Abstract
The objective of the present study was to develop a combination of fluorescent stains that would allow visualisation of the network of mitochondria and lipid droplets (intramyocellular lipids or IMCL) in human skeletal muscle fibres by means of conventional and confocal microscopy. Muscle biopsies were taken from the vastus lateralis of three lean, healthy and physically active male subjects. Frozen muscle sections were stained for mitochondria using antibodies against three mitochondrial proteins; porin, cytochrome c oxidase (COX) and NADH-ubiquinol oxidoreductase and neutral lipids were stained with oil red O. Anti-COX staining produced images with the strongest fluorescence signal and the highest resolution of the mitochondrial network and this stain was successfully combined with the antibody against type I fibre myosin. A highly organised matrix arrangement of mitochondria within the sarcomeres (in pairs at the I-band) was observed in the oxidative type I fibres. The density of mitochondria was the highest in the subsarcolemmal region. Anti-COX staining was combined with oil red O demonstrating that in type I fibres lipid droplets are mainly located in the space between the mitochondria.
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Bergstrom J (1975) Percutaneous needle biopsy of skeletal muscle in physiological and clinical research. Scand J Clin Lab Invest 35:609–616
De Bock K, Dresselaers T, Kiens B, Richter EA, van Hecke P, Hespel P (2007) Evaluation of intramyocellular lipid breakdown during exercise by biochemical assay, NMR spectroscopy, and oil-red-oil staining. Am J Physiol Endocrinol Metab doi:10.1152/ajpendo.00112.2007
De Pinto V, Ludwig O, Krause J, Benz R, Palmeri F (1987) Porin pores of mitochondrial outer membranes from high and low eukaryotic cells: biochemical and biophysical characterization. Biochim Biophys Acta 894:109–119
Granneman JG, Moore HPH, Granneman RL, Greenberg AS, Obin MS, Zhu Z (2007) Analysis of lipolytic protein trafficking and interactions in adipocytes. J Biol Chem 282:5726–5735
Hashimoto T, Masuda S, Taguchi S, Brooks GA (2005) Immunohistochemical analysis of MCT1, MCT2, and MCT 4 expression in rat plantaris muscle. J Physiol 567:121–129
He J, Goodpaster BH, Kelley DE (2004) Effects of weight loss and physical activity on muscle lipid content and droplet size. Obese Res 12:761–769
Hoppeler H (1999) Skeletal muscle substrate metabolism. Int J Obes Relat Metab Disord 23:S7–S10
Holloway GP, Thrush AB, Heigenhauser GJF, Tandon NN, Dyck DJ, Bonen A, Spriet LL (2007) Skeletal muscle mitochondrial FAT/CD36 content and palmitate oxidation are not decreased in obese women. Am J Physiol Endocrinol Metab 292:E1782–E1789
Howald H, Hoppeler H, Claassen H, Mathieu O, Straub R (1985) Influences of endurance training on the ultrastructural composition of the different muscle fiber types in humans. Pflugers Arch 403:369–376
Itani SI, Pories WJ, MacDonald KG, Dohm GL (2001) Increased protein kinase C theta in skeletal muscle of diabetic patients. Metabolism 50:553–557
Itani SI, Ruderman NB, Schmeider F, Boden G (2002) Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IκB-α. Diabetes 51:2005–2011
Kayar SR, Hoppeler H, Essen-Gustavsson B, Schwerzmann K (1988a) The similarity of mitochondrial distribution in equine skeletal muscles of differing oxidative capacity. J Exp Biol 137:253–263
Kayar SR, Hoppeler H, Mermod L, Weibel ER (1988b) Mitochondrial size and shape in equine skeletal muscle: a three-dimensional reconstruction study. Anat Rec 222:333–339
Kim JY, Hickner RC, Cortright RL, Dohm GL, Houmard JA (2000) Lipid oxidation is reduced in obese human skeletal muscle. Am J Physiol Endocrinol Metab 279:E1039–E1044
Koopman R, Schaart G, Hesselink MKC (2001) Optimisation of oil red O staining permits combination with immunofluorescence and automated quantification of lipids. Histochem Cell Biol 116:63–68
Kuznetsov AV, Troppmair J, Sucher R, Hermann M, Saks V, Margreiter R (2006) Mitochondrial subpopulations and heterogeneity revealed by confocal imaging: possible physiological role? Biochimica Biophysica Acta 1757:686–691
Malenfant P, Joanisse DR, Theriault R, Goodpaster BH, Kelley DE, Simoneau JA (2001) Fat content in individual muscle fibers of lean and obese subjects. Int J Obes Relat Metab Disord 25:1316–1321
Ogata T, Yamasaki Y (1997) Ultra-high-resolution scanning electron microscopy of mitochondria and sarcoplasmic reticulum arrangement in human red, white and intermediate muscle fibres. Anatomical Rec 248:214–223
Petersen KF, Shulman GI (2006) Etiology of insulin resistance. Am J Med 119:10S–16S
Prats C, Donsmark M, Qvortrup K, Londos C, Sztalryd C, Holm C, Galbo H, Ploug T (2006) Decrease in intramuscular lipid droplets and translocation of HSL in response to muscle contraction and epinephrine. J Lipid Res 47:2392–2399
Ritov VB, Menshikova EV, He J, Ferrell RE, Goodpaster BH, Kelley DE (2005) Deficiency of subsarcolemmal mitochondria in obesity and type 2 diabetes. Diabetes 54:8–14
Schrauwen-Hinderling VB, van Loon LJC, Koopman R, Nicolay K, Saris WHM, Kooi ME (2003) Intramyocellular lipid content is increased after exercise in nonexercising human skeletal muscle. J Appl Physiol 95:2328–2332
Shulman GI (2000) Cellular mechanisms of insulin resistance. J Clin Invest 106:171–176
Stellingwerf T, Boon H, Jonkers RAM, Senden JM, Spriet LL, Koopman R, van Loon LJC (2007) Significant intramyocellular lipid use during prolonged cycling in endurance trained males as assessed by three different methodologies. Am J Physiol Endocrinol Metab 292:E1715–E1723
Tarnopolsky MA, Rennie CD, Robertshaw HA, Fedak-Tarnopolsky SN, Brose AN, Devries MC, Hamadeh MJ (2006) The influence of endurance exercise training and sex on intramyocellular lipid and mitochondrial ultrastructure. Am J Physiol Regul Integr Comp Physiol 292:R1271–R1278
van Loon LJC (2004) Use of intramuscular triacylglycerol as a substrate source during exercise in humans. J Appl Physiol 97:1170–1187
van Loon LJC, Greenhaff PL, Constantin-Teodosiu D, Saris WHM, Wagenmakers AJM (2001) The effects of increasing exercise intensity on muscle fuel utilisation in humans. J Physiol 536:295–304
van Loon LJC, Koopman R, Stegen JHCH, Wagenmakers AJM, Keizer HA, Saris WHM (2003) Intramyocellular lipids form an important substrate source during moderate intensity exercise in endurance-trained males in a fasted state. J Physiol 553:611–625
van Loon LJC, Manders RJF, Koopman R, Kaastra B, Stegen JHCH, Gijsen AP, Saris WHM, Keizer HA (2005) Inhibition of adipose tissue lipolysis increases intramuscular lipid use in type 2 diabetic patients. Diabetologia 48:2097–2107
Vendelin M, Beraud N, Guerrero K, Andrienko T, Kuznetsov AV, Olivares J, Kay L, Saks VA (2005) Mitochondrial regular arrangement in muscle cells: a “crystal-like” pattern. Am J Physiol Cell Physiol 288:C757–C767
Vock R, Hoppeler H, Claasen H, Wu DXY, Billeter R, Weber J, Taylor CR, Weibel ER (1996) Design of the oxygen substrate pathways. VI. Structural basis of intracellular substrate supply to mitochondria in muscle cells. J Exp Biol 199:1689–1697
Watt MJ, Heigenhauser GJF, Spriet LL (2002) Intramuscular triacylglycerol utilization in human skeletal muscle during exercise: is there a controversy? J Appl Physiol 93:1185–1195
Yu C, Chen Y, Cline GW, Zhang D, Zong H et al (2002) Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem 277:50230–50236
Acknowledgments
The antibody against myosin (human slow fibres, A4.840) used in the study was developed by Dr. Blau and was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.
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Shaw, C.S., Jones, D.A. & Wagenmakers, A.J.M. Network distribution of mitochondria and lipid droplets in human muscle fibres. Histochem Cell Biol 129, 65–72 (2008). https://doi.org/10.1007/s00418-007-0349-8
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DOI: https://doi.org/10.1007/s00418-007-0349-8