AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.

The pH dependence of proton uptake and electron transfers during the reaction between fully reduced cytochrome c oxidase and oxygen has been studied using the flow-flash method. Proton uptake was monitored using different pH indicators. We have also investigated the effect of D2O on the electron-transfer reactions. Proton uptake was biphasic throughout the pH range studied (6.3-9.3), and the decrease of the observed rate constants at increasing pH could be described by titration curves with pKa values of 8-8.5. Of the four phases resolved in the redox reaction, the rate constants for the first two were independent of pH, whereas that of the third decreased at increasing pH with a pKa of 7.9. All phases except the first were slower in D2O than in H2O. The values obtained for kH/kD were 1.0 for the first phase, 1.4 for the second and third phases, and 2.5 for the fourth phase. We suggest from these results that the fast phase of proton uptake is initiated by the second phase of the redox reaction and that this step includes a partially rate-limiting internal proton transfer. The third and fourth phases of the redox reaction are suggested to be rate limited by proton uptake from the medium. The pH dependencies of the proton uptake reactions are consistent with the participation of a titrable group in the protein in proton transfer from the medium to the oxygen-binding site.

Highlights d Mature adipocytes cultured as MAAC preserve cellular identity and function d Subcutaneous and visceral adipocytes can be cultured as MAAC d Adipocytes from lean and obese donors can be cultured d Human mature white adipocytes can transdifferentiate into brown-like adipocytes

We describe multiwell assays for detecting the accumulation as well as the subsequent oxidation of 14

Changes in pH during the reactions of the fully reduced and mixed-valence cytochrome oxidase with molecular oxygen have been followed in flow-flash experiments, using the pH indicator phenol red. Solubilized enzyme as well as enzyme reconstituted into phospholipid vesicles has been studied. With the solubilized enzyme, a biphasic uptake of one proton from the medium was observed, whereas the reconstituted enzyme gave release of 1.3 protons to the extravesicular medium. It is concluded from these results that a total of two to three protons are taken up during oxidation of the fully reduced enzyme. Kinetic analysis suggests that the proton uptake is initiated by the transfer of the third electron to the oxygen binding site. A reaction scheme that integrates proton transfers and oxygen chemistry is presented.

AMPK (AMP-activated protein kinase) is an attractive therapeutic drug target for treating metabolic disorders. We studied the effects of an AMPK activator developed by Merck (ex229 from patent application WO2010036613), comparing chemical activation with contraction in intact incubated skeletal muscles. We also compared effects of ex229 with those of the Abbott A769662 compound and AICAR (5-amino-4-imidazolecarboxamide riboside). In rat epitrochlearis muscle, ex229 dose-dependently increased AMPK activity of α1-, α2-, β1- and β2-containing complexes with significant increases in AMPK activity seen at a concentration of 50 μM. At a concentration of 100 μM, AMPK activation was similar to that observed after contraction and importantly led to an ~2-fold increase in glucose uptake. In AMPK α1-/α2-catalytic subunit double-knockout myotubes incubated with ex229, the increases in glucose uptake and ACC (acetyl-CoA carboxylase) phosphorylation seen in control cells were completely abolished, suggesting that the effects of the compound were AMPK-dependent. When muscle glycogen levels were reduced by ~50% after starvation, ex229-induced AMPK activation and glucose uptake were amplified in a wortmannin-independent manner. In L6 myotubes incubated with ex229, fatty acid oxidation was increased. Furthermore, in mouse EDL (extensor digitorum longus) and soleus muscles, ex229 increased both AMPK activity and glucose uptake at least 2-fold. In summary, ex229 efficiently activated skeletal muscle AMPK and elicited metabolic effects in muscle appropriate for treating Type 2 diabetes by stimulating glucose uptake and increasing fatty acid oxidation.

Absorbance changes following CO dissociation by flash photolysis from mixed-valence cytochrome oxidase have been followed in the Soret and alpha regions. Apart from CO dissociation and recombination, three kinetic phases with rate constants in the range 10(5)-10(3) s-1 at pH 7.5 can be resolved in both spectral regions. The slowest one of these phases, which had earlier only been observed in the alpha region, has now been detected in the Soret region by the use of a low CO concentration to slow down the recombination reaction. This phase had been assigned to a structural change, but a kinetic difference spectrum demonstrates that it represents electron transfer from cytochrome a3 to cytochrome a. A kinetic deuterium isotope effect of 2-3 at pH 7.5 suggests that it involves proton transfer as well. The temperature dependence of the reaction gives an Arrhenius activation energy of 42 kJ.mol-1. The reaction is faster at low pH, and the equilibrium is shifted toward cytochrome a as the pH is raised. The rate and equilibrium changes can be described as involving acid-base groups with pKa values of approximately 7.7 and 8.7, respectively. The kinetic results can be simulated on the basis of a model in which one acid-base group interacts with cytochrome a3, so that its pKa drops on oxidation of this center. The group is in proton equilibrium with the solvent via a proton pathway, suggested to be a proton channel. The rate of a shift in the redox equilibrium between the two cytochromes reaches a high limit at low pH, where the channel is saturated with protons.(ABSTRACT TRUNCATED AT 250 WORDS)

Heat-producing beige/brite (brown-in-white) adipocytes in white adipose tissue have the potential to suppress metabolic disease in mice and hold great promise for the treatment of obesity and type 2 diabetes in humans. Here, we demonstrate that human adipose-derived stromal/progenitor cells (hASCs) from subcutaneous white adipose tissue can be efficiently converted into beige adipocytes. Upon pharmacological activation of peroxisome proliferator-activated receptor-γ, hASC-derived adipocytes activated beige fat-selective genes and a brown/beige fat-selective electron transport chain gene program. Importantly, hASC-derived beige fat cells displayed the bioenergetic characteristics of genuine brown fat cells, including a capacity for increased respiratory uncoupling in response to β-adrenergic agonists. Furthermore, knock-down experiments reveal that the thermogenic capacity of human beige fat cells was entirely dependent on the presence of Uncoupling protein 1. In summary, this study reveals that hASCs can be readily differentiated into beige adipocytes that, upon activation, undergo uncoupling protein 1-dependent thermogenesis.