Lysine suppresses protein degradation through autophagic–lysosomal system in C2C12 myotubes

Muscle mass is determined between protein synthesis and protein degradation. Reduction of muscle mass leads to bedridden condition and attenuation of resistance to diseases. Moreover, bedridden condition leads to additional muscle loss due to disuse muscle atrophy. In our previous study (Sato et al. 2013), we showed that administered lysine (Lys), one of essential amino acid, suppressed protein degradation in skeletal muscle. In this study, we investigated that the mechanism of the suppressive effects of Lys on skeletal muscle proteolysis in C2C12 cell line. C2C12 myotubes were incubated in the serum-free medium containing 10 mM Lys or 20 mM Lys, and myofibrillar protein degradation was determined by the rates of 3-methylhistidine (MeHis) release from the cells. The mammalian target of rapamycin (mTOR) activity from the phosphorylation levels of p70-ribosormal protein S6 kinase 1 and eIF4E-binding protein 1 and the autophagic–lysosomal system activity from the ratio of LC3-II/I in C2C12 myotubes stimulated by 10 mM Lys for 0–3 h were measured. The rates of MeHis release were markedly reduced by addition of Lys. The autophagic–lysosomal system activity was inhibited upon 30 min of Lys supplementation. The activity of mTOR was significantly increased upon 30 min of Lys supplementation. The suppressive effect of Lys on the proteolysis by the autophagic–lysosomal system was maintained partially when mTOR activity was inhibited by 100 nM rapamycin, suggesting that some regulator other than mTOR signaling, for example, Akt, might also suppress the autophagic–lysosomal system. From these results, we suggested that Lys suppressed the activity of the autophagic–lysosomal system in part through activation of mTOR and reduced myofibrillar protein degradation in C2C12 myotubes.     

Neuromodulatory State and Sex Specify Alternative Behaviors through Antagonistic Synaptic Pathways in C. elegans

Pheromone responses are highly context dependent. For example, the C.?elegans pheromone ascaroside C9 (ascr#3) is repulsive to wild-type hermaphrodites, attractive to wild-type males, and usually neutral to "social" hermaphrodites with reduced activity of the npr-1 neuropeptide receptor gene. We show here that these distinct behavioral responses arise from overlapping push-pull circuits driven by two classes of pheromone-sensing neurons. The ADL sensory neurons detect C9 and, in?wild-type hermaphrodites, drive C9 repulsion through their chemical synapses. In npr-1 mutant hermaphrodites, C9 repulsion is reduced by the recruitment of a gap junction circuit that antagonizes ADL chemical synapses. In males, ADL sensory responses are diminished; in addition, a second pheromone-sensing neuron, ASK, antagonizes C9 repulsion. The additive effects of these antagonistic circuit elements generate attractive, repulsive, or neutral pheromone responses. Neuronal modulation by circuit state and sex, and flexibility in synaptic output pathways, may permit small circuits to maximize their adaptive behavioral outputs.     

Exercise Protects against Diet-Induced Insulin Resistance through Downregulation of Protein Kinase Cβ in Mice

Physical exercise is an important and effective therapy for diabetes. However, its underlying mechanism is not fully understood. Protein kinase Cβ (PKCβ) has been suggested to be involved in the pathogenesis of obesity and insulin resistance, but the role of PKCβ in exercise-induced improvements in insulin resistance is completely unknown. In this study, we evaluated the involvement of PKCβ in exercise-attenuated insulin resistance in high-fat diet (HFD)-fed mice. PKCβ^-/- and wild-type mice were fed a HFD with or without exercise training. PKC protein expression, body and tissue weight change, glucose and insulin tolerance, metabolic rate, mitochondria size and number, adipose inflammation, and AKT activation were determined to evaluate insulin sensitivity and metabolic changes after intervention. PKCβ expression decreased in both skeletal muscle and liver tissue after exercise. Exercise and PKCβ deficiency can alleviate HFD-induced insulin resistance, as evidenced by improved insulin tolerance. In addition, fat accumulation and mitochondrial dysfunction induced by HFD were also ameliorated by both exercise and PKCβ deficiency. On the other hand, exercise had little effect on PKCβ^-/- mice. Further, our data indicated improved activation of AKT, the downstream signal molecule of insulin, in skeletal muscle and liver of exercised mice, whereas PKCβ deficiency blunted the difference between sedentary and exercised mice. These results suggest that downregulation of PKCβ contributes to exercise-induced improvement of insulin resistance in HFD-fed mice.     

Sensory regulation of the C. elegans germline through TGF-β-dependent signaling in the niche

The proliferation/differentiation balance of stem and progenitor cell populations must respond to the physiological needs of the organism [1, 2]. Mechanisms underlying this plasticity are not well understood. The C. elegans germline provides a tractable system to study the influence of the environment on progenitor cells (stem cells and their proliferative progeny). Germline progenitors accumulate during larval stages to form an adult pool from which gametes are produced. Notch pathway signaling from the distal tip cell (DTC) niche to the germline maintains the progenitor pool [3-5], and the larval germline cell cycle is boosted by insulin/IGF-like receptor signaling [6]. Here we show that, independent of its role in the dauer decision, TGF-β regulates the balance of proliferation versus differentiation in the C. elegans germline in response to sensory cues that report population density and food abundance. Ciliated ASI sensory neurons are required for TGF-β-mediated expansion of the larval germline progenitor pool, and the TGF-β receptor pathway acts in the germline stem cell niche. TGF-β signaling thereby couples germline development to the quality of the environment, providing a novel cellular and molecular mechanism linking sensory experience of the environment to reproduction.     

TMC Proteins Modulate Egg Laying and Membrane Excitability through a Background Leak Conductance in C. elegans

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N − 3PUFA differentially modulate palmitate-induced lipotoxicity through alterations of its metabolism in C2C12 muscle cells

Excessive energy intake leads to fat overload and the formation of lipotoxic compounds mainly derived from the saturated fatty acid palmitate (PAL), thus promoting insulin resistance (IR) in skeletal muscle. N − 3 polyunsaturated fatty acids (n − 3PUFA) may prevent lipotoxicity and IR. The purpose of this study was to examine the differential effects of n − 3PUFA on fatty acid metabolism and insulin sensitivity in muscle cells. C2C12 myotubes were treated with 500 μM of PAL without or with 50 μM of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for 16 h. PAL decreased insulin-dependent AKT activation and glucose uptake and increased the synthesis of ceramides and diglycerides (DG) derivatives, leading to protein kinase Cθ activation. EPA and DHA, but not ALA, prevented PAL-decreased AKT activation but glucose uptake was restored to control values by all n − 3PUFA vs. PAL. Total DG and ceramide contents were decreased by all n − 3PUFA, but only EPA and DHA increased PAL β-oxidation, decreased PAL incorporation into DG and reduced protein kinase Cθ activation. EPA and DHA emerge as better candidates than ALA to improve fatty acid metabolism in skeletal muscle cells, notably via their ability to increase mitochondrial β-oxidation.     

Methyl-β-cyclodextrin restores impaired autophagy flux in Niemann-Pick C1-deficient cells through activation of AMPK

The drug 2-hydroxypropyl-β-cyclodextrin (HPβCD) reduces lysosomal cholesterol accumulation in Niemann-Pick disease, type C (NPC) and has been advanced to human clinical trials. However, its mechanism of action for reducing cholesterol accumulation in NPC cells is uncertain and its molecular target is unknown. We found that methyl-β-cyclodextrin (MβCD), a potent analog of HPβCD, restored impaired macroautophagy/autophagy flux in Niemann-Pick disease, type C1 (NPC1) cells. This effect was mediated by a direct activation of AMP-activated protein kinase (AMPK), an upstream kinase in the autophagy pathway, through MβCD binding to its β-subunits. Knockdown of PRKAB1 or PRKAB2 (encoding the AMPK β1 or β2 subunit) expression and an AMPK inhibitor abolished MβCD-mediated reduction of cholesterol storage in NPC1 cells. The results demonstrate that AMPK is the molecular target of MβCD and its activation enhances autophagy flux, thereby mitigating cholesterol accumulation in NPC1 cells. The results identify AMPK as an attractive target for drug development to treat NPC.     

Neurotrophins Induce Neuregulin Release through Protein Kinase C�� Activation

Proper, graded communication between different cell types is essential for normal development and function. In the nervous system, heart, and for some cancer cells, part of this communication requires signaling by soluble and membrane-bound factors produced by the NRG1 gene. We have previously shown that glial-derived neurotrophic factors activate a rapid, localized release of soluble neuregulin from neuronal axons that can, in turn promote proper axoglial development (Esper, R. M., and Loeb, J. A. (2004) J. Neurosci. 24, 6218–6227). Here we elucidate the mechanism of this localized, regulated release by implicating the delta isoform of protein kinase C (PKC). Blocking the PKC delta isoform with either rottlerin, a selective antagonist, or small interference RNA blocks the regulated release of neuregulin from both transfected cells and primary neuronal cultures. PKC activation also leads to the rapid phosphorylation of the pro-NRG1 cytoplasmic tail on serine residues adjacent to the membrane-spanning segment, that, when mutated markedly reduce the rate of NRG1 activity release. These findings implicate this specific PKC isoform as an important factor for the cleavage and neurotrophin-regulated release of soluble NRG1 forms that have important effects in nervous system development and disease.The neuregulins (NRGs)² are a family of growth and differentiation factors with a broad range of functions during development and in the adult. NRGs are necessary for glial and cardiac development and participate in a wide range of biologic processes ranging from proper formation of peripheral nerves and the neuromuscular junction to tumor growth (2–9). The NRGs have also been implicated as both potential mediators and therapeutic targets for a number of human diseases including cancer, schizophrenia, and multiple sclerosis (10–12). NRGs function as mediators of cell-to-cell communication through a multitude of alternatively spliced isoforms arising from at least four distinct genes that bind to and activate members of the epidermal growth factor receptor family HER-2/3/4 (ErbB-2/3/4) (13–19).Although all known isoforms of the NRG1 gene have an epidermal growth factor-like domain sufficient to bind to and activate its receptors (20), products of this gene are divided into three classes based on structurally and functionally different N-terminal regions (21) The type I and II forms have a unique N-terminal, heparin-binding Ig-like domain (22–26). This Ig-like domain potentiates the biological activities of soluble NRG1 forms and leads to their highly selective tissue distributions through its affinity for specific cell-surface heparan sulfates (12, 20, 27, 28). These forms are first expressed as transmembrane precursors (pro-NRG1) that undergo proteolytic cleavage to release their soluble ectodomains. The type III NRG1 forms, on the other hand, are not typically released from cells, because their N-terminal domain consists of a cysteine-rich domain that can serve as a membrane tether making this form ideal for juxtacrine signaling. This form has been strongly implicated to be important peripheral nerve myelination (29–31).While many of the biological functions of type I/II NRG1 forms are less clear, their ability to be released from axons in the peripheral and central nervous systems in a regulated manner provides the potential for long range cell-cell communication not possible from membrane-bound forms. Studies examining the regulation of type I NRG1 release from neuronal axons have implicated protein kinase C (PKC) as a mediator of NRG1 release from pro-NRG1 in transfected cell lines (32). Subsequent studies in intact neurons found that PKC activation was sufficient to release NRG1 from sensory and motor neuron axons and that NRG1 could also be released by Schwann cell-derived neurotrophic factors, such as BDNF and GDNF (1). Recently, the β-secretase protease BACE1 has been suggested to cleave these NRG1 forms so that when it is knocked out in mice, deficits similar to those seen in NRG1 knockouts are seen (33, 34). These findings suggest that reciprocal communication between NRG1s and neurotrophins could be an important mechanisms for local axoglial communication that is critical for normal peripheral nerve development. Consistently, PKC has been implicated as a key mediator for the electrically mediated release of NRG1 from cultured cerebellar granule cells and pontine nucleus neurons (35).The PKC family consists of 10 serine/threonine kinases isoforms (α, βI, βII, γ, δ, ϵ, ζ, θ, λ, and η) each with a unique cellular distribution, target specificity, mechanism of activation, and function (36). One of these functions promotes the cleavage and release of soluble signaling proteins that are initially synthesized as membrane-spanning precursors. In addition to NRG1, other proteins released upon PKC activation include epidermal growth factor, transforming growth factor-α, amyloid precursor protein, l-selectin, and interleukins (1, 37–43). We hypothesize that neurotrophic factors induce the cleavage and release of NRG1 from pro-NRG1 through PKC activation. This hypothesis seems reasonable, because neurotrophin binding to the Trk family of neurotrophin receptor tyrosine kinases, but not the low affinity neurotrophin receptor p75 (44), activates phospholipase Cγ-mediated conversion of membrane-bound phosphatidylinositol bisphosphate to inositol triphosphate and diacylglycerol, which in turn, can activate PKC (45–48). Although this can be achieved using phorbol 12-myristate 13-acetate (PMA), a diacylglycerol analog sufficient to activate most PKC isozymes (48), the exact PKC isoform and mechanism by which this occurs is not known. Here, we demonstrate NRG1 is released from cells through direct activation of the PKCδ isoform using siRNA and PKC isoform-specific inhibitors in transfected Chinese hamster ovary (CHO) cells, PC12, and primary neuronal cultures. We further demonstrate that PKC activation induces rapid phosphorylation of the cytoplasmic tail of pro-NRG1 on specific serine residues that are required for efficient NRG1 activity release. These findings provide mechanistic insights into how highly localized, reciprocal signaling occurs along neuronal axons, which has important implications for normal development and disease.     

ApoA-I enhances generation of HDL-like lipoproteins through interaction between ABCA1 and phospholipase Cγ in rat astrocytes

In the previous paper, we reported that apolipoprotein (apo) A-I enhances generation of HDL-like lipoproteins in rat astrocytes to be accompanied with both increase in tyrosine phosphorylation of phospholipase Cγ (PL-Cγ) and PL-Cγ translocation to cytosolic lipid-protein particles (CLPP) fraction. In this paper, we studied the interaction between apoA-I and ATP-binding cassette transporter A1 (ABCA1) to relate with PL-Cγ function for generation of HDL-like lipoproteins in the apoA-I-stimulated astrocytes. ABCA1 co-migrated with exogenous apoA-I with apparent molecular weight over 260kDa on SDS-PAGE when rat astrocytes were treated with apoA-I and then with a cross-linker, BS3. The solubilized ABCA1 of rat astrocytes was associated with the apoA-I-immobilized Affi-Gel 15. An LXR agonist, To901317, increased the cellular level of ABCA1, association of apoA-I with ABCA1 and apoA-I-mediated lipid release in rat astrocytoma GA-1/Mock cells where ABCA1 expression at baseline is very low. PL-Cγ was co-isolated by apoA-I-immobilized Affi-Gel 15 and co-immunoprecipitated by anti-ABCA1 antibody along with ABCA1 from the solubilized membrane fraction of rat astrocytes. The SiRNA of ABCA1 suppressed not only the PL-Cγ binding to ABCA1 but also the tyrosine phosphorylation of PL-Cγ. A PL-C inhibitor, U73122, prevented generation of apoA-I-mediated HDL-like lipoproteins in rat astrocytes. To901317 increased the association of PL-Cγ with ABCA1 in GA-1/Mock cells dependently on the increase of cellular level of ABCA1 without changing that of PL-Cγ. These findings suggest that the exogenous apoA-I augments the interaction between PL-Cγ and ABCA1 to stimulate tyrosine phosphorylation and activation of PL-Cγ for generation of HDL-like lipoproteins in astrocytes.     

Interleukin-1β Reduces L-type Ca2+ Current through Protein Kinase C? Activation in Mouse Heart

Inflammation is now widely recognized as a key component of heart disease. Patients suffering from arrhythmias and heart failure have increased levels of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β). Evidence suggests that these cytokines are important mediators of cardiac remodeling; however, their effects on ion channels and arrhythmogenesis remain incompletely understood. The L-type Ca²⁺ current (I_CaL) is a major determinant of the plateau phase of cardiac action potential and has a critical excitation-contraction coupling role. Thus, altering its properties could have detrimental effects on cardiac electrical and contractile functions. Accordingly, the objective of this study was to elucidate the effect of TNFα and IL-1β on I_CaL, while exploring the underlying regulatory mechanisms. Neonatal mouse ventricular myocytes were treated with a pathophysiological concentration (30 pg/ml) of TNFα and IL-1β for 24 h. Voltage-clamp recordings showed that TNFα had no effect on I_CaL, whereas IL-1β decreased the current density by 36%. Although both IL-1β- and TNFα-treated myocytes showed significant increase in reactive oxidative species (ROS), Western blot experiments revealed that only IL-1β increased PKCϵ membrane translocation. The antioxidant N-acetyl-l-cysteine normalized ROS levels and restored I_CaL density. Furthermore, the PKCϵ translocation inhibitor ϵ-V1-2 blocked the effect of IL-1β on I_CaL. The reduction of I_CaL by IL-1β was also seen in cultured adult ventricular myocytes. Overall, chronic IL-1β treatment decreased I_CaL density in cardiomyocytes. These effects implicated ROS signaling and PKCϵ activation. These findings could contribute to explain the role of IL-1β in the development of arrhythmia and heart failure.     

C5a/CD88 signaling alters blood-brain barrier integrity in lupus through nuclear factor-κB

Inflammation is a key factor in a number of neurodegenerative diseases including systemic lupus erythematosus. The complement system is an important mechanism in initiating and amplifying inflammation. Our recent studies demonstrate that C5a, a protein fragment generated during complement activation could alter the blood-brain barrier integrity, and thereby disturb the brain microenvironment. To understand the mechanism by which this occurs, we examined the effects of C5a on apoptosis, translocation of nuclear factor-κB (NF-κb) and the expression of Iκbα, MAPK, CREB and TJ protein, zona occludens (ZO-1) in mouse brain endothelial cells. Apoptosis was examined by DNA laddering and caspase 3 activity and the distribution of the ZO-1 and the p65 subunit of NF-κB were determined by immunofluorescence. Inhibition of CD88 reduced translocation of NF-κb into the nucleus, altered ZO-1 at the interfaces of neighboring cells, decreased caspase 3 activity and prevented apoptosis in these cells. Our results indicate that signaling through CD88 regulates the blood-brain barrier in a NF-κb-dependent manner. These studies suggest that the C5a receptor, CD88 is a promising therapeutic target that will reduce NF-κb-signaling cascades in inflammatory settings.     

Individual recognition through olfactory–auditory matching in lemurs

Individual recognition can be facilitated by creating representations of familiar individuals, whereby information from signals in multiple sensory modalities become linked. Many vertebrate species use auditory–visual matching to recognize familiar conspecifics and heterospecifics, but we currently do not know whether representations of familiar individuals incorporate information from other modalities. Ring-tailed lemurs (Lemur catta) are highly visual, but also communicate via scents and vocalizations. To investigate the role of olfactory signals in multisensory recognition, we tested whether lemurs can recognize familiar individuals through matching scents and vocalizations. We presented lemurs with female scents that were paired with the contact call either of the female whose scent was presented or of another familiar female from the same social group. When the scent and the vocalization came from the same individual versus from different individuals, females showed greater interest in the scents, and males showed greater interest in both the scents and the vocalizations, suggesting that lemurs can recognize familiar females via olfactory–auditory matching. Because identity signals in lemur scents and vocalizations are produced by different effectors and often encountered at different times (uncoupled in space and time), this matching suggests lemurs form multisensory representations through a newly recognized sensory integration underlying individual recognition.     

Over-expression of l-galactono-γ-lactone dehydrogenase increases vitamin C, total phenolics and antioxidant activity in lettuce through bio-fortification

Epidemiological studies have shown that regular consumption of fruits and vegetables is associated with reduced risk of chronic diseases. Vegetables can provide vitamins, phenolics, flavonoids, minerals and dietary fibers for optimal health benefits. However, some nutrients contained in many fruits and vegetables cannot meet of the complete nutrition need in the human body. Biotechnology has the potential to improve the nutritional value of crops. Considering the high consumption of romaine lettuce in human diet worldwide, the objective of study is to enhance the contents of vitamin C, phenolics and antioxidant activity in lettuce leaves by genetic engineering techniques. The gene expression level, vitamin C content, total phenolics, as well as total and cellular antioxidant activities were analyzed by real-time PCR, HPLC, Folin–Ciocalteu, Hydro-PSC and CAA methods, respectively. The bio-fortification of genetically engineered lettuce increased vitamin C up to 48.94 ± 1.34 mg/100 g FW following the increased over-expression of _At GLDH. This is almost a 3.2-fold increase as the content when compared with wild type lettuce (p < 0.05). In addition, phenolic compounds in transgenic lettuce contained 120.4 ± 1.62 mg GA equiv./100 g FW, almost double the phenolic content of the wild type. Total antioxidant activities were 735.4 ± 47.7 μmol vitamin C equiv./100 g FW, cellular antioxidant activities were 7.33 ± 0.86 μmol quercetin equiv./100 g FW (PBS wash) and 18.14 ± 0.68 μmol quercetin equiv./100 g FW (No PBS wash) in transgenic lettuce, respectively, 1.5, 4 and twofold increases when compared with the wild type. This study suggests that bio-fortification by genetic engineering has great potential to improve vitamin C, phenolic contents and antioxidant activity in lettuce.     

The Small G Protein Rac1 Activates Phospholipase Cδ1 through Phospholipase Cβ2

Rac1, which is associated with cytoskeletal pathways, can activate phospholipase Cβ2 (PLCβ2) to increase intracellular Ca²⁺ levels. This increased Ca²⁺ can in turn activate the very robust PLCδ1 to synergize Ca²⁺ signals. We have previously found that PLCβ2 will bind to and inhibit PLCδ1 in solution by an unknown mechanism and that PLCβ2·PLCδ1 complexes can be disrupted by Gβγ subunits. However, because the major populations of PLCβ2 and PLCδ1 are cytosolic, their regulation by Gβγ subunits is not clear. Here, we have found that the pleckstrin homology (PH) domains of PLCβ2 and PLCβ3 are the regions that result in PLCδ1 binding and inhibition. In cells, PLCβ2·PLCδ1 form complexes as seen by Förster resonance energy transfer and co-immunoprecipitation, and microinjection of PHβ2 dissociates the complex. Using PHβ2 as a tool to assess the contribution of PLCβ inhibition of PLCδ1 to Ca²⁺ release, we found that, although PHβ2 only results in a 25% inhibition of PLCδ1 in solution, in cells the presence of PHβ2 appears to eliminates Ca²⁺ release suggesting a large threshold effect. We found that the small plasma membrane population of PLCβ2·PLCδ1 is disrupted by activation of heterotrimeric G proteins, and that the major cytosolic population of the complexes are disrupted by Rac1 activation. Thus, the activity of PLCδ1 is controlled by the amount of bound PLCβ2 that changes with displacement of the enzyme by heterotrimeric or small G proteins. Through PLCβ2, PLCδ1 activation is linked to surface receptors as well as signals that mediate cytoskeletal pathways.