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Christensen M.E.,Materials Medical Research Institute | Christensen M.E.,The Queensland Institute for Medical Research | Jansen E.S.,Materials Medical Research Institute | Sanchez W.,The Translational Research Institute | And 5 more authors.
Methods | Year: 2013

Mitochondria play a pivotal role in life and death of the cell because they produce the majority of energy required for survival and also regulate the intrinsic pathway to apoptosis. The involvement of mitochondria in cell death is generally measured by following mitochondrial membrane depolarisation or mitochondrial outer membrane permeabilisation (MOMP). These events can be assayed using cationic dyes that are attracted to the negative charge across the inner membrane of healthy mitochondria or by following translocation of cytochrome c from the mitochondria to the cytoplasm respectively. These events progress rapidly in individual cells but are observed as bi-phasic peaks in flow cytometry assays because cell death generally occurs asynchronously in a population. This allows researchers to use flow cytometry to easily distinguish healthy cells with intact mitochondria healthy from dying cells with permeabilised mitochondria. This article will therefore review methods using flow cytometry to follow mitochondrial membrane depolarisation and cytochrome c release during apoptosis, and will highlight some studies that resulted in development of these assays. © 2013 Elsevier Inc.

Barclay J.L.,University of Queensland | Barclay J.L.,The Translational Research Institute | Agada H.,University of Queensland | Agada H.,The Translational Research Institute | And 8 more authors.
Journal of Endocrinology | Year: 2015

Clinical cases of glucocorticoid (GC) excess are characterized by increased fat mass and obesity through the accumulation of white adipocytes. The effects of GCs on growth and function of brown adipose tissue are unknown and may contribute to the negative energy balance observed clinically. This study aims to evaluate the effect of GCs on proliferation, differentiation, and metabolic function of brown adipocytes. Human brown adipocytes sourced from supraclavicular fat biopsies were grown in culture and differentiated to mature adipocytes. Human white adipocytes sourced from subcutaneous abdominal fat biopsies were cultured as controls. Effects of dexamethasone on growth, differentiation (UCP1, CIDEA, and PPARGC1A expression), and function (oxygen consumption rate (OCR)) of brown adipocytes were quantified. Dexamethasone (1 μM) significantly stimulated the proliferation of brown preadipocytes and reduced that of white preadipocytes. During differentiation, dexamethasone (at 0.1, 1, and 10 μM) stimulated the expression of UCP1, CIDEA, and PPARGC1A in a concentration-dependent manner and enhanced by fourfold to sixfold the OCR of brown adipocytes. Isoprenaline (100 nM) significantly increased (P<0.05) expression of UCP1 and OCR of brown adipocytes. These effects were significantly reduced (P<0.05) by dexamethasone. Thus, we show that dexamethasone stimulates the proliferation, differentiation, and function of human brown adipocytes but inhibits adrenergic stimulation of the functioning of brown adipocytes. We conclude that GCs exert complex effects on development and function of brown adipocytes. These findings provide strong evidence for an effect of GCs on the biology of human brown adipose tissue (BAT) and for the involvement of the BAT system in the metabolic manifestation of Cushing's syndrome. © 2015 Society for Endocrinology.

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