Red Skeletal Muscle Research Articles

Chronic Leptin Administration Decreases Fatty Acid Uptake and Fatty Acid Transporters in Rat Skeletal Muscle

Chronic leptin administration reduces triacylglycerol content in skeletal muscle. We hypothesized that chronic leptin treatment, within physiologic limits, would reduce the fatty acid uptake capacity of red and white skeletal muscle due to a reduction in transport protein expression (fatty acid translocase (FAT/CD36) and plasma membrane-associated fatty acid-binding protein (FABPpm)) at the plasma membrane. Female Sprague-Dawley rats were infused for 2 weeks with leptin (0.5 mg/kg/day) using subcutaneously implanted miniosmotic pumps. Control and pair-fed animals received saline-filled implants. Leptin levels were significantly elevated (approximately 4-fold; p < 0.001) in treated animals, whereas pair-fed treated animals had reduced serum leptin levels (approximately -2-fold; p < 0.01) relative to controls. Palmitate transport rates into giant sarcolemmal vesicles were reduced following leptin treatment in both red (-45%) and white (-84%) skeletal muscle compared with control and pair-fed animals (p < 0.05). Leptin treatment reduced FAT mRNA (red, -70%, p < 0.001; white, -48%, p < 0.01) and FAT/CD36 protein expression (red, -32%; p < 0.05) in whole muscle homogenates, whereas FABPpm mRNA and protein expression were unaltered. However, in leptin-treated animals plasma membrane fractions of both FAT/CD36 and FABPpm protein expression were significantly reduced in red (-28 and -34%, respectively) and white (-44 and -56%, respectively) muscles (p < 0.05). Across all experimental treatments and muscles, palmitate uptake by giant sarcolemmal vesicles was highly correlated with the plasma membrane FAT/CD36 protein (r = 0.88, p < 0.01) and plasma membrane FABPpm protein (r = 0.94, p < 0.01). These studies provide the first evidence that protein-mediated long chain fatty acid transport is subject to long term regulation by leptin.

The two myostatin genes of Atlantic salmon (Salmo salar) are expressed in a variety of tissues.

Two myostatin isoforms were identified in Atlantic salmon (Salmo salar) by RT-PCR, and genomic sequences encoding this negative muscle growth factor were for the first time isolated from a nonmammalian species. Salmon myostatin isoform I is transcribed in white skeletal muscle as a 2346-nucleotide mRNA species that encodes a precursor protein of 373 amino acids. Salmon myostatin I shows 93% sequence identity with isoform II which was isolated from white muscle as a partial cDNA sequence of 1409 nucleotides. In contrast to the restricted gene expression of myostatin in mammals, salmon myostatin I and II mRNAs were identified by RT-PCR in multiple tissues, including white muscle, intestine, brain, gills, tongue and eye. In addition, isoform I mRNA was found in red skeletal muscle, heart, spleen, and ovarian tissue. Using polyclonal antibodies against both isoforms, a 55-kDa precursor protein was detected by Western blot analysis in the red and white skeletal muscle, heart, intestine, and brain. Immunoreactive peptides of 35-40 kDa were identified in the gills, tongue, spleen, and head kidney, while the 25-kDa mature myostatin was found in the eye and serum, and in vitro expressed in rabbit reticulocyte lysate. Salmon myostatin was immunohistochemically localized in the sarcoplasma of red and white muscle fibres, in intestinal epithelial cells, at the basis of the branchial primary lamellae, and in odontoblasts and ameloblasts of the tongue teeth. The results indicate that the role of fish myostatin may not be restricted to muscle growth regulation, but may have additional functions similar to the growth/differentiation factor-11 in mammals.

Effects of anoxia exposure and aerobic recovery on metabolic enzyme activities in the freshwater turtleTrachemys scripta elegans

The effects of anoxic submergence (20 h at 7°C in nitrogen-bubbled water) and subsequent aerobic recovery (24 h at 7°C) on the maximal activities of 21 metabolic enzymes were assessed in liver, kidney, heart, brain, and red and white skeletal muscle of an anoxia-tolerant freshwater turtle, the red-eared slider, Trachemys scripta elegans. Anoxia exposure affected the activities of only a few enzymes; for example, it reduced the activity of phosphofructokinase in liver and brain, hexokinase in kidney, glycerol-3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase in heart, glutamate dehydrogenase and serine dehydratase in brain, and 3-hydroxyacyl-CoA dehydrogenase in red muscle. During aerobic recovery, activities of most of these enzymes rebounded and activities of 10 others that were not affected by anoxia rose during recovery. Anoxia-induced changes in selected enzymes appear to meet very specific needs such as glycolytic-rate depression, regulation of glycolytic versus gluconeogenic flux in liver, or alterations in amino acid neurotransmitter levels in brain. Overall, the data demonstrate that the enzymatic make-up of turtle organs undergoes very few changes during anoxia exposure and recovery, which shows that the constitutive activities of enzymes are well designed to meet the metabolic demands of anoxic excursions.

Interstitial adenosine concentration in rat red or white skeletal muscle during systemic hypoxia or contractions.

Interstitial adenosine concentrations in red soleus (SL) or white extensor digitorum longus (EDL) muscles of anaesthetised rats were determined using microdialysis and HPLC. Systemic hypoxia was induced by ventilating the animals with 10% oxygen in nitrogen for 15 min: arterial PO2 decreased from 111.8 +/- 10.9 to 42.2 +/- 4.3 mmHg (n = 4; P < 0.01) and mean systemic arterial blood pressure from 97.6 +/- 4.9 to 59.0 +/- 3.6 mmHg (n = 22; P < 0.001). The interstitial adenosine concentration was not significantly changed from its control values of 294 +/- 44 nM (n = 20) in EDL and 302 +/- 36 nM (n = 20) in SL during hypoxia or the recovery period. The interstitial lactate concentration did not change in the early part of the hypoxia but increased from 1.0 +/- 0.2 to 1.4 +/- 0.3 mM (n = 6; P < 0.05) in SL and from 2.0 +/- 0.4 to 2.4 +/- 0.4 mM (n = 6; P < 0.05) in EDL during the later part of the hypoxia, and remained elevated in the recovery period. Muscle contractions (2 Hz for 15 min) produced a transient increase in the interstitial adenosine concentration of SL from 150 +/- 35 to 244 +/- 75 nM (n = 10; P < 0.05) during the first 5 min of stimulation. In EDL the interstitial adenosine concentration increased from 145 +/- 50 to 435 +/- 144 nM (n = 10; P < 0.05) in the later part of the contraction and remained elevated in the early part of the recovery period. These data suggest that: (i) in systemic hypoxia adenosine does not appear in the interstitial space, which rules out its release from skeletal muscle, although it may be formed by the vascular tissues in this condition; (ii) adenosine is formed in the interstitial space of skeletal muscle during muscle contractions; (iii) there is slow clearance of adenosine from the interstitial space of white muscle, perhaps due to the low vascularity of the tissue.

ENHANCED SKELETAL MUSCLE HEALING AND REGENERATION BY RAT SKELETAL MUSCLE EXTRACT

One of the most challenging problems in muscular skeletal trauma is the limited capacity of regeneration of the damaged skeletal muscle fibers after injury and the inevitable replacement by fast growing fibrous tissue compromising function. To date, there is no effective therapeutic measure available in clinical practice. In the present study, red skeletal muscle aqueous extract has been prepared from female adult rats. This muscle extract could strikingly trigger the proliferation and differentiation of myoblasts whose progeny subsequently fuse to preexisting partially damaged muscle fibers to repair the damaged muscle fibers or to form new myotubes to replace the completely damaged muscle fibers during the process of muscle healing and regeneration in vivo. The active myogenic components are probably polypeptides since trypsin or heat treatment could abolish their activity in myogenesis.

Oxygen Diffusion Coefficient in Isolated Chicken Red and White Skeletal Muscle Fibers in Ontogenesis

Oxygen diffusion from medium to cultured isolated muscle fibers from red gastrocnemius muscle (deep part) (RGM) and white pectoralis muscle (WPM) of embryonic and postnatal chickens (about 6 months) was explored. The intracellular effective O2 diffusion coefficient (Di) in muscle fiber was calculated from a model of a cylindrical fiber with a uniform distribution of an oxygen sink based on these experimentally measured parameters: critical tension of O2 (PO2) on the surface of a fiber, specific rate of O2 consumption by a weight unit of muscle fibers (VO2), and average diameter of muscle fibers. The results document the rapid hypertrophic growth of RGM fibers when compared to WPM fibers in the second half of the embryonic period and the higher values of VO2 and critical PO2 during the ontogenetic period under study. The oxygen Di in RGM fibers of embryos and 1-day chickens was two to three times higher than observed for WPM fibers. For senior chickens, the oxygen Di value in RGM and WPM fibers does not differ. The Di of O2 in both RGM and WPM fibers increased from 1.4–2.7 × 10−8 to 90–95 × 10−8 cm2/s with an ontogenetic increase in fiber diameter from 7.5 to 67.0 μm. At all stages the oxygen Di values in RGM and WPM fibers are significantly lower than the O2 diffusion coefficient in water: for 11-day embryos they are 889 and 1714 times lower and for adult individuals 25 and 27 times lower, respectively. Why oxygen Di values in RGM and WPM fibers are so low and why they are gradually increasing during the course of hypertrophic ontogenetic growth are still unclear.

GLP-l effectively reverts insulin resistance in rats with CCL 4-induced liver cirrhosis

Introduction: Insulin resistance is a characteristic feature of patients with liver cirrhosis. To characterize and quantify the extent as well as the site of impaired glucose utilization and to elucidate the possible potency of GLP-l to revert the impaired glucose utilization, both healthy and cirrhotic rats underwent euglycemic hyperinsulinemic damp studies. Methods: In male Lewis rats, liver cirrhosis was induced by inhalation of CCI4 for 10 weeks and phenobarbital (PB)-enriched drinking water. Using the euglycemic hyperinsulinemic clamp technique coupled with isotopic measurements of glucose uptake in individual tissues, insulin action was studied in the presence and absence of 3.0 pmol GLP-l/kgBW/min during clamping. Clamp studies were performed by continuous insulin infusion in doses ranging from 1 to 10 nmol/kg BW/h. Blood samples were obtained and glucose infusion was adjusted accordingly to maintain euglycemia allowing determination of insulin action on whole body glucose disposal. During euglycemia, insulin action on individual tissues was measured with 2-deoxy-D-[1-3H]glucose and [U-1 Results: After 10 weeks, a CCli PB-induced and histologically-validated liver cirrhosis was found in 98 % of treated rats. In cirrhotic rats, glucose utilization was impaired and resulted in a significant reduction of total body glucose disposal by 30 %. In these animals, glycogen formation was significantly reduced in the diaphragm and red skeletal muscle. Administration of GLP-I fully reverted the impaired glucose utilization. Conclusion: In rats with a CCI4/PBinduced liver cirrhosis, insulin resistance is predominantly marked by an impaired glucose utilization located in the diaphragm and red skeletal muscle which can be completely reverted by GLP-l. These results stress the potential pharmacological properties of GLP-l in the treatment of NIDDM in humans.

Alterations in the expression and activity of creatine kinase-M and mitochondrial creatine kinase subunits in skeletal muscle following prolonged intense exercise in rats.

Creatine kinase (CK) isoenzymes are important structural and energy metabolism components in skeletal muscle. In this study, CK isoenzyme alterations were examined in male rats, with an 8% body mass weight attached to their tail. The rats were either forced to swim for 5 h (5S, n = 51), or were pre-trained for 8 days and then forced to swim for 5 h (T5S, n = 48). Rats were sacrificed either immediately (0 h PS), 3 h (3 h PS), or 48 h post-swimming (48 h PS). Serum CK was increased significantly (P < 0.01) 6.2- and 2.0-fold at 0 h PS following the 5S and T5S protocols, respectively. However, training (T5S protocol) significantly (P < 0.01) decreased CK release. Soleus and white gastrocnemius (WG) CK activity was significantly decreased following the 5S protocol (P < 0.05), but not following the T5S protocol. The CK-M activity of the soleus muscle was significantly (P < 0.05) decreased at 0 h PS following both the 5S and T5S protocols, and returned to control values at 3 h PS. The CK-M activity of the WG was significantly (P < 0.05) decreased at 0 h PS following the 5S protocol. Sarcomeric mitochondrial CK (sCK-Mit) was decreased significantly (P < 0.01) at 0 h PS (20%), 3 h PS (14%), 24 h PS (22%), and 48 h PS (15%) following the 5S protocol. However, sCK-Mit was decreased significantly (P < 0.01) only at 0 h PS (7%) following the T5S. The results of this study demonstrate that prolonged intense exercise causes a loss of skeletal muscle CK-M and sCK-Mit activity and that training prior to the prolonged intense exercise attenuates the exercise-induced CK-M and sCK-Mit loss in both red and white skeletal muscles.

Co-expression in rat heart and skeletal muscle of four genes coding for proteins implicated in long-chain fatty acid uptake

It has been suggested that specific membrane-associated and cytoplasmic proteins cooperate in the uptake of long-chain fatty acids by cardiac and skeletal muscle cells. A prerequisite for this hypothesis would be the co-occurrence of these proteins in muscle. Thus, we studied the possible co-expression in rat muscles of the genes coding for the integral membrane proteins fatty acid transport protein (FATP) and fatty acid translocase (FAT), the membrane-associated plasmalemmal fatty acid-binding protein (FABPpm) and the cytoplasmic heart-type fatty acid-binding protein (H-FABPc). The transcripts of the four proteins were assessed in heart and skeletal muscles of adult Wistar rats, in isolated cells and cell lines from rat heart and also in rat heart during development and upon streptozotocin-induced diabetes. All four genes showed high expression levels in heart, somewhat lower in red skeletal muscle (soleus) and appreciably lower in white skeletal muscle (extensor digitorum longus). FATP, FAT and H-FABPc showed a 3- to 5-fold increase in mRNA expression during maturational growth of the heart, while the FABPpm expression remained virtually constant. In the heart, streptozotocin-diabetes induced a slight, but statistically not significant, increase in the expression of all four genes. In conclusion, this study shows the co-expression of FATP, FAT, FABPpm and H-FABPc in rat muscles. This finding supports the possible cooperation of these proteins in the uptake of long-chain fatty acids by muscle cells.

A sensitive immunoassay for rat fatty acid translocase (CD36) using phage antibodies selected on cell transfectants: abundant presence of fatty acid translocase/CD36 in cardiac and red skeletal muscle and up-regulation in diabetes

The rat membrane protein fatty acid translocase (FAT), which shows sequence similarity to human CD36 (a membrane protein supposedly involved in a variety of membrane processes), is implicated in the transport of long-chain fatty acids across cellular membranes. To set up an immunoassay for quantification of FAT in different tissues, we isolated a series of anti-FAT antibodies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted specifically with FAT, and most likely recognize the same or closely located immunodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-IV7) completely blocked the binding of all these phage antibodies to cells. A sandwich ELISA was set up using mAb 131.4 (directed against purified CD36 from human platelets) as capture antibody and phage antibodies and anti-phage sera as detector. With this ELISA (sensitivity 0.05 µg/ml), the FAT content in isolated cardiomyocytes was found to be comparable with that of total heart (≈ 3 mg/g of protein), while liver tissue and endothelial cells were below the detection limit (< 0.1 mg of FAT/g of protein). During rat heart development, protein levels of FAT rose from 1.7±0.7 mg/g of protein on the day before birth to 3.6±0.4 mg/g of protein on day 70. Comparing control with streptozotocin-induced diabetic rats, a statistically significant (P< 0.05) 2–4-fold increase of FAT was seen in heart (from 4.2±2.3 to 11.0±5.7 mg/g of protein), soleus (from 0.6±0.1 to 1.4±0.5 mg/g of protein) and extensor digitorum longus (EDL) muscle (from 0.3±0.1 to 1.2±0.8 mg/g of protein). In addition, the FAT contents of each of these muscles were found to be of similar magnitude to the contents of cytoplasmic heart-type fatty-acid-binding protein in both diabetic rats and controls, supporting the suggested roles of these two proteins in cellular fatty acid metabolism. This is the first time phage display technology has been succesfully applied for direct selection, from a large naive antibody library, of antibodies that recognize selected membrane proteins in their natural context.

Diffusional anisotropy is induced by subcellular barriers in skeletal muscle.

The time- and orientational-dependence of phosphocreatine (PCr) diffusion was measured using pulsed-field gradient nuclear magnetic resonance (PFG-NMR) as a means of non-invasively probing the intracellular diffusive barriers of skeletal muscle. Red and white skeletal muscle from fish was used because fish muscle cells are very large, which facilitates the examination of diffusional barriers in the intracellular environment, and because they have regions of very homogeneous fiber type. Fish were cold-acclimated (5 degrees C) to amplify the contrast between red and white fibers. Apparent diffusion coefficients, D, were measured axially, D(axially) and radially, D(radially), in small muscle strips over a time course ranging from 12 to 700 ms. Radial diffusion was strongly time dependent in both fiber types, and D decreased with time until a steady-state value was reached at a diffusion time approximately 100 ms. Diffusion was also highly anisotropic, with D(axially) being higher than D(radially) for all time points. The time scale over which changes in D(radially) occurred indicated that the observed anisotropy was not a result of interactions with the thick and thin filament lattice of actin and myosin or restriction within the cylindrical sarcolemma, as has been previously suggested. Rather, the sarcoplasmic reticulum (SR) and mitochondria appear to be the principal intracellular structures that inhibit mobility in an orientation-dependent manner. This work is the first example of diffusional anisotropy induced by readily identifiable intracellular structures.

A sensitive immunoassay for rat fatty acid translocase (CD36) using phage antibodies selected on cell transfectants: abundant presence of fatty acid translocase/CD36 in cardiac and red skeletal muscle and up-regulation in diabetes

The rat membrane protein fatty acid translocase (FAT), which shows sequence similarity to human CD36 (a membrane protein supposedly involved in a variety of membrane processes), is implicated in the transport of long-chain fatty acids across cellular membranes. To set up an immunoassay for quantification of FAT in different tissues, we isolated a series of anti-FAT antibodies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted specifically with FAT, and most likely recognize the same or closely located immunodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-IV7) completely blocked the binding of all these phage antibodies to cells. A sandwich ELISA was set up using mAb 131. 4 (directed against purified CD36 from human platelets) as capture antibody and phage antibodies and anti-phage sera as detector. With this ELISA (sensitivity 0.05 microgram/ml), the FAT content in isolated cardiomyocytes was found to be comparable with that of total heart ( approximately 3 mg/g of protein), while liver tissue and endothelial cells were below the detection limit (<0.1 mg of FAT/g of protein). During rat heart development, protein levels of FAT rose from 1.7+/-0.7 mg/g of protein on the day before birth to 3.6+/-0.4 mg/g of protein on day 70. Comparing control with streptozotocin-induced diabetic rats, a statistically significant (P<0.05) 2-4-fold increase of FAT was seen in heart (from 4.2+/-2.3 to 11.0+/-5.7 mg/g of protein), soleus (from 0.6+/-0.1 to 1.4+/-0.5 mg/g of protein) and extensor digitorum longus (EDL) muscle (from 0.3+/-0.1 to 1. 2+/-0.8 mg/g of protein). In addition, the FAT contents of each of these muscles were found to be of similar magnitude to the contents of cytoplasmic heart-type fatty-acid-binding protein in both diabetic rats and controls, supporting the suggested roles of these two proteins in cellular fatty acid metabolism. This is the first time phage display technology has been succesfully applied for direct selection, from a large naive antibody library, of antibodies that recognize selected membrane proteins in their natural context.

Role of local contractile activity and muscle fiber type on LPL regulation during exercise.

The purpose of this study was to determine the influence of local contractile activity on lipoprotein lipase (LPL) regulation in skeletal muscle. Short-term voluntary run training increased LPL mRNA concentration and LPL immunoreactive mass about threefold in white skeletal muscles of the rat hindlimb (all P < 0.01). Training also increased total and heparin-releasable LPL enzyme activity in white hindlimb muscles and in postheparin plasma (P < 0.05). Training did not enhance LPL regulation in a white muscle that was not recruited during running (masseter). LPL levels were already high in red skeletal muscles of control rats, and training did not result in a further rise. In resting rats, local electrical stimulation of a motor nerve to a predominantly white muscle caused a significant rise in LPL mRNA, immunoreactive mass, and enzyme activity relative to the contralateral control muscle of the same animals (all P < 0.01). Finally, LPL expression was several times greater in a red muscle (soleus) of rats with normal postural activity than rats with immobilized hindlimbs (P < 0.01). In summary, these studies support the hypothesis that local contractile activity is required for increasing LPL expression during exercise training and for maintaining a high level of LPL expression in postural muscles.

Inhibition and Kinetic Properties of Membrane-Bound Carbonic Anhydrases in Rabbit Skeletal Muscles

It was the aim of this study to investigate whether the carbonic anhydrases associated with the sarcoplasmic reticulum (SR) and sarcolemmal membranes differ in their kinetic and inhibitory properties. To this end, sarcolemmal and SR membrane vesicle fractions were prepared from rabbit white and red skeletal muscles, the white muscle sarcolemmal fraction (WSL), the red muscle sarcolemmal fraction (RSL), the white muscle SR fraction (WSR), and the red muscle SR fraction (RSR). WSL displayed a specific carbonic anhydrase activity of 22.1 U · ml/mg and RSL of 7.5 U · ml/mg, whereas the SR fractions showed a much lower activity of 0.5 U · ml/mg for WSR and of 2.4 U · ml/mg for RSR. In both SR fractions phase separation experiments with Triton X-114 demonstrated that the carbonic anhydrase activity is due to a membrane-bound enzyme and not due to a cytosolic isozyme. The kinetic properties of carbonic anhydrase from the four distinct membane fractions were evaluated by determination of the Michaelis constant,Km, and of the catalytic centre activitykcat.Kmappears to be somewhat lower for SR than for SL. Inhibition constants of SR and SL carbonic anhydrases were determined applying six carbonic anhydrase inhibitors: chlorzolamide, ethoxzolamide, methazolamide, benzolamide, and acetazolamide, and also cyanate. The inhibition constants of the SR fractions were significantly different from those of the corresponding sarcolemmal fractions, indicating that the carbonic anhydrase measured in the SR fractions does not originate from contaminating sarcolemmal membrane vesicles, but appears to represent a distinct carbonic anhydrase associated with the SR membrane.

Glycemia-lowering effect of cobalt chloride in the diabetic rat: role of decreased gluconeogenesis.

Results of previous studies indicated that treatment of diabetic rats (induced by streptozotocin) with cobalt chloride (CoCl2) resulted in a significant decrement in serum glucose concentration. The present study was designed to determine the potential role of enhanced glucose uptake vs. decreased glucose production in the above response. The rate of systemic appearance of glucose, measured under fasting conditions using [3-3H]glucose tracer, was reduced from 35.5 +/- 2.5 to 17.5 +/- 1.8 micromol . kg-1 . min-1 in diabetic rats treated with 2 mM CoCl2 added to the drinking water for 10-14 days (P < 0.01). Tissue accumulation of intravenously administered 2-deoxy-[14C]glucose was significantly reduced in kidney and eye of diabetic rats treated with CoCl2, whereas the uptake remained unchanged in several other tissues including cerebrum, red and white skeletal muscle, heart, and liver. The relative content of phosphoenolpyruvate carboxykinase (PEPCK) mRNA was increased 3.1-fold in livers of diabetic compared with normal rats (P < 0.001), and treatment of diabetic rats with CoCl2 decreased hepatic PEPCK mRNA levels to normal. The content of PEPCK mRNA in the liver was decreased by 33% in CoCl2-treated normal rats (P < 0.05). Treatment with CoCl2 resulted in no change in cAMP levels in the livers of either diabetic or normal rats. These results suggest that the glycemia-lowering effect of CoCl2 is mediated by reductions in the rate of systemic appearance of glucose and hepatic gluconeogenesis.

Intravenous iron supplementation effect on tissue iron and hemoproteins in chronically phlebotomized lambs.

Chronic phlebotomy is an important mechanism of iron loss in premature infants. We studied inter- and intraorgan iron allocation in 10 twin lamb pairs undergoing an acute 40-50% reduction in red cell volume followed by smaller intermittent phlebotomies over an 11-day period. One twin received no supplemental iron sucrose, while the other received an average daily intravenous dose of iron sucrose of either 1 (n = 3), 2 (n = 3), 5 (n = 3), or 15 (n = 1) mg.kg-1.day-1. The total iron content of the red blood cells, liver, skeletal muscle, heart, and brain was directly related to iron dose up to 2 mg.kg-1.day-1. Tissue iron concentrations remained stable until liver iron was < 200 g/g dry wt, after which iron was preferentially directed to red blood cells over skeletal muscle, heart, and brain. Hemoprotein concentrations decreased proportionately to tissue iron, except myocardial cytochrome c, which remained preserved. Any available iron in phlebotomized, rapidly growing lambs is preferentially directed to red blood cells, and lambs require iron supplementation to maintain tissue iron and hemoprotein concentrations. A decrease in nonheme tissue iron results in the high prioritization of iron among iron-containing proteins.

Reversal of chronic alterations of skeletal muscle protein kinase C from fat-fed rats by BRL-49653.

We have recently shown that the reduction in insulin sensitivity of rats fed a high-fat diet is associated with the translocation of the novel protein kinase C epsilon (nPKC epsilon) from cytosolic to particulate fractions in red skeletal muscle and also the downregulation of cytosolic nPKC theta. Here we have further investigated the link between insulin resistance and PKC by assessing the effects of the thiazolidinedione insulin-sensitizer BRL-49653 on PKC isoenzymes in muscle. BRL-49653 increased the recovery of nPKC isoenzymes in cytosolic fractions of red muscle from fat-fed rats, reducing their apparent activation and/or downregulation, whereas PKC in control rats was unaffected. Because BRL-49653 also improves insulin-stimulated glucose uptake in fat-fed rats and reduces muscle lipid storage, especially diglyceride content, these results strengthen the association between lipid availability, nPKC activation, and skeletal muscle insulin resistance and support the hypothesis that chronic activation of nPKC isoenzymes is involved in the generation of muscle insulin resistance in fat-fed rats.

Chronic electrical stimulation increases MCT1 and lactate uptake in red and white skeletal muscle.

We examined whether chronic stimulation of red and white rat muscles increased the concentrations of the monocarboxylate transporter MCT1. Red and white tibialis anterior (RTA and WTA, respectively) and extensor digitorum longus (EDL) muscles were chronically stimulated via the peroneal nerve for 7 days. Stimulated and contralateral control muscles were examined for MCT1 content, L-lactate uptake, lactate dehydrogenase (LDH) isoforms, and muscle fiber composition. MCT1 was 1.5 times greater in stimulated RTA, 3 times greater in stimulated WTA, and 1.9 times greater in stimulated EDL compared with respective control muscles (P < 0.05). L-Lactate uptake increased in all stimulated muscles (P < 0.05), and this was highly correlated with the increase in MCT1 (r = 0.96). The heart-type LDH (H-LDH) subunits also increased in all stimulated muscles (P < 0.05). The H-LDH subunits correlated highly with MCT1 in the muscles (r = 0.83). There was no change in muscle-type LDH subunits (P > 0.05). There were negligible alterations in muscle fiber composition in the stimulated muscles, suggesting that the increase in MCT1 was independent of changes in muscle fiber composition. These studies are the first to demonstrate that chronic muscle contraction increases MCT1 concentrations in both red and white skeletal muscles.

Synthesis and decay of calmodulin-ubiquitin conjugates in cell-free extracts of various rabbit tissues

Calmodulin is the natural substrate for ubiquitin-ligation by the enzyme ubiquitin-calmodulin ligase (uCaM-synthetase; EC 6.3.2.21). The activity of this ligase is regulated by the binding of the second messenger Ca 2+ to the substrate calmodulin, which increases the activity ca. 10-fold. Up till now, two components of the ligase could be identified: uCaM Syn-F1 and uCaM Syn-F2, the first of which binds to ubiquitin and the second which binds to calmodulin. Since the physiological role of this enzyme is still unclear, this study was designed to examine whether the activity of uCaM-Synthetase in 40 000×g tissue supernatants correlates with the calmodulin content in the various tissues. In reticulocytes, spleen, erythrocytes, testis and brain, which are rich in uCaM synthetase, the tissue contents calculated on the basis of activity measurements were between 4–80-fold higher than in red and white skeletal muscle. These activities did not correlate with the respective calmodulin contents of the tissues indicating that other factors were determining these enzyme levels. A second aim was to gain information on the role of the ATP-ubiquitin-dependent proteolytic pathway in those tissues displaying uCaM synthetase activity. In the reticulocyte system which contains the classical ATP-ubiquitin-dependent proteolytic pathway as measured with 125I-BSA, no ubiquitin-dependent degradation of calmodulin could be detected. We therefore examined the other tissues of the rabbit with the substrate 125I-BSA and succeeded in finding a ubiquitin-independent ATP-dependent proteolytic activity in every case but no ubiquitin-dependent activity. The ubiquitin-independent activity was highest in smooth muscle and red skeletal muscle being ca. 3–4-fold higher than in lung and testis. In 50% of the tissue crude extracts the time curve of calmodulin ubiquitylation progressed through a maximum indicating a dynamic steady state based on conjugate synthesis and decay. If a ubiquitylation pulse of 30 min was followed in liver crude extracts by the addition of EGTA, which specifically inhibits ubiquityl-calmodulin synthesis, a half-life of calmodulin-conjugate decay of 15–20 min is observed. A similar conjugate half-life of ca. 30 min was observed after addition of EDTA excluding that conjugate decay is due to an ATP-dependent proteolytic process. Studying the decay of purified ubiquitin- 125I-BH-calmodulin conjugates in cell-free reticulocyte extracts led to the discovery of an ATP-independent isopeptidase activity which splits ubiquitin-calmodulin conjugates without leading to detectable calmodulin fragments. The rapid decay of ubiquitin-calmodulin conjugates in tissue extracts can therefore be plausibly explained by a ubiquityl-calmodulin splitting isopeptidase activity.

The molar ratios of alpha and beta subunits of the Na+-K+-ATPase differ in distinct subcellular membranes from rat skeletal muscle.

The Na+-K+-ATPase consists of alpha and beta subunits proposed to function as an alpha-beta heterodimer. Skeletal muscle is characterized by expression of alpha1, alpha2, beta1, and beta2 subunit isoforms. The relative molar proportions of each subunit or each protein isoform are not known, yet their subcellular distribution and expression in muscles of different fiber types are markedly different. In this study, the molar ratio of each pump subunit isoform was measured in purified membranes from skeletal muscle and compared with those in kidney and brain microsomes. Recombinant proteins were used as standards to quantitate each isoform by immunoblotting in combination with measurements of [3H]ouabain binding. The results indicate that in kidney microsomes, which express predominantly alpha1 and beta1 isoforms, the alpha:beta subunit molar ratio is approximately 1:1. In brain microsomes, the sum of all alpha (alpha1, alpha2, and alpha3) and all beta (beta1 and beta2) subunits also yielded a molar ratio of approximately 1:1. In contrast, in red (oxidative) skeletal muscles, the all alpha:beta subunit ratio was 0.2 in plasma membranes and 0.4 in intracellular membranes. The ratio of alpha2 subunits to alpha1 subunits ranged from 1.6 in surface membranes to up to 7 in internal membranes, while the beta1 subunits exceeded the beta2 subunits by approximately 4-fold in all membrane fractions. Thus, intracellular membranes of red skeletal muscles contain primarily alpha2 and beta1 subunits. When these intracellular membranes were further subfractionated by velocity gradient centrifugation, the alpha2:beta1 subunit ratio was 0.5 in the faster migrating (larger) membranes and 1.0 in the slower migrating (smaller) ones. This was due to a progressive decrease in abundance of the beta1 subunits without a change in the concentration of alpha2 subunits per unit protein. The Na+-K+-ATPase hydrolytic activity was higher in the larger vesicles than in the smaller ones along the sucrose gradient. These results suggest that the ratio of beta to alpha subunits may serve to regulate the catalytic activity of the Na+-K+-ATPase in skeletal muscle.