Changes In AMP Concentration Research Articles

AMPD1: a novel therapeutic target for reversing insulin resistance.

BackgroundInsulin resistance is one of the hallmark manifestations of obesity and Type II diabetes and reversal of this pathogenic abnormality is an attractive target for new therapies for Type II diabetes. A recent report that metformin, a drug known to reverse insulin resistance, demonstrated in vitro the metformin can inhibit AMP deaminase (AMPD) activity. Skeletal muscle is one of the primary organs contributing to insulin resistance and that the AMPD1 gene is selectively expressed at high levels in skeletal muscle.MethodsRecognizing the background above, we asked if genetic disruption of the AMPD1 gene might ameliorate the manifestations of insulin resistance. AMPD1 deficient homozygous mice and control mice fed normal chow diet or a high-fat diet, and blood analysis, glucose tolerance test and insulin tolerance test were performed. Also, skeletal muscle metabolism and gene expression including nucleotide levels and activation of AMP activated protein kinase (AMP kinase) were evaluated in both conditions.ResultsDisruption of the AMPD1 gene leads to a less severe state of insulin resistance, improved glucose tolerance and enhanced insulin clearance in mice fed a high fat diet. Given the central role of AMP kinase in insulin action, and its response to changes in AMP concentrations in the cell, we examined the skeletal muscle of the AMPD1 deficient mice and found that they have greater AMP kinase activity as evidenced by higher levels of phosphorylated AMP kinase.ConclusionsTaken together these data suggest that AMPD may be a new drug target for the reversal of insulin resistance and the treatment of Type II diabetes.Electronic supplementary materialThe online version of this article (doi:10.1186/1472-6823-14-96) contains supplementary material, which is available to authorized users.

Structural insight into the autoinhibition mechanism of AMP-activated protein kinase

The AMP-activated protein kinase (AMPK) is characterized by its ability to bind to AMP, which enables it to adjust enzymatic activity by sensing the cellular energy status and maintain the balance between ATP production and consumption in eukaryotic cells. It also has important roles in the regulation of cell growth and proliferation, and in the establishment and maintenance of cell polarity. These important functions have rendered AMPK an important drug target for obesity, type 2 diabetes and cancer treatments. However, the regulatory mechanism of AMPK activity by AMP binding remains unsolved. Here we report the crystal structures of an unphosphorylated fragment of the AMPK alpha-subunit (KD-AID) from Schizosaccharomyces pombe that contains both the catalytic kinase domain and an autoinhibitory domain (AID), and of a phosphorylated kinase domain from Saccharomyces cerevisiae (Snf1-pKD). The AID binds, from the 'backside', to the hinge region of its kinase domain, forming contacts with both amino-terminal and carboxy-terminal lobes. Structural analyses indicate that AID binding might constrain the mobility of helix alphaC, hence resulting in an autoinhibited KD-AID with much lower kinase activity than that of the kinase domain alone. AMP activates AMPK both allosterically and by inhibiting dephosphorylation. Further in vitro kinetic studies demonstrate that disruption of the KD-AID interface reverses the autoinhibition and these AMPK heterotrimeric mutants no longer respond to the change in AMP concentration. The structural and biochemical data have shown the primary mechanism of AMPK autoinhibition and suggest a conformational switch model for AMPK activation by AMP.

5′-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C α and native bovine protein phosphatase-2A c

Human protein phosphatase-2C α (PP2C α) was purified to homogeneity after expression in Escherichia coli. AMP inhibited the dephosphorylation of AMP-activated protein kinase (AMPK), but not phosphocasein, by PP2C α. The concentration dependence and the effects of other nucleotides (ATP and formycin A-5′-monophosphate) suggest that AMP acts by binding to the same site which causes direct allosteric activation of AMPK. A similar, although less pronounced, effect was observed with another protein phosphatase (PP2A c). We have now shown that AMPK activates the AMPK cascade by four mechanisms, which should make the system exquisitely sensitive to changes in AMP concentration.

Adenosine production and energy metabolism in ischaemic and metabolically stimulated rat heart

Adenosine may modulate blood flow and electrical activity in heart in response to changes in myocardial energy metabolism. In the present study, 31P NMR spectroscopy was used to examine the relation between cytosolic phosphate metabolite levels and release of adenosine into the venous effluent of isovolumic heart during graded low-flow ischaemia or metabolic stimulation with isoproterenol. When coronary flow rate was varied in steps between 1.6 and 12 ml/min/g, cytosolic ATP levels did not change significantly but the phosphorylation potential exhibited a linear correlation with flow rate below approximately 7 ml/min/g. Purine release (adenosine and inosine) correlated linearly with the cytosolic phosphorylation potential and free AMP concentration. Metabolic stimulation of hearts with isoproterenol (0.4, 3.0, and 60 n m), produced a significant fall in cytosolic ATP levels and decreased the cytosolic phosphorylation potential. Purine release in these hearts increased exponentially as the cytosolic phosphorylation potential dropped, and as cytosolic free AMP increased. These results support a link between the phosphorylation potential and the mechanism of adenosine production during ischaemia and metabolic stimulation. Presumably, this link is the activity of the enzyme 5′-nucleotidase, which is responsible for converting AMP to adenosine, together with the concentration of its substrate, AMP. In low-flow ischaemia, cytosolic AMP may control adenosine formation. With isoproterenol stimulation, a more complex relationship exists, indicating possible allosteric regulation of the enzyme(s) responsible for adenosine formation, in addition to changes in AMP concentration.

Platelet AMP deaminase. Regulation by Mg-ATP2- and inorganic phosphate and inhibition by the transition state analog coformycin.

Kinetic studies with platelet AMP deaminase, at pH 7.0 and 100 mM NaC1, gave cooperative initial velocity curves with AMP as substrate, with Mg-ATP2- as an activator, and with Pi as an inhibitor. In the absence of Mg-ATP2-, the s0.5 for AMP was 4.5 mM with a Hill coefficient approaching 2.0. In the presence of saturating Mg-ATP2-, the s0.5 for AMP was reduced to 0.18 mM, the maximum velocity was increased by about 35%, and the Hill coefficient was 1.0. The half-activation constant for Mg-ATP2- varied from 0.7 to 0.07 mM as the concentration of AMP was varied from 0.1 to 5.0 mM and the Hill coefficient for Mg-ATP activation changed from 2.0 to 1.0 over the same range. Phosphate inhibition was competitive with AMP and with Mg-ATP2- (Ki = 2.0 mM) and reversed the activation by Mg-ATP2-. Coformycin inhibited the Mg-ATP-activated enzyme with a Ki less than 0.25 microM. Coformycin inhibition was slow, with a second order rate constant of 6.0 X 10(4) M-1 min-1, suggesting that the compound acts as a transition state analog according to Frieden, C., Kurz, L. C., and Gilbert, H. R. (1980) Biochemistry 19, 5303-5309. The kinetic properties of the enzyme indicate that substantial regulation can occur through changes in AMP concentration acting synergistically to enhance Mg-ATP2- binding and displace Pi from a single type of regulatory site.