Hypothalamic SLC7A14 accounts for aging-reduced lipolysis in white adipose tissue of male mice

Abstract

The central nervous system has been implicated in the age-induced reduction in adipose tissue lipolysis. However, the underlying mechanisms remain unclear. Here, we show the expression of SLC7A14 is reduced in proopiomelanocortin (POMC) neurons of aged mice. Overexpression of SLC7A14 in POMC neurons alleviates the aging-reduced lipolysis, whereas SLC7A14 deletion mimics the age-induced lipolysis impairment. Metabolomics analysis reveals that POMC SLC7A14 increased taurochenodeoxycholic acid (TCDCA) content, which mediates the SLC7A14 knockout- or age-induced WAT lipolysis impairment. Furthermore, SLC7A14-increased TCDCA content is dependent on intestinal apical sodium-dependent bile acid transporter (ASBT), which is regulated by intestinal sympathetic afferent nerves. Finally, SLC7A14 regulates the intestinal sympathetic afferent nerves by inhibiting mTORC1 signaling through inhibiting TSC1 phosphorylation. Collectively, our study suggests the function for central SLC7A14 and an upstream mechanism for the mTORC1 signaling pathway. Moreover, our data provides insights into the brain-gut-adipose tissue crosstalk in age-induced lipolysis impairment.

Fig. 1. Overexpression of SLC7A14 in POMC neurons is resistant to reduction in lipolysis with age.

a SLC7A14 and TUBULIN proteins in hypothalamus. b Immunofluorescence (IF) staining for SLC7A14 ARC sections (left), and quantification of SLC7A14 cell numbers (right). c, d IF staining for tdTomato, SLC7A14 and merge in ARC sections (left), and quantification of SLC7A14 and tdTomato colocalized cell numbers (right). e Fat mass by NMR. f Lean mass by NMR. g Tissue weight. h Representative images of hematoxylin and eosin (H&E) staining of sWAT. i Cell size of sWAT quantified by Image J analysis of H&E images. j The DNA content of sWAT. k P-HSL, t-HSL, p-PKA substrates, FAS, CPT1α, CD36 and TUBULIN proteins in sWAT by western blotting (left) and quantified by densitometric analysis (right). n = 6 mice. l Glycerol release assays in sWAT. m The cAMP levels of sWAT. Studies for a–c were conducted using 2-month-old mice (2 M) or 22-month-old mice (22 M); d–m were conducted using 20-month-old POMC Cre mice receiving AAVs expressing mCherry (- AAV-SLC7A14) or SLC7A14 (+AAV-SLC7A14) in ARC. Metabolic tests were performed 1 month after adeno-associated virus injection. For (a), n = 6. For (b), n = 6 (2 M) and 5 (22 M). For (c), n = 4 (2 M) and 5 (22 M). For (d–j and m), n = 5. For k, n = 6. For l, n = 6 (-AAV-SLC7A14) and 5 (+AAV-SLC7A14). Data are expressed as the mean ± SEM, with individual data points. Data were analyzed by two-tailed unpaired Student’s t-test (a–g, i–m). Source data are provided as a Source Data file.

Fig. 2. The absence of SLC7A14 in POMC neurons mimic age-induced lipolysis impairment.

a IF staining for tdTomato, SLC7A14 and merge in ARC sections (left), and quantification of SLC7A14 and tdTomato colocalized cell numbers (right). b Fat mass by NMR. c Tissue weight. d Representative images of H&E staining of sWAT. e Cell size of sWAT quantified by Image J analysis of H&E images. f The DNA content of sWAT. g P-HSL, t-HSL, p-PKA substrates, FAS, CPT1α, CD36 and TUBULIN proteins in sWAT by western blotting (left) and quantified by densitometric analysis (right). h Glycerol release assays in sWAT. i The cAMP levels of sWAT. Studies for (a–i) were conducted using control mice (- SPKO) or mice with SLC7A14 deletion in POMC neurons (+ SPKO), and at age of 6-month-old in (a), (c–i). For (a and h), n = 5. For (b), n = 5 (- SPKO) and 6 (+SPKO). For (c), n = 7 (- SPKO) and 6 (+SPKO). For (d, e, g and i), n = 6. For (e), n = 6. For (f), n = 7 (- SPKO) and 6 (+SPKO). Data are expressed as the mean ± SEM, with individual data points. Data were analyzed by two-tailed unpaired Student’s t-test (a, c, e–i), or two-way RM ANOVA with Geisser-Greenhouse’s correction (b). Source data are provided as a Source Data file.

Fig. 3. The decreased TCDCA mediates SPKO-induced WAT lipolysis impairment.

a Metabolite set enrichment analysis. b–d The TCDCA levels of serum. e The TCDCA levels of WAT. f Fat mass by NMR. g Tissue weight. h Representative images of H&E staining of sWAT. i Cell size of sWAT quantified by Image J analysis of H&E images. j P-HSL and t-HSL proteins in sWAT. k Glycerol release assays in sWAT. l The cAMP levels of sWAT. Studies for a and b were conducted using 20-month-old POMC Cre mice receiving AAVs expressing mCherry (-AAV-SLC7A14) or SLC7A14 (+AAV-SLC7A14) in ARC. c–l were conducted using 5-month-old control mice (-SPKO) or mice with SLC7A14 deletion in POMC neurons (+ SPKO). Mice were intraperitoneally (i.p.) injected with PBS (-TCDCA) or 50 mg/kg body weight TCDCA (+TCDCA) every other day. On the fifteenth day, mice were i.p. injected with a single dose of TCDCA before euthanized in (d–l). For (a, b), n = 5 (- AAV-SLC7A14) and 4 (+AAV-SLC7A14). For (c), n = 7 (- SPKO) and 4 (+SPKO). For (d), n = 6 (- SPKO - TCDCA), 5 (+SPKO - TCDCA), 7 (- SPKO + TCDCA) and 7 (+SPKO + TCDCA). For (e), n = 6 (- SPKO - TCDCA), 7 (+SPKO - TCDCA), 7 (- SPKO + TCDCA) and 6 (+SPKO + TCDCA). For (f and h–j), n = 5. For (g), n = 6. For k, n = 5 (−SPKO - TCDCA), 5 (+SPKO - TCDCA), 5 (−SPKO + TCDCA) and 6 (+SPKO + TCDCA). For l, n = 5 (−SPKO - TCDCA), 6 (+SPKO - TCDCA), 6 (- SPKO + TCDCA) and 5 (+SPKO + TCDCA). Data are expressed as the mean ± SEM, with individual data points. Data were analyzed by Fisher’s exact test (a) or two-tailed unpaired Student’s t-test (b, c), or ordinary two-way ANOVA with Tukey’s multiple comparisons test (d–g, i–l). Source data are provided as a Source Data file.

Fig. 4. TGR5 mediates TCDCA-stimulated lipolysis via regulating cAMP levels.

a P-HSL and t-HSL proteins in primary white adipocytes. b, d, g and i Glycerol release assays in primary white adipocytes. c, f and h The cAMP levels of primary white adipocytes. e The mRNA levels of TGR5 in primary white adipocytes. Studies for a were conducted using primary white adipocytes treated with control vehicle (-TCDCA) or TCDCA (+TCDCA) at the concentration as indicated for 4 h. b Were conducted using primary white adipocytes treated with control vehicle (-TCDCA) or 100 μM TCDCA (+TCDCA) for 2 h, followed by incubation with Krebs-Ringer buffer, with or without 10 µM FSK, with or without 100 μM TCDCA at 37 °C for 2 h. c, d were conducted using primary white adipocytes treated with or without 100 µM TCDCA in the presence or absence of 100 µM SQ22536 for 4 h; e–g were conducted using primary white adipocytes transfected with double-stranded siRNA targeting TGR5 for 72 h, followed by treating with control vehicle (-TCDCA) or 100 μM TCDCA (+TCDCA) for 4 h; h, i were conducted using primary white adipocytes treated with or without 30 µM INT777 in the presence of control vehicle or 100 µM SQ22536 for 2 h. For (a, c, d, f, h and i), n = 5. For (b), n = 7 (- TCDCA - FSK), 6 (+TCDCA - FSK), 6 (-TCDCA + FSK) and 5 (+TCDCA + FSK). For (e), n = 6 (-TCDCA – siTGR5), 6 (+TCDCA - siTGR5), 5 (- TCDCA + siTGR5) and 6 (+TCDCA + siTGR5). For (g), n = 6 (-TCDCA – siTGR5), 5 (+TCDCA - siTGR5), 5 (- TCDCA + siTGR5) and 6 (+TCDCA + siTGR5). Data are expressed as the mean ± SEM, with individual data points. Data were analyzed by ordinary one-way ANOVA with Dunnett’s multiple comparisons test (a), or two-way RM ANOVA with Geisser-Greenhouse’s correction (b, d, g, i) or ordinary two-way ANOVA with Tukey’s multiple comparisons test (c, e, f, and h). Source data are provided as a Source Data file.

Fig. 5. The intestinal ASBT mediates POMC SLC7A14-induced TCDCA levels and WAT lipolysis.

a and e The TCDCA levels of ileum. b and f The TCDCA levels of feces. c, g, o and t The mRNA levels of Asbt in ileum by RT-PCR. d and h ASBT and TUBULIN proteins in ileum. i SLC7A14 and TUBULIN proteins in ARC. j The TCDCA levels of serum. k P-HSL and t-HSL proteins in sWAT. l Glycerol release assays in sWAT. m and s The Pparα mRNA levels in ileum by RT-PCR. n PPARα and TUBULIN proteins in ileum. p–r The OEA levels of ileum. Studies for a-d, q and i-o were conducted using POMC Cre mice receiving AAVs expressing mCherry (- AAV-SLC7A14) or SLC7A14 (+AAV-SLC7A14) in ARC, and treated twice daily with control vehicle (- linerixibat) or 0.05 mg/kg linerixibat (+ linerixibat) via oral gavage in i–l, or the ileum of mice was injected with AAVs expressing shPPARα (+ AAV-shPPARα) or EGFP (−AAV-shPPARα) in m–o. e–h, p and r–t were conducted using 6-month-old control mice (- SPKO) or mice with SLC7A14 deletion in POMC neurons (+ SPKO), and mice were given OEA (10 mg/kg, aladdin) in (r–t). For a, n = 7 (- AAV-SLC7A14) and 6 (+AAV-SLC7A14). For b, n = 6 (- AAV-SLC7A14) and 5 (+AAV-SLC7A14). For (c), n = 7. For (d, i–k, n, and p–t), n = 5. For e and g, n = 5 (- SPKO) and 6 (+SPKO). For (f), n = 6 (- SPKO) and 5 (+SPKO). For (h) and m–o, n = 6. For l, n = 5 (- AAV-SLC7A14 - Linerixibat), 5 (+AAV-SLC7A14 - Linerixibat), 6 (- AAV-SLC7A14 + Linerixibat) and 5 (+AAV-SLC7A14 + Linerixibat). Data are expressed as the mean ± SEM, with individual data points. Data were analyzed by two-tailed unpaired Student’s t-test (a–h, p, q), or ordinary two-way ANOVA with Tukey’s multiple comparisons test (i–o, r–t). Source data are provided as a Source Data file.

Fig. 6. POMC SLC7A14 regulates ASBT expression via intestinal sympathetic afferent nerves.

a Representative micrographs of the ileum wall demonstrating neurons expressing PRV. b Representative micrographs of the CG-SMG demonstrating neurons expressing PRV. c Representative micrographs of the brain demonstrating neurons expressing PRV, tdTomato and merge. d–g, k TH and TUBULIN proteins in ileum. e–h IF staining for DAPI, TH and merge in ileum. (f, i and l) The NE levels of ileum. j IF staining for tdTomato, SLC7A14 and merge in ARC. m The OEA levels of ileum. n The Pparα mRNA levels in ileum. o The Asbt mRNA levels in ileum. p ASBT and TUBULIN proteins in ileum. q The TCDCA levels of serum. Studies for a-c were conducted using 5-month-old POMC Cre mice, which carrying Ai9 (tdTomato) reporter, injected with pseudorabies virus (BC-PRV-531-PRV-CAG-EGFP) for 6 days in ileum. d–f were conducted using 5-month-old control mice (- SPKO) or mice with SLC7A14 deletion in POMC neurons (+ SPKO); (g–i, j–q) were conducted using 20 month-old POMC Cre mice receiving AAVs expressing mCherry (- AAV-SLC7A14) or SLC7A14 (+AAV-SLC7A14) in ARC, and treated with sham surgery (- CG/SMG ganglionectomy) or surgical removal of celiac-superior mesenteric ganglion (+ CG/SMG ganglionectomy) in j-q. For a–c, n = 7. For (d–g, n) n = 6. For (e) and (h) n = 3. For (f–i, k–m) and (p-q) n = 5. For (j) n = 4 (−AAV-SLC7A14 - CG/SMG ganglionectomy), 4 (+AAV-SLC7A14 - CG/SMG ganglionectomy), 5 (- AAV-SLC7A14 + CG/SMG ganglionectomy) and 5 (+AV-SLC7A14 + CG/SMG ganglionectomy). For (o) n = 6 (−AAV-SLC7A14 - CG/SMG ganglionectomy), 6 (+AAV-SLC7A14 - CG/SMG ganglionectomy), 7 (−AAV-SLC7A14 + CG/SMG ganglionectomy) and 5 (+AAV-SLC7A14 + CG/SMG ganglionectomy). Data are expressed as the mean ± SEM, with individual data points. Data were analyzed by two-tailed unpaired Student’s t-test (d–f, g–i) or ordinary two-way ANOVA with Tukey’s multiple comparisons test (j–q). Source data are provided as a Source Data file.

Fig. 7. The SLC7A14-induced intestinal sympathetic afferent nerves change is regulated by POMC mTORC1 signaling.

a, b P-S6K, t-S6K, p-S6, t-S6, SLC7A14 and TUBULIN proteins in primary hypothalamic cells. c, d mTORC1 kinase assays in primary hypothalamic cells. e IF staining for tdTomato, p-S6 and merge in ARC sections. f IF staining for tdTomato, RAPTOR and merge in ARC sections. g Fat mass by NMR. h Lean mass by NMR. i IF staining for TH. j The NE levels of ileum. k The mRNA levels of Asbt in ileum by RT-PCR. l The TCDCA levels of serum. Studies for (a–d) were conducted using primary hypothalamic cells, infected with adenovirus expressing green fluorescent protein (- Ad-SLC7A14) or Ad-SLC7A14 (+ Ad-SLC7A14) for 48 h in (a) and (c), or transfected with double-stranded siRNA targeting mouse SLC7A14 for 72 h in b and d; e-l were conducted using 5 month-old control mice (- SPKO) or mice with SLC7A14 deletion in POMC neurons (+ SPKO), and injected with AAVs expressing mCherry (- AAV-RAPTOR) or shRAPTOR (+ AAV- shRAPTOR) in (f–l). For a-b, n = 6. For c-d and i, n = 3. For e, n = 6 (- SPKO) and 5 (+SPKO). For (f) n = 5. For (g, h), n = 5 (- SPKO - shRAPTOR), 5 (+SPKO - shRAPTOR), 5 (- SPKO + shRAPTOR) and 6 (+SPKO + shRAPTOR). For (j) and (l) n = 5 (- SPKO - shRAPTOR), 6 (+SPKO - shRAPTOR), 5 (- SPKO + shRAPTOR) and 6 (+SPKO + shRAPTOR). For (k), n = 6 (- SPKO - shRAPTOR), 6 (+SPKO - shRAPTOR), 5 (- SPKO + shRAPTOR) and 5 (+SPKO + shRAPTOR). Data are expressed as the mean ± SEM, with individual data points. Data were analyzed by two-tailed unpaired Student’s t-test (a, b, and e) or ordinary two-way ANOVA with Tukey’s multiple comparisons test (f–h, j–l). Source data are provided as a Source Data file.

Fig. 8. POMC SLC7A14 regulates mTORC1 signaling via TSC1.

a, b P-TSC1, t-TSC1, SLC7A14 and TUBULIN proteins in primary hypothalamic cells. c P-S6K, t-S6K, p-S6, t-S6, t-TSC1, SLC7A14 and TUBULIN proteins in primary hypothalamic cells. d P-TSC1, t-TSC1, IKKβ, SLC7A14 and TUBULIN proteins in primary hypothalamic cells. e, g-h Immunoblotting and co-immunoprecipitation in primary hypothalamic cells. f Venn chart of interactions for SLC7A14, IKKβ, and TSC1 in HitPredict. i Working model. Studies for (a–e, g) and h were conducted using primary hypothalamic cells, infected with adenovirus expressing green fluorescent protein (- Ad-SLC7A14) or Ad-SLC7A14 (+ Ad-SLC7A14) for 48 h in a, or transfected with double-stranded siRNA targeting mouse SLC7A14 for 72 h in b, or transfected with double-stranded shRNA targeting mouse TSC1 (+ Sh-TSC1) or without TSC1 shRNA (- Sh-TSC1) for 72 h and infected with adenovirus expressing green fluorescent protein (- Ad-SLC7A14) or Ad-SLC7A14 (+ Ad-SLC7A14) for 48 h in (c), or infected with adenovirus expressing green fluorescent protein (- Ad-SLC7A14) or Ad-SLC7A14 (+ Ad-SLC7A14) and transfected with (+ IKKβ−3xflag) or without (- IKKβ−3xflag) FLAG-tagged IKKβ plasmids for 48 h in d; or infected with adenovirus expressing green fluorescent protein (- Ad-SLC7A14) or Ad-SLC7A14 (+ Ad-SLC7A14) for 48 h. Immunoprecipitation (IP) and immunoblotting (IB) were performed using the antibodies indicated in (e–h). For (a-d) and (g), n = 6. For e and h, n = 3. Data are expressed as the mean ± SEM, with individual data points. Data were analyzed by two-tailed unpaired Student’s t-test (a and b), or ordinary two-way ANOVA with Tukey’s multiple comparisons test (c, d). Source data are provided as a Source Data file.