The Role Of Microbiota And Macrophage Lipolysis In Regulation Of Blood Lipid Levels

BackgroundCoronary heart disease(CHD) is the most common heart disease in middle-aged people in developed and now many developing countries. The underlying pathological basis of CHD is atherosclerosis(AS). Basic and clinical studies have demonstrated that dyslipidemia and inflammation contribute significantly to atherogenesis. For example, epidemiological studies have shown thatthe increased blood low-density lipoprotein-cholesterol(LDL-C) level is an independent risk factor for AS. The mechanisms responsible for dyslipidemia are likely multifactorial. In addition to common genetic and environmental factors, microbiota has recently been implicated in metabolic cardiovascular diseases. It has been reported that manipulations of microbiota can prevent or even cure some metabolic cardiovascular diseases. Howeverit remains largely unknown how microbiota exert these effects. Studies have shown that microbiota affect lipid and bile acid metabolism, suggesting that microbiota may function through regulation of lipid metabolism, but its underlying mechanismsremain unknown. The whole body cholesterol homeostasis is governed via three major metabolic pathways: de novo biosynthesis, biliary/fecal excretion, and intestinal absorption. Intestinal cholesterol absorption is an active process mediated by a protein named Niemann-Pick C1-Like 1(NCP1L1). Our previous studies have shown that NPC1L1 knockout mice display not only a deficiency in intestinal cholesterol absorption, but also a decreasein fecal mass. Given the significant contribution of gut microbiota to feces, our observation may imply that NPC1L1 may interact with gut microbiota to modulate whole-body cholesterol homeostasis. The goal of this study was to explore these interactions and the underlying mechanisms.Early atherosclerotic lesions are characterized by deposition of macrophage foam cells beneath the intima of arteries. The formation of macrophage foam cells is attributable to over-accumulation of triglycerides and cholesterol esters. We found that macrophagespecific inactivation of comparative gene identification-58(CGI-58) causes massive accumulation of triglycerides and cholesterol esters in both peritoneal and bone marrow-derived macrophages. CGI-58 is a lipolytic activator that serves as a coactivator of adipose triglyceride lipase(ATGL) to promote intracellular triglyceride hydrolysis. CGI-58-deficient macrophages also exert proinflammatory phenotypes, secreting more proinflammatory cytokines when compared to wild-type controls. Considering important roles of CGI-58 in limiting macrophage lipid accumulation and inflammation, two major factors promoting atherogenesis, a major goal of our project wasto examine whether macrophage CGI-58 regulates atherosclerosis development and progression.Objectives1. To determine whether microbiota affectregulatory mechanisms of cholesterol metabolism, especially NPC1L1-dependent cholesterol absorption. A long-term goal is toidentify gut microbiota that modulateintestinal cholesterol absorption.2. To establish a unique AS animal model, i.e., apolipoprotein E(apo E)-knockout mice expressing no CGI-58 in macrophages, to determine whether macrophage CGI-58 regulates whole-body lipid metabolism, inflammation and atherosclerosis.Methods1. To determine whether microbiota affectregulatory mechanisms of cholesterol metabolism1.1 Germ-free(GF) mice, an essential animal model for studying the relationship between microbiota and diseases, and the controlspecific pathogen-free(SPF) mice were fed a Western diet supplemented with or without a cholesterol absorption inhibitorezetimibe.1.2 A mixture of [14C]-cholesterol and [3H]-Sitosterol was used to detecte cholesterol absorption rate by being gavaged into mice. 5-Cholestene, β-Coprostanl, α-Cholestanone, Cholesterol were also used as standards in Gas Chromatography for measuring fecal neutral sterol excretion.1.3 Reliable enzymatic and ELISA methods were chosen for analyses of blood/bile/tissue lipids in blood and liver.1.4 A series of methods were used for analyzing the relationship between microbiota and cholesterol metabolisms, such as q PCR method for tissue gene expression levels, immunoblotting for tissue protein expression.1.5 To determine the details of gut microbiota relevant to cholesterol absorption, metagenomics was chosen for analyses of gut microbiota.2. To establish the effect on macrophage lipolysis in AS development2.1 Different macrophages, including peritoneal macrophages and bone marrow-derived macrophages isolated from macrophage CGI-58 knockout mice and wide type mice, were treated with several AS risk factors, such as oxidized LDL, to determine whether these factors interact with CGI-58 to regulate macrophager foam cell formation.2.2 A unique AS model wasestablishedand fed a western-type die to determine whether macrophage CGI-58 influences blood lipids and AS development.Results1. Microbiota prevents cholesterol loss from the body by regulating host gene expression in mice.1.1 A hallmark of GF mice is the giant cecum. Consistently we observed that the weight of cecum was significantly increased in GF relative to SPF mice. GF versus SPF mice had reducedbody weight, inguinal fat, and subcutaneous fat. Treatments with the cholesterol absorption inhibitor ezetimibe further reduced these measures. In addition, ezetimibe treatment decreasedfecal weight only in SPF, but not GF mice, indicating that ezetimibe needs microbiota to lower fecal output.1.2 There was a significant decrease in intestinal cholesterol absorption and a significant increase in fecal cholesterol excretion in GF mice relative to SFP mice. Ezetimibe treatment exerts stronger effects on cholesterol absorption and excretion in GF mice compared to SPF mice.1.3 We examined gallbladder biliary concentrations of cholesterol, phospholipids and bile acids, and found that biliary cholesterol was decreased in GF versus SPF mice on western diet. Ezetimibe further reduced biliary cholesterol in both GF and SPF mice and abolished the difference in biliary cholesterol between the two types of mice.1.4 The concentrations of plasma cholesterol, triglycerides and phospholipids were all lower in GF mice than SPF mice. Ezetimibe treatment had a greater effect on plasma cholesterol and triglycerides in GF than SPF mice. Hepatic contents of free cholesterol, 5 total cholesterol, cholesterol ester, triglycerides and phospholipids were also lower in GF mice compared to SPF mice. Ezetimibe treatment significantly reduced these lipids in both GF and SPF mice, and the reduction was greater in GF than SPF mice overall.1.5 We found that jejunal expression levels of genes implicated in liogenesis, such as SREBP-1C, FAS and SCD-1, were lower in GF mice relative to SPF mice. The expression levels of genes related to bile acids sensing and transport, such as farnesoid x receptor(FXR), OST-βand FGF-15, were also lower in GF mice than in SPF mice. In addition, jejunal expression levels of genes related to lipoprotein metabolism and transport were also altered in GF mice. For example, HDL receptor SR-BI expression was much higher in GF mice than SPF mice. Ezetimibe treatment further reduced SR-BI expression. The expression levelsof m RNA for LDL receptor did not differ between GF and SPF mice. Ezetimibe treatment significantly increased LDLR m RNA levels in both types of mice. Meanwhile, the m RNA and protein levels of genes related to cholesterol transport and absorption were also altered. For example, the m RNA and protein levels of ABCA1 and NPC1L1 were significantly reduced, and those of ABCG5 and ABCG8 were increased, in GF mice compared to SPF mice. Ezetimibe had no further impact on expression levels of these genes except further reducing ABCA1.1.6 The results showed that the m RNA expression levels of cholesterol synthetic enzymes, such as HMG-Co A, HMGCR, HMGCS and FPPS, were all higher in GF mice than SPF mice. Ezetimibe treatment further increased these levels. Expression levels of lipoprotein receptors, such as SR-BI and LDLR, were also higher in GF mice. Expression levels of ABCA1, SREBP-1C and SCD-1, which are the targets of cholesterol sensor liver x receptor(LXR), significantly lower in GF mice than SPF mice. Mixed changes were observed for genes related to bile acids metabolism. While FXR expression was lower, CYP7A1 and CYP7B1 expression levels were higher in GF mice compared to SPF mice.1.7 The results showed that NPC1L1 knckout-induced inhibition of intestinal cholesterol absorption caused dramatic changes in gut microbiota, especially under the high-fat diet condition.2. The effects of macrophage lipolysis on the development and progression of AS.2.1 We found that ox-LDL treatmentsignificantly down- regulated CGI-58 protein expression in THP-1 cells, primary peritoneal macrophages and bone marrow-derived macrophages. To assess foam cell formation, we stained intracellular lipid droplets with BODYPI dye in peritoneal macrophages. Consistently with our previous findings, lipid droplets were increased in macrophages isolated from macrophage-specific CGI-58 knockout mice. After ox-LDL treatment, lipid droplets were increased in peritoneal macrophages isolated from bothmacrophage-specific CGI-58 knockout mice and controls with more lipid droplets seen in the knockout group.2.2 We genetically inactivated macrophage CGI-58 in Apo E knockout mice. The genotype of our animal model was Apo E-/-CGI-58f/f/Lyso Cre+(DKO).DKO mice showed no alterations in weight gainand fertility. Interestinly, their blood levels of cholesterol and triglycerides were significantly increased after 6 weeks ona western diet when compared to control mice(Apo E-/-CGI-58f/f/). A pilot AS study showed that the plaques on aorta wereincreased in the DKO mice compared to the controls. Although further studies are needed to reach a conclusion, significant increases in blood lipids imply that macrophage CGI-58-deficient apo E-KO mice may develop more atherosclerotic plaques.2.3 Through glucose and insulin tolerance tests, we found that the DKO mice exhibited impaired glucose and insulin tolerance, suggesting that macrophage CGI-58 also regulates glucose metabolism.Conclusion1. Microbiota may be the important element of body cholesterol regulation.2. Microbiota may increase intestinal cholesterol absorption and reduce fecal cholesterol excretion by regulating expression levels of genes related to cholesterol metabolism and transport in intestine and liver of the host.3. Firmicutes and Bacterium may relevant to cholesterol regulation mediated by NPC1L1.4. AS risk factors may promote the formation of foam cells by inhibiting macrophage CGI-58 expression5. Macrophage CGI-58-dependent lipolysis is an important regulator of blood lipid levels..