Maternal high-fat diet and fetal programming: increased proliferation of hypothalamic peptide-producing neurons that increase risk for overeating and obesity

Abstract

Recent studies in adult and weanling rats show that dietary fat, in close association with circulating lipids, can stimulate expression of hypothalamic peptides involved in controlling food intake and body weight. In the present study, we examined the possibility that a fat-rich diet during pregnancy alters the development of these peptide systems in utero, producing neuronal changes in the offspring that persist postnatally in the absence of the diet and have long-term consequences. The offspring of dams on a high-fat diet (HFD) versus balanced diet (BD), from embryonic day 6 to postnatal day 15 (P15), showed increased expression of orexigenic peptides, galanin, enkephalin, and dynorphin, in the paraventricular nucleus and orexin and melanin-concentrating hormone in the perifornical lateral hypothalamus. The increased density of these peptide-expressing neurons, evident in newborn offspring as well as P15 offspring cross-fostered at birth to dams on the BD, led us to examine events that might be occurring in utero. During gestation, the HFD stimulated the proliferation of neuroepithelial and neuronal precursor cells of the embryonic hypothalamic third ventricle. It also stimulated the proliferation and differentiation of neurons and their migration toward hypothalamic areas where ultimately a greater proportion of the new neurons expressed the orexigenic peptides. This increase in neurogenesis, closely associated with a marked increase in lipids in the blood, may have a role in producing the long-term behavioral and physiological changes observed in offspring after weaning, including an increase in food intake, preference for fat, hyperlipidemia, and higher body weight.

Figure 1.

Prenatal HFD in both the HFD and HFD-BD groups compared with the BD group (n = 6–8/diet group) produced behavioral, physiological, and neurochemical changes (mean ± SEM) in the male offspring after weaning, as indicated by a significant increase (*p < 0.05) in measures of body weight or daily caloric intake on day 30 (D30) and day 70 (D70) and of preference (% of total diet) for fat versus carbohydrate on D50–D60 (A); triglyceride, NEFA, galanin mRNA, and peptide levels in the PVN, and body fat pad weights on D70 (B); and leptin and insulin levels, with no change in glucose, CORT, and NPY on D70 (C).

Figure 2.

Prenatal HFD in both the HFD and HFD-BD groups compared with the BD group (n = 4–6/age/diet group) produced changes in gene expression (mean ± SD) of orexigenic peptides in the PVN and PFLH on postnatal day 15 (P15), as measured by real-time quantitative PCR. This is indicated by a significant increase (*p < 0.05) in both prenatal HFD groups of GAL, ENK, and DYN mRNA levels in the PVN and ORX and MCH mRNA in the PFLH, along with a decrease in peptide mRNA in the ARC (A); and circulating levels of triglycerides, with no change in body weight or leptin and increased levels only in the HFD group of NEFA, glucose, insulin, and CORT (B).

Figure 3.

Prenatal HFD in both the HFD and HFD-BD groups compared with the BD group (n = 4–6/diet group) produced changes in the orexigenic peptides (mean ± SD) in the PVN and PFLH of postnatal offspring, as measured by different techniques. This is indicated by a significant increase (*p < 0.05) in mRNA levels (% of BD group) of GAL, ENK, and DYN in the PVN and ORX and MCH in the PFLH at P15, as measured by radiolabeled in situ hybridization with ³⁵S-labeled probes (A); photomicrographs illustrating this effect in the PVN and PFLH of the HFD-BD offspring compared with the BD (B); and a significant increase (*p < 0.05) in peptide levels in the PVN, as measured by radioimmunoassay in P15 offspring, or in the PFLH, as measured by immunofluorescence in P21 offspring (C).

Figure 4.

Prenatal HFD compared with BD offspring (n = 4–6/diet group) produced changes in orexigenic peptide mRNA in the PVN and PFLH at birth (P0), as measured using in situ hybridization with digoxigenin-labeled probes. This is indicated by a significant increase (*p < 0.01) in mRNA levels (mean ± SEM) of GAL, ENK, and DYN in the PVN and of ORX and MCH in the PFH and LH (A); and photomicrographs of GAL mRNA in the anterior PVN outlined by a dash line and ORX mRNA in the PFLH just dorsal and lateral to the fornix (F) (scale bar: 100 μm) (B).

Figure 5.

Prenatal HFD stimulated cell proliferation and neurogenesis in the PVN and PFLH of postnatal offspring compared with BD (n = 4–6/diet group). This is indicated by a significant increase (*p < 0.01) in BrdU⁺ cell density in the PVN and PFLH of the HFD versus BD offspring at P0, from dams receiving i.p. injections of BrdU from E11 to E13, E13 to E14, and E14 to E15 (mean ± SEM) (A); photomicrographs illustrating BrdU⁺ cells in the PVN (dash line) and PFLH around the fornix (F) of P0 offspring (B); and a significant increase (*p < 0.01) in double-labeled BrdU⁺/NeuN⁺ cells in the PVN (scale bar: 100 μm) and PFLH (scale bar: 50 μm) of HFD and HFD-BD offspring at P8, presented as percentage of double-labeled neurons relative to total single-labeled BrdU⁺ cells or NeuN⁺ neurons (mean ± SEM) (C).

Figure 6.

Prenatal HFD increased neurogenesis in the PVN, specifically neurons that express orexigenic peptides. This is shown by an increase (*p < 0.01) in the HFD and HFD-BD versus BD offspring at P8 in double labeling of GAL, ENK, and DYN mRNA with BrdU in PVN neurons, as revealed by peptide digoxigenin-labeled in situ hybridization histochemistry and BrdU immunofluorescence [data (mean ± SEM) are expressed as the percentage of double-labeled cells relative to the total single-labeled BrdU⁺ cells (left) or peptide⁺ neurons (right)] (A); and photomicrographs of HFD-BD versus BD P8 offspring illustrating single-labeled GAL-expressing neurons (black), single-labeled BrdU-immunoreactive cells (red), and double-labeled GAL⁺ + BrdU⁺ neurons (red + black) in the PVN (scale bars: 50 μm for columns 1–3; 100 μm for column 4) (B). Insets (scale bar: 200 μm) provide examples of double-labeled GAL + BrdU neurons, identified by an arrow.

Figure 7.

Prenatal HFD increased neurogenesis in the PFLH, specifically neurons that express orexigenic peptides. This is shown by an increase (*p < 0.01) in the HFD and HFD-BD versus BD offspring at P21 in double labeling of ORX and MCH peptide with BrdU in PFLH neurons, as revealed by double-labeling immunofluorescence [data (mean ± SEM) are expressed as the percentage of double-labeled cells relative to the total single-labeled BrdU⁺ cells (left) or peptide⁺ neurons (right)] (A); and photomicrographs of HFD-BD versus BD P21 offspring illustrating single-labeled ORX-synthesizing neurons (green), single-labeled BrdU-immunoreactive cells (red), and double-labeled ORX⁺ + BrdU⁺ neurons (green + red) in the PFLH (scale bar: 100 μm), in the area of the fornix (F) (B). Examples of double-labeled neurons are indicated by arrows.

Figure 8.

Embryos of HFD dams at E14 exhibited an increase in neurogenesis compared with embryos of BD dams (n = 4–6/diet group). A, The dash box in the coronal section shows the ventral neuroepithelial lobe (VL) of the hypothalamic third ventricle (3v) and the surrounding hypothalamic area (HYP). B, C, The enhanced neurogenesis in HFD embryos is demonstrated by double-labeling immunofluorescence revealing an increase (*p < 0.05) in the percentage of BrdU⁺/NeuN⁺ newborn neurons relative to the total number of single-labeled BrdU⁺ cells (left) or NeuN⁺ neurons (right) in the VL and HYP (mean ± SEM) (B); and immunofluorescence photomicrographs of HFD versus BD embryos illustrating an increase in BrdU⁺ cells (red, top panel), NeuN⁺ neurons (green, middle panel), and double-labeled BrdU⁺ + NeuN⁺ newborn neurons (yellow, bottom panel) in the VL, with a small effect in the HYP adjacent to the VL (scale bar: 100 μm) (C). The inset in bottom panel for HFD embryo, showing an enlargement of the area indicated by a box, illustrates examples of double-labeled neurons (yellow) detected in the VL.

Figure 9.

Embryos of HFD dams at E14 exhibited an increase in the development of neuronal precursor cell phenotype compared with embryos of BD dams (n = 4–6/diet group). This is demonstrated by single-labeling immunofluorescence, which revealed the following in the HFD versus BD embryos: an increased density (*p < 0.05) in the HFD embryos of Dcx⁺ neurons in the ventral neuroepithelial lobe (VL) of the third ventricle (3v) and surrounding hypothalamic (HYP) area (left) [this is illustrated to the right by photomicrographs of Dcx⁺ neurons (green) that are considerably more dense in the HYP while still evident in the VL of HFD embryos but not BD embryos (scale bar: 200 μm)] (A); and an increased density (*p < 0.05) of TuJ1⁺ neuronal precursor cells in the VL and HYP (left), as illustrated to the right by photomicrographs of TuJ1⁺ cells (red) in these areas surrounding the 3v (scale bar: 100 μm) (B). C, Analyses of serum from pregnant dams (E14–E18) and newborn offspring (P0) revealed significantly elevated (*p < 0.01) circulating levels of triglycerides and NEFA. Data are mean ± SEM.

Figure 10.

This diagram provides a schematic representation of the effects that prenatal HFD exposure has on circulating lipids, neuronal proliferation, differentiation, and migration, peptide gene expression in the PVN and PFLH, and the physiological and behavioral phenotype of the male offspring after weaning through adolescence. 3v, Third ventricle; AH, anterior hypothalamus; IL, inferior neuroepithelial lobule; PH, posterior hypothalamus.