Reproducibility of an HPLC-ESI-MS/MS Method for the Measurement of Stable-Isotope Enrichment of in Vivo-Labeled Muscle ATP Synthase Beta Subunit

We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R2 = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase134-143 peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase134-143 peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R2 = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase134-143 peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase134-143 peptide

SARS-CoV-2 infection produces chronic pulmonary epithelial and immune cell dysfunction with fibrosis in mice

A subset of individuals who recover from coronavirus disease 2019 (COVID-19) develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal tissue samples. The mouse-adapted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute to clinical recovery phases. At 15 to 120 days post-virus clearance, pulmonary histologic findings included subpleural lesions composed of collagen, proliferative fibroblasts, and chronic inflammation, including tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal up-regulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC., After recovery from acute SARS-CoV-2 infection, mice exhibit chronic lung disease similar to some humans, allowing for testing of therapeutics

Effects of acute exposure to increased plasma branched-chain amino acid concentrations on insulin-mediated plasma glucose turnover in healthy young subjects.

Plasma branched-chain amino acids (BCAA) are inversely related to insulin sensitivity of glucose metabolism in humans. However, currently, it is not known whether there is a cause-and-effect relationship between increased plasma BCAA concentrations and decreased insulin sensitivity.To determine the effects of acute exposure to increased plasma BCAA concentrations on insulin-mediated plasma glucose turnover in humans.Ten healthy subjects were randomly assigned to an experiment where insulin was infused at 40 mU/m2/min (40U) during the second half of a 6-hour intravenous infusion of a BCAA mixture (i.e., BCAA; N = 5) to stimulate plasma glucose turnover or under the same conditions without BCAA infusion (Control; N = 5). In a separate experiment, seven healthy subjects were randomly assigned to receive insulin infusion at 80 mU/m2/min (80U) in association with the above BCAA infusion (N = 4) or under the same conditions without BCAA infusion (N = 3). Plasma glucose turnover was measured prior to and during insulin infusion.Insulin infusion completely suppressed the endogenous glucose production (EGP) across all groups. The percent suppression of EGP was not different between Control and BCAA in either the 40U or 80U experiments (P > 0.05). Insulin infusion stimulated whole-body glucose disposal rate (GDR) across all groups. However, the increase (%) in GDR was not different [median (1st quartile - 3rd quartile)] between Control and BCAA in either the 40U ([199 (167-278) vs. 186 (94-308)] or 80 U ([491 (414-548) vs. 478 (409-857)] experiments (P > 0.05). Likewise, insulin stimulated the glucose metabolic clearance in all experiments (P 0.05).Short-term exposure of young healthy subjects to increased plasma BCAA concentrations does not alter the insulin sensitivity of glucose metabolism

Plasma glucose and insulin concentrations in the study associated with the infusion of insulin at 80 mU/m²/min.

<p>Values are means ± SE and median (1^st quartile—3^rd quartile); Time 0, prior to the initiation of any infusions; Basal Period, infusion of saline (Control, N = 3) or branched-chain amino acids (BCAA, N = 4); Hyperinsulinemic-euglycemic Clamp, infusion of 80 mU/m²/min insulin together with variable rate of 20% dextrose to maintain the plasma glucose concentrations at those measured at the end of the Basal Period;</p><p>*<i>P</i> ≤0.05 when compared to Time 0 and Basal Period</p><p>^&<i>P</i> = 0.09 when compared to Time 0 and Basal Period</p><p>Plasma glucose and insulin concentrations in the study associated with the infusion of insulin at 80 mU/m²/min.</p

Physical and clinical characteristics of the subjects.

<p>Values are means ± SE; BMI, body mass index, ALT, alanine aminotransferase; AST, aspartate aminotransferase; BCAA, branched-chain amino acids; HDL-C, High Density Lipoprotein-Cholesterol; HOMA-IR, Homeostatic model assessment of insulin resistance; Body fat was determined using bioimpedance; There were no significant differences between groups (<i>P</i> >0.05).</p><p>Physical and clinical characteristics of the subjects.</p

Plasma branched-chain amino acid concentrations in the study associated with the infusion of insulin at 40 mU/m²/min.

<p>Values are means ± SE and median (1^st quartile—3^rd quartile); Time 0, prior to the initiation of any infusions; Basal Period, infusion of saline (Control, N = 5) or branched-chain amino acids (BCAA, N = 5); Hyperinsulinemic-euglycemic Clamp, infusion of 40 mU/m²/min insulin together with variable rate of 20% dextrose to maintain the plasma glucose concentrations at those measured at the end of the Basal Period.</p><p>*Statistically different compared to Time 0 (<i>P</i> ≤0.05)</p><p>^#Statistically different compared to Time 0 and Basal Period (<i>P</i> ≤0.05)</p><p>†Statistically different between Control and BCAA groups (<i>P</i> ≤0.05)</p><p>Plasma branched-chain amino acid concentrations in the study associated with the infusion of insulin at 40 mU/m²/min.</p

Experimental design.

<p>Infusion protocol depicting the “basal” and “hyperinsulinemic-euglycemic clamp” study periods described in the text. [6,6–²H₂]glucose, branched-chain amino acids (BCAA; i.e., BranchAmin), saline, insulin and glucose were infused as indicated; [BCAA], total branched-chain amino acids concentration.</p

Plasma branched-chain amino acid concentrations in the study associated with the infusion of insulin at 80 mU/m²/min.

<p>Values are means ± SE and median (1^st quartile—3^rd quartile); Time 0, prior to the initiation of any infusions; Basal Period, infusion of saline (Control, N = 3) or branched-chain amino acids (BCAA, N = 4); Hyperinsulinemic-euglycemic Clamp, infusion of 80 mU/m²/min insulin together with variable rate of 20% dextrose to maintain the plasma glucose concentrations at those measured at the end of the Basal Period.</p><p>*Statistically different compared to Time 0 (<i>P</i> ≤0.05)</p><p>†Statistically different between Control and BCAA groups (<i>P</i> ≤0.05).</p><p>Plasma branched-chain amino acid concentrations in the study associated with the infusion of insulin at 80 mU/m²/min.</p

Plasma glucose turnover.

<p>Rates of endogenous glucose production (EGP) and whole-body glucose disposal (GDR) in the basal period (i.e., Basal) and following insulin infusion (i.e., Insulin). Insulin was infused at either 40 mU/m²/min in a control group (A) and a group with increased plasma branched-chain amino acid concentrations (B) or 80 mU/m²/min in a control group (C) and a group with increased plasma branched-chain amino acid concentrations (D). Boxes describe interquartile range (IQR; 1^st quartile—3^rd quartile) with the horizontal line in the box representing the median value. <i>P</i> values are for the comparison of the corresponding medians.</p