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Pyruvate carboxylase (PC; EC 6.4.1.1) is critical in gluconeogenesis from lactate and maintenance of tricarboxylic acid cycle intermediates. Whereas increases in PC mRNA have been observed during feed restriction, the mechanism of regulation is unknown; however, coinciding increases in circulating NEFA concentrations suggests that fatty acids may contribute to regulation of gene expression during feed restriction. The objective of this study was to examine the direct effect of exposure to serum from full-fed control cows with serum from cows that were restricted to 50% of ad libitum intake for 5 d on PC expression in vitro. Rat hepatoma (H4IIE) cells were transiently transfected with bovine promoter-luciferase constructs containing bovine PC promoter 1 and treated with serum from control cows, serum from feed-restricted cows, or modified serum. Modified serum pools were generated by supplemented serum from control cows with C14:0, C16:0, C18:0, C18:1n-9 cis, C18:2n-6 cis, and C18:3n-3 cis to match the total NEFA in serum from feed-restricted cows (1.3 mM) in the relative proportion found in serum from control or feed-restricted cows. Exposure of cells to serum from feed-restricted cows increased (P < 0.05) PC promoter 1 activity 2.2-fold compared with cells exposed to control cow serum. Exposure to serum from control cows with fatty acids added to a NEFA concentration of 1.3 mM to reflect the fatty acid profile of control and feed-restricted cows increased (P < 0.05) promoter 1 activity 2.1- and 2.5-fold, respectively, compared with cells incubated with control cow serum. There was no difference (P ≥ 0.05) in promoter 1 activity in cells treated with modified serum compared with serum from feed-restricted cows. These data indicate that promoter 1 is activated by fatty acids found in serum of feed-restricted cows. These data suggest a role of NEFA to regulate expression of bovine PC mRNA through specific activation of PC promoter 1.

Pyruvate carboxylase (PC; EC 6.4.1.1) is a critical enzyme for gluconeogenesis and maintenance of tricarboxylic acid (TCA) cycle intermediates, and expression of PC mRNA is increased at calving and during feed restriction. The bovine PC gene contains 3 promoters (3, 2, and 1 from 5' to 3') that produce 6 mRNA 5' variants (A through F). Products of promoter 1, untranslated region (UTR) variants A, B, C, and F are specifically expressed in glucogenic and lipogenic tissues. The objective of this study was to develop a quantitative PCR-based assay for bovine PC 5' UTR variants that would permit simultaneous characterization of PC variant expression and to determine the pattern of PC variant expression during the transition to lactation and during feed restriction. Primer combinations specific to the coding region of PC and the 5' UTR for variants D, E, and F were used with Taqman probes in a real-time PCR multiplex assay to simultaneously determine total PC mRNA and expression of each UTR variant. The intraassay and interassay CV were less than 2 and 10%, respectively. Total PC mRNA and PC 5' UTR variant profile was determined for liver biopsy samples collected from Holstein cows (n = 8) at -28, +1, and +28 d relative to calving (DRTC) and from mid-lactation Holstein cows subjected to either feed restriction (n = 8) or fed for ad libitum intake (n = 8). The expression of PC mRNA corresponding to the coding region of PC and PC 5' UTR variant regions A, B, C, and F increased (P < 0.05) 4-fold with feed restriction and 6-fold at calving. Nuclei isolated from liver biopsy samples and used to determine the rates of PC gene transcription indicate changes in the abundance of PC 5' mRNA variants A, B, C, and F that are due to corresponding changes in the rate of transcription of the bovine PC gene. The data support the use of the multiplex assay described here as a proxy measure of the activity of bovine PC promoter 1. The increased PC mRNA expression observed at calving and during feed restriction is the result of specific increases in 5' UTR variants A, B, C, and F due to increased transcriptional activity of promoter 1 of the PC gene.

Gluconeogenesis is a crucial process to support glucose homeostasis when nutritional supply with glucose is insufficient. Because ingested carbohydrates are efficiently fermented to short-chain fatty acids in the rumen, ruminants are required to meet the largest part of their glucose demand by de novo genesis after weaning. The qualitative difference to nonruminant species is that propionate originating from ruminal metabolism is the major substrate for gluconeogenesis. Disposal of propionate into gluconeogenesis via propionyl-CoA carboxylase, methylmalonyl-CoA mutase, and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) has a high metabolic priority and continues even if glucose is exogenously supplied. Gluconeogenesis is regulated at the transcriptional and several posttranscriptional levels and is under hormonal control (primarily insulin, glucagon, and growth hormone). Transcriptional regulation is relevant for regulating precursor entry into gluconeogenesis (propionate, alanine and other amino acids, lactate, and glycerol). Promoters of the bovine pyruvate carboxylase (PC) and PEPCK genes are directly controlled by metabolic products. The final steps decisive for glucose release (fructose 1,6-bisphosphatase and glucose 6-phosphatase) appear to be highly dependent on posttranscriptional regulation according to actual glucose status. Glucogenic precursor entry, together with hepatic glycogen dynamics, is mostly sufficient to meet the needs for hepatic glucose output except in high-producing dairy cows during the transition from the dry period to peak lactation. Lactating cows adapt to the increased glucose requirement for lactose production by mobilization of endogenous glucogenic substrates and increased hepatic PC expression. If these adaptations fail, lipid metabolism may be altered leading to fatty liver and ketosis. Increasing feed intake and provision of glucogenic precursors from the diet are important to ameliorate these disturbances. An improved understanding of the complex mechanisms underlying gluconeogenesis may further improve our options to enhance the postpartum health status of dairy cows.

Growth of the corn ethanol industry has created a need for alternatives to corn for lactating dairy cows. Concurrent expansion in soydiesel production is expected to increase availability and promote favorable pricing for glycerol, a primary co-product material. The objective of this study was to determine the feeding value of glycerol as a replacement for corn in diets fed to lactating dairy cattle. Sixty lactating Holstein cows housed in individual tie stalls were fed a base diet consisting of corn silage, legume forages, corn grain, soyhulls, roasted soybeans, and protein supplements. After a 2-wk acclimation period, cows were fed diets containing 0, 5, 10, or 15% refined glycerol for 56 d. Cows were milked twice daily and weekly milk samples were collected. Milk production was 36.3, 37.2, 37.9, and 36.2 +/- 1.6 kg/d and feed intake was 23.8, 24.6, 24.8, and 24.0 +/- 0.7 kg/d for 0, 5, 10, and 15% glycerol treatments, respectively, and did not differ except for a modest reduction in feed intake during the first 7 d of the trial for 15% glycerol (treatment x time effect). Milk composition was not altered by glycerol feeding except that milk urea nitrogen was decreased from 12.5 +/- 0.4 to 10.2 +/- 0.4 mg/dL with glycerol addition. Cows fed diets containing 10 and 15% glycerol gained more weight than those fed rations containing 0 or 5% glycerol but body condition scores did not differ with glycerol feeding. The data indicate that glycerol is a suitable replacement for corn grain in diets for lactating dairy cattle and that it may be included in rations to a level of at least 15% of dry matter without adverse effects on milk production or milk composition.

Previous reports suggest that parathyroid hormone (PTH) is associated with insulin resistance. This research investigated the effects of PTH on insulin signaling in differentiated 3T3-L1 adipocytes. PTH (10 nM, 24 h) treatment induced a reduction in insulin-stimulated glucose uptake, AKT activity (phosphorylated AKT/total AKT protein expression) and a decrease in GLUT4 and IRS-1 protein expression compared to vehicle treated controls in differentiated adipocytes. PTH treatment also induced increased phosphorylation of IRS-1 on serine 307, which suppresses insulin signaling. In addition, treatment of cells with adenyl cyclase inhibitor SQ52236 ameliorated the effects of PTH on insulin-stimulated glucose uptake, whereas inhibition of phospholipase C alpha (U73122) did not significantly alter the effects of PTH. Thus, PTH treatment of differentiated 3T3-L1 adipocytes suppresses insulin-stimulated glucose uptake and insulin signaling via cAMP pathway, potentially through the phosphorylation of IRS-1 at serine 307.

The growing incidence of prediabetes and clinical type 2 diabetes, in part characterised by insulin resistance, is a critical health problem with consequent devastating personal and health-care costs. Vitamin D status, assessed by serum 25-hydroxyvitamin D levels, is inversely associated with diabetes in epidemiological studies. Several clinical intervention studies also support that vitamin D, or its active metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D), improves insulin sensitivity, even in subjects with glucose metabolism parameters classified within normal ranges. The mechanisms proposed which may underlie this effect include potential relationships with improvements in lean mass, regulation of insulin release, altered insulin receptor expression and specific effects on insulin action. These actions may be mediated by systemic or local production of 1,25(OH)2D or by suppression of parathyroid hormone, which may function to negatively affect insulin sensitivity. Thus, substantial evidence supports a relationship between vitamin D status and insulin sensitivity; however, the underlying mechanisms require further exploration.

The bovine pyruvate carboxylase (PC) gene is expressed as 6 alternatively spliced variants that share a common open reading frame but that differ within their 5' untranslated regions (UTR). The PC 5' UTR variants (A through F) contain 6 combinations of 5 exons and are 68, 253, 363, 89, 226, 178 bp in length, respectively. The objective of this experiment was to determine whether or not the bovine PC mRNA variants exhibit different translational efficiencies. Each bovine PC 5' UTR variant was linked to the firefly luciferase coding region, and the resulting constructs were transcribed and translated in a rabbit reticulocyte lysate assay. All constructs resulted in synthesis of luciferase protein. The abundance of luciferase protein synthesized from the UTR of bovine PC 5' D was greater (P < 0.05) than synthesis from either PC 5' UTR C or E, and the abilities of UTR D, A, B, and F to drive protein translation were similar. The disproportionate contribution to protein synthesis of the PC 5' D UTR compared with UTR variant C or E indicates a complexity of control for PC enzyme synthesis in the bovine that is dependent on the profile of PC variants. These observations are consistent with differences in PC variant expression that have been observed in vivo and indicate that when PC mRNA is elevated, the pattern of variants directs an increase in PC activity through augmented PC enzyme synthesis.

High vitamin D intake is associated with reduced insulin resistance. Expression of extra-renal 1α,25-dihydroxyvitamin D hydroxylase (1α-hydroxylase) has been reported in several tissues and contributes to local synthesis of 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D) from the substrate 25-hydroxyvitamin D (25OHD). Expression and dietary regulation of 1α-hydroxylase in tissues associated with energy metabolism, including adipose tissue, has not been assessed. Male Wistar rats were fed a high calcium (1.5%) and high vitamin D (10,000 IU/kg) or a low calcium (0.25%), low vitamin D (400 IU/kg) with either a high fat (40% energy) or high sucrose (66% energy) dietary background for 14 weeks. Expression of 1α-hydroxylase, assessed by real time PCR, was detected in adipose tissue and did not differ with dietary level of calcium and vitamin D. 1α-Hydroxylase mRNA was also detected in 3T3-L1 preadipocytes and 25OHD treatment at 10 nM levels induced 1,25(OH) 2 D responsive gene, CYP24, and this response was reduced in the presence of the p450 inhibitor, ketoconazole. In addition, 3 H 25OHD was converted to 3 H 1,25(OH) 2 D in intact 3T3-L1 preadipocytes. Cumulatively, these results demonstrate that 1α-hydroxylase is expressed in adipose tissue and is functional in cultured adipocytes. Thus, the capacity for local production may play a role in regulating adipocyte growth and metabolism.

The aim of the current study was to determine the effects and the mechanisms of inclusion of dietary whey protein, high calcium, and high vitamin D intake with either a high-sucrose or high-fat base diets on body composition of rodents. Male Wistar rats were assigned to either no whey protein, suboptimal calcium (0.25%), and vitamin D (400 IU/kg) diet (LD), or a diet containing whey protein, high calcium (1.5%), and vitamin D (10 000 IU/kg) diet (HD), and either high-fat (40% of energy) or high-sucrose (60%) base diets for 13 weeks. Liver tissue homogenates were used to determine [(14)C]glucose and [(14)C]palmitate oxidation. mRNA expression of enzymes related to energy metabolism in liver, adipose, and muscle, as well as regulators of muscle mass and insulin receptor was assessed. The results demonstrated that there was reduced accumulation of body fat mass (P = .01) and greater lean mass (P = .03) for the HD- compared to LD-fed group regardless of the background diet. There were no consistent differences between the LD and HD groups across background diets in substrate oxidation and mRNA expression for enzymes measured that regulate energy metabolism, myostatin, or muscle vascular endothelial growth factor. However, there was an increase in insulin receptor mRNA expression in muscle in the HD compared to the LD groups. In conclusion, elevated whey protein, calcium, and vitamin D intake resulted in reduced accumulation of body fat mass and increased lean mass, with a commensurate increase in insulin receptor expression, regardless of the level of calories from fat or sucrose.

This study was conducted to determine the effect of experimentally increased glucose demand on voluntary consumption of molasses by dairy calves. Three-week-old calves received 0.365 g of phlorizin by s.c. injection. Urinary output and molasses consumption were measured hourly, and urinary glucose concentration was screened. Molasses consumption for the 24 h after treatment was (mean +/- SE) 72.0 g (+/-7) for the control group and 142 g (+/-1) for the phlorizin-treated group. Urinary output for the 8-h test period was 1.13 kg for the control group and 1.67 kg for the phlorizin-treated calves. Mean urinary glucose peaked at 10 g/L by 4 h after treatment for calves given phlorizin, whereas the concentration for the control group remained close to 0 g/L. Phlorizin treatment increased voluntary consumption of molasses in 3-wk-old Holstein calves.

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.

Maternal nutritional stress during pregnancy acts to program offspring metabolism. We hypothesized that the nutritional stress caused by maternal fructose or low protein intake during pregnancy would program the offspring to develop metabolic aberrations that would be exacerbated by a diet rich in fructose or fat during adult life. The objective of this study was to characterize and compare the fetal programming effects of maternal fructose with the established programming model of a low-protein diet on offspring. Male offspring from Sprague-Dawley dams fed a 60% starch control diet, a 60% fructose diet, or a low-protein diet throughout pregnancy and lactation were weaned onto either a 60% starch control diet, 60% fructose diet, or a 30% fat diet for 15 weeks. Offspring from low-protein and fructose-fed dam showed retarded growth (P.05) at weaning (50.3, 29.6 vs 59.1±0.8 g) and at 18 weeks of age (420, 369 vs 464±10.9 g). At 18 weeks of age, offspring from fructose dams expressed greater quantities (P.05) of intestinal Pgc1a messenger RNA compared with offspring from control or low-protein dams (1.31 vs 0.89, 0.85; confidence interval, 0.78-1.04). Similarly, maternal fructose (P=.09) and low-protein (P.05) consumption increased expression of Pgc1a in offspring liver (7.24, 2.22 vs 1.22; confidence interval, 2.11-3.45). These data indicate that maternal fructose feeding is a programming model that shares some features of maternal protein restriction such as retarded growth, but is unique in programming of selected hepatic and intestinal transcripts.

Transition heifers face multiple stressors during the periparturient period, including first exposure to milking, that may adversely impact dry matter intake (DMI), reduce milk production, compromise immune function, and increase susceptibility to disease. It was hypothesized that reducing the combined stressors experienced at calving would improve the periparturient performance, health, and well-being of heifers. The objective of this study was to determine the effect of initiating the milking procedure 3 wk before expected calving on production, DMI, body weight, energy balance, udder health, calving traits, and health status, as indicated by plasma acute phase protein concentrations. Twenty-two primigravid heifers, blocked by expected calving date, were assigned randomly either to a prepartum milking (PM) group or control group. The PM heifers were milked twice daily beginning at 21 d before expected calving, and control heifers were not milked until after calving. All heifers had access to the same precalving and post-calving diets. Results indicated that PM heifers produced more milk during the first 2 wk after calving and had greater DMI as a percentage of body weight during the first month after calving than did control heifers, although energy balance was more negative for PM heifers. The PM heifers had reduced somatic cell counts through the first month after calving and lower average somatic cell scores during lactation despite having more quarters with mastitis infection at calving. The PM heifers had less udder edema at the third milking postcalving, and had reduced concentrations of haptoglobin in blood sooner than did control heifers. These results indicate that prepartum milking is an alternative management practice that has beneficial effects on the production, health, and well-being of first-lactation cows.

Thirty-four multiparous Holstein cows were used in a randomized block design to evaluate the effects of feeding nonforage fiber sources (NFFS), monensin, or their combination on expression of gluconeogenic enzymes in the liver during the transition to lactation. The addition of 0 or 300 mg/d of monensin to a conventional (CONV) or NFFS prepartum diet was evaluated in a 2 x 2 factorial arrangement of treatments. The NFFS diet was formulated by replacing 30% of the forage component of the CONV diet with cottonseed hulls and soyhulls. The CONV and NFFS basal diets were fed at dry-off and continued through parturition. Monensin was fed from -28 d relative to calving (DRTC) through parturition. At calving, all cows were placed on the same diet. Liver biopsy samples obtained at -28, -14, +1, +14, and +28 DRTC were used to determine pyruvate carboxylase (PC) and cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) mRNA expression. Feeding NFFS resulted in greater (P < 0.05) prepartum DMI compared with the CONV diet. There was no effect of prepartum diets on postpartum DMI or average milk production to 56 d of lactation. Expression of PC mRNA was elevated (P < 0.05) at 1 d postpartum, but there was no effect of NFFS or monensin on PC mRNA abundance. Expression of PEPCK-C mRNA at calving was increased (P < 0.05) with prepartum monensin feeding. The data indicate that feeding monensin to transition cows induces hepatic PEPCK-C mRNA expression before calving. The increased expression of hepatic PEPCK-C mRNA with monensin feeding suggests a feed-forward mechanism of metabolic control in ruminants that links molecular control of gluconeogenesis with the profile of rumen fermentation end products.

Hepatocytes monolayers from neonatal calves were used to determine the responses of the cytosolic phosphoenolpyruvate carboxykinase (PCK1) mRNA expression to propionate and direct hormonal cues including cyclic AMP (cAMP), dexamethasone, and insulin. The responses of other key gluconeogenic genes, including mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphotase (G6PC), were also measured. Expression of PCK1 was linearly induced with increasing propionate concentrations in media and 2.5 mM propionate increased PCK1 mRNA at 3 and 6h of incubation; however, the induction disappeared at 12 and 24 h. The induction of PCK1 mRNA by propionate was mimicked by 1 mM cAMP, or in combination with 5 µM dexamethasone, but not by dexamethasone alone. The induction of PCK1 mRNA by propionate or cAMP was eliminated by addition of 100 nM insulin. Additionally, expression of PCK2 and PC mRNA was also induced by propionate in a concentration-dependent manner. Consistent with PCK1, propionate-stimulated PCK2 and PC mRNA expression was inhibited by insulin. Expression of G6PC mRNA was neither affected by propionate nor cAMP, dexamethasone, insulin, or their combinations. These findings demonstrate that propionate can directly regulate its own metabolism in bovine calf hepatocytes through upregulation of PCK1, PCK2, and PC mRNA expression.

Fundamental nutrition seeks to describe the complex biochemical reactions involved in assimilation and processing of nutrients by various tissues and organs, and to quantify nutrient movement (flux) through those processes. Over the last 25 yr, considerable progress has been made in increasing our understanding of metabolism in dairy cattle. Major advances have been made at all levels of biological organization, including the whole animal, organ systems, tissues, cells, and molecules. At the whole-animal level, progress has been made in delineating metabolism during late pregnancy and the transition to lactation, as well as in whole-body use of energy-yielding substrates and amino acids for growth in young calves. An explosion of research using multicatheterization techniques has led to better quantitative descriptions of nutrient use by tissues of the portal-drained viscera (digestive tract, pancreas, and associated adipose tissues) and liver. Isolated tissue preparations have provided important information on the interrelationships among glucose, fatty acid, and amino acid metabolism in liver, adipose tissue, and mammary gland, as well as the regulation of these pathways during different physiological states. Finally, the last 25 yr has witnessed the birth of "molecular biology" approaches to understanding fundamental nutrition. Although measurements of mRNA abundance for proteins of interest already have provided new insights into regulation of metabolism, the next 25 yr will likely see remarkable advances as these techniques continue to be applied to problems of dairy cattle biology. Integration of the "omics" technologies (functional genomics, proteomics, and metabolomics) with measurements of tissue metabolism obtained by other methods is a particularly exciting prospect for the future. The result should be improved animal health and well being, more efficient dairy production, and better models to predict nutritional requirements and provide rations to meet those requirements.

Somatotropin (ST) increases milk production and through coordinated changes in hepatic glucose synthesis and amino acid metabolism in dairy cows. The objective of this study was to determine the effects of ST on hepatic mRNA expression for phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC), enzymes that are critical to the synthesis of glucose in liver and hepatic mRNA expression for carbamylphosphate synthetase I (CPS-I), argininosuccinate synthetase (AS), and ornithine transcarbamylase (OTC), critical enzymes of the urea cycle. Eighteen cows were randomly allocated to 2 treatment groups and received either recombinant bovine ST (Posilac; Monsanto, St. Louis, MO) or saline injections at 14-d intervals during a 42-d period. Expression of mRNA was determined using Northern blot analysis. Nuclei, isolated from liver biopsy samples, were used to determine effects of ST on transcription rate of PEPCK. Milk production was increased with ST (37.3 vs. 35.1±0.6 kg/d). Plasma NEFA was increased with ST (299 vs. 156±34 μM ). There were no differences in the expression of CPS-I, AS, and OTC mRNA with ST. Expression of PEPCK and IGF-I mRNA were increased with ST but PC mRNA was unchanged. The data indicate increased PEPCK mRNA in cows given ST and indicates a greater capacity for gluconeogenesis from gluconeogenic precursors that form oxaloacetate. The effects of ST to elevate PEPCK mRNA expression require chronic administration and involve increased transcription of the PEPCK gene.

Cattle are exposed to growth hormone stimulants and to stressors that cause cortisol release. Both of these hormones affect immune responses which may reduce disease resistance. Toll-like receptors are the pattern recognition molecules of pathogens that are on immune cells. They then orchestrate the induction of the appropriate acute phase cytokines of the early innate response. The objective of this study was to determine changes in toll-like receptors and acute phase cytokines following treatment with a synthetic glucocorticoid (dexamethasone) and growth hormone (GH). Twenty-eight calves were given the control (Cnt), dexamethasone (DEX), GH, or dexamethasone and GH (Both) treatments from 3 until 56 days of age. Blood was collected by jugular venipuncture on days 14, 28, 42, and 56. On day 56, a lung lavage was performed and spleen and thymus tissues collected. Total RNA was extracted from blood leukocytes, lung lavage cells, spleen and thymus cells. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to quantify interleukin-1 (IL-1), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor (TNF)-alpha, toll-like receptor 2 (TLR2), and toll-like receptor 4 (TLR4). Blood leukocytes had a time effect for IL-1Ra (P < 0.01), with a trend for a treatment effect (P = 0.07) and had a treatment by time interaction (P < 0.05). IL-1, TNF, and TLR2 and TLR4 were greatest (P < 0.05) for Cnt only at day 14. IL-1 expression of lung lavage cells was greatest (P < 0.05) for calves on the Both treatment compared to the other three treatments. However, IL-1Ra was not different among the treatments. Toll-like receptor 2 expression was enhanced with Both compared to either DEX (P < 0.05) or GH (P < 0.05) and tended to be greater than Cnt expression (P = 0.07). Expression of TLR4 tended to be reduced by Both compared to Cnt (P = 0.06). Tumor necrosis factor-alpha was greatly enhanced by Both compared to the other three treatments (P < 0.05). Spleen cell tended to have different IL-1 expression between GH and Both (P < 0.10). Interleukin-1 receptor antagonist and TLR2 and TLR4 were not different among treatments. However, TNF-alpha expression was enhanced by the DEX treatment alone compared to the GH treatment (P < 0.05), and tended (P < 0.10) to be greater than Cnt expression. None of the gene expressions were different among treatments for thymus cells. Lung lavage cell expression appears to be most susceptible to these hormones while blood leukocyte expression was only slightly affected, and thymus cells were not affected at all. These data demonstrate that TLR2 and TLR4 and acute phase cytokine expression can be altered by stress and growth hormones, which may decrease resistance of those animals to disease.

Research has revealed the role of adverse behaviors during pregnancy on metabolic and cardiovascular health outcomes in offspring. However, minimal attention has been focused on positive prenatal behaviors, such as exercise, and the effect on offspring health outcomes. The objective of this investigation was to test the hypothesis that mothers who voluntarily exercise during pregnancy would improve endothelial function in offspring from exercising compared with sedentary mothers.Female Sprague Dawley rats were divided into sedentary (n = 10) or exercise (n = 9) groups with, the exercise group having voluntary access to a running wheel throughout gestation. Litter characteristics (weight and size) were taken 1 d after birth. After weaning, offspring were placed in sedentary cages where they remained until 4 or 8 months of age. Offspring food consumption and cage activity were assessed during a 72-h interval at 2, 4, 6, and 8 months of age. The abdominal aortas of offspring were harvested at 4 or 8 months of age, and vascular function was assessed using cumulative doses of endothelium-dependent (acetylcholine/10(-10)-10(-4) M) and independent (sodium nitroprusside/10(-10)-10(-4) M) vasodilators.There were no significant differences in litter size and litter weight at weaning between the sedentary and exercise groups. Food consumption and wheel running activity did not differ between the sedentary and exercise offspring at 2, 4, 6, or 8 months of age. Vasorelaxation response to acetylcholine or sodium nitroprusside did not differ between the offspring from sedentary and exercise dams.Maternal voluntary wheel running during pregnancy does not significantly alter behavior or vascular function in adult offspring.

Photoperiod impacts feeding behavior, health, growth and milk production of dairy cattle. In humans behaviors that are asynchronous with the natural light-dark cycle, such as shift work or jet lag, are strongly associated with metabolic disorders as well as impaired reproductive performance. The objective of this study was to determine the effects of exposing mid-lactation dairy cows to chronically shifting 8h light (L)-8h dark (D) cycles on feed intake, milk yield, milk composition and mammary gene expression. Six first lactation Holsteins ∼90d in milk were maintained on a 16 h L: 8 h D cycle and milked at 5AM and 4PM for 7d (control, Period 1). During Period 2, 7 d immediately following Period 1, cows were exposed to continuous cycles of 8 h L: 8 h D, but maintained on the same milking and feeding schedule. Exposure to chronic 8 h LD cycles significantly depressed milk yield (P

Breast cancer is the most common cancer among women. Emerging research indicates that modifying lifestyle factors during pregnancy may convey long-term health benefits to offspring. This study was designed to determine whether maternal exercise during pregnancy leads to reduced mammary tumorigenesis in female offspring. Pregnant rats were randomly assigned to exercised and sedentary groups, with the exercised group having free access to a running wheel and the sedentary group housed with a locked wheel during pregnancy. Female pups from exercised or sedentary dams were weaned at 21 days of age and fed a high fat diet without access to a running wheel. At 6 weeks, all pups were injected with the carcinogen N-methyl-N-nitrosourea (MNU). Mammary tumor development in all pups was monitored for 15 weeks. Pups from exercised dams had a substantially lower tumor incidence (42.9%) compared to pups from sedentary dams (100%). Neither tumor latency nor histological grade differed between the two groups. These data are the first to demonstrate that exercise during pregnancy potentiates reduced tumorigenesis in offspring. This study provides an important foundation towards developing more effective modes of behavior modification for cancer prevention.

Studies were designed to investigate the effect of 1,25 dihydroxyvitamin D (1,25D) on glutamine metabolism during cancer progression employing untransformed (10A) and ras oncogene transformed MCF10A human breast epithelial cells (ras). Glutamine restriction reduced cell number in both cell lines, and 1,25D ameliorated these reductions. Intracellular glutamine and glutamate levels, determined by NMR, were increased 25% in ras cells compared to 10A cells, and 1,25D reduced these levels by 23% in ras cells. Fractional glutamine and glutamate flux, utilizing U13C5-L-glutamine, into the TCA cycle was increased in ras cells by 21% and 18%, respectively, compared to 10A cells. 1,25D decreased glutamine flux into the TCA cycle by 18% and 31% in 10A and ras cells, respectively, and decreased glutamate flux into the TCA cycle by 13% and 17% in 10A and ras cells, respectively. The mRNA expression of the glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was decreased by 34% in ras compared to ...

Breast cancer is the most common cancer among women. Emerging research indicates that modifying lifestyle factors during pregnancy may convey long-term health benefits to offspring. This study was designed to determine whether maternal exercise during pregnancy leads to reduced mammary tumorigenesis in female offspring. Pregnant rats were randomly assigned to exercised and sedentary groups, with the exercised group having free access to a running wheel and the sedentary group housed with a locked wheel during pregnancy. Female pups from exercised or sedentary dams were weaned at 21 days of age and fed a high fat diet without access to a running wheel. At 6 weeks, all pups were injected with the carcinogen N-methyl-N-nitrosourea (MNU). Mammary tumor development in all pups was monitored for 15 weeks. Pups from exercised dams had a substantially lower tumor incidence (42.9%) compared to pups from sedentary dams (100%). Of the pups with tumors, the pups from exercised dams had a higher tumor frequency (1.7±0...

The inability to markedly attenuate cholesterol levels in chicken eggs has led to speculation that cholesterol is essential for yolk formation and that egg production would cease when yolk cholesterol deposition was inadequate for embryonic survival. However, this critical level hypothesis remains unproven. Here, we determine the relative responsiveness of laying hens to three select inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the rate-limiting enzyme of cholesterol biosynthesis. A control diet, either alone or supplemented with one of two dietary levels (0.03 or 0.06%) of atorvastatin, lovastatin, or simvastatin, was fed to White Leghorn hens for 5 wk. Liver cholesterol concentrations (mg/g tissue) were decreased (P/= 0.05) by each HMGR inhibitor; however, total liver cholesterol (mg) did not differ among treatments. Microsomal hepatic HMGR activities were increased one- to twofold in all HMGR inhibitor-treated groups, while HMGR mRNA levels were unaffected. Diameters of plasma VLDL particles, the main cholesterol-carrying yolk precursor macromolecules, were reduced (P/= 0.05) only in hens fed 0.06% atorvastatin, and the particles contained 38% less total cholesterol (P/= 0.05) than controls. Plasma total cholesterol concentrations were lowered (P/= 0.05) by both doses of atorvastatin (-56, -63%) and simvastatin (-36,-45%). Egg cholesterol contents were maximally reduced by 46% (P/= 0.05), 7% (P0.05), and 22% (P/= 0.05) in hens fed the 0.06% level of atorvastatin, lovastatin, and simvastatin, respectively, while overall egg production [-19% (P/= 0.05), +4% (P0.05), and -3% (P0.05)], was much less affected. We concluded that cholesterol per se may not be an obligatory component for yolk formation in chickens and, as such, may be amenable to further pharmacological manipulation

Psyllium, a source of dietary fiber rich in soluble components results in lower serum cholesterol concentration in several species. Suggested mechanisms for the hypocholesterolemic effect include a greater excretion of fecal bile acids and total steroids, and up-regulation of bile acid biosynthesis. The activity of cholesterol 7alpha-hydroxylase (7alphaOHase), the rate limiting enzyme in bile acid biosynthesis, is higher in rats fed 5% psyllium. Whether this higher activity corresponds to an increase in mRNA levels has not been determined. Four groups of 10 rats were fed a semipurified diet containing 5% cellulose (CEL; control), 5% cellulose plus 1% cholic acid (CCA), 5% cellulose plus 2% cholestyramine (CHY) or 5% psyllium hydrocolloid (PSY) for 3 wk. Liver cholesterol concentration, fecal bile acid and total steroid excretion, 7alphaOHase activity and 7alphaOHase mRNA levels were measured. Liver cholesterol content in rats fed CCA was significantly higher than in all other groups. Rats fed CHY and PSY had significantly lower liver cholesterol content than those fed CEL. Total fecal steroid and bile acid excretions were significantly greater in rats fed CCA, CHY and PSY than in those fed CEL. Activities and mRNA levels of 7alphaOHase in rats fed CHY and PSY were significantly higher than in rats fed CEL or CCA. These data indicate that feeding psyllium to rats increases fecal bile acid and total steroid excretion as well as 7alphaOHase activity and 7alphaOHase mRNA levels.

Patatin-like phospholipase domain-containing protein 3 (PNPLA3), commonly known as adiponutrin, is part of a novel subfamily of triglyceride lipase enzymes with potential effects on triglyceride metabolism in adipose and hepatic tissues. The predicted bovine PNPLA3 sequence has been identified, but expression of the gene had not been examined. The objectives of this study were to confirm the predicted bovine PNPLA3 gene sequence, determine expression of the bovine PNPLA3 gene in response to whole-animal energy balance, identify single nucleotide polymorphisms present in dairy cows, and verify the presence of the protein in the liver. Using liver biopsy samples collected from cows at +28d relative to calving (DRTC), RNA was isolated and used to generate a cDNA template for amplification of the entire predicted coding sequence of PNPLA3 via PCR. To determine if energy balance alters the expression of PNPLA3, RNA was isolated and mRNA expression quantified in liver samples from mid-lactation cows after a 5-d ad libitum period (n=5) and after a subsequent 5-d 50% feed restriction period (n=5), and in samples collected from cows at -14, +1, +14, and +28 DRTC (n=16). The presence of PNPLA3 protein was detected by Western blot in liver protein samples collected at +28 DRTC. Expression of hepatic PNPLA3 was decreased after a period of feed restriction (8.14 vs. 1.08±2.17 arbitrary units, ad libitum vs. fasted). Expression of PNPLA3 mRNA was decreased at +1 and +14 DRTC compared with -14 DRTC (23.35, 7.28, 10.17, and 14.5±4.9 arbitrary units, -14, +1, +14, and +28 DRTC, respectively). The presence of PNPLA3 protein was detected as a 55-kDa band in hepatic protein isolations from liver tissue collected at +28 DRTC. These data confirm the presence and sequence of the bovine hepatic PNPLA3 gene and single nucleotide polymorphisms. Furthermore, these data indicate responsiveness of bovine hepatic PNPLA3 to energy balance.

Changes to milking frequency (MF) affect the metabolic and energetic status of dairy cows. However, the duration of altered MF necessary to modify hepatic transcription during early lactation is less clear. Additionally, long-term responses to short-term alterations in MF have not been established. Holstein-Friesian dairy cows (n=120) were allocated to 3 or 6 wk of either once-daily (1×) or thrice-daily (3×) milking, immediately postpartum. Following treatment, cows were switched to twice-daily (2×) milking. These 4 treatment groups were compared with cows milked 2× (n=30) for the whole lactation. Liver tissue was collected by biopsy at 1, 3, 6, and 9 wk postpartum from 12 cows per treatment, RNA was extracted, and transcript abundance of genes involved in hepatic metabolism was quantified. Milking frequency altered the expression of most of the genes measured; however, we observed no effects caused by the length of time on the alternative milking frequency and no interactions between MF and length. During the MF treatment, mRNA expression of some, but not all, genes involved in gluconeogenesis ( G6PC , PCK1 ), fatty acid β-oxidation ( CPT1A , CPT2 ), ketogenesis ( HMGCS2 ), lipid transport ( APOA1 ), and lipolysis ( PNPLA2 ) were lower for cows milked 1× and plasma glucose and insulin concentrations were greater. Cows milked 3× had reduced mRNA expression for some of the genes involved in fatty acid synthesis ( ACACA ) and lipid transport ( APOB ) and had greater plasma NEFA concentrations at wk 1. At 9 wk postpartum, expression data indicated that cows previously milked 3× had a greater capacity for gluconeogenesis ( PCK1 ), ketogenesis ( HMGCS2 ), and urea cycling ( ASL , CPS1 ) and lower glucose concentrations than cows previously milked 1×, because some of the genes involved in these processes were still altered. Milking cows 1× relative to 2×, however, did not result in significant carryover effects on the expression of the genes measured in this study, indicating that metabolic changes are not sustained beyond the period of reduced MF. Changes to MF altered the hepatic response during early lactation; however, this was not dependent on the duration of MF change. Although we observed only minimal carryover effects on hepatic metabolism from short periods of reduced MF postpartum, there may be long-term effects on urea cycling ( ASL , CPS1 ) and ketogenesis ( HMGCS2 ) when 3× milking occurs immediately postpartum.

This study was designed to investigate the impact of 1,25 dihydroxyvitamin D (1,25(OH)2D) on glucose metabolism during early cancer progression. Untransformed and ras-oncogene transfected (ras) MCF10A human breast epithelial cells were employed to model early breast cancer progression. 1,25(OH)2D modified the response of the ras cells to glucose restriction, suggesting 1,25(OH)2D may reduce the ras cell glucose addiction noted in cancer cells. To understand the 1,25(OH)2D regulation of glucose metabolism, following four-day 1,25(OH)2D treatment, metabolite fluxes at the cell membrane were measured by a nanoprobe biosensor, [13C6]glucose flux by 13C-mass isotopomer distribution analysis of media metabolites, intracellular metabolite levels by NMR, and gene expression of related enzymes assessed. Treatment with 1,25(OH)2D reduced glycolysis as flux of glucose to 3-phosphoglycerate was reduced by 15% (P = 0.017) and 32% (P < 0.003) in MCF10A and ras cells respectively. In the ras cells, 1,25(OH)2D reduced lactate dehydrogenase activity by 15% (P < 0.05) with a concomitant 10% reduction in the flux of glucose to lactate (P = 0.006), and reduction in the level of intracellular lactate by 55% (P = 0.029). Treatment with 1,25(OH)2D reduced flux of glucose to acetyl-coA 24% (P = 0.002) and 41% (P < 0.001), and flux to oxaloacetate 34% (P = 0.003) and 33% (P = 0.027) in the MCF10A and ras cells, respectively, suggesting a reduction in tricarboxylic acid (TCA) cycle activity. The results suggest a novel mechanism involving the regulation of glucose metabolism by which 1,25(OH)2D may prevent breast cancer progression.

International audience; The present study was conducted to determine whether porcine somatotropin (pST) differentially regulates expression of the GLUT4 and fatty acid synthase (FAS) genes in pig adipose tissue. Three different experiments were conducted in which pigs were treated daily with different doses of pST for different time periods (7 or 14 d and from 60 to 90 kg of body wt). In these experiments, pST significantly and consistently decreased FAS mRNA levels (80%, 66% and 85%, respectively); however, GLUT4 mRNA was not affected by pST in two of the three experiments, and in the one showing an effect (Experiment 2), the decrease was less than observed for FAS (44%). Because of these results, we conducted subsequent experiments to see if the effects of pST on glucose metabolism in cultured pig adipose tissue (48 h) differed when glucose concentrations were changed from 1 to 5 mmol/L. These studies revealed that the antagonistic effect of pST on insulin action was more potent when glucose transport was saturated (5 mmol/L) than when glucose concentration limited glucose entry into the cell (1 mmol/L). In summary, these results suggest that the effects of pST on glucose transport in pig adipocytes are secondary to changes elicited by the hormone on intracellular glucose use for lipogenesis. When considered in the context of the decrease previously observed in glucose transport in pig adipocytes, the findings reported herein suggest that pST acts to decrease GLUT4 protein activity and/or distribution between the plasma membrane and the intracellular pool with little alteration in GLUT4 gene expression or total cell GLUT4 protein.

Hepatocyte monolayers were established from young preruminating (7 to 14 d of age) or older ruminating (11 to 12 wk of age) calves and used to evaluate the effects of insulin and glucagon on incorporation of carbon from 2.5 mM [2-14C]propionate and 2.0 mM [U-14C]lactate into glucose and glycogen. Developmental state (young preruminating vs older ruminating) of the donor calf did not affect the rate of gluconeogenesis from propionate in the absence of hormones. Insulin decreased (P < .05) gluconeogenesis and increased (P < .05) glycogenesis from propionate and lactate in hepatocytes from preruminating calves but had no effect on hepatocytes from ruminating calves. Lactate was poorly metabolized to glucose and was not responsive to glucagon in hepatocytes from ruminating calves compared with hepatocytes from preruminating calves. Hepatocytes responded to glucagon by increasing (P < .05) gluconeogenesis from propionate. Maximal responsiveness to glucagon did not differ between ruminating and preruminating calves, but hepatocytes from preruminating calves responded at lower glucagon concentrations (P < .05). These data demonstrate a similar capacity of hepatocytes from preruminating and ruminating calves to metabolize propionate to glucose, but there was a seven- to eightfold decrease in gluconeogenesis from lactate in ruminating calves that was accompanied by a decreased response to acute changes in insulin and glucagon.

Condensed Distillers Solubles (CDS) and corn stover mixed at a 3:1 or 2:1 ratio has been shown to have potential as a competitive feedstuff for beef and dairy cattle producers. Through lab scale silo tests, the aerobic stability, pH, mold and nutrient values suggest that these blends can partially replace corn silage. Cost projections were modeled for 8 processes of mixing the CDS and stover for storage in bag silos and were calculated using published custom rates, machinery prices, fuel price, machinery capacity, power requirements, transportation, and material costs. The processes focused on strategies of baling or chopping the stover, while shredding and processing were additional options. The Chop/Mix/Bag model was considered best with currently available technology, costing $63.6/Mg ($58.4/ton). Decreasing the price of corn by 33% saved the user $11.1/Mg or 16% per unit of the CDS and stover mix. Corn silage may be currently preferred due to its close proximity to the farm, relatively easy handling and lower fat content compared to the CDS and stover blend, but optimal inclusion rates in livestock diets may change with increased grain prices.

The impact of vitamin D supplementation in overweight and obese adults during resistance training on body composition, muscle function, and glucose tolerance was investigated.Twenty-three overweight and obese (age: 26.1±4.7 y; BMI: 31.3±3.2 kg/m(2); 25-hydroxyvitamin D: 19.3±7.2 ng/mL) adults were recruited for participation in a double-blind, placebo-controlled trial. Participants were randomly divided into vitamin D (VitD, 4000 IU/d; 5 females, 5 males) and placebo (PL; 7 females, 6 males) groups. Both groups completed 12 weeks of resistance training. 25-hydroxyvitamin D, parathyroid hormone, body composition, and glucose tolerance were assessed at baseline and 12 weeks. Muscle function (strength and power) was assessed at baseline, 4, 8, and 12 weeks.During the intervention, 25-hydroxyvitamin D increased and parathyroid hormone decreased in the VitD group (P0.05). Peak power was significantly increased at 4 weeks in the VitD group only (P0.05). Regression analysis revealed an inverse association between the change in 25-hydroxyvitamin D with the change in waist-to-hip ratio (R(2)=0.205, P=0.02). No other improvements were observed with supplementation.Vitamin D supplementation in overweight and obese adults during resistance training induced an early improvement in peak power, and elevated vitamin D status was associated with reduced waist-to-hip ratio.NCT01199926.

Continuous selection of dairy cows for production traits may alter the regulation of metabolic pathways. High-producing North American (NA) cows produce more milk and have a larger degree of somatotropic axis uncoupling than less intensively selected New Zealand (NZ) cows. The objective of this study was to determine if production-based selection priorities (i.e., NA cows) have altered the regulation of the gluconeogenic pathway relative to selection priorities based on production traits (i.e., NZ cows). In this study conducted in New Zealand, NZ (n=27) and NA cows (n=27) were monitored from 1 wk before calving to 12 wk post-calving. Cows were pasture-fed and supplemented with 0, 3, or 6 kg of concentrate DM/d. Liver biopsy samples were collected at 0, +1, and +4 wk relative to calving (WRTC) for mRNA analysis. Milk production of NA cows was greater during wk 5 to 11 postpartum and concentrate supplementation increased milk production for both NA and NZ cows. No genotype (NA vs. NZ) by diet interaction occurred for blood glucose, NEFA, or insulin. Expression of pyruvate carboxylase (PC) mRNA was increased at +1 and +4 WRTC compared with 0 WRTC (3.04 and 2.42 vs. 1.25±0.13 arbitrary units, respectively: mean ± standard error of the means) and expression of cytosolic phosphoenolpyruvate carboxykinase mRNA was increased at +4 compared with calving and +1 WRTC (4.78 vs. 2.18 and 2.48±1.41 arbitrary units, respectively). Expression of PC mRNA tended to be greater in NZ cows and tended to decrease with concentrate supplementation in both NZ and NA cows. The responses of NZ and NA cows to the transition to lactation and concentrate supplementation appeared to be similar; however, NZ cattle had a higher basal expression of PC.

Hepatocytes isolated from male dairy calves were used in monolayer culture or in suspension culture to determine their suitability for the study of hormonal regulation of hepatic gluconeogenesis. The rate of gluconeogenesis (nanomoles of 2.5 mM [2-14C]propionate incorporated into glucose.microgram of DNA-1.hour-1) was higher for monolayers than for suspension cultures. Gluconeogenesis and ureagenesis (nanomoles of urea N formed.microgram of DNA-1.3 hours-1) were similar in monolayers cultured for 24 and 48 h but declined by 120 h. Ureagenesis was barely detectable in suspension cultures. Glucagon (10 nM) increased gluconeogenesis from propionate in monolayers but was without effect on suspension cultures. Actinomycin D (800 nM) and cycloheximide (200 microM) abolished glucagon stimulation of gluconeogenesis, suggesting that glucagon acts to mediate gene expression. Prolonged exposure (45 h) of monolayers to insulin (1,000 nM) decreased basal gluconeogenic rates but did not affect glucagon-stimulated gluconeogenesis. Prior incubation with glucose or valerate did not affect gluconeogenesis. Cells can be successfully maintained in serum-free media for 41 h at the expense of diminished basal gluconeogenic activity. Culture of bovine hepatocytes as monolayers provides a useful tool for the study of chronic and acute hormonal regulation of specific liver functions in the bovine.

The objective of this study was to compare the standard methodology with an alternative method to determine feed sorting in dairy cows during the transition period. Twenty-six Holstein multiparous cows were paired by expected calving date and fed diets containing either glycerol or high moisture corn from -28 through +56 days relative to calving (DRTC). Feed sorting was determined on -16, -9, +9, +15 and +51 DRTC in two different ways. Firstly, it was determined as the actual intake of each screen of the Penn State Particle Separator (PSPS) consumed between 0-4, 4-8, 8-12 and 12-24 hours post feeding, and expressed as a percentage of the predicted intake of that correspondent screen. Secondly, by measuring the particle size distribution of feed consumed between 0-4, 4-8, 8-12 and 12-24 hours post feeding. The total mixed ration (TMR) at feeding and at each time post feeding was separated by size using the 3-screen (19, 8, and 1.18 mm) Penn State Particle Separator (PSPS) to yield long (>19 mm), medium ( 8 mm), short ( 1.18 mm), and fine particles ( 19 mm) and reduced (P 1.18 mm) and fine particles ( 0.05) the proportion of DM% retained as medium particles ( 8 mm). Cows fed prepartum glycerol increased (P 19 mm) according to the standard methodology (77.2 vs. 101.5%, control vs. glycerol) and also in the alternative methodology (9.2 vs. 17.8%, control vs. glycerol). Cows fed prepartum glycerol discriminated against (P 1.18 mm) in the standard methodology (102.6 vs. 94.2%, control vs. glycerol) as well as in the alternative methodology (42 vs. 37.3%, control vs. glycerol). There was no response (P>0.05) of diet on feed sorting of fine particles ( 8 mm) according to the standard methodology (108.6 vs. 116.5%, control vs. glycerol), but did not (P>0.05) according to the alternative methodology. Cows fed postpartum glycerol discriminated against (P 1.18 mm) according to the standard methodology (100.6 vs. 96.6%, control vs. glycerol), but did not (P>0.05) according to the alternative methodology. Feeding prepartum glycerol to transition dairy cows increases the preference for the long-stem forage particles of the diet. The alternative methodology proposed in this study is more reliable than the standard methodology to determine feed sorting. KEYWORDS: Biodiesel, byproduct, particle size, preference.

Dietary etiologies or treatments for complex mental disorder are highly controversial in psychiatry. Nevertheless, diet affects brain chemistry (particularly serotonin), and can reduce abnormal behavior in humans and animals. We formulated a diet that elevated brain serotonin and tested whether it would reduce hair pulling in a mouse model of trichotillomania. In a double-blind crossover trial, dietary elevation of brain serotonin unexpectedly increased hair pulling (P = 0.0006) and induced ulcerative dermatitis (UD; P = 0.001). The causative agent for UD is unknown. Therefore, we fed the treatment diet to a second group of mice to test whether UD is behavioral in origin. The diet increased scratching behavior (P < 0.0001). However, high scratching behavior (P = 0.027) and low barbering (P = 0.040) prior to treatment predicted the development of UD. Thus diet can trigger the onset of a complex disorder in the absence of an underlying metabolic deficit. Furthermore, we propose UD as model of compulsive skin-picking.

Pyruvate carboxylase (PC) is a critical enzyme in supplying carbon for gluconeogenesis and oxaloacetate for the tricarboxylic acid cycle. The bovine PC (EC 6.4.1.1) gene contains 3 promoter sequences (P3, P2, and P1 from 5' to 3'). Physiological stressors, including the onset of calving and feed restriction, lead to elevated nonesterified fatty acids and glucocorticoid levels that coincide with an increase in PC mRNA expression. The effects of elevated fatty acids on bovine PC mRNA expression and promoter function have not been determined. The objective of this experiment was to determine the direct effects of stearic, oleic, and linoleic acids, dexamethasone, and Wy14643 (a peroxisome proliferator-activated receptor-α agonist) on bovine PC promoter activity. Promoter-luciferase constructs, containing 1,005 bp of P1, 1,079 bp of P2, or 1,010 bp of P3, were transiently transfected into rat hepatoma (H4IIE) cells. Cells were then treated with 1mM stearic, oleic, or linoleic acids, 1 μM dexamethasone, or 10 μM Wy14643 for 23 h. Activity of P1 was suppressed with exposure to stearic acid (1.58 vs. 6.19±0.81 arbitrary units for stearic vs. control, respectively) and enhanced with exposure to Wy14643 (9.26 vs. 6.19±0.81 arbitrary units for Wy14643 vs. control, respectively). Conversely, stearic acid enhanced P3 activity (2.55 vs. 0.40±0.33 arbitrary units for stearic vs. control, respectively). Dexamethasone, linoleic acid, and oleic acid failed to elicit a response from any of the promoters tested. These data demonstrate the direct role of fatty acids in regulating PC expression and indicate that fatty acids provide promoter-specific regulation of PC promoters.

Glucose is the central molecule in many biochemical pathways, and numerous approaches have been developed for fabricating micro biosensors designed to measure glucose concentration in/near cells and/or tissues. An inherent problem for microsensors used in physiological studies is a low signal-to-noise ratio, which is further complicated by concentration drift due to the metabolic activity of cells. A microsensor technique designed to filter extraneous electrical noise and provide direct quantification of active membrane transport is known as self-referencing. Self-referencing involves oscillation of a single microsensor via computer-controlled stepper motors within a stable gradient formed near cells/tissues (i.e., within the concentration boundary layer). The non-invasive technique provides direct measurement of trans-membrane (or trans-tissue) analyte flux. A glucose micro biosensor was fabricated using deposition of nanomaterials (platinum black, multiwalled carbon nanotubes, Nafion) and glucose oxidase on a platinum/iridium microelectrode. The highly sensitive/selective biosensor was used in the self-referencing modality for cell/tissue physiological transport studies. Detailed analysis of signal drift/noise filtering via phase sensitive detection (including a post-measurement analytical technique) are provided. Using this highly sensitive technique, physiological glucose uptake is demonstrated in a wide range of metabolic and pharmacological studies. Use of this technique is demonstrated for cancer cell physiology, bioenergetics, diabetes, and microbial biofilm physiology. This robust and versatile biosensor technique will provide much insight into biological transport in biomedical, environmental, and agricultural research applications. (C) 2010 Elsevier B.V. All rights reserved.