Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • Based on both the observational and theoretical

    2020-02-17

    Based on both the observational and theoretical data generated in this study, it fosaprepitant dimeglumine appears that the mechanism(s) underlying lower IFM CPT1 activity is complex. Modeling enzyme activity versus alterations in catalytic efficiency (Fig. 3A) suggests that the observed age-associated changes in Km and Vmax for palmitoyl-CoA account for essentially all of the observed loss of CPT1 activity. Thus, even though there is a general age-dependent decrease in myocardial l-carnitine levels, its decline has little consequence to enzyme activity. These rather surprising results actually reinforce the concept that CPT1 activity loss is localized to a specific mitochondrial subpopulation as diminished l-carnitine content would adversely affect CPT1 activity in the SSM fraction as well. Considering that we also previously showed no age-associated alterations to malonyl-CoA levels in the aging rat fosaprepitant dimeglumine (Moreau et al., 2004), it now becomes clear that the age-specific loss of IFM CPT1 catalytic efficiency stems from modifications to the enzyme and is not because of alterations to substrate levels or allosteric effectors. CPT1 follows a bi–bi ordered mode of catalysis where binding of a long-chain fatty acyl-CoA molecule to the enzyme active site initiates the reaction (McGarry and Brown, 1997, Ramsay et al., 2001). Mutations of glycine residues in human hepatic CPT1 specifically affect the palmitoyl-CoA binding pocket and lead to loss of enzyme function (Gobin et al., 2003, Morillas et al., 2004). Furthermore, enzyme activity was completely abolished in mitochondria from yeast expressing the mutant liver-specific CPT1 protein where Gly710 was changed to a Glu residue in the protein hydrophobic core (Gobin et al., 2003). We therefore propose that the loss of catalytic efficiency for palmitoyl-CoA utilization stems from limitations in its binding to the enzyme, thereby lowering overall catalytic activity even when l-carnitine levels are saturating. This type of altered enzyme kinetics is akin to an uncompetitive mode of inhibition where alterations of the enzyme substrate complex adversely affect overall catalytic efficiency of the enzyme (Fig. 6). The precise cause(s) leading to this uncompetitive mode of IFM CPT1 inhibition was not directly explored in the present study, and may be multifactorial. A plausible hypothesis is that age-associated oxidative modifications that selectively affect IFM CPT1 alter its catalytic activity by destabilization of palmitoyl-CoA, and that long-term supplementation of ALCAR reverses such alterations (see below). This concept is buttressed by previous reports showing that both CPT1 and carnitine octanoyltransferase contain specific binding sites for l-carnitine, acyl-CoA, and CoASH where amino acid residues in or proximal to the acyl-CoA binding region are key to stabilizing the acyl-CoA–enzyme complex during catalysis (Jogl et al., 2005, Morillas et al., 2004). Thus, reactive oxygen and nitrogen species, electrophiles, or small molecule conjugation, may alter critical amino acid residues at or near the palmitoyl-CoA binding pocket and limit formation of the initial enzyme–palmitoyl-CoA complex (Fig. 6). While characterization of specific protein modification(s) that lead to the aging lesion in IFM CPT1 catalysis is beyond the scope of the present study, there is literature precedent suggesting that IFM are particularly susceptible to protein modification. First, we previously showed that aging leads to higher rates of IFM oxidant appearance versus SSM (Suh et al., 2003), which could promote oxidative modifications that selectively affect that mitochondrial subpopulation. Second, Lesnefsky et al. (2001b) further reported that the cytochrome c binding site of complex III was specifically altered with age, but only in the IFM subpopulation. On the other hand, Sharma et al. (2010) recently showed that incubating cardiac mitochondria with peroxynitrite (ONOO−) initiated post-translational modifications of specific cysteine and tyrosine residues of CPT1, which in turn modulated enzyme activity. Taken together, these results suggest that aging causes an enhanced pro-oxidant milieu that is specific for IFM, thereby initiating conditions that adversely affect CPT1 catalysis in that mitochondrial subpopulation. We are currently examining the linkage between protein modification and the specific alterations to CPT1 activity seen in IFM of aging rat hearts.