NAT8L (N-Acetyltransferase 8-Like) Accelerates Lipid Turnover and Increases Energy Expenditure in Brown Adipocytes

NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes.     

Association/Dissociation of the Nucleotide-binding Domains of the ATP-binding Cassette Protein MsbA Measured during Continuous Hydrolysis

In ATP-binding cassette proteins, the two nucleotide-binding domains (NBDs) work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. It is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or partial opening of the dimers such that the NBDs remain in contact during the hydrolysis cycle. We studied the bacterial lipid flippase MsbA by luminescence resonance energy transfer (LRET). The LRET signal between optical probes reacted with single-cysteine mutants was employed to follow NBD association/dissociation in real time. The intermonomer distances calculated from LRET data indicate that the NBDs separate completely following ATP hydrolysis, even in the presence of mm MgATP, and that the dissociation occurs following each hydrolysis cycle. The results support association/dissociation, as opposed to constant contact models, for the mode of operation of ATP-binding cassette proteins.     

Role of AKAP79/150 Protein in β1-Adrenergic Receptor Trafficking and Signaling in Mammalian Cells

Protein kinase A-anchoring proteins (AKAPs) participate in the formation of macromolecular signaling complexes that include protein kinases, ion channels, effector enzymes, and G-protein-coupled receptors. We examined the role of AKAP79/150 (AKAP5) in trafficking and signaling of the β₁-adrenergic receptor (β₁-AR). shRNA-mediated down-regulation of AKAP5 in HEK-293 cells inhibited the recycling of the β₁-AR. Recycling of the β₁-AR in AKAP5 knockdown cells was rescued by shRNA-resistant AKAP5. However, truncated mutants of AKAP5 with deletions in the domains involved in membrane targeting or in binding to calcineurin or PKA failed to restore the recycling of the β₁-AR, indicating that full-length AKAP5 was required. Furthermore, recycling of the β₁-AR in rat neonatal cardiac myocytes was dependent on targeting the AKAP5-PKA complex to the C-terminal tail of the β₁-AR. To analyze the role of AKAP5 more directly, recycling of the β₁-AR was determined in ventricular myocytes from AKAP5^−/− mice. In AKAP5^−/− myocytes, the agonist-internalized β₁-AR did not recycle, except when full-length AKAP5 was reintroduced. These data indicate that AKAP5 exerted specific and profound effects on β₁-AR recycling in mammalian cells. Biochemical or real time FRET-based imaging of cyclic AMP revealed that deletion of AKAP5 sensitized the cardiac β₁-AR signaling pathway to isoproterenol. Moreover, isoproterenol-mediated increase in contraction rate, surface area, or expression of β-myosin heavy chains was significantly greater in AKAP5^−/− myocytes than in AKAP5^+/+ myocytes. These results indicate a significant role for the AKAP5 scaffold in signaling and trafficking of the β₁-AR in cardiac myocytes and mammalian cells.     

Visualizing and Manipulating Focal Adhesion Kinase Regulation in Live Cells

Focal Adhesion Kinase (FAK) is essential for cell migration and plays an important role in tumor metastasis. However, the complex intermolecular and intramolecular interactions that regulate FAK activity at the focal adhesion remain unresolved. We have engineered a toolbox of FRET sensors that retain all of the individual FAK domains but modulate a key intramolecular regulatory interaction between the band 4.1/ezrin/radixin/moesin (FERM) and kinase domains of FAK. We demonstrate systematic control and quantitative measurement of the FERM-kinase interaction at focal adhesions, which in turn allows us to control cell migration. Using these sensors, we find that Tyr-397 phosphorylation, rather than kinase activity of FAK, is the key determinant of cell migration. Our sensors directly demonstrate, for the first time, a pH-dependent change in a protein-protein interaction at a macromolecular structure in live cells. The FERM-kinase interaction at focal adhesions is enhanced at acidic pH, with a concomitant decrease in Tyr-397 phosphorylation, providing a potential mechanism for enhanced migration of cancer cells.     

Seasonal variations in the energy budget of Elliot’s pheasant (Syrmaticus ellioti) in cage

This study aimed to discuss the energy budget of Elliot’s pheasant Syrmaticus ellioti in different seasons, with life and health, good growth and normal digestion of Elliot’s pheasant as the tested objects, The energy budget of Elliot’s pheasant was measured by daily collection of the trial pheasants’ excrement in the biological garden of Guangxi Normal University from March 2011 to February 2012. The results showed that the gross energy consumption, metabolic energy and excrement energy varied by season, increasing as temperature decreased. There was significant difference in gross energy consumption, metabolic energy, excrement energy between adults and nonages. There was also a trend that food digestibility of pheasants increases as temperature increases. In the same season, the food digestibility of adults was better than that of nonages. Throughout spring, summer, autumn and winter, the metabolic energy of 4-year adults were 305.77±13.40 kJ/d, 263.67±11.89 kJ/d, 357.23±25.49 kJ/d and 403.12±24.91 kJ/d, respectively, and the nonages were 284.86±17.22 kJ/d, 284. 66±15.16 kJ/d, 402. 26±31.46 kJ/d and 420. 30±31.98 kJ/d, respectively. The minimum metabolic energies were 247.65±21.81 g, 265.86±26.53 g, respectively for each group, detected between 4-year adults and 1-year nonages. Further study is needed to determine whether 29.6 C is the optimal temperature for the Elliot’s pheasant.     

Potential energy expenditure by litter-roosting bats associated with temperature under leaf litter during winter

In temperate portions of North America, some bats that remain active during winter undergo short periods of hibernation below leaf litter on the forest floor during episodes of below-freezing weather. These winter roosts may provide above-freezing conditions, but the thermal conditions under leaf litter are unclear. Further, little is known of the relationship between temperatures under litter and potential energy expenditure by bats. Therefore, I characterized thermal conditions below leaf litter, compared temperatures encountered under different litter depths, and evaluated the quality of these sites as hibernacula based on potential energy use by eastern red bats (Lasiurus borealis) during winter in forests of the Ouachita Mountains, Arkansas, USA. Over an averaged 24-h period, there was no significant difference in temperature among different depths of leaf litter, but temperatures under litter remained significantly warmer than air temperatures, especially during nighttime and under snow cover. Temperatures below leaf litter were significantly warmer on south-facing slopes than north-facing slopes, but predicted metabolic rates did not differ among aspects. Predicted metabolic rates of eastern red bats were lowest under the deepest leaf litter measured (8 cm) and highest under ambient air conditions. Depending on depth of leaf litter cover, predicted energy savings based on O₂ consumption from roosting under litter were 1.9 to 3.1 times greater than remaining in ambient air during periods of freezing weather and around 5.6 times greater when roosting under leaf litter with snow cover. A model for predicted total energy consumption (estimated as the total oxygen consumption during a 24-h period) by eastern red bats indicated that when roosting below leaf litter, energy consumption would be reduced with greater ground temperatures, greater leaf litter moisture, and when located on south-facing slopes. Predicted metabolic rates and total energy consumption may provide more insight on the quality of roost sites for wintering bats than temperature of roost sites alone.     

Optimising settlement tiles: The effects of surface texture and energy,orientation and deployment duration upon the fouling community

The aim of this study was to produce a set of tiles for field studies on settlement and biofouling with carefully controlled surface characteristics and practical design, and test them under field conditions. Impressions of precisely defined surface textures were made in silicone. Double sided tiles in epoxy, polyester and silanised epoxy resins were cast from the impressions. Tile characteristics tested were texture (R_a = 0, 0.19, 0.62, 1.1, 2.2 mm), surface free energy (60, 52, 24 mN m^{m 2}), and surface orientation (up, down, into, away). Tiles were deployed in the Red Sea for 4 and 7 months. Measures of community cover, dominance and richness were all significantly affected by each of the factors. The tiles proved durable and robust during the 7 month deployment with no observable changes in surface characteristics and none were lost or broken. These settlement tiles have a wide applicability for both biofouling and ecological studies. The field test demonstrated the complexities of the interactions between just four surface characteristics. This study has also underlined the need for multidimensional analysis of fouling communities for applied and basic research.     

Free Energy Simulations Over Creation/Annihilation Paths for a Flexible Solute

The inclusion of molecular flexibility into free energy simulations over creation/annihilation paths has been analyzed. A new formalism is presented for such simulations with the intramolecular degrees of freedom being active during the simulation and a recently introduced path is reviewed that allows the inclusion of the flexibility using separate simulations.     

Application of the Free Energy Calculations to Study Drug-enzyme and Drug-dna Complexes

Applications of two free energy calculation approaches are presented to study drug-biomolecule complexes. The first method, the free energy perturbation (FEP) method and molecular dynamics simulations has been applied to study the JG-365 inhibitor bound to the HIV-aspartic protease. The FEP method has been applied to predict the consequence of replacing each of the seven peptide bonds in the JG-365 by trans-ethylene or fluoroethylene units. The necessary initial conformations of the inhibitor for "in water" perturbations have been found using neural network clustering approach applied to the long molecular dynamics trajectory of the inhibitor in water solution. The second method is applied to study binding free energies of some DNA-drug complexes and is based on analysis of long molecular dynamics trajectories by continuum solvent approach (MM/PBSA).