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ISSN 1330-7142 UDK = 633.11:581.1 INFLUENCE OF TEMPERATURE AND RELATIVE HUMIDITY ON GRAIN MOISTURE, GERMINATION AND VIGOUR OF THREE WHEAT CULTIVARS DURING ONE YEAR STORAGE I. Strelec (1), Ruža Popović (2), Ilonka Ivanišić (2), Vlatka Jurković (2), Zorica Jurković (3), Žaneta Ugarčić-Hardi (1), Mirjana Sabo (1) Preliminary communication Prethodno priop}enje SUMMARY Changes in grain moisture, germination and vigour of three wheat cultivars packed in paper bags and stored for one year under four different conditions of environmental temperature and relative humidity (RH) were investigated. During the first ninety days of storage significant reduction in grain moisture content of 4, 2.5 and 0.9 %, respectively, under 40 °C, 25 °C and 4 °C and RH of 45 % occurred. Subsequently grain moisture remained constant until the end of storage. Seeds of examined cultivars lost their germination ability and vigour only under elevated storage temperatures. Germination and vigour loss after one year of storage differed between cultivars being higher for seeds kept under 40°C, RH = 45 % (35-85 % and 55-94 %, respectively), than under 25°C, RH = 45 % (10-20 % and 15-22 %, respectively). Obtained data indicate significant influence of storage conditions on moisture content, germination and vigour changes during storage of wheat seeds, as well as varietal dependence of seed viability. Key-words: grain moisture, germination, relative humidity, temperature, vigour, wheat INTRODUCTION Environmental temperature and relative humidity are two most important factors influencing seed viability and longevity during the storage. Interdependence of these two factors during seed storage and their subsequent effect on grain moisture has been recognized by Harrington who suggested the three “rules of thumbs” regarding optimal seed storage. The first one which can be applied to seeds with grain moisture from 5 to 14 % states that each 1 % reduction of seed moisture doubles the storage life of the seeds; the second one states that for each 10 °F (5.6 °C) decrease in seed storage temperature storage life of seed is doubled; and the third one states that arithmetic sum of relative humidity and storage temperature should not exceed 100 for safe seed storage, or 120 as later reported (Bewley and Black, 1985; Copeland and McDonald, 1999; Harrington, 1973). POLJOPRIVREDA 16:2010 (2) 20-24 Temperatures below 21 °C and relative humidity values no higher than 60 % or 70 % for seeds kept at 4 °C should be used in implementation of these rules for long-term storage of crops, including wheat seeds, from 3 to 10 years. However, if the purpose of seed storage is to preserve planting stocks from one season to the next, implementations of suggested temperatures and humidity values for seed producers are impractical due to substantial costs regarding installation and maintenance of refrigeration and dehumidification equipment. Therefore, crop seeds and among them wheat, are in most cases stored under (1) DSc Ivica Strelec (, DSc @aneta Ugar~i}-Hardi, DSc Mirjana Sabo – Faculty of Food Technology, Franje Kuha~a 20, HR-31000 Osijek, (2) MSc Ru`a Popovi}, Ilonka Ivani{i}, BSc, Vlatka Jurkovi}, BSc – Agricultural Institute Osijek, Ju`no predgra|e 17, HR-31000 Osijek, (3) DSc Zorica Jurkovi} – Croatian Food Agency, Ivana Gunduli}a 36b, HR-31000 Osijek I. Strelec et al.: INFLUENCE OF TEMPERATURE AND RELATIVE HUMIDITY ON GRAIN. normal warehouse conditions. Since normal warehouse conditions fluctuate dependent on the season of the year, seeds in storage are exposed to low temperatures and humidity during winter, moderate temperatures and humidity during spring and autumn, and elevated during summer. Under such conditions seeds kept in cloths or paper bags temperate and easily exchange moisture with environmental air of defined relative humidity and temperature depending on species (Copeland and McDonald, 1999; Volenik et al., 2006). If moisture equilibration between seeds and environmental air result in an increase in seed moisture, deteriorative process increases with concomitant temperature within seeds speeds up, consequently leading to germination and vigour decrease. Seed deterioration physiology and dynamics of seed mortality during storage have been reviewed by a number of authors (Bernal-Lugo and Leopold, 1998; Bewley and Black, 1985; Copeland and McDonald, 1999; McDonald, 1999; Walters, 1998). However, much less is known influence of storage temperature and relative humidity on possible differences in germination and vigour between wheat cultivars. Therefore, the aim of this study was to monitor changes in grain moisture, germination and vigour of three wheat cultivar seeds packed in paper bags and stored at four different storage conditions. 21 Water Management of the Republic of Croatia (Narodne Novine, 04/2005). Filter paper was used as a sprouting base. Seed vigour was determined 4, and germination 7 days after germination test has been done. Temporal changes in grain moisture content, seed vigour and germination at various conditions of temperature and relative humidity were analysed by Friedman ANOVA and Kendall coefficient of concordance, while differences among cultivars in viability curves at the same storage conditions were examined by Wilcoxon t-test (p < 0.05) using statistical software Statistica (Stat Soft Inc., USA). RESULTS AND DISCUSSION Hygroscopic nature of seeds allows them to maintain equilibrium moisture content with any given relative humidity of storage air, which may lead to decrease or increase in initial seed moisture content during seed storage until equilibrium has been reached. During the first 90 days of wheat seeds storage moisture equilibration between grains and storage air occurred as shown in Figure 1. MATERIAL AND METHODS Seeds of wheat cultivars Žitarka and Srpanjka harvested in 2005 were generously supplied by Agricultural Institute in Osijek, and Divana cultivar by Visoko gospodarsko učilište, Križevci. Seeds of examined cultivars containing 13.7 % of moisture, were divided in batches (4 ageing conditions x 12 months; 1 kg of seed per batch), packed in paper bags, bags sealed, and storage of wheat seeds was done at four different conditions of environmental temperature and relative humidity (% RH): (1) 40 °C, RH = 45 %; (2) 25 °C, RH = 45 %; (3) 4 °C, RH = 45 %; (4) the range of warehouse conditions on the basis of local environmental conditions, varying from 2 to 25 °C and RH = 40-74 %. Storage was carried out in thermostatic incubator Heraeus (Heraeus, Germany) (1), in a storage box placed in conditioned warehouse (2), in a refrigerator (3), and on a shelf positioned 20 cm from the floor of unconditioned warehouse (4). Relative humidity of 45 % during storage was adjusted using saturated solutions of calcium chloride or potassium nitrite (Copeland and McDonald, 1999). Samples were taken monthly over a period of 12 months and moisture content, germination and seed vigour were examined. Grain moisture was measured in whole grains by Near Infrared Transmission (NIT) using Foss Tecator 1241 Grain Analyzer. Seed vigour and germination of examined cultivars were determined by the standard method of examination according to The Ministry of Agriculture, Forestry and Figure 1. Grain moisture change of Divana wheat cultivar during storage at different temperatures and relative humidity values Slika 1. Promjene vla`nosti zrna sorte Divana tijekom skladi{tenja pri razli~itim uvjetima temperature i relativne vla`nosti zraka Grain moisture of examined cultivars decreased: (i) from 3.7 to 4 % for seeds kept at 40 °C, RH = 45 %, (ii) for 2.6 % at 25 °C, RH = 45 %, and (iii) from 0.6 to 0.9 % at 4 °C, RH = 45 %, while increase in grain moisture between 0.2 to 0.4 % was detected for seeds kept in warehouse under uncontrolled storage conditions (2-25 °C, RH = 40-74 %). After equilibration, grain moisture of POLJOPRIVREDA 16:2010 (2) 20-24 22 I. Strelec et al.: INFLUENCE OF TEMPERATURE AND RELATIVE HUMIDITY ON GRAIN. Table 1. Average grain moisture content of different wheat cultivars equilibrated with air after 90 days of storage Tablica 1. Prosje~ne uravnote`ene vla`nosti zrna sorti p{enica koje zrna poprimaju nakon 90 dana skladi{tenja Storage conditions Divana Žitarka Srpanjka Grain moisture content/% 40 °C, RH = 45 % 10.0 ± 0.3 9.8 ± 0.3 9.7 ± 0.4 25 °C, RH = 45 % 11.1 ± 0.3 11.1 ± 0.2 11.0 ± 0.3 4 °C, RH = 45 % 12.9 ± 0.2 12.8 ± 0.1 13.1 ± 0.2 2-25°C, RH = 40-74 % 13.9 ± 0.3 13.8 ± 0.4 14.1 ± 0.3 examined cultivars remained at a constant level (Table 1) until the end of storage period. Analysis of differences in equilibrium grain moisture content (g.m.c.) by Wilcoxon t-test showed that significant differences in equilibrated g.m.c. exists between all applied storage conditions for each cultivar, while there were no significant differences in grain moisture content between cultivars kept at the same storage conditions. Decrease or increase in grain moisture in relation to relative humidity and temperature of storage air has been well documented (Bewley and Black, 1985; Brown, 2009; Chrastil, 1990; Copeland and McDonald, 1999; McDonald, 1999; Rehman and Shah, 1999; Volenik, 2006; Walters, 1998). The g.m.c. changes of examined wheat cultivars during moisture equilibration, observed in this work, as well as length of moisture equilibration period, and equilibrated g.m.c. are consistent with literature data (Brown, 2009; Copeland and McDonald, 1999). The decrease in wheat seed moisture content during the first 90 days of storage of approximately 4, 2.5 and 1 % at 40, 25 and 4 °C, respectively, and relative humidity of 45 % should lead to 8, 5 or 2-fold prolonged seed viability, since each 1% decrease in grain moisture content doubles the viability of seeds (Copeland and McDonald, 1999). However, viability of seeds is not only influenced by relative humidity and consequently grain moisture content, but also with seed temperature. According to the second Harrington’s “rule of thumb” each 5.6 °C reduction in seed temperature doubles the viability of the seed (Copeland and McDonald, 1999; Harrington, 1973), implying that seeds kept at 25 °C should have viability prolonged by factor 6 compared to seeds at 40 °C. When these factors of prolonged viability due to decrease in seed moisture content and temperature reduction are multiplied and compared, it becomes obvious that seeds kept at 25 °C should have 1.5-fold prolonged viability than seeds kept at 40 °C. This has been partially confirmed by observed changes in seed vigour and germination of examined wheat seeds. During one year of storage at 40 °C, RH = 45 % and 25 °C, RH = 45 % there was significant loss of germination and vigour in all examined cultivars. On the other hand, seeds kept at 4 °C, RH = 45 % and in warehouse with fluctuating environmental conditions Figure 2. Changes of germination and vigour of different wheat cultivars during storage at different environmental temperatures and relative humidity values. Wheat cultivars: (a) Divana, (b) @itarka, and (c) Srpanjka; Full markers and lines represent germination, and empty markers and dotted lines vigour changes Slika 2. Promjena klijavosti i energije klijanja sorti p{enica tijekom skladi{tenja pri razli~itim uvjetima temperature i relativne vla`nosti zraka. Sorte: (a) Divana, (b) @itarka i (c) Srpanjka; Punim markerima i linijama ozna~ena je klijavost, a praznim markerima i isprekidanim linijama energija klijanja POLJOPRIVREDA 16:2010 (2) 20-24 I. Strelec et al.: INFLUENCE OF TEMPERATURE AND RELATIVE HUMIDITY ON GRAIN. (2-25 °C and RH = 40-74 %) did not show significant changes in either germination or vigour (Figure 2). Decrease in germination and vigour was much greater for seeds kept at 40 °C than at 25 °C (Figure 2). Significant decrease in germination has been observed for Divana and Srpanjka at 240 days, and at 270 days of storage at 40 °C, RH = 45 % for Žitarka cultivar. For wheat seeds kept at 25 °C, RH = 45 % significant decrease started at 300 days of storage for Žitarka, at 330 days for Srpanjka, and at 360 days for Divana cultivar. At the end of storage period at 40 °C, RH = 45 % germination percentage of cultivar Divana was the lowest, only 15 %, compared to cultivars Žitarka and Srpanjka which had significantly higher germination percentage of 65 and 56 %, respectively. On the contrary, germination percentage of Žitarka and Srpanjka stored at 25 °C, RH = 45 % was lower, 80 and 82 %, compared to Divana (90 %). Similar decrease was observed for the seed vigour, which only in the case of Divana started to decrease 30-60 days earlier than germination. All cultivars had lower vigour than germination percentage (Figure 2). Cultivar Divana had vigour of 6 and 85 %, Žitarka 38 and 78 %, while Srpanjka cultivar 45 and 80 % at the end of storage period at 40 °C and 25 °C, respectively (RH = 45 %). Due to lower values of vigour compared to germination percentage, seed viability curves of vigour changes during storage at 40 °C and 25 °C, always preceded the ones representing germination changes (Figure 2). Such differences in viability curves were expected since vigour was being determined fourth and germination percentage seventh day of the germination test. Therefore, incompletely developed seeds fourth day of germination, unaccounted to vigour, had additional three days for development, and if developed properly they are accounted for germination percentage. This implies vigour as better indicator of seed deterioration. Viability curves of germination or vigour changes during storage of seeds at 40 °C, RH = 45 % as well as at 25 °C, RH = 45 % slightly differed between the examined cultivars. Analysis of differences among cultivars in viability curves at the same storage condition by Wilcoxon t-test showed that there was significant difference in viability curves of germination changes during storage at 40 °C, RH = 45 % between all cultivars, while no significant differences between cultivars could be found for seeds kept at 25 °C, RH = 45 %. Similarly, for viability curves of vigour changes during storage there were no significant differences for seeds kept at 25 °C, RH = 45 % while for seeds kept at 40 °C, RH = 45 % only cultivar Divana significantly differed from Žitarka and Srpanjka (p < 0.05). Differences in viability curves between the wheat cultivars point to that seed viability during storage was not only influenced by storage temperature, relative humidity and grain moisture content, but also by varietal characteristics. Seed viability differences between cultivars of the same species have been reported for maize, barley, soybeans and rice (Ali et al., 2003; Bewley and Black, 1985; Copeland and McDonald, 1999; Moller and Munck, 23 2002; Ramanadane and Ponnuswamy, 2004). The most significant reasons for variety differences in seed viability as well as dynamics of seed mortality are: (a) amount of soluble sugars in seeds that influence glassy state of living parts of grains and prevents deterioration reaction within seeds during storage; (b) antioxidants potential of seeds dependent on the level of endogenous antioxidants such as carotenoids, tocoferols, reduced glutathione, ascorbic acid, polyphenols, flavonoids and free amino acids which prevent oxidation and nonenzymatic glycolysation of proteins, lipids and nucleic acids within seeds during storage, and (c) endogenous level of proteolytic and antioxidant enzymes that are capable to fix cellular impairments caused by storage at onset and during germination process (Bernal-Lugo and Leopold, 1998; Calucci et al., 2004; McDonald, 1999; Pinzino, 1999; Steadman et al., 1996; Strelec et al., 2008; Sun and Leopold, 1993). Lower levels of soluble sugars, antioxidants, or endogenous enzymes in Divana cultivar could be the reasons for diminished viability of Divana seeds compared to Žitarka and Srpanjka cultivars. Further research my reveal probable culprit and give insights into desirable compositional qualities of the wheat seeds. CONCLUSION Wheat seeds packed in paper bags and stored during one year at four different storage conditions change moisture content, germination and vigour dependent on applied temperature and relative humidity values of environmental air. Significant decrease in grain moisture content of 4 %, 2.5 and 1 % at relative humidity of 45 % and temperature of 40 °C, 25 °C and 4 °C, respectively, occured during first ninety days of seed storage. Seed germination and vigour are significantly reduced during one year storage only at elevated temperatures. Storage of seeds at 40 °C, RH = 45 % caused 35-85 % decreased germination and 55-94 % reduced vigour; at 25 °C, RH = 45 %, 10-20 % decrease in germination and 15-22 % reduced vigour were detected. Significant differences between cultivars in viability curves of germination and vigour changes during storage at 40 °C, RH = 45 % indicate that wheat seeds viability may be highly dependent on cultivar probably due to variety differences in soluble sugar content, antioxidant potential, and endogenous oxido-reductive and proteolytic enzyme levels. REFERENCES 1. 2. Ali, M.G., Naylor, R.E.R., Matthews, S. (2003): The effect of aging (using controlled deterioration) on the germination at 21°C as an indicator of physiological quality of seed lots of fourteen Bangladesi rice (Oryza sativa L.) cultivars. Pakistan Journal of Biological Sciences, 6(10): 910-917. Bernal-Lugo, I., Leopold, A.C. (1998): The dynamics of seed mortality. Journal of Experimental Botany, 49: 1455-1461. POLJOPRIVREDA 16:2010 (2) 20-24 24 I. Strelec et al.: INFLUENCE OF TEMPERATURE AND RELATIVE HUMIDITY ON GRAIN. 3. Bewley, J.D., Black, M. (1985): Seeds: Physiology of Development and Germination. Plenum Press, New York. 4. Brown, C. (2009): Agronomy Guide for Field Crops Publication 811. Ministry of equilibrium value of MeCpG steps (,+14 deg.) [31,44]. In comparison, methylation has a significantly lower stability cost when happening at major groove positions, such as 211 and 21 base pair from dyad (mutations 9 and 12), where the roll of the nucleosome bound conformation (+10 deg.) is more compatible with the equilibrium geometry of MeCpG steps. The nucleosome destabilizing effect of cytosine methylation increases with the number of methylated cytosines, following the same position dependence as the single methylations. The multiple-methylation case reveals that each major groove meth- PLOS Computational Biology | 3 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning ylation destabilizes the nucleosome by around 1 kJ/mol (close to the average estimate of 2 kJ/mol obtained for from individual methylation studies), while each minor groove methylation destabilizes it by up to 5 kJ/mol (average free energy as single mutation is around 6 kJ/mol). This energetic position-dependence is the reverse of what was observed in a recent FRET/SAXS study [30]. The differences can be attributed to the use of different ionic conditions and different sequences: a modified Widom-601 sequence of 157 bp, which already contains multiple CpG steps in mixed orientations, and which could assume different positioning due to the introduction of new CpG steps and by effect of the methylation. The analysis of our trajectories reveals a larger root mean square deviation (RMSD) and fluctuation (RMSF; see Figures S2– S3 in Text S1) for the methylated nucleosomes, but failed to detect any systematic change in DNA geometry or in intermolecular DNA-histone energy related to methylation (Fig. S1B, S1C, S4–S6 in Text S1). The hydrophobic effect should favor orientation of the methyl group out from the solvent but this effect alone is not likely to justify the positional dependent stability changes in Figure 2, as the differential solvation of the methyl groups in the bound and unbound states is only in the order of a fraction of a water molecule (Figure S5 in Text S1). We find however, a reasonable correlation between methylation-induced changes in hydrogen bond and stacking interactions of the bases and the change in nucleosome stability (see Figure S6 in Text S1). This finding suggests that methylation-induced nucleosome destabilization is related to the poorer ability of methylated DNA to fit into the required conformation for DNA in a nucleosome. Changes in the elastic deformation energy between methylated and un-methylated DNA correlate with nucleosomal differential binding free energies To further analyze the idea that methylation-induced nucleosome destabilization is connected to a worse fit of methylated DNA into the required nucleosome-bound conformation, we computed the elastic energy of the nucleosomal DNA using a harmonic deformation method [36,37,44]. This method provides a rough estimate of the energy required to deform a DNA fiber to adopt the super helical conformation in the nucleosome (full details in Suppl. Information Text S1). As shown in Figure 2, there is an evident correlation between the increase that methylation produces in the elastic deformation energy (DDE def.) and the free energy variation (DDG bind.) computed from MD/TI calculations. Clearly, methylation increases the stiffness of the CpG step [31], raising the energy cost required to wrap DNA around the histone octamers. This extra energy cost will be smaller in regions of high positive roll (naked DNA MeCpG steps have a higher roll than CpG steps [31]) than in regions of high negative roll. Thus, simple elastic considerations explain why methylation is better tolerated when the DNA faces the histones through the major groove (where positive roll is required) that when it faces histones through the minor groove (where negative roll is required). Nucleosome methylation can give rise to nucleosome repositioning We have established that methylation affects the wrapping of DNA in nucleosomes, but how does this translate into chromatin structure? As noted above, accumulation of minor groove methylations strongly destabilizes the nucleosome, and could trigger nucleosome unfolding, or notable changes in positioning or phasing of DNA around the histone core. While accumulation of methylations might be well tolerated if placed in favorable positions, accumulation in unfavorable positions would destabilize the nucleosome, which might trigger changes in chromatin structure. Chromatin could in fact react in two different ways in response to significant levels of methylation in unfavorable positions: i) the DNA could either detach from the histone core, leading to nucleosome eviction or nucleosome repositioning, or ii) the DNA could rotate around the histone core, changing its phase to place MeCpG steps in favorable positions. Both effects are anticipated to alter DNA accessibility and impact gene expression regulation. The sub-microsecond time scale of our MD trajectories of methylated DNAs bound to nucleosomes is not large enough to capture these effects, but clear trends are visible in cases of multiple mutations occurring in unfavorable positions, where unmethylated and methylated DNA sequences are out of phase by around 28 degrees (Figure S7 in Text S1). Due to this repositioning, large or small, DNA could move and the nucleosome structure could assume a more compact and distorted conformation, as detected by Lee and Lee [29], or a slightly open conformation as found in Jimenez-Useche et al. [30]. Using the harmonic deformation method, we additionally predicted the change in stability induced by cytosine methylation for millions of different nucleosomal DNA sequences. Consistently with our calculations, we used two extreme scenarios to prepare our DNA sequences (see Fig. 3): i) all positions where the minor grooves contact the histone core are occupied by CpG steps, and ii) all positions where the major grooves contact the histone core are occupied by CpG steps. We then computed the elastic energy required to wrap the DNA around the histone proteins in unmethylated and methylated states, and, as expected, observed that methylation disfavors DNA wrapping (Figure 3A). We have rescaled the elastic energy differences with a factor of 0.23 to match the DDG prediction in figure 2B. In agreement with the rest of our results, our analysis confirms that the effect of methylation is position-dependent. In fact, the overall difference between the two extreme methylation scenarios (all-in-minor vs all-in-major) is larger than 60 kJ/mol, the average difference being around 15 kJ/ mol. We have also computed the elastic energy differences for a million sequences with CpG/MeCpG steps positioned at all possible intermediate locations with respect to the position (figure 3B). The large differences between the extreme cases can induce rotations of DNA around the histone core, shifting its phase to allow the placement of the methylated CpG steps facing the histones through the major groove. It is illustrative to compare the magnitude of CpG methylation penalty with sequence dependent differences. Since there are roughly 1.5e88 possible 147 base pairs long sequence combinations (i.e., (4n+4(n/2))/2, n = 147), it is unfeasible to calculate all the possible sequence effects. However, using our elastic model we can provide a range of values based on a reasonably large number of samples. If we consider all possible nucleosomal sequences in the yeast genome (around 12 Mbp), the energy difference between the best and the worst sequence that could form a nucleosome is 0.7 kj/mol per base (a minimum of 1 kJ/mol and maximum of around 1.7 kJ/mol per base, the first best and the last worst sequences are displayed in Table S3 in Text S1). We repeated the same calculation for one million random sequences and we obtained equivalent results. Placing one CpG step every helical turn gives an average energetic difference between minor groove and major groove methylation of 15 kJ/ mol, which translates into ,0.5 kJ/mol per methyl group, 2 kJ/ mol per base for the largest effects. Considering that not all nucleosome base pair steps are likely to be CpG steps, we can conclude that the balance between the destabilization due to CpG methylation and sequence repositioning will depend on the PLOS Computational Biology | 4 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning Figure 3. Methylated and non-methylated DNA elastic deformation energies. (A) Distribution of deformation energies for 147 bplong random DNA sequences with CpG steps positioned every 10 base steps (one helical turn) in minor (red and dark red) and major (light and dark blue) grooves respectively. The energy values were rescaled by the slope of a best-fit straight line of figure 2, which is 0.23, to source of circulating FGF-21. The lack of association between circulating and muscle-expressed FGF-21 also suggests that muscle FGF-21 primarily works in a local manner regulating glucose metabolism in the muscle and/or signals to the adipose tissue in close contact to the muscle. Our study has some limitations. The number of subjects is small and some correlations could have been significant with greater statistical power. Another aspect is that protein levels of FGF-21 were not determined in the muscles extracts, consequently we cannot be sure the increase in FGF-21 mRNA is followed by increased protein expression. In conclusion, we show that FGF-21 mRNA is increased in skeletal muscle in HIV patients and that FGF-21 mRNA in muscle correlates to whole-body (primarily reflecting muscle) insulin resistance. These findings add to the evidence that FGF-21 is a myokine and that muscle FGF-21 might primarily work in an autocrine manner. Acknowledgments We thank the subjects for their participation in this study. 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