Donald R. Trout

Nutritional Stimulation of Milk Protein Yield of Cows Is Associated with Changes in Phosphorylation of Mammary Eukaryotic Initiation Factor 2 and Ribosomal S6 Kinase 1

Production of protein by the lactating mammary gland is stimulated by intake of dietary energy and protein. Mass-action effects of essential amino acids (EAA) cannot explain all of the nutritional response. Protein synthesis in tissues of growing animals is regulated by nutrients through the mammalian target of rapamycin (mTOR) and integrated stress response (ISR) networks. To explore if nutrients signal through the mTOR and ISR networks in the mammary gland in vivo, lactating cows were feed-deprived for 22 h and then infused i.v. for 9 h with EAA+ glucose (Glc), Glc only, l-Met+l-Lys, l-His, or l-Leu. Milk protein yield was increased 33 and 27% by EAA+Glc and Glc infusions, respectively. Infusions of Met+Lys and His generated 35 and 41%, respectively, of the EAA+Glc response. Infusion of EAA+Glc reduced phosphorylation of the ISR target, eukaryotic initiation factor(eIF) 2, in mammary tissue and increased phosphorylation of the mTOR targets, ribosomal S6 kinase 1 (S6K1) and S6. Both responses are stimulatory to protein synthesis. Glucose did not significantly increase mammary S6K1 phosphorylation but reduced eIF2 phosphorylation by 62%, which implicates the ISR network in the stimulation of milk protein yield. In contrast, the EAA infusions increased (P < 0.05) or tended to increase (P < 0.1) mammary mTOR activity and only His, like Glc, decreased eIF2 phosphorylation by 62%. Despite activation of these protein synthesis signals to between 83 and 127% of the EAA+Glc response, EAA infusions produced less than one-half of the milk protein yield response generated by EAA+Glc, indicating that ISR and mTOR networks exert only a portion of the control over protein yield.

Effect of Supplemental Chromium on Whole-Body Kinetics of Glucose, Lactate, and Propionate in Rams Fed a High Grain Diet

The isotope dilution technique of [6-3H]glucose, [U-14C]lactate and [l-14C]propionate was used to evaluate the effect of dietary chromium (Cr) supplementation on whole-body kinetics of glucose, lactate, and propionate in rams. Rams were fed a high grain diet at 2% of body weight with or without 0.5 ppm of supplemental Cr from chelated Cr for the initial 14 days, and then intake was increased to 2.5% at body weight for the last 9 days. Weight gain was enhanced (P < 0.01) with Cr supplementation. Plasma concentrations of glucose, lactate, and propionate were not influenced by Cr supplementation. Turnover rates of glucose and lactate, and their interconversion were also not influenced. Propionate turnover rate tended to increase (P = 0.11) and the conversion of propionate to glucose increased (P < 0.05) with Cr supplementation, leading the increased proportional contribution of propionate to glucose turnover rate (P < 0.05). Chromium supplementation may influence the contribution of each glucogenic substrate for glucose production in rams fed a high grain diet.

Mammary blood flow and metabolic activity are linked by a feedback mechanism involving nitric oxide synthesis

To test which, if any, of the major milk precursors can elicit a rapid change in the rate of mammary blood flow (MBF) and to define the time course and magnitude of such changes, 4 lactating cows were infused with glucose, amino acids, or triacylglycerol into the external iliac artery feeding one udder half while iliac plasma flow (IPF) was monitored continuously by dye dilution. Adenosine and saline were infused as positive and negative controls, respectively, and insulin was infused to characterize the response to a centrally produced anabolic hormone. To test the roles of cyclooxygenase, NO synthase and ATP-sensitive K (KATP) channels in nutrient-mediated changes in blood flow, their respective inhibitors-indomethacin, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), and glibenclamide-were infused simultaneously with glucose. Each day, 1 infusate was given twice to each cow, over a 20-min period each time, separated by a 20-min washout period. In addition, each treatment protocol was administered on 2 separate days. A 73% increase in IPF during adenosine infusion showed that the mammary vasodilatory response was quadratic in time, with most changes occurring in the first 5min. Glucose infusion decreased IPF by 9% in a quadratic manner, most rapidly in the first 5min, indicating that a feedback mechanism of local blood flow control, likely through adenosine release, was operative in the mammary vasculature. Amino acid infusion increased IPF 9% in a linear manner, suggesting that mammary ATP utilization was stimulated more than ATP production. This could reflect a stimulation of protein synthesis. Triacylglycerol only tended to decrease IPF and insulin did not affect IPF. A lack of IPF response to glibenclamide indicates that KATP channels are not involved in MBF regulation. Indomethacin and L-NAME both depressed IPF. In the presence of indomethacin, glucose infusion caused a quadratic 9% increase in IPF. Indomethacin is an inhibitor of mitochondrial function, so the glucose-induced increase in IPF was interpreted as feedback on mammary adenosine release from an anabolic response to glucose. Because NO synthase was not inhibited during indomethacin infusion, the feedback system is postulated to act through endothelial NO synthase. In the presence of L-NAME, glucose infusion had no effect on IPF, indicating that endothelial cyclooxygenase is not involved in glucose-induced changes in MBF.

Xenogeneic (bovine) peripheral blood leukocytes engrafted into severe combined immunodeficient mice retain primary immune function

The immune responsiveness of xenogeneic PBL engrafted into SCID mice was investigated using the bovine PBL-reconstituted SCID mouse model system (PBL-SCID-bo). Bovine PBL-reconstitution and B-cell activity were monitored by bovine serum Ig production. Bovine T-cell function was demonstrated by an antigen-specific immune response to bovine transplantation antigens provided by bovine skin allografts. Bovine allograft rejection was clearly evident in > 65% PBL-SCID-bo that received a bovine PBL inoculum either 30 days after bovine skin grafting, or 7-52 days before bovine skin grafting. Bovine allograft rejection was confirmed via histological examination and was characterized primarily by a band of infiltrating bovine lymphocytes at the periphery of the graft and tissue necrosis. A secondary immune response could be elicited if bovine cells in the PBL inoculum were presensitized to Ag from the bovine skin allograft donor. This study is the first to show that bovine cells engrafted in SCID mice after i.p. injection of bovine PBL retain some aspects of immune competency. These results confirm the value of the xenogeneic PBL-reconstituted SCID mouse model in the study of primary immunity.

Inhibition of local blood flow control systems in the mammary glands of lactating cows affects uptakes of energy metabolites from blood

To test the effect of mammary blood flow on net uptakes of milk precursors by the mammary glands, inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX) were infused into the mammary circulation of 4 lactating cows. Inhibitors were infused in a 4×4 Latin square design, where treatments were infusion for 1 h of saline, NOS inhibitor (Nω-nitro-l-arginine methyl ester hydrochloride), COX inhibitor (indomethacin), or both NOS + COX inhibitors into one external iliac artery. Para-aminohippuric acid was also infused to allow for estimation of iliac plasma flow (IPF), of which approximately 80% flows to the mammary glands. Blood samples were collected before, during, and after inhibitor infusion from the contralateral external iliac artery and ipsilateral mammary vein. Inhibition of COX and NOS each produced a decrease in IPF, although the NOS effect was smaller and IPF continued to be depressed throughout the recovery period. The combination of COX and NOS inhibition produced a 50% depression in IPF and there was no carryover into the recovery period. Treatments that depressed IPF also increased arterial concentrations of acetate, β-hydroxybutyrate (BHBA), and glucose. Similarly, arteriovenous differences of acetate, BHBA, and glucose were all increased during IPF depression. To correct for a potential effect of arterial concentration, arteriovenous differences were normalized to arterial concentration, producing an extraction percentage. Inhibition of COX increased glucose extraction and tended to increase acetate and BHBA extraction. Dual inhibition only increased BHBA extraction and had no effect on mammary extraction of other metabolites. These extractions did not increase because clearances of glucose and TAG decreased as IPF decreased, and clearances of acetate and BHBA tended to decrease. Net uptake of TAG was depressed by dual NOS/COX inhibition, whereas uptakes of acetate, BHBA, and glucose were not affected by any of the treatments. To separate effects of flow from effects of arterial concentration, uptakes were regressed against IPF and arterial concentration simultaneously. According to the slopes of the regressions, a 10% decrease in IPF from the mean observed during saline infusion resulted in 3.8, 7.3, and 10.4% decreases in uptakes of acetate, glucose, and triacylglycerol, respectively. These findings indicate that mammary blood flow affects milk precursor uptake, and that clearance should not be assumed constant to predict mammary uptakes of milk precursors in situations where blood flow is changing.

The effect of short-term hyperammonaemia on milk synthesis in dairy cows.

To test the hypothesis that ammonia detoxification in ruminants consumes amino acids to the detriment of milk protein production, we infused four lactating dairy cows with ammonium acetate or sodium acetate in switchback experiments. Plasma ammonia concentrations increased to 411 microm within 1 h of the start of infusion of ammonium acetate at 567 mmol/h. The rate constant for ammonia clearance from plasma was 0 x 054/min and the half-life was 12 x 9 min. Infusion at 567 mmol/h for 1 h followed by 1 h without infusion, repeated four times between am- and pm-milking, caused a decrease in feed intake. Compared with sodium acetate, continuous infusion of ammonium acetate at 360 mmol/h throughout an entire 10-h milking interval increased plasma ammonia concentrations to 193 microm and caused a 20% decrease in milk, protein and lactose production with no effect on percentage composition of milk or the yield of milk fat. Arterial concentrations of glucose and non-esterified fatty acids tended to increase; there was no effect on arterial acetate, beta-hydroxybutyrate or triacylglcerol, and branched-chain amino acids, Lys and Thr decreased. Mammary plasma flow, estimated by assuming 100% uptake/output of Phe+Tyr, was significantly correlated with milk yield. Mammary uptakes of acetate tended to be reduced by hyperammonaemia, but uptakes of other energy metabolites and amino acids were not affected. Thus, while an increase in amino acid consumption during hyperammonaemia was apparent from the drop in circulating concentrations of Leu, Ile, Val, Lys and Thr, there was no evidence to support the hypothesis that milk yield is affected by the lower concentrations. An ammonia-induced depression in feed intake may have caused the decrease in milk synthesis.

The Histamine H1 Receptor Is Not Involved in Local Control of Mammary Blood Flow in Dairy Cows

Low concentrations of the essential amino acid histidine in circulation have been shown to increase mammary blood flow and it has been suggested that this effect is mediated by histamine. The hypotheses tested in this experiment were that interstitial histamine concentrations in the mammary gland are related to arterial His concentrations and that mammary blood flow is reduced by extracellular histamine via H(1) receptors. The hypotheses were tested by infusing saline or chlorpheniramine, a blocker of the H(1) histamine receptor, into the arterial supply of the mammary glands of lactating cows infused with 44 g/h of amino acid mixtures with or without His for 10 h. Infusates were administered in a 2 x 2 factorial arrangement within a 4 x 4 Latin square to 4 multiparous Holstein cows in mid lactation. Exclusion of His from the infusate decreased protein content in milk from the infused udder half from 3.98 to 3.77%, and increased arterial alpha-aminonitrogen concentration from 3.2 to 3.4 mM. Neither the decreased arterial His concentration nor the H(1) blocker affected plasma flow to the infused udder half. We conclude that histamine is not involved in the regulation of mammary blood flow. The H(1) blocker decreased milk production in the infused udder half from 4.6 to 3.5 kg without affecting protein, fat, and lactose percentages, suggesting an inhibition of milk ejection. Cows on chlorpheniramine ate less feed during the infusion than saline-infused cows, which resulted in lower arterial concentrations and mammary uptakes of acetate. The efficiency of plasma triacylglycerol uptake across the mammary glands was decreased by chlorpheniramine but net uptake of long-chain fatty acids was not affected. The mechanism by which an amino acid deficiency influences mammary blood flow does not involve histamine signaling through the H(1) receptor and remains unidentified.