Hepatic and muscular effects of different dietary fat content in VLCAD deficient mice

Molecular Genetics and Metabolism 104 (2011) 546–551

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Hepatic and muscular effects of different dietary fat content in VLCAD deficient mice Sonja Primassin ⁎, Sara Tucci, Ute Spiekerkoetter Department of General Pediatrics, University Children's Hospital, Duesseldorf, D-40225, Germany

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Article history: Received 25 July 2011 Received in revised form 8 September 2011 Accepted 8 September 2011 Available online 16 September 2011 Keywords: VLCAD Acylcarnitines Fatty acid oxidation Fat restriction Myopathy

a b s t r a c t Background: Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common long-chain fatty acid oxidation defect presenting with heterogeneous clinical phenotypes. Dietary fat plays a crucial role in disease pathogenesis and fat restriction is a common treatment measure. We here investigate the hepatic and muscular effects of a fat-enriched and a fat-restricted diet. Methods: VLCAD knock-out (KO) and wild-type (WT) mice are subjected to a fat-rich (10.6%), a fat-reduced (2.6%) or a regular mouse diet (5.1%) for 5 weeks. Analyses are performed at rest and after one hour exercise on a treadmill. Acylcarnitines in muscle as well as lipid and glycogen content in muscle and liver are quantified. Expression of genes involved in lipogenesis is measured by Real-Time-PCR. Results: At rest, VLCAD KO mice develop no clinical phenotype with all three diets, but importantly VLCAD KO mice cannot perform one hour exercise as compared to WT, this is especially apparent in mice with a fatreduced diet. Moreover, changes in dietary fat content induce a significant increase in muscular long-chain acylcarnitines and hepatic lipid content in VLCAD KO mice after exercise. A fat-reduced diet up-regulates hepatic lipogenesis at rest. At the same time, muscular glycogen is significantly lower than in WT. Conclusions: We here demonstrate that a fat-reduced and carbohydrate-enriched diet does not prevent the myopathic phenotype in VLCAD KO mice. An increase in dietary fat is safe at rest with respect to the muscle but results in a significant muscular acylcarnitine increase after exercise. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Mitochondrial β-oxidation defects are inherited in an autosomal recessive manner. Very long-chain acyl-coenzyme A dehydrogenase (VLCAD; EC 1.3.99.3) initializes the mitochondrial β-oxidation and is responsible for the first oxidation step of long-chain fatty acids. Deficiency of VLCAD leads to different clinical symptoms including cardiomyopathy, hepatic encephalopathy, hepatic steatosis and rhabdomyolysis under variable catabolic stress situations. VLCAD deficiency occurs with an incidence of up to 1:31.500 births [1]. There is a great heterogeneity of symptoms, severe and less severe forms are reported [2] as well as age- and stress-dependency [3]. The role of environmental factors is discussed but remains unclear. Newborn screening for many disorders of mitochondrial β-oxidation including VLCAD deficiency is performed in several countries worldwide. Presymptomatic identification can prevent catastrophic events of affected individuals, especially sudden death [4–7].

Abbreviations: ACC1α, acetyl CoA carboxylase 1 α; ESI-MS/MS, electron spray ionization tandem mass spectrometry; FAO, fatty acid oxidation; FASN, fatty acid synthase; FFA, free fatty acids; KO, knock-out; LCT, long-chain triglycerides; TAG, triglycerides; VLCAD, very-long-chain acyl-CoA dehydrogenase; WT, wild-type. ⁎ Corresponding author at: Department of General Pediatrics, University Children's Hospital, Moorenstraße 5, D-40225 Duesseldorf, Germany. Fax: + 49 211 811 6969. E-mail address: [email protected] (S. Primassin). 1096-7192/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2011.09.011

Dietary treatment is a widely discussed topic to prevent clinical symptoms in long-chain fatty acid oxidation (FAO) disorders [1]. In particular, fat restriction and fat modification with medium-chain triglycerides have been reported to be important treatment interventions [8] and several studies have proven their effectiveness [9,10]. However, treatment recommendations have in part been loosened in asymptomatic patients with expected mild deficiency identified by newborn screening with special respect to the initially suggested fat restriction. Important questions for clinicians are how many dietary longchain triglycerides (LCT) should be allowed and which effect may an LCT overload have on hepatic and muscular fat metabolism. We here studied the effects of an isocaloric fat-enriched diet and an isocaloric fat-reduced, carbohydrate-enriched diet on hepatic and muscular fat metabolism at rest and after exercise in the VLCAD knock-out (KO) mouse model. Special focus of our study was laid on the question whether a fat-reduced, carbohydrate-enriched diet is able to prevent myopathic symptoms as patients with milder phenotypes are often treated only by dietary fat restriction. 2. Materials and methods 2.1. Animals VLCAD KO mice were generated as previously described [11–13] and were provided by Prof. Dr. Arnold Strauss (former Vanderbilt

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University Hospital, Nashville, TN, USA, now University of Cincinnati, College of Medicine, Cincinnati, OH, USA). Genotypes were determined by duplicate polymerase chain reaction (PCR) analysis as previously described [13]. Experiments were performed on fourth- to fifth-generation intercrosses of C57BL6 + 129sv VLCAD genotypes. Littermates served as controls. At the age of 10–12 weeks, mice of both genotypes were analyzed under well-fed, resting conditions and after short-term exercise. They received their diet ad libitum. Animals were divided into three differently treated groups, each receiving a diet with a different amount of fat. Each group consisted of five mice (n = 5). All mice were weighed and sacrificed by cervical dislocation, the mice in the “exercise group” immediately after termination of the running protocol. Liver and skeletal muscle samples were rapidly removed and frozen immediately in liquid nitrogen. All animal studies were performed with the approval of the Heinrich-HeineUniversity Institutional Animal Care and Use Committee. Care of the animals was in accordance with the Heinrich-Heine-University Medical Centre and Institutional Animal Care and Use Committee guidelines. 2.2. Diet composition After being weaned, the first group of wild-type (WT) and VLCAD KO mice received a purified regular mouse diet containing 5.1% crude fat (ssniff® EF R/M Control, ssniff Spezialdiäten GmbH), corresponding to 13% of metabolizable energy from fat calculated with Atwater factors. This diet is subsequently referred to as control diet (Table 1). The second group of WT and VLCAD KO mice received a diet containing 10.6% of crude fat (ssniff® EF R/M control, ssniff GmbH) a two-fold amount of fat compared with the control diet corresponding to 24% of total metabolizable energy from fat calculated with Atwater factors. The protein content remained unchanged, but the starch content was reduced. This diet is subsequently referred to as fat-rich diet (Table 1). The third group of WT and VLCAD KO mice was fed with a diet containing 2.6% crude fat (ssniff® EF R/M control, ssniff GmbH), corresponding to 7% of metabolizable energy from fat calculated with Atwater factors. The fat content has been reduced to 50% of the control diet. The protein content remained unchanged and the starch content was increased. This diet is subsequently referred to as fat-reduced diet (Table 1). The diets were almost isocaloric and fed for 5 weeks ad libitum. 2.3. Exercise protocol As mice are nocturnal animals, treadmill running was performed during the dark cycle. Three-month-old WT and VLCAD KO animals were exercised 60 min on a Columbus Instruments Simplex II metabolic rodent treadmill consisting of four individual lanes without inclination and an electric shock grid (10 mAmp, frequency of 10 Hz). Mice were placed in an exercise chamber; after an adaptation period of 15 min initial belt speed was set to 4 m/min and increased every 5 min by 2 m/min to a maximum of 16 m/min. Mice were exercised until they either displayed signs of exhaustion or the exercise was Table 1 Nutrient content of the diets. Metabolizable energy (ME) is shown as percentage (%) of fat, carbohydrates and protein. Crude nutrients are shown as% of the total diet. Control

Fat-rich

Fat-reduced

ME [%] Fat Carbohydrates Protein ME [MJ/kg]

13 65 22 15.4

24 55 21 16.7

7 70 23 15

Crude nutrients [%] Crude fat Crude protein Starch Sugar

5.1 20.7 46.5 11.7

10.6 20.8 41.4 11.6

2.6 20.9 49.2 11.6

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terminated after 60 min. Exhaustion was defined as resting more than 15 s ∗ min − 1 on the electric shock grid or as falling back on the electric shock grid more than 15 times ∗ min − 1 [14]. 2.4. Analysis of acylcarnitines Analysis of long-chain acylcarnitines was performed in skeletal muscle as previously described [15]. Briefly, acylcarnitines were extracted from skeletal muscle in the presence of an internal standard ([ 2H3] C16 carnitine, for C14–C18 carnitines) with acetonitrile/water (ACN/H2O) (80/20% v/v). The extracted supernatant was dried and the butylated acylcarnitines were analyzed by electron spray ionization tandem mass spectrometry (ESI-MS/MS). All even-chain C14– C18 acylcarnitines (saturated and unsaturated) were measured. 2.5. Lipid analysis Free fatty acid (FFA) concentrations in serum samples and liver and serum triglycerides (TAGs) were measured as duplicates by using enzymatic kits (EnzyChrom™ Free Fatty Acid Assay Kit and EnzyChrom™ Triglyceride Assay Kit, BioTrend, Cologne, respectively) on an Infinite M200 Tecan (Crailsheim, Germany) plate reader. The assays were performed following the manufacturer's instructions. The intrahepatic lipid content was measured gravimetrically according to a method by Folch et al. [16] modified as previously reported [17]. 2.6. Glycogen analysis Glycogen concentrations in muscle and liver samples were measured as duplicates by using an enzymatic kit (EnzyChrom™ Glycogen Assay Kit, BioTrend, Cologne) on an Infinite M200 Tecan (Crailsheim, Germany) plate reader. The assays were performed following the manufacturer's instructions. 2.7. Real-time-PCR analysis Total liver RNA was isolated with the RNeasy mini kit (Qiagen, Hilden Germany). Forward and reverse primers for β-actin, fatty acid synthase (FASN) and acetyl-CoA carboxylase 1α (ACC1α) were designed with the FastPCR program (R. Kalendar, Institute of Biotechnology, Helsinki) and are available on request. Real-Time-PCR was performed in a single step procedure with QuantiTect SYBR Green™ RT-PCR (Qiagen, Hilden, Germany) on an Applied Biosystems 7500HT Sequence Detection System in Micro Amp 96-well optical reaction plates capped with MicroAmp optical caps (Applied Biosystems, Foster City, CA, USA) as previously described [18]. The values in all samples were normalized to the expression level of the internal standard. 2.8. Statistical analysis ESI-MS/MS-Data were acquired and analyzed using MassLynx NT v4.0 (Micromass, UK). All Data are presented as the mean± standard error of the mean (SEM). Statistical analysis of differences between two means and multiple means was compared by a two-way analysis of the variance (ANOVA) with a Bonferroni post hoc test (GraphPad Prism 5.0, San Diego, CA, USA). A probability level of p b 0.05 was regarded as significant. 3. Results 3.1. Clinical phenotype In this study we could demonstrate that in the VLCAD KO mouse model a high-fat diet and a low-fat diet were not associated with development of clinical symptoms when applied for 5 weeks under

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non stressed conditions. Mean body weights were not significantly different between VLCAD KO mice and WT littermates (Supplemental Table). Importantly, also the mean body weights were not significantly different between WT and VLCAD KO mice after modification of the fat content of the diet for 5 weeks (Supplemental Table). Food intake, behavior and activity level also remained unchanged under different dietary protocols. We do not have exact data on how much the mice consumed, but the similar body weights obtained after dietary modification suggest that the energy consumed was similar as the diets were iso-caloric. All WT mice independent of the dietary group were able to complete the running protocol at 16 m/min over the maximum running time of 60 min. Running time was reduced in VLCAD KO mice compared with WT mice in each dietary group due to exhaustion (Table 2). Running times were not significantly different between VLCAD KO mice in the different groups suggesting that a carbohydrate-enriched, lower fat diet did not improve their physical capability. On the other hand, as shown in Table 2, a high fat diet did not worsen myopathy clinically. 3.2. Acylcarnitines in skeletal muscle as parameters of energy deficiency Accumulation of long-chain acylcarnitines is an efficient parameter reflecting energy deficiency as it demonstrates increased energy demand that cannot be sufficiently provided from fat in long-chain FAOD. Under resting conditions the long-chain acylcarnitine concentrations in skeletal muscle of VLCAD KO mice remained unchanged and were not significantly different compared to WT mice under all dietary regimens (Fig. 1). Changes in long-chain acylcarnitine concentrations were most significant in skeletal muscle after exercise. In that, the fatrich diet resulted in an enormous increase of long-chain acylcarnitines with values of 250 (±80) nmol/g wet weight. A similar acylcarnitine accumulation was also observed in VLCAD KO mice fed with the fatreduced diet (Fig. 1), whereas muscular acylcarnitines increased much less after exercise under a regular diet.

Fig. 1. Acylcarnitine concentrations in skeletal muscle of WT (n = 5) and VLCAD KO mice (n = 5). Acylcarnitine concentrations are presented in nmol/g wet weight. White bars and black bars represent WT and VLCAD KO mice, respectively. Values are mean ± SEM. *p b 0.05 indicates significant differences between WT and VLCAD KO mice performed by Student's t test, #p b 0.05 indicates significant differences between VLCAD KO mice in different dietary groups performed by two-way ANOVA and §p b 0.05 indicates significant differences between resting and exercised VLCAD KO mice within one dietary treatment group. Samples were analyzed in duplicate.

Real-time PCR revealed that at rest under a low fat, high carbohydrate diet the expression of lipogenic genes was significantly up-regulated at mRNA level in both genotypes compared to mice under the control

3.3. Hepatic lipid accumulation After exercise, intrahepatic lipid content significantly increased in VLCAD KO mice under both dietary regimens (fat-reduced and fat-rich) in contrast to VLCAD KO mice fed with the control diet (Fig. 2A). This was in line with significantly increased TAG concentrations in liver after exercise in VLCAD KO mice fed with both the fat-rich and the fat-reduced diet. The liver TAG content in WT and VLCAD KO mice fed with the fat-rich diet after exercise corresponded to 184.52 ± 34.29 nmol/mg and 350.34 ±20.83 nmol/mg, respectively (Fig. 2B). The fat reduced diet resulted after exercise in a much higher TAG accumulation with values of 273.64 ±69.86 nmol/mg and 615.5± 61.86 nmol/mg for WT and VLCAD KO mice, respectively (Fig. 2B). Signs of liver dysfunction were not observed with increased intrahepatic lipid content. 3.4. Fat-reduced diet results in up-regulation of lipogenic genes Two hepatic lipogenic genes namely ACC1α and FASN involved in de novo biosynthesis and elongation of fatty acids were studied.

Table 2 Running time [min] of VLCAD KO mice and their WT littermates after 5 weeks of dietary application with an average speed of 16 m/min. Diets

n

WT

KO

Control Fat-rich Fat-reduced

5 5 5

60 (± 0.0) 60 (± 0.0) 60 (± 0.0)

55 (± 1.2)* 54 (± 3.2)* 49 (± 5.1)*

Values are mean ± SEM. *Values were considered significant if p b 0.05. *Indicates significant differences between WT and VLCAD−/− mice within one dietary group.

Fig. 2. Intrahepatic lipid content (A) and triglyceride (TAG) content (B). Mean concentrations are expressed in dry weight (% dw) for the lipid content and nmol/mg for TAG content. The values represent mean ± SEM for WT (n = 5) and VLCAD KO (n = 5) mice per dietary group under resting conditions and after exercise. White bars and black bars represent WT and VLCAD KO mice, respectively. *p b 0.05 indicates significant differences between WT and VLCAD KO mice performed by Student's t test, #p b 0.05 indicates significant differences between VLCAD KO mice in different dietary groups performed by two-way ANOVA and §p b 0.05 indicates significant differences between resting and exercised VLCAD KO mice within one dietary treatment group. Samples were analyzed in duplicate.

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diet (Figs. 3A/B). Moreover, up-regulation was significantly higher in VLCAD KO mice as compared to WT mice.

3.5. Serum lipid profile Under a high-fat intake, serum TAG concentrations were strongly increased in VLCAD KO mice after exercise as compared to WT mice under the same dietary regimen and as compared to mice under other diets (Supplemental Fig. A). In addition, FFAs were significantly elevated in VLCAD KO mice at rest under the fat-rich diet. Here, serum FFA concentrations did not further increase after exercise. An interesting observation was also, that KO mice under a fat-reduced diet also presented with significantly higher FFA concentrations after exercise as compared to WT mice (Supplemental Fig. B).

3.6. Glycogen as alternative energy source At rest both genotypes displayed significantly higher glycogen concentrations in skeletal muscle under the fat-rich diet as compared to mice with a regular diet (Fig. 4A). Interestingly, WT mice fed with the fat-reduced diet also displayed significantly higher glycogen levels in skeletal muscle, whereas this was not observed in VLCAD KO mice. After exercise, muscle glycogen stores were depleted in all dietary groups. In a similar manner, liver glycogen significantly decreased after exercise in all dietary groups compared to glycogen values at rest, as shown in Fig. 4B. Here, WT but not VLCAD KO mice fed with the fat-rich diet displayed significantly higher glycogen concentrations after exercise than control mice.

Fig. 4. Glycogen concentrations in skeletal muscle (A) and liver (B). Mean concentrations are expressed in mg/g dry weight. The values are mean ± SEM for WT (n = 5) and VLCAD KO (n = 5) mice per dietary group under resting conditions and after exercise. White bars and black bars represent WT and VLCAD KO mice, respectively. Values were considered significant if p b 0.05. *Indicates significant differences between WT and VLCAD KO mice within a group. #Indicates significant differences between WT or VLCAD KO mice under different dietary conditions. §Indicates significant differences between resting and exercised under the same dietary regimen. Significances were calculated by Student's t test and two-way ANOVA with Bonferroni post-test.

4. Discussion In the present study, we demonstrate that an isocaloric fatreduced and carbohydrate-enriched diet cannot prevent the skeletal muscle phenotype in VLCAD-deficient mice after physical exercise. In fact, despite carbohydrate enrichment the increased muscular energy demand during exercise cannot be supplied. Moreover, this dietary modification induces hepatic lipogenesis during a feeding period of 5 weeks. Since dietary fat restriction is part of the recommended treatment in long-chain fatty acid oxidation defects [8], this observation is of great clinical relevance. In fact, many VLCAD-deficient patients receive a fat-reduced, carbohydrate-enriched diet, but they still suffer from myopathic symptoms despite treatment [8]. With respect to this observation, an important question is whether a higher fat intake would be harmful, which has been proposed for a long time. 4.1. Low fat, carbohydrate-enriched diet

Fig. 3. Relative expression of FASN (A) and ACC-1α (B) genes at mRNA level. Mean concentrations are expressed as % and control WT mice were set as 100%. White bars and black bars represent WT and VLCAD KO mice, respectively. Values are mean ± SEM for WT (n = 5) and VLCAD KO (n = 5) under resting conditions. *p b 0.05 indicates significant differences between WT and VLCAD KO mice performed by Student's t test, #p b 0.05 indicates significant differences between VLCAD KO mice in different dietary groups performed by two-way ANOVA. Samples were analyzed in triplicate.

An excessive supplementation of carbohydrates is known to stimulate hepatic lipogenesis [19,20]. Accordingly, we observed a strong upregulation of the lipogenic genes ACC1α and FASN in the liver of VLCAD KO mice with the fat-reduced diet. These results confirm that the overload of carbohydrates is not used but will be converted and stored as fatty acids or TAGs [21,22]. Importantly, also during exercise these carbohydrates do not supply sufficient energy resulting in significant lipolysis and significant accumulation of liver lipids and TAGs in VLCAD KO mice. Other signs of insufficient energy supply from fat are a marked accumulation of acylcarnitines in skeletal muscle [14] coupled with a depletion of muscular glycogen. Whereas high dietary

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carbohydrate content is known to result in increased hepatic glycogen stores [23] as also observed in our WT mice, we here demonstrate significantly lower levels in VLCAD KO mice at rest suggesting that these mice already rely on glucose oxidation during daily life and, thereby may compensate the enzyme defect. For endurance exercise, however, an intact FAO would be essential to contribute to energy production resulting in clinical symptoms as observed by a reduced running capability in our KO mice. 4.2. High-fat diet Despite defective FAO, it is surprising, that a dietary fat overload for 5 weeks does not induce an obvious clinical phenotype in VLCAD KO mice under resting conditions. Whereas, a previous study in WT mice demonstrated that a short-term high fat diet up to 28 days has a deep impact on metabolic response in skeletal muscle [24]. According to this cited study, besides metabolic adaptations, muscle undergoes morphological changes with promotion of more oxidative type I fibers rich in mitochondria in the beginning followed by a strong decrease in oxidative capacity during further follow-up [24]. We, therefore, may suspect that a fat-enriched diet has similar morphological effects in VLCAD KO mice, promoting more severe muscular symptoms during exercise with a high fat diet as being reflected by the tremendous increase in muscular acylcarnitines after exercise. This hypothesis would be supported by a higher muscle glycogen content under the fat-enriched diet as compared to the control diet suggesting reduced use of glucose for energy production with increased concentration of type I fibers. Moreover, liver glycogen, an important energy supplier during intensive physical activity [25], was not completely depleted in both genotypes. In fact, glycogen was six-fold higher in the mean under a fat-rich diet (Fig. 4B) as compared to the control diet. After performance of running, liver lipid and triglyceride content strongly increased confirming a high lipolytic rate and energy deficiency during exercise [26]. Overall, fat restriction and fat modification are important interventions in the treatment of VLCAD deficiency. Studies in VLCAD KO mice as well as in VLCAD-deficient patients already suggest that the treatment should be individually adapted especially with respect to the energy demand [10,27]. Furthermore, it has been reported that changes in the daily fat or carbohydrate content do not improve physical capabilities as demonstrated by no beneficial effects in athletes [29]. Since a chronic intake of an imbalanced macronutrient diet may result in severe life-style-related diseases [28], long-term studies need to prove the clear efficacy of these dietary modifications in VLCAD deficient patients. 5. Conclusions In summary, we here demonstrate that the application of either a fat-rich or a fat-reduced, carbohydrate-enriched diet for 5 weeks does not have negative biochemical and clinical effects at rest in the VLCAD KO mouse. However, the fat overload induces a dramatic worsening of the biochemical phenotype after exercise with significant intramuscular acylcarnitine elevation. Running capacity, however, is not worsening with a fat rich diet. More important, a fat-reduced, carbohydrate-enriched diet, although recommended as therapy, is not able to provide the required energy during exercise and also results in significant intramuscular acylcarnitine accumulation as sign of activated fatty acid oxidation. Supplementary materials related to this article can be found online at doi:10.1016/j.ymgme.2011.09.011. Acknowledgments The study was financially supported by grants from the Deutsche Forschungsgemeinschaft (DFG: SFB 575 and SFB 612).

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