Long-chain fatty acid (LCFA) oxidation has been shown to play an important role in interleukin-4 (IL-4)-mediated macrophage polarization (M(IL-4)). However, many of these conclusions are based on the ...inhibition of carnitine palmitoyltransferase-1 with high concentrations of etomoxir that far exceed what is required to inhibit enzyme activity (EC90 < 3 μM). We employ genetic and pharmacologic models to demonstrate that LCFA oxidation is largely dispensable for IL-4-driven polarization. Unexpectedly, high concentrations of etomoxir retained the ability to disrupt M(IL-4) polarization in the absence of Cpt1a or Cpt2 expression. Although excess etomoxir inhibits the adenine nucleotide translocase, oxidative phosphorylation is surprisingly dispensable for M(IL-4). Instead, the block in polarization was traced to depletion of intracellular free coenzyme A (CoA), likely resulting from conversion of the pro-drug etomoxir into active etomoxiryl CoA. These studies help explain the effect(s) of excess etomoxir on immune cells and reveal an unappreciated role for CoA metabolism in macrophage polarization.
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•Alternative macrophage activation by IL-4 does not require fatty acid oxidation•Etomoxir concentrations ≥10 μM inhibit the adenine nucleotide translocase•OXPHOS is dispensable for many markers of IL-4-activated macrophages (M(IL-4))•200 μM etomoxir blocks M(IL-4) by perturbing intracellular coenzyme A homeostasis
The CPT-1 inhibitor etomoxir has been used to suggest long-chain fatty acid (LCFA) oxidation is necessary for alternative macrophage activation. Divakaruni and colleagues now show that LCFA oxidation is dispensable. They demonstrate multiple off-target effects of etomoxir and show that depletion of coenzyme A by etomoxir blocks M(IL-4) differentiation.
In the mammalian retina, a metabolic ecosystem exists in which photoreceptors acquire glucose from the choriocapillaris with the help of the retinal pigment epithelium (RPE). While the photoreceptor ...cells are primarily glycolytic, exhibiting Warburg-like metabolism, the RPE is reliant on mitochondrial respiration. However, the ways in which mitochondrial metabolism affect RPE cellular functions are not clear. We first used the human RPE cell line, ARPE-19, to examine mitochondrial metabolism in the context of cellular differentiation. We show that nicotinamide induced rapid differentiation of ARPE-19 cells, which was reversed by removal of supplemental nicotinamide. During the nicotinamide-induced differentiation, we observed using quantitative PCR, Western blotting, electron microscopy, and metabolic respiration and tracing assays that (1) mitochondrial gene and protein expression increased, (2) mitochondria became larger with more tightly folded cristae, and (3) mitochondrial metabolism was enhanced. In addition, we show that primary cultures of human fetal RPE cells responded similarly in the presence of nicotinamide. Furthermore, disruption of mitochondrial oxidation of pyruvate attenuated the nicotinamide-induced differentiation of the RPE cells. Together, our results demonstrate a remarkable effect of nicotinamide on RPE metabolism. We also identify mitochondrial respiration as a key contributor to the differentiated state of the RPE and thus to many of the RPE functions that are essential for retinal health and photoreception.
Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions ...in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.
Glioblastoma (GBM) metabolism has traditionally been characterized by a primary dependence on aerobic glycolysis, prompting the use of the ketogenic diet (KD) as a potential therapy. In this study we ...evaluated the effectiveness of the KD in GBM and assessed the role of fatty acid oxidation (FAO) in promoting GBM propagation. In vitro assays revealed FA utilization throughout the GBM metabolome and growth inhibition in nearly every cell line in a broad spectrum of patient-derived glioma cells treated with FAO inhibitors. In vivo assessments revealed that knockdown of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme for FAO, reduced the rate of tumor growth and increased survival. However, the unrestricted ketogenic diet did not reduce tumor growth and for some models significantly reduced survival. Altogether, these data highlight important roles for FA and ketone body metabolism that could serve to improve targeted therapies in GBM.
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•Glioblastoma cells can use fatty acid oxidation and ketones for energy requirements•Inhibition of fatty acid oxidation reduces glioblastoma growth in vivo and in vitro•Unrestricted ketogenic diet does not reduce tumor growth in multiple models of GBM•IDH1 mutant glioblastomas may use FAO differently than IDH wild-type tumors
Pathophysiology; Cancer; Diet
The basolateral amygdala (BLA) is critical for associating initially neutral cues with appetitive and aversive stimuli and receives dense neuromodulatory acetylcholine (ACh) projections. We measured ...BLA ACh signaling and activity of neurons expressing CaMKIIα (a marker for glutamatergic principal cells) in mice during cue-reward learning using a fluorescent ACh sensor and calcium indicators. We found that ACh levels and nucleus basalis of Meynert (NBM) cholinergic terminal activity in the BLA (NBM-BLA) increased sharply in response to reward-related events and shifted as mice learned the cue-reward contingency. BLA CaMKIIα neuron activity followed reward retrieval and moved to the reward-predictive cue after task acquisition. Optical stimulation of cholinergic NBM-BLA terminal fibers led to a quicker acquisition of the cue-reward contingency. These results indicate BLA ACh signaling carries important information about salient events in cue-reward learning and provides a framework for understanding how ACh signaling contributes to shaping BLA responses to emotional stimuli.
There is a growing call for civic engagement, largely in the form of formal volunteering, among older adults in America. This call is a response to the aging of the baby boom population, believed to ...be the healthiest and wealthiest cohort of older adults to date. It also coincides with the devolution of welfare programs. We argue that current discussions of civic engagement are too narrow and may exclude important informal contributions that older adults make to civic society, and put undue stress on, and devalue those who may not contribute to society due to poor health, poverty or other barriers. We draw on data collected from older adults of lower socio-economic status and diverse ethnic backgrounds in Baltimore City using focus groups to explore their definitions of volunteering and barriers which they face. Through a discussion of existing barriers and motivators for engagement, we critically assess the use of these terms and advance discussions on how to facilitate and value contributions of all older adults. We conclude that civic engagement includes more than formal volunteering and that significant barriers need to be removed to facilitate greater participation of all elders in both formal and informal activities.
Students may need to decide whether to invest limited resources evenly across all courses and thus end with moderate grades in all, or focus on some of the courses and thus end with variable grades. ...This study examined which pattern of grades is perceived more favorably. When judging competency, people give more weight to positive than negative information, in which case heterogeneous grades would be perceived more favorably as they have more positive grades than homogeneous moderate grades. Furthermore, high school students are told to demonstrate their passion in college applications. Nonetheless, people generally overweigh negative information, which can result in a preference for a student with homogeneous grades lacking extremely negative grades. The college admissions decisions in particular may also involve emphasis on long-term stable, consistent, and responsible character, which the homogeneous grades may imply. Study 1 found that laypeople, undergraduate students, and admissions officers preferred to admit a student with homogeneous grades to a college than a student with heterogeneous grades even when their GPAs were the same. Study 2 used a heterogeneous transcript signaling a stereotypic STEM or humanities student, and found that while undergraduate students were more split in their choices, laypeople and admissions officers still preferred a student with homogeneous grades. Study 3 further replicated the preference for a student with homogeneous grades by using higher or lower average GPAs and wider or narrower range of grades for the heterogeneous grades. Possible reasons and limitations of the studies are discussed.
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