Background

Developmental programming of disease is now a well-recognized phenomenon. Our findings show that early life exposure to a low n6/n3 polyunsaturated fatty acid (PUFA) ratio programs neonatal adipose tissue expansion (ATE) and adult resistance to diet induced obesity. Very little is known about the mRNA patterns and cellular diversity of neonatal adipocyte precursor cells (APCs) that establish the fat depots during development.

Methods

Unrelated wildtype (WT) pups were cross-fostered onto WT or fat-1 damsoverexpressing an n3 PUFA desaturase that depletes n6 and bolsters n3 PUFA in milk. APCs were sorted by flow cytometry for single cell RNA sequencing (scRNA-seq). scRNA-seq data was visualized by t-distributed Stochastic Neighbor Embedding (t-SNE), density-based clustering, and Discriminative Dimensionality Reduction Tree (DDRTree). Differential genes were identified by two-sided Student’s t-test. Stable isotope incorporation quantified in vivo APC proliferation and live cell imaging was used to quantify in vitro proliferation/differentiation.

Results

scRNA-seq analysis revealed differential expression of principle regulators of adipogenesis (KLF2, SREBP1, CEBPa, and PPARg) by low n6/n3 exposure group. Low n6/n3 exposure over 12-days of life increased the fraction of committed preadipocytes (P=0.05), with no difference for in vivo AP cell proliferation, and made smaller and more numerous mature adipocytes. Low n6/n3 exposure induced insulin sensitive (IR, IGFR2, Akt1, PKA and RXR/RAR) and antiadipogenic (WNT/CTNN2, NR2F2, FGF1) mRNAs. In vitro, flow sorted APCs from low n6/n3 pups had significantly slower proliferation (P=0.001) and differentiation (P<0.001).

Conclusions

Our findings indicate that low n6/n3 ratio exposure via milk tempers offspring adipogenic potential in APCs by differential regulation of key adipogenic regulators. By upregulating both pro- and antiadipogenic mRNA programs, low n6/n3 exposure changes the APC subtype diversity in offspring.