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. 2023 Jun 15;24(6):485-495.
doi: 10.1631/jzus.B2200562.

Fibroblast growth factor 21 (FGF21) attenuates tacrolimus-induced hepatic lipid accumulation through transcription factor EB (TFEB)-regulated lipophagy

[Article in English, Chinese]
Zhensheng Zhang  1   2   3   4   5 , Li Xu  6   3   4   5 , Xun Qiu  1   2   3   4   5 , Xinyu Yang  1   2   3   4   5 , Zhengxing Lian  1   2   3   4   5 , Xuyong Wei  1   2   3   4   5 , Di Lu  1   2   3   4   5 , Xiao Xu  7   8   9   10   11
Affiliations

Fibroblast growth factor 21 (FGF21) attenuates tacrolimus-induced hepatic lipid accumulation through transcription factor EB (TFEB)-regulated lipophagy

[Article in English, Chinese]
Zhensheng Zhang et al. J Zhejiang Univ Sci B. .

Abstract
in English, Chinese, Chinese

Tacrolimus (TAC), also called FK506, is one of the classical immunosuppressants to prevent allograft rejection after liver transplantation. However, it has been proved to be associated with post-transplant hyperlipemia. The mechanism behind this is unknown, and it is urgent to explore preventive strategies for hyperlipemia after transplantation. Therefore, we established a hyperlipemia mouse model to investigate the mechanism, by injecting TAC intraperitoneally for eight weeks. After TAC treatment, the mice developed hyperlipemia (manifested as elevated triglyceride (TG) and low-density lipoprotein cholesterol (LDL-c), as well as decreased high-density lipoprotein cholesterol (HDL-c)). Accumulation of lipid droplets was observed in the liver. In addition to lipid accumulation, TAC induced inhibition of the autophagy-lysosome pathway (microtubule-associated protein 1 light chain 3β (LC3B) II/I and LC3B II/actin ratios, transcription factor EB (TFEB), protein 62 (P62), and lysosomal-associated membrane protein 1 (LAMP1)) and downregulation of fibroblast growth factor 21 (FGF21) in vivo. Overexpression of FGF21 may reverse TAC-induced TG accumulation. In this mouse model, the recombinant FGF21 protein ameliorated hepatic lipid accumulation and hyperlipemia through repair of the autophagy-lysosome pathway. We conclude that TAC downregulates FGF21 and thus exacerbates lipid accumulation by impairing the autophagy-lysosome pathway. Recombinant FGF21 protein treatment could therefore reverse TAC-caused lipid accumulation and hypertriglyceridemia by enhancing autophagy.

他克莫司（TAC），也称为FK506，是预防肝移植后同种异体移植排斥反应的经典免疫抑制剂之一。然而，它已被证明与移植后高脂血症有关。但其背后的机制尚不清楚，因此迫切需要探索移植后高脂血症的预防策略。我们通过腹腔注射8周TAC建立了一个高脂血症小鼠模型来研究其机制。TAC处理后，小鼠发生高脂血症（表现为甘油三酯（TG）和低密度脂蛋白胆固醇（LDL-c）升高，以及高密度脂蛋白胆固醇（HDL-c）降低）以及肝脏脂质的累积。除脂质积累外，TAC还抑制了自噬-溶酶体途径（LC3B II/I和LC3BII/actin比值、转录因子EB（TFEB）、P62和LAMP1），并下调成纤维细胞生长因子21（FGF21）的表达。而FGF21的过表达可逆转TAC诱导的TG积累。在该小鼠模型中，重组FGF21蛋白通过修复自噬-溶酶体途径改善肝脏脂质积累和高脂血症。综上所述，TAC下调FGF21，从而通过抑制自噬-溶酶体途径来加剧脂质积累。此外，重组FGF21蛋白处理可以通过增强自噬来逆转TAC引起的脂质积累和高甘油三酯血症。.

他克莫司（TAC），也称为FK506，是预防肝移植后同种异体移植排斥反应的经典免疫抑制剂之一。然而，它已被证明与移植后高脂血症有关。但其背后的机制尚不清楚，因此迫切需要探索移植后高脂血症的预防策略。我们通过腹腔注射8周TAC建立了一个高脂血症小鼠模型来研究其机制。TAC处理后，小鼠发生高脂血症（表现为甘油三酯（TG）和低密度脂蛋白胆固醇（LDL-c）升高，以及高密度脂蛋白胆固醇（HDL-c）降低）以及肝脏脂质的累积。除脂质积累外，TAC还抑制了自噬-溶酶体途径（LC3B II/I和LC3BII/actin比值、转录因子EB（TFEB）、P62和LAMP1），并下调成纤维细胞生长因子21（FGF21）的表达。而FGF21的过表达可逆转TAC诱导的TG积累。在该小鼠模型中，重组FGF21蛋白通过修复自噬-溶酶体途径改善肝脏脂质积累和高脂血症。综上所述，TAC下调FGF21，从而通过抑制自噬-溶酶体途径来加剧脂质积累。此外，重组FGF21蛋白处理可以通过增强自噬来逆转TAC引起的脂质积累和高甘油三酯血症。

Keywords: Autophagy; Fibroblast growth factor 21 (FGF21); Lipid; Lipophagy; Lysosome; Tacrolimus; Transcription factor EB (TFEB).

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Figures

Fig. 1
Fig. 1. Induction of hepatic lipid accumulation and hypertriglyceridemia by TAC. (a, b) Representative ORO and HE staining results of liver sections after TAC treatment for eight weeks. (c‒f) TG, TC, HDL-c, and LDL-c levels in mouse plasma. (g‒i) TG contents of the three cell lines (HepLi-2, HepG2, and AML12) after TAC treatment for 48 h. (j) Representative ORO staining results of the three cell lines. Scale bar=20 μm. Data are shown as mean±SD, n=3. * P<0.05, ** P<0.01, *** P<0.001; n.s.: not significant. HE: hematoxylin-eosin; ORO: Oil Red O; TAC: tacrolimus; NC: negative control; TC: total cholesterol; TG: triglyceride; LDL-c: low-density lipoprotein cholesterol; HDL-c: high-density lipoprotein cholesterol; SD: standard deviation.
Fig. 2
Fig. 2. Induction of lipid accumulation by TAC through autophagy inhibition. (a) Immunoblot detection of LAMP1, TFEB, P62, LC3B, and nTFEB in HepG2 and AML12 cell lines. (b) Representative TFEB staining results of the two cell lines. (c) Representative LC3B staining results of the two cell lines. (d) Representative LAMP1 and lipid-droplet staining results of the two cell lines. Scale bar=10 μm. TAC: tacrolimus; NC: negative control; LAMP1: lysosomal-associated membrane protein 1; TFEB: transcription factor EB; P62: protein 62; LC3B: microtubule-associated protein 1 light chain 3β; nTFEB: nuclear TFEB; H3: histone 3; DAPI: 4',6-diamidino-2-phenylindole.
Fig. 3
Fig. 3. Reversion of TAC-induced lipid accumulation by overexpression of FGF21. (a) Immunoblot detection of FGF21 expression in the liver. (b) qPCR-assisted detection of FGF21 in the liver. (c) FGF21 serum level. (d) Immunoblot detection of FGF21 expression in three cell lines. (e) qPCR-assisted detection of FGF21 in three cell lines. (f) Immunoblot detection of FGF21 overexpression in three cell lines. (g) qPCR-assisted detection of FGF21 overexpression in three cell lines. (h) TG contents after DMSO or TAC treatment in three cell lines with or without FGF21 overexpression. (i) Representative ORO staining results of three cell lines. Scale bar=20 μm. Data are shown as mean±SD, n=3. * P<0.05, ** P<0.01, *** P<0.001. ORO: Oil Red O; FGF21: fibroblast growth factor 21; TAC: tacrolimus; NC: negative control; OE: overexpression; qPCR: quantitative real-time polymerase chain reaction; TG: triglyceride; DMSO: dimethyl sulfoxide; mRNA: messenger RNA; SD: standard deviation.
Fig. 4
Fig. 4. Reversion of defective lipid homeostasis by recombinant FGF21 protein. (a) The experimental process of in vivo study. (b) Representative ORO and HE staining results of liver sections after co-treatment with TAC and recombinant FGF21 protein. (c‒e) TG, HDL-c, and LDL-c levels in mouse plasma. (f) Representative ORO staining results in HepG2 and AML12 cells after TAC treatment for 48 h with or without recombinant FGF21 co-treatment. (g) TG contents in HepG2 and AML12 cells. Scale bar=20 μm. Data are shown as mean±SD, n=3. * P<0.05, ** P<0.01, *** P<0.001; n.s.: not significant. FGF21: fibroblast growth factor 21; ORO: Oil Red O; HE: hematoxylin-eosin; TAC: tacrolimus; NC: negative control; TG: triglyceride; LDL-c: low-density lipoprotein cholesterol; HDL-c: high-density lipoprotein cholesterol; rmFGF21: recombinant mouse FGF21; SD: standard deviation.
Fig. 5
Fig. 5. Effect of recombinant FGF21 protein on TAC-impaired autophagy. (a) Immunoblot detection of LAMP1, TFEB, P62, LC3B, and nTFEB in mouse livers. (b) Representative TFEB staining results of liver sections. (c) Representative LC3B staining results of liver sections. (d) Representative LAMP1 and lipid-droplet staining results of liver sections. (e) Immunoblot detection of LAMP1, TFEB, LC3B, and nTFEB in HepG2 and AML12 cell lines. (f) Representative LC3B staining results of AML12 cell line. (g) Representative LAMP1 and lipid-droplet staining results of the two cell lines. Scale bar=10 μm. FGF21: fibroblast growth factor 21; LAMP1: lysosomal associated membrane protein 1; TFEB: transcription factor EB; P62: protein 62; LC3B: microtubule-associated protein 1 light chain 3β; TAC: tacrolimus; NC: negative control; nTFEB: nuclear TFEB; rh(m)FGF21: recombinant human (mouse) FGF21; DAPI: 4',6-diamidino-2-phenylindole; H3: histone 3.
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