Influence of chrysin on D-galactose induced-aging in mice: Up regulation of AMP kinase/liver kinase B1/peroxisome proliferator-activated receptor-γ coactivator 1-α signaling pathway.

Adenosine monophosphate kinase/liver kinase B1/peroxisome proliferator-activated receptor-γ coactivator 1-α (AMPK/LKB1/PGC1α) pathway has a vital role in regulating age-related diseases. It controls neurogenesis, cell proliferation, axon outgrowth, and cellular energy homeostasis. AMPK pathway also regulates mitochondrial synthesis. The current study evaluated the effect of chrysin on D-galactose (D-gal) induced-aging, neuron degeneration, mitochondrial dysfunction, oxidative stress, and neuroinflammation in mice. The mice were allocated randomly into four groups (10 each group): Group 1: normal control group, Group 2: D-gal group, Groups 3 and 4: chrysin (125 and 250 mg/kg, respectively). Groups 2-4 were injected with D-gal (200 mg/kg/day; s.c) for 8 weeks to induce aging. Groups 3 and 4 were orally gavaged every day concurrent with D-gal. At the end of experiment, behavioral, brain biochemical and histopathological changes were monitored. Chrysin administration elevated discrimination ratio in object recognition, Y Maze percentage alternation, locomotor activity and brain contents of AMPK, LKB1, PGC1α, NAD (P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), nerve growth factor (NGF) (neurotrophin-3; NT-3), and seretonin as well as reduced brain contents of tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), advanced glycation end products (AGEs) and glial fibrillary acidic protein (GFAP) compared to D-gal-treated mice. Chrysin also alleviated cerebral cortex and white matter neurons degeneration. Chrysin protects against neurodegeneration, improves mitochondrial autophagy and biogenesis as well as activates antioxidant genes expression. In addition, chrysin ameliorates neuroinflammation and stimulates the release of NGF and serotonin neurotransmitter. So, chrysin has a neuroprotective effect in D-gal induced-aging in mice.

Aspirin Protects against UVB-Induced DNA Damage through Activation of AMP Kinase.

The anti-inflammatory and chemopreventive activities of aspirin (ASA) may be mediated through its cyclooxygenase inhibitor function. We have previously shown that ASA can protect against UVR-induced skin inflammation and DNA damage; however, the role of inflammation in UV-induced DNA damage and the mechanism underlying ASA protection are poorly characterized. Using immunodeficient NOD scid gamma mice and immunocompetent C57BL/6 mice treated with immune cell‒depleting antibodies, we found that inflammation was not required for UVB-induced 8-oxoguanine and cyclobutane pyrimidine dimers in vivo. Unlike ASA, neither its immediate metabolite salicylate nor the cyclooxygenase inhibitor indomethacin reduced UVB-induced 8-oxoguanine or cyclobutane pyrimidine dimers in melanocyte Melan-a or keratinocyte HaCat cells in vitro. Moreover, addition of prostaglandin E2 did not reverse the protective effect of ASA on UVB-treated cells. Phosphorylation of the 5' AMP protein kinase, observed in ASA-treated cells, could be blocked by the 5' AMP protein kinase inhibitor compound C. Compound C or 5' AMP protein kinase knockdown partially reduced ASA-mediated protection against UVB-induced DNA damage. Finally, injection of compound C partially reversed the protective effect of ASA on UVB-treated mouse skin in vivo. These studies suggest that ASA confers protection against UVB-induced DNA damage through the activation of 5' AMP protein kinase rather than through cyclooxygenase inhibition.

Metformin Protects Against Sunitinib-induced Cardiotoxicity: Investigating the Role of AMPK.

ABSTRACT: Sunitinib is associated with cardiotoxicity through inhibition of AMP-protein kinase (AMPK) signaling. By contrast, the common antidiabetic agent metformin has demonstrated cardioprotection through indirect AMPK activation. In this study, we investigate the effects of metformin during sunitinib-induced cytotoxicity. Left ventricular developed pressure, coronary flow, heart rate, and infarct size were measured in Langendorff-perfused rat hearts treated with 1 µM sunitinib ±50 µM metformin ±1 µM human equilibrative nucleoside transporter inhibitor S-(4-Nitrobenzyl)-6-thioinosine (NBTI). Western blot analysis was performed for p-AMPKα levels. Primary isolated cardiac myocytes from the left ventricular tissue were used to measure live cell population levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess adjunctive treatment of and metformin in human hepatoma G2 and promyelocytic leukemia (HL-60) cells treated with 0.1-100 µM sunitinib ±50 µM metformin. In the perfused hearts, coadministration of metformin attenuated the sunitinib-induced changes to left ventricular developed pressure, infarct size, and cardiac myocyte population. Western blot analysis revealed a significant decrease in p-AMPKα during sunitinib treatment, which was attenuated after coadministration with metformin. All metformin-induced effects were attenuated, and NBTI was coadministered. The MTT assay demonstrated an increase in the EC50 value during coadministration of metformin with sunitinib compared with sunitinib monotherapy in hepatoma G2 and HL-60 cell lines, demonstrating the impact and complexity of metformin coadministration and the possible role of AMPK signaling. This study highlights the novel cardioprotective properties of metformin and AMPK activation during sunitinib-induced cardiotoxicity when administered together in the Langendorff heart model.

Ilexgenin A restrains CRTC2 in the cytoplasm to prevent SREBP1 maturation via AMP kinase activation in the liver.

BACKGROUND AND PURPOSE: Ilexgenin A is a triterpenoid from ShanLv Cha with beneficial effects on metabolic homeostasis. We investigated whether ilexgenin A could inhibit hepatic de novo fatty acid synthesis via the interfering with SREBP1 maturation. EXPERIMENTAL APPROACH: The effects of Ilexgenin A on CRTC2 translocation and SREBP1 maturation were investigated in the liver of fasted mice and hepatocytes exposed to saturated fatty acids. The effect of Iilexgenin A on hepatic lipid accumulation was also observed in high-fat diet fed mice. KEY RESULTS: Sec23A and Sec31A are two subunits of COPII complex and their interaction is essential for the processing of SREBP1 maturation. Ilexgenin A activates AMPK by reducing cellular energy and preventing cytoplasmic CRTC2 to compete with Sec23A for binding to Sec31A under nutrient-rich conditions. Consequently, ilexgenin A impaired COPII-dependent SREBP1 maturation via disrupting Sec31A-Sec23A interaction, leading to the inhibition of de novo fatty acid synthesis in the liver. In contrast, mTORC1 phosphorylated Ser136 of CRTC2, facilitating the formation of Sec31A-Sec23A interaction to promote SREBP1 maturation, whereas this action was reversed by ilexgenin A in an AMPK-dependent manner. Ilexgenin A protected CRTC2 function and restrained hepatic lipogenic response in high fat diet-fed mice, providing in vivo evidence to support the beneficial effects of ilexgenin A on lipid metabolism. CONCLUSIONS AND IMPLICATIONS: Ilexgenin A activated AMPK and restrained CRTC2 to the cytoplasm to prevent SREBP1 maturation via impairing COPII function in the liver. This suggests that CRTC2 might be a potential target for pharmacological intervention to prevent hepatic lipid deposition. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.

Beta-3 agonist-induced lipolysis and nitric oxide production: relationship to PPARgamma agonist/antagonist and AMP kinase modulation.

PPARgamma receptor agonist -troglitazone increases insulin sensitivity in visceral adipocytes and also increases fat mass. Beta-3 adrenergic receptor agonists mediate lipolysis and NO production (iNOS transcription) in visceral adipocytes. Troglitazone could possibly interfere with Beta-3-triggered lipolysis. We tested the crosstalk between PPARgamma agonist and Beta-3 agonist pathways on lipolysis and NO production in first 24 hours of treatment. Isolated epididymal rat adipocytes were cultivated in DMEM for 24 hours with treatment with Beta-3 agonist - BRL-37344, PPARgamma agonist - troglitazone, PPARgamma antagonist - SR-202 and AMPK blocker - compound C alone as well as in combinations. After 24 hours, lipolysis was measured by free glycerol, NO production by Griess reagent and iNOS mRNA by qRT-PCR. BRL-37344 increased lipolysis and NO production with iNOS transcription. Troglitazone increased all the three parameters as well but less than BRL-37344. Combination of troglitazone or SR-202 with BRL-37344 decreased NO production, iNOS transcription and lipolysis triggered before adding of BRL-37344. Compound C completely blocked the effect of troglitazone (and SR-202 as well) on BRL-37344. PPARgamma agonist/antagonist interferes with Beta-3 agonist activity in 24 hours. Troglitazone/SR-202 effect on Beta-3 triggered lipolysis and iNOS mRNA production is probably not PPAR gamma- but rather AMPK-dependent in first 24 hours (AMPK blocker - compound C blocked the effect).

AK2 activates a novel apoptotic pathway through formation of a complex with FADD and caspase-10.

Mitochondrial proteins function as essential regulators in apoptosis. Here, we show that mitochondrial adenylate kinase 2 (AK2) mediates mitochondrial apoptosis through the formation of an AK2-FADD-caspase-10 (AFAC10) complex. Downregulation of AK2 attenuates etoposide- or staurosporine-induced apoptosis in human cells, but not that induced by tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) or Fas ligand (FasL). During intrinsic apoptosis, AK2 translocates to the cytoplasm, whereas this event is diminished in Apaf-1 knockdown cells and prevented by Bcl-2 or Bcl-X(L). Addition of purified AK2 protein to cell extracts first induces activation of caspase-10 via FADD and subsequently caspase-3 activation, but does not affect caspase-8. AFAC10 complexes are detected in cells undergoing intrinsic cell death and AK2 promotes the association of caspase-10 with FADD. In contrast, AFAC10 complexes are not detected in several etoposide-resistant human tumour cell lines. Taken together, these results suggest that, acting in concert with FADD and caspase-10, AK2 mediates a novel intrinsic apoptotic pathway that may be involved in tumorigenesis.

Several dehydrogenases and kinases compete for endocytosis from plasma by rat tissues.

Plasma contains many enzymes that are probably derived from damaged cells. These enzymes are cleared at characteristic rates. We showed previously that in rats the rapid clearance of alcohol dehydrogenase, lactate dehydrogenase M4 and the mitochondrial and cytosolic isoenzymes of malate dehydrogenase is largely due to endocytosis by macrophages in liver, spleen and bone marrow. We now demonstrate that uptake of each of the enzymes by these tissues is in general decreased by simultaneous injection of a high dose of one of the other dehydrogenases or a high dose of adenylate kinase or creatine kinase. A similar dose of colloidal albumin did not significantly decrease uptake of the four dehydrogenases. Nor was uptake of colloidal albumin, apo-peroxidase from horseradish or multilamellar liposomes influenced by a high dose of mitochondrial malate dehydrogenase. These results indicate that the four dehydrogenases and the two kinases are specifically endocytosed via the same receptor. We suggest that this receptor contains a group, possibly a nucleotide, with affinity for the nucleotide-binding sites of the enzymes.

Regulation of ternary (Met-tRNAf - GTP - eukaryotic initiation factor 2) protein synthesis initiation complex formation by the adenylate energy charge.

Formation of the ternary [Met-tRNAf - GTP - eukaryotic initiation factor 2] protein synthesis initiation complex in rabbit reticulocyte ribosomal eluates is dependent on the GTP: GDP ratio and on the adenylate energy charge. The elements controlling ternary initiation complex formation have been studied in a reconstituted system contianing eukaryotic initiation factor 2 and nucleoside diphosphate kinase purified from the ribosomal eluate. The concentration of GTP required for half maximal formation of the ternary initiation complex is 2.5 - 10(6) M; GDP is a potent competitive inhibitor with Ki equals 3.4 - 10(7) M. Sensitive control of ternary initiation complex formation by the adenylate energy charge occurs through nucleoside diphosphate kinase regulation of the GTP : GDP ratio. Over a wide range of GTP : GDP ratios, 50% of maximal ternary initiation complex formation is observed at an adenylate energy charge of 0.85-0.90 resembling that seen in the unfractionated system. Small changes in adenylate energy charge near this value result in significant changes in the extent of ternary initiation complex formation. Since GTP is continually hydrolyzed to GDP during protein synthesis and since GDP is a competitive inhibitor of GTP binding to several of the protein factors necessary for mRNA translation, the synthetic process provides sensitive control by product inhibition. Ribosome-associated nucleoside diphosphate kinase control of GTP regeneration in response to the adenylate energy charge provides one mechanism for linking protein synthesis to the nutrient state and energy charge of the cell.