Gut-derived serotonin is a multifunctional determinant to fasting adaptation

Abstract

Energy release from cellular storage is mandatory for survival during fasting. This is achieved through lipolysis and liver gluconeogenesis. We show here that in the mouse, gut-derived serotonin (GDS) is upregulated during fasting and that it favors both mechanisms. In adipocytes, GDS signals through the Htr2b receptor to favor lipolysis by increasing phosphorylation and activity of hormone-sensitive lipase. In hepatocytes, GDS signaling through Htr2b promotes gluconeogenesis by enhancing activity of two rate-limiting gluconeogenic enzymes, FBPase and G6Pase. In addition, GDS signaling in hepatocytes prevents glucose uptake in a Glut2-dependent manner, thereby further favoring maintenance of blood glucose levels. As a result, inhibition of GDS synthesis can improve glucose intolerance caused by high-fat diet. Hence, GDS opposes deleterious consequences of food deprivation by favoring lipolysis and liver gluconeogenesis while preventing glucose uptake by hepatocytes. As a result, pharmacological inhibition of its synthesis may contribute to improve type 2 diabetes.

Fig. 1. GDS is required for fasting induced lipolysis

(A) Relative expression levels of Tph1 in duodenum from mice fasted for indicated times (n ≥ 5). (B) Plasma levels of serotonin and (C) glucagon in mice fasted for indicated times (n ≥ 5). Plasma levels of (D) glycerol, (E) FFAs, (F) β-hydroxybutyrate and (G) triglycerides in mice of indicated genotypes fasted for indicated times (n ≥ 7). (H) % of fat content in fed and fasted mice of indicated genotypes measured by magnetic resonance imaging (MRI) (n ≥ 5). (I) % of fat content loss during 48h fasting in relation to initial fat content of each animal, average of 5 mice of Tph1f/f and Tph1_gut∆/∆ genotype. (J) Epigonadal fat pad weight to body weight ratio in fed and fasted Tph1f/f and Tph1_gut∆/∆ mice (n ≥ 6). (K) Histology of fat pad from fed and 48h fasted Tph1f/f and Tph1_gut∆/∆ mice (n ≥ 5). (L) Average size of adipocytes from fat pad of fed and 48h fasted Tph1f/f and Tph1_gut∆/∆ mice (n ≥ 5). Plasma levels of (M) glycerol and (N) FFAs in mice with implanted placebo or serotonin-releasing pellets fasted for indicated time (n ≥ 8). Plasma levels of (O) glycerol and (P) FFAs at indicated time points after i.p. injection of serotonin (doses in mg/kg BW) (n ≥ 5). Data represented as mean +/− SEM, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 2. GDS favors lipolysis through Htr2b receptor expressed in adipocytes

(A) Glycerol and (B) FF As release from mouse epigonadal fat explants stimulated with indicated doses of serotonin (n > 5). (C) Relative expression of indicated serotonin receptors in adipose tissue of fed and fasted mice. Plasma levels of (D) glycerol, (E) FF As and (F) β-hydroxybutyrate in mice lacking Htr2b in adipose tissue (deletion by aP2-Cre – indicated on graphs Htr2bf_atΔ/Δ (aP2)) fasted for indicated times (n ≥ 20). Plasma levels of (G) glycerol and (H) FFAs in mice lacking Htr2b in adipose tissue (deletion by Adiponectin-Cre – indicated on graphs Htr2bf_atΔ/Δ (Aq) fasted for indicated times (n > 6). (I) Epigonadal fat pad weight to body weight ratio in fed and fasted Htr2bf/f and Htr2bf_atΔ/Δ(aP2) mice (n > 12). (J) Glycerol and (K) FFAs levels in Htr2bf/f and Htr2bf_atΔ/Δ(aP2) mice implanted with serotonin-releasing pellets. (L) Glycerol and (M) FFAs release from epigonadal fat explants isolated from Htr2bf/f and Htr2bf_atA/A(aP2) mice and stimulated with 50μM serotonin or 1μM isoproterenol (n = 5). (N) Western blot analysis of expression and activation of indicated proteins in fat explants isolated from Htr2bf/f and Htr2bf_atΔ/Δ(aP2) mice and stimulated with 50μM serotonin. (O) Western blot analysis of expression and activation of indicated proteins in fat pads isolated from Htr2bf/f and Htr2bf_atΔ/Δ(aP2) mice fasted for 48h. Data represented as mean +/− SEM, ^*P < 0.05, ^***P < 0.001.

Fig. 3. GDS promotes gluconeogenesis in liver

(A) Glycerol-evoked glucose production in Tph1f/f and Tph1_gut∆/∆ mice (n ≥ 8). (B) Pyruvate tolerance test in Tph1f/f and Tph1_gut∆/∆ mice (n ≥ 8). (C) Glycerol tolerance test in mice with implanted placebo or serotonin-releasing pellets (n ≥ 8). (D) Glycerol-evoked glucose production in Tph1f/f and Tph1_gut∆/∆ mice injected with serotonin (10 mg/kg BW) 90min. before the test (n ≥ 7). Hepatic glucose production (E) and (F) glucose infusion rate during hyperinsulinemic-euglycemic clamp in Tph1f/f and Tph1_gut∆/∆ mice (n ≥ 7). (G) Production of glucose from indicated substrates by primary hepatocytes stimulated with indicated doses of serotonin or 100nM glucagon (n = 5). (H) Glucose tolerance test in Tph1f/f and Tph1_gut∆/∆ mice (n ≥ 7). (I) Plasma insulin levels during glucose tolerance test in Tph1f/f and Tph1_gut∆/∆ mice (n ≥ 7). (J) Glucose tolerance test in mice with implanted placebo or serotonin-releasing pellets (n ≥ 8). (K) Glucose tolerance test in Tph1f/f and Tph1_gut∆/∆ mice injected with serotonin (10 mg/kg BW) 90min. before the test (n ≥ 7). (L) Glucose uptake in primary hepatocytes stimulated with indicated doses of serotonin. (M) Glucose tolerance test in Tph1+/+ and Tph1−/− mice (n ≥ 3). Data represented as mean +/− SEM, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 4. GDS promotes gluconeogenesis in liver by signaling through the Htr2b receptor

(A) Relative expression of indicated serotonin receptors in primary mouse hepatocytes. Red color indicates receptors for which the transcript was detected. (B) Relative expression of indicated serotonin receptors in liver of fed and fasted mice. (C) Glycerol tolerance test in Htr2bf/f and Htr2b_liver∆/∆ mice (n ≥ 7). (D) Pyruvate tolerance test in Htr2bf/f and Htr2b_liver∆/∆ mice (n ≥ 7). (E) Production of glucose from indicted substrates by hepatocytes isolated from Htr2bf/f and Htr2b_liver∆/∆ mice and stimulated by 50μM serotonin (n = 5). (F) Glycerol tolerance test in Htr2bf/f and Htr2b_liver∆/∆ mice implanted with placebo or serotonin-releasing pellets (n ≥ 6). Normalized (G) FBPase and (H) G6Pase activity in livers of fasted Htr2bf/f and Htr2b_liver∆/∆ mice (n ≥ 6). Normalized (I) FBPase and (J) G6Pase activity in isolated hepatocytes stimulated with indicated doses of serotonin (n ≥ 3). Data represented as mean +/− SEM, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 5. GDS inhibits glucose uptake in liver by signaling through the Htr2b receptor

(A) Glucose tolerance test in Htr2bf/f and Htr2b_liver∆/∆ mice (n ≥ 8). (B) Glucose tolerance test in Htr2bf/f and Htr2b_liver∆/∆ mice implanted with placebo or serotonin-releasing pellets (n ≥ 6). (C) Relative glucose uptake in Htr2b-deficient hepatocytes stimulated with 50 μM serotonin. (D) Immunofluorescence with antibodies against Glut1 and Glut2 on primary hepatocytes of indicated genotypes stimulated with 50μM serotonin. (E) Western blot analysis of expression of indicated proteins in livers from fasted Htr2bf/f and Htr2b_liver∆/∆ mice. (F) Normalized glucokinase activity in livers of fasted Htr2bf/f and Htr2b_liver∆/∆ mice. Data represented as mean +/− SEM, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 6. Inhibition of GDS synthesis protects from type 2 diabetes

(A) Insulin tolerance test in Tph1f/f and Tph1_gut∆/∆ mice (n ≥ 7). (B) Insulin tolerance test in Htr2bf/f and Htr2b_fat∆/∆ mice (n ≥ 12). (C) Body weight gain of Tph1f/f and Tph1_gut∆/∆ mice fed normal or high fat diet (n ≥ 5). Plasma levels of (D) glycerol and (E) FFAs in mice of indicated genotypes fed normal (ND) or high fat diet (HFD), fasted for 4h (n ≥ 5). (F) Pyruvate, (G) insulin and (H) glucose tolerance tests in Tph1f/f and Tph1_gut∆/∆ mice on indicated diets (n ≥ 5). (I) Plasma serotonin concentrations in mice treated with vehicle or LP533401 inhibitor (n ≥ 8). (J) Body weight gain of mice treated with vehicle or LP533401 inhibitor, fed ND or HFD (n ≥ 8). Plasma levels of (K) glycerol and (L) FFAs in mice treated with vehicle or LP533401 inhibitor fed ND or HFD and fasted for 4h (n ≥ 7). (M) Pyruvate, (N) insulin and (O) glucose tolerance tests in mice treated with vehicle or LP533401 inhibitor fed ND or HFD (n > 7). Data represented as mean +/− SEM, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.