Maternal obesity increases hypothalamic miR-505-5p expression in mouse offspring leading to altered fatty acid sensing and increased intake of high-fat food

Abstract

In utero exposure to maternal obesity programs increased obesity risk. Animal models show that programmed offspring obesity is preceded by hyperphagia, but the mechanisms that mediate these changes are unknown. Using a mouse model of maternal obesity, we observed increased intake of a high-fat diet (HFD) in offspring of obese mothers that precedes the development of obesity. Through small RNA sequencing, we identified programmed overexpression of hypothalamic miR-505-5p that is established in the fetus, lasts to adulthood and is maintained in hypothalamic neural progenitor cells cultured in vitro. Metabolic hormones and long-chain fatty acids associated with obesity increase miR-505-5p expression in hypothalamic neurons in vitro. We demonstrate that targets of miR-505-5p are enriched in fatty acid metabolism pathways and overexpression of miR-505-5p decreased neuronal fatty acid metabolism in vitro. miR-505-5p targets are associated with increased BMI in human genetic studies. Intra-cerebroventricular injection of miR-505-5p in wild-type mice increased HFD intake, mimicking the phenotype observed in offspring exposed to maternal obesity. Conversely, maternal exercise intervention in an obese mouse pregnancy rescued the programmed increase of hypothalamic miR-505-5p in offspring of obese dams and reduced HFD intake to control offspring levels. This study identifies a novel mechanism by which maternal obesity programs obesity in offspring via increased intake of high-fat foods.

Fig 1. Offspring of obese mothers eat more of a high-fat pellet and develop increased adiposity compared to offspring of lean mothers fed the same diet.

(a) Diagram depicting mouse model of maternal obesity. At weaning offspring are weaned onto either a chow or HFD, and food intake, body weight, and adiposity are measured weekly. (b) Normal chow daily kilocalorie intake of male offspring born to control (Off-C; n = 21) and obese mothers (Off-Ob; n = 15). (c) HFD pellet daily kilocalorie intake of male offspring born to control (Off-C; n = 22) and obese mother (Off-Ob; n = 19). (d) Sucrose preference of male offspring during a two-bottle choice test (Off-C; n = 6 and Off-Ob; n = 4). (e) Intake (g) of sucrose solution during two-bottle choice test (Off-C; n = 6 and Off-Ob; n = 4). (f) Body weight and (g) fat mass from 4 to 8 weeks of age in offspring weaned onto a chow diet (Off-C; n = 12 and Off-Ob; n = 21). (h) Body weight and (i) fat mass from 4 to 8 weeks of age in offspring weaned onto an HFD (Off-C; n = 12 and Off-Ob; n = 13). **P < 0.01, ***P < 0.001. Statistical significance was determined using mixed effects analysis with Sidak’s post hoc test. The underlying data are provided as S1 Data file. HFD, high-fat diet.

Fig 2. Hypothalamic miR-505-5p expression is increased in offspring of obese mothers from fetal to adult life and in vitro in neurons by exposure to metabolic hormones and fatty acids.

(a) Sequence overlap of mature miR-505-5p in mice and humans. (b, c) Volcano plot of small RNA sequencing results showing significant overexpression of miR-505-5p (red dot surrounded by blue diamond) in the ARC (b) and PVH (c) of 8-week-old offspring born to obese mothers compared to offspring of lean mothers. (d–f) qPCR analysis of miR-505-5p expression in (d) ARC at 8 weeks of age; (e) in whole hypothalamus at E13; (f) in hypothalamic neural progenitor cells extracted at E13 and cultivated as neurospheres for 8 days in vitro. (g) Atp11c (miR-505-5p host gene) mRNA levels in E13 whole hypothalamus. (h) miR-505-5p relative expression of mHypoE-N46 cells exposed to insulin or leptin. (i) miR-505-5p relative expression of mHypoE-N46 cells exposed to oleic acid, palmitic acid, stearic acid, linoleic acid, and glucose for 24 h. (j) miR-505-5p relative expression of C8-D1A (astrocyte) cells exposed to oleic acid, palmitic acid, stearic acid, and glucose for 24 h. (k) miR-505-5p relative expression in MBH in lean, diet-induced obese or genetically obese (Ob/Ob) male mice.* P < 0.05, **P < 0.01. Statistical significance was determined with unpaired t test. Outliers were excluded from (g) for obese group (outlier excluded value 0.878) and (j) for oleic acid treatment (outlier excluded value 1.554). The underlying data are provided as S1 Data file. ARC, arcuate nucleus of hypothalamus; DIO, diet-induced obese; MBH, mediobasal hypothalamus; NPC, neural progenitor cell; PVH, paraventricular nucleus of hypothalamus.

Fig 3. miR-505-5p targets are involved in FA metabolism and genetically associated with BMI in humans.

(a) Diagram of pulsed SILAC experiment in GT1-7 cells to identify targets of miR-505-5p. (b) Cumulative fraction analysis of lys8-labelled: lys4-labelled proteins ratio according to occurrence and type of mRNA binding site for miR-505-5p, as predicted by TargetScan (v. 7.2). (c) Workflow used to identify direct targets of miR-505-5p. (d) Top regulated pathways among miR-505-50 direct targets as reported in IPA. (e) miR-505-5p target proteins of interest identified through IPA and manual search of pSILAC data. (f) Radiolabelled oleic acid metabolism into acid-soluble metabolites and oleic acid uptake in mHypoE-N46 following transfection with either a miR-505-5p mimic or control for 48 h. (g) Heatmap showing the overlap between human orthologs of identified miR-505-5p FA metabolism-related target genes in mice and human genetic datasets. Target genes were annotated on the basis of (i) proximity to GWAS signals, (ii) aggregate gene-level associations to the trait, (iii) colocalization between the GWAS and eQTL data, and (iv) the presence of known enhancers within the GWAS signals. Total number of the observed concordant predictors (out of 4) is displayed in the summary panel. Expanded results can be found in S4 Table. *p < 0.05, ** p < 0.01, *** p < 0.001. Statistical significance was determined by unpaired T test. The underlying data are provided as S1 Data file. BMI, body mass index; eQTL, expression quantitative trait loci; FA, fatty acid; GWAS, genome-wide association study; IPA, Ingenuity Pathway Analysis.

Fig 4. miR-505-5p target genes related to FA sensing are down-regulated after treatment with miR-505-5p in vitro and in fetal hypothalamus of offspring from obese mothers.

(a) Relative mRNA expression of genes related to lipid metabolism in mHypoE-N46 cells after treatment with a miR-505-5p mimic and (b) in the hypothalamus of fetuses from control or obese pregnancy at E13 * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance was determined by unpaired T test. Outliers were excluded from (a) for miR-505-5p mimic (Cpt1a outlier excluded value 1.19, Soat1 outlier excluded value 1.71). The underlying data are provided as S1 Data file. FA, fatty acid.

Fig 5. ICV injection of miR-505-5p in adult mice increases intake of an HFD, while a maternal exercise intervention corrects offspring hypothalamic miR-505-5p levels and reduces intake of an HFD.

(a) Diagram depicting surgical schedule. Mice were allowed to recover for 5 days post-implanted of an ICV cannula into the lateral ventricle. Mice then received injection of either a vehicle or miR-505-5p mimic into the ICV cannula and food intake was measured over 3 days. (b) qPCR confirming overexpression of miR-505-5p in the hypothalamus after ICV injection of miR-505-5p mimic. (c) Chow diet cumulative food intake after 3 consecutive days of treatment (24–1 dose, 48–2 doses, and 72 h—3 doses—after first treatment). (d) HFD cumulative food intake after 3 consecutive days of treatment (24–1 dose, 48–2 doses, and 72 h—3 doses—after first treatment). (e) Graphical depiction of maternal obesity model with third group of dams that underwent moderate exercise intervention during obese pregnancy. (f) Relative expression of miR-505-5p in the arcuate nucleus of 8-week-old male offspring born to control, obese or obese-exercised dams. (g) HFD pellet daily kilocalorie intake of male offspring born to control, obese or obese-exercised dams. * p < 0.05, ** p < 0.01. Statistical significance was determined by unpaired T test (c and d), one-way ANOVA (f), and mixed effects analysis with Sidak post hoc test (g). The underlying data are provided as S1 Data file. HFD, high-fat diet; ICV, intra-cerebroventricular.