dorsomorphin and Disease-Models--Animal

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by progressive heterotopic bone formation in skeletal muscle tissue. In patients with FOP, heterozygous mutations have been identified in the BMP type I receptor, ALK2. This finding stimulates establishment of some animal models of FOP and development of novel treatments for FOP.

Topics: Activin Receptors, Type I; Animals; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Disease Models, Animal; Drug Design; Genes, Dominant; Humans; Muscles; Mutation; Myositis Ossificans; Pyrazoles; Pyrimidines; Regeneration; Signal Transduction

Glucosamine hydrochloride (GAH), one of the most basic and important derivatives of chitin, is obtained by hydrolysis of chitin in concentrated hydrochloric acid. At present, little is known about how GAH functions in skeletal development. In this report, we demonstrate that GAH, extracted from the cell wall of

Topics: Agaricus; Animals; Bone and Bones; Bone Morphogenetic Proteins; Disease Models, Animal; Glucosamine; Larva; Osteoporosis; Regeneration; Signal Transduction; Skeleton; Zebrafish

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Rosmarinic acid (RA) is extensively utilized in herbal medicine in China. The AMP-activated protein kinase (AMPK) signaling can be activated by RA and inhibited by the synthetic, reversible AMP-competitive inhibitor, Compound C (CC). The objective of this study was to investigate the role of AMPK signaling involving the protective effects of RA on concanavalin A (Con A)-induced autoimmune hepatitis (AIH) in mice. BALB/c mice were treated with RA, with or without CC, followed by the pretreatment with Con A. Analysis of serum aminotransferases and cytokines were conducted and liver tissue histology was performed to evaluate hepatic injury. Cytokine levels in serum and hepatic tissue were respectively measured by enzyme-linked immunoassay (ELISA) and used quantitative (q)PCR. Levels of phosphorylated acetyl CoA carboxylase in the liver, representing AMPK activation, were detected by Western blotting. Compared with the Con A group, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in RA group (100 and 150 mg/kg/d) were significantly reduced. RA also reduced hepatocyte swelling, cell death, and infiltration of leukocytes in the liver of Con A-treated mice. Serum levels of cytokines, such as interferon-γ (IFN-γ), interleukin-2 (IL-2) and interleukin-1β (IL-1β), were reduced by RA pretreatment, while the levels of serum interleukin-10 (IL-10), an anti-inflammatory cytokine, was elevated. These protective effects were reversed by treatment with CC. RA treatment reduced the hepatic damage via the activation of AMPK in the mice of Con A-induced. So RA acts as a potential part in the therapy of autoimmune hepatitis.

Topics: Alanine Transaminase; AMP-Activated Protein Kinases; Animals; Aspartate Aminotransferases; Cinnamates; Concanavalin A; Depsides; Disease Models, Animal; Drug Evaluation, Preclinical; Hepatitis, Autoimmune; Humans; Liver; Male; Mice; Mice, Inbred BALB C; Protective Agents; Pyrazoles; Pyrimidines; Rosmarinic Acid; Signal Transduction

Metformin has been shown to have anti-cytokine property. Lipopolysaccharide (LPS)-induced or repeated water avoidance stress (WAS)-induced visceral allodynia and increased gut permeability were pro-inflammatory cytokine-dependent responses, which were considered to be animal models of irritable bowel syndrome (IBS). We hypothesized that metformin improves symptoms in the patients with IBS by attenuating these visceral changes and tested the hypothesis in rats.. The threshold of the visceromotor response induced by colonic balloon distention was measured. Colonic permeability was determined in vivo by quantifying the absorbed Evans blue for 15 min spectrophotometrically.. Subcutaneously injected LPS (1 mg/kg) reduced the threshold of visceromotor response, and metformin (5-50 mg/kg for 3 days) intraperitoneally attenuated this response in a dose-dependent manner. Repeated WAS (1 h daily for 3 days) induced visceral allodynia, which was also blocked by metformin. The antinociceptive effect of metformin on the LPS-induced allodynia was reversed by compound C, an adenosine monophosphate-activated protein kinase inhibitor or N. Metformin attenuated the visceral allodynia and increased gut permeability in animal IBS models. These actions may be evoked via activation of adenosine monophosphate-activated protein kinase, nitric oxide, and central dopamine D2 pathways. These results indicate the possibility that metformin can be useful for treating IBS.

Topics: AMP-Activated Protein Kinases; Animals; Colon; Disease Models, Animal; Domperidone; Dopamine Antagonists; Evans Blue; Hyperalgesia; Hypoglycemic Agents; Intestinal Mucosa; Irritable Bowel Syndrome; Lipopolysaccharides; Male; Metformin; Naloxone; Narcotic Antagonists; NG-Nitroarginine Methyl Ester; Nociception; Permeability; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Rats; Rats, Sprague-Dawley; Stress, Physiological; Sulpiride

Topics: Animals; Aorta; Atherosclerosis; Biomarkers; Bone Morphogenetic Protein 2; Cells, Cultured; Chondrogenesis; Disease Models, Animal; Extracellular Matrix Proteins; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout, ApoE; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphates; Plaque, Atherosclerotic; Primary Cell Culture; Protein Binding; Proteins; Pyrazoles; Pyrimidines; Signal Transduction; Smad Proteins; Vascular Calcification

Various studies proved spinal AMPA receptors were involved in the formation of neuropathic pain. In this study, we investigated the effect of methyl cinnamate (MC), a flavoring agent widely used in food and commodity industry, on CCI-induced upregulation of spinal AMPARs and pain hypersensitive behaviors. Results indicated that MC treatment dosage-dependently inhibited CCI-induced mechanical and thermal hypersensitivity. To further investigate the effect of MC after the formation of neuropathic pain, MC at the dosage of 100 mg/kg was administrated on day 7-14 on CCI rats. Results showed that MC treatment for seven days alleviated CCI-induced pain hypersensitivity after the formation of neuropathic pain. MC treatment reversed CCI-induced upregulation of GluR2, GluR3 and phosphorylation of GluR1. Further, MC dosage-dependently alleviated CCI-induced activation of mTOR and the downstream p70s6k. MC dosage-dependently induced activation of AMPK. All the MC-induced effects in CCI rats were completely reversed by Compound C, a AMPK inhibitor. These results meant MC treatment mitigated CCI-induced upregualtion of spinal AMPA receptors and pain hypersensitive behaviors through actviation of AMPK.

Topics: AMP-Activated Protein Kinases; Animals; Cinnamates; Constriction, Pathologic; Disease Models, Animal; Dose-Response Relationship, Drug; Humans; Hyperalgesia; Male; Neuralgia; Phosphorylation; Pyrazoles; Pyrimidines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Spinal Cord; Up-Regulation

5'-AMP-activated protein kinase (AMPK) is an energy sensor that controls cell metabolism, and it has been related to apoptosis and cell-cycle arrest. Although its role in metabolic homeostasis is well documented, its function in cancer is much less clear. In this study, we examined the role of AMPK in a mouse model of astrocytoma driven by oncogenic H-Ras(V12) and/or with PTEN deletion based on the common constitutive activation of the Raf/MEK/ERK and PI3K/AKT cascades in human astrocytomas. We also evaluated the activity and role of AMPK in human glioblastoma cells and xenografts. AMPK was constitutively activated in astrocytes expressing oncogenic H-Ras(V12) in parallel with high cell division rates. Genetic deletion of AMPK or attenuation of its activity in these cells was sufficient to reduce cell proliferation. The levels of pAMK were always related to the levels of phosphorylated retinoblastoma (Rb) at Ser804, which may indicate an AMPK-mediated phosphorylation of Rb. We confirmed this AMPK-Rb relationship in human glioblastoma cell lines and xenografts. In clinical specimens of human glioblastoma, elevated levels of activated AMPK appeared especially in areas of high proliferation surrounding the blood vessels. Together, our findings indicate that the initiation and progression of astrocytic tumors relies upon AMPK-dependent control of the cell cycle, thereby identifying AMPK as a candidate therapeutic target in this setting.

Topics: AMP-Activated Protein Kinases; Animals; Astrocytes; Astrocytoma; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Enzyme Activation; Gene Expression; Glioblastoma; Humans; Mice; Protein Transport; Proto-Oncogene Proteins p21(ras); Pyrazoles; Pyrimidines; Xenograft Model Antitumor Assays

Since AMP-activated protein kinase (AMPK) activation in skeletal muscle of obese rodents stimulates fatty acid oxidation, it is reasonable to hypothesize that pharmacological activation of AMPK might be of therapeutic benefit in obesity.. To investigate the effects of the traditional Korean anti-obesity drug GGEx18, a mixture of three herbs, Laminaria japonica Aresch (Laminariaceae), Rheum palmatum L. (Polygonaceae), and Ephedra sinica Stapf (Ephedraceae), on obesity and the involvement of AMPK in this process.. After high fat diet-induced obese mice were treated with GGEx18, we studied the effects of GGEx18 on body weight, fat mass, skeletal muscle lipid accumulation, and the expressions of AMPK, peroxisome proliferator-activated receptor ά (PPARα), and PPARα target genes. The effects of GGEx18 and/or the AMPK inhibitor compound C on lipid accumulation and expression of the above genes were measured in C2C12 skeletal muscle cells.. Administration of GGEx18 to obese mice for 9 weeks significantly (p < 0.05) decreased body and adipose tissue weights compared with obese control mice (p < 0.05). Lipid accumulation in skeletal muscle was inhibited by GGEx18. GGEx18 significantly (p < 0.05) increased skeletal muscle mRNA levels of AMPKα1 and AMPKα2 as well as PPARα and its target genes. Consistent with the in vivo data, GGEx18 inhibited lipid accumulation, and similar activation of genes was observed in GGEx18-treated C2C12 cells. However, compound C inhibited these effects in C2C12 cells.. These results suggest that GGEx18 improves obesity through skeletal muscle AMPK and AMPK-stimulated expression of PPARα and its target enzymes for fatty acid oxidation.

Topics: Adiposity; AMP-Activated Protein Kinases; Animals; Anti-Obesity Agents; Cell Line; Diet, High-Fat; Disease Models, Animal; Dose-Response Relationship, Drug; Ephedra sinica; Gene Expression Regulation; Laminaria; Lipid Metabolism; Male; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Obesity; Plant Extracts; Plant Preparations; Plants, Medicinal; PPAR alpha; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Rheum; RNA, Messenger; Signal Transduction; Time Factors; Weight Loss

Chlorogenic acid (CGA) has been shown to delay intestinal glucose absorption and inhibit gluconeogenesis. Our aim was to investigate the role of CGA in the regulation of glucose transport in skeletal muscle isolated from db/db mice and L6 skeletal muscle cells. Oral glucose tolerance test was performed on db/db mice treated with CGA and soleus muscle was isolated for 2-deoxyglucose transport study. 2DG transport was also examined in L6 myotubes with or without inhibitors such as wortmannin or compound c. AMPK was knocked down with AMPKα1/2 siRNA to study its effect on CGA-stimulated glucose transport. GLUT 4 translocation, phosphorylation of AMPK and Akt, AMPK activity, and association of IRS-1 and PI3K were investigated in the presence of CGA. In db/db mice, a significant decrease in fasting blood sugar was observed 10 minutes after the intraperitoneal administration of 250 mg/kg CGA and the effect persisted for another 30 minutes after the glucose challenge. Besides, CGA stimulated and enhanced both basal and insulin-mediated 2DG transports in soleus muscle. In L6 myotubes, CGA caused a dose- and time-dependent increase in glucose transport. Compound c and AMPKα1/2 siRNA abrogated the CGA-stimulated glucose transport. Consistent with these results, CGA was found to phosphorylate AMPK and ACC, consistent with the result of increased AMPK activities. CGA did not appear to enhance association of IRS-1 with p85. However, we observed activation of Akt by CGA. These parallel activations in turn increased translocation of GLUT 4 to plasma membrane. At 2 mmol/l, CGA did not cause any significant changes in viability or proliferation of L6 myotubes. Our data demonstrated for the first time that CGA stimulates glucose transport in skeletal muscle via the activation of AMPK. It appears that CGA may contribute to the beneficial effects of coffee on Type 2 diabetes mellitus.

Topics: AMP-Activated Protein Kinases; Animals; Blood Glucose; Cell Proliferation; Cell Survival; Chlorogenic Acid; Coffee; Diabetes Mellitus, Type 2; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Fasting; Gene Silencing; Glucose Transporter Type 4; Humans; Insulin Receptor Substrate Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Phosphatidylinositol 3-Kinases; Protein Transport; Pyrazoles; Pyrimidines; Signal Transduction

Hypothermia is robustly protective in pre-clinical models of both global and focal ischemia, as well as in patients after cardiac arrest. Although the mechanism for hypothermic neuroprotection remains unknown, reducing metabolic drive may play a role. Capitalizing on the beneficial effects of hypothermia while avoiding detrimental effects such as infection will be the key to moving this therapy forward as a treatment for stroke. AMPK is a master energy sensor that monitors levels of key energy metabolites. AMPK is activated via phosphorylation (pAMPK) when cellular energy levels are low, such as that seen during ischemia. AMPK activation appears to be detrimental in experimental stroke, likely via exacerbating ischemia-induced metabolic failure. We tested the hypothesis that hypothermia reduces AMPK activation. First, it was found that hypothermia reduced infarct after middle cerebral artery occlusion. Second, induced hypothermia reduced brain pAMPK in both sham control and stroke mice. Third, hypothermic neuroprotection was ameliorated after administration of compound C, an AMPK inhibitor. Finally, deletion of one of the catalytic isoforms of AMPK completely reversed the effect of hypothermia on stroke outcome after both acute and chronic survival. These effects were mediated by a reduction in AMPK activation rather than a reduction in LKB1, an upstream AMPK kinase. In summary, these studies provide evidence that hypothermia exerts its protective effect in part by inhibiting AMPK activation in experimental focal stroke. This suggests that AMPK represents a potentially important biological target for stroke treatment.

Topics: Acute Disease; AMP-Activated Protein Kinases; Animals; Chronic Disease; Disease Models, Animal; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuroprotective Agents; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Pyrimidines; Stroke

Increased blood-brain barrier (BBB) Na-K-Cl cotransporter activity appears to contribute to cerebral edema formation during ischemic stroke. We have shown previously that inhibition of BBB Na-K-Cl cotransporter activity reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. We have also shown that the BBB cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), although the mechanisms responsible are not well understood. AMP-activated protein kinase (AMPK), a key mediator of cell responses to stress, can be activated by a variety of stresses, including ischemia, hypoxia, and aglycemia. Previous studies have shown that the AMPK inhibitor Compound C significantly reduces infarct in mouse MCAO. The present study was conducted to evaluate the possibility that AMPK participates in ischemic factor-induced stimulation of the BBB Na-K-Cl cotransporter. Cerebral microvascular endothelial cells (CMEC) were assessed for Na-K-Cl cotransporter activity as bumetanide-sensitive (86)Rb influx. AMPK activity was assessed by Western blot analysis and immunofluorescence methods using antibodies that detect total versus phosphorylated (activated) AMPK. We found that hypoxia (7% and 2% O(2)), aglycemia, AVP, and oxygen-glucose deprivation (5- to 120-min exposures) increase activation of AMPK. We also found that Compound C inhibition of AMPK reduces hypoxia-, aglycemia-, and AVP-induced stimulation of CMEC Na-K-Cl cotransporter activity. Confocal immunofluorescence of perfusion-fixed rat brain slices revealed the presence of AMPK, both total and phosphorylated kinase, in BBB in situ of both control and ischemic brain. These findings suggest that ischemic factor stimulation of the BBB Na-K-Cl cotransporter involves activation of AMPK.

Topics: Adenylate Kinase; Animals; Arginine Vasopressin; Blood-Brain Barrier; Blotting, Western; Bumetanide; Cattle; Cell Hypoxia; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Fluorescent Antibody Technique; Glucose; Infarction, Middle Cerebral Artery; Male; Microvessels; Phosphorylation; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Rats; Rats, Sprague-Dawley; Signal Transduction; Sodium Potassium Chloride Symporter Inhibitors; Sodium-Potassium-Chloride Symporters; Time Factors; Up-Regulation

Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor (PPAR)-γ agonist, has been demonstrated to possess cardioprotective properties during ischemia-reperfusion. However, this notion remains controversial as recent evidence has suggested an increased risk in cardiac events associated with long-term use of RGZ in patients with type 2 diabetes. In this study, we tested the hypothesis that acute RGZ treatment is beneficial during I/R by modulating cardioprotective signaling pathways in a nondiabetic mouse model. RGZ (1 μg/g) was injected intravenously via the tail vein 5 min before reperfusion. Myocardial infarction was significantly reduced in mice treated with RGZ compared with vehicle controls (8.7% ± 1.1% vs. 20.2% ± 2.5%, P < 0.05). Moreover, isolated hearts were subjected to 20 min of global, no-flow ischemia in an ex vivo heart perfusion system. Postischemic recovery was significantly improved with RGZ treatment administered at the onset of reperfusion compared with vehicle (P < 0.001). Immunoblot analysis data revealed that the levels of both phospho-AMP-activated protein kinase (Thr(172)) and phospho-Akt (Ser(473)) were significantly upregulated when RGZ was administered 5 min before reperfusion compared with vehicle. On the other hand, inflammatory signaling [phospho-JNK (Thr(183)/Tyr(185))] was significantly downregulated as a result of RGZ treatment compared with vehicle (P < 0.05). Intriguingly, pretreatment with the selective PPAR-γ inhibitor GW-9662 (1 μg/g iv) 10 min before reperfusion significantly attenuated these beneficial effects of RGZ on the ischemic heart. Taken together, acute treatment with RGZ can reduce ischemic injury in a nondiabetic mouse heart via modulation of AMP-activated protein kinase, Akt, and JNK signaling pathways, which is dependent on PPAR-γ activation.

Topics: AMP-Activated Protein Kinases; Anilides; Animals; Cardiotonic Agents; Disease Models, Animal; Drug Administration Schedule; Enzyme Activation; Injections, Intravenous; JNK Mitogen-Activated Protein Kinases; Male; Mice; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphorylation; PPAR gamma; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyrimidines; Rosiglitazone; Signal Transduction; Thiazolidinediones; Time Factors; Ventricular Function, Left; Ventricular Pressure

Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation.. Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A(2A) receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A(2A) receptor or alpha1-AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-kappaB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation.. Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis.

Topics: Adenosine A2 Receptor Antagonists; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Apolipoproteins E; Atherosclerosis; Blood Coagulation; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Activation; Fibrinolytic Agents; Humans; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Monocytes; NF-kappa B; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyrimidines; Receptor, Adenosine A2A; Ribonucleosides; RNA, Messenger; Sepsis; Signal Transduction; Thromboplastin; Transcription Factor AP-1; Triazines; Triazoles; Up-Regulation; Venous Thrombosis

Adenosine monophosphate activated protein kinase (AMPK) has been identified as a regulator of vascular function via the preservation of endothelial cell (EC) function. In this study, we examined whether the beneficial effects of AMPK on ECs are dependent on its involvement in cholesterol efflux and its impact on hypercholesterolemia-induced endothelial dysfunction.. Using human aortic ECs and bovine aortic ECs, we show that AMPK activation upregulates ATP binding cassette G1 (ABCG1) expression independently of liver X receptor alpha (LXR alpha) transcriptional activity but through a posttranscriptional mechanism that increases mRNA stability. Using a heterologous system and a luciferase reporter, we further identify that the 3'-untranslated region of the ABCG1 mRNA is responsible for the regulatory effects of AMPK activation. 5-Aminoimidazole-4-carboxamide-1-beta-D-riboside treatment promotes endothelial 7-ketocholesterol efflux and prevents 7-ketocholesterol (7-KC)-induced reactive oxygen species production in an ABCG1-dependent manner, thus preserving endothelial nitric oxide synthase activity and nitric oxide bioavailability. Notably, in vivo studies using C57BL/6J mice receiving a high-cholesterol diet revealed that the infusion of 5-aminoimidazole-4-carboxamide-1-beta-d-riboside increases vascular ABCG1 expression and improves vascular reactivity. These effects are abrogated by the AMPK antagonist compound C and by the vascular gene transfer of ABCG1 small interfering RNA.. Our current findings uncover a novel mechanism by which AMPK protects against hypercholesterolemia-mediated endothelial dysfunction.

Topics: 3' Untranslated Regions; Acetylcholine; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; ATP Binding Cassette Transporter, Subfamily G, Member 1; ATP-Binding Cassette Transporters; Biological Transport; Cattle; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Endothelium, Vascular; Enzyme Activation; Enzyme Activators; Genes, Reporter; Humans; Hypercholesterolemia; Ketocholesterols; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Nitric Oxide Synthase Type III; Nitroprusside; Oxidative Stress; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Reactive Oxygen Species; Ribonucleotides; RNA Interference; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger; Transfection; Up-Regulation; Vasodilation; Vasodilator Agents

Macrophages (MPhis) utilize macropinocytosis to integrate immune and metabolic signals in order to initiate an effective immune response. Diabetes is characterized by metabolic abnormalities and altered immune function. Here we examine the influence of diabetes on macropinocytosis in primary mouse macrophages and in an in vitro diabetes model.. The data demonstrate that peritoneal MPhis from diabetic (db/db) mice had reduced macropinocytosis when compared to MPhis from non-diabetic (db/+) mice. Additionally, MPhis cultured in hyperglycemic conditions were less adept at macropinocytosis than those cultured in low glucose. Notably, AMP-activated protein kinase (AMPK) activity was decreased in MPhis cultured in hyperglycemic conditions. Activation of AMPK with leptin or 5-aminoimidazole-4-carboxamide-1-beta-riboside (AICAR) increased macropinocytosis and inhibition of AMPK with compound C decreased macropinocytosis.. Taken together, these findings indicate that MPhis from diabetic mice have decreased macropinocytosis. This decrease appears dependent on reduced AMPK activity. These results demonstrate a previously unrealized role for AMPK in MPhis and suggest that increasing AMPK activity in diabetic MPhis could improve innate immunity and decrease susceptibility to infection.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Cell Culture Techniques; Cell Line, Tumor; Diabetes Mellitus, Type 2; Disease Models, Animal; Energy Metabolism; Glucose; Hyperglycemia; Immunity; Leptin; Macrophage Activation; Macrophages, Peritoneal; Mice; Pinocytosis; Pyrazoles; Pyrimidines; Ribonucleosides

Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2). Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I receptor kinases, LDN-193189 (ref. 6), inhibits activation of the BMP signaling effectors SMAD1, SMAD5 and SMAD8 in tissues expressing caALK2 induced by adenovirus specifying Cre (Ad.Cre). This treatment resulted in a reduction in ectopic ossification and functional impairment. In contrast to localized induction of caALK2 by Ad.Cre (which entails inflammation), global postnatal expression of caALK2 (induced without the use of Ad.Cre and thus without inflammation) does not lead to ectopic ossification. However, if in this context an inflammatory stimulus was provided with a control adenovirus, ectopic bone formation was induced. Like LDN-193189, corticosteroid inhibits ossification in Ad.Cre-injected mutant mice, suggesting caALK2 expression and an inflammatory milieu are both required for the development of ectopic ossification in this model. These results support the role of dysregulated ALK2 kinase activity in the pathogenesis of FOP and suggest that small molecule inhibition of BMP type I receptor activity may be useful in treating FOP and heterotopic ossification syndromes associated with excessive BMP signaling.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type I; Cell Line; Disease Models, Animal; Mice; Mice, Inbred C57BL; Molecular Structure; Myositis Ossificans; Ossification, Heterotopic; Pyrazoles; Pyrimidines; Signal Transduction; Tomography, X-Ray Computed

p38 mitogen-activated protein kinase (p38 MAPK) and AMP-activated protein kinase (AMPK) are activated by, and influence sensitivity to, myocardial ischemia. Recently a number of studies have suggested that AMPK may participate in the activation of p38 MAPK. We therefore examined whether AMPK may be the principal "ischemia sensor" responsible for p38 MAPK activation during myocardial ischemia.. We used a variety of approaches to alter AMPK activity during ischemia and studied the repercussions on p38 MAPK activation.. The activities of AMPK and p38 MAPK were temporally related in adult rat ventricular myocytes (ARVM) subjected to simulated ischemia and in isolated mouse hearts subjected to no-flow ischemia. However p38 MAPK activation was unaltered in mouse hearts lacking the predominant or minor myocardial isoforms, AMPKalpha2 or AMPKalpha1 respectively. Likewise, in ARVM, adenoviral-driven expression of the minor myocardial isoform AMPKalpha1, in a constitutively active or dominant negative form reducing AMPK activity, did not alter p38 MAPK activation under basal conditions or during simulated ischemia. Finally, pharmacological inhibition of AMPK during ischemia with compound C did not attenuate the coincident activation of p38 MAPK.. Although AMPK and p38 MAPK are both activated during myocardial ischemia, the activation of p38 MAPK occurs independently of AMPK.

Topics: AMP-Activated Protein Kinases; Animals; Cells, Cultured; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardial Ischemia; Myocytes, Cardiac; p38 Mitogen-Activated Protein Kinases; Pyrazoles; Pyrimidines; Rats