Cell culture models of fatty acid overload: Problems and solutions

Chronic exposure of pancreatic β cells to high concentrations of free fatty acids leads to lipotoxicity (LT)‐mediated suppression of glucose‐stimulated insulin secretion. This effect is in part caused by a decline in mitochondrial function as well as by a reduction in lysosomal acidification. Because both mitochondria and lysosomes can alter one another's function, it remains unclear which initiating dysfunction sets off the detrimental cascade of LT, ultimately leading to β‐cell failure. Here, we investigated the effects of restoring lysosomal acidity on mitochondrial function under LT. Our results show that LT induces a dose‐dependent lysosomal alkalization accompanied by an increase in mitochondrial mass. This increase is due to a reduction in mitochondrial turnover as analyzed by MitoTimer, a fluorescent protein for which the emission is regulated by mitochondrial clearance rate. Mitochondrial oxygen consumption rate, citrate synthase activity, and ATP content are all reduced by LT. Restoration of lysosomal acidity using lysosome‐targeted nanoparticles is accompanied by stimulation of mitochondrial turnover as revealed by mitophagy measurements and the recovery of mitochondrial mass. Remarkably, re‐acidification restores citrate synthase activity and ATP content in an insulin secreting β‐cell line (INS‐1). Furthermore, nanoparticle‐mediated lysosomal reacidification rescues mitochondrial maximal respiratory capacity in both INS‐1 cells and primary mouse islets. Therefore, our results indicate that mitochondrial dysfunction is downstream of lysosomal alkalization under lipotoxic conditions and that recovery of lysosomal acidity is sufficient to restore the bioenergetic defects.—Assali, E. A., Shlomo, D., Zeng, J., Taddeo, E. P., Trudeau, K. M., Erion, K. A., Colby, A. H., Grinstaff, M. W., Liesa, M., Las, G., Shirihai, O. S. Nanoparticle‐mediated lysosomal reacidification restores mitochondrial turnover and function in β cells under lipotoxicity. FASEB J. 33, 4154–4165 (2019). http://www.fasebj.org

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“…5A). This lipid combination is more physiologically relevant than palmitate alone, because chronic high-dose palmitate can be toxic (21,22).…”

Section: Panps Increase Atp Content and Improve Mitochondrial Respiramentioning

confidence: 99%

Nanoparticle‐mediated lysosomal reacidification restores mitochondrial turnover and function in β cells under lipotoxicity

et al. 2018

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“…This further supports the notion that dietary interventions may not only affect physiological Ca 2+ handling but also modulate damaging effects of supra-physiological Ca 2+ accumulation. Interestingly, caloric restriction and nutrient starvation also modulate mitochondrial morphology, stimulating mitochondrial fusion (Rambold et al, 2011;Khraiwesh et al, 2014;Cerqueira et al, 2016), while nutrient overload is often associated with mitochondrial fission (Molina et al, 2009;Liesa and Shirihai, 2013;Alsabeeh et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial Morphology Regulates Organellar Ca²⁺Uptake and Changes Cellular Ca²⁺Homeostasis

Kowaltowski

Menezes-Filho

Assali

et al. 2019

Preprint

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Changes in mitochondrial size and shape have been implicated in several physiological processes, but their role in mitochondrial Ca 2+ uptake regulation and overall cellular Ca 2+ homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics towards increased fusion through expression of a dominant negative form of the fission protein DRP1 (DRP1-DN) markedly increased both mitochondrial Ca 2+ retention capacity and Ca 2+ uptake rates in permeabilized C2C12 cells. Similar results were seen using the pharmacological fusion-promoting M1 molecule. Conversely, promoting a fission phenotype through the knockdown of the fusion protein mitofusin 2 (MFN2) strongly reduced mitochondrial Ca 2+ uptake speed and capacity in these cells. These changes were not dependent on modifications in inner membrane potentials or the mitochondrial permeability transition. Implications of mitochondrial morphology modulation on cellular calcium homeostasis were measured in intact cells; mitochondrial fission promoted lower basal cellular calcium levels and lower endoplasmic reticulum (ER) calcium stores, as measured by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium-replenishing process of store-operated calcium entry (SOCE) was impaired in MFN2 knockdown cells, while DRP1-DN-promoted fusion resulted in faster cytosolic Ca 2+ increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca 2+ uptake, which impacts on cellular Ca 2+ homeostasis.

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“…We first reasoned that, if increased energy expenditure caused by UK5099 requires a rise in intracellular levels of fatty acids, then their sequestration would prevent the boost on respiratory rates upon MPC inhibition. To test this prediction, we supplemented the respirometry media with 0.1 % BSA-FAF (fatty acid free), which would remove the excess intracellular free fatty acids [22]. Figure 1E shows that supplementation with BSA-FAF reversed the stimulatory effects of UK5099 on basal respiration, when compared to control media.…”

Section: Pharmacological Inhibition Of the Mpc In Non-stimulated Browmentioning

confidence: 97%

Blocking mitochondrial pyruvate import causes energy wasting via futile lipid cycling in brown fat

Veliova

Ferreira

Benador

et al. 2019

Preprint

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Futile lipid cycling is an ATP-wasting process proposed to participate in energy expenditure of mature fat-storing white adipocytes, given their inability to oxidize fat. The hallmark of activated brown adipocytes is to increase fat oxidation by uncoupling respiration from ATP synthesis. Whether ATPconsuming lipid cycling can contribute to BAT energy expenditure has been largely unexplored. Here we find that pharmacological inhibition of the mitochondrial pyruvate carrier (MPC) in brown adipocytes is sufficient to increase ATP-synthesis fueled by fatty acid oxidation, even in the absence of adrenergic stimulation. We find that elevated ATP-demand induced by MPC inhibition results from activation of futile lipid cycling. Furthermore, we identify that glutamine consumption and the Malate-Aspartate Shuttle are required for the increase in Energy Expenditure induced by MPC inhibition in Brown Adipocytes (MAShEEBA). These data demonstrate that futile energy expenditure through lipid cycling can be activated in BAT by altering fuel availability to mitochondria. Therefore, we identify a new mechanism to increase fat oxidation and energy expenditure in BAT that bypasses the need for adrenergic stimulation of mitochondrial uncoupling.Conceptualization; M.V., M.L.

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Cell culture models of fatty acid overload: Problems and solutions

Cited by 114 publications

References 139 publications

Nanoparticle‐mediated lysosomal reacidification restores mitochondrial turnover and function in β cells under lipotoxicity

Nanoparticle‐mediated lysosomal reacidification restores mitochondrial turnover and function in β cells under lipotoxicity

Mitochondrial Morphology Regulates Organellar Ca²⁺Uptake and Changes Cellular Ca²⁺Homeostasis

Blocking mitochondrial pyruvate import causes energy wasting via futile lipid cycling in brown fat

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Cell culture models of fatty acid overload: Problems and solutions

Cited by 114 publications

References 139 publications

Nanoparticle‐mediated lysosomal reacidification restores mitochondrial turnover and function in β cells under lipotoxicity

Nanoparticle‐mediated lysosomal reacidification restores mitochondrial turnover and function in β cells under lipotoxicity

Mitochondrial Morphology Regulates Organellar Ca2+Uptake and Changes Cellular Ca2+Homeostasis

Blocking mitochondrial pyruvate import causes energy wasting via futile lipid cycling in brown fat

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Mitochondrial Morphology Regulates Organellar Ca²⁺Uptake and Changes Cellular Ca²⁺Homeostasis