Appetite regulation is independent of the changes in ghrelin levels in pregnant rats fed low-protein diet

Abstract

Gestational protein restriction causes hypertension in the adult offspring. Very little is known about the food intake regulation and ghrelin signaling in pregnant dams fed a low-protein (LP) diet. We hypothesized that diet intake and ghrelin signaling are altered in pregnant rats fed the low-protein diet. Sprague-Dawley rats were fed a control (CT) or LP diet from Day 3 of pregnancy. Diet intake and body weight were monitored daily. Expression of ghrelin production-related genes in the stomach and appetite-related genes in the hypothalamus was analyzed by real-time PCR. Plasma levels of total and active ghrelin, growth hormone and leptin were measured by ELISA. Main results include: (1) Daily diet intake was greater in the LP group than in the CT group in early pregnancy, but substantially lower in late pregnancy; (2) Daily gain in body weight was substantially lower in the LP group in late pregnancy; (3) Expression of ghrelin production-related genes in the stomach and plasma total ghrelin levels were increased in LP group in late pregnancy; (4) Plasma active ghrelin levels were elevated in the LP group at mid-late pregnancy, but growth hormone and leptin levels were uncorrelated with active ghrelin in late pregnancy; and (5) Hypothalamic expression of ghrelin-stimulated genes in LP rats was unassociated with the changes in both plasma ghrelin levels and the diet intake. Taken together, the appetite in LP rats is greater in early pregnancy but reduced at late pregnancy, possibly due to ghrelin insensitivity in appetite regulation.

Keywords: Diet intake; ghrelin; low‐protein diet; pregnancy; rat.

Figure 1

Daily diet intake. CT: control; LP, low-protein diet. The bar represents the mean ± SEM expressed as the ratio of diet intake (gram) to body weight (gram) of pregnant rats on each day of pregnancy (n = 8–10). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Daily gain in body weight during Day 3–20 of gestation (A) and cumulative gain in body weight on Days 10, 14, 18, and 21 of gestation (B). CT: control; LP, low-protein diet. The bar represents the mean ± SEM (n = 8–10). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Quantitative real-time PCR analysis of Ghrl (A), Mboat4 (B), and Bche (C) in the stomach on Days 10, 14, 18, and 21 of gestation. CT: control; LP, low-protein diet. The bar represents the mean ± SEM expressed as relative units of mRNA standardized against Actb (n = 8–10). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Quantitative real-time PCR analysis of Npy (A), Agrp (B), Pomc (C), and Cart (D) in the hypothalamus on Days 10 and 21 of gestation. CT: control; LP, low-protein diet. The bar represents the mean ± SEM expressed as relative units of mRNA standardized against Actb (n = 8–10). *P < 0.05.

Figure 5

Quantitative real-time PCR analysis of Ghsr (A), Fasn (B), Cpt1a (C), Cpt1c (D), and Ucp2 (E) in the hypothalamus on Days 10 and 21 of gestation. CT: control; LP, low-protein diet. The bar represents the mean ± SEM expressed as relative units of mRNA standardized against Actb (n = 8–10). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

Quantitative real-time PCR analysis of Npy (A), Agrp (B), Pomc (C), and Cart (D) in arcuate nucleus region of the hypothalamus on Days 19 and 22 of gestation. CT: control; LP, low-protein diet; ARC, arcuate nucleus. The bar represents the mean ± SEM expressed as relative units of mRNA standardized against Actb (n = 8–10). *P < 0.05.

Figure 7

ELISA assays on plasma total (A) and active (B) ghrelin on Days 10, 14, 18, and 21 of gestation. CT: control; LP, low-protein diet; the error bar represents the mean ± SEM (n = 8–10). **P < 0.01; ***P < 0.001.

Figure 8

ELISA assay on plasma growth hormone on Days 14, 18, and 21 of gestation. CT: control; LP, low-protein diet. The bar represents the mean ± SEM (n = 8–10). *P < 0.05; ^#P = 0.08.

Figure 9

ELISA assay on plasma leptin on Days 14, 19, and 22 of gestation. CT: control; LP, low-protein diet. The bar represents the mean ± SEM (n = 8–10). ***P < 0.001.