To test the hypothesis that Aloxe3 is induced by acute fasting or pseudo-fasting conditions, we subjected WT mice to 48-hour fasting. During acute fasting, hepatic Aloxe3 expression was increased and sustained at least through 48 hours (Figure 1A). Oral administration of the fasting-mimetic hepatic glucose transport inhibitor trehalose (3% PO, ad libitum), similarly induced Aloxe3 expression after 24-hour and 48-hour trehalose feeding (Figure 1B). The hepatic Aloxe3 response to fasting contrasted with fasting Aloxe3 expression in white adipose tissue (WAT), wherein Aloxe3 mediates fat differentiation and lipid storage. Accordingly, Aloxe3 was significantly suppressed during both fasting and trehalose feeding in epididymal WAT (Figure 1, A and B, respectively). We then modeled Aloxe3 expression in vitro to determine whether fasting-induced Aloxe3 expression is cell-autonomously regulated. Isolated primary murine hepatocytes were subjected to treatment by either low serum and glucose (0.5% serum, 1 g/l glucose) or trehalose (Figure 1C). In addition, we characterized fasting-mimetic effects of a trehalose analogue that resists enzymatic degradation by trehalases (21–23), called lactotrehalose (α-D-glucopyronosyl-(1,1)-α-D-galactopyranoside; Figure 1C and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.120794DS1). In each case, Aloxe3 expression was significantly increased, most potently by trehalose and lactotrehalose (Figure 1C and Supplemental Figure 1).

Figure 1 Aloxe3 is induced in response to fasting and pseudofasting. (A) Aloxe3 expression in liver and white adipose tissue (WAT) from fasting WT mice (0–48 hours [h]). n = 6 each of fed, 12 h fasting, and 24 h fasting mice. For 48 h fasting mice, n = 7. (B) Aloxe3 expression in WT mice fed oral trehalose (3% in sterile water fed ad libitum, 0–48 h). For control and trehalose (24 h) mice, n = 5 and 6. For 48 h trehalose-treated mice, n = 6. (C) Aloxe3 expression in isolated primary murine hepatocytes treated with 0.5% FCS/1 g/l glucose media, 100 mM trehalose, or 100 mM lactotrehalose (24 h). n = 4 per group. (D) Aloxe3 expression in trehalose-treated isolated primary hepatocytes pretreated with or without 5 mM pyruvate. n = 4 per group. (E) Aloxe3 expression in trehalose-treated WT and Atg16l1-mutant mice in vivo (24 h). n = 6 per group. (F) Aloxe3 expression in response to trehalose in the presence or absence of kinase-dead AMPK overexpression or siRNA-mediated AMPK knockdown. n = 4 per group. (G) Aloxe3 expression in response to trehalose in the presence or absence of antisense oligonucleotide (ASO) directed against PGC1, or FGF21. FGF receptor 1–4 inhibitor (LY2874455) was included as a control to demonstrate Aloxe3 blockade in context. n = 4 per group. (H) Fasting-responsive marker gene expression in the presence or absence of trehalose following treatment with or without Aloxe3-directed siRNA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by 2-tailed t test with Bonferroni-Dunn post hoc correction versus untreated controls group or versus the bracketed comparison group where indicated.

In light of evidence that Aloxe3 is a potentially novel hepatocyte fasting-responsive factor in mice and in isolated murine hepatocytes, we evaluated whether this factor is expressed in isolated human hepatocytes and in Huh7 and HepG2 human hepatoma cell lines. Consistent with immunoblot and immunohistochemical eLOX3 protein detection in human liver tissue (24), quantitative PCR (qPCR) analysis demonstrated ALOXE3 expression in both HepG2 and in primary human hepatocytes (Supplemental Figure 2).

To determine whether Aloxe3 induction depended upon energy substrate deficit, we treated primary hepatocytes with trehalose in the presence or absence of pyruvate. This provided energy substrate for the cell, independently of glucose transporter blockade. Pyruvate reversed trehalose-induced Aloxe3 induction by ~60% (P < 0.0001), suggesting that Aloxe3 expression is at least partly energy substrate dependent (Figure 1D). In light of our prior data in which we demonstrated that the autophagy complex protein ATG16L1 was required for autophagic and antisteatotic effects of trehalose in hepatocytes (9, 25), we examined whether ATG16L1 is required for Aloxe3 induction. We treated primary hepatocytes from WT littermates or from mice with homozygous hypomorphic Atg16l1 alleles (Atg16l1HM) with or without trehalose (24 hours). Aloxe3 mRNA quantification revealed robust Aloxe3 induction in WT hepatocytes, which was not suppressed in Atg16l1HM hepatocytes (Figure 1E). Similarly, AMPK inhibition (by kinase-dead AMPK overexpression or by Aloxe3 siRNA transfection; Figure 1F) and PPARα, PGC1α, and FGF21 knockdown each failed to reverse Aloxe3 induction after trehalose treatment or glucose and serum withdrawal (Figures 1G and 1H). Accordingly, moderate hepatocyte-specific overexpression of the fasting-induced transcription factor SIRT1 did not induce Aloxe3 expression (Supplemental Figure 3). In contrast, siRNA-based Aloxe3 knockdown attenuated PPARα and PGC1α at baseline and during glucose and serum withdrawal from hepatocytes in vitro, without effects on FGF21 expression (Figure 1H). Together, our data demonstrate that Aloxe3 is a hepatocyte lipoxygenase that is induced by glucose transporter blockade and energetic deficit via a mechanism that does not require canonical ATG16L1-dependent and AMPK-PGC1α/PPARα-FGF21 fasting mechanisms.

eLOX3 generates the PPARγ ligand 12-KETE and an epoxyalcohol from 12-HpETE in the metabolism of plasma membrane arachidonic acid (Figure 2A) (26–30). We tested whether Aloxe3 mediates similar lipid metabolism in hepatocytes upon forced Aloxe3 expression. We first evaluated the effect of Aloxe3 overexpression on hepatocyte lipoxygenase activity by measuring LOX substrate oxidation in cell extracts from hepatocytes overexpressing β galactosidase (β-gal) or Aloxe3. Consistent with prior data demonstrating intrinsic, latent dioxygenase activity of eLOX3 (31), Aloxe3 overexpression significantly increased total oxidized LOX substrate accumulation (Figure 2B) and 12-KETE accumulation in primary hepatocytes (Figure 2C). This effect of Aloxe3 overexpression on 12-KETE accumulation was indeed phenocopied by trehalose treatment (Figure 2C). Moreover, Aloxe3 overexpression decreased the abundance of alternate arachidonic acid metabolic pathway products 5-HETE and 12-HETE (Figure 2D).

Figure 2 Aloxe3 and trehalose induce the PPARγ ligand 12-KETE in murine hepatocytes. (A) Arachidonic acid metabolism mediated by lipoxygenases 12-LOX, 5-LOX, and eLOX3 (adapted from refs. 26, 29, 58, 59). (B) Lipoxygenase activity in primary murine hepatocytes upon overexpression of β galactosidase or Aloxe3. n = 3 from 1 representative experiment of 2 experiments with similar results. (C and D) Quantitative GC-MS analysis of the stable lipoxygenase reaction products 12-KETE, 5-HETE, and 12-HETE. Data represent 4 independent cultures per group from 1 experiment, representing 2 independent experiments with similar results. **P < 0.01 and ***P < 0.001 by 2-tailed t test with Bonferroni-Dunn post hoc correction versus untreated controls group or versus the bracketed comparison group where indicated.

To ascertain transcriptome-wide effects of forced Aloxe3 overexpression, we next treated primary hepatocytes with adenovirus encoding β-gal or Aloxe3 prior to RNA sequencing (RNA-seq) analysis. Pathway analysis revealed that 5 of the 10 most downregulated processes in Aloxe3-overexpressing cells were devoted to inflammatory signaling, including TNFα, NF-κB, chemokine signaling, cytokine receptor signaling, and MAPK signaling (Figure 3A). Given these findings, and in light of the fact that Aloxe3 upregulation by trehalose correlates with reduced diet- and genetically induced steatosis (9, 25), we examined whether Aloxe3 attenuates fat-induced inflammatory signaling and triglyceride (TG) accumulation. Primary hepatocytes treated with BSA-conjugated fatty acids (FA) induced Il1b and Tnfa gene expression, as well as TG accumulation (Figure 3, B and C). These FA-induced effects on TG accumulation and Il1b and Tnfa gene expression effects were reversed in FA-treated cultures overexpressing Aloxe3.

Figure 3 Aloxe3 reduces inflammatory signaling and steatosis in hepatocytes. (A) RNA-seq analysis of the top 9 downregulated genes in primary hepatocytes expressing either β galactosidase or Aloxe3. n = 3 from a single RNA-seq run performed once. The P value for significantly downregulated pathways is demonstrated as (–Log[P value]). (B) Hepatic TG accumulation and (C) IL-1β or TNFα expression in hepatocytes treated with BSA-conjugated fatty acids with or without Aloxe3 expression. n = 4 in B and n = 4 in C, single representative experiments, which were repeated twice with similar results. ***P < 0.001 and ****P < 0.0001 by 2-tailed t testing with Bonferroni-Dunn post hoc correction versus the bracketed comparison group as indicated.

Aloxe3-deficient mice are not amenable to in vivo metabolic studies because germline Aloxe3 deficiency results in postnatal mortality secondary to massive skin permeability and water loss (32). To evaluate the in vivo metabolic consequences of hepatocyte Aloxe3 activation, we tested the in vivo effects of hepatocyte-directed Aloxe3 expression. We first confirmed overexpression of the Aloxe3 transgene in liver (Figure 4A) without changes in Aloxe3 expression in skeletal muscle, WAT, or brown adipose tissue (not shown). This correlated with upregulation of fasting and oxidative genes in unperturbed Aloxe3-overexpressing mice, including Aloxe3 (Figure 4A), Ppargc1a (PGC1), hepatocyte nuclear factor 4α (Hnf4A/HNF4α), and phosphoenolpyruvate carboxykinase 1 (Pck1, Figure 4A.).

Figure 4 Enhanced whole-body metabolism in mice ALOXE3-overexpressing mice. (A) qPCR quantification of expression for oxidative and fasting-response genes in unperturbed mice expressing hepatocyte GFP or Aloxe3. (B) Body weight over time in low-fat or high–trans fat/cholesterol diet–fed mice expressing hepatocyte GFP or Aloxe3. (C) Body fat content in mice fed HTFC or LFD with or without hepatic Aloxe3 overexpression. (D) LDL-C and total cholesterol in mice fed HTFC or LFD with or without hepatic Aloxe3 overexpression. (E–G) Circulating insulin, glucose, and calculated HOMA-IR in LFD- and HTFC-fed mice overexpressing hepatocyte GFP or Aloxe3. Number of mice in each group is: 5, AAV8GFP LFD; 10, AAV8GFP HTFC; 5, AAV8ALOXE3 LFD; and 10, AAV8ALOXE3 HTFC. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by 2-tailed t test with Bonferroni-Dunn post hoc correction versus the bracketed comparison group as indicated.

We next evaluated the effect of hepatic Aloxe3 expression in mice fed low-fat diet (LFD) or steatogenic, high trans-fat/cholesterol diet (HTFC) (12 weeks). Mice overexpressing Aloxe3 fed HTFC gained significantly less weight, had lower total body mass (Figure 4B), and had lower body fat mass (Figure 4C) without changes in lean mass (not shown) when compared with Gfp-overexpressing mice fed HTFC. Accordingly, low density lipoprotein-cholesterol (LDL-C) and total cholesterol were significantly lowered in HTFC-fed mice overexpressing Aloxe3 (Figure 4D). Indices of glucose homeostasis were also improved by Aloxe3 overexpression in HTFC-fed mice (Figure 4, E–G). Specifically, HTFC feeding increased circulating insulin and homeostatic model assessment– insulin resistance (HOMA-IR) in GFP-overexpressing mice (Figure 4, E and G), without altering fasting plasma glucose (Figure 4F). In contrast, HTFC-fed mice overexpressing hepatic Aloxe3 were protected from HTFC-induced hyperinsulinemia and insulin resistance (Figure 4, E and G). Indeed, fasting glucose was also lowered in Aloxe3-overexpressing, HTFC-fed mice when compared with GFP-expressing, HTFC-fed mice (Figure 4F). Together, hepatocyte Aloxe3 expression was sufficient to reduce diet-induced weight gain, body fat accumulation, dyslipidemia, and insulin resistance.

We next examined hepatic lipid metabolism in GFP- and Aloxe3-overexpressing mice fed LFD or HTFC. Frozen liver sections from HTFC-fed mice exhibited increased Oil Red O staining and macrosteatosis when compared with LFD-fed mice. Consistent with prior models of PPARγ activation (33–35), Aloxe3 expression in LFD-fed mice resulted in a mild basal TG accumulation (Figure 5, A and B). However, we did not observe evidence of hepatic inflammation or fibrosis by histological analysis of H&E- or trichrome-stained liver sections in LFD-fed mice after 12 weeks Aloxe3 overexpression (Supplemental Figure 4). Also consistent with prior reports on hepatic PPARγ activation, Aloxe3 overexpression modestly but significantly protected from HTFC-induced TG accumulation that took on a microsteatotic staining pattern (Figure 5, A and B). No changes in hepatic LDL-C or total cholesterol were observed (Figure 5, C and D) upon hepatic Aloxe3 overexpression in LFD- or HTFC-fed mice. None of our genetic or dietary manipulations had any effect on hepatic synthetic function, as ascertained by quantitative circulating albumin levels (Figure 5E).

Figure 5 Reduced hepatic steatosis correlates with Aloxe3-induced fasting responses. (A) Oil Red O staining in livers from low-fat or high–trans fat/cholesterol–fed mice overexpressing GFP or Aloxe3. (B–D) Hepatic tissue quantification of triglycerides, LDL-C, total cholesterol in low-fat or high–trans fat/cholesterol–fed mice overexpressing empty vector or Aloxe3. (E) Serum albumin measurements in mice analyzed in A–D Number of mice in each group is: 5, AAV8GFP LFD; 10, AAV8GFP HTFC; 5, AAV8ALOXE3 LFD; and 10, AAV8ALOXE3 HTFC. Scale bar: 100 μm. **P < 0.01 and ****P < 0.0001 by 2-tailed t test with Bonferroni-Dunn post hoc correction versus untreated controls group or versus the bracketed comparison group where indicated.

To examine mechanistically how Aloxe3 might activate hepatocyte-starvation–like responses, we evaluated the effects of Aloxe3 overexpression on mitochondrial respiratory function at the cellular and molecular levels. RNA-seq analysis of primary hepatocytes overexpressing Aloxe3 revealed that 6 of the 10 most downregulated molecular processes encompassed mitochondrial electron transport function, including proton transport, ATPase activity, transmembrane ion transport, and hydrogen export (Figure 6A). We therefore tested functionally the hypothesis that Aloxe3 mitigates ATP production by inducing hepatocyte mitochondrial uncoupling. Seahorse analysis of hepatocytes overexpressing Aloxe3 exhibited elevated proton leak, ATP production, and coupling efficiency, concomitant with suppressed basal oxygen consumption rate and enhanced glycolytic rate when compared with hepatocytes expressing β-gal (Figure 6B). Each of these parameters was partly or fully reversed in the presence of the PPARγ inhibitor GW9662 (Figure 6B). No changes in nonmitochondrial oxygen consumption were observed (Figure 6C), suggesting that Aloxe3 specifically affected mitochondrial energy metabolism.

Figure 6 Hepatic PPARγ is required for Aloxe3 to induce hepatic energetic inefficiency. (A) RNA-seq analysis of the top 5 downregulated molecular processes in hepatocytes overexpressing Aloxe3. Highlighted are ATPase-related and mitochondrial coupling processes. Graphed is logFC relative to cultures expressing β galactosidase. n = 3 from a single RNA sequencing run performed once. (B and C) Seahorse XF96 analysis of proton leak, basal OCR, ATP production, coupling efficiency, and nonmitochondrial oxygen consumption in AML12 cells overexpressing Aloxe3 with or without GW9662 (PPARγ inhibitor) treatment. n = 8 independent cultures combined from 2 distinct experimental runs. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by 2-tailed t testing with Bonferroni-Dunn post hoc correction versus the bracketed comparison group as indicated.

Mechanistic interrogation in vivo was executed to determine whether hepatocyte Aloxe3 mediated peripheral insulin and glucose homeostasis in a leptin-dependent manner. To that end, we overexpressed Aloxe3 or GFP in db/db mice, which lack the leptin receptor. Again, in the liver, we observed modest, statistically significant attenuation of hepatic TG accumulation in db/db mice overexpressing Aloxe3 when compared with db/db controls (Figure 7, A and B). We confirmed Aloxe3 overexpression correlated with increases in oxidative and fasting-response genes Ppargc1a, Hnf4a, and Pc1 (Figure 7C). Moreover, Aloxe3 overexpression reduced genetic markers of de novo lipogenesis in db/db mice, including Fsp27, Scd1, and Fasn (Figure 7D). Concomitant PPARγ inhibition by the inhibitor GW9662 produced either statistically significant or trends toward significant reversal in each of these Aloxe3-altered marker genes (Figure 7, C and D).

Figure 7 Reduced hepatic steatosis correlates with Aloxe3-induced fasting and reduction of de novo lipogenesis. (A) H&E staining in livers from low-fat or high–trans fat/cholesterol–fed mice overexpressing empty vector or Aloxe3. (B) Hepatic tissue triglyceride quantification in db/db mice overexpressing GFP or Aloxe3 in the presence or absence of GW9662. (C and D) qPCR quantification of expression for oxidative and fasting-response genes (C) and de novo lipogenic genes (D) in db/db mice expressing GFP or Aloxe3 with or without GW9662 administration. Number of mice in each group is: 10, db/db AAV8GFP; 10, db/db AAV8ALOXE3; 10, AAV8ALOXE3 + GW9662. Scale bar: 100 μm. **P < 0.01, or ****P < 0.0001 by 2-tailed t testing with Bonferroni-Dunn post hoc correction versus the bracketed comparison group as indicated.

Although baseline weights were not statistically different, db/db mice overexpressing Aloxe3 gained significantly less weight and had a lower end-of-trial weight than db/db mice over the 28-day trial (Figure 8A). Indirect calorimetry revealed that the attenuated weight gain in aloxe3 db/db mice was associated with enhanced light- and dark-cycle heat generation and O2-CO2 exchange (Figure 8, B-D). Neither heat generation nor O 2 -CO 2 exchange in Aloxe3db/db was affected by GW9662 coadministration (Figure 8, B–D).

Figure 8 Enhanced whole-body energy metabolism in db/db diabetic mice overexpressing Aloxe3. (A) Body weight over time in db/db mice expressing empty vector or Aloxe3 in the presence or absence of GW9662. (B) Heat generation over time in db/db mice expressing GFP or Aloxe3. (C and D) Indirect calorimetric quantification of light- and dark-cycle heat generation in db/db mice expressing Aloxe3 or GFP treated with or without GW9662. (E) Fasting serum insulin determined by ELISA, serum glucose determined by colorimetric assay, and calculated HOMA-IR index based on glucose and insulin data. (F–I) Serum TG, cholesterol, LDL-C, and FFA content in db/db mice with or without hepatic Aloxe3 overexpression and with or without GW9662 treatment. Number of mice in each group is: 10, db/db AAV8GFP; 10, db/db AAV8ALOXE3; 10, AAV8ALOXE3 + GW9662. *P < 0.05, **P < 0.01, ***P < 0.001, and < 0.0001 by 2-tailed t test with Bonferroni-Dunn post hoc correction versus bracketed comparison groups.

Because hepatic Aloxe3 expression enhanced peripheral insulin sensitivity in HTFC-fed mice (Figure4), and because PPARγ agonism by thiazolidinediones (TZDs) enhances peripheral insulin sensitivity (36, 37), we evaluated whether Aloxe3 enhances peripheral insulin and glucose homeostasis dependent on PPARγ and yet independent of the leptin receptor. Fasting circulating insulin and HOMA-IR were significantly lower in Aloxe3db/db mice without changes in circulating glucose when compared with db/db mice expressing GFP. The reduction in circulating insulin and HOMA-IR were reversed in mice concomitantly treated with the PPARγ inhibitor GW9662, suggesting that Aloxe3 improves glucose and insulin homeostasis via a PPARγ-dependent mechanism. In contrast with our diet-induced model, however, targeted hepatic Aloxe3 expression did not reduce circulating lipids in a leptin receptor–deficient mice. Together, the data in our leptin-deficient model elucidate leptin- and PPARγ-dependent functions by Aloxe3.

To gain further specificity regarding the role of hepatocyte PPARγ in Aloxe3-enhanced insulin sensitivity, we generated mice harboring a hepatocyte-specific PPARG deletion (hereafter referred to as PPARγ-LKO mice) by crossing mice with homozygous floxed Pparg alleles with mice expressing Cre recombinase driven by the albumin promoter. Mice were placed on a chow or Western diet (WD) to induce insulin resistance over 12 weeks; they were then subjected to insulin tolerance testing (ITT, Figure 9, A and B). Area under the ITT curve analysis revealed that WD feeding increased AUC in GFP-overexpressing animals, whereas AUC was reduced in Aloxe3-overexpressing animals. In striking contrast, WD-fed Aloxe3-overexpressing PPARγ-LKO mice had a significantly elevated AUC when compared with WD-fed Aloxe3-overexpressing mice harboring WT hepatic PPARγ.