Microbial products linked to steatohepatitis are reduced by deletion of nuclear hormone receptor SHP in mice.

Deletion of the nuclear hormone receptor small heterodimer partner (Shp) ameliorates the development of obesity and nonalcoholic steatohepatitis (NASH) in mice. Liver-specific SHP plays a significant role in this amelioration. The gut microbiota has been associated with these metabolic disorders, and the interplay between bile acids (BAs) and gut microbiota contributes to various metabolic disorders. Since hepatic SHP is recognized as a critical regulator in BA synthesis, we assessed the involvement of gut microbiota in the antiobesity and anti-NASH phenotype of Shp-/- mice. Shp deletion significantly altered the levels of a few conjugated BAs. Sequencing the 16S rRNA gene in fecal samples collected from separately housed mice revealed apparent dysbiosis in Shp-/- mice. Cohousing Shp-/- mice with WT mice during a Western diet regimen impaired their metabolic improvement and effectively disrupted their distinctive microbiome structure, which became indistinguishable from that of WT mice. While the Western diet challenge significantly increased lipopolysaccharide and phenylacetic acid (PAA) levels in the blood of WT mice, their levels were not increased in Shp-/- mice. PAA was strongly associated with hepatic peroxisome proliferator-activated receptor gamma isoform 2 (Pparg2) activation in mice, which may represent the basis of the molecular mechanism underlying the association of gut bacteria and hepatic steatosis. Shp deletion reshapes the gut microbiota possibly by altering BAs. While lipopolysaccharide and PAA are the major driving forces derived from gut microbiota for NASH development, Shp deletion decreases these signaling molecules via dysbiosis, thereby partially protecting mice from diet-induced metabolic disorders.

Synthesis, nanostructuring and in silico studies of a new imine bond containing a macroheterocycle as a promising PBP-2a non-ß-lactam inhibitor.

This study is devoted to the synthesis of a 40-membered macroheterocycle with its further nanostructuring by magnetite nanoparticles. The mentioned macroheterocycle was synthesized by the [2+2] cyclocondensation of the oxygen-containing diamine with an aromatic dialdehyde in a non-catalytic medium and with no work-up procedure. The structure of the obtained macroheterocycle was studied by 1H and 13C nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Furthermore, the nanosupramolecular complex of macroheterocycles with magnetite nanoparticles was obtained and investigated by Fourier-transform infrared and ultraviolet-visible spectroscopy methods. Shifts in the infrared spectra of the nanosupramolecular complex indicate the interaction through metal-aromatic ring non-covalent bonding. The shift is also observed for the C-O-C stretching band of ether bonds. The loading rate of macroheterocycles on magnetite nanoparticles was 18.6%. The morphology of the ensemble was studied by transmission electron microscopy, which confirmed the synthesis of nanospherical particles with a diameter range of 10-20 nm. Powder X-ray diffraction analysis showed patterns of cubic Fe3O4 nanoparticles with a crystallite size equal to 9.1 nm. The macroheterocycle and its nanosupramolecular complex were tested against Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. The results have shown that the created complex has shown 64 times better activity against Staphylococcus aureus in comparison with the individual macroheterocycle and 32 times better activity in comparison with the pristine antibiotic Ampicillin as a control. In addition, computational analysis of the macroheterocycle was performed at the B3LYP/6-31G level in water. Molecular docking analyses for the macroheterocycle revealed Penicillin-binding protein PBP2a (5M18) from the transpeptidase family as a target protein in Staphylococcus aureus.

A high-fat diet increases hepatic mitochondrial turnover through restricted acetylation in a NAFLD mouse model.

Lysine acetylation of proteins has emerged as a key posttranslational modification (PTM) that regulates mitochondrial metabolism. Acetylation may regulate energy metabolism by inhibiting and affecting the stability of metabolic enzymes and oxidative phosphorylation (OxPhos) subunits. Although protein turnover can be easily measured, due to the low abundance of modified proteins, it has been difficult to evaluate the effect of acetylation on the stability of proteins in vivo. We applied 2H2O-metabolic labeling coupled with immunoaffinity and high-resolution mass spectrometry method to measure the stability of acetylated proteins in mouse liver based on their turnover rates. As a proof-of-concept, we assessed the consequence of high-fat diet (HFD)-induced altered acetylation in protein turnover in LDL receptor-deficient (LDLR-/-) mice susceptible to diet-induced nonalcoholic fatty liver disease (NAFLD). HFD feeding for 12 wk led to steatosis, the early stage of NAFLD. A significant reduction in acetylation of hepatic proteins was observed in NAFLD mice, based on immunoblot analysis and label-free quantification with mass spectrometry. Compared with control mice on a normal diet, NAFLD mice had overall increased turnover rates of hepatic proteins, including mitochondrial metabolic enzymes (0.159 ± 0.079 vs. 0.132 ± 0.068 day-1), suggesting their reduced stability. Also, acetylated proteins had slower turnover rates (increased stability) than native proteins in both groups (0.096 ± 0.056 vs. 0.170 ± 0.059 day-1 in control, and 0.111 ± 0.050 vs. 0.208 ± 0.074 day-1 in NAFLD). Furthermore, association analysis revealed a relationship between the HFD-induced decrease in acetylation and increased turnover rates for hepatic proteins in NAFLD mice. These changes were associated with increased expressions of the hepatic mitochondrial transcriptional factor (TFAM) and complex II subunit without any changes to other OxPhos proteins, suggesting that enhanced mitochondrial biogenesis prevented restricted acetylation-mediated depletion of mitochondrial proteins. We conclude that decreased acetylation of mitochondrial proteins may contribute to adaptive improved hepatic mitochondrial function in the early stages of NAFLD.NEW & NOTEWORTHY This is the first method to quantify acetylome dynamics in vivo. This method revealed acetylation-mediated altered hepatic mitochondrial protein turnover in response to a high-fat diet in a mouse model of NAFLD.

Metabolic labeling unveils alterations in the turnover of HDL-associated proteins during diabetes progression in mice.

Several aspects of diabetes pathophysiology and complications result from hyperglycemia-induced alterations in the structure and function of plasma proteins. Furthermore, insulin has a significant influence on protein metabolism by affecting both the synthesis and degradation of proteins in various tissues. To understand the role of progressive hyperglycemia on plasma proteins, in this study, we measured the turnover rates of high-density lipoprotein (HDL)-associated proteins in control (chow diet), prediabetic [a high-fat diet (HFD) for 8 wk] or diabetic [HFD for 8 wk with low-dose streptozotocin (HFD + STZ) in weeks 5-8 of HFD] C57BL/6J mice using heavy water (2H2O)-based metabolic labeling approach. Compared with control mice, HFD and HFD + STZ mice showed elevations of fasting plasma glucose levels in the prediabetic and diabetic range, respectively. Furthermore, the HFD and HFD + STZ mice showed increased hepatic triglyceride (TG) levels, total plasma cholesterol, and plasma TGs. The kinetics of 40 proteins were quantified using the proteome dynamics method, which revealed an increase in the fractional synthesis rate (FSR) of HDL-associated proteins in the prediabetic mice compared with control mice, and a decrease in FSR in the diabetic mice. The pathway analysis revealed that proteins with altered turnover rates were involved in acute-phase response, lipid metabolism, and coagulation. In conclusion, prediabetes and diabetes have distinct effects on the turnover rates of HDL proteins. These findings suggest that an early dysregulation of the HDL proteome dynamics can provide mechanistic insights into the changes in protein levels in these conditions.NEW & NOTEWORTHY This study is the first to examine the role of gradual hyperglycemia during diabetes disease progression on HDL-associated protein dynamics in the prediabetes and diabetic mice. Our results show that the fractional synthesis rate of HDL-associated proteins increased in the prediabetic mice whereas it decreased in the diabetic mice compared with control mice. These kinetic changes can help to elucidate the mechanism of altered protein levels and HDL dysfunction during diabetes disease progression.

Measuring acetyl-CoA and acetylated histone turnover in vivo: Effect of a high fat diet.

Cellular availability of acetyl-CoA, a central intermediate of metabolism, regulates histone acetylation. The impact of a high-fat diet (HFD) on the turnover rates of acetyl-CoA and acetylated histones is unknown. We developed a method for simultaneous measurement of acetyl-CoA and acetylated histones kinetics using a single 2H2O tracer, and used it to examine effect of HFD-induced perturbations on hepatic histone acetylation in LDLR-/- mice, a mouse model of non-alcoholic fatty liver disease (NAFLD). Mice were given 2H2O in the drinking water and the kinetics of hepatic acetyl-CoA, histones, and acetylated histones were quantified based on their 2H-labeling. Consumption of a high fat Western-diet (WD) for twelve weeks led to decreased acetylation of hepatic histones (p< 0.05), as compared to a control diet. These changes were associated with 1.5-3-fold increased turnover rates of histones without any change in acetyl-CoA flux. Acetylation significantly reduced the stability of histones and the turnover rates of acetylated peptides were correlated with the number of acetyl groups in neighboring lysine sites. We conclude that 2H2O-method can be used to study metabolically controlled histone acetylation and acetylated histone turnover in vivo.

Early Pro-Inflammatory Remodeling of HDL Proteome in a Model of Diet-Induced Obesity: 2H2O-Metabolic Labeling-Based Kinetic Approach.

Mice fed a high-fat diet for 12 weeks or longer develop hyperglycemia, insulin resistance, dyslipidemia, and fatty liver. Additionally, a high-fat diet induces inflammation that remodels and affects the anti-inflammatory and antiatherogenic property of the high-density lipoprotein (HDL). However, the precise time course of metabolic disease progression and HDL remodeling remains unclear. Short-term (four weeks) high-fat feeding (60% fat calories) was performed in wild-type male C57BL/6J mice to gain insights into the early metabolic disease processes in conjunction with a HDL proteome dynamics analysis using a heavy water metabolic labeling approach. The high-fat diet-fed mice developed hyperglycemia, impaired glucose tolerance, hypercholesterolemia without hypertriglyceridemia or hepatic steatosis. A plasma HDL proteome dynamics analysis revealed increased turnover rates (and reduced half-lives) of several acute-phase response proteins involved in innate immunity, including complement C3 (12.77 ± 0.81 vs. 9.98 ± 1.20 h, p < 0.005), complement factor B (12.71 ± 1.01 vs. 10.85 ± 1.04 h, p < 0.05), complement Factor H (19.60 ± 1.84 vs. 16.80 ± 1.58 h, p < 0.05), and complement factor I (25.25 ± 1.29 vs. 19.88 ± 1.50 h, p < 0.005). Our findings suggest that an early immune response-induced inflammatory remodeling of the plasma HDL proteome precedes the diet-induced steatosis and dyslipidemia.