Myostatin regulates glucose metabolism via the AMP-activated protein kinase pathway in skeletal muscle cells

Myostatin (Mstn) is a secreted growth factor predominately expressed in skeletal muscle that negatively regulates skeletal muscle mass. Recent studies have indicated that loss function of myostatin not only increases muscle mass but also improves insulin sensitivity in vivo. In the present report, we demonstrated that myostatin regulates glucose metabolism by promoting glucose consumption and glucose uptake, increasing glycolysis, and inhibiting glycogen synthesis in skeletal muscle cells. Microarray analysis revealed that myostatin upregulates several genes involved in regulating glucose metabolism such as Glut1, Glut4, Hk2, and IL-6. Further investigation of the molecular basis of these phenomena revealed that AMP-activated protein kinase (AMPK), a key component for maintaining energy homeostasis, was activated by myostatin for promotion of glycolysis. Taken together, these findings provide the first experimental evidence that myostatin regulates glucose metabolism through the AMPK signal pathway in muscle cells. Importantly, our findings highlight that continued investigation of the metabolic function of myostatin is necessary for a comprehensive understanding of its active role in the regulation of skeletal muscle energy metabolism.     

Calcium-dependent regulation of NAD kinase.

An activator protein of NAD kinase from the pea, $\text{Pisum}\text{satavum}$ L., has been shown to be Ca²⁺-dependent. This plant activator protein also stimulates the activity of modulator protein dependent-cyclic nucleotide phosphodiesterase from porcine brain. This stimulation is similar to that observed with modulator protein isolated from animal sources. Furthermore, Ca²⁺-dependent modulator proteins isolated from porcine brain, bovine brain, and the coelenterate, $\text{Renilla}$, will regulate the NAD kinase activity of peas. Other common properties of the plant activator protein and animal modulator proteins are their acidic nature, heat stabilities, similar Stokes' radii, and their interactions with troponin I.     

Inhibition of a signaling pathway in cardiac muscle cells by active mitogen-activated protein kinase kinase.

Signaling via the Ras pathway involves sequential activation of Ras, Raf-1, mitogen-activated protein kinase kinase (MKK), and the extracellular signal-regulated (ERK) group of mitogen-activated protein (MAP) kinases. Expression from the c-Fos, atrial natriuretic factor (ANF), and myosin light chain-2 (MLC-2) promoters during phenylephrine-induced cardiac muscle cell hypertrophy requires activation of this pathway. Furthermore, constitutively active Ras or Raf-1 can mimic the action of phenylephrine in inducing expression from these promoters. In this study, we tested whether constitutively active MKK, the molecule immediately downstream of Raf, was sufficient to induce expression. Expression of constitutively active MKK induce ERK2 kinase activity and caused expression from the c-Fos promoter, but did not significantly activate expression of reporter genes under the control of either the ANF or MLC-2 promoters. Expression of CL100, a phosphatase that inactivates ERKs, prevented expression from all of the promoters. Taken together, these data suggest that ERK activation is required for expression from the Fos, ANF, and MLC-2 promoters but MKK and ERK activation is sufficient for expression only from the Fos promoter. Constitutively active MKK synergized with phenylephrine to increase expression from a c-Fos- or an AP1-driven reporter. However, active MKK inhibited phenylephrine- and Raf-1-induced expression from the ANF and MLC-2 promoters. A DNA sequence in the MLC-2 promoter that is a target for inhibition by active MKK, but not CL100, was mapped to a previously characterized DNA element (HF1) that is responsible for cardiac specificity. Thus, activation of cardiac gene expression during phenylephrine-induced hypertrophy requires ERK activation but constitutive activation by MKK can inhibit expression by targeting a DNA element that controls the cardiac specificity of gene expression.     

Developmental regulation of calmodulin gene expression in rat brain and skeletal muscle.

Three different calmodulin genes that encode the identical protein have been identified in the rat (Nojima, 1989); however, calmodulin gene expression at the various stages of tissue differentiation and maturation has not been previously determined. We have quantitated the content of mRNAs encoding calmodulin in the developing brain and skeletal muscle using RNA blot analysis with three specific cDNA probes. Our results show that five species of calmodulin mRNAs: 4.0 and 1.7 kb for CaM I, 1.4 kb for CaM II, and 2.3 and 0.8 kb for CaM III are detectable at all ages in the brain as well as in skeletal muscle but exhibit a tissue-specific developmental pattern of expression. The comparison of the temporal pattern of calmodulin gene expression with both mitotic activity, as demonstrated by cyclin A mRNA levels, and differentiation and maturation of specific brain or muscle regions is consistent with calmodulin involvement in development.     

Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter

Protein kinase D (PKD) regulates many diverse cellular functions in response to diacylglycerol. To monitor PKD signaling in live cells, we generated a genetically encoded fluorescent reporter for PKD activity, DKAR (D kinase activity reporter). DKAR expressed in mammalian cells undergoes reversible fluorescence resonance energy transfer changes upon activation and inhibition of endogenous PKD. Surprisingly, we find that agonist-evoked activation of PKD is driven not only by diacylglycerol production, but by Ca(2+). Furthermore, elevation of intracellular Ca(2+), in the absence of any other stimulus, is sufficient to activate PKD. Concurrent imaging of Ca(2+), diacylglycerol, and PKD activity reveals that thapsigargin-mediated elevation of intracellular Ca(2+) is closely followed by a robust increase in diacylglycerol production, in turn followed by PKD activation. The Ca(2+)-induced production of diacylglycerol and accompanying PKD activation is dependent on phospholipase C activity. These data reveal that Ca(2+) is a major contributor to the initiation of PKD signaling through positive feedback regulation of diacylglycerol production, unveiling a new mechanism in PKD activation.     

Mining frequent patterns for AMP-activated protein kinase regulation on skeletal muscle

Background  

AMP-activated protein kinase (AMPK) has emerged as a significant signaling intermediary that regulates metabolisms in response to energy demand and supply. An investigation into the degree of activation and deactivation of AMPK subunits under exercise can provide valuable data for understanding AMPK. In particular, the effect of AMPK on muscle cellular energy status makes this protein a promising pharmacological target for disease treatment. As more AMPK regulation data are accumulated, data mining techniques can play an important role in identifying frequent patterns in the data. Association rule mining, which is commonly used in market basket analysis, can be applied to AMPK regulation.     

Thalidomide induces apoptosis in human monocytes by using a cytochrome c-dependent pathway

Thalidomide has been shown to be an effective treatment in various immunologic diseases such as Crohn's disease and rheumatoid arthritis. Its major effect is thought to be mediated by the inhibition of TNF-alpha, but the exact mechanism of action is still uncertain. Recent observations could demonstrate that the induction of monocyte apoptosis is a common feature of a variety of anti-inflammatory agents. Therefore, we investigated the role of thalidomide on monocyte apoptosis. Treatment with thalidomide resulted in apoptosis of human peripheral blood monocytes in a time- and dose-dependent manner as demonstrated by annexin V staining. Monocyte apoptosis required the activation of caspases, as combined stimulation by thalidomide together with the broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone markedly prevented monocyte cell death. Apoptosis was triggered by a CD95/CD95 ligand, TNF-RI, and TRAIL-R1 independent pathway with an inhibition of AKT-1 kinase and consecutive mitochondrial release of cytochrome c, followed by the proteolytic activation of initiator caspase-9 and effector caspase-3. Our data suggest that thalidomide-induced monocyte apoptosis is at least partially mediated by a mitochondrial signaling pathway and might contribute to the complex immunomodulatory properties of the drug.     

Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of creatine kinase in skeletal muscle.

The AMP-activated protein kinase (AMPK) is activated by a fall in the ATP:AMP ratio within the cell in response to metabolic stresses. Once activated, it phosphorylates and inhibits key enzymes in energy-consuming biosynthetic pathways, thereby conserving cellular ATP. The creatine kinase-phosphocreatine system plays a key role in the control of ATP levels in tissues that have a high and rapidly fluctuating energy requirement. In this study, we provide direct evidence that these two energy-regulating systems are linked in skeletal muscle. We show that the AMPK inhibits creatine kinase by phosphorylation in vitro and in differentiated muscle cells. AMPK is itself regulated by a novel mechanism involving phosphocreatine, creatine and pH. Our findings provide an explanation for the high expression, yet apparently low activity, of AMPK in skeletal muscle, and reveal a potential mechanism for the co-ordinated regulation of energy metabolism in this tissue. Previous evidence suggests that AMPK activates fatty acid oxidation, which provides a source of ATP, following continued muscle contraction. The novel regulation of AMPK described here provides a mechanism by which energy supply can meet energy demand following the utilization of the immediate energy reserve provided by the creatine kinase-phosphocreatine system.     

5'-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gamma(3)) AMPK subunits and exercise-induced AMPK activity are influenced by exercise training status, muscle biopsies were obtained from seven endurance exercise-trained and seven sedentary young healthy men. The alpha(1)- and alpha(2)-AMPK mRNA contents in trained subjects were both 117 +/- 2% of that in sedentary subjects (not significant), whereas mRNA for gamma(3) was 61 +/- 1% of that in sedentary subjects (not significant). The level of alpha(1)-AMPK protein in trained subjects was 185 +/- 34% of that in sedentary subjects (P < 0.05), whereas the levels of the remaining subunits (alpha(2), beta(1), beta(2), gamma(1), gamma(2), gamma(3)) were similar in trained and sedentary subjects. At the end of 20 min of cycle exercise at 80% of peak O(2) uptake, the increase in phosphorylation of alpha-AMPK (Thr(172)) was blunted in the trained group (138 +/- 38% above rest) compared with the sedentary group (353 +/- 63% above rest) (P < 0.05). Acetyl CoA-carboxylase beta-phosphorylation (Ser(221)), which is a marker for in vivo AMPK activity, was increased by exercise in both groups but to a lower level in trained subjects (32 +/- 5 arbitrary units) than in sedentary controls (45 +/- 1 arbitrary units) (P < 0.01). In conclusion, trained human skeletal muscle has increased alpha(1)-AMPK protein levels and blunted AMPK activation during exercise.     

Flux control of cytochrome c oxidase in human skeletal muscle

In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the flux control coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 +/- 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 +/- 0.2-fold excess capacity of COX was determined. In agreement with these findings, the flux control of COX increased, in the presence of the three substrates, from 0.27 +/- 0.03 to 0.36 +/- 0.08. These results indicate a tight in vivo control of respiration by COX in human skeletal muscle. This tight control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased flux control coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme.     

Identification of a eukaryotic-like protein kinase gene in Archaebacteria.

Primary sequence patterns based on known conserved sites in eukaryotic protein kinases were used to search for eukaryotic-like protein kinase sequences in a six-frame translation of the bacterial subsection of GenBank. This search identified a previously unrecognized eukaryotic-like protein kinase gene in three related methanogenic archaebacteria, Methanococcus vannielii, M. voltae, and M. thermolithotrophicus. The proposed coding sequences are located in orthologous open reading frames (ORFs): ORF547, ORF294, and ORF114, respectively. The C-terminus of the ORFs contains 9 of the 11 subdomains characteristically conserved within the eukaryotic protein kinase catalytic domain. The N-terminus of the ORFs is similar to a putative glycoprotease in Pasteurella haemolytica and its homologue in Escherichia coli, the orfX gene. This is the first report of a eukaryotic-like protein kinase sequence observed in Archaebacteria.     

Neural regulation of parvalbumin expression in mammalian skeletal muscle.

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.     

An alternative non-tyrosine protein kinase product of the c-src gene in chicken skeletal muscle.

While the c-src locus is expressed as a 4.0-kilobase (kb) mRNA coding for pp60c-src in various chicken tissues, including embryonic muscle, it is expressed as a novel 3.0-kb mRNA in adult skeletal muscle. We have analyzed the primary structure of this alternatively transcribed and spliced c-src mRNA. The sequence revealed three open reading frames, with the previously defined c-src exons 1 through 5 or 6 comprising the third, on the 3' untranslated region of this 3-kb mRNA. The exons coding for the tyrosine kinase domain of pp60c-src were excluded. On the 5' side, 2 kb of sequence upstream from the previously defined exon 1 of the c-src gene was included in this mRNA. The start site for the 3-kb mRNA probably lies downstream of that for the 4-kb mRNA. The first reading frame of the 3.0-kb mRNA, called sur (for src upstream region), encoded a 24-kilodalton (kDa) protein product rich in cysteine and proline residues. In vitro analysis indicated that the 24-kDa sur protein was membrane associated. Antibodies to sur protein detected in vivo a 24-kDa muscle-specific protein which was developmentally regulated and corresponded to the switch from the 4-kb to the 3-kb c-src mRNA. A striking kinetic pattern of appearance of sur protein and disappearance of pp60c-src suggests that the expression of these two proteins is inversely related.     

Identification of a high molecular weight actin-binding protein in skeletal muscle.

A large polypeptide having a molecular weight of 240,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate has been identified in whole cell homogenates from chick skeletal muscle myoblasts and the rat myoblast L6 cell line. A similar polypeptide was identified in both thigh and breast chicken skeletal muscle, but the latter contained less of this protein per g of tissue. Antibodies made to gizzard filamin (an actin-binding protein having a molecular weight of 240,000) cross-reacted with the partially purified Mr = 240,000 protein from chicken skeletal muscle. With use of the indirect immunofluorescence technique, the filamin antibody localized in the Z-line region of chicken skeletal muscle myofibrils. These results indicate that skeletal muscle contains a filamin-like protein that may form an integral part of the myofibril structure.     

Regulation of contractile protein gene expression in unloaded mouse skeletal muscle

Hindlimb unloading was performed on mice in an effort to study the regulation of contractile protein genes. In particular, the regulation of myosin heavy chain IIb was examined. During unloading, muscle fibers undergo a type conversion. Preliminary data from this study does not support the hypothesis that the fiber type conversion is due to an increase in promoter activity of fast isoform genes, such as myosin heavy chain IIb. The consequences of this finding are examined, with particular focus on other factors controlling gene regulation.