Relationship between insulin and Netrin-1/DCC guidance cue pathway regulation in the prefrontal cortex of rodents exposed to prenatal dietary restriction

Abstract

Fetal restriction (FR) alters insulin sensitivity, but it is unknown how the metabolic profile associated with restriction affects development of the dopamine (DA) system and DA-related behaviors. The Netrin-1/DCC guidance cue system participates in maturation of the mesocorticolimbic DA circuitry. Therefore, our objective was to identify if FR modifies Netrin-1/DCC receptor protein expression in the prefrontal cortex (PFC) at birth and mRNA in adulthood in rodent males. We used cultured HEK293 cells to assess if levels of miR-218, microRNA regulator of DCC, are sensitive to insulin. To assess this, pregnant dams were subjected to a 50% FR diet from gestational day 10 until birth. Medial PFC (mPFC) DCC/Netrin-1 protein expression was measured at P0 at baseline and Dcc/Netrin-1 mRNA levels were quantified in adults 15 min after a saline/insulin injection. miR-218 levels in HEK-293 cells were measured in response to insulin exposure. At P0, Netrin-1 levels are downregulated in FR animals in comparison to controls. In adult rodents, insulin administration results in an increase in Dcc mRNA levels in control but not FR rats. In HEK293 cells, there is a positive correlation between insulin concentration and miR-218 levels. Since miR-218 is a Dcc gene expression regulator and our in vitro results show that insulin regulates miR-218 levels, we suggest that FR-induced changes in insulin sensitivity could be affecting Dcc expression via miR-218, impacting DA system maturation and organization. As fetal adversity is linked to nonadaptive behaviors later in life, this may contribute to early identification of vulnerability to chronic diseases associated with fetal adversity.

Keywords: Axonal guidance cues; Food restriction; Insulin; Intrauterine growth restriction; Mesocorticolimbic dopamine pathway; Prenatal adversity.

Figure 1.

PFC Netrin-1 and DCC expression in PND0 males. Levels of Netrin-1 and DCC receptors in the PFC were measured in AdLib and FR animals of age PND 0. (a) Netrin-1 protein levels were found to be downregulated in the PFC of FR animals when compared to AdLib animals (W(8) = 51; p = 0.0499). (b) DCC protein expression was not significantly different in the PFC of FR animals when compared to AdLib animals (t(14) = −1.25; p = 0.231).

Figure 2.

PFC Netrin-1 and Dcc mRNA expression in PND 100 males. Levels of Netrin-1 and Dcc in the PFC were measured 15 min after a peripheral saline or insulin injection in AdLib and FR animals. (a) There were no significant differences in Netrin-1 mRNA levels across the four groups: AdLib Saline, AdLib Insulin, FR Saline, and FR Insulin (F(3,33) = 0.066, p = 0.98). (b) There was a significant difference in Dcc mRNA levels over groups (F(3,33) = 4.41, p = 0.0103). There is a significant upregulation of Dcc mRNA in AdLib animals treated with an insulin injection when comparing to AdLib animals administered a saline injection (p = 0.008) and a significant downregulation in Dcc mRNA levels in FR animals treated with the saline injection when compared to the AdLib animals given the insulin injection (p = 0.032).

Figure 3.

Insulin regulates miR-218 expression in a concentration-dependent manner. miR-218 expression was measured in response to four different insulin concentrations in addition to the control group (UNT): 0 nM, 50 nM, 100 nM, 200 nM. There was a significant difference in miR-218 expression across groups (F(4,10) = 4.394, p = 0.0262). miR-218 levels were upregulated at 100 nM of insulin when compared to the control group.

Figure 4.

Effects of insulin signaling on miR-218 levels over time. Fold change in miR-218 were measured in response to 100 nM of insulin over a time curve at five different time points in addition to the control group (UNT): 0 min, 10 min, 30 min, 60 min, and 360 min. There was a significant difference in miR-218 expression across groups (F(5,12) = 27.26, p < 0.001). miR-218 levels were upregulated at 10 min, 30 min, 60 min, and 360 min in comparison to the control group.