M. J. Zhu

Maternal obesity up-regulates inflammatory signaling pathways and enhances cytokine expression in the mid-gestation sheep placenta

Obesity in pregnant women is a growing public health concern. The placenta is a source of cytokines which can induce maternal gestational insulin resistance and alter nutrient transport to the fetus. Obesity induces placental inflammation at term, but the impact of obesity on placental inflammation earlier in pregnancy has not been defined. Using sheep as an experimental model, we hypothesized that maternal obesity (MO) would induce inflammation in the cotyledonary (COT) tissue of the placentome by mid-gestation. Nonpregnant ewes were randomly assigned to a control (C, 100% of NRC recommendations) or obese (OB, 150% of NRC) group from 60 days before conception to 75 day of gestation (dG), when ewes were necropsied and placental COT tissue collected for analyses. Free fatty acids content, triglyceride and cholesterol content were higher (P < 0.05) in the fetal plasma of OB compared to C ewes on day 75. MO increased mRNA levels of toll-like receptor (TLR) 2 (P < 0.05) and TLR4 (P = 0.06), macrophage markers cluster of differentiation (CD)11b (P = 0.06), CD14 and CD68 (P < 0.05), and proinflammatory cytokines tumor necrosis factor (TNF)alpha (P < 0.01), interleukin (IL)-6 (P < 0.05), IL-8(P < 0.01) and IL-18 (P = 0.06), in COT tissue. Inflammatory c-Jun N-terminal kinase (JNK)/c-Jun and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) signaling pathways were up-regulated (P < 0.05) in COT of OB ewes. In conclusion, MO enhanced the placental inflammatory response in OB ewes at mid-gestation, possibly as a result of increased TLR4 and free fatty acids.

AMP-activated protein kinase and adipogenesis in sheep fetal skeletal muscle and 3T3-L1 cells.

Marbling, or i.m. fat, is an important factor determining beef quality. Both adipogenesis and hypertrophy of existing adipocytes contribute to enhanced marbling. We hypothesized that the fetal stage is important for the formation of i.m. adipocytes and that AMP-activated protein kinase (AMPK) has a key role in adipogenesis during this stage. The objective of this study was to assess the role of AMPK in adipogenesis in fetal sheep muscle and 3T3-L1 cells. Nonpregnant ewes were randomly assigned to a control (Con, 100% of NRC recommendations, n = 7) or overfed (OF, 150% of NRC, n = 7) diet from 60 d before to 75 d after conception, when the ewes were killed. The fetal LM was collected at necropsy for biochemical analyses. The activity of AMPK was less in the fetal muscle of OF sheep. The expression of peroxisome proliferator-activated receptor (PPAR)gamma, a marker of adipogenesis, was greater in OF fetal muscle compared with Con fetal muscle. To further show the role of AMPK in adipogenesis, we used 3T3-L1 cells. The 3T3-L1 cells were incubated in a standard adipogenic medium for 24 h and 10 d. Activation of AMPK by 5-aminoimidazole-4-car-boxamide-1-beta-d-ribonucleoside dramatically inhibited the expression of PPARgamma and reduced the presence of adipocytes after 10 d of differentiation. Inhibition of AMPK by compound C enhanced the expression of PPARgamma. In conclusion, these data show that AMPK activity is inversely related to adipogenesis in fetal sheep muscle and 3T3-L1 cells.

Maternal obesity upregulates fatty acid and glucose transporters and increases expression of enzymes mediating fatty acid biosynthesis in fetal adipose tissue depots1

Maternal nutrient restriction leads to alteration in fetal adipose tissue, and offspring from obese mothers have an increased risk of developing obesity. We hypothesized that maternal obesity increases fetal adipogenesis. Multiparous ewes (Columbia/Rambouillet cross 3 to 5 yr of age) carrying twins were assigned to a diet of 100% (Control; CON; n = 4) or 150% (Obese; OB, n = 7) of NRC maintenance requirements from 60 d before conception until necropsy on d 135 of gestation. Maternal and fetal plasma were collected and stored at -80°C for glucose and hormone analyses. Fetal measurements were made at necropsy, and perirenal, pericardial, and subcutaneous adipose tissues were collected from 7 male twin fetuses per group and snap frozen at -80°C. Protein and mRNA expression of fatty acid translocase [cluster of differentiation (CD) 36], fatty acid transport proteins (FATP) 1 and 4, insulin-sensitive glucose transporter (GLUT-4), fatty acid synthase (FASN), and acetyl-coA carboxylase (ACC) was evaluated. Fetal weight was similar, but fetal carcass weight (FCW) was reduced (P < 0.05) in OB versus CON fetuses. Pericardial and perirenal adipose tissue weights were increased (P < 0.05) as a percentage of FCW in OB versus CON fetuses, as was subcutaneous fat thickness (P < 0.001). Average adipocyte diameter was greater (P < 0.01) in the perirenal fat and the pericardial fat (P = 0.06) in OB fetuses compared with CON fetuses. Maternal plasma showed no difference (P > 0.05) in glucose or other hormones, fetal plasma glucose was similar (P = 0.42), and cortisol, IGF-1, and thyroxine were reduced (P ≤ 0.05) in OB fetuses compared with CON fetuses. Protein and mRNA expression of CD 36, FATP 1 and 4, and GLUT-4 were increased (P ≤ 0.05) in all fetal adipose depots in OB versus CON fetuses. The mRNA expression of FASN and ACC was increased (P < 0.05) in OB vs. CON fetuses in all 3 fetal adipose tissue depots. Fatty acid concentrations were increased (P = 0.01) in the perirenal depot of OB versus CON fetuses, and specific fatty acid concentrations were altered (P < 0.05) in subcutaneous and pericardial adipose tissue because of maternal obesity. In conclusion, maternal obesity was associated with increased fetal adiposity, increased fatty acid and glucose transporters, and increased expression of enzymes mediating fatty acid biosynthesis in adipose depots. These alterations, if maintained into the postnatal period, could predispose the offspring to later obesity and metabolic disease.

Fetal muscle development, mesenchymal multipotent cell differentiation, and associated signaling pathways.

Enhancing muscle growth while reducing fat accumulation improves the efficiency of animal production. The fetal stage is crucial for skeletal muscle development. Fetal muscle development involves myogenesis, adipogenesis, and fibrogenesis from mesenchymal multipotent cells (MC), which are negatively affected by maternal nutrient deficiencies. Enhancing myogenesis increases the lean-to-fat ratio of animals, enhancing intramuscular adipogenesis increases intramuscular fat that is indispensible for the superior eating properties of meat because fat is the major contributor to meat flavor. The promotion of fibrogenesis leads to the accumulation of connective tissue, which contributes to the background toughness of meat and is undesirable. Thus, it is essential to regulate MC differentiation to enhance lean growth and improve meat quality. To date, our understanding of mechanisms regulating the lineage commitment of MC is limited. In this review, we first discuss the impact of maternal nutrient deficiency on fetal development, offspring body composition, and meat quality. Because maternal nutrition affects fetal muscle through altering MC differentiation, we then review several important extracellular morphogens regulating MC differentiation, including hedgehog, Wingless and Int (Wnt), and bone morphogenic proteins. Possible involvement of epigenetic modifications associated with histone deacetylases class IIa and histone acetyltransferase, p300, in MC differentiation is also discussed.

Enhanced transforming growth factor-β signaling and fibrogenesis in ovine fetal skeletal muscle of obese dams at late gestation

Maternal obesity (MO) is increasing at an alarming rate. The objective of this study was to evaluate the effect of MO on fibrogenesis in fetal skeletal muscle during maturation in late gestation. Nonpregnant ewes were assigned to a control diet (Con; fed 100% of NRC nutrient recommendations, n = 6) or obesogenic diet (OB; fed 150% of NRC recommendations, n = 6) from 60 days before conception, and fetal semitendenosus (St) muscle was sampled at 135 days of gestation (term 148 days). Total concentration and area of collagen in cross-sections of muscle increased by 27.0 +/- 6.0 (P < 0.05) and 105.1 +/- 5.9% (P = 0.05) in OB compared with Con fetuses. The expression of precursor TGF-beta was 177.3 +/- 47.6% higher, and concentration of phospho-p38 74.7 +/- 23.6% was higher (P < 0.05) in OB than in CON fetal muscle. Increases of 327.9 +/- 168.0 (P < 0.05) and 188.9 +/- 82.1% (P < 0.05), respectively, were observed for mRNA expression of Smad7 and fibronectin in OB compared with Con muscles. In addition, enzymes involved in collagen synthesis, including lysyl oxidase, lysyl hydroxylase 2b, and prolyl 4-hydroxylase-alpha1, were increased by 350.2 +/- 90.0 (P < 0.05), 236.5 +/- 25.2 (P < 0.05), and 82.0 +/- 36.2% (P = 0.05), respectively, in OB muscle. In conclusion, MO-enhanced fibrogenesis in fetal muscle in late gestation was associated with upregulation of the TGF-beta/p38 signaling pathway. Enhanced fibrogenesis at such an early stage of development is expected to negatively affect the properties of offspring muscle because muscle fibrosis is a hallmark of aging.

Effects of maternal plane of nutrition and increased dietary selenium in first-parity ewes on inflammatory response in the ovine neonatal gut

Many areas of the western United States have soils that have increased Se content, and ruminants grazing these rangelands may ingest increased quantities of Se. In addition, high-energy diets or increased Se intake may induce gut inflammation. The objective of this study was to evaluate the effects of maternal plane of nutrition and increased dietary Se during gestation on inflammatory responses in neonatal lamb ileal tissue, a major immune organ. Rambouillet ewes (age = 240 ± 17 d; initial BW = 52.1 ± 6.2 kg) were allocated to 4 treatments arranged in a 2 × 2 factorial. Factors included Se [adequate Se (ASe, 11.5 µg/kg of BW) or high Se (HSe, 77.0 µg/kg of BW)] initiated at breeding, and nutritional plane [100% (CON) or 140% (HIH) of requirements] initiated at d 40 of gestation. Ewes were fed individually from d 40, and lambs were removed at parturition and fed artificial colostrum and milk replacer. Lambs were necropsied at 20 d of age, and ileal tissues were sampled for immunoblotting and real-time quantitative reverse-transcription PCR analyses. The ASe-HIH and HSe-CON treatments had no effect (P = 0.179) on inflammatory signaling compared with ASe-CON. However, greater inflammatory signaling was detected in the HSe-HIH group, as shown by increased (P < 0.05) mRNA expression of tumor necrosis factor-α and chemotaxis IL-8. Consistently, phosphorylation of c-Jun N-terminal kinase, a primary inflammatory signaling mediator, was greater (P < 0.05) in the HSe-HIH group compared with other treatments. Consistent with cytokine expression, mast cell density was less in the HSe-CON group than in other treatments. The expression of transforming growth factor β mRNA was greater (P < 0.05) in the HSe-HIH group; consistently, collagen content was increased in the HSe-HIH group compared with the ASe-CON group (P < 0.05). In conclusion, independently, neither HSe nor HIH had major effects on inflammation, but in combination, these maternal treatments induced an inflammatory response in the neonatal intestine.

Mouse AMP-activated protein kinase γ3 subunit R225Q mutation affecting mouse growth performance when fed a high-energy diet.

The Rendement Napole (RN) genotype widely exists in Hampshire pigs. Recently, RN gene was identified as a R200Q mutation in AMP-activated protein kinase (AMPK) gamma3 subunit. The effect of RN genotype on the growth performance of animals and the underlying mechanisms remain controversial. Using transgenic mice carrying an analogous R225Q mutation, the objective of this study was to study the role of RN gene in the growth performance of animals at different energy levels. Wild-type (WT) mice and those with the RN mutation were assigned to 4 groups: 1) WT plus normal diet, 2) RN plus normal diet, 3) WT plus high-energy diet, and 4) RN plus high-energy diet. Mice were weaned at 21 d old and fed the trial diets for 1 mo and then killed for carcass measurements. The pH of postmortem muscle from RN mice was less (P < 0.01) than that from WT mice. No difference in growth performance was observed when mice were fed a normal diet. When fed a high-energy diet, RN mice showed a greater fat accumulation (WT vs. RN, 1.11 vs. 1.63 g for gonadal fat and 1.40 vs. 1.84 g for subcutaneous fat; P < 0.05). Muscle weight was also increased (WT vs. RN, 0.27 vs. 0.30 g for gastrocnemius muscle; P < 0.05). The food consumption was greater in RN compared with WT mice (2.95 vs. 2.49 g; P < 0.05). The AMPK content and its downstream target, acetyl-CoA carboxylase (ACC), content were greater in RN mice (P < 0.05). The phosphorylation of ACC at Ser 79, a site exclusively phosphorylated by AMPK, was increased (P < 0.05), showing greater AMPK activity in RN mouse muscle. No difference in muscle fiber composition and mitochondrial content was observed between WT and RN mice. High fat diet downregulates protein kinase B but upregulates extracellular signal-regulated kinase signaling. In conclusion, the R225Q mutation has no major effect on the growth performance of animals fed a normal diet; a high-energy diet increased fatness in RN mice, likely due to their greater consumption of feed compared with WT mice.