l-phenylalanine modulates gut hormone release and glucose tolerance, and suppresses food intake through the calcium-sensing receptor in rodents

Abstract

Objective: High-protein diets (HPDs) are associated with greater satiety and weight loss than diets rich in other macronutrients. The exact mechanisms by which HPDs exert their effects are unclear. However, evidence suggests that the sensing of amino acids produced as a result of protein digestion may have a role in appetite regulation and satiety. We investigated the effects of l-phenylalanine (L-Phe) on food intake and glucose homeostasis in rodents.

Methods: We investigated the effects of the aromatic amino-acid and calcium-sensing receptor (CaSR) agonist l-phenylalanine (L-Phe) on food intake and the release of the gastrointestinal (GI) hormones peptide YY (PYY), glucagon-like peptide-1 (GLP-1) and ghrelin in rodents, and the role of the CaSR in mediating these effects in vitro and in vivo. We also examined the effect of oral l-Phe administration on glucose tolerance in rats.

Results: Oral administration of l-Phe acutely reduced food intake in rats and mice, and chronically reduced food intake and body weight in diet-induced obese mice. Ileal l-Phe also reduced food intake in rats. l-Phe stimulated GLP-1 and PYY release, and reduced plasma ghrelin, and also stimulated insulin release and improved glucose tolerance in rats. Pharmacological blockade of the CaSR attenuated the anorectic effect of intra-ileal l-Phe in rats, and l-Phe-induced GLP-1 release from STC-1 and primary L cells was attenuated by CaSR blockade.

Conclusions: l-Phe reduced food intake, stimulated GLP-1 and PYY release, and reduced plasma ghrelin in rodents. Our data provide evidence that the anorectic effects of l-Phe are mediated via the CaSR, and suggest that l-Phe and the CaSR system in the GI tract may have therapeutic utility in the treatment of obesity and diabetes. Further work is required to determine the physiological role of the CaSR in protein sensing in the gut, and the role of this system in humans.

Figure 1

The effect of l-Phe on food intake in rodents. (a) The effect of intra-ileal administration of 10 mm l-Phe on food intake in ad libitum-fed rats at 0–1 h post administration during early dark phase (n=9 crossover, P<0.01 vs control). The effect of OG of control (water) and 3 mmol kg⁻¹ l-Phe on food intake in (b) male rats following an overnight fast (n=13, vehicle; 11, L-Phe; *P<0.05, **P<0.01, ***P<0.001 vs control) and in (c) fasted male mice following an overnight fast receiving an OG of vehicle (10% TWEEN20 in water) and 12 mmol kg⁻¹ l-Phe (n=5, **P<0.01, ***P<0.001 vs control). (d) The effect of OG of vehicle (10% TWEEN20 in water), and 3 and 6 mmol kg⁻¹ l-Phe in ad libitum-fed rats at the beginning of dark phase (n=10, *P<0.05, **P<0.01; 6 mmol kg⁻¹ l-Phe vs control). (e) The effect of OG of vehicle (10% TWEEN20 in water), and 6 and 12 mmol kg⁻¹ l-Phe in ad libitum chow-fed mice at the beginning of dark phase (n=10; *P<0.05, 6 mmol kg⁻¹ l-Phe vs control; **P<0.01, 12 mmol kg⁻¹ l-Phe vs control; ^#P<0.05, 6 vs 12 mmol kg⁻¹ l-Phe), and in (f) ad libitum high-fat diet-fed DIO mice (n=10; *P<0.05, 12 mmol kg⁻¹ l-Phe vs control). All figures show food intake at 0–1, 1–2, 0–2 (except a), 2–4, 4–8 and 0–24 h post administration. All data presented as mean±s.e.m.

Figure 2

The effect of oral administration of l-Phe on food intake, energy expenditure and activity in rats. The effect of OG administration of vehicle (10% TWEEN20 in water; control) or 6 mmol kg⁻¹ l-Phe on (a) the timeline of cumulative food intake and the AUC at 0–120 min, (b) the timeline of cumulative food intake at 0–12 h, (c) the timeline of VO₂ and the AUC at 0–120 min; (d) the timeline of VCO₂ and the AUC at 0–120 min; (e) the timeline of respiratory exchange ratio (RER) and the AUC at 0–120 min, (f) the timeline of activity (XTOT) and the AUC at 0–120 min in rats injected at the onset of the dark phase and placed in comprehensive laboratory animal-monitoring system cages for 12 h. Recordings were taken over a period of 12 h at 16 min intervals following administration. High-resolution food intake recordings were taken every minute for 120 min. Dotted lines on graphs represent the 0–120 min interval. Data presented as mean±s.e.m. and AUC. n=8, *P<0.05.

Figure 3

The effect of l-Phe on gut hormone release. (a) The effect of 1, 10, 30, 50 and 100 mm l-Phe on GLP-1 release from STC-1 cells. Cells were incubated with each treatment for 2 h. n=9 independent plates. *P<0.05, **P<0.01, P***<0.001 vs control. (b) The effect of l-Phe on GLP-1 release from primary mice colonic L cells incubated with 1, 10 and 100 mm l-Phe for 2 h. n=6 plates from 6 mice; and (c) the effect of l-Phe on PYY release from primary mice colonic L cells incubated with 10, 50 and 100 mm l-Phe for 2 h. n=9 plates from 9 mice. *P<0.05, **P<0.01 vs control. The effect of OG of water (control) or 3 mmol kg⁻¹ l-Phe on (d) plasma CCK and (e) plasma acylated ghrelin 30 min after administration in rats following an overnight fast (n=11, *P<0.05 vs control). (f) The effect of intra-ileal administration of 10 mm l-Phe on PYY levels 15 min following administration in overnight-fasted rats (n=7, vehicle; 10, L-Phe; P=0.06 vs control). The effect of OG of water (control) or 3 mmol kg⁻¹ l-Phe on (g) plasma GLP-1 and (h) plasma insulin 30 min after administration in rats following an overnight fast (n=11, **P<0.01 vs control).

Figure 4

The effect of l-Phe on glucose homeostasis in rats. The effect of oral l-Phe on (a) glucose tolerance test and (b) AUC for glucose in overnight-fasted male rats that received an intraperitoneal injection of 20% glucose solution (2 g kg⁻¹ body weight) followed by an immediate OG of 6 mmol kg⁻¹ l-Phe. (c) Plasma insulin levels measured at t=30 during IPGTT (n=15, *P<0.05, **P<0.01 vs control). All data are presented as mean±s.e.m. and AUC.

Figure 5

The role of CaSR in mediating the anorectic effect of l-Phe. (a) The effect of oral administration vehicle (1.85% dimethyl sulfoxide (DMSO) in water) and 1.1 mg kg⁻¹ R568.HCl in overnight-fasted rats at 0–1 h following administration during early hours of light phase (n=8, vehicle; 9, L-Phe; P<0.05 vs control); and (b) the effect of intra-ileal administration of vehicle (0.25 DMSO, 0.9% saline), 1 μm NPS.2143.HCl, 10 mm l-Phe, or 10 mm l-Phe and 1 μm NPS2143.HCl on food intake in rats with ad libitum access to food injected at the beginning of dark phase at 0–1 h post administration (n=18, crossover). (c) The effect of 50 mm l-Phe in presence or absence of 10 μm NPS2143.HCl on GLP-1 release from STC-1 cells following 2 h incubation, n=8 independent plates. (d) The effect of 30 mm l-Phe in presence or absence of 15 μm NPS2390 on GLP-1 release from primary mice colonic L cells following 2 h incubation, n=5 independent plates. (e) The effect of 30 mm l-Phe in presence or absence of 15 μm NPS2390 on GLP-1 release from mouse ileal organoids following 24 h incubation, n=8, L-Phe; 9, NPS2390, control; 10, L-Phe+NPS2390, independent organoid cultures. **P<0.01, ***P<0.001, ****P<0.0001 vs control; ^##P<0.01, ^###P<0.001 vs L-Phe. All data presented as mean±s.e.m.

Figure 6

The effect of repeated OG administration of l-Phe on food intake, body weight and gut hormones in DIO mice. The effect of repeated OG of vehicle (10% TWEEN20 in water) or 12 mmol kg⁻¹ l-Phe on (a) cumulative food intake, (b) body weight change, (c) plasma leptin, (d) acylated ghrelin, (e) GLP-1 and (f) PYY following twice daily OG during days 0–3 and three times daily OG during 4–7 days of chronic study. Dotted line highlights the start of three times daily injection. Plasma gut hormones and leptin levels were measured 24 h following the completion of the chronic administration in ad libitum-fed DIO mice. n=10 per group. *P<0.05, ***P<0.001 vs vehicle. All data expressed as mean±s.e.m.