Lipocalin 2 regulates retinoic acid-induced activation of beige adipocytes

in Journal of Molecular Endocrinology
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Lipocalin-2 (LCN2) has been previously characterized as an adipokine regulating thermogenic activation of brown adipose tissue and retinoic acid (RA)-induced thermogenesis in mice. The objective of this study was to explore the role and mechanism for LCN2 in the recruitment and retinoic acid-induced activation of brown-like or ‘beige’ adipocytes. We found LCN2 deficiency reduces key markers of thermogenesis including uncoupling protein-1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) in inguinal white adipose tissue (iWAT) and inguinal adipocytes derived from Lcn2 −/− mice. Lcn2 −/− inguinal adipocytes have attenuated insulin-induced upregulation of thermogenic gene expression and p38 mitogen-activated protein kinase (p38MAPK) signaling pathway activation. This is accompanied by a lower basal and maximal oxidative capacity in Lcn2 −/− inguinal adipocytes, indicating mitochondrial dysfunction. Recombinant Lcn2 was able to restore insulin-induced p38MAPK phosphorylation in both WT and Lcn2 −/− inguinal adipocytes. Rosiglitazone treatment during differentiation of Lcn2 −/− adipocytes is able to recruit beige adipocytes at a normal level, however, further activation of beige adipocytes by insulin and RA is impaired in the absence of LCN2. Further, the synergistic effect of insulin and RA on UCP1 and PGC-1α expression is markedly reduced in Lcn2 −/− inguinal adipocytes. Most intriguingly, LCN2 and the retinoic acid receptor-alpha (RAR-α) are concurrently translocated to the plasma membrane of adipocytes in response to insulin, and this insulin-induced RAR-α translocation is absent in adipocytes deficient in LCN2. Our data suggest a novel LCN2-mediated pathway by which RA and insulin synergistically regulates activation of beige adipocytes via a non-genomic pathway of RA action.

 

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    Thermogenic gene expression and mitochondrial function in Lcn2 −/− WAT and inguinal adipocytes. (A) Ucp1 gene expression in inguinal WAT (iWAT) and epididymal WAT (eWAT) from WT and Lcn2 −/− mice on high-fat diet for 12 weeks, n = 3–5/group. (B) Thermogenic gene expression in primary inguinal adipocytes isolated from WT and Lcn2 −/− mice, n = 3–5/group. (C and D) Oxygen consumption rate in differentiated inguinal adipocytes isolated from WT and Lcn2 −/− mice, n = 8/treatment. (E) NAD+/NADH in differentiated inguinal adipocytes isolated from WT and Lcn2 −/− mice, n = 3/treatment. (F) mtDNA relative to nuclear DNA in inguinal differentiated adipocytes from WT and Lcn2 −/− mice, n = 3/treatment. The cell culture experiments were repeated 2–3 times yielding similar results. The data are represented as mean ± s.e.m. *P < 0.05 vs WT; **P < 0.01 vs WT.

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    β-adrenergic signaling in Lcn2 −/− inguinal adipocytes. p38MAPK (A and B) and HSL (C and D) phosphorylation following treatment with 1 µM norepinephrine (NE) ± insulin (Ins). (E) Glycerol release into cell supernatant from differentiated inguinal adipocytes treated with 1 µM isoproterenol (Iso). (F) Gene expression in differentiated inguinal adipocytes treated with 1 µM norepinephrine (NE) for 18 h, n = 3/treatment. Experiments were repeated two times yielding similar results. The data are represented as mean ± s.e.m. For B and D, *P < 0.05 vs Control of same genotype; **P < 0.01 vs Control of same genotype; #P < 0.01 vs WT of same group. For E and F, *P < 0.05 vs WT of same group.

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    Thermogenic gene expression and p38MAPK signaling by recombinant LCN2 in Lcn2 −/− inguinal adipocytes. (A) Gene expression of Ucp1, Pgc1a, and Cidea following 18 h treatment with or without insulin (INS), n = 3/group. (B) p38MAPK phosphorylation in differentiated inguinal adipocytes following 18 h of treatment with 500 ng/mL recombinant LCN2 ± insulin (INS). (C) Quantification of band intensity of phosphorylated p38MAPK normalized to total p38MAPK for 18 h treatment, n = 3 independent experiments. (D, E) Gene expression of Ucp1 following 18 h treatment with or without 1 µM norepinephrine (NE) in the presence or absence of 100 µM Propranolol, n = 3/group. (F) Gene expression of Ucp1 following 18 h treatment with or without 500 ng/mL recombinant LCN2 in the presence or absence of 100 µM Propranolol, the data are mean from three independent experiments, n = 3/treatment for each experiment. (G) p38MAPK phosphorylation following 18 h treatment with or without 500 ng/mL recombinant LCN2 in the presence or absence of 100 µM Propranolol. (H) Quantification of band intensity of phosphorylated p38MAPK normalized to total p38MAPK for Western blots in G, n = 2 independent experiments. For A, B, C, D, E, F, G and H, the experiments were repeated 2–3 times yielding similar results. The data are represented as mean ± s.e.m. *P < 0.05 vs WT of same group; **P < 0.01 vs WT of same group; ***P < 0.001 vs WT of same group; #P < 0.05 vs Control of same genotype; ##P < 0.01 vs Control of same genotype; ###P < 0.001 vs Control of same genotype; $P < 0.05 vs WT control.

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    RA-induced thermogenesis in Lcn2 −/− iWAT and adipocytes. (A, B and C) UCP1 protein levels (A and B) (n = 3–4 mice/group) and Ucp1 and Pgc1a gene expression (C) (n = 4–6 mice/group) in iWAT from WT and Lcn2 −/− mice following oral gavage with RA for 14 days. *P < 0.05 vs WT; #P < 0.05 vs WT RA. (D and E) Ucp1 (D) and Pgc1a (E) gene expression in differentiated inguinal adipocytes following 24 h treatment with insulin and 1 µM RA, n = 3/treatment. *P < 0.05 vs WT; #P < 0.05 vs WT-Con. (F and G) p38MAPK phosphorylation in differentiated inguinal adipocytes following 30 min treatment with 1 µM RA, n = 4 independent experiments. For cell culture studies, experiments were repeated 2–3 times yielding similar results. The data are represented as mean ± s.e.m. For B and C, *P < 0.05 vs RA; **P < 0.01 vs RA; #P < 0.05 vs WT of same group; ##P < 0.01 vs WT of same group. For D, E, and G, *P < 0.05 vs WT; #P < 0.05 vs WT of same group.

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    Effect of rosiglitazone on thermogenesis in Lcn2 −/− inguinal adipocytes. (A) Oil red O Staining in differentiated inguinal adipocytes treated with or without 1 µM rosiglitazone (Rosi-7d) during the 7 days of differentiation to mature adipocytes. (B) Adipogenic gene expression in differentiated inguinal adipocytes treated with or without 1 µM rosiglitazone (Rosi-7d) during the 7 days of differentiation to mature adipocytes, n = 3/treatment. (C and D) Ucp1 gene expression (C) and protein levels (D) in differentiated inguinal adipocytes treated with 7 days of 1 µM rosiglitazone (Rosi) during differentiation followed by 24 h treatment with 1 µM RA, n = 3/treatment. (E) Quantification of band intensity of UCP1 protein for western blots in D. (F) Quantification of band intensity of PPARγ for western blots in D. (G) Levels of proteins involved in insulin signaling in differentiated inguinal adipocytes treated with 1 µM rosiglitazone (Rosi) during the 7 day differentiation to adipocytes followed by 1 µM RA or insulin for 30 min. Experiments were repeated 2–3 times yielding similar results. The data are represented as mean ± s.e.m. **P < 0.01 vs WT from similar treatment group; #P < 0.05 vs no rosi treatment; ##P < 0.01 vs no rosi treatment. A full color version of this figure is available at https://doi.org/10.1530/JME-18-0017.

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    Plasma membrane localization of RAR-α in Lcn2 −/− brown and inguinal adipocytes. (A) Levels of RAR-α and LCN2 in plasma membrane isolated from WT brown adipocytes and WT inguinal adipocytes following 45 min treatment with insulin. (B) Levels of RAR-α and LCN2 in plasma membrane isolated from WT and Lcn2 −/− inguinal adipocytes following 45 min treatment with insulin ± 1 µM RA. (C) Rara gene expression in differentiated inguinal adipocytes treated with 24 h of 1 µM RA, n = 3/treatment. (D) Quantification of basal and insulin-stimulated levels of RAR-α in plasma membrane isolated from WT and Lcn2 −/− adipocytes following 45 min treatment ± insulin, n = 4–5 independent experiments. (E) RAR-α protein levels in differentiated inguinal adipocytes treated with 1 µM rosiglitazone during the differentiation to adipocytes followed by 24 h treatment with 1 µM RA (F). The data are represented as mean ± s.e.m. *P < 0.05 vs control.

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    A model for a potential mechanism of LCN2 role in the synergistic action of insulin and retinoic acid on beiging of inguinal adipocytes. A full color version of this figure is available at https://doi.org/10.1530/JME-18-0017.

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