High-intensity interval training (HIT) is known to increase mitochondrial content in a similar way as endurance training [60-90% of maximal oxygen uptake (VO2peak)]. Whether HIT increases the mitochondria's ability to oxidize lipids is currently debated. We investigated the effect of HIT on mitochondrial fat oxidation in skeletal muscle and adipose tissue. Mitochondrial oxidative phosphorylation (OXPHOS) capacity, mitochondrial substrate sensitivity (K(m)(app)), and mitochondrial content were measured in skeletal muscle and adipose tissue in healthy overweight subjects before and after 6 weeks of HIT (three times per week at 298 ± 21 W). HIT significantly increased VO2peak from 2.9 ± 0.2 to 3.1 ± 0.2 L/min. No differences were seen in maximal fat oxidation in either skeletal muscle or adipose tissue. K(m)(app) for octanoyl carnitine or palmitoyl carnitine were similar after training in skeletal muscle and adipose tissue. Maximal OXPHOS capacity with complex I- and II-linked substrates was increased after training in skeletal muscle but not in adipose tissue. In conclusion, 6 weeks of HIT increased VO2peak. Mitochondrial content and mitochondrial OXPHOS capacity were increased in skeletal muscle, but not in adipose tissue. Furthermore, mitochondrial fat oxidation was not improved in either skeletal muscle or adipose tissue.
Reference proteins (RP) or the total protein (TP) loaded is used to correct for uneven loading and/or transfer in Western blotting. However, the signal sensitivity and the influence of physiological conditions may question the normalization methods. Therefore, three widely used reference proteins [β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and α-tubulin], as well as TP loaded measured by Stain-Free technology (SF) as normalization tool were tested. This was done using skeletal muscle samples from men subjected to physiological conditions often investigated in applied physiology where the intervention has been suggested to impede normalization (ageing, muscle atrophy, and different muscle fiber type composition). The linearity of signal and the methodological variation coefficient was obtained. Furthermore, the inter- and intraindividual variation in signals obtained from SF and RP was measured in relation to ageing, muscle atrophy, and different muscle fiber type composition, respectively. A stronger linearity of SF and β-actin compared with GAPDH and α-tubulin was observed. The methodological variation was relatively low in all four methods (4-11%). Protein level of β-actin and GAPDH was lower in older men compared with young men. In conclusion, β-actin, GAPDH, and α-tubulin may not be used for normalization in studies that include subjects with a large age difference. In contrast, the RPs may not be affected in studies that include muscle wasting and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared with the existing methods for correcting for loading inaccuracy in Western blotting of human skeletal muscle in applied physiology.
Key pointsr This study aimed to provide molecular insight into the differential effects of age and physical inactivity on the regulation of substrate metabolism during moderate-intensity exercise.r Using the arteriovenous balance technique, we studied the effect of immobilization of one leg for 2 weeks on leg substrate utilization in young and older men during two-legged dynamic knee-extensor moderate-intensity exercise, as well as changes in key proteins in muscle metabolism before and after exercise.r Age and immobilization did not affect relative carbohydrate and fat utilization during exercise, but the older men had higher uptake of exogenous fatty acids, whereas the young men relied more on endogenous fatty acids during exercise.r Using a combined whole-leg and molecular approach, we provide evidence that both age and physical inactivity result in intramuscular lipid accumulation, but this occurs only in part through the same mechanisms.Abstract Age and inactivity have been associated with intramuscular triglyceride (IMTG) accumulation. Here, we attempt to disentangle these factors by studying the effect of 2 weeks of unilateral leg immobilization on substrate utilization across the legs during moderate-intensity exercise in young (n = 17; 23 ± 1 years old) and older men (n = 15; 68 ± 1 years old), while the contralateral leg served as the control. After immobilization, the participants performed two-legged isolated knee-extensor exercise at 20 ± 1 W (ß50% maximal work capacity) for 45 min with catheters inserted in the brachial artery and both femoral veins. Biopsy samples obtained from vastus lateralis muscles of both legs before and after exercise were used for analysis of substrates, protein content and enzyme activities. During exercise, leg substrate utilization (respiratory quotient) did not differ between groups or legs. Leg fatty acid uptake was greater in older than in young men, and although young men demonstrated net leg glycerol release during exercise, older men showed net glycerol uptake. At baseline, IMTG, muscle pyruvate dehydrogenase complex activity and the protein content of adipose triglyceride lipase, acetyl-CoA carboxylase 2 and AMP-activated protein kinase (AMPK)γ3 were higher in young than in older men. Furthermore, adipose triglyceride lipase, plasma membrane-associated fatty acid binding protein and AMPKγ3 subunit protein contents were lower and IMTG was higher in the immobilized than the contralateral leg in young and older men. Thus, immobilization and age did not affect substrate choice (respiratory quotient) during moderate exercise, but the whole-leg and molecular differences in fatty acid mobilization could explain the age-and immobilization-induced IMTG accumulation.
Despite the frequent expression of N-terminally truncated ErbB2 (DNErbB2/p95HER2) in breast cancer and its association with Herceptin resistance and poor prognosis, it remains poorly understood how DNErbB2 affects chemotherapy-induced cell death. Previously it was shown that DNErbB2 upregulates acid extrusion from MCF-7 breast cancer cells and that inhibition of the Na þ /H þ exchanger (SLC9A1/NHE1) strongly sensitizes DNErbB2-expressing MCF-7 cells to cisplatin chemotherapy. The aim of this study was to identify the mechanism through which DNErbB2 regulates cisplatin-induced breast cancer cell death, and determine how NHE1 regulates this process. Cisplatin treatment elicited apoptosis, ATM phosphorylation, upregulation of p53, Noxa (PMAIP1), and PUMA (BBC3), and cleavage of caspase-9, -7, fodrin, and PARP-1 in MCF-7 cells. Inducible DNErbB2 expression strongly reduced cisplatin-induced ATM-and p53-phosphorylation, augmented Noxa upregulation and caspase-9 and -7 cleavage, doubled p21 WAF1/Cip1 (CDKN1A) expression, and nearly abolished Bcl-2 expression. LC3-GFP analysis demonstrated that autophagic flux was reduced by cisplatin in a manner augmented by DNErbB2, yet did not contribute to cisplatin-induced death. Using knockdown approaches, it was shown that cisplatininduced caspase-7 cleavage in DNErbB2-MCF-7 cells was Noxaand caspase-9 dependent. This pathway was augmented by NHE1 inhibition, while the Na þ /HCO 3 À cotransporter (SLC4A7/ NBCn1) was internalized following cisplatin exposure.Implications: This work reveals that DNErbB2 strongly affects several major pro-and antiapoptotic pathways and provides mechanistic insight into the role of NHE1 in chemotherapy resistance. These findings have relevance for defining therapy regimens in breast cancers with DNErbB2 and/or NHE1 overexpression.
Many recent studies of ecological speciation have focused on "magic trait" scenarios, in which divergent selection on viability traits leads inextricably to corresponding divergence in mechanisms, especially mate recognition systems, that facilitate assortative mating. Speciation however may also proceed via other scenarios, such as when populations experience directly selected or random divergence in mate recognition systems. The relative contributions of magic trait versus other scenarios for speciation remain virtually unexplored. The present study aims to test the relative contribution of the magic trait scenario in the divergence of populations of the medium ground finch Geospiza fortis of Santa Cruz Island, Galapagos. First, we assess differences in G. fortis song between a northern population (Borrero Bay) and a southeastern population (El Garrapatero), differences that we propose (along with other within-island geographic song variations) have arisen via scenarios that do not involve a magic trait scenario. Pairwise comparisons of raw and composite (PC) song parameters, as well as discriminant functions analyses, reveal significant patterns of song divergence between sites. Second, we test the ability of territorial males at Borrero Bay to discriminate songs from the two sites. We find that G. fortis males can discriminate within-island song variants, responding more strongly to local than to "foreign" songs, along 3 raw and 1 composite response measures. Third, we compare these findings to prior data sets on song divergence and discrimination in Santa Cruz G. fortis. These comparisons suggest that song divergence and discrimination are shaped less strongly by geographic sources than by morphological (beak-related) sources. We thus argue that interpopulation song divergence and discrimination, fundamental elements of assortative mating in Darwin's finches, can be fostered in early stages of divergence under magic trait as well as alternative scenarios for speciation, but with more emphasis on the magic trait scenario, at least for this species on this island [Current Zoology 59 (1): [8][9][10][11][12][13][14][15][16][17][18][19] 2013].
<p>Supplementary figure 1: Effects of cisplatin treatment on expression of ΔNErbB2 and NHE1, and subcellular localization of cytochrome c. MCF-7 vector (V) and ΔNErbB2 (ΔN) cells were washed free of tetracycline 48 h before the experiment, and exposed to cisplatin (25 μM) or control conditions for 18 h. Cells were subsequently processed for immunoblotting (A), immunofluorescence analysis (B) or cell surface biotinylation (C-D). A. ΔNErbB2, top panel shows representative immunoblots, lower panel shows ΔNErbB2 protein level relative to that in vector cells under control conditions. B. Subcellular localization of cytochrome c. Nuclei are stained with DAPI. C. Representative immunoblots of NHE1 in total lysate fractions (TLF), intracellular fractions (ITF) and biotinylated fractions (BF) in MCF-7 vector (V) and ΔNErbB2 (ΔN) cells. F. Top panel shows representative immunoblots of NHE1 in TLF and BF in native MCF-7 cells, lower panel shows NHE1 protein level relative to that in native MCF-7 cells under control conditions. Data are representative of 3-5 independent experiments for each condition. Western blot data are shown as means with SEM error bars. * indicates comparison of the value in cisplatin treated to that in the respective cell line control, # indicate comparison of values in vector- and ΔNErbB2 cell lines under same treatment. */#, **/##, ***/### and ****/#### indicate p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively (C, one-way ANOVA with Bonferroni post-test) (A, two-way ANOVA with Bonferroni post-test).</p>
<p>Supplementary figure 3: Working model. Pathways involved in cisplatin-induced death in MCF-7 breast cancer cells, and their relation to ΔNErbB2 expression.</p>
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