Famsin, a novel gut-secreted hormone, contributes to metabolic adaptations to fasting via binding to its receptor OLFR796

Abstract

The intestine is responsible for nutrient absorption and orchestrates metabolism in different organs during feeding, a process which is partly controlled by intestine-derived hormones. However, it is unclear whether the intestine plays an important role in metabolism during fasting. Here we have identified a novel hormone, famsin, which is secreted from the intestine and promotes metabolic adaptations to fasting. Mechanistically, famsin is shed from a single-pass transmembrane protein, Gm11437, during fasting and then binds OLFR796, an olfactory receptor, to activate intracellular calcium mobilization. This famsin-OLFR796 signaling axis promotes gluconeogenesis and ketogenesis for energy mobilization, and torpor for energy conservation during fasting. In addition, neutralization of famsin by an antibody improves blood glucose profiles in diabetic models, which identifies famsin as a potential therapeutic target for treating diabetes. Therefore, our results demonstrate that communication between the intestine and other organs by a famsin-OLFR796 signaling axis is critical for metabolic adaptations to fasting.

Fig. 1. Famsin is secreted after cleavage from Gm11437 by furin.

a Immunostaining showing the orientation of FLAG-tagged Gm11437 on the cell membrane in Cos7 cells. The signals from anti-Famsin antibody were observed in both unpermeabilized and permeabilized cells, while the signals from anti-FLAG were only detected in permeabilized cells. These results indicate that the N-terminus of Gm11437 has an extracellular orientation. DAPI, 4,6-diamidino-2-phenylindole; TM, transmembrane. Scale bars, 10 μm. b Immunoblots showing the glycosylation status of famsin in HEK293T cells. The protein extracts from culture medium or whole cell lysate were treated with or without deglycosylation enzymes. The blue arrowheads indicate full-length Gm11437-FLAG and the red arrowheads indicate famsin. Deglyco, deglycosylation. c Left panel, schematic overview showing the biotin labeling of Gm11437 located at the plasma membrane and its subsequent detection by anti-Famsin antibody. O/N, overnight; IP, immunoprecipitation; IB, immunoblotting. Right panel, immunoblots showing cleavage of famsin from plasma membrane-localized Gm11437 in HEK293T cells. The blue arrowheads indicate full-length Gm11437-FLAG and the red arrowheads indicate famsin. d Left panel, schematic diagram showing the subcellular fractionation of HEK293T cells and subsequent detection of famsin by anti-Famsin antibody. Right panel, immunoblots showing cleavage of famsin from cytoplasm-localized Gm11437 in HEK293T cells. The proteins were treated by the deglycosylation mix. The blue arrowheads indicate full-length Gm11437-FLAG and the red arrowheads indicate famsin. TCL, total cell lysate; L, light fraction that includes plasma membranes; P, pellet that includes cellular vesicles. ATP1A1 (Na⁺/K⁺-ATPase) is a marker of the plasma membrane fraction. e–g Effect of proprotein convertase inhibitors (5 μM, e) or overexpression of different proprotein convertases (f) on Gm11437 cleavage, and effect of furin on cleavage of WT Gm11437 or Gm11437/AA (g) in HEK293T cells. The blue arrowheads indicate full-length Gm11437-FLAG and the red arrowheads indicate famsin. AA, K190A/R191A.

Fig. 2. Famsin is secreted from the intestine and promotes fasting-induced metabolism.

a Effect of ad lib feeding, fasting, and refeeding 1.5 h after fasting on plasma famsin levels. Fasting was started at Zeitgeber time 0 (ZT0). At each indicated time point, plasma famsin was measured by sandwich ELISA assay in six 12-week-old male mice. Data are shown as means ± SEM. b qPCR results showing relative mRNA levels of Gm11437 in different tissues from 8–10-week-old male mice. BAT, brown adipose tissue; WAT, white adipose tissue. Data are shown as means ± SEM. n = 6 mice. c Plasma famsin levels measured by sandwich ELISA assay in WT and Gm11437 IKO 8–10-week-old male mice with ad lib feeding or overnight fasting. Data are shown as means ± SEM. Comparison of different groups was carried out using two-way analysis of variance (ANOVA) followed by Tukey’s test. **P < 0.01, ***P < 0.001. n = 6 mice. d Immunoblots (top panel) and quantification of serum famsin from immunoblots (bottom panel) showing the effect of ad lib feeding or overnight fasting on plasma famsin and intestine Gm11437 levels of 8–10-week-old male mice. Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test. **P < 0.01. NS no statistical significance. n = 3. e–g Blood glucose (e), plasma β-hydroxybutyrate (f) and hepatic acetyl-CoA (g) levels from overnight fasted 8-10-week-old WT male mice and Gm11437 IKO male mice. Data are shown as means ± SEM. Comparison of different groups was carried out using unpaired two-tailed Student’s t-test. **P < 0.01, ***P < 0.001. n = 12 mice (e), n = 10 mice (f, g). h qPCR results showing relative mRNA levels of genes involved in lipolysis (Pnlip, Pnliprp2, Clps, Cel), lipid oxidation (Cpt1a), ketogenesis (Hmgcs2) and gluconeogenesis (G6pc, Pck1) from liver extracts of overnight fasted 8–10-week-old WT and IKO male mice. Data are shown as means ± SEM. Comparison of different groups was carried out using unpaired two-tailed Student’s t-test. **P < 0.01, ***P < 0.001. n = 7 mice. i Immunoblots showing the effect of Gm11437 deficiency on hepatic lipolysis and gluconeogenesis from liver extracts of 8–10-week-old male mice. j–l Pyruvate tolerance test (j), glucose tolerance test (k) and insulin tolerance test (l) results from 8–10-week-old WT and Gm11437 IKO male mice. For each test, the relative area under the curve (AUC) is shown in the inset of each panel. Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test (curve data, ^##P < 0.01, ^###P < 0.001) or unpaired two-tailed Student’s t-test (AUC data, ***P < 0.001). n = 8 mice.

Fig. 3. Famsin promotes torpor and starvation resistance.

a–h Effect of Gm11437 IKO and famsin on torpor evaluated by Tb (a), torpor frequency (number of torpor bouts, b), time for Tb < 35 °C (c), torpor entry time (d), minimum Tb (e), locomotor activity (f), relative total locomotor activity (g) and starvation resistance (h) of fasted 8-week-old male mice. Failure of starvation resistance was judged as Tb < 28 °C following a quick decrease in Tb below the environmental temperature. 400 μg/kg famsin was intraperitoneally injected after 4 h fasting. The gray and white backgrounds (a, f) indicate 12-h periods of darkness and light, respectively. Data are shown as means ± SEM. Comparison of different groups was carried out using one-way ANOVA followed by Tukey’s test (b–e, g) or log-rank test (h). *P < 0.05, **P < 0.01. NS no statistical significance. n = 7 mice. i–p Effect of adenoviral-expressed WT Gm11437 or Gm11437/AA on torpor evaluated by plasma famsin level (i), Tb (j), torpor frequency (k), time for Tb < 35 °C (l), torpor entry time (m), locomotor activity (n), relative total locomotor activity (o) and starvation resistance (p) of fasted 8-week-old male mice. Failure of starvation resistance was judged as Tb < 28 °C following a quick decrease in Tb below the environmental temperature. The gray and white backgrounds (j, n) indicate 12-h periods of darkness and light, respectively. Data are shown as means ± SEM. Comparison of different groups was carried out using one-way ANOVA followed by Tukey’s test (i, k–m, o) or log-rank test (p). *P < 0.05, **P < 0.01, ***P < 0.001. n = 7 mice.

Fig. 4. OLFR796 is a receptor of famsin.

a Binding assay on frozen tissue sections from 8–10-week-old Olfr796^+/+ and Olfr796^–/– male mice. 100 nM GST or GST-famsin was incubated with frozen tissue slices. For competition binding, tissues were pretreated with 5 μM His-ProS2 or His-ProS2-famsin for 30 min. Scale bars, 20 μm. b Binding assay on mouse primary hepatocytes. 100 nM GST or GST-famsin was incubated with hepatocytes. For competition binding, cells were pretreated with 5 μM His-ProS2 or His-ProS2-famsin for 30 min. Scale bars, 10 μm. c Effect of WT OLFR796 or its mutant (Mut, R187D/R195D/E197A) on famsin binding in mouse primary hepatocytes. Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test. ^###P < 0.001. n = 3. d, e qPCR results (d) and in situ hybridization (e) showing relative mRNA levels of Olfr796 in different tissues from 8–10-week-old male mice. In the pancreas sections, the islets are outlined in red. OE, olfactory epithelium; OB, olfactory bulb. Data are shown as means ± SEM. n = 6 mice (d). f GST pull-down assay showing the interaction between GST-famsin and OLFR796-Flag or its mutant (Mut, R187D/R195D/E197A) in Hi-5 cells. g Quantification of the binding affinity between famsin and WT OLFR796 or its mutant (Mut, R187D/R195D/E197A) by MST. ΔF_norm indicates the change in the normalized fluorescence. Data are shown as means ± SEM. n = 3.

Fig. 5. Famsin activates OLFR796-coupled calcium signaling.

a Schematic diagram showing the activation of OLFR796-coupled calcium signaling by famsin, and the targets of different chemicals used in this study. b, c Effect of famsin (30 nM) on calcium mobilization (b) and relative mRNA levels of G6pc (c) in the presence or absence of YM-254890 (YM, 2 μM), U73122 (10 μM) or Xestospongin C (XC, 2 μM) in mouse primary hepatocytes. Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test. ^###P < 0.001 (b), ***P < 0.001 (c). n = 24–48 cells (b), n = 6 (c). d Co-IP showing the association of GNAQ-Myc with OLFR796-Flag or its mutant (Mut, R187D/R195D/E197A) in HEK293T cells treated with or without famsin (30 nM) for 10 min. e, f Effect of WT OLFR796 or its mutant (Mut, R187D/R195D/E197A) on calcium mobilization (e) and relative mRNA levels of G6pc (f) in mouse primary hepatocytes in the presence or absence of famsin (30 nM). Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test. ^###P < 0.001 (e), ***P < 0.001 (f). n = 18–48 cells (e), n = 6 (f).

Fig. 6. Olfr796 knockout attenuates metabolic adaptations to fasting.

a–c Blood glucose (a), plasma β-hydroxybutyrate (b) and hepatic acetyl-CoA (c) levels from overnight fasted 8–10-week-old Olfr796^+/+ and Olfr796^–/– male mice. Data are shown as means ± SEM. Comparison of different groups was carried out using unpaired two-tailed Student’s t-test (a–c). n = 8 mice. d Immunoblots showing the effect of Olfr796 knockout on hepatic lipolysis and gluconeogenesis from liver extracts of 8–10-week-old male mice. e–g Pyruvate tolerance test (e), glucose tolerance test (f) and insulin tolerance test (g) results from 8–10-week-old Olfr796^+/+ and Olfr796^–/– male mice. The relative AUC is shown in the inset of each panel. Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test (curve data, ^#P < 0.05, ^##P < 0.01) or unpaired two-tailed Student’s t-test (AUC data, ***P < 0.001). n = 8 mice. h qPCR results showing relative mRNA levels of genes involved in lipolysis (Pnlip, Pnliprp2, Clps, Cel), lipid oxidation (Cpt1a), ketogenesis (Hmgcs2) and gluconeogenesis (G6pc, Pck1) from liver extracts of overnight fasted 8–10-week-old Olfr796^+/+ and Olfr796^–/– male mice treated with or without famsin. Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001. NS no statistical significance. n = 6 mice. i–o Effect of Olfr796 knockout on torpor evaluated by Tb (i), torpor frequency (j), time for Tb < 35 °C (k), torpor entry time (l), locomotor activity (m), relative total locomotor activity (n) and starvation resistance (o) of fasted 8-week-old male mice. Failure of starvation resistance was judged as Tb < 28 °C following a quick decrease in Tb below the environmental temperature. 400 μg/kg famsin was intraperitoneally injected after 4 h fasting. The gray and white backgrounds (i, m) indicate 12-h periods of darkness and light, respectively. Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test (j–l, n) or log-rank test (o). *P < 0.05, **P < 0.01. NS no statistical significance. n = 6–8 mice.

Fig. 7. Neutralization of famsin reduces blood glucose levels in mice.

a, b Plasma famsin levels (a) and plasma furin activity (b) in 8–10-week-old lean, db/db and ob/ob male mice with ad lib feeding or overnight fasting. Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test. **P < 0.01, ***P < 0.001. NS no statistical significance. n = 5 mice (a), n = 6 mice (b). c, d Plasma famsin levels (c) and plasma furin activity (d) in regular diet (RD) or HFD-fed 16-week-old male mice with ad lib feeding or overnight fasting. Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test. **P < 0.01, ***P < 0.001. NS no statistical significance. n = 6 mice. e, f Plasma famsin levels (e) and plasma furin activity (f) in normal people or patients with type 2 diabetes after overnight fasting or 2 h after eating (postprandial). Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test. ***P < 0.001. NS no statistical significance. n = 15 humans. g Effect of anti-Famsin antibody (200 μg/kg) on blood glucose in Olfr796^+/+ and Olfr796^–/– male mice fed a HFD for 16 weeks. Data are shown as means ± SEM. Comparison of different groups was carried out using two-way ANOVA followed by Tukey’s test. ***P < 0.001. NS no statistical significance. n = 8 mice. h Famsin, an intestine-derived hormone, binds OLFR796 and promotes energy mobilization via gluconeogenesis and ketogenesis in the liver, and energy conservation via torpor in the hypothalamus during fasting. Famsin activates calcium mobilization in the liver, but the downstream signaling of famsin in the hypothalamus is unclear (indicated with a question mark). Thus, a famsin-OLFR796 signaling axis promotes energy mobilization and conservation, and thereby enhances mouse metabolic adaptations to fasting.