Adipose tissue dysfunction signals progression of hepatic steatosis towards nonalcoholic steatohepatitis in C57BL/6 mice

Abstract

Objective: Nonalcoholic fatty liver disease (NAFLD) is linked to obesity and diabetes, suggesting an important role of adipose tissue in the pathogenesis of NAFLD. Here, we aimed to investigate the interaction between adipose tissue and liver in NAFLD and identify potential early plasma markers that predict nonalcoholic steatohepatitis (NASH).

Research design and methods: C57Bl/6 mice were chronically fed a high-fat diet to induce NAFLD and compared with mice fed a low-fat diet. Extensive histological and phenotypical analyses coupled with a time course study of plasma proteins using multiplex assay were performed.

Results: Mice exhibited pronounced heterogeneity in liver histological scoring, leading to classification into four subgroups: low-fat low (LFL) responders displaying normal liver morphology, low-fat high (LFH) responders showing benign hepatic steatosis, high-fat low (HFL) responders displaying pre-NASH with macrovesicular lipid droplets, and high fat high (HFH) responders exhibiting overt NASH characterized by ballooning of hepatocytes, presence of Mallory bodies, and activated inflammatory cells. Compared with HFL responders, HFH mice gained weight more rapidly and exhibited adipose tissue dysfunction characterized by decreased final fat mass, enhanced macrophage infiltration and inflammation, and adipose tissue remodeling. Plasma haptoglobin, IL-1β, TIMP-1, adiponectin, and leptin were significantly changed in HFH mice. Multivariate analysis indicated that in addition to leptin, plasma CRP, haptoglobin, eotaxin, and MIP-1α early in the intervention were positively associated with liver triglycerides. Intermediate prognostic markers of liver triglycerides included IL-18, IL-1β, MIP-1γ, and MIP-2, whereas insulin, TIMP-1, granulocyte chemotactic protein 2, and myeloperoxidase emerged as late markers.

Conclusions: Our data support the existence of a tight relationship between adipose tissue dysfunction and NASH pathogenesis and point to several novel potential predictive biomarkers for NASH.

FIG. 1.

A subpopulation of mice fed an HFD develops NASH. A: Changes in body weight in C57Bl/6 mice fed an LFD (□; n = 10) or HFD (■; n = 8). B: Mean energy intake of mice fed an LFD or HFD during 21 weeks of dietary intervention. C: Weight of epididymal fat pad after 21 weeks of dietary intervention. Error bars reflect SD. *Significantly different from mice fed an LFD according to Student's t test (P < 0.05). H-E staining (D) and oil red O staining (E) of representative liver sections of the four subgroups (LFL, LFH, HFL, and HFH). F: liver triglyceride concentration. G: Liver weight (expressed as percentage of total body weight [BW]). H: Activity of alanine aminotransferase (ALT) (glutamate pyruvate transaminase) in plasma. Error bars reflect SD. Bars with different letters are statistically different (P < 0.05 according to Student's t test). n = 4 mice per group for LFL, HFL, and HFH, and n = 6 mice per group for LFH. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Upregulation of inflammatory and fibrotic gene expression in HFH responder mice. A: Number of genes up- or downregulated in the various subgroups in comparison with the LFL mice as determined by Affymetrix GeneChip analysis. Genes with a P value <0.05 were considered significantly regulated. B: Heat map showing changes in expression of selected genes involved in lipid metabolism, inflammation, and fibrosis in liver. Mean expression in LFL mice was set at 1. Gene expression changes in individual mice within the HFH group are shown on the right. C: Changes in gene expression of selected genes as determined by real-time quantitative PCR. Mean expression in LFL mice was set at 100%. Error bars reflect SD. Bars with different letters are statistically different (P < 0.05 according to Student's t test). Number of mice per group: n = 4 for the LFL, HFL, and HFH groups and n = 6 for the LFH group.

FIG. 3.

(Immuno)histochemical staining confirms enhanced inflammation and early fibrosis in HFH mice. A: Immunohistochemical staining of macrophage activation in representative liver sections of HFL (left panel) and HFH (right panel) mice using antibody against the specific macrophage marker Cd68. B: Collagen staining using fast green FCF/sirius red F3B. C: Staining of stellate cell activation using antibody against GFAP. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Adipose dysfunction in HFH mice. A: Body weight changes in the four subgroups during the 21-week dietary intervention. White squares, LFL; light-gray squares, LFH; dark-gray squares, HFL; black squares, HFH. B: Mean daily energy intake. C: Positive correlation between final body weight and liver triglyceride concentration (P < 0.05). D: Weight of epididymal fat depot. E: Adipose tissue leptin mRNA expression as determined by quantitative PCR. Mean expression in LFL mice was set at 100%. F: Plasma free fatty acid levels. Error bars reflect SD. *Significantly different from HFL mice according to Student's t test (P < 0.05). Number of mice per group: n = 4 for LFL, HFL, and HFH and n = 6 for LFH. G: H-E staining of representative adipose tissue sections. H: Immunohistochemical staining of macrophages using antibody against Cd68. I: Collagen staining using fast green FCF/sirius red F3B. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Changes in adipose gene expression indicates adipose tissue dysfunction. Adipose tissue mRNA expression of a selected group of genes was determined by quantitative real-time PCR after 21 weeks of dietary intervention. Mean expression in LFL mice was set at 100%. Error bars reflect SD. *Significantly different from HFL mice according to Student's t test (P < 0.05). Number of mice per group: n = 4 for LFL, HFL, and HFH and n = 6 for LFH.

FIG. 6.

Plasma proteins as early predictive biomarker for NASH in C57Bl/6 mice. A: Plasma concentration of haptoglobin, TIMP-1, IL-1β, leptin, and insulin were determined by multiplex assay at specific time points during the 21 weeks of dietary intervention after a 6-h fast. White squares, LFL; light-gray squares, LFH; dark-gray squares, HFL; black squares, HFH. Error bars reflect SD. *Significantly different from HFL mice according to Student's t test (P < 0.05). Number of mice per group: n = 4 for LFL, HFL, and HFH and n = 6 for LFH. B: Graphs illustrating the result of multivariate analysis showing the association of protein plasma concentrations at various time points with final liver triglyceride content. The absRSD is the absolute relative standard deviation: the standard deviation of the regression coefficients divided by the absolute mean value of the regression coefficients. Significant proteins display an inverse absRSD value higher than two (bold line indicates the inverse absRSD threshold value of 2). w, weeks.