The endocannabinoid system:Overview of an emerging multi-faceted therapeutic target

The endocannabinoids anandamide (AEA) and 2-arachidonoylglyerol (2-AG) are endogenous lipid mediators that exert protective roles in pathophysiological conditions, including cardiovascular diseases. In this brief review, we provide a conceptual framework linking endocannabinoid signaling to the control of the cellular and molecular hallmarks, and categorize the key components of endocannabinoid signaling that may serve as targets for novel therapeutics. The emerging picture not only reinforces endocannabinoids as potent regulators of cellular metabolism but also reveals that endocannabinoid signaling is mechanistically more complex and diverse than originally thought

Co-expression of LKB1, MO25α and STRADα in bacteria yield the functional and active heterotrimeric complex

The tumour suppressor LKB1 plays a critical role in cell proliferation, polarity and energy metabolism. LKB1 is a Ser/Thr protein kinase that is associated with STRAD and MO25 invivo. Here, we describe the individual expression of the three components of the LKB1 complex using monocistronic vectors and their co-expression using tricistronic vectors that were constructed from monocistronic vectors using a fully modular cloning approach. The data show that among the three individually expressed components of the LKB1 complex, only MO25α can be expressed in soluble form, whereas the other two, LKB1 and STRADα are found almost exclusively in inclusion bodies. However, using the tricistronic vector system, functional LKB1-MO25α-STRADα complex was expressed and purified from soluble extracts by sequential immobilized-metal affinity and heparin chromatography, as shown by Western blotting using specific antibodies. In size exclusion chromatography, MO25α and STRADα exactly co-elute with LKB1 with an apparent molecular weight of the heterotrimeric complex of 160kDa. The specific activity in the peak fraction of the size exclusion chromatography was 250U/mg at approximately 25% purity. As shown by autoradiography, LKB1 and STRADα, both strongly autophosphorylate in vitro. Moreover, recombinant LKB1 complex activates AMPK by phosphorylation of the α-subunit at the Thr-172 site as shown (i) by Western blotting using phospho-specific antibodies after LKB1-dependent phosphorylation, (ii) by LKB1-dependent incorporation of radioactive phosphate into the α-subunit of kinase dead AMPK heterotrimer, and (iii) by activity determination of AMPK. Functional mammalian LKB1 complex is constitutively active, and when enriched from bacteria should prove to be a valuable tool for studying its molecular function and regulatio

Phosphocreatine interacts with phospholipids, affects membrane properties and exerts membrane-protective effects

A broad spectrum of beneficial effects has been ascribed to creatine (Cr), phosphocreatine (PCr) and their cyclic analogues cyclo-(cCr) and phospho-cyclocreatine (PcCr). Cr is widely used as nutritional supplement in sports and increasingly also as adjuvant treatment for pathologies such as myopathies and a plethora of neurodegenerative diseases. Additionally, Cr and its cyclic analogues have been proposed for anti-cancer treatment. The mechanisms involved in these pleiotropic effects are still controversial and far from being understood. The reversible conversion of Cr and ATP into PCr and ADP by creatine kinase, generating highly diffusible PCr energy reserves, is certainly an important element. However, some protective effects of Cr and analogues cannot be satisfactorily explained solely by effects on the cellular energy state. Here we used mainly liposome model systems to provide evidence for interaction of PCr and PcCr with different zwitterionic phospholipids by applying four independent, complementary biochemical and biophysical assays: (i) chemical binding assay, (ii) surface plasmon resonance spectroscopy (SPR), (iii) solid-state (31)P-NMR, and (iv) differential scanning calorimetry (DSC). SPR revealed low affinity PCr/phospholipid interaction that additionally induced changes in liposome shape as indicated by NMR and SPR. Additionally, DSC revealed evidence for membrane packing effects by PCr, as seen by altered lipid phase transition. Finally, PCr efficiently protected against membrane permeabilization in two different model systems: liposome-permeabilization by the membrane-active peptide melittin, and erythrocyte hemolysis by the oxidative drug doxorubicin, hypoosmotic stress or the mild detergent saponin. These findings suggest a new molecular basis for non-energy related functions of PCr and its cyclic analogue. PCr/phospholipid interaction and alteration of membrane structure may not only protect cellular membranes against various insults, but could have more general implications for many physiological membrane-related functions that are relevant for health and disease

The interaction between AMPK beta 2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content

11 páginas, 7 figuras.AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at threonine-148 (Thr-148), which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. Here, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid analyses and co-immunoprecipitation of the overexpressed proteins in HEK293T cells revealed that both AMPKβ1 and β2 wild-type (WT) isoforms bind to R6. The AMPKβ/R6 interaction was stronger with the muscle-specific β2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced AMPKβ2/R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous to increased AMPKβ2 Thr-148 autophosphorylation. Such model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle.DN is recipient of a VIDI-Innovational Research Grant from the Netherlands Organization of Scientific Research (NWO-ALW Grant no. 864.10.007). This work has further been supported by grants from the Spanish Ministry of Education and Science SAF2014-54604-C3-1-R and a grant from Generalitat Valenciana (PrometeoII/2014/029) to PS.Peer reviewe

Palmitate-Induced Vacuolar-Type H(+)-ATPase Inhibition Feeds Forward Into Insulin Resistance and Contractile Dysfunction

Dietary fat overconsumption leads to myocardial lipid accumulation through mechanisms that are incompletely resolved. Previously, we identified increased translocation of the fatty acid transporter CD36 from its endosomal storage compartment to the sarcolemma as the primary mechanism of excessive myocellular lipid import. Here, we show that increased CD36 translocation is caused by alkalinization of endosomes resulting from inhibition of proton pumping activity of vacuolar-type H+-ATPase (v-ATPase). Endosomal alkalinization was observed in hearts from rats fed a lard-based high-fat diet and in rodent and human cardiomyocytes upon palmitate overexposure, and appeared as an early lipid-induced event preceding the onset of insulin resistance. Either genetic or pharmacological inhibition of v-ATPase in cardiomyocytes exposed to low palmitate concentrations reduced insulin sensitivity and cardiomyocyte contractility, which was rescued by CD36 silencing. The mechanism of palmitate-induced v-ATPase inhibition involved its dissociation into two parts: the cytosolic V-1 and the integral membrane V-0 subcomplex. Interestingly, oleate also inhibits v-ATPase function, yielding triacylglycerol accumulation but not insulin resistance. In conclusion, lipid oversupply increases CD36-mediated lipid uptake that directly impairs v-ATPase function. This feeds forward to enhanced CD36 translocation and further increased lipid uptake. In the case of palmitate, its accelerated uptake ultimately precipitates into cardiac insulin resistance and contractile dysfunction

Is TAK1 a Direct Upstream Kinase of AMPK?

Alongside Liver kinase B1 (LKB1) and Ca2+/Calmodulin-dependent protein kinase kinase 2 (CaMKK2), Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) has been suggested as a direct upstream kinase of AMP-activated protein kinase (AMPK). Several subsequent studies have reported on the TAK1-AMPK relationship, but the interpretation of the respective data has led to conflicting views. Therefore, to date the acceptance of TAK1 as a genuine AMPK kinase is lagging behind. This review provides with argumentation, whether or not TAK1 functions as a direct upstream kinase of AMPK. Several specific open questions that may have precluded the consensus are discussed based on available data. In brief, TAK1 can function as direct AMPK upstream kinase in specific contexts and in response to a subset of TAK1 activating stimuli. Further research is needed to define the intricate signals that are conditional for TAK1 to phosphorylate and activate AMPKα at T172