Brief Summary of Minutes of Annual Meeting

Washington Station (Dodson). 2010 was an interesting year for me. I spent considerable time working on collaborative projects, writing survey/review-type papers as a direct result of simply thinking about the "status" of the research field, and/or generating information about the productivity/impact of my research program for internal assessment. Considering the change in direction of some federal granting agencies, re-strategizing about where/how to submit grant proposals to a variety of granting agencies occupied much of my remaining time. Moreover, I tried to assemble interested individuals to work with me on a variety of collaborative projects in 2010--carried over to 2011. For the most part, however, I would consider my data-generating efforts to be minimal in 2010...especially considering that I was away from the laboratory for 2 months due to medical leave. Regardless, perhaps my best 2010/2011 accomplishment was to work with Sylvia, Gary and Werner to nominate specific members of this committee for ASAS awards for 2011. Georgia Station (Azain). Increased consumption of fructose has been suggested as a basis for increased rates of obesity in humans. Rodent studies have shown an increase in de novo lipogenesis and decreased insulin sensitivity in response to feeding high levels of fructose, but it is unclear if these effects occur in the same progression in humans. The use of the pig as a model for the human may be more appropriate than the rodent, as pigs and humans have similar GIT physiology and nutrient metabolism. Two experiments were conducted to determine if the source of dietary carbohydrate, with or without added fat, had an effect on body weight gain, glucose metabolism, or insulin response in growing pigs. In the first experiment, pigs (24 barrows, initial body weight 28 kg) were fed one of four diets where the source of carbohydrate was varied: 1) 20% starch; 2) 10% glucose + 10% starch; 3) 10% fructose + 10% starch; and 4) 20% fructose for 9 weeks. An intravenous glucose tolerance test (i.v.GTT) and response to meal (RTM) test were conducted at the end of 9 wks. Blood was analyzed for glucose, fructose, insulin, leptin, TG, and cholesterol concentrations. Dietary treatment had no effect on growth performance or carcass characteristics. Pigs fed the 10% fructose diet tended to have the lowest glucose area under the curve during the GTT (P=0.07). Otherwise, there were no differences in glucose tolerance, insulin, or other metabolites. Serum fructose concentrations after the RTM test were elevated in pigs fed fructose diets. Experiment 2 was conducted as a 3 x 2 factorial with main effects of carbohydrate source (Starch, Glucose, Fructose) and fat level (0 vs 10%). Pigs (24 barrows, initial body weight 71 kg) were fed one of six experimental diets for 9 weeks. As in the first experiment, there were no differences in growth rate or carcass characteristics. As would be expected, there was a main effect of dietary fat to reduce feed intake. There was a CHO x Fat interaction for serum glucose and insulin during the GTT. Compared to the other dietary treatments, pigs fed fructose with high fat had an elevated glucose area under the curve during the GTT (CHO x Fat interaction, P< 0.01). This same group had a lower insulin response (CHO x Fat, P < 0.05). As in experiment 1, serum fructose after the RTM test was elevated in the fructose fed pigs. It is likely that studies of longer duration are needed to determine if these changes are indicative of insulin resistance. The work demonstrates that pigs can be a viable model to test long-term effects of dietary carbohydrates on metabolism and body composition. Alabama Station (Bergen). During the past year we have continued our focus on the molecular determinants of depot specific adipose accretion in beef cattle. We have continued to analyze expression patterns of putative and novel regulatory genes responsible for lipid and protein metabolism in economically relevant tissues. In one study weve examined the effect of days on feed and Beta-agonist administration on gene expression patterns in finishing heifers (see abstract below). In another study we looked at the effects of forage finishing on the expression of key regulatory factors in beef steers using serial adipose and skeletal muscle biopsies during the finishing phase. In general we have determined that depot-specific, temporal changes in gene expression may contribute to phenotypic variation in overall and intramuscular adiposity. The Effect Of Days On Feed and Beta-Agonist Administration On The Expression Of Adipocyte-Specific Regulatory Genes in Skeletal Muscle and Adipose Tissue of Finishing Heifers. FASEB J. April 2011 25 (Meeting Abstract Supplement) 985.4 The development of marbling (Intramuscular fat; IMF) is an economically relevant trait that is mediated at the molecular level of adipose tissue metabolism. The objective of the current research was to evaluate adipose-specific regulatory genes in skeletal muscle IMF, and adipose depots of finishing heifers. Gastrocnemius Muscle (GM), Longissimus Dorsi Muscle (LM), and subcutaneous adipose tissue (AT) were collected after heifers were on feed for 79, 121, and 163 days and treated with (BAA) or without (Control) beta-agonist for the final 30d of feeding, respectively. Quantitative Real-Time PCR was used for gene expression analysis. Pref-1 expression was highest in the initial slaughter group (P<0.05), and decreased as time on feed increased. LM showed decreased Pref-1 expression relative to GM indicating an increased IMF adipogenic capacity in this muscle depot. BAA treatment appeared to decrease Pref-1 expression in both LM and GM. Subcutaneous AT showed increased Pref-1 expression relative to LM (P<0.05) when treatments within slaughter groups were combined, but when individual tissues and treatments were compared, there was no difference between LM and AT (P>0.05), while the GM showed higher expression (P<0.05) relative to AT and LM. This study showed a temporal Pref-1 expression pattern that appears to be consistent with the paradigm of late development of the IMF depot. The effects of compensatory growth on gene expression patterns in the loin muscle and subcutaneous adipose tissue of forage fed beef. The purpose of this project was to determine the effects of different levels of growth on beef steers. To this end 48 steers were subjected to one of four feeding regimens consisting of either dormant pasture or growing ryegrass for different durations during the trial period (See figure below). Skeletal muscle (Longissimus dorsi) and adipose tissue biopsy samples were collected every 42 days (d42, d84, and d126) for gene expression analysis. Loin muscle, gastrocnemius muscle, and subcutaneous fat samples were also collected at slaughter to determine depot specific patterns of gene expression as affected by different levels of available energy during the forage-finishing period. Data analysis for this study is currently underway. Ohio Station (Lee). We found that amounts of ATGL protein could not perfectly reflect lipolytic activities, suggesting other regulatory mechanisms are involved in these processes. We demonstrated that ATGL-medicated lipolysis is regulated by modulation of other proteins, an activator and inhibitor of ATGL protein in chicken adipose tissue. The genes encoding activator and inhibitor proteins (CGI-58 and G0S2 genes) were successfully cloned for chickens, turkeys and quail and reported in the Genbank. The hormonal, developmental and nutritional regulation of these genes in adipose tissue was performed to relate the function of these genes in lipolysis of the avian species (JAS papers). We are currently working on generation of transgenic quail expressing these genes under the adipose-specific promoter to study interaction of these genes in the regulation of lipolysis. We recently cloned bovine ATGL cDNA for several different breeds and characterize the ATGL gene expression in various cattle tissues. We also investigated the expression of genes involved in lipid metabolism in the muscle of cattle fed a flaxseed supplement (FS). We found supplemented animals had a greater triglyceride content in the muscle compared with unsupplemented, increasing marbling. Dietary supplementation, with flaxseed meal containing a greater amount of omega-3 fatty acids, alters muscle fat content with associated changes in the expression of genes (A-FABP, ATGL and SCD1) involved in lipid metabolism within the skeletal muscle. The results from these studies were published in Lipids. In general, genomic imprinting is an important regulatory mechanism of gene expression either from the maternal or paternal allele. Dlk1, a gene responsible for muscle hypertrophy of callipyge sheep, is an imprinted gene that is expressed from the paternal allele in mammals. Like other imprinted genes found in the cluster, Dlk1 is found in the conserved synteny of the imprinted gene cluster, called the Dlk1-Dio3 cluster in mammals. However, the imprinting status of Dlk1 in the avian species has not been reported and conservation of the chicken orthologue of the mammalian imprinted gene cluster has not been studied yet. The objective of this experiment was to clone Dlk1 cDNAs for turkeys and quail, and to determine the genomic structure of the cluster in the chicken and the imprinting status of avian Dlk1 genes. Our data clearly demonstrated the avian Dlk1 genes are expressed from both the paternal and maternal allele and are not subject to genomic imprinting. We provide evidence supporting the evolvement of genomic imprinting only in mammals after the divergence of mammals and birds (PS paper). In addition, transgene expression between different founders of quail can be regulated by methylation status of the transgene (Mol. Biotechnol.) I have been leading the collaborative work in the area of brown adipose tissue metabolism during cold stress. This study focuses on the cellular signaling events, as well as the gene expression changes associated with the alterations to lipid metabolism in the brown adipose tissue of mice when they are exposed to extreme cold. This research has significant implications in the study of human obesity and diabetes; activation of the heat generating system within mammals increases metabolic inefficiency, thus, allowing the burning of fat to create heat (Lipids paper). West Virginia Station (Barnes). The primary goal of our laboratory is to understand the mechanism(s) of action of dietary conjugated linoleic acid-induced body fat loss. We have utilized a model of enhanced CLA responsiveness in mice raised from weaning, for 6 weeks prior to CLA supplementation, on diets deficient in polyunsaturated fatty acids (coconut oil). As well, we have investigated the effect of different sources of DHA on body composition and serum lipids and the effect of CLA on pork marbling. Major findings from this years work include: 1. CLA increases lipolysis in coconut oil-fed mice more rapidly than in soy oil-fed mice, 2 Algal oil must be fed at a greater dietary DHA concentration than fish oil to achieve equal tissue DHA concentrations. And a dose of algal oil that resulted in greater tissue DHA concentration was required to achieve similar reductions in serum triacylglycerol and cholesterol, 3. Increases in loin lipid content in CLA-fed pigs may involve increased intramuscular adipocyte size. Time-dependent effect of conjugated linoleic acid-induced body fat loss and lipolysis in coconut oil fed mice. SiriManasa Ippagunta and Kimberly M. Barnes To be presented at Experimental Biology, April 10, 2011 Abst #109.2. Dietary conjugated linoleic acid (CLA) causes a body fat loss that is enhanced when mice are fed coconut oil (CO). The objective was to determine if there is a time-dependent effect of CLA feeding on lipolysis. Male mice (ICR; n=80; 3wk-old) were fed 7% SO or CO diets for 6wk then 0 or 0.5% CLA for 3, 7, 10 or 14d. A body fat index (BFI) was calculated and lipolysis was determined ex vivo by NEFA and glycerol release from adipose tissue. The relative expression of perilipin and phosphorylated perilipin (P-perilipin) were determined by western blotting. The BFI was reduced by CO on d7 (P<0.01), and by both CLA (P<0.05) and CO (P<0.05) on d14. NEFA release was increased by CLA in CO-fed mice (1.84 vs 7.93 µmol/g; P<0.01) but not in SO-fed mice (1.64 vs 2.03 µmol/g) on d7 but on d14 CLA increased NEFA release in both CO (2.83 vs 6.16 µmol/g) and SO-fed mice (2.01 vs 4.99 µmol/g). Glycerol release was increased by CLA in CO-fed mice but not in SO-fed mice on d3 and d7 (P<0.05). P-perilipin was not altered by diet but total perilipin tended to be increased by CLA in CO-fed mice (P=0.055) on d7. Therefore CLA-induced lipolysis may occur more rapidly in CO vs SO-fed mice but appears to be transitory. Effect of high docosahexaenoic acid-algal oil on body fat and serum lipids in mice Abigail G. Shelton, Ralph A. Pietrofesa, Michael Azain, and Kimberly M. Barnes To be presented at Experimental Biology, April 10, 2011 Abst #586.5. Algal oil (AO) contains a high content of docosahexaenoic acid (DHA) but no eicosapentaenoic acid (EPA), unlike fish oil (FO) which has both DHA and EPA. AO, fed at equal DHA levels, was not as effective as FO at reducing body fat or serum lipids. Our objective was to determine the dose response of AO vs FO on body fat and serum lipids. Male mice (ICR; n=94; 9-wk-old) were fed a 12% lipid diet containing 0 (soy oil; SO), 10 (FO or AO-10), 20 (AO-20), 35 (AO-35), or 50 (AO-50) g/kg DHA for 2 or 4 wks. A body fat index (BFI) was calculated, serum lipids, adipose fatty acid profile, and proximate composition of the carcasses were determined. At 2 wk FO-fed mice were leaner (p<0.01) than SO-fed mice, with all AO-fed mice intermediate. No differences in body composition were observed at 4 wk. FO and all AO doses reduced (p<0.05) serum triglycerides and cholesterol at 2 wk but only AO-35, AO-50, and FO reduced (p<0.001) cholesterol at 4 wk. Tissue DHA was similar between FO, AO-10, and AO-20-fed mice, with AO-35 and AO-50-fed mice having greater (p<0.001) tissue DHA. Therefore, algal oil appears to not be as effective as fish oil in decreasing body fat and serum lipids. Iowa Station (Beitz). The current study was designed to identity polymorphisms in the genes involved in milk lipid biosynthesis to provide animal breeders with tools that allow selection of animals producing milk with healthier fatty acid composition. High concentrations of saturated fatty acids (SFA) in human diets are known to increase plasma cholesterol concentrations and, as a result, increase the risk of developing cardiovascular diseases (CVD), the number one cause of death worldwide. Because bovine milk is one of the primary sources of SFA and individual atherogenic fatty acids such as palmitic (16:0) and myristic (14:0) in human diets the improvement of the healthfulness of milk through selection becomes one of the primary measures that has been taken with the intention of decreasing the incidence of CVD among humans. The candidate gene approach was used to address the objectives of the study. Genes involved in milk triacylglycerol (TAG) biosynthesis, fatty acid uptake into mammary gland and fatty acid transport inside the mammary epithelial cells, and transcriptional regulation of some lipogenic genes were investigated. DNA sequencing was used to discover single nucleotide polymorphisms (SNPs) in the genes of interest. After genotyping animals on the study for the discovered SNPs, the intragenic haplotypes were reconstructed and tested for the association with milk fatty acid composition. The glycerol-3-phosphate acyltransferases-1 and -4 (GPAT1 and GPAT4), 1-acylglycerol-3-phosphate acyltransferase-1 (AGPAT1), and phosphatidate phosphatase (LPIN1) genes from the TAG biosynthetic pathway were studied in the first set of experiments to test the associations of the polymorphisms in those genes with milk fatty acid composition. The polymorphisms in GPAT4 were associated with large differences in atherogenic index (AI), concentrations of SFA, unsaturated fatty acids (UFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), SFA/UFA, concentrations of capric (10:0), lauric (12:0), palmitic (16:0), and oleic (18:1 c9 ) acids, CLA (18:2 c9, t11 ), C16 and C18 desaturation indices in milk. The size of the effects of GPAT4 polymorphisms for some of the traits was numerically at least the same or larger compared with the effect of DGAT1 A232K mutation, making the polymorphisms in GPAAT4 to be a very valuable tool for the improvement of the healthfulness of milk. Other polymorphisms significantly associated with the studied traits in the first set of experiments were in GPAT1 for milk fat percentage, concentrations of short- and medium-chain SFA, and myristoleic (14:1 c9 ) acid concentration, and in AGPAT1 for the concentrations of linoleic (18:2 c9, c12 ) acid and other UFA. The polymorphisms in GPAT1 can be used to select for animals producing milk with higher percentage of fat and desirable concentrations of short- and medium-chain SFA. The polymorphisms in AGPAT1 can be used to select for animals producing milk with higher concentration of UFA and linoleic (18:2 c9, c12 ) acid, in particular. In the second set of experiments, the polymorphisms in the solute carrier family 27 (SLC27A6), isoform A6 and fatty acid binding proteins-3 and -4 (FABP3 and FABP4) genes involved in fatty acid uptake into mammary gland and fatty acid transport inside the mammary epithelial cells were tested for the association with milk fatty acid composition. The haplotype effects of SLC27A6 were associated significantly with milk fat percentage, AI, the concentrations of SFA, UFA, MUFA, SFA/UFA, the concentrations of capric (10:0), lauric (12:0), myristic (14:0), and palmitic (16:0) acids. The size of the haplotype effects of SLC27A6 on the studied traits was large and numerically similar to the size of allelic effects of DGAT1 A232K mutation that makes the polymorphisms in SLC27A6 as valuable as the of DGAT1 A232K mutation to select for animals producing milk with higher fat percentage and healthier fatty acid composition. The haplotype effects of FABP4 were associated significantly with the concentrations of SFA, UFA, MUFA, PUFA, SFA/UFA, the concentrations of linoleic (18:2 c9, c12 ) acid, CLA (18:2 c9, t11 ), and C18 desaturation index. The sterol regulatory element binding protein-1c (SREBP-1c) is involved in the transcriptional regulation of lipogenesis and its proteolytic activation is controlled by SREBP cleavage-activating protein (SCAP) and insulin-induced genes (Insig) that are all part of the SREBP pathway. In the third set of experiments, the significant association of the overall haplotype effect of SREBP1 with the concentrations of myristic (14:0), myristoleic (14:1 c9 ) acids, and C14 desaturation index were detected. The overall haplotype effect of Insig1 was associated with the concentrations of PUFA and linoleic (18:2 c9, c12 ) acid. There were no significant associations with milk fatty acid composition determined for SCAP. In conclusion, we were able to identify polymorphisms in a number of genes that were associated significantly with milk fat percentage and fatty acid composition. The information about those polymorphisms can be used to select for animals producing healthier milk. Wyoming Station (Du). AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; it is inhibited under obese conditions and is activated by exercise and by many anti-diabetic drugs. Emerging evidence also suggests that AMPK regulates cell differentiation including adipogenesis, but the underlying mechanisms are unclear. ²-Catenin is a key mediator of Wingless and Int (Wnt)/²-catenin signaling pathway, which is required for early embryonic development, cell proliferation, and differentiation, and activation of b-catenin signaling pathway inhibits adipogenesis. We postulate that AMPK regulates adipogenesis through cross-talk with Wnt/²-catenin signaling pathway. We previously observed that AMPK phosphorylates b-catenin, which enhances b-catenin stability. Here, we further show that AMPK also regulates b-catenin expression. Epigenetic modifications including histone acetylation and methylation, and DNA methylation, regulate gene transcription. Histone acetylation is regulated by histone acetyltransferase (HAT) and histone deacetylase (HDAC). HDAC5 belongs to the class IIa HDAC family, and acts as a conserved transcriptional repressor. HDAC5 interacts with myocyte enhancer factor-2 (MEF2) to target specific gene promoters. We identified a MEF2 binding site on the b-catenin promoter. These data prompted us to hypothesize that AMPK regulates b-catenin expression through phosphorylation of HDAC5. Here, we present data showing that AMPK phosphorylates HDAC5, which promotes its nuclear export, leading to the acetylation of histones that are bound to the b-catenin promoter and enhanced b-catenin expression In both C3H10T1/2 cells and mouse embryonic fibroblasts (MEFs), AMPK activity was positively correlated with b-catenin content. Chemical inhibition of HDAC5 increased b-catenin mRNA expression. HDAC5 over-expression reduced and HDAC5 knockdown increased H3K9 acetylation and cellular b-catenin content. HDAC5 formed a complex with myocyte enhancer factor-2 (MEF2) to down-regulate b-catenin mRNA expression. AMPK phosphorylated HDAC5, which promoted HDAC5 exportation from the nucleus; mutation of two phosphorylation sites in HDAC5, Ser 259 and 498, abolished the regulatory role of AMPK on b-catenin expression. In conclusion, AMPK promotes b-catenin expression through phosphorylation of HDAC5, which reduces HDAC5 interaction with the b-catenin promoter via MEF2. Thus, the data indicate that AMPK regulates cell differentiation and adipogenesis via cross-talk with the wingless and Int (Wnt)/b-catenin signaling pathway. Wyoming Station (Rule). Our hypothesis was dietary rumen-protected n -3 PUFA will increase concentrations of these fatty acids in tissues of grass-fed beef cattle. Forty half-blood LowLine Angus steers (290.5 ± 6.6 kg initial BW) were allotted to either a control (CON; no supplemental fat), saturated fatty acid Ca salt (SAT), or fish oil fatty acid Ca salt (N3) treatment in a completely randomized designed experiment. Beet pulp supplements that contained 7.6% molasses, 4.0% CaCO3 for CON, 4.4% mineral mix, and 1.8% Poloxalene were individually fed and formulated to provide 0.25% of BW as supplement and 2.0% of DM as fat for SAT and N3. Irrigated pasture consisted of 25% bromegrass, 25% wheatgrass, and 50% alfalfa (CP = 20.9%; 36.7 kg DM " head-1 " d-1), and was rotated weekly from June 1 through October 15, 2008 when steers were fed forage harvested from the same pastures until December 8. Blood was sampled at 45 and 93 d. Steers were shipped 137 km for slaughter at a commercial plant; liver was sampled upon evisceration. Twelve days post mortem, 100 g each of longissimus (12th rib), supraspinatus, and semitendinosus muscles were obtained. Fatty acids extracted from serum, liver, and muscles were analyzed by GLC with C13:0 as internal standard. In liver compared with CON, SAT caused increased (P < 0.01) C16:0, C18:0, C18:1 n-9, C18:2 n-6, C20:3 n-3, and C20:4 n-6, and decreased (P = 0.01) C18:3 n-3. Compared with CON, N3 supplementation resulted in greater (P < 0.01) C18:1 t-11, C20:5 n-3 (eicospentaenoic acid, EPA) and C22:6 n-3 (docoshexaenoic acid, DHA), and less (P < 0.01) C18:1 n-9, C18:2 n-6, C20:3 n-3, C20:4 n-6 in liver. In muscle, concentrations of C18:2 n-6 and C20:4 n-6 increased (P < 0.01) for SAT compared with CON. For each muscle, N3 resulted in greater (P < 0.01) EPA and DHA compared with CON. Serum concentrations of fatty acids reflected differences in supplemental intake of C16:0 and C18:1 n-9 of SAT, as well as EPA and DHA of N3. Overall, supplementation of N3 resulted in 86.4% and 85.6% increases in concentration of EPA + DHA in liver and muscle, respectively. Taiwan Station (Ding). My group emphasized on the regulation of genes related to adipose function and obesity. We have established the nutritional regulation of DHA on the expression of metabolism related genes. We have also delineated the mechanism by which PUFA regulate fat deposition. Several regulation cascades, ie., serum amyloid protein, FoxO, and C/EBPbeta were found to mediate the PUFA effects. Indiana Station (Purdue- Ajuwon). In 2010 our research team continued our work on the role of adipose tissue extracellular matrix in the regulation of adipose tissue function and on the factors that regulate the turnover of adipose tissue extracellular matrix during diet induced obesity. In 2010, we determined the changes in adipose tissue matrix proteoglycans and collagens in response to high fat diet, acute or chronic inflammation. Our finding is that acute inflammation does not play a major role in regulation ECM gene expression, even though it does lead to significant upregulation of inflammatory genes. However, obesity induced by high fat diet feeding leads to induction of extracellular matrix gene expression determined by RT-PCR. However, western blot analysis of collagen 1a1, a major adipose tissue collagen shows reduction in this protein in obesity in humans and mice, despite an upregulation of its mRNA level. This discordance between mRNA abundance and protein level suggests a high turnover of the protein that cannot be matched by the increased mRNA abundance. This study demonstrates the importance of ECM breakdown machinery, mediated by matrix metalloproteases and other matrix degrading enzymes, in regulating the abundance of ECM proteins. We have also determined that ECM proteoglycans such as decorin and biglycan play important roles in the regulation of preadipocyte proliferation and apoptosis. Both decorin and biglycan inhibit adipocyte proliferation. However, biglycan is more proapototic than decorin. The implication of this finding is that the abundance of specific ECM proteins may determine the overall ability of adipose tissue to enlarge via the regulation of the proliferation and apoptotic rates of preadipocytes. Finally, we determined the anti-inflammatory function of secretory leucocyte protease inhibitor (SLPI) in adipose tissue. We found out the SLPI is regulated by obesity and exerts anti-inflammatory function in adipocytes.