Mary-Ellen Harper

SirT1 Regulates Energy Metabolism and Response to Caloric Restriction in Mice

The yeast sir2 gene and its orthologues in Drosophila and C. elegans have well-established roles in lifespan determination and response to caloric restriction. We have studied mice carrying two null alleles for SirT1, the mammalian orthologue of sir2, and found that these animals inefficiently utilize ingested food. These mice are hypermetabolic, contain inefficient liver mitochondria, and have elevated rates of lipid oxidation. When challenged with a 40% reduction in caloric intake, normal mice maintained their metabolic rate and increased their physical activity while the metabolic rate of SirT1-null mice dropped and their activity did not increase. Moreover, CR did not extend lifespan of SirT1-null mice. Thus, SirT1 is an important regulator of energy metabolism and, like its orthologues from simpler eukaryotes, the SirT1 protein appears to be required for a normal response to caloric restriction.

Physiological Role of UCP3 May Be Export of Fatty Acids from Mitochondria When Fatty Acid Oxidation Predominates: An Hypothesis

This hypothesis proposes a physiological role for uncoupling protein-3 (UCP3) in the export of fatty acid anions from muscle and brown adipose tissue (BAT) mitochondria when fatty acids are the predominant substrate being used. It proposes that excess acyl CoA within the mitochondria is hydrolyzed by a mitochondrial acyl CoA thioesterase, yielding fatty acid anion and CoASH. The fatty acid anion is exported to the cytosol by being carried across the inner mitochondrial membrane by UCP3. The CoASH is conserved within the mitochondrion to participate in other reactions for which it is needed during fatty acid oxidation in the β-oxidation cycle and in the tricarboxylic acid cycle. The export of the fatty acid anion thus permits continued rapid fatty acid oxidation in the face of an oversupply. The hypothesis provides a logical explanation for the observed up-regulation of gene expression for UCP3 in muscle when there is a switch to fatty acid oxidation, as during fasting, and in BAT when fatty acid oxidation is stimulated, as during exposure to cold. It provides a plausible physiological role for UCP3 as a transporter protein, not as an uncoupling protein.

Uncoupling proteins and the control of mitochondrial reactive oxygen species production

Reactive oxygen species (ROS), natural by-products of aerobic respiration, are important cell signaling molecules, which left unchecked can severely impair cellular functions and induce cell death. Hence, cells have developed a series of systems to keep ROS in the nontoxic range. Uncoupling proteins (UCPs) 1-3 are mitochondrial anion carrier proteins that are purported to play important roles in minimizing ROS emission from the electron transport chain. The function of UCP1 in this regard is highly contentious. However, UCPs 2 and 3 are generally thought to be activated by ROS or ROS by-products to induce proton leak, thus providing a negative feedback loop for mitochondrial ROS production. In our laboratory, we have not only confirmed that ROS activate UCP2 and UCP3, but also demonstrated that UCP2 and UCP3 are controlled by covalent modification by glutathione. Furthermore, the reversible glutathionylation is required to activate/inhibit UCP2 and UCP3, but not UCP1. Hence, our findings are consistent with the notion that UCPs 2 and 3 are acutely activated by ROS, which then directly modulate the glutathionylation status of the UCP to decrease ROS emission and participate in cell signaling mechanisms.

Restriction of energy intake, energy expenditure, and aging

Energy restriction (ER), without malnutrition, increases maximum life span and retards the development of a broad array of pathophysiological changes in laboratory rodents. The mechanism responsible for the retardation of aging by ER is, however, unknown. One proposed explanation is a reduction in energy expenditure (EE). Reduced EE may increase life span by decreasing the number of oxygen molecules interacting with mitochondria, thereby lowering reactive oxygen species (ROS) production. As a step toward testing this hypothesis, it is important to determine the effect of ER on EE. Several whole-body, organ, and cellular studies have measured the influence of ER on EE. In general, whole-body studies have reported an acute decrease in mass-adjusted EE that disappears with long-term ER. Organ-specific studies have shown that decreases in EE of liver and gastrointestinal tract are primarily responsible for initial reductions in EE with ER. These data, however, do not determine whether cellular EE is altered with ER. Three major processes contributing to resting EE at the cellular level are mitochondrial proton leak, Na(+)-K(+)-ATPase activity, and protein turnover. Studies suggest that proton leak and Na(+)-K(+)-ATPase activity are decreased with ER, whereas protein turnover is either unchanged or slightly increased with ER. Thus, two of the three major processes contributing to resting EE at the cellular level may be decreased with ER. Although additional cellular measurements are needed, the current results suggest that a lowering of EE could be a mechanism for the action of ER.

Characterization of a novel metabolic strategy used by drug-resistant tumor cells

Acquired or inherent drug resistance is the major problem in achieving successful cancer treatment. However, the mechanism(s) of pleiotropic drug resistance remains obscure. We have identified and characterized a cellular metabolic strategy that differentiates drug‐resistant cells from drug‐sensitive cells. This strategy may serve to protect drug‐resistant cells from damage caused by chemotherapeutic agents and radiation. We show that drug‐resistant cells have low mitochondrial membrane potential, use nonglucose carbon sources (fatty acids) for mitochondrial oxygen consumption when glucose becomes limited, and are protected from exogenous stress such as radiation. In addition, drug‐resistant cells express high levels of mitochondrial uncoupling protein 2 (UCP2). The discovery of this metabolic strategy potentially facilitates the design of novel therapeutic approaches to drug resistance.—Harper, M.‐E., Antoniou, A., Villalobos‐Menuey, E., Russo, A., Trauger, R., Vendemelio, George, A. M., Bartholomew, R., Carlo, D., Shaikh, A., Kupperman, J., Newell, E. W., Bespalov, I. A., Wallace, S. S., Liu, Y., Rogers, J. R., Gibbs, G. L., Leahy, J. L., Camley, R. E., Melamede, R., Newell, M. K. Characterization of a novel metabolic strategy used by drug‐resistant tumor cells. FASEB J. 16, 1550–1557 (2002)

Electron transport chain dependent and independent mechanisms of mitochondrial H2O2 emission during long-chain fatty acid oxidation

Oxidative stress in skeletal muscle is a hallmark of various pathophysiologic states that also feature increased reliance on long-chain fatty acid (LCFA) substrate, such as insulin resistance and exercise. However, little is known about the mechanistic basis of the LCFA-induced reactive oxygen species (ROS) burden in intact mitochondria, and elucidation of this mechanistic basis was the goal of this study. Specific aims were to determine the extent to which LCFA catabolism is associated with ROS production and to gain mechanistic insights into the associated ROS production. Because intermediates and by-products of LCFA catabolism may interfere with antioxidant mechanisms, we predicted that ROS formation during LCFA catabolism reflects a complex process involving multiple sites of ROS production as well as modified mitochondrial function. Thus, we utilized several complementary approaches to probe the underlying mechanism(s). Using skeletal muscle mitochondria, our findings indicate that even a low supply of LCFA is associated with ROS formation in excess of that generated by NADH-linked substrates. Moreover, ROS production was evident across the physiologic range of membrane potential and was relatively insensitive to membrane potential changes. Determinations of topology and membrane potential as well as use of inhibitors revealed complex III and the electron transfer flavoprotein (ETF) and ETF-oxidoreductase, as likely sites of ROS production. Finally, ROS production was sensitive to matrix levels of LCFA catabolic intermediates, indicating that mitochondrial export of LCFA catabolic intermediates can play a role in determining ROS levels.

Uncoupling protein-3: clues in an ongoing mitochondrial mystery

Uncoupling protein (UCP) 3 (UCP3) is a mitochondrial anion carrier protein with highly selective expression in skeletal muscle. Despite a great deal of interest, to date neither its molecular mechanism nor its biochemical and physiological functions are well understood. Based on its high degree of homology to the original UCP (UCP1), early studies examined a role for UCP3 in thermogenesis. However, evidence for such a function is lacking. Recent studies have focused on two distinct, but not mutually exclusive, hypotheses: 1) UCP3 mitigates reactive oxygen species (ROS) production, and 2) UCP3 is somehow involved in fatty acid (FA) translocation. While supportive evidence exists for both hypotheses, the interpretation of the corresponding evidence has created some controversy. Mechanistic studies examining mitigated ROS production have been largely conducted in vitro, and the physiological significance of the findings is questioned. Conversely, while physiological evidence exists for FA translocation hypotheses, the evidence is largely correlative, leaving causal relationships unexplored. This review critically assesses evidence for the hypotheses and attempts to link the outcomes from mechanistic studies to physiological implications. Important directions for future studies, using current and novel approaches, are discussed.—Bézaire V., Seifert E. L., Harper M‐E. Uncoupling protein‐3: clues in an ongoing mitochondrial mystery. FASEB J. 21, 312–324 (2007)

Glutathionylation acts as a control switch for uncoupling proteins UCP2 and UCP3

The mitochondrial uncoupling proteins 2 and 3 (UCP2 and -3) are known to curtail oxidative stress and participate in a wide array of cellular functions, including insulin secretion and the regulation of satiety. However, the molecular control mechanism(s) governing these proteins remains elusive. Here we reveal that UCP2 and UCP3 contain reactive cysteine residues that can be conjugated to glutathione. We further demonstrate that this modification controls UCP2 and UCP3 function. Both reactive oxygen species and glutathionylation were found to activate and deactivate UCP3-dependent increases in non-phosphorylating respiration. We identified both Cys25 and Cys259 as the major glutathionylation sites on UCP3. Additional experiments in thymocytes from wild-type and UCP2 null mice demonstrated that glutathionylation similarly diminishes non-phosphorylating respiration. Our results illustrate that UCP2- and UCP3-mediated state 4 respiration is controlled by reversible glutathionylation. Altogether, these findings advance our understanding of the roles UCP2 and UCP3 play in modulating metabolic efficiency, cell signaling, and oxidative stress processes.

Constitutive UCP3 overexpression at physiological levels increases mouse skeletal muscle capacity for fatty acid transport and oxidation

Uncoupling protein 3 (UCP3) expression is directly correlated to fatty acid oxidation in skeletal muscle. UCP3 has been hypothesized to facilitate high rates of fatty acid oxidation, but evidence thus far is lacking. Our aim was to investigate the effects of UCP3 overexpression and ablation on fatty acid uptake and metabolism in muscle of mice having congenic backgrounds. In mice constitutively expressing the UCP3 protein (human form) at levels just over twofold higher than normal (230% of wild‐type levels), indirect calorimetry demonstrated no differences in total energy expenditure (VO2), but a shift toward increased fat oxidation compared with wild‐type (WT) mice. Metabolic efficiency (gram weight gain/kcal ingested) was similar between Ucp3 overexpressors, WT and Ucp3 (−/−) mice. In muscle of Ucp3‐tg mice, plasma membrane fatty acid binding protein (FABPpm) content was increased compared with WT mice. Although hormone‐sensitive lipase activity was unchanged across the genotypes, there were increases in carnitine palmitoyltransferase I, β‐hydroxyacylCoA dehydrogenase, and citrate synthase activities and decreases in intramuscular triacylglycerol in muscle of Ucp3‐tg mice. There were no differences in muscle mitochondrial content. High‐energy phosphates and total muscle carnitine and CoA were also greater in Ucp3‐tg compared with WT mice. Taken together, the findings demonstrate an increased capacity for fat oxidation in the absence of significant increases in thermogenesis in Ucp3‐tg mice. Findings from Ucp3 (−/−) mice revealed few differences compared with WT mice, consistent with the possibility of compensatory mechanisms. In conjunction with our observed increases in CoA and carnitine in muscle of Ucp3 overexpressors, the findings support the hypothesized role for Ucp3 in facilitating fatty acid oxidation in muscle.

OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand

Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1s role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation‐induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein‐dependent manner.