Chronic Treatment with Azide in Situ Leads to an Irreversible Loss of Cytochrome c Oxidase Activity via Holoenzyme Dissociation

Leary, Scot C.; Hill, Bruce C.; Lyons, C. N.; Carlson, Christopher G.; Michaud, Denise; Kraft, Claudia S.; Ko, Kenton; Glerum, D. Moira; Moyes, Christopher D.

doi:10.1074/jbc.m112303200

Cited by 78 publications

(76 citation statements)

References 64 publications

(55 reference statements)

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“…For all further experiments, azide concentrations of 50 and 75 M were used. Previous studies have demonstrated that, at micromolar concentrations of azide, the activity of cytochrome c oxidase (COX) is specifically reduced without affecting either mRNA or protein expression levels of COX or the activity of other mitochondrial enzymes (14,15). Assessment of cell viability by trypan blue exclusion also demonstrated that, in this study, treatment with micromolar concen-trations of azide did not have a detrimental effect on cell viability.…”

Section: Measurement Of Cellular Respiratory Chain Function In Azidetsupporting

confidence: 49%

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Does impaired mitochondrial function affect insulin signaling and action in cultured human skeletal muscle cells?

Brown

Elstner²,

Yeaman³

et al. 2008

American Journal of Physiology-Endocrinology and Metabolism

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To address this, a model of impaired cellular respiratory function was established by incubating human skeletal muscle cell cultures with the mitochondrial inhibitor sodium azide and examining the effects on insulin action. Incubation of human skeletal muscle cells with 50 and 75 M azide resulted in 48 Ϯ 3% and 56 Ϯ 1% decreases, respectively, in respiration compared with untreated cells mimicking the level of impairment seen in type 2 diabetes. Under conditions of decreased respiratory chain function, insulin-independent (basal) glucose uptake was significantly increased. Basal glucose uptake was 325 Ϯ 39 pmol/min/mg (mean Ϯ SE) in untreated cells. This increased to 669 Ϯ 69 and 823 Ϯ 83 pmol/min/mg in cells treated with 50 and 75 M azide, respectively (vs. untreated, both P Ͻ 0.0001). Azide treatment was also accompanied by an increase in basal glycogen synthesis and phosphorylation of AMP-activated protein kinase. However, there was no decrease in glucose uptake following insulin exposure, and insulin-stimulated phosphorylation of Akt was normal under these conditions. GLUT1 mRNA expression remained unchanged, whereas GLUT4 mRNA expression increased following azide treatment. In conclusion, under conditions of impaired mitochondrial respiration there was no evidence of impaired insulin signaling or glucose uptake following insulin exposure in this model system.

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Section: Measurement Of Cellular Respiratory Chain Function In Azidetsupporting

confidence: 49%

“…To address this specific question, we studied insulin action in primary human skeletal muscle cultures and titrated the concentration of azide, a specific inhibitor of cytochrome c oxidase (complex IV) in the mitochondrial respiratory chain (14,15), to regulate the degree of suppression of mitochondrial respiration.…”

mentioning

confidence: 99%

Does impaired mitochondrial function affect insulin signaling and action in cultured human skeletal muscle cells?

Brown

Elstner²,

Yeaman³

et al. 2008

American Journal of Physiology-Endocrinology and Metabolism

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“…30 It is well established that NaN 3 is a good quencher of singlet oxygen. Quenching rate constants, k q , over the range ~ 3-6 × 10 8 s -1 M -1 have been reported for experiments performed in aqueous systems, and a value as large as 5 × 10 9 s -1 M -1 has been reported for CH 3 CN.…”

Section: Singlet Oxygen Quenching By Sodium Azide In Sucrose Solutionmentioning

confidence: 99%

Singlet Oxygen in a Cell: Spatially Dependent Lifetimes and Quenching Rate Constants

2008

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Singlet molecular oxygen, O 2 (a 1 ∆ g ), can be created in a single cell from ground state oxygen, O 2 (X 3 Σ g -), upon focused laser irradiation of an intracellular sensitizer. This cytotoxic species can subsequently be detected by its 1270 nm phosphorescence (a 1 ∆ g → X 3 Σ g -) with subcellular spatial resolution. The singlet oxygen lifetime determines its diffusion distance and, hence, the intracellular volume element in which singlet-oxygen-initiated perturbation of the cell occurs. In this study, the time-resolved phosphorescence of singlet oxygen produced by the sensitizers chlorin (Chl) and 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) was monitored. These molecules localize in different domains of a living cell. The data indicate that (i) the singlet oxygen lifetime and (ii) the rate constant for singlet oxygen quenching by added NaN 3 depend on whether Chl or TMPyP was the photosensitizer.These observations likely reflect differences in the chemical and physical constituency of a given subcellular domain (e.g., spatially-dependent oxygen and NaN 3 diffusion coefficients) and, as such, are evidence that singlet oxygen responds to the inherent heterogeneity of a cell.Thus, despite a relatively long intracellular lifetime, singlet oxygen does not diffuse a great distance from its site of production. This is a consequence of an apparent intracellular viscosity that is comparatively large.3

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“…We investigated how myogenesis was affected by the inhibition of mitochondrial activity or by glucose deprivation. NaN 3 can irreversibly inhibit cytochrome c oxidase (50% inhibitory concentration, Ͻ10 M) in C2C12 cells (31), thus blocking the oxidative phosphorylation in mitochondria. However, myogenic differentiation of C2C12 myoblasts did not change significantly when chronically treated with 100 M NaN 3 in the presence of 4.5 g of glucose per liter (Fig.…”

Section: Myogenesis Under Hypoxiamentioning

confidence: 99%

Adaptive Myogenesis under Hypoxia

Yun

Lin

Giaccia

2005

Molecular and Cellular Biology

128

171

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Previous studies have indicated that myoblasts can differentiate and repair muscle injury after an ischemic insult. However, it is unclear how hypoxia or glucose deprivation in the ischemic microenvironment affects myoblast differentiation. We have found that myogenesis can adapt to hypoxic conditions. This adaptive mechanism is accompanied by initial inhibition of the myoD, E2A, and myogenin genes followed by resumption of their expression in an oxygen-dependent manner. The regulation of myoD transcription by hypoxia is correlated with transient deacetylation of histones associated with the myoD promoter. It is noteworthy that, unlike the differentiation of other cell types such as preadipocytes or chondroblasts, the effect of hypoxia on myogenesis is independent of HIF-1, a ubiquitous regulator of transcription under hypoxia. While myogenesis can also adapt to glucose deprivation, the combination of severe hypoxia and glucose deprivation found in an ischemic environment results in pronounced loss of myoblasts. Our studies indicate that the ischemic muscle can be repaired via the adaptive differentiation of myogenic precursors, which depends on the levels of oxygen and glucose in the ischemic microenvironment.Skeletal muscle possesses the remarkable ability to withstand chronic ischemia. However, the newly developed muscle fibers tend to be smaller in diameter than those of healthy, nonischemic myofibers (46). Interestingly, chronic exposure to high altitude can also result in smaller myofiber area and lower muscle mass in mountaineers (25). Similar myofiber differences are also observed in healthy people who live at high altitude for generations (25). Taken together, these observations suggest that decreased oxygen tension or hypoxia alone may be sufficient to alter the differentiation of myoblasts.Ischemic muscle damage is repaired by myogenic satellite cells, a small population of precursor cells located between myofibers (12,23,47). When stimulated, the otherwise quiescent satellite cells undergo terminal differentiation into myocytes to regenerate damaged myofibers. It has been found that cultured satellite cells form new muscle fibers when transplanted into ischemic muscle (12). The myogenic differentiation of myoblasts is highly orchestrated by a family of myogenic regulatory factors (MRFs), such as myoD, myogenin, myf5, and MRF4 in collaboration with the E2A gene products (E12 or E47) and/or the myogenic enhancing factors (6, 41, 53). Although a plethora of information on the embryonic development of muscle exists, there is still a lack of clear understanding of how postnatal myogenic satellite cells are regulated during muscle regeneration, especially by their microenvironment.Tissue ischemia in myocardium and skeletal muscle results in hypoxia and elevates the expression of the hypoxia-inducible transcription factor HIF-1␣ and its downstream gene, VEGF (32,40). Although ischemic tissue is exposed to hypoxia and decreased nutritional supply, cells will alter their metabolism, proliferation, and differe...

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Chronic Treatment with Azide in Situ Leads to an Irreversible Loss of Cytochrome c Oxidase Activity via Holoenzyme Dissociation

Cited by 78 publications

References 64 publications

Does impaired mitochondrial function affect insulin signaling and action in cultured human skeletal muscle cells?

Does impaired mitochondrial function affect insulin signaling and action in cultured human skeletal muscle cells?

Singlet Oxygen in a Cell: Spatially Dependent Lifetimes and Quenching Rate Constants

Adaptive Myogenesis under Hypoxia

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