Single muscle fiber enzyme shifts with hindlimb suspension and immobilization

1989 ◽  
Vol 256 (5) ◽  
pp. C1082-C1091 ◽  
Author(s):  
R. H. Fitts ◽  
C. J. Brimmer ◽  
A. Heywood-Cooksey ◽  
R. J. Timmerman
Keyword(s):  
Citrate Synthase ◽  
Fiber Type ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Glycolytic Enzymes ◽  
Hindlimb Suspension ◽  
Metabolic Enzyme ◽  
Fiber Types ◽  
Freeze Dried ◽  
Fast Fiber

The purpose of this investigation was to determine how models of weightlessness, hindlimb suspension (HS), and hindlimb immobilization (HI) affect the metabolic enzyme profile in the slow oxidative (SO), fast oxidative glycolytic (FOG), and fast glycolytic (FG) fibers of rat hindlimb. After 1, 2, or 4 wk of HS or HI, single fibers were isolated from freeze-dried soleus and gastrocnemius muscles; a small section of each fiber was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels to identify fiber type, and the remaining piece was assayed for either lactate dehydrogenase (LDH) and citrate synthase (CS) or phosphofructokinase (PFK) and beta-hydroxyacyl-CoA dehydrogenase (beta-OH-acyl-CoA). Two weeks of HS induced an almost twofold increase in the activity of CS (2.13 +/- 0.13 vs. 3.60 +/- 0.26 mol.kg dry wt-1.h-1) in the SO fiber of the soleus, and the activity stayed high at 4 wk. Although the FOG fiber had significantly higher CS activity (3.85 +/- 0.29) than either the SO or FG (1.59 +/- 0.16 mol.kg dry wt-1.h-1) fiber, neither fast fiber type was altered by HS. The glycolytic enzymes LDH and PFK were both elevated in the SO fiber after HS. The increase in LDH occurred by 1 wk (14.80 +/- 1.51 vs. 8.83 +/- 0.78), whereas the activity of PFK was not significantly changed until 4 wk (1.16 +/- 0.13 vs. 0.68 +/- 0.05 mol.kg dry wt-1.h-1). The control FG fiber had the highest LDH (44.30 +/- 2.29) and PFK (2.40 +/- 0.16) activities, followed by the FOG fiber (LDH, 34.10 +/- 2.83; PFK, 1.62 +/- 0.17 mol.kg dry wt-1.h-1); however, the activities of these glycolytic enzymes in the fast fiber types were unaltered by HS. The activity of beta-OH-acyl-CoA was not affected by HS in either the slow or fast fiber types. HI showed qualitatively similar changes to those observed with HS; however, the enzyme shifts developed with a slower time course. In conclusion, both HS and HI shifted the SO fiber enzyme pattern toward that of the control FOG fiber; however, a complete conversion from the SO to FOG fiber did not occur within the 4-wk treatment period.

Shortening velocity and ATPase activity of rat skeletal muscle fibers: effects of endurance exercise training

1994 ◽  
Vol 266 (6) ◽  
pp. C1699-C1713 ◽  
Author(s):  
J. M. Schluter ◽  
R. H. Fitts
Keyword(s):  
Atpase Activity ◽  
Endurance Exercise ◽  
Citrate Synthase ◽  
Fiber Type ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Treadmill Running ◽  
Type I ◽  
Marker Enzyme ◽  
Shortening Velocity

Mechanical properties were measured in single skinned fibers from rat hindlimb muscle to test the hypothesis that the fast type IIb fiber exhibits a higher maximal shortening velocity (Vo) than the fast type IIa fiber and that the difference is directly attributable to a higher myofibrillar adenosinetriphosphatase (ATPase) activity in the type IIb fiber. Additional measurements were made to test the hypotheses that regular endurance exercise increases and decreases the Vo of the type I and IIa fiber, respectively, and that the altered Vo is associated with a corresponding change in the fiber ATPase activity. Rats were exercised by 8-12 wk of treadmill running for 2 h/day, 5 day/wk, up a 15% grade at a speed of 27 m/min. Fiber Vo was determined by the slack test, and the ATPase was measured fluorometrically in the same fiber. The myosin isozyme profile of each fiber was subsequently determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mean +/- SE Vo (7.9 +/- 0.22 fiber lengths/s) of the type IIb fiber was significantly greater than the type IIa fiber (4.4 +/- 0.21 fiber lengths/s), and the higher Vo was associated with a higher ATPase activity (927 +/- 70 vs. 760 +/- 60 microM.min-1.mm-3). The exercise program induced cardiac hypertrophy and an approximately twofold increase in the mitochondrial marker enzyme citrate synthase. Exercise had no effect on fiber diameter or peak tension per cross-sectional area in any fiber type, but, importantly, it significantly increased (23%) both the Vo and the ATPase activity of the slow type I fiber of the soleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Inducible isoform of HSP70 is constitutively expressed in a muscle fiber type specific pattern

1991 ◽  
Vol 261 (5) ◽  
pp. C774-C779 ◽  
Author(s):  
M. Locke ◽  
E. G. Noble ◽  
B. G. Atkinson
Keyword(s):  
Muscle Fiber ◽  
Fiber Type ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Constitutive Expression ◽  
Muscle Fiber Types ◽  
Type I ◽  
Fiber Types ◽  
Nylon Membrane ◽  
White Gastrocnemius

The most prominent group of stress or heat-shock proteins (HSPs) has an Mr of approximately 70,000 and is collectively referred to as the HSP70 family. The extent of stress inducibility and subcellular location of the various HSP70 isoforms differ, but all appear to be involved with ATP-dependent stabilization or solubilization of proteins. One isoform, termed the inducible isoform of HSP70 (HSP72i), is normally absent in unstressed cells. In a previous study, we detected a protein corresponding in Mr and pI to HSP72i in unstressed rat muscle. Therefore, it was of interest to determine if this expression in unstressed muscle cells is general or confined to specific muscle fiber types. To answer this question we have employed various rat hindlimb muscles that differ in fiber type proportion from predominantly type I (soleus) to predominantly type IIB (white gastrocnemius). Proteins from muscle homogenates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to a nylon membrane, probed with a monoclonal antibody for HSP72i, and visualized using an alkaline phosphatase-conjugated secondary antibody. Immunoblot analyses demonstrate the constitutive expression of HSP72i in rat muscles comprised primarily of type I muscle fibers (soleus), but not in muscles comprised primarily of type IIB fibers (white gastrocnemius). In muscles of mixed fiber type, HSP72i content is roughly proportional to the percentage of type I fibers. These results substantiate that unstressed rat muscles express the inducible HSP72 isoform and demonstrate that its constitutive expression is proportional to the type I muscle fiber composition.

scholarly journals Force-velocity and power characteristics of rat soleus muscle fibers after hindlimb suspension

1994 ◽  
Vol 77 (4) ◽  
pp. 1609-1616 ◽  
Author(s):  
K. S. McDonald ◽  
C. A. Blaser ◽  
R. H. Fitts
Keyword(s):  
Isometric Force ◽  
Fiber Type ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hindlimb Suspension ◽  
Type I ◽  
Shortening Velocity ◽  
Power Characteristics ◽  
Sds Page ◽  
Force Velocity

The effects of 1, 2, and 3 wk of hindlimb suspension (HS) on force-velocity and power characteristics of single rat soleus fibers were determined. After 1, 2, or 3 wk of HS, small fiber bundles were isolated, placed in skinning solution, and stored at -20 degrees C until studied. Single fibers were isolated and placed between a motor arm and force transducer, functional properties were studied, and fiber protein content was subsequently analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Additional fibers were isolated from soleus of control and after 1 and 3 wk of HS, and fiber type distribution and myosin light chain stoichiometry were determined from SDS-PAGE analysis. After 1 wk of HS, percent type I fibers declined from 82 to 74%, whereas hybrid fibers increased from 10 to 18%. Percent fast type II fibers increased from 8% in control and 1 wk of HS to 26% by 3 wk of HS. Most fibers showed an increased unloaded maximal shortening velocity (Vo), but myosin heavy chain remained entirely slow type I. The mechanism for increased Vo is unknown. There was a progressive decrease in fiber diameter (14, 30, and 38%) and peak force (38, 56, and 63%) after 1, 2, and 3 wk of HS, respectively. One week of HS resulted in a shift of the force-velocity curve, and between 2 and 3 wk of HS the curve shifted further such that Vo was higher than control at all relative loads < 45% peak isometric force. Peak absolute power output of soleus fibers progressively decreased through 2 wk of HS but showed no further change at 3 wk.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme activities of FT and ST muscle fibers in heavy-resistance trained athletes

1989 ◽  
Vol 67 (1) ◽  
pp. 83-87 ◽  
Author(s):  
P. A. Tesch ◽  
A. Thorsson ◽  
B. Essen-Gustavsson
Keyword(s):  
Resistance Training ◽  
Enzyme Activities ◽  
Citrate Synthase ◽  
Fiber Type ◽  
Vastus Lateralis ◽  
Vastus Lateralis Muscle ◽  
Fiber Types ◽  
Tissue Samples ◽  
Fiber Area ◽  
Freeze Dried

Tissue samples were obtained from the vastus lateralis muscle of elite olympic weight and power lifters (OL/PL, n = 6), bodybuilders (BB, n = 7), and sedentary men (n = 7). Enzyme activities of citrate synthase (CS), lactate dehydrogenase (LD), 3-OH-acyl-CoA-dehydrogenase (HAD), and myokinase (MK) were assayed on freeze-dried dissected pools of slow-twitch (ST) and fast-twitch (FT) fiber fragments by fluorometric means. Histochemical analyses were carried out to assess fiber type composition and fiber area. CS and HAD activities were lower (P less than 0.05), and LD and MK were higher (P less than 0.05) in FT than ST fibers in the entire subject pool (n = 20). CS of FT fibers and HAD of ST fibers were lower in athletes (P less than 0.05–0.01) compared with nonathletes, whereas LD of both fiber types was higher (P less than 0.05–0.001) in athletes. CS activity of ST fibers and MK activity of FT fibers were higher (P less than 0.05) in BB compared with OL/PL. FT and ST fiber area was greater (P less than 0.05) in athletes than in nonathletes. BB displayed greater (P less than 0.05) fiber size than OL/PL. FT/ST area was greater (P less than 0.05) in OL/PL than BB. It is suggested that long-term heavy-resistance training results in specific metabolic adaptations of FT and ST fiber types. These changes appear to be influenced by the type of resistance training.

Development of metabolic enzyme activity in locomotor and cardiac muscles of the migratory barnacle goose

1995 ◽  
Vol 269 (1) ◽  
pp. R64-R72 ◽  
Author(s):  
C. M. Bishop ◽  
P. J. Butler ◽  
S. Egginton ◽  
A. J. el Haj ◽  
G. W. Gabrielsen
Keyword(s):  
Plasma Glucose ◽  
Citrate Synthase ◽  
Specific Activity ◽  
Glycolytic Enzymes ◽  
Metabolic Enzyme ◽  
Mitochondrial Enzymes ◽  
Pectoralis Muscle ◽  
Barnacle Goose ◽  
Average Maximum ◽  
Cardiac Muscles

Preflight development of the goslings was typified by rapid increases in the mitochondrial enzymes of the semimembranosus and heart ventricular muscles resulting in near-adult values by 3 wk of age. In contrast, aerobic capacity of the pectoralis muscle initially developed slowly but showed a rapid increase between 5 and 7 wk of age, in preparation for becoming airborne. Activities of glycolytic enzymes in the pectoralis muscle showed similar patterns of development as those found for the aerobic enzymes, except for hexokinase, which was low at all ages, indicating an adaptation for catabolism of both intracellular glycogen and plasma fatty acids in preference to plasma glucose. Muscle mass specific activity of citrate synthase in the pectoralis increased by only 33% from goslings during the first few days of flight, compared with premigratory geese. Activities of anaerobic glycolytic enzymes in the ventricles were low, but values for hexokinase, which is involved in the phosphorylation of plasma glucose, developed rapidly. Values for lactate dehydrogenase were also high, reflecting the capacity of the heart to catabolize plasma lactate. Substrate flux supplied by carnitine palmitoyltransferase and oxoglutarate dehydrogenase (OGD), in the pectoralis muscles of the premigratory geese, appears to have the smallest excess capacities to meet the requirements of sustained aerobic flight. The average maximum oxygen uptake for premigratory geese during flight, as indicated by values for OGD, is calculated to be 484 ml O2/min (or 208 ml O2.min-1.kg-1).

Size and metabolic properties of fibers in rat fast-twitch muscles after hindlimb suspension

1987 ◽  
Vol 62 (6) ◽  
pp. 2348-2357 ◽  
Author(s):  
R. R. Roy ◽  
M. A. Bello ◽  
P. Bouissou ◽  
V. R. Edgerton
Keyword(s):  
Fiber Type ◽  
Female Rats ◽  
Medial Gastrocnemius ◽  
Hindlimb Suspension ◽  
Staining Reaction ◽  
Fiber Types ◽  
Image Analysis System ◽  
Cross Sectional ◽  
Glycerophosphate Dehydrogenase ◽  
Metabolic Marker

Hindlimb suspension (HS) results in whole muscle atrophic and metabolic changes that vary in magnitude in different hindlimb muscles. The present study was designed to investigate these effects in single fibers. Fiber type and size and the activities of two metabolic marker enzymes were determined in a deep (close to the bone) and a superficial (away from the bone) region of the medial gastrocnemius (MG) and the tibialis anterior (TA) of control (CON) and 28-day HS adult female rats. Fibers were classified as dark or light adenosinetriphosphatase (ATPase) based on their qualitative staining reaction for myosin ATPase following alkaline preincubation. Fiber area and succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities were determined in tissue sections by use of an image analysis system. After 28 days of HS, the mean body weights of the CON and HS were similar. MG atrophied 28%, whereas TA weight was maintained in the HS. Both dark and light ATPase fibers in the deep region of the MG had smaller cross-sectional areas following HS, with the atrophic response being approximately twice as great in the light ATPase fibers. No significant changes in fiber type composition in either muscle or in fiber sizes in the superficial region of the MG or in either region of the TA were observed. Mean SDH activities of both fiber types were significantly lower in the MG and TA following HS. In contrast, mean GPD activities were either increased or maintained in light and dark ATPase fibers of both muscles in HS. Changes in SDH and GPD activity could not be directly linked to changes in fiber cross-sectional area. In summary, these data suggest an independence of the mechanisms determining muscle fiber size and metabolic adaptations associated with HS.

16 BIOCHEMICAL ANALYSIS OF COMPONENT IN SEMINAL GEL SECRETED WITH BOAR SEMEN

2015 ◽  
Vol 27 (1) ◽  
pp. 100
Author(s):  
G. Takahashi ◽  
M. Maeda ◽  
Y. Kimura ◽  
H. Funahashi
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Sugar ◽  
Polyacrylamide Gel ◽  
High Fertility ◽  
Rp Hplc ◽  
Ms Analysis ◽  
Freeze Dried

Seminal gel (SG), a part of semen, of the boar originates from secretions from the Cowper's gland and has a high viscosity and water-holding capacity, preventing backflow of semen at natural mating. However, there are is little information available about biochemical and functional characteristics of boar SG. In this study, as a first step to elucidate the chemical features of the SG, we examined the structure of O-glycans and the primary structure of protein from the boar SG. Seminal gel was collected from ejaculated semen of a Berkshire boar with high fertility and freeze-dried. Samples were preserved in a refrigerator until experiments were conducted. For Exp. 1 the presence of O-glycans in SG was confirmed by detection of the amino sugar, galactosamine (GalNH2), from acid hydrolysis of GalNAc. The freeze-dried SG (1 mg) was hydrolyzed with 4N trifluoroacetic acid at 110°C for 2 h. The resulting amino sugar was labelled with phenyl isothiocyanate (PITC) and then analysed by RP-HPLC. The GalNAc was detected as a main amino sugar, suggesting that the SG contains O-glycosylated glycoprotein. For Exp. 2 the O-glycans were prepared from the freeze-dried SG (5 mg) by hydrazinolysis at 100°C for 2 h. After N-acetylation, the O-glycans were pyridylaminated. The structures were identified by anion-exchange HPLC, size-fractionation HPLC, glycosidase digestion, and ESI-MS and MS/MS analysis. Almost all glycans were digested by α2–3,6-sialidasae, indicating that these O-glycans are sialylated and give the glycoproteins viscosity. Furthermore, the MS analysis showed that the de-sialylated O-glycans consist of HexNAc-PA (m/z 300.0) and Hex-HexNAc-PA (m/z 462.0) and major glycans are di- or tri-saccharides. For Exp. 3 proteins in the SG were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition with 5% 2-mercaptoethanol. Proteins were stained with Coomassie Brilliant Blue R-250. Three bands (~160, 140, and 70 kDa) were found on 7.5% polyacrylamide gel, but two bands (160, 140 kDa) were converted to ~130 kDa after the sialidase digestion, indicating that native two proteins (160 and 140 kDa) may be highly sialylated. For Exp. 4 internal amino acid sequence was analysed using one of the peptic peptides. The freeze-dried SG (5 mg) was digested with porcine pepsin in 5% formic acid at 37°C for 3 h. The resulting peptides were separated by RP-HPLC. N-terminal sequence of one of the peptic peptides was WSEKYGIPGGKAH. The amino acid sequence showed a high homology with tyrosine-protein kinase ZAP-70. These results suggest that boar SG contains mucin-like glycoproteins carrying heavily sialylated O-glycans. Additionally, the current study suggests a possibility that some protein components of the boar SG derive from high concentration of the kinase in (dead) sperms.

Lower activity of oxidative key enzymes and smaller fiber areas in skeletal muscle of postobese women

1998 ◽  
Vol 275 (3) ◽  
pp. E487-E494 ◽  
Author(s):  
Anne Raben ◽  
Elsebeth Mygind ◽  
Arne Astrup
Keyword(s):  
Skeletal Muscle ◽  
Citrate Synthase ◽  
Fiber Type ◽  
Normal Weight ◽  
Fiber Types ◽  
Fiber Morphology ◽  
Key Enzymes ◽  
Control Subjects ◽  
Type Composition ◽  
And Control

Muscle fiber morphology and activities of four key enzymes, as well as energy metabolism, were determined in nine normal-weight postobese women and nine matched control subjects. No differences in fiber type composition, but a smaller mean fiber area and area of fiber types I and IIb, were found in postobese compared with control subjects ( P < 0.05). The activities of β-hydroxyacyl-CoA dehydrogenase (HADH) and citrate synthase (CS) were 20% lower in postobese than in control subjects ( P < 0.05). However, the activities of lactate dehydrogenase and lipoprotein lipase were not significantly different between postobese and control subjects. Basal metabolic rate and respiratory exchange ratio were also similar, but maximal oxygen uptake (V˙o 2 max) tended to be lower in postobese than in control subjects ( P = 0.06). When adjustments were made for differences inV˙o 2 max, HADH and CS were not different between postobese and control subjects. In conclusion, these data suggest that smaller fiber areas and lower enzyme activities, i.e., markers of aerobic capacity of skeletal muscle, but not fiber composition, may be factors predisposing to obesity.

Changes in the metabolic profile of equine muscle from birth through 1 yr of age

1990 ◽  
Vol 68 (4) ◽  
pp. 1399-1404 ◽  
Author(s):  
K. H. Kline ◽  
P. J. Bechtel
Keyword(s):  
Lactate Dehydrogenase ◽  
Citrate Synthase ◽  
Fiber Type ◽  
Gluteus Medius ◽  
Fiber Types ◽  
Superficial Fascia ◽  
Muscle Enzyme ◽  
Lactate Dehydrogenase Activity ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Fast Twitch

The purpose of this study was to investigate metabolic changes in equine muscle from birth to 1 yr of age. Duplicate biopsies from the middle portion of the gluteus medius were obtained from a depth of 2 cm beneath the superficial fascia at 1 day, 7 days, 1 mo, 3 mo, 6 mo, and 1 yr of age in 11 quarter horses and at 1 day, 3 mo, 6 mo, and 1 yr of age in 5 Standardbreds. Muscle enzyme activities determined were citrate synthase, 3-hydroxyacyl-CoA dehydrogenase, phosphorylase, and lactate dehydrogenase. Percent fast-twitch, fast-twitch high oxidative, and slow-twitch oxidative fiber types were determined using succinate dehydrogenase and myosin adenosinetriphosphatase (pH 9.4) histochemical stains. Histochemically determined muscle fiber-type percents did not change dramatically with increasing age. However, lactate dehydrogenase activity increased threefold in quarter horses and twofold in Standardbreds, and phosphorylase activity increased sixfold in quarter horses and sevenfold in Standardbreds from 1 day to 6 mo of age. Citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities decreased during the first 3 mo of age in quarter horses.

scholarly journals Ractopamine-induced fiber type-specific gene expression in porcine skeletal muscles is independent of growth

2020 ◽  
Vol 98 (11) ◽  
Author(s):  
Andrea M Gunawan ◽  
Con-Ning Yen ◽  
Brian T Richert ◽  
Allan P Schinckel ◽  
Alan L Grant ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscles ◽  
Muscle Hypertrophy ◽  
Citrate Synthase ◽  
Fiber Type ◽  
Muscle Growth ◽  
Specific Gene ◽  
Type I ◽  
Fiber Types ◽  
Adequate Diet

Abstract Feeding ractopamine (RAC), a β-adrenergic agonist (BAA), to pigs increases type IIB muscle fiber type-specific protein and mRNA expression. However, increases in the abundance of these fast-twitch fiber types occur with other forms of muscle hypertrophy and thus BAA-induced changes in myosin heavy chain (MyHC) composition may simply be associated with increased muscle growth known to occur in response to BAA feeding. The objective of this study was to determine whether RAC feeding could change the MyHC gene expression in the absence of maximal muscle growth. Pigs were fed either an adequate diet that supported maximal muscle hypertrophy or a low nutrient diet that limited muscle growth. RAC was included in diets at 0 or 20 mg/kg for 1, 2, or 4 wk. Backfat depth was less (P &lt; 0.05) in pigs fed the low nutrient diet compared with the adequate diet but was not affected by RAC. Loin eye area was greater (P &lt; 0.05) in pigs fed an adequate diet plus RAC at 1 wk but did not differ among remaining pigs. At 2 and 4 wk, however, pigs fed the adequate diet had greater loin eye areas (P &lt; 0.05) than pigs fed the low nutrient diet regardless of RAC feeding. Gene expression of the MyHC isoforms, I, IIA, IIX, and IIB, as well as glycogen synthase, citrate synthase, β 1-adrenergic receptor (AR), and β 2-AR were determined in longissimus dorsi (LD) and red (RST) and white (WST) portions of the semitendinosus muscles. MyHC type I gene expression was not altered by RAC or diet. Feeding RAC decreased (P &lt; 0.01) MyHC type IIA gene expression in all muscles, but to a greater extent in WST and LD. MyHC type IIX gene expression was lower (P &lt; 0.05) in WST and LD muscles in response to RAC but was not altered in RST muscles. RAC increased (P &lt; 0.05) MyHC type IIB gene expression in all muscles, but to a greater extent in RST. β 1-AR gene expression was unaffected by RAC or diet, whereas the expression of the β 2-AR gene was decreased (P &lt; 0.001) by RAC. No significant RAC * diet interactions were observed in gene expression in this study, indicating that RAC altered MyHC and β 2-AR gene expression in porcine skeletal muscles independent of growth.

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