ABSTRACT
SCOPE: Piperonal is an aromatic compound found in vanilla and has a floral odor resembling vanillin. This study was aimed to test whether piperonal attenuates visceral adiposity induced by a high-fat diet (HFD) in mice and to explore the underlying molecular mechanisms. METHODS AND RESULTS: Male C57BL/6N mice were fed a normal diet, HFD, or 0.05% piperonal-supplemented HFD (PSD) for 10 weeks. PSD-fed mice showed attenuation of body weight gain, total visceral fat pad weights, and plasma lipid levels compared to HFD-fed mice. Piperonal supplementation of the HFD increased the mRNA expression of certain isotypes of adenylate cyclase (Adcy) and protein kinase A (PKA) in the white adipose tissue (WAT) of mice. The adipogenesis-related genes were downregulated, whereas fatty acid oxidation- and thermogenesis-related genes were upregulated in the WAT of PSD-fed mice compared to those in HFD-fed mice. Piperonal directly activated Adcy by decreasing the Km for its substrate (ATP) in plasma membranes prepared from the WAT of mice. Furthermore, piperonal-induced inhibition of adipocyte differentiation and elevation of Adcy and PKA activities in 3T3-L1 cells were abrogated by an Adcy inhibitor. CONCLUSION: The anti-adipogenic effect of piperonal in mice fed the high-fat diet appears to be associated with increased Adcy-PKA signaling in WAT.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Adenylyl Cyclases/metabolism , Adiposity , Anti-Obesity Agents/therapeutic use , Benzaldehydes/therapeutic use , Benzodioxoles/therapeutic use , Intra-Abdominal Fat/pathology , Obesity, Abdominal/prevention & control , 3T3-L1 Cells , AMP-Activated Protein Kinases/genetics , Adenylyl Cyclase Inhibitors/pharmacology , Adenylyl Cyclases/chemistry , Adenylyl Cyclases/genetics , Adipogenesis/drug effects , Adiposity/drug effects , Animals , Anti-Obesity Agents/metabolism , Benzaldehydes/metabolism , Benzodioxoles/metabolism , Diet, High-Fat/adverse effects , Dietary Supplements , Gene Expression Regulation, Enzymologic/drug effects , Intra-Abdominal Fat/drug effects , Intra-Abdominal Fat/enzymology , Intra-Abdominal Fat/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Mice , Mice, Inbred C57BL , Obesity, Abdominal/etiology , Obesity, Abdominal/metabolism , Obesity, Abdominal/pathology , Random Allocation , Signal Transduction/drug effects , Specific Pathogen-Free Organisms , Thermogenesis/drug effectsABSTRACT
Adenylyl cyclase 2 (AC2) is one of nine membrane-bound isoforms of adenylyl cyclase that converts ATP into cyclic AMP (cAMP), an important second messenger molecule. Upregulation of AC2 is linked to cancers like pancreatic and small intestinal neuroendocrine tumors (NETs). The structures of the various isoforms of adenylyl cyclases are highly homologous, posing a significant challenge to drug discovery efforts for an effective, isoform-selective modulator of AC2. In a previous study, a screen identified a potential isoform-selective and noncompetitive inhibitor of AC2, SKF83566. In the present study, molecular modeling is used to explore the mode of inhibition of AC2 by SKF83566 and to investigate the active enantiomer of SKF83566. Homology models of hAC2 were built based on canine AC5-C1a and rat AC2-C2a templates. With these models, a combination of flexible docking, molecular dynamics simulations, and free energy calculations using the MM/GBSA methodology suggested an allosteric mechanism in which (S)-SKF83566 binds to an allosteric site near ATP and alters the protein conformation of the ATP binding site, potentially preventing the adenosine moiety of ATP from forming an archlike shape to form cAMP. The predicted binding preference for the (S)-SKF83566 enantiomer and the predicted free energy are consistent with the experimental data.
Subject(s)
Adenylyl Cyclases/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Molecular Docking Simulation , Molecular Dynamics Simulation , Adenylyl Cyclases/chemistry , Drug Evaluation, Preclinical , Enzyme Inhibitors/metabolism , Humans , Protein Conformation , Sequence Homology, Amino Acid , StereoisomerismABSTRACT
SCOPE: Resveratrol (RSV), a natural polyphenol, has been reported to attenuate nonalcoholic fatty liver disease (NAFLD); however, its underlying mechanism is unclear. Autophagy was recently identified as a critical protective mechanism during NAFLD development. Therefore, we investigated the role of autophagy in the beneficial effects of RSV on hepatic steatosis. METHODS AND RESULTS: Via Oil red O staining, triglyceride, and ß-hydroxybutyrate detection, we found that RSV decreased palmitate-induced lipid accumulation and stimulated fatty acid ß-oxidation in hepatocytes. Based on Western blot assay, confocal microscopy and transmission electron microscopy, we found that RSV induced autophagy in hepatocytes, whereas autophagy inhibition markedly abolished RSV-mediated hepatic steatosis improvement. Moreover, RSV increased cAMP levels and the levels of SIRT1 (sirtuin 1), pPRKA (phosphorylated protein kinase A), and pAMPK (phosphorylated AMP-activated protein kinase), as well as SIRT1 activity in HepG2 cells. Incubation with inhibitors of AC (adenylyl cyclase), PRKA, AMPK, SIRT1, or with AC, PRKA, AMPK, or SIRT1 siRNA abolished RSV-mediated autophagy. Similar results were obtained in mice with hepatic steatosis. CONCLUSION: RSV improved hepatic steatosis partially by inducing autophagy via the cAMP-PRKA-AMPK-SIRT1 signaling pathway, which provides new evidence regarding RSV's effects on NAFLD treatment.
Subject(s)
Antioxidants/therapeutic use , Autophagy , Cyclic AMP/agonists , Dietary Supplements , Liver/metabolism , Non-alcoholic Fatty Liver Disease/diet therapy , Second Messenger Systems , Stilbenes/therapeutic use , Adenylyl Cyclases/chemistry , Adenylyl Cyclases/genetics , Adenylyl Cyclases/metabolism , Animals , Antioxidants/metabolism , Autophagy/drug effects , Cyclic AMP/antagonists & inhibitors , Cyclic AMP/metabolism , Enzyme Induction/drug effects , Enzyme Inhibitors/pharmacology , Fatty Acids, Nonesterified/adverse effects , Fatty Acids, Nonesterified/antagonists & inhibitors , Fatty Acids, Nonesterified/metabolism , Hep G2 Cells , Humans , Lipid Metabolism/drug effects , Liver/drug effects , Liver/pathology , Liver/ultrastructure , Mice, 129 Strain , Microscopy, Electron, Transmission , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , RNA Interference , Resveratrol , Second Messenger Systems/drug effects , Sirtuin 1/antagonists & inhibitors , Sirtuin 1/chemistry , Sirtuin 1/genetics , Sirtuin 1/metabolism , Stilbenes/metabolismABSTRACT
The catalytic efficiency of adenylyl cyclase activity of edema factor (EF) from Bacillus anthracis is enhanced by approximately 1000-fold upon its binding to mammalian protein calmodulin (CaM). A tandem cell-based and protein binding-based screen of a 10,000 member library identified a molecule that inhibits the EF-CaM interaction and therefore the adenylyl cyclase activity. A combination of fluorescence spectroscopy and photolabeling studies showed that the molecule targets the CaM binding region of EF. A series of related compounds were synthesized and evaluated to identify one compound, 4-[4-(4-nitrophenyl)-thiazolylamino]-benzenesulfonamide, that maintained activity against EF but showed minimal toxicity to two cultured cell lines. This compound represents an important reagent to study the role of EF in anthrax pathology and may represent a drug lead against anthrax infection.
Subject(s)
Adenylyl Cyclase Inhibitors , Adenylyl Cyclases/metabolism , Calmodulin/antagonists & inhibitors , Calmodulin/metabolism , Drug Evaluation, Preclinical/methods , Sulfonamides/chemistry , Sulfonamides/pharmacology , Thiazoles/chemistry , Thiazoles/pharmacology , Adenylyl Cyclases/chemistry , Animals , Anthrax/drug therapy , Antigens, Bacterial , Bacillus anthracis/chemistry , Bacillus anthracis/metabolism , Bacterial Toxins , Calmodulin/chemistry , Cell Line , Cricetinae , Ligands , Mice , Models, Molecular , Protein Binding/drug effects , Protein Conformation , Structure-Activity Relationship , Substrate Specificity , Sulfonamides/toxicity , Surface Plasmon Resonance , Thiazoles/toxicityABSTRACT
The retinal pigment epithelium (RPE) fulfills important supporting tasks to maintain the visual functions of the sensorineural retina. One major signalling mechanism by which adjacent tissues impinge on the RPE is the adenylyl cyclase (AC)/cAMP pathway. In the RPE, cAMP seems to modulate unique functions such as the phagocytosis of discs shed from the rod outer segments, transport of vitamin A or the ion and fluid control in the subretinal space. We analyzed the AC expression pattern in the retina and the RPE and found AC type 7 to be almost the only isoform expressed in the RPE. We cloned AC type 7 from a cDNA library established with fresh bovine RPE, expressed this isoform in eukaryotic cells and characterized some of its properties.
Subject(s)
Adenylyl Cyclases/metabolism , Isoenzymes/metabolism , Pigment Epithelium of Eye/enzymology , Adenylyl Cyclases/chemistry , Adenylyl Cyclases/genetics , Amino Acid Sequence , Animals , Blotting, Northern , COS Cells/enzymology , Cattle , DNA, Complementary/chemistry , Glucagon/pharmacology , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Kidney , Mice , Molecular Sequence Data , Norepinephrine/pharmacology , Pigment Epithelium of Eye/drug effects , Polymerase Chain Reaction , Signal Transduction , Vasoactive Intestinal Peptide/pharmacologyABSTRACT
We have cloned a cDNA that encodes a novel Xenopus laevis oocyte adenylyl cyclase (xlAC) using oligonucleotides against conserved mammalian adenylyl cyclase regions. The isolated cDNA is 4372 bp long with an open reading frame of 4065 nucleotides which encodes a protein of 1355 amino acids. Comparison of the deduced amino acid sequence with previously cloned mammalian adenylyl cyclases shows a low identity, 19.7% with type 2 rat adenylyl cyclase and 24.2% with type 4 rat adenylyl cyclase, indicating that this Xenopus isoform represents a new member of this protein family. Gene expression studies of the xlAC by reverse PCR showed that this gene is expressed in all oogenesis stages but not during early embryogenesis. Expression of the xlAC in COS-7 cells resulted in increased basal AC activity, that was stimulated by forskolin, Gpp(NH)p and aluminium fluoride, and was insensitive to calcium and calcium-calmodulin (Ca2(+)-CaM).