Loss of lysosomal acid lipase results in mitochondrial dysfunction and fiber switch in skeletal muscles of mice

Objective Lysosomal acid lipase (LAL) is the only enzyme known to hydrolyze cholesteryl esters (CE) and triacylglycerols in lysosomes at an acidic pH. Despite the importance of lysosomal hydrolysis in skeletal muscle (SM), research in this area is limited. We hypothesized that LAL may play an important role in SM development, function, and metabolism as a result of lipid and/or carbohydrate metabolism disruptions. Results Mice with systemic LAL deficiency (Lal−/−) had markedly lower SM mass, cross-sectional area, and Feret diameter despite unchanged proteolysis or protein synthesis markers in all SM examined. In addition, Lal−/− SM showed increased total cholesterol and CE concentrations, especially during fasting and maturation. Regardless of increased glucose uptake, expression of the slow oxidative fiber marker MYH7 was markedly increased in Lal−/−SM, indicating a fiber switch from glycolytic, fast-twitch fibers to oxidative, slow-twitch fibers. Proteomic analysis of the oxidative and glycolytic parts of the SM confirmed the transition between fast- and slow-twitch fibers, consistent with the decreased Lal−/− muscle size due to the “fiber paradox”. Decreased oxidative capacity and ATP concentration were associated with reduced mitochondrial function of Lal−/− SM, particularly affecting oxidative phosphorylation, despite unchanged structure and number of mitochondria. Impairment in muscle function was reflected by increased exhaustion in the treadmill peak effort test in vivo. Conclusion We conclude that whole-body loss of LAL is associated with a profound remodeling of the muscular phenotype, manifested by fiber type switch and a decline in muscle mass, most likely due to dysfunctional mitochondria and impaired energy metabolism, at least in mice.


INTRODUCTION
Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triacylglycerols (TG) at acidic pH [1,2].Congenital mutations of the LALencoding LIPA gene cause a substantial reduction in enzyme activity, resulting in a massive accumulation of neutral lipids in lysosomes [3,4], ultimately leading to cellular dysfunction and damage of various cells and organs.The main organs affected by LAL deficiency (LAL-D) are the liver, spleen, adrenal glands, small intestine, and the vasculature [5].The infantile form of LAL-D, formerly known as Wolman disease, is a severe and rapidly progressive condition that develops within the first few weeks of life.In these patients, LAL activity is 1e2% of mean normal levels, and patients die at a median age of 3.7 months [6].The disease manifests with nausea, vomiting, intestinal malabsorption, impaired growth and development, and severe liver damage [7], rapidly progressing to fibrosis and cirrhosis [8].In contrast, lateonset LAL-D, formerly known as CE storage disease, occurs between 2 and 60 years of age and may be latent and asymptomatic; thus, it is often not diagnosed until a routine physical examination and biochemical blood tests are performed.Symptoms include liver damage such as hepatomegaly, elevated alanine and aspartate aminotransferase, steatosis, liver fibrosis, and cirrhosis.In addition, splenomegaly and concomitant pathologies such as anemia and thrombocytopenia may be observed, as well as early development of atherosclerosis [5,9].
In contrast to humans with complete loss of LAL, Lal-deficient (LalÀ/ À) mice [10] are viable with a median lifespan of almost one year.Similar to LAL-D patients, they exhibit ectopic CE and TG accumulation, particularly affecting the liver, spleen, adrenal glands, and small intestine [11].Moreover, the animals suffer from growth retardation and progressive loss of white and brown adipose tissue [10].LalÀ/À mice represent a genetic model for early-onset LAL-D but phenotypically and histopathologically more closely resemble patients suffering from late-onset LAL-D [12,13].Reduced intestinal lipid absorption [14,15], decreased plasma leptin concentrations, and lipodystrophy lead to impaired lipid metabolism in LalÀ/À mice, resulting in reduced circulating TG levels in the fasted state [16].In addition, TG and CE cannot be hydrolyzed in the absence of LAL and remain entrapped in the lysosomes of the LalÀ/À liver.To adapt to the resulting reduced availability of fatty acids (FA), glucose consumption is increased, leading to a decrease in plasma and liver glucose levels as well as hepatic glycogen content [16].The SM is a remarkably malleable organ that undergoes substantial remodeling in response to a wide range of different stimuli such as nutrient content or physical activity [17e19].Recent studies have suggested that lysosomes play a critical role in maintaining SM mass, particularly as an intracellular signaling hub for activation of the mechanistic target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB)/signaling in conjunction with lysosome biogenesis in regulating mTORC1-mediated protein synthesis [20].However, how impaired lipid processing in lysosomes affects SM has not yet been well described.As SM is not able to store excess lipids like adipose tissue, FA that enter the SM are either oxidized for energy production or used for other metabolic processes.For this reason, lipid degradation by autophagy may be difficult to detect under these conditions [21].However, FA exported from the lysosome are known to be intensively utilized by the SM for mitochondrial uptake, oxidation, and ATP synthesis to maintain cellular bioenergetics during stress such as physical activity [22].Previous reports suggested decreased SM activity [23,24] and mass [25e28] in LAL-D patients, but the role of LAL in the metabolic balance of SM has never been thoroughly investigated.Given the high energy demand of SM, we hypothesized that changes in energy homeostasis caused by the loss of LAL might affect muscle biology.To test our hypothesis, we compared SM from LalÀ/À mice with those of their wild-type (Wt) littermates.We found that muscles from LalÀ/À mice exhibited morphological and biochemical abnormalities that affected SM phenotype, fiber type distribution, proteomic profiles, and mitochondrial functions, which were associated with compromised exercise performance on a treadmill.

Animals
Male young (8e12 or 12e17 weeks of age) and mature (40e55 weeks of age), as well as female young (19e21 weeks of age) and mature (33e44 weeks of age) LalÀ/À mice and their corresponding Wt littermates on a C57BL/6J background were used for the experiments.We analyzed quadriceps (QU), gastrocnemius (GA), tibialis anterior (TA) (higher percentage of fast-twitch glycolytic fibers), and soleus (SO) (higher percentage of slow-twitch oxidative fibers) harvested under fed and 6-or 12-h fasted conditions.Mice were maintained in a clean, temperature-controlled (22 AE 1 C) environment on a regular lightedark cycle (12-h light, 12-h dark).Animals had unlimited access to chow diet (4 % fat and 19 % protein; Altromin 1324, Lage, Germany).All animal experiments were performed according to the European Directive 2010/63/EU in compliance with national laws and were approved by the Austrian Federal Ministry of Education, Science and Research, Vienna, Austria (2020-0.129.904;2022-0.121.513;BMWFW-66.010/0081-WF/V/3b/2017).

Proteasome activity assay
To determine chymotrypsin-like, trypsin-like, and caspase-like activities in SM, we used the Proteasome Activity Fluorometric Assay Kit II (UBPBio, Aurora, CO) according to the manufacturer's protocol.The standard curve was used to calculate the absolute amounts of released 7-amino-4-methylcoumarin (AMC) fluorescence in each sample.

Amino acid (AA) quantification
Plasma samples (70 mL) from fed and fasted female mice (aged 33e 44 weeks) were mixed with 50 mL of 1.5 M perchloric acid, vortexed, and kept at RT for 2 min before adding 1.125 mL of water and 25 mL of 2 M K 2 CO 3 .The tubes were again vortexed, centrifuged at 3,000Âg for 5 min, and the supernatant was collected and stored at À80 C until analysis.AA were separated using a high-performance liquid chromatography (HPLC) system equipped with an LC20AD pump (Shimadzu, Kyoto, Japan Snap-frozen GA samples from young male mice were processed as described previously [29].NMR spectra were processed and analyzed as recently described [30]. 2.8.Measurement of ATP by mass spectrometry SM samples from young male mice (w25 mg) were transferred to

Measurement of mitochondrial respiration and fatty acid oxidation (FAO)
Oxygen consumption for estimation of mitochondrial respiration and FA oxidation (FAO) in permeabilized SM fibers was measured with a highresolution Oxygraph-2k respirometer (Oroboros Instruments, Innsbruck, Austria) as described previously [33].Briefly, the predominantly oxidative part of GA was separated into small bundles and permeabilized with saponin (50 mg/mL).One to 3 mg of the fibers were transferred to the calibrated respirometer, which contained 2 mL of respiration medium (MiRO6; 110 mM D-sucrose, 60 mM potassium lactobionate, 0.5 mM EGTA, 3 mM MgCl 2 , 20 mM taurine, 10 mM KH 2 PO 4 , 20 mM HEPES, 1 g/L bovine serum albumin, and w280 U/mL catalase) in each chamber.Substrate/uncoupler/inhibitor (SUIT) protocol 11 was used to examine mitochondrial respiration.The following substrates and inhibitors were added sequentially after the oxygen slope was stable: 10 mM malate followed by 10 mM glutamate (basal respiration), 5 mM ADP (active ADP-stimulated state), 10 mM cytochrome C, 10 mM succinate, 1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP), 0.5 mM rotenone, and 2.5 mM antimycin.To analyze FAO, we used the SUIT-005 protocol with some modifications.The following substrates and inhibitors were added sequentially after the oxygen slope was stable: 100 mM and 2 mM malate (basal respiration and respiratory stimulation of FAO) (Fpathway), followed by 25 mM ADP, 10 mM cytochrome C, 5 mM pyruvate (respiratory stimulation by simultaneous action of the F-pathway and NADH electron transfer pathway (ETP, N-pathway) together with convergent electron), 10 mM succinate, 1 mM CCCP, 0.5 mM rotenone, and 2.5 mM antimycin.Respiration signals were analyzed using DatLab O2k-6 software.
2.13.Electron microscopy GA was collected from LalÀ/À and corresponding control mice perfused with 4 % paraformaldehyde.The muscles were then processed as previously described [31].

Quantification of the number of mitochondria
The concentration of extracted DNA was estimated using Nanodrop (Peqlab, Darmstadt, Germany) and diluted to 10 ng/mL for qPCR amplification to compare the expression of 16S (mitochondrial gene) with that of hexokinase 2 (hk2, nuclear gene).Primer sequences are listed in Table S1.The number of mitochondria was determined from the ratio of mitochondrial (mt) to nuclear (n)DNA as previously described [34].
2.15.Measurement of maximum O 2 consumption (VO 2 max ) and peak effort testing using treadmills Effort tolerance, peak effort, and maximal oxygen consumption (VO 2 max ) of Wt and LalÀ/À mice were studied using a motorized treadmill coupled to a calorimetric unit with gas analyzer (CaloTreadmill, TSE Systems GmbH, Bad Homburg, Germany) as described previously [35].Briefly, mice were subjected to a ramp running protocol with an initial adaptation velocity of 3 m/min for 60 s, followed by a constant acceleration of 3 m/min without inclination.The exercise session ended at maximal exhaustion, defined as the animal's inability to maintain running speed despite being in contact with the electrical grid for more than 5 s.VO 2 max and run distance were determined at the point at which oxygen uptake reached a plateau during exhaustive exercise.Maximum workload was calculated as the final running distance multiplied by body weight and divided by 1,000.

Sample preparation and processing for proteomics analysis
The "red" deep proximal and medial and the "white" most superficial parts of GA were lysed in 100 mM TriseHCl (pH 8.

Reduced cross-sectional area and SM mass in Lal À/À mice
To study the consequences of LAL loss in SM, we isolated gastrocnemius (GA), quadriceps (QU), tibialis anterior (TA), and soleus (SO) and compared the mass between male Wt and LalÀ/À mice in the fed (15e16 weeks of age, designated young) and fasted (40e55 weeks of age, designated mature) states.The absolute (Figure 1A) and relative weights (Figs.S1B and C) of QU, TA, and GA were drastically reduced in LalÀ/À mice at young and mature ages, whereas the body weights of LalÀ/À mice were approx.30 % lower compared to controls (Fig. S1A).The weight of SO remained unchanged and comparable with those of Wt mice (Figure 1A, S1B,C).Of note, the muscles from LalÀ/À mice were paler than the muscles from Wt mice (Fig. S1D).To quantitatively and qualitatively assess muscle fibers and estimate the muscular phenotype, we determined the cross-sectional area (CSA) and minimum Feret diameter of myofibers by quantifying the laminin-stained area with Image J. Consistent with the reduced muscle mass in LalÀ/À mice, the mean fiber CSA (Figure 1B,C) and Feret diameter (Figure 1B,D) of QU, TA, and GA were significantly lower in LalÀ/À mice compared to their Wt littermates.Examination of the SM sections and use of the "Muscle morphometry" plugin of Image J failed to reveal signs of myopathy, such as centronucleated myofibers, in either genotype.In summary, these findings demonstrate reduced muscle size in LalÀ/À mice.
3.2.Unaffected protein turnover in SM of LalÀ/À mice During cold exposure, chow diet-fed LalÀ/À mice utilize their muscle AA as an additional energy sources, as indicated by the upregulation of the muscle proteolysis markers Murf1 and Atrogin1 in GA [41].In contrast to the cold-exposed animals, Murf1 and Atrogin1 mRNA levels were downregulated in GA (Figure 2A), QU (Fig. S2A), and TA (Fig. S2B) but not in SO (Fig. S2C) of LalÀ/À mice in the fed state, whereas no difference was evident in the fasted state in GA (Figure 2B), QU (Fig. S2D), TA (Fig. S2E), and SO (Fig. S2F).One of the key metabolic mechanisms controlling muscle wasting is the ubiquitin proteasome system [42].In GA (Figure 2C), QU (Fig. S2G), and TA (Fig. S2H) from LalÀ/À mice, chymotrypsin-like activity was reduced, whereas trypsin-like and caspase-like proteasome activities were comparable between the genotypes, arguing against activation of the ubiquitin proteasome system to degrade proteins for energy supply in LalÀ/À SM.Analysis of plasma AA concentrations revealed decreased abundance of the glucogenic AA glutamine (Gln) in the fed condition (Figure 2D).Increased concentrations of branched-chain AA such as valine (Val) (Figure 2D), glucogenic/ketogenic isoleucine (Ile) (Fig. S2I), and the ketogenic AA leucine (Leu) (Fig. S2J) in fasted LalÀ/ À mice indicate minor alterations in AA metabolism, however, may not be associated with muscle fiber degradation in LalÀ/À mice.The IGF-1/Akt/mTOR pathway is a crucial intracellular regulator of muscle mass [43e45].However, we failed to detect any significant changes in the ratio of pAkt/Akt and p4eBP1/4eBP1 protein expression in LalÀ/À QU (Figure 2E,F) and GA (Figs.S2K and L) as well as Igf1 (Fig. S2M) gene expression in QU and GA from fed LalÀ/À and Wt mice.

Loss of LAL impacts SM energy metabolism
The intramuscular stores of ATP are generally relatively small [46].The markedly decreased ATP concentrations in QU of LalÀ/À mice (Figure 3A) would have to be compensated by activation of metabolic pathways such as phosphocreatine and muscle glycogen degradation followed by anaerobic glycolysis and aerobic carbohydrate and lipid catabolism [46,47].Creatine and glycogen concentrations were slightly increased in the GA of LalÀ/À mice (Figs.S3A and B).Consistent with our previous observation of reduced circulating glucose levels and increased glucose uptake in SM [16], we observed increased [ 3 H]2deoxy-D-glucose uptake in all SM examined (Figure 3B).Surprisingly, glucose uptake was increased not only in SM rich in glycolytic fibers but also in the highly oxidative SO.LalÀ/À mice accumulate lipid-laden lysosomes in ectopic tissues such as the liver, spleen, and small intestine [12,15].In contrast to these tissues, we found increased CE concentrations in ad libitum -fed LalÀ/À mice only in GA (Figure 3C), whereas in SO we found only a tendency toward increased CE but elevated concentration of FC (Fig. S3C).Levels of cholesterols in QU and TA (Figs.S3D and E) as well as TG were comparable in all SM (Fig. 3C, S3C-E).In the fasted state, mature LalÀ/À mice showed a drastic decrease in TG concentrations in QU (Figure 3D) and trends to decreased concentrations in TA (Figure S3F), GA (Fig. S3G), and SO (Fig. S3H).We additionally observed higher concentrations of CE in QU (Figure 3D), TA (Fig. S3F), and GA (Fig. S3G) in LalÀ/À mice compared to the corresponding littermates, apparently due to the increased TC concentrations also in SO (Fig. S3H).During fasting, lipid oxidation is the predominant source of energy in the resting SM [48].Since LalÀ/À mice suffer from loss of adipose tissue [12] and have reduced circulating TG levels in the fasted state [16], we next analyzed whether FAO was affected in LalÀ/À SM.Despite comparable mRNA expression of genes involved in FA transport and oxidation, with the exception of slightly increased Cpt1b expression (Fig. S3K), total acyl-carnitine (Figure 3E) and individual acyl-carnitine concentrations were markedly reduced in QU (Figure 3F), SO (Figure S3I), and TA (Fig. S3J).Respiratory stimulation of the F-pathway at the non-phosphorylating resting state (PctM L(n) ) and active OXPHOS state (PctM P ) were reduced in permebealized muscle fibers from LalÀ/À mice (Figure 3G).In addition, respiratory stimulation by simultaneous action of F-and Npathways with convergent electron flow (PctPM P ), respiratory stimulation by simultaneous action of the F-, N-, succinate pathway (S-, PctPMS P ), and electron transfer capacity (PctPMS E ) were decreased (Figure 3G), confirming decreased FA utilization in the SM of fasted LalÀ/À mice.Despite the insufficient FA supply and acyl-CoA in the liver, hepatic mitochondrial function and energy production of LalÀ/À mice were unaltered [16].To assess SM mitochondrial function, we determined the oxygen consumption rate in permeabilized SM fibers by highresolution respirometry.Loss of LAL resulted in a slight decrease in basal respiration (GM L ) driven by complex I substrates (glutamate and malate) and the active ADP-stimulated state (GM P ).Maximal respiration driven by complexes I and II was significantly reduced upon the addition of succinate (GMS P ) and in the presence of the uncoupler CCCP (GMS C ) in myofibers from LalÀ/À mice, indicating general consequences on mitochondrial coupling (Figure 3H).This result suggests impaired mitochondrial function in LalÀ/À SM, regardless of Original Article 6 the number or structure of mitochondria, as morphology (Figure 3I) and mtDNA (Fig. S3L) remained unaltered between the genotypes.

Reduced exercise capacity in LalÀ/À mice
To investigate whether the observed changes in fiber types translate into reduced physical performance, we performed an exercise tolerance test using the treadmill peak effort test coupled with indirect calorimetry.We found that LalÀ/À mice ran significantly  shorter distances (Figure 5A) and exhibited a lower total workload (Figure 5B) than Wt mice.Consistent with this observation, LalÀ/À mice had lower maximal aerobic capacity (VO 2 max ) compared to Wt mice (Figure 5C).These findings possibly indicate that remodeling of the muscular phenotype translates into impaired exercise capacity in vivo.
3.6.Altered protein expression patterns in the SM of LalÀ/À mice Expression of Lipa in SM is low compared with organs with high lipoprotein turnover such as the liver, but is approximately 2-fold higher in the highly oxidative SO than in QU, TA, and GA (Fig. S5).These observed differences between the SM prompted us to gain more insight into the protein abundance between more oxidative and more glycolytic SM parts of Wt and LalÀ/À mice.Thus, we divided the GA into "red" (enriched with oxidative fibers) and "white" (enriched with glycolytic ones) parts and performed proteomic analyses.After filtering for a minimum of four valid values out of six in at least one group and imputing missing values, we quantified 3917 proteins in red and 3852 proteins in white muscle fibers.Statistical analysis of the muscle proteome of LalÀ/À mice (FDR <0.01, S0 ¼ 0.1) revealed 567 (300 down-and 267 upregulated) and 430 (215 downand 215 upregulated) significantly changed proteins in red and white muscle fibers, respectively (Table S3).Principal component analysis  showed a clear separation between Wt and LalÀ/À samples in oxidative (Fig. S6A) and glycolytic (Fig. S6B) parts of GA, with significant up-or downregulation of various proteins in both the red and white parts of the LalÀ/À GA, as shown in the volcano plots (Figure 4A,B).The highly upregulated proteins found in both red and white muscle fibers included myosin-7 (MYH7), ceruloplasmin (CERU), cathepsin S (CATS), haptoglobin (HPT), alpha-1-acid glycoprotein 1 and 2 (A1AG1, A1AG2), glutamine synthetase (GLNA), and several types of troponins specific to slow SM (troponin I (TNNI1), troponin C (TNNC1)) (Figure 6A,B; Table S3).Some of the highly downregulated proteins observed in both parts of GA included insulin-like growth factor-binding protein 5 (IBP5), mitochondrial creatine kinase S-type (KCRS), collagen alpha-1(III) chain (CO3A1), and NADH dehydrogenase [ubiquinone] 1 alpha subcomplex assembly factor 2 (NDUF2) (Figure 6A,B; Table S3).A substantial number of the upregulated proteins were related to muscle structure and function, whereas the downregulated proteins were associated with mitochondria and metabolism.We next performed PANTHER enrichment analysis to identify up-and downregulated GOBP and GOCC terms as well as Reactome pathways (Table S3).Consistent with reduced mitochondrial respiration (Figure 3G), highly significant GOBP terms downregulated in the oxidative part of LalÀ/À GA were associated with mitochondrial function and structure, whereas upregulated terms involved RNA processing (Figure 6C).In contrast, the upregulated GOBP terms in the glycolytic part of LalÀ/À GA mainly involved various metabolic and catabolic processes (Figure 6D).We also found that protein folding is a significantly downregulated GOBP in LalÀ/À white GA (Figure 6D).We next examined how the loss of LAL affects the expression of proteins annotated to various cellular components.GOCC terms downregulated in the oxidative part of LalÀ/À GA included respirasome, mitochondrial membrane, and mitochondrial respiratory chain complex I (Fig. S6C), whereas no GOCC terms were downregulated in the glycolytic part (Fig. S6D).Nucleosome and myosin filament were among the highly significant upregulated GOCC terms in the glycolytic LalÀ/À GA part (Fig. S6D).Similarly, in the oxidative part of LalÀ/À GA, nuclear protein-containing complex, myosin complex, and spliceosomal complex were upregulated (Fig. S6C).To investigate metabolic clustering of regulated proteins in more detail, we performed Reactome pathway enrichment analysis.Selected highly upregulated Reactome pathways in the oxidative part of LalÀ/À GA were strongly associated with RNA splicing, gene expression, and muscle contraction, whereas the downregulated terms included translation and citric acid cycle (Figure 6E).In the glycolytic part of LalÀ/À GA, upregulated Reactome pathways included transport of small molecules, lipid and energy metabolism, whereas the downregulated terms were related to respiratory electron transport, endosomal sorting complex, and mitochondrial iron-sulfur cluster biogenesis (Figure 6F).Network analysis using Cytoscape based on proteineprotein interactions represented by the significantly differentially expressed proteins revealed comparable interacting clusters in both parts of GA.In particular, we observed clusters formed by several downregulated proteins that play a role in oxidative phosphorylation and mitochondria (Figure 6G and S6E), consistent with reduced ATP concentrations (Figure 3A) and the mitochondrial dysfunction in LalÀ/À SM determined by high-resolution respirometry (Figure 3G).Another group of downregulated proteins was clustered in protein processing in the ER and protein folding, whereas upregulated proteins formed clusters related to muscle contraction and muscle development (Figure 6G and   S6E).This finding further confirmed the dysfunction of SM mitochondria and fiber switch in LalÀ/À mice.

DISCUSSION
The impact of LAL-D on the pathophysiology of SM is still unclear, although decreased muscle size and muscle weakness were reported as characteristic features of LAL-D patients [23e26,28].Compromised lipid homeostasis in LalÀ/À mice, arising from impaired hepatic and intestinal lipid metabolism as well as lipodystrophy, leads to increased glucose consumption despite unaltered insulin levels, which ultimately results in reduced glucose concentrations in plasma and liver [16].Since SM require a considerable amount of energy to function efficiently, alterations in lipid and/or glucose metabolism may influence the metabolic state of the muscle and thus the proper functionality of the organ.We therefore aimed to characterize muscle structure, function, and metabolism in an animal model of LAL-D.Our findings demonstrate that the phenotype and metabolism of LalÀ/À SM are substantially impaired most likely due to an energy deficit and impaired energy metabolism associated with mitochondrial dysfunction.Muscles from LalÀ/À mice are smaller and exhibit diminished myofiber CSA and Feret diameter.However, preliminary data from our laboratory failed to reveal any differences in SM mass (QU) between 2week-old LalÀ/À and Wt mice or any alterations in muscle differentiation and myogenesis marker genes (data not presented), despite the crucial role of LAL during early development [31].Thus, we assumed that the reduced SM mass and size worsened with age in LalÀ/À mice, reflecting either growth retardation [50] or progressive loss of SM mass due to muscle proteolysis [51].Plasma AA could serve as a substrate for the translation of muscle proteins [52].The concentrations of glucogenic AA, which were expected to be increased during starvation due to muscle proteolysis, especially in mature mice, were unchanged or even reduced (Gln and Arg) in LalÀ/À compared to Wt mice.Elevated concentrations of the branched-chain AA Leu, Ile, and Val in fasted LalÀ/À mice could meet the elevated body demand for Ala and Gln due to increased muscle proteolysis or reduced muscle protein synthesis [53,54].However, unaltered proteasomal activity and expression of muscle proteolysis markers in the SM of fasted LalÀ/À mice argued against muscle wasting being responsible for the smaller size and mass of LalÀ/À SM.PI3K/Akt, activated by either insulin or IGF-1, is critical for controlling protein production in SM [43,44,55].Other key regulatory proteins involved in translation such as 4E-BP-1 are downstream targets of the mammalian target of rapamycin (mTOR) kinase, which is phosphorylated and triggered by activation of Akt [45,56].Despite unaltered expression of these markers, translation itself was one of the significantly downregulated terms revealed in Reactome pathways in red muscle fibers, suggesting altered protein synthesis by various signaling pathways, e.g. the nuclear factor kappa B (NF-kB)-dependent pathway [45].Of note, LalÀ/À mice suffer from systemic inflammation, as demonstrated by elevated levels of pro-inflammatory cytokines and macrophage infiltration in the liver, spleen, lung, and small intestine [12,15,57,58].However, despite unaltered Igf1 and Igf1r (data not shown) expression, IGFBP5, which generally enhances the effect of IGF-1, was strongly downregulated in both parts of GA.The Igfbp5 gene encoding IGFBP5 is low expressed during fasting, cachexia, diabetes, and other conditions that cause skeletal muscle atrophy [59].Recent results also suggested that IGFBP5 modulates the action of autocrine IGF-2 in promoting myogenic differentiation [60], however, they contradict the finding that overexpression of IGFBP-5 under a constitutive promoter impairs myogenic differentiation [61,62].Nevertheless, a decrease in IGFBP5 expression was described upon muscle denervation or unloading [63], which is in line with decreased locomotor activity [41] and physical capacity in LalÀ/À mice during the treadmill peak performance test.It is important to mention that cardiac abnormalities have not been described in LalÀ/À mice.Whether the pathophysiological phenotype of LalÀ/À lungs [57] contributes to physical performance of LalÀ/À mice is currently unknown.LalÀ/À mice display reduced muscle ATP concentrations, resulting in compensatory activation of various metabolic pathways, including increased glucose uptake and degradation of phosphocreatine, glycogen, and lactate [16].While TG levels remain unchanged in LalÀ/ À mice in the fed state, fasting leads to reduced TG concentrations in different muscles.The significant decrease in total acyl-carnitine levels and the confirmed reduced FA utilization further underscore the complex metabolic dysregulations in LalÀ/À mice that affect both energy production and substrate utilization.The distribution and abundance of specific MyHC isoforms dictate the predominance of "red" oxidative (slow-twitch) or "white" glycolytic (fast-twitch) fibers, thereby influencing the functional properties and metabolic features of the SM.Myofibers of vertebrate SM differ in their contractile properties, mitochondrial density, and metabolic characteristics, with different SM exhibiting a combination of various fibers.Slow-twitch fibers are characterized by the presence of type I MyHC expression and high mitochondrial density, which is predominantly associated with an oxidative metabolism.Fast-twitch fibers express type II MyHCs subdivided into IIa, IIx, and IIb [49].Thus, QU, TA, and GA are mainly composed of MyHCIIb (fast glycolytic fibers) but in varying proportions, whereas SO has a combination of MyHCI and MyHCIIa [64].Changes in muscle size may also be associated with a switch in muscle fiber types, since slow (type I) fibers have a smaller CSA than fast (type II) fibers, which was termed the "muscle fiber type e fiber size paradox" [65].When we analyzed gene expression and proteome signature changes, MYH7 and other specific slow oxidative muscle markers such as TNNC1 and TNNI1 were upregulated in GA of LalÀ/À mice, suggesting that loss of LAL leads to a switch from fast type II to slow type I myofibers with reduced CSA and Feret diameter.However, the mechanism underlying the increased expression of proteins specific to type I fibers remains unclear, especially since glucose consumption was elevated in SM of LalÀ/À mice.SM with fast-twitch glycolytic fibers are more sensitive to energy substrate deprivation than slowtwitch oxidative fibers under atrophic conditions [66].Thus, LalÀ/ ÀSM may adapt to the dysfunctional whole-body energy homeostasis, as slow fibers work sufficiently but consume less ATP with lower power utilization than fast fibers [67].An increased number of oxidative fibers should be associated with more mitochondria, contributing to enhanced cellular respiration [68].However, we observed decreased oxidative capacity in permeabilized fibers isolated from LalÀ/À GA, indicating mitochondrial dysfunction in LalÀ/À mice, although the number and morphology of mitochondria remained unchanged.Proteomic analyses confirmed that pathways associated with oxidative phosphorylation, electron transport chain, and ATP synthesis were the most dysregulated biological processes in the red but also the white part of LalÀ/À GA.Downregulated proteins included NDUF, SDH, UQCR, and ATP5 family members representing complexes I, II, III, and V, respectively, which were previously described to be strongly reduced in sarcopenic muscles [69].ATP plays an essential role in preventing protein aggregation and is a major energy source required for most energy-dependent cellular functions such as protein synthesis (about 30 % of available ATP [56]), folding, translocation to the ER, and protein degradation [70].Thus, insufficient ATP supply for protein processing may also explain the reduced muscle size in LalÀ/À mice.It is worth noting that the downregulated GOBP terms and protein interaction network generated by Cytoscape confirmed impaired protein folding in both the red and white parts of GA.Interestingly, among the most downregulated proteins, we found major urinary protein 11 (MUP11) in both the oxidative and glycolytic parts of LalÀ/À GA, and MUP3 in addition in the red part.The SM was reported to be one of the major metabolic target tissues of MUP1, a close homolog of MUP3 [71].Low concentrations of circulating MUP1 contribute to the metabolic dysregulation in obese and diabetic mice, which was markedly ameliorated by MUP1 replenishment by increasing the expression of genes involved in mitochondrial biogenesis and enhancing mitochondrial oxidative capacity, predominantly in SM [71].Our findings may indicate a potential role of other MUPs as potential regulators in the diminished mitochondrial function in LalÀ/À mice.Loss of LAL is associated with ectopic accumulation of lipids, especially CE [12,16,58,72,73], also in SM [74].Cholesterol accumulation may contribute to mitochondrial dysfunction, including reduced respiration and decreased ATP production [75,76] in LalÀ/À SM.The previously described rapid development of fatigue and the reduction in ATP turnover of the SM [77] is consistent with the LalÀ/À SM mitochondrial dysfunction and the reduction in ATP concentration.The decreased mitochondrial respiration may also result from an overall reduction in the import of components of the mitochondrial oxidative phosphorylation system and/or substrate transporters throughout the body as a consequence of the inaccessibility of lipids, the absence of white adipose tissue, and the rapid glucose consumption in various organs of LalÀ/À mice.

CONCLUSION
Taken together, our data provide conclusive evidence that whole-body loss of LAL affects the phenotype and, most probably, functions of SM, due to insufficient ATP production associated with dysfunctional mitochondria and impaired energy metabolism.The described alterations result in SM fiber switch, and LalÀ/À mice may serve as a model to study the complex molecular mechanisms of muscle remodeling under conditions of impaired lipid metabolism.However, it is still elusive whether the reduction in muscle mass and increased muscle fatigue are attributable to the global loss of LAL activity in SM or to the systemic loss of the enzyme, which mainly affects the liver, small intestine, and adipose tissue and is associated with severe macrophage infiltration and systemic inflammation.

Figure 2 :
Figure 2: Reduced muscle size in LalL/L mice is independent of muscle degradation.Relative mRNA expression of genes encoding the proteolysis markers Murf and Atrogin1 in the gastrocnemius (GA) of (A) ad libitum-fed young male mice (n ¼ 3) and (B) young female mice fasted for 12 h (n ¼ 3e4) relative to cyclophilin A expression as reference gene.(C) Chymotrypsin-like (Che-like), trypsin-like (Try-like), and caspase-like proteasome activities in frozen GA of 6-h fasted young female Wt and LalÀ/À mice (n ¼ 3) using 7-amino-4-methylcoumarin (AMC) as fluorescence reference standard.(D) Plasma glucogenic amino acid (AA) concentrations quantified using AA separation by high-performance liquid chromatography (HPLC) of mature female ad libitum-fed and 12-h fasted Wt and LalÀ/À mice (n ¼ 5).(E, F) Representative Western blotting experiments of (E) pAkt, Akt, (F) p4eBP1, and 4eBP1 protein expression in QU and densitometric quantification of (E) pAkt/Akt and (F) p4eBP1/4eBP1 ratios (n ¼ 4) from young male LalÀ/À and Wt mice in the fed state.a-Tubulin expression was used as loading control.Data represent mean AE SD. *p < 0.05, **p 0.01.(AeC) Unpaired Student's t test.(D) Oneway ANOVA.

Figure 4 :
Figure 4: Increased formation of oxidative fibers in skeletal muscles of LalL/L mice.(A) Relative mRNA expression of genes encoding different isoforms of myosin heavy chain in gastrocnemius (GA) of male mice in the fed state (n ¼ 3) relative to cyclophilin A expression as reference gene.(B) Representative Western blotting experiment of MYH7 protein expression and its densitometric quantification relative to GAPDH (n ¼ 3) in quadriceps (QU) from young 6-h fasted male LalÀ/À and Wt mice.GAPDH expression was used as loading control.(C) Representative images of immunofluorescence co-staining for MYH7 (red) and laminin (green) in GA and tibialis anterior (TA) cross sections from ad libitumfed Wt and LalÀ/À mice.Nuclei are stained with DAPI (blue).Scale bar, 100 mm.(D) Percentage of MYH7-stained area quantified using Image J (2e3 technical replicates from 3 mice).Data represent mean AE SD. *p 0.05.Unpaired Student's t test.

Figure 5 :
Figure 5: Decreased treadmill exercise performance in LalL/L mice.Male Wt and LalÀ/À mice aged 15e16 weeks were subjected to peak effort testing on a treadmill coupled to indirect calorimetry to determine (A) maximal running distance, (B) workload, and (C) VO 2 max normalized to body weight.Data show mean AE SD (n ¼ 9e10).*p < 0.05, **p 0.01, ***p 0.001 compared to Wt mice.Unpaired Student's t test.

Figure 6 :
Figure 6: Proteome changes in the oxidative and glycolytic parts of gastrocnemius (GA) in LalL/L mice.Volcano plots of protein abundance from (A) oxidative and (B) glycolytic parts of the GA from Wt or LalÀ/À young male mice in the fed state (blue, downregulated in LalÀ/À; red, upregulated in LalÀ/À; grey, unchanged) (n ¼ 5e6) (S0 ¼ 0.1, FDR <0.01).Analyses of the (C, E) oxidative (red) and (D, F, G) glycolytic (white) parts of GA in Wt and LalÀ/À mice.(C, D) Downregulated (blue) and upregulated (red) GO terms associated with enriched proteins in SM of LalÀ/À mice classified into biological processes (GOBP).(E, F) Significantly enriched Reactome pathways in red and white muscle fibers, respectively.(G) Proteineprotein interaction network represented by the modulation of the significantly changed proteins in the glycolytic (white) part of GA.The interaction network was generated using Cytoscape.
[32]d extraction and biochemical analysisLipid extraction and quantification of TG, total cholesterol (TC), free cholesterol (FC), and CE concentrations were performed as previously described[31].2.11.Analysis of acylcarnitines by mass spectrometry SM samples from overnight fasted young (8e12 weeks old) male mice were pulverized in liquid nitrogen and 5e15 mg were transferred to 2 mL Safe-Lock PP-tubes.Lipids were extracted according to Matyash et al.[32].In brief, samples were homogenized with two 6-mm steal beads on a Mixer Mill (Retsch, Haan, Germany; 2 Â 10 s, frequency 30/s) in 700 mL MTBE/MeOH (3:1) containing 500 pmol butylated hydroxytoluene, 1 % acetic acid, and 3 pmol palmitoyl-1,2,3,4e 13 C4-L-carnitine (SigmaeAldrich, St. Louis, MO) as internal standards.Lipids were extracted under constant shaking for 30 min at RT.After addition of 140 mL dH 2 O, samples were vigorously vortexed (3 Â 10 s) and centrifuged at 1,000Âg for 15 min.Thereafter, 500 mL of the 2-deoxy-D-glucose/30 g body weight.Animals were sacrificed 60 min post-injection, and liver and SM were isolated, lyophilized for 48 h, and dry weight was measured.Tissues were then digested in 1 mL of 1 M NaOH overnight at 65 C, transferred to a scintillation vial containing 8 mL scintillation cocktail, mixed properly, and stored overnight at 4 C. Radioactivity was determined by liquid scintillation counting and normalized to dry tissue weight.2.10.upper,organic phase was collected and dried under a stream of nitrogen.Lipids were dissolved in 500 mL MTBE/methanol (3:1) and diluted 1:5 in 2-propanol/methanol/dH 2 O (7:2.5:1) for UHPLC-QqQ analysis.The remaining protein slurry was dried and used for BCA protein determination after lysis in 300 mL of 0.3 N NaOH at 60 C.Chromatographic separation was performed on a 1290 Infinity II LC system (Agilent, Santa Clara, CA) equipped with a Zorbax RRHD Eclipse Plus C18 column (2.1 Â 50 mm, 1.8 mm; Agilent) and a 10-min gradient of 95 % solvent A (H 2 O; 10 mM ammonium acetate, 0.1 % formic acid, 8 mM phosphoric acid) to 100 % solvent B (2-propanol; 10 mM ammonium acetate, 0.1 % formic acid, 8 mM phosphoric acid) at a flow rate of 500 mL/min.The column compartment was kept at 50 C. Lipids were detected in positive mode using a 6470 triple quadrupole mass spectrometer (Agilent, Santa Clara, CA) equipped with an ESI source.Acylcarnitine species were analyzed by dynamic multiple reaction monitoring ([MþH] þ to m/z 84.9, CE 28, Fragmentor 164, CAV 5).Data acquisition and processing were performed by MassHunter Data Acquisition software (Version 10.0 SR1, Agilent, Santa Clara, CA) and MassHunter Workstation Quantitative Analysis for QQQ (Version 10.0, Agilent, Santa Clara, CA), respectively.Data were normalized for recovery, extraction, and ionization efficacy by calculating analyte/internal standard ratios (AU) and expressed as AU/mg protein.
[39,40]ocessing using protein group quantities and functional analysis were done with Perseus software version 1.6.15.0,JupyterNotebook using Python version 3.9 and Cytoscape.Protein intensities were log2-transformed before filtering the data.To avoid exclusion of relevant proteins that were not detected because of low expression in one of the conditions (either Wt or LalÀ/À), data were filtered for at least 4 valid values from 5 to 6 samples in at least one group and missing values were replaced by random values from the Gaussian distribution (width of 0.3, downshift of 1.8).Principal component analyses were performed on standardized data (z-scored) and visualized with Jupyter Notebook using the Pandas, Numpy, Matplotlib, Sklearn, Seaborn, and Bioinfokit packages.Two-sample t-tests followed by correction of multiple testing by the permutation-based FDR method were used to identify altered protein groups (S0 ¼ 0.1, FDR <0.01).Enrichment analysis for Gene Ontology Biological Process (GOBP), GO Cellular Component (GOCC), and Reactome pathways was performed using the PANTHER enrichment test for log2-fold changes of proteins[39,40].Significantly changed pathways are shown in Supplementary TableS3.
[37,38]ed to a final concentration of 1 % trifluoroacetic acid and desalted using self-made stage-tips with styrenedivinylbenzenereversed phase sulfonate as material.2.17.Proteome analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) Peptides were separated on the UltiMateÔ 3000 RSLCnano Dionex system (ThermoFisher Scientific, Waltham, MA) using an IonOpticks Aurora Series UHPLC C18 column (250 mm Â 75 mm, 1.6 mm) 2.18.LC-MS/MS proteomics data processing, bioinformatics, and statistical analysis Raw data files were analyzed and proteins were quantified using DIA-NN software (version 1.8.1)[37,38].The SwissProt Mus musculus proteome database in fasta format (containing common contaminants; downloaded on 2021/08/17, 17,219 sequences) was used for a library-free search with false discovery rate (FDR) set to 1 %.Deep learning-based spectra and retention time prediction was enabled, minimum fragment m/z was set to 200 and maximum fragment m/z to 1800.N-terminal methionine excision was enabled, and the maximum number of trypsin missed cleavages was set to 2. The minimum peptide length was set to 7 and the maximum to 30 AA. Cysteine carbamidomethylation was used as fixed modification and methionine oxidation as variable modification.DIA-NN optimized the mass accuracy automatically using the first run in the experiment.2.19.Statistical analysesStatistical analyses and graphs were performed using GraphPad Prism 9 software.Significance was calculated by unpaired Student's t-test and analysis of variance (one-way ANOVA) followed by Bonferroni posttest.Data are shown as mean AE SD or þ SD.The following levels of statistical significance were used: *p < 0.05, **p 0.01, ***p 0.001.Bioinformatical and statistical analysis of -omics analyses are described in section 2.18.