Figure 1.
(A) The mRNA was measured via qRT-PCR in the PVN of P15 offspring prenatally exposed to high-fat diet (HFD) or chow, and a statistically significant decrease was found in YAP expression from offspring that were prenatally exposed to high-fat diet (HFD) compared to chow. (B) The mRNA was measured in whole hypothalamic tissue of E19 embryos prenatally exposed to HFD or chow, and a statistically significant decrease was observed for both TEF and YAP expression from HFD embryos compared to chow. The mRNA levels were normalized to cyclophilin and calculated using the CT comparative method. Results are expressed as means ± SEM (n = 6–9). *p < 0.05, compared with chow. YAP = Yes-associated protein; TEF = transcription enhancing factor.
Figure 2.
Protein levels in P15 offspring.
(A, C) TEF protein level was measured via western blot in the PVN of high-fat diet (HFD) compared to chow P15 offspring, and a statistically significant increase in total TEF was found. (B, C) Further immunoprecipitation (IP) of total TEF protein from the PVN revealed a statistically significant increase in levels of phosphorylated TEF and ubiquitin-tagged TEF. (D, E) YAP protein level was measured via western blot in the PVN of high-fat diet (HFD) compared to chow P15 offspring, and a statistically significant decrease in total YAP and phosphorylated YAP protein was found. Results are expressed as means ± SEM (n = 4) *p < 0.05. The β-actin bands were used to ensure that all samples had the same concentration of protein; they were not used to normalize TEF and YAP bands. The change in TEF and YAP band densities from the HFD group was determined relative to the chow control group. p-TEF = phosphorylated TEF; ub-TEF = ubiquitin-tagged TEF.
Figure 3.
Protein levels in E19 embryos.
(A, C) TEF protein level was measured via western blot in the PVN of high-fat diet (HFD) compared to chow E19 embryos, and a statistically significant increase was found for total TEF protein levels in whole hypothalamus. (B, C) Further immunoprecipitation (IP) of total TEF protein from whole hypothalamus revealed a statistically significant increase in levels of phosphorylated TEF and ubiquitin-tagged TEF. (D, E) YAP protein level was measured via western blot in whole hypothalamus of high-fat diet (HFD) compared to chow E19 embryos, and a statistically significant decrease in total YAP and phosphorylated YAP protein was found. Results are expressed as means ± SEM (n = 4). *p < 0.05. The β-actin bands were used to ensure that all samples had the same concentration of protein; they were not used to normalize TEF and YAP bands. The change in TEF and YAP band densities from the HFD group was determined relative to the chow control group.
Figure 4.
Isolated hypothalamic neurons from E19 embryos.
Single-labeling immunocharacterization of dissociated hypothalamic neurons from chow embryos expressing (A) TEF, (B) YAP, or (C) ENK; and from HFD embryos expressing (D) TEF, (E) YAP, or (F) ENK. (G). In the HFD compared to chow embryos, there was a statistically significant decrease in the number of YAP-labeled neurons and an increase in the number of TEF- and ENK-labeled neurons. Top set of images is neurons from chow embryos, bottom set is neurons from HFD embryos. Arrows point to the same cell in the top and bottom image (DIC and fluorescence). Insert shows larger image of neuron with either cytoplasmic or nuclear localization of TEF or YAP. Results are expressed as means ± SEM (n = 4). *p < 0.05, compared with chow. ENK = enkephalin; scale bar is 25 µM.
Figure 5.
Probability density histogram.
The fluorescence intensity for each cell from chow (top) or HFD (bottom) embryos was normalized by subtracting the mean background intensity of a nearby area of comparable size and multiplying the mean intensity within the region by its area, as described in the methods, plotted as a histogram, and fitted to a probability density function. The area under the curve represents the density of cells within each population and is portrayed as a percentage of the total area. (A) Two populations of TEF-expressing neurons were found from chow embryos, arbitrarily assigned as L and M representing low and medium peptide levels, and two were found from HFD embryos, assigned as M and H representing medium and high peptide levels. This shift in fluorescence intensity suggests that HFD increased the levels of TEF in each individual neuron. (B) Two populations of YAP-expressing neurons were found from chow embryos, arbitrarily assigned as H1 and H2 representing high levels of peptides, and two populations were found in neurons from HFD embryos, L1 and L2, representing low levels of peptides. A shift from higher to lower intensity was found in HFD compared with chow embryos, suggesting decreased YAP levels in individual neurons; (C) Three populations of ENK-expressing neurons with the same intensity, L, M and H, were found in both HFD and chow embryos, with a shift in the percentage of neurons exhibiting low intensity to medium and high intensity in the HFD embryos, suggesting increased ENK levels in individual neurons. Goodness of fit was determined based on X2 values corresponding to p < 0.05.
Figure 6.
Co-localization of TEF or YAP with ENK.
Immunocharacterization of dissociated hypothalamic neurons from E19 embryos co-labeled with (A) TEF and ENK and (B) YAP and ENK. (C) In HFD embryos, there was a significant decrease in the density of co-labeled TEF and ENK neurons but no change in the density of co-labeled YAP and ENK neurons. (D) Knockdown of either TEF or YAP with siRNA results in a significant increase in ENK expression. Representative images of neurons are from HFD embryos. Arrows point to the same cell from images left to right (DIC, fluorescence and merged images). Results are expressed as means ± SEM (n = 4). *p < 0.05, compared with chow. TF = transcription factor; KD = knockdown; scale bar is 25 µM.