Serum starvation is one of the most frequently performed procedures in molecular biology and there are literally thousands of research papers reporting its use. In fact, this method has become so ingrained in certain areas of research that reports often simply state that cells were serum starved without providing any factual details as to how the procedure was carried out. Even so, we quite obviously lack unequivocal terminology, standard protocols, and perhaps most surprisingly, a common conceptual basis when performing serum starvation. Such inconsistencies not only hinder interstudy comparability but can lead to opposing and inconsistent experimental results. Although it is frequently assumed that serum starvation reduces basal activity of cells, available experimental data do not entirely support this notion. To address this important issue, we studied primary human myotubes, rat L6 myotubes and human embryonic kidney (HEK)293 cells under different serum starvation conditions and followed time-dependent changes in important signaling pathways such as the extracellular signal-regulated kinase 1/2, the AMP-activated protein kinase, and the mammalian target of rapamycin. Serum starvation induced a swift and dynamic response, which displayed obvious qualitative and quantitative differences across different cell types and experimental conditions despite certain unifying features. There was no uniform reduction in basal signaling activity. Serum starvation clearly represents a major event that triggers a plethora of divergent responses and has therefore great potential to interfere with the experimental results and affect subsequent conclusions.

Skeletal muscle contains one of the largest and the most dynamic pools of Na,K-ATPase (NKA) in the body. Under resting conditions, NKA in skeletal muscle operates at only a fraction of maximal pumping capacity, but it can be markedly activated when demands for ion transport increase, such as during exercise or following food intake. Given the size, capacity, and dynamic range of the NKA pool in skeletal muscle, its tight regulation is essential to maintain whole body homeostasis as well as muscle function. To reconcile functional needs of systemic homeostasis with those of skeletal muscle, NKA is regulated in a coordinated manner by extrinsic stimuli, such as hormones and nerve-derived factors, as well as by local stimuli arising in skeletal muscle fibers, such as contractions and muscle energy status. These stimuli regulate NKA acutely by controlling its enzymatic activity and/or its distribution between the plasma membrane and the intracellular storage compartment. They also regulate NKA chronically by controlling NKA gene expression, thus determining total NKA content in skeletal muscle and its maximal pumping capacity. This review focuses on molecular mechanisms that underlie regulation of NKA in skeletal muscle by major extrinsic and local stimuli. Special emphasis is given to stimuli and mechanisms linking regulation of NKA and energy metabolism in skeletal muscle, such as insulin and the energy-sensing AMP-activated protein kinase. Finally, the recently uncovered roles for glutathionylation, nitric oxide, and extracellular K(+) in the regulation of NKA in skeletal muscle are highlighted.

Besides being a neuronal fuel, L-lactate is also a signal in the brain. Whether extracellular L-lactate affects brain metabolism, in particular astrocytes, abundant neuroglial cells, which produce L-lactate in aerobic glycolysis, is unclear. Recent studies suggested that astrocytes express low levels of the L-lactate GPR81 receptor (EC50 ≈ 5 mM) that is in fat cells part of an autocrine loop, in which the Gi-protein mediates reduction of cytosolic cyclic adenosine monophosphate (cAMP). To study whether a similar signaling loop is present in astrocytes, affecting aerobic glycolysis, we measured the cytosolic levels of cAMP, D-glucose and L-lactate in single astrocytes using fluorescence resonance energy transfer (FRET)-based nanosensors. In contrast to the situation in fat cells, stimulation by extracellular L-lactate and the selective GPR81 agonists, 3-chloro-5-hydroxybenzoic acid (3Cl-5OH-BA) or 4-methyl-N-(5-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-4-(2-thienyl)-1,3-thiazol-2-yl)cyclohexanecarboxamide (Compound 2), like adrenergic stimulation, elevated intracellular cAMP and L-lactate in astrocytes, which was reduced by the inhibition of adenylate cyclase. Surprisingly, 3Cl-5OH-BA and Compound 2 increased cytosolic cAMP also in GPR81-knock out astrocytes, indicating that the effect is GPR81-independent and mediated by a novel, yet unidentified, excitatory L-lactate receptor-like mechanism in astrocytes that enhances aerobic glycolysis and L-lactate production via a positive feedback mechanism.

Methotrexate (MTX) is a widely used anticancer and antirheumatic drug that has been postulated to protect against metabolic risk factors associated with type 2 diabetes, although the mechanism remains unknown. MTX inhibits 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) and thereby slows the metabolism of 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranosyl-59-monophosphate (ZMP) and its precursor AICAR, which is a pharmacological AMPK activator. We explored whether MTX promotes AMPK activation in cultured myotubes and isolated skeletal muscle. We found MTX markedly reduced the threshold for AICAR-induced AMPK activation and potentiated glucose uptake and lipid oxidation. Gene silencing of the MTX target ATIC activated AMPK and stimulated lipid oxidation in cultured myotubes. Furthermore, MTX activated AMPK in wildtype HEK-293 cells. These effects were abolished in skeletal muscle lacking the muscle-specific, ZMP-sensitive AMPK-g3 subunit and in HEK-293 cells expressing a ZMP-insensitive mutant AMPK-g2 subunit. Collectively, our findings underscore a role for AMPK as a direct molecular link between MTX and energy metabolism in skeletal muscle. Cotherapy with AICAR and MTX could represent a novel strategy to treat metabolic disorders and overcome current limitations of AICAR monotherapy.Chronic therapy with methotrexate (MTX), a broadly used anticancer and antirheumatic drug, may protect rheumatic patients against metabolic risk factors associated with cardiovascular disease, obesity (1), and type 2 diabetes (2). This notion is supported by recent observations that MTX alleviates hyperglycemia and insulin resistance in diabetic (db/db) mice (3) and obese mice fed a high-fat diet (4). MTX is a folate antagonist and inhibits DNA replication (5), which explains its anticancer action but the not improvements in glucose homeostasis in type 2 diabetes or obesity. Suppression of chronic inflammation, thought to arise from MTX-stimulated adenosine release (6), may improve glucose homeostasis indirectly (7). Because antirheumatic drugs are not invariably associated with metabolic protection (1,8), other mechanisms are likely. Although MTX-induced adenosine release may have direct metabolic effects (4), its exact role is ambiguous, because adenosine receptor activation (9) and blockage (10) both improve insulin sensitivity. Clearly, the molecular underpinnings of MTX action in relation to metabolic disease remain undefined.MTX has several pharmacological targets, including 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) (11). ATIC is essential for the conversion of 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranosyl-59-monophosphate (ZMP) to inosine monophosphate in the final two steps of the de novo purine synthesis pathway. Thus, congenital ATIC deficiency or MTX therapy elevates intracellular ZMP content and excretion of its metabolites (6,12,13). The purine precursor ZMP is also an AMP mimetic and activa...

Benziane B, Widegren U, Pirkmajer S, Henriksson J, Stepto NK, Chibalin AV. Effect of exercise and training on phospholemman phosphorylation in human skeletal muscle.

Triple negative breast cancer, characterised by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER-2), has a poor prognosis mostly due to increased rate of distant metastases 1,2 . During the process of metastasation, cancer cells in primary tumour locally invade the tumour-associated stroma, detach from the invasion front of the tumour, and enter the lymphatic and/or blood vessels. Circulating cancer cells ultimately migrate through the capillary wall in distant tissues, re-attach to the extracellular matrix, and proliferate in a new microenvironment 3 . Once cancer cells detach from the main tumour mass, they must resist anoikis, a programmed cell death induced by extracellular matrix detachment 4 . MDA-MB-231 cells, the most commonly used in vitro model of triple negative breast cancer 5 , are highly metastatic and tumorigenic 5 . They form colonies in an anchorage-independent condition 6 , and are resistant to anoikis 7 . Albeit breast cancer cells must detach from extracellular matrix in order to metastasise in vivo [8][9][10][11] , floating MDA-MB-231 cells in vitro are commonly thought to be dead. Only a few studies investigated the viability of floating MDA-MB-231 cells in vitro [12][13][14] . Metabolic adaptations enable survival of cancer cells in an anchorage-independent condition 6,[15][16][17][18] . By stimulating glucose uptake, oncogenes restore redox and energy balance and prevent anoikis in breast cancer cells 18 .

Background: Contractions activate the sodium pump, Na ϩ ,K ϩ -ATPase, and the energy sensor, AMP-activated protein

Boon H, Kostovski E, Pirkmajer S, Song M, Lubarski I, Iversen PO, Hjeltnes N, Widegren U, Chibalin AV. Influence of chronic and acute spinal cord injury on skeletal muscle Na ϩ -K ϩ -ATPase and phospholemman expression in humans. Am J Physiol Endocrinol Metab 302: E864 -E871, 2012. First published January 24, 2012; doi:10.1152/ajpendo.00625.2011.-Na ϩ -K ϩ -ATPase is an integral membrane protein crucial for the maintenance of ion homeostasis and skeletal muscle contractibility. Skeletal muscle Na ϩ -K ϩ -ATPase content displays remarkable plasticity in response to long-term increase in physiological demand, such as exercise training. However, the adaptations in Na ϩ -K ϩ -ATPase function in response to a suddenly decreased and/or habitually low level of physical activity, especially after a spinal cord injury (SCI), are incompletely known. We tested the hypothesis that skeletal muscle content of Na ϩ -K ϩ -ATPase and the associated regulatory proteins from the FXYD family is altered in SCI patients in a manner dependent on the severity of the spinal cord lesion and postinjury level of physical activity. Three different groups were studied: 1) six subjects with chronic complete cervical SCI, 2) seven subjects with acute, complete cervical SCI, and 3) six subjects with acute, incomplete cervical SCI. The individuals in groups 2 and 3 were studied at months 1, 3, and 12 postinjury, whereas individuals with chronic SCI were compared with an ablebodied control group. Chronic complete SCI was associated with a marked decrease in [ 3 H]ouabain binding site concentration in skeletal muscle as well as reduced protein content of the ␣ 1-, ␣2-, and ␤ 1-subunit of the Na ϩ -K ϩ -ATPase. In line with this finding, expression of the Na ϩ -K ϩ -ATPase ␣1-and ␣2-subunits progressively decreased during the first year after complete but not after incomplete SCI. The expression of the regulatory protein phospholemman (PLM or FXYD1) was attenuated after complete, but not incomplete, cervical SCI. In contrast, FXYD5 was substantially upregulated in patients with complete SCI. In conclusion, the severity of the spinal cord lesion and the level of postinjury physical activity in patients with SCI are important factors controlling the expression of Na ϩ -K ϩ -ATPase and its regulatory proteins PLM and FXYD5. FXYD proteins; sodium pump; physical inactivity; paralysis THE Na ϩ -K ϩ -ATPase IS AN INTEGRAL MEMBRANE PROTEIN that is crucial for the maintenance of ion homeostasis, cell volume, and muscle contractibility. It is composed of two polypeptide subunits, a catalytic 112-kDa ␣-subunit (␣ 1 -, ␣ 2 -, and ␣ 3 -isoforms) and a 35-to 60-kDa glycosylated ␤-subunit (␤ 1 -, ␤ 2 -, and ␤ 3 -isoforms) (6, 35). The relative abundance of each isoform and Na ϩ -K ϩ -ATPase activity is regulated in a muscleand fiber type-specific manner (29,59,62