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Circadian disruption increased insulin resistance and decreased mammary development in late gestation, nonlactating (dry) cows. The objective was to measure the effect of circadian disruption on transcriptomes of the liver and mammary gland. At 35 days before expected calving (BEC), multiparous dry cows were assigned to either control (CON) or phase-shifted treatments (PS). CON was exposed to 16-h light and 8-h dark. PS was exposed to 16-h light to 8-h dark, but phase of the light-dark cycle was shifted 6 h every 3 days. On day 21 BEC, liver and mammary were biopsied. RNA was isolated ( n = 6 CON, n = 6 PS per tissue), and libraries were prepared and sequenced using paired-end reads. Reads mapping to bovine genome averaged 27 ± 2 million and aligned to 14,222 protein-coding genes in liver and 15,480 in mammary analysis. In the liver, 834 genes, and in the mammary gland, 862 genes were different (nominal P < 0.05) between PS and CON. In the liver, genes upregulated in PS functioned in cholesterol biosynthesis, endoplasmic reticulum stress, wound healing, and inflammation. Genes downregulated in liver function in cholesterol efflux. In the mammary gland, genes upregulated functioned in mRNA processing and transcription and downregulated genes encoded extracellular matrix proteins and proteases, cathepsins and lysosomal proteases, lipid transporters, and regulated oxidative phosphorylation. Increased cholesterol synthesis and decreased efflux suggest that circadian disruption potentially increases the risk of fatty liver in cows. Decreased remodeling and lipid transport in mammary may decrease milk production capacity during lactation.

Sensors in and around the environment becoming ubiquitous has ushered in the concept of smart animal agriculture which has the potential to greatly improve animal health and productivity using the concepts of remote health monitoring which is a necessity in times when there is a great demand for animal products. The data from in and around animals gathered from sensors dwelling in animal agriculture settings have made farms a part of the Internet of Things space. This has led to active research in developing efficient communication methodologies for farm networks. This study focuses on the first hop of any such farm network where the data from inside the body of the animals is to be communicated to a node dwelling outside the body of the animal. In this paper, we use novel experimental methods to calculate the channel loss of signal at sub-GHz frequencies of 100 - 900 MHz to characterize the in-body to out-of-body communication channel in large animals. A first-of-its-kind 3D bovine modeling is done with computer vision techniques for detailed morphological features of the animal body is used to perform Finite Element Method based Electromagnetic simulations. The results of the simulations are experimentally validated to come up with a complete channel modeling methodology for in-body to out-of-body animal body communication. The experimentally validated 3D bovine model is made available publicly on https://github.com/SparcLab/Bovine-FEM-Model.git GitHub. The results illustrate that an in-body to out-of-body communication channel is realizable from the rumen to the collar of ruminants with ≤ 90 dB path loss at sub-GHz frequencies ( 100-900 MHz) making communication feasible. The developed methodology has been illustrated for ruminants but can also be used for other related in-body to out-of-body studies. Using the developed channel modeling technique, an efficient communication architecture can be formed for in-body to out-of-body communication in animals which paves the way for the design and development of future smart animal agriculture systems.

Cows experience a significant negative protein balance during the first 30 d of lactation. Given the functional effects of AA on health, especially in challenging periods such as calving, higher levels of protein and specific AA in the diet may act to improve health and feed intake. The response of dairy cows to 3 protein supplementation strategies during the transition period and through the first 45 d in milk was evaluated. The final data set had 39 Holstein cows blocked based on parity (primiparous vs. multiparous) and expected calving and randomly assigned within each block to one of 3 dietary treatments: low protein (LP), high protein (HP), or high protein plus rumen-protected methionine (HPM). Treatments were offered from d -18 ± 5 to 45 d relative to parturition. Pre- and postpartum diets were formulated for high metabolizable protein (MP) supply from soybean meal, and HP and HPM provided higher MP balance than LP. Preplanned contrasts were LP versus HP+HPM and HP versus HPM. Significance was declared at P ≤ 0.05 and trends at 0.05

Metabolizable protein supply is a limiting factor for milk production in dairy cows, and the availability of AA is a function of the quantity of the metabolizable protein available and of hepatic AA catabolism. This study aimed to evaluate the effect of postruminal protein infusion on key genes for ureagenesis and AA catabolism. Six multiparous Holstein cows in early lactation were used in a replicated crossover design. Cows were fed a TMR and infused postruminally with either 0 or 600 g/d of milk protein isolate. Periods were 21 d long, consisting of 14 d of adjustment to surroundings, followed by 7 d of protein infusion. On the last day of each infusion, liver samples were collected for mRNA analysis and explant culture, milk samples were collected for mRNA analysis, and blood samples were collected for plasma metabolite analysis. Postruminal infusion of protein increased milk yield by 10.5%, milk fat yield by 12.5%, milk protein yield by 20%, milk lactose yield by 11%, and total solids yield by 15.5%. Postruminal infusion of protein increased milk urea N by 23.5%, blood urea N by 18.6%, and the abundance of hepatic ornithine transcarbamoylase mRNA by 52.8%. Postruminal infusion of protein did not alter the mRNA abundance of hepatic argininosuccinate synthase, α-aminoadipate semialdehyde synthase, cysteine sulfinic acid decarboxylase, or cystathionase. The abundance of RNA for milk proteins was unchanged with postruminal protein infusion. Metabolism of l -[U 14C] Lys to CO2 was increased by 127% (0.143 vs. 0.063 ± 0.04 nmol product·mg tissue−1·h−1), and the metabolism of l -[U 14C] Ala to CO2 increased by 40.5% (0.52 vs. 0.37 ± 0.06 nmol product·mg tissue−1·h−1) with postruminal protein infusion. The rate of l -[1-14C] Met oxidation did not differ. These data indicate increased ureagenesis matched by upregulation of nonessential AA catabolism and a disproportional increase in Lys oxidation in response to increased postruminal protein infusion.

An increase in bovine pyruvate carboxylase (PC; EC 6.4.1.1) at calving and during feed restriction corresponds with increased circulating nonesterified fatty acids as a consequence of negative energy balance. Regulation of PC mRNA and effect of specific combinations of saturated and unsaturated fatty acid profiles has yet to be explored. Our objective was to determine the effects of chain length, degree of saturation, and copresence of saturated and unsaturated fatty acids on activity of bovine PC promoter 1 (PCP1). For these experiments, Madin-Darby bovine kidney cells were transfected with a full-length bovine PCP1 construct from -1002 to +3 bp relative to the bovine PC gene transcription start site (bovine PCP1(-1002_+3)) ligated to a Firefly luciferase reporter, or with one of a series of nested 5' serial truncations (bovine PCP1(-773_+3), bovine PCP1(-494_+3), or bovine PCP1(-222_+3)). Cells were exposed for 23 h to either individual fatty acids (C16:0, C18:0, or C18:3n-3 cis) bound to BSA or to fatty acid mixtures in ratios of 90:10, 75:25, 50:50, or 25:75, corresponding to combinations of C16:0: C18:3n-3 cis or C18:0: C18:3n-3 cis. Total fatty acid concentration was 1.00 mM. Exposure to either C16:0 or C18:3n-3 cis alone elicited a significant increase in capacity to drive bovine PCP1(-1002_+3) activity compared with 1% BSA in Dulbecco's Modified Eagle's Medium control treatment (2.29, 2.89, and 1.00 ± 0.26 fold of promoter induction for C16:0, C18:3n-3 cis, and control, respectively). Treatment with C18:3n-3 cis alone caused a greater increase in promoter activity compared with C16:0 alone, indicating a lesser response to C16:0 alone for bovine PCP1(-1002_+3). Interestingly, inclusion of C18:3n-3 cis, at any level of fatty acid ratios examined, in combination with C16:0 increased promoter activity of bovine PCP1(-773_+3) or bovine PCP1(-222_+3) compared with treatment with C16:0 alone or control. Data from the bovine PCP1 truncation and fatty acid copresence experiments reveal the potential for response elements of unsaturated fatty acids or fatty acid ligands in several bovine PCP1 promoter regions. In silico analysis of bovine PCP1 identified putative peroxisome proliferator-activated receptor α and sterol regulatory element binding protein binding sites which may be implicated in fatty acid signaling to alter bovine PCP1 activity. Pyruvate carboxylase promoter 1 activity that is mediated by unsaturated fatty acids acting through elements within -1002 and -222 bp of bovine PCPI may determine PC response during periods of negative energy balance in dairy cows.

Metabolic fates of fatty acids in tissue may be influenced by extracellular concentration and profile of fatty acids. Previous work has demonstrated the ability of C18:3n-3 cis to ameliorate the effects of C16:0- or C18:0-induced depression of pyruvate carboxylase (PC) mRNA expression. Pyruvate carboxylase catalyzes oxaloacetate synthesis and connects gluconeogenesis from lactate and fatty acid metabolism. Our objective was to determine the effects of co-presence of saturated and unsaturated fatty acids on cellular partitioning of [1-14C]C16:0 metabolism to CO2 or acid-soluble products (ASP) in Madin-Darby bovine kidney cells and the role of PC in this relationship. We hypothesized that the ratio of saturated to unsaturated fatty acid pretreatments regulates [1-14C]C16:0 partitioning to CO2 or ASP. Cells were exposed for 21 h to either individual fatty acids, C16:0, C18:0, C18:1n-9 cis, or C18:3n-3 cis, or to fatty acid combinations in 10:90, 25:75, 50:50, 75:25, or 90:10 ratios for 3 combinations: C16:0/C18:3n-3 cis, C18:0/C18:3n-3 cis, or C18:1n-9 cis/C18:3n-3 cis. Total fatty acid concentration was 1.0 mM during the 21-h pretreatment phase. Following the 21-h incubation phase with fatty acid combinations, cells were incubated in the presence of 1.0 mM [1-14C]C16:0 for 3 h to determine the rate of metabolism to CO2 and ASP collection (per µg DNA-1·h-1). Pretreatment with either C16:0 or C18:0 alone significantly depressed subsequent oxidation of [1-14C]C16:0 to ASP by 62.7 and 41.2%, respectively, compared with C18:3n-3 cis pretreatment. Similar patterns were observed for [1-14C]C16:0 oxidation to CO2. Expression of PC mRNA was significantly decreased with exposure to either C16:0 or C18:0 compared with expression after exposure to either C18:3n-3 cis or control 1% BSA in Dulbecco's modified Eagle's medium. Expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) mRNA followed a similar pattern. Fatty acid treatments containing C18:1n-9 cis did not alter PC or PCK1 expression from control or C18:3n-3 cis results. Pearson coefficient correlations were determined for PC mRNA expression and rate of [1-14C]C16:0 metabolism to CO2 or ASP, including ketones, and for PCK1 mRNA expression and rate of [1-14C]C16:0 metabolism to CO2 or ASP. Production of CO2 from [1-14C]C16:0 was positively correlated (r = 0.63) with PC expression, whereas ASP production from [1-14C]C16:0 only tended to positively correlate (r = 0.51) with PC mRNA expression. Production of CO2 or ASP from [1-14C]C16:0 were both positively correlated (r = 0.80 and r = 0.69, respectively) with PCK1 expression. Results show a regulation of ketone production by Madin-Darby bovine kidney cells in response to saturated and unsaturated fatty acid pretreatments.

An emerging hallmark of cancer is cellular metabolic reprogramming to adapt to varying cellular environments. Throughout the process of metastasis cancer cells gain anchorage independence which confers survival characteristics when detached from the extracellular matrix (ECM). Previous work demonstrates that the bioactive metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25[OH]

Obesity and metabolic disorders caused by energy surplus pose an increasing concern within the global population. Brown adipose tissue (BAT) dissipates energy through mitochondrial non-shivering thermogenesis, thus representing a powerful agent against obesity. Here we explore the novel role of a mitochondrial outer membrane protein, LETM1-domain containing 1 (LETMD1), in BAT. We generated a knockout (Letmd1KO ) mouse model and analyzed BAT morphology, function and gene expression under various physiological conditions. While the Letmd1KO mice are born normally and have normal morphology and body weight, they lose multilocular brown adipocytes completely and have diminished mitochondrial abundance, DNA copy number, cristae structure, and thermogenic gene expression in the intrascapular BAT, associated with elevated reactive oxidative stress. In consequence, the Letmd1KO mice fail to maintain body temperature in response to acute cold exposure without food and become hypothermic within 4 h. Although the cold-exposed Letmd1KO mice can maintain body temperature in the presence of food, they cannot upregulate expression of uncoupling protein 1 (UCP1) and convert white to beige adipocytes, nor can they respond to adrenergic stimulation. These results demonstrate that LETMD1 is essential for mitochondrial structure and function, and thermogenesis of brown adipocytes.

Background: Throughout metastatic progression, cancer cells acquire anchorage independence, or the ability to survive detached from the extracellular matrix (ECM). While untransformed epithelial cells reduce energy metabolism when detached, cancer cells display metabolic flexibility to continue important metabolic processes. Glucose and glutamine are predominant nutrients utilized for energy as well as other purposes, and their metabolism is regulated by cancer cells.Methods: The purpose of the current studies was to determine the effects of detachment on glucose and glutamine metabolism in human breast epithelial MCF10A cells transfected with the Harvey-ras oncogene (MCF10A-ras), a model of early-stage cancer. Detachment was simulated with poly-HEMA coated plates, and intracellular metabolic flux was determined using stably labeled 13C5-glutamine and 13C6-glucose tracers.Results: Results show reduced glutamine flux in detached cells as determined by reduced accumulation of label in glutamate (21%), malate (30%), and aspartate (23%) from 13C5-glutamine. Detachment also reduced flux of 13C6-glucose to pyruvate and lactate pools by 51% and 29%, respectively. Similarly, detachment reduced total intracellular pool sizes of pyruvate (51%), lactate (49%), α-ketoglutarate (43%), fumarate (32%), malate (19%), alanine (35%), serine (35%), and glutamate (28%) compared to attached cells, but citrate and aspartate pool sizes were unchanged. Compared to attached cells, detachment increased pyruvate carboxylase (PC) mRNA abundance and protein expression by 131% and 190%, respectively. In detachment, PC activity, determined by 13C6-glucose derived M + 3 isotopomers, was shown to preferentially replenish malate and aspartate, but not citrate pools. In addition, doxycycline-inducible shRNA depletion of PC significantly decreased, while doxycycline-inducible PC overexpression significantly increased, detached cell viability. Further, a switch from glutamine to PC activity for anaplerosis was demonstrated, as supplementation with the cell permeable analog of the tricarboxylic acid cycle intermediate, α-ketoglutarate, a downstream metabolite of glutamine, decreased PC mRNA abundance in detached cells.Conclusion: Collectively, these results suggest that detached breast cancer cells increase PC activity in response to decreased glutamine-derived anaplerosis to promote cell survival.

Our objective was to determine the effects of uncouplers of oxidative phosphorylation on the metabolism of propionate in liver tissue of dairy cows in the postpartum period. A total of 8 primiparous dairy cows were biopsied for liver tissue explants in 2 block-design experiments. Cows were 5.9 ± 2.8 (mean ± SD) days in milk, and the 2 experiments were run concurrently. Treatments for experiment 1 were 10 μM 2,4-dinitrophenol methyl ether (DNPME) or propylene carbonate (vehicle control). Treatments for experiment 2 were 5 mM sodium salicylate (SAL) or no treatment (control). Explants were incubated in 2.5 mM [13C3]propionate with treatments and terminated after 0.5, 15, and 60 min of exposure to tracer. Treatment with DNPME had no effects on measured metabolites compared with control. Treatment with SAL increased total 13C% enrichment of succinate (3.03 vs. 2.45%), but tended to decrease total 13C% enrichment of fumarate (2.86 vs. 3.10%) and decreased total 13C% enrichment of malate (3.96 vs. 4.58%) compared with the control. Treatment with DNPME appeared to have no effects on hepatic propionate metabolism, and treatment with SAL may impair the succinate dehydrogenase reaction.

A more complete understanding of the molecular mechanisms that support milk synthesis is needed to develop strategies to efficiently and sustainably meet the growing global demand for dairy products. With the postulate that coding gene transcript abundance reflects relative importance in supporting milk synthesis, we analyzed the global transcriptome of early lactation cows across magnitudes of normalized RNA-Seq read counts. Total RNA was isolated from milk samples collected from early-lactation cows ( n = 6) following two treatment periods of postruminal lysine infusion of 0 or 63 g/day. Twelve libraries were prepared and sequenced on an Illumina NovaSeq6000 platform using paired end reads. Normalized read counts were averaged across both treatments, because EBseq analysis found no significant effect of lysine infusion. Approximately 10% of the total reads corresponded to 12,730 protein coding transcripts with a normalized read count mean ≥5. For functional annotation analysis, the protein coding transcripts were divided into nine categories by magnitude of reads. The 13 most abundant transcripts (≥50K reads) accounted for 67% of the 23M coding reads and included casein and whey proteins, regulators of fat synthesis and secretion, a ubiquitinating protein, and a tRNA transporter. Mammalian target of rapamycin, JAK/STAT, peroxisome proliferator-activated receptor alpha, and ubiquitin proteasome pathways were enriched with normalized reads ≥100 counts. Genes with ≤100 reads regulated tissue homeostasis and immune response. Enrichment in ontologies that reflect maintenance of translation, protein turnover, and amino acid recycling indicated that proteostatic mechanisms are central to supporting mammary function and primary milk component synthesis.

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a key enzyme for gluconeogenesis that is positively regulated by propionate in bovines at the transcription level. The specific elements that determine propionate responsiveness within the bovine PCK1 promoter are unknown. In silico promoter analysis of the bovine PCK1 gene revealed several clusters of transcription factor binding sites. In the present study, we determined the essentiality of the putative cyclic AMP response element (CRE) at -94 through -87 bp and the 2 putative hepatic nuclear factor 4α (HNF4α) binding elements at +68 through +72 and -1,078 through -1,074, respectively, in mediating bovine PCK1 promoter responses to propionate and other regulators, including butyrate, cyclic AMP (cAMP), and glucocorticoids. The wild-type bovine PCK1 promoter [PCK1(WT)] was ligated to a luciferase reporter gene and transfected into rat hepatoma (H4IIE) cells. Activities of PCK1(WT) were induced by approximately 2-, 2-, 4-, 8-, 9-, 18-, and 16-fold respectively when exposed to cAMP (as 1.0 mM 8-Br-cAMP), 5.0 μM dexamethasone, cAMP + dexamethasone, 2.5 mM propionate, cAMP + propionate, cAMP + dexamethasone + propionate, and 2.5 mM butyrate. Seven mutants lacking either one single site, 2 of the 3 sites, or all 3 sites, generated by site-directed mutagenesis, were tested. Responses to propionate and all other treatments were completely abolished when CRE at -94 through -87 bp and HNF4α at +68 through +72 bp were both deleted. Our data indicate that these 2 regulatory elements act synergistically to mediate the bovine PCK1 promoter responses to propionate as well as butyrate, cAMP, and dexamethasone. The activation of PCK1 through these regulatory elements serves to activate the metabolic potential of bovine toward gluconeogenesis when the primary substrate for gluconeogenesis, propionate, is also present.

Our objective was to determine the temporal effects of increasing supply of propionate on propionate metabolism in liver tissue of dairy cows in the postpartum (PP) period. A total of 6 dairy cows [primiparous: n = 3, 9.00 ± 1.00 d PP (mean ± SD) and multiparous: n = 3; 4.67 ± 1.15 d PP] were biopsied for liver explants in a block-design experiment. Explants were treated with 3 concentrations of [13C3]sodium propionate of 1, 2, or 4 mM. Explants were incubated in 2 mL of Medium 199 supplemented with 1% BSA, 0.6 mM oleic acid, 2 mM sodium l-lactate, 0.2 mM sodium pyruvate, and 0.5 mMl-glutamine at 38°C and sampled at 0.5, 15, and 60 min. Increasing the concentration of [13C3]propionate increased total 13C% enrichment of propionyl coenzyme A (CoA), succinate, fumarate, malate, and citrate with time. Concentration of propionate did not affect total 13C% enrichment of hepatic glucose or acetyl CoA, but total 13C% enrichment increased with time for hepatic glucose. The 13C labeling from propionate was incorporated into acetyl CoA, but increased concentrations of propionate did not result in greater labeling of acetyl CoA. However, increases in 13C% enrichment of [M+4]citrate and [M+5]citrate concentrations of [13C3]propionate indicate propionate conversion to acetyl CoA and subsequent entry of acetyl CoA into the tricarboxylic acid cycle in dairy cows in the PP period. This research presents evidence that despite an increase in hepatic acetyl CoA concentration and general consensus on the upregulation of gluconeogenesis of dairy cows during the PP period, carbon derived from propionate contributes to the pool of acetyl CoA, which increases as concentration of propionate increases, in addition to stimulating oxidation of acetyl CoA from other sources. Because of the hypophagic effects of propionate, but importance of propionate as a glucose precursor, a balance of propionate supply to dairy cows could lead to improvements in dry matter intake, and subsequently, health and production in dairy cows.

It is well established that adipose tissue can both store and metabolize vitamin D. The active form of vitamin D, 1,25 dihydroxyvitamin D [1,25(OH)2D], regulates adipocyte differentiation and inflammation, highlighting the multifaceted role that vitamin D plays in adipose tissue physiology. However, there is limited evidence regarding vitamin D regulation of mature adipocyte lipid metabolism. We hypothesize that 1,25(OH)2D alters lipid and glucose metabolism in differentiated 3T3-L1 adipocytes to reduce triacylglycerol (TAG) accumulation. In this study, 1,25(OH)2D (10 nmol/L) stimulated a 21% reduction in TAG accumulation in differentiated 3T3-L1 adipocytes after 4 days (P = .01) despite a significant increase in fatty acid uptake (P

The Earth is at a sociotechnical crossroads with humanity hanging in the balance – and high-tech agriculture can help bail us out. Human population growth, increasing urbanization and rising incomes is likely to drastically increase demand for animal agriculture in the coming decades. The US Department of Agriculture (USDA) predicts the need to double global food production by 2050 as the global population increases from 7.3 billion in 2015 to 9.7 billion in 2050 as shown in Fig 1. Much of this growth will be concentrated in the world’s poorest countries where standards of living are set to rise rapidly, increasing the demand for resource-intensive meat and dairy products which has been the historical trend. At the same time, agriculture in the 21st century faces multiple challenges: it must produce more food and fiber to feed a growing population with a smaller rural labor force, produce additional feedstocks for a potentially huge bioenergy market, contribute to overall development in the many agriculture-dependent developing countries, adopt more efficient and sustainable production methods, and adapt to climate change. Additionally, the world’s arable land is already fully employed and shrinking -- the world has lost a third of its arable land due to erosion or pollution in the past 40 years. All these factors put enormous pressure on improving the production efficiency of the world’s supply of food to meet the demand.

Maintaining reductive-oxidative (redox) balance is an essential feature in breast cancer cell survival, with cellular metabolism playing an integral role in maintaining redox balance through its supply of reduced NADPH. In the present studies, the effect of 1,25-dihydroxyvitamin D (1,25(OH)2D) on redox balance was investigated in early stages of breast cancer. Treatment with 1,25(OH)2D promoted oxidative stress in MCF10A-ras and MCF10A-ErbB2 breast epithelial cells, as measured by the decreased ratios of NADPH/NADP+ and reduced to oxidized glutathione (GSH/GSSG). The mRNA and protein expression of the enzyme pyruvate carboxylase (PC) was downregulated with 1,25(OH)2D treatment, suggesting a potential mechanism. Genetic depletion of PC in MCF10A-ras cells resulted in a decreased ratio of NADPH/NADP+ and GSH/GSSG, with 1,25(OH)2D treatment having no further effect. Mutation analysis confirmed the presence and functionality of a vitamin D response element in the PC gene promoter region. Collectively, these results provide evidence that 1,25(OH)2D promotes oxidative stress in early breast cancer progression through transcriptional downregulation of PC.

The objective for this study was to determine the effect of glucose dose and days following peak milk yield on plasma glucose, serum insulin, and plasma nonesterified fatty acids (NEFA) kinetics during an intravenous glucose tolerance test (IVGTT) in lactating dairy cattle. Six lactating Holstein dairy cows (3 primiparous and 3 multiparous) were assigned to 2 squares and received 0.092, 0.15, or 0.3 g of glucose/kg of body weight (BW) during an IVGTT at 74 and 221 d in milk (DIM), representing early (post-peak) lactation and mid lactation, respectively. Treatments were applied in a replicated Latin square design using contiguous 7-d periods within each stage of lactation. Milk production and dry matter intake were determined daily during the first 6 d of each period. The IVGTT was performed on d 7. For the IVGTT, cows were prepared with indwelling catheters in each jugular vein, and blood samples were collected at −15, −10, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min relative to the glucose infusion. Samples were analyzed for plasma glucose, serum insulin, and plasma NEFA concentrations. Increasing the glucose dose during the IVGTT increased plasma glucose area under the curve (AUC), decreased glucose half-life, and increased maximal plasma glucose concentrations in plasma during the IVGTT. Greater glucose dose during the IVGTT elevated serum insulin AUC and increased nadir NEFA concentrations. Maximal plasma glucose concentration during the IVGTT was lower, whereas maximum NEFA concentration, NEFA AUC, and NEFA clearance rate were greater at 74 than at 221 DIM. Only glucose half-life was responsive to stage of lactation × glucose dose effects during the IVGTT, and the decrease in glucose half-life with increasing glucose dose was greater at 74 than at 221 DIM. Glucose AUC was greater and NEFA AUC lower for cows at 74 than at 221 DIM. For the doses tested, a glucose dose greater than 0.092 g/kg of BW resulted in peak blood glucose concentration that exceeded the previously reported renal glucose excretion threshold of 8.3 mM. There is a need for accompanying data to determine if this is the case for the glucose doses evaluated in this experiment. Based on maximal peak glucose concentrations and effects on glucose half-life, we identify 0.092 g of glucose/kg of BW (0.46 g/kg of metabolic body weight) as the preferred dose for the IVGTT for cows at 74 and 221 DIM in this study.

Introduction Meat consumption has been steadily increasing since the 1960s1. As living standards and income levels rise, more people include meat into their diet. Although an important source of nutrients, livestock production has a negative influence on greenhouse gas emissions, water consumption, water pollution, and more, with cattle being the main culprits2. However, while farming practices have improved over the last 60 years resulting in improved efficiencies in water, land, and crop use, studies suggest that cattle farmers can do more to reduce their overall environmental impact towards more sustainable production of animal protein3. The primary value of ruminants in the food chain is their ability to convert non-protein nitrogen, cellulose-rich carbohydrates, and other precursors to a digestible form of protein for human consumption. This is achieved through bacterial fermentation in the rumen that converts nitrogen (ammonia) to bacterial protein, which provides the animal with an essential protein supply to support growth and thereby meat and milk production for human consumption. Too little ammonia in the rumen limits bacterial protein synthesis. However, excess ammonia is converted to urea, which is excreted urine, and is an environmental burden. Excess nitrogen in manure is released to the atmosphere and is part of the issue with nitrogen runoff4. Our group is currently developing robotic technologies, termed RUMENS (Rumen Understanding through Millipede-Engineered Navigation and Sensing), to enable live, in vivo mapping of the ruminal environment, based on a suite of commercial and custom built sensors, of various compounds present in rumen5. Here, we propose a miniature, rapid update zinc oxide (ZnO) film-based sensor for real-time liquid ammonia detection in the rumen. Method Zinc acetate (ZnAc) and 2-methoxyethanol (2ME) were purchased from VWR, ethanolamine and ammonium hydroxide solution (28%) were purchased from Sigma Aldrich. All chemicals were used as received without any purification. Planar electrodes (inset of Figure 1), with dimensions of 1-mm-long and 2.54-mm-wide, were obtained from thermally evaporated Cr and Au on cleaned glass substrate, with the thicknesses of 3 nm and 50 nm, respectively. For ZnO layer deposition, 140 mg of ZnAc was dissolved in 1 mL of 2ME and 45 μL of ethanolamine at 60 °C for about 8 hours. The solution was spin cast at 600 rpm for 60 seconds. After manually patterning between two adjacent devices (to avoid crosstalk), the sample was then placed on a hot plate set to 300 °C in air for 10 minutes. After removal and cooling, the samples were rinsed consecutively in deionized H2O, acetone, ethanol, and IPA for 10 seconds each and then dried on a hotplate in air for 10 minutes at 200 °C6. An impedance measurement, using RLC meter (NF ZM 2372), was used to test the resistance with different ammonium concentrations. Ammonia of 10-1, 10-2, 10-3 and 10-4 M were prepared by diluting a known amount of ammonium hydroxide solution in DI water. A 3 μL of analyte was dropped onto the ZnO layer, directly between two planar gold electrodes. Devices were thoroughly (5 minutes) washed in DI water between measurements. Results and Conclusions Figure 1 shows the relationship between channel impedance (Z’) and concentration of ammonia at different scan frequencies, ranging from 1 to 100 kHz. The measurements reveal that at a fixed frequency, the impedance decreases with increased ammonia concentration. Also, at a single concentration, at low frequencies ( A possible sensing mechanism is the nitrification of NH4 + into nitrate ions, NO3 -, with nitrification reaction: NH4 + + 2O2 → NO3 - + H2O + 2H+. The energy released during oxidation should be sufficient for electrons to jump to the conduction band, resulting in decreasing of resistivity proportional to concentration of ammonia7. These results indicate a suitable device for aqueous ammonia detection in rumen. References Gerten, D.; Heck, V.; Jägermeyr, J.; Bodirsky, B. L.; Fetzer, I.; Jalava, M.; Kummu, M.; Lucht, W.; Rockström, J.; Schaphoff, S.; Schellnhuber, H. J., Feeding ten billion people is possible within four terrestrial planetary boundaries. Nature Sustainability 2020, 3 (3), 200-208. Gerber, P. J.; Steinfeld, H.; Henderson, B.; Mottet, A.; Opio, C.; Dijkman, J.; Falcucci, A.; Tempio, G. Tackling Climate Change Through Livestock - A Global Assessment of Emissions And Mitigation Opportunities; Food and Agriculture Organization of the United Nations: Rome, 2013. González, N.; Marquès, M.; Nadal, M.; Domingo, J. L., Meat consumption: Which are the current global risks? A review of recent (2010–2020) evidences. Food Research International 2020, 137, 109341. Schwab, C. G.; Broderick, G. A., A 100-Year Review: Protein and amino acid nutrition in dairy cows. Journal of Dairy Science 2017, 100 (12), 10094-10112. Balakuntala, M. V.; Ayad, M.; Voyles, R. M.; White, R.; Nawrocki, R.; Sundaram, S.; Priya, S.; Chiu, G.; Donkin, S.; Min, B.-C.; Daniels, K., Global Sustainability through Closed-Loop Precision Animal Agriculture. Mechanical Engineering 2018, 140 (06), S19-S23. Nawrocki, R. A.; Galiger, E. M.; Ostrowski, D. P.; Bailey, B. A.; Jiang, X.; Voyles, R. M.; Kopidakis, N.; Olson, D. C.; Shaheen, S. E., An inverted, organic WORM device based on PEDOT:PSS with very low turn-on voltage. Organic Electronics 2014, 15 (8), 1791-1798. Franco, F. F.; Manjakkal, L.; Shakthivel, D.; Dahiya, R. In ZnO based Screen Printed Aqueous Ammonia Sensor for Water Quality Monitoring, 2019 IEEE Sensors, 2019; pp 1-4. Fig 1. Impedance vs frequency of ammonia sensor as characterized for different concentrations. Figure 1

Both increased de novo fatty acid synthesis and higher neutral lipid accumulation are a common phenotype observed in aggressive breast cancer cells, making lipid metabolism a promising target for breast cancer prevention. In the present studies we demonstrate a novel effect of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)2D) on lipid metabolism in malignant breast epithelial cells. Treatment of MCF10CA1a breast epithelial cells with 1,25(OH)2D (10 nM) for five and seven days decreased the level of triacylglycerol, the most abundant form of neutral lipids, by 20%(±3.9) and 50%(±5.9), respectively. In addition, 1,25(OH)2D treatment for five days decreased palmitate synthesis from glucose, the major fatty acid synthesized de novo (48%±5.5 relative to vehicle). We have further identified the anaplerotic enzyme pyruvate carboxylase (PC) as a target of 1,25(OH)2D mediated regulation and hypothesized that 1,25(OH)2D regulates breast cancer cell lipid metabolism through inhibition of PC. PC mRNA expression was downregulated with 1,25(OH)2D treatment at two (73%±6 relative to vehicle) and five (56%±8 relative to vehicle) days. Decrease in mRNA abundance corresponded with a decrease in PC protein expression at five days of treatment (54%±12 relative to vehicle). Constitutive overexpression of PC in MCF10CA1a cells using a pCMV6-PC plasmid inhibited the effect of 1,25(OH)2D on both TAG accumulation and de novo palmitate synthesis from glucose. Together, these studies demonstrate a novel mechanism through which 1,25(OH)2D regulates lipid metabolism in malignant breast epithelial cells.

Lysine supply is potentially limiting for milk production in dairy cows. The availability of Lys to the mammary gland and other tissues is a function of the quantity of metabolizable Lys supplied and Lys catabolism by the liver. Likewise, Lys catabolism may be influenced by Lys supply. This study evaluated the effect of increased postruminal Lys supply on the expression of aminoadipate semialdehyde synthase (AASS, a committing step in Lys catabolism in the liver) and ornithine transcarbamoylase and argininosuccinate synthase (key urea cycle enzymes that are responsive to protein supply). Eight multiparous peak Holstein cows were used in a replicated 4 × 4 Latin square. Cows were fed a Lys-limiting ration and infused postruminally with 0, 9, 27, or 63 g/d of Lys. The study consisted of 10 d of pretreatment followed by 10 d of Lys infusion. On the last day of each period, liver and milk samples were collected for mRNA analysis, and blood samples were collected for analysis of amino acids and Lys metabolites. Milk protein percent increased by 5.9%, plasma Lys increased by 74%, and α-aminoadipic acid increased by 51% with postruminal infusion of 63 g/d Lys compared with 0 g/d. Expression of AASS, ornithine transcarbamoylase, and argininosuccinate synthase mRNA in liver did not differ with postruminal infusion of Lys. Milk fat globule mRNA for major milk proteins and AASS were not affected by Lys infusion. Postruminal infusion of Lys resulted in an 86% greater increase in AASS mRNA in the liver compared with mammary mRNA. These changes suggest that hepatic Lys metabolism is not responsive to Lys supply at the transcription level, and that the availability of Lys to extrahepatic tissue may be determined by hepatic Lys metabolism.

Breast cancer is the second most common cancer among women in the US. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is proposed to inhibit cellular processes and to prevent breast cancer. The current studies investigated the effect of 1,25(OH)2D on glutamine metabolism during cancer progression employing Harvey-ras oncogene transformed MCF10A human breast epithelial cells (MCF10A-ras). Treatment with 1,25(OH)2D significantly reduced intracellular glutamine and glutamate levels measured by nuclear magnetic resonance (NMR) by 23 ± 2% each. Further, 1,25(OH)2D treatment reduced glutamine and glutamate flux, determined by [U-13C5] glutamine tracer kinetics, into the TCA cycle by 31 ± 0.2% and 17 ± 0.4%, respectively. The relative levels of mRNA and protein abundance of the major glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was significantly decreased by 1,25(OH)2D treatment in both MCF10A-ras cells and MCF10A which overexpress ErbB2 (HER-2/neu). Consistent with these results, glutamine uptake was reduced by 1,25(OH)2D treatment and the impact was eliminated with the SLC1A5 inhibitor L-γ-Glutamyl-p-nitroanilide (GPNA). A consensus sequence to the vitamin D responsive element (VDRE) was identified in silico in the SLC1A5 gene promoter, and site-directed mutagenesis analyses with reporter gene studies demonstrate a functional negative VDRE in the promoter of the SLC1A5 gene. siRNA-SLC1A5 transfection in MCF10A-ras cells significantly reduced SLC1A5 mRNA expression as well as decreased viable cell number similar to 1,25(OH)2D treatment. SLC1A5 knockdown also induced an increase in apoptotic cells in MCF10A-ras cells. These results suggest 1,25(OH)2D alters glutamine metabolism in MCF10A-ras cells by inhibiting glutamine uptake and utilization, in part through down-regulation of SLC1A5 transcript abundance. Thus, 1,25(OH)2D down-regulation of the glutamine transporter, SLC1A5, may facilitate vitamin D prevention of breast cancer.

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a critical enzyme within the metabolic networks for gluconeogenesis, hepatic energy metabolism, and tricarboxylic acid cycle function, and is controlled by several transcription factors including hepatic nuclear factor 4α (HNF4α). The primary objective of the present study was to determine whether propionate regulates bovine PCK1 transcription. The second objective was to determine the action of cyclic AMP (cAMP), glucocorticoids, and insulin, hormonal cues known to modulate glucose metabolism, on bovine PCK1 transcriptional activity. The proximal promoter of the bovine PCK1 gene was ligated to a Firefly luciferase reporter and transfected into H4IIE hepatoma cells. Cells were exposed to treatments for 23 h and luciferase activity was determined in cell lysates. Activity of the PCK1 promoter was linearly induced by propionate, and maximally increased 7-fold with 2.5 mM propionate, which was not muted by 100 nM insulin. Activity of the PCK1 promoter was increased 1-fold by either 1.0 mM cAMP or 5.0µM dexamethasone, and 2.2-fold by their combination. Induction by cAMP and dexamethasone was repressed 50% by 100 nM insulin. Propionate, cAMP, and dexamethasone acted synergistically to induce the PCK1 promoter activity. Propionate-responsive regions, identified by 5' deletion analysis, were located between -1,238 and -409 bp and between -85 and +221 bp. Deletions of the core sequences of the 2 putative HNF4α sites decreased the responsiveness to propionate by approximately 40%. These data indicate that propionate regulates its own metabolism through transcriptional stimulation of the bovine PCK1 gene. This induction is mediated, in part, by the 2 putative HNF4α binding sites in the bovine PCK1 promoter.

Mounting evidence indicates that maternal exercise confers protection to adult offspring against various diseases. Here we hypothesized that maternal exercise during gestation would reduce high-fat diet (HFD)-induced hepatic steatosis in adult rat offspring.Following conception, pregnant dams were divided into either voluntary wheel running exercise (GE) or wheel-locked sedentary (GS) groups throughout gestation (days 4-21). Post-weaning, offspring received either normal chow diet (CD; 10% fat, 70% carbohydrate, 20% protein) or HFD (45% fat, 35% carbohydrate, and 20% protein) until sacrificed at 4- or 8-months of age.GE did not affect offspring birth weight or litter size. HFD feeding in offspring increased weight gain, body fat percentage, and glucose tolerance test area under the curve (GTT-AUC). Male offspring from GE dams had reduced body fat percentage across all ages (p0.05). In addition, 8-month male offspring from GE dams were protected against HFD-induced hepatic steatosis, which was associated with increased markers of hepatic mitochondrial biogenesis (PGC-1α and TFAM), autophagic potential (ATG12:ATG5 conjugation) and hepatic triacylglycerol secretion (MTTP).The current study provides the first evidence that gestational exercise can reduce susceptibility to HFD-induced hepatic steatosis in adult male offspring.

Nutritional status and glucose precursors are known regulators of gluconeogenic gene expression. Glycerol can replace corn in diets fed to dairy cows and use of glycerol is linked to increased rumen propionate production. The effect of dietary glycerol on the regulation of gluconeogenic enzymes is unknown. The objective of this study was to examine the effect of glycerol on expression of pyruvate carboxylase (PC), cytosolic and mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-C and PEPCK-M), and glucose-6-phosphatase. Twenty-six multiparous Holstein cows were fed either a control diet or a diet where high-moisture corn was replaced by glycerol from -28 through +56 d relative to calving (DRTC). Liver tissue was collected via percutaneous liver biopsy at -28, -14, +1, +14, +28, and +56 DRTC for RNA analysis. Expression of PC mRNA increased 6-fold at +1 and 4-fold at +14 DRTC relative to precalving levels. Dietary glycerol did not alter expression of PC mRNA expression. Expression of PEPCK-C increased 2.5-fold at +14 and 3-fold at +28 DRTC compared with +1 DRTC. Overall, dietary glycerol increased PEPCK-C expression compared with that of cows fed control diets. The ratio of PC to PEPCK-C was increased 6.3-fold at +1 DRTC compared with precalving and tended to be decreased in cows fed glycerol. We detected no effect of diet or DRTC on PEPCK-M or glucose-6-phosphatase mRNA, and there were no interactions of dietary treatment and DRTC for any transcript measured. Substituting corn with glycerol increased the expression of PEPCK-C mRNA during transition to lactation and suggests that dietary energy source alters hepatic expression. The observed increase in PEPCK-C expression with glycerol feeding may indicate regulation of hepatic gene expression by changes in rumen propionate production.

Elevated needs for glucose in lactating dairy cows are met through a combination of increased capacity for gluconeogenesis and increased supply of gluconeogenic precursors, primarily propionate. This study evaluated the effects of propionate on mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC), key gluconeogenic enzymes, and capacity for glucose synthesis in liver of dairy cattle. In experiment 1, six multiparous mid-lactation Holstein cows were used in a replicated 3×3 Latin square design consisting of a 6-d acclimation or washout phase followed by 8h of postruminal infusion of either propionate (1.68mol), glucose (0.84mol), or an equal volume (10mL/min) of water. In experiment 2, twelve male Holstein calves [39±4 kg initial body weight (BW)] were blocked by birth date and assigned to receive, at 7d of age, either propionate [2mmol·h(-1)·(BW(0.75))(-1)], acetate [3.5mmol·h(-1)·(BW(.75))(-1)], or an equal volume (4mL/min) of saline. In both experiments, blood samples were collected at 0, 2, 4, 6, and 8h relative to the start of infusion and liver biopsy samples were collected at the end of the infusion for mRNA analysis. Liver explants from experiment 1 were used to measure tricarboxylic acid cycle flux and gluconeogenesis using (13)C mass isotopomer distribution analysis from (13)C3 propionate. Dry matter intake and milk yield were not altered by infusions in cows. Serum insulin concentration in cows receiving propionate was elevated than cows receiving water, but was not different from cows receiving glucose. Hepatic expression of PCK1 and G6PC mRNA and glucose production in cows receiving propionate were not different from cows receiving water, but tended to be higher compared with cows receiving glucose. Hepatic expression of PCK2 and PC mRNA was not altered by propionate infusion in cows. Blood glucose, insulin, and glucagon in calves receiving propionate were not different than controls. Calves receiving propionate had increased PCK1 mRNA, tended to have increased G6PC mRNA, and had similar PC mRNA compared with saline controls. These data indicate a tendency for in vivo effects of propionate to alter hepatic gene expression in mid-lactation cows and neonatal calves, which are consistent with a feed-forward effect of propionate to regulate its own metabolism toward gluconeogenesis through changes in hepatic PCK1 mRNA.

Heat stress (HS) results from excessive heat load on animals such that all adaptive mechanisms used to dissipate the heat do not return the body to normal body temperature. In pigs, HS results in increased fat deposition compared with pair-fed animals in a thermoneutral environment. Although there is evidence that HS increases activity of lipoprotein lipase (LPL) in adipose tissue of heat stressed pigs, the fundamental causes of the increased adiposity are still unknown. It remains unclear whether HS directly alters metabolism in adipocytes. Therefore, to understand the mechanism of HS effects on porcine adipocytes, we used an in vitro adipocyte differentiation model to characterize cellular responses that occur during differentiation of pig adipocytes. Preadipocytes (stromovascular cells) were differentiated for 9 d at a normal (37°C) or HS (41.5°C) temperature under 5% CO. Expressions of HS genes such as heat shock proteins (HSP; HSP27, HSP60, HSP70, and HSP90), adipogenic markers peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), fatty acid synthase (FAS), adipocyte protein 2 (aP2), fatty acid translocase 36 (CD36), fatty acid transport protein 4 (FATP4), fatty acid transport protein 6 (FATP6), LPL, glucose transporter protein type 4 (GLUT4), phosphoenolpyruvate carboxykinase 1 (PCK1 or PEPCK-C), and glycerol kinase (GK) and adipokines (adiponectin and leptin) were determined by real-time-PCR and immunoblotting or ELISA. Cellular triglyceride (TAG) and ATP concentrations were also determined. As expected, HS increased ( < 0.05) the expressions of HSP genes. There was no HS treatment effect on the level of PPARγ, although C/EBPα was induced ( < 0.05) in HS. So it remains unclear whether HS affects adipocyte differentiation. However, HS leads to increased expressions of genes involved in fatty acid uptake and TAG synthesis (FAS, aP2, CD36, FATP4, FATP6, LPL, GLUT4, PCK1, and GK). This is supported by increased cellular TAG under HS. Therefore, HS promotes increased adipocyte TAG storage, perhaps through upregulation of genes involved in fatty acid uptake and TAG synthesis.

Chemical treatment may improve the nutritional value of corn crop residues, commonly referred to as corn stover, and the potential use of this feed resource for ruminants, including lactating dairy cows. The objective of this study was to determine the effect of prestorage chopping, hydration, and treatment of corn stover with Ca(OH)2 on the feeding value for milk production, milk composition, and dry matter intake (DMI). Multiparous mid-lactation Holstein cows (n = 30) were stratified by parity and milk production and randomly assigned to 1 of 3 diets. Corn stover was chopped, hydrated, and treated with 6% Ca(OH)2 (as-fed basis) and stored in horizontal silo bags. Cows received a control (CON) total mixed ration (TMR) or a TMR in which a mixture of treated corn stover and distillers grains replaced either alfalfa haylage (AHsub) or alfalfa haylage and an additional portion of corn silage (AH+CSsub). Treated corn stover was fed in a TMR at 0, 15, and 30% of the diet DM for the CON, AHsub, and AH+CSsub diets, respectively. Cows were individually fed in tiestalls for 10 wk. Milk production was not altered by treatment. Compared with the CON diet, DMI was reduced when the AHsub diet was fed and tended to be reduced when cows were fed the AH+CSsub diet (25.9, 22.7, and 23.1 ± 0.88 kg/d for CON, AHsub, and AH+CSsub diets, respectively). Energy-corrected milk production per unit of DMI (kg/kg) tended to increase with treated corn stover feeding. Milk composition, energy-corrected milk production, and energy-corrected milk per unit of DMI (kg/kg) were not different among treatments for the 10-wk feeding period. Cows fed the AHsub and AH+CSsub diets had consistent DMI over the 10-wk treatment period, whereas DMI for cows fed the CON diet increased slightly over time. Milk production was not affected by the duration of feeding. These data indicate that corn stover processing, prestorage hydration, and treatment with calcium hydroxide can serve as an alternative to traditional haycrop and corn silage in diets fed to mid-lactation dairy cows.

The greatest risk of metabolic and infectious disease in dairy cows is during the transition from pregnancy to lactating (i.e., the transition period). The objective of this experiment was to determine the effects of extracellular vesicles (microvesicles involved in cell-to-cell signaling) isolated from transition cows on target cell function. We previously identified differences in the protein profiles of exosomes isolated from cows divergent in metabolic health status. Therefore, we hypothesized that these exosomes would affect target tissues differently. To investigate this, 2 groups of cows (n = 5/group) were selected based on the concentration of β-hydroxybutyrate and fatty acids in plasma and triacylglycerol concentration in liver at wk 1 and 2 postcalving. Cows with high concentrations of β-hydroxybutyrate, fatty acids, and triacylglycerol were considered at increased risk of clinical disease during the transition period (high-risk group; n = 5) and were compared with cows that had low concentrations of the selected health indicators (low-risk group; n = 5). At 2 time points during the transition period (postcalving at wk 1 and 4), blood was sampled and plasma exosomes were isolated from the high-risk and low-risk cows. The exosomes were applied at concentrations of 10 and 1 µg/mL to 5 × 103 Madin-Darby bovine kidney cells grown to 50% confluence in 96-well plates. Results indicate a numerical increase in cell proliferation when exosomes from high-risk cows were applied compared with those from low-risk cows. Consistent with an effect on cell proliferation, quantitative reverse transcriptase PCR indicated a trend for upregulation of 3 proinflammatory genes (granulocyte colony-stimulating factor, ciliary neurotrophic factor, and CD27 ligand) with the application of high-risk exosomes, which are involved in cellular growth and survival. Proteomic analysis indicated 2 proteins in the low-risk group that were not identified in the high-risk group (endoplasmin and catalase), which may also be indicative of the metabolic state of origin. It is likely that the metabolic state of the transition cow affects cellular function through exosomal messaging; however, more in-depth research into cross-talk between exosomes and target cells is required to determine whether exosomes influence Madin-Darby bovine kidney cells in this manner.

BACKGROUND: Many pregnancies in the United States are associated with maternal calorie overconsumption. Few data exist that track the impact of maternal and offspring calorie consumption on the risks for obesity development. METHODS: To determine the effects of maternal calorie intake during gestation on programming for adiposity in the offspring, pregnant gilts were fed either a normal (NE) or high (HE) energy diet to induce higher than normal (30 % increase) pregnancy weight gain and the profile of genes related to adipose tissue development was determined in the subcutaneous adipose tissue of the offspring. Gilts were fed the same lactation diet after farrowing and piglets were allowed to suckle from their mothers. Offspring were also fed either a normal energy (NE) or a high energy (HE) diet after weaning (3 weeks of age). Offspring were sacrificed at 48 h, 3 weeks and 3 months of age and the subcutaneous adipose tissue obtained for gene expression analysis by RT-PCR. RESULTS: Gilts on the HE diet had higher pregnancy weight gain and backfat thickness than those on the NE diet. Expression of adipogenic genes, such as peroxisome proliferator activated receptor (PPAR) γ and CCAAT enhancer binding protein (CEBP) α was not different between offspring from NE and HE mothers at 48 h after birth, but they were higher (P

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.

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.

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.

Breast cancer is the second most common cancer among women in the US. Genetics, environment, as well as dietary factors are thought to have significant impacts on breast cancer risk. Epidemiological evidence supports a role for vitamin D in protection against breast cancer. 1,25 dihydroxyvitamin D (1,25(OH)2D), the active form of vitamin D, is proposed to regulate cellular processes that are involved in breast cancer progression. Metabolic reprogramming of glucose, termed the Warburg effect, as well as increased glutamine uptake and metabolism for energy use is a characteristic of many cancer cells. The current studies were designed to investigate the effect of 1,25(OH)2D on glutamine metabolism in mammary cells during an early stage of cancer progression. We employed untransformed (MCF10A) and ras oncogene transformed MCF10A human breast epithelial cells (MCF10A-ras). Intracellular glutamine and glutamate levels, determined by nuclear magnetic resonance, were both reduced with 1,25(OH)2D by 23% in MCF10A-ras cells. However, 1,25(OH)2D decreased intracellular glutamine level in MCF10A cells by only 9%, with no effect on intracellular glutamate level in MCF10A cells. Glutamine and glutamate flux into the TCA cycle were determined using [U-13C5] L-glutamine and gas chromatography-mass spectrometry. Treatment with 1,25(OH)2D decreased glutamine flux into the TCA cycle by 18% and 31% in MCF10A and MCF10A-ras cells, respectively, and decreased glutamate flux into the TCA cycle by 13% and 17% in MCF10A and MCF10A-ras cells, respectively. The mRNA and protein expression of the major glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was significantly decreased by 1,25(OH)2D in MCF10A-ras cells. Consistent with these results, glutamine uptake was reduced by 1,25(OH)2D, and the SLC1A5 inhibitor L-γ-Glutamyl-p-nitroanilide (GPNA) inhibited the effect of 1,25(OH)2D on glutamine uptake in both MCF10A and MCF10A-ras cells. The mRNA level of the glutamine transporter, SLC1A5, was also decreased by 1,25(OH)2D in MCF10A which overexpress ErbB2 (HER-2/neu) and in malignant MCF10CA1a cells. These results suggest 1,25(OH)2D may alter glutamine metabolism in MCF10A-ras cells by inhibiting glutamine uptake and utilization, in part through down-regulation of SLC1A5 transcript abundance. The 1,25(OH)2D down-regulation of the glutamine transporter, SLC1A5, may play a role in vitamin D prevention of breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Xuanzhu Zhou, Wei Zheng, Fariba Tayyari, G.A.Nagana Gowda, Daniel Raftery, Shawn S. Donkin, Brian Bequette, Dorothy Teegarden. 1,25-Dihydroxyvitamin D regulation of glutamine metabolism in Harvey-ras transformed MCF10A human breast epithelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1200. doi:10.1158/1538-7445.AM2015-1200