Figure 1.
Role of α-gustducin in the effect of a glyceryl trioctanoate-enriched diet (OD) on ghrelin levels.
(A) Octanoyl ghrelin levels in stomach extracts from WT (n = 8–15) and gust−/− (n = 8–13) mice fed a control diet (CD) (white bars) or OD (black bars) for two weeks. (B) Octanoyl to desoctanoyl ghrelin ratio in stomach extracts of WT and gust−/− mice. (C) Octanoyl ghrelin and (D) total ghrelin levels in plasma of WT (n = 16–20) and gust−/− (n = 17–19) mice fed a CD or OD for two weeks. (E) Ghrelin mRNA and (F) GOAT mRNA expression in stomach of WT (n = 13–17) and gust−/− (n = 14–18) mice fed a CD or OD for two weeks. All values are means ± SEM’s. *: P<0.05, ***: P<0.0005 CD vs. OD, ###: P<0.001 WT vs. gust−/−.
Figure 2.
Role of α-gustducin in the effect of a diet enriched with glyceryl trioctanoate on body weight, food intake, hypothalamic AgRP mRNA expression and gastric emptying.
(A, B) Time-dependent changes in body weight of WT (A) or gust−/− (B) mice fed a CD (open symbols) or OD (filled symbols) (n = 18 mice per group) for two weeks. Results are expressed as a % of body weight before the start of the experiments. (C) 24-h food intake measured in WT and gust−/− at day 14 on a CD or OD (n = 8 mice per group). Results are expressed as % of 24-h food intake measured before the start of the experiment. (D) Hypothalamic AgRP mRNA levels in WT or gust−/− mice after two weeks on CD or OD (n = 10–14 mice per group). (E) Gastric emptying measured before the start of the experiment and at day 7 and 14 after feeding an OD in WT (open circles), gust−/− (filled circles) or GHS-R−/− (filled triangles) (n = 8 per genotype). All values are means ± SEM’s and are expressed as a % of their respective Thalf value at day 0 when mice were on the control diet. *: P<0.05, **: P<0.001 vs. day 0, #: P<0.05 WT vs. GHS-R−/−, ##: P<0.01 WT vs. gust−/−.
Figure 3.
Immunofluorescence colocalization studies between GPR40 and ghrelin or the gustatory G-proteins in sections of the mouse stomach and duodenum.
(A) Double-immunofluorescence staining showing colocalization between anti-GPR40 staining (red) and anti-total ghrelin staining (green) in endocrine cells. (B) GPR40 (red) immunoreactive endocrine cells did not colocalize with octanoyl ghrelin (green) immunoreactive endocrine cells, but some GPR40 positive cells were in close proximity with octanoyl ghrelin positive cells as pointed by the arrow. (C) No colocalization of GPR40 (red) and α-transducin (green) in stomach endocrine cells. (D) Double staining of GPR40 (red) and α-gustducin (green) in endocrine cells. (E) Double staining between GPR40 (red) and total ghrelin (green) in mouse duodenum. No colocalization is detected. Bar = 25 µm.
Figure 4.
Immunofluorescence colocalization studies between GPR120 and ghrelin in sections of the mouse stomach and duodenum.
(A) Double immunofluorescence staining showing no colocalization between anti-GPR120 (green) and anti-total ghrelin (red) staining in endocrine cells of the stomach. (B) GPR120 (green) immunoreactive staining was present in the brush cells at the limiting ridge. No colocalization was observed with ghrelin positive cells, but some were in close proximity. (C) GPR120 (red) positive cells did colocalize with total ghrelin (green) in sections of the mouse duodenum. Bar = 25 µm.
Figure 5.
Effect of FFA stimulation on ghrelin secretion in the ghrelinoma cell line.
MGN3-1 cells were stimulated with different concentrations of octanoic acid (A), α-linolenic acid (B), MEDICA16 (D) or grifolic acid (E). Both octanoyl (filled circles) and total ghrelin (open circles) levels were measured in the medium 4 h after administration of the FFA. (C) Immunofluorescence staining for DAPI (left), GPR120 (middle) and negative control (right) in the ghrelinoma cell line. Bar = 100 µm. All values are means ± SEM’s of 4 independent experiments performed in triplicate and are expressed as a % of ghrelin secretion obtained after stimulation with vehicle.
Figure 6.
Role of GPR120 in the effect of octanoic acid (A–B) and α-linolenic acid (C–D) on ghrelin levels in the ghrelinoma cell line.
Octanoyl and total ghrelin secretion was measured 4 h after administration of vehicle, octanoic acid (10−4 M), or α-linolenic acid (10−5 M) to MGN3-1 cells transfected with GPR120 siRNA or a non-targeting siRNA pool as negative control. All values are means ± SEM’s of 4 independent experiments performed in sevenfold. *: P<0.05, **: P<0.01 vehicle vs. octanoic acid or α-linolenic acid.
Figure 7.
Effect of MEDICA16 and grifolic acid on ghrelin secretion in vivo.
Octanoyl ghrelin (left) and total ghrelin (right) levels were measured in plasma (A) or in stomach extracts (B) 40 min after gavage of 0,18% DMSO (vehicle), 10 nmol/kg MEDICA16 or grifolic acid. All values are means ± SEM’s of experiments performed in 8 mice. *: P<0.05 vs. vehicle.