An orally active plant Rubisco-derived peptide increases neuronal leptin responsiveness

Abstract

Nutrient excess, such as the intake of a high-fat diet, reduces hypothalamic responses to exogenously administered leptin and induces dietary obesity; however, orally active components that attenuate neural leptin dysregulation have yet to be identified. We herein demonstrated that YHIEPV, derived from the pepsin-pancreatin digestion of the green leaf protein Rubisco, increased the leptin-induced phosphorylation of STAT3 in ex vivo hypothalamic slice cultures. We also showed that YHIEPV mitigated palmitic acid-induced decreases in leptin responsiveness. Furthermore, orally administered YHIEPV promoted leptin-induced reductions in body weight and food intake in obese mice. In addition, dietary-induced body weight gain was significantly less in mice orally or centrally administered YHIEPV daily than in saline-control mice. Cellular leptin sensitivity and the levels of proinflammatory-related factors, such as IL1β and Socs-3, in the hypothalamus of obese mice were also restored by YHIEPV. YHIEPV blocked cellular leptin resistance induced by forskolin, which activates Epac-Rap1 signaling, and reduced the level of the GTP-bound active form of Rap1 in the brains of obese mice. Collectively, the present results demonstrated that the orally active peptide YHIEPV derived from a major green leaf protein increased neural leptin responsiveness and reduced body weight gain in mice with dietary obesity.

Figure 1

YHIEPV promotes intracellular leptin sensitivity. (A, B) Effects of YHIEPV on cellular leptin sensitivity in organotypic hypothalamus slices. Hypothalamic slices were made from C57BL/6 pups. Slices were incubated with YHIEPV (100 μM, 24 h) and then stimulated with leptin (30 nM, 60 min). (A) Representative immunohistochemistry images for pSTAT3 in fixed slices. Scale bar: 200 μm. (B) Quantification of hypothalamic pSTAT3 (n = 6–8/group) in organotypic hypothalamus slices. (C, D) Effects of YHIEPV on palmitic acid-induced cellular leptin resistance in organotypic hypothalamus slices. Slices were incubated with palmitic acid (30 μM) in the presence or absence of YHIEPV (100 μM) for 6 h and then stimulated with leptin (60 nM, 60 min). (C) A leptin-induced pSTAT3 image is shown. Scale bar, 200 μm. (D) Quantification of hypothalamic pSTAT3 (n = 4–5/group) in organotypic hypothalamus slices. *p < 0.05, **p < 0.01, ***p < 0.001 for a one-way ANOVA followed by Tukey’s multiple comparisons tests in (C and D). All error bars are SEM.

Figure 2

Orally administered YHIEPV increases leptin sensitivity in HFD-fed mice. (A, B) The oral administration of YHIEPV increased leptin-induced reductions in body weight (A) and food intake (B). YHIEPV oral injections (0.3 mg/kg) were started 3 days before leptin injections. Leptin (0.5 μg) or vehicle was i.c.v. infused with or without YHIEPV (0.3 mg/kg, p.o.) to HFD-fed ddY mice (HFD for 5 weeks, control: 48.30 ± 1.4 g, YHIEPV: 47.29 ± 1.8 g, Leptin: 49.68 ± 1.8 g, YHIEPV/Leptin: 49.32 ± 2.1 g, on the leptin experimental day) (n = 10–12/group). (C, D) Leptin (0.5 μg, i.c.v., 60 min) was administered to the indicated ddY mice (n = 3/group). YHIEPV was orally injected for 3 days. (C) Representative immunohistochemistry images for pSTAT3. Scale bar, 100 μm. (D) Quantification of immunohistochemistry. *p < 0.05, **p < 0.01, ***p < 0.001 for a two-way ANOVA followed by Tukey’s multiple comparisons tests in (A) and (B) or a one-way ANOVA followed by Tukey’s multiple comparison test in (D). All error bars are SEM.

Figure 3

YHIEPV protects against dietary obesity. (A–D) Effects of YHIEPV on body weight and food intake in HFD-induced obese ddY mice. Body weight changes (A) and cumulative food intake or weekly average food intake (B). HFD-fed obese ddY mice (HFD for 2 months, n = 9/group) were orally administered YHIEPV (0.3 mg/kg for 35 days) once a day. Epididymal and mesenteric fat masses (C) and plasma leptin, insulin, and glucose levels (D) were measured on day 35. Mice were fasted for 3 h (from 8 to 11AM) before the collection of tissue and blood samples. (E, F) YHIEPV was centrally infused (1 μg, every day) into C57BL/6 mice (n = 7). HFD was initiated on day 0. Body weight (E) was measured daily. Plasma levels of leptin and insulin and blood glucose levels (F) were measured on day 30. Mice were fasted for 3 h (from 8 to 11AM) before the collection of blood samples. *p < 0.05, **p < 0.01, ***p < 0.001 for a two-way ANOVA followed by Bonferroni multiple comparisons tests in (A–C) or t-tests in (D) and (F). All error bars are SEM.

Figure 4

YHIEPV reduces central responses of proinflammatory-related factors and Rap1 activation. (A, B) Hypothalamic expression of genes involved in decreased leptin responsiveness in YHIEPV-treated and control C57BL/6 mice. The hypothalamus was collected from 4-month-old HFD-fed male mice after 5 days of the YHIEPV treatment (0.3 mg/kg, p.o.). YHIEPV reduced the hypothalamic mRNA expression of Socs-3, Tcptp (A), and IL1β (B). Mice were fasted for 3 h (from 8 to 11AM) before the collection of hypothalami. (C) Effects of YHIEPV on forskolin-induced cellular leptin resistance in organotypic hypothalamus slices. Slices were incubated with forskolin (Fsk, 20 μM) in the presence or absence of YHIEPV (100 μM) for 6 h and then stimulated with leptin (60 nM, 60 min). Leptin-induced pSTAT3 is shown (Left). Scale bar, 200 μm. Quantification of hypothalamic pSTAT3 (Right) (n = 5–9/group) in slices. (D) Western blot images (left) and quantification (right) of the amount of the active form of Rap1 in the brains of HFD-induced obese C57BL/6 mice treated with YHIEPV (0.3 mg/kg, p.o., for 5 days) or vehicle (n = 4/group). *p < 0.05, **p < 0.01, ***p < 0.001 for the t-test in (A), (B), and (D) or a one-way ANOVA followed by Tukey’s multiple comparison test in (C). All error bars are SEM.