3.1. The Effects of Clofibrate and Dietary Treatments (Succ, TC5, TC6 and TMPA) on Carnitine Status
Carnitine status has been studied extensively in numerous species including pigs [
6,
27,
28], rodents [
6,
29], humans [
10,
30], felids [
31] and canines [
32], and in different tissues such as liver, muscle, kidney, heart [
33,
34,
35], and plasma [
36,
37]. However, very limited information is available on carnitine status in intestine, especially in neonates. In this study, we measured carnitine and acyl-carnitine concentrations in intestine mucosa isolated from 5 d-old piglets receiving a milk replacer containing different MCTs and with or without supplementation of clofibrate, a PPARα agonist. The results showed that neither dietary MCTs nor supplementation of clofibrate had an impact on small intestinal carnitine status. The concentration of free carnitine and acyl-carnitine in intestinal mucosa of 5 d-old piglets were similar as those observed in 3-day old guinea pigs, but much higher than young guinea pigs and adult rats [
38]. Concentrations measured in intestinal mucosa were also higher than in plasma and liver from fetal pigs [
33], neonatal pigs [
27] and those in our study. Intestinal concentrations were also higher than levels measured in skeletal muscle [
39], and even higher than carnivore species (neonatal cats) [
31], but similar to concentrations in the liver and muscle of neonatal dogs [
32]. These results suggest that carnitine status varies from species to species and tissue to tissue.
Supplementation of clofibrate significantly increased plasma acyl-carnitine and hepatic free and acyl-carnitines (
Table 1 and
Table 2) although it had no impact on intestinal carnitine and acyl-carnitines. Similar results were observed previously in liver of clofibrate treated adult rats [
40], and in liver and plasma of clofibrate treated hens [
41]. The increase in liver but not in plasma was also observed in rats fed a diet with oxidized fat [
30]. The increase in hepatic carnitines was likely associated with an increase in carnitine synthesis [
42] by increased BBOX activity [
43]. BBOX activity also increases with age and predominates in carnitine synthesis in pigs by 7 days of age [
44]. These results suggest that the increase in carnitine in liver could be due to the increased enzyme activity the carnitine synthetic pathway. Although the activity of BBOX or the carnitine synthesis pathway was not determined in this study, we found that the expression of the gene encoding TMLHE, the first enzyme of the carnitine biosynthesis pathway, and ALDH9A1, the third enzyme in the carnitine synthesis pathway, both increased in liver of clofibrate treated pigs. An increase in ALDH9A1 expression was observed in previous studies with mice [
8,
30,
45] but not in rats [
42]. Although the effect of clofibrate on TMLHE and ALDH9A1 was not detected in rats, an increase in N-6-trimethyllysine, the carnitine metabolic precursor, was observed in previous studies with rats [
46,
47]. In addition to TMLHE and ALDH9A, we also found that the mRNA abundance of organic transporter-2 (OCTN2) increased in the liver of clofibrate treated pigs. This was consistent with the results observed previously in liver of mice [
17,
18,
45,
48,
49], rats [
50] and pigs [
51,
52], demonstrating that PPARα plays an important role in carnitine homeostasis by regulating OCTN2 expression. However, clofibrate administration had no impacts on the expression of BBOX in liver. A similar result was reported in rats [
42] but not in mice. The expression of BBOX was increased in clofibrate treated mice [
8,
30,
45]. These results indicate that the regulatory role and mechanism of PPARα activity in carnitine biosynthesis is likely both specie- and tissue- specific as well as impacted by physiological status such as age and development.
An association of carnitine status and regulation with development and dietary carnitine levels was observed in rats [
53]. Moreover, this observation was consistent with the development and activity of the CPT system, the enzymes that require carnitine as a substrate (cofactor) for fatty acid mitochondrial transfer and oxidation. Indeed, supplementation of carnitine in the diet of young pigs resulted in a dose-dependent increase in free carnitine, acetyl and total carnitine concentrations in plasma, liver, kidney, heart, and skeletal muscle [
34]. Results from a previous study in our laboratory also showed that supplementation of carnitine to sows during gestation increased the deposition of carnitine in liver, heart, and skeletal muscle of fetal piglets [
33,
39], and of carnitine in term fetuses in a similar study by Birkenfeld et al. [
37]. It was also observed that carnitine and acyl-carnitine concentration in small intestinal mucosa in rats increased with dietary carnitine supplementation, and the concentrations of free carnitine and acyl-carnitine decreased with age from d 1 to d 29 in intestinal mucosa of developing guinea pigs [
38]. Therefore, the carnitine status in intestinal mucosa in the neonate is associated with mother’s carnitine status and the carnitine concentration in the diet after birth. In our study, all pigs were from sows fed the same standard diet and pigs received the same milk replacer (the concentration of carnitine in the milk replacer was approximately 2 nmol/mg), and thus the concentration in mucosa could reflect the carnitine concentration in the milk replacer and carnitine deposited at birth and not due to the dietary supplementation of succinate and the MCFAs. Although it has been reported that dietary levels of fat, carbohydrate and protein could decrease the efficiency of carnitine absorption [
54,
55], the level of energy in all diets was isocaloric. In support of the observations in intestine, the concentrations of free carnitine and acyl-carnitine in plasma and liver measured in this study were not affected by the dietary energy source.
3.2. The Effects of Clofibrate and Dietary Treatments (Succ, TC5, TC6 and TMPA) on Fatty Acid Oxidation in Intestine
An interaction was detected between clofibrate and dietary treatment on palmitic acid oxidation in intestinal mucosa in this study. Administration of clofibrate increased the
14C accumulation in CO
2, ASP and total (CO
2 + ASP) in pigs receiving diets containing Succ, TC5 and TMPA, and the increases were consistent with the results previously observed in liver and kidney of neonatal pigs receiving dietary clofibrate [
19,
21,
56,
57,
58] and by oral gavage [
19,
21]. This result suggests that the response of fatty acid oxidation in intestinal mucosa to PPARα activation is similar to other tissues in neonatal pigs. In previous studies, the increased fatty acid oxidation induced by clofibrate in liver and kidney was accompanied with an increase in CPT I activity and expression [
19,
59]. Similar to previous studies, CPT I and II activities in the intestinal mucosa were increased but changes in gene expression of CPT I and II were not detected. The same results were observed also in liver and kidney tissues [
58]. A significant difference in gene expression of CPTI was observed between intestine and liver in rats during the pre and postnatal periods [
59]. The expression of CPT I in intestine increased quickly after birth and reached maximum levels at 3 days, which was maintained until 12 days, while the expression in liver increased after birth and also reached maximum levels at 3 days, but then decreased quickly [
59]. We examined gene expression at d 5 in this study, where the mRNA enrichment of CPT I in intestine of the piglets, like in the intestine of rat during the postnatal period, probably was at a peak value and could not be elevated any further. The difference in CPT I expression between liver and intestine was also observed in adult rats that received the PPARα agonist Wy-14643 via intraperitoneal injection [
60]. Whether the lack of response of CPT I to clofibrate in intestine in this study is due to a development stage or tissue difference is not known. However, fatty acid oxidation in mitochondria is directly related to the enzyme activity that can be regulated by changing (1) the expression of CPT I, (2) the concentration of malonyl-CoA via acetyl-CoA carboxylase (ACC) and MCD; [
23], and/or (3) the sensitivity of CPT I to malonyl-CoA inhibition [
24]. Thus, the increase in fatty acid oxidation in intestine may be due to the increase in activity of CPT I and possibly by a reduced sensitivity to malonyl-CoA inhibition and/or malonyl-CoA concentration in the clofibrate treated pigs.
In addition, the effect of clofibrate on fatty acid oxidation in intestinal mucosa appeared to be affected by the dietary energy source. We found that dietary supplementation of MCTs increased palmitic acid oxidation by either increasing
14C accumulation in CO
2 or ASP compared to diets containing Succ. This implies that dietary MCT may modify the rate of LCFA oxidation. The products of dietary TC5 from β-oxidation, acetyl-CoA and propionyl-CoA, are considered as ketogenic and anaplerotic carbon sources, respectively, [
21] for potentially increasing ketone body production and CAC capacity. Compared to TC5, TC6 can only generate acetyl-CoA, and TMPA can only produce propionyl-CoA via β-oxidation. Interestingly, dietary supplementation TC5 and TMPA not only increased the rate of LCFA oxidation but also increased the stimulation of clofibrate on fatty acid oxidation compared to the Succ diet. Supplementation with TC6 increased the rate of LCFA oxidation as well but attenuated clofibrate stimulation of fatty acid oxidation (especially
14C accumulation in CO
2) compared to TC5 and TMPA. This result suggests that propionyl-CoA from odd- or branch-chain MCFA, as an anaplerotic carbon source, stimulates CAC activity in intestinal tissue. Again, the effect of dietary MCTs on fatty acid oxidation with or without induction by clofibrate apparently was not associated with activity or gene expression of CPI I and II, as the dietary MCT treatments were without effect. This probably was due to the oxidation of MCFA being independent of CPT I system. Compared with CPT I, the gene expression of FABP was modified by dietary MCT, especially dietary TC5. This is similar to the results reported by Poirier et al. [
61], in which a weak effect of MCFA on FABP expression was observed in the jejunum of rats. The extent of the effect might be associated with an affinity of FABP for LCFA and the assimilation of lipid in the intestine [
62]. FABP as a LCFA transporter targets fatty acid to β-oxidation in liver. Significant direct interactions between FABP and PPARα in liver were reported [
63,
64]. As in liver, MCFA may promote intestinal LCFA to catabolic and anabolic pathways by modifying FABP gene expression.
Furthermore, we noticed that dietary supplementation of TC5, TC6 and TMAP had different effects on palmitic acid oxidation in clofibrate treated and untreated pigs, and the effects were not associated with citrate synthase. The production of CO
2 from palmitic acid oxidation in untreated pigs receiving TC6 and TMPA was higher than in pigs receiving Succ and TC5, while the production of ASP was higher in untreated pigs receiving TC5 and TC6 than those receiving Succ and TMPA. Combining CO
2 and ASP together, we found that the pigs not given clofibrate but receiving TC6 had the highest palmitic acid oxidation rate. This probably was related to acetyl-CoA metabolic fate and CoA availability as we speculated in kidney, in which an increased palmitic acid oxidation was only observed in pigs fed a diet with TC6 [
21]. The extent of the effect might be associated with an affinity of FABP for MCFA and the structure of MCFA, because the effect of MCTs on fatty acid oxidation as the effect of even-chain MCFAs was greater than odd-chain and branch-chain MCFAs in this study. Compared to the dietary MCTs, specific transporters are needed for succinate to pass through the plasma or mitochondria membrane [
65,
66]. Metabolic studies showed that catabolism of ketone bodies is balanced by the ratio of succinate and succinyl-CoA [
67]. The ketone body catabolic pathway uses succinyl-CoA as CoA donor, while excess of succinate inhibits ketone body utilization [
67]. Therefore, the difference between dietary succinate and MCTs in CO
2 and/or ASP is probably due to the difference in their metabolic fate.
Interestingly, not only was the total fatty acid oxidation modified but also the distribution of 14C accumulation (%) between CO2 and ASP was altered by the dietary treatments. Compared to the effect of dietary MCT, the effect of clofibrate on fatty acid oxidation did not change the % of 14C accumulation in CO2 and ASP even though the stimulation of clofibrate on fatty acid oxidation was higher from TC5 and TMPA than Succ and TC6. However, we found that TC5 reduced the % of CO2 and increased ASP, while TMPA increased the % of CO2 and decreased ASP. Because the % of CO2 and ASP reflects the capacity of the CAC and the activity of the ketogenic pathways, these results imply that the differences in the effects of dietary TC5 and TMPA on the CAC and ketogenic pathway were different under the current conditions. The product of β-oxidation, propionyl-CoA from TMPA could increase the CAC capacity via providing anaplerotic carbon (as discussed above), while acetyl-CoA and propionyl-CoA generated from TC5 could increase the activity of ketogenic pathway or the production of acetyl-carnitine which is part of ASP.
3.3. The Effects of Carnitine and Ketogenic Pathway Inhibitors on Intestinal Fatty Acid Oxidation
Milk fat is the primary energy source for neonates to grow and develop after birth. The exogenous carnitine requirement is greater for neonates than adults [
68]. The effects of carnitine on fatty acid oxidation and ketogenic capacity have been well studied and documented in liver, kidney, and muscle during postnatal period [
69,
70,
71,
72,
73,
74]. However, to our knowledge, this is the first study to determine the effects of supplemental carnitine on intestinal fatty acid oxidation. The results from our study confirmed that supplementation of carnitine in intestinal mucosa isolated from 5-day-old pigs significantly increased fatty acid oxidation to both CO
2 and ASP, suggesting that carnitine was a limiting factor for intestinal fatty acid oxidation. As discussed previously, carnitine status in intestinal mucosa should be associated with the dietary level of carnitine and the capability of carnitine synthesis [
21,
35]. Although both CO
2 and ASP were increased by the carnitine supplementation, the % of CO
2 was significantly reduced, suggesting that the overflow of acetyl-CoA generated from the β-oxidation can be converted to acetyl-carnitine. This result was consistent with the increase in acyl-carnitine in plasma from the clofibrate treated pigs.
Supplementation of iodoacetamide, an inhibitor of AACD for acetoacetate synthesis [
75] acid oxidation or the oxidative metabolite distribution between CO
2 and ASP. This was similar to results we obtained in kidney and liver, suggesting that the ketogenic pathway via AACD is negligible in intestine as well [
21,
58]. Unlike in kidney, supplementation of the L659699, an inhibitor of both mitochondrial HMGCS and cytosolic HMGCS [
76], significantly reduced palmitic acid oxidation and this reduction was related to a decrease in ASP production, implying that cholesterol synthesis and/or ketogenesis might be impacted by reducing the precursor 3-hydroxy-3-methylglutaryl-CoA. Studies on ketogenic capacity in intestine are very limited, and it has been reported that the key enzyme mHMGCS is expressed in intestine of rodent species and humans [
77,
78,
79] especially in the large intestine in which ketogenesis plays an important role in intestinal cell differentiation [
80]. However, mHMGCS expression was not detected in small intestine in pigs during development [
81], suggesting that the effect of L659699 on ASP observed in this study could be primarily associated with cytosolic HMGCS. In addition, the activity of HMGCS needs to be determined, as it has not been measured in either mitochondria or cytoplasm of small intestine of pigs.