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Mitochondrial 2,4-dienoyl-CoA reductase deficiency in mice results in severe hypoglycemia with stress intolerance and unimpaired ketogenesis.

Miinalainen IJ, Schmitz W, Huotari A, Autio KJ, Soininen R, Ver Loren van Themaat E, Baes M, Herzig KH, Conzelmann E, Hiltunen JK - PLoS Genet. (2009)

Bottom Line: Enzyme defects in this pathway cause fatty acid oxidation disorders.Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure.This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biocenter Oulu, University of Oulu, Oulu, Finland.

ABSTRACT
The mitochondrial beta-oxidation system is one of the central metabolic pathways of energy metabolism in mammals. Enzyme defects in this pathway cause fatty acid oxidation disorders. To elucidate the role of 2,4-dienoyl-CoA reductase (DECR) as an auxiliary enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids, we created a DECR-deficient mouse line. In Decr(-/-) mice, the mitochondrial beta-oxidation of unsaturated fatty acids with double bonds is expected to halt at the level of trans-2, cis/trans-4-dienoyl-CoA intermediates. In line with this expectation, fasted Decr(-/-) mice displayed increased serum acylcarnitines, especially decadienoylcarnitine, a product of the incomplete oxidation of linoleic acid (C(18:2)), urinary excretion of unsaturated dicarboxylic acids, and hepatic steatosis, wherein unsaturated fatty acids accumulate in liver triacylglycerols. Metabolically challenged Decr(-/-) mice turned on ketogenesis, but unexpectedly developed hypoglycemia. Induced expression of peroxisomal beta-oxidation and microsomal omega-oxidation enzymes reflect the increased lipid load, whereas reduced mRNA levels of PGC-1alpha and CREB, as well as enzymes in the gluconeogenetic pathway, can contribute to stress-induced hypoglycemia. Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure. This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.

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Effect of fasting on hepatic expression levels of genes for mitochondrial and extramitochondrial fatty acid metabolism.Quantitative real-time PCR analysis was used to determine changes in hepatic gene expression in Decr−/− mice (solid bars) after 24 h of dietary stress compared with wild type mice (open bars). (A) Relative expression levels of genes involved in mitochondrial β-oxidation; CPT-1, LCAD, and VLCAD. (B) Relative expression levels of genes involved in the peroxisomal β-oxidation pathway; Acox, MFE1 and ECI. (C) Relative expression levels of genes involved in fatty acid synthesis, desaturation and microsomal ω-oxidation; Acaca, SCD1 and Cyp4A10, respectively. (D) Relative expression levels of genes involved in the gluconeogenetic pathway and ketone body synthesis; PEPCK, G-6Pase and HMGCS, respectively. (E) Relative expression levels of genes encoding transcriptional factors; PPARα, Srebp1, chREBP, CREB, and the co-activator PGC-1α. For relative quantification of gene expression, the results were normalized using GAPDH as an endogenous control for each sample, and the data obtained for wild type samples were set to 1. Results represent means±SE of 5 mice of each genotype per group. Statistically significant differences in expression levels between wild type and Decr−/− mice are indicated by asterisks (* p<0.05, ** p<0.01, *** p<0.001).
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pgen-1000543-g008: Effect of fasting on hepatic expression levels of genes for mitochondrial and extramitochondrial fatty acid metabolism.Quantitative real-time PCR analysis was used to determine changes in hepatic gene expression in Decr−/− mice (solid bars) after 24 h of dietary stress compared with wild type mice (open bars). (A) Relative expression levels of genes involved in mitochondrial β-oxidation; CPT-1, LCAD, and VLCAD. (B) Relative expression levels of genes involved in the peroxisomal β-oxidation pathway; Acox, MFE1 and ECI. (C) Relative expression levels of genes involved in fatty acid synthesis, desaturation and microsomal ω-oxidation; Acaca, SCD1 and Cyp4A10, respectively. (D) Relative expression levels of genes involved in the gluconeogenetic pathway and ketone body synthesis; PEPCK, G-6Pase and HMGCS, respectively. (E) Relative expression levels of genes encoding transcriptional factors; PPARα, Srebp1, chREBP, CREB, and the co-activator PGC-1α. For relative quantification of gene expression, the results were normalized using GAPDH as an endogenous control for each sample, and the data obtained for wild type samples were set to 1. Results represent means±SE of 5 mice of each genotype per group. Statistically significant differences in expression levels between wild type and Decr−/− mice are indicated by asterisks (* p<0.05, ** p<0.01, *** p<0.001).

Mentions: When the expression levels of several mitochondrial β-oxidation enzymes in the liver were compared between wild type and Decr−/− mice (Figure 8), a 2-fold increase was observed in the expression level of the rate-limiting enzyme carnitine palmitoyltransferase (CPT-1) in Decr−/− mice. Expression levels of other studied mitochondrial β-oxidation enzymes, long chain acyl-CoA dehydrogenase (LCAD) and very long chain acyl-CoA dehydrogenase (VLCAD) were comparable, but were slightly increased in Decr−/− mice (Figure 8A). A greater change was observed in the expression levels of genes associated with peroxisomal β-oxidation, because the expression levels of acyl-CoA oxidase (Acox) and peroxisomal multifunctional enzyme 1 (MFE1) were 2.3- and 3.4-fold higher in Decr−/− mice, respectively (Figure 8B). In addition, the expression of ECI, which is one of the auxiliary enzymes that functions in the oxidation of polyunsaturated fatty acids with double bonds in odd-numbered positions, was slightly upregulated (1.5-fold). A significant 2.1-fold increase was also observed in the expression level of cytochrome P450 IVA1 (CYP 4A10), which is a key enzyme in microsomal ω-oxidation (Figure 8C). Normally, enzymes involved in fatty acid synthesis and desaturation are downregulated during fasting. The expression level of acetyl-CoA carboxylase (Acaca), which catalyzes the first step in the fatty acid synthesis pathway, was lower in Decr−/− mice, although it was not significant (Figure 8C). However, the messenger RNA level of the enzyme responsible for synthesis of monounsaturated fatty acids, stearoyl-CoA desaturase 1 (SCD1), was markedly lower in Decr−/− mice (Figure 8C). During fasting, glucose homeostasis is maintained in part by the production and utilization of ketone bodies and in part by the production of glucose via gluconeogenesis. The hypoglycemic response to fasting prompted us to study the expression of phosphoenoylpyruvate carboxykinase (PEPCK-C) and glucose-6-phosphatase (G6Pase), key enzymes in the gluconeogenesis and glyceroneogenesis pathway, as well as mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS), an enzyme required for ketone body synthesis (Figure 8D). We found that the levels of PEPCK and G6Pase were decreased (2- and 2.2-times, respectively) in Decr−/− mice, whereas no differences were detected in the levels of HMGCS. Glucose homeostasis is regulated systemically by hormones such as insulin and glucagon and at the cellular level by energy status. During fasting, glucagon enhances glucose output from the liver via a PKA signal transduction pathway by activating cyclic AMP-responsive element binding protein (CREB), which in turn activates the expression of PPARγ coactivator-1α (PGC-1α) [22]. PGC-1α has been ascribed a central role in controlling the transcription of genes involved in major metabolic pathways in the liver (mitochondrial biogenesis, fatty acid catabolism, oxidative phosphorylation, and mitochondrial biogenesis) through the coactivation of several nuclear receptors and other transcription factors [23]. During fasting, increased PGC-1α levels in the liver induce gluconeogenesis by activating PEPCK and G6Pase promoters through direct interaction with hepatic nuclear factor 4α (HNF4α) and forkhead box transcription factor, FOXO1. Interestingly, we observed significantly decreased expression levels of CREB and PGC-1α (2.1 and 2.8-times, respectively) in Decr−/− mice compared with wild type mice after fasting (Figure 8E). Although peroxisome proliferator activated receptor α (PPARα) has a central role in the transcriptional control of genes encoding fatty acid oxidation enzymes, further transcription factors are responsible for the regulation of other metabolic pathways (e.g., sterol regulatory element-binding protein (SREBP), which regulates genes involved in lipogenesis, cholesterogenesis, and glucose metabolism and carbohydrate responsive element binding protein (chREBP), which mediates the transcriptional effects of glucose on glycolytic and lipogenic genes). Fasting produced no differences in the expression level of PPARα between wild type and Decr−/− mice; however, the expression levels of SREBP1 and chREBP in Decr−/− mice were significantly repressed (0.3 and 0.25 times the level observed in wild type mice after fasting) (Figure 8E).


Mitochondrial 2,4-dienoyl-CoA reductase deficiency in mice results in severe hypoglycemia with stress intolerance and unimpaired ketogenesis.

Miinalainen IJ, Schmitz W, Huotari A, Autio KJ, Soininen R, Ver Loren van Themaat E, Baes M, Herzig KH, Conzelmann E, Hiltunen JK - PLoS Genet. (2009)

Effect of fasting on hepatic expression levels of genes for mitochondrial and extramitochondrial fatty acid metabolism.Quantitative real-time PCR analysis was used to determine changes in hepatic gene expression in Decr−/− mice (solid bars) after 24 h of dietary stress compared with wild type mice (open bars). (A) Relative expression levels of genes involved in mitochondrial β-oxidation; CPT-1, LCAD, and VLCAD. (B) Relative expression levels of genes involved in the peroxisomal β-oxidation pathway; Acox, MFE1 and ECI. (C) Relative expression levels of genes involved in fatty acid synthesis, desaturation and microsomal ω-oxidation; Acaca, SCD1 and Cyp4A10, respectively. (D) Relative expression levels of genes involved in the gluconeogenetic pathway and ketone body synthesis; PEPCK, G-6Pase and HMGCS, respectively. (E) Relative expression levels of genes encoding transcriptional factors; PPARα, Srebp1, chREBP, CREB, and the co-activator PGC-1α. For relative quantification of gene expression, the results were normalized using GAPDH as an endogenous control for each sample, and the data obtained for wild type samples were set to 1. Results represent means±SE of 5 mice of each genotype per group. Statistically significant differences in expression levels between wild type and Decr−/− mice are indicated by asterisks (* p<0.05, ** p<0.01, *** p<0.001).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2697383&req=5

pgen-1000543-g008: Effect of fasting on hepatic expression levels of genes for mitochondrial and extramitochondrial fatty acid metabolism.Quantitative real-time PCR analysis was used to determine changes in hepatic gene expression in Decr−/− mice (solid bars) after 24 h of dietary stress compared with wild type mice (open bars). (A) Relative expression levels of genes involved in mitochondrial β-oxidation; CPT-1, LCAD, and VLCAD. (B) Relative expression levels of genes involved in the peroxisomal β-oxidation pathway; Acox, MFE1 and ECI. (C) Relative expression levels of genes involved in fatty acid synthesis, desaturation and microsomal ω-oxidation; Acaca, SCD1 and Cyp4A10, respectively. (D) Relative expression levels of genes involved in the gluconeogenetic pathway and ketone body synthesis; PEPCK, G-6Pase and HMGCS, respectively. (E) Relative expression levels of genes encoding transcriptional factors; PPARα, Srebp1, chREBP, CREB, and the co-activator PGC-1α. For relative quantification of gene expression, the results were normalized using GAPDH as an endogenous control for each sample, and the data obtained for wild type samples were set to 1. Results represent means±SE of 5 mice of each genotype per group. Statistically significant differences in expression levels between wild type and Decr−/− mice are indicated by asterisks (* p<0.05, ** p<0.01, *** p<0.001).
Mentions: When the expression levels of several mitochondrial β-oxidation enzymes in the liver were compared between wild type and Decr−/− mice (Figure 8), a 2-fold increase was observed in the expression level of the rate-limiting enzyme carnitine palmitoyltransferase (CPT-1) in Decr−/− mice. Expression levels of other studied mitochondrial β-oxidation enzymes, long chain acyl-CoA dehydrogenase (LCAD) and very long chain acyl-CoA dehydrogenase (VLCAD) were comparable, but were slightly increased in Decr−/− mice (Figure 8A). A greater change was observed in the expression levels of genes associated with peroxisomal β-oxidation, because the expression levels of acyl-CoA oxidase (Acox) and peroxisomal multifunctional enzyme 1 (MFE1) were 2.3- and 3.4-fold higher in Decr−/− mice, respectively (Figure 8B). In addition, the expression of ECI, which is one of the auxiliary enzymes that functions in the oxidation of polyunsaturated fatty acids with double bonds in odd-numbered positions, was slightly upregulated (1.5-fold). A significant 2.1-fold increase was also observed in the expression level of cytochrome P450 IVA1 (CYP 4A10), which is a key enzyme in microsomal ω-oxidation (Figure 8C). Normally, enzymes involved in fatty acid synthesis and desaturation are downregulated during fasting. The expression level of acetyl-CoA carboxylase (Acaca), which catalyzes the first step in the fatty acid synthesis pathway, was lower in Decr−/− mice, although it was not significant (Figure 8C). However, the messenger RNA level of the enzyme responsible for synthesis of monounsaturated fatty acids, stearoyl-CoA desaturase 1 (SCD1), was markedly lower in Decr−/− mice (Figure 8C). During fasting, glucose homeostasis is maintained in part by the production and utilization of ketone bodies and in part by the production of glucose via gluconeogenesis. The hypoglycemic response to fasting prompted us to study the expression of phosphoenoylpyruvate carboxykinase (PEPCK-C) and glucose-6-phosphatase (G6Pase), key enzymes in the gluconeogenesis and glyceroneogenesis pathway, as well as mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS), an enzyme required for ketone body synthesis (Figure 8D). We found that the levels of PEPCK and G6Pase were decreased (2- and 2.2-times, respectively) in Decr−/− mice, whereas no differences were detected in the levels of HMGCS. Glucose homeostasis is regulated systemically by hormones such as insulin and glucagon and at the cellular level by energy status. During fasting, glucagon enhances glucose output from the liver via a PKA signal transduction pathway by activating cyclic AMP-responsive element binding protein (CREB), which in turn activates the expression of PPARγ coactivator-1α (PGC-1α) [22]. PGC-1α has been ascribed a central role in controlling the transcription of genes involved in major metabolic pathways in the liver (mitochondrial biogenesis, fatty acid catabolism, oxidative phosphorylation, and mitochondrial biogenesis) through the coactivation of several nuclear receptors and other transcription factors [23]. During fasting, increased PGC-1α levels in the liver induce gluconeogenesis by activating PEPCK and G6Pase promoters through direct interaction with hepatic nuclear factor 4α (HNF4α) and forkhead box transcription factor, FOXO1. Interestingly, we observed significantly decreased expression levels of CREB and PGC-1α (2.1 and 2.8-times, respectively) in Decr−/− mice compared with wild type mice after fasting (Figure 8E). Although peroxisome proliferator activated receptor α (PPARα) has a central role in the transcriptional control of genes encoding fatty acid oxidation enzymes, further transcription factors are responsible for the regulation of other metabolic pathways (e.g., sterol regulatory element-binding protein (SREBP), which regulates genes involved in lipogenesis, cholesterogenesis, and glucose metabolism and carbohydrate responsive element binding protein (chREBP), which mediates the transcriptional effects of glucose on glycolytic and lipogenic genes). Fasting produced no differences in the expression level of PPARα between wild type and Decr−/− mice; however, the expression levels of SREBP1 and chREBP in Decr−/− mice were significantly repressed (0.3 and 0.25 times the level observed in wild type mice after fasting) (Figure 8E).

Bottom Line: Enzyme defects in this pathway cause fatty acid oxidation disorders.Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure.This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biocenter Oulu, University of Oulu, Oulu, Finland.

ABSTRACT
The mitochondrial beta-oxidation system is one of the central metabolic pathways of energy metabolism in mammals. Enzyme defects in this pathway cause fatty acid oxidation disorders. To elucidate the role of 2,4-dienoyl-CoA reductase (DECR) as an auxiliary enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids, we created a DECR-deficient mouse line. In Decr(-/-) mice, the mitochondrial beta-oxidation of unsaturated fatty acids with double bonds is expected to halt at the level of trans-2, cis/trans-4-dienoyl-CoA intermediates. In line with this expectation, fasted Decr(-/-) mice displayed increased serum acylcarnitines, especially decadienoylcarnitine, a product of the incomplete oxidation of linoleic acid (C(18:2)), urinary excretion of unsaturated dicarboxylic acids, and hepatic steatosis, wherein unsaturated fatty acids accumulate in liver triacylglycerols. Metabolically challenged Decr(-/-) mice turned on ketogenesis, but unexpectedly developed hypoglycemia. Induced expression of peroxisomal beta-oxidation and microsomal omega-oxidation enzymes reflect the increased lipid load, whereas reduced mRNA levels of PGC-1alpha and CREB, as well as enzymes in the gluconeogenetic pathway, can contribute to stress-induced hypoglycemia. Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure. This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.

Show MeSH
Related in: MedlinePlus