Metabolic Depletion of Atp by Fructose Inversely Controls Cd95- and Tumor Necrosis Factor Receptor 1–Mediated Hepatic Apoptosis

Hepatocyte apoptosis is crucial in several forms of liver disease. Here, we examined in different models of murine liver injury whether and how metabolically induced alterations of hepatocyte ATP levels control receptor-mediated apoptosis. ATP was depleted either in primary hepatocytes or in vivo by various phosphate-trapping carbohydrates such as fructose. After the activation of the tumor necrosis factor (TNF) receptor or CD95, the extent of hepatocyte apoptosis and liver damage was quantified. TNF-induced cell death was completely blocked in ATP-depleted hepatocyte cultures, whereas apoptosis mediated by CD95 was enhanced. Similarly, acute TNF-induced liver injury in mice was entirely inhibited by ATP depletion with ketohexoses, whereas CD95-mediated hepatotoxicity was enhanced. ATP depletion prevented mitochondrial cytochrome c release, loss of mitochondrial membrane potential, activation of type II caspases, DNA fragmentation, and cell lysis after exposure to TNF. The extent of apoptosis inhibition correlated with the severity of ATP depletion, and TNF-induced apoptosis was restored when ATP was repleted by increasing the extracellular phosphate concentration. Our study demonstrates that TNF-induced hepatic apoptosis can be selectively and reversibly blocked upstream of mitochondrial dysfunction by ketohexose-mediated ATP depletion

Die Rolle von ATP in der Zytokin-induzierten Leberschädigung in der Maus

Die Todesrezeptoren TNF-R1 und CD95 sind an der Leberschädigung durch Hyperinflammation beteiligt. Diese Arbeit untersucht die Rolle von intrazellulärem ATP in der Leberzellschädigung. Dazu wurde der ATP-Gehalt der Zelle mit Phosphat-konsumierenden Ketohexosen depletiert. Die differentiellen Konsequenzen der ATP-Depletion wurden in vivo für die Todesrezeptor-vermittelte Leberschädigung in der Maus untersucht, sowie in vitro in primären Maushepatozyten hinsichtlich des molekularen Mechanismus der Inhibition der TNF-R1-Signaltransduktion. Dieser experimentelle Ansatz sollte einerseits zu einem besseren Verständnis von pathologischem Zelltod beitragen und andererseits mögliche selektive Therapiestrategien aufzeigen. 1) Fruktose und weitere Ketohexosen depletierten zeit- und dosisabhängig ATP in der Leber. 2) Nach Fruktose-Vorbehandlung war die Aktivierung von Caspasen sowie die DNA-Fragmentierung nach TNF gehemmt. Eine TNF-induzierte lethale Leberschädigung wurde verhindert. Die CD95-vermittelte Leberschädigung wurde nach ATP-Depletion verstärkt. 3) Unter ATP-depletierten Bedingungen, wurde auch die Caspase-unabhängige, aber dennoch TNF-R1-vermittelte Nekrose inhibiert. 4) In den LPS-Modellen war nach ATP-Depletion die systemische Freisetzung von proinflammatorischen Zytokinen unterdrückt, die Freisetzung des anti-inflammatorischen Zytokins IL-10 hingegen verstärkt. Die ATP-Depletion war spezifisch auf Parenchymzellen beschränkt; als Mediator für die suppressive Interaktion zwischen ATP-depletierten Hepatozyten und Zytokin-produzierenden Makrophagen wurde wahrscheinlich Adenosin erzeugt. 5) In primären Hepatozyten verhinderte die ATP-Depletion die TNF-induzierte Apoptose. Mechanistische Untersuchungen ergaben, dass die Inhibition der Signaltransduktion vom TNF-Rezeptor-1 am Signalkomplex des Rezeptors (DISC) erfolgte. 6) Die ATP-Depletion verhinderte zudem die TNF-vermittelte Hepatotoxizität des Topoisomerase-Inhibitors Camptothecin

Defined inflammatory states in astrocyte cultures: correlation with susceptibility towards CD95-driven apoptosis

A complete cytokine mix (CCM) or its individual components tumour necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and interferon-gamma (IFN-γ) were used to switch resting murine astrocytes to reactive states. The transformation process was characterized by differential up-regulation of interleukin-6 (IL-6), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) mRNA and protein and a subsequent release of prostaglandin E2, nitric oxide (NO) and IL-6. Both CD95L and anti-CD95 antibodies triggered caspase activation followed by apoptotic death in fully pro-inflammatory astrocytes, whereas resting cells were totally resistant. Two other death-inducing ligands, TNF and TNF-related apoptosis-inducing ligand (TRAIL) did not induce apoptosis in reactive astrocytes. The switch in astrocyte sensitivity was accompanied by up-regulation of caspase-8 and CD95 as well as the capacity to recruit Fas-associated death domain (FADD) to the activated death receptor complex. Neither CD95-mediated death, nor other inflammatory parameters were affected by inhibition of iNOS or COX, respectively. Accordingly, IFN-γ was absolutely essential for up-regulation of iNOS, but not for the switch in apoptosis sensitivity. In contrast, p38 kinase activity was identified as an important controller of both the inflammatory reaction and apoptosis both in astrocytes stimulated with CCM and in glia exposed to TNF and IL-1 only

Strontium is a biased agonist of the calcium-sensing receptor in rat medullary thyroid carcinoma 6-23 cells

The calcium-sensing receptor (CaSR)-specific allosteric mod-ulator cinacalcet has revolutionized the treatment of secondary hyperparathyroidism in patients with chronic kidney disease. However, its application is limited to patients with end-stage renal disease because of hypocalcemic side effects presum-ably caused by CaSR-mediated calcitonin secretion from thy-roid parafollicular C-cells. These hypocalcemic side effects might be dampened by compounds that bias the signaling of CaSR, causing similar therapeutic effects as cinacalcet without stimulating calcitonin secretion. Because biased signaling of CaSR is poorly understood, the objective of the present study was to investigate biased signaling of CaSR by using rat med-ullary thyroid carcinoma 6-23 cells as a model of thyroid para-follicular C-cells. By doing concentration-response experi