Neonatal intake of Omega-3 fatty acids enhances lipid oxidation in adipocyte precursors

Abstract

Establishing metabolic programming begins during fetal and postnatal development, and early-life lipid exposures play a critical role during neonatal adipogenesis. We define how neonatal consumption of a low omega-6 to -3 fatty acid ratio (n6/n3 FA ratio) establishes FA oxidation in adipocyte precursor cells (APCs) before they become adipocytes. In vivo, APCs isolated from mouse pups exposed to the low n6/n3 FA ratio had superior FA oxidation capacity, elevated beige adipocyte mRNAs Ppargc1α, Ucp2, and Runx1, and increased nuclear receptor NR2F2 protein. In vitro, APC treatment with NR2F2 ligand-induced beige adipocyte mRNAs and increased mitochondrial potential but not mass. Single-cell RNA-sequencing analysis revealed low n6/n3 FA ratio yielded more mitochondrial-high APCs and linked APC NR2F2 levels with beige adipocyte signatures and FA oxidation. Establishing beige adipogenesis is of clinical relevance, because fat depots with energetically active, smaller, and more numerous adipocytes improve metabolism and delay metabolic dysfunction.

Keywords: Biological sciences; Endocrine regulation; Endocrinology.

Graphical abstract

Figure 1

Cross-fostered pups have reduced n6/n3 PUFA ratios, activated Acylation Stimulating Protein (ASP), and Retinol Binding Protein 4 (RBP4) in circulation (A) Study design to test milk-specific n6/n3 PUFA ratio effects on adipogenesis. (B) Milk and serum levels of fatty acids and their ratios were quantified by lipid mass spectrometry collected from individual dam’s milk (n = 5/group) and the pooled serum of their cross-fostered litters (n = 5 litter dyads per n6/n3 FA ratio exposure) (See also Figure S1). (C) Circulating factors in PND12 serum that stimulate differential adipogenic responses based on high or low n6/n3 PUFA in vivo exposure (See also Data S1). ELISAs for activated Acylation Stimulating Protein (ASP), Retinol Binding Protein 4 (RBP4), and insulin in an independent cohort of cross-fostered PND12 pup sera (n = 5-8 litter dyads per n6/n3 FA ratio exposure). Data are represented as mean ± SEM.

Figure 2

Cross-fostered pups have lower percent body fat and smaller, multilocular adipocyte cellularity (A) Body fat mass, lean mass, and total body mass of cross-fostered litters containing male and female pups following neonatal adipogenesis in the presence of high or low n6/n3 PUFA in circulation (n = 6-8 litters per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter). (B) Representative H & E images of SWAT depots following 10 days of exposure to either high or low n6/n3 PUFA ratio. (C) Scale weights of the dissected SWAT following of 12-day od cross-fostered neonates from high or low n6/n3 PUFA ratio exposure, and D) quantification of the adipocyte cellularity within the SWAT following 10-day of n6/n3 PUFA exposure (n = 5 neonatal SWAT sections from independent dam/litter dyads per n6/n3 group). Data are represented as mean ± SEM.

Figure 3

Primary flow-sorted APCs programmed by low n6/n3 FA ratio exposure have reduced white adipose differentiation in vitro, greater FA oxidation, and increased mitochondrial potential (A) Subcutaneous WAT was enzymatically digested to produce a stromal vascular pellet and Fluorescence Assisted Cell Sorting (FACS) was used to isolate CD24⁺ progenitor from CD24⁻preadipocyte APCs. Deuterium incorporation into the DNA in vivo indicated the rate of APC proliferation is not different, whole SWAT tended to be lower (p = 0.07), and a large variance was observed in the whole blood DNA (n = 5-7 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter). APC population analysis indicated no overt difference in CD24⁺ progenitors and CD24⁻preadipocytes, and a trend toward the fraction of committed preadipocytes in the low n6/n3 PUFA group (n = 7-8 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter). (B) Sca-1+ APCs plated for live cell imaging using Incucyte grew more slowly and accumulated less neutral lipid when differentiated to white adipocytes (n = 3 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter), suggesting the potential for a soluble inhibitory factor that curtails white adipocyte differentiation under low n6/n3 in vivo programming. (C-F) Seahorse substrate oxidation assays for primary Sca1+ APCs isolated from low or high n6/n3 ratio exposure (n = 3 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter). Maximal and spare respiration rates from C) exogenous palmitate oxidation, D) glucose, and E) glutamine in low and high n6/n3 in vivo programmed primary APCs measured by Seahorse. F) Kinetic data of exogenous palmitate, glucose, and glutamine oxidation assays in C, D, and E. (G) TMRE live cell mitochondrial potential dye in primary APCs programmed in vivo by low and high n6/n3 FA ratio exposure at 24 h post plating (n = 3 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter). Scale bar represents 75 μm. Data are represented as mean ± SEM.

Figure 4

Bulk RNA-seq of flow-sorted APC CD24⁺ progenitors and CD24⁻preadipocytes reveals transcriptomic signatures for adipogenic, mitochondrial, and energetics pathways by low n6/n3 FA ratios (A) Differential gene expression by n6/n3 exposure group within either progenitor or preadipocyte APC subtypes was analyzed using comparative analysis in Ingenuity Pathway Analysis. Significantly enriched canonical pathways in common between CD24⁺ progenitor and CD24⁻preadipocyte APCs were ordered by FDR p value (≤0.01) and colored by the predicted activation Z score. Coloring indicates common pathways predicted to be “on” (yellow), “off” (navy), or “no prediction” (white) due to low n6/n3 exposure. Pathway activation Z score values are presented for either APC subtype, and values ≥2.0 or ≤ −2.0 are considered significant. (B) Significantly different genes in either CD24⁺ progenitor or CD24⁻preadipocyte relative to the high n6/n3 FA ratio group colored by Log2 fold change and grouped into transcription factor genes (Regulatory) and signal transduction pathway genes (Ligands/Kinases). (C) Significantly different lipid metabolism, mitochondrial, and glycolytic genes indicating that the low n6/n3 FA ratio changes oxidative capacity potentially through aldehyde dehydrogenases (Aldh1a1 and Aldh1a3), uncoupling protein 2 (Ucp2), citrate synthase (CS), isocitrate dehydrogenase (Idh1 and 2), malic enzyme (Me1), malate dehydrogenase (Mdh1), phosphofructokinases (Pfkm, Pfkp, and Pfkfb1), and transketolase (Tk) (See also Figure S2). (D) Canonical Pathway enrichment within either CD24⁺ progenitor or CD24⁻preadipocyte APCs plotted by the percentage DE-Gs within a pathway (%) by the pathway enrichment significance (-log(p value)) and colored by the predicted pathway activation (Z score, >2.0 and < −2.0 was considered significant) (See also Data S3 for all pathways). (E) Immunoblots for NR2F2, UCP2, C/EBPα, and DNMT1 from all lineage negative, CD29+/CD34+, and Sca1+ APCs isolated by flow cytometry from pooled litters (n = 3 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter). (F) EVC005 murine APC cell line was treated with and without NR2F2 ligand 1-DSO for 48 h, which induced gene expression of Prdm16, P2rx5, Runx1, Ucp2, Dnmt1, Pparγ2, and C/ebpα. (G) TMRE live cell mitochondrial potential dye following 48 h of 1-DSO treatment in EVC005 cells, indicating an increased mitochondrial potential. Panels F and G are n = 3 wells per treatment group. Scale bar represents 50 μm. Data are represented as mean ± SEM.

Figure 5

Sca1+ primary APCs single-cell RNA sequencing reveals population differences in APC subtypes (A) UMAP of the scRNA-seq dataset indicating that exposure to a low n6/n3 ratio produced overlapping populations of primary APCs. (B) Cluster markers were calculated and principal genes within each cluster were compared to the literature to identify the APC subtypes present (See also Figures S3 and S4, and Data S4). (C) The distribution of APC subtype within each cluster by n6/n3 FA ratio treatment UMAPs. (D) The percent difference of APC subtypes within each cluster was calculated (relative to high n6/n3) and indicates populational differences in progenitor cluster 2, regulatory cluster 6, and mitochondrial high cluster 13. (E) Pseudotime analysis indicated two branchpoints (a and b) that lead to a small pool of immature adipocytes (cluster 10 in panel B), and the other that branches three ways within the regulatory APCs leading to the preadipocyte APCs. (F) RNA expression trendlines for key adipogenic regulators along pseudotime analysis, indicating a bimodal regulation of Pparγ and C/Ebpα, while adipogenic inhibitor, Klf2, is more highly expressed in the low n6/n3 APCs along pseudotime.

Figure 6

Differential gene expression within clusters by n6/n3 FA ratio group in Sca1+ adipocyte precursors Genes that were significantly different by n6/n3 exposure were plotted by dot plot for each cluster. The dot size represents the percentage of cells within a cluster expressing a given gene, and the coloring of each dot represents the mean expression of a given cluster relative to the maximum expression for each gene. The canonical Wnt signaling inhibitor Secreted frizzled-related protein 4 (Sfrp4) and an inactivator of lipoprotein lipase Angiopoietin-related protein 4 (Angptl4) were consistently downregulated by low n6/n3 exposure in clusters 1, 3, 5, 6, 8, and 9. Adipogenic transcriptional regulators, Krueppel-like factors 2 and 4, were significantly induced by low n6/n3 exposure but differences were confined to the progenitor clusters (cluster 1 and 3, respectively). No DE-Gs by n6/n3 FA ratio were observed in clusters 10, 11, 13, or 13, which are not shown. (See also Figure S5).

Figure 7

Model of molecular and cellular diversity of APC subtypes regulated by low n6/n3 exposure during postnatal development (A) Integration of bulk- and scRNA-sequencing observations for adipogenesis-related genes that are up- (red) or down- (blue) regulated in vivo by low n6/n3 FA ratio exposure. This transcriptomic signature may establish a cellular metabolism with a greater fatty acid oxidation capacity prior to functional differentiation into a mature adipocyte. (B) Populations of APC subtypes, such as the balance among regulatory APCs and the number of mitochondrial high APCs, may influence the types of functional mature adipocytes that arise from the APCs. Additionally, the overall proportion of the committed CD24⁻preadipocyte population that expands from the CD24⁺ APCs provides a ready supply of preadipocytes that can functionally differentiate into smaller, more numerous mature adipocytes. Artwork created with BioRender.com.