Metabolic maturation of differentiating cardiosphere-derived cells.

Cardiosphere-derived cells (CDCs) can be expanded in vitro and induced to differentiate along the cardiac lineage. To recapitulate the phenotype of an adult cardiomyocyte, differentiating progenitors need to upregulate mitochondrial glucose and fatty acid oxidation. Here we cultured and differentiated CDCs using protocols aimed to maintain stemness or to promote differentiation, including triggering fatty acid oxidation using an agonist of peroxisome proliferator-activated receptor alpha (PPARα). Metabolic changes were characterised in undifferentiated CDCs and during differentiation towards a cardiac phenotype. CDCs from rat atria were expanded on fibronectin or collagen IV via cardiosphere formation. Differentiation was assessed using flow cytometry and qPCR and substrate metabolism was quantified using radiolabelled substrates. Collagen IV promoted proliferation of CDCs whereas fibronectin primed cells for differentiation towards a cardiac phenotype. In both populations, treatment with 5-Azacytidine induced a switch towards oxidative metabolism, as shown by changes in gene expression, decreased glycolytic flux and increased oxidation of glucose and palmitate. Addition of a PPARα agonist during differentiation increased both glucose and fatty acid oxidation and expression of cardiac genes. We conclude that oxidative metabolism and cell differentiation act in partnership with increases in one driving an increase in the other.

Exploiting the metabolic energy demands of drug efflux pumps provides a strategy to overcome multidrug resistance in cancer.

BACKGROUND: P-glycoprotein (P-gp) is a prevalent resistance mediator and it requires considerable cellular energy to ensure ATP dependent efflux of anticancer drugs. The glycolytic pathway generates the majority of catabolic energy in cancer cells; however, the high rates of P-gp activity places added strain on its inherently limited capacity to generate ATP. This is particularly relevant for compounds such as verapamil that are believed to trap P-gp in a futile transport process that requires continuing ATP consumption. Ultimately, this leads to cell death and the hypersensitivity of resistant cells to verapamil is termed collateral sensitivity. RESULTS: We show that the addition of verapamil to resistant cells produces a prominent reduction in ATP levels that supports the idea of disrupted energy homeostasis. Even in the absence of verapamil, P-gp expressing cells display near maximal rates of glycolysis and oxidative phosphorylation, which prevents an adequate response to the demand for ATP to sustain transport activity. Moreover, the near perpetually maximal rate of oxidative phosphorylation in the presence of verapamil resulted in elevated levels of reactive oxygen species that affect cell survival and underscore collateral sensitivity. CONCLUSIONS: Our results demonstrate that the strained metabolic profiles of P-gp expressing resistant cancer cells can be overwhelmed by additional ATP demands. GENERAL SIGNIFICANCE: Consequently, collateral sensitising drugs may overcome the resistant phenotype by exploiting, rather than inhibiting, the energy demanding activity of pumps such as P-gp.

The utilisation of glutamine and glucose by a 3-D tumour model trapped in quiescence.

Solid tumours modify their metabolic strategy to ensure sufficient biomass and energy to maintain a high rate of proliferation. However, solid tumours are characterised by a high proportion of quiescent cells and little is known about their metabolic profile. A tumour spheroid model with DLD1 cells was used to investigate the influence of a quiescent state on the cellular utilisation of glucose and glutamine. Quiescent DLD1 spheroids displayed increased depletion of both nutrients from the bathing medium compared to their proliferative counterparts and displayed highly active overall metabolism. A combination of biochemical and metabolomics approaches demonstrated that glucose utilisation resulted in an increased production of the 3-carbon intermediates lactate and alanine in quiescent spheroids. In addition, glutamine metabolism was directed to anabolic pathways; including the "reverse TCA cycle" to produce citrate for fatty-acid synthesis. These adaptations in DLD1 spheroids may propose a metabolic altruism of quiescent regions in solid tumours to provide biosynthetic intermediates required to sustain tumour growth, angiogenesis and metastasis.

Regulation of Postabsorptive and Postprandial Glucose Metabolism by Insulin-Dependent and Insulin-Independent Mechanisms: An Integrative Approach.

Glucose levels in blood must be constantly maintained within a tight physiological range to sustain anabolism. Insulin regulates glucose homeostasis via its effects on glucose production from the liver and kidneys and glucose disposal in peripheral tissues (mainly skeletal muscle). Blood levels of glucose are regulated simultaneously by insulin-mediated rates of glucose production from the liver (and kidneys) and removal from muscle; adipose tissue is a key partner in this scenario, providing nonesterified fatty acids (NEFA) as an alternative fuel for skeletal muscle and liver when blood glucose levels are depleted. During sleep at night, the gradual development of insulin resistance, due to growth hormone and cortisol surges, ensures that blood glucose levels will be maintained within normal levels by: (a) switching from glucose to NEFA oxidation in muscle; (b) modulating glucose production from the liver/kidneys. After meals, several mechanisms (sequence/composition of meals, gastric emptying/intestinal glucose absorption, gastrointestinal hormones, hyperglycemia mass action effects, insulin/glucagon secretion/action, de novo lipogenesis and glucose disposal) operate in concert for optimal regulation of postprandial glucose fluctuations. The contribution of the liver in postprandial glucose homeostasis is critical. The liver is preferentially used to dispose over 50% of the ingested glucose and restrict the acute increases of glucose and insulin in the bloodstream after meals, thus protecting the circulation and tissues from the adverse effects of marked hyperglycemia and hyperinsulinemia.

UCP2 - Taking the heat out of P-glycoprotein?

Cancer cells are highly proliferative, invasive, metastatic and initiate angiogenesis. These activities demand plentiful energy and bountiful stores of anabolic precursors, a situation that puts significant strain on metabolic pathways and necessitates juggling of finite resources. However, the location and erratic structural organisation of tumours means they reside in a nutrient-poor environment. The glycolytic phenotype has evolved in cancer cells to provide a suitable balance between bioenergetic and biosynthetic pathways. Does this adopted strategy also support the overexpression of an ATP-dependent transporter (P-glycoprotein) to maintain resistance against chemotherapy? This article highlights the metabolic adaptations used by cancer cells to maintain both a glycolytic phenotype and sustain the activity of P-glycoprotein. We argue that these cells negotiate an energy precipice to achieve these adaptations. Finally, we advocate the use of compounds that place resistant cells expressing P-glycoprotein under further metabolic strain and how uncoupling protein-2 may provide an ideal target for them.

Context-dependent regulation of endothelial cell metabolism: differential effects of the PPARß/δ agonist GW0742 and VEGF-A.

Peroxisome proliferator activated receptor ß/δ (PPARß/δ) has pro-angiogenic functions, but whether PPARß/δ modulates endothelial cell metabolism to support the dynamic phenotype remains to be established. This study characterised the metabolic response of HUVEC to the PPARß/δ agonist, GW0742, and compared these effects with those induced by VEGF-A. In HUVEC monolayers, flux analysis revealed that VEGF-A promoted glycolysis at the expense of fatty acid oxidation (FAO), whereas GW0742 reduced both glycolysis and FAO. Only VEGF-A stimulated HUVEC migration and proliferation whereas both GW0742 and VEGF-A promoted tubulogenesis. Studies using inhibitors of PPARß/δ or sirtuin-1 showed that the tubulogenic effect of GW0742, but not VEGF-A, was PPARß/δ- and sirtuin-1-dependent. HUVEC were reliant on glycolysis and FAO, and inhibition of either pathway disrupted cell growth and proliferation. VEGF-A was a potent inducer of glycolysis in tubulogenic HUVEC, while FAO was maintained. In contrast, GW0742-induced tubulogenesis was associated with enhanced FAO and a modest increase in glycolysis. These novel data reveal a context-dependent regulation of endothelial metabolism by GW0742, where metabolic activity is reduced in monolayers but enhanced during tubulogenesis. These findings expand our understanding of PPARß/δ in the endothelium and support the targeting of PPARß/δ in regulating EC behaviour and boosting tissue maintenance and repair.

Acetoacetate is a more efficient energy-yielding substrate for human mesenchymal stem cells than glucose and generates fewer reactive oxygen species.

Stem cells have been assumed to demonstrate a reliance on anaerobic energy generation, suited to their hypoxic in vivo environment. However, we found that human mesenchymal stem cells (hMSCs) have an active oxidative metabolism with a range of substrates. More ATP was consistently produced from substrate oxidation than glycolysis by cultured hMSCs. Strong substrate preferences were shown with the ketone body, acetoacetate, being oxidised at up to 35 times the rate of glucose. ROS-generation was 45-fold lower during acetoacetate oxidation compared with glucose and substrate preference may be an adaptation to reduce oxidative stress. The UCP2 inhibitor, genipin, increased ROS production with either acetoacetate or glucose by 2-fold, indicating a role for UCP2 in suppressing ROS production. Addition of pyruvate stimulated acetoacetate oxidation and this combination increased ATP production 27-fold, compared with glucose alone, which has implications for growth medium composition. Oxygen tension during culture affected metabolism by hMSCs. Between passages 2 and 5, rates of both glycolysis and substrate-oxidation increased at least 2-fold for normoxic (20% O2)- but not hypoxic (5% O2)-cultured hMSCs, despite declining growth rates and no detectable signs of differentiation. Culture of the cells with 3-hydroxybutyrate abolished the increased rates of these pathways. These findings have implications for stem cell therapy, which necessarily involves in vitro culture of cells, since low passage number normoxic cultured stem cells show metabolic adaptations without detectable changes in stem-like status.

The Effects of Severe Hypoxia on Glycolytic Flux and Enzyme Activity in a Model of Solid Tumors.

Solid tumors contend with, and adapt to, a hostile micro-environment that includes limited availability of nutrient fuels and oxygen. The presence of hypoxia (O2 <5%) stabilizes the transcription factor Hif1 and results in numerous cellular adaptations including increased flux of glucose through glycolysis. Increasingly, more sophisticated analysis of tumor oxygenation has revealed large gradients of oxygen tension and significant regions under severe hypoxia (O2 ∼0.1%). The present investigation has demonstrated a significant increase in the glycolytic flux rate when tumor spheroids were exposed to 0.1% O2 . The severe hypoxia was associated with uniform pimonidazole adduct formation and elevated levels of Hif1α and c-Myc. This resulted in elevated expression of GLUT and MCT transporters, in addition to increased activity of PFK1 in comparison to that observed in normoxia. However, the protein expression and enzymatic capacity of HK2, G6PDH, PK, and LDH were all reduced by severe hypoxia. Clearly, the effects of exposure to severe hypoxia lead to a significantly abridged Hif1 response, yet one still able to elevate glycolytic flux and prevent loss of intermediates to anabolism. J. Cell. Biochem. 117: 1890-1901, 2016. © 2016 Wiley Periodicals, Inc.

Essential role of excessive tryptophan and its neurometabolites in fatigue.

PURPOSE: Serotonin, a neurotransmitter synthesized from tryptophan, has been proposed to play a key role in central fatigue. In this study, we examined whether tryptophan itself and/or its two metabolites, kyneurenic acid (KYNA) and quinolinic acid (QUIN), are involved in central fatigue. MATERIALS AND METHODS: Experiments were conducted using Sprague-Dawley rats (SDR) and Nagase analbuminemic rats (NAR). Central fatigue was assessed by treadmill running and a Morris water maze test. Microdialysis was used to collect samples for measurement of extracellular concentration of tryptophan, serotonin and 5-hydroxyindoleacetic acid (5-HIAA) and to infuse test agents. To examine the kinetics of release, synaptosomes in the striatum were prepared in vitro to measure intra- and extrasynaptosomal concentration of tryptophan, serotonin and 5-HIAA. RESULTS: The concentration of tryptophan secreted into the extracellular space of the striatum was higher during fatigue only, and quickly returned to basal levels with recovery from fatigue. Running time to exhaustion was reduced by activation of tryptophan receptors. Time to exhaustion was shorter in NAR, which maintain a higher extracellular level of striatum tryptophan than SDR. Impaired memory performance in a water maze task after tryptophan treatment was attributable to high levels of KYNA and QUIN in the hippocampus acting synergistically on N-methyl-D-aspartic acid receptors. When branched-chain amino acids were administered, tryptophan transport to the extracellular space of the striatum was drastically inhibited. CONCLUSION: Our findings demonstrate that the increase in fatigue which occurs because of excessively elevated brain tryptophan can be further amplified by the use of synthetic KYNA and QUIN.