Browsing by Author "Muoio, Deborah M"

Acetyl-CoA holds a prominent position as the common metabolic intermediate of glucose, amino acid and fatty acid oxidation. Because acetyl-CoA fuels the tricarboxylic acid (TCA) cycle, the primary source of reducing equivalents that drives mitochondrial oxidative phosphorylation, understanding acetyl-CoA pool regulation becomes imperative to understanding mitochondrial energetics. Carnitine acetyltransferase (CrAT), a muscle-enriched mitochondrial enzyme, catalyzes the freely reversible conversion of acetyl-CoA to its membrane permeant carnitine ester, acetylcarnitine. Because CrAT has long been thought to regulate the acetyl-CoA metabolite pool, we investigated the role of CrAT in acetyl-CoA regulation. Although the biochemistry and enzymology of the CrAT reaction has been well studied, its physiological role remains unknown. Investigations herein suggest that CrAT-mediated maintenance of the mitochondrial acetyl-CoA pool is imperative for preservation of energy homeostasis. We provide compelling evidence that CrAT is critical for fine-tuning acetyl-CoA balance during the fasted to fed transition and during exercise. These studies suggest that compromised CrAT activity results in derangements in mitochondrial homeostasis.

In chapter 3, we examined the effects of obesity and lipid exposure on CrAT activity. Recent studies have shown that acetyl-CoA-mediated inhibition of pyruvate dehydrogenase (PDH), the committed step in glucose oxidation, is modulated by the CrAT enzyme. Because PDH and glucose oxidation are negatively regulated by high fat feeding and obesity, we reasoned that nutritional conditions that promote lipid availability and fat oxidation might likewise compromise CrAT activity. We report an accumulation of long chain acylcarnitines and acyl-CoAs but a decline in the acetylcarnitine/acetyl-CoA ratio in obese and diabetic rodents. This reduction in the skeletal muscle acetylcarnitine/acetyl-CoA ratio was accompanied by a decrease in CrAT specific activity, despite increased protein abundance. Exposure to long chain acyl-CoAs in vitro demonstrated that palmitoyl-CoA acts as a mixed model inhibitor of CrAT. Furthermore, primary human skeletal muscle myocytes exposed to fatty acid and or CPT1b overexpression had elevated long chain acylcarnitines but decreased production and efflux of CrAT-derived short chain acylcarnitines. These data suggest that exposure to fatty acids in obesity and diabetes can counter-regulate the CrAT enzyme leading to decreased activity.

Alternatively, chapter 4 addresses the importance of acetyl-CoA buffering during exercise and suggests that a deficit in CrAT activity leads to fatigue. Because CrAT is highly expressed in tissues specifically designed for work and because acetylcarnitine, the primary product of the CrAT reaction, is increased during contraction, we reasoned that CrAT could play an important role in exercise. To investigate this possibility, we employed exercise intervention and ex-vivo analysis on a genetically novel mouse model of skeletal muscle CrAT deficiency (CrATSM-/-). Though resting acetyl-CoA levels were elevated in CrATSM-/- mice, these levels dropped significantly after intense exercise while acetylcarnitine content followed the opposite pattern. This contraction-induced acetyl-CoA deficit in CrATSM-/- mice was coupled with compromised performance and diminished whole body glucose oxidation during high intensity exercise. These results imply that working muscles clear and consume acetylcarnitine in order to maintain acetyl-CoA buffering during exercise. Importantly, provision of acetylcarnitine enhanced force generation, delayed fatigue and improved mitochondrial energetics in muscles from CrATfl/fl controls but not CrATSM-/- littermates, emphasizing the importance of acetyl-CoA maintenance. In aggregate, these data demonstrate a critical role for CrAT-mediated acetyl-CoA buffering in exercise tolerance and suggest its involvement in energy metabolism during skeletal muscle contraction and fatigue. These findings could have important clinical implications for individuals with muscle weakness and fatigue due to multiple conditions, such as peripheral vascular or cardiometabolic disease.

In summary, data herein emphasize the role of CrAT in regulation of mitochondrial acetyl-CoA pool. We demonstrate that CrAT is critical for fine-tuning acetyl-CoA balance both during the fasted to fed transition and during exercise. These data suggest that a deficit in CrAT activity leads to glucose intolerance and exercise fatigue. We examine these studies and suggest future areas of study.

Objective

Methods

Results

Conclusion

Heart failure is a worldwide public health problem with substantial clinical burden and economic costs. In the progression into failure, the heart undergoes dramatic alterations in mitochondrial fuel metabolism and bioenergetics. As such, there is considerable interest in the delineation of regulatory events involved in the metabolic dysfunction of heart failure. Previous collaborative work identified three metabolic signatures associated with early stage heart failure: 1) accumulation of acylcarnitine metabolites; 2) mitochondrial hyperacetylation; and 3) elevated ketone catabolism. The goal of this dissertation was to explore the role of these metabolic signatures in the pathogenesis of heart failure.

Tissue accumulation of acylcarnitine metabolites is characteristic of mitochondrial dysfunction and indicative of incomplete β-oxidation. This occurs when a large portion of the fatty acids (i.e., acyl groups) within the mitochondria are not fully catabolized and the resulting intermediates are transferred to carnitine esters, enabling the traversal of biological membranes and departure from the mitochondrial matrix.

Nϵ-acetylation in the mitochondrial matrix is a non-enzymatic, post-translational modification (PTM) that spontaneously arises from the relatively basic pH and abundance of acetyl-CoA. Accumulation of this PTM has been observed in other tissues and disease states with evidence suggesting it impairs mitochondrial metabolism and causes dysfunction. However, convincing studies are lacking to establish a direct causal connection between dysfunction and acetylation. To address this shortcoming, a novel assay platform for the comprehensive assessment of mitochondrial bioenergetic transduction was developed and validated. Next, we generated and validated a novel mouse model of cardiac mitochondrial hyperacetylation and utilized the bioenergetic assay platform to test the hypothesis that it causes metabolic perturbations. Surprisingly, these hyperacetylated mitochondria exhibited almost no deficits in mitochondrial oxidative metabolism. To determine if hyperacetylation causes mitochondrial dysfunction in vivo under pathologic stimuli, the mouse model and littermate controls were subjected to transaortic constriction, a surgical method to induce pressure-overload heart failure. The hyperacetylated animals did not exhibit enhanced sensitivity toward cardiac dysfunction relative controls. With these results, we concluded that mitochondrial hyperacetylation does not contribute to the pathogenesis of heart failure.

Elevated ketone catabolism was observed in early stage failing hearts. Through a series of murine and canine heart failure models, ketone catabolism was shown to be adaptive in response to pathological stress. Additionally, the mitochondrial bioenergetic assay platform was applied to cardiac mitochondria under substrate limited-conditions. These results indicate that ketone catabolism improves bioenergetic efficiency under constraints which mimic the failing heart. With these results, we conclude ketone catabolism is an important metabolic defense in response to the dysfunction of the failing heart.

The Muoio lab studies the interplay between lipid whole body energy balance,

mitochondrial function and insulin action in skeletal muscle. Data from our lab suggests that lipid-induced insulin resistance in skeletal muscle may stem from excessive incomplete oxidation of fatty acids, which occurs when high rates of β-­oxidation exceed TCA cycle flux (Koves et al., 2005; Koves et al., 2008). Most notably, we have shown that mice with a genetically engineered decrease in mitochondrial uptake and oxidation of fatty acids are protected against diet-­induced insulin resistance (Koves et al., 2008). This

suggests that an excessive and/or inappropriate metabolic burden on muscle

mitochondria provokes insulin resistance. Our working model predicts that: 1) high rates of incomplete β-oxidation reflect a state of ”mitochondrial stress,” and 2) that energy-overloaded mitochondria generate a yet unidentified signal that mediates insulin

resistance. One possibility is that this putative mitochondrial-derived signal stems from redox imbalance and disruptions in redox sensitive signaling cascades. Therefore, we are interested in identifying molecules that link redox balance, mitochondrial function and insulin action in skeletal muscle. The work described herein identifies thioredoxin-interacting protein (TXNIP) as an attractive candidate that regulates both glucose homeostasis and mitochondrial fuel selection.

TXNIP is a redox sensitive, α-arrestin protein that has been implicated as a negative regulator of glucose control. Mounting evidence suggested that TXNIP might play a key role in regulating mitochondrial function; however, the molecular nature of this relationship was poorly defined. Previous studies in TXNIP knockout mice reported that deficiency of this protein compromises oxidative metabolism, increases glycolytic activity and promotes production of reactive oxygen species (ROS), while also affording protection against insulin resistance. Therefore, we hypothesized that TXNIP might serve as a nutrient sensor that couples cellular redox status to the adjustments in mitochondrial function. We tested this hypothesis by exploiting loss of function models to evaluate the effects of TXNIP deficiency on mitochondrial metabolism and respiratory function.

In chapter 3, we comprehensively evaluated oxidative metabolism, substrate

selection, respiratory kinetics and redox balance in mice with total body and skeletal muscle-­specific TXNIP deficiency. Targeted metabolomics, comprehensive bioenergetics analysis, whole-body respirometry and conventional biochemistry showed that TXNIP deficiency results in reduced exercise tolerance with marked impairments in skeletal muscle oxidative metabolism. The deficits in substrate oxidation were not secondary to decreased mitochondrial mass or increased H₂O₂ emitting potential from the electron transport chain. Instead, the activities of several mitochondrial dehydrogenases involved in branched-chain amino acid and ketone catabolism, the tricarboxylic acid (TCA) cycle and fatty acid β-oxidation were significantly diminished in TXNIP null muscles. These deficits in mitochondrial enzyme activities were accompanied by decreased protein abundance without changes in mRNA expression. Taken together, these results suggest that in skeletal muscle TXNIP plays an essential role in maintaining protein synthesis and/or stability of a subset of mitochondrial dehydrogenase enzymes that permit muscle use of alternate fuels under conditions of glucose deprivation.

Based on these conclusions, we questioned whether additional regulatory

mechanisms could contribute to the reduced oxidative metabolism in the absence ofTXNIP. Several metabolic enzymes of the TCA cycle have been shown to be redox-sensitive protein targets regulated by the thioredoxin (TRX1/TRX2) and glutathione (GSH) redox-mediated circuits. TXNIP has been shown to respond to oxidative stress by shuttling to the mitochondria where it binds to TRX2 and/or other proteins, thus affecting downstream signaling pathways, such as the apoptotic cascade. Therefore, we speculated whether there was a role for redox imbalance in mediating the mitochondrial phenotype of the TXNIP knockout (TKO) mice. In chapter 4, we present preliminary evidence that increased glucose uptake promotes non-mitochondrial ROS production, causing a shift in redox balance, decreased GSH/GSSG, and S-glutathionylation of α-­ketoglutarate dehydrogenase (&alpha-KGD). This post-translational modification protects the protein from permanent oxidative damage, but at the cost of reversible loss of activity and subsequent disruption of TCA cycle flux that contributes, in part, to the diminished oxidative metabolism observed in the TXNIP deficient mice.

In aggregate, this work sheds new light onto the physiological role of TXNIP in

skeletal muscle as it pertains to substrate metabolism and fuel switching in response to nutrient availability. This work has important implications for metabolic diseases such as obesity and type 2 diabetes, which are characterized by marked disruptions in fuel selection.