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Response of CPT1A, CPT1B, CPT2, and CRAT mRNA levels to carnitine supplementation after cultivation of cells in dialysed foetal calf serum (FCS); HEPG2 cells were cultivated in medium with non-dialysed 10% (v=v) FCS or dialysed 10% (v=v) FCS for 48 h and subsequently incubated without or with L-carnitine for 4 h; the final concentrations of L-carnitine were 10 M, 40, 80, 120, and 160 M, respectively; steady state level mRNA analysis and quantification by real-time RT-PCR were performed as described in the experimental section; data shown are mean values of triplicate independent experiments; to allow direct comparison between different transcripts, data are expressed relative to the mRNA levels detected in cells cultivated in FCS (1.0); error bars are standard deviations
Source publication
In order to analyze the biological influence of carnitine on the transcriptional regulation of mitochondrial carnitine acetyltransferase (CRAT) and carnitine palmitoyltransferases (CPT1A and CPT2) in conditions simulating carnitine deficiency in vivo, we cultured mouse (rat) fibroblasts and human liver cells with dialysed serum (carnitine deficient...
Contexts in source publication
Context 1
... CS (1.0); error bars are standard deviations In analogy to the experimental procedure administrated for Fig. 1, we deter- mined the steady state mRNA levels of CRAT, CPT1A, CPT1B, and CPT2 in human liver cells (HEPG2) treated with non-dialysed foetal calf serum (FCS) and dialysed FCS with or without L-carnitine supplementation by real time RT-PCR (Fig. 2). The influence of the dialysis and supplementation on transcript levels of the carnitine acyltransferases in HEPG2 cells is very similar to the patterns observed in NIH3T3 and rat-1 fibroblasts shown in Fig. 1. Fig. 3. Time dependent response of CPT1A, CRAT, and CPT2 mRNA levels to carnitine supple- mentation in NIH3T3 cells grown in ...
Context 2
... mRNA levels reached its peak value after 24 h, although in the previous experiments ( Figs. 1 and 2) the mRNA levels were enhanced with twice the con- centration of carnitine (80 M) already after 4 h incubation. The mRNA levels of CPT2 exhibited an interesting pattern with more than a five-fold induction after 48 h, at the time point when the other two carnitine acyltransferases transcripts already declined. ...
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Citations
... Moreover, FAS catalyzes the synthesis of fat (TG) from acetyl coenzyme A, malonyl monoacyl coenzyme A, and NADPH, thus playing an important role in animal body fat deposition (Smith et al., 2003). Furthermore, CPT1 catalyzes the generation of acyl-CoA to carnitine forming acyl-carnitine esters, in the inner mitochondrial membrane facilitating fatty acids β-oxidation process (Morash et al., 2008) and lipolysis (Godarova et al., 2005). Therefore, the increased mRNA expression of acc, fas and cpt1 genes enhanced fatty acids catabolism, which resulted in reduced TG content. ...
... This hypothesized delay in L-carnitine biosynthesis, and hence the rate of FAO, appeared to be more pronounced in gilt oocytes in contrast to CCs, with a difference in BBOX1 and CPT2 transcript abundance still being present in gilt oocytes after maturation, relative to sows. This hypothesis is supported by the study of Godárová et al. [78], which demonstrated that insufficient levels of L-carnitine caused a reduction in CPT2 transcript abundance, while the abundance of CPT2 increased significantly within 48 h of L-carnitine supplementation. The results from the current study indicate that BBOX1 encodes the more rate-limiting enzyme for L-carnitine biosynthesis than TMLHE in both CCs and oocytes as more significant differences in transcript abundance were observed for BBOX1. ...
Cumulus cells (CCs) are pivotal during oocyte development. This study aimed to identify novel marker genes for porcine oocyte quality by examining the expression of selected genes in CCs and oocytes, employing the model of oocytes from prepubertal animals being of reduced quality compared to those from adult animals. Total RNA was extracted either directly after follicle aspiration or after in vitro maturation, followed by RT-qPCR. Immature gilt CCs accumulated BBOX1 transcripts, involved in L-carnitine biosynthesis, to a 14.8-fold higher level (p < 0.05) relative to sows, while for CPT2, participating in fatty acid oxidation, the level was 0.48 (p < 0.05). While showing no differences between gilt and sow CCs after maturation, CPT2 and BBOX1 levels in oocytes were higher in gilts at both time points. The apparent delayed lipid metabolism and reduced accumulation of ALDOA and G6PD transcripts in gilt CCs after maturation, implying downregulation of glycolysis and the pentose phosphate pathway, suggest gilt cumulus–oocyte complexes have inadequate ATP stores and oxidative stress balance compared to sows at the end of maturation. Reduced expression of BBOX1 and higher expression of CPT2 in CCs before maturation and higher expression of G6PD and ALDOA after maturation are new potential markers of oocyte quality.
... Acyl-CoAs participate in the synthesis of cell lipids or in degradation via β-oxidation for energy production (Coleman and Lee, 2004). CPT1 and CPT2 are key enzymes in the carnitine system located on the mitochondrial membrane (Godarova et al. 2005). CPT1Ab, also called CPT1, is highly expressed in the liver (Longo et al. 2016). ...
... CPT1Ab, also called CPT1, is highly expressed in the liver (Longo et al. 2016). CPT1Ab, located in the outer located within the outer membrane of mitochondria, initiated the transport of the long-chain fatty acid into mitochondria for β-oxidation in the liver (Godarova et al. 2005;Moody et al. 2019). CPT1 is considered to be a key regulatory enzyme in mitochondrial fatty acid oxidation (Dong et al. 2014). ...
... CPT1 is considered to be a key regulatory enzyme in mitochondrial fatty acid oxidation (Dong et al. 2014). After translocation, carnitine palmitoyltransferase 2 (CPT2) catalyzed acyl-CoAs to produce fatty acyl-CoA for the next oxidation (Godarova et al. 2005). The degradation of acyl-CoAs in mitochondria is catalyzed by a variety of enzymes, such as ACOX1, ECHS1, HADH, and ACAA2. ...
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... ACAD11 and ACADM are two members of fatty acyl-CoA dehydrogenases that catalyze the first step in each cycle of fatty acid beta oxidation [45]. CRAT catalyzes the reversible transfer of an acetyl group from acyl-CoA to carnitine [46]. ECI2 is involved in the beta oxidation of unsaturated fatty acids, catalyzing the formation of 2-trans-enoyl-CoA from 3-cis or trans-enoyl-CoA [47]. ...
Background
Pantothenic acid deficiency (PAD) results in growth depression and intestinal hypofunction of animals. However, the underlying molecular mechanisms remain to be elucidated. Mucosal proteome might reflect dietary influences on physiological processes.
Results
A total of 128 white Pekin ducks of one-day-old were randomly assigned to two groups, fed either a PAD or a pantothenic acid adequate (control, CON) diet. After a 16-day feeding period, two ducks from each replicate were sampled to measure plasma parameters, intestinal morphology, and mucosal proteome. Compared to the CON group, high mortality, growth retardation, fasting hypoglycemia, reduced plasma insulin, and oxidative stress were observed in the PAD group. Furthermore, PAD induced morphological alterations of the small intestine indicated by reduced villus height and villus surface area of duodenum, jejunum, and ileum. The duodenum mucosal proteome of ducks showed that 198 proteins were up-regulated and 223 proteins were down-regulated (> 1.5-fold change) in the PAD group compared to those in the CON group. Selected proteins were confirmed by Western blotting. Pathway analysis of these proteins exhibited the suppression of glycolysis and gluconeogenesis, fatty acid beta oxidation, tricarboxylic acid cycle, oxidative phosphorylation, oxidative stress, and intestinal absorption in the PAD group, indicating impaired energy generation and abnormal intestinal absorption. We also show that nine out of eleven proteins involved in regulation of actin cytoskeleton were up-regulated by PAD, probably indicates reduced intestinal integrity.
Conclusion
PAD leads to growth depression and intestinal hypofunction of ducks, which are associated with impaired energy generation, abnormal intestinal absorption, and regulation of actin cytoskeleton processes. These findings provide insights into the mechanisms of intestinal hypofunction induced by PAD.
... Although multiple lines of evidence suggest that Lcarnitine plays an important role in modulating metabolic functions in humans, few studies have elucidated the regulatory role of L-carnitine on the molecular level. Artificial carnitine depletion in vitro has been shown to decrease mRNA expression of carnitine acetyltransferase (CrAT) and carnitine palmitoyl transferases (CPT1A and CPT2), whereas subsequent carnitine supplementation leads to a full reversal of the downregulation [8] . In addition to this effect, L-carnitine has been hypothesized to interact with several members of the nuclear hormone receptor family [9 ,10] . ...
L-carnitine is an indispensable metabolite facilitating the transport of fatty acids into the mitochondrial matrix and has been previously postulated to exert a nutrigenomic effect. However, the underlying molecular mechanisms remain mostly unclear. We hypothesized that L-carnitine interacts with nuclear receptors involved in metabolic regulation, thereby modulating downstream targets of cellular metabolism. Therefore, we investigated the effect of L-carnitine supplementation on protein activity, mRNA expression, and binding affinities of nuclear receptors as well as mRNA expression of downstream targets in skeletal muscle cells, hepatocytes, and differentiated adipocytes. L-carnitine supplementation to hepatocytes increased the protein activity of multiple nuclear receptors (RAR, RXR, VDR, PPAR, HNF4, ER, LXR). Diverging effects on the mRNA expression of PPAR-α, PPAR-δ, PPAR-γ, RAR-β, LXR-α, and RXR-α were observed in adipocytes, hepatocytes, and skeletal muscle cells. mRNA levels of PPAR-α, a key regulator of lipolysis and β-oxidation, were significantly upregulated, emphasizing a role of L-carnitine as a promoter of lipid catabolism. L-carnitine administration to hepatocytes modulated the transcription of key nuclear receptor target genes, including ALDH1A1, a promoter of adipogenesis, and OGT, a contributor to insulin resistance. Electrophoretic mobility shift assays proved L-carnitine to increase binding affinities of nuclear receptors to their promoter target sequences, suggesting a molecular mechanism for the observed transcriptional modulation. Overall, these findings indicate that L-carnitine modulates the activity and expression of nuclear receptors, thereby promoting lipolytic gene expression and decreasing transcription of target genes linked to adipogenesis and insulin resistance.
... ACAD11 and ACADM belong to the family of fatty acyl-CoA dehydrogenases that catalyze the initial step in each cycle of fatty acid beta-oxidation [45]. CRAT catalyzes the reversible transfer of an acetyl group from acyl-CoA to carnitine [46]. ECI2 is involved in the beta oxidation of unsaturated fatty acids, converting 3-cis or trans-enoyl-CoA to 2-trans-enoyl-CoA [47]. ...
Background: Pantothenic acid deficiency (PAD) results in growth depression and intestinal hypofunction of animals. However, the underlying mechanisms remain to be established. We investigated intestinal mucosal proteome changes induced by PAD in ducks to explain its effects on growth and intestine.
Results: A total of 128 one-day-old Pekin ducks were divided into two groups, with 8 replicates and 8 birds per replicate. All the ducks were fed either a PAD or a pantothenic acid adequate (control, CON) diet for 16 days. High mortality, growth retardation, fasting hypoglycemia, reduced plasma insulin, and oxidative stress were observed in the PAD group compared to the CON group. Furthermore, PAD induced morphological alterations of the small intestine indicated by reduced villus height and villus surface area of duodenum, jejunum, and ileum. The duodenum mucosal proteome of ducks showed that 198 proteins were upregulated and 223 proteins were downregulated (> 1.5-fold change) in the PAD group compared to those in the CON group. Pathway analysis of these proteins exhibited the suppression of glycolysis and gluconeogenesis, fatty acid beta oxidation, tricarboxylic acid cycle, oxidative phosphorylation, oxidative stress, and intestinal absorption in the PAD group, indicating impaired energy generation and abnormal intestinal absorption. We also show that nine out of eleven proteins involved in regulation of actin cytoskeleton were upregulated by PAD, probably indicates reduced intestinal integrity.
Conclusion: PAD leads to growth depression and intestinal hypofunction of ducks, which are associated with impaired in energy generation, abnormal intestinal absorption, and regulation of actin cytoskeleton processes. These findings have important implications for understanding the effect of PAD on intestines.
... ACAD11 and ACADM belong to the family of fatty acyl-CoA dehydrogenases that catalyze the initial step in each cycle of fatty acid beta-oxidation [45]. CRAT catalyzes the reversible transfer of an acetyl group from acyl-CoA to carnitine [46]. ECI2 is involved in the beta oxidation of unsaturated fatty acids, converting 3-cis or trans-enoyl-CoA to 2-trans-enoyl-CoA [47]. ...
Background: Pantothenic acid deficiency (PAD) results in growth depression and intestinal hypofunction of animals. However, the underlying mechanisms remain to be established. Mucosal proteome might reflect dietary influences on physiological processes.
Results: A total of 128 one-day-old Pekin ducks were divided into two groups, with 8 replicates and 8 birds per replicate. All the ducks were fed either a PAD or a pantothenic acid adequate (control, CON) diet. After a 16-day feeding period, two ducks from each replicate were sampled to measure plasma parameters, intestinal morphology, and mucosal proteome. High mortality, growth retardation, fasting hypoglycemia, reduced plasma insulin, and oxidative stress were observed in the PAD group compared to the CON group. Furthermore, PAD induced morphological alterations of the small intestine indicated by reduced villus height and villus surface area of duodenum, jejunum, and ileum. The duodenum mucosal proteome of ducks showed that 198 proteins were upregulated and 223 proteins were downregulated (> 1.5-fold change) in the PAD group compared to those in the CON group. Selected proteins were confirmed by Western blotting. Pathway analysis of these proteins exhibited the suppression of glycolysis and gluconeogenesis, fatty acid beta oxidation, tricarboxylic acid cycle, oxidative phosphorylation, oxidative stress, and intestinal absorption in the PAD group, indicating impaired energy generation and abnormal intestinal absorption. We also show that nine out of eleven proteins involved in regulation of actin cytoskeleton were upregulated by PAD, probably indicates reduced intestinal integrity.
Conclusion: PAD leads to growth depression and intestinal hypofunction of ducks, which are associated with impaired energy generation, abnormal intestinal absorption, and regulation of actin cytoskeleton processes. These findings provide insights into the mechanisms of intestinal mucosa metabolic disorders due to PAD.
... ACAD11 and ACADM belong to the family of fatty acyl-CoA dehydrogenases that catalyze the initial rate-limiting step of the beta-oxidation cycle [50]. CRAT catalyzes the reversible transfer of acyl-CoA from carnitine to free CoA [51]. ECI2 is an auxiliary enzyme in the beta oxidation of unsaturated fatty acids that converts 3-cis or trans-enoyl-CoA to 2-trans-enoyl-CoA [52]. ...
Background
Pantothenic acid deficiency (PAD) results in growth depression and intestinal hypofunction of animals. The underlying mechanisms, however, remain to be established and an overview of molecular alterations is still lacking. We investigated intestinal mucosal proteome changes induced by PAD in ducks to explain its effects on growth and intestine.
Methods
A total of 128 one-day-old Pekin ducks were divided into two groups, with 8 replicates and 8 birds per replicate. All the ducks were fed either a PAD or a pantothenic acid adequate (control, CON) diet for 16 days.
Results
High mortality, growth retardation, fasting hypoglycemia, reduced plasma insulin, and oxidative stress were observed in the PAD group compared to the CON group. Furthermore, PAD induced morphological alterations of the small intestine indicated by reduced villus height and villus surface area of duodenum, jejunum, and ileum. The duodenum mucosal proteome of ducks showed that 198 proteins were upregulated and 223 proteins were downregulated (> 1.5-fold change) in the PAD group compared to those in the CON group. Pathway analysis of these proteins exhibited the suppression of glycolysis and gluconeogenesis, fatty acid beta oxidation, tricarboxylic acid cycle, oxidative phosphorylation, oxidative stress, and intestinal absorption in the PAD group, indicating impaired energy generation and abnormal intestinal absorption. We also show that nine out of eleven proteins involved in regulation of actin cytoskeleton were upregulated by PAD, probably indicates reduced intestinal integrity.
Conclusion
PAD leads to growth depression and intestinal hypofunction of ducks, which are associated with impaired in energy generation, abnormal intestinal absorption, and regulation of actin cytoskeleton processes. These findings contribute to our understanding of the mechanisms of intestinal mucosa metabolic disorders due to PAD.
... ACAD11 and ACADM belong to the family of fatty acyl-CoA dehydrogenases that catalyze the initial rate-limiting step of the beta-oxidation cycle [50]. CRAT catalyzes the reversible transfer of acyl-CoA from carnitine to free CoA [51]. ECI2 is an auxiliary enzyme in the beta oxidation of unsaturated fatty acids that converts 3-cis or trans-enoyl-CoA to 2-trans-enoyl-CoA [52]. ...
Background: Pantothenic acid deficiency (PAD) results in growth depression and intestinal hypofunction of animals. The underlying mechanisms, however, remain to be established and an overview of molecular alterationsis still lacking.We investigated intestinal mucosalproteome changes induced by PADin ducks to explain its effects on growth and intestine.
Methods:A total of128 one-day-oldPekinducks were divided into two groups,with 8 replicates and 8 birds per replicate. All the ducks were fed either a PAD or a pantothenic acid adequate (control, CON) dietfor 16 days.
Results:High mortality, growthretardation,fasting hypoglycemia,reduced plasma insulin, and oxidative stresswere observed in the PAD group compared to the CON group. Furthermore, PAD induced morphological alterationsof the small intestine indicated by reduced villus height and villus surface area of duodenum, jejunum, and ileum.The duodenum mucosal proteome of ducks showed that 198 proteins were upregulated and 223 proteins were downregulated (> 1.5-fold change) in the PAD group compared to those in the CON group. Pathway analysis of these proteins exhibited the suppression of glycolysis and gluconeogenesis, fatty acid beta oxidation, tricarboxylic acid cycle, oxidative phosphorylation, oxidative stress, and intestinal absorption in the PAD group, indicating impaired energy generation and abnormal intestinal absorption.Wealso show that nine out of eleven proteins involved in regulation of actin cytoskeleton were upregulated by PAD, probably indicates reduced intestinal integrity.
Conclusion:PAD leads to growth depression and intestinal hypofunctionof ducks, which are associated with impaired in energy generation, abnormal intestinal absorption, and regulation of actin cytoskeletonprocesses. These findings contribute to our understanding of the mechanisms of intestinal mucosa metabolic disorders due to PAD.
... CPT2, an inner membrane protein, catalyses the formation of acyl-CoA from acylcarnitine and CoA [34]. CRAT catalyses the reversible transfer of acyl-CoA from carnitine to free CoA [35]. SLC25A20 facilitates the transfer of acylcarnitine esters in exchange for free carnitine across the mitochondrial membrane [36]. ...
Background:
Maternal riboflavin deficiency (RD) induces embryonic death in poultry. The underlying mechanisms, however, remain to be established and an overview of molecular alterations at the protein level is still lacking. We investigated embryonic hepatic proteome changes induced by maternal RD to explain embryonic death.
Methods:
A total of 80 45-week-old breeding female ducks were divided into two groups of 40 birds each, and all birds were raised individually for 8 weeks. All the female ducks received either a RD or a riboflavin adequate (control, CON) diet, which supplemented the basal diet with 0 or 10 mg riboflavin /kg of diet respectively.
Results:
The riboflavin concentrations of maternal plasma and egg yolk, as well as egg hatchability declined markedly in the RD group compared to those in the CON group after 2 weeks, and declined further over time. The hepatic proteome of E13 viable embryos from 8-week fertile eggs showed that 223 proteins were upregulated and 366 proteins were downregulated (> 1.5-fold change) in the RD group compared to those in the CON group. Pathway analysis showed that differentially expressed proteins were mainly enriched in the fatty acid beta-oxidation, electron transport chain (ETC), and tricarboxylic acid (TCA) cycle. Specifically, all the proteins involved in the fatty acid beta-oxidation and ETC, as well as six out of seven proteins involved in the TCA cycle, were diminished in the RD group, indicating that these processes could be impaired by RD.
Conclusion:
Maternal RD leads to embryonic death of offspring and is associated with impaired energy generation processes, indicated by a number of downregulated proteins involved in the fatty acid beta-oxidation, ETC, and TCA cycle in the hepatic of duck embryos. These findings contribute to our understanding of the mechanisms of liver metabolic disorders due to maternal RD.
