Effects of serine palmitoyltransferase inhibition by myriocin in ad libitum-fed and nutrient-restricted ewes.

The fungal isolate myriocin inhibits serine palmitoyltransferase and de novo ceramide synthesis in rodents; however, the effects of myriocin on ceramide concentrations and metabolism have not been previously investigated in ruminants. In our study, 12 non-lactating crossbred ewes received an intravenous bolus of myriocin (0, 0.1, 0.3, or 1.0 mg/kg/body weight [BW]; CON, LOW, MOD, or HIGH) every 48 h for 17 d. Ewes consumed a high-energy diet from day 1 to 14 and were nutrient-restricted (straw only) from day 15 to 17. Blood was collected preprandial and at 1, 6, and 12 h relative to bolus and nutrient restriction. Tissues were collected following euthanasia on day 17. Plasma was analyzed for free fatty acids (FFAs), glucose, and insulin. Plasma and tissue ceramides were quantified using mass spectrometry. HIGH selectively decreased metabolizable energy intake, BW, and plasma insulin, and increased plasma FFA (Dose, P < 0.05). Myriocin linearly decreased plasma very-long-chain (VLC) ceramide and dihydroceramide (DHCer) by day 13 (Linear, P < 0.05). During nutrient restriction, fold-change in FFA was lower with increasing dose (P < 0.05). Nutrient restriction increased plasma C16:0-Cer, an effect suppressed by MOD and HIGH (Dose × Time, P < 0.05). Myriocin linearly decreased most ceramide and DHCer species in the liver and omental and mesenteric adipose, VLC ceramide and DHCer in the pancreas, and C18:0-Cer in skeletal muscle and subcutaneous adipose tissue (Linear, P ≤ 0.05). We conclude that the intravenous delivery of 0.3 mg of myriocin/kg of BW/48 h decreases circulating and tissue ceramide without modifying energy intake in ruminants.

Intravenous trimethylamine N-oxide infusion does not modify circulating markers of liver health, glucose tolerance, and milk production in early-lactation cows.

In rodents and humans, the gut bacteria-derived metabolite trimethylamine N-oxide (TMAO) has been implicated in the progression of cardiovascular disease, chronic kidney disease, fatty liver, and insulin resistance; however, the effects of TMAO on dairy cattle health and milk production have not been defined. We aimed to determine whether intravenous TMAO infusion modifies measures of liver health, glucose tolerance, and milk production in early-lactation cows. Eight early-lactation Holstein cows (30.4 ± 6.41 d in milk; 2.88 ± 0.83 lactations) were enrolled in a study with a replicated 4 × 4 Latin square design. Cows were intravenously infused TMAO at 0 (control), 20, 40, or 60 g/d for 6 d. Washout periods lasted 9 d. Intravenous glucose tolerance tests (GTT) occurred on d 5. Blood was collected daily. Milk was collected on d -1, 0, 5, and 6. Urine was collected on d -1 and 6. Circulating metabolites, milk components, and TMAO concentrations in milk, urine, and plasma were quantified. Data were analyzed using a mixed model that included the fixed effects of treatment. Concentrations of TMAO in plasma, milk, and urine increased linearly with increasing dose. Dry matter intake and milk production were not modified by treatment. Daily plasma triacylglycerol, fatty acid (FA), and glucose concentrations were not modified. Serum albumin, total protein, globulin, total bilirubin, direct bilirubin, aspartate aminotransferase, γ-glutamyl transferase, and glutamate dehydrogenase concentrations were also not modified by treatment. Serum GTT glucose, FA, and insulin concentrations were not modified by treatment. Plasma total, reduced, and oxidized glutathione concentrations were also not modified by treatment. We conclude that a 6-d intravenous infusion of TMAO does not influence measures of liver health, glucose tolerance, or milk production in early-lactation dairy cows.

Impact of Local Electrostatics on the Redox Properties of Tryptophan Radicals in Azurin: Implications for Redox-Active Tryptophans in Proton-Coupled Electron Transfer.

Tyrosine and tryptophan play critical roles in facilitating proton-coupled electron transfer (PCET) processes essential to life. The local protein environment is anticipated to modulate the thermodynamics of amino acid radicals to achieve controlled, unidirectional PCET. Herein, square-wave voltammetry was employed to investigate the electrostatic effects on the redox properties of tryptophan in two variants of the protein azurin. Each variant contains a single redox-active tryptophan, W48 or W108, in a unique and buried protein environment. These tryptophan residues exhibit reversible square-wave voltammograms. A Pourbaix plot, representing the reduction potentials versus pH, is presented for the non-H-bonded W48, which has potentials comparable to those of tryptophan in solution. The reduction potentials of W108 are seen to be increased by more than 100 mV across the same pH range. Molecular dynamics shows that, despite its buried indole ring, the N-H of W108 hydrogen bonds with a water cluster, while W48 is completely excluded from interactions with water or polar groups. These redox properties provide insight into the role of the protein in tuning the reactivity of tryptophan radicals, a requirement for controlled biological PCET.

Nutrient Restriction Increases Circulating and Hepatic Ceramide in Dairy Cows Displaying Impaired Insulin Tolerance.

The progression of insulin resistance in dairy cows represents a maternal adaptation to support milk production during heightened energy demand; however, excessive adipose tissue lipolysis can develop. In diabetic non-ruminants, the mechanisms that mediate insulin resistance involve the sphingolipid ceramide. We tested the hypothesis that ceramide accumulates in dairy cows experiencing lipolysis and insulin resistance. Nine dairy cows were utilized in a replicated 3 × 3 Latin square design. Cows were ad libitum fed, nutrient-restricted (NR), or NR with nicotinic acid (NA; 5 mg of NA/h per kg BW; delivered i.v.) for 34 h. When provided access, cows were ad libitum fed a mixed ration of grass hay and ground corn to meet requirements. Intake for NR cows was limited to vitamins and minerals. Nicotinic acid was administered to suppress lipolysis. Saline was infused in cows not provided NA. At 32 and 33 h of treatment, a liver biopsy and insulin tolerance test were performed, respectively. Samples were analyzed using colorimetry, immunoassay, and mass spectrometry. Nutrient restriction increased serum fatty acids and ceramide levels, and impaired insulin sensitivity; however, NA infusion was unable to prevent these responses. We also show that NR increases hepatic ceramide accumulation, a response that was positively associated with serum ceramide supply. Our data demonstrate that circulating and hepatic 24:0-Cer are inversely associated with systemic insulin tolerance, an effect not observed for the 16:0 moiety. In conclusion, our results suggest that ceramide accrual represents a metabolic adaptation to nutrient restriction and impaired insulin action in dairy cows.