Abstract 1

NOVEL CAUSATIVE RELATIONSHIP BETWEEN LOW HDL AND DIET-INDUCED NONALCOHOLIC FATTY LIVER DISEASE

低HDL和饮食诱导的非酒精性脂肪肝之间的新因果关系

Background and Aims: During the biogenesis of HDL, lipid free or minimally lipidated apoA-I interacts functionally with the lipid transporter ABCA1 to form immature discoidal HDL which are then converted into mature spherical particles by the action of lecithin:cholesterol acyl transferase (LCAT). Here we investigated the mechanistic relationship between low and dysfunctional HDL and diet-induced NAFLD development using mouse models.

Methods: We employed male apoA-I-deficient (apoA-I−/−) mice that lack classical apoA-I containing HDL and male deficient (LCAT−/−)mice that have immature discoidal HDL.

Results: Mice were fed the standard western-type diet for 24 weeks and then histological and biochemical analyses were performed.ApoA-I−/− mice showed increased diet-induced hepatic triglyceride deposition and disturbed hepatic histology while they exhibited reduced glucose tolerance and insulin sensitivity. Quantification of FASN-1, DGAT-1, and PPARg mRNA expression suggested that the increased hepatic triglyceride content of the apoA-I−/− mice was not due to de novo synthesis of triglycerides. Similarly, metabolic profiling did not reveal differences in the energy expenditure between the two mouse groups. However, apoA-I−/− mice exhibited enhanced intestinal absorption of dietary triglycerides, accelerated clearance of postprandial triglycerides, and a reduced rate of hepatic very low density lipoprotein triglyceride secretion. In agreement with these findings, adenovirus-mediated gene transfer of apoAI Milano in apoA-I−/− mice fed western-type diet for 12 weeks resulted in a significant reduction in hepatic triglyceride content and an improvement of hepatic histology and architecture. Similar to apoAI−/−mice, LCAT−/− mice were characterized by increased diet-induced hepatic triglyceride deposition and impaired hepatic histology and architecture. Adenovirus-mediated gene transfer of LCAT in LCAT−/−mice that were fed western-type diet for 12 weeks resulted in a significant reduction in hepatic triglyceride content and a great improvement of hepatic histology and architecture.

Results: Taken together, our data establish that the HDL metabolic pathway is a central contributor to the deposition of dietary triglycerides to the liver and the development of NAFLD. Our data further support that the coexistence of reduced HDL levels and NAFLD in an individual with metabolic syndrome may not be a mere coincidence, rather it underlays a strong causative relationship between these two conditions.

背景与目的：在高密度脂蛋白（HDL）生物合成过程中，无脂质或轻度脂化apoA-I与脂质转运体ABCA1相互作用，形成未成熟盘状的HDL，然后在卵磷脂胆固醇酰基转移酶（LCAT）的作用下转变为成熟的球形颗粒。本研究通过小鼠模型从机制上研究了低水平和功能异常的HDL与饮食诱导的非酒精性脂肪肝形成之间的关系。

方法：采用apoA-I缺陷（apoA-I-/-）雄性小鼠和（LCAT-/-）缺陷雄性小鼠，上述两种小鼠分别缺乏含HDL的经典apoA-I和LCAT。

结果：小鼠喂食标准西方型饮食24周，然后进行组织学和生化分析。结果发现apoA-I-/-小鼠饮食诱导的肝甘油三酯沉积增加，同时出现肝组织学异常，而且糖耐量和胰岛素敏感性降低。FASN-1，DGAT-1和PPARγmRNA表达定量分析表明，apoA-I-/-小鼠肝脏中甘油三酯含量的增加并非从头合成甘油三酯所致。同样，代谢分析也没有发现两组小鼠间能量消耗的差异。然而，apoA-I-/-小鼠肠道吸收的膳食甘油三酯增加，餐后甘油三酯清除加快，并且肝极低密度脂蛋白甘油三酯分泌速率降低。与这些发现一致的是，喂食西方型饮食12周的腺病毒介导apoA-IMilano转基因的apoA-I-/-小鼠，肝脏甘油三酯含量显著减少，肝组织学和结构明显改善。与apoA-I-/-小鼠相似，LCAT-/-小鼠也出现特征性饮食诱导的肝脏甘油三酯沉积增加以及肝组织学和结构受损。喂食西方型饮食12周的腺病毒介导LCAT转基因的LCAT-/-小鼠，肝脏甘油三酯含量显著减少，肝组织学和结构明显改善。

结论：综上所述，该研究结果表明HDL代谢途径是饮食甘油三酯在肝脏沉积和非酒精性脂肪肝发生的关键因素。我们的数据进一步支持代谢综合征患者HDL水平下降和NAFLD并存可能不是偶然的巧合，而是这两者之间有很强的因果关系。

Abstract 2

INTRAHEPATIC CHANGES IN BILE ACID COMPOSITION PROTECTS BSEP (ABCB11) KO MICE FROM HEPATIC INFLAMMATION IN METHIONINE CHOLINE DEFICIENT DIET INDUCED NASH

Background: Bile acids (BAs) activate G-coupled and nuclear receptors controlling hepatic glucose and lipid metabolism as well as inflammation. Non-alcoholic-steatohepatitis (NASH) is characterized by increased hepatic lipid storage and inflammation. BAs are transported into bile via the bile salt export pump (BSEP;ABCB11). BSEP transgenic mice display less fat accumulation but severe inflammation. We therefore hypothesized that absence of BSEP may sensitize to hepatic steatosis while at the same time reducing inflammation in a mouse model of NASH.

Methods: Wildtype (WT) and BSEP knockout (KO) mice were fed a methionine-choline-deficient (MCD) diet for 5 weeks to induce NASH. Liver RNA profile analysis was performed by RT-PCR. Serum biochemistry, hepatic TG, BA content/composition as well as liver histology were assessed.

Results: MCD feeding induced hepatic TG accumulation (1.8-fold) in WT mice and to a lower extent in BSEP KO mice. In line, mRNA expression of de novo lipogenesis and fatty acid transport markers was repressed to a higher extent in MCD fed BSEP KO mice. In contrast to WT animals, BSEP KO were protected from hepatic inflammation (reflected by Tnfa, F4/80, Mcp1 and iNOS expression) induced by MCD feeding. mRNA expression of the BA-importer Ntcp was down-regulated by 60% in WT mice upon MCD but to a greater extent in BSEP KO mice. Conversely, expression of Mrp4 and Ostb was increased (10-fold; 2.8-fold) only in BSEP KO mice. Measurement of hepatic bile acid composition uncovered a distinct increase in hepatic CDCA (3-fold) and CA (3-fold) concentration (both BAs are known FXR agonists) in BSEP KO MCD mice compared toWT controls, whereas amounts of hepatic UDCA and b-muricholic acid were decreased.

Conclusion: Absence of BSEP protects from hepatic inflammation and reduces fatty acid storage by modulating hepatic bile acid transport and intrahepatic bile acid composition. These effects are most likely mediated via FXR induction. Thus, pharmacological modulation of bile acid transport/metabolism could constitute a new therapeutic option for modulating inflammation during progression from fatty liver to NASH.

Abstract 3

CARDIOTROPHIN-1 INHIBITS LIPOGENESIS AND STIMULATES FATTY ACID OXIDATION IN THE LIVER RESOLVING HEPATIC STEATOSIS

Background and Aims: Cardiotrophin-1 (CT-1) is a member of the interleukin (IL)-6 family of cytokines which signals through the gp130/leukemia inhibitory factor receptor (LIFR) heterodimer. Recently, we have reported an important role for CT-1 in energy balance. In the present work, we have studied the effect of CT-1 on liver lipid metabolism.

Methods: We have analyzed the liver of CT-1 knock-out mice at different ages as well as the role of obesogenic diet in the development of fatty liver in CT-1−/− young animals. The effect of recombinant CT-1 (rCT-1) has been analyzed in two models of hepatic steatosis: ob/ob mice and normal mice fed with high fat diet (HFD) for 12 weeks. Histological studies and expression of genes and proteins involved in lipid metabolism were quantified by PCR and Western blots. In vitro studies were conducted to assess the effect of CT-1 on lipogenesis and fatty acid oxidation (FAO) in isolated hepatocytes.

Results: We observed that CT-1−/− mice under chow diet showed marked steatosis at 6 months of age. Also, younger CT-1 deficient animals subjected to HFD developed more severe fatty liver than wild type mice. These findings were accompanied by decreased expression of genes involved in FAO and increased expression of lipogenic genes in CT-1 null mice. The administration of rCT-1 to ob/ob mice or to HFD-fed mice resulted in a complete remission of liver steatosis. This was associated with down-regulation of genes involved in lipogenesis and fatty acid import to liver cells and up-regulation of those implicated in FAO. Mechanistically, rCT-1 increased the ratio AMP/ATP resulting in phosphorylation of AMPactivated kinase (AMPK), a key suppressor of lipogenesis and activator of FAO.We found that rCT1 induced vigorous upregulation of SIRT1, PGC1a deacetylation and stimulation of FAO. Functional studies in primary hepatocytes showed that the inhibitory effect of rCT-1 on lipogenesis and its stimulatory activity on FAO were abolished by dominant negative AMPK.

Conclusions: CT-1 plays a key role in hepatic lipid metabolism. This cytokine strongly inhibits lipogenesis and fatty acid import to the liver and activates FAO. In steatotic livers CT-1 administration efficiently eliminates fat accumulation.

Abstract 4

MAPPING EXPRESSION QUANTITATIVE TRAITS LOCI (eQTL) FOR PNPLA3 GENE IDENTIFIES ADDITIONAL SNPS ASSOCIATED WITH NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD) INDEPENDENT OF rs738409

Background: The PNPLA3 I148M (rs738409) polymorphism is associated with NAFLD. However, questions remain how PNPLA3 is regulated and whether additional polymorphisms contribute to its function or confer susceptibility to advanced NAFLD.

Aim: To identify genetic polymorphisms affecting hepatic PNPLA3 expression, and to investigate their effect on hepatic fat accumulation and NAFLD phenotype.

Methods: Using genome-wide hepatic eQTL mapping, we assessed for SNPs significantly associated with PNPLA3 mRNA expression. Identified SNPs were genotyped in 27 healthy liver donors, and correlated with hepatic total fat (HTF), triglyceride (HTG)and cholesterol (H-Chol) levels. In addition, we examined the association between these SNPs and histological features of NAFLD in the recent GWAS data generated by the FLIP (Fatty Liver:Inhibition of Progression) consortium.

Results: A genome-wide significant (p < 10−5) eQTL locus was mapped to the PNPLA3-SAMM50 region. Detailed linkage disequilibrium (LD) analysis revealed two major groups of SNPs, represented by rs139051 (p = 7.2×10−9) and rs2294918 (p = 3.5×10−7), independently affected PNPLA3 expression (LD R2=0.3). The previously identified rs738409 was not strongly associated with PNPLA3 expression (p = 0.01). LD level between rs738409 and rs139051 or rs2294918 was relatively weak (R2 < 0.15),suggesting that these loci independently influenced PNPLA3 function.The SNP rs139051 was significantly associated with HTF (r2 = 0.33,p = 0.002), THG (r2 = 0.25, p = 0.008) and H-Chol (r2 = 0.14, p = 0.05) in the liver samples. There is also a significant association between rs738409 and increased HTF (r2 = 0.21, p = 0.01), HTG (r2 = 0.21,p = 0.02) and H-Chol (r2 = 0.20, p = 0.02).In the FLIP GWAS, rs2294918 was significantly associated with NAFLD (p = 1.9×10−9), severe steatosis (1.0×10−9), steatohepatitis(1.5×10−7), and fibrosis (7.4×10−7). Neither rs738409 nor rs139051 were represented on the GWAS chip. No SNPs were in strong LD with rs139051. However rs2896019, a SNP in strong LD (R2 = 0.68) with rs738409, had the strongest genome-wide association with NAFLD (p = 4.1×10−30), severe steatosis (7.7×10−27), steatohepatitis (2.3×10−24), and fibrosis (1.2×10−15).

Conclusions: We identified SNPs, independent of I148M, affecting hepatic PNPLA3 expression and associated with hepatic lipid accumulation and advanced NAFLD. These data suggest a combination of qualitative (rs738409 as a missense, protein function-altering allele) and quantitative (rs139051 and rs2294918 as strong eQTLs) regulation of PNPLA3 function and play a role in the pathogenesis of NAFLD.

Abstract 5

KRüPPEL LIKE FACTOR 6 (KLF6) PROTECTS FROM NAFLD PROGRESSION THROUGH REGULATION OF ADIPOSE TISSUE INSULIN RESISTANCE

KLF6通过调节脂肪组织胰岛素抵抗阻止NAFLD进展

Background and Aims: We have previously shown that adipose tissue insulin resistance index (Adipo-IR), which reflects impaired insulin action in inhibiting peripheral lipolysis, is associated with higher degree of liver fibrosis and hepatic insulin resistance in patients with NAFLD. The intronic polymorphism in the zinc finger transcription factor Kruppel-like Factor 6 (KLF6), KLF6-IVS1–27G>A, is associated with lower NAFLD progression and lower hepatic insulin resistance (Hep-IR). KLF6 is also involved with adipogenesis that is promoted by insulin. Thus, the aim of this study was to evaluated if KLF6-IVS1–27G>A was associated with lower Adipo_IR and better lipid metabolism in previously genotyped 1218 healthy subjects of European ancestry recruited from 19 centers (RISC consortium).

Methods: RISC data collected included measures of body mass index (BMI), fat mass, fasting free fatty acids (FFA), insulin(INS), lipid profile (total cholesterol, HDL, LDL, triglycerides, apolipoprotein A-I, A-II, B, C-III and E), beta hydroxybutyrate (BHB)a marker of hepatic beta oxidation, Adipo-IR (fasting FFAxIns),Hep-IR (EGPxINS in a subgroup of n = 368). Genotype/phenotype relationships were studied by linear trend analysis corrected for age, sex and recruitment center.

Results: In the whole dataset we observed a stepwise increase in Adipo-IR in association with KLF6 genotype (KLF6_AA 10.7±2.6 n=12; KLF6_GA 16.7±1.1 n=151; KLF6_GG 17.2±0.4 n = 1055) that was statistically significant by linear regression analyses corrected for age, sex, recruitment centre and fat mass (p = 0.023) as well as a stepwise increase in Hep-IR (KLF6_AA 343±102 n=5; KLF6_GA 422±44 n = 38; KLF6_GG 496±19 n = 325, p = 0.02). Increased Adipo-IR was associated with Hep-IR and increased concentrations of TG,BHB, LDL, Apo-AII, Apo-B, Apo-CIII and fatty liver index, indicating hepatic fat overload. However, no significant linear trend was observed between KLF6 and increased cholesterol, triglyceride, Apolipoproteins or BHB plasma concentrations.

Conclusions: KLF6-IVS-27G, the genotype associated with NAFLD progression is linked to an increase in Adipo-IR. We propose that KLF6 plays a central role also in regulating peripheral lipid metabolism in response to insulin, contributing not only to the development and progression of NAFLD, but also to the pathogenesis of the metabolic syndrome itself.

背景与目的：先前研究已经表明，反映胰岛素抑制外周脂肪降解作用受损的脂肪组织胰岛素抵抗指数（Adipo-IR），与非酒精性脂肪肝患者高程度肝纤维化、以及肝胰岛素抵抗有关。KLF6的内含子具有多态性，KLF6-IVS1-27G>A与更少的脂肪肝进展和更低的肝胰岛素抵抗（HEP-IR）有关。KLF6也参与了由胰岛素促进的脂肪细胞分化。因此，本研究的目的是评估KLF6-IVS1-27G>A是否与Adipo-IR的降低和脂质代谢过程有关。研究对象为来自19个中心已进行基因分型的1218名欧洲血统的健康受试者。

方法：收集的数据包括身体质量指数（BMI）、脂肪量、空腹游离脂肪酸（FFA）、胰岛素（INS）、血脂（总胆固醇[TG]、高密度脂蛋白、低密度脂蛋白[LDL]、甘油三酯、载脂蛋白A-I、A-II、B、C-III和E）、肝β氧化标志物β-羟基丁酸酯（BHB）、Adipo-IR（空腹FFAxIns）、以及Hep-IR（一个亚组EGPxINS，n=368）。对基因型/表型之间的关系进行线性趋势分析。

结果：通过分析所有数据，我们观察到Adipo-IR逐步增加与KLF6的基因型有关（KLF6_AA：10.7±2.6，n=12；KLF6_GA：16.7±1.1，n=151；KLF6_GG：17.2±0.4，n=1055），并且在校正年龄、性别等因素后，线性回归分析有显著统计学意义（P=0.023）。此外，Hep-IR也逐步增加（KLF6_AA：343±102，n=5；KLF6_GA：422±44，n=38；KLF6_GG：496±19，n=325，P=0.02）。Adipo-IR增加与Hep-IR和TG、BHB，LDL，Apo-AII，Apo-B，Apo-CIII含量增加及脂肪肝指数有关，提示肝脂肪过量。然而，KLF6和TG、甘油三酯、载脂蛋白的升高或BHB血浆浓度之间未观察到显著线性趋势。

结论：与非酒精性脂肪肝进展相关的基因型KLF6 IVS-27G与Adipo-IR增加有关。我们推测KLF6在调节外周脂质代谢对胰岛素反应中起关键作用，不仅促进了非酒精性脂肪肝的发生和发展，也是代谢综合征自身的发病机制。