12-Lipoxygenase Regulates Cold Adaptation and Glucose Metabolism by Producing the Omega-3 Lipid 12-HEPE from Brown Fat

Abstract

Distinct oxygenases and their oxylipin products have been shown to participate in thermogenesis by mediating physiological adaptations required to sustain body temperature. Since the role of the lipoxygenase (LOX) family in cold adaptation remains elusive, we aimed to investigate whether, and how, LOX activity is required for cold adaptation and to identify LOX-derived lipid mediators that could serve as putative cold mimetics with therapeutic potential to combat diabetes. By utilizing mass-spectrometry-based lipidomics in mice and humans, we demonstrated that cold and β3-adrenergic stimulation could promote the biosynthesis and release of 12-LOX metabolites from brown adipose tissue (BAT). Moreover, 12-LOX ablation in mouse brown adipocytes impaired glucose uptake and metabolism, resulting in blunted adaptation to the cold in vivo. The cold-induced 12-LOX product 12-HEPE was found to be a batokine that improves glucose metabolism by promoting glucose uptake into adipocytes and skeletal muscle through activation of an insulin-like intracellular signaling pathway.

Keywords: 12-HEPE; adipocytes; brown adipose tissue; diabetes; eicosapentaenoic acid; fat; lipidomic; lipokine; obesity; thermogenesis.

Figure 1:. Cold exposure induces 12-LOX derived lipid secretion into the circulation

A) Volcano plot of the lipids profiled in serum showing fold change between male C57BL6/J mice exposed to 5°C or 30°C for 7 days, co mpared to the p-value of this comparison. B) Serum levels of the 12-LOX products 12-HEPE, 14-HDHA and 12-HETE from male mice exposed to 30°C or 5°C for 7 days (n = 5). C) Volcano plot of the serum lipids from C57BL6/J female mice exposed to 30°C or 5°C for 7 days. D) Serum levels of the 12-LOX products from female mice exposed to 30°C or 5°C for 7 days (n = 5). E) Volcano plot of the lipids profiled in serum showing fold change between male C57BL6/J mice exposed to 5°C for 1 hour and mice kept at room temperature (22°C) compared to the p-value of this comparison. F) Circulating levels of the 12-LOX products from male mice exposed to 5°C or 22°C for 1 hour (n = 5). G) Alox12 mRNA expression in differentiated WT-1 brown adipocytes stimulated with cyclic AMP (1mM), norepinephrine (1μM) or CL316,243 (1μM) for 4 hours. H) Western blotting for 12-LOX protein expression in iBAT and ingWAT from mice housed at 5°C or 30°C for 7 days (n = 4). I) Scheme illustrating the biosynthetic pathways by which 12-HEPE, 14-HDHA and 12-HETE are derived from their polyunsaturated fatty-acid precursors EPA, DHA and AA. For all panels, *P<0.05, unpaired student’s t-test. Data are represented as mean ± S.E.M. See also Figure S1. All the lipid quantification data were detected using non-targeted lipidomics, thus relative values are shown.

Figure 2:. Mirabegron treatment increases the secretion of 12-LOX metabolites in human subjects

A) Volcano plot of the lipids profiled in plasma showing fold change between human subjects treated with a single oral dose of the β3-adrenergic agonist mirabegron (200mg) and subjects treated with placebo compared to the p-value of this comparison, (n = 10-11). p values determined by multiple t-tests. B) Circulating levels of the 12-LOX derived lipids from human subjects treated with placebo or mirabegron (n = 10-11). **p<0.01, ***p<0.001. Data are represented as paired individual values. C-E) Spearman correlation between circulating 12-LOX metabolites levels and BAT glucose uptake measured by PET-CT, and expressed as standardized uptake values (SUV), obtained from human subjects treated with placebo or mirabegron. See also Figure S2. All the lipid quantification data were detected using non-targeted lipidomics, thus relative values are shown.

Figure 3:. 12-HEPE is a cold-inducible lipid that is biosynthesized in BAT

A) Schematic panel illustrating the experimental design of the lipidomics performed in serum from Bmpr1a^flox (WT) and Myf5^CRE/Bmpr1a^flox (mice with iBAT paucity) male mice exposed to 30°C or 5°C for 2 days. B) Serum lipid levels in male Bmpr1a^flox (WT) and Myf5^CRE/Bmpr1a^flox mice exposed to 30°C or 5°C (n = 4-5). C) Schematic panel illustrating the experimental design of the lipidomics performed in iBAT and ingWAT harvested from male C57BL6/J mice exposed to 30°C or 5°C for 7 days. D) Lipid levels in iBAT and ingWAT from C57BL6/J male mice exposed to 30°C or 5°C for 7 days (n = 5-6). Targeted lipidomics was used for lipid quantification. E) Schematic panel illustrating the experimental design of the lipidomics performed in conditioned medium incubated with ingWAT or iBAT explants, harvested from C57BL6/J mice exposed to 30°C or 5°C for 7 days. F) Medium lipid levels in Krebs Ringer buffer after its incubation (1 hour) with iBAT or ingWAT harvested from C57BL6/J male mice exposed to 30°C or 5°C for 7 days. G) Schematic panel illustrating the experimental design of the lipidomics performed in media conditioned by in vitro differentiated human brown adipocytes stimulated with forskolin or vehicle for 4 hours. H) 12-HEPE, 14-HDHA and 12-HETE levels in conditioned media of human brown adipocytes stimulated with forskolin or vehicle. *P<0.05, **p<0.01, ***p<0.001. Unpaired Student’s t-test. N=5 per group. Data are represented as mean ± S.E.M. See also Figure S3. All the lipid quantification data (except for Figure 3D) were detected using non-targeted lipidomics, thus relative values are shown.

Figure 4:. 12-LOX ablation in brown adipocytes impairs glucose utilization and cold adaptation

A) Serum lipid levels in C57BL6/J male mice i.p. injected with LOXblock-1 for 15 min or its vehicle DMSO, and then exposed to 5°C for 4h. (n = 6-7). B) Rectal temperature during cold tolerance test in C57BL6/J male mice i.p. injected with LOXblock-1 or vehicle (DMSO) in the presence or absence of 12(S)-HEPE (200μg/kg via i.p.) and then exposed to 5°C. (n = 5-6). C) Schematic panel illustrating the strategy for the generation of Ucp1^CRE/Alox12 KO mice from the Ucp1^CRE and CRISPR/Cas9 knock-in mice. D) Serum 12-LOX-derived lipid concentrations from the empty vector (EV) mice and Ucp1^CRE/Alox12 KO mice, exposed to 22°C or 5°C for 1 hour. (n = 6). E) Rectal temperature during cold tolerance test in EV and Ucp1^CRE/Alox12 KO male mice, by measuring rectal temperature under 5°C (n = 12). F) Oxygen consumption measured by CLAMS in EV and Ucp1^CRE/Alox12 KO mice i.p. injected with norepinephrine (n = 12). G) Glucose uptake in wild-type, Alox12 KO and 12-LOX re-expressed Alox12 KO differentiated brown adipocytes in vitro. H) Extracellular acidification rate (ECAR) during the Seahorse glycolysis stress test in Alox12-KO and 12-LOX re-expressed Alox12 KO differentiated brown adipocytes. I) Oxygen consumption ratio (OCR) during the Seahorse mitochondrial stress test in EV, Alox12-KO and 12-LOX re-expressed Alox12 KO differentiated brown adipocytes. *p<0.05, **p<0.01, ***p<0.001. Data are represented as mean ± S.E.M. See also Figure S4. The lipid quantification data were detected using non-targeted lipidomics, thus relative values are shown.

Figure 5:. Regulation of 12-LOX metabolite levels in human and murine obesity

A - C) 12-HEPE, 14-HDHA and 12-HETE serum levels in serum samples from lean (BMI<25), overweight (BMI>25, <30) and obese (BMI>30) subjects (n = 60). **P<0.01, ***P<0.001. Data are represented as mean ± S.E.M. See also Figure S4. D-F) Spearman correlation between the serum levels of 12-LOX products and insulin resistance measured by HOMA-IR index. G-I) Spearman correlation between the serum levels of 12-LOX products and serum leptin concentration. See also Figure S5. The lipid quantification data were detected using non-targeted lipidomics, thus relative values are shown.

Figure 6:. 12-HEPE serves as a glucose uptake mediator

A) Body weight in 12(S)-HEPE and Vehicle treated DIO male mice (n = 7). B) Fasting serum glucose measured in 12(S)-HEPE and Vehicle treated DIO mice (n = 7). C) Glucose tolerance test (GTT) performed in 6h fasted 12(S)-HEPE and Vehicle treated DIO mice. (n = 7). D) Glycemia during the insulin tolerance test (ITT) performed in 6h fasted 12(S)-HEPE and Vehicle treated DIO mice (n = 7). E) Ratio of tissue weight to body weight calculated for pgWAT, ingWAT, liver and BAT harvested from 12(S)-HEPE and Vehicle treated DIO mice (n = 7). F-H) mRNA expression measured by qPCR of genes associated with the thermogenic program associated, glucose transport and de novo lipogenesis in BAT from 12(S)-HEPE and Vehicle treated DIO mice (n = 7). I) Glucose uptake into BAT, ingWAT, pgWAT, quadriceps, gastrocnemius, soleus and liver of C57BL6/J lean mice i.v. injected with 12(S)-HEPE or vehicle, 30 minutes before an [³H]2-deoxyglucose i.v. injection (n = 6-8). J-N) In vitro glucose uptake into murine brown adipocytes, human brown adipocytes, murine myocytes (C2C12), murine white adipocytes (3T3-F442A cells) and human white adipocytes (hWAT) treated with 12(S)-HEPE or vehicle for 30 minutes before adding [³H]2-deoxyglucose. (n = 4-6 experimental replicates from at least 2 biological replicates). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean ± S.E.M. See also Figure S6.

Figure 7:. 12-HEPE promotes glucose uptake via a G_sPCR/PI3K/AKT axis

A) Western blotting for phospho-AKT (Ser473 and Th308), Akt, phospho-mTORC2 (Ser2481), mTOR, phospho-AS160 (Th642), and β-tubulin (loading control) in mouse brown adipocytes treated with 12(S)-HEPE or vehicle. B) In vitro glucose uptake into murine brown adipocytes treated with 12(S)-HEPE or vehicle in the presence or absence of pre-treatment with rapamycin, Torin 1 or Wortmannin. C) Immunofluorescent staining for Glut-4 in murine brown adipocytes treated with PBS, insulin or 12(S)-HEPE. The white arrows indicate Glut-4 staining in the plasma membrane. D-F) In vitro glucose uptake into murine brown adipocytes treated with 12(S)-HEPE or vehicle in the presence or absence of pre-treatment with YM254890, Pertussis Toxin or Melittin. G) In vitro glucose uptake into Scramble (control) or Gnas KD murine brown adipocytes treated with 12(S)-HEPE or vehicle. *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean ± S.E.M. See also Figure S7.