Effect of the lysosomotropic agent chloroquine on mTORC1 activation and protein synthesis in human skeletal muscle

Background Previous work in HEK-293 cells demonstrated the importance of amino acid-induced mTORC1 translocation to the lysosomal surface for stimulating mTORC1 kinase activity and protein synthesis. This study tested the conservation of this amino acid sensing mechanism in human skeletal muscle by treating subjects with chloroquine—a lysosomotropic agent that induces in vitro and in vivo lysosome dysfunction. Methods mTORC1 signaling and muscle protein synthesis (MPS) were determined in vivo in a randomized controlled trial of 14 subjects (10 M, 4 F; 26 ± 4 year) that ingested 10 g of essential amino acids (EAA) after receiving 750 mg of chloroquine (CHQ, n = 7) or serving as controls (CON, n = 7; no chloroquine). Additionally, differentiated C2C12 cells were used to assess mTORC1 signaling and myotube protein synthesis (MyPS) in the presence and absence of leucine and the lysosomotropic agent chloroquine. Results mTORC1, S6K1, 4E-BP1 and rpS6 phosphorylation increased in both CON and CHQ 1 h post EAA ingestion (P \u3c 0.05). MPS increased similarly in both groups (CON, P = 0.06; CHQ, P \u3c 0.05). In contrast, in C2C12 cells, 1 mM leucine increased mTORC1 and S6K1 phosphorylation (P \u3c 0.05), which was inhibited by 2 mg/ml chloroquine. Chloroquine (2 mg/ml) was sufficient to disrupt mTORC1 signaling, and MyPS. Conclusions Chloroquine did not inhibit amino acid-induced activation of mTORC1 signaling and skeletal MPS in humans as it does in C2C12 muscle cells. Therefore, different in vivo experimental approaches are required for confirming the precise role of the lysosome and amino acid sensing in human skeletal muscle

NSAID use and clinical outcomes in COVID-19 patients: a 38-center retrospective cohort study.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used to reduce pain, fever, and inflammation but have been associated with complications in community-acquired pneumonia. Observations shortly after the start of the COVID-19 pandemic in 2020 suggested that ibuprofen was associated with an increased risk of adverse events in COVID-19 patients, but subsequent observational studies failed to demonstrate increased risk and in one case showed reduced risk associated with NSAID use. METHODS: A 38-center retrospective cohort study was performed that leveraged the harmonized, high-granularity electronic health record data of the National COVID Cohort Collaborative. A propensity-matched cohort of 19,746 COVID-19 inpatients was constructed by matching cases (treated with NSAIDs at the time of admission) and 19,746 controls (not treated) from 857,061 patients with COVID-19 available for analysis. The primary outcome of interest was COVID-19 severity in hospitalized patients, which was classified as: moderate, severe, or mortality/hospice. Secondary outcomes were acute kidney injury (AKI), extracorporeal membrane oxygenation (ECMO), invasive ventilation, and all-cause mortality at any time following COVID-19 diagnosis. RESULTS: Logistic regression showed that NSAID use was not associated with increased COVID-19 severity (OR: 0.57 95% CI: 0.53-0.61). Analysis of secondary outcomes using logistic regression showed that NSAID use was not associated with increased risk of all-cause mortality (OR 0.51 95% CI: 0.47-0.56), invasive ventilation (OR: 0.59 95% CI: 0.55-0.64), AKI (OR: 0.67 95% CI: 0.63-0.72), or ECMO (OR: 0.51 95% CI: 0.36-0.7). In contrast, the odds ratios indicate reduced risk of these outcomes, but our quantitative bias analysis showed E-values of between 1.9 and 3.3 for these associations, indicating that comparatively weak or moderate confounder associations could explain away the observed associations. CONCLUSIONS: Study interpretation is limited by the observational design. Recording of NSAID use may have been incomplete. Our study demonstrates that NSAID use is not associated with increased COVID-19 severity, all-cause mortality, invasive ventilation, AKI, or ECMO in COVID-19 inpatients. A conservative interpretation in light of the quantitative bias analysis is that there is no evidence that NSAID use is associated with risk of increased severity or the other measured outcomes. Our results confirm and extend analogous findings in previous observational studies using a large cohort of patients drawn from 38 centers in a nationally representative multicenter database

Protein Requirements in Critically Ill Older Adults

Critically ill elderly patients’ nutritional needs are not well understood and vary with the phase of illness and recovery. Patients’ nutritional needs should be assessed early in hospitalization and re-assessed throughout the stay with additional attention during the transitions from critical illness, to severe illness, to post-hospital rehabilitation. In this review, we summarize recent findings and highlight recommendations for protein supplementation in critically ill geriatric patients throughout the stages of recovery. Future research specifically focusing on protein dose, its relationship with caloric needs, and delivery modality must be conducted to provide more specific guidelines for clinical practice

In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish

Oxidative modification of LDL is an early pathological event in the development of atherosclerosis. Oxidation events such as malondialdehyde (MDA) formation may produce specific, immunogenic epitopes. Indeed, antibodies to MDA-derived epitopes are widely used in atherosclerosis research and have been demonstrated to enable cardiovascular imaging. In this study, we engineered a transgenic zebrafish with temperature-inducible expression of an EGFP-labeled single-chain human monoclonal antibody, IK17, which binds to MDA-LDL, and used optically transparent zebrafish larvae for imaging studies. Feeding a high-cholesterol diet (HCD) supplemented with a red fluorescent lipid marker to the transgenic zebrafish resulted in vascular lipid accumulation, quantified in live animals using confocal microscopy. After heat shock–induced expression of IK17-EGFP, we measured the time course of vascular accumulation of IK17-specific MDA epitopes. Treatment with either an antioxidant or a regression diet resulted in reduced IK17 binding to vascular lesions. Interestingly, homogenates of IK17-EGFP–expressing larvae bound to MDA-LDL and inhibited MDA-LDL binding to macrophages. Moreover, sustained expression of IK17-EGFP effectively prevented HCD-induced lipid accumulation in the vascular wall, suggesting that the antibody itself may have therapeutic effects. Thus, we conclude that HCD-fed zebrafish larvae with conditional expression of EGFP-labeled oxidation-specific antibodies afford an efficient method of testing dietary and/or other therapeutic antioxidant strategies that may ultimately be applied to humans