Pyruvate dehydrogenase activation and kinase expression in human skeletal muscle during fasting

Fasting forces adaptive changes in whole body and skeletal muscle metabolism that increase fat oxidation and decrease the oxidation of carbohydrate. We tested the hypothesis that 40 h of fasting would decrease pyruvate dehydrogenase (PDH) activity and increase PDH kinase (PDK) isoform mRNA expression in human skeletal muscle. The putative transcriptional activators of PDK isozymes, peroxisome proliferator-activated receptor-α (PPAR-α) protein, and forkhead homolog in rhabdomyosarcoma (FKHR) mRNA were also measured. Eleven healthy adults fasted after a standard meal (25% fat, 60% carbohydrate, 15% protein) with blood and skeletal muscle samples taken at 3, 15, and 40 h postprandial. Fasting increased plasma free fatty acid, glycerol, and &beta;-hydroxybutyrate concentrations and decreased glucose and insulin concentrations. PDH activity decreased from 0.88 ± 0.11 mmol acetyl-CoA · min^-1 · kg wet muscle wt^-1 at 3 h to 0.62 ± 0.10 (P = not significant) and 0.39 ± 0.06 (P < 0.05) mmol · min^-1 · kg wet mass^-1 after 15 and 40 h of fasting. Although all four PDK isoforms were expressed in human skeletal muscle, PDK^-2 and -4 mRNA were the most abundant. PDK^-1 and ^-3 mRNA abundance was ~1 and 15% of the PDK^-2 and 4^- levels, respectively. The 40-h fast had no effect on PDK^-1, ^-2, and ^-3 mRNA expression. PDK-4 mRNA was significantly increased ~3-fold after 15 h and ~14-fold after 40 h of fasting. Skeletal muscle PPAR-α protein and FKHR mRNA abundance were unaffected by the fast. The results suggest that decreased PDH activation after 40 h of fasting may have been a function of the large increase in PDK-4 mRNA expression and possible subsequent increase in PDK protein and activity. The changes in PDK-4 expression and PDH activity did not coincide with increases in the transcriptional activators PPAR-α and FKHR.

Altering dietary nutrient intake that reduces glycogen content leads to phosphorylation of nuclear p38 MAP kinase in human skeletal muscle: association with IL-6 gene transcription during contraction

To determine the effect of glycogen availability and contraction on intracellular signaling and IL-6 gene transcription, eight males performed 60 min of exercise on two occasions: either with prior ingestion of a normal (Con) or low carbohydrate (LCHO) diet that reduced pre-exercise muscle glycogen content. Muscle biopsies were obtained and analyzed for IL-6 mRNA. In addition, nuclear proteins were isolated from the samples and analyzed for the mitogen- activated protein kinases (MAPK) c-jun amino-terminal kinase (JNK) 1 and 2 and p38 MAPK. Nuclear fractions were also analyzed for the phosphorylated forms of JNK (p-JNK) and p38 MAPK (p-p38 MAPK) and the abundance of the nuclear transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-&beta; (NF-κ&beta;). No differences were observed in the protein abundance of total JNK 1/2, p38 MAPK, NFAT, or NF-κ&beta; before exercise, but the nuclear abundance of p-p38 MAPK was higher (P<0.05) in LCHO. Contraction resulted in an increase (P<0.05) in nuclear p-JNK 1/2, but there were no differences when comparing CON with LCHO. The fold increase in IL-6 mRNA with contraction was potentiated (P<0.05) in LCHO. A correlation between pre-exercise nuclear phosphorylated p38 MAPK and contraction-induced fold increase in IL-6 mRNA was performed, revealing a highly significant correlation (r=0.96; P<0.01). We next incubated L6 myotubes in ionomycin (a compound known to induce IL-6 mRNA) with or without the pyridinylimidazole p38 MAPK inhibitor SB203580. Treatments did not affect total nuclear p38 MAPK, but ionomycin increased (P<0.05) both nuclear p-p38 MAPK and IL-6 mRNA. The addition of SB203580 to ionomycin decreased (P<0.05) nuclear p-p38 MAPK and totally abolished (P<0.05) the ionomycin- induced increase in IL-6 mRNA. These data suggest that reduced carbohydrate intake that results in low intramuscular glycogen leads to phosphorylation of p38 MAPK at the nucleus. Furthermore, phosphorylation of p38 MAPK in the nucleus appears to be an upstream target for IL-6, providing new insights into the regulation of IL-6 gene transcription.

Inorganic-organic hybrids of the p,p'-diphenylmethylenediphosphinate ligand with bivalent metals: a new 2D-layered phenylphosphinate zinc(II) complex

By reaction of Zn(CH₃COO)₂ with p,p′-diphenylmethylenediphosphinic acid in water a new inorganic–organic polymeric hybrid of formula [Zn(CH₂(P(Ph)O₂)₂)] has been synthesized and completely characterized. The X-ray analysis established that the structure consists of 2D-layered polymeric array, the 2D-sheets being built up through strong covalent linkages between the zinc metal and the oxygen donors of the phenylphosphinate ligand. The 2D-layers, which are featuring a mesh-net fashion, present voids of various dimensionality, up to 24-membered rings. The organic parts of the hybrid ligand, namely the phenyl rings, are shielding the inorganic skeleton of the layers, preventing the propagation of the polymer in the third dimension. No water molecules are present in the lattice, both of coordination and crystallization. Crystal data are: monoclinic, P2₁Ic, a=11.840(2), b=9.646(9), c=12.516(5) Å, &beta;=95.03(2), V=1423.9(15) ų, Z=4. The solid material has been characterized by ³¹P MAS NMR spectroscopy and thermogravimetric analysis.

Characterisation of proteins in the milk of fur seals

Milk protein composition was investigated throughout the lactation periods of the Australian fur seal (Arctocephalus pusillus doriferus) and Antarctic fur seal (Arctocephalus gazella). The mean protein content of the milk was found to be 10.9% and 10.6% respectively. The concentration of total protein did not change during lactation, although a decline in casein content of the milk in late lactation was apparent. Milk protein concentration during a foraging/suckling cycle of the Antarctic fur seal analysed at the time of arrival on shore, and 24 h and 72 h after arrival was 12.8%, 11.4% and 12.5% respectively. Re-feeding animals at 72 h resulted in a significant increase in milk protein content to 14.9%. Characterisation of milk protein by SDS-PAGE analysis revealed 5 casein and 10 major whey protein bands. Amino-terminal sequencing indicated that the majority of the whey fraction of the milk is &beta;-lactoglobulin (&beta;-LG). The limited amino acid sequence indicated 3 different &beta;-LGs were secreted in the milk. Subsequently, RT-PCR was used to extend the sequence of one of the &beta;-LGs and translation of the 464 bp fragment indicated that it shared 79% sequence identity with feline &beta;-LG II.

A pseudosymmetric cell adhesion regulatory domain in the β7 tail of the integrin α4β7 that interacts with focal adhesion kinase and src

The &beta;7 integrins α4&beta;7 and E&beta;7 play key roles in forming the gut-associated lymphoid tissue, and contribute to chronic inflammation. The α4&beta;7 integrin-mediated adhesion of activated lymphocytes is largely due to a transient increase in avidity from ligand-induced clustering of α4&beta;7 at the cell-surface. Here, we report that L and D enantiomers of a cell-permeable peptide YDRREY encompassing residues 735-740 of the cytoplasmic tail of the &beta;7 subunit inhibit the adhesion of T cells to &beta;7 integrin ligands. The YDRREY peptide abrogated mucosal addressin cell adhesion molecule-1-induced clustering of α4&beta;7 on the surface of activated T cells. A mutated form of the YDRREY peptide carrying either single or double conservative mutations at Tyr⁷³⁵Phe and Tyr⁷⁴⁰Phe was unable to inhibit T cell adhesion, suggesting that both tandem tyrosines are critical for activity. The YDRREY peptide was bound and phosphorylated by focal adhesion kinase and src, which may serve to sequester cytoskeletal proteins to the cytoplasmic domain of 4&beta;7. The quasi-palindromic sequence YDRREY within the &beta;7 cytoplasmic tail constitutes a cell adhesion regulatory domain that modulates the interaction of &beta;7-expressing leukocytes with their endothelial and epithelial ligands. Cell-permeable peptidomimetics based on this motif have utility as anti-inflammatory reagents for the treatment of chronic inflammatory disease.

Adiponectin decreases pyruvate dehydrogenase kinase 4 gene expression in obese- and diabetic derived

Aim: To investigate the effects of globular adiponectin (gAd) on gene expression and whether these effects are mediated through 3',5'-cyclic monophosphate-activated protein kinase in skeletal muscle myotubes obtained from lean, obese and obese diabetic individuals.

Methods: Rectus abdominus muscle biopsies were obtained from surgical patients to establish primary skeletal muscle cell cultures. Three distinct primary cell culture groups were established (lean, obese and obese diabetic; n = 7 in each group). Once differentiated, these cultures were then exposed to gAd or 5-aminoimidazole-4-carboxamide-1-&beta;-d-ribofuranoside (AICAR) for 6 h.

Results: Stimulation with gAd decreased pyruvate dehydrogenase kinase 4 (PDK4) gene expression in the obese and diabetic samples (p ≤ 0.05) and increased cytochrome c oxidase (COX) subunit 4 (COXIV) gene expression in the myotubes derived from lean individuals only (p < 0.05). AICAR treatment also decreased PDK4 gene expression in the obese- and diabetic-derived myotubes (p ≤ 0.05) and increased the gene expression of the mitochondrial gene, COXIII, in the lean-derived samples only (p < 0.05).

Conclusions: This study demonstrated distinct disparity between myotubes derived from lean compared with obese and obese diabetic individuals following gAd and AICAR treatment. Further understanding of the regulation of PDK4 in obese and diabetic skeletal muscle and its interaction with adiponectin signalling is required as this appears to be an important early molecular event in these disease states that may improve blood glucose control and metabolic flux.

AMP-activated protein kinase regulates GLUT4 transcription by phosphorylating histone deacetylase 5

Objective: Insulin resistance associated with obesity and diabetes is ameliorated by specific overexpression of GLUT4 in skeletal muscle. The molecular mechanisms regulating skeletal muscle GLUT4 expression remain to be elucidated. The purpose of this study was to examine these mechanisms.

Research Design and Methods and Results: Here, we report that AMP-activated protein kinase (AMPK) regulates GLUT4 transcription through the histone deacetylase (HDAC)5 transcriptional repressor. Overexpression of HDAC5 represses GLUT4 reporter gene expression, and HDAC inhibition in human primary myotubes increases endogenous GLUT4 gene expression. In vitro kinase assays, site-directed mutagenesis, and site-specific phospho-antibodies establish AMPK as an HDAC5 kinase that targets S259 and S498. Constitutively active but not dominant-negative AMPK and 5-aminoimidazole-4-carboxamide-1-&beta;-d-ribonucleoside (AICAR) treatment in human primary myotubes results in HDAC5 phosphorylation at S259 and S498, association with 14-3-3 isoforms, and H3 acetylation. This reduces HDAC5 association with the GLUT4 promoter, as assessed through chromatin immunoprecipitation assays and HDAC5 nuclear export, concomitant with increases in GLUT4 gene expression. Gene reporter assays also confirm that the HDAC5 S259 and S498 sites are required for AICAR induction of GLUT4 transcription.

Conclusions: These data reveal a signal transduction pathway linking cellular energy charge to gene transcription directed at restoring cellular and whole-body energy balance and provide new therapeutic targets for the treatment and management of insulin resistance and type 2 diabetes.

Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase α1 following overload in LKB1 knockout mice

The activation of the AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1) is hypothesized to underlie the fact that muscle growth following resistance exercise is decreased by concurrent endurance exercise. To directly test this hypothesis, the capacity for muscle growth was determined in mice lacking the primary upstream kinase for AMPK in skeletal muscle, LKB1. Following either 1 or 4 weeks of overload, there was no difference in muscle growth between the wild type (wt) and LKB1−/− mice (1 week: wt, 38.8 ± 7.75%; LKB1−/−, 27.8 ± 12.98%; 4 week: wt, 75.8 ± 15.2%; LKB1−/−, 85.0 ± 22.6%). In spite of the fact that the LKB1 had been knocked out in skeletal muscle, the phosphorylation and activity of the α1 isoform of AMPK were markedly increased in both the wt and the LKB1−/− mice. To identify the upstream kinase(s) responsible, we studied potential upstream kinases other than LKB1. The activity of both Ca2+–calmodulin-dependent protein kinase kinase α(CaMKKα) (5.05 ± 0.86-fold) and CaMKK&beta; (10.1 ± 2.59-fold) increased in the overloaded muscles, and this correlated with their increased expression. Phosphorylation of TAK-1 also increased 10-fold following overload in both the wt and LKB1 mice. Even though the α1 isoform of AMPK was activated by overload, there were no increases in expression of mitochondrial proteins or GLUT4, indicating that the α1 isoform is not involved in these metabolic adaptations. The phosphorylation of TSC2, an upstream regulator of the TORC1 pathway, at the AMPK site (Ser1345) was increased in response to overload, and this was not affected by LKB1 deficiency. Taken together, these data suggest that the α1 isoform of AMPK is preferentially activated in skeletal muscle following overload in the absence of metabolic adaptations, suggesting that this isoform might be important in the regulation of growth but not metabolism.

Characterisation of monotreme caseins reveals lineage-specific expansion of an ancestral casein locus in mammals

Using a milk-cell cDNA sequencing approach we characterised milk-protein sequences from two monotreme species, platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus) and found a full set of caseins and casein variants. The genomic organisation of the platypus casein locus is compared with other mammalian genomes, including the marsupial opossum and several eutherians. Physical linkage of casein genes has been seen in the casein loci of all mammalian genomes examined and we confirm that this is also observed in platypus. However, we show that a recent duplication of &beta;-casein occurred in the monotreme lineage, as opposed to more ancient duplications of α-casein in the eutherian lineage, while marsupials possess only single copies of α- and &beta;-caseins. Despite this variability, the close proximity of the main α- and &beta;-casein genes in an inverted tail-tail orientation and the relative orientation of the more distant kappa-casein genes are similar in all mammalian genome sequences so far available. Overall, the conservation of the genomic organisation of the caseins indicates the early, pre-monotreme development of the fundamental role of caseins during lactation. In contrast, the lineage-specific gene duplications that have occurred within the casein locus of monotremes and eutherians but not marsupials, which may have lost part of the ancestral casein locus, emphasises the independent selection on milk provision strategies to the young, most likely linked to different developmental strategies. The monotremes therefore provide insight into the ancestral drivers for lactation and how these have adapted in different lineages.

Effect of training on activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in rat soleus muscle

1. The effect of a chronic programme of either low- or moderate-to-high-intensity treadmill running on the activation of the extracellular-signal regulated protein kinase (ERK1/2) and the p38 mitogen-activated protein kinase (MAPK) pathways was determined in rat muscle. 2. Sprague-Dawley rats were assigned to one of three groups: (i) sedentary (NT; n = 8); (ii) low-intensity training (8 m/min; LIT; n = 16); and (iii) moderate-to-high-intensity training (28 m/min; HIT;n = 16). The training regimens were planned so that animals covered the same distance and had similar glycogen utilization for both LIT and HIT exercise sessions. 3. A single bout of LIT or HIT following 8 weeks of training led to a twofold increase in the phosphorylation of ERK1/2 (P = 0.048) and a two- to threefold increase in p38 MAPK (P = 0.005). Extracellular signal-regulated kinase 1/2 phosphorylation in muscle sampled 48 h after the last exercise bout was similar to sedentary values, while p38 MAPK phosphorylation was 70-80% lower than sedentary. One bout of LIT or HIT increased total ERK1/2 and p38 MAPK expression, with the magnitude of this increase being independent of prior exercise intensity or duration. Extracellular signal-regulated kinase 1/2 expression was increased three- to fourfold in muscle sampled 48 h after the last exercise bout irrespective of the prior training programme (P = 0.027), but p38 MAPK expression was approximately 90% lower than sedentary values. 4. In conclusion, exercise-training of different intensities/durations results in selective postexercise activation of intracellular signalling pathways, which may be one mechanism regulating specific adaptations induced by diverse training programmes.

Epidermal growth factor receptor-tyrosine kinase inhibitors in advanced squamous cell carcinoma of the lung: a meta-analysis.

Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) are well established in treating metastatic pulmonary adenocarcinoma, especially patients with activating EGFR mutations. EGFR mutations are rare in pulmonary squamous cell carcinomas (SCCs). There are conflicting data supporting the efficacy of EGFR-TKIs in advanced lung SCC. We analyzed the impact of EGFR-TKIs on progression-free survival (PFS) and overall survival (OS) in unselected patients with lung SCC.

The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition and small interfering RNA-mediated knockdown of Erk1/2 also remarkably increased the level of LC3-II in MCF7 cells. Moreover, Danu inhibited EMT in both MCF7 and MDA-MB-231 cells with upregulated E-cadherin and zona occludens protein 1 (ZO-1) but downregulated N-cadherin, zinc finger E-box-binding homeobox 1 (TCF8/ZEB1), snail, slug, vimentin, and &beta;-catenin. Notably, Danu showed lower cytotoxicity toward normal breast epithelial MCF10A cells. These findings indicate that Danu promotes cellular apoptosis and autophagy but inhibits EMT in human breast cancer cells via modulation of p38 MAPK/Erk1/2/Akt/mTOR signaling pathways. Danu may represent a promising anticancer agent for breast cancer treatment. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Danu in breast cancer therapy.

Hydrotelluration of acetylenic esters: structural characterization of stereoisomers of methyl/ethyl beta-(aryltelluro)acrylates

Synthesis and complete characterization of some ester functionalized vinylic tellurides bearing an aryl ligand with varying steric and electronic effects bound to tellurium is described. Hydrotelluration of methyl propiolate using Ar2Te2/NaBH4 in methanol results in a mixture of stereoisomers of methyl &beta;-(aryltelluro)acrylates, ArTeCH[double bond, length as m-dash]CHCOOMe (Ar = 4-MeOC6H4, 1A; 1-C10H7, 2A; 2,4,6-Me3C6H2, 3A; C5H5FeC5H4, 4A; 4-Me2NC6H4, 5A; and 2-C4H3S, 6A). The same reaction in ethanol provides isomeric mixtures of the ethyl esters ArTeCH[double bond, length as m-dash]CHCOOEt (1B–6B). However, in the reactions between methyl propiolate and Ar2Te2 (Ar = 2,4,6-Me3C6H2, 4-Me2NC6H4) in isopropanol or t-butanol, no exchange of alkyl groups between the parent ester and the solvent is observed, instead detelluration of the Ar2Te2 to Ar2Te is a competing reaction along with almost exclusive formation of the (Z)-isomers (3Aa, 5Aa). The geometry of the separated stereoisomers is established in solution, with the help of 1H, 13C and 125Te NMR spectrometry. Of particular interest is the observation that 125Te chemical shifts {deshielded in (Z) compared to (E); Δδ = 106–136 ppm} and the geminal heteronuclear coupling constants {2J(1H–125Te) values for (E) are more than seven times that of the corresponding (Z) isomer} can be used to distinguish between liquid isomers. Structural characterization in the solid state by single-crystal X-ray diffraction for the 2Ba, 3Aa, 3Ba, 5Aa, 8 (Z)-isomers as well as for both stereoisomers of 4-Me2NC6H4TeCH[double bond, length as m-dash]CHCOOEt (5Ba and 5Bb) is also presented. The carbonyl O atom of the ester group is invariably involved, at least in the solid state, in a secondary bonding interaction with the Te(II) atom. While an intermolecular Te⋯O interaction gives rise to one-dimensional supramolecular arrays in the crystal lattice of 5Bb with (E) configuration, it is realized intramolecularly in the case of the (Z)-isomers due to the cis position of the chalcogen atoms.