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Skeletal muscle remodeling is a critical component of an organism's response to environmental changes. Exercise causes structural changes in muscle and can induce phase shifts in circadian rhythms, fluctuations in physiology and behavior with a period of around 24 hours that are maintained by a core clock mechanism. Both exercise-induced remodeling and circadian rhythms rely on the transcriptional regulation of key genes.
We used DNA microarrays to determine the effects of resistance exercise (RE) on gene regulation in biopsy samples of human quadriceps muscle obtained 6 and 18 hours after an acute bout of isotonic exercise with one leg. We also profiled diurnal gene regulation at the same time points (2000 and 0800 hours) in the non-exercised leg. Comparison of our results with published circadian gene profiles in mice identified 44 putative genes that were regulated in a circadian fashion. We then used quantitative PCR to validate the circadian expression of selected gene orthologs in mouse skeletal muscle.
The coordinated regulation of the circadian clock genes Cry1, Per2, and Bmal1 6 hours after RE and diurnal genes 18 hours after RE in the exercised leg suggest that RE may directly modulate circadian rhythms in human skeletal muscle.
Resistance exercise (RE) can improve the overall quality of life and reduce the symptoms of many clinical disorders, including obesity, type II diabetes mellitus , coronary heart disease, and stroke . The effects of RE on skeletal muscle are mediated by activation of muscle-specific signaling cascades that increase muscle mass , cytoskeletal protein levels, and the force of contraction without increasing the number of myofibers . Exercise-induced transcription of genes involved in growth, vascularization, and metabolism [5-8] indicates that large-scale changes in transcriptional regulation play a key role in muscle remodeling, inducing growth responses and metabolic shifts.
Exercise also appears to help reset circadian rhythms in shift-workers, travelers who have changed time zones, and people with sleep disorders [9-13]. Circadian rhythms are approximately 24-hour fluctuations in gene regulation, physiology, and behavior that have evolved to optimize daily cycles of sleep, activity, feeding, and metabolism . Exercise influences both the phase  and the amplitude  of circadian rhythms in mice, but this phenomenon is largely unexplored in human tissues.
Circadian rhythms are controlled by a clock mechanism located in central and peripheral tissues. The mechanism comprises an autoregulatory transcriptional feedback loop that includes the circadian-clock genes Clock, Bmal1, Period (Per) and Cryptochrome (Cry). Clock and Bmal1 constitute the positive arm of the feedback loop. These proteins form heterodimers, bind to specific DNA regulatory elements (E-boxes), and initiate transcription of Per 1, 2, and 3 and Cry 1 and 2. In mammals, the Per and Cry gene products, which constitute the negative arm of the transcriptional feedback loop, form homo- and/or heteromeric complexes, translocate to the nucleus, and repress Clock/Bmal1-mediated transcription [17,18] in a temporal manner. Although most studies have focused on the rhythmic expression of these few core clock genes, recent evidence suggests that genes regulated in a circadian fashion (or circadian output genes) constitute 8-10% of all genes expressed in mouse tissues [19,20].
Classically, peripheral clocks are thought to be controlled by a central clock located in the suprachiasmatic nucleus (SCN), which is believed to also synchronize clocks in other brain regions . However, under certain conditions (such as restricted feeding), peripheral tissue clocks can be regulated independently of the SCN [22,23]. The phase-shifting effects of exercise on mammalian circadian rhythms are thought to be mediated by inputs to SCN neurons (these include serotonin , neuropeptide Y , and melatonin [13,26]) and ultimately lead to acute changes in SCN Per1 and Per2 expression . The extent to which RE may be able to directly affect circadian-regulated genes in peripheral tissues such as human muscle is not known, however.
To understand the global transcriptional effects of RE and time of day on gene expression in human skeletal muscle (hSkM), we used DNA microarrays to analyze biopsies of exercised and non-exercised hSkM collected at different times of day. This study was designed to answer three specific questions. What genes and biological processes are regulated in hSkM by RE and time of day? Which orthologs of genes that undergo temporal regulation in hSkM also undergo circadian regulation in mouse skeletal muscle (mSkM), liver (mLvr), heart (mHrt) or SCN (mSCN)? Are diurnally regulated genes expressed in skeletal muscle directly affected by exercise? To answer these questions, we compared gene expression in the exercised and non-exercised legs of four human subjects after an acute bout of RE. We then filtered and annotated (by biological process) the significantly changed genes and compared these genes to orthologs regulated in microarray studies of rodent models of exercise and circadian gene regulation. Finally, we used quantitative reverse-transcription polymerase chain reaction (RT-PCR) to validate the circadian regulation of selected diurnally regulated hSkM gene orthologs in mSkM.
Comparison of gene expression profiles of the exercised and non-exercised legs showed that 704 genes were differentially regulated at 6 hours and 1,479 genes at 18 hours after RE (p < 0.05). In the non-exercised leg, comparison of gene expression at 0800 and 2000 hours with the same statistical criteria showed that 608 genes were differentially regulated.
We used MAPPFinder  to link gene expression data to the Gene Ontology (GO) hierarchy (Table (Table1).1). The program computes a significance score (Z score) that is useful for ranking GO terms by their relative amounts of gene expression changes (see Materials and methods). With 40% of the circadian-rhythm genes classified by the GO hierarchy significantly changed at 6 hours after RE, circadian rhythm displayed the highest Z score (Z = 7.17) in this comparison, indicating that RE may regulate circadian genes directly in the exercised leg (see below). As expected, RE also upregulated a variety of genes involved in intracellular responses (nucleic acid metabolism, G2/M transition of mitotic cell cycle, anti-apoptosis, and transcription) and downregulated genes involved in oxygen and calcium transport, DNA repair, regulation of translational initiation, and glycogen metabolism (Table (Table1).1). Many of these processes were similarly regulated in a rat model of RE .
MAPPFinder analysis identified several biological processes influenced by diurnal gene regulation in hSkM, including transcription, cell differentiation, response to stress, hemopoiesis, oncogenesis, protein biosynthesis, and metabolism (carbohydrate metabolism and tricarboxylic acid cycle) (Table (Table1).1). These findings provide human gene targets that correlate with the circadian regulation of metabolism and cancer recently reported in mouse models [19,20,29-31].
The significant upregulation of Per1 and Per2 in our diurnal comparison provided the first indication that the 608 genes that changed significantly with time contained known circadian-regulated genes. To filter out potential noise in this comparison and to identify genes with the highest likelihood of being regulated in a circadian fashion, we compared our results to published data on circadian gene regulation in mouse peripheral tissues [19,20]. These comparisons resulted in a list of 44 'putative circadian genes' that were significantly regulated in both our human diurnal dataset and in the mouse circadian studies (Figures (Figures1,1, ,2).2). We then used quantitative RT-PCR to analyze mRNA expression levels of 12 mouse orthologs in mSkM isolated every 4 hours for 24 hours (Figure (Figure33).
Quantitative RT-PCR indicated that the core circadian-clock genes Bmal1, Per1, and Per2 and selected mouse orthologs of the 44 putative circadian genes exhibited circadian regulation in mSkM (Figure (Figure3).3). Quantitative RT-PCR also provided a potential explanation for our inability to detect a significant change in the expression of the circadian-regulated core clock genes Cry1 and Bmal1 in hSkM [19,20], as these genes cycle out of phase with Per1 and Per2 in mSkM (Figure (Figure3)3) and in many other tissues, including mLvr  and mHrt . As only two time points were measured in the human samples, genes whose peak and trough times are out of phase with our sampling times (for example, Cry1 and Bmal1) would not be detected.
An interesting finding is that Per1 and Per2 are upregulated in the morning in hSkM (Figure (Figure2),2), and downregulated in the morning in mSkM (Figure (Figure3b).3b). We cannot determine the exact phase of the potentially circadian-regulated genes in hSkM with only two time points of sampling. However, the observed opposite regulation may reflect opposing phases of core clock gene expression between human and mouse peripheral tissues. Further examination of this phenomenon may provide a molecular explanation for the opposite activity/rest cycles in diurnal versus nocturnal mammals.
To identify conserved circadian-regulated genes, three peripheral tissues were selected for comparison: mHrt , mLvr [19,20], and hSkM. Four genes were significantly regulated in all three tissues: Per2, Nr1d2 (nuclear receptor subfamily 1, group D, member 2), Herpud1 (homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1), and Oazi (ornithine decarboxylase antizyme inhibitor) (Figures (Figures1,1, ,2).2). Per2 is a well-characterized conserved core clock element ]. Human Nr1d2 (Rev-Erbβ) is 90% identical to mouse Rev-Erbα, a newly identified component of the circadian clock that represses the transcription of Bmal . Herpud1 appears to be a membrane-bound endoplasmic reticulum protein induced by stress . It contains a ubiquitin-like domain at the amino terminus, indicating its involvement in a protein degradation pathway, a biological process important for maintaining circadian rhythms. The Oazi gene product is an inhibitor of antizyme, which inhibits ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis that is essential for normal cell growth . The discovery of common circadian genes in multiple tissues suggests that these genes and their patterns of expression are important in a variety of tissue pathways.
We hypothesized that RE affects circadian-regulated gene expression directly in skeletal muscle. In support of this, we found that three core circadian clock genes, Cry1, Per2, and Bmal1, were upregulated 6 hours after RE in the exercised leg (1.5-fold, 1.2-fold, and 1.2-fold, respectively; Figure Figure2b).2b). Although the RE-induced changes of these circadian clock genes were modest in magnitude, they are statistically significant, coordinated, and precede RE-mediated changes in diurnal genes 18 hours after RE in the exercised leg.
RE appeared to shift the expression patterns of diurnal-regulated genes by upregulating genes (n = 12, p < 0.1) that normally were repressed in the morning (n = 29, p < 0.05) or by downregulating genes (n = 5, p < 0.1) normally induced in the morning (n = 15, p < 0.05) (Figure (Figure2c).2c). If we extend this analysis to all the diurnal genes that are significantly changed (p < 0.05) 18 hours after RE, we find all (64 genes) but one reflect this potential phase-shifting effect.
Although we cannot determine whether RE induces a phase advance or phase delay in these genes, our data are consistent with previous studies that show that physical exercise during the day (similar to our study) can induce a circadian phase advance in humans  as measured by circulating hormone levels. Our studies now indicate that this phase advance may occur at the level of gene expression in muscle.
To validate our experimental protocol and to identify key genes that may regulate the effects of RE in both humans and rodents, we compared human genes that were regulated by our isotonic RE protocol at 6 hours with rat orthologs that were regulated either transcriptionally or translationally in a published rat eccentric RE study  (Figure (Figure4).4). The rat eccentric exercise protocol induced skeletal muscle hypertrophy  and consisted of titanic contractions that were electrically evoked with multistrand electrodes implanted on both sides of the sciatic nerve. Contractions consisted of 10 sets of six repetitions with each repetition lasting 3 seconds. Contractions were stimulated at a frequency of 100 Hz, 6-12 V, 1 msec duration, 9 msec delay. Rat muscles were then harvested 6 hours after the acute bout of exercise . Lists of rat 6-hour RE genes were downloaded from the supplemental data at . Human and rat gene orthologs that were regulated in the same directions are shown in Figure Figure44.
Grouping the orthologs by biological function revealed that two genes, Rrad (Ras associated with diabetes) and G6pt1 (glucose-6-phosphatase, transport protein 1), were regulated in directions suggesting decreased glucose transport, and one gene, Gpd2 (glycerol-3-phosphate dehydrogenase 2 (mitochondrial)) was regulated in a direction that suggested a decrease in glucose metabolism. Other genes involved in the metabolism of glycogen (the cellular storage form of glucose) were also downregulated (Table (Table11).
Interestingly, all three of these genes have been implicated in human diseases. Rrad was cloned because it was upregulated in patients with type II diabetes . In cultured muscle and fat cells, overexpression of Rrad decreases insulin-stimulated glucose uptake . The upregulation of Rrad provides a potential mechanism for previous reports that acute RE reduces insulin-stimulated glucose uptake [40-42]. Mutations in Gpd2 were found in a family of type II diabetics , and mutations in G6pt1 were found in patients with glycogen storage disease .
Two potent muscle-remodeling genes, myogenin and myostatin, were regulated in opposite directions in response to RE in humans, providing a potential mechanism for exercise-induced muscle hypertrophy. Myogenin was upregulated in the 6-hour comparison, and this observation was validated in the published  rat RE study (Figure (Figure4).4). Myogenin, a muscle-specific transcription factor containing a basic helix-loop-helix domain, is important for muscle development and differentiation . Myostatin, a member of the TGF-β family, is a negative regulator of skeletal muscle size and was downregulated in our study. It is a potent inhibitor of muscle development and proliferation . Downregulation of myostatin may be associated with skeletal muscle growth  and is likely to have a major role in muscle remodeling in response to exercise.
The interleukin-1 gene (IL-1) was upregulated 6 hours after RE (Figure (Figure4).4). The potential importance of IL-1 regulation in response to RE is indicated by the fact that two genes regulated by IL-1, Nr4a3  and Carp , had the highest fold changes (3.5-fold and 2.3-fold, respectively, p < 0.05) 6 hours after RE. These two genes were also regulated in the rat exercise study (Figure (Figure4).4). Furthermore, the vascular endothelial growth factor gene (Vegf) , which is also regulated by IL-1, had the highest fold change at 18 hours after RE (1.6-fold). Vegf is believed to be an important mediator of endurance exercise-induced angiogenesis in skeletal muscle . However, resistance exercise protocols do not result in increases in capillaries per muscle area but do result in a redistribution of blood flow to hypertrophied muscle fiber types .
To identify potential co-regulated genes, we performed hierarchical clustering analysis  with expression values in the non-exercised leg at 2000 hours as the baseline. Two clusters included genes upregulated at 6 hours after RE (Figure (Figure5),5), one centered on Nr4a3 (Figure (Figure5a)5a) and the other on Rrad (Figure (Figure5b).5b). These two genes displayed the highest fold changes 6 hours after RE. Cry1, a member of the core clock mechanism (Figure (Figure2),2), was found in the Nr4a3 cluster.
Two clusters contained core circadian genes Per1 (Figure (Figure6a)6a) and Per2 (Figure (Figure6b)6b) which were upregulated at 0800 hours. A third cluster (Figure (Figure6c)6c) contained genes downregulated at 0800 but not at 2000 hours. The Per2 cluster contained genes that were upregulated both at 0800 hours and in response to exercise (2000 hours + RE).
Analysis of our data in the context of biological processes and pathways allowed us to define physiologically the transcriptional basis of muscle remodeling induced by RE and its potential circadian gene regulation. This large-scale expression analysis of acute RE and circadian gene regulation in hSkM suggests that RE can directly regulate circadian clock genes (Per2, Cry1, and Bmal1) and circadian output genes. Our findings support the emerging idea that peripheral clocks can regulate themselves independently of the SCN [22,23]. If the SCN were responsible for the phase shifting, the same changes in gene expression would have occurred in both the control and exercised legs, which was not observed. However, acute phase advances in SCN Per1 and Per2 expression in response to exercise have been observed in hamsters . Whereas the SCN is still probably involved in the long-term effects of clock phase shifting in peripheral tissue, our evidence suggests that the skeletal muscle clock responds quickly to RE by transcriptional regulation of specific clock genes.
Although circadian studies in human tissue are critical for understanding the effects of circadian gene regulation on human physiology, access to ample tissue samples is difficult, costly, and painful. We therefore found it valuable to validate and compare our results with those of similar experiments in rodents, in which tissue can be harvested at multiple time points to establish the circadian cycle. Such comparisons were also valuable in identifying gene orthologs regulated 6 hours after RE in both humans and rats. Cross-species, cross-tissue comparisons are essential for defining transcriptional regulatory pathways of key genes and will allow us to define new genes and pathways responsible for tissue regulation and remodeling.
Four healthy men, 31-51 years old, were recruited for study. The subjects had not carried out RE training for at least 3 months before enrolment, had no history of chronic illnesses, and showed no abnormalities on the screening physical examination or routine hematology and chemistry tests. Subjects were admitted to the General Clinical Research Center at San Francisco General Hospital, where they were fed a constant metabolic diet that provided 1.2 g of protein/kg body weight and 35 kcal/kg body weight per day for nine days before the bout of exercise. Equilibration on the diet was evidenced by constancy of urine urea nitrogen excretion. The study protocol was approved by the Committee on Human Research of the University of California, San Francisco, and informed consent was obtained from each subject.
Maximum strength (one-repetition maximum) during isotonic knee extension from 90° to 170° was tested in the right leg 8 days before the study exercise session. Subjects did not perform any RE between the testing and the study session. On the ninth hospital day, beginning at 1330 h, subjects performed a vigorous bout of RE with the right leg. Exercise consisted of 10 sets of eight repetitions of isotonic knee extension (Cybex 4850 leg extension, Ronkonkoma, NY) at 80% of the predetermined one-repetition maximum, with 3-min rest periods between sets, over 30-45 min. The isotonic knee extensions include both concentric and eccentric phases.
Muscle biopsies were performed 6 hours (between 1930 and 2000 hours) and 18 hours (between 0730 and 0800 hours the next day) after RE in both the exercised and non-exercised leg. Tissue (200-300 mg) was obtained from the lateral portion of each vastus lateralis muscle approximately 20 cm above the knee with a 4-mm Bergstrom needle (Stille, Stockholm, Sweden) under local anesthesia with 1% lidocaine. Blood and visible fat were removed, and the tissue was immediately frozen in liquid nitrogen and stored at -80°C for later analysis. Although relatively homogeneous when compared to other tissues, skeletal muscle is a complex tissue consisting of many cell types, and thus our results must be interpreted in that context.
Total RNA was extracted from frozen tissue with a polytron homogenizer and Trizol (Invitrogen, Carlsbad, CA). Fragmented, biotin-labeled cRNA samples were generated from 5-14 μg total RNA and hybridized to Affymetrix human U95Av2 arrays. For each array, the .cel files were generated with Affymetrix Microarray Suite 5.0 and analyzed with Robust Microarray Analysis (RMA) .
Total RNA was extracted from 5-100 mg frozen tissue with a polytron homogenizer and Trizol (Invitrogen) and purified with an RNEasy kit (Qiagen, Santa Clara, CA). Depending on the amount of starting material, 5-14 μg total RNA was reverse transcribed with an oligo-dT primer containing a T7 RNA polymerase promoter (Affymetrix) and then converted into double-stranded cDNA (ds cDNA) with a ds cDNA synthesis kit (Invitrogen). After the second-strand synthesis, ds cDNA was extracted with phenol-chloroform-isoamyl alcohol and recovered by ethanol precipitation. Biotinylated cRNA was generated from ds cDNA by in vitro transcription (IVT) with an Enzo BioArray high-yield RNA transcript labeling kit (Affymetrix). After a further round of purification with the Qiagen RNEasy kit, IVT reactions yielded 30-70 μg biotinylated cRNA, which was fragmented into lengths of around 100 base-pairs (bp) before hybridization.
IVT reaction products (5 μg) were hybridized to Affymetrix Test2 arrays before hybridization to the HG-U95Av2 chip (Affymetrix) to ensure full-length transcript representation of GAPDH. Each chip was hybridized to 15 μg fragmented cRNA. Arrays were hybridized and scanned with a GeneArray Scanner (Hewlett-Packard/Affymetrix) at the Genomics Core Facility of the General Clinical Research Center at San Francisco General Hospital.
Three comparisons were made (Figure (Figure7).7). Gene expression in the exercised leg was compared with that in the non-exercised leg at 6 and 18 hours after RE. The diurnal effects on gene expression were assessed by comparing gene transcript levels in the non-exercised leg at 2000 and 0800 hours. Two-tailed paired t tests were used to compare each sample with its respective baseline value. These t tests were validated using permutation t tests. Human U95Av2 chip probe set to gene annotations were obtained from NetAffx .
Genes that were significantly upregulated or downregulated (p < 0.05) were annotated with GO  information with the MAPPFinder 1.0 program  (Table (Table1).1). MAPPFinder is an accessory program to GenMAPP , a freely available software tool that colors biological pathways with gene expression data . MAPPFinder Z-scores, a statistical measure of significance for gene expression in a given group, were calculated by subtracting the number of genes expected to be randomly changed in a GO term from the observed number of changed genes in that GO term. This value was then divided by the standard deviation of the observed number of genes under a hypergeometric distribution. Output from the MAPPFinder analysis was manually filtered to remove processes that represented the same genes (typically parent-child processes). The top six biological processes for each comparison are listed in Table Table1.1. For a biological process to be included in the table, it was required that at least three genes changed significantly (nested results) and the Z-score was > 2.
Adult male C57BL/6J mice were subjected to a 12-hour light/12-hour dark cycle for 2 weeks before tissue collection (n = 3 per time point). Mice were sacrificed every 4 hours for 24 hours, and quadriceps muscle was dissected and rapidly frozen on dry ice. Total RNA was extracted from frozen samples with Trizol (Sigma) and diluted to 0.1 mg/ml. TaqMan real-time RT-PCR assays were performed using the comparative amplification detection threshold of target gene expression (CT) method, an ABI 7700 Sequence Detector, and TaqMan EZ RT-PCR kit reagents (Applied Biosystems, Foster City, CA). Probe and primer sets were designed with Primer Express software (Applied Biosystems). mRNA levels were measured by determining the cycle number at which CT was reached. In each sample, CT was normalized to GAPDH expression, performed in parallel (ΔCT). Normalized ΔCT values from each time point were then subtracted from the average ΔCT value at all time points (ΔΔCT) to determine the relative abundance values (2-ΔΔCT).
To relate diurnally regulated hSkM genes to known circadian-regulated genes (potential hSkM circadian genes), we linked human U95Av2 probe sets to mouse U74Av2 ortholog probe sets using three public databases: NCBI Homologene , TIGR Resourcerer , and NetAffx. A similar approach was used to link the human and rat orthologs regulated 6 hours after RE (U95Av2 to U34A probe sets). 'Overlap genes' were defined as those exhibiting a fold change greater than 20% at a significance level of p < 0.05 that corresponded to significantly changed rodent orthologs based on the statistical filters used in the published studies [8,19,20].
For hierarchical clustering, we used data from the 3,260 genes (of 12,626 examined) that resulted from a p < 0.05 in any of three comparisons. The analysis was performed with Cluster and TreeView . As input for Cluster, the average (N = 4) log2 truncated value of the unexercised 2000-hour biopsies (control leg) was used as the baseline. All 3,260 genes were clustered by correlation uncentered similarity metric, using complete linking clustering. Figures were generated with TreeView.
The following additional data files are available with the online version of this article: two Excel sheets containing the Affymetrix data (MAS 4.0, target intensity 800) from rat 6 hours after exercise  total and polysomal (Additional data file 1); two Excel sheets containing log2 RMA signal values, fold changes, P values and probe level annotation, and descriptions of column titles, respectively (Additional data file 2); two sheets with information for linking orthologous probe sets between Affymetrix Hs. U95A, Mm. U74A and Rn. U34A arrays (Additional data file 3); two sheets (Hs RE/rat total RE and Hs RE/rat polysomal RE) of 6 h after exercise-regulated gene orthologs regulated in this study and those published by Chen et al.  (Additional data file 4); four Excel sheets containing the putative circadian overlap genes (Additional data file 5); and, finally, five sheets each with the unfiltered output results from the MAPPFinder analysis (Additional data file 6). All of the data, including RMA expression values, annotated chip information, GenMAPP expression dataset , MAPPFinder results , lists of links between the human and rodent probe sets, and full lists of ortholog matches, are also available for download from the GenMAPP site .
The Affymetrix data (MAS 4.0, target intensity 800) from rat 6 hours after exercise
Log2 RMA signal values, fold changes, P values and probe level annotation, and descriptions of column titles
Information for linking orthologous probe sets between Affymetrix Hs. U95A, Mm. U74A and Rn. U34A arrays
Hs RE/rat total RE and Hs RE/rat polysomal RE of 6 h after exercise-regulated gene orthologs regulated in this study and those published by Chen et al.
The putative circadian overlap genes
The unfiltered output results from the MAPPFinder analysis
We thank Karyn A. Esser for insightful discussions and help in revising the manuscript, Bethany Taylor for assistance with manuscript preparation and submission, Stephen Ordway and Gary Howard for editorial assistance, and Chandi Griffith for hybridizing and scanning arrays. J.S.T. is an Investigator and E.L.M. is a Research Associate in the Howard Hughes Medical Institute. This work is supported by the J. David Gladstone Institutes, the San Francisco General Hospital General Clinical Research Center (RR-00083), the National Heart, Lung, and Blood Institute (HL61689, HL60664), the NHLBI Programs for Genomic Applications (BayGenomics HL66621), and Diabetes, Endocrinology & Metabolism Training Grant (DK07418).