Maternal Obesity Reduces Milk Lipid Production in Lactating Mice by Inhibiting Acetyl-CoA Carboxylase and Impairing Fatty Acid Synthesis

<div><p>Maternal metabolic and nutrient trafficking adaptations to lactation differ among lean and obese mice fed a high fat (HF) diet. Obesity is thought to impair milk lipid production, in part, by decreasing trafficking of dietary and <i>de novo</i> synthesized lipids to the mammary gland. Here, we report that <i>de novo</i> lipogenesis regulatory mechanisms are disrupted in mammary glands of lactating HF-fed obese (HF-Ob) mice. HF feeding decreased the total levels of acetyl-CoA carboxylase-1 (ACC), and this effect was exacerbated in obese mice. The relative levels of phosphorylated (inactive) ACC, were elevated in the epithelium, and decreased in the adipose stroma, of mammary tissue from HF-Ob mice compared to those of HF-fed lean (HF-Ln) mice. Mammary gland levels of AMP-activated protein kinase (AMPK), which catalyzes formation of inactive ACC, were also selectively elevated in mammary glands of HF-Ob relative to HF-Ln dams or to low fat fed dams. These responses correlated with evidence of increased lipid retention in mammary adipose, and decreased lipid levels in mammary epithelial cells, of HF-Ob dams. Collectively, our data suggests that maternal obesity impairs milk lipid production, in part, by disrupting the balance of <i>de novo</i> lipid synthesis in the epithelial and adipose stromal compartments of mammary tissue through processes that appear to be related to increased mammary gland AMPK activity, ACC inhibition, and decreased fatty acid synthesis.</p></div

Effects of HF feeding and obesity on perilipin 2 levels in mammary glands of lactating dams.

<p>Perilipin 2 levels in mammary glands of LF-fed, HF-Ln and HF-Ob dams at L10. (A) Representative immunoblot images of Plin2 and β-actin in total mammary gland extracts. (B) Chemiluminescence quantification of Plin2 relative to β-actin in mammary gland extracts. The left (HF Feeding) panel compares relative Plin2 levels in mammary gland extracts of LF- and HF-fed dams. The right (Obesity) panel compares relative Plin2 levels in mammary gland extracts of HF-Ln and HF-Ob dams. The values are means ± SEM for Plin2 normalized to β-actin (N = 5). (C) Representative images of mammary gland sections from LF-Fed, HF-Ln and HF-Ob dams at L10 immunostained for Plin2 (green), Plin1 (red, to identify mammary adipose), and DAPI (blue, to identify nuclei). Boxed areas show regions of the mammary epithelium indicated by the white arrows at higher magnification. White arrowheads indicate Plin2 coated CLD in mammary epithelial cells. Yellow stars indicate regions of the mammary gland containing adipose. (D) Quantification of the effects of HF feeding and obesity on Plin2 immunofluorescence in the mammary epithelium. The left (HF Feeding) panel compares average relative Plin2 IF levels in mammary epithelium of LF- and HF-fed (HF-Ln and HF-Ob) dams. The right (Obesity) panel compares average relative Plin2 IF levels in mammary epithelium of HF-Ln and HF-Ob dams. Plin2 IF intensities in mammary alveoli were quantified in 5 randomly chosen sections from each animal and normalized to the percentage of gland within each section. The values are means ± SEM (N = 5). Statistically significant differences are indicate by the following symbols: *<i>p</i><0.05; ♦♦p<0.02.</p

Effects of HF feeding and obesity on milk lipid production.

<p>The graphs show the effects of HF feeding (LF-fed vs HF-Ln+HF-Ob) (A, C, E, G) and obesity (B, D, F, H) on 24 h production of total lipids (A, B); de novo synthesized lipids (C, D); and medium chain fatty acids (MCFA) (E, F) in milk of dams at L10; and the content of de novo synthesized lipids in mammary glands of L10 dams (G, H). (A, B) Total milk lipid produced over 24 h. Values were extrapolated from milk lipid measured at the end of the study multiplied by the total milk produced over 24 h as presented previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098066#pone.0098066-Wahlig1" target="_blank">[19]</a>. (C, D) The incorporation of <i>de novo</i> synthesized lipids into milk lipid was estimated from the net retention of ³H content within milk lipid over 24 hours, the graph was adapted from Table 2 in reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098066#pone.0098066-Wahlig1" target="_blank">[19]</a>. (E, F) The quantity of medium chain fatty acids in milk produced over 24 hours, measured via GC mass spectrometry, and expressed as mg/day. (G, H) Mammary gland content of <i>de novo</i> synthesized lipids estimated from the retention of ³H in the lipid fraction of mammary gland extracts. Values are expressed as nCi/g tissue. All data are expressed as means ± SEM for 5 animals per group. Statistically significant differences are indicate by the following symbols: *<i>p</i><0.05; **p<0.01; †p<0.003; ‡p<0.002.</p

Effects of HF feeding and obesity on ACC and P-ACC levels in mammary glands of lactating dams.

<p>ACC and P-ACC levels in mammary glands of LF-fed, HF-Ln and HF-Ob dams at L10. (A) Representative immunoblots of ACC, P-ACC and β-actin in mammary gland extracts. (B–G) Chemiluminescence quantification of ACC (B, C) and P-ACC (D, E) relative to β-actin, and the P-ACC/ACC ratio (F, G) in mammary gland extracts. The values are means ± SEM (N = 5). Panels B, D and F compare the effects of HF feeding (LF-fed vs HF-Ln+HF-Ob) on relative mammary gland levels of ACC (B), P-ACC (D) and P-ACC/ACC (F). Panels C, E and G compare the effects of obesity (HF-Ln vs HF-Ob) on relative mammary gland levels of ACC (C), P-ACC (E) and P-ACC/ACC (G). Statistically significant differences are indicate by the following symbols: *<i>p</i><0.05; ♦p<0.03; ‡‡p<0.004.</p

Effects of HF feeding and obesity on mammary gland tissue morphology and composition.

<p>Glandular morphologies, and relative epithelial and adipose contents of mammary glands are shown for LF-fed, HF-Ln and HF-Ob dams at L10. (A) Representative images of H&E stained mammary glands. Higher magnification images taken from areas outlined by the black boxes are shown in the lower panels. Arrowheads in higher magnification images indicate mammary epithelium and stars indicate adipocytes. Graphs in B–D compare the effects of HF feeding (LF-fed vs HF-Ln+HF-Ob) and obesity (HF-Ln vs HF-Ob) on mammary gland weights (B), epithelial (white bars) and adipose (grey bars) compositions of mammary glands (C), and adipocyte diameters (D). (E) Shows the size distribution range of mammary gland adipocytes expressed as percentage of adipocytes with diameters in the ranges of 0–20 µm, 20–40 µm, 40–60 µm, 60–80 µm, and 80–100 µm. For panel B, values are means ± SEM for 5 mice per group. For panels C and D, values are means ± SEM, of 5 sections per animal and 5 animals per group. Statistically significant differences are indicate by the following symbols: ♦<i>p</i><0.03; **p<0.01; ††p<0.001.</p

Effects of HF feeding and obesity on P-ACC and ACC in epithelial and adipose compartments.

<p>Panels A and B show representative images of mammary gland sections from LF-fed, HF-Ln and HF-Ob dams at L10 immunostained for ACC (green, A) or P-ACC (green, B). Mammary adipose was identified by immunostaining with Plin1 (red) and nuclei were identified by DAPI staining (blue). Panel C shows representative higher (600X) magnification monochrome images of mammary tissue immunostained for P-ACC and Plin1. Dashed white lines outline epithelial compartments; yellow stars indicate areas of adipose, and white arrowheads indicate ACC or P-ACC localization in mammary epithelial cells. Panels D-I show quantification of relative ACC (D, G) and P-ACC (E, H) IF levels and P-ACC/ACC IF ratios (F, I) in epithelial (white bars) and adipose (grey bars) compartments of mammary glands from LF-fed, HF-Ln and HF-Ob dams at L10. Panels D-F compare the effects of HF feeding (LF-fed vs HF-Ln+HF-Ob) on ACC and P-ACC immunofluorescence. Panels G-I compare the effects of obesity (HF-Ln vs HF-Ob) on ACC and P-ACC immunofluorescence. Relative ACC and P-ACC IF levels in each compartment were measured in 5 randomly chosen mammary gland sections and normalized to the respective total ACC or P-ACC IF in each section. The values are means ± SEM for sections from 5 animals per group. Statistically significant differences are indicate by the following symbols: *<i>p</i><0.05; ♦♦p<0.02; ▴p<0.009.</p

Milk fatty acid composition.

<p>LF-fed: Low fat lean dams; HF-fed: HF-Ln+HF-Ob dams; HF-Ln: High fat lean dams; HF-Ob: High fat obese dams.</p><p>Data expressed as % of the total milk lipid.</p><p>The effect of HF feeding = LF-fed vs. HF-fed and the effect of obesity = HF-Ln vs. HF-Ob.</p><p>Significance was assumed when p value ≤0.05.</p

Room calorimeter results (mean±SE).

1<p>OB > LN.</p>2<p>LF > HF.</p>3<p>LF < HF.</p>4<p>PAL  = 24 h EE/resting metabolic rate.</p><p>RQ = respiratory quotient.</p><p>FFM = fat free mass.</p><p>FM = fat free mass.</p

Mean (± SE) Protein (Top), carbohydrate (Middle), and fat (Bottom) oxidation (g/kg FFM/day) measured using room calorimetry during LF (black bars) and HF (grey bars) in LN (N = 10) and OB (N = 9).

<p>* LF significantly different from HF (P<0.05).</p

Mean (± SE) total (Top) and phosphorylated (Bottom) AMPK protein during LF (black bars) and HF (grey bars) in LN (N = 5) and OB (N = 6).

<p>Representative blots are show above the graphs. * LF significantly different from HF (P<0.05).</p