Low-protein diet in puberty impairs testosterone output and energy metabolism in male rats

in Journal of Endocrinology
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We examined the long-term effects of protein restriction during puberty on the function of hypothalamic–pituitary–adrenal (HPA) and hypothalamic–pituitary–gonadal (HPG) axes in male rats. Male Wistar rats from the age of 30 to 60 days were fed a low-protein diet (4%, LP). A normal-protein diet (20.5%) was reintroduced to rats from the age of 60 to 120 days. Control rats were fed a normal-protein diet throughout life (NP). Rats of 60 or 120 days old were killed. Food consumption, body weight, visceral fat deposits, lipid profile, glycemia, insulinemia, corticosteronemia, adrenocorticotropic hormone (ACTH), testosteronemia and leptinemia were evaluated. Glucose-insulin homeostasis, pancreatic-islet insulinotropic response, testosterone production and hypothalamic protein expression of the androgen receptor (AR), glucocorticoid receptor (GR) and leptin signaling pathway were also determined. LP rats were hypophagic, leaner, hypoglycemic, hypoinsulinemic and hypoleptinemic at the age of 60 days (P < 0.05). These rats exhibited hyperactivity of the HPA axis, hypoactivity of the HPG axis and a weak insulinotropic response (P < 0.01). LP rats at the age of 120 days were hyperphagic and exhibited higher visceral fat accumulation, hyperleptinemia and dyslipidemia; lower blood ACTH, testosterone and testosterone release; and reduced hypothalamic expression of AR, GR and SOCS3, with a higher pSTAT3/STAT3 ratio (P < 0.05). Glucose-insulin homeostasis was disrupted and associated with hyperglycemia, hyperinsulinemia and increased insulinotropic response of the pancreatic islets. The cholinergic and glucose pancreatic-islet responses were small in 60-day-old LP rats but increased in 120-day-old LP rats. The hyperactivity of the HPA axis and the suppression of the HPG axis caused by protein restriction at puberty contributed to energy and metabolic disorders as long-term consequences.


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    Body weight gain (A) and relative food intake (B). Data are presented as the means ± s.e.m. of 20 rats from 4 different litters. The left panels, as an inset to each figure, depict the area under the curve (AUC) for both periods of dietary treatment. Bars on the left in each panel depict the AUC for body weight gain and relative food intake during the protein-calorie restriction period (30–60 days old). Bars on the right depict the AUC during the period of dietary reestablishment (60–120 days old). ***P < 0.001 using Student’s t test.

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    Insulinemia and glycemia during the intravenous glucose tolerance test (ivGTT). Data are presented as the means ± s.e.m. of 10 rats from 4 different litters. Insulinemia (A) and glycemia (B) during the ivGTT at the end of protein-calorie restriction (60 days old). Insulinemia (C) and glycemia (D) during the ivGTT after the period of dietary reestablishment (120 days old). The upper panel of each figure represents the area under the curve (AUC). *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

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    Short- and long-term effects of pubertal protein-calorie restriction on insulin secretion. Data are presented as the means ± s.e.m. of insulin secretion from the pancreatic islets of 6 rats from 3 different litters. Insulinotropic effect of different glucose (A and C) and acetylcholine (B and D) concentrations refers to data obtained from rats at the end of protein-calorie restriction (60 days old) and after dietary reestablishment (120 days old). *P < 0.05, **P < 0.01, ***P < 0.001 using Student’s t test.

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    Short- and long-term effects of pubertal protein-calorie restriction on testosterone output and the correlation between corticosteronemia and testosteronemia. Data are presented as the means ± s.e.m. of testosterone output for isolated testicles of 6 rats from 3 different litters. Testosterone output without (–) and with (+) hCG 100 IU/mL at the end of protein-calorie restriction (60 days old, A) and after the dietary reestablishment (120 days old, B). Correlation between plasma levels of corticosterone and testosterone in rats of 60 days old (C) and those of 120 days old (D). Symbols over the bars depict significant differences between NP and LP values of testosterone output with and without hCG, as determined using a one-way ANOVA, in which *P < 0.01, **P < 0.001 compared to NP without hCG; Ф P < 0.001 compared to LP without hCG; and # P < 0.001 compared to NP with hCG.

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    Hypothalamic measurement of AR (A) and GR (B) protein expression. Data are presented as the means ± s.e.m. of AR and GR protein expression in hypothalamus samples of 6 rats from 3 different litters. Representative blots of AR, GR and β-actin (control load) are shown in (C). Mean values from the hypothalamic protein expression of AR and GR from LP rats at the end of protein-calorie restriction (60 days old) or after dietary reestablishment (120 days old) were significantly different from those in the NP group: *P < 0.01 and ***P < 0.001 using Student’s t test.

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    Hypothalamic measurement of protein expression of the leptin signaling pathway. Data are presented as the means ± s.e.m. of ObR-b (A), pJAK2/JAK2 (B), pSTAT3/STAT3 (C) and SOCS3 (D) protein expression in hypothalamus samples of 6 rats from 3 different litters of each group. Representative blots of ObR-b, pJAK2, JAK2, pSTAT3, STAT3, SOCS3 and β-actin (control load) are shown in (E). Significant differences between the LP and NP values of hypothalamic protein expression were determined using Student’s t test. *P < 0.01 and ***P < 0.001.


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