Brief Summary of Minutes of Annual Meeting

Summary of minutes of annual meeting: Dr. Rule opened the meeting by introducing Drs. Kinder and Mirando, then new members, graduate students and visitors. Dr. Hausman and Dr. Novakofski were elected as co-chairs for the 2009 meeting. The committee decided to continue meeting in conjunction with Experimental Biology, which will be in New Orleans for 2009. The committee discussed hosting a symposium at EB in 2010 or at ASAS in 2009. Additional outreach in the form of a JAS review article with Hausman as lead author was discussed. The issue of attendance was discussed and the committee agreed to send an e-mail before the meeting stressing the importance of attendance, and to revisit the issue of members that were not attending at the 2009 business meeting. Non-attending members with no other evidence of interest or commitment would be administratively removed in the future. Dr. Kinder gave a brief administrative update, and stressed the importance of communicating the value of the group activities, especially impacts and output, in annual reports and summaries. Dr. Kinder reminded the committee that the project renewal was due before December 1, 2008 and stressed the involvement of pos-docs and grad students and the balance between research, teaching and outreach activities. Dr. Mirando discussed potential factors that might impact funding opportunities for NCCC097 members, as well as current and projected CSREES budgets. Dr. Mirando also discussed the potential to use outreach activities as research. Project Directors, graduate students, and postdoctoral researchers provided research update (see Accomplishments) followed by discussion and questions for each project. Thirteen research updates were presented by committee members with a guest presentation on from Xing Yu, Isis Pharmaceuticals, who had attended NCCC097 meetings as a student.

Accomplishments: The research objectives for NCCC097, October 2004 to September 2009, are available at the NCRA website: (http://lgu.umd.edu/lgu_v2/homepages/outline.cfm?trackID=3848) 1. Share and critique new techniques, experimental designs, and new unpublished data regarding adipocyte biology. 2. Through the interactive process at the meeting and afterward, elicit input to improve experimental design and to develop mutual interests toward joint projects. 3. Study biological regulation of catabolic (fatty acid mobilization and oxidation) and anabolic (fatty acid synthesis, elongation and desaturation, and triacylglycerol synthesis) lipid metabolism in domestic species at the molecular, cellular, tissue, and organismal level 4. Study cellular and molecular controls of adipocyte hyperplasia, differentiation and growth in domestic species. 5. Study nutrient determinants of excess fat deposition (diet composition, and amount and source of dietary fat, calories, and protein). 6. Study mechanisms associated with genetic variation in excess fat deposition and the post-genomic variation of individual phenotypes (determinants of cellularity, receptor populations, alternative metabolic signalling pathways, alternative gene expression and promoter function) 7. Establish approaches to maintain or enhance intramuscular fat concentration, but at the same time maintain or decrease subcutaneous and intermuscular fat. Intramuscular fat (marbling) is an important contributor to the organoleptic properties of meat, but the molecular and cellular mechanisms that retard intramuscular fat deposition until late in development are not understood. 8. Investigate the interplay between adipose tissue, liver, gut, and skeletal muscle lipid metabolism. The growth of adipose tissue is regulated, not only within the adipocyte, but also by the the interorgan interchange of metabolites, various lipoproteins, and numerous hormones and signalling molecules. 9. Investigate the role of endocrine factors produced by the adipocyte in co-ordinate regulation of feed intake, reproductive competency, energy expenditure, and immune function. 10. Study mechanisms for intervention strategies to decrease excess fat deposition (exercise, caloric restriction, pharmacotherapy, selective control of gene expression through transcription factors and modulation of promoter function). NCCC097 scientists conducted the following work over the past year. The Experiment Station and project member, followed by the objective number(s) met by the work, are provided for each entry. 1. Experiment Station: West Virginia. Representative: Kimberly M. Barnes. Objectives 1, 2 and 10. This year has been spent primarily setting up our laboratory and acquiring instruments. We have two primary projects planned for initiation this summer: The effect of oil source on conjugated linoleic acid-induced lipolysis in mice and The effect of feeding high-DHA Crypthecodinium cohnii on body composition in mice. Previously, we demonstrated that mice fed coconut oil from weaning for 6 weeks lost more body fat when supplemented with CLA than did mice fed soy oil. Part of this greater response appears to be due to increased basal lipolysis observed in CLA-fed mice raised on coconut oil but not soy oil. In cell culture models increased lipolysis in response to CLA has been observed and associated with increased activation of perilipin. This is followed by de-differentiation of the adipocytes, marked by decreased perilipin expression and increased adipose differentiation-related protein (ADRP). We plan to measure the phosphorylation (indicating activation) and expression of perilipin and hormone sensitive lipase and the expression of ADRP in reteroperitoneal fat pads of mice fed soy oil, soy oil + CLA, coconut oil, or coconut oil + CLA. Further studies on the activation of signaling molecules and a time-course of lipolysis activation are also planned. A colleague at West Virginia University, Dr. Kristen Matak, has developed a continuous culture system to grow Crypthecodinium cohnii. This algae has the potential to accumulate a large percentage of its biomass as lipid and to be highly enriched for the omega-3 fatty acid DHA. In Dr. Mataks system the algae has been determined to be over 18% lipid, with almost 30% of that lipid as DHA. We have determined that fish oil has the potential to lower body fat percentage alone but to inhibit CLA-induced body fat loss. 2. Experiment Station: North Carolina State University. Representative: Jack Odle. Objectives 1, 2, 3 and 8. Infant formula companies began fortifying formulas with long-chain polyunsaturated fatty acids (PUFA) in 2002. Long-chain PUFAs are precursors for the synthesis of prostanoids which stimulate recovery of intestinal barrier function. The objective of these experiments was to investigate the effect of higher supplementation of AA on intestinal barrier repair in ischemic-injured porcine ileum. One day-old pigs (n=24) were fed a milk-based formula for 10 d. Diets contained either no PUFA (0%AA), 0.5%AA, 5%AA, or 5% eicosapentaenoic acid (EPA) of total fatty acids. Following dietary enrichment, ileum from piglets were subjected to in vivo ischemia injury by clamping the local mesenteric blood supply for 45 min. Following the ischemic period, control (non-ischemic) and ischemic loops were harvested and transepithelial electrical resistance (TER) was measured over a 240 min recovery period. Ischemia-injured ileal tissues from piglets fed the 5% AA diet and 5% EPA exhibited enhanced recovery (% increase in TER = 13±13, 21.6±12 59.1±12, 50.8±13, for 0%AA, 0.5%AA, 5%AA, and 5%EPA, respectively, p<0.05). Enhanced TER recovery response observed with 5% AA supplementation was attenuated by in vitro application of indomethacin suggesting responses were mediated by prostaglandins. Histological evaluation of piglets fed the 5% AA formula showed reduced lesions after ischemia compared to other dietary treatments (P<0.05). Long-chain PUFAs enhances recovery of TER in ischemic injured porcine ileum mediated via endogenous prostaglandins. Objectives 1, 2, 3, 5 and 8. Another study examined the effect of maternal feeding of 0.8% clofibrate for 7 days on fatty acid oxidation in newborn pigs. The maternal-fed clofibric acid had no impact on the liver weight of the newborn pigs, but hepatic fatty acid oxidation examined in fresh homogenates showed that clofibrate significantly increased 14C-accumulation in CO2 and ASP by 2.9-fold from [1-14C]-oleic acid and 1.6-fold from [1-14C]-lignoceric acid. Correspondingly, hepatic carnitine palmitoyltransferase (CPT) and fatty acid oxidase (FAO) activities were increased also by 36% and 42% compared to the newborn pigs from the control sow. Clofibrate crosses the placental membrane of the sow and enters fetal circulation. Thirty four colostrum-deprived newborn pigs were fed milk replacer and orally gavaged with either vehicle (2 % Tween 80) or clofibrate (75 mg /kg body weight) +/- etomoxir (5 mg/ kg body weight) once daily for up to 7 days, and the sensitivity of liver CPT I to malonyl-CoA inhibition and fatty acid oxidation were determined using 1-14C-octanoic, oleic and erucic acids. Administration of clofibrate significantly increased liver CPT I activity (60%) and malonyl-CoA IC50 values (56%). Addition of etomoxir potently inhibited CPT I activity, but malonyl-CoA IC50 remained unchanged. Consistent with CPT I activity, clofibrate increased mitochondrial fatty acid oxidation by 53%, but did not affect the rate of peroxisomal beta-oxidation. Etomoxir did not alter the rate of fatty acid oxidation despite a 30% reduction in CPT I activity. Fatty acid oxidation induced by clofibrate is due to both an increase in CPT I activity and a decrease in sensitivity of CPT I to malonyl-CoA inhibition. Objectives 1, 2, 8 and 9. Another research area was safety assessments of novel infant formula ingredients in neonatal pig models. One-day old pigs were fed a cows milk-based formula supplemented with PDX (0, 2, 5, 10 or 20 g/L) for 18 days (n=6/formula group) to further substantiate the efficacy and safety of the ingredient. Additional reference groups included 6 pigs sampled at day 0 and 12 sow-reared pigs. Growth rate, formula intake, stool consistency, behavior score, blood metabolites and relative organ weights (i.e. liver, spleen and kidney as % of body weight) did not differ among formula-fed pigs (P > 0.1). Among PDX-fed pigs, digesta pH decreased linearly (P < 0.05; ileum, cecum, colon) and quadratically (P < 0.05; ileum, cecum) as dietary PDX increased, with a maximal reduction approaching 0.5 pH units in those fed 20 g/L. Cecal and colonic pH of sow-reared pigs were similar to pigs fed 20 g/L PDX. Polydextrose shows promise as a prebiotic additive in infant formula. 3. Experiment Station: Washington Station. Representative: M.V. Dodson. Objectives 1, 3, 4 and 7. Cell culture model of adipocytes has proven to be both a boon and a bust to scientists. If the cell culture system is well developed, and time is not required to establish the limits of the in vitro system, then relatively large amounts of research data may be generated in relatively short timeframes and researchers may make steady research gains and publish above average numbers of papers with minimal delays. However, cell culture systems and conditions differ between laboratories. In many cases data generated under similar conditions but in different laboratories are not consistent. For adipogenesis studies, cell lines and stromal vascular cells are commonly used, even though the value of each system is limited. Between-laboratory comparisons may NOT occur if the cultures are not maintained similarly/properly, are/become transformed, or if the entire cellular system is ill-defined (cells such as SV cell cultures can NEVER be enumerated for consistent subpopulation components between laboratories). Vigilance is required to obtain repeatable data from these systems, especially if mechanistic or developmental experimental designs are attempted. We suggest that all aspects of basic cell culture are as important as growing cells. Our progeny cell cultures are not like SV cells or cell lines in dynamics of re-differentiation and formation of lipid-filled adipocytes. Objectives 1, 3, 4 and 6. Another project examined gene regulation of bovine-derived adipofibroblasts. It is important to understand mechanisms of fat metabolism in beef cattle as the fat component and distribution are directly associated with the quality and the value of the meat. The discovery of gene markers associated with various carcass traits in beef cattle has created links between the genetic background of the animal and its performance. Animal performance is directly associated with the end products of gene expression and the functional proteins expressed. Beef-derived adipocytes (derived from mature adipocyte dedifferentiation in Dr. Dodson's laboratory) were analyzed by Dr. Leluo Guan (University of Alberta), with genomic/proteomic tools. Using perimuscular-derived adipocyte-derived preadipocytes, Dr. Guan showed that no C/EBP± was expressed by differentiating beef-derived cells in vitro. As C/EBP± operates with other nuclear signaling agents (especially PPAR³), either PPAR³ functions alone in regulating adipogenesis in the perimuscular adipose tissue depot of beef steers, or the expression of C/EBP± is transitory. Adipocytes from one adipose tissue depot in beef cattle are different to other types of fat cell systems, or other adipose depots. Objectives 1, 3 and 7. Adipose tissue contains a large portion of stem cells and it is important toidentify transdifferentiated cells from progeny cultures derived from dedifferentiation of mature adipocytes in vitro. These cells appear morphologically like fibroblasts, and are primarily derived from the stromal cell fraction. Mature (lipid-filled) adipocytes possess the ability to become proliferative cells and have been shown to produce progeny cells that possess the same morphological (fibroblast-like) appearance as the stem cells from the stromal fraction. This simple fact may prove to provide a novel mechanism via which we can learn about the stem-cell nature of the progeny cells, determine if the proliferative-competent progeny cells are capable of becoming other cell types, and capitalize on the properties of the progeny cells for tissue engineering purposes. Objective 1 and 2. Many dynamics of domestic animal physiology, adipogenesis and assimilation of energy are different from rodents and/or humans. We periodically submit scientific papers to help teach scientists in other disciplines about domestic animal-derived adipocyte research. Historical research with adipose tissue/adipocytes proved that tissue/cells actually played a large role in the outcome of any given experiment. Much of today's research with adipose tissue/cells, however, is focused on subcomponents of the cell (DNA/RNA), intracellular regulatory pathway (signaling mechanism) of cell morphogenesis, or the possible identification of adipose tissue-derived stem cells for tissue engineering purposes. Years ago, both tissue characteristics and the source of the cell were quite important. Today, what is important is the molecular mechanism being defined. Considering the molecular emphasis placed on graduate training in this field, it is easy to suggest that new investigators might be incapable of extrapolating their molecular data to the cell, tissue or live animal in any practical manner. One goal of my work is to provide historical reference to those interested in adipose tissue/cells in animal sciences. 4. Experiment Station: Western Human Nutrition Research Center. Representative: Sean H. Adams. Objectives 1, 2, 4, 5 and 6. We introduced our initial characterization of tumor suppressor candidate 5 (Tusc5) at the 2006 and 2007 NCCC097 meetings, and published on this gene in 2007. We have extended those findings by identifying and characterizing a second gene-protein with unique robust expression in both afferent neurons and adipocytes: g-synuclein (SNCG). Searching databases for expression similar to Tusc5, and follow up by RT-PCR, confirmed high expression in these sites in humans, mice, and rats. This protein has been previously implicated in cancer tissue and its knockdown limits cancer cell line proliferation. With James Pan, Toronto we found that the gene is downregulated by PPARg agonists and is induced during 3T3-L1 differentiation. Our model is that SNCG is involved with fat tissue (and neuron) plasticity in response to metabolic cues. SNCG mRNA expression is significantly increased in the WAT of obese humans and is highly expressed in subcutaneous vs. visceral/omental fat (confirmed in several human cohorts). Its expression patterns are closely correlated with those of leptin in both cell culture and human WAT, indicating shared regulatory elements. Since SNCG was reported to physically interact with TIMP-2, a regulator of extracellular matrix remodeling, future studies will examine how SNCG influences the adipose ECM in response to obesity and PPAR modulation. Objectives 1, 2, 6, 9 and 10. Low muscle fatty acid catabolism is associated with insulin resistance. Identification of biomarkers of muscle fat combustion could prove useful to predict disease or monitor efficacy of interventions. No metabolite or metabolite pattern has specifically been tracked to muscle fat catabolism. Comprehensive acylcarnitine analyses and clinical chemistry were conducted to compare plasma of controls vs. persons harboring a missense mutation in UCP3 (a muscle-specific protein in humans; the missense yields 50% lower fat metabolism and a higher CHO catabolism), in collaboration with Drs. Tim Garvey (UAB) and Chuck Hoppel (Case Western). Few differences in metabolites were observed when comparing wildtype vs. UCP3 polymorphic subjects, with the exception of butyrylcarnitine and lactate that were lower in the latter. Comparing diabetics vs. non-diabetics across genotypes, diabetics displayed significantly increased acylcarnitine:free carnitine ratio, higher acetylcarnitine, and reduced propionylcarnitine. Current results are consistent with the hypotheses that: (a) persons with dysfunctional UCP3 in muscle improve efficiency of pyruvate flux toward the TCA cycle (hence, lower lactates), (b) C4-carnitine may be a sensitive marker of ß-oxidation/CHO oxidation dynamics, and (c) type 2 diabetics may have more limited TCA cycle function reflected in higher acetylcarnitine levels in plasma resulting from reduced capacity to dispose of acetyl-CoA. Lower C3-carnitine in diabetics might mark a more limited generation of propionyl-CoA, an important anaplerotic carbon source for replenishment of the TCA, which if true could contribute to compromised TCA cycle function. 5. Experiment Station: University of Geogia. Representative: M. J. Azain. Objectives 1, 2, 7 and 10. The 14 wk study was conducted as a 2 x 2 factorial with main effects of consuming an omega-3 supplemented diet during growing and/or early finishing phase. The n-6/n-3 ratios of the control and fish oil diets were in the range of 10-12 and 2-3, for the soy and fish oil diets, respectively. There was no effect of dietary fat source on growth performance. There were only minimal effects of diet on carcass characteristics. There was a trend for greater lipid content in the loin muscle from pigs fed fish oil for the first 5 weeks of the study. At slaughter, average lipid % was 4.23% in pigs fed the control diet for the first 5 weeks as compared to 5.04% in pigs fed fish oil (P < 0.10). As expected, dietary fat affected tissue fatty acid profiles. The n-6/n-3 ratio in loin muscle of control pigs was 18.7, 17.7 and 20.9 in tissues sampled on days 35, 70 and 98. In contrast, the ratio was 4.4, 3.7 and 5.8 on days 35, 70 and 98, respectively, in pigs fed fish oil from day 0-70. Feeding fish oil for only the first or second 5 week periods resulted in intermediate ratios. Calculated long-chain omega-3 content was increased from less than 5 mg per serving of pork to 30-60 mg/serving in pigs fed fish oil for 5 to10 weeks. Although a taste panel test was not conducted, there were no obvious fishy odors in any of the samples on day 98 (4-9 week withdrawal period). The increase in intramuscular fat and changes in fatty acid profiles in response to feeding fish oil in the grower period, are worth of further investigation. Objectives 1, 2, 3 and 7. A significant portion of the US corn crop is currently going into ethanol production. This has resulted in greater feed costs, causing producers to examine alternative feed ingredients. Distillers dried grains with solubles (DDGS) is a co-product of ethanol production from corn and has received considerable attention with expansion of ethanol plants. DDGS is essentially corn without the starch and thus, is enriched in protein, ether extract and fiber. Fermentation converts much of the phytin phosphorous in corn to a more readily available forms. One issue with use of DDGS in swine diets is the higher level of ether extract in the product. Since this is essentially corn oil, it is an unsaturated fat source and feeding may result in higher iodine values which reduces the acceptability of pork by the packing industry and reduces shelf-life in the display case. A means to offset the negative effect of DDGS on pork carcasses would be advantageous. We determined if addition of a saturated fat to the diet counteracts the negative effects of the unsaturated fat in DDGS on pork quality. The study was conducted as a replicated 2 x 2 design with main effects of saturated or hydrogenated fat addition (0 and 4% Fat Pak 100, Milk Specialties), and DDGS ( 0 vs 30% ) inclusion. Pigs fed diets with 30% DDGS had reduced intake during the first 2 weeks of the study and overall. Intake was not different from week 2-6, but pigs did not recover from the reduction in the first 2 weeks. As a result, overall growth rate and final body weight was reduced in pigs fed DDGS. Tissue fatty acid profiles were altered by DDGS feeding. Calculated iodine value in subcutaneous, belly and leaf fat, but not loin muscle, was increased with DDGS. This was associated with a greater linoleic acid content in tissues from pigs fed DDGS. The addition of hydrogenated fat product, Fat Pak, had minimal effects on fatty acid profiles. The lack of effect of this fat source on tissue profiles is accounted for by the lack of digestibility of the product. 6. Experiment Station: University of California Davis. Representative: Dr. Roberto D. Sainz. Objectives 1, 2, 4, 5, 6 and 7. The beef and other meat industries are becoming more competitive and more uncertain, due to environmental concerns, increases in commodity prices and diversity in feed availability for animals. Prices received by producers are based upon the carcass value of their animals (quality and yield grades for beef cattle). The primary determinants of carcass value are the amount and distribution of carcass fat. The key to maximizing carcass value and profit is to feed animals to their optimal endpoint with adequate marbling and not excessive fat trim. Thus there is a need for tools to guide producers in their management decisions. Mathematical models of animal growth and body composition have been shown to accurately predict performance and composition of animals of different genotypes under different management scenarios. To predict the optimal slaughter endpoint, a model must be dynamic, i.e., the model should predict gain and composition over a period of time using known relationships between state variables. The best models currently available are also deterministic (there are no random elements) and mechanistic, which means that equation forms and parameter values are based on knowledge of the underlying biology. Current models (e.g., NRC, 2000) are static and empirical (relationships are based on regressions), so cannot be applied to production scenarios beyond the range of values used in their development. The Davis Growth Model (DGM) is a dynamic, deterministic and mechanistic model of beef cattle growth and body composition (Oltjen et. al., 1986; Sainz and Hasting, 2000). The DGM is based on fundamental biological concepts of tissue growth, such as hyperplasia and hypertrophy, as well as net energy. Body protein accretion is calculated based on the difference between the rates of protein synthesis (determined by total DNA content, hormonal status and plane of nutrition) and protein degradation. Accumulation of total body fat is determined from the net energy available after accounting for maintenance and protein gain. Body fat is further partitioned among four depots. The DGM predicts overall growth and body composition of cattle of different genotypes, implant status and feed regimes with excellent accuracy and precision. The equations to partition fat gain among of adipose depots, however, was based upon only two data sets (Robelin, 1981; Cianzio et al., 1985), as these were the only publications found in which fat depot weights, cell numbers and sizes were reported. As a result, the DGM can be used to accurately forecast body composition over time, but predictions of fat distribution are inadequate. To improve these equations, more data on adipose cellularity in animals of different genotypes, ages, and growth trajectories will be required. Objectives 1 and 2. The objective of this research is to investigate the effect of breed, sex, implant status and energy level of the diet on adipocyte development and distribution in beef cattle. Animals will be harvested at different ages and fat depot weights will be determined by dissection. Samples from each the four fat depots will be collected, and prepared for histological examination of cell numbers and sizes. Another goal is to develop a faster method to make those measurements in the cold carcass, in order to enable data acquisition on a larger scale in the future. In addition to measurements of adipocyte cellularity, transcriptional factors involved in regulation of adipogenesis will be determined. These will include C/EBP, PPAR³, and ADD1/SREBP. Other factors may include adiponectin, leptin, and other adipocyte-specific markers. The exact nature of the molecular analyses will depend on consultations and collaborations with other members of the committee. The relationships and equations will be specific to beef cattle, but the underlying biology should be applicable to other species including humans. 7. Experiment Station: Auburn University/ Alabama Agricultural Experiment Station. Representative: Werner G. Bergen. Objectives 1, 2, 3,4, and 6. We haveshown that Duroc and Pietrain F1 pigs differ in lipid deposition and fatty acid oxidation in liver, adipose and skeletal muscle. Finishing Duroc pigs had a greater propensity for de novo fatty acid synthesis and a lesser propensity for fatty acid oxidation than finishing Pietrain pigs based on mRNA abundance for key lipogenic and beta oxidation enzymes. We have continued to explore metabolic differences in fat vs. lean type pigs by assessing differential expression of PGC-1± and UCP-3 in skeletal muscle. The expression results below are based on qRT-PCR assays. For both PGC-1± and UCP-3 there are no apparent differences in mRNA abundance in control pigs; however the beta adrenergic agonist marginally enhanced UCP-3 expression in both pig genotypes while was PCG-1± slightly enhanced in both breeds. We are presently also evaluating the relationship between residual feed efficiency (RFI), feed efficiency and expression of trans- acting factors, coactivators and lipid anabolic and oxidative genes in skeletal muscle and adipose tissue of beef cattle. Tissue samples were secured during the last week of a finishing trial by biopsy and gene expression was assessed using qRT-PCR. There were 8 lower RFI bulls and 8 higher RFI steers (Angus sired from Angus X Simmental dams). The RFI values are not yet available; however the bulls had a lower F/G (5.82) than the steers (7.20). The mRNA abundance in adipose biopsies for PGC-1± were not different between the bulls and steers. 8. Experiment Station: University of New Hampshire. Representative: Gale B. Carey. Objectives 1, 4, 6, 8, and 9 Our recent publication (Obesity 15:2942-2950, 2007) describes the effect of administering polybrominated diphenyl ethers (PBDEs), a family of flame retardants, to rats daily for 4 weeks, on isolated adipocyte metabolism. These lipid-soluble, flame-retarding environmental chemicals will deposit in adipose tissue, and our findings demonstrate that adipocytes isolated from treated rats are 30% more sensitive to epinephrine and 60% less sensitive to insulin, compared to adipocytes from control rats. This adipocyte metabolic obesity occurs despite similar body weight and adipocyte size between treated and control rats. Our next step was to investigate two possible sites of insulin hormone disruption due to PBDE treatment of rats: insulin-stimulated glucose transport and GLUT4 levels, and insulin-stimulated glycolytic and TCA-cycle flux, in adipocytes from PBDE-treated vs. control rats. To our surprise  but, as weve since learned  not uncommon to adipocyte researchers - our first few experiments revealed that the insulin-stimulation of glucose oxidation (measured by 14C-glucose conversion to 14CO2 over a 90-minute incubation), in adipocytes from control rats (~350 gms), had vanished. Over the next eight months, experiments were conducted testing different amounts and sources of albumin, collagenase, adenosine, new plastic ware, all new reagents, a different source of water, different ages of rats, even conducting the experiment in a different lab. Thirty experiments later, there was still no reproducible insulin effect. One variable, identified in literature published ~40 years ago, that we did not control was the timing and amount of food consumed. Therefore, we conducted a meal-feeding experiment, in which rats were adapted to eating their food in 2 hours/day. We measured insulin-stimulated glucose oxidation in adipocytes from 180 grams rats immediately after eating (n=2), 5 hours later (n=2) and 10 hours later (n=2). Weve had 6 successful experiments with a 5-fold stimulation of glucose oxidation in response to insulin at each time point. 9. Experiment Station: Iowa State University. Representative: Donald Beitz. Objectives 1, 2, 3, 5, 6, and 9. Elevated blood cholesterol and low-density lipoprotein-cholesterol are the main factors contributing to the CVD development in humans. The concentration of those metabolites in blood is increased when foods rich in saturated fatty acids are consumed. The major sources of those fatty acids are animal products that provide collectively 56% of total lipids, 74% of saturated fatty acids, and 100% of cholesterol consumed by humans. Decreasing the percentage of saturated fatty acids in milk and other animal products can lead to the improvement of the healthfulness of those foods and to decrease incidence of CVD in humans. Contributions of individual fatty acids to atherogenic potential for a lipid source or a diet are summarized by an atherogenic index (AI) that was developed by Ulbricht and Southgate in 1991. The AI is calculated as a ratio of the sum of concentrations of lauric (12:0), four times myristic (14:0), and palmitic (16:0) acids to the sum of concentrations of mono- and polyunsaturated fatty acids. The objective of this study was to determine if variations in single nucleotide polymorphism (SNP) in thioesterase domain of the fatty acid synthase (g.17924 A>G Threonine>Alanine) and diacylglycerol acyltransferase-1 (g.10433/10434 GC/AA Alanine>Lysine) genes would explain variations in milk fatty acid composition among Holstein dairy cattle. About 200 cows participated in the study. Milk samples were collected monthly throughout the first ten months of lactation and analyzed for milk fatty acid composition by gas chromatography. Blood samples were used to obtain a DNA sample for each animal. Cows with g.17924GG genotype had lower AI compared with cows of g.17924AG genotype (P=0.007). Likewise, cows with g.10433/10434GC genotype had lower AI compared with cows of g.10433/10434AA genotype (P=0.016). The decrease in AI for cows with g.17924GG and g.10433/10434GC genotypes was achieved by the decrease in the concentration of palmitic acid (P=0.06 and P<0.0001, respectively) and by the increase in the concentration of mono- and polyunsaturated fatty acids for both genotypes. The results of this study indicate the potential of using SNPs as DNA markers to select breeding stocks for milk fatty acid composition. 10. Experiment station: USDA, ARS, Richard B. Russell Res. Center Agricultural Research Center. Representative: G. J. Hausman. Objectives 1, 2, 3 and 4. The expression of many genes encoding transcription factors, nuclear receptors, enzymes and other regulatory proteins have been studied in human and rodent adipose tissue with cDNA microarrays, but comparable studies in adipose tissue from neonatal and growing pigs have not been reported. In the first study, total RNA was isolated from 90 d fetal stromal-vascular (SV) cell cultures (n = 4; 2 arrays, 2 cultures / array) and adipose tissue from 105 d fetuses (n =2) and neonatal (5 d old) pigs (n=2). In the second study, total RNA was collected at slaughter from outer s.c. adipose tissue (OSQ) and middle s.c. adipose tissue (MSQ) samples from gilts at 90, 150, and 210 d ( n = 5 / age). In both studies, dye-labeled cDNA probes were hybridized to custom microarrays (70 mer oligonucleotides) representing about 600 pig genes involved in growth and reproduction. In the first study, microarray data analysis showed significant expression of 179 genes encoding transcription factors, nuclear receptors, enzymes and other regulatory proteins in fetal SV cell cultures and fetal and neonatal adipose tissue. In the second study, expression intensity ratios revealed little change with age in gene expression overall of 100 transcription factors, nuclear receptors, enzymes and other regulatory proteins in OSQ and MSQ from pigs between 90 - 210 d of age. However, distinct patterns of relative gene expression were evident within adipose tissue from growing pigs (90, 150, and 210 d old pigs). Furthermore, the relative expression of 13 genes distinguished OSQ and MSQ depots in growing pigs. With microarray and RT-PCR assays we showed that the expression of several regulatory genes did not change with age in OSQ and MSQ depots. However, the expression of several genes were influenced by age including an increase in HSF1 and 15-oxoprostaglandin 13-reductase expression in MSQ and a decrease in UCP2 and prohibitin -2 expression in OSQ. These studies demonstrate the expression of several genes in pig adipose tissue that may influence or be associated with leptin expression and adipose tissue metabolism. Objectives 1, 2 , 3, 4, 6 and 8. In this study, transcriptional profiling was used to identify genetic mechanisms that respond to alpha-MSH, an MC3/4-R agonist. Three MC4R genotypes (2 homozygous and the heterozygous for MC4R) were selected from the UGA swine herd (PIC composite). Thirty six pigs, 6 per genotype per treatment were randomly assigned to one of the following treatments: ICV administration of 150 ul 0.9% saline, or 10 µg NDP-MSH (agonist) in 150 ul of 0.9% saline. Feed intake was measure at 12 and 24 hous after treatment (time 0). All pigs were sacrificed 24 hours post-injection and hypothalamus, liver and middle layer of back fat was collected and mRNA was hybridized to 24,123 probe set Affymetrix Porcine Genome Arrays. MSH suppressed (P< 0.04) feed intake in all animals at 12 and 24 hr after treatment regardless of genotype with no teatment x genotype interaction detected (P > 0.8). A mixed linear model was fit to each tissue and gene using SAS Proc Mixed and interested contrasts were tested. In response to MSH injection, 5070 genes in adipose tissue were declared differentially expressed with q-value<0.07. This criterion was satisfied for 290 adipose tissue genes for the MC4R genotype effect and 1724 adipose genes for the genotype x treatment interaction. Genes representing lipid biosynthesis such as LPL, fatty acid synthase, aconitase-1 and acetyl CoA synthase were down regulated by MSH treatment as were leptin and heat shock protein 70.2 genes. Genes upregulated in adipose tissue tissue by MSH treatment included angiopoietin-like 4, pyruvate dehydrogenase kinase 4 isoform 1, UCP-3 and IGFBP- 3. Angiopoietin-like 4 inhibits LPL activity in rodent adipose tissue and was upregulated more than any other probe in the probe set. Along with blood hormone assays, confirmation of expression of additional candidate genes will be performed to determine key pathways that responded to MSH treatment in pigs. These results demonstrate that the MC4R genotype did not effect the feeding behavior reponse to MSH but did affect adipose tissue gene expression.

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