A high-throughput respirometric assay for mitochondrial biogenesis and toxicity

Abstract

Mitochondria are a common target of toxicity for drugs and other chemicals, and results in decreased aerobic metabolism and cell death. In contrast, mitochondrial biogenesis restores cell vitality and there is a need for new agents to induce biogenesis. Current cell-based models of mitochondrial biogenesis or toxicity are inadequate because cultured cell lines are highly glycolytic with minimal aerobic metabolism and altered mitochondrial physiology. In addition, there are no high-throughput, real-time assays that assess mitochondrial function. We adapted primary cultures of renal proximal tubular cells (RPTC) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions and used the Seahorse Biosciences analyzer to measure mitochondrial function in real time in multi-well plates. Using uncoupled respiration as a marker of electron transport chain (ETC) integrity, the nephrotoxicants cisplatin, HgCl 2 and gentamicin exhibited mitochondrial toxicity prior to decreases in basal respiration and cell death. Conversely, using FCCP-uncoupled respiration as a marker of maximal ETC activity, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), SRT1720, resveratrol, daidzein, and metformin produced mitochondrial biogenesis in RPTC. The merger of the RPTC model and multi-well respirometry results in a single high throughput assay to measure mitochondrial biogenesis and toxicity, and nephrotoxic potential.

CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability

“…Agents that induce mitochondrial biogenesis have been of interest for treatment of numerous pathologies (38). Our findings indicate that drugs that induce mitochondrial biogenesis could hold promise as immunotherapeutics to improve vaccination strategies.…”

Tissue-specific metabolic reprogramming drives nutrient flux in diabetic complications

“…Additionally, FCCP-uncoupled respiration, a marker for total mitochondrial capacity (25), was decreased in diabetic mitochondria, and expression of mitochondrial uncoupling protein 2 was increased 4-fold (Supplemental Figure 4B). The results of these studies suggest that electron transport chain dysfunction, leading to less efficient ATP production and compensatory increases in glucose and fatty acid metabolic flux, may be the primary metabolic abnormality in the diabetic kidney cortex.…”

Metabolic profile analysis of a single developing zebrafish embryo via monitoring of oxygen consumption rates within a microfluidic device

“…16 Moreover, the lowest respiration due to proton leak (black) was found at 3 hpf, which lies just in the beginning of the process by which new mitochondria are formed in massive quantities in the cell (termed mitochondrial biogenesis). 17 The proton leak through the fully developed mitochondrial inner membrane to the matrix can prompt mitochondrial respiration. Thus, the minimum in the leak levels at 3 hpf might be because these newly nascent mitochondria might not FIG.…”

“…The high proton leak levels after 7 hpf, during the segmentation stage (10-24 hpf) of the embryo, indicate that the mitochondria are now fully functional. 17 The moderately high proton leak could reduce free radical production and increase the sensitivity of metabolic reactions to effectors, which would benefit a developing embryo during the segmentation stage. 18 Prior to hatching, increased cardiac activity at 48 hpf could result in the increase in both mitochondrial and non-mitochondrial respiration by decreasing the respiration due to proton leak.…”