Maternal overweight programs insulin and adiponectin signaling in the offspring

Abstract

Gestational exposure to maternal overweight (OW) influences the risk of obesity in adult life. Male offspring from OW dams gain greater body weight and fat mass and develop insulin resistance when fed high-fat diets (45% fat). In this report, we identify molecular targets of maternal OW-induced programming at postnatal d 21 before challenge with the high-fat diet. We conducted global transcriptome profiling, gene/protein expression analyses, and characterization of downstream signaling of insulin and adiponectin pathways in conjunction with endocrine and biochemical characterization. Offspring born to OW dams displayed increased serum insulin, leptin, and resistin levels (P < 0.05) at postnatal d 21 preceding changes in body composition. A lipogenic transcriptome signature in the liver, before development of obesity, was evident in OW-dam offspring. A coordinated locus of 20 sterol regulatory element-binding protein-1-regulated target genes was induced by maternal OW. Increased nuclear levels of sterol regulatory element-binding protein-1 and recruitment to the fatty acid synthase promoter were confirmed via ELISA and chromatin immunoprecipitation analyses, respectively. Higher fatty acid synthase and acetyl coenzyme A carboxylase protein and pAKT (Thr(308)) and phospho-insulin receptor-beta were confirmed via immunoblotting. Maternal OW also attenuated AMP kinase/peroxisome proliferator-activated receptor-alpha signaling in the offspring liver, including transcriptional down-regulation of several peroxisome proliferator-activated receptor-alpha-regulated genes. Hepatic mRNA and circulating fibroblast growth factor-21 levels were significantly lower in OW-dam offspring. Furthermore, serum levels of high-molecular-weight adiponectin (P < 0.05) were decreased in OW-dam offspring. Phosphorylation of hepatic AMP-kinase (Thr(172)) was significantly decreased in OW-dam offspring, along with lower AdipoR1 mRNA. Our results strongly suggest that gestational exposure to maternal obesity programs multiple aspects of energy-balance regulation in the offspring.

Figures

Figure 1

A, Hierarchical clustering of 147 transcripts altered by maternal OW in offspring liver. Gene expression was assessed in offspring liver at PND21 using Rat Genome 230 2.0 microarrays (Affymetrix) (n = 3 microarrays per group). Genes were filtered based on a minimum ±1.8-fold change (OW vs. lean) and P value ≤0.05 using Student's t test. B, Correlation-based clustering of genes regulated by SREBP-1 with functions in carbohydrate or lipid metabolism derived from the list of genes altered by maternal OW. Heat maps were generated using GeneSpring Gx. Orange, yellow, and blue represent up-regulation, no relative effect, and down-regulation of transcripts, respectively. C, IPA gene network of highest significance identified using IPA software from the list of altered genes. A set of SREBP-1-regulated lipogenic genes and those involved in fatty acid/cholesterol metabolism is evident. Colors green and red represent down-regulation and up-regulation respectively. D, Representative photomicrographs of liver tissues from offspring of lean and OW dams at PND21. Top panel, H&E-stained sections; bottom panel, Oil Red O-stained sections. Magnification, ×400.

Figure 2

A, Hepatic mRNA expression of genes from offspring of lean and OW dams at PND21 (n = 8 per group). B–D, Gene expression was assessed via real-time RT-PCR. Hepatic SREBP-1 in nuclear extracts using Western blots (n = 5 per group) (B and C) and TransAM ELISA (n = 8 per group) (D) from offspring lean and OW dams at PND21. E, ChIP analyses of SREBP-1 recruitment on FASN promoter. Statistical differences were determined using a Student's t test. *, P < 0.05. ADU, Arbitrary density units.

Figure 3

A, Expression of lipogenic enzymes and signaling proteins in total lysates from livers of offspring from lean and OW dams at PND21 by Western blotting (n = 6 per group). B, Immunoprecipitation (IP) of phosphotyrosine in total liver lysates from offspring at PND21 (n = 3 pools representing a total of eight animals per group). Immunoblotting (IB) was performed using anti-IR-β antibody. C, Densitometric quantitation of immunoblots from offspring at PND21. Statistical differences were determined using a Student's t test. *, P < 0.05. ADU, Arbitrary density units.

Figure 4

A, Correlation-based clustering of genes regulated by PPAR-α from the list of transcripts altered by maternal OW. B, mRNA expression of PPAR-α-regulated genes in livers of offspring from lean and OW dams at PND21 (n = 8 per group). Gene expression was assessed via real-time RT-PCR. C, Serum FGF21 levels determined by RIA in offspring of lean and OW dams at PND21 (n = 8 per group). D, Mitochondrial Cpt-1a protein levels and densitometric quantitation in liver of offspring at PND21 (n = 4 per group, each pool represents two separate animals). E, ChIP analyses showing PPAR-α recruitment on FGF21 promoter. Statistical differences were determined using a Student's t test. *, P < 0.05. ADU, Arbitrary density units.

Figure 5

A and B, Expression and densitometric quantitation of hepatic phosphorylated (Thr172) and total AMPK in total liver lysates from offspring of lean and OW dams at PND21. C and D, Analyses of adiponectin monomers by immunoblotting after FPLC-based separation of serum from offspring at PND21. Thirty fractions (0.215 ml) from over the retention volume of the complexes (7–14 ml) were collected. Aliquots of each fraction were denatured and separated by SDS-PAGE. E, Percentage of HMW adiponectin in serum of offspring from lean or OW dams at PND21 (n = 8 per group). F, mRNA expression of AdipoR1 and AdipoR2 in liver and adiponectin in retroperitoneal white adipose tissue in offspring at PND21 (n = 8 per group). Gene expression was assessed via real-time RT-PCR. Statistical differences were determined using Student's t test. *, P < 0.05. ADU, Arbitrary density units.

Figure 6

Schematic summarizing changes in genes regulating lipid biosynthesis and oxidation in offspring liver at PND21. Genes represented in black and gray boxes are up-regulated and down-regulated, respectively (either transcriptionally or via phosphorylation). Overall, genes involved in lipid biosynthesis and insulin signaling are up-regulated, and lipid oxidation regulating genes via PPAR-α and AMPK are down-regulated.