Dual role of protein tyrosine phosphatase 1B in the progression and reversion of non-alcoholic steatohepatitis

Abstract

Objectives: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. Protein tyrosine phosphatase 1B (PTP1B), a negative modulator of insulin and cytokine signaling, is a therapeutic target for type 2 diabetes and obesity. We investigated the impact of PTP1B deficiency during NAFLD, particularly in non-alcoholic steatohepatitis (NASH).

Methods: NASH features were evaluated in livers from wild-type (PTP1BWT) and PTP1B-deficient (PTP1BKO) mice fed methionine/choline-deficient diet (MCD) for 8 weeks. A recovery model was established by replacing MCD to chow diet (CHD) for 2-7 days. Non-parenchymal liver cells (NPCs) were analyzed by flow cytometry. Oval cells markers were measured in human and mouse livers with NASH, and in oval cells from PTP1BWT and PTP1BKO mice.

Results: PTP1BWT mice fed MCD for 8 weeks exhibited NASH, NPCs infiltration, and elevated Fgf21, Il6 and Il1b mRNAs. These parameters decreased after switching to CHD. PTP1B deficiency accelerated MCD-induced NASH. Conversely, after switching to CHD, PTP1BKO mice rapidly reverted NASH compared to PTP1BWT mice in parallel to the normalization of serum triglycerides (TG) levels. Among NPCs, a drop in cytotoxic natural killer T (NKT) subpopulation was detected in PTP1BKO livers during recovery, and in these conditions M2 macrophage markers were up-regulated. Oval cells markers (EpCAM and cytokeratin 19) significantly increased during NASH only in PTP1B-deficient livers. HGF-mediated signaling and proliferative capacity were enhanced in PTP1BKO oval cells. In NASH patients, oval cells markers were also elevated.

Conclusions: PTP1B elicits a dual role in NASH progression and reversion. Additionally, our results support a new role for PTP1B in oval cell proliferation during NAFLD.

Keywords: Inflammation; Oval cells; PTP1B; Steatohepatitis; Steatosis.

Figure 1

Lack of PTP1B accelerated MCD-induced NASH. A. Representative images of Hematoxylin & Eosin staining, and NAFLD score (NAS). B. Serum ALT and hepatic Fgf21 mRNA levels. C. Serum TG levels. D. Hepatic TG content and Cd36 mRNA levels. Experimental groups: PTP1BWT and PTP1BKO mice fed chow (CHD) or methionine/choline-deficient diet for 4 (4wMCD) or 8 weeks (8wMCD) (n = 8–12 animals/group). *P < 0.05, **P < 0.01 and ***P < 0.005, PTP1BKO vs. PTP1BWT; #P < 0.05, ##P < 0.01 and ###P < 0.005, 4wMCD or 8wMCD vs. CHD. E. Bromopalmitate uptake and Nile Red staining in PTP1BWT and PTP1BKO hepatocytes. (n = 4 independent experiments performed in duplicate). ***P < 0.005, KO vs. WT.

Figure 2

PTP1B deficiency enhanced an inflammatory response during the MCD diet challenge. A. Hepatic mRNA levels of inflammatory markers. B, C. Analysis of immune NPCs populations. Experimental groups: PTP1BWT and PTP1BKO mice fed chow (CHD) or methionine/choline-deficient diet for 4 (4wMCD) or 8 weeks (8wMCD) (n = 8–12 animals/group). *P < 0.05, **P < 0.01 and ***P < 0.001, PTP1BKO vs. PTP1BWT; #P < 0.05, ##P < 0.01 and ###P < 0.005, 4wMCD or 8wMCD vs. CHD.

Figure 3

Differential effects on neutrophils and natural killer cells during the MCD challenge between genotypes. A, B, C. Analysis of immune NPCs populations. Experimental groups: PTP1BWT and PTP1BKO mice fed chow (CHD) or methionine/choline-deficient diet for 4 (4wMCD) or 8 weeks (8wMCD) (n = 8–12 animals/group). *P < 0.05, **P < 0.01 and ***P < 0.005, PTP1BKO vs. PTP1BWT; #P < 0.05, ##P < 0.01 and ###P < 0.005, 4wMCD or 8wMCD vs. CHD.

Figure 4

PTP1B deficiency increased the proinflammatory response in macrophages treated with palmitate. A. Nitrites accumulation. B, C. mRNA levels of inflammatory cytokines markers. Peritoneal macrophages were isolated from wild-type and PTP1B-deficient mice treated with palmitate 500 μM. (n = 3 independent experiments performed in triplicate). *P < 0.05, **P < 0.01 and ***P < 0.005, P vs. B; #P < 0.05, ##P < 0.01 and ###P < 0.005, KO vs. WT.

Figure 5

Histological and molecular characterization of livers from wild-type and PTP1B-deficient mice re-fed chow diet (CHD) after 8 weeks of methionine/choline-deficient diet (MCD). A. Representative images of Hematoxylin & Eosin staining, and NAFLD score (NAS). B. Serum ALT and hepatic Fgf21 mRNA levels. C. Serum TG levels. D. Hepatic TG content and Cd36 mRNA levels. E. Hepatic mRNA levels of inflammatory markers. Experimental groups: PTP1BWT and PTP1BKO re-fed chow diet for 2 (8wMCD+2dCHD), 4 (8wMCD+4dCHD) or 7 days (8wMCD+7dCHD) after 8 weeks of methionine/choline-deficient diet (8wMCD). (n = 8–12 animals/group) *P < 0.05, **P < 0.01 and ***P < 0.005, PTP1BKO vs. PTP1BWT; #P < 0.05, ##P < 0.01 and ###P < 0.005, 8wMCD+CHD vs. 8wMCD.

Figure 6

Analysis of NPCs population during the recovery phase in livers from PTP1BWT and PTP1BKO mice. A, B, C. Analysis of immune NPCs populations. Experimental groups: PTP1BWT and PTP1BKO re-fed chow diet for 4 (8wMCD+4dCHD) after 8 weeks of methionine/choline-deficient diet (8wMCD). (n = 8–12 animals/group) **P < 0.01 and ***P < 0.005, PTP1BKO vs. PTP1BWT; ###P < 0.005, 8wMCD+4dCHD vs. 8wMCD.

Figure 7

Enhanced proliferative capacity and expression of oval cell markers in livers from PTP1BKO mice. A. Representative images of Ki67 immunostaining. B. Quantification of Ki67-positive cells. C. Hepatic mRNA levels of oval cell markers. Experimental groups: PTP1BWT and PTP1BKO fed chow (CHD) or methionine/choline-deficient diet for 4 (4wMCD) or 8 weeks (8wMCD), or re-fed chow diet for 2 (8wMCD+2dCHD), 4 (8wMCD+4dCHD) or 7 days (8wMCD+7dCHD) after 8 weeks of MCD. (n = 8–12 animals/group) *P < 0.05, **P < 0.01 and ***P < 0.005, PTP1BKO vs. PTP1BWT; #P < 0.05, ##P < 0.01 and ###P < 0.005, 8wMCD+CHD vs. 8wMCD. D. Hepatic mRNA levels of oval cell markers in livers from NAFL and NASH patients. *P < 0.05, **P < 0.01 and ***P < 0.005, NAFL or NASH vs. NL.

Figure 8

Characterization and responsiveness to HGF of oval cell isolated from wild-type and PTP1B-deficient mice treated with DDC. A. Representative images of cytokeratin 19 (CK19) immunostaining. B. Hepatic mRNA levels of oval cell markers. Experimental groups: PTP1BWT and PTP1BKO treated with DDC for 14 days. (n = 6–8 animals/group) *P < 0.05, PTP1BKO vs. PTP1BWT; ##P < 0.01 and ###P < 0.005, DDC vs. CHD. C. Representative images of immunofluorescence of oval cells markers. D. Representative blots with the indicated antibodies and quantification of blots corresponding to all experiments performed. E. Crystal violet assay. PTP1BWT and PTP1BKO oval cells were isolated from wild-type and PTP1B-deficient mice treated with DDC (n = 3 independent experiments performed in triplicate). **P < 0.01 and ***P < 0.005, KO vs. WT; ##P < 0.01 and ###P < 0.005, vs. basal condition.

Supplementary Figure 1A. Body weight curves. B. Liver weight. C. Epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) weight. *P < 0.05, PTP1BKO vs. PTP1BWT; #P < 0.05, ##P < 0.01 and ###P < 0.005, 4wMCD or 8wMCD vs. CHD.

Supplementary Figure 2A. Body weight curves. B. Liver weight. C. Epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) weight. *P < 0.05, **P < 0.01 and ***P < 0.005, PTP1BKO vs. PTP1BWT; #P < 0.05, ##P < 0.01 and ###P < 0.005, 8wMCD+CHD vs. 8wMCD.