 mRNA.However,how the two factors coordinate to regulate cholesterol uptake and outflow needs to be further confirmed in vivo and in vitro.Recent studies have found that temperature is also closely related to fat metabolism.Adipose tissue stores more than 50%of the body’s cholesterol and is an crucial metabolic organ.It is mainly divided into two types:white adipose tissue(WAT),which is mainly used for energy storage,secretion of hormones and cytokines,and regulation of metabolism;and brown adipose tissue(BAT),whose main function is to maintain body temperature(thermogenesis)and basic/induced energy output(productivity).The thermogenesis of fat metabolism activated under cold conditions is mainly completed by BAT,while cold stimulation can also effectively activate BAT and transform WAT into BAT.This intermediate adipocyte is called beige or brite(brown-in-white)adipocyte.Evidence suggested that BAT,activated by cold stimulation,can clear TG in circulation and improve insulin resistance.However,it has also been found that stress can induce BAT to secrete a large amount of inflammatory factor Interleukin-6(IL-6),which affects metabolism and leads to obesity.Therefore,whether BAT is friend or foe needs further study.In our previous study,we found that cold stimulation activated BAT,TC and LDL-C in the animal model of atherosclerosis(ApoE-/-mice).Interestingly,wild type mice showed no significant change in TC,while LDL-C showed a downward trend.The mechanism behind this seemingly "contradictory" phenomenon has yet to be elucidated.We speculated that under different pathophysiological conditions,different adipose tissues would have different lipid scavenging abilities under cold stimulation.In our research,the expression of PCSK9 in human and animal models at different temperatures and its role in AS process were studied.Cell experiments confirmed that cold stimulation mediated cholesterol clearance through the mTORC1-SERBP-PCSK9 pathway in brown adipocytes.We explored the molecular mechanism of the high incidence of AS disease in winter and provided a new target for prevention and treatment.Objectives1.To investigate the effects of cold stimulation and lipid concentration on metabolism and AS process;2.To observe the changes of PCSK9 in different adipose tissues after cold stimulation;3.To explore the regulatory pathway of PCSK9 in brown adipocytes.Methods1.Detection of PCSK9 content in subjectsThe case information and blood samples of coronary heart disease patients and normal subjects in winter(January-March)and summer(July-September)were collected,and the plasma PCSK9 content of each group was detected by ELISA kit.2.Construct animal model in cold environmentMale C57BL/6 mice(n=20)and ApoE-/-mice(n=20)aged 6-8 weeks were selected.After adapting to 18℃ environment for 1 week,they were transferred to 4℃environment for next 7 weeks,with high fat and high cholesterol diet(0.25%cholesterol+15%fat).Male C57BL/6 mice(n=20)and ApoE-/-mice(n=20)aged 6-8 weeks were also selected as the control group and placed in a thermoneutral environment at 30℃ for 8 weeks.3.To investigate the effect of cold stimulation on BAT of C57BL/6 and ApoE-/-miceAfter 8 weeks of cold stimulation,mice in each group were anesthetized and placed in the respiratory chamber of small animal respiratory metabolism measuring instrument.Oxygen consumption,carbon dioxide generation,temperature,humidity and pressure were continuously recorded for 30 minutes,which was used as the basal metabolic rate of mice.Norepinephrine was injected subcutaneously(0.2mg/ml norepinephrine at 0.05ml per 10g body weight),and respiratory metabolism parameters were continuously measured for 45-60 min as an indicator of BAT activity in vivo mice.4.To investigate the effect of cold stimulation on browning in C57BL/6 and ApoE-/miceInterscapular BAT(iBAT)was taken from each group and weighed.HE staining:to observe the number and volume of adipocytes in each field.Immunohistochemical staining:To observe the expression of UCP-1.5.To investigate the effects of cold stimulation on food intake,body weight and body mass index(BMI)of C 57BL/6 and ApoE-/-miceThe feeding conditions of mice in each group were recorded every day.The feed was weighed by cage,and the number of mice in each cage and the feed weight were recorded.Each mouse was weighed weekly and the change in body length was measured.After anesthesia,we weighed the weight,measured body length,and calculated the BMI.6.To investigate the effect of cold stimulation on glucose tolerance of C57BL/6 and ApoE-/-miceThe mice were starved overnight and tested the next day.Weighed and recorded the weight of the mice.The tip of the tail was subtracted with sterilized scissors.Wiped the first drop of blood away.Mice were intraperitoneally injected 2g/kg body weight glucose solution(0.1ml 20%glucose per 10g body weight)according to their body weight,and blood glucose values were recorded after 15min,30min,60min,90min and 120min.7.To investigate the effect of cold stimulation on lipid degradation in C57BL/6 and ApoE-/-miceVAT was reserved for each group of mice,part of which was homogenized,and cAMP content was measured by ELISA kit.The other part was separated and digested and placed in adipocyte incubation solution with or without isoproterenol[125mM NaCl,5mM KCl,1mM CaCL2,2.5mM MgC12,1mM KH2PO4,2%BSA,4mM Glucose,125mm NaCl,5mm KCL,1mm cacL2,2.5 mm MgCl2,1mm KH2PO4,2%BSA,4mm glucose,25mM Tris(pH 7.4)].Medium was collected 2 hours later and glycerol levels were measured using an ELISA kit.8.To investigate the effect of cold stimulation on lipid level of C57BL/6 and ApoE-/miceThe blood of mice was collected and plasma was separated.The contents of TC,TG,LDL-C,Lp(a),sdLDL and HDL-C in plasma of mice in each group were detected by ELISA.9.To investigate the effect of cold stimulation on aortic plaque area and stability in ApoE-/-miceAorta oil red O staining:to observe the plaque area and proportion.HE staining:to observe plaque morphology and area.Oil red O staining:to observe the composition and proportion of lipids in plaques.Sirius scarlet staining:to observe the collagen composition and proportion in the plaques.Immunohistochemistry and immunofluorescence staining:to observe the expression ofα-SMA and MOMA-2 in plaques.10.To investigate the effects of cold stimulation on the expression of SREBP2,PCSK9 and LDLR in C57BL/6 and ApoE-/-micePlasma,liver,BAT,VAT and other tissues were collected,plasma or tissue homogenate was tested by ELISA.Part of the tissue was analyzed by Western Blot and qRT-PCR,and part of the tissue was analyzed by paraffin or frozen section for chemical study.The expression of SREBP2,PCSK9 and LDLR in each tissue was observed.11.To explore the pathway regulating SREBP2-PCSK9/LDLR expression in brown adipose cellsAfter differentiating 3T3-L1 cell lines into mature adipocytes,β-agonist CL-316243(10-6 mM)was used to induce browning,and mTORCl pathway was inhibited by mTORC1 inhibitor Rapamycin(50 mM).The expression of SREBP2 and PCSK9 was detected by WB.12.To investigate the effects of cold stimulation and different concentrations of ox-LDL on the expression of SREBP2,PCSK9 and LDLR in adipocytesAdd β-agonist CL-316243(10-6 mM)or equal volume PBS into mature adipocytes for 48 h,followed by ox-LDL(0 mM,20 mM,50 mM)for 48 h.The cells were divided into the following 6 groups:Control group,20 mM ox-LDL group,50 mM ox-LDL group,CL-316243 group,CL-316243+20 mM ox-LDL group,CL-316243+50 mM ox-LDL group.After stimulation,cell proteins were collected and the expressions of SREBP2,PCSK9 and LDLR in each group were detected by Western Blot.Results1.PCSK9 is correlated with season and CVDThe plasma PCSK9 level in patients with CVD was higher than that in normal subjects.The plasma PCSK9 level of winter subjects was significantly higher than that of summer subjects.2.Cold stimulation promotes BAT activationEffect of cold stimulation on non-shivering thermogenesis(NST)in C57BL/6 and ApoE-/-mice.The oxygen consumption and carbon dioxide generation of C57BL/6 and ApoE-/-mice were increased after cold stimulation,indicating the activation of BAT and the increase of non-shivering thermogenesis(NST).After cold stimulation,iBAT weight of C57BL/6 and ApoE-/-mice was decreased,but cell volume and density were increased.Immunohistochemical staining showed that UCP1 expression was increased and BAT was activated.After cold stimulation,in the VAT of C57BL/6 and ApoE-/-mice,cells decreased in volume and increased in density.Immunohistochemical staining showed that UCP1 expression increased and browning occurred.3.The influence of cold stimulation on metabolic condition1.1.Cold increases energy intakeCold stimulation increased food intake.1.2.The influence of cold stimulation on body weight is related to metabolic phenotypeCold-exposed ApoE-/-mice lost weight,cold-exposed C57BL/6 mice grew slightly longer,and BMI of both mice decreased.1.3.Cold exacerbates impaired glucose tolerance caused by high cholesterolAfter cold stimulation,the area under curve of glucose tolerance(AUC of GTT)of C57BL/6 mice decreased,while that of ApoE-/-mice increased.The AUC of GTT of ApoE-/mice was higher than that of C57BL/6 mice regardless of cold stimulation.1.4.Cold increases lipid degradation in the presence of high cholesterolThere was no significant difference in cAMP in VAT homogenate of C57BL/6 mice after cold stimulation,but cAMP in VAT homogenate of ApoE-/-mice increased.After cold stimulation,glycerol level of C57BL/6 mice in basal state had no significant change,while glycerol level in stimulation state showed a downward trend.Glycerol levels in ApoE-/-mice were increased in basal state and stimulated state.1.5.Cold exacerbates high cholesterolAfter cold stimulation,TC remained unchanged,TG,HDL-C,LDL-C,sdLDL and Lp(a)were decreased in C57BL/6 mice.After cold stimulation,TC and LDL-C increased TG,sdLDL remained unchanged and HDL-C as well as Lp(a)decreased in ApoE-/-mice.4.Cold speeds up AS progressionGeneral oil red O staining showed increased aortic plaques in ApoE-/-mice after cold stimulation.HE staining showed increased aortic plaque area in ApoE-/-mice after cold stimulation.Oil red O staining showed increased lipids in aortic plaques of ApoE-/-mice after cold stimulation.Sirius scarlet staining showed decreased collagen content in aortic plaques of ApoE-/-mice after cold stimulation.Immunohistochemical staining showed that macrophages increased and smooth muscle cells decreased in aortic plaques of ApoE-/-mice after cold stimulation.Plaque instability index was calculated according to the formula[(oil red O+area plus MOMA-2+area)/(α-SMA+area plus collagen I+area)],which showed decreased aortic plaque stability in ApoE-/-mice after cold stimulation.5.Cold inhibits lipid clearance in the presence of high cholesterolAfter cold stimulation,the expression of SREBP2 protein in VAT,BAT and liver of C57BL/6 mice was increased,and the mRNA level was also increased.There was no significant difference in SREBP2 protein expression in VAT after cold stimulation in ApoE-/mice,while SREBP2 protein expression in BAT increased significantly and decreased in liver.PCSK9 levels in plasma,BAT and liver of C57BL/6 mice were increased after cold stimulation,but there was no significant difference in VAT.PCSK9 levels in plasma and BAT increased after cold stimulation of ApoE-/-mice,but decreased in VAT and liver.In BAT,PCSK9 level of ApoE-/-mice increased more significantly after cold stimulation than that of C57BL/6 mice.There was no significant difference in LDLR levels in C57BL/6 and ApoE-/-BAT mice after cold stimulation,while LDLR levels in liver decreased significantly.6.Cold promotes SREBP2 and PCSK9 expression through mTORC1When CL-316243 was used to simulate cold stimulation,SREBP2 and PCSK9 levels were increased in cells,while the stimulative effect of cold on SREBP2 and PCSK9 expression was suppressed after the addition of Rapamycin.7.Cold and high cholesterol synergistically promote the expression of PCSK9There was no difference in the levels of SREBP2 and PCSK9 in ox-LDL stimulated cells at different concentrations without CL-316243.When CL-316243 was added,high concentration of ox-LDL stimulated the expression of SREBP2 and PCSK9 sharply.Conclusion1.Cold greatly increased TC and LDL-C and aggravated AS under the condition of high cholesterol level;2.Cold activated BAT and increased PCSK9 level in BAT,which was more obvious when the level of cholesterol was high;3.Cold regulated the expression of SREBP2 and PCSK9 in brown adipocytes through mTORC1... |
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