Metabolic Flexibility and the Freedom of Choice
Cellular rates of CO2 production relative to oxygen consumption, or the respiratory quotient (RQ), fluctuate between 0.7 and 1.0 and provide an approximation of mitochondrial fuel use under typical conditions in which amino acids contribute only minimally as an oxidative substrate. A high RQ is indicative of glucose oxidation, whereas a low RQ reflects predominately fat oxidation.
Normal physiology is characterized by diurnal oscillations in whole-body RQ, reflective of a metabolically flexible state in which mitochondria switch freely between substrates (fat and sugar) based on nutritional and physiological cues. For the purpose of this discussion, a metabolically sensitive and flexible system is defined as one in which nutrient and energetic signals are rapidly propagated and appropriately interpreted to elicit finely tuned adjustments in fuel partitioning.
The physiological importance of metabolic plasticity cannot be understood without first considering the evolutionary pressure for mitochondria to choose fat as a fuel source when systemic glucose reserves are threatened. Whereas lipids provide an abundant, carbon-rich energy source for most tissues, the brain has limited capacity for fat catabolism and therefore relies heavily on a continuous supply of glucose. During periods of food restriction or sustained exercise, protection against hypoglycemia is accomplished by having more versatile tissues (e.g., cardiac, skeletal muscle, liver) convert to a lipid-based respiratory economy. At a systemic level, this switch in metabolic currency is mediated in large part by the counter-regulatory hormones, insulin and glucagon, both of which exert strong influence on adipose tissue lipolysis. Fasting lowers the insulin:glucagon ratio, which stimulates hydrolysis of adipose tissue triacylglycerol and thereby increases delivery of free fatty acids to the periphery. Lipolysis also occurs in tissues such as muscle and liver, further facilitating rapid provision of lipid fuel.
As first recognized by Randle (Randle, 1998), systemic changes in energy supply and demand are also monitored and controlled locally, such that glucose consumption is suppressed when fat oxidation increases. Randle’s glucose-fatty acid cycle hypothesis further proposed that a rise in cellular citrate inhibits phosphofructokinase-1 and that lowering of glycolysis and pyruvate oxidation results in accumulation of glucose-6-phosphate (G6P), leading to allosteric inhibition of hexokinase 2 (HK2) in muscle and heart and diminished glucose uptake.
Another important alternative fuel source during starvation comes as a result of proteolysis and amino acid catabolism, which is regulated, in part, by the mitochondrial branched-chain ketoacid dehydrogenase (BCKD) complex. Like PDH, this complex is allosterically inhibited by NADH and the acyl-CoA esters that arise during branched-chain amino acid catabolism and is covalently inactivated by phosphorylation via BCKD kinase.
By contrast, the nutrient and hormonal milieu elicited by the postprandial state favors glucose uptake, glycolysis, and pyruvate oxidation and a corresponding suppression of fatty acid catabolism. The mechanisms governing the meal-induced switch from fatty acid to glucose oxidation first came to light through the elegant work of McGarry and colleagues (McGarry, 2002), who discovered that feeding increases tissue levels of a precursor for de novo lipogenesis, malonyl-CoA, that also serves as a potent allosteric inhibitor of carnitine palmitoyltransferase-1 (CPT-1) (Figure 1). Positioned on the outer mitochondrial membrane, CPT-1 converts long-chain fatty acyl-CoAs to long-chain acylcarnitines that are able to traverse the inner membrane.
Additionally, the glucose-induced rise in pyruvate inhibits the PDKs and thereby favors dephosphorylation and activation of the PDH complex. This step results in refilling of the TCAC and net export of citrate. Upon delivery to the cytoplasm, citrate is cleaved by ATP-citrate lyase to oxaloacetate and acetyl-CoA, the latter of which is then carboxylated and converted to malonyl-CoA by one of two isoforms of acetyl CoA carboxylase (ACC). Citrate also acts as an allosteric activator of ACC in a feedforward manner.
Collectively, the mechanisms originally described by Randle and McGarry represent key components of a sophisticated metabolic network that monitors and responds to the local and systemic nutrient environments to maintain glucose homeostasis. Transitions between fasting and feeding trigger a wave of metabolite signals that guide mitochondrial fuel choice and regulate carbon trafficking (Figure 1).
Metabolic Congestion Results in Mitochondrial Indecision
It is important to consider that human physiology evolved to cope with dramatic fluctuations in energy supply and demand during periods of feast and famine or hunting/gathering. Thus, episodes of refueling were typically preceded by a sustained period of energy deficit. By contrast, physiology in the modern era is characterized by a steady influx of competing fuels. A large body of evidence suggests that overnutrition and unabated substrate competition lead to a state of metabolic insensitivity and inflexibility, characterized by distorted nutrient sensing, blunted substrate switching, and impaired energy homeostasis.
The concept of metabolic inflexibility was first introduced by Kelley and colleagues, who monitored gas exchange across the leg to examine substrate switching in healthy compared to obese and diabetic subjects (Figure 2) (Kelley et al., 1999). Lean, healthy subjects shifted from a low RQ in the fasted state to a high RQ during a hyperinsulinemiceuglycemic clamp, an infusion procedure that mimics the fed state by increasing plasma insulin concentrations while holding blood glucose constant at basal levels. When this same test was applied to obese and type 2 diabetic subjects, the transition to the “fed” state was accompanied by only a marginal change in RQ. Therefore, the insulin-resistant individuals continued to oxidize a fixed mixture of fats and carbohydrates regardless of the nutritional context. Additionally, emerging evidence suggests that increased amino acid supply and catabolism (which elicits an intermediate RQ) might also contribute to obesity-related perturbations in fuel use (Newgard, 2012). Thus, in the context of chronic over-feeding, competition between substrates escalates, cooperation is lost, and the mitochondria are left in a state of indecision characterized by persistent oxidation of all three major fuels.
Whereas most of these studies evaluated substrate use by skeletal muscle, heart, and/or liver, emerging evidence shows that similar perturbations in fuel switching and nutrient responsiveness manifest in other organs and cell types, including adipose tissue (Sparks et al., 2009), macrophages (Asterholm et al., 2012a), and monocytes (Liu et al., 2012).
Mitochondrial Overload Leads to Metabolic Gridlock
Research to delineate the molecular origins of metabolic inflexibility has focused largely on the glucose-fatty acid cycle and/or aberrant production of malonyl-CoA. Although these mechanisms certainly weigh heavily on fuel selection, the finding that inflexibility is associated with or can be provoked by a broad range of clinical and experimental circumstances suggests that the molecular basis of this condition reaches beyond dysregulation of PDH and/or CPT-1. It is important to consider that: (1) fuel selection occurs at the level of the mitochondrion, (2) carbon substrates are the source of the electrons that feed the ETC, and (3) oxidative phosphorylation satisfies 70%–90% of ATP demand in most cells. Accordingly, this organelle is ideally positioned to monitor and transmit energy and nutrient status throughout the metabolic network (Anderson et al., 2009; Mailloux et al., 2013; Muoio and Neufer, 2012; Newman et al., 2012). This mitocentric model of nutrient sensing and partitioning suggests that cellular energy charge and shifts in flux control are integrated and executed as a function of mitochondrial carbon load.
To conceptualize a network model of metabolic inflexibility, the spatiotemporal features of carbon flux through the metabolic interstates of an organism transitioning from fasting to feeding can be viewed in a manner analogous to the onset of rush hour traffic. When volume is light, free-flowing traffic remains highly responsive to internal cues based on operator decisions and mechanical function, as well as external inputs from traffic signs and signals. Under these circumstances, distance between vehicles remains generous, buffering capacity is robust, and abrupt fluctuations in traffic density and speed can occur with minimal risk of collision. As volume expands, bottlenecks at highly traveled intersections impede flow, the buffering capacity of the network diminishes, and the probability of random collision mounts. An incident at a critical node can lead to systemic paralysis, such that traffic flow remains unresponsive even when a major signal changes from red to green. The roadways reach a state of grid-lock, and the time and energy required to restore normal flow depends on the extent of the impasse and the severity of the damage.
When electron supply to the Q cycle exceeds demand for ATP synthesis, mitochondrial membrane potential rises and proton pumping at complexes I, III, and IV is met with increasing back pressure. Under these circumstances, the main escape route for incoming electrons occurs via the reduction of molecular oxygen and generation of the superoxide anion, followed by its rapid conversion to hydrogen peroxide (H2O2) by superoxide dismutase. Recent studies also identify PDH, BCKD, and α-ketoglutarate dehydrogenase as important sites of ROS production (Fisher-Wellman et al., 2013).
Hydrogen peroxide and other reactive oxygen species are now well recognized as bona fide signaling molecules that modulate reversible oxidation/reduction of sulfur atoms within critical cysteine residues of numerous proteins. The interconversion of these so-called “sulfur switches” from protein thiols (reduced form) to their corresponding disulfides (oxidized form) affects the activities and/or functions of an expansive network of redox-sensitive metabolic enzymes and signaling proteins (Brandes et al., 2009). This link implies that perturbations in the frequency, amplitude, and/or duration of the mitochondrial H2O2 pulse could have far-reaching effects on redox circuitry, nutrient flux, and energy homeostasis (Mailloux et al., 2013).
Meanwhile, as delivery of carbons persists and the amount of excess fuel mounts, the NADH/NAD(+) ratio within the mitochondrial lumen increases and redox inhibition of several TCAC enzymes limits flux through this major metabolic beltway. The ensuing mismatch between the early steps of carbon degradation and TCAC flux can lead to intramitochondrial accumulation of acetyl-CoA and other acyl-CoAs at several crucial bottlenecks, particularly at sites where catabolism of the three fuels converge (Figure 3) (Koves et al., 2008; Newgard, 2012).
Metabolic Countermeasures and Mitochondrial Damage Control
Considering that most routes connecting food consumption to ATP production travel through the mitochondria and because reactive lysine and cysteine residues of mitochondrial proteins are more vulnerable to nucleophilic attack due to the alkaline environment of the matrix (Ghanta et al., 2013; Mailloux et al., 2013; Wagner and Payne, 2013), it is not surprising that this organelle has developed various countermeasures to buffer excess traffic and repair the damage caused by inadvertent molecular collisions. For example, the redox circuits modulated by H2O2 are buffered by the interdependent glutathione- and thioredoxin-reducing systems (Mailloux et al., 2013). Both use the reducing power of NADPH to mitigate oxidative stress and to modulate reversible oxidation/reduction of protein thiols/disulfides.
Another buffering system that plays a key role in energy homeostasis utilizes a family of mitochondrial carnitine acyltransferase (CAT) enzymes that catalyze the exchange of acyl groups between CoA and L-carnitine (Ramsay and Zammit, 2004). Conversely, dietary L-carnitine supplementation promotes glucose tolerance and enhances metabolic flexibility in concert with increased circulating levels of the main CrAT product, acetylcarnitine (Muoio et al., 2012; Noland et al., 2009). Likewise, muscle CrAT activity correlates positively with insulin sensitivity in rodents and humans (Lindeboom et al., 2014).
Whereas the carnitine system relieves substrate push on mitochondrial protein acylation, the sirtuin family of NAD+-dependent deacylases performs damage control by removing acyl groups from lysine residues. Importantly, however, the sirtuins do not act on all acyl-lysine residues; thus, it appears that only a specific subset of these PTMs is reversible. SIRT3, SIRT4, and SIRT5 are found in the mitochondrial matrix. SIRT3 is the main mitochondrial deacetylase and the best characterized of the mitochondrial sirtuins (Newman et al., 2012).
Insulin Resistance Viewed as a Case of Mixed Signals
Aberrant transitions between fuel types at both the cellular and systemic levels link to phenotypes associated with metabolic co-morbidities, including insulin resistance. An important question is whether insulin resistance causes metabolic inflexibility or vice versa. One view suggests that blunted glucose oxidation in response to a meal simply reflects a consequence of impaired insulin signaling. On the other hand, studies in rodents show that metabolic inflexibility occurs early in the course of glucose intolerance, and obesity-induced perturbations in substrate switching are evident in isolated mitochondria and tissue homogenates (Muoio et al., 2012; Noland et al., 2009). These findings suggest that derangements in fuel selection are at least partly independent of and might actually precede and contribute to insulin resistance.
It is important to reiterate that the models developed by Randle and McGarry center on the concepts of reciprocation, cooperation, and communication between substrates. In a healthy state, crosstalk between metabolic pathways is mediated by robust, concise, and decisive changes in cellular levels of metabolites such as fatty acids, pyruvate, citrate, and malonyl-CoA, which in turn regulate incoming mitochondrial traffic (Figure 1). By contrast, chronic overnutrition leads to a state of metabolic confusion, wherein excessive carbon supply and heightened substrate competition give rise to a set of muted and/or conflicting signals. As a result, the gateways that control mitochondrial traffic are never fully open or shut, and the continuous influx of carbon fuel from multiple sources and directions interferes with efficient substrate switching. Thus, metabolic inflexibility can be viewed as both a cause and indicator of mitochondrial congestion, which in turn influences insulin action.
These observations underscore two important points. First, obesity does not cause insulin resistance. Instead, increased adiposity is merely a symptom of chronic (positive) energy imbalance, presumably the true culprit. Second, even brief episodes of increased energy expenditure and accelerated carbon combustion can reset the energy charge of the muscle and enhance insulin action. The precise molecular mechanisms underlying exercise-mediated enhancement of insulin responsiveness have evaded scientists for decades. Interestingly, however, recent studies have established a strong positive association between exercise training and metabolic flexibility (Bergouignan et al., 2013; Koves et al., 2013), suggesting that physical activity enhances mitochondrial traffic control and that persistent substrate competition is a key component of insulin resistance.
Strategies for Decongestion
The idea that mitochondrial carbon overload and metabolic inflexibility lie at the core of insulin resistance implies that maneuvers to prevent oxidative catabolism of at least one of the three major fuel sources might alleviate substrate competition and restore glucose control.
Numerous provocative studies in animals and humans show that very low-carbohydrate (ketogenic) diets produce favorable metabolic outcomes when compared to a traditional mixed macronutrient diet (Hu et al., 2012). Whereas these findings have sparked intense controversy over dietary recommendations for disease prevention, perhaps the salient observation is that a dietary regimen that effectively limits mitochondrial substrate competition produces marked improvements in metabolic control.
Although blocking mitochondrial influx of carbons and/or building buffering capacity might alleviate the metabolic load on this particular organelle, these strategies do not address the fundamental problem of energy imbalance and the broader implications of nutrient surplus. Thus, approaches targeting the cause rather than the symptoms of nutrient overload should yield more desirable outcomes. Moving beyond the standard recommendation to “eat less and move more,” recent studies employing unconventional behavior modification interventions have produced intriguing results, including intermittent fasting regimens in which rodents or human subjects fast for extended periods followed by ad libitum eating (Azevedo et al., 2013; Trepanowski et al., 2011), as well as “exercise snacking” regimens in which individuals exercise vigorously for brief (~5 min) periods before consuming a standard-size meal (Francois et al., 2014). - 'Exercise snacks' before meals: a novel strategy to improve glycaemic control in individuals with insulin resistance
Referência:
Cell. 2014 Dec 4;159(6):1253-62.
