Prenatal Nicotine Exposure Induces Low Birthweight and Hyperinsulinemia in Male Rats

Abstract

Smoking during pregnancy is one of the causes of low birthweight. Ingestion of nicotine during pregnancy has various metabolic impacts on the fetus and offspring. According to the developmental origins of health and disease theory, low birthweight is a risk factor for developing various non-communicable diseases, including diabetes. We hypothesized that when nicotine-induced low-birthweight rats, when exposed to a high-fat diet (HFD) after growth, are predisposed to glucose intolerance as a result of a mismatch between the eutrophic environment and small body size. Therefore, we investigated whether hyperinsulinemia was caused by exposure of nicotine-induced low-birthweight rats to HFD, including whether this phenomenon exhibited possible sex differences. The average birthweight and body weight at weaning day of offspring from nicotine-administered dams was lower than those of controls. The offspring from nicotine-administered dams did not show rapid fat accumulation after exposure to HFD, and weight and body fat ratio of these animals did not differ from those of the controls. Blood glucose levels did not differ between the groups, but insulin levels increased only in male HFD-exposed offspring from nicotine-administered dams. Similarly, only in HFD-exposed male from nicotine-administered dams showed decreases in the insulin receptor expression in the liver. We conclude that male rats subjected to prenatal nicotine exposure develop hyperinsulinemia when exposed to HFD after growth. Our results suggest that decreased expression of insulin receptors in the liver may be involved in the mechanism underlying hyperinsulinemia in low-birthweight offspring, a phenomenon that appeared to exhibit a sex-specific bias.

Keywords: DOHaD (development origins of health and disease); insulin; nicotine; prenatal; sex-specificity.

Figure 1

Body size and plasma concentrations of GH and IGF-1, and expression of IGF-1 mRNA in the liver. Bodyweight at birthday (A), body length (B) and bodyweight at weaning day (3-week-old) (C) were measured. Plasma concentrations of growth hormone (GH) (D) and insulin-like growth factor (IGF)-1 (E) of offspring from nicotine-administered dams (NIC) and saline-treated dams (Sal) were measured. The mRNA expression levels of Igf1 in the liver of offspring from nicotine-administered dams (NIC) and saline-treated dams (Sal) were quantified (F). The mRNA expression levels were normalized to Gapdh levels and then to that in Sal, which was defined as 100%. Values are presented as means ± SEM (n=8). Statistical analysis was performed using unpaired-T test (A) and one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons (B–F). n.s., not significant (p ≥ 0.05).

Figure 2

Bodyweights and body fat ratio. Plots of the bodyweight before (5-week-old) (A for males and B for females) and after (13-week-old) (C for males and D for females) exposure to the high-fat diet (HFD) or standard chow (SC), and the body fat percentage after exposure to the HFD or SC (13-week-old) (E for males and F for females) are shown. Values are presented as means ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. n.s., not significant (p ≥ 0.05).

Figure 3

Fasting blood sugar and insulin levels. Plasma concentrations of glucose (A for males and B for females) and insulin (C for males and D for females) of standard chow-fed (SC) offspring or high fat diet (HFD)-exposed offspring from nicotine-administered dams (NIC) or saline-treated dams (Sal) were measured (13-week-old). Values are presented as means ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. n.s., not significant (p ≥ 0.05).

Figure 4

Blood sugar and insulin levels after oral glucose challenge. Blood samples were collected 15 min after glucose solution (2g/100g b.w.) ingestion. Their plasma concentrations of glucose (A for males and B for females) and insulin (C for males and D for females) of standard chow-fed (SC) offspring or high fat diet (HFD)-exposed offspring from nicotine-administered dams (NIC) or saline-treated dams (Sal) were measured. Values are presented as means ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. n.s., not significant (p ≥0.05).

Figure 5

Expression of insulin receptor (Insr) in the liver. Insr mRNA expression levels (A for males and B for females) and Insr protein expression levels (C for males and D for females) in the liver of standard chow-fed (SC) offspring or high fat diet (HFD)-exposed offspring from nicotine-administered dams (NIC) or saline-treated dams (Sal) were quantified. The mRNA expression levels were normalized to Gapdh levels and then to that in Sal, which was defined as 100%. The protein expression levels were normalized to that of ß-actin and then to that in Sal, which was defined as 100%. Values are presented as means ± SEM (n=8). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. n.s., not significant (p ≥ 0.05).

Figure 6

Conceptual diagram of the onset of hyperinsulinemia due to embryonic nicotine exposure. Exposure to nicotine in dams causes a decrease in uterine blood flow, and metabolic programming due to hypoxia and malnutrition in the fetus is assumed. Nicotine may directly affect the egg via cord blood, but the effect is unknown in this study. According to DOHaD theory, the mismatch between the predispositions acquired by low-birthweight offspring and the post-growth environment increases the risk of developing the disease. Female hormones (estrogens) may act protectively in the development of diabetes. In our experimental model, it is possible that the decrease in the expression of insulin receptors in the liver was blocked in the female rats compared to that of male rats, but since there was no difference in the DNA methylation of the insulin receptor gene, further analyses are needed to clarify the mechanism underlying the sex-specificity in the future.