Short chain acyl-CoA dehydrogenase deficiency and short-term high-fat diet perturb mitochondrial energy metabolism and transcriptional control of lipid-handling in liver

The BALB/cByJ (Acads−/−) and BALB/cByKZ (Acads+/+) mice were obtained from breeding colonies maintained at the Pennington Biomedical Research Center (PBRC). Mice were singly housed in filter-top cages and kept in a specific-pathogen free facility, under 12 h light/12 h dark cycle and at an ambient temperature of 22–23 °C. All animals were fed standard rodent chow (no. 5001, LabDiets, Richmond, IN) until initiation of experimental diets. All animal experiments were approved by the Institutional Animal Care and Use Committee of the PBRC. The BALB/cByJ mouse strain carries a spontaneous 278-bp deletion at the 3’ end of the structural gene for Acads [6, 7], resulting in missplicing of RNA and a truncated, unstable SCAD enzyme with no residual activity [8]. This mutation occurred in the BALB/cByJ subline around 1982 [9] after it was separated from the BALB/cBy subline. Although the BALB/cByJ or Acads−/− mice have no detectable butyryl-CoA dehydrogenase activity [8], they appear to be normal [8, 10]. Their primary clinical phenotype is an impaired adaptation to prolonged fasting, due to the rapid depletion of hepatic glycogen stores [8]. The BALB/cBy line is considered the best Acads+/+ control for the Acads−/− BALB/cByJ [11]. Our BALB/cByKZ.Acads+/+ substrain was separated from the research colonies at The Jackson Laboratory in 1996.

Several days prior to each experiment, bedding was removed and replaced with stainless steel wire floor inserts, to permit measurement of spilled food. Polyvinylchloride nesting tubes were provided to reduce time spent on wire flooring. The experimental diets were equivalent for both micronutrients and for protein (16.4 % of energy). The balance of calories was contributed by 58 % fat and 25.5 % carbohydrate in D12331 and by 10.5 % fat and 73.1 % carbohydrate in D12329 (Research Diets, Inc., New Brunswick, NJ). The diets obtain most of their fat content from medium chain fatty acids. Tissues were harvested on day 2 after initiation of the high-fat (D12331) or low-fat (D12329) diet. Thus, there was no diet choice in the current study design. We selected the day 2 time point for tissue harvest to coincide with the time at which animals begin to avoid eating fat in a diet selection paradigm [1].

Twelve week old male BALB/cByJ (Acads−/−) and BALB/cByKZ (Acads+/+) mice were fed the experimental high- (HF) and low-fat (LF) diets for two days and then killed by CO₂ inhalation followed by cervical dislocation. The liver was rapidly excised, frozen in liquid nitrogen, then powdered under liquid nitrogen and stored at −80 °C. Measurement of liver acylcarnitine content was performed by the Metabolomics Core of the University of Michigan. Liver samples were extracted and proteins precipitated by addition of solvent containing labeled internal standards (Cambridge Isotopes, labeled carnitine standards set B, cat#NSK-B1). Weighed samples were sonicated briefly (10 % duty cycle, 2 min, on ice) in the extraction solvent (Methanol-Chloroform-Water (8:1:1). Acylcarnitine species were measured by MRM transitions using an Agilent 1260 HPLC and 6430 Triple Quadrupole LC/MS system (Agilent Technologies, Santa Clara, CA). All data were acquired and analyzed using Agilent Masshunter Quantitative software, version 6.0. Standard curves were generated and acylcarnitines were measured using isotope dilution internal standards. Data were normalized to wet tissue weight and analyzed using 2-way analysis of variance in SAS v9.4 (SAS Institute Inc., Cary, NC, USA).

Male, Acads−/− and Acads+/+ mice were fed the HF and LF experimental diets for 0, 1, or 2 days. On the day of tissue harvest, mice were euthanized with CO₂ between 10:00 and 11:00 am; the liver was quickly removed, frozen in liquid nitrogen and stored at −80 °C. Total protein was isolated using Lysis buffer (25 mM HEPES pH 7.8, 1 % Nonidet P-40, 50 mM KCl, 125 μM DTT, 1 mM PMSF, 5 mM NaPPi, 1 mM EDTA, 50 mM NaF including protease inhibitor cocktail). The samples were centrifuged and protein concentrations of the supernatants were determined using the BCA Protein Assay (Pierce Biotechnology, Rockford, IL). Electrophoretic separations were carried out on Mini-Protean II electrophoresis cells (Bio-Rad Laboratories, Inc., Hercules, CA). Supernatants (45 μg) were resolved by 10 % SDS-PAGE and subjected to immunoblotting. The protein was detected with antibodies against AMPKp(Thr¹⁷²), AMPK (Abcam Inc., Cambridge, MA) and beta Actin (HRP) (Abcam) using Chemiluminescence Reagent Plus (Perkin-Elmer Life Sciences). Signal intensity was quantified by densitometry analysis using ImageJ Software [12].

Male Acads−/− and Acads+/+ mice were fed the high fat (HF) diet for two days, as described above. On the morning of the experiment, mice were euthanized using CO₂ inhalation and liver tissue was collected in ice cold isolation buffer and subsequently homogenized according to the detailed method of Frezza et al. [13]. Mitochondrial protein content was measured using the BCA Protein Assay (Pierce Biotechnology, Rockford, IL). The preparation was kept on ice at all times and used within 6 h of isolation for mitochondrial bioenergetic measurements.

The oxygen consumption rate (OCR) of mitochondria during respiration was measured in the assay solution (MAS-1, 1X): 70 mM sucrose, 220 mM mannitol, 10 mM KH₂PO₄, 5 mM MgCl₂, 2 mM HEPES, 1 mM EGTA and 0.2 % BSA, using a Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica MA, USA) [14]. Ten (10) μg of freshly prepared mitochondria were added to each well in an initial volume of 50ul, and the plate was centrifuged at 1800 g, 4 °C for 10 min to insure that mitochondria adhered to the bottom of the well. Substrate mix was added and the plate was brought to equilibrium at 37 °C for 10 min before loading it into the XF24-3 and initiating the experiment. State 4 respiration was determined in the presence of either succinate/rotenone (5.5 mM/2.2uM), butyrate/malate (900uM/2 mM), or palmitoylcarnitine/malate (80uM/2 mM) as substrates. The substrates butyrate/malate and palmitoylcarnitine/malate were used to involve short- and long-chain acyl-CoA dehydrogenases, respectively. First, two baseline measurements of oxygen consumption rate (OCR) were obtained. Next, ADP (4 mM) was injected to determine state 3 respiration, followed by sequential injections of oligomycin (2uM), FCCP (4uM) and antimycin A (4uM). Collectively, this injection series allowed for determination of ATP-linked OCR (ADP), proton leak (oligomycin), maximal respiration (FCCP) and non-mitochondrial or residual oxygen consumption (antimycin A). Samples from individual animals were run in 2–3 replicates using typical mix and measurement cycle times [14]. Respiratory control ratios (RCR) were calculated as the state 3 respiratory rate divided by the state 4 respiratory rate, obtained from the ADP-linked/oligomycin OCR readings [15]. Data are displayed as absolute, point-to-point oxygen consumption rates (pmol/min/well) and were analyzed using the XF software and Microsoft Excel. OCR data represent the mean of 2–3 replicate wells ± SE. Differences in OCR between genotype groups were tested using a two-tailed Student’s t-test, with P values less than 0.05 considered to be statistically significant.

Mitochondrial DNA was quantified by determining the ratios of encoded mitochondrial cytochrome c oxidase (mt-Co2) and cytochrome b (mt-Cybt) to nuclear intron of hemoglobin beta (Hbb) and glucagon (Gcg), respectively, by qRT–PCR of isolated DNA. Total DNA was extracted from liver using the DNeasy Qiagen Kit (Qiagen, Germantown, MD) and amplified using published primer sequences [16] and quantitative PCR (7900HT Sequence Detection System; Life Technologies, Carlsbad, CA). All primers were synthesized by Sigma-Aldrich (St. Louis, MO, USA).

Mice used for tissue harvest in the microarrays showed no statistically significant differences in baseline body weight or total energy intake between the genotype (Acads−/−, Acads+/+) or diet (HF, LF) groups (Additional file 1: Table S1). Animals were euthanatized with CO₂ between 10:00 and 11:00 am on the morning of dissection. The liver was quickly removed, frozen in liquid nitrogen and stored at −80 °C. Total RNA from 12 animals (3 biological replicates in each of four diet-genotype comparisons) was isolated from liver using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA), and assessed for quality in an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). One μg of total RNA was used to transcribe DIG-labeled cRNA using AB Chemiluminescent RT-IVT Kit v2.0. Hybridization of 10 μg fragmented DIG-labeled cRNA to AB Mouse Genome Survey microarray (version 2.0), processing, chemiluminescence detection, imaging, auto-gridding, and image analysis were performed according to AB protocols, using the 1700 Chemiluminescent Microarray Analyzer Software v. 1.0.3. The AB Expression system software V2.0 was used to extract assay signal and signal-to-noise ratios from the images as previously described [17].

Signal intensities across microarrays were log transformed (base 2) and quantile normalized using the BRB-Array Tools software [18]. Statistical significance of the differentially expressed (DE) genes was ascertained via a regularized t-test based on the Bayesian statistical framework [19]. The magnitude of gene over-expression or under-expression was quantified by the difference in the log₂ average signals between the treatment and control groups. Using analysis of variance, gene expression profiles in liver were analyzed for main effects involving diet and genotype, as well as diet by genotype interactions [18]. The MIAME guidelines compliant microarray data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) repository, under accession number GSE35180.

Over-representation analysis (ORA) of canonical pathways was carried out by subjecting a pre-filtered list of differentially expressed genes with P < 0.01 and absolute fold-change ≥1.5-fold to Ingenuity Pathway Analysis (IPA). The Ingenuity Knowledge Base was used as the source for the reference genes. Fisher’s exact test p-values were used to estimate the significance of over-representation and corrections for multiple testing were made using the Benjamini-Hochberg procedure for false discovery rate [20]. Significantly enriched pathways were compared across treatments by 2-way clustering and then color coded according to the negative logarithm of their Fisher’s exact P-value.

IPA was further utilized to identify ‘upstream regulators’ whose activation/inhibition could explain the observed gene expression results. An overrepresentation analysis (Fisher’s exact test) was first performed to determine whether a regulator was enriched for differential expression of its target genes (the list of regulators and their target genes were obtained from the Ingenuity Knowledge Base). The overall activation/inhibition status of the regulator was inferred from the degree of consistency (up- or down-regulation) in the expression patterns of its target genes, expressed as a z-score. Regulators with z ≥ 2 or z ≤ −2 were considered to be activated or inhibited, respectively. A new score (C), combining the magnitude of the z-score with significance of overlap (P-value), was created according to the relationship: C = z-score * -logP. The C score was used to summarize the predicted effects from the upstream regulators.

Complementary DNA (cDNA) was obtained by reverse transcription (SuperScript III First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA, USA) of 4 μg of RNA from each liver sample. cDNA was subjected to QIAquick PCR purification columns (Qiagen, Valencia, CA, USA). For qRT-PCR, samples from 6–12 individuals were tested for the Acads−/− and Acads+/+ strains, respectively, using the microarray samples (3 per strain and condition) and additional samples collected under the same conditions.

Primers for amplification were designed using gene sequences obtained from the National Center for Biotechnology Information (NCBI) and Primer Express Software v3.0 (Life Technologies). Primers were synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA). The locations and sequences of primers are listed in Additional file 2: Table S2.

Gene expression levels were measured using the ABI PRISM 7900HT Sequence Detection System. Individual samples were run in triplicate. As an endogenous reference for normalization, we used the measurement of Cyclophilin (Ppia) cDNA in the same sample; Ppia levels were unaffected by diet and genotype on the arrays. Analysis was performed using SDS Software v2.3 for the 7900HT (Life Technologies). Relative quantification was calculated using the comparative C_T method (AB: User Bulletin #2: Relative quantification of gene expression. P/N 4303859 [21]. A two-tailed Student’s t-test was performed on ΔC_T values to evaluate strain differences in gene expression. P values of ≤ 0.05 were considered statistically significant.

Acylcarnitines are intermediary metabolites derived from mitochondrial acyl-CoA metabolism. To examine the biochemical effects of SCAD deficiency and dietary fat on lipid substrate metabolism in the liver, we measured acylcarnitines of Acads−/− and Acads+/+ mice fed either HF or LF fat diet for 2 days. The analysis of variance (ANOVA) revealed significant main effects of diet on twenty (20) acylcarnitines, and of genotype on twenty-five (25) acylcarnitines (Additional file 3: Table S3). For example, on HF diet, the liver content of C4 (butyryl) and C5 (isovaleryl) was 30-fold (P < 0.0001) and 2-fold (P < 0.0001) higher, respectively, in Acads−/− compared with Acads+/+ mice (Fig. 1), demonstrating the metabolic block. On LF diet however, the liver C4 content (P < 0.01) was only 4-fold higher in mutant animals. Six acylcarnitine species showed a significant diet-by-genotype interaction (see Additional file 3: Table S3). The accumulation of excess medium-chain and long-chain lipid byproducts, e.g., 3-fold higher levels of C12:0 and C14:0 acylcarnitines in HF-fed Acads−/− compared to HF-fed Acads+/+ could have resulted, in part, from the enrichment of medium chain fatty acids from coconut oil in the HF diet. In liver, 20–25 acylcarnitine species were affected by genotype or diet, compared to only 5 species in plasma, as shown previously [17], suggesting a dysregulation of hepatic mitochondrial beta oxidation.

Phosphorylation of AMP-kinase (pAMPK) is a key signal in the response to changes in energy balance including consumption of HF diet [22]. Cellular conditions that inhibit ATP production will result in a change in the AMP:ATP ratio and thus activate the AMPK signaling pathway. We therefore examined the effects of SCAD deficiency on hepatic pAMPK in Acads−/− mice fed HF diet for 2 d. Immunoblot analysis of liver tissue showed enhanced pAMP-kinase, compared to wild type controls, as evidenced by the ~40 % increase in (Thr-172) phosphorylation of the AMPK protein (Fig. 2). The adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylates numerous intracellular proteins and alters the transcription of genes involved in the regulation of energy metabolism including the transcriptional co-activation of PPAR-alpha. The net effect of AMPK activation is to turn off biosynthetic pathways such as lipogenesis and to activate catabolic pathways that produce ATP (e.g., fatty acid oxidation) [23]. This finding is consistent with a cellular “switch” from energy storage to energy release/use, suggesting that ATP availability is reduced under the conditions of impaired short chain fatty acid oxidation and high-fat diet, and agrees with our previous observation of AMPK activation in the hypothalamus of high-fat fed Acads−/− animals [17]. Finally, in light of previous reports that SCAD-deficient (also known as Acads−/−) mice are prone to fasting induced hypoglycemia [8], the animals in this experiment were tested a few hours after their most recent meal, when plasma glucose concentrations were similar between genotypes (data not shown). The absence of hypoglycemia in this study suggests that the observed AMPK activation in liver results from the metabolic effects of impaired short-chain fatty acid catabolism.

We found that pAMPK was significantly elevated in the liver of HF-fed, Acads−/− mice, suggesting a depletion of ATP. Mitochondria are the main sites of cellular ATP production from substrate oxidative phosphorylation [24] and play a critical role in fatty acid oxidation. Thus, we investigated the effects of SCAD deficiency on mitochondrial respiration in isolated hepatic mitochondria from Acads−/− mice fed HF diet. Because the SCAD protein is the first enzyme involved in the short-chain fatty acid beta oxidation spiral, we tested mitochondria for their ability to oxidize exogenously added fatty acids.

Energy balance is maintained by changes in feeding behavior and metabolism which can be regulated by gene expression. To identify the transcriptional responses to Acads inactivation and dietary fat, we used microarrays to profile the expression of 32,381 transcripts in the liver of Acads−/− and Acads+/+ mice fed either high- or low-fat diet. We found more pronounced differences in transcript abundance (absolute fold-change ≥ 1.5 and nominal P-value of <0.01) between the two diet comparisons than between the two genotype comparisons (Table 1).

Microarray analysis of gene expression in the liver, under the HF diet condition, revealed changes in 182 genes due to genotype, of which 108 genes showed increased levels in Acads−/− mice (Table 1). LF diet resulted in a considerably weaker transcriptional response, as only 72 genes were differentially expressed, with 31 increased in Acads−/− mice. Of these, 20 were common to both the HF and LF diets, with an equal number of up- and down-regulated transcripts (Fig. 4a and b). The extent of this overlap was statistically significant, based on the hypergeometric test (p < 2.92e-13). For example, we uncovered the genotype-regulated induction of Glo1, encoding glyoxalase 1, with both the HF and LF diets, by a factor of 2.0 and 2.5, respectively. The glyoxalase system acts to degrade endogenous reactive dicarbonyls such as glyoxal formed by lipid peroxidation and/or methylglyoxal derived from glycolysis [31]. Glyoxal and methylglyoxal are toxic byproducts of metabolism, which if not neutralized, can produce deleterious modifications of protein and DNA through glycation [32]. Activation of the hepatic glyoxalase pathway in Acads mutants, compared to wild type controls, suggests an enzymatic response to the accumulation of intermediates brought about by SCAD deficiency. The observed Glo1 induction in this model is consistent with the inferred activation of Nfe2l2/Nrf2 (see “Predicted Upstream Regulators” below), an oxidative stress response gene and transcriptional regulator of Glo1 [32]. A more detailed list of the top 25 up- and down-regulated genes for each of the above treatments is provided in Additional file 5: Table S4.

Two hundred and eighty-seven (287) diet-responsive genes were differentially expressed in Acads−/− mice, with 142 of them decreased in the HF fed group. By contrast in wild type controls, two hundred and eighty-four genes (284) were differentially expressed, with 187 genes down-regulated in the HF fed group. Eighty-three (83) genes were altered by high-fat diet in both Acads−/− and Acads+/+ mice (Fig. 4a) and of these, 50 were down-regulated. The overlap in these 83 genes was highly significant by the hypergeometric test (P < 9.87e-12). Diet-modified genes involved in fatty acid (FA) synthesis and elongation were strongly decreased by HF (vs. LF) diet in both mouse strains, usually with greater fold changes noted in Acads−/− animals. For example, Me1 (malic enzyme; also known as Mod1) was decreased by 5.8-fold in Acads−/− and by 3.5-fold in Acads+/+ mice. Me1 induction by carbohydrate consumption in the LF diet groups, independent of genotype, may be responsible for this outcome (Fig. 4b), producing enzymatic action that links the glycolytic and citric acid cycles. As another example, Elovl6 (elongation of very long chain fatty acids) was reduced by 25-fold in Acads−/− but only by 3.3-fold in wild type controls, suggesting a consequence of impaired SCFA oxidation in the mutants.

A total of 137 genes demonstrated significant diet x genotype interactions at the P < 0.01 level (Additional file 6: Table S5). For nearly all of these transcripts, the interaction model explained substantially more of the variation in expression than did a model based only on main effects. Twenty-nine (29) of these transcripts encode for mitochondrial proteins, e.g., Acsl3 (activates LCFAs for both synthesis and beta oxidation), Cyp27a1 (drug metabolism, cholesterol synthesis), Gpx1 (glutathione peroxidase), Ndufc2 (complex I mitochondrial enzyme), Uqcrfs1 (complex III), as well as three cytochrome c oxidase (Cox) components of complex IV: Cox5b, Cox7c and Cox11. Notably, genes in three complexes of the electron transport chain were affected by a genotype x diet interaction, and of these, Ndufc2, Uqcrfs1, and Cox11 were more responsive to genotype. The possibility that these specific results may reflect a secondary defect in oxidative phosphorylation, and thus ATP production, in the Acads−/− mice [33], remains speculative.

Canonical pathway analysis using IPA highlighted key alterations associated with short term high-fat diet and/or genotype (Fig. 5a), e.g., a significant inhibition of fatty acid and triacylglycerol synthesis in both Acads+/+ and Acads−/− liver. This process was reflected by the decreased expression of Scd1, Fasn, Srebf1, and Mod1 (fatty acid synthesis), as well as Gpam and Elovl6 (fatty acid storage and elongation). IPA analysis also found strong enrichment of “Fatty Acid Beta Oxidation” in HF-fed Acads−/− mice, e.g., genes involved in long-chain FA degradation (Acsl3, Acsl5, Slc17a2, Slc17a5) and in peroxisomal beta oxidation (see below).

In Acads−/− mice fed HF diet for 2 days, all three top enriched pathways involved Rxra (retinoid X receptor alpha), which forms heterodimers with other nuclear receptors such as PPARs [34], and is required for Ppara transcriptional activity on fatty acid oxidation genes [34, 35]. A potential mechanism for the induction of RXRA/PPARA pathways is butyryl CoA buildup in the cell [36], which is expected to occur with SCAD deficiency. Additionally, the transcriptional regulation of other genes is consistent with activation of alternate lipid handling pathways in the liver, including both beta- (mitochondria, peroxisomes) and omega-oxidation (microsomes). Long-chain fatty acids (C14-C20) are catabolized by either mitochondrial or peroxisomal beta-oxidation pathways, whereas very long-chain fatty acids (C22) are chain-shortened through peroxisomal beta-oxidation before being completely oxidized in the mitochondria [37]. The increased expression of Acaa1a, Ehhadh and Decr2, genes encoding key enzymes of peroxisomal beta-oxidation [38], provides evidence that peroxisomal oxidation was enhanced in Acads−/− mice, but not in wild type. Further support for alternate lipid handling was indicated by the 3-fold induction of Acot1 (acyl-CoA thioesterase 1) which predominantly hydrolyzes long-chain (C12-C16) acyl-CoA molecules. Evidence for an adaptive metabolic response was also observed in the 5 to 6-fold induction in Cyp4a14 and Cyp4a10 in Acads−/− liver by HF diet. The CYP4A subfamily of cytochrome P450 enzymes, located in the endoplasmic reticulum, function in microsomal omega oxidation, i.e., they metabolize medium and long chain length fatty acids at their omega-carbon atom to produce dicarboxylates as an alternative route to mitochondrial beta oxidation [39]. Normally, omega oxidation accounts for less than 10 % of total fatty acid oxidation in the liver [40], but when beta oxidation is defective, microsomal omega-oxidation may provide a means for eliminating toxic levels of free fatty acids [41]. For example, this pathway includes mitochondrial aldehyde dehydrogenase (Aldh2), encoding an antioxidant defense protein, which was induced by 5-fold in Acads−/− animals.

A comprehensive list of all the significantly enriched IPA pathways in each experimental comparison is provided in Additional file 7: Table S6.

To better understand the possible molecular basis for the observed transcriptomic changes, we investigated the predicted activation or inhibition of upstream transcription factors (TF) with at least 5 target genes in the list of differentially expressed genes (absolute fold change ≥ 1.5 and nominal p-value of <0.01) for a given diet or genotype comparison. In the diet comparison involving Acads−/− mice, we noted the significant, predicted activation of Nfe2l2 (nuclear factor, erythroid derived 2, like 2) as well as the significant inhibition of Mlx (MAX-like protein X), a transcriptional activator of glycolytic target genes (Fig. 6). The predicted activation or inhibition status for the full list of upstream regulators (beyond TFs) is shown in Additional file 8: Table S7. The similarities and dissimilarities in upstream regulator activation or inhibition across the 4 treatment groups are shown via hierarchical clustering in Additional file 9: Figure S2. 

Using differentially expressed genes obtained from the diet comparison (HF vs. LF) in Acads−/− animals, upstream regulator analysis revealed 24 significantly differentially expressed genes whose direction of expression was consistent with activation of the transcription factor Nfe2l2 (Fig. 7a). Nfe2l2 (synonymous with Nrf2) binds to antioxidant response elements (ARE) in the upstream promoter region and initiates the transcription of a variety of cyto-protective target genes involved in the response to oxidative stress caused by xenobiotic exposure and other factors [42]. Several of these genes (e.g., Gsta4, Cyp4a14) are also known to regulate various aspects of lipid metabolism, e.g., the alpha class of glutathione S-transferases, e.g., Gsta4, functions to detoxify products of lipid peroxidation, as illustrated by significant enrichment of the “Glutathione-mediated Detoxification” pathway, when both SCAD deficiency and HF diet are involved (Fig. 5a). As well, dysregulation of the cytochrome P450 (Cyp) 4a subfamily of genes by HF diet may be related to the demands for transformation of excess fatty acids which are substrates for Cyp4 isoforms. Overall, the inferred activation of Nfe2l2/ARE and the explicit induction of the Cyp4a subfamily in Acads−/− liver, appear to be directed at processing the incomplete products of mitochondrial beta oxidation [43]. Confirmation of this result will require a direct test of NFE2L2 activity or studies using Nfe2l2−/− mice.

Upstream regulator analysis also was performed on differentially expressed transcripts obtained by comparing genotypes (Acads−/− vs. Acads+/+) in HF-fed animals. Fourteen (14) gene targets were identified whose direction of expression suggested PPARA activation, including Cyp4a14 whose mRNA was elevated by nearly 9-fold (Fig. 7b). The predicted target genes represent the diverse functions of Ppara, including lipoprotein metabolism (Apoa1), microsomal omega oxidation (Cyp4a10, Cyp4a14), peroxisomal beta oxidation (Ehhadh), hydrolysis of fatty acyl-CoAs by thioesterases (Acot1), and biotransformation (Gstt2). Overall, these effects are consistent with the regulatory role of PPAR-alpha in energy metabolism, including all three hepatic fatty acid oxidation systems, i.e., mitochondrial and peroxisomal beta oxidation, as well as microsomal omega oxidation in the endoplasmic reticulum [38].

Both chronic high-fat feeding and fasting activate PPARA signaling in mouse liver, as part of the physiological response to an increased need for fatty acid oxidation [44]. In our short-term feeding experiment, the inferred PPARA activation may have been triggered by accumulated acylcarnitines, both in the circulation [17] and in liver tissue [45]. This result follows the pattern of PPARA induction reported previously for both the Lcad−/− and Vlcad−/− mouse strains with chow feeding [46]. It is also possible that PPARA signaling and fatty acid oxidation pathways were activated by a low energy state, as indicated by the enhanced hepatic pAMPK (Fig. 2) and reduced mitochondrial OCR in HF-fed Acads−/− mice (Fig. 3b).

To investigate the potential evidence for RXR-alpha activation and determine the effects of high-fat diet and SCAD deficiency on common partners of Rxr, we used quantitative RT-PCR to measure the expression of eleven nuclear receptor genes, as well as some glucose metabolism genes, in all four diet and genotype comparisons (total of 44 tests) (Table 2). Statistical significance for differential expression in microarray analysis was confirmed in 36/44 or 82 % of the tests. In addition to this validation study, we selected a set of 27 transcripts to represent metabolic processes and examined their expression as a function of diet in Acads−/− mice. The results showed significant (P < 0.05) differences between high- and low-fat diets in 23/27 or 85 % of the genes (Table 3). We observed 100 % agreement in the direction of expression changes between the two platforms, and thus have confidence that the qRT-PCR and microarray results are congruent, and that they reveal a predominant effect of diet, but not of genotype.

The transcriptional changes in hepatic fatty acid and glucose metabolism in Acads−/− mice fed HF or LF diet are summarized in Fig. 8, in a manner similar to that of Zhang et al. [47].

In general, fatty acids are catabolized in the mitochondria and/or peroxisomes to produce energy through beta oxidation while excess lipids are stored as triglycerides. We observed the increased expression of genes encoding enzymes involved in peroxisomal beta oxidation and in cytosolic hydrolysis of C12-C16 acyl-CoAs, as well as increased expression of genes involved in routing lipids for metabolism. These include FA transport genes (Abcc3, Slc27a2, Slc27a5) and peroxisomal Eci2 (formerly Peci) localized in both mitochondria and peroxisomes. In addition, acyl-CoA synthetase gene expression was decreased (e.g., Acsl5), which may promote the induction of peroxisomes as an alternate route of lipid disposal [48]. The increased expression of Rxra indicates activation of the PPAR/RXR signaling pathway, as do the upstream regulator analyses, particularly the increased expression of PPARA-target gene Cyp4a14 (Fig. 7b). The expression of genes encoding proteins involved in lipid biogenesis, as well fatty acid elongation and desaturation, was decreased in Acads−/− mice fed HF vs. LF diet, (Fig. 8).

The observed changes in gene expression are consistent with limited hepatic glucose utilization. For example, the expression of genes involved in glucose transport, as well as glycolysis and glycogen synthesis, including rate-limiting enzymes Pklr (−5.3-fold) and Gys2, respectively, was decreased. Furthermore, the expression of gluconeogenesis genes G6pc and Gpd2, together with rate-limiting enzyme fructose-1,6-bisphosphatase (Fbp1), was much lower in Acads−/− liver. Notably, we also observed a ~4-fold reduction in mitochondrial Pdk4, validated by qRT-PCR, along with a decrease in Pdk1 (Table 3), suggesting increased pyruvate dehydrogenase complex (PDC) activity. This result appears paradoxical, based on what is known about the transcriptional regulation of pyruvate dehydrogenase kinase, and the prediction of an up-regulation of Pdk4 in response to increased fat supply from the HF diet [49]. However, the decreased Pdk4 gene expression may reflect the short-term nature of diet exposure (2 days), possibly indicating that the metabolically active liver tissue has not yet “switched” on its PDC activity to maintain ATP levels, i.e., by regulating entry of glycolytic products into the tricarboxylic acid cycle. These results suggest that the altered fuel utilization in Acads−/− animals differs from that of the classic fed-fasted cycle [49].