Pharmacological promotion of autophagy alleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditions in mice

Abstract

Background & aims: Pharmacological approaches can potentially improve fatty liver condition in alcoholic and non-alcoholic fatty liver diseases. The salutary effects of reducing lipid synthesis or promoting lipid oxidation have been well reported, but the benefits of increasing lipid degradation have yet to be well explored. Macroautophagy is a cellular degradation process that can remove subcellular organelles including lipid droplets. We thus investigated whether pharmacological modulation of macroautophagy could be an effective approach to alleviate fatty liver condition and liver injury.

Methods: C57BL/6 mice were given ethanol via intraperitoneal injection (acute) or by a 4-week oral feeding regime (chronic), or high fat diet for 12 weeks. An autophagy enhancer, carbamazepine or rapamycin, or an autophagy inhibitor, chloroquine, was given before sacrifice. Activation of autophagy, level of hepatic steatosis, and blood levels of triglycerides, liver enzyme, glucose and insulin were measured.

Results: In both acute and chronic ethanol condition, macroautophagy was activated. Carbamazepine, as well as rapamycin, enhanced ethanol-induced macroautophagy in hepatocytes in vitro and in vivo. Hepatic steatosis and liver injury were exacerbated by chloroquine, but alleviated by carbamazepine. The protective effects of carbamazepine and rapamycin in reducing steatosis and in improving insulin sensitivity were also demonstrated in high fat diet-induced non-alcoholic fatty liver condition.

Conclusions: These findings indicate that pharmacological modulation of macroautophagy in the liver can be an effective strategy for reducing fatty liver condition and liver injury.

Figure 1. Carbamazepine induces autophagy in hepatocytes and alleviates acute ethanol-induced liver steatosis and liver injury whereas chloroquine inhibits both

(A–B). GFP-LC3 transgenic mice were treated with CBZ with or without pretreatment of CQ for 16 hours. GFP-LC3 puncta were quantified. (C–D) Primary hepatocytes were pre-incubated with adenoviral GFP-LC3 overnight and then cultured in complete medium (CM), or in EBSS, or in CM with CBZ, rapamycin or 3-MA for 6 hours followed by fluorescence microscopy for LC3 dot formation (C) or for 16 hours for long-lived protein degradation assay (D). (E). Wild type mice were given ethanol (E) with or without rapamycin, or CBZ. Mice were sacrificed 16 hours later and the numbers of Bodipy-positive LDs were quantified. (F–H). The levels of hepatic (F) and plasma (G) TG, and plasma ALT (H) were measured. *: p<0.01 (n=3–7).

Figure 2. Autophagy helps to control hepatic steatosis and liver injury under chronic ethanol condition

(A–B) GFP-LC3 mice were given control diet (CD) or 29% ethanol diet (ED) for 4 weeks, followed by CQ administrated 16 hr before sacrifice. (C) Wild type mice were fed with CD or 36% ED for 4 weeks. A subgroup of mice was given CQ before sacrifice. The levels of LC3-II quantified by densitometry (normalized to the loading level and to the CD without CQ) were shown below the blot. (D-–E). Wild type mice were treated with CD or 29% ED for 4 weeks and given CQ 16 hr before sacrifice. Liver cryosections were stained with Bodipy (D) and the number of LDs were quantified (E). (F–H). Mice were treated as indicated and analyzed for hepatic TG (F), blood TG (G) and blood ALT (H) levels. #: p<0.05, *: p<0.01 (n=3–5 for A–E, 2–10 for F–H).

Figure 3. Enhanced autophagy alleviated chronic ethanol-induced liver steatosis and liver injury

Mice were treated with control diet, 29% (A–E) or 36% (B–E) ethanol diet and then given rapamycin every other day for three times in the week before sacrifice, or CBZ (12.5 mg/kg) once on the day before sacrifice. Cryosections of the livers were stained with Bodipy 493/503 (A). The numbers of LDs were quantified (B). Hepatic TG (C), blood TG (D) and ALT (E) levels were determined. *: p<0.01 (n=3–13).

Figure 4. Enhanced autophagy alleviated HFD-induced steatosis

Wild type mice were fed on a regular chew diet or a HFD for 12 weeks. CBZ or Rapamycin was injected every other day for two or three times, respectively, in the week before sacrifice. (A–B) Cryosections of the livers were stained with Bodipy 493/503 and the numbers of LDs were quantified. Hepatic (C) and blood (D) TG and blood ALT levels (E) were determined. (F–G) Blood glucose and insulin levels were measured in mice fed with HFD with or without rapamycin or CBZ treatment (F), from which the HOMA values were calculated (G). #: p<0.05, *: p<0.01(n=3–7).