Maternal melatonin programs the daily pattern of energy metabolism in adult offspring

Abstract

Background: Shift work was recently described as a factor that increases the risk of Type 2 diabetes mellitus. In addition, rats born to mothers subjected to a phase shift throughout pregnancy are glucose intolerant. However, the mechanism by which a phase shift transmits metabolic information to the offspring has not been determined. Among several endocrine secretions, phase shifts in the light/dark cycle were described as altering the circadian profile of melatonin production by the pineal gland. The present study addresses the importance of maternal melatonin for the metabolic programming of the offspring.

Methodology/principal findings: Female Wistar rats were submitted to SHAM surgery or pinealectomy (PINX). The PINX rats were divided into two groups and received either melatonin (PM) or vehicle. The SHAM, the PINX vehicle and the PM females were housed with male Wistar rats. Rats were allowed to mate and after weaning, the male and female offspring were subjected to a glucose tolerance test (GTT), a pyruvate tolerance test (PTT) and an insulin tolerance test (ITT). Pancreatic islets were isolated for insulin secretion, and insulin signaling was assessed in the liver and in the skeletal muscle by western blots. We found that male and female rats born to PINX mothers display glucose intolerance at the end of the light phase of the light/dark cycle, but not at the beginning. We further demonstrate that impaired glucose-stimulated insulin secretion and hepatic insulin resistance are mechanisms that may contribute to glucose intolerance in the offspring of PINX mothers. The metabolic programming described here occurs due to an absence of maternal melatonin because the offspring born to PINX mothers treated with melatonin were not glucose intolerant.

Conclusions/significance: The present results support the novel concept that maternal melatonin is responsible for the programming of the daily pattern of energy metabolism in their offspring.

Figure 1:. Body weight and basal glycemia in male and female SHAM-P1, PINX-P1 and PM-P1 rats.

Male (A) and female (B) SHAM-P1, PINX-P1 and PM-P1 rats were weighed at the second day of life and at the end of the 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th, 10^th, 16^th and 18^th weeks of life at ZT10 (n=38 to 49 for males and n=35 to 41 for females). Glycemia of the male (C) and female (D) SHAM-P1, PINX-P1 and PM-P1 rats was assessed at the end of the 4^th, 8^th and 18^th weeks of life at ZT10 after a 12h fasting (n=11 to 16 for males and n=9 to 12 for females). SHAM-P1 are dotted lines with open circles, PINX-P1 are continuous lines with closed squares and PM-P1 are dashed lines with closed triangles. The data are presented as the mean ± SE. *P<0.05 vs. week 0 within the same group.

Figure 2:. Glucose tolerance in male and female SHAM-P1, PINX-P1 and PM-P1 rats at different moments of the light/dark cycle.

Fasting glycemia was assessed in the male and female SHAM-P1, PINX-P1 and PM-P1 rats. Next, the animals received an i.p. injection of glucose (2 g/kg), and glycemia was assessed at 10, 15, 30, 60 and 120 min after injection. The glycemic values were plotted vs. time after the injection, and the AUC was calculated. Tests were performed in male offspring at the end of the 8^th (A and B) and 18^th (C and D) weeks of life at ZT10. Tests were also performed in female offspring at the end of the 18^th week of life at ZT10 (E and F) and in male offspring at the end of the 18^th week of life at ZT3 (G and H). SHAM-P1 are dotted lines with open circles, PINX-P1 are continuous lines with closed squares and PM-P1 are dashed lines with closed triangles. The data are presented as the mean ± SE. *P<0.05 vs. SHAM-P1 at the same time after glucose injection; #P<0.05 vs. SHAM-P1 (n=6).

Figure 3:. Whole body insulin sensitivity in male and female SHAM-P1, PINX-P1 and PM-P1 rats.

Fasting glycemia was assessed in male and female SHAM-P1, PINX-P1 and PM-P1 rats. Next, rats received an i.p. injection of insulin (2 IU/kg), and glycemia was assessed at 5, 10, 15, 20, 25 and 30 min after injection. The glycemic values were plotted vs. time after the injection, and the K_ITT was calculated. Tests were performed in male (A and B) and female (C and D) offspring at the 18^th week of life at ZT10. SHAM-P1 are dotted lines with open circles, PINX-P1 are continuous lines with closed squares and PM-P1 are dashed lines with closed triangles. The data are presented as the mean ± SE (n=6).

Figure 4:. Glucose production from pyruvate in male and female SHAM-P1, PINX-P1 and PM-P1 at different moments of the light/dark cycle.

Fasting glycemia was assessed in male and female SHAM-P1, PINX-P1 and PM-P1 rats. Next, the rats received an i.p. injection with sodium pyruvate (2 g/kg), and glycemia was assessed at 15, 30, 60, 90, 120 and 150 min after injection. The values of glycemia were plotted vs. time after injection, and the AUC was calculated. Tests were performed in male (A and B) and female (C and D) offspring at the 18^th week of life at ZT10. Tests were also performed in male offspring at 18 weeks of life at ZT3 (E and F). SHAM-P1 are dotted lines with open circles, PINX-P1 are continuous lines with closed squares and PM-P1 are dashed lines with closed triangles. The data are presented as the mean ± SE. *P<0.05 vs. SHAM-P1 at the same time after pyruvate injection; #P<0.05 vs. SHAM-P1 (n=6).

Figure 5:. Insulin levels and insulin secretion by pancreatic islets of male SHAM-P1, PINX-P1 and PM-P1 rats.

Male offspring were decapitated at ZT10, the pancreata were perfused with collagenase and the islets were isolated. After isolation, groups of five islets were initially incubated for 45 min at 37°C in Krebs–bicarbonate buffer containing 5.6 mM glucose and equilibrated with 95% O₂–5% CO₂, pH 7.4. The solution was then replaced with fresh Krebs–bicarbonate buffer, and the islets were incubated for an additional 1 h with medium containing 5.6, 8.3, 11.1 or 16.7 mM glucose. Insulin was quantified by RIA in the supernatant (A). Serum from the rats was used to determine circulating insulin levels (B). Open bars are SHAM-P1, black bars are PINX-P1 and grey bars are PM-P1. The data represent the cumulative insulin secretion over 1 h and are given as the mean ± SE. *P<0.05 vs. SHAM-P1 with 5.6 mM glucose; #P<0.05 vs. PM-P1 with 5.6 mM glucose; &P<0.05 vs. SHAM-P1 (n=5 for insulin secretion and insulin levels).

Figure 6:. Insulin signaling in the livers of male and female SHAM-P1, PINX-P1 and PM-P1 rats.

Male and female offspring at the 18^th week of life were anesthetized at ZT10, and a fragment of the liver was removed to detect basal phosphorylation. An additional fragment of the liver was removed 30 seconds after an intravenous insulin injection. The samples were processed by protein extraction and western blot detection of pAKT and GAPDH in male (A) and in female (F) offspring. The values obtained from male and female pAKT were normalized to GAPDH (B and G, respectively). The samples used for basal phosphorylation were run on a separate gel and transferred to membranes for the western blot detection of AKT, PEPCK and GAPDH in male (C) and in female (H) offspring. The values of AKT and PEPCK obtained from male (D and E, respectively) and from female offspring (I and J, respectively) were normalized to GAPDH. Open bars are SHAM-P1, black bars are PINX-P1 and grey bars are PM-P1. The data are presented as the mean ± SE. *P<0.05 vs. non-stimulated within the same group; #P<0.05 vs. insulin-stimulated SHAM-P1; &P<0.05 vs. non-stimulated SHAM-P1; @P<0.05 vs. non-stimulated PINX-P1 (n=6).

Figure 7:. Insulin signaling in the skeletal muscle of male SHAM-P1, PINX-P1 and PM-P1 rats.

Male offspring at the 18^th week of life were anesthetized at ZT10, and a soleus skeletal muscle was removed to detect basal phosphorylation. The remaining soleus muscle was removed 90 seconds after an intravenous insulin injection. The samples were processed by protein extraction and western blot detection of p-Tyr (A), pAKT and GAPDH (D). The values for tyrosine-phosphorylated pp95 (B) and pp185 (C) and pAKT (E) were normalized to GAPDH. Open bars are SHAM-P1, black bars are PINX-P1 and grey bars are PM-P1. The data are presented as the mean ± SE. *P<0.05 vs. non-stimulated within the same group (n=6).