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

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.

Figure 1. β-oxidation of fatty acids with double bonds at even- or odd-numbered positions in mitochondria.

Degradation of fatty acids with even-numbered double bonds results in 2,4-dienoyl-CoA esters, which are oxidized as shown on the left. 2,5-dienoyl-CoA esters arising from odd-numbered double bonds can be oxidized either via an isomerase-dependent pathway (middle) or via a reductase-dependent pathway (right). AD, acyl-CoA dehydrogenase (EC 1.3.3.6, EC 1.3.99.3, EC 1.3.99.13 or EC 1.3.99.-); EH, enoyl-CoA hydratase (EC 4.1.2.17 or EC 4.2.1.74); HD, 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 or EC 1.1.1.211); KT, 3-ketoacyl thiolase (EC 2.3.1.16) ; ECI, Δ³,Δ²-enoyl isomerase (EC 5.3.3.8); DECI, Δ^3,5,Δ^2,4-dienoyl-CoA isomerase (no EC number available); DECR (shown in bold), 2,4-dienoyl-CoA reductase (EC 1.3.1.34).

Figure 2. Targeting of the Decr locus and verification of gene inactivation.

(A) Schematic drawing of the targeting strategy showing the wild type allele, targeting vector and targeted allele after homologous recombination. The targeted allele depicts the strategy used to delete exon 1, in which the endogenous sequence is replaced by a neomycin-positive selection cassette (neo). A thymidine kinase cassette (tk) was used for negative selection. Exons are denoted as numbered solid bars (1–9). Recognition sites for restriction endonucleases are marked as B = BamHI, H = HindIII, E = EcoRI, S = SmalI, EV = EcoRV. The genomic fragment used as an external probe for Southern analysis is marked with an “X”, and the expected fragments for the wild type and targeted allele after BamHI digestion are indicated with arrows. (B) Southern blot analysis of mouse liver DNA. Genomic DNA was digested with BamHI and detected using probe X to yield the expected fragments of 5.8 kb for the wild type allele in Decr^+/+ mice (+/+), 4.7 kb for the targeted allele in Decr^−/− mice (−/−), and both fragments in heterozygous Decr^+/− mice (+/−). (C) Mouse genotypes were determined from tail samples using PCR with primers denoted with arrows a, b, and c in the schematic drawing. The amplified fragment for wild type Decr^+/+ mice was 382 bp, 280 bp for homozygous Decr^−/− mice, and both fragments were amplified from heterozygous Decr^+/− mice. (D) Western blot analysis of mitochondrial homogenates from liver, muscle and heart using an antibody against rat DECR showing the presence or absence of the 33-kDa band corresponding to DECR in Decr^+/+ and Decr^−/− mice, respectively. Twenty micrograms of protein was loaded in each lane.

Figure 3. Effects of fasting on organ weights in wild-type and Decr ^−/− mice.

Age-matched male mice were fasted for 24 h, after which they were sacrificed. The wet weight of selected organs (heart, liver, kidney and fat) was determined and compared between wild type and Decr^−/− mice. Weights were calculated as a percentage of body weight and are expressed as means±SE of 4–6 mice of each genotype per group. Asterisks (*) denote significant differences (p<0.01) between wild type and Decr^−/− mice.

Figure 4. Histological assessment of liver morphology in fasted and non-fasted wild-type and Decr ^−/− mice.

Light microscopic images of representative paraffin-embedded liver sections from wild type (+/+) and Decr^−/− mice (−/−) stained with hematoxylin and eosin (A–D) and cryosections stained with Oil red O (E,F). Liver morphology of non-fasted animals showed no differences between wild type and Decr^−/− mice (A,B). Fasting for 24 h revealed no apparent changes in the liver morphology of wild type mice, but induced microvesicular steatosis in Decr^−/− mice, as observed by the appearance of foamy hepatocytes with centralized nuclei (C,D). Oil red O staining of neutral fat in representative liver cryosections revealed small, lightly stained vacuoles in the wild type sample (E), whereas a large number of intensively stained vacuoles of varying size can be seen in Decr^−/− mice (F), indicating the accumulation of fat. Magnification×20.

Figure 5. Effect of fasting on serum NEFA, glucose, and OH–BUT levels and liver and muscle glycogen content in wild-type and Decr^−/− mice.

Age-matched male wild type (open boxes/bars) and Decr^−/− mice (solid boxes/bars) were fasted for 0, 24, and 48 h, after which the serum levels of non-esterified fatty acids (A) and glucose (B) were determined. Glycogen content of liver (C) and muscle (D) tissue from wild type (open bars) and Decr^−/− mice (solid bars) in the fed state and after mice were fasted for 6 h and/or 15 h was analyzed using the phenol-sulfuric acid method. Serum β-hydroxybutyric acid levels were measured in the fed state and after 24 h of fasting (E). At each time point, the results are expressed as means±SE of 5–6 mice of each genotype per group. Significant differences in glucose and NEFA concentrations between wild type and Decr^−/− mice are indicated by asterisks (* p<0.05, ** p<0.01).

Figure 6. Fatty acid pattern of total liver lipids in fasted and non-fasted wild-type and Decr^−/− mice.

Total fatty acids were isolated from pooled liver homogenate samples of 5–6 mice per genotype and analyzed using positive ion mass spectrometry. (A) Fatty acid profile of total liver fatty acids under normal fed state conditions showing the concentrations of different fatty acids for wild type (open bars) and Decr^−/− (solid bars) mice. (B) Fatty acid profile of liver total fatty acids after fasting for 24 h showing increased concentrations of unsaturated fatty acids in Decr^−/− mice when compared with wild type mice. (C) Proportions of saturated fatty acids (SAFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) among the total liver fatty acids in wild type (WT) and Decr^−/− (KO) mice in the fed state and after 24 h of fasting (fasted).

Figure 7. Serum acylcarnitine analysis under normal and fasted conditions.

Serum acylcarnitines were analyzed for wild type (solid line) and Decr^−/− mice (dotted line) by mass spectrometry. (A) Serum acylcarnitine profile under normal conditions, as determined from the mass spectral data. (B) To determine the serum acylcarnitine profile under fasted conditions mice were subjected to a 24 h fast prior to serum collection. The concentrations of acylcarnitine are expressed as means±SE of 5–6 mice of each genotype per group.

Figure 8. 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).

Figure 9. Changes in body temperature during cold exposure with prior fasting.

Cold tolerance was tested after 20 h of fasting by exposing individually housed wild type mice (open boxes) and Decr^−/− mice (solid boxes) to a +4°C environment for a maximum of 4 hours or until body temperature dropped below 25°C. Temperature was measured from the shaved mid-dorsal body surface using an infrared thermometer. At each time point, body temperatures are presented as mean values of three measurements from the same individual.

Figure 10. Effect of fasting and cold exposure on physical activity and heat production.

To assess physical activity, Decr^−/− mice (solid boxes and bars) and wild type mice (open boxes and bars) were continuously monitored with the LabMaster system. (A) Activity pattern during 48 hours of fasting. Fasting was started at 8 am and continued for 48 hours, with data collected every 30 min. Group means±SEM (n = 4) are shown. (B) Average total activity (counts/30 min) during the 48 h fast. Group means±SEM (n = 4) are shown. (C) Total activity during cold exposure. Decr^−/− and wild type mice were fasted 20 h and then exposed to cold for 2 h (+9.6°C). Total activity was measured continuously and data were collected every 15 minutes. (D) Average total activity (counts/15 min) during cold exposure. Group means±SEM (n = 4) are shown. (E) Average heat production (kcal/h/kg) during cold exposure. Group means±SEM (n = 4) are shown. Student's t-test was used for statistical analysis, and p-values below 0.05 were considered statistically significant. Data for total activity during cold exposure were analyzed by two-way ANOVA followed by Bonferroni's post-test.

Figure 11. PUFA and activation of gluconeogenesis.

Simplified schematic presentation of the identified kinase cascades regulating gluconeogenesis. The activation cascade leads to phosphorylation of CREB (cAMP-responsive element binding protein), which, together with TORC2 (transducer of regulated CREB activity 2), drives the expression of coactivator PGC-1α. Transcription of the key gluconeogenic enzymes PEPCK (phosphoenoylpyruvate carboxykinase) and G6Pase (glucose-6-phosphatase) is induced when PGC-1α associates with HNF4α (hepatic nuclear factor 4α) and FOXO1 (forkhead box transcription factor). Nuclear translocation of TORC2, which is needed for activation of the gluconeogenic program, is controlled by phosphorylation by activated AMPK (AMP–activated protein kinase) and SIK (salt-inducible kinase). Observed changes in Decr^−/− mice are indicated with red arrows. Possible targets of accumulated PUFA or their derivatives are also indicated.