Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy

Abstract

For more than a century, thyroid hormones (THs) have been known to exert powerful catabolic effects, leading to weight loss. Although much has been learned about the molecular mechanisms used by TH receptors (TRs) to regulate gene expression, little is known about the mechanisms by which THs increase oxidative metabolism. Here, we report that TH stimulation of fatty acid β-oxidation is coupled with induction of hepatic autophagy to deliver fatty acids to mitochondria in cell culture and in vivo. Furthermore, blockade of autophagy by autophagy-related 5 (ATG5) siRNA markedly decreased TH-mediated fatty acid β-oxidation in cell culture and in vivo. Consistent with this model, autophagy was altered in livers of mice expressing a mutant TR that causes resistance to the actions of TH as well as in mice with mutant nuclear receptor corepressor (NCoR). These results demonstrate that THs can regulate lipid homeostasis via autophagy and help to explain how THs increase oxidative metabolism.

Figures

Figure 1. T3 induces autophagy in hepatic cells expressing TRα.

(A–C) Immunoblot and densitometric analysis of LC3-II levels in T3-treated HepG2 cells expressing TRα1 (HepG2/TRα) (n = 4; *P < 0.05). (D and E) Time course of LC3-II accumulation in HepG2/TRα cells treated with 1 μM T3 (n = 3; *P < 0.05). (F and G) Dose response of LC3-II accumulation in HepG2/TRα cells treated with indicated concentrations of T3 for 72 hours (n = 3; *P < 0.05). Results are expressed as mean ± SEM.

Figure 2. T3 stimulates autophagic flux and also induces autophagy in multiple hepatic cell lines.

(A–C) Immunoblot and densitometric for LC3-II showing autophagic flux using HepG2/TRα cells treated with 1 μM T3 and 50 μM CQ for 72 hours (n = 4; *P < 0.05). (D and E) Immunoblot analysis of LC3-II levels in AML-12, Hep3B, and Huh7 cells upon 1 μM T3 treatment for 72 hours showing increased autophagy (n = 3; *P < 0.05). Results are expressed as mean ± SEM.

Figure 3. T3 stimulates autophagosomal and lysosomal activity in hepatic cells.

(A) LC3 immunostaining. Punctate staining shows autophagosome formation in cells treated with 1 μM T3 for 72 hours. (B) Lysosomal acidification visualized using MDC and AO in HepG2/TRα cells treated with 1 μM T3 for 72 hours.

Figure 4. T3 induces “lipophagy” in hepatic cells.

Electron micrograph of untreated control (A) and T3-treated HepG2/TRα cells (B–F) cells. Note the increased number of lipid droplets and autophagosomes in T3-treated cells. (D) Higher magnification images of the boxed area in B showing autophagosomes with lipids (red arrows). (E and F) Magnified images of the boxed parts in C showing autolysosomes (white arrows) containing lipids (red arrows) and double-membrane autophagosomes (yellow arrows). Scale bars: 5 μm (A and B), 2 μm (C and E); 1 μm (D and F). (G) LC3 (red) with BODIPY 493/503 (green) staining in HepG2/TRα cells. Colocalization of LC3 (autophagosomes) and BODIPY (lipid droplets) is shown by white arrow pointing to yellow spots in the digitally enlarged boxed area (×2.5 digital zoom). Original magnification, ×40.

Figure 5. T3 induces hepatic autophagy in vivo.

(A and B) Immuno­blot and densitometric analysis of LC3-II levels in euthyroid and hyperthyroid mice injected with 10 μg T3/100 g BW for 3 days (n = 3; *P < 0.05). (C and D) Immunoblot and densitometric analysis of p62 levels in euthyroid and hyperthyroid mice (n = 3; *P < 0.05). Results are expressed as mean ± SEM. EM showing livers obtained from control (E) and hyperthyroid (F–I) mice injected with 10 μg T3/100 g BW for 3 days. White arrowheads in F denote increased number of mitochondria in hyperthyroid mouse liver, and white arrows show autolysosomes. (G) Magnified image of the boxed area in F showing double-membrane lipid droplets inside autolysosomes. (H) Autophagic vesicle containing several lysosomes (white arrows) and lipid droplets (black arrow). (I) Autophagosome inside a large lipid droplet in hepatocyte from hyperthyroid mouse liver. Scale bars: 2 μm (E and F); 0.2 μm (G and H); 0.1 μm (I).

Figure 6. In vivo regulation of hepatic autophagy is TR dependent.

(A) Immuno­blot showing LC3-II levels in WT euthyroid and TRβPV/PV mice. (B) Immunoblot showing LC3-II and p62 levels in (2 to 3 months old) euthyroid mice, hypothyroid mice (fed with a low-iodine diet supplemented with 0.15% propylthiouracil [Harlan Teklad] for 35 days, and TRβPV/PV mice.

Figure 7. Autophagy mediates fatty acid oxidation and ketosis by T3 in hepatic cells.

HepG2/TRα cells were transfected with either control siRNA or ATG5-specific siRNA. Cells then were cultured with oleic acid (0.5 mM) in the absence or presence of T3 for the next 48 hours before harvesting. (A) β-Hydroxybutyrate concentrations in culture medium were measured along with (B) ATG5 and LC3-II protein levels, which were measured by Western blotting (n = 3/each group). Results are expressed as mean ± SEM.

Figure 8. T3-induced autophagy is tightly coupled with fatty acid β-oxidation in mouse liver in vivo.

(A) Immunoblot of ATG5 and LC3-II from livers of representative mice treated with control siRNA or ATG5 siRNA in the absence or presence of T3. (B) Densitometric analyses of immunoblots of ATG5 and LC3-II in livers of mice treated with control siRNA or ATG5 siRNA in the absence or presence of T3 (n = 4–5; *P < 0.05). Note that T3 stimulation of LC3-II was blocked in the ATG5-knockdown mice. (C) Serum β-hydroxybutyrate levels from mice treated with control siRNA or ATG5 siRNA in the absence or presence of T3 (n = 4–5; *P < 0.05). Note that β-hydroxybutyrate levels in ATG5-knockdown mice treated with T3 returned to the same levels as those in mice treated with control siRNA alone. Results are expressed as mean ± SEM.

Figure 9. NCoR modulates T3-mediated autophagy and acylcarnitine levels in vivo.

(A and B) Immunoblot and densitometric analysis of LC3-II levels in WT and NCoR DADm mice. Female WT and NCoR DADm mice (19 weeks old) were used for the experiment. Fresh drinking water containing 1% perchlorate and 0.05% methimazole was provided daily for 2 weeks to induce hypothyroidism. Fourteen hours before they were killed, all animals were given a subcutaneous injection of vehicle (0.9% saline in 100 μl volume for control and hypothyroid groups or 40.0 μg/100 g T4 with 4.0 μg/100 g T3 for hyperthyroid group). *P < 0.05; n = 3 animals in each group. (C) Metabolomic analysis of medium-chain acylcarnitines in the above-described animal groups (n = 5; *P < 0.05). (D) Metabolomic analysis of long-chain acylcarnitines in the above-described animal groups (n = 5, *P < 0.05; **P < 0.01; ***P < 0.001). Results are expressed as mean ± SEM.