sto-609 and Diabetes-Mellitus--Type-2

Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

Topics: Adaptor Proteins, Signal Transducing; Air Pollutants; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Benzimidazoles; Calcium-Calmodulin-Dependent Protein Kinases; Carbon-Oxygen Lyases; Carrier Proteins; Cell Cycle Proteins; Cells, Cultured; Cystathionine beta-Synthase; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Eukaryotic Initiation Factors; Extracellular Matrix Proteins; Glucose; Hydrogen Sulfide; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Multiprotein Complexes; Naphthalimides; Peptide Chain Elongation, Translational; Peptide Chain Initiation, Translational; Phosphoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Proteins; Rats; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Sweetening Agents; TOR Serine-Threonine Kinases

AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK alpha1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in alpha subunits, we screened a chemical library with inactive human alpha1(394) (alpha1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK alpha1(394), alpha1(335), alpha2(398), and even heterotrimer alpha1beta1gamma1. Based on PT1-docked AMPK alpha1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 with-out an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.

Topics: Acetyl-CoA Carboxylase; Adenosine Monophosphate; Adenosine Triphosphate; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Benzimidazoles; Diabetes Mellitus, Type 2; Energy Metabolism; Enzyme Activation; Enzyme Activators; HeLa Cells; Humans; Multienzyme Complexes; Myoblasts; Naphthalimides; Obesity; Phosphorylation; Protein Serine-Threonine Kinases; Protein Structure, Quaternary; Pyrazoles; Pyrimidines; Time Factors