Hepatocyte specific TIMP3 expression prevents diet dependent fatty liver disease and hepatocellular carcinoma

Abstract

Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of conditions, ranging from non-progressive bland steatosis to hepatocarcinoma. Tissue inhibitor of metalloproteinase 3 (Timp3) has a role in the pathogenesis of fatty liver disease associated with obesity and is silenced during metabolic disorders and liver cancer. We generated an hepatocyte-specific TIMP3 'gain-of-function' mouse model under the control of the Albumin promoter (AlbT3) and investigated its effects during high-fat diet (HFD). After 16 weeks of HFD, TIMP3 overexpression significantly improved glucose metabolism, hepatic fatty acid oxidation and cholesterol homeostasis. In AlbT3 mice CYP7A1, MDR3 and MRP2 gene expressions were observed, consistent with higher bile acid synthesis and export. Next, to evaluate the role of A Disintegrin and Metalloproteinase 17 (ADAM17), a crucial target of TIMP3, in these processes, we created mice deficient in Adam17 specifically in hepatocyte (A17LKO) or in myeloid lineage (A17MKO), founding that only A17LKO showed improvement in liver steatosis induced by HFD. Moreover, both, AlbT3 and A17LKO significantly reduced diethylnitrosamine-initiated, HFD-promoted hepatic tumorigenesis assessed by tumor multiplicity and total tumor area. Taken together, these data indicate that hepatic TIMP3 can slow progression of NAFLD, and tumorigenesis, at least in part, through the regulation of ADAM17 activity.

Figure 1

Generation and characterization of AlbT3 transgenic mice. (A) Relative amount of Timp3 mRNA expression in liver, WAT (with adipose tissue), kidney, muscle and heart of wild-type (wt) and AlbT3 mice. (B) TIMP3 and albumin (hepatocyte marker) mRNA expression in purified hepatocytes (Hep) and non-parenchymal cells (NPCs) from AlbT3 and wt mouse livers. Expression of mRNA was determined by qRT-PCR and values normalized to the endogenous control (β-actin). (n = 3 per group, *p < 0.03, **p < 0.01, Student’s t test, data are means ± SEM). (C) Weight and (D) blood glucose concentration. (E) Intraperitoneal glucose tolerance test (IPGTT). (n = 14 per group, data are means ± SEM). a.u.: arbitrary units.

Figure 2

Metabolic effect in AlbT3 mice after 16 weeks of high-fat diet (HFD). (A) glucose (n = 20 per group); insulin levels and HOMA-IR (n = 8 per group). (B) IPGTT and intraperitoneal insulin tolerance test (IPITT) (n = 13 per group). (C) Timp3, Adam17 and Tnf-α mRNA expression (normalized to β-actin) and (D) TIMP3, ADAM17 and TNF-α protein levels in livers of AlbT3 and wt mice. (E) ADAM17 activity in livers and TNF-α levels in serum of wt and AlbT3 (n = 7 per group). (F) Akt phosphorylation (pSer473-Akt) in liver, skeletal muscle and WAT (n = 5 per group). A representative cropped image of 3 mice per group is shown. (*p < 0.05, **p ≤ 0.02, ***p ≤ 0.005, ****p < 0.002; Student’s t test, data are means ± SEM). AUC: area under the curve.

Figure 3

Reduced hepatic steatosis and cholesterol content in wt and AlbT3 mice after 16 weeks of HFD. (A) Serum biochemical analytes. ALT: alanine transaminase; AST: aspartate transaminase; LDH: lactate dehydrogenase (p values are shown). (B) Liver cholesterol and triglycerides content. (C) Representative pictures (×4 and ×10 magnification) of liver stained with hematoxylin/eosin (H&E) and Oil Red O (ORO). Quantification of the area occupied by lipid in ORO staining. (D) Liver mRNA expression of genes involved in de novo lipogenesis, fatty acids uptake and oxidative metabolism. (normalized to β-actin). (E) Representative cropped image of Western blots for AMPKα phosphorylation (pThr172-AMPKα) in liver extract. (n = 7 per group; *p < 0.05, **p ≤ 0.02, ***p ≤ 0.005; Student’s t test, data are means ± SEM). (F) Heatmap of acyl-carnitines in serum. Regions of red or blue indicate that the metabolite content is increased or decreased, respectively (n = 5; ^§indicates significant difference with p ≤ 0.05 between the groups, metabolite ratio of <1.00; Welch’s Two-Sample t-Test).

Figure 4

Bile acids (BAs) synthesis and transport regulation in wt and AlbT3 mice after 16 weeks of HFD. (A,B,C) Liver mRNA expression of genes involved in cholesterol export, BAs synthesis and secretion. (normalized to β-actin). (D) Plasmatic and hepatic total BAs levels. (E) mRNA expression of genes related to ileal BAs transporting, (normalized to β-actin). (F) Livers from wt and AlbT3 mice were analyzed for NRG1 in total extract (normalized to Actin), ErbB4 in plasmatic membrane extract (normalized to plasmatic membrane protein loading) and SREBP2 in nuclear extract (normalized to Lamin A/C) by Western blot; a representative cropped image of 3 mice per group is shown. (n = 7 per group; *p ≤ 0.05, **p ≤ 0.02, ***p < 0.01; Student’s t test, data are means ± SEM).

Figure 5

Effect of hepatocyte and myeloid specific Adam17 deletion on glucose metabolism and liver steatosis after 16 weeks of HFD. (A) Fasting blood glucose levels (n = 20 per group) and insulin levels (n = 6 per group). (B) IPGTT and ITT (n = 20 per group). (C) ELISA of serum TNF-α; (D) representative sections (×4 and ×10 magnification) of liver stained with H&E and ORO and quantification of the area occupied by lipid in ORO staining and (E) liver cholesterol content (n = 8 per group). (*p < 0.05, **p ≤ 0.02, ***p < 0.0005, ****p < 0.0001, φ = 0.0641; one-way ANOVA with Dunnett’s Multiple Comparison Test, data are means ± SEM).

Figure 6

BAs regulation and ErbB4 signaling in A17LKO mice fed a HFD for 16 weeks. (A) Liver gene expression normalized to β-actin. (B) Plasma and liver content of total BAs. (C) Representative cropped image of Western blot for NRG1 in total extract (normalized to Actin), ErbB4 in plasmatic membrane extract (normalized to plasmatic membrane protein loading) and SREBP2 in nuclear extract (normalized to Lamin A/C) from livers of Ct and A17LKO mice. (n = 8 per group; *p < 0.05, **p < 0.02; Student’s t test, data are means ± SEM).

Figure 7

Timp3 overexpression or Adam17 selective deletion in hepatocyte reduced DEN-induced liver tumorigenesis during HFD. (A) Representative cropped images of wt and AlbT3 livers. The number and size of tumors in livers were counted. (B) NRG1 and Actin in total extract and (C) ErbB4 in plasmatic membrane extract analyzed by Western blot. (wt n = 10, AlbT3 n = 7). (D) Representative cropped images of Ct and A17LKO livers. The number and size of tumors in livers were counted. (E) NRG1 and Actin in total extract and (F) ErbB4 in plasmatic membrane extract analyzed by Western blot. (Ct n = 9, A17LKO n = 8). (*p < 0.05, **p < 0.02, ***p < 0.01; Student’s t test, data are means ± SEM).

Figure 8

TIMP3 downregulates NRG1/ErbB4/SREBP2 signaling by the inhibition of ADAM17 activity, resulting in increased BA synthesis through enhancing Cyp7A1 expression.