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 exercise on protein kinase C activity and localization in human skeletal muscle

To investigate the effect of exercise on protein kinase C (PKC) activity and localization in human skeletal muscle, eight healthy men performed cycle  ergometer exercise for 40 min at 76±1% the peak pulmonary O₂ uptake (V_O2peak), with muscle samples obtained at rest and after 5 and 40 min of exercise. PKC expression, phosphorylation and activities were examined by immunoblotting and in vitro kinase assays of fractionated and whole tissue preparations. In response to exercise, total PKC activity was slightly higher at 40 min in an enriched membrane fraction, and using a pSer-PKC-substrate motif antibody it was revealed that exercise increased the serine phosphorylation of a ∼50 kDa protein. There were no changes in conventional PKC (cPKC) or PKCθ activities; however, atypical PKC (aPKC) activity was ∼70% higher at 5 and 40 min, and aPKC expression and Thr^410/403 phosphorylation were unaltered by exercise. There were no effects of exercise on the abundance of PKCα, PKCδ, PKCθ and aPKC within cytosolic or enriched membrane fractions of skeletal muscle. These data indicate that aPKC, but not cPKC or PKCθ, are activated by exercise in contracting muscle suggesting a potential role for aPKC in the regulation of skeletal muscle function and metabolism during exercise in humans.

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.

Alteration of copper physiology in mice overexpressing the human menkes protein ATP7A

The Menkes protein (ATP7A) is defective in the Cu deficiency disorder Menkes disease and is an important contributor to the maintenance of physiological Cu homeostasis. To investigate more fully the role of ATP7A, transgenic mice expressing the human Menkes gene ATP7A from chicken beta-actin composite promoter (CAG) were produced. The transgenic mice expressed ATP7A in lung, heart, liver, kidney, small intestine, and brain but displayed no overt phenotype resulting from expression of the human protein. Immunohistochemical analysis revealed that ATP7A was found primarily in the cardiac muscle, smooth muscle of the lung, distal tubules of the kidney, intestinal enterocytes, and patches of hepatocytes, as well as in the hippocampus, cerebellum, and choroid plexus of the brain. In 60-day- and 300-day-old mice, Cu concentrations were reduced in most tissues, consistent with ATP7A playing a role in Cu efflux. The reduction in Cu was most pronounced in the hearts of older T22#2 females (24%), T22#2 males (18%), and T25#5 females (23%), as well as in the brains of 60-day-old T22#2 females and males (23% and 30%, respectively).

UCP3 protein regulation in human skeletal muscle fibre types I, IIa andIIx is dependent on exercise intensity.

It has been proposed that mitochondrial uncoupling protein 3 (UCP3) behaves as an uncoupler of oxidative phosphorylation. In a cross-sectional study, UCP3 protein levels were found to be lower in all fibre types of endurance-trained cyclists as compared to healthy controls. This decrease was greatest in the type I oxidative fibres, and it was hypothesised that this may be due to the preferential recruitment of these fibres during endurance training. To test this hypothesis, we compared the effects of 6 weeks of endurance (ETr) and sprint (STr) running training on UCP3 mRNA expression and fibre-type protein content using real-time PCR and immunofluorescence techniques, respectively. UCP3 mRNA and protein levels were downregulated similarly in ETr and STr (UCP3 mRNA: by 65 and 50 %, respectively; protein: by 30 and 27 %, respectively). ETr significantly reduced UCP3 protein content in type I, IIa and IIx muscle fibres by 54, 29 and 16 %, respectively. STr significantly reduced UCP3 protein content in type I, IIa and IIx muscle fibres by 24, 31 and 26 %, respectively. The fibre-type reductions in UCP3 due to ETr, but not STr, were significantly different from each other, with the effect being greater in type I than in type IIa, and in type IIa than in type IIx fibres. As a result, compared to STr, ETr reduced UCP3 expression significantly more in fibre type I and significantly less in fibre types IIx. This suggests that the more a fibre is recruited, the more it adapts to training by a decrease in its UCP3 expression. In addition, the more a fibre type depends on fatty acid beta oxidation and oxidative phosphorylation, the more it responds to ETr by a decrease in its UCP3 content.

Increased insulin-stimulated Akt pSer473 and cytosolic SHP2 protein abundance in human skeletal muscle following acute exercise and short-term training.

The purpose of the present study was to determine in human skeletal muscle whether a single exercise bout and 7 days of consecutive endurance (cycling) training 1) increased insulin-stimulated Akt pSer473and 2) altered the abundance of the protein tyrosine phosphatases (PTPases), PTP1B and SHP2. In healthy, untrained men (n = 8; 24 ± 1 yr), glucose infusion rate during a hyperinsulinemic euglycemic clamp, when compared with untrained values, was not improved 24 h following a single 60-min bout of endurance cycling but was significantly increased (~30%; P < 0.05) 24 h following completion of 7 days of exercise training. Insulin-stimulated Akt pSer473was ~50% higher (P < 0.05) 24 h following the acute bout of exercise, with this effect remaining after 7 days of training (P < 0.05). Insulin-stimulated insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation were not altered 24 h after acute exercise and short-term training. Insulin did not acutely regulate the localization of the PTPases, PTP1B or SHP2, although cytosolic protein abundance of SHP2 was increased (P < 0.05; main effect) 24 h following acute exercise and short-term training. In conclusion, insulin-sensitive Akt pSer473and cytosolic SHP2 protein abundance are higher after acute exercise and short-term training, and this effect appears largely due to the residual effects of the last bout of prior exercise. The significance of exercise-induced alterations in cytosolic SHP2 and insulin-stimulated Akt pSer473on the improvement in insulin sensitivity requires further elucidation.

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.

Prolonged submaximal exercise induces isoform-specific Na+-K+-ATPase mRNA and protein responses in human skeletal muscle

This study investigated effects of prolonged submaximal exercise on Na⁺-K⁺-ATPase mRNA and protein expression, maximal activity, and content in human skeletal muscle. We also investigated the effects on mRNA expression of the transcription initiator gene, RNA polymerase II (RNAP II), and key genes involved in protein translation, eukaryotic initiation factor-4E (eIF-4E) and 4E-binding protein 1 (4E-BP1). Eleven subjects (6 men, 5 women) cycled at 75.5% (SD 4.8%) peak O₂ uptake and continued until fatigue. A vastus lateralis muscle biopsy was taken at rest, fatigue, and 3 and 24 h postexercise. We analyzed muscle for Na⁺-K⁺-ATPase α1, α2, α3, β1, β2, and β3, as well for RNAP II, eIF-4E, and 4E-BP1 mRNA expression by real-time RT-PCR and Na⁺-K⁺-ATPase isoform protein abundance using immunoblotting. Muscle homogenate maximal Na⁺-K⁺-ATPase activity was determined by 3-O-methylfluorescein phosphatase activity and Na⁺-K⁺-ATPase content by [³H]ouabain binding. Cycling to fatigue [54.5 (SD 20.6) min] immediately increased {alpha}3 (P = 0.044) and {beta}2 mRNA (P = 0.042) by 2.2- and 1.9-fold, respectively, whereas {alpha}1 mRNA was elevated by 2.0-fold at 24 h postexercise (P = 0.036). A significant time main effect was found for α3 protein abundance (P = 0.046). Exercise transiently depressed maximal Na⁺-K⁺-ATPase activity (P = 0.004), but Na⁺-K⁺-ATPase content was unaltered throughout recovery. Exercise immediately increased RNAP II mRNA by 2.6-fold (P = 0.011) but had no effect on eIF-4E and 4E-BP1 mRNA. Thus a single bout of prolonged submaximal exercise induced isoform-specific Na⁺-K⁺-ATPase responses, increasing α1, α3, and β2 mRNA but only α3 protein expression. Exercise also increased mRNA expression of RNAP II, a gene initiating transcription, but not of eIF-4E and 4E-BP1, key genes initiating protein translation.

Human multidrug resistance associated protein 4 resitance to camtothecins

Purpose The multidrug resistance associated protein (MRP) 4 is a member of the adenosine triphosphate (ATP)-binding cassette transporter family. Camptothecins (CPTs) have shown substantial anticancer activity against a broad spectrum of tumors by inhibiting DNA topoisomerase I, but tumor resistance is one of the major reasons for therapeutic failure. P-glycoprotein, breast cancer resistance protein, MRP1, and MRP2 have been implicated in resistance to various CPTs including CPT-11 (irinotecan), SN-38 (the active metabolite of CPT-11), and topotecan. In this study, we explored the resistance profiles and intracellular accumulation of a panel of CPTs including CPT, CPT-11, SN-38, rubitecan, and 10-hydroxy-CPT (10-OH-CPT) in HepG2 cells with stably overexpressed human MRP4. Other anticancer agents such as paclitaxel, cyclophosphamide, and carboplatin were also included.
Methods HepG2 cells were transfected with an empty vehicle plasmid (V/HepG2) or human MRP4 (MRP4/HepG2). The resistance profiles of test drugs in exponentially growing V/HepG2 and MRP4/HepG2 cells were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay with 4 or 48 h exposure time of the test drug in the absence or presence of various MRP4 inhibitors. The accumulation of CPT-11, SN-38, and paclitaxel by V/HepG2 and MRP4/HepG2 cells was determined by validated high-performance liquid chromatography methods.
Results Based on the resistance folds from the MTT assay with 48 h exposure time of the test drug, MRP4 conferred resistance to CPTs tested in the order 10-OH-CPT (14.21) > SN-38 carboxylate (9.70) > rubitecan (9.06) > SN-38 lactone (8.91) > CPT lactone (7.33) > CPT-11 lactone (5.64) > CPT carboxylate (4.30) > CPT-11 carboxylate (2.68). Overall, overexpression of MRP4 increased the IC50 values 1.78- to 14.21-fold for various CPTs in lactone or carboxylate form. The resistance of MRP4 to various CPTs tested was significantly reversed in the presence of dl-buthionine-(S,R)-sulfoximine (BSO, a γ-glutamylcysteine synthetase inhibitor), MK571, celecoxib, or diclofenac (all MRP4 inhibitors). In addition, the accumulation of CPT-11 and SN-38 over 120 min in MRP4/HepG2 cells was significantly reduced compared to V/HepG2 cells, whereas the addition of celecoxib, MK571, or BSO significantly increased their accumulation in MRP4/HepG2 cells. There was no significant difference in the intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells, indicating that P-glycoprotein was not involved in the observed resistance to CPTs in this study. MRP4 also conferred resistance to cyclophosphamide and this was partially reversed by BSO. However, MRP4 did not increase resistance to paclitaxel, carboplatin, etoposide (VP-16), 5-fluorouracil, and cyclosporine.
Conclusions Human MRP4 rendered significant resistance to cyclophosphamide, CPT, CPT-11, SN-38, rubitecan, and 10-OH-CPT. CPT-11 and SN-38 are substrates for MRP4. Further studies are needed to explore the role of MRP4 in resistance, toxicity, and pharmacokinetics of CPTs and cyclophosphamide.

Copper transport during lactation in transgenic mice expressing the human ATP7A protein

Both copper transporting ATPases, ATP7A and ATP7B, are expressed in mammary epithelial cells but their role in copper delivery to milk has not been clarified. We investigated the role of ATP7A in delivery of copper to milk using transgenic mice that over-express human ATP7A. In mammary gland of transgenic mice, human ATP7A protein was 10- to 20-fold higher than in control mice, and was localized to the basolateral membrane of mammary epithelial cells in lactating mice. The copper concentration in the mammary gland of transgenic dams and stomach contents of transgenic pups was significantly reduced compared to non-transgenic mice. The mRNA levels of endogenous Atp7a, Atp7b, and Ctr1 copper transporters in the mammary gland were not altered by the expression of the ATP7A transgene, and the protein levels of Atp7b and ceruloplasmin were similar in transgenic and non-transgenic mice. These data suggest that ATP7A plays a role in removing excess copper from the mammary epithelial cells rather than supplying copper to milk.

Reduced glycogen availability is associated with increased AMPKα2 activity, nuclear AMPKα2 protein abundance, and GLUT4 mRNA expression in contracting human skeletal muscle

Glycogen availability can influence glucose transporter 4 (GLUT4) expression in skeletal muscle through unknown mechanisms. The multisubstrate enzyme AMP-activated protein kinase (AMPK) has also been shown to play an important role in the regulation of GLUT4 expression in skeletal muscle. During contraction, AMPK [alpha]2 translocates to the nucleus and the activity of this AMPK isoform is enhanced when skeletal muscle glycogen is low. In this study, we investigated if decreased pre-exercise muscle glycogen levels and increased AMPK [alpha]2 activity reduced the association of AMPK with glycogen and increased AMPK [alpha]2 translocation to the nucleus and GLUT4 mRNA expression following exercise. Seven males performed 60 min of exercise at ~70% [VO.sub.2] peak on 2 occasions: either with normal (control) or low (LG) carbohydrate pre-exercise muscle glycogen content. Muscle samples were obtained by needle biopsy before and after exercise. Low muscle glycogen was associated with elevated AMPK [alpha]2 activity and acetyl-CoA carboxylase [beta] phosphorylation, increased translocation of AMPK [alpha]2 to the nucleus, and increased GLUT4 mRNA. Transfection of primary human myotubes with a constitutively active AMPK adenovirus also stimulated GLUT4 mRNA, providing direct evidence of a role of AMPK in regulating GLUT4 expression. We suggest that increased activation of AMPK [alpha]2 under conditions of low muscle glycogen enhances AMPK [alpha]2 nuclear translocation and increases GLUT4 mRNA expression in response to exercise in human skeletal muscle.

Molecular analysis of the human copper uptake protein hCTR1

This study examined the role of the high-affinity copper uptake protein hCTR1 in cellular copper homeostasis and found hCTR1 was internalized in response to raised copper levels. The work from this thesis supports a model in which the regulation of hCTR1 is partially or wholly dependent upon internal copper levels.

Glycogen-protein associations in human skeletal muscle

The collective findings of this dissertation demonstrated little effect of exercise on the absolute or relative expression of glycogen regulatory proteins associated with a glycogen enriched fraction in human skeletal muscle. However the findings of this thesis help inform methodological approaches to future investigations into glycogen-protein associations.