CC3/TIP30 regulates metabolic adaptation of tumor cells to glucose limitation

CC3/TIP30 is a metastasis and tumor suppressor, with reduced or absent expression in a variety of aggressive tumors. Overexpression of CC3 in tumor cells predisposes them to apoptosis in response to different death signals. We found that silencing of CC3 expression does not increase apoptotic resistance of cells. However, it strongly improves survival of tumor cells in response to glucose limitation. HeLa cells with silenced CC3 survive long-term in low glucose, and, in comparison to control HeLa cells, show superior metabolic adaptation to glucose limitation. First, unlike the parental HeLa cells, HeLa with silenced CC3 activate and maintain high levels of mitochondrial respiration that is critical for their ability to thrive in low glucose. Second, silencing of CC3 leads to higher expression levels of mitochondrial proteins in respiration complexes when cells are continuously cultured in limiting glucose. Third, HeLa cells with silenced CC3 maintain higher levels of c-MYC and the M2 isoform of pyruvate kinase in low glucose, contributing to more efficient glycolysis. Fourth, HeLa cells with silenced CC3 fail to fully activate AMPK in response to glucose limitation. Inhibition of AMPK, either pharmacologic or via siRNA, protects control HeLa cells from death in low glucose. The metabolic flexibility acquired by cells after silencing of CC3 could be directly relevant to the development of metastatic and aggressive human tumors that frequently have low or absent expression of CC3.     

Control of Glycolytic Flux by AMP-Activated Protein Kinase in Tumor Cells Adapted to Low pH

Tumor cells grow in nutrient- and oxygen-deprived microenvironments and adapt to the suboptimal growth conditions by altering their metabolic pathways. This adaptation process commonly results in a tumor phenotype that displays a high rate of aerobic glycolysis and aggressive tumor characteristics. The glucose regulatory molecule, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), is a bifunctional enzyme that is central to glycolytic flux and is downstream of the metabolic stress sensor AMP-activated protein kinase (AMPK), which has been suggested to modulate glycolysis and possibly activate isoforms of PFKFB, specifically PFKFB3 expressed in tumor cells. Our results demonstrated that long-term low pH exposure induced AMPK activation, which resulted in the up-regulation of PFKFB3 and an increase in its serine residue phosphorylation. Pharmacologic activation of AMPK resulted in an increase in PFKFB3 as well as an increase in glucose consumption, whereas in contrast, inhibition of AMPK resulted in the down-regulation of PFKFB3 and decreased glycolysis. PFKFB3 overexpression in DB-1 tumor cells induced a high rate of glycolysis and inhibited oxygen consumption, confirming its role in controlling glycolytic flux. These results show that low pH is a physiological stress that can promote a glycolytic phenotype commonly associated with tumorigenesis. The implications are that the tumor microenviroment contributes to tumor growth and treatment resistance.     

AMPK protects proximal tubular cells from stress-induced apoptosis by an ATP-independent mechanism: potential role of Akt activation

We examined the role of AMP-activated protein kinase (AMPK) in modulating the viability of cultured kidney proximal tubular cells subjected to metabolic stress induced by either dextrose deprivation, inhibition of glycolysis, or inhibition of mitochondrial respiration. We used BU.MPT cells, a conditionally immortalized kidney epithelial cell line derived from the proximal tubules of transgenic mice bearing a temperature-sensitive mutation of the simian virus 40 large-tumor antigen. All three forms of metabolic stress increased the phosphorylation and activity of AMPK. Activation of AMPK led to changes in the phosphorylation of two downstream targets of AMPK, acetyl coenzyme A carboxylase and p70 S6 kinase. Inhibition of AMPK, either pharmacologically with compound C (CC) or by gene silencing, significantly increased the amount of apoptosis in response to all three forms of metabolic stress. Although the amount of apoptosis was directly related to the severity of ATP depletion, inhibition of AMPK had no effect on cellular ATP levels. Notably, metabolic stress increased the phosphorylation and activity of Akt. Furthermore, inhibition of AMPK, with CC or gene silencing, abrogated the ability of metabolic stress to activate Akt. The augmentation of apoptosis induced by inhibition of AMPK was comparable to that induced by inhibition of Akt. We conclude that activation of AMPK following acute metabolic stress plays a major role in promoting the viability of cultured proximal tubular cells. Protection by AMPK appears to be due not to AMPK-mediated conservation of cell energy stores, but rather, at least in part, to AMPK-mediated activation of Akt.     

Carcinoma cells activate AMP-activated protein kinase-dependent autophagy as survival response to kaempferol-mediated energetic impairment

Kaempferol, a dietary cancer chemopreventive polyphenol, has been reported to trigger apoptosis in several tumor histotypes, but the mechanism underlying this phenomenon is not fully understood. Here, we demonstrate that, in HeLa cells, kaempferol induces energetic failure due to inhibition of both glucose uptake and Complex I of the mitochondrial respiratory chain. As adaptive response, cells activate autophagy, the occurrence of which was established cytofluorometrically, upon acridine orange staining, and immunochemically, by following the increase of the autophagolysosome-associated form of the microtubule-associated protein light chain 3 (LC3-II). Autophagy is an early and reversible process occurring as survival mechanisms against apoptosis. Indeed, chemical inhibition of autophagy, by incubations with monensin, wortmannin, 3-methyladenine, or by silencing Atg5, significantly increases the extent of apoptosis, which takes place via the mitochondrial pathway, and shortens the time at which the apoptotic markers are detectable. We also demonstrate that autophagy depends on the early activation of the AMP-activated protein kinase (AMPK)/mTOR-mediated pathway. The over-expression of dominant negative AMPK results in a decrease of autophagic cells, a decrement of LC3-II levels, and a significant increase of apoptosis. Experiments performed with another carcinoma cell line yielded the same results, suggesting for kaempferol a unique mechanism of action.     

5′-AMP-activated Protein Kinase (AMPK) Supports the Growth of Aggressive Experimental Human Breast Cancer Tumors

Rapid tumor growth can establish metabolically stressed microenvironments that activate 5′-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [¹³C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism.     

Overexpression of Mitochondrial Sirtuins Alters Glycolysis and Mitochondrial Function in HEK293 Cells

SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.     

Glycolysis–respiration relationships in a neuroblastoma cell line

Background

Although some reciprocal glycolysis–respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized.

Methods

We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions.

Results

Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased.

Conclusions

These data document a novel intracellular glycolysis–respiration effect in which restricting glycolysis flux increases mitochondrial respiration.

General significance

Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis–respiration effect can practically inform the development of new mitochondrial medicine approaches.     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

Regulation of autophagy and apoptosis by Dp44mT-mediated activation of AMPK in pancreatic cancer cells

Upon activation, the 5′-adenosine monophosphate-activated protein kinase (AMPK) increases catabolism, while inhibiting anabolism. The anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), activates AMPK in multiple tumor cell-types (Biochim. Biophys Acta 2016;1863:2916–2933). This acts as an initial cell “rescue response” after iron-depletion mediated by Dp44mT. Considering Dp44mT-mediated AMPK activation, the role of AMPK on Dp44mT cytotoxicity was examined. Dp44mT increased the p-AMPK/AMPK ratio in multiple tumor cell-types over short (24 h) and longer (72 h) incubations. Notably, Dp44mT was more effective in inhibiting tumor cell proliferation after AMPK silencing, potentially due to the loss of AMPK-mediated metabolic plasticity that protects cells against Dp44mT cytotoxicity. The silencing of AMPK-increased cellular cholesterol and stabilized lysosomes against Dp44mT-mediated lysosomal membrane permeabilization. This was substantiated by studies demonstrating that the cholesterol-depleting agent, methyl-β-cyclodextrin (MβCD), restores Dp44mT-mediated lysosomal membrane permeabilization in AMPK silenced cells. The increased levels of cholesterol after AMPK silencing were independent of the ability of AMPK to inhibit the rate-limiting step of cholesterol synthesis via the inactivating phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) at Ser872. In fact, Dp44mT did not increase phosphorylation of HMGCR at (Ser872), but decreased total HMGCR expression similarly in both the presence or absence of AMPK silencing. Dp44mT was demonstrated to increase autophagic initiation after AMPK silencing via an AMPK- and Beclin-1-independent mechanism. Further, there was increased cleaved caspase 3 and cleaved PARP after incubation of AMPK silenced cells with Dp44mT. Overall, AMPK silencing promotes Dp44mT anti-proliferative activity, suggesting a role for AMPK in rescuing its cytotoxicity by inhibiting autophagy and also apoptosis.     

Changes in the expression of the human adenine nucleotide translocase isoforms condition cellular metabolic/proliferative status

Human cells express four mitochondrial adenine nucleotide translocase (hANT) isoforms that are tissue-specific and developmentally regulated. hANT1 is mainly expressed in terminally differentiated muscle cells; hANT2 is growth-regulated and is upregulated in highly glycolytic and proliferative cells; and hANT3 is considered to be ubiquitous and non-specifically regulated. Here, we studied how the expression of hANT isoforms is regulated by proliferation and in response to metabolic stimuli, and examined the metabolic consequences of their silencing and overexpression. In HeLa and HepG2 cells, expression of hANT3 was upregulated by shifting metabolism towards oxidation or by slowed growth associated with contact inhibition or growth-factor deprivation, indicating that hANT3 expression is highly regulated. Under these conditions, changes in hANT2 mRNA expression were not observed in either HeLa or HepG2 cells, whereas in SGBS preadipocytes (which, unlike HeLa and HepG2 cells, are growth-arrest-sensitive cells), hANT2 mRNA levels decreased. Additionally, overexpression of hANT2 promoted cell growth and glycolysis, whereas silencing of hANT3 decreased cellular ATP levels, limited cell growth and induced a stress-like response. Thus, cancer cells require both hANT2 and hANT3, depending on their proliferation status: hANT2 when proliferation rates are high, and hANT3 when proliferation slows.     

Inhibitory effects of Bcl-2 on mitochondrial respiration

In contrast to the well-established anti-apoptotic effect of Bcl-2 protein, we have recently demonstrated that Bcl-2 overexpression by vaccinia virus causes apoptosis in BSC-40 cells, while it prevents apoptosis in HeLa G cells. Given the key role of mitochondria in the process of apoptosis, we focused on effects of Bcl-2 expression on mitochondrial energetics of these two cell lines. In this study we present data indicating that BSC-40 cells derive their ATP mainly from oxidative phosphorylation whereas HeLa G cells from glycolysis. More importantly, we show that in both cell lines, Bcl-2 inhibits mitochondrial respiration and causes a decrease of the ATP/ADP ratio. However, it appears that BSC-40 cells cannot sustain this decrease and die, while HeLa G cells survive, being adapted to the low ratio of ATP/ADP maintained by glycolysis. Based on this observation, we propose that the outcome of Bcl-2 expression is determined by the type of cellular ATP synthesis, namely that Bcl-2 causes apoptosis in cells relying on oxidative phosphorylation.     

Alterations in Cellular Energy Metabolism Associated with the Antiproliferative Effects of the ATM Inhibitor KU-55933 and with Metformin

KU-55933 is a specific inhibitor of the kinase activity of the protein encoded by Ataxia telangiectasia mutated (ATM), an important tumor suppressor gene with key roles in DNA repair. Unexpectedly for an inhibitor of a tumor suppressor gene, KU-55933 reduces proliferation. In view of prior preliminary evidence suggesting defective mitochondrial function in cells of patients with Ataxia Telangiectasia (AT), we examined energy metabolism of cells treated with KU-55933. The compound increased AMPK activation, glucose uptake and lactate production while reducing mitochondrial membrane potential and coupled respiration. The stimulation of glycolysis by KU-55933 did not fully compensate for the reduction in mitochondrial functions, leading to decreased cellular ATP levels and energy stress. These actions are similar to those previously described for the biguanide metformin, a partial inhibitor of respiratory complex I. Both compounds decreased mitochondrial coupled respiration and reduced cellular concentrations of fumarate, malate, citrate, and alpha-ketogluterate. Succinate levels were increased by KU-55933 levels and decreased by metformin, indicating that the effects of ATM inhibition and metformin are not identical. These observations suggest a role for ATM in mitochondrial function and show that both KU-55933 and metformin perturb the TCA cycle as well as oxidative phosphorylation.     

Metabolic interplay between glycolysis and mitochondrial oxidation: The reverse Warburg effect and its therapeutic implication

Aerobic glycolysis, i.e., the Warburg effect, may contribute to the aggressive phenotype of hepatocellular carcinoma. However, increasing evidence highlights the limitations of the Warburg effect, such as high mitochondrial respiration and low glycolysis rates in cancer cells. To explain such contradictory phenomena with regard to the Warburg effect, a metabolic interplay between glycolytic and oxidative cells was proposed, i.e., the "reverse Warburg effect". Aerobic glycolysis may also occur in the stromal compartment that surrounds the tumor; thus, the stromal cells feed the cancer cells with lactate and this interaction prevents the creation of an acidic condition in the tumor microenvironment. This concept provides great heterogeneity in tumors, which makes the disease difficult to cure using a single agent. Understanding metabolic flexibility by lactate shuttles offers new perspectives to develop treatments that target the hypoxic tumor microenvironment and overcome the limitations of glycolytic inhibitors.     

Inhibition of mitochondrial complex I improves glucose metabolism independently of AMPK activation

Accumulating evidences showed metformin and berberine, well‐known glucose‐lowering agents, were able to inhibit mitochondrial electron transport chain at complex I. In this study, we aimed to explore the antihyperglycaemic effect of complex I inhibition. Rotenone, amobarbital and gene silence of NDUFA13 were used to inhibit complex I. Intraperitoneal glucose tolerance test and insulin tolerance test were performed in db/db mice. Lactate release and glucose consumption were measured to investigate glucose metabolism in HepG2 hepatocytes and C2C12 myotubes. Glucose output was measured in primary hepatocytes. Compound C and adenoviruses expressing dominant negative AMP‐activated protein kinase (AMPK) α1/2 were exploited to inactivate AMPK pathway. Cellular NAD⁺/NADH ratio was assayed to evaluate energy transforming and redox state. Rotenone ameliorated hyperglycaemia and insulin resistance in db/db mice. It induced glucose consumption and glycolysis and reduced hepatic glucose output. Rotenone also activated AMPK. Furthermore, it remained effective with AMPK inactivation. The enhanced glycolysis and repressed gluconeogenesis correlated with a reduction in cellular NAD⁺/NADH ratio, which resulted from complex I suppression. Amobarbital, another representative complex I inhibitor, stimulated glucose consumption and decreased hepatic glucose output in vitro, too. Similar changes were observed while expression of NDUFA13, a subunit of complex I, was knocked down with gene silencing. These findings reveal mitochondrial complex I emerges as a key drug target for diabetes treatment. Inhibition of complex I improves glucose homoeostasis via non‐AMPK pathway, which may relate to the suppression of the cellular NAD⁺/NADH ratio.     

Tyrosine phosphorylation of lactate dehydrogenase A is important for NADH/NAD(+) redox homeostasis in cancer cells

The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD(+). LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD(+). However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD(+) redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.     

Mitochondrial Bioenergetics of Metastatic Breast Cancer Cells in Response to Dynamic Changes in Oxygen Tension: Effects of HIF-1α

Solid tumors are characterized by regions of low oxygen tension (OT), which play a central role in tumor progression and resistance to therapy. Low OT affects mitochondrial function and for the cells to survive, mitochondria must functionally adapt to low OT to maintain the cellular bioenergetics. In this study, a novel experimental approach was developed to examine the real-time bioenergetic changes in breast cancer cells (BCCs) during adaptation to OT (from 20% to <1% oxygen) using sensitive extracellular flux technology. Oxygen was gradually removed from the medium, and the bioenergetics of metastatic BCCs (MDA-MB-231 and MCF10CA clones) was compared with non-tumorigenic (MCF10A) cells. BCCs, but not MCF10A, rapidly responded to low OT by stabilizing HIF-1α and increasing HIF-1α responsive gene expression and glucose uptake. BCCs also increased extracellular acidification rate (ECAR), which was markedly lower in MCF10A. Interestingly, BCCs exhibited a biphasic response in basal respiration as the OT was reduced from 20% to <1%. The initial stimulation of oxygen consumption is found to be due to increased mitochondrial respiration. This effect was HIF-1α-dependent, as silencing HIF-1α abolished the biphasic response. During hypoxia and reoxygenation, BCCs also maintained oxygen consumption rates at specific OT; however, HIF-1α silenced BCC were less responsive to changes in OT. Our results suggest that HIF-1α provides a high degree of bioenergetic flexibility under different OT which may confer an adaptive advantage for BCC survival in the tumor microenvironment and during invasion and metastasis. This study thus provides direct evidence for the cross-talk between HIF-1α and mitochondria during adaptation to low OT by BCCs and may be useful in identifying novel therapeutic agents that target the bioenergetics of BCCs in response to low OT.     

Fatty acids revert the inhibition of respiration caused by the antidiabetic drug metformin to facilitate their mitochondrial β-oxidation

While metformin has been widely used to treat type 2 diabetes for the last fifty years, its mode of action remains unclear. Hence, we investigated the short-term alterations in energy metabolism caused by metformin administration in 3T3-L1 adipocytes. We found that metformin inhibited mitochondrial respiration, although ATP levels remained constant as the decrease in mitochondrial production was compensated by an increase in glycolysis. While AMP/ATP ratios were unaffected by metformin, phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase augmented. The inhibition of respiration provoked a rapid and sustained increase in superoxide levels, despite the increase in UCP2 and superoxide dismutase activity. The inhibition of respiration was rapidly reversed by fatty acids and thus respiration was lower in treated cells in the presence of pyruvate and glucose while rates were identical to control cells when palmitate was the substrate. We conclude that metformin reversibly inhibits mitochondrial respiration, it rapidly activates AMPK without altering the energy charge, and it inhibits fatty acid synthesis. Mitochondrial β-oxidation is facilitated by reversing the inhibition of complex I and, presumably, by releasing the inhibition of carnitine palmitoyltransferase. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).