Novel Roles Of LXR In Lipid Metabolism Of Liver And Brain

Liver X Receptors (LXR) are members of the nuclear receptor superfamily. LXR has two subtypes: LXRαand LXRβ. LXRαmainly expresses in intestine, liver, kidney and macrophage. The highest mRNA expression of LXRαwas in liver. LXRβis expressed in all tissues. The highest mRNA expression of LXRβis in brain. It is well known that LXRαand LXRβhave same ligands. So far, a number of cholesterol derivatives-oxysterol have been identified as natural ligand for LXR, such as 24(s), 25-epoxycholesterol, 24(s)-hydroxycholesterol, 22(R)-hydroxycholesterol and so on .The synthetic LXR agonists are T0901317 and GW3965. As a transcription factor, LXR plays an important role in keeping cholesterol homeostasis by regulating expression of target genes after activated by ligand and binding to response element of related genes. For example, LXR can promote the efflux of cholesterol from neurocyte in brain(ABCA1 and ABCG1), promote cholesterol reverse transport (ABCA1, ABCG1 and apoE), promote the conversion of cholesterol to bile acids (CYP7A1), limit intestinal absorption of cholesterol (ABCG5 and ABCG8), promote biliary excretion of cholesterol (ABCG5 and ABCG8), increase the fatty acids synthesis (FAS and SREBP1-c).Conversion of cholesterol to bile acids in the liver is an important pathway for elimination of cholesterol from the body. LXRαis the major regulator for this pathway. microsomal CYP7A1 (cholesterol 7α-hydroxylase) is the rate-limiting enzyme. The activity and expression level of CYP7A1 determin the rate of bile acid synthesis. peroxisomal D-3-Hydroxyacyl-CoA dehydratase /D-3- Hydroxyacyl-CoA dehydrogenase (D-bifunctional protein, DBP) also is a necessary enzyme for bile acid synthesis. The side chain of bile acid intermediates must be shortened via DBP. After activating LXR, enhancing the expression of CYP7A1 and increasing bile acid synthesis, the expression change of DBP is not well known .There are plenty of cholesterol in brain. Cholesterol plays an important role in determining the fluidity and permeability of plasma membrane, increase the velocity of movement of the action potential down the axon, minimize the energy requirements to maintain the potential differences across the plasma membranes of axons, keep the insulation character of myelin, promote the synapse formation and keep the stability of structure. The cholesterol in brain is synthesized de novo in situ. HMG-CoA Reductase is the limiting-speed enzyme in the course of synthesis of cholesterol. ABCA1 can promote the efflux of cholesterol from neurocyte. In addtion, brain cholesterol can be converted to 24S-hydroxycholesterol by cholesterol 24S-hydroxylase (CYP46) . 24S-hydroxycholesterol can across blood brain barrier easily and metabolism in liver. But many studies had showed that there is a close link between cholesterol and Alzheimer disease (AD). Accumulation and aggregation of beta-amyloid peptides (Aβ) is considered a pivotal fator associated with the pathogenesis of AD. When the intracellular cholesterol content was increased, the generation of Aβalso increase, then promote the aggregation of Aβ. When inhibit the intracellular cholesterol synthesis or decrease the cholesterol content in plasma membranes, the production of Aβin neuron also decrease. After inhibiting the expression of LXRβwhich high express in brain and decrease the expression of genes with respect to the efflux of cholesterol to change the cholesterol metabolism state, The chang of Aβproduction and expression of genes which is related to the generation of Aβis unclear.Docosahexaenoic acid (DHA) plays an important role in normal neurological development, especially in brain. The last step of DHA biosynthetic pathway is completed through peroxisomesβ-oxidation, DBP take part in the process. In central nervous system, LXRβnot only keep the cholesterol homeostasis, but also promote fatty acid synthesis by inducing the expression of fatty acid synthetase (FAS) and SREBP1-c (sterol regulatory element binding protein 1c ). It is unclear Whether DHA synthesis content and expression level of genes involved in last step of DHA synthesis will be affected after inhibiting the expression of LXRβand decrease fatty acid synthesis.In the present work, we have studied the novel roles of LXR in lipid metabolism from three different aspects. Firstly, to investigate the relationship between DBP and bile acid biosynthesis through determining the chang of expression and activity of DBP when activated-LXR increase bile acid biosynthesis by inducing the expression of CYP7A1. Secondly, to investigate the role of LXRβin Aβproduction through determining the chang of cholesterol, Aβcontent and expression of genes which are relevant to cholesterol synthesis and APP metabolism, respectively, after inhibiting LXRβexpression and decrease the expression of ABCA1, CYP46 in primary cultured rat cortical neurons. Thirdly, to investigate the role of LXRβin DHA synthesis through determining the chang of DHA content and expression level of genes (ACOX, DBP, SCPX) involved in peroxisomesβ-oxidation in primary cultured rat cortical neurons, after inhibiting LXRβexpression and decrease fatty acids synthesis.Partâ… The activated-LXR increase bile acid synthesis by enhancing the expression of DBPObjective: To investigate the relationship between DBP and bile acid biosynthesis through determining the chang of expression and activity of DBP when activated-LXR increase bile acid biosynthesis by inducing the expression of CYP7A1.Methods: Male C57BL/6J mouse were divided randomly into control group (Con) and agonist group (To). Con group and To group were administeed orally with vehicle solution and LXR agongist T0901317 at 20mg/kg/day respectively for 1 week. Collect the feces of day 7 for bile acids detection. All animals were sacrified at day 8. liver was immediately submerged in liquid Nitrogen then stored at - 70℃for the extraction of total RNA and measurement of DBP activity and protein.Male Wistar rats were divided randomly into two groups:â‘ control group (Con), fed common rodent diet.â‘¡cholesterol group (CH), fed 2% cholesterol plus 10%corn oil diet. Rats were given free access to food and water for 2 wks. When serum cholesterol level of CH group (2.729±0.757 mmol/L) was significantly higher than that of control group (1.426±0.257 mmol/L). Rats were killed. Liver was immediately submerged in liquid Nitrogen then stored at - 70℃for the extraction of total RNA and measurement of DBP activity.Results:1 The content of bile acids in fecal (determined by Auto-biochemical-analyst apparatus)Fecal bile acids of To group and Con group were respectively 1.323±0.277, 1.008±0.205. Fecal bile acids of CH group and Con group were respectively 5.539±1.710, 0.575±0.125. Both of fecal bile acids of To group and CH group were significantly higher than that of their control group (Pmice<0.05,Prat<0.01). The result showed that bile acids synthesis increase after activating LXR.2 The mRNA level of CYP7A1 and DBP in liver (determined by RT-PCR)Compared with the mRNA level of CYP7A1 (0.635±0.107) and DBP (0.671±0.049) of Con group, the mRNA level of CYP7A1 (0.764±0.067) and DBP (0.806±0.060) of To group were significantly increased (PCYP7A1<0.05,PDBP<0.01). Compared with the mRNA level of CYP7A1 (0.473±0.0646) and DBP ( 0.721±0.0635 ) of Con group, the mRNA level of CYP7A1 (0.877±0.0664) and DBP (0.881±0.0482) of CH group was also significantly increased (P<0.01). The results showed that the expression of DBP was enhanced when activated-LXR increase bile acid synthesis by inducing the expression of CYP7A1.3 The activity of DBP in liver (determined by Spectrophotometry)Compared with control group (11.1±2.4), the activity of To group (15±2.7) was significantly increased (P < 0.01). Compared with control group (8.811±2.52), the activity of CH group (11.563±1.911) was significantly increased (P<0.05). The results showed that the activity of DBP was also enhanced when activated-LXR increase bile acid synthesis by inducing the expression of CYP7A1.4 The protein level of DBP in liver of C57BL/6J mice (determined by Western Blotting)Compared with Con group (10.75±0.866), the DBP protein level of To group(12±1.0) was significantly increased (P<0.05). The results showed that the protein level of DBP was also increased when activated-LXR increase bile acid synthesis by inducing the expression of CYP7A1.5 The correlation of DBP and CYP7A1 mRNA level (determined by SAS software)The mRNA level of CYP7A1 and DBP in mice showed positive correlation (r=0.6555,P<0.05). The mRNA level of CYP7A1 and DBP in rat also showed positive correlation (r=0.74, P<0.05). The results showed that Enhancement of hepatic DBP expressiom is related to the increase of bile acid synthesis.Conclusion:The increase of bile acid synthesis by activated-LXR is relevant to the enhancement of hepatic DBP mRNA level and activity.Partâ…¡The role of LXRβin AβProduction in cortical neuronsObjective: To investigate the role of LXRβin Aβproduction through determining the chang of Aβcontent and expression of genes which are relevant to Aβgeneration after inhibiting LXRβexpression and change the cholesterol metabolism state in primary cultured rat cortical neurons.Methods: Primary cultured rat cortical neurons were divided randomly into antisense group (Anti), control group (Con) and mismatch group (Mis). Anti group and Mis group were respectively administered with 600μl culture medium plus 500pmol oligodeoxyribonucleotides and 5μl Lipofectamine^TM2000 for 24h. Con group was only administered with 600μl culture medium plus 5ul Lipofectamine^TM2000 for 24h. Collect neurons and culture medium to observe the expression change of genes with respect to cholesterol metabolism (ABCA1, HMG CoA R, CYP46) and Aβgeneration (APP, BACE1, ADAM10) by RT-PCR, the protein level of ABCA1 and HMG CoA R by Western blotting, Aβcontent in medium by ELISA, total cholesterol in medium and neurons by the kits of enzymic method.Results:1 Results with respect to cholesterol metabolism1.1 The mRNA (RT-PCR) and protein (Western Blotting) level of ABCA1The ABCA1 mRNA level of Anti group (1.311±0.205) was singnificantly lower than that of Con (2.395±0.317) and Mis group (2.196±0.397,P<0.01). There was no statistical difference in ABCA1 mRNA level between Con group and Mis group (P>0.05). The ABCA1 protein level of Anti group (9.55±2.5) was singnificantly lower than that of Con (79.5±15) and Mis group (78.3±15,P<0.05). There was no statistical difference in ABCA1 protein level between Con group and Mis group (P>0.05). The results showed that the expression of ABCA1 which is involved in the efflux of cholesterol from neuron decreased after inhibiting the expression of LXRβ.1.2 Total cholesterol in medium (determined by the kits of enzymic method)The total cholesterol in medium of Anti, Con and Mis group were respectively 299.487±47.034 mmol/L, 356.102±20.430 mmol/L, 374.700±44.479 mmol/L. The total cholesterol of Anti group was singnificantly lower than that of Con and Mis group (P<0.05). There was no statistical difference in total cholesterol between Con group and Mis group (P>0.05). The results showed that the down-regulated expression of ABCA1 and the cholesterol secreted from neuron into medium decrease lead to the decrease of total cholesterol in medium after inhibiting the expression of LXRβ.1.3 Cellular cholesterol in cortical neurons (determined by the kits of enzymic method)The cellular cholesterol of Anti, Con and Mis group were respectively 0.622±0.052 mmol/μg, 0.875±0.082 mmol/μg, 0.879±0.209 mmol/μg. The cellular cholesterol of Anti group was singnificantly lower than that of Con and Mis group (P<0.05). There was no statistical difference in total cholesterol between Con group and Mis group (P>0.05). The results showed that the cellular cholesterol in cortical neurons decreased after inhibiting the expression of LXRβ.1.4 The mRNA(RT-PCR) and protein(Western Blotting) level of HMG CoA RThe HMG CoA R mRNA level of Anti group (0.603±0.108) was singnificantly lower than that of Con (0.849±0.201) and Mis group (0.888±0.153,P<0.05). There was no statistical difference in HMG CoA R mRNA level between Con group and Mis group (P>0.05). The HMG CoA R protein level of Anti group (0.840±0.162) was singnificantly lower than that of Con (1.371±0.096) and Mis group (1.513±0.164,P<0.01). There was no statistical difference in HMG CoA R protein level between Con group and Mis group (P>0.05). The results showed that the transcript and translation levele of HMG CoA R (rate-limiting enzyme in the course of synthesis of cholesterol) was down-regulated which leaded to the decrease of celluar cholesterol synthesis after inhibiting the expression of LXRβ.1.5 The mRNA level of CYP46 (determined by RT-PCR method)The CYP46 mRNA level of Anti, Con and Mis group were respectively 0.637±0.063, 1.025±0.175, 1.050±0.182. The CYP46 mRNA level of Anti group was singnificantly lower than that of Con and Mis group (P<0.01). There was no statistical difference between Con and Mis group (P>0.05). The results showed that mRNA level of CYP46 which involed in efflux of cholesterol from Brain decreased after inhibiting the expression of LXRβ.2 Results with respect to Aβgeneration2.1 The mRNA expression of APP (determined by RT-PCR method)APP695 and APP751+770 mRNA level of Anti group were respectively 0.642±0.114, 0.392±0.072. APP695 and APP751+770 mRNA level of Con group were respectively 0.663±0.072, 0.727±0.164. APP695 and APP751+770 mRNA level of Mis group were respectively 0.575±0.088, 0.841±0.161. APP751+770 mRNA level of Anti group was singnificantly lower than that of Con and Mis group (P<0.01). There was no statistical difference in APP751+770 mRNA level between Con group and Mis group (P>0.05). In addition, there was no statistical difference in APP695 mRNA level among Anti group, Con group and Mis group (P>0.05). The results showed that the expression of APP as Aβprecurosor were down-regulated after inhibiting the expression of LXRβ.2.2 The mRNA level of BACE1 and ADAM10 mRNA (determined by RT-PCR method)The BACE1 and ADAM10 mRNA level of Anti group were respectively 0.534±0.098, 0.471±0.051. The BACE1 and ADAM10 mRNA level of Con group were respectively 0.876±0.095, 0.870±0.124. The BACE1 and ADAM10 mRNA level of Mis group were respectively 0.819±0.105, 0.862±0.119. The BACE1 and ADAM10 mRNA level of Anti group was singnificantly lower than that of Con and Mis group (P<0.01). There was no statistical difference between Con group and Mis group (P>0.05). The results showed that theα-secretase pathway andβ-secretase pathway of APP processing were down-regulated after inhibiting the expression of LXRβ.2.3 Aβcontent in medium (determined by ELISA method)The Aβcontent in medium of Anti group (101.140±16.057 pg/ml) was singnificantly lower than that of Con (157.189±32.603 pg/ml) and Mis group (145.724±37.743 pg/ml) (P < 0.01). There was no statistical difference between Con group and Mis group (P>0.05).The results showed that not only the transcription level of APP and BACE1 decrease, but also the Aβproduction of neuron decreased, after inhibiting the expression of LXRβ.Conclusion:1 LXRβcan affect the expression of genes with respect to APP metabolism and then affect the generation of Aβin cortical neurons.2 LXRβcan affect the expression of non target genes and cholesterol production.Partâ…¢The role of LXRβin DHA synthesis in cortical neuronsObjective: To investigate the role of LXRβin DHA synthesis through determining the chang of DHA content and expression level of genes involved in last step of DHA synthesis in primary cultured rat cortical neurons, after inhibiting LXRβexpression and decrease expression level of genes involved in fatty acids synthesis.Methods: Primary cultured rat cortical neurons were divided randomly into antisense group (Anti), control group (Con) and mismatch group (Mis). Anti group and Mis group were respectively administered with 600μl culture medium plus 500 pmol oligodeoxyribonucleotides and 5μl LipofectamineTM2000 for 24h. Con group was only administered with 600μl culture medium plus 5μl LipofectamineTM2000 for 24h .Collect neurons to observe the change of DHA content (gas chromatography) and mRNA expression of FAS, SREBP1-c, PPARα, ACOX1, ACOX3, DBP, SCPx and LBP ( RT-PCR).Results:1 The mRNA level of FAS and SREBP1-c (determined by RT-PCR method)The FAS and SREBP1-c mRNA level of Anti group were respectively 0.531±0.034, 0.547±0.130. The FAS and SREBP1-c mRNA level of Con group were respectively 0.701±0.105, 1.031±0.163. The FAS and SREBP1-c mRNA level of Mis group were respectively 0.681±0.126, 1.118±0.204. The FAS and SREBP1-c mRNA level of Anti group was singnificantly lower than that of Con and Mis group (P<0.01). There was no statistical difference between Con group and Mis group (P>0.05). The results showed that mRNA expression of FAS and SREBP1-c related to fatty acids synthesis were down-regulated after inhibiting the expression of LXRβ.2 The mRNA level of ACOX1,DBP and SCPx mRNA (determined by RT-PCR method)The ACOX1 mRNA level of Anti group were 0.798±0.145, on the same experiment condition, we failed to detect mRNA level of DBP and SCPx of Anti group. The ACOX1, DBP and SCPx mRNA level of Con group were respectively 1.332±0.217, 0.560±0.114, 0.599±0.077. The ACOX1, DBP and SCPx mRNA level of Mis group were respectively 1.447±0.130, 0.477±0.094, 0.615±0.183. The mRNA level of ACOX1, DBP and SCPx mRNA of Anti group was singnificantly lower than that of Con and Mis group (P<0.05). There was no statistical difference between Con group and Mis group (P> 0.05). The results showed that mRNA expression of ACOX1, DBP and SCPx involved in DHA synthesis were down-regulated after inhibiting the expression of LXRβ.3 The DHA content in neuron (%) (determined by gas chromatography) The sum of C_16:0, C₁₈, C_20:0 and C_22:6 was regard as total fatty acid in neuron, the C_22:6/ total fatty acid was regard as the DHA content in neuron. The DHA content in neuron of Anti group (3.989±1.193) was significantly lower than that of Con group (6.195±1.273) and Mis group (6.55±0.644, P<0.05). There was no statistical difference between Con group and Mis group (P>0.05). The results showed that the transcription level of ACOX1, DBP and SCPx involved in DHA synthesis were decreased which lead to the decrease of DHA generation after inhibiting the expression of LXRβ.4 The mRNA level of PPARα, ACOX3 and LBP (determined by RT-PCR method)On the same experiment condition, we failed to detect mRNA level of PPARαof Anti group. The ACOX3 and LBP mRNA level of Anti group were respectively 0.341±0.069, 0.235±0.025. The mRNA level of PPARα, ACOX3 and LBP of Con group were respectively 0.806±0.157, 0.563±0.142, 0.210±0.038. The mRNA level of PPARα, ACOX3 and LBP of Mis group were respectively 0.692±0.117, 0.623±0.094, 0.207±0.043. The ACOX3 and PPARαmRNA level of Anti group were singnificantly lower than that of Con and Mis group (P<0.01). There was no statistical difference in PPARαand ACOX3 mRNA level between Con group and Mis group (P>0.05). There was no statistical difference in LBP mRNA level among Anti group, Con group and Mis group (P>0.05). The results showed that mRNA expression of PPARαand ACOX3 related to peroxisomesβ-oxidation were down-regulated, but expression of LXRβwas not affected, after inhibiting the expression of LXRβ.Conclusion:1 LXRβcan affect the transcription of genes involved in the last step of DHA synthesis and then affect the generation of DHA in neurons. 2 LXRβcan affect the transcription of genes involved in peroxisomal fatty acidβ-oxidation.Summary:1 The increase of bile acid synthesis by activated-LXR is relevant to the enhancement of hepatic DBP mRNA level and activity.2 LXRβplay an important role in keep the cholesterol balance in neuron. The expression of genes with respect to APP metabolism and peroxisomal fatty acidsβ-oxidation in the process of Aβand DHA production were down-regulated after inhibiting the expression of LXRβ...