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Open Access Publications from the University of California

Scholarly Works (47 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2022)

Macrophages are innate immune cells that execute a variety of functions including microbial clearance, antigen presentation, and tissue repair. When exposed to the cytokine interleukin 4 (IL-4), these cells express genes associated with alternative activation. Previous work had shown that etomoxir-mediated disruption of coenzyme A homeostasis decreases the macrophage IL-4 response, while exogenous addition of CoA enhances the presence IL-4-associated cell-surface markers. However, the mechanism by which etomoxir decreased intracellular CoA levels and how this ubiquitous metabolic cofactor could instruct alternative activation was entirely unclear. In this work, I show that etomoxir likely decreases intracellular CoA levels by thioester-mediated pantothenate kinase inhibition. Further, I demonstrate that exogenous CoA enhances the IL-4 response in vitro and in vivo. I determined that CoA acts as a weak toll-like receptor 4 agonist, which enhances the IL-4 response through activation of the MyD88 signaling cascade. Finally, I show that activation of the MyD88 pathway is sufficient to enhance alternative activation both in vitro and in vivo.

    Cover page: Determining the Role of Coenzyme A in Alternative Macrophage Activation
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2022)
    Measurement of oxygen consumption is a powerful and uniquely informative experimental technique. It can help identify mitochondrial mechanisms of action following pharmacologic and genetic interventions, and characterize energy metabolism in physiology and disease. The conceptual and practical benefits of respirometry have made it a frontline technique to understand how mitochondrial function can interface with-and in some cases control-cell physiology. Nonetheless, an appreciation of the complexity and challenges involved with such measurements is required to avoid common experimental and analytical pitfalls. Here we provide a practical guide to oxygen consumption measurements covering the selection of experimental models and instrumentation, as well as recommendations for the collection, interpretation and normalization of data. These guidelines are provided with the intention of aiding experimental design and enhancing the overall reputability, transparency and reliability of oxygen consumption measurements.
      • Thesis
      • Peer Reviewed
      UCLA Electronic Theses and Dissertations (2023)

      Elevated levels of BCAAs have been associated with heart failure and metabolic disease. The branched-chain ketoacid dehydrogenase kinase (BCKDK) inhibitor BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid) was designed to induce branched-chain amino acid (BCAA) oxidation to restore levels of BCAAs. BT2 treatment confers cardioprotection and protects from metabolic disease in preclinical models including mice subjected to trans-aortic constriction or LAD ligation, db/db mice, ob/ob mice, and Zucker fatty rats. However, mice with the BCKDK knocked out specifically in the heart or skeletal muscle does not protect from heart failure, suggesting an alternative molecular mechanism by which BT2 is conferring its protective effects. In this study, we provide evidence characterizing BT2 as a mitochondrial uncoupler. Using reductionist systems including respirometry, mitochondrial membrane potential, and patch-clamp electrophysiology, we demonstrate that BT2 is indeed uncoupling mitochondria and is approximately five-fold milder than the well-known chemical uncoupler DNP. Functional assays expose BT2’s protective effects originate from diminishing ROS production and reducing de novo lipogenesis. The evidence provided here more likely solves the long-standing question on BT2’s mechanism by which it is conferring therapeutic effects. Furthermore, these studies establish mild uncouplers of mitochondria as promising pharmacological agents for the treatment of cardiovascular and metabolic disease.

        Cover page: The BCKDK inhibitor BT2 is a chemical uncoupler that lowers mitochondrial ROS production and de novo lipogenesis
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2020)
        Mitochondria are metabolic organelles essential not only for energy transduction, but also a range of other functions such as biosynthesis, ion and metal homeostasis, maintenance of redox balance, and cell signaling. A hallmark example of how mitochondria can rebalance these processes to adjust cell function is observed in macrophages. These innate immune cells are responsible for a remarkable breadth of processes including pathogen elimination, antigen presentation, debris clearance, and wound healing. These diverse, polarized functions often include similarly disparate alterations in the metabolic phenotype associated with their execution. In this chapter, mitochondrial bioenergetics and signaling are viewed through the lens of macrophage polarization: both classical, pro-inflammatory activation and alternative, anti-inflammatory activation are associated with substantive changes to mitochondrial metabolism. Emphasis is placed on recent evidence that aims to clarify the essential - rather than associative - mitochondrial alterations, as well as accumulating data suggesting a degree of plasticity within the metabolic phenotypes that can support pro- and anti-inflammatory functions.
          Cover page: Macrophage activation as an archetype of mitochondrial repurposing
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2021)
          Carnitine palmitoyltransferase-1 (CPT-1) is a rate-controlling enzyme for long-chain fatty acid oxidation. This manuscript provides protocols for measuring CPT-1-mediated respiration in permeabilized, adherent cell monolayers and mitochondria freshly isolated from tissue, along with examples to assess the potency and specificity of interventions targeting CPT-1. Strengths of the approach include ease, speed, and breadth of analysis, whereas drawbacks include loss of physiological regulation in reductionist systems and indirect assessment of CPT-1 enzymatic activity. For complete details on the use and execution of this protocol, please refer to Divakaruni et al. (2018).
            Cover page: Measuring CPT-1-mediated respiration in permeabilized cells and isolated mitochondriaCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2018)
            Activities of enzymes localized to the mitochondrial matrix of mammalian cells are often critical regulatory steps in cellular metabolism. As such, measurement of matrix enzyme activities in response to genetic modifications or drug interventions is often desired. However, measurements in intact cells are often hampered by the presence of other isozymes in the cytoplasm as well as the inability to deliver enzyme substrates across cellular membranes. Classic approaches to liberate matrix enzymes utilize harsh treatments that disrupt intracellular architecture or require significant starting material to allow mitochondrial isolation prior to sample extraction. We describe a method using permeabilization reagents for both the plasma and mitochondrial membranes to allow in situ measurement of matrix enzyme activities. It is applied to adherent cell monolayers in 96-well plates treated with perfringolysin O to permeabilize the plasma membrane and alamethicin to permeabilize the mitochondrial inner membrane. We present three examples validated with inhibitor sensitivity: (i) Complex I-mediated oxygen consumption driven by NADH, (ii) ATP hydrolysis by the F1FO complex measuring pH changes in an Agilent Seahorse XF Analyzer, and (iii) Mitochondrial glutaminase (GLS1) activity in a coupled reaction monitoring NADH fluorescence in a plate reader.
              Cover page: In situ measurements of mitochondrial matrix enzyme activities using plasma and mitochondrial membrane permeabilization agents
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2021)
              Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels.
                Cover page: A Single LC-MS/MS Analysis to Quantify CoA Biosynthetic Intermediates and Short-Chain Acyl CoAsCreative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2016)
                Sestrin2 is a member of a family of stress responsive proteins, which controls cell viability via antioxidant activity and regulation of the mammalian target of rapamycin protein kinase (mTOR). Sestrin2 is induced by different stress insults, which diminish ATP production and induce energetic stress in the cells. Glucose is a critical substrate for ATP production utilized via glycolysis and mitochondrial respiration as well as for glycosylation of newly synthesized proteins in the endoplasmic reticulum (ER) and Golgi. Thus, glucose starvation causes both energy deficiency and activation of ER stress followed by the unfolding protein response (UPR). Here, we show that UPR induces Sestrin2 via ATF4 and NRF2 transcription factors and demonstrate that Sestrin2 protects cells from glucose starvation-induced cell death. Sestrin2 inactivation sensitizes cells to necroptotic cell death that is associated with a decline in ATP levels and can be suppressed by Necrostatin 7. We propose that Sestrin2 protects cells from glucose starvation-induced cell death via regulation of mitochondrial homeostasis.
                  Cover page: Sestrin2 is induced by glucose starvation via the unfolded protein response and protects cells from non-canonical necroptotic cell deathCreative Commons 'BY' version 4.0 license
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2023)
                  Methods for isolating mitochondria from different rodent tissues have been established for decades. Although the general principles for crude mitochondrial preparations are largely shared across tissues - tissue disruption followed by differential centrifugation - critical differences exist for isolation from different tissues to optimize mitochondrial yield and function. This protocol offers a unified resource for preparations of isolated mitochondria from mouse liver, kidney, heart, brain, skeletal muscle, and brown and white adipose tissue suitable for functional analysis.
                    Cover page: Isolation of Mitochondria from Mouse Tissues for Functional Analysis.
                    • Article
                    • Peer Reviewed
                    UC San Diego Previously Published Works (2020)

                    Objectives

                    Cerebral ischemia/reperfusion (IR) drives oxidative stress and injurious metabolic processes that lead to redox imbalance, inflammation, and tissue damage. However, the key mediators of reperfusion injury remain unclear, and therefore, there is considerable interest in therapeutically targeting metabolism and the cellular response to oxidative stress.

                    Methods

                    The objective of this study was to investigate the molecular, metabolic, and physiological impact of itaconate treatment to mitigate reperfusion injuries in in vitro and in vivo model systems. We conducted metabolic flux and bioenergetic studies in response to exogenous itaconate treatment in cultures of primary rat cortical neurons and astrocytes. In addition, we administered itaconate to mouse models of cerebral reperfusion injury with ischemia or traumatic brain injury followed by hemorrhagic shock resuscitation. We quantitatively characterized the metabolite levels, neurological behavior, markers of redox stress, leukocyte adhesion, arterial blood flow, and arteriolar diameter in the brains of the treated/untreated mice.

                    Results

                    We demonstrate that the "immunometabolite" itaconate slowed tricarboxylic acid (TCA) cycle metabolism and buffered redox imbalance via succinate dehydrogenase (SDH) inhibition and induction of anti-oxidative stress response in primary cultures of astrocytes and neurons. The addition of itaconate to reperfusion fluids after mouse cerebral IR injury increased glutathione levels and reduced reactive oxygen/nitrogen species (ROS/RNS) to improve neurological function. Plasma organic acids increased post-reperfusion injury, while administration of itaconate normalized these metabolites. In mouse cranial window models, itaconate significantly improved hemodynamics while reducing leukocyte adhesion. Further, itaconate supplementation increased survival in mice experiencing traumatic brain injury (TBI) and hemorrhagic shock.

                    Conclusions

                    We hypothesize that itaconate transiently inhibits SDH to gradually "awaken" mitochondrial function upon reperfusion that minimizes ROS and tissue damage. Collectively, our data indicate that itaconate acts as a mitochondrial regulator that controls redox metabolism to improve physiological outcomes associated with IR injury.
                      Cover page: Itaconate modulates tricarboxylic acid and redox metabolism to mitigate reperfusion injuryCreative Commons 'BY-NC-ND' version 4.0 license
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