scholarly journals Plate-Based Respirometry to Assess Thermal Sensitivity of Zebrafish Embryo Bioenergetics in situ

2021 ◽  
Vol 12 ◽  
Author(s):  
Erik Rollwitz ◽  
Martin Jastroch
Keyword(s):  
Oxygen Consumption ◽  
Thermal Sensitivity ◽  
Coupling Efficiency ◽  
Temperature Acclimation ◽  
Effect Of Temperature ◽  
Proton Leak ◽  
Zebrafish Embryos ◽  
Atp Production ◽  
Extracellular Flux

Oxygen consumption allows measuring the metabolic activity of organisms. Here, we adopted the multi-well plate-based respirometry of the extracellular flux analyzer (Seahorse XF96) to investigate the effect of temperature on the bioenergetics of zebrafish embryos (Danio rerio) in situ. We show that the removal of the embryonic chorion is beneficial for oxygen consumption rates (OCR) and penetration of various mitochondrial inhibitors, and confirm that sedation reduces the variability of OCR. At 48h post-fertilization, embryos (maintained at a routine temperature of 28°C) were exposed to different medium temperatures ranging from 18°C to 37°C for 20h prior OCR measurement. Measurement temperatures from 18°C to 45°C in the XF96 were achieved by lowering the room temperature and active in-built heating. At 18°C assay temperature, basal OCR was low due to decreased ATP-linked respiration, which was not limited by mitochondrial power, as seen in substantial spare respiratory capacity. Basal OCR of the embryos increased with assay temperature and were stable up to 37°C assay temperature, with pre-exposure of 37°C resulting in more thermo-resistant basal OCR measured at 41°C. Adverse effects of the mitochondrial inhibitor oligomycin were seen at 37°C and chemical uncouplers disrupted substrate oxidation gradually with increasing assay temperature. Proton leak respiration increased at assay temperatures above 28°C and compromised the efficiency of ATP production, calculated as coupling efficiency. Thus, temperature impacts mitochondrial respiration by reduced cellular ATP turnover at lower temperatures and by increased proton leak at higher temperatures. This conclusion is coherent with the assessment of heart rate, an independent indicator of systemic metabolic rate, which increased with exposure temperature, peaking at 28°C, and decreased at higher temperatures. Collectively, plate-based respirometry allows assessing distinct parts of mitochondrial energy transduction in zebrafish embryos and investigating the effect of temperature and temperature acclimation on mitochondrial bioenergetics in situ.

scholarly journals Mitochondrial Respiration in Female Zucker Rats: Effects of Obesity and Short-Term Metformin Treatment

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1682-1682
Author(s):  
Shannon Rose ◽  
Eugenia Carvalho ◽  
David Irby ◽  
Sirish Bennuri ◽  
Alexandria Beebe ◽  
...  
Keyword(s):  
Mitochondrial Respiration ◽  
Coupling Efficiency ◽  
Zucker Rats ◽  
Proton Leak ◽  
Acid Oxidation ◽  
Flux Analysis ◽  
Metformin Treatment ◽  
Short Term ◽  
Funding Sources ◽  
Extracellular Flux

Abstract Objectives As childhood obesity rates climb, so has the incidence of type 2 diabetes among children and adolescents. Metformin, an FDA approved anti-hyperglycemic drug, is thought to target the mitochondria, but its effects on mitochondrial function in obesity have not been well studied. We used an obese Zucker rat model to investigate the effects of obesity and short-term metformin treatment on mitochondrial respiration. Methods Five-week old female Zucker rats (n = 16 lean, n = 16 obese) were fed AIN-93 G diet for 8 weeks before equally randomized to receive metformin (mixed in diet at 1 g/kg of feed); thus forming 4 groups with 8 rats each: lean +/– metformin and obese +/– metformin. Rats were sacrificed 10 weeks post-metformin treatment and spleens, perigonadal visceral adipose tissue (VAT) and skeletal muscle (SM; gracilis) collected. Mitochondrial respiration was measured in splenocytes by extracellular flux analysis and in VAT and SM fibers by high resolution respirometry. Results Effects of obesity on mitochondrial respiration were found in VAT and SM, but not splenocytes. In VAT, obese rats exhibited increased OXPHOS capacity over lean rats when octanoylcarnitine and malate were provided as substrates (obese vs lean: 1.33 vs 0.76 pmol O2/s/mg; SEdiff = 0.18, P = .005), and after subsequent additions of pyruvate (P = .012), glutamate (P = .009), and succinate (P = .045). In SM, OXPHOS capacity was increased when octanoylcarnitine and malate were provided as substrates (obese vs lean: 12.18 vs 5.45 pmol O2/s/mg; SEdiff = 2.31, P = .011) in obese vs lean rats. Metformin effects were observed only in splenocytes: coupling efficiency was decreased (metformin vs no metformin; 56.2% vs 69.8%; SEdiff = 4.1%, P = 0.005) and proton leak was increased (P < .001) in metformin treated rats as compared to rats not treated with metformin. Conclusions We found obesity was associated with increased mitochondrial respiration, particularly fatty acid oxidation, in VAT and SM. Short-term metformin treatment did not alter mitochondrial respiration in VAT or SM, but was found to increase proton leak and reduce coupling efficiency in splenocytes. Funding Sources Arkansas Children's Research Institute, Arkansas Biosciences Institute (R.H). S.R. and E.C. are also supported by the National Institute of General Medical Sciences of the National Institutes of Health.

scholarly journals THERMAL ACCLIMATION ALTERS BOTH ADRENERGIC SENSITIVITY AND ADRENOCEPTOR DENSITY IN CARDIAC TISSUE OF RAINBOW TROUT

1993 ◽  
Vol 181 (1) ◽  
pp. 27-48 ◽  
Author(s):  
J. E. Keen ◽  
D. M. Vianzon ◽  
A. P. Farrell ◽  
G. F. Tibbits
Keyword(s):  
Rainbow Trout ◽  
Cardiac Tissue ◽  
Radioligand Binding ◽  
Temperature Acclimation ◽  
Effect Of Temperature ◽  
Acclimation Temperature ◽  
Binding Studies ◽  
Tension Development

We examined the effect of temperature acclimation on the sensitivity of the rainbow trout heart to adrenaline and on the density of beta-adrenoceptors. The sensitivity of the heart was assessed using in situ working perfused heart and in vitro isometric ventricular strip preparations. When tested in situ and at acclimation temperature, hearts from fish acclimated to 8°C were approximately 10-fold more sensitive to adrenaline-supplemented perfusate than were hearts from fish acclimated to 18°C. The concentrations required for half-maximal stimulation (EC50) of myocardial power output were 1.9×10- 8 mol l-1 adrenaline and 1.7×10-7 mol l-1 adrenaline for hearts acclimated to 8°C and 18°C, respectively. In vitro, isometric ventricular strip preparations demonstrated a similar increase in adrenergic sensitivity with cold-acclimation. The EC50 values for maximal tension development were 2.7×10-7 mol l-1 adrenaline (8°C-acclimated) and 1.1×10-6 mol l-1 adrenaline (18°C-acclimated) when tested at acclimation temperature. This shift in adrenergic sensitivity was a function of the temperature acclimation because changes in bath temperature per se, either from 8°C to 18°C for 8°C- acclimated hearts or from 18°C to 8°C for 18°C-acclimated hearts, had no significant effect on the concentration-response curve for adrenaline. We conducted radioligand binding studies with [125I]iodocyanopindolol and propranolol to quantify the beta-adrenoceptor density (Bmax) of both homogenates and isolated sarcolemmal fractions of ventricles from rainbow trout acclimated to either 8°C or 18°C. The Bmax for isolated sarcolemmal fractions was significantly higher in the 8°C-acclimated group, but the Bmax of ventricular homogenates was not significantly different in the two acclimation groups. No significant differences in dissociation constant (Kd) were apparent in either the homogenates or sarcolemmal fractions. These results suggest that cardiac tissue from rainbow trout acclimated to 8°C has a greater cell surface adrenoceptor population available for beta-antagonist binding. This might explain the heightened cardiac sensitivity to adrenaline observed in situ and in vitro in 8°C-acclimated fish.

scholarly journals Temperature affects the diffusion of small molecules through cytosol of fish muscle

1987 ◽  
Vol 129 (1) ◽  
pp. 191-203 ◽  
Author(s):  
B. D. Sidell ◽  
J. R. Hazel
Keyword(s):  
Small Molecules ◽  
Diffusion Coefficients ◽  
White Perch ◽  
Thermal Sensitivity ◽  
Fish Muscle ◽  
Temperature Acclimation ◽  
Effect Of Temperature ◽  
Relative Temperature ◽  
D Values

Undiluted cytosolic extracts were prepared from fast glycolytic muscle tissue of white perch (Morone americanus). Diffusion coefficients (D) through the cytosol preparations were estimated in vitro for a series of selected low molecular weight compounds using an experimental diffusion chamber. Determinations were made at 5 degrees and 25 degrees C to assess thermal sensitivity of the process. Non-metabolizable analogues of naturally occurring compounds were employed to avoid chemical alteration of solutes by the catalytically competent preparations during diffusion experiments. Kinematic viscosity of cytosolic extracts, which is a major determinant of diffusive resistance, increases from 2.94 +/− 0.06 to 5.35 +/− 0.02 X 10(−2) cm2 s-1 between temperatures of 25 degrees and 5 degrees C (Q10 = 1.35 +/− 0.01). The diffusion coefficients (D) of D-lactic acid are 2.26 +/− 0.84 and 0.79 +/− 0.15 X 10(−6) cm2s-1 at 25 degrees and 5 degrees C, respectively (Q10 = 1.84 +/− 0.36). The D values of 2-deoxyglucose are 2.87 +/− 1.01 and 1.22 +/− 0.36 X 10(−6) cm2s-1 at 25 degrees and 5 degrees C (Q10 = 1.75 +/− 0.54). The D values of Ca2+ are 2.47 +/− 0.28 and 1.09 +/− 0.36 X 10(−6) cm2s-1 at 25 degrees and 5 degrees C (Q10 = 2.04 +/− 0.36). The D values for the ATP analogue, AMP-PNP, are 0.87 +/− 0.33 and 0.81 +/− 0.15 X 10(−6) cm2s-1 at 25 degrees and 5 degrees C (Q10 = 0.98 +/− 0.12). AMP-PNP is the only compound tested which did not show significant thermal sensitivity of diffusion. Recently reported changes in muscle cell ultrastructure induced by temperature acclimation of fishes may serve to counteract the effect of temperature change on diffusion of key small molecules through the aqueous cytoplasm, thus maintaining flux rates between cellular compartments. These mechanisms may be of considerable import in achieving relative temperature independence of cellular function that is characteristic of many eurythermal aquatic animals.

scholarly journals Cell-Permeable Succinate Rescues Mitochondrial Respiration in Cellular Models of Statin Toxicity

2021 ◽  
Vol 22 (1) ◽  
pp. 424
Author(s):  
Vlad F. Avram ◽  
Imen Chamkha ◽  
Eleonor Åsander-Frostner ◽  
Johannes K. Ehinger ◽  
Romulus Z. Timar ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Respiration ◽  
Complex I ◽  
Ex Vivo ◽  
Coupling Efficiency ◽  
Human Platelets ◽  
Lipid Lowering ◽  
Lipid Lowering Therapy ◽  
Cellular Models

Statins are the cornerstone of lipid-lowering therapy. Although generally well tolerated, statin-associated muscle symptoms (SAMS) represent the main reason for treatment discontinuation. Mitochondrial dysfunction of complex I has been implicated in the pathophysiology of SAMS. The present study proposed to assess the concentration-dependent ex vivo effects of three statins on mitochondrial respiration in viable human platelets and to investigate whether a cell-permeable prodrug of succinate (complex II substrate) can compensate for statin-induced mitochondrial dysfunction. Mitochondrial respiration was assessed by high-resolution respirometry in human platelets, acutely exposed to statins in the presence/absence of the prodrug NV118. Statins concentration-dependently inhibited mitochondrial respiration in both intact and permeabilized cells. Further, statins caused an increase in non-ATP generating oxygen consumption (uncoupling), severely limiting the OXPHOS coupling efficiency, a measure of the ATP generating capacity. Cerivastatin (commercially withdrawn due to muscle toxicity) displayed a similar inhibitory capacity compared with the widely prescribed and tolerable atorvastatin, but did not elicit direct complex I inhibition. NV118 increased succinate-supported mitochondrial oxygen consumption in atorvastatin/cerivastatin-exposed platelets leading to normalization of coupled (ATP generating) respiration. The results acquired in isolated human platelets were validated in a limited set of experiments using atorvastatin in HepG2 cells, reinforcing the generalizability of the findings.

scholarly journals The Role of the Pathogen Dose and PI3Kγ in Immunometabolic Reprogramming of Microglia for Innate Immune Memory

2021 ◽  
Vol 22 (5) ◽  
pp. 2578
Author(s):  
Trim Lajqi ◽  
Christian Marx ◽  
Hannes Hudalla ◽  
Fabienne Haas ◽  
Silke Große ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Immune Tolerance ◽  
Immune Cells ◽  
Low Dose ◽  
Innate Immune ◽  
Immune Memory ◽  
Innate Immune Cells ◽  
Atp Production ◽  
Dose Dependent

Microglia, the innate immune cells of the CNS, exhibit long-term response changes indicative of innate immune memory (IIM). Our previous studies revealed IIM patterns of microglia with opposing immune phenotypes: trained immunity after a low dose and immune tolerance after a high dose challenge with pathogen-associated molecular patterns (PAMP). Compelling evidence shows that innate immune cells adopt features of IIM via immunometabolic control. However, immunometabolic reprogramming involved in the regulation of IIM in microglia has not been fully addressed. Here, we evaluated the impact of dose-dependent microglial priming with ultra-low (ULP, 1 fg/mL) and high (HP, 100 ng/mL) lipopolysaccharide (LPS) doses on immunometabolic rewiring. Furthermore, we addressed the role of PI3Kγ on immunometabolic control using naïve primary microglia derived from newborn wild-type mice, PI3Kγ-deficient mice and mice carrying a targeted mutation causing loss of lipid kinase activity. We found that ULP-induced IIM triggered an enhancement of oxygen consumption and ATP production. In contrast, HP was followed by suppressed oxygen consumption and glycolytic activity indicative of immune tolerance. PI3Kγ inhibited glycolysis due to modulation of cAMP-dependent pathways. However, no impact of specific PI3Kγ signaling on immunometabolic rewiring due to dose-dependent LPS priming was detected. In conclusion, immunometabolic reprogramming of microglia is involved in IIM in a dose-dependent manner via the glycolytic pathway, oxygen consumption and ATP production: ULP (ultra-low-dose priming) increases it, while HP reduces it.

Effect of temperature on streambed vertical hydraulic conductivity

2013 ◽  
Vol 45 (1) ◽  
pp. 89-98 ◽  
Author(s):  
Weihong Dong ◽  
Gengxin Ou ◽  
Xunhong Chen ◽  
Zhaowei Wang
Keyword(s):  
Hydraulic Conductivity ◽  
Water Temperature ◽  
Sediment Cores ◽  
Effect Of Temperature ◽  
In Situ Tests ◽  
Effect Of Water ◽  
Vertical Hydraulic Conductivity ◽  
Increasing Trend ◽  
Streambed Vertical Hydraulic Conductivity

In this study, in situ and on-site permeameter tests were conducted in Clear Creek, Nebraska, USA to evaluate the effect of water temperature on streambed vertical hydraulic conductivity Kv. Fifty-two sediment cores were tested. Five of them were transferred to the laboratory for a series of experiments to evaluate the effect of water temperature on Kv. Compared with in situ tests, 42 out of the 52 tests have higher Kv values for on-site tests. The distribution of water temperature at the approximately 50 cm depth of streambed along the sand bar was investigated in the field. These temperatures had values in the range 14–19 °C with an average of 16 °C and had an increasing trend along the stream flow. On average, Kv values of the streambed sediments in the laboratory tests increase by 1.8% per 1 °C increase in water temperature. The coarser sandy sediments show a greater increase extent of the Kv value per 1 °C increase in water temperature. However, there is no distinct increasing trend of Kv value for sediment containing silt and clay layers.

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