<p>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.</p><p>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. </p><p>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. </p><p>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.</p> / Dissertation

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Kalp ve Damar Cerrahisi Anabilim Dalı, 2006. / Bibliyografya var.

Decline in cardiac pump function is a hallmark of aging where mitochondrial decay is an important underlying cause. Although certainly multifactorial in nature, both dysfunction of the machinery involved in the chemiosmotic process of energy transduction and lower capacity to maintain fatty acid-driven respiration are identified as intrinsic factors of mitochondrial decay in the aged myocardium. Age-associated destabilization of electron transport supercomplexes as a potential factor of mitochondrial decay in the rat heart. Defective operation of the electron transport chain (ETC) constitutes a key mechanism involved in the age-associated loss of mitochondrial energy metabolism. Nevertheless, the molecular events underlying inefficient electron flux that ultimately leads to higher superoxide appearance and impaired respiration are not fully known. As recent biophysical evidence shows that the ETC may form large macromolecular assemblies (i.e. supercomplexes) that disintegrate in certain pathologies (e.g. heart failure or Barth syndrome) reminiscent of aging, we investigated the hypothesis that alterations in supercomplexes are partly responsible for the age-related loss of cardiac ETC function. In this dissertation, age-associated changes in supercomplex organization and stability were investigated in subsarcolemmal (SSM) and interfibrillary (IFM) mitochondria isolated from cardiac tissue from young (3-5 months) and old (24-28 months) male Fischer 344 rats. Blue native-PAGE (BN-PAGE) analysis of digitonin-solubilized mitochondrial membranes coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to investigate supercomplex organization. Results show that both SSM and IFM display supercomplexes comprised of various stoichiometries of complexes I, III and IV (never complex II), which typically organize as high mass (1500-2300 kDa) assemblies containing up to four copies of complex IV (i.e. I���III���IV[subscript N]-type supercomplexes). Interestingly, analysis of IFM proteins showed that, in general, supercomplex levels declined by up to 15 % (p < 0.05) with age; however, different degrees of supercomplex deterioration were observed, depending on the particular supercomplex investigated. Supercomplexes of the highest molecular weights (i.e. 1900-2300 kDa), which were also composed of the most complex stoichiometries (i.e. I1III2IVN, N ��� 2), were primarily lost with age. In particular, I���III���IV���, I���III���IV��� and I���III���IV��� supercomplexes were found to decline by 13% (p < 0.05), 30% (p < 0.05) and 45% (p < 0.05), respectively, on an age basis. Therefore, the age-associated loss of supercomplexes in IFM stems from destabilization of the assemblies that comprise several copies of complex IV, which could partially limit proper electron transfer to O��� for its reduction, affecting mitochondrial respiratory capacity. In contrast to IFM, the aging defects of SSM supercomplexes appeared to be confined to the assembly comprised of only one copy of complex IV (I���III���IV���, 1700 kDa) (37% loss; p = 0.06), while the higher molecular weight supercomplex sub-types that were most affected in IFM (i.e. I���III���IV[subscript N], N ��� 2) were not significantly altered with age. Thus, the results from this dissertation indicate that mitochondria from different subcellular locations in the myocyte show different degrees of supercomplex destabilization in the aging rat heart. The more robust supercomplex deficits noted for IFM fit well with previous observations that electron transport characteristics of this subpopulation are more adversely affected with age than SSM. Although the underlying factor(s) of supercomplex deterioration are not fully known, the hypothesis that age-related alterations of certain constituents of the IMM (e.g. cardiolipin) may be important factors of supercomplex destabilization in cardiac mitochondria was investigated in this dissertation. To this end, LC-MS/MS characterization of supercomplex proteins and HPLC analysis of cardiolipin were used as approaches to elucidate potential factor(s) of supercomplex destabilization in the aging rat heart. Age-related alterations of cardiolipin levels and its acyl-chain content showed a strong parallel to the age-associated destabilization of supercomplexes. Specifically, cardiolipin levels declined by 10% (p < 0.05) in IFM, the mitochondrial subpopulation displaying the highest degree of supercomplex deterioration. In addition, the content of (18:2)���-cardiolipin, the predominant species in the heart, was found to decline by 50% (p < 0.05) on average in both populations of cardiac mitochondria. Therefore, the data presented in this dissertation indicate that changes in cardiolipin may be at least one of the factors involved in supercomplex destabilization in the aging heart. Age-related decline in carnitine palmitoyltransferase I (CPT1) activity as a mitochondrial lesion that limits fatty acid catabolism in the rat heart. Loss of fatty acid utilization, another intrinsic factor of mitochondrial decay in the aged myocardium, has been associated with age-related alterations in the activity of carnitine palmitoyltransferase 1 (CPT1), the rate-controlling enzyme for overall fatty acid ��-oxidation. Nevertheless, the exact molecular mechanism involved in the age-related loss of fatty acid-driven bioenergetics is not fully understood. In this dissertation, it was also investigated whether the aging lesion for fatty oxidation lies in a particular mitochondrial subpopulation or more generally results from cardiac decrements in L-carnitine levels. In order to clarify the role of each one of these factors, the effect of long-term dietary supplementation with the L-carnitine analogue, acetyl-L-carnitine (ALCAR), was also investigated. Results show that aging selectively decreases CPT1 activity in IFM by reducing enzyme catalytic efficiency for palmitoyl-CoA. IFM displayed a 28% (p < 0.05) loss of CPT1 activity, which correlated with a decline (41%, p < 0.05) in palmitoyl-CoA-driven state 3 respiration. Interestingly, SSM had preserved enzyme function and efficiently utilized palmitate. Analysis of IFM CPT1 kinetics showed both diminished V[subscript max] and K[subscript m] (60% and 49% respectively, p < 0.05) when palmitoyl-CoA was the substrate. However, no age-related changes in enzyme kinetics were evident with respect to L-carnitine. ALCAR supplementation restored CPT1 activity in heart IFM, but not apparently through remediation of L-carnitine levels. Rather, ALCAR influenced enzyme activity over time, potentially by modulating conditions in the aging heart that ultimately affect palmitoyl-CoA binding and CPT1 kinetics. In conclusion, this dissertation presents a characterization of age-associated alterations in the macromolecular organization of the IMM components that could partly explain the loss of mitochondrial oxidative capacity that affects the aging heart. In addition, the characterization of an age-related lesion of the controlling enzyme for ��-oxidation is presented as another important factor that limits mitochondrial function and energy metabolism in cardiac mitochondria. / Graduation date: 2013