Acetyl-CoA carboxylase 2 inhibition reduces skeletal muscle bioactive lipid content and attenuates progression of type 2 diabetes in Zucker diabetic fatty rats.

Intramyocellular lipid (IMCL) accumulation in skeletal muscle is closely associated with development of insulin resistance. In particular, diacylglycerol and ceramide are currently considered as causal bioactive lipids for impaired insulin action. Recently, inhibition of acetyl-CoA carboxylase 2 (ACC2), which negatively modulates mitochondrial fatty acid oxidation, has been shown to reduce total IMCL content and improve whole-body insulin resistance. This study aimed to investigate whether ACC2 inhibition-induced compositional changes in bioactive lipids, especially diacylglycerol and ceramide, within skeletal muscle contribute to the improved insulin resistance. In skeletal muscle of normal rats, treatment of the ACC2 inhibitor compound 2e significantly decreased both diacylglycerol and ceramide levels while having no significant impact on other lipid metabolite levels. In skeletal muscle of Zucker diabetic fatty (ZDF) rats, which exhibited greater lipid accumulation than that of normal rats, compound 2e significantly decreased diacylglycerol and ceramide levels corresponding to reduced long chain acyl-CoA pools. Additionally, in the lipid metabolomics study, ZDF rats treated with compound 2e also showed improved diabetes-related metabolic disturbance, as reflected by delayed hyperinsulinemia as well as upregulated gene expression associated with diabetic conditions in skeletal muscle. These metabolic improvements were strongly correlated with the bioactive lipid reductions. Furthermore, long-term treatment of compound 2e markedly improved whole-body insulin resistance, attenuated hyperglycemia and delayed insulin secretion defect even at severe diabetic conditions. These findings suggest that ACC2 inhibition decreases diacylglycerol and ceramide accumulation within skeletal muscle by enhancing acyl-CoA breakdown, leading to attenuation of lipid-induced insulin resistance and subsequent diabetes progression.

In Vivo Pharmacodynamic Study of Cefiderocol, a Novel Parenteral Siderophore Cephalosporin, in Murine Thigh and Lung Infection Models.

The pharmacokinetic (PK) and pharmacodynamic (PD) parameters which correlated with the in vivo efficacy of cefiderocol were evaluated using neutropenic murine thigh and lung infection models in which the infections were caused by a variety of Gram-negative bacilli. The dose fractionation study using the thigh infection model in which the infection was caused by Pseudomonas aeruginosa showed that the cumulative percentage of a 24-h period that the free drug concentration in plasma exceeds the MIC (%fT>MIC) rather than the free peak level divided by the MIC (fCmax/MIC) and the area under the free concentration-time curve over 24 h divided by the MIC (fAUC/MIC) was the PK/PD parameter that best correlated with efficacy. The study with multiple carbapenem-resistant strains revealed that the %fT>MIC determined in iron-depleted cation-adjusted Mueller-Hinton broth (ID-CAMHB) better reflected the in vivo efficacy of cefiderocol than the %fT>MIC determined in cation-adjusted Mueller-Hinton broth (CAMHB). The mean %fT>MIC of cefiderocol required for a 1-log10 reduction against 10 strains of Enterobacteriaceae and 3 strains of Pseudomonas aeruginosa in the thigh infection models were 73.3% and 77.2%, respectively. The mean %fT>MIC for Enterobacteriaceae, P. aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia in the lung infection model were 64.4%, 70.3%, 88.1%, and 53.9%, respectively. These results indicate that cefiderocol has potent efficacy against Gram-negative bacilli, including carbapenem-resistant strains, irrespective of the bacterial species, in neutropenic thigh and lung infection models and that the in vivo efficacy correlated with the in vitro MIC under iron-deficient conditions.

ACC2 Deletion Enhances IMCL Reduction Along With Acetyl-CoA Metabolism and Improves Insulin Sensitivity in Male Mice.

Intramyocellular lipid (IMCL) accumulation in skeletal muscle greatly contributes to lipid-induced insulin resistance. Because acetyl-coenzyme A (CoA) carboxylase (ACC) 2 negatively modulates mitochondrial fatty acid oxidation (FAO) in skeletal muscle, ACC2 inhibition is expected to reduce IMCL via elevation of FAO and to attenuate insulin resistance. However, the concept of substrate competition suggests that enhanced FAO results in reduced glucose use because of an excessive acetyl-CoA pool in mitochondria. To identify how ACC2-regulated FAO affects IMCL accumulation and glucose metabolism, we generated ACC2 knockout (ACC2-/-) mice and investigated skeletal muscle metabolites associated with fatty acid and glucose metabolism, as well as whole-body glucose metabolism. ACC2-/- mice displayed higher capacity of glucose disposal at the whole-body levels. In skeletal muscle, ACC2-/- mice exhibited enhanced acylcarnitine formation and reduced IMCL levels without alteration in glycolytic intermediate levels. Notably, these changes were accompanied by decreased acetyl-CoA content and enhanced mitochondrial pathways related to acetyl-CoA metabolism, such as the acetylcarnitine production and tricarboxylic acid cycle. Furthermore, ACC2-/- mice exhibited lower levels of IMCL and acetyl-CoA even under HFD conditions and showed protection against HFD-induced insulin resistance. Our findings suggest that ACC2 deletion leads to IMCL reduction without suppressing glucose use via an elevation in acetyl-CoA metabolism even under HFD conditions and offer new mechanistic insight into the therapeutic potential of ACC2 inhibition on insulin resistance.

Gene Silencing Activity and Hepatic Accumulation of Antisense Oligonucleotides Bearing Cholesterol-Conjugated Thiono Triester at the Gap Region.

Cholesterol (Chol) conjugation to the 5' or 3' end of antisense oligonucleotide (ASO) enables delivery to the liver, and Chol conjugation at the gap region can also be expected to improve delivery to the liver. In this study, we synthesized ASOs bearing the Chol-conjugated thiono triester and evaluated their activity and hepatic accumulation. We found that Chol conjugations at the gap region improved in vitro activity and hepatic accumulation when compared to unconjugated ASOs. However, Chol conjugation with phosphorothioate linkage did not improve in vivo activity in the liver, suggesting the importance of cleaving the phosphodiester between ASO and Chol. These results offer useful information for tuning the oligonucleotide structure to improve pharmaceutical properties and designing ASOs for multiple ligand conjugations and combinations with end modification.

Comparative Characterization of Hepatic Distribution and mRNA Reduction of Antisense Oligonucleotides Conjugated with Triantennary N-Acetyl Galactosamine and Lipophilic Ligands Targeting Apolipoprotein B.

TriantennaryN-acetyl galactosamine (GalNAc, GN3) and lipophilic ligands such as cholesterol andα-tocopherol conjugations dramatically improve the distribution and efficacy of second-generation antisense oligonucleotides (ASOs) in the whole liver. To characterize ligands for delivery to liver cells based on pharmacokinetics and efficacy, we used a locked nucleic acid gapmer of ASO targeting apolipoprotein B as a model compound and evaluated the amount of ASO and apolipoprotein B mRNA in the whole liver, hepatocytes, and nonparenchymal (NP) cells as well as plasma total cholesterol after administration of ASO conjugated with these ligands to mice. Compared with unconjugated ASO, GN3 conjugation increased the amount (7-fold) and efficacy (more than 10-fold) of ASO in hepatocytes only and showed higher efficacy than the increased rate of the amount of ASO. On the other hand, lipophilic ligand conjugations led to increased delivery (3- to 5-fold) and efficacy (5-fold) of ASO to both hepatocytes and NP cells. GN3 and lipophilic ligand conjugations increased the area under the curve of ASOs and the pharmacodynamic duration but did not change the half-life in hepatocytes and NP cells compared with unconjugated ASO. In the liver, the phosphodiester bond between ASO and these ligands was promptly cleaved to liberate unconjugated ASO. These ligand conjugations reduced plasma total cholesterol compared with unconjugated ASO, although these ASOs were well tolerated with no elevation in plasma transaminases. These findings could facilitate ligand selection tailored to liver cells expressed in disease-related genes and could contribute to the discovery and development of RNA interference-based therapy.

Reconstruction of localized fluorescent target from multi-view continuous-wave surface images of small animal with lp sparsity regularization.

Fluorescence diffuse optical tomography using a multi-view continuous-wave and non-contact measurement system and an algorithm incorporating the lp (0 < p ≤ 1) sparsity regularization reconstructs a localized fluorescent target in a small animal. The measurement system provides a total of 25 fluorescence surface 2D-images of an object, which are acquired by a CCD camera from five different angles of view with excitation from five different angles. Fluorescence surface emissions from five different angles of view are simultaneously imaged on the CCD sensor, thus leading to fast acquisition of the 25 images within three minutes. The distributions of the fluorophore are reconstructed by solving the inverse problem based on the photon diffusion equations. In the reconstruction process incorporating the lp sparsity regularization, the regularization term is reformulated as a differentiable function for gradient-based non-linear optimization. Numerical simulations and phantom experiments show that the use of the lp sparsity regularization improves the localization of the target and quantitativeness of the fluorophore concentration. A mouse experiment demonstrates that a localized fluorescent target in a mouse is successfully reconstructed.

Dynamics of light-induced conformational changes of the phoborhodopsin/transducer complex formed in the n-dodecyl beta-D-maltoside micelle.

A complex of photoreceptor phoborhodopsin (ppR; also called sensory rhodopsin II) and its cognate halobacterial transducer II (pHtrII) existing in the plasma membrane mediates the light signal to the cytoplasm in the earliest step of negative phototaxis in Natronomonas pharaonis. We have investigated the dynamics of the light-induced conformational changes of the ppR/pHtrII(1-159) complex formed in the presence of 0.1% n-dodecyl beta-d-maltoside (DDM) by a fluorescence resonance energy transfer (FRET) based method. Fluorescence donor and acceptor dyes were linked to cysteine residues genetically introduced at given positions in pHtrII and ppR. The light-induced FRET efficiency changes for various pairs of dye-labeled cysteine residues were determined to examine dynamics of movements of given residues in the transmembrane and the linker region including the HAMP domain in pHtrII induced by photoexcitation of ppR. Upon flash excitation of ppR, FRET efficiency changed depending on pairs of the labeled cysteine residues. The distances between V185 in ppR and the five given residues (102 through 141) in the pHtrII linker region estimated from the FRET efficiency increased by 0.3-0.8 A; on the other hand, the distances between S31 in ppR and the five residues in pHtrII decreased. The changes arose within 70 ms (the dead time of instrument) and decayed at a rate of 1.1 +/- 0.2 s. Azide significantly increased the decay rate of light-induced FRET efficiency changes by accelerating the decay of the M state of ppR. The decay rate of FRET efficiency changes coincided with the rate of recovery of the ppR to the initial state but not the decay of the M state. We conclude that the light-induced conformational change of pHtrII occurs before, at the formation or during the M state, and its relaxation is coupled tightly with the decay of the O state of ppR in the 1:1 complex formed in the DDM micelle.

Application of fluorescence resonance energy transfer (FRET) to investigation of light-induced conformational changes of the phoborhodopsin/transducer complex.

The photoreceptor phoborhodopsin (ppR; also called sensory rhodopsin II) forms a complex with its cognate the Halobacterial transducer II (pHtrII) in the membrane, through which changes in the environmental light conditions are transmitted to the cytoplasm in Natronomonas pharaonis to evoke negative phototaxis. We have applied a fluorescence resonance energy transfer (FRET)-based method for investigation of the light-induced conformational changes of the ppR/pHtrII complex. Several far-red dyes were examined as possible fluorescence donors or acceptors because of the absence of the spectral overlap of these dyes with all the photointermediates of ppR. The flash-induced changes of distances between the donor and an acceptor linked to cysteine residues which were genetically introduced at given positions in pHtrII(1-159) and ppR were determined from FRET efficiency changes. The dye-labeled complex was studied as solubilized in 0.1% n-dodecyl-beta-D-maltoside (DDM). The FRET-derived changes in distances from V78 and A79 in pHtrII to V185 in ppR were consistent with the crystal structure data (Moukhametzianov, R. et al. [2006] Nature, 440, 115-119). The distance from D102 in pHtrII linker region to V185 in ppR increased by 0.33 angstroms upon the flash excitation. These changes arose within 70 ms (the dead time of instrument) and decayed with a rate of 1.1 +/- 0.2 s. Thus, sub-angstrom-scale distance changes in the ppR/pHtrII complex were detected with this FRET-based method using far-red fluorescent dyes; this method should be a valuable tool to investigate conformation changes in the transducer, in particular its dynamics.

The elution profile of immobilized liposome chromatography: determination of association and dissociation rate constants.

The interaction of lipophilic cations, tetraphenylphosphonium and triphenylphosphonium homologues with liposomes was investigated using immobilized liposome chromatography (ILC). Large unilamellar liposomes with a mean diameter of 100 nm were stably immobilized in chromatographic gel beads by avidin-biotin. The distribution coefficient calculated from (Ve-V0)/Vs (Ve, retention volume; V0, the void volume; Vs, the stationary phase volume) was found to be independent of flow rate, injection amount and gel bed volume, which is consistent with chromatograph theory. The relationship between the bandwidth and solvent flow rate did not follow band-broadening theories reported thus far. We hypothesized that the solvent might be forced to produce large eddies, spirals or turbulent flow due to the presence of liposomes fixed in the gel. Therefore, we developed a new theory for ILC elution: The column is composed of a number of thin disks containing liposomes and solution, and within each disk the solution is well mixed. This theory accounts for our results, and we were able to use it to estimate the rate constants of association and dissociation of the phosphonium to/from liposomes.