A greater carnitine availability due to dietary carnitine supplementation was associated with altered hepatic metabolite profiles of the cows, including an increased hepatic concentration of short- and long-chain acyl-carnitines and their derivatives. The concentrations of all these acyl-carnitines (C2, C3, C4, C5, C14, C16, and C18) were 3 to sixfold higher in the CAR group than in the CON group, confirming our hypothesis that these intermediary products would accumulate in the liver in relation to enhanced carnitine availability. In the same trial, cows treated with carnitine had significantly higher expression of hepatic very long-chain acyl-CoA dehydrogenase mRNA (P < 0.01) , suggesting an upregulated utilization of fatty acids with 14–18 carbons in the first step of beta-oxidation. Further, our metabolomics assay revealed the perturbations of hepatic acyl-carnitine, biogenic amine, and amino acid concentrations in association with a LPS challenge in mid-lactation cows. Although previous studies have used metabolomics to study hepatic metabolite profiles of dairy cows [22, 23], the current study was the first to report the alterations of hepatic metabolite profiles associated with supplementary carnitine and LPS injection. Many aspects of the metabolic disturbances that commonly occur in cows during the transition from gestation to lactation are tracked back to an unresolved and often subclinical inflammation, which can involve disturbed mitochondrial function [8, 9]. The metabolic stress of early lactation cows often derives from Gram-negative bacterial mastitis when the blood-milk barrier is disrupted by inflammatory mediators, or from the gastrointestinal bacterial load when the epithelial barrier is disrupted due to subacute ruminal acidosis or “leaky gut” induced by heat stress or other challenges . In addition to these sources of LPS load, the increased circulating concentration of saturated fatty acids, common in early lactation cows, can also activate Toll-like receptor 4 (also known as the LPS receptor), initiating nuclear factor kappa B inflammatory signaling . We chose to use mid-lactation cows in our current study to avoid the common metabolic disturbances of cows directly after calving and during early lactation, so that we can attribute the observed metabolic alterations to the LPS challenge with higher confidence.
Our findings of increased hepatic carnitine concentration were consistent with our previous report where plasma and milk carnitine concentrations of the same cows increased after carnitine supplementation . The uptake of carnitine from the gastrointestinal tract can first increase the carnitine concentration in the hepatic tissues, followed by blood and subsequently milk concentration changes. Supplementary carnitine was shown to enhance fatty acid oxidation in the liver, and decrease BHB and glucose output as well as triglyceride accumulation in the liver of cows . Acyl-carnitines are metabolic byproducts of mitochondrial fatty acid, glucose, and amino acid oxidation ; hence they were characterized as biomarkers of mitochondrial functionality in humans . Moreover, circulating concentrations of acyl-carnitines were also linked to insulin resistance in humans  and mitochondrial haplotypes of greater oxidative capacity in cows . The liver is discussed to be the main source of circulating acyl-carnitines, and, commonly, studies reporting circulating concentrations interpret their findings as an indication of altered hepatic mitochondrial function . The mitochondrial carnitine shuttle system transports activated long-chain fatty acids from the cytosol inside the mitochondria for subsequent beta-oxidation . The liver can also re-partition the excess of acyl-carnitines to other peripheral tissues, such as to the mammary gland, supporting effective oxidative energy production . In our previous study, cows with improved health status had a higher concentration of carnitine and several acyl-carnitines, such as valeryl-carnitine and hexadecadienyl-carnitine, compared with cows of shorter productive life span . The greater availability of hepatic acyl-carnitines can reduce oxidative stress by avoiding the accumulation of reactive oxygen species. The synthesis of acyl-carnitines prevents CoA trapping, which causes the damage of hepatic cells by the accumulation of fatty acid degradation intermediates, therefore, acyl-carnitine efflux may serve as a detoxification process to protect hepatic mitochondria . As a rate-limiting step, additional carnitine was hypothesized to enhance the transport capacity of long-chain fatty acids into the mitochondria. However, not all transported fatty acids can be fully oxidized in the mitochondria if the transport capacity exceeds oxidation capacity. We found greater hepatic C16 and C18 acyl-carnitine concentrations compared with lower chain lengths such as C14, which can indicate that palmitic acid (16 carbons) and stearic acid (18 carbons) were preferentially transported into the mitochondria by carnitine. This was in line with previous studies showing that the majority of palmitate was esterified in cows treated with supplementary carnitine . The enhanced beta-oxidation of palmitic acid can increase the concentration of acetyl-CoA in mitochondria, stimulating the flux of lactate and alanine through pyruvate carboxylase rather than pyruvate dehydrogenase . The final products of beta-oxidation are acetyl groups that are used in the TCA cycle for energy production. The concentration of acetyl-carnitine (C2) in the CAR group was 4 times higher than its concentration in the CON group, indicating a maximum load of TCA cycle in the mitochondria in the CAR group. Consequently, the concentration of intermediate metabolites related to the rate-limiting steps in the TCA cycle was possibly higher in the CAR group than the CON group too, however, these metabolites were not covered in our metabolomics analysis. Our metabolomics data show that the systemic effect of supplementary carnitine extends to mitochondrial oxidative capacity, even if this could not be reflected by circulating concentrations of conventional biomarkers, such as FFAs, BHB, and TG . In our mid-lactation cows, the concluding results were that the serum concentrations of FFAs, BHB, and TG 24 h after LPS injection were not altered by dietary carnitine supplementation. However, 48–72 h after LPS injection, the concentrations of both serum FFAs and BHB were different between CON and CAR groups . Even if continuous carnitine supplementation does not alter the concentration of these three serum metabolites (FFAs, BHB, and TG) during mid-lactation, their concentrations seem to be affected by the association between lipid metabolism and inflammatory response. Although the stressed condition of hepatic lipid metabolism of cows in early lactation is typically diminished in mid-lactation, the inflammatory response caused by the LPS challenge could still affect the general metabolism in the liver and peripheral tissues, which explained the significant effect of LPS on serum FFAs, BHB, and TG, and hepatic TG .
Compared with the significant effect of the carnitine supplementation on acyl-carnitines, its effect on biogenic amines was limited. In fact, only dopamine was affected by the carnitine supplementation, having a decreased concentration after the LPS challenge. Dopamine is primarily synthesized from phenylalanine and tyrosine ; however, the concentration of phenylalanine and tyrosine were not affected in our study. Higher dopamine concentrations can reduce prolactin levels, and thereby reduce milk production . A decrease in hepatic dopamine concentration in the carnitine supplemented cows herein is proposed to be a potentially beneficial mechanism to support milk production .
The purpose of the intravenously administered LPS was to provoke an inflammatory response, which is a widely used technique to mimic the clinical symptoms caused by an increased LPS load derived from Gram-negative bacteria [31, 32]. The short-term effects of the LPS injection (2.5 to 12 h) on hepatic inflammation and lipid metabolism-related mRNA expressions, such as tumor necrosis factor-alpha and acetyl-CoA carboxylase, were reported previously [14, 33]. On a molecular signaling level, a LPS load above a certain threshold activates inflammatory signaling through a positive feedback loop of nuclear factor kappa B amplification activated via pattern recognition receptors such as Toll-like receptor 4 expressed on Kupffer cells in the liver . In addition, there are also other converging signaling pathways, such as c-Jun NH2-terminal kinase and cluster of differentiation 14 mediated signaling, that trigger the release of inflammatory cytokines such as tumor necrosis factor-alpha and interleukin 1 beta from the Kupffer cells as well as the production of acute-phase proteins in the hepatocytes . There are numerous ways in which inflammatory signals can interact with metabolic, translational, and cell cycle regulatory mechanisms , which includes potential interactions with liver homeostasis as reviewed by Robinson et al. . Further, we characterized the changes of plasma and milk metabolite profiles 72 h after the LPS injection, and found that the LPS challenge altered plasma and milk short-chain acyl-carnitine concentrations as well as plasma sarcosine, glutamine, and isoleucine concentrations . Our previous studies used serum metabolite profiles to explore the metabolic pathways related to an enhanced productive life span  and dietary carnitine supplementation  in dairy cows. Both productive life span and carnitine treatment were suggested to be associated with hepatic lipid metabolism and mitochondrial function. At the onset of lactation, the liver plays a central role in the respective homeorhetic adaptations , and is also involved in important immune and endocrine functions [37, 38]. The metabolic response to the LPS challenge was different between the carnitine supplemented and the controls cows. In the CON group, acyl-carnitines (mainly C14, C16, C18, and their derivatives) dominated the list of most affected metabolites. Their decreased hepatic concentrations after the LPS challenge likely reflected an enhanced utilization of fatty acids shuttled into the mitochondria for oxidation, and also an increased release of acyl-carnitines from the liver into the circulation, as reflected by increased plasma and milk acyl-carnitine concentrations in these cows reported previously . The latter could reflect passive leak from hepatocytes as well as an active process aiming at the trafficking of acyl-carnitines as either substrates or signal molecules to peripheral tissues.
In contrast, the CAR group showed increased concentrations of C16 and C18 acyl-carnitines after the LPS challenge, indicating well-functioning mitochondria that are in excess of oxidative substrates. Further, the concentrations of biogenic amines (including putrescine and spermine) increased in the liver of carnitine supplemented cows after the LPS challenge, most likely representing an improved anti-oxidant and anti-inflammatory status.
Compared with the effect of the LPS challenge on plasma metabolite profile in our previous study , the effects on hepatic metabolite profiles were less prominent herein. The possible explanation is that the LPS challenge likely affected the metabolism of peripheral tissues, such as the mammary gland and skeletal muscle, in addition to the liver. The acute LPS challenge may also affect the interaction between the inflammatory response and oxidative stress response , however, reactive oxygen metabolites were not assessed in our analyses. According to our results, the significant effect of the LPS challenge and supplementary carnitine on the majority of acyl-carnitines indicate the potential association between inflammatory response and mitochondrial fatty acid metabolism. In previous studies, the greater extent of lipid mobilization and oxidation for energy production was associated with an increase in the inflammatory response in cows during early lactation . The LPS injection affected the hepatic mRNA expression of genes involved in fatty acid metabolism . In bovine endothelial cells, palmitic acid and stearic acid were shown to promote inflammation by enhancing the production of inflammatory cytokines and reactive oxygen species . The relationship between the inflammatory response triggered by the acute LPS load and the modulation of hepatic metabolic pathways might also be reflected by the observed perturbations in biogenic amine and amino acid metabolism.
Spermine, a polycationic aliphatic amine (polyamine), is synthesized by somatic cells and also by gut microbiota. Polyamines were suggested to increase longevity by decreasing low-grade inflammation in the gut and other organs . The capability of spermidine to modulate autophagy, a mechanism responsible for the regeneration of cellular components and organelles, is discussed to be the major underlying pathway for longevity and other beneficial effects of spermidine . Since polyamines can be also modulated by dietary factors directly or indirectly by modulating microbial populations and their polyamine production, these metabolites are not only proposed as candidates as biomarkers for metabolic balance but also as a target for nutraceutical approaches to improve anti-inflammation and to extend productive life span in dairy cows. As an essential amino acid, threonine could be limiting for optimal protein synthesis in dairy cows . The decrease in hepatic threonine concentrations after the LPS challenge can indicate that interventions supporting protein synthesis might be of interest in an attempt to attenuate inflammatory challenges in cows. This is also supported by the decrease in hepatic ornithine and acetyl-ornithine concentrations, which also point to enhanced proteolysis after LPS challenge, associated with increased urea production and increased metabolism of ornithine to avoid a toxic accumulation of ammonia . The LPS challenge also increased the concentration of conventional blood metabolites, including FFAs, BHB, and triglycerides. Our results were consistent with previous studies in which increased circulating concentrations of FFAs and BHB were observed in cows infused with LPS during mid-lactation , which can be explained by the enhanced adipose tissue lipolysis triggered by the inflammatory response .
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