Obeticholic Acid Modulates Serum Metabolites and Gene Signatures Characteristic of Human NASH and Attenuates Inflammation and Fibrosis Progression in Ldlr-/-.Leiden Mice

Abstract

Concerns have been raised about whether preclinical models sufficiently mimic molecular disease processes observed in nonalcoholic steatohepatitis (NASH) patients, bringing into question their translational value in studies of therapeutic interventions in the process of NASH/fibrosis. We investigated the representation of molecular disease patterns characteristic for human NASH in high-fat diet (HFD)-fed Ldlr-/-.Leiden mice and studied the effects of obeticholic acid (OCA) on these disease profiles. Multiplatform serum metabolomic profiles and genome-wide liver transcriptome from HFD-fed Ldlr-/-.Leiden mice were compared with those of NASH patients. Mice were profiled at the stage of mild (24 weeks HFD) and severe (34 weeks HFD) fibrosis, and after OCA intervention (24-34 weeks; 10 mg/kg/day). Effects of OCA were analyzed histologically, biochemically, by immunohistochemistry, using deuterated water technology (de novo collagen formation), and by its effect on the human-based transcriptomics and metabolomics signatures. The transcriptomics and metabolomics profile of Ldlr-/-.Leiden mice largely reflected the molecular signature of NASH patients. OCA modulated the expression of these molecular profiles and quenched specific proinflammatory-profibrotic pathways. OCA attenuated specific facets of cellular inflammation in liver (F4/80-positive cells) and reduced crown-like structures in adipose tissue. OCA reduced de novo collagen formation and attenuated further progression of liver fibrosis, but did not reduce fibrosis below the level before intervention. Conclusion: HFD-fed Ldlr-/-.Leiden mice recapitulate molecular transcriptomic and metabolomic profiles of NASH patients, and these signatures are modulated by OCA. Intervention with OCA in developing fibrosis reduces collagen deposition and de novo synthesis but does not resolve already manifest fibrosis in the period studied. These data show that human molecular signatures can be used to evaluate the translational character of preclinical models for NASH.

Figure 1

Representation of human metabolome in 24‐week HFD‐fed Ldlr‐/‐.Leiden mice. (A) Heatmap showing HFD‐induced alterations in serum metabolome in Ldlr‐/‐.Leiden mice as compared with chow‐fed Ldlr‐/‐.Leiden mice. Green indicates down‐regulation; red indicates up‐regulation. (B) Heatmap representation of the serum metabolomic profile from 535 biopsy‐confirmed NAFLD/NASH patients. Each data point corresponds to the relative ion abundance of a given metabolite (horizontal axis) in an individual patient’s serum (vertical axis), showing the top 50 metabolites that differentiated HFD‐fed Ldlr‐/‐.Leiden mice from chow. Blue indicates down‐regulation and red indicates up‐regulation relative to the level in the rest of the study population (mean value: 0). Hierarchical clustering based on the optimum average silhouette width divided the patients into two groups: the Ldlr‐/‐.Leiden subtype (serum metabolome resembles that observed in HFD‐fed Ldlr‐/‐.Leiden mice; right‐hand cluster) and the non‐Ldlr‐/‐.Leiden subtype (patients that have a different serum metabolome; left‐hand cluster). Abbreviations: AA, amino acid; AC, acyl carnitine; BA, bile acid; CE, cholesteryl ester; Cer, ceramide; CMH, monohexosylceramide; DG, diglyceride; FAA, fatty acyl amide; FSB, free sphingoid base; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; MG, monoglyceride; MUFA, monounsaturated fatty acid; NAE, N‐acylethanolamine; oxFA, oxidized fatty acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; SM, sphingomyelin; and TG, triglyceride.

Figure 2

Representation of human pathophysiological processes in 24‐week HFD‐fed Ldlr‐/‐.Leiden mice. (A) Heatmap showing significantly regulated genes in human NASH livers versus normal controls10 are largely represented in 24‐week HFD‐fed Ldlr‐/‐.Leiden mice. (B) Heatmap diagram showing recapitulation of hepatic gene expression profile that differentiates mild (fibrosis stage 0‐1) from severe (fibrosis stage 3‐4) NASH patients9 in 24‐week HFD‐fed Ldlr‐/‐.Leiden mice. Green indicates down‐regulation; red indicates up‐regulation. (C) Enrichment of the top 15 human NASH‐related processes10 in 24‐week HFD‐fed Ldlr‐/‐.Leiden mice. P values equal to or smaller than −log(P value) of 5 are shown as −log(P value) of 5.

Figure 3

OCA treatment reduces adipocyte hypertrophy and adipose tissue inflammation in the epidydimal depot. Mice were treated with OCA (10 mg/kg/day) from t = 24 weeks to t = 34 weeks. (A) OCA treatment reduced the average adipocyte size in the eWAT, reducing the presence of large adipocytes while tending to increase the presence of smaller adipocytes. (B) Adipose tissue inflammation, expressed as the number of CLS/1,000 adipocytes, was significantly reduced by OCA. Data are mean ± SD. *P < 0.05 HFD at 34 weeks.

Figure 4

OCA treatment reduces hepatic steatosis in 34‐week HFD‐fed Ldlr‐/‐.Leiden mice. Mice were treated with OCA (10 mg/kg/day) from t = 24 weeks to t = 34 weeks. (A) Representative images of hematoxylin and eosin–stained liver cross‐sections showing reduced hepatic steatosis in OCA‐treated mice. (B) Biochemically determined liver lipid content revealed a reduction in hepatic triglycerides and hepatic cholesteryl esters in OCA‐treated animals, whereas free cholesterol levels were not affected. Data are mean ± SD. *P < 0.05; ***P < 0.001 versus HFD at 34 weeks.

Figure 5

OCA treatment reduces hepatic inflammation in 34‐week HFD‐fed Ldlr‐/‐.Leiden mice. Mice were treated with OCA (10 mg/kg/day) from t = 24 weeks to t = 34 weeks. (A) OCA‐treatment tended to reduce the number of inflammatory clusters in liver. (B) The number of F4/80+ CLS was reduced by OCA. (C) OCA significantly increased the ratio of Arg1 to CD68 mRNA expression. The ratio of CD11c to CD68 mRNA expression (D) and CCR2 to CD68 mRNA expression (E) tended to be reduced in HFD+OCA relative to HFD. OCA tended to enhance the disappearance of CD4+ (F) and CD8+ (G) cells. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 versus HFD at 34 weeks.

Figure 6

OCA treatment reduces hepatic fibrosis in 34‐week HFD‐fed Ldlr‐/‐.Leiden mice. Mice were treated with OCA (10 mg/kg/day) from t = 24 weeks to t = 34 weeks. (A) Picrosirius red–stained liver cross‐sections showing reduced fibrosis in OCA‐treated mice. (B) Pathologist’s assessment of perihepatocellular fibrosis revealed reduced progression of fibrosis in HFD+OCA. (C) Biochemically determined hepatic collagen was reduced in HFD+OCA. OCA reduced the collagen area and degree of reticulation of thin‐fiber collagen (D) and aggregated‐fiber collagen (E) (multiphoton and SHG imaging). Data are mean ± SD. *P < 0.05; **P < 0.01 versus HFD at 34 weeks.

Figure 7

OCA treatment reduces de novo collagen synthesis in 34‐week HFD‐fed Ldlr‐/‐.Leiden mice. Mice were treated with OCA (10 mg/kg/day) from t = 24 weeks to t = 34 weeks. (A) OCA reduced de novo total collagen synthesis assessed by stable‐isotope labeling and in parallel reduced de novo synthesis of individual collagen chains in the guanidine‐insoluble (B) and guanidine‐soluble (C) fractions. Data are mean ± SD. *P < 0.05; **P < 0.01 versus HFD at 34 weeks.

Figure 8

OCA treatment modulates human‐based omics signatures. (A) Heatmap showing HFD‐induced alterations in Ldlr‐/‐.Leiden metabolome at 34 weeks (relative to chow) and effects of OCA thereupon. (B) Heatmap showing expression of genes differentially regulated in human NASH livers versus normal controls10 in 34‐week HFD‐fed Ldlr‐/‐.Leiden mice relative to chow and OCA‐treated mice relative to HFD. (C) Heatmap showing recapitulation of hepatic gene expression profile that differentiates mild (fibrosis stage 0‐1) from severe (fibrosis stage 3‐4) NASH patients9 in 34‐week HFD‐fed Ldlr‐/‐.Leiden mice relative to chow and modulation of this profile in OCA‐treated mice. Green indicates down‐regulation; red indicates up‐regulation. Abbreviations: CE, cholesteryl ester; Cer, ceramide; CMH, monohexosylceramide; DG, diglyceride; LPC, lyso‐phosphatidylcholine; LPE, lysophosphatidylethanolamine; MG, monoglyceride; PC, phosphatidylcholine; PE, phosphatidyl‐ethanolamine; SM, sphingomyelin; and TG, triglyceride.