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Fetuses with intrauterine growth restriction (FGR) have impaired oxidative and energy metabolism, with persistent consequences on their postnatal development. In this study, we test the hypothesis that FGR skeletal muscle has lower mitochondrial respiration rate and alters the transcriptomic profiles associated with energy metabolism in an ovine model. At late gestation, mitochondrial oxygen consumption rates (OCRs) and transcriptome profiles were evaluated in the skeletal muscle collected from FGR and control fetuses. The ex vivo mitochondrial OCRs were reduced (p < 0.01) in permeabilized FGR soleus muscle compared to the control muscle but only with pyruvate as the metabolic substrate. Mitochondrial OCRs were similar between the FGR and control groups for palmitoyl-carnitine (fatty acid-driven) or pyruvate plus palmitoyl-carnitine metabolic substrates. A total of 2284 genes were differentially expressed in the semitendinosus muscle from growth restricted fetuses (false discovery rate (FDR) ≤ 0.05). A pathway analysis showed that the upregulated genes (FGR compared to control) were overrepresented for autophagy, HIF-1, AMPK, and FOXO signaling pathways (all with an FDR < 0.05). In addition, the expression of genes modulating pyruvate's entry into the TCA cycle was downregulated, whereas the genes encoding key fatty acid oxidation enzymes were upregulated in the FGR muscle. These findings show that FGR skeletal muscle had attenuated mitochondrial pyruvate oxidation, possibly associated with the inability of pyruvate to enter into the TCA cycle, and that fatty acid oxidation might compensate for the attenuated energy metabolism. The current study provided phenotypic and molecular evidence for adaptive deficiencies in FGR skeletal muscle.

Intrauterine growth restriction (IUGR) increases the risk of developing diabetes in later life, which indicates developmental programming of islets. IUGR fetuses with placental insufficiency develop hypoxemia, elevating epinephrine and norepinephrine (NE) concentrations throughout late gestation. To isolate the programming effects of chronically elevated catecholamines, NE was continuously infused into normally grown sheep fetuses for 7 days. High plasma NE concentrations suppress insulin, but after the NE infusion was terminated, persistent hypersecretion of insulin occurred. Our objective was to identify differential gene expression with RNA sequencing (RNAseq) in fetal islets after chronic adrenergic stimulation. After determining the NE-regulated genes, we identified the subset of differentially expressed genes that were common to both islets from NE fetuses and fetuses with IUGR to delineate the adrenergic-induced transcriptional responses. A portion of these genes were investigated in mouse insulinoma (Min6) cells chronically treated with epinephrine to better approximate the β-cell response. In islets from NE fetuses, RNAseq identified 321 differentially expressed genes that were overenriched for metabolic and hormone processes, and the subset of 96 differentially expressed genes common to IUGR islets were overenriched for protein digestion, vitamin metabolism, and cell replication pathways. Thirty-eight of the 96 NE-regulated IUGR genes changed similarly between models with functional enrichment for proliferation. In Min6 cells, chronic epinephrine stimulation slowed proliferation and augmented insulin secretion after treatment. These data establish molecular mechanisms underlying persistent adrenergic stimulation in hyperfunctional fetal islets and identify a subset of genes dysregulated by catecholamines in IUGR islets that may represent programming of β-cell proliferation capacity.

Background: In food animal agriculture, there is a need to identify the mechanisms that can improve the efficiency of muscle growth and protein accretion. Callipyge sheep provide excellent machinery since the up-regulation of DLK1 and RTL1 results in extreme postnatal muscle hypertrophy in distinct muscles. The aim of this study is to distinguish the genes that directly respond to DLK1 and RTL1 signaling from the genes that change as the result of muscle specific effects., Results: The quantitative PCR results indicated that DLK1 expression was significantly increased in hypertrophied muscles but not in non-hypertrophied muscles. However, RTL1 was up-regulated in both hypertrophied and non-hypertrophied muscles. Five genes, including PARK7, DNTTIP1, SLC22A3, METTL21E and PDE4D, were consistently co-expressed with DLK1, and therefore were possible transcriptional target genes responding to DLK1 signaling. Treatment of myoblast and myotubes with DLK1 protein induced an average of 1.6-fold and 1.4-fold increase in Dnttip1 and Pde4d expression respectively. Myh4 expression was significantly elevated in DLK1-treated myotubes, whereas the expression of Mettl21e was significantly increased in the DLK1-treated myoblasts but reduced in DLK1-treated myotubes. DLK1 treatment had no impact on Park7 expression. In addition, Park7 and Dnttip1 increased Myh4 and decreased Myh7 promoter activity, resemble to the effects of Dlk1. In contrast, expression of Mettl21e increased Myh7 and decreased Myh4 luciferase activity., Conclusion: The study provided additional supports that RTL1 alone was insufficient to induce muscle hypertrophy and concluded that DLK1 was likely the primary effector of the hypertrophy phenotype. The results also suggested that DNTTIP1 and PDE4D were secondary effector genes responding to DLK1 signaling resulting in muscle fiber switch and muscular hypertrophy in callipyge lamb.

The risk of type 2 diabetes is increased in children and adults who exhibited fetal growth restriction. Placental insufficiency and intrauterine growth restriction (IUGR) are common obstetrical complications associated with fetal hypoglycemia and hypoxia that reduce the β-cell mass and insulin secretion. In the present study, we have defined the underlying mechanisms of reduced growth and proliferation, impaired metabolism, and defective insulin secretion previously established as complications in islets from IUGR fetuses. In an IUGR sheep model that recapitulates human IUGR, high-throughput RNA sequencing showed the transcriptome of islets isolated from IUGR and control sheep fetuses and identified the transcripts that underlie β-cell dysfunction. Functional analysis expanded mechanisms involved in reduced proliferation and dysregulated metabolism that include specific cell cycle regulators and growth factors and mitochondrial, antioxidant, and exocytotic genes. These data also identified immune responses, wnt signaling, adaptive stress responses, and the proteasome as mechanisms of β-cell dysfunction. The reduction of immune-related gene expression did not reflect a change in macrophage density within IUGR islets. The present study reports the islet transcriptome in fetal sheep and established processes that limit insulin secretion and β-cell growth in fetuses with IUGR, which could explain the susceptibility to premature islet failure in adulthood. Islet dysfunction formed by intrauterine growth restriction increases the risk for diabetes., (Copyright © 2017 Endocrine Society.)

Liposarcomas (LPSs) are the most common soft-tissue cancer. Because of the lack of animal models, the cellular origin and molecular regulation of LPS remain unclear. Here, we report that mice with adipocyte-specific activation of Notch signaling (Ad/N1ICD) develop LPS with complete penetrance. Lineage tracing confirms the adipocyte origin of Ad/N1ICD LPS. The Ad/N1ICD LPS resembles human dedifferentiated LPS in histological appearance, anatomical localization, and gene expression signature. Before transformation, Ad/N1ICD adipocytes undergo dedifferentiation that leads to lipodystrophy and metabolic dysfunction. Although concomitant Pten deletion normalizes the glucose metabolism of Ad/N1ICD mice, it dramatically accelerates the LPS prognosis and malignancy. Transcriptomes and lipidomics analyses indicate that Notch activation suppresses lipid metabolism pathways that supply ligands to Pparγ, the master regulator of adipocyte homeostasis. Accordingly, synthetic Pparγ ligand supplementation induces redifferentiation of Ad/N1ICD adipocytes and tumor cells, and prevents LPS development in Ad/N1ICD mice. Importantly, the Notch target HES1 is abundantly expressed in human LPS, and Notch inhibition suppresses the growth of human dedifferentiated LPS xenografts. Collectively, ectopic Notch activation is sufficient to induce dedifferentiation and tumorigenic transformation of mature adipocytes in mouse., (© 2016 Bi et al.)

Callipyge sheep exhibit postnatal muscle hypertrophy due to the up-regulation of DLK1 and/or RTL1. The up-regulation of PARK7 was identified in hypertrophied muscles by microarray analysis and further validated by quantitative PCR. The expression of PARK7 in hypertrophied muscle of callipyge lambs was confirmed to be up-regulated at the protein level. PARK7 was previously identified to positively regulate PI3K/AKT pathway by suppressing the phosphatase activity of PTEN in mouse fibroblasts. The purpose of this study was to investigate the effects of PARK7 in muscle growth and protein accretion in response to IGF1. Primary myoblasts isolated from Park7 (+/+) and Park7 (-/-) mice were used to examine the effect of differential expression of Park7. The Park7 (+/+) myotubes had significantly larger diameters and more total sarcomeric myosin expression than Park7 (-/-) myotubes. IGF1 treatment increased the mRNA abundance of Myh4, Myh7 and Myh8 between 20-40% in Park7 (+/+) myotubes relative to Park7 (-/-). The level of AKT phosphorylation was increased in Park7 (+/+) myotubes at all levels of IGF1 supplementation. After removal of IGF1, the Park7 (+/+) myotubes maintained higher AKT phosphorylation through 3 hours. PARK7 positively regulates the PI3K/AKT pathway by inhibition of PTEN phosphatase activity in skeletal muscle. The increased PARK7 expression can increase protein synthesis and result in myotube hypertrophy. These results support the hypothesis that elevated expression of PARK7 in callipyge muscle would increase levels of AKT activity to cause hypertrophy in response to the normal IGF1 signaling in rapidly growing lambs. Increasing expression of PARK7 could be a novel mechanism to increase protein accretion and muscle growth in livestock or help improve muscle mass with disease or aging.

The intrauterine environment is influenced by maternal behaviour and programmes atherosclerotic disease susceptibility in offspring. The aim of this investigation was to test the hypothesis that mothers' exercise during pregnancy improves endothelial function in 3-, 5- and 9-month-old porcine offspring. The pregnant sows in the exercise group ran for an average of 39.35 ± 0.75 min at 4.81 ± 0.35 km h(-1) each day for 5 days per week for all but the last week of gestation. This induced a significant reduction in resting heart rate (exercised group, 89.3 ± 3.5 beats min(-1); sedentary group, 102.1 ± 3.1 beats min(-1); P < 0.05) but no significant differences in gestational weight gain (65.8 ± 2.1 versus 63.3 ± 1.9%). No significant effect on bradykinin-induced vasorelaxation with and without l-NAME was observed. A significant main effect was identified on sodium nitroprusside-induced vasorelaxation (P = 0.01), manifested by a reduced response in femoral arteries of all age groups from exercised-trained swine. Nitric oxide signalling was not affected by maternal exercise. Protein expression of MYPT1 was reduced in femoral arteries from 3-month-old offspring of exercised animals. A significant interaction was observed for PPP1R14A (P < 0.05) transcript abundance and its protein product CPI-17. In conclusion, pregnant swine are able to complete an exercise-training protocol that matches the current recommendations for pregnant women. Gestational exercise is a potent stimulus for programming vascular smooth muscle relaxation in adult offspring. Specifically, exercise training for the finite duration of pregnancy decreases vascular smooth muscle responsiveness in adult offspring to an exogenous nitric oxide donor.

Background: The heterogeneous progression of atherosclerotic disease in the peripheral arteries is currently not well understood. In humans, artery specific disease progression is partly attributed to the local hemodynamic environments. However, despite similar hemodynamic environments, porcine brachial arteries are protected while femoral arteries are highly susceptible to advanced lesion formation. The aim of this investigation was to determine whether artery specific gene expression patterns contribute to the uneven distribution of peripheral arterial disease (PAD) in Rapacz Familial-Hypercholesterolemic (FHC) swine., Results: Histological results confirmed rapid atherosclerotic disease progression in femoral but not brachial arteries. A total of 18,922 probe sets had sufficient signal abundance. A main effect for age and artery was observed for 1784 and 1256 probe sets, respectively. A significant age x artery interaction was found for 184 probe sets. Furthermore, comparison between arteries found a decrease from 714 to 370 differentially expressed transcripts from nine months to two years of age. Gene ontology analysis of the 56 genes with a main effect for artery and an age x artery interaction identified vascular smooth muscle contraction as enhanced biological signaling pathway., Conclusion: This is the first investigation to report that the total number of differential genes decreases with diverging atherosclerotic disease pattern between porcine brachial and femoral arteries.

Selective breeding programs aiming to increase the productivity and profitability of the sheep meat industry use elite, progeny tested sires. The broad genetic traits of primary interest in the progeny of these sires include skeletal muscle yield, fat content, eating quality, and reproductive efficiency. Natural mutations in sheep that enhance muscling have been identified, while a number of genome scans have identified and confirmed quantitative trait loci (QTL) for skeletal muscle traits. The detailed phenotypic characteristics of sheep carrying these mutations or QTL affecting skeletal muscle show a number of common biological themes, particularly changes in developmental growth trajectories, alterations of whole animal morphology, and a shift toward fast twitch glycolytic fibers. The genetic, developmental, and biochemical mechanisms underpinning the actions of some of these genetic variants are described. This review critically assesses this research area, identifies gaps in knowledge, and highlights mechanistic linkages between genetic polymorphisms and skeletal muscle phenotypic changes. This knowledge may aid the discovery of new causal genetic variants and in some cases lead to the development of biochemical and immunological strategies aimed at enhancing skeletal muscle.

The mechanisms underlying the unequal distribution of atherosclerotic disease in the peripheral arteries are currently unclear. Gene expression differences in healthy arteries may influence the heterogeneous distribution of atherosclerosis. Therefore, this investigation compares gene expression in healthy atheroprotected brachial and atherosusceptible femoral arteries of young and disease free Rapacz familial hypercholesterolemic (FHC) swine. We hypothesized that transcripts related to atherosusceptibility would be differentially expressed between these arteries prior to the onset of disease. Femoral and brachial arteries were harvested from four 13-day-old Rapacz FHC swine. No atherosclerotic disease was detected using Sudan IV, Verhoeff-van Gieson, and hematoxylin-eosin staining. Gene expression was quantified using Affymetrix GeneChip Porcine Genome Arrays. An average of 15,552 probe sets had detectable transcripts, while 430 probe sets showed a significant differential expression between arteries (false discovery rate < 0.05). The human orthologs of 63 probe sets with differential expression and a 1.5-fold or greater transcript abundance between arteries are associated with Wnt/β-catenin, lysophospholipid, and Ca-signaling, as well as apoptosis. This is the first investigation reporting that differences in relative abundance of gene expression exist between brachial and femoral arteries in young Rapacz FHC swine prior to the development of atherosclerotic lesions.

Delta-like 1homolog (Dlk1) is an imprinted gene encoding a transmembrane protein whose increased expression has been associated with muscle hypertrophy in animal models. However, the mechanisms by which Dlk1 regulates skeletal muscle plasticity remain unknown. Here we combine conditional gene knockout and over-expression analyses to investigate the role of Dlk1 in mouse muscle development, regeneration and myogenic stem cells (satellite cells). Genetic ablation of Dlk1 in the myogenic lineage resulted in reduced body weight and skeletal muscle mass due to reductions in myofiber numbers and myosin heavy chain IIB gene expression. In addition, muscle-specific Dlk1 ablation led to postnatal growth retardation and impaired muscle regeneration, associated with augmented myogenic inhibitory signaling mediated by NF-κB and inflammatory cytokines. To examine the role of Dlk1 in satellite cells, we analyzed the proliferation, self-renewal and differentiation of satellite cells cultured on their native host myofibers. We showed that ablation of Dlk1 inhibits the expression of the myogenic regulatory transcription factor MyoD, and facilitated the self-renewal of activated satellite cells. Conversely, Dlk1 over-expression inhibited the proliferation and enhanced differentiation of cultured myoblasts. As Dlk1 is expressed at low levels in satellite cells but its expression rapidly increases upon myogenic differentiation in vitro and in regenerating muscles in vivo, our results suggest a model in which Dlk1 expressed by nascent or regenerating myofibers non-cell autonomously promotes the differentiation of their neighbor satellite cells and therefore leads to muscle hypertrophy.

Background: The developmental transition between the late fetus and a newborn animal is associated with profound changes in skeletal muscle function as it adapts to the new physiological demands of locomotion and postural support against gravity. The mechanisms underpinning this adaption process are unclear but are likely to be initiated by changes in hormone levels. We tested the hypothesis that this developmental transition is associated with large coordinated changes in the transcription of skeletal muscle genes., Results: Using an ovine model, transcriptional profiling was performed on Longissimus dorsi skeletal muscle taken at three fetal developmental time points (80, 100 and 120 d of fetal development) and two postnatal time points, one approximately 3 days postpartum and a second at 3 months of age. The developmental time course was dominated by large changes in expression of 2,471 genes during the interval between late fetal development (120 d fetal development) and 1-3 days postpartum. Analysis of the functions of genes that were uniquely up-regulated in this interval showed strong enrichment for oxidative metabolism and the tricarboxylic acid cycle indicating enhanced mitochondrial activity. Histological examination of tissues from these developmental time points directly confirmed a marked increase in mitochondrial activity between the late fetal and early postnatal samples. The promoters of genes that were up-regulated during this fetal to neonatal transition were enriched for estrogen receptor 1 and estrogen related receptor alpha cis-regulatory motifs. The genes down-regulated during this interval highlighted de-emphasis of an array of functions including Wnt signaling, cell adhesion and differentiation. There were also changes in gene expression prior to this late fetal--postnatal transition and between the two postnatal time points. The former genes were enriched for functions involving the extracellular matrix and immune response while the latter principally involved functions associated with transcriptional regulation of metabolic processes., Conclusions: It is concluded that during late skeletal muscle development there are substantial and coordinated changes in the transcription of a large number of genes many of which are probably triggered by increased estrogen levels. These changes probably underpin the adaption of muscle to new physiological demands in the postnatal environment.

Members of the Ty3-Gypsy retrotransposon family are rare in mammalian genomes despite their abundance in invertebrates and some vertebrates. These elements contain a gag-pol-like structure characteristic of retroviruses but have lost their ability to retrotranspose into the mammalian genome and are thought to be inactive relics of ancient retrotransposition events. One of these retrotransposon-like elements, PEG11 (also called RTL1) is located at the distal end of ovine chromosome 18 within an imprinted gene cluster that is highly conserved in placental mammals. The region contains several conserved imprinted genes including BEGAIN, DLK1, DAT, GTL2 (MEG3), PEG11 (RTL1), PEG11as, MEG8, MIRG and DIO3. An intergenic point mutation between DLK1 and GTL2 causes muscle hypertrophy in callipyge sheep and is associated with large changes in expression of the genes linked in cis between DLK1 and MEG8. It has been suggested that over-expression of DLK1 is the effector of the callipyge phenotype; however, PEG11 gene expression is also strongly correlated with the emergence of the muscling phenotype as a function of genotype, muscle type and developmental stage. To date, there has been no direct evidence that PEG11 encodes a protein, especially as its anti-sense transcript (PEG11as) contains six miRNA that cause cleavage of the PEG11 transcript. Using immunological and mass spectrometry approaches we have directly identified the full-length PEG11 protein from postnatal nuclear preparations of callipyge skeletal muscle and conclude that its over-expression may be involved in inducing muscle hypertrophy. The developmental expression pattern of the PEG11 gene is consistent with the callipyge mutation causing recapitulation of the normal fetal-like gene expression program during postnatal development. Analysis of the PEG11 sequence indicates strong conservation of the regions encoding the antisense microRNA and in at least two cases these correspond with structural or functional domains of the protein suggesting co-evolution of the sense and antisense genes.

Callipyge sheep exhibit extreme postnatal muscle hypertrophy in the loin and hindquarters as a result of a single nucleotide polymorphism (SNP) in the imprinted DLK1-DIO3 domain on ovine chromosome 18. The callipyge SNP up-regulates the expression of surrounding transcripts when inherited in cis without altering their allele-specific imprinting status. The callipyge phenotype exhibits polar overdominant inheritance since only paternal heterozygous animals have muscle hypertrophy. Two studies were conducted profiling gene expression in lamb muscles to determine the down-stream effects of over-expression of paternal allele-specific DLK1 and RTL1 as well as maternal allele-specific MEG3, RTL1AS and MEG8, using Affymetrix bovine expression arrays. A total of 375 transcripts were differentially expressed in callipyge muscle and 25 transcripts were subsequently validated by quantitative PCR. The muscle-specific expression patterns of most genes were similar to DLK1 and included genes that are transcriptional repressors or affect feedback mechanisms in beta-adrenergic and growth factor signaling pathways. One gene, phosphodiesterase 7A had an expression pattern similar to RTL1 expression indicating a biological activity for RTL1 in muscle. Only transcripts that localize to the DLK1-DIO3 domain were affected by inheritance of a maternal callipyge allele. Callipyge sheep are a unique model to study over expression of both paternal allele-specific genes and maternal allele-specific non-coding RNA with an accessible and nonlethal phenotype. This study has identified a number of genes that are regulated by DLK1 and RTL1 expression and exert control on postnatal skeletal muscle growth. The genes identified in this model are primary candidates for naturally regulating postnatal muscle growth in all meat animal species, and may serve as targets to ameliorate muscle atrophy conditions including myopathic diseases and age-related sarcopenia.

Pyruvate carboxylase (PC) catalyzes a pivotal reaction in gluconeogenesis and lipid metabolism in liver. In bovine the PC gene is expressed as six 5' untranslated region (UTR) mRNA variants. The objectives for this study were to clone and sequence the bovine PC gene, determine the intron and exon organization and identify PC promoter region(s). Oligonucleotide sequences that corresponded to the 5' UTR mRNA variants and coding sequence of bovine PC were used to isolate 2 clones from the RPCI-42 bovine bacterial artificial chromosome (BAC) library. Sequencing data confirmed the presence of regions for the 5' UTR for bovine PC mRNA. The exon arrangement from 5' to 3' is 48 (exon I), 41 (exon II), 178 (exon IIIA and IIIB), and 185 (exon IV) bp. Three promoter regions, P3, P2, and P1, adjacent to exon I, II, and IIIA, respectively, were identified based on computer analysis of sequence data. Putative promoters were cloned into a firefly luciferase vector and transiently transfected into H4IIE rat hepatoma cells. All PC promoters demonstrated luciferase activity comparable with the minimal promoter luciferase vector and higher than the promoterless luciferase vector. In addition, PC promoter 1 exhibited greater luciferase activity compared with PC promoter 2 or 3. These data provide information about the arrangement of the 4 bovine PC 5' UTR exons, the identity of the promoter regions for the bovine PC gene, and indicate differences in relative basal activity of the promoter regions.

Genetic strategies to improve the profitability of sheep operations have generally focused on traits for reproduction. However, natural mutations exist in sheep that affect muscle growth and development, and the exploitation of these mutations in breeding strategies has the potential to significantly improve lamb-meat quality. The best-documented mutation for muscle development in sheep is callipyge (CLPG), which causes a postnatal muscle hypertrophy that is localized to the pelvic limbs and loin. Enhanced skeletal muscle growth is also observed in animals with the Carwell (or rib-eye muscling) mutation, and a double-muscling phenotype has been documented for animals of the Texel sheep breed. However, the actual mutations responsible for these muscular hypertrophy phenotypes in sheep have yet to be identified, and further characterization of the genetic basis for these phenotypes will provide insight into the biological control of muscle growth and body composition.

Background: The callipyge mutation is located within an imprinted gene cluster on ovine chromosome 18. The callipyge trait exhibits polar overdominant inheritance due to the fact that only heterozygotes inheriting a mutant paternal allele (paternal heterozygotes) have a phenotype of muscle hypertrophy, reduced fat and a more compact skeleton. The mutation is a single A to G transition in an intergenic region that results in the increased expression of several genes within the imprinted cluster without changing their parent-of-origin allele-specific expression., Results: There was a significant effect of genotype (p < 0.0001) on the transcript abundance of DLK1, PEG11, and MEG8 in the muscles of lambs with the callipyge allele. DLK1 and PEG11 transcript levels were elevated in the hypertrophied muscles of paternal heterozygous animals relative to animals of the other three genotypes. The PEG11 locus produces a single 6.5 kb transcript and two smaller antisense strand transcripts, referred to as PEG11AS, in skeletal muscle. PEG11AS transcripts were detectable over a 5.5 kb region beginning 1.2 kb upstream of the PEG11 start codon and spanning the entire open reading frame. Analysis of PEG11 expression by quantitative PCR shows a 200-fold induction in the hypertrophied muscles of paternal heterozygous animals and a 13-fold induction in homozygous callipyge animals. PEG11 transcripts were 14-fold more abundant than PEG11AS transcripts in the gluteus medius of paternal heterozygous animals. PEG11AS transcripts were expressed at higher levels than PEG11 transcripts in the gluteus medius of animals of the other three genotypes., Conclusions: The effect of the callipyge mutation has been to alter the expression of DLK1, GTL2, PEG11 and MEG8 in the hypertrophied skeletal muscles. Transcript abundance of DLK1 and PEG11 was highest in paternal heterozygous animals and exhibited polar overdominant gene expression patterns; therefore, both genes are candidates for causing skeletal muscle hypertrophy. There was unique relationship of PEG11 and PEG11AS transcript abundance in the paternal heterozygous animals that suggests a RNA interference mechanism may have a role in PEG11 gene regulation and polar overdominance in callipyge sheep.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the PPAR subfamily of nuclear hormone receptors. In rodents and humans, expression of PPARgamma is predominantly found in adipose tissue where it regulates adipocyte differentiation and the expression of multiple adipocyte genes. The primary aim of this work was to clone the porcine PPARgamma cDNA and examine the regulation of gene expression in porcine subcutaneous adipose tissue. The porcine PPARgamma gene encodes a 1.8-kb mRNA transcript and shares 99, 96 and 97% amino acid sequence identity to the human, mouse and cow PPARgamma molecules, respectively. Both isoforms of PPARgamma (gamma1 and gamma2) are highly expressed in porcine adipose tissue. The gamma2 isoform is expressed in low abundance in porcine spleen, whereas the gamma1 isoform is highly expressed in spleen and lung and at a low abundance in several other tissues. Western blot analysis confirmed a high level of PPARgamma protein expression in porcine adipose tissue compared to other tissues. Both caloric restriction and fasting significantly reduced PPARgamma2 but not gamma1 mRNA and PPARgamma protein abundance in subcutaneous adipose tissue compared to ad-libitum fed controls. We provide the first evidence that PPARgamma is abundantly expressed in porcine subcutaneous adipose tissue, and that expression is regulated by caloric intake. Thus, PPARgamma may play an important role in adipogenesis and hormone action in porcine adipocytes.

The relationship between obese gene expression and energy intake was determined in pigs of various body weights. With ad libitum consumption, expression increased (P < 0.001) with body weight from 55 to 163 kg. Obese mRNA relative abundance was correlated with fat mass (r = 0.74, P < 0.0001) and percentage of fat (r = 0.72, P < 0. 0001). Obese expression was also evaluated at 159 kg (initial weight) and ad libitum, maintenance or 23% of maintenance intake for 28 d. Obese mRNA was independent of treatment (P > 0.78) despite considerable weight differences. Obese mRNA abundance was then compared at 136 kg (initial weight) and ad libitum or maintenance intake for 3 or 28 d. Abundance was not influenced by either duration of treatment or intake, despite a small increase (P < 0.01) in serum nonesterified fatty acids (NEFA) and a reduction (P < 0.02) in insulin attributable to maintenance intake. Finally, mRNA abundance was determined at 60 and 136 kg and conditions of food deprivation or ad libitum intake for 3 d. Food deprivation reduced (P < 0.01) serum insulin and increased (4- to 5-fold) NEFA concentrations. Obese mRNA abundance was greater (P < 0.01) in the heavier pigs and was reduced (P < 0.01) by food deprivation. We conclude that obese mRNA abundance in pigs correlates with fat mass and percentage of body fat under conditions of ad libitum intake. Furthermore, obese mRNA abundance is reduced by food deprivation, whereas lesser degrees of intake restriction do not change obese mRNA abundance, even when accompanied by appreciable weight loss.

The postnatal increase in skeletal alpha-actin (Sk-alpha-Act) synthesis in pigs is due, in part, to increased transcription. To characterize the factors responsible for its transcriptional regulation, we have cloned and determined the nucleotide sequence of a 5.2-kb HindIII genomic DNA fragment which contains the complete coding region of Sk-alpha-Act distributed over seven exons, plus 1.9 kb of 5' flanking region and 0.5 kb of 3' flanking sequence. The major transcription start point (tsp) of Sk-alpha-Act was determined to be 840 bp 5' to the ATG start codon by primer extension and RNase protection analysis. To demonstrate that the Sk-alpha-Act promoter was functional, L6 myoblasts, C2C12 myoblasts and HeLa cells were transfected with a construct (pPSKAFL-CAT) linking the 5' Sk-alpha-Act promoter to the chloramphenicol acetyltransferase reporter gene (cat). Cell lysates from L6 myoblasts, L6 myotubes, C2C12 myoblasts, C2C12 myotubes, and HeLa cells were analyzed for CAT activity. CAT activity was detected only in C2C12 myotubes. Thus, the porcine Sk-alpha-Act promoter is regulated in a developmental and cell-type specific manner.

Sturgeon are an ancient family (Acipenseridae) of fishes that lie close to the divergence of fish that eventually evolved into terrestrial animals and those that evolved into modern teleost species. Therefore, white sturgeon vitellogenin sequences fill a gap in the current understanding of the functional domains of this protein family. Vitellogenin cDNA was sequenced and used to investigate gene expression in white sturgeon, Acipenser transmontanus. Estrogen-induced vitellogenin mRNA was detected in the livers of both males and females and was also detected in undifferentiated gonads of estrogen-treated fish. Low levels of vitellogenin mRNA were also detected in the testis of both control and estrogen-treated males. The cDNA encoded a 186-kDa protein that was missing only six to seven of the amino-terminal amino acids. Comparisons to silver lamprey, Xenopus, and chicken vitellogenin sequences indicate that the overall structure of the yolk protein domains were highly conserved. There was considerable homology in three regions of the lipovitellin I domain. These conserved sequences are likely to be involved in vitellogenin receptor binding. The phosvitin domain of white sturgeon vitellogenin contained fewer and shorter serine repeats as predicted from yolk protein phosphate content of fish compared to Xenopus and chicken. However, the vitellogenin of white sturgeon had a lower serine content as compared with silver lamprey, indicating that an increased serine content is not strictly a function of evolutionary age.