Perinatal overnutrition exacerbates adipose tissue inflammation caused by high-fat feeding in C57BL/6J mice

Abstract

Obesity causes white adipose tissue (WAT) inflammation and insulin resistance in some, but not all individuals. Here, we used a mouse model of early postnatal overfeeding to determine the role of neonatal nutrition in lifelong WAT inflammation and metabolic dysfunction. C57BL/6J mice were reared in small litters of 3 (SL) or normal litters of 7 pups (NL) and fed either regular chow or a 60% high fat diet (HFD) from 5 to 17 weeks. At weaning, SL mice did not develop WAT inflammation despite increased fat mass, although there was an up-regulation of WAT Arg1 and Tlr4 expression. On HFD, adult SL mice had greater inguinal fat mass compared to NL mice, however both groups showed similar increases in visceral fat depots and adipocyte hypertrophy. Despite the similar levels of visceral adiposity, SL-HFD mice displayed greater impairments in glucose homeostasis and more pronounced hepatic steatosis compared to NL-HFD mice. In addition, WAT from SL mice fed a HFD displayed greater crown-like structure formation, increased M1 macrophages, and higher cytokine gene expression. Together, these data suggest that early postnatal overnutrition may be a critical determinant of fatty liver and insulin resistance in obese adults by programming the inflammatory capacity of adipose tissue.

Fig 1. Neonatal overfeeding promotes rapid weight gain and increases adiposity without causing WAT inflammation at weaning.

A: Pre-weaning growth curves (body weights) of mice raised in normal litters (NL) or small litters (SL) ( n = 19–25 per group from ≥ 9 litters). B-C: Mass (B) and mean adipocyte areas (C) of epididymal (eWAT) and subcutaneous (sWAT) adipose tissue of P21 SL and NL mice ( n = 4–5 per group from ≥ 5 litters). D-E: Representative images showing adipocyte morphology (immunostained for perilipin, green fluorescence) and F4/80-immunoreactive cells (red fluorescence) in eWAT (D) and sWAT (E) of NL and SL mice at P21. F: Quantification of F4/80-immunoreactive cells in eWAT (D) and sWAT (E) of NL and SL mice at P21 ( n = 4–5 per group from ≥ 4 litters). G-H: Relative gene expression of macrophage markers (G) and cytokines (H) in sWAT at P21. I-K: Plasma levels of TNF-α (I), glucose (J), and insulin (K) in NL and SL mice at P21 ( n = 4–5 per group from ≥ 4 litters). *P<0.05 and **P<0.01 versus NL. Scale bar, 100 μm.

Fig 2. Neonatal overfeeding increases HFD-induced weight gain and alters fat distribution.

A-B: Post-weaning bodyweights (A) and naso-anal length (B) of mice raised in normal litters (NL) or small litters (SL) and fed a chow or a high-fat diet (HFD) starting at 5 weeks of age ( n = 9–13 per group from ≥ 5 litters). C-E: Mass (C) and mean adipocyte areas (E) of epididymal (eWAT), retroperitoneal (rWAT), and inguinal (iWAT) adipose tissue in adult SL and NL mice fed a chow or a HFD ( n = 7–8 per group from ≥ 4 litters). Representative images illustrating adipocyte morphology (immunostained for perilipin, green fluorescence) in adult NL and SL mice fed a chow or a HFD (D). *P<0.05 and ***P<0.001 versus NL matched for diet; Δ P<0.05, ΔΔΔ P<0.001 for Diet main-effect; Σ P<0.05, ΣΣ P<0.01 for Litter Size main-effect. Scale bar, 100 μm.

Fig 3. Neonatal overnutrition perturbs glucose homeostasis and causes hepatic steatosis in diet-induced obesity.

A-B: Glucose and (A) insulin (B) tolerance tests and incremental area under the curves/inverted incremental area under the curves of adult SL and NL mice fed a chow or a high-fat diet (HFD) (n = 4–10 per group from ≥ 4 litters). C-E: Plasma levels of glucose (C), insulin (D), and hepatic triglyceride content (E) of adult SL and NL mice fed a chow or a HFD (n = 5–11 per group from ≥ 5 litters). F: Representative images showing Oil Red-O stain in the liver of adult SL and NL mice fed a chow or a HFD. *P<0.05, **P<0.01 and ***P<0.001 versus NL matched for diet; ΔΔ P<0.01 for diet main-effect. Scale bar, 100 μm.

Fig 4. Neonatal overnutrition exacerbates HFD-induced adipose tissue inflammation.

A: Representative images of F4/80 immunoreactivity (red fluorescence) in the epididymal adipose tissue of NL and SL mice fed a chow diet. B: Representative images of F4/80 immunoreactivity (red fluorescence) in the epididymal, retroperitoneal, and inguinal adipose tissue of NL and SL mice fed a high-fat diet (HFD). C: Quantification of crown-like structures (CLS) in the epididymal (eWAT), retroperitoneal (rWAT), and inguinal (iWAT) adipose tissue of adult NL and SL mice fed a HFD (n = 9–13 per group from ≥ 6 litters). D: Representative scatterplots showing CD301 and CD11c heterogeneity in CD45⁺CD64⁺ macrophages from eWAT of adult NL and SL fed a chow or a HFD. E: Histological illustration of antibodies used for flow cytometry. F: Quantification of CD45⁺CD64⁺ total macrophages, CD45⁺CD64⁺CD11c⁺ M1 macrophages and CD45⁺CD64⁺CD301⁺ M2 macrophages in eWAT of SL and NL fed a chow or a HFD (n = 7–8 per group from ≥ 4 litters). *P<0.05, **P<0.01 and ***P<0.001 versus NL matched for diet; Δ P<0.05 for diet main-effect. Scale bar, 100 μm.

Fig 5. Neonatal overnutrition exacerbates HFD-induced expression of pro-inflammatory genes.

A-B: Relative gene expression pro-inflammatory (A) and anti-inflammatory (B) markers in eWAT of NL and SL fed a high-fat diet (HFD) (n = 5–8 per group from 5–8 litters). C: Serum concentrations of adipokines/cytokines (n = 4–8 per group from ≥ 4 litters). *P<0.05 and **P<0.01 versus NL-HFD; ΔΔ P<0.01, ΔΔΔ P<0.001 for Diet main-effect.