Yap/Taz activity is associated with increased expression of phosphoglycerate dehydrogenase that supports myoblast proliferation.

In skeletal muscle, the Hippo effector Yap promotes satellite cell, myoblast, and rhabdomyoblast proliferation but prevents myogenic differentiation into multinucleated muscle fibres. We previously noted that Yap drives expression of the first enzyme of the serine biosynthesis pathway, phosphoglycerate dehydrogenase (Phgdh). Here, we examined the regulation and function of Phgdh in satellite cells and myoblasts and found that Phgdh protein increased during satellite cell activation. Analysis of published data reveal that Phgdh mRNA in mouse tibialis anterior muscle was highly expressed at day 3 of regeneration after cardiotoxin injection, when markers of proliferation are also robustly expressed and in the first week of synergist-ablated muscle. Finally, siRNA-mediated knockdown of PHGDH significantly reduced myoblast numbers and the proliferation rate. Collectively, our data suggest that Phgdh is a proliferation-enhancing metabolic enzyme that is induced when quiescent satellite cells become activated.

Myotube growth is associated with cancer-like metabolic reprogramming and is limited by phosphoglycerate dehydrogenase.

The Warburg effect links growth and glycolysis in cancer. A key purpose of the Warburg effect is to generate glycolytic intermediates for anabolic reactions, such as nucleotides → RNA/DNA and amino acids → protein synthesis. The aim of this study was to investigate whether a similar 'glycolysis-for-anabolism' metabolic reprogramming also occurs in hypertrophying skeletal muscle. To interrogate this, we first induced C2C12 myotube hypertrophy with IGF-1. We then added 14C glucose to the differentiation medium and measured radioactivity in isolated protein and RNA to establish whether 14C had entered anabolism. We found that especially protein became radioactive, suggesting a glucose → glycolytic intermediates → non-essential amino acid(s) → protein series of reactions, the rate of which was increased by IGF-1. Next, to investigate the importance of glycolytic flux and non-essential amino acid synthesis for myotube hypertrophy, we exposed C2C12 and primary mouse myotubes to the glycolysis inhibitor 2-Deoxy-d-glucose (2DG). We found that inhibiting glycolysis lowered C2C12 and primary myotube size. Similarly, siRNA silencing of PHGDH, the key enzyme of the serine biosynthesis pathway, decreased C2C12 and primary myotube size; whereas retroviral PHGDH overexpression increased C2C12 myotube size. Together these results suggest that glycolysis is important for hypertrophying myotubes, which reprogram their metabolism to facilitate anabolism, similar to cancer cells.

Genes controlling skeletal muscle glucose uptake and their regulation by endurance and resistance exercise.

Exercise improves the insulin sensitivity of glucose uptake in skeletal muscle. Due to that, exercise has become a cornerstone treatment for type 2 diabetes mellitus (T2DM). The mechanisms by which exercise improves skeletal muscle insulin sensitivity are, however, incompletely understood. We conducted a systematic review to identify all genes whose gain or loss of function alters skeletal muscle glucose uptake. We subsequently cross-referenced these genes with recently generated data sets on exercise-induced gene expression and signaling. Our search revealed 176 muscle glucose-uptake genes, meaning that their genetic manipulation altered glucose uptake in skeletal muscle. Notably, exercise regulates the expression or phosphorylation of more than 50% of the glucose-uptake genes or their protein products. This included many genes that previously have not been associated with exercise-induced insulin sensitivity. Interestingly, endurance and resistance exercise triggered some common but mostly unique changes in expression and phosphorylation of glucose-uptake genes or their protein products. Collectively, our work provides a resource of potentially new molecular effectors that play a role in the incompletely understood regulation of muscle insulin sensitivity by exercise.

PKM2 Determines Myofiber Hypertrophy In Vitro and Increases in Response to Resistance Exercise in Human Skeletal Muscle.

Nearly 100 years ago, Otto Warburg investigated the metabolism of growing tissues and discovered that tumors reprogram their metabolism. It is poorly understood whether and how hypertrophying muscle, another growing tissue, reprograms its metabolism too. Here, we studied pyruvate kinase muscle (PKM), which can be spliced into two isoforms (PKM1, PKM2). This is of interest, because PKM2 redirects glycolytic flux towards biosynthetic pathways, which might contribute to muscle hypertrophy too. We first investigated whether resistance exercise changes PKM isoform expression in growing human skeletal muscle and found that PKM2 abundance increases after six weeks of resistance training, whereas PKM1 decreases. Second, we determined that Pkm2 expression is higher in fast compared to slow fiber types in rat skeletal muscle. Third, by inducing hypertrophy in differentiated C2C12 cells and by selectively silencing Pkm1 and/or Pkm2 with siRNA, we found that PKM2 limits myotube growth. We conclude that PKM2 contributes to hypertrophy in C2C12 myotubes and indicates a changed metabolic environment within hypertrophying human skeletal muscle fibers. PKM2 is preferentially expressed in fast muscle fibers and may partly contribute to the increased potential for hypertrophy in fast fibers.

Genes Whose Gain or Loss-of-Function Increases Endurance Performance in Mice: A Systematic Literature Review.

Endurance is not only a key factor in many sports but endurance-related variables are also associated with good health and low mortality. Twin and family studies suggest that several endurance-associated traits are ≈50% inherited. However, we still poorly understand what DNA sequence variants contribute to endurance heritability. To address this issue, we conducted a systematic review to identify genes whose experimental loss or gain-of-function increases endurance capacity in mice. We found 31 genes including two isoforms of Ppargc1a whose manipulation increases running or swimming endurance performance by up to 1800%. Genes whose gain-of-function increases endurance are Adcy5, Adcy8, Hk2, Il15, Mef2c, Nr4a3, Pck1 (Pepck), Ppard, Ppargc1a (both the a and b isoforms of the protein Pgc-1α), Ppargc1b, Ppp3ca (calcineurin), Scd1, Slc5a7, Tfe3, Tfeb, Trib3 & Trpv1. Genes whose loss-of-function increases endurance in mice are Actn3, Adrb2, Bdkrb2, Cd47, Crym, Hif1a, Myoz1, Pappa, Pknox1, Pten, Sirt4, Thbs1, Thra, and Tnfsf12. Of these genes, human DNA sequence variants of ACTN3, ADCY5, ADRB2, BDKRB2, HIF1A, PPARD, PPARGC1A, PPARGC1B, and PPP3CA are also associated with endurance capacity and/or VO2max trainability suggesting evolutionary conservation between mice and humans. Bioinformatical analyses show that there are numerous amino acid or copy number-changing DNA variants of endurance genes in humans, suggesting that genetic variation of endurance genes contributes to the variation of human endurance capacity, too. Moreover, several of these genes/proteins change their expression or phosphorylation in skeletal muscle or the heart after endurance exercise, suggesting a role in the adaptation to endurance exercise.

Genes Whose Gain or Loss-Of-Function Increases Skeletal Muscle Mass in Mice: A Systematic Literature Review.

Skeletal muscle mass differs greatly in mice and humans and this is partially inherited. To identify muscle hypertrophy candidate genes we conducted a systematic review to identify genes whose experimental loss or gain-of-function results in significant skeletal muscle hypertrophy in mice. We found 47 genes that meet our search criteria and cause muscle hypertrophy after gene manipulation. They are from high to small effect size: Ski, Fst, Acvr2b, Akt1, Mstn, Klf10, Rheb, Igf1, Pappa, Ppard, Ikbkb, Fstl3, Atgr1a, Ucn3, Mcu, Junb, Ncor1, Gprasp1, Grb10, Mmp9, Dgkz, Ppargc1a (specifically the Ppargc1a4 isoform), Smad4, Ltbp4, Bmpr1a, Crtc2, Xiap, Dgat1, Thra, Adrb2, Asb15, Cast, Eif2b5, Bdkrb2, Tpt1, Nr3c1, Nr4a1, Gnas, Pld1, Crym, Camkk1, Yap1, Inhba, Tp53inp2, Inhbb, Nol3, Esr1. Knock out, knock down, overexpression or a higher activity of these genes causes overall muscle hypertrophy as measured by an increased muscle weight or cross sectional area. The mean effect sizes range from 5 to 345% depending on the manipulated gene as well as the muscle size variable and muscle investigated. Bioinformatical analyses reveal that Asb15, Klf10, Tpt1 are most highly expressed hypertrophy genes in human skeletal muscle when compared to other tissues. Many of the muscle hypertrophy-regulating genes are involved in transcription and ubiquitination. Especially genes belonging to three signaling pathways are able to induce hypertrophy: (a) Igf1-Akt-mTOR pathway, (b) myostatin-Smad signaling, and (c) the angiotensin-bradykinin signaling pathway. The expression of several muscle hypertrophy-inducing genes and the phosphorylation of their protein products changes after human resistance and high intensity exercise, in maximally stimulated mouse muscle or in overloaded mouse plantaris.

Kinesin's step dissected with single-motor FRET.

The motor protein Kinesin-1 drives intracellular transport along microtubules, with each of its two motor domains taking 16-nm steps in a hand-over-hand fashion. The way in which a single-motor domain moves during a step is unknown. Here, we use Förster resonance energy transfer (FRET) between fluorescent labels on both motor domains of a single kinesin. This approach allows us to resolve the relative distance between the motor domains and their relative orientation, on the submillisecond timescale, during processive stepping. We observe transitions between high and low FRET values for certain kinesin constructs, depending on the location of the labels. These results reveal that, during a step, a kinesin motor domain dwells in a well-defined intermediate position for approximately 3 ms.

Novel ways to determine kinesin-1's run length and randomness using fluorescence microscopy.

The molecular motor protein Kinesin-1 drives intracellular transport of vesicles, by binding to microtubules and making hundreds of consecutive 8-nm steps along them. Three important parameters define the motility of such a linear motor: velocity, run length (the average distance traveled), and the randomness (a measure of the stochasticity of stepping). We used total internal reflection fluorescence microscopy to measure these parameters under conditions without external load acting on the motor. First, we tracked the motility of single motor proteins at different adenosine triphosphate (ATP) concentrations and determined both velocity and (for the first time, to our knowledge, by using single-molecule fluorescence assays) randomness. We show that the rate of Kinesin-1 at zero load is limited by two or more exponentially distributed processes at high ATP concentrations, but that an additional, ATP-dependent process becomes the sole rate-limiting process at low ATP concentrations. Next, we measured the density profile of moving Kinesin-1 along a microtubule. This allowed us to determine the average run length in a new way, without the need to resolve single-molecules and to correct for photobleaching. At saturating ATP concentration, we measured a run length of 1070 +/- 30 nm. This value did not significantly change for different ATP concentrations.

Alternating-site mechanism of kinesin-1 characterized by single-molecule FRET using fluorescent ATP analogues.

Kinesin-1 motor proteins move along microtubules in repetitive steps of 8 nm at the expense of ATP. To determine nucleotide dwell times during these processive runs, we used a Förster resonance energy transfer method at the single-molecule level that detects nucleotide binding to kinesin motor heads. We show that the fluorescent ATP analog used produces processive motility with kinetic parameters altered <2.5-fold compared with normal ATP. Using our confocal fluorescence kinesin motility assay, we obtained fluorescence intensity time traces that we then analyzed using autocorrelation techniques, yielding a time resolution of approximately 1 ms for the intensity fluctuations due to fluorescent nucleotide binding and release. To compare these experimental autocorrelation curves with kinetic models, we used Monte-Carlo simulations. We find that the experimental data can only be described satisfactorily on the basis of models assuming an alternating-site mechanism, thus supporting the view that kinesin's two motor domains hydrolyze ATP and step in a sequential way.

Kinesin moving through the spotlight: single-motor fluorescence microscopy with submillisecond time resolution.

Kinesin-1 is one of the motor proteins that drive intracellular transport in eukaryotes. This motor makes hundreds of 8-nm steps along a microtubule before releasing. Kinesin-1 can move at velocities of up to approximately 800 nm/s, which means that one turnover on average takes 10 ms. Important details, however, concerning the coordination between the two motor domains have not been determined due to limitations of the techniques used. In this study, we present an approach that allows the observation of fluorescence intensity changes on individual kinesins with a time resolution far better than the duration of a single step. In our approach, the laser focus of a confocal fluorescence microscope is pointed at a microtubule and the photons emitted by fluorescently labeled kinesin motors walking through the spot are detected with submicrosecond accuracy. We show that the autocorrelation of a fluorescence time trace of an individual kinesin motor contains information at time lags down to 0.1 ms. The quality and time resolution of the autocorrelation is primarily determined by the amount of signal photons used. By adding the autocorrelations of several tens of kinesins, fluorescence intensity changes can be observed at a timescale below 100 micros.

Dual architectural roles of HU: formation of flexible hinges and rigid filaments.

The nucleoid-associated protein HU is one of the most abundant proteins in Escherichia coli and has been suggested to play an important role in bacterial nucleoid organization and regulation. Although the regulatory aspects of HU have been firmly established, much less is understood about the role of HU in shaping the bacterial nucleoid. In both functions (local) modulation of DNA architecture seems an essential feature, but information on the mechanical properties of this type of sequence-independent nucleoprotein complex is scarce. In this study we used magnetic tweezers and atomic force microscopy to quantify HU-induced DNA bending and condensation. Both techniques revealed that HU can have two opposing mechanical effects depending on the protein concentration. At concentrations <100 nM, individual HU dimers induce very flexible bends in DNA that are responsible for DNA compaction up to 50%. At higher HU concentrations, a rigid nucleoprotein filament is formed in which HU appears to arrange helically around the DNA without inducing significant condensation.