Human skeletal muscle creatine transporter mRNA and protein expression in healthy, young males and females

The present study investigated whether there were any differences between males and females in respect to creatine transporter (CreaT) gene expression and/or total creatine (TCr) content in human vastus lateralis muscle. Skeletal muscle obtained from young healthy male (n = 13, age: 23.2 ± 5.0 years) and female subjects (n = 12, age: 21.7 ± 4.3 years) was analyzed for CreaT mRNA, CreaT protein and TCr content. Total CreaT protein content in the muscle was similar (p > 0.05) between the sexes. Two bands (~ 55 and 73 kDa) of the CreaT protein were detected in all muscle samples. Both the 55 and the 73 kDa bands were present in similar (p > 0.05) amounts in males compared with females. The 73 kDa band was in greater abundance (p < 0.05) than the 55 kDa band, irrespective of gender. In addition, CreaT mRNA expression relative to ß-actin mRNA and the TCr content (males: 117.8 ± 2.2, females: 125.3 ± 4.3 mmol.kg^–1 dry mass) were also unaffected (p > 0.05) by gender. These data demonstrate that gender does not influence skeletal muscle TCr content and CreaT gene expression in young human subjects.

Effect of short-term training on GLUT-4 mRNA and protein expression in human skeletal muscle

Six untrained, male subjects (23 ± 1 years old, 84 ± 5 kg, V_O2peak= 3.7 ± 0.8 l min^–1) exercised for 60 min at 75 ± 1%V_O2peak on 7 consecutive days.  Muscle samples were obtained before the start of cycle exercise training and 24 h after the first and seventh exercise sessions and analysed for citrate synthase activity, glycogen and glucose transporter 4 (GLUT-4) mRNA and protein expression. Exercise training increased (P < 0.05) citrate synthase by ~20% and muscle glycogen concentration by ~40%. GLUT-4 mRNA levels 24 h after the first and seventh exercise sessions were similar to those  measured before the start of exercise training. In contrast, GLUT-4 protein expression was increased after 7 days of exercise training (12.4 ± 1.5 versus 3.4 ± 1.0 arbitray units (a.u.), P < 0.05) and although it tended to be higher 24 h after the first exercise session (6.0 ± 3.0 versus 3.4 ± 1.0 a.u.), this was not significantly different (P= 0.09). These results support the suggestion that the adaptive increase in skeletal muscle GLUT-4 protein expression with short-term exercise training arises from the repeated, transient increases in GLUT-gene transcription following each exercise bout leading to a gradual accumulation of GLUT-4 protein, despite GLUT-4 mRNA returning to basal levels between exercise stimuli.

UCP3 protein expression is lower in type I, IIa and IIx muscle fiber types of endurance-trained compared to untrained subjects

Uncoupling protein 3 (UCP3) is a muscle mitochondrial protein believed to uncouple the respiratory chain, producing heat and reducing aerobic ATP production. Our aim was to quantify and compare the UCP3 protein levels in type I, IIa and IIx skeletal muscle fibers of endurance-trained (Tr) and healthy untrained (UTr) individuals. UCP3 protein content was quantified using Western blot and immunofluorescence. Skeletal muscle fiber type was determined by both an enzymatic ATPase stain and immunofluorescence. UCP3 protein expression measured in skeletal muscle biopsies was 46% lower ( P=0.01) in the Tr compared to the UTr group. UCP3 protein expression in the different muscle fibers was expressed as follows; IIx>IIa>I in the fibers for both groups ( P<0.0167) but was lower in all fiber types of the Tr when compared to the UTr subjects ( P<0.001). Our results show that training status did not change the skeletal muscle fiber hierarchical UCP3 protein expression in the different fiber types. However, it affected UCP3 content more in type I and type IIa than in the type IIx muscle fibers. We suggest that this decrease may be in relation to the relative improvement in the antioxidant defense systems of the skeletal muscle fibers and that it might, as a consequence, participate in the training induced improvement in mechanical efficiency.

Intense exercise up-regulates Na+, K+ -ATPase isoform mRNA, but not protein expression in human skeletal muscle

Characterization of expression of, and consequently also the acute exercise effects on, Na+,K+-ATPase isoforms in human skeletal muscle remains incomplete and was therefore investigated. Fifteen healthy subjects (eight males, seven females) performed fatiguing, knee extensor exercise at 40% of their maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue and 3 and 24 h postexercise, and analysed for Na+,K+-ATPase 1, 2, 3, ß1, ß2 and ß3 mRNA and crude homogenate protein expression, using Real-Time RT-PCR and immunoblotting, respectively. Each individual expressed gene transcripts and protein bands for each Na+,K+-ATPase isoform. Each isoform was also expressed in a primary human skeletal muscle cell culture. Intense exercise (352 ± 69 s; mean ±S.E.M.) immediately increased 3 and ß2 mRNA by 2.4- and 1.7-fold, respectively (P < 0.05), whilst 1 and 2 mRNA were increased by 2.5- and 3.5-fold at 24 h and 3 h postexercise, respectively (P < 0.05). No significant change occurred for ß1 and ß3 mRNA, reflecting variable time-dependent responses. When the average postexercise value was contrasted to rest, mRNA increased for 1, 2, 3, ß1, ß2 and ß3 isoforms, by 1.4-, 2.2-, 1.4-, 1.1-, 1.0- and 1.0-fold, respectively (P < 0.05). However, exercise did not alter the protein abundance of the 1–3 and ß1–ß3 isoforms. Thus, human skeletal muscle expresses each of the Na+,K+-ATPase 1, 2, 3, ß1, ß2 and ß3 isoforms, evidenced at both transcription and protein levels. Whilst brief exercise increased Na+,K+-ATPase isoform mRNA expression, there was no effect on isoform protein expression, suggesting that the exercise challenge was insufficient for muscle Na+,K+-ATPase up-regulation.

Anti-metastatic and differential effects on protein expression of epigallocatechin-3-gallate in HCCLM6 hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third highest cause of cancer-related mortality in humans. Epigallocatechin-3-gallate (EGCG) has been shown to inhibit the metastatic activity of certain cancer cells. The aim of this study was to determine the effects and molecular mechanism(s) of action of EGCG in human HCC cells. A migration and invasion assay for the metastatic behavior of HCCLM6 cells was performed. The anti-metastatic effects of EGCG were investigated by RT-PCR and gelatin zymography. A total cellular protein profile was obtained using 2-dimensional gel electrophoresis (2-DE), followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analyses of proteins with significant differences in expression following treatment with EGCG. The results revealed that EGCG induced apoptosis and inhibited the metastasis of HCCLM6 cells. The anti-metastatic effects of EGCG were associated with the inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 activity. The expression levels of far upstream element (FUSE) binding protein 1 (FUBP1), heat shock protein beta 1 (HSPB1), heat shock 60 kDa protein 1 (chaperonin) (CH60) and nucleophosmin (NPM) proteins, which are associated with metastasis, were significantly altered in the EGCG-treated HCCLM6 cells. The data from the present study suggest that EGCG has potential as a therapeutic agent for the treatment of HCC.

Changes in mitochondrial function and mitochondria associated protein expression in response to 2-weeks of high intensity interval training

PURPOSE: High-intensity short-duration interval training (HIT) stimulates functional and metabolic adaptation in skeletal muscle, but the influence of HIT on mitochondrial function remains poorly studied in humans. Mitochondrial metabolism as well as mitochondrial-associated protein expression were tested in untrained participants performing HIT over a 2-week period. METHODS: Eight males performed a single-leg cycling protocol (12 × 1 min intervals at 120% peak power output, 90 s recovery, 4 days/week). Muscle biopsies (vastus lateralis) were taken pre- and post-HIT. Mitochondrial respiration in permeabilized fibers, citrate synthase (CS) activity and protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) and respiratory complex components were measured. RESULTS: HIT training improved peak power and time to fatigue. Increases in absolute oxidative phosphorylation (OXPHOS) capacities and CS activity were observed, but not in the ratio of CCO to the electron transport system (CCO/ETS), the respiratory control ratios (RCR-1 and RCR-2) or mitochondrial-associated protein expression. Specific increases in OXPHOS flux were not apparent after normalization to CS, indicating that gross changes mainly resulted from increased mitochondrial mass. CONCLUSION: Over only 2 weeks HIT significantly increased mitochondrial function in skeletal muscle independently of detectable changes in mitochondrial-associated and mitogenic protein expression.

Upregulation of sodium iodide symporter (NIS) protein expression by an innate immunity component: promising potential for targeting radiosensitive retinoblastoma

Retinoblastoma (RB), a malignant tumour of the eye arising from developing retina, is the most frequent primary intraocular malignancy of childhood. Its primary management with chemotherapy involves combination regimen of etoposide, vincristine and carboplatin and intra vitreal chemotherapy using melphalan when vitreous seeds develop. Radiotherapy is another effective mode in treating RB. We recently explored the notion if radiotherapy in RB can be mediated via Sodium Iodide Symporter (NIS), an intrinsic membrane glycoprotein which is a key regulator of iodide access to thyroid gland. Its expression has been exploited successfully for diagnostic imaging and molecular radionuclide-based therapy of thyroid cancer. We determined that NIS is expressed endogenously in RB tumour tissues, and in retinoblastoma cell lines Y79 and Weri-Rb-1, and therefore made an attempt to enhance the endogenously low expression of NIS protein in both Y79 and Weri-Rb-1 cells. Here we report about the potential of bovine lactoferrin (bLf) which is a known chemo preventive and emerging safe anti-cancer bio drug, as well as a natural transcriptional activator of genes, to enhance the endogenous expression of NIS in Y79 and Weri-Rb-1 cells. Real time PCR revealed that both cell lines express mRNA of lactoferrin receptors while flow cytometry and confocal microscopy showed the cells efficiently internalize bLf which upregulates NIS expression. These findings highlight an important step that could be taken towards the development of less harmful approaches for the treatment of RB by employing natural supplement bLf (with its clinically proven safe profile), and warrants further studies in future, focussing on enhancing NIS expression in RB cells and NIS functional assays in these cells.

Interaction of exercise and diet on GLUT-4 protein and gene expression in type I and type II rat skeletal muscle

We determined the interaction of exercise and diet on glucose transporter (GLUT-4) protein and mRNA expression in type I (soleus) and type II [extensor digitorum longus (EDL)] skeletal muscle. Forty-eight Sprague Dawley rats were randomly assigned to one of two dietary conditions: high-fat (FAT, n =24) or high-carbohydrate (CHO, n =24). Animals in each dietary condition were allocated to one of two groups: control (NT, n =8) or a group that performed 8 weeks of treadmill running (4 sessions week^–1 of 1000 m @ 28 m min^–1 , RUN, n =16). Eight trained rats were killed after their final exercise bout for determination of GLUT-4 protein and mRNA expression: the remainder were killed 48 h after their last session for measurement of muscle glycogen and triacylglycerol concentration. GLUT-4 protein expression in NT rats was similar in both muscles after 8 weeks of either diet. However, there was a main effect of training such that GLUT-4 protein was increased in the soleus of rats fed with either diet (P < 0.05) and in the EDL in animals fed with CHO (P < 0.05). There was a significant diet–training interaction on GLUT-4 mRNA, such that expression was increased in both the soleus (100% ↑P < 0.05) and EDL (142% ↑P < 0.01) in CHO-fed animals. Trained rats fed with FAT decreased mRNA expression in the EDL (↓ 45%, P < 0.05) but not the soleus (↓ 14%, NS). We conclude that exercise training in CHO-fed rats increased both GLUT-4 protein and mRNA expression in type I and type II skeletal muscle. Despite lower GLUT-4 mRNA in muscles from fat-fed animals, exercise-induced increases in GLUT-4 protein were largely preserved, suggesting that control of GLUT-4 protein and gene expression are modified independently by exercise and diet.

Expression of uncoupling protein-3 subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its moduclation by fasting

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differentially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully validated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxidative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mitochondria. Taken together, these results suggest that oxidative muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24-h fast, which greatly increases UCP3 mRNA (16.4-fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5-fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes.

Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli

The HIV-1 Gag precursor protein, Pr55(Gag), is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55(Gag) assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55(Gag) has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55(Gag) for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag's natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55(Gag) to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55(Gag) using a bacterial heterologous expression system. Recombinant HIV-1 Pr55(Gag) was expressed with a C-terminal His×6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55(Gag) that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.

Statin-induced increases in atrophy gene expression occur independently of changes in PGC1α protein and mitochondrial content

One serious side effect of statin drugs is skeletal muscle myopathy. Although the mechanism(s) responsible for statin myopathy remains to be fully determined, an increase in muscle atrophy gene expression and changes in mitochondrial content and/or function have been proposed to play a role. In this study, we examined the relationship between statin-induced expression of muscle atrophy genes, regulators of mitochondrial biogenesis, and markers of mitochondrial content in slow- (ST) and fast-twitch (FT) rat skeletal muscles. Male Sprague Dawley rats were treated with simvastatin (60 or 80 mg·kg(-1)·day(-1)) or vehicle control via oral gavage for 14 days. In the absence of overt muscle damage, simvastatin treatment induced an increase in atrogin-1, MuRF1 and myostatin mRNA expression; however, these were not associated with changes in peroxisome proliferator gamma co-activator 1 alpha (PGC-1α) protein or markers of mitochondrial content. Simvastatin did, however, increase neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and AMPK α-subunit protein expression, and tended to increase total NOS activity, in FT but not ST muscles. Furthermore, simvastatin induced a decrease in β-hydroxyacyl CoA dehydrogenase (β-HAD) activity only in FT muscles. These findings suggest that the statin-induced activation of muscle atrophy genes occurs independent of changes in PGC-1α protein and mitochondrial content. Moreover, muscle-specific increases in NOS expression and possibly NO production, and decreases in fatty acid oxidation, could contribute to the previously reported development of overt statin-induced muscle damage in FT muscles.

Exercise, diet and skeletal muscle gene expression

Skeletal muscle, as a consequence of its mass and great capacity for altered metabolism, has a major impact on whole-body metabolic homeostasis and is capable of remarkable adaptation in response to various physiological stimuli, including exercise and dietary intervention. Exercise-induced increases in skeletal muscle mRNA levels of a number of genes have been reported, due to transcriptional activation and/or increased mRNA stability. The cellular adaptations to exercise training appear to be due to the cumulative effects of transient increases in gene transcription after repeated exercise bouts. The relative importance of transcriptional (mRNA synthesis) and translational (mRNA stability or translational efficiency) mechanisms for the training-induced increases in skeletal muscle protein abundance remains to be fully elucidated. Dietary manipulation, and the associated alterations in nutrient availability and hormone levels, can also modify skeletal muscle gene expression, although fewer studies have been reported. A major challenge is to understand how exercise and diet exert their effects on gene and protein expression in skeletal muscle. In relation to exercise, potential stimuli include stretch and muscle tension, the pattern of motor nerve activity and the resultant calcium transients, the energy charge of the cell and substrate availability, oxygen tension and circulating hormones. These are detected by various cellular signaling mechanisms, acting on a range of downstream targets and a wide range of putative transcription factors. A key goal in the years ahead is to identify how alterations at the level of gene expression are coupled to the changes in skeletal muscle phenotype. It is clear that gene expression, although representing a specific site of regulation, is only one step in a complex cascade from the initial stimulus to the final phenotypic adaptation and integrated physiological response.