Micro-respirometry of whole cells and isolated mitochondria

Levitsky, Yan; Pegouske, David; Hammer, Sandra S; Frantz, Nathan L.; Fisher, Kiera; Muchnik, Artem; Saripalli, Anand; Kirschner, Philip; Bazil, Jason N.; Busik, Julia V.; Proshlyakov, Denis A.

doi:10.1039/c9ra05289e

Cited by 9 publications

(12 citation statements)

References 44 publications

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“…R e s p i r a t i o n o f B R E C s w a s m e a s u r e d u s i n g a microrespirometer as previously described [38]. Briefly, On the day of measurements, the chips were closed, mounted in a microrespirometer, and immediately perfused at a constant 10 μl min −1 flow rate.…”

Section: Mitochondrial Respiration Measurementsmentioning

confidence: 99%

Fasting and fasting-mimicking treatment activate SIRT1/LXRα and alleviate diabetes-induced systemic and microvascular dysfunction

et al. 2021

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Aims/hypothesis Homo sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting-mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage. Methods The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as liver X receptor alpha [LXRα]) acetylation was measured via western blot analysis. Results IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression. Sandra S. Hammer and Cristiano P. Vieira contributed equally as first authors. Maria B. Grant and Julia V. Busik contributed equally as senior authors.

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Section: Mitochondrial Respiration Measurementsmentioning

confidence: 99%

Fasting and fasting-mimicking treatment activate SIRT1/LXRα and alleviate diabetes-induced systemic and microvascular dysfunction

et al. 2021

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Section: Diabetes Induces Acid Sphingomyelinase (Asm)-mediated Changes In Mitochondrial Function Of Human Rpe Cellsmentioning

confidence: 99%

“…To determine whether the structural changes of mitochondria were correlated with detectable functional differences, we used microrespirometry to examine oxidative phosphorylation in controland diabetic-derived RPE cells [28]. In this approach, the flow of oxygenated medium over adherent cells is intermittently stopped and respiration leads to a steady consumption of oxygen, seen as periodic downward slopes in the O 2 concentration traces (Figure 4A).…”

Section: Diabetes Induces Acid Sphingomyelinase (Asm)-mediated Changes In Mitochondrial Function Of Human Rpe Cellsmentioning

confidence: 99%

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Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes

Levitsky

Hammer

Fisher

et al. 2020

IJMS

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Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood–retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1β, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 μM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

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“…Respiration of BREC's was measured using an open-shell microrespirometer as previously described (41). Briefly, BREC at passage 4 were seeded and cultured on fibronectin-coated chips overnight at 37° C, 95% RH, 5% CO2.…”

Section: Mitochondrial Respiration Measurementsmentioning

confidence: 99%

Pharmacological and fasting-induced activation of SIRT1/LXRα signaling alleviates diabetes-induced retinopathy

McFarland

et al. 2019

Preprint

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In diabetes, the retina, a tissue with unique metabolic needs, demonstrates dysregulation of the intricate balance between nutrient availability and utilization. This results in cholesterol accumulation, pro-inflammatory and pro-apoptotic changes, and consequently neurovascular damage. Sirtuin 1 (SIRT1), a nutrient sensing deacetylase, is downregulated in the diabetic retina. In this study, the effect of SIRT1 stimulation by fasting or by pharmacological activation using SRT1720, was evaluated on retinal cholesterol metabolism, inflammation and neurovascular damage. SIRT1 activation, in retinal endothelial cells (REC) and neuronal retinal progenitor cells (R28), led to Liver X Receptor alpha (LXRα) deacetylation and subsequent increased activity, as measured by increased ATP-binding cassette transporter (ABC) A1 and G1 mRNA expression. In turn, increased cholesterol export resulted in decreased REC cholesterol levels. SIRT1 activation also led to decreased inflammation. SIRT1 activation, in vivo, prevented diabetes-induced inflammation and vascular and neural degeneration.Diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response, was significantly improved as a result of SIRT1 activation. Taken together, activation of SIRT1 signaling is an effective therapeutic strategy that provides a

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Micro-respirometry of whole cells and isolated mitochondria

Abstract: 3D printed microfluidic respirometer allows for quantitative investigation of biological energy transduction in adherent and suspension samples.

Cited by 9 publications

References 44 publications

Fasting and fasting-mimicking treatment activate SIRT1/LXRα and alleviate diabetes-induced systemic and microvascular dysfunction

Fasting and fasting-mimicking treatment activate SIRT1/LXRα and alleviate diabetes-induced systemic and microvascular dysfunction

Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes

Pharmacological and fasting-induced activation of SIRT1/LXRα signaling alleviates diabetes-induced retinopathy

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