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

Duval C, Thissen U, Keshtkar S, Accart B, Stienstra R, Boekschoten MV, Roskams T, Kersten S, Müller M - Diabetes (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands.

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.

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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.)
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Figure 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.)

Mentions: Mice classified as high responders also gained the most body weight (Fig. 4A), likely related to increased food intake (Fig. 4B). Indeed, a positive correlation was found between final body weight and hepatic triglycerides (Fig. 4C). Remarkably, despite increased weight gain, weight of the epididymal fat pad at sacrifice was markedly lower in HFH compared with that in HFL mice (Fig. 4D). As expected, leptin expression in adipose tissue mirrored adiposity (Fig. 4E), which was also true for the plasma free fatty acids (Fig. 4F). Evaluation of the morphology of the epididymal fat pad in HFH mice after H-E staining revealed atrophied adipocytes surrounded by inflammatory cells, which were hardly observed in HFL responders (Fig. 4G). Cd68 immunostaining indicated increased presence of macrophages in HFH mice (Fig. 4H), which was supported by gene expression of F4/80 and Cd68 (Fig. 5). In contrast, expression of the anti-inflammatory adipokine adiponectin was markedly reduced in HFH mice, as was resistin (Fig. 5). Interestingly, expression of adipogenic (Pparγ and Fabp4) and lipogenic (Dgat2, Srebp-1, and fatty acid synthase, Fasn) marker genes was significantly downregulated in HFH mice compared with HFL mice, suggesting adipose tissue dysfunction. Finally, collagen staining revealed fibrotic adipose tissue in HFH mice (Fig. 4I), which was supported by increased expression of tissue inhibitor of matrix metalloproteinases (Timp-1) (Fig. 5). These data suggest that HFH responders, classification of which is entirely determined by liver histology, exhibit adipose tissue dysfunction characterized by decreased fat mass, enhanced macrophage infiltration, inflammation, and adipose tissue remodelling.


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

Duval C, Thissen U, Keshtkar S, Accart B, Stienstra R, Boekschoten MV, Roskams T, Kersten S, Müller M - Diabetes (2010)

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.)
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Figure 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.)
Mentions: Mice classified as high responders also gained the most body weight (Fig. 4A), likely related to increased food intake (Fig. 4B). Indeed, a positive correlation was found between final body weight and hepatic triglycerides (Fig. 4C). Remarkably, despite increased weight gain, weight of the epididymal fat pad at sacrifice was markedly lower in HFH compared with that in HFL mice (Fig. 4D). As expected, leptin expression in adipose tissue mirrored adiposity (Fig. 4E), which was also true for the plasma free fatty acids (Fig. 4F). Evaluation of the morphology of the epididymal fat pad in HFH mice after H-E staining revealed atrophied adipocytes surrounded by inflammatory cells, which were hardly observed in HFL responders (Fig. 4G). Cd68 immunostaining indicated increased presence of macrophages in HFH mice (Fig. 4H), which was supported by gene expression of F4/80 and Cd68 (Fig. 5). In contrast, expression of the anti-inflammatory adipokine adiponectin was markedly reduced in HFH mice, as was resistin (Fig. 5). Interestingly, expression of adipogenic (Pparγ and Fabp4) and lipogenic (Dgat2, Srebp-1, and fatty acid synthase, Fasn) marker genes was significantly downregulated in HFH mice compared with HFL mice, suggesting adipose tissue dysfunction. Finally, collagen staining revealed fibrotic adipose tissue in HFH mice (Fig. 4I), which was supported by increased expression of tissue inhibitor of matrix metalloproteinases (Timp-1) (Fig. 5). These data suggest that HFH responders, classification of which is entirely determined by liver histology, exhibit adipose tissue dysfunction characterized by decreased fat mass, enhanced macrophage infiltration, inflammation, and adipose tissue remodelling.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands.

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.

Show MeSH
Related in: MedlinePlus