👤 Lars Verschuren

Non-alcoholic steatohepatitis (NASH) is the most rapidly growing liver disease that is nevertheless without approved pharmacological treatment. Despite great effort in developing novel NASH therapeutics, many have failed in clinical trials. This has raised questions on the adequacy of preclinical models. Elafibranor is one of the drugs currently in late stage development which had mixed results for phase 2/interim phase 3 trials. In the current study we investigated the response of elafibranor in APOE*3Leiden.CETP mice, a translational animal model that displays histopathological characteristics of NASH in the context of obesity, insulin resistance and hyperlipidemia. To induce NASH, mice were fed a high fat and cholesterol (HFC) diet for 15 weeks (HFC reference group) or 25 weeks (HFC control group) or the HFC diet supplemented with elafibranor (15 mg/kg/d) from week 15-25 (elafibranor group). The effects on plasma parameters and NASH histopathology were assessed and hepatic transcriptome analysis was used to investigate the underlying pathways affected by elafibranor. Elafibranor treatment significantly reduced steatosis and hepatic inflammation and precluded the progression of fibrosis. The underlying disease pathways of the model were compared with those of NASH patients and illustrated substantial similarity with molecular pathways involved, with 87% recapitulation of human pathways in mice. We compared the response of elafibranor in the mice to the response in human patients and discuss potential pitfalls when translating preclinical results of novel NASH therapeutics to human patients. When taking into account that due to species differences the response to some targets, like PPAR-α, may be overrepresented in animal models, we conclude that elafibranor may be particularly useful to reduce hepatic inflammation and could be a pharmacologically useful agent for human NASH, but probably in combination with other agents. Show less

While fibrosis stage predicts liver-associated mortality, cardiovascular disease (CVD) is still the major overall cause of mortality in patients with NASH. Novel NASH drugs should thus ideally reduce both liver fibrosis and CVD. Icosabutate is a semi-synthetic, liver-targeted eicosapentaenoic acid (EPA) derivative in clinical development for NASH. The primary aims of the current studies were to establish both the anti-fibrotic and anti-atherogenic efficacy of icosabutate in conjunction with changes in lipotoxic and atherogenic lipids in liver and plasma respectively. The effects of icosabutate on fibrosis progression and lipotoxicity were investigated in amylin liver NASH (AMLN) diet (high fat, cholesterol and fructose) fed ob/ob mice with biopsy-confirmed steatohepatitis and fibrosis and compared with the activity of obeticholic acid. APOE*3Leiden.CETP mice, a translational model for hyperlipidaemia and atherosclerosis, were used to evaluate the mechanisms underlying the lipid-lowering effect of icosabutate and its effect on atherosclerosis. In AMLN ob/ob mice, icosabutate significantly reduced hepatic fibrosis and myofibroblast content in association with downregulation of the arachidonic acid cascade and a reduction in both hepatic oxidised phospholipids and apoptosis. In APOE*3Leiden.CETP mice, icosabutate reduced plasma cholesterol and TAG levels via increased hepatic uptake, upregulated hepatic lipid metabolism and downregulated inflammation pathways, and effectively decreased atherosclerosis development. Icosabutate, a structurally engineered EPA derivative, effectively attenuates both hepatic fibrosis and atherogenesis and offers an attractive therapeutic approach to both liver- and CV-related morbidity and mortality in NASH patients. Show less

Icosabutate is a structurally engineered eicosapentaenoic acid derivative under development for nonalcoholic steatohepatitis (NASH). In this study, we investigated the absorption and distribution properties of icosabutate in relation to liver targeting and used rodents to evaluate the effects of icosabutate on glucose metabolism, insulin resistance, as well as hepatic steatosis, inflammation, lipotoxicity, and fibrosis. The absorption, tissue distribution, and excretion of icosabutate was investigated in rats along with its effects in mouse models of insulin resistance ( Show less

Epidemiological studies have reported positive associations between serum perfluorooctanoic acid (PFOA) and total and non-high-density lipoprotein cholesterol (non-HDL-C) although the magnitude of effect of PFOA on cholesterol lacks consistency. The objectives of this study were to evaluate the effect of PFOA on plasma cholesterol and triglyceride metabolism at various plasma PFOA concentrations relevant to humans, and to elucidate the mechanisms using APOE*3-Leiden.CETP mice, a model with a human-like lipoprotein metabolism. APOE*3-Leiden.CETP mice were fed a Western-type diet with PFOA (10, 300, 30 000 ng/g/d) for 4-6 weeks. PFOA exposure did not alter plasma lipids in the 10 and 300 ng/g/d dietary PFOA dose groups. At 30 000 ng/g/d, PFOA decreased plasma triglycerides (TG), total cholesterol (TC), and non-HDL-C, whereas HDL-C was increased. The plasma lipid alterations could be explained by decreased very low-density lipoprotein (VLDL) production and increased VLDL clearance by the liver through increased lipoprotein lipase activity. The concomitant increase in HDL-C was mediated by decreased cholesteryl ester transfer activity and changes in gene expression of proteins involved in HDL metabolism. Hepatic gene expression and pathway analysis confirmed the changes in lipoprotein metabolism that were mediated for a major part through activation of the peroxisome proliferator-activated receptor (PPAR)α. Our data confirmed the findings from a phase 1 clinical trial in humans that demonstrated high serum or plasma PFOA levels resulted in lower cholesterol levels. The study findings do not show an increase in cholesterol at environmental or occupational levels of PFOA exposure, thereby indicating these findings are associative rather than causal. Show less

Treatment with the second and third generation BCR-ABL1 tyrosine kinase inhibitors (TKIs) increases cardiovascular risk in chronic myeloid leukemia (CML) patients. We investigated the vascular adverse effects of three generations of TKIs in a translational model for atherosclerosis, the APOE*3Leiden.CETP mouse. Mice were treated for sixteen weeks with imatinib (150 mg/kg BID), nilotinib (10 and 30 mg/kg QD) or ponatinib (3 and 10 mg/kg QD), giving similar drug exposures as in CML-patients. Cardiovascular risk factors were analyzed longitudinally, and histopathological analysis of atherosclerosis and transcriptome analysis of the liver was performed. Imatinib and ponatinib decreased plasma cholesterol (imatinib, -69%, Show less

Salsalate (salicylsalicylic acid) is an anti-inflammatory drug that was recently found to exert beneficial metabolic effects on glucose and lipid metabolism. Although its utility in the prevention and management of a wide range of vascular disorders, including type 2 diabetes and metabolic syndrome has been suggested before, the potential of salsalate to protect against non-alcoholic steatohepatitis (NASH) remains unclear. The aim of the present study was therefore to ascertain the effects of salsalate on the development of NASH. Transgenic APOE*3Leiden.CETP mice were fed a high-fat and high-cholesterol diet with or without salsalate for 12 and 20 weeks. The effects on body weight, plasma biochemical variables, liver histology and hepatic gene expression were assessed. Salsalate prevented weight gain, improved dyslipidemia and insulin resistance and ameliorated diet-induced NASH, as shown by decreased hepatic microvesicular and macrovesicular steatosis, reduced hepatic inflammation and reduced development of fibrosis. Salsalate affected lipid metabolism by increasing β-oxidation and decreasing lipogenesis, as shown by the activation of PPAR-α, PPAR-γ co-activator 1β, RXR-α and inhibition of genes controlled by the transcription factor MLXIPL/ChREBP. Inflammation was reduced by down-regulation of the NF-κB pathway, and fibrosis development was prevented by down-regulation of TGF-β signalling. Salsalate exerted a preventive effect on the development of NASH and progression to fibrosis. These data suggest a clinical application of salsalate in preventing NASH. Show less

Plasma total cholesterol (TC) levels are highly genetically determined. Although ample evidence of genetic determination of separate lipoprotein cholesterol levels has been reported, using TC level directly as a phenotype in a relatively large broad-gene based association study has not been reported to date. We genotyped 361 single nucleotide polymorphisms (SNPs) across 243 genes based on pathways potentially relevant to cholesterol metabolism in 3575 subjects that were examined thrice over 11 years. Twenty-three SNPs were associated with TC levels after adjustment for multiple testing. We used 12 of them (rs7412 and rs429358 in APOE, rs646776 in CELSR2, rs1367117 in APOB, rs6756629 in ABCG5, rs662799 in APOA5, rs688 in LDLR, rs10889353 in DOCK7, rs2304130 in NCAN, rs3846662 in HMGCR, rs2275543 in ABCA1, rs7275 in SMARCA4) that were confirmed in previous candidate association or genome-wide-association studies to define a gene risk score (GRS). Average TC levels increased from 5.23 ± 0.82 mmol/L for those with 11 or less cholesterol raising alleles to 6.03 ± 1.11 mmol/L for those with 18 or more (P for trend<0.0001). The association with TC levels was slightly stronger when the weighted GRS that weighted the magnitude of allelic effects was used. A panel of common genetic variants in the genes pivotal in cholesterol metabolism could possibly help identify those people who are at risk of high cholesterol levels. Show less

The delta-5 and delta-6 desaturases, encoded by the FADS1 and FADS2 genes, are rate-limiting enzymes in polyunsaturated fatty acid (PUFA) biosynthesis. Single nucleotide polymorphisms in the FADS gene cluster region have been associated with both PUFA concentrations in plasma or erythrocyte membrane phospholipids and cholesterol concentrations in recent genome-wide association studies. We examined whether genetic variations in the FADS gene cluster region interact with dietary intakes of n-3 (omega-3) and n-6 (omega-6) PUFAs to affect plasma total, HDL-, and non-HDL-cholesterol concentrations. Dietary intakes of n-3 and n-6 PUFAs, plasma concentrations of total and HDL cholesterol, and rs174546, rs482548, and rs174570 in the FADS gene cluster region were measured in 3575 subjects in the second survey of the Doetinchem Cohort Study. Significant associations between rs174546 genotypes and total and non-HDL-cholesterol concentrations were observed in the group with a high intake of n-3 PUFAs (> or =0.51% of total energy; P = 0.006 and 0.047, respectively) but not in the low-intake group (P for interaction = 0.32 and 0.51, respectively). The C allele was associated with high total and non-HDL-cholesterol concentrations. Furthermore, the C allele was significantly associated with high HDL-cholesterol concentrations in the group with a high intake of n-6 PUFAs (> or =5.26% of total energy, P = 0.004) but not in the group with a low intake (P for interaction = 0.02). Genetic variation in the FADS1 gene potentially interacts with dietary PUFA intakes to affect plasma cholesterol concentrations, which should be investigated further in other studies. Show less

The known genetic variants determining plasma HDL cholesterol (HDL-C) levels explain only part of its variation. Three hundred eighty-four single nucleotide polymorphisms (SNPs) across 251 genes based on pathways potentially relevant to HDL-C metabolism were selected and genotyped in 3,575 subjects from the Doetinchem cohort, which was examined thrice over 11 years. Three hundred fifty-three SNPs in 239 genes passed the quality-control criteria. Seven SNPs [rs1800777 and rs5882 in cholesteryl ester transfer protein (CETP); rs3208305, rs328, and rs268 in LPL; rs1800588 in LIPC; rs2229741 in NRIP1] were associated with plasma HDL-C levels with false discovery rate (FDR) adjusted q values (FDR_q) < 0.05. Five other SNPs (rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, rs6060717 near SCAND1, and rs3213451 in MBTPS2 in women) were associated with plasma HDL-C levels with FDR_q between 0.05 and 0.2. Two less well replicated associations (rs3135506 in APOA5 and rs1800961 in HNF4A) known from the literature were also observed, but their significance disappeared after adjustment for multiple testing (P = 0.008, FDR_q = 0.221 for rs3135506; P = 0.018, FDR_q = 0.338 for rs1800961, respectively). In addition to replication of previous results for candidate genes (CETP, LPL, LIPC, HNF4A, and APOA5), we found interesting new candidate SNPs (rs2229741 in NRIP1, rs3213451 in MBTPS2, rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, and rs6060717 near SCAND1) for plasma HDL-C levels that should be evaluated further. Show less