JMJD3 epigenetically activates liver autophagy gene networks

To explore epigenetic regulation of hepatic autophagy, we first examined whether selected histone modifications were altered by fasting in mouse liver. Levels of histone H3K27-me3 were markedly decreased in fasted mice, and levels of JMJD3, which activates genes by demethylating H3K27-me313, were increased after fasting, whereas expression of UTX H3K27 demethylase and EZH2 H3K27 methyltransferase was unchanged (Supplementary Fig. 1). These results suggest that JMJD3 may epigenetically activate hepatic autophagy in response to nutrient deficiency.

To examine the role of JMJD3 in global regulation of hepatic genes, including autophagy genes, JMJD3 was downregulated specifically in the liver by infection of JMJD3-floxed mice with hepatocyte-targeting AAV-TBG-Cre15,16 (Fig. 1a), and effects of the downregulation on global gene expression and H3K27-me3 levels were examined by RNA-seq and ChIP-seq, respectively. Expression of 846 genes was decreased by downregulation of JMJD3 (Fig. 1b, Supplementary Fig. 2a), which include genes involved in autophagy, Ulk1, Atg3, Atg7, and Lc3; a transcriptional activator of autophagy, Tfeb7; a lipase important for lipophagy, Atgl17; and a fasting-induced hepatokine promoting lipid catabolism, Fgf2118,19, and H3K27-me3 levels detected by ChIP-seq at nearly all of these genes were increased (Fig. 1c, Supplementary Fig. 2c). Remarkably, gene ontology (GO) analysis of 564 potential JMJD3 target genes with both decreased expression and increased levels of H3K27-me3 after downregulation of JMJD3, revealed that autophagy and lysosomal function are potentially regulated by JMJD3 in fasted mice (Fig. 1d, Supplementary Fig. 2b–d). Analysis of mRNA and H3K27-me3 levels for selected autophagy genes validated these genomic results (Fig. 1e, f) and occupancy of JMJD3 at these genes in fasted mice was decreased as expected by liver-specific downregulation of JMJD3 (Fig. 1g). These global analyses reveal a potential role for JMJD3 in epigenetic induction of hepatic autophagy.

Fig. 1: JMJD3 epigenetically activates hepatic autophagy-network genes. JMJD3-floxed mice were infected with AAV-TBG-Cre or control AAV-GFP for 12 weeks, and then, fasted for 16 h. a Experimental outline (top) and the levels of JMJD3 detected by IB in the liver and intestine (bottom). b RNA-seq: heat maps of changes in mRNA levels of autophagy-related genes (n = 3 mice). c ChIP-seq: normalized H3K27-me3 peaks at hepatic genes involved in autophagic pathways (UCSC genome browser). d Venn diagram (top) of downregulated genes and genes with increased H3K27-me3 levels after liver-specific downregulation of JMJD3 and G/O analysis (bottom) of the hepatic genes that show both increased H3K27-me3 levels and decreased expression. e The mRNA levels of the indicated genes in liver of mice fasted for 16 h (Fs) or refed (Fd) for 6 h after fasted for 10 h were measured by q-RTPCR (n = 5–10 mice). f, g ChIP assays: effects of the downregulation of JMJD3 on f H3K27-me3 levels and g occupancy of JMJD3 at the indicated genes (n = 3 mice). Source data are provided as a Source Data file. All values are presented as mean ± SD. Statistical significance was measured using the e–g two-way ANOVA with the Bonferroni post-test. *P < 0.05, **P < 0.01, and NS statistically not significant. Full size image

JMJD3 activates hepatic autophagy under nutrient deficiency

To examine whether induction of autophagy-network genes by JMJD3 actually leads to autophagy, we examined the effects of liver-specific downregulation or overexpression of JMJD3 on autophagic markers, the ratio of lipidated LC3-II to non-lipidated LC3-I, the levels of the autophagosome adapter p62, and lysosome-associated membrane protein (Lamp)1 and Lamp220. The ratio of hepatic LC3-II/I, the number of LC3 puncta, and Lamp1 and 2 levels were decreased, and p62 levels were increased by downregulation of JMJD3 in mice (Fig. 2a), indicative of decreased autophagy20. Conversely, adenoviral-mediated liver-specific expression of JMJD3 in mice resulted in the opposite effects (Fig. 2b). Similar effects of overexpression or downregulation of JMJD3 on the number of GFP-LC3 puncta were observed in Hepa1c1c7 cells (Fig. 2c).

Fig. 2: JMJD3 promotes hepatic autophagy, including lipophagy. a The indicated hepatic proteins were detected by IB in JMJD3-floxed mice infected with AAV-TBG-Cre or AAV-GFP for 12 weeks and fasted for 16 h. Relative band intensities for p62 and the LC3-II/LC3-I ratios are below the blots (left, n = 5 mice). LC3 and p62 in representative images of liver sections detected by IHC (middle) and numbers of LC3 puncta/cell (right, n = 10 hepatocytes) (scale bar = 10 μm for LC3, 50 μm for p62). b Levels of the indicated hepatic proteins were determined by IB in C57BL/6 mice infected with Ad-empty (GFP) or Ad-JMJD3 for 4 weeks and fasted for 8 h (n = 3 mice). c, d Hepa1c1c7 cells were transfected with expression plasmids or JMJD3 siRNA as indicated for 72 h and cultured in HBSS for 2 h. c Representative confocal images and the average number of GFP-LC3-II puncta/cell (right, n = 10 cells) are shown (scale bar = 5 μm). d Cells were stained for lipid droplets with BODIPY (red) and imaged by confocal microscopy. Co-localization of GFP-LC3 puncta and BODIPY in the merged image is indicated by white arrows. The number of fluorescent puncta that co-localized with lipid staining (right, n = 20 cells) are shown (scale bar = 5 μm). e–g C57BL/6 mice were fed a normal chow diet (ND) or high-fat diet (HFD) for 8 weeks and then were injected with adenoviruses as indicated for 4 weeks. e Hepatic levels of the indicated proteins were measured by IB (n = 3 mice). The ratios of band intensities for the LC3-I/LC3-II relative to those in the first lane are shown below the blot. f Lipids in liver sections stained with Oil Red O (ORO) and H&E (scale bar = 50 μm). g Levels of hepatic triglycerides (TG) and serum β-hydroxybutyrate (β-HDB) (n = 5 mice). Source data are provided as a Source Data file. All values are presented as mean ± SD. Statistical significance was measured using the (a, c, d) Mann–Whitney test or g two-way ANOVA with the Bonferroni post-test. *P < 0.05, **P < 0.01, and NS statistically not significant. Full size image

Downregulation of JMJD3 also decreased the LC3-II/I ratio and decreased the mRNA levels of genes involved in autophagy and lysosomal functions in primary mouse hepatocytes (PMH) (Supplementary Fig. 3a, b). Furthermore, while treatment with a lysosomal inhibitor, bafilomycin-A1, increased autophagy detected by increased LC3 puncta or ratios of LC3-II to LC3-I, overexpression of JMJD3 further increased autophagy while downregulation decreased it (Supplementary Fig. 3c–e). Similarly, treatment with an mTOR inhibitor increased the LC3-II/I ratio, and downregulation of JMJD3 decreased autophagy without reversing the phosphorylation of the mTOR target, pS6 (Supplementary Fig. 3f). These results suggest that JMJD3 activates autophagy independent of either lysosomal or mTOR action. Overall, these results, together with global studies (Fig. 1), demonstrate that JMJD3 transcriptionally activates hepatic autophagy under nutrient deprivation.

JMJD3 promotes autophagy-mediated degradation of lipids

As key genes important for lipophagy, including Atgl, Tfeb, Pgc-1α, and Fgf217,17,21, are direct targets of JMJD3 (Fig. 1, Supplementary Fig. 2), and JMJD3 promotes fatty acid β-oxidation15, we further examined whether JMJD3 activates hepatic lipophagy.

Overexpression of JMJD3 in Hepa1c1c7 cells increased the number of GFP-LC3 puncta that co-localized with BODIPY-stained lipids and the opposite effects were observed after downregulation of JMJD3 (Fig. 2d). Furthermore, in electron microscopy studies, autophagic vesicles within lipid droplets were observed in mouse liver after adenoviral-mediated expression of JMJD3 (Supplementary Fig. 4a). Exogenous expression of JMJD3 in livers of mice fed a normal chow diet (ND) increased the LC3-II/I ratio (Fig. 2e), decreased hepatic lipid (Fig. 2f, Supplementary Fig. 4b) and triglyceride (TG) levels, and increased serum ketone body levels (Fig. 2g). In mice fed a high-fat diet (HFD), expression of JMJD3 resulted in increased autophagy and decreased hepatic TG levels, but these JMJD3-mediated effects were blunted in autophagy-defective Atg7-downregulated mice (Fig. 2e–g). These results suggest that JMJD3 promotes lipophagy, which contributes to decreased liver TG levels and that autophagy is important for the JMJD3-mediated lipid-lowering effects.

Fasting-induced autophagy is blunted in FGF21-LKO mice

Fasting increased JMJD3 occupancy and decreased histone H3K27-me3 levels at selected genes promoting lipophagy, including Tfeb, Ulk1, Atgl, and Fgf21, in mice (Supplementary Fig. 5). As the fasting-induced FGF21 promotes lipid catabolism18,19 and lysosomal function21, we examined whether FGF21 has a role in JMJD3-induced autophagy using liver-specific FGF21-knockout (FGF21-LKO) mice22.

The increased ratio of LC3-II/I, decreased p62 levels, and increased LC3 puncta (Fig. 3a,b), and induction of Tfeb, Pgc-1α, Ulk1, Atg7, Atgl, and JMJD3 (Fig. 3c) observed after fasting of control mice were attenuated in FGF21-LKO mice. These results suggest that physiological levels of endogenous FGF21 induced by fasting promote hepatic autophagy in an autocrine-manner. Consistent with these results, treatment with FGF21 increased JMJD3 occupancy and demethylation of H3K27-me3 at numerous autophagy genes, and increased expression of these genes (Fig. 3e, f).

Fig. 3: Fasting-induced FGF21 promotes hepatic autophagy. a–c FGF21 floxed or FGF21-LKO mice were fasted (Fs) for 24 h, or refed (Fd) for 24 h after fasting. a LC3 and p62 levels in liver extracts detected by IB. The ratios of the LC3-II/I and p62 band intensities are shown below the blot. (n = 3 mice). b LC3 or p62 was detected by IHC analysis. Representative images of liver sections and the average number of LC3-II puncta/cell (right, n = 10 hepatocytes) are shown (scale bar = 10 μm for LC3, 50 μm for p62). c The mRNA levels of the indicated genes measured by q-RTPCR (n = 5–8 mice). d The indicated hepatic proteins detected by IB with the ratios of the LC3-II/I with the band intensities of p62 relative to the first lane shown below the blot (n = 3 mice). e, f C57BL/6 mice were treated with FGF21 (0.1 mg/kg) for 3 h. e Effects of FGF21 on occupancy of JMJD3 (left) and H3K27-me3 levels (right) at the indicated genes (n = 3 mice). f Hepatic mRNA levels measured by q-RTPCR (n = 6–8 mice). g Effects of siRNA-mediated downregulation of FGF21 on levels of indicated proteins in PMH incubated with M199 or HBSS medium. h PMH were transfected with siFGF21 or control RNA for 48 h and infected with Ad-JMJD3 or Ad-empty for 24 h. Cells were cultured in serum-free or complete M199 medium for 12 h and levels of the indicated proteins were determined by IB (n = 2 culture dishes). Source data are provided as a Source Data file. All values are presented as mean ± SD. Statistical significance was measured using the f Mann–Whitney test or b, c, e two-way ANOVA with the Bonferroni post-test. *P < 0.05, **P < 0.01, and NS statistically not significant. Full size image

In hepatocytes, the increased levels of LC3, Lamp1/2, and JMJD3 expression in nutrient-deprived medium were attenuated by downregulation of FGF21 (Fig. 3g). Moreover, expression of JMJD3 increased the LC3-II/I ratio but this JMJD3-mediated effect was also blunted by FGF21 downregulation (Fig. 3h). These findings reveal a critical role for hepatic FGF21 in hepatic autophagy induced either by nutrient deprivation or overexpression of JMJD3.

JMJD3 has a critical role in FGF21-induced hepatic autophagy

As JMJD3 epigenetically induces hepatic expression of Fgf21 in response to fasting15, we further examined whether JMJD3 is also important for FGF21-induced hepatic autophagy. FGF21 treatment in mice increased the LC3II/I ratio and Lamp1/2 levels (Fig. 4a), LC3 puncta (Fig. 4b), and expression of Tfeb, Ulk1, and Atg7 (Fig. 4c), but these FGF21-mediated effects on autophagy were markedly blunted by downregulation of hepatic JMJD3. Consistent with these findings in mice, in primary mouse hepatocytes (PMH), FGF21 treatment increased p-ERK levels and the LC-II/I ratio and decreased p62 levels (Supplementary Fig. 6a). Furthermore, treatment with FGF21, but not rapamycin, increased expression of JMJD3 (Supplementary Fig. 6b). These results indicate that JMJD3 is important for hepatic autophagy induced by FGF21. As JMJD3 induces FGF21 expression and FGF21 signaling activates JMJD3, which is required for FGF21 induction of autophagy, these findings reveal an intriguing feedforward loop between FGF21 and JMJD3 to activate hepatic lipophagy upon nutrient deprivation.

Fig. 4: FGF21-induced hepatic autophagy is largely dependent on JMJD3. a–c JMJD3-floxed mice were infected with AAV-TBG-Cre or AAV-GFP for 12 weeks (n = 6 mice/group), and injected i.v. with vehicle or FGF21 (0.1 mg/kg) for 3 h. a The indicated hepatic proteins were detected by IB (n = 3 mice). b LC3 or p62 was detected in liver sections by IHC and the number of LC3-II puncta/cell were quantified (right, n = 10 hepatocytes) (scale bar = 10 μm for LC3, 50 μm for p62). c Hepatic mRNA levels of the indicated autophagy-related genes measured by q-RTPCR (n = 5 mice). d, e PMH from JMJD3-floxed mice were infected with AAV-TBG-GFP or AAV-TBG-Cre for 72 h, and treated with vehicle or FGF21 (100 ng/ml) for 12 h. d Levels of the indicated proteins measured by IB (n = 3 culture dishes). e Levels of cellular triglycerides (TG) (n = 10 culture dishes). f Hepa1c1c7 cells were transfected with GFP-LC3 plasmid and with control (siC) or JMJD3 siRNA as indicated. After 72 h, cells were supplemented with 400 μM oleic acid for 6 h before incubation in serum-free DMEM containing vehicle or 100 ng/ml FGF21 for 12 h. Cells were stained for lipid droplets with BODIPY (red) and imaged by confocal microscopy. Co-localization of GFP-LC3 puncta (green) and BODIPY in the merged images is indicated by white arrows. The average number/cell (right, n = 20 cells) of fluorescent puncta that co-localized with lipid staining is shown (scale bar = 5 μm). Source data are provided as a Source Data file. All values are presented as mean ± SD. Statistical significance was measured using the (b, c, e, f) two-way ANOVA with the Bonferroni post-test. **P < 0.01, and NS statistically not significant. Full size image

FGF21 can directly act on hepatocytes and induce lipophagy

FGF21 lowers TG levels in liver, but it remains controversial whether the liver is a direct target organ for the FGF21 action or indirectly targeted through the CNS18,19,23,24. We, thus, examined whether FGF21-mediated autophagy observed in mice can also occur in isolated hepatocytes. FGF21 treatment increased autophagy and decreased cellular TG levels in PMH (Fig. 4d, e), and increased autophagy gene expression (Supplementary Fig. 6c). Each of these FGF21-mediated effects was blunted by downregulation of JMJD3. Furthermore, FGF21 treatment increased co-localization of LC3 puncta and lipids in Hepa1c1c7 cells, but these effects were blunted by JMJD3 downregulation (Fig. 4f). These results suggest that FGF21 can directly act on hepatocytes and induce lipophagy, which is associated with decreased TG levels.

PPARα is a key component of the FGF21-JMJD3-autophagy axis

We next examined the mechanism by which JMJD3 transmits the FGF21 signal to epigenetically activate autophagy genes. We first identified transcriptional factors that might recruit JMJD3 to autophagy genes by examining candidate factors that are known to promote autophagy, PPARα7,9, CREB8, and FOXO112. The increase in the LC3-II/I ratio induced by JMJD3 expression was largely blocked by downregulation of PPARα, while a substantial increase was still observed after downregulation of CREB or FOXO1 (Supplementary Fig. 7a). Comparative analyses of our RNA-seq data (Fig. 1b, Supplementary Fig. 2a) with published PPARα microarray25 or ChIP-seq9 data revealed that ~70% of hepatic genes downregulated in JMJD3-depleted mice had binding peaks for PPARα (Fig. 5a) and ~70% of the hepatic genes downregulated in PPARα-KO mice also had increased levels of histone H3K27-me3 (Supplementary Fig. 7b). These genes, potentially regulated by both PPARα and JMJD3, were involved in autophagy, lysosome, and lipid catabolism based on GO analysis (Fig. 5a).

Fig. 5: JMJD3 coactivates PPARα to induce hepatic autophagy. a Venn diagram (top) for genes with PPARα cistrome detected by ChIP-seq and hepatic genes inhibited by liver JMJD3 downregulation (as shown in Fig. 1b). G/O analysis (bottom) of the overlapping genes. b WT or PPARα-KO mice were fasted (Fs) for 24 h or refed for 24 h (Fd) after fasting. Levels of LC3 in liver extracts measured by IB with the LC3-II/I ratios shown below the blot (top, n = 3 mice). The mRNA levels of indicated genes (bottom, n = 5 mice). c PMH from JMJD3-floxed mice were infected with AAV-GFP or AAV-Cre for 72 h and treated with vehicle or WY14643 for 12 h. The indicated proteins were detected by IB with the LC3-II/I ratios shown below the blot (top, n = 3 culture dishes). The mRNA levels of indicated genes (bottom, n = 5 mice). d C57BL/6 or PPARα-KO mice were fasted for 1 h and treated with vehicle or 0.1 mg/kg FGF21 for 3 h. Hepatic levels of LC3 measured by IB with the LC3-II/I ratios shown below the blot (top, n = 3). The mRNA levels of indicated genes (bottom, n = 5 mice). e LC3 and p62 detected by IHC. Representative images of liver sections (left) and the average number of puncta/cell (right, n = 10 hepatocytes) are shown (scale bar = 10 μm for LC3, 50 μm for p62). f re-ChIP: Hepatocytes were transfected with PPARα siRNA, 72 h later, cells were treated with FGF21 for 2 h. PPARα was immunoprecipitated followed by immunoprecipitation of JMJD3, and enrichment of Tfeb, Atg7, and Atgl sequences was determined (n = 3 culture dishes). g Hepa1c1c7 cells were transfected with a luciferase reporter containing the PPARα binding site or mutated site from Tfeb or Atg7 and with plasmids and siRNAs as indicated. After 36 h, the cells were treated with 50 μM WY14643 and 100 ng/ml FGF21 overnight. Luciferase activities were normalized to β-galactosidase activities (n = 4 culture dishes). Source data are provided as a Source Data file. b–g Values are presented as mean ± SD. Statistical significance was measured using the g one- or b–f two-way ANOVA with the Bonferroni post-test. **P < 0.01. Full size image

To determine the importance of PPARα in the FGF21-JMJD3-autophagy axis, we utilized PPARα-KO mice. Fasting increased LC3-II/I ratios and autophagy gene expression in control mice, but these effects were blunted in PPARα-KO mice (Fig. 5b). Conversely, activation of PPARα by treatment with an agonist, WY14643, increased LC3-II/I ratios and expression of autophagy genes in a JMJD3-dependent manner in PMH (Fig. 5c) and increased the interaction of JMJD3 with PPARα (Supplementary Fig. 7c). The effects of FGF21 treatment (Fig. 5d) or JMJD3 expression (Fig. 5e, Supplementary Fig. 7d) on autophagy were also blunted in PPARα-KO mice. Further, FGF21 treatment increased co-occupancy of JMJD3 and PPARα at Tfeb, Atg7, and Atgl genes (Fig. 5f). Consistent with these results, the interaction of JMJD3 with PPARα was increased after treatment with FGF21, but not with rapamycin (Supplementary Fig. 7e, f). In reporter assays, expression of JMJD3 enhanced PPARα-mediated transactivation of Tfeb-luc and Atg7-luc (Fig. 5g). Collectively, these findings demonstrate that PPARα is a key component of the induction of autophagy network genes mediated by the fasting-triggered FGF21-JMJD3 axis.

JMJD3 phosphorylation at T1044 is critical for its function

We further investigated the mechanism by which FGF21 signaling activates JMJD3. In mass spectrometry analysis, Thr-1044 was the only phosphorylation site detected in JMJD3 from FGF21-treated hepatocytes (Fig. 6a, Supplementary Fig. 8a). Indeed, p-Thr JMJD3 levels for WT-JMJD3, but not T1044A-JMJD3, were increased by FGF21 treatment in PMH (Fig. 6b, Supplementary Fig. 8b). These results indicate that JMJD3 is phosphorylated at Thr-1044 in response to FGF21.

Fig. 6: Phosphorylation of JMJD3 by FGF21-activated PKA is critical for autophagy induction. a Experimental outline (top) and spectrum from LC-MS/MS analysis identifying a JMJD3 peptide containing phosphorylated Thr-1044 (bottom). b PMH were transfected with plasmids as indicated, and after 48 h, were treated with vehicle or FGF21 (100 ng/ml) for 30 min. p-Thr-JMJD3 levels were detected by IP/IB and input protein by IB (n = 3 culture dishes). c Hepa1c1c7 cells were transfected with JMJD3 expression plasmids and treated with vehicle or FGF21 for 30 min. Flag-JMJD3 (green) was detected by immunofluorescence (scale bar = 5 μm). d PMH were transfected with JMJD3 expression plasmids as indicated and treated with vehicle or FGF21 for 30 min for CoIP (left) or 2 h for qRT-PCR (right). PPARα in flag-JMJD3 immunoprecipitates or in input detected by IB (left) and the mRNA levels of the indicated genes (right, n = 3 culture dishes). e FGF21-floxed or -LKO mice were fasted for 24 h or refed for 24 h after fasting. Levels of proteins were detected by IB (left) and p-Thr JMJD3 were detected by IP/IB (right) (n = 3 mice). f C57BL/6 mice were treated with 0.1 mg/kg FGF21 for 3 h and then, phosphorylated levels of PKA, ERK, and JMJD3 were detected by IB (n = 3 mice). g Immunoprecipitated flag-JMJD3 was incubated with ATP, PKA, or ERK1/2 as indicated and levels of p-Thr-JMJD3 were detected by IB. h Schematic of fragments of JMJD3 that were fused to GST (top). Binding of PKA or ERK1/2 to GST-JMJD3 proteins was detected by IB (bottom). i PMH were treated with FGF21 and with vehicle or a PKA (H89,10 μM) or MEK/ERK (PD98059, 40 μM) inhibitor for 30 min. Levels of p-Thr-JMJD3 were detected by IP/IB (top) and quantified (bottom, n = 4 culture dishes). Source data are provided as a Source Data file. d, i Values are presented as mean ± SD. Statistical significance was measured using (d, i) two-way ANOVA with the Bonferroni post-test. **P < 0.01, and NS statistically not significant. Full size image

We next examined the role of the FGF21 signal-induced JMJD3 phosphorylation in induction of autophagy genes. JMJD3 was detected in the cytoplasmic and mitochondrial fractions and FGF21 treatment increased nuclear localization of JMJD3 in PMH (Supplementary Fig. 8c, d) and also in Hepa1c1c7 cells (Fig. 6c). Further, FGF21 treatment increased the interaction of JMJD3 with PPARα, the expression of Atgl, Atg7, and Tfeb and the LC3 II/I ratio (Fig. 6d, Supplementary Fig. 8e, f). These FGF21-mediated effects were blocked by the p-defective T1044A mutation of JMJD3, while effects similar to those in FGF21-treated cells were observed even in vehicle-treated cells with a p-mimic T1044E mutation (Fig. 6c, d, Supplementary Fig. 8d–f). Notably, FGF21-induced phosphorylation of JMJD3 was detected in both the cytoplasm and nucleus of FGF21-treated cells, while the interaction of JMJD3 with PPARα was detected only in the nucleus (Supplementary Fig. 8g). Collectively, these results indicate that FGF21-induced phosphorylation of JMJD3 is critical for its activation and for the induction of autophagy genes.

FGF21-activated PKA mediates the phosphorylation of JMJD3

Analysis of JMJD3 sequence adjacent of Thr-1044 revealed motifs for several kinases, including PKA, as well as, a well-known FGF21 signaling kinase, ERK18,19 (Supplementary Fig. 9a). Fasting overnight increased the phosphorylation of JMJD3 and both PKA and ERK in control mice, but not in FGF21-LKO mice (Fig. 6e), consistent with mediation of the phosphorylation by fasting-induced FGF21. Indeed, FGF21 treatment in mice increased phosphorylation of JMJD3 and both PKA and ERK (Fig. 6f) and increased the interaction of JMJD3 with PKA, but not with ERK (Supplementary Fig. 9b). In in vitro kinase assays, PKA, but not ERK, phosphorylated WT-JMJD3, but not T1044A-JMJD3 (Fig. 6g) and in GST pull down assays, PKA directly interacted with JMJD3 through the domain containing Thr-1044 (Fig. 6h, Supplementary Fig. 9c). Importantly, in PMH, FGF21-induced phosphorylation of JMJD3 was significantly blunted by treatment with an inhibitor of PKA, but not of MEK/ERK (Fig. 6i). These findings indicate that FGF21 signal-activated PKA mediates the phosphorylation of JMJD3, which is important for induction of autophagy.

Beneficial FGF21 effects on fatty liver are JMJD3-dependent

FGF21 and its analogs have beneficial lowering lipid effects in obese animals and humans26,27,28. Knowing that JMJD3 promotes hepatic autophagy, including lipophagy (Fig. 2), and mitochondrial fatty acid oxidation15, and importantly, FGF21-induced autophagy is dependent on JMJD3 (Fig. 4), we next asked whether JMJD3 has a role in mediating the lipid-lowering effects of FGF21. Administration of FGF21 to high-fat diet (HFD) obese mice decreased body weight without significant changes in food intake and decreased the size of liver (Fig. 7a). Levels of hepatic lipids and TG (Fig. 7b) and long-chain acylcarnitine were decreased, and levels of serum ketone bodies (Fig. 7c) and glucose tolerance (Fig. 7d) were increased. O 2 consumption and CO 2 production (Fig. 7e, Supplementary Fig. 10) were increased, indicating increased energy expenditure. FGF21 treatment also increased autophagy gene expression and autophagy (Fig. 7f, g). Each of these FGF21-mediated effects was blunted in hepatic JMJD3-downregulated mice (Fig. 7a–g). These results demonstrate that JMJD3 is required for FGF21-induced autophagy and beneficial outcomes, particularly lowering lipids, in obese mice.

Fig. 7: FGF21-mediated reversal of defective autophagy and hepatosteatosis in obese mice is dependent on JMJD3. JMJD3-floxed mice fed a HFD for 4 weeks were injected with the indicated viruses and then treated with vehicle or FGF21 every 2 days for 4 weeks with continued feeding of a HFD. a–f n = 5 mice/group, g n = 3 mice/group. a Experimental outline (top), body weights, food intakes, and images of liver. b Neutral lipids in liver sections stained with H&E and Oil Red O detected by IHC (left) and liver TG levels (right) (scale bar = 50 μm). c Levels of liver acylcarnitine species (left) and serum β-hydroxybutyric acid (β-HDB) (right). d Glucose tolerance test (GTT). e O 2 consumption (left) and CO 2 production (right) rates measured by indirect calorimetry. f Relative mRNA levels of the indicated hepatic proteins. g Levels of the indicated proteins in liver extracts from mice measured by IB with the LC3-II/I ratio and p62 band intensities shown below the blot (left), liver sections with LC3 detected by IHC (middle), and the number of LC3-II puncta/cell (right, n = 10 hepatocytes) (scale bar = 10 μm). Source data are provided as a Source Data file. a–g Values are presented as mean ± SD. Statistical significance was measured using the (b, c, e line graph, f, g) one-way or d two-way ANOVA with the Bonferroni post-test, and (e, bar graph) Student’s t-test. *P < 0.05, **P < 0.01, and NS statistically not significant. Full size image

Expression of KLB in fatty livers improves FGF21 signaling

Circulating FGF21 levels are highly elevated in non-alcoholic fatty liver disease (NAFLD) patients, as well as, in obese animals, suggestive of impaired FGF21 signaling18,29,30. Further, decreased autophagic flux and defective autophagy have been implicated in the development of NAFLD5,6,31. We, therefore, examined the effect of a HFD on FGF21 signaling and hepatic autophagy in mice.

Responsiveness to FGF21, as measured by increased p-PKA and p-ERK levels, was impaired, and FGF21-mediated induction of autophagy was absent in HFD obese mice compared to lean mice (Fig. 8a). Since β-Klotho (KLB) is the essential coreceptor for FGF21 action18,19, and its hepatic expression is downregulated in obesity32, we examined the effect of feeding a HFD for different lengths of time on expression of JMJD3, KLB, and FGFR. The mRNA levels of JMJD3 and KLB were decreased, whereas those of Fgfr1 and Fgfr4 were increased, after feeding a HFD (Fig. 8b), suggesting that decreased expression of KLB may contribute to FGF21 resistance in obesity.

Fig. 8: Expression of KLB in obese mice restores FGF21 signaling and autophagy. a Mice that had been fed a ND or HFD for 12 weeks were treated i.v. for 3 h with 0.1 mg/kg FGF21, and hepatic levels of the indicated proteins were detected by IB (left, n = 3 mice). The ratios of band intensities for the indicated proteins are shown (right). b Mice were fed a HFD for the times indicated and mRNA levels of Klb, JMJD3, Fgfr1, and Fgfr4 were measured by q-RTPCR (n = 3 mice). c Mice fed a HFD for 8 weeks were injected with AAV-TBG-KLB or AAV-TBG-GFP. Four weeks later, the mice were treated i.v. with vehicle or 0.1 mg/kg FGF21 for 3 h, and hepatic protein levels of the indicated proteins were detected by IB (n = 2 mice). Source data are provided as a Source Data file. a, b Values are presented as mean ± SD. Statistical significance was measured using the a two-way ANOVA with the Bonferroni or b one-way ANOVA with the Dunnett post-test *P < 0.05, **P < 0.01, and NS, statistically not significant. Full size image

We, therefore, further tested whether restoring KLB levels in obese mice can rescue FGF21 signaling and subsequently, expression of JMJD3 and hepatic autophagy (Fig. 8c, top). Viral-mediated liver-specific expression of KLB in HFD obese mice resulted in increased p-ERK and p-PKA levels, increased JMJD3 protein levels, and increased ratios of LC3II/I proteins (Fig. 8c). These results suggest that restoring expression of KLB in obese mice improves FGF21 signaling and consequently, FGF21-induced autophagy.

Expression of JMJD3 and autophagy genes is reduced in NAFLD

To assess potential human relevance of our findings, we examined the expression of JMJD3 and key autophagy genes in livers of human NAFLD patients. Autophagy has been reported to be defective in these patients5,6,31. Intriguingly, hepatic mRNA levels of JMJD3, TFEB, ULK1, ATG7, and ATGL, were all decreased in both simple steatosis and advanced NASH-fibrosis patients compared to normal subjects (Fig. 9a). The mRNA levels of KLB was also decreased in the patients (Fig. 9a) with decreased p-ERK levels, suggesting impaired FGF21 signaling18,29,30 (Fig. 9b). Protein levels of JMJD3, TFEB, ATG7, ULK1, LC3-II, and p-ERK, detected by IB of liver extracts (Fig. 9b) or IHC of liver sections (Fig. 9c), were decreased in the patients. While these results in humans are only correlative and do not necessarily link a defective FGF21-JMJD3 axis with defective autophagy in the patients, these findings are consistent with results in obese mice (Figs. 7 and 8), suggesting that the FGF21-JMJD3-autophagy axis is dysregulated in NAFLD.