Influence of mitochondrial content on the sensitivity of respiratory control

This study evaluated the sensitivity of mitochondrial respiratory control as a function of tissue oxidative capacity. The mitochondrial content of rat skeletal muscle was increased by exercise training or decreased by hypothyroidism. Muscles of the lower hindlimb were stimulated to tetanically contract in situ for 3 min at one of four frequencies to elicit a 30-fold range of oxygen consumption rates. Freeze-clamped sections of fast-twitch red gastrocnemius muscle were extracted and analyzed for metabolite levels. The sensitivity of respiratory control was examined for three models of cytosolic respiratory control (ADPf, ATP/ADPf, and ATP/(ADPf X Pi]; for each proposed model, sensitivity went up as mitochondrial content increased. Thus, a smaller change in cytosolic modulator (e.g., ADPf) is required as oxidative capacity increases. Increases in the sensitivity of cytosolic respiratory control resulted in lower flux through the near-equilibrium energy exchange reactions of creatine kinase and myokinase such that calculated free concentrations of ADP and AMP were less. Other energetically important reactions/pathways were also affected. Accumulation of lactate and the deamination of AMP to IMP were lower in tissues with higher mitochondrial content. In summary, changes in oxidative capacity directly influence the sensitivity of cytosolic respiratory control and this, in turn, has important consequences for maintenance of cellular energy balance.     

Molecular and kinetic alterations of muscle AMP deaminase during chronic creatine depletion

We examined apossible mechanism to account for the maintenance of peak AMPdeamination rate in fast-twitch muscle of rats fed the creatine analog-guanidinopropionic acid (-GPA), in spite of reduced abundance ofthe enzyme AMP deaminase (AMPD). AMPD enzymatic capacity (determined atsaturating AMP concentration) and AMPD protein abundance (Western blot)were coordinately reduced ~80% in fast-twitch white gastrocnemiusmuscle by -GPA feeding over 7 wk. Kinetic analysis of AMPD in thesoluble cell fraction demonstrated a single Michaelis-Menten constant(K_m; ~1.5 mM) in control muscle extracts. An additional high-affinityK_m (~0.03 mM)was revealed at low AMP concentrations in extracts of -GPA-treated muscle. The kinetic alteration in AMPD reflects increased molecular activity at low AMP concentrations; this could account for high ratesof deamination in -GPA-treated muscle in situ, despite the loss ofAMPD enzyme protein. The elimination of this kinetic effect bytreatment of -GPA-treated muscle extracts with acid phosphatase invitro suggests that phosphorylation is involved in the kinetic controlof skeletal muscle AMPD in vivo.

     

Increased adenine nucleotides in liver mitochondria after mannoheptulose injection in vivo

In adult rats, mannoheptulose injection causes a transient decrease in the serum insulin-to-glucagon ratio and a concomitant increase in serum glucose concentration. These effects attain a maximum 1 h after the injection and then decline toward normal. Correlated with the hormone changes is a dramatic increase in the adenine nucleotide content (ATP + ADP + AMP) of liver mitochondria, which peaks to over 50% of control values at 1 h. The increase in mitochondrial adenine nucleotides must occur by uptake from the cytosol, because the adenine nucleotide content of the whole tissue remains constant. The accumulation of adenine nucleotides by the mitochondria probably occurs over the recently characterized carboxyatractyloside-insensitive transport pathway that allows exchange of ATP-Mg for Pi. The actual mechanism by which net uptake is regulated after mannoheptulose injection has not yet been elucidated; however, changes in the Km or Vmax of the carrier and an increase in the tissue ATP/ADP ratio were eliminated as possibilities. The increase in matrix adenine nucleotide content in response to hormone changes brought about by mannoheptulose was much greater and more reproducible than what is achieved with glucagon injection. Mannoheptulose treatment may therefore be preferable as a model for further study of hormone effects on mitochondrial function.     

Alterations in AMP deaminase activity and kinetics in skeletal muscle of creatine kinase-deficient mice

Alterations in the competency of the creatine kinase systemelicit numerous structural and metabolic compensations, including changes in purine nucleotide metabolism. We evaluated molecular andkinetic changes in AMP deaminase from skeletal muscles of micedeficient in either cytosolic creatine kinase alone(M-CK^/) or alsodeficient in mitochondrial creatine kinase(CK^/) comparedwith wild type. We found that predominantly fast-twitch muscle, but notslow-twitch muscle, from bothM-CK^/ andCK^/ mice had muchlower AMP deaminase; the quantity of AMP deaminase detected by Westernblot was correspondingly lower, whereas AMP deaminase-1(AMPD1) gene expressionwas unchanged. Kinetic analysis of AMP deaminase from mixed musclerevealed negative cooperativity that was significantly greater increatine kinase deficiencies. Treatment of AMP deaminase with acidphosphatase abolished negative cooperative behavior, indicating that aphosphorylation-dephosphorylation cycle may be important in theregulation of AMP deaminase.