Low Neonatal Plasma n-6/n-3 PUFA Ratios Regulate Offspring Adipogenic Potential and Condition Adult Obesity Resistance

Abstract

Adipose tissue expansion progresses rapidly during postnatal life, influenced by both prenatal maternal factors and postnatal developmental cues. The ratio of omega-6 (n-6) relative to n-3 polyunsaturated fatty acids (PUFAs) is believed to regulate perinatal adipogenesis, but the cellular mechanisms and long-term effects are not well understood. We lowered the fetal and postnatal n-6/n-3 PUFA ratio exposure in wild-type offspring under standard maternal dietary fat amounts to test the effects of low n-6/n-3 ratios on offspring adipogenesis and adipogenic potential. Relative to wild-type pups receiving high perinatal n-6/n-3 ratios, subcutaneous adipose tissue in 14-day-old wild-type pups receiving low n-6/n-3 ratios had more adipocytes that were smaller in size; decreased Pparγ2, Fabp4, and Plin1; several lipid metabolism mRNAs; coincident hypermethylation of the PPARγ2 proximal promoter; and elevated circulating adiponectin. As adults, offspring that received low perinatal n-6/n-3 ratios were diet-induced obesity (DIO) resistant and had a lower positive energy balance and energy intake, greater lipid fuel preference and non-resting energy expenditure, one-half the body fat, and better glucose clearance. Together, the findings support a model in which low early-life n-6/n-3 ratios remodel adipose morphology to increase circulating adiponectin, resulting in a persistent adult phenotype with improved metabolic flexibility that prevents DIO.

Figure 1

Experimental study designs for perinatal programming and adult obesogenic diet. A: Study 1 was of WT (high n-6/n-3 PUFA ratio) or n-3 fat-1 (low n-6/n-3 PUFA ratio) mothers maintained on chow and bred at 10 weeks old. Dams had normal gestation and reared their biological litters during lactation, and litters were standardized with six to eight pups per dam. Pups and mothers were assessed for body composition before sacrifice at PND14. Offspring outcomes were adipose morphology and cellularity, adipose gene and protein expression, and circulating hormone, glucose, and fatty acid composition. B: In study 2, litters born to and reared by WT or fat-1 dams (high and low n-6/n-3 ratio exposures, respectively) were weaned and maintained on chow until 17 weeks old. At 17 weeks, adults were assessed for body composition, placed into the indirect calorimeter for 1 week, assessed for body composition and switched to an HFD for 1 week, and assessed for body composition at week 19. Adults were maintained on an HFD for an additional 3 weeks, when a final body composition and oral glucose tolerance test (OGTT) were taken before sacrifice (Sac).

Figure 2

Offspring reared by fat-1 dams have lower n-6 PUFAs and greater n-3 PUFAs in circulation. A: Milk fatty acid composition quantified by lipid mass spectrometry indicates that fat-1 dams synthesize milk containing significantly lower n-6/n-3 ratios for both LC-PUFA and AA/DHA + EPA (P < 0.001) without significantly changing amounts of essential dietary fatty acids (LA/LNA) or levels of de novo fatty acids, SFAs, MUFAs, or PUFAs (n = 5 dams/genotype). B: Offspring serum lipid composition quantified by lipid mass spectrometry mirrored the milk composition with significantly lower LC-PUFA and AA/DHA + EPA ratios (P < 0.001) and identified significantly reduced n-6 PUFAs (20:2 and 20:4; P < 0.01) with significantly enriched n-3 PUFAs (22:6; P = 0.038) (n = 5 pups/condition from five independent dams). *P < 0.05; **P < 0.01; ***P < 0.001. n/s, not significant.

Figure 3

Reduced n-6/n-3 PUFA ratio exposure leads to smaller and more numerous subcutaneous adipocytes and higher systemic adiponectin. A: Quantification of enzymatically digested sWAT from PND14 offspring. Exposure to a low n-6/n-3 PUFA ratio results in an adipocyte cellularity profile with a greater percentage of small-sized and fewer large-sized adipocytes (n = 29 WT pups from five WT dams; n = 18 WT pups from five fat-1 dams). B: Total adipocyte number per gram of sWAT and overall sWAT weight were both greater in low n-6/n-3 PUFA ratio offspring (P = 0.01 and 0.005, respectively; same number of pups and dams as in A). C: Systemic adiponectin, specifically the low-molecular-weight (LMW) isoforms, was significantly increased in response to a low n-6/n-3 PUFA ratio in PND14 pups (P < 0.001), whereas systemic glucose, insulin, and leptin levels were equivalent (n = 8 WT pups from four WT dams; n = 8 WT pups from five fat-1 dams). *P < 0.05. HMW, high molecular weight.

Figure 4

PPARγ2 proximal promoter is hypermethylated in sWAT under low n-6/n-3 PUFA ratios. A: Heat map of significant differentially expressed genes by high and low n-6/n-3 PUFA ratio exposure in PND14 pup sWAT for lipid metabolism, signal transduction, and adipocyte-specific categories (P < 0.05 by using one-way ANOVA with a Bonferroni false discovery rate of 0.1; n = 4 pups/condition from four independent dams). B: Quantitative levels of PPARγ2 mRNA and downstream target genes by quantitative RT-PCR by using a different subset of offspring than for the microarray analysis, indicating significant decreases and decreasing trends as a result of low n-6/n-3 PUFA ratio exposure in sWAT of PND14 offspring (P < 0.05, n = 6 pups/condition from five independent dams). C: Significant reductions in protein levels of PPARγ2, FASN, and PLIN1 in the sWAT of PND14 offspring relative to CypA loading control (n = 6 pups/condition from five independent dams). D: The proximal promoter of PPARγ2 is hypermethylated at positions −3,195 and −322 upstream from the transcriptional start site in sWAT of PND14 pups exposed to a low n-6/n-3 PUFA ratio (n = 8 pups/condition from four independent dams). *P < 0.01. DMR, differentially methylated region; Kb, kilobase.

Figure 5

Adult DIO is programmed by early-life n-6/n-3 PUFA ratio exposures. A: Body composition of adult animals exposed to high or low n-6/n-3 PUFA ratios during the perinatal window. Both groups initially underwent significantly increased adipose deposition in response to the HF/HS diet between weeks 18 and 19 (P < 0.05; n = 8/condition). The low n-6/n-3 PUFA exposure group was resistant to additional adipose deposition during HF/HS diet maintenance (weeks 19–21), whereas the high n-6/n-3 PUFA exposure group continued to accumulate adipose tissue as reflected by the total mass increase by 21 weeks. B: Indirect calorimetry of adult offspring exposed to high or low n-6/n-3 PUFA ratios during the perinatal window. The low n-6/n-3 exposure group had a lower overall energy balance, with reduced energy intake on days 2 and 5 after challenge with the HF/HS diet. For the low n-6/n-3 exposure group, the respiratory exchange ratio was significantly lower during the lead-in period and days 1–3 of the HF/HS diet, total energy expenditure was significantly greater during the lead-in period, and non–resting energy expenditure was significantly greater during the lead-in period and on day 3 of the HF/HS challenge. C: Oral glucose tolerance was improved in adult offspring in the low n-6/n-3 exposure group relative to the high n-6/n-3 exposure group (area under the curve P < 0.05). *P < 0.05; **P < 0.01; ***P < 0.001. Acc, acclimation.