Loss of UCP1 function augments recruitment of futile lipid cycling for thermogenesis in murine brown fat.

OBJECTIVE: Classical ATP-independent non-shivering thermogenesis enabled by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) is activated, but not essential for survival, in the cold. It has long been suspected that futile ATP-consuming substrate cycles also contribute to thermogenesis and can partially compensate for the genetic ablation of UCP1 in mouse models. Futile ATP-dependent thermogenesis could thereby enable survival in the cold even when brown fat is less abundant or missing. METHODS: In this study, we explore different potential sources of UCP1-independent thermogenesis and identify a futile ATP-consuming triglyceride/fatty acid cycle as the main contributor to cellular heat production in brown adipocytes lacking UCP1. We uncover the mechanism on a molecular level and pinpoint the key enzymes involved using pharmacological and genetic interference. RESULTS: ATGL is the most important lipase in terms of releasing fatty acids from lipid droplets, while DGAT1 accounts for the majority of fatty acid re-esterification in UCP1-ablated brown adipocytes. Furthermore, we demonstrate that chronic cold exposure causes a pronounced remodeling of adipose tissues and leads to the recruitment of lipid cycling capacity specifically in BAT of UCP1-knockout mice, possibly fueled by fatty acids from white fat. Quantification of triglyceride/fatty acid cycling clearly shows that UCP1-ablated animals significantly increase turnover rates at room temperature and below. CONCLUSION: Our results suggest an important role for futile lipid cycling in adaptive thermogenesis and total energy expenditure.

The lipidome of primary murine white, brite, and brown adipocytes-Impact of beta-adrenergic stimulation.

Lipid species patterns are conserved within cells to maintain physicochemical properties of membranes and cellular functions. We present the lipidome, including sterols, glycerolipids (GLs), glycerophospholipids (GPLs), and sphingolipids (SLs), of primary ex vivo differentiated (I) white, (II) brite, and (III) brown adipocytes derived from primary preadipocytes isolated from (I) epididymal white, (II) inguinal white, and (III) intrascapular brown adipose tissue. Quantitative lipidomics revealed significantly decreased fractions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), with longer (C > 36) and more polyunsaturated species, as well as lower levels of cardiolipin (CL) in white than in brite and brown adipocytes. Together, the brite and brown lipidome was comparable and indicates differences in membrane lipid packing density compared with white adipocytes. Changes in ceramide species profile could be related to the degree of browning. Beta-adrenergic stimulation of brown adipocytes led to generation of saturated lyso-PC (LPC) increasing uncoupling protein (UCP) 1-mediated leak respiration. Application of stable isotope labeling showed that LPC formation was balanced by an increased de novo synthesis of PC.

Quantification of Fecal Short Chain Fatty Acids by Liquid Chromatography Tandem Mass Spectrometry-Investigation of Pre-Analytic Stability.

Short chain fatty acids (SCFAs) are generated by the degradation and fermentation of complex carbohydrates, (i.e., dietary fiber) by the gut microbiota relevant for microbe⁻host communication. Here, we present a method for the quantification of SCFAs in fecal samples by liquid chromatography tandem mass spectrometry (LC-MS/MS) upon derivatization to 3-nitrophenylhydrazones (3NPH). The method includes acetate, propionate, butyrate, and isobutyrate with a run time of 4 min. The reproducible (coefficients of variation (CV) below 10%) quantification of SCFAs in human fecal samples was achieved by the application of stable isotope labelled internal standards. The specificity was demonstrated by the introduction of a quantifier and qualifier ions. The method was applied to investigate the pre-analytic stability of SCFAs in human feces. Concentrations of SCFA may change substantially within hours; the degree and kinetics of these changes revealed huge differences between the donors. The fecal SCFA level could be preserved by the addition of organic solvents like isopropanol. An analysis of the colon content of mice either treated with antibiotics or fed with a diet containing a non-degradable and -fermentable fiber source showed decreased SCFA concentrations. In summary, this fast and reproducible method for the quantification of SCFA in fecal samples provides a valuable tool for both basic research and large-scale studies.

Substrate fluxes in brown adipocytes upon adrenergic stimulation and uncoupling protein 1 ablation.

Brown adipocytes are highly specialized cells with the unique metabolic ability to dissipate chemical energy in the form of heat. We determined and inferred the flux of a number of key catabolic metabolites, their changes in response to adrenergic stimulation, and the dependency on the presence of the thermogenic uncoupling protein 1 and/or oxidative phosphorylation. This study provides reference values to approximate flux rates from a limited set of measured parameters in the future and thereby allows to evaluate the plausibility of claims about the capacity of metabolic adaptations or manipulations. From the resulting model, we delineate that in brown adipocytes (1) free fatty acids are a significant contributor to extracellular acidification, (2) glycogen is the dominant glycolytic substrate source in the acute response to an adrenergic stimulus, and (3) the futile cycling of free fatty acids between lipolysis and re-esterification into triglyceride provides a mechanism for uncoupling protein 1-independent, non-shivering thermogenesis in brown adipocytes.

The gut microbiota promotes hepatic fatty acid desaturation and elongation in mice.

Interactions between the gut microbial ecosystem and host lipid homeostasis are highly relevant to host physiology and metabolic diseases. We present a comprehensive multi-omics view of the effect of intestinal microbial colonization on hepatic lipid metabolism, integrating transcriptomic, proteomic, phosphoproteomic, and lipidomic analyses of liver and plasma samples from germfree and specific pathogen-free mice. Microbes induce monounsaturated fatty acid generation by stearoyl-CoA desaturase 1 and polyunsaturated fatty acid elongation by fatty acid elongase 5, leading to significant alterations in glycerophospholipid acyl-chain profiles. A composite classification score calculated from the observed alterations in fatty acid profiles in germfree mice clearly differentiates antibiotic-treated mice from untreated controls with high sensitivity. Mechanistic investigations reveal that acetate originating from gut microbial degradation of dietary fiber serves as precursor for hepatic synthesis of C16 and C18 fatty acids and their related glycerophospholipid species that are also released into the circulation.

Taking control over intracellular fatty acid levels is essential for the analysis of thermogenic function in cultured primary brown and brite/beige adipocytes.

Thermogenesis in brown adipocytes, conferred by mitochondrial uncoupling protein 1 (UCP1), is receiving great attention because metabolically active brown adipose tissue may protect humans from metabolic diseases. In particular, the thermogenic function of brown-like adipocytes in white adipose tissue, known as brite (or beige) adipocytes, is currently of prime interest. A valid procedure to quantify the specific contribution of UCP1 to thermogenesis is thus of vital importance. Adrenergic stimulation of lipolysis is a common way to activate UCP1. We here report, however, that in this frequently applied setup, taking control over intracellular fatty acid levels is essential for the analysis of thermogenic function in cultured brown and brite adipocytes. By the application of these findings, we demonstrate that UCP1 is functionally thermogenic in intact brite adipocytes and adrenergic UCP1 activation is largely dependent on adipose triglyceride lipase (ATGL) rather than hormone sensitive lipase (HSL).

Sensitivity of ab Initio vs Empirical Methods in Computing Structural Effects on NMR Chemical Shifts for the Example of Peptides.

The structural sensitivity of NMR chemical shifts as computed by quantum chemical methods is compared to a variety of empirical approaches for the example of a prototypical peptide, the 38-residue kaliotoxin KTX comprising 573 atoms. Despite the simplicity of empirical chemical shift prediction programs, the agreement with experimental results is rather good, underlining their usefulness. However, we show in our present work that they are highly insensitive to structural changes, which renders their use for validating predicted structures questionable. In contrast, quantum chemical methods show the expected high sensitivity to structural and electronic changes. This appears to be independent of the quantum chemical approach or the inclusion of solvent effects. For the latter, explicit solvent simulations with increasing number of snapshots were performed for two conformers of an eight amino acid sequence. In conclusion, the empirical approaches neither provide the expected magnitude nor the patterns of NMR chemical shifts determined by the clearly more costly ab initio methods upon structural changes. This restricts the use of empirical prediction programs in studies where peptide and protein structures are utilized for the NMR chemical shift evaluation such as in NMR refinement processes, structural model verifications, or calculations of NMR nuclear spin relaxation rates.

Ketoamide resistance and hepatitis C virus fitness in val55 variants of the NS3 serine protease.

Drug-resistant viral variants are a major issue in the use of direct-acting antiviral agents in chronic hepatitis C. Ketoamides are potent inhibitors of the NS3 protease, with V55A identified as mutation associated with resistance to boceprevir. Underlying molecular mechanisms are only partially understood. We applied a comprehensive sequence analysis to characterize the natural variability at Val55 within dominant worldwide patient strains. A residue-interaction network and molecular dynamics simulation were applied to identify mechanisms for ketoamide resistance and viral fitness in Val55 variants. An infectious H77S.3 cell culture system was used for variant phenotype characterization. We measured antiviral 50% effective concentration (EC50) and fold changes, as well as RNA replication and infectious virus yields from viral RNAs containing variants. Val55 was found highly conserved throughout all hepatitis C virus (HCV) genotypes. The conservative V55A and V55I variants were identified from HCV genotype 1a strains with no variants in genotype 1b. Topology measures from a residue-interaction network of the protease structure suggest a potential Val55 key role for modulation of molecular changes in the protease ligand-binding site. Molecular dynamics showed variants with constricted binding pockets and a loss of H-bonded interactions upon boceprevir binding to the variant proteases. These effects might explain low-level boceprevir resistance in the V55A variant, as well as the Val55 variant, reduced RNA replication capacity. Higher structural flexibility was found in the wild-type protease, whereas variants showed lower flexibility. Reduced structural flexibility could impact the Val55 variant's ability to adapt for NS3 domain-domain interaction and might explain the virus yield drop observed in variant strains.

Effects of terminal functional groups on the stability of the polyproline II structure: a combined experimental and theoretical study.

The conformational stability of the polyproline II (PPII) helix with respect to the functional groups at the C- and N-termini was examined both experimentally and theoretically. Oligoprolines AcN-[Pro](12)-CONH(2) (1), HN-[Pro](12)-CONH(2) (2), AcN-[Pro](12)-CO(2)H (3), and HN-[Pro](12)-CO(2)H (4) with charged and capped termini served as model compounds, and the relative ease with which they switch from the PPII to the polyproline I (PPI) helix was used as a measure to analyze their conformational stabilities. CD spectroscopic studies demonstrate that a positively charged N-terminus and a negatively charged C-terminus destabilize the PPII helix and favor the PPI helix, whereas capped termini favor the PPII over the PPI helix. These experimental findings are supported by the energy differences between the PPII and PPI helices of oligoprolines 1-4 computed by ab initio methods including electron-correlation effects (second-order Møller-Plesset perturbation theory, MP2). Furthermore, these quantum-chemical calculations show that differences in charge-dipole interactions are responsible for the experimentally and computationally observed relative stabilities. Although these electrostatic interactions between the terminal charges and the amide dipoles stabilize both helices, they are significantly stronger in the PPI helix where the amide bonds are oriented almost linear to the helix axis as compared to the PPII helix in which the amides are nearly perpendicular to the axis. Moreover, we demonstrate that a negative charge at the C-terminus has a more pronounced effect on the relative stability as compared to a positive charge at the N-terminus due to destabilization of the PPII helix by repulsive interaction between the C-terminal carboxylate with the neighboring amide bond. Studies at different pH values verified the electrostatic nature of the observed effects and demonstrate how changes in the protonation state can be used to deliberately stabilize the PPII helix over the PPI helix or vice versa.

An arginine switch in the species B adenovirus knob determines high-affinity engagement of cellular receptor CD46.

Adenoviruses (Ads) are icosahedral, nonenveloped viruses with a double-stranded DNA genome. The 51 known Ad serotypes exhibit profound variations in cell tropism and disease types. The number of observed Ad infections is steadily increasing, sometimes leading to fatal outcomes even in healthy individuals. Species B Ads can cause kidney infections, hemorrhagic cystitis, and severe respiratory infections, and most of them use the membrane cofactor protein CD46 as a cellular receptor. The crystal structure of the human Ad type 11 (Ad11) knob complexed with CD46 is known; however, the determinants of CD46 binding in related species B Ads remain unclear. We report here a structural and functional analysis of the Ad11 knob, as well as the Ad7 and Ad14 knobs, which are closely related in sequence to the Ad11 knob but have altered CD46-binding properties. The comparison of the structures of the three knobs, which we determined at very high resolution, provides a platform for understanding these differences and allows us to propose a mechanism for productive high-affinity engagement of CD46. At the center of this mechanism is an Ad knob arginine that needs to switch its orientation in order to engage CD46 with high affinity. Quantum chemical calculations showed that the CD46-binding affinity of Ad11 is significantly higher than that of Ad7. Thus, while Ad7 and Ad14 also bind CD46, the affinity and kinetics of these interactions suggest that these Ads are unlikely to use CD46 productively. The proposed mechanism is likely to determine the receptor usage of all CD46-binding Ads.

Thin-film properties of DNA and RNA bases: a combined experimental and theoretical study.

The structures of the DNA and RNA bases cytosine, uracil, and thymine in thin films with a nominal film thickness of about 20 nm are studied by using X-ray photoemission spectroscopy (XPS) and Fourier-transform infrared spectroscopy. The molecules are evaporated in situ from powder on a gold foil. The experimental results indicate that cytosine is composed of two energetically close tautomeric forms, whereas uracil and thymine exist in only one tautomeric form. Additionally, quantum chemical calculations are performed to complement the experimental results. The relative energies of the tautomeric forms of cytosine, uracil, and thymine are calculated using Hartree-Fock (HF), density functional theory (DFT), and post-HF methods. Furthermore, the assignment of the XPS spectra is supported by using simple model considerations employing Koopmans ionization energies and Mulliken net atomic charges.

An atomic orbital-based reformulation of energy gradients in second-order Møller-Plesset perturbation theory.

A fully atomic orbital (AO)-based reformulation of second-order Møller-Plesset perturbation theory (MP2) energy gradients is introduced, which provides the basis for reducing the computational scaling with the molecular size from the fifth power to linear. Our formulation avoids any transformation between the AO and the molecular orbital (MO) basis and employs pseudodensity matrices similar to the AO-MP2 energy expressions within the Laplace scheme for energies. The explicit computation of perturbed one-particle density matrices emerging in the new AO-based gradient expression is avoided by reformulating the Z-vector method of Handy and Schaefer [J. Chem. Phys. 81, 5031 (1984)] within a density matrix-based scheme.

Linear-scaling Cholesky decomposition.

We present linear-scaling routines for the calculation of the Cholesky decomposition of a symmetric positive-definite matrix and its inverse. As an example, we consider the inversion of the overlap matrix of DNA and amylose fragments as well as of linear alkanes, where the largest system corresponds to a 21,442 x 21,442 matrix. The efficiency and the scaling behavior are discussed and compared to standard LAPACK routines. Our Cholesky routines are publicly available on the web.

The "azido gauche effect"-implications for the conformation of azidoprolines.

The "azido gauche effect" was examined both experimentally and theoretically and was found to determine the conformation of, for example, (4R)- and (4S)-azidoproline (Azp) derivatives. For (4R)Azp derivatives, the azido gauche effect induces a preferred C(4)-exo conformation of the pyrrolidine ring, which leads to stabilization of the s-trans amide conformer of, e.g., Ac-(4R)Azp-OCH(3) (5R) via an n-->pi interaction between the nonbonding electrons of the oxygen of the acetyl group and the carbonyl group of the ester. For (4S)Azp derivatives, the azido gauche effect results in a C(4)-endo conformation of the pyrrolidine ring that does not allow for this stabilizing n-->pi interaction of the s-trans conformer. Consequently, a significantly higher s-trans:s-cis amide conformer ratio is observed for (4R)Azp compared to (4S)Azp derivatives (e.g., 6.1:1 versus 2.6:1 in D(2)O for Ac-(4R)Azp-OCH(3) (5R) compared to Ac-(4S)Azp-OCH(3) (5S)). These conformational preferences are reflected in the higher tendency of (4S)Azp-containing peptides to form cyclic peptides with all-cis amide bonds compared to (4R)Azp derivatives. Ab initio calculations demonstrate that the strength of the azido gauche effect is comparable to that of the well-known "fluorine gauche effect". For azidoethane derivatives N(3)-CH(2)CH(2)-X (X = N(3), NHCOH, NHAc, or N(CH(3))Ac), the ab initio calculations revealed energy differences of 5-13 kJ mol(-)(1) between the anti and gauche conformations in favor of the gauche conformer. Calculations were also performed for the (4R)Azp and (4S)Azp derivatives 5R and 5S, supporting the experimentally observed data.