Neurotensin-neurotensin receptor 2 signaling in adipocytes suppresses food intake through regulating ceramide metabolism

Abstract

Neurotensin (NTS) is a secretory peptide produced by lymphatic endothelial cells. Our previous study revealed that NTS suppressed the activity of brown adipose tissue via interactions with NTSR2. In the current study, we found that the depletion of Ntsr2 in white adipocytes upregulated food intake, while the local treatment of NTS suppressed food intake. Our mechanistic study revealed that suppression of NTS-NTSR2 signaling enhanced the phosphorylation of ceramide synthetase 2, increased the abundance of its products ceramides C20-C24, and downregulated the production of GDF15 in white adipose tissues, which was responsible for the elevation of food intake. We discovered a potential causal and positive correlation between serum C20-C24 ceramide levels and human food intake in four populations with different ages and ethnic backgrounds. Together, our study shows that NTS-NTSR2 signaling in white adipocytes can regulate food intake via its direct control of lipid metabolism and production of GDF15. The ceramides C20-C24 are key factors regulating food intake in mammals.

Fig. 1. Depletion of Ntsr2 in the adipocytes induced body weight elevation.

a The expression of Ntsr2 in the adipocyte-specific transcriptome of lean and obese mice (n = 4). b Scheme of the establishment of adipocyte-specific Ntsr2 KO mouse strain. c KO efficiency and specificity of the Ntsr2 gene (n = 3–6). d–f The body weight (d), GTT (e) and ITT (f) of mice fed by HFD (n = 5–7). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2. NTS-NTSR2 signaling in the adipose tissue regulated food intake.

a Food intake of control and Ntsr2 AKO mice (n = 7–8). b–d Changes of body weight (b), GTT (c) and ITT (d) of mice pair-fed by HFD (n = 6–10). e Schematic diagram of local NTS treatment. f, g Food intake of WT lean (f) or obese (g) mice upon NTS treatment (n = 6–12). h Food intake of control and Ntsr2 AKO mice upon NTS treatment (n = 7–8). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 3. NTS-NTSR2 signaling regulated ceramide metabolism in WATs.

a A volcano plot of phospho-proteomics data. b Relative abundance of p-CerS2 (n = 3). c p-CerS2 levels detected by pull-down and western blot analysis in the primary adipocytes (n = 5–6). d, e Ceramide C16-C24 levels (d) or individual ceramide levels (e) in eWATs of control and Ntsr2 AKO mice (n = 6). f Individual ceramide levels in the serum of control and Ntsr2 AKO mice (n = 6). g Scheme of the establishment of CerS2^+/– mouse strain. h, i Individual ceramide levels in the eWAT (h) and serum (i) of control and CerS2^+/– mice (n = 3). j, k Food intake of control and CerS2^+/– mice (j), and vehicle vs ceramide C22-treated mice (k); n = 6. l Food intake of control and Ntsr2 AKO mice with CerS2 knockdown (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 4. NTS-NTSR2 signaling regulated UPR via CerS2.

a–f Expression levels of UPR-related genes (a, b) and proteins (c–f, n = 3) in the adipose tissues of control and Ntsr2 AKO mice; n = 3–6. g Expression levels of UPR-related genes upon NTS treatment in vivo (n = 4). h, i Expression levels of UPR-related genes in the primary adipocytes upon the combinational treatment of NTS and RhoAa (h) or RhoAi (i); n = 4. j p-CerS2 levels in the primary adipocytes upon the combinational treatment of NTS and RhoAi or RhoAa (n = 2–3). k, l Expression levels of UPR-related genes in the primary adipocytes upon knockdown (k) or overexpression of CerS2 (l); n = 4–5. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5. Decreasing GDF15 levels led to an increase in food intake in Ntsr2 AKO mice.

a Expression levels of Gdf15 in the adipose tissues (n = 4–7). b–d GDF15 protein concentrations in the serum of control and Ntsr2 AKO mice fed by a chow diet (b, n = 8–11), HFD (c, n = 8–12) or treated by NTS (d, n = 5). e Illustration of the experimental design. f Food intake of control and Ntsr2 AKO mice with or without knockdown of Gfral (n = 8). g Food intake of mice treated by NTS in iWATs with Gfral knockdown (n = 5). *P < 0.05; ***P < 0.001; ns, not significant.

Fig. 6. NTS-NTSR2 signaling regulated the production of GDF15 via CerS2.

a Expression level of Gdf15 upon NTS treatment in the primary adipocytes of WT mice (n = 3–4). b–d Expression levels of GDF15 protein upon CerS2 knockdown (b, n = 3–5), CerS2 overexpression (c, n = 6) and ceramide C22 treatment (d, n = 3–4) in primary adipocytes. e–g Serum concentrations of GDF15 (e, n = 6), mRNA expression levels of Gdf15 (f, n = 3) and GDF15 protein abundance in adipose tissues (g, n = 3) of control and CerS2^+/– mice. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7. The correlation between ceramides C20–C24 and food intake in humans.

a, b Basic information of child (a) and adult (b) participants in the study. c–f A positive correlation was obtained based on multivariable linear regression models between the serum concentration of ceramides C20–C24 and energy intake in the cohorts of children (c, d) and adults (e, f). g Summary of Mendelian randomization analysis in adults. h Illustration of the mechanism identified in this study.