Inhibiting peripheral serotonin synthesis reduces obesity and metabolic dysfunction by promoting brown adipose tissue thermogenesis

Abstract

Mitochondrial uncoupling protein 1 (UCP1) is enriched within interscapular brown adipose tissue (iBAT) and beige (also known as brite) adipose tissue, but its thermogenic potential is reduced with obesity and type 2 diabetes for reasons that are not understood. Serotonin (5-hydroxytryptamine, 5-HT) is a highly conserved biogenic amine that resides in non-neuronal and neuronal tissues that are specifically regulated via tryptophan hydroxylase 1 (Tph1) and Tph2, respectively. Recent findings suggest that increased peripheral serotonin and polymorphisms in TPH1 are associated with obesity; however, whether this is directly related to reduced BAT thermogenesis and obesity is not known. We find that Tph1-deficient mice fed a high-fat diet (HFD) are protected from obesity, insulin resistance and nonalcoholic fatty liver disease (NAFLD) while exhibiting greater energy expenditure by BAT. Small-molecule chemical inhibition of Tph1 in HFD-fed mice mimics the benefits ascribed to Tph1 genetic deletion, effects that depend on UCP1-mediated thermogenesis. The inhibitory effects of serotonin on energy expenditure are cell autonomous, as serotonin blunts β-adrenergic induction of the thermogenic program in brown and beige adipocytes in vitro. As obesity increases peripheral serotonin, the inhibition of serotonin signaling or its synthesis in adipose tissue may be an effective treatment for obesity and its comorbidities.

Figure 1

Tph1^−/− mice are protected from obesity, chronic low-grade inflammation, NALFD and insulin resistance. (a,b) Body mass (n = 9 Tph1^+/+ and n = 19 Tph1^−/−) (a) and adiposity (b) of HFD–fed Tph1^+/+ and Tph1^−/− mice (n = 5 per group). Right (b), representative CT image. (c,d) eWAT inflammatory mRNA (n = 8) (c), liver weights (n = 9 Tph1^+/+ and n = 19 Tph1^−/−) and liver lipid area of HFD-fed Tph1^+/+ and Tph1^−/− mice (n = 5 each group) (d). Right (d), representative liver cross-sections stained with H&E; scale bar is 100 μm. (e–h) Fed blood glucose over the course of the diet intervention (n = 9 Tph1^+/+ and n = 19 Tph1^−/−) (e), fasting serum insulin concentrations (n = 6 Tph1^+/+ and n = 5 Tph1^−/−) (f) and glucose tolerance test (GTT) (g) and insulin tolerance test (ITT) (h) performed after 10–12 weeks of HFD in Tph1^+/+ and Tph1^−/− mice (n = 9 Tph1^+/+ and n = 19 Tph1^−/−). AUC area under the curve. (i) Akt^S473 phosphorylation relative to total Akt in liver, eWAT and mixed gastrocnemius muscle from Tph1^+/+ and Tph1^−/− mice killed 15 min following an injection of 0.5 U kg⁻¹ insulin (n = 4 per group). AU, arbitrary units. (j) Oxygen consumption (VO₂) during light and dark cycles (left) and in the absence of movement (right) in HFD-fed Tph1^+/+ and Tph1^−/− mice (n = 12 per group). Data are expressed as means ± s.e.m. *P < 0.05 relative to Tph1^+/+ mice as determined using a Student’s t-test or two-way repeated measures analysis of variance (ANOVA) and Bonferroni post hoc test.

Figure 2

Mice lacking Tph1 have increased metabolic rate and brown adipose tissue activity due to an inhibition of β–adrenergic signaling by serotonin. (a) Tissue FDG uptake in Tph1^+/+ and Tph1^−/− mice fed a HFD (n = 5 per group). SUV, standardized uptake values. (b,c) iBAT serotonin content determined by ELISA (n = 5 per group) (b) and UCP1 protein content (n = 8 Tph1^+/+ and n = 7 Tph1^−/−) (left, c) from iBAT of Tph1^+/+ and Tph1^−/− mice fed a HFD as assessed by western blot (top, c) and tissue sections stained with H&E (right, c; scale bar is 50 μm). AU, arbitrary units. (d,e) Oxygen consumption (n = 6 per group) (d) and dorsal interscapular surface temperature (e) of anesthetized HFD-fed Tph1^+/+ and Tph1^−/− mice following injection with saline or CL-316,243 (n = 5 per group). Right (e), representative thermal images of mice injected with saline or CL-316,243. (f) cAMP levels determined by ELISA in iBAT of HFD–fed Tph1^+/+ and Tph1^−/− mice (n = 11 per group). (g) Relative PKA substrate phosphorylation in iBAT of HFD-fed Tph1^+/+ and Tph1^−/− mice 15 min following an injection of saline or CL-316,243 (n = 5 per group, right: representative western blot). (h–j) cAMP (n = 3 per treatment in two separate experiments) (h) HSL phosphorylation (S660, n = 5 per treatment in two separate experiments) (i) and Ucp1 mRNA (j) in control and isoproterenol-stimulated brown adipocytes (n = 4 per treatment in two separate experiments). Data are expressed as means ± s.e.m. *P < 0.05 relative to Tph1^+/+ mice or Ctl, ^#P < 0.05 relative to saline or ^†P < 0.05 versus Tph1^+/+ as determined using a Student’s t-test or, where appropriate, a two-way analysis of variance (ANOVA) and Bonferroni post hoc test.

Figure 3

Chemical inhibition of Tph1 prevents obesity and insulin resistance and increases brown adipose tissue activity and UCP1 expression in HFD-fed C57BL/6 mice. (a) Plasma serotonin concentrations of HFD-fed C57BL/6 mice after 8 weeks of LP533401 treatment (n = 5 vehicle and n = 6 LP533401 treated). (b) Body mass of HFD-fed C57BL/6 mice over 14 weeks treated with vehicle or LP533401 for the last 12 weeks (n = 9 vehicle and n = 8 LP533401). (c) Adiposity of vehicle- and LP533401-treated mice (n = 6 vehicle and LP533401). Top right, H&E-stained sections of eWAT, scale bar is 100 μm; bottom right, representative images of mice from each treatment group. (d) Liver weight (n = 9 vehicle and n = 8 LP533401) and lipid area fraction (n = 3 vehicle and LP533401) in vehicle- and LP533401-treated, HFD-fed mice. Right, H&E-stained sections of liver from vehicle- and LP533401-treated, HFD-fed mice; scale bar is 100 μm. (e,f) Fed blood glucose over the treatment period (e) and GTT (f) of vehicle- and LP533401-treated, HFD-fed mice and AUC. n = 9 vehicle and n = 8 LP533401 for all three graphs. (g) Akt^S473 phosphorylation relative to total Akt in liver, eWAT and mixed gastrocnemius muscle from vehicle- and LP533401-treated mice 15 min following an injection of 0.5 U kg⁻¹ insulin (n = 9 vehicle and n = 8 LP533401). (h,i) Oxygen consumption during light and dark cycles (n = 7 per group) (h) and tissue FDG uptake (n = 6 per group) (i) in vehicle- and LP533401-treated mice. Right (i), representative PET-CT images from each group. (j) Representative immunohistochemical staining of UCP1 (top, scale bar is 100 μm) and H&E (bottom, scale bar is 50 μm) representative of 8 vehicle- and 6 LP533401-treated mice and lipid area fraction in iBAT (right) from mice treated with vehicle or LP533401 (n = 8 vehicle and n = 6 LP533401). Data are expressed as means ± s.e.m. *P < 0.05 compared to vehicle as determined using a Student’s t-test or, where appropriate, using a two-way repeated measures ANOVA.

Figure 4

UCP1 expression is required for the metabolic benefits of Tph1 inhibition. (a,b) Change in body mass after 6 weeks of HFD (a) and adiposity (b) from vehicle- and LP533401- treated Ucp1^+/+ and Ucp1^−/− mice. (c,d) Liver mass (c) and lipid content (d; right, representative H&E cross-sections) in vehicle- and LP533401- treated mice. Scale bar is 100 μm. (e) GTT and AUC of vehicle- and LP533401-treated Ucp1^+/+ and Ucp1^−/− mice. For a–e, n = 9 for Ucp1^+/+ vehicle and LP533401, n = 7 for Ucp1^−/− vehicle and n = 6 for Ucp1^−/− LP533401. (f,g) Change in oxygen uptake (f) and dorsal interscapular surface temperature (g) in vehicle- and LP533401-treated mice acutely injected with saline or CL-316,243 (n = 4 for Ucp1^+/+ and Ucp1^−/− vehicle, n = 5 for Ucp1^+/+ LP533401 and n = 3 for Ucp1^−/− LP533401). Right (g), representative thermal images of all mice in a given group. Data are expressed as means ± s.e.m. *P < 0.05 relative to indicated groups or versus saline condition. ^#P < 0.05 relative to wild-type and ^†P < 0.05 relative to all other groups as determined by two-way ANOVA and Bonferroni post hoc test.