Addition of liraglutide in patients with Type 2 diabetes well controlled on metformin monotherapy improves several markers of vascular function.

AIMS: The aim of this study was to investigate the vascular effects of liraglutide in patients well controlled on metformin monotherapy. METHODS: Forty-four patients with Type 2 diabetes were included in the study. Main inclusion criteria were: pretreatment with metformin on a stable dosage, HbA(1c) < 53 mmol/mol (7.0%), age 30-65 years. Patients were randomized to receive additional liraglutide or to remain on metformin monotherapy. After 6 weeks (1.2 mg) and after 12 weeks (1.8 mg), venous blood was taken for the measurement of several laboratory markers characterizing vascular and endothelial function. In addition, retinal microvascular endothelial function and arterial stiffness were measured. RESULTS: HbA(1c) levels declined from 45 ± 4 mmol/mol (6.3 ± 0.4%; mean ± SD) to 40 ± 3 mmol/mol (5.8 ± 0.3%) during liraglutide treatment. Asymmetric dimethylarginin was reduced by liraglutide treatment from 0.39 ± 0.08 to 0.35 ± 0.06 µmol/l, E-selectin from 43.6 ± 15.4 to 40.8 ± 15.1 ng/ml, plasminogen activator inhibitor 1 from 861.6 ± 584.3 to 666.1 ± 499.4 ng/ml and intact proinsulin from 9.0 ± 7.2 to 7.0 ± 4.8 pmol/l at 12 weeks of treatment. The microvascular response to flicker light increased from 7.0 ± 15.1 to 15.4 ± 11.5% after 6 weeks and to 11.1 ± 9.9% after 12 weeks. No change could be observed for high-sensitivity C-reactive protein, monocyte chemotactic protein 1, vascular cell adhesion molecule or arterial stiffness parameters. CONCLUSIONS: In patients with Type 2 diabetes, well controlled with metformin monotherapy, addition of liraglutide improves several cardiovascular risk markers beyond glycaemic control.

Inhibition of nitric-oxide-mediated apoptosis in Jurkat leukemia cells despite cytochrome c release.

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.

Nitric oxide-mediated apoptosis in human breast cancer cells requires changes in mitochondrial functions and is independent of CD95 (APO-1/Fas).

We have previously shown that nitric oxide (NO) induces apoptosis in different human neoplastic lymphoid cells through caspase activation. Here we studied the NO-mediated apoptosis in human breast cancer cell lines derived from primary tumor (BT-20) or from metastasis (MCF-7). NO donor glycerol trinitrate (GTN) induced apoptosis in both cell lines which was completely abrogated after pretreatment with the broad spectrum caspase inhibitor zVAD-fmk. NO triggered also a time-dependent activation of caspase-1, caspase-3, and caspase-6 in these cells. Moreover, NO caused a release of mitochondrial protein cytochrome c into the cytosol, an increase in the number of cells with low mitochondrial transmembrane potential and with high level of reactive oxygen species production. However, NO did not induce mRNA expression of CD95 (APO-1/Fas) ligand. FAS-associated phosphatase-1 (FAP-1) molecule was constitutively expressed at the mRNA level and did not show any changes upon NO treatment in both breast cancer cell lines. The expression of the pro-apoptotic protein Bax and of the anti-apoptotic protein Bcl-2 remained unchanged in MCF-7 and BT-20 cells upon GTN treatment. We suggest that the mechanism of NO-mediated activation of the caspase cascade and subsequent apoptosis in human breast cancer cells required mitochondrial damage (in particular, cytochrome c release, disruption of mitochondrial transmembrane potential and generation of reactive oxygen species) but not the activation of the CD95/CD95L pathway.

Inhibition of tyrosine phosphatases antagonizes CD95-mediated apoptosis.

Ligation of the CD95 receptor resulted in a transient increase of cellular tyrosine phosphorylation. The inhibition of protein tyrosine phosphatases by pervanadate, a potent activator of B cells and T cells through the induction of tyrosine phosphorylation and downstream signaling events in the activation cascade, antagonized CD95-triggered apoptosis. Pervanadate exerted its inhibitory effect only during the early phase of apoptosis prior to the CD95-induced decrease of the mitochondrial transmembrane potential. Inhibition of tyrosine phosphatases delayed the cleavage and activation of caspase-8 and caspase-3 and antagonized the tyrosine dephosphorylation of the CD95 receptor-associated phosphoproteins p61 and p89/92. In contrast, ligation of the tumor necrosis factor (TNF) receptor resulted in a continuous tyrosine dephosphorylation of cellular proteins. Pervanadate-induced tyrosine phosphorylation increased the TNF-alpha-induced cytotoxicity and NF-kappaB activation, suggesting that it stimulates early signaling events prior to the separation of the two signaling pathways.

Modulation of tumor necrosis factor-alpha-mediated cytotoxicity by changes of the cellular methylation state: mechanism and in vivo relevance.

A combination of adenosine (Ado) and homocysteine (Homo) enhances tumor necrosis factor (TNF)-alpha cytotoxicity in vitro and in vivo in several tumor cells. Ado and Homo at concentrations that enhanced TNF-alpha-mediated cytotoxicity accumulated S-adenosylhomocysteine (AdoHcy) and as consequence decreased the cellular methylation state, i.e. the ratio of S-adenosylmethionine to AdoHcy. This decrease led to inhibition of the isoprenylcysteine carboxyl methyltransferase (MTase), an enzyme that catalyzes carboxyl methylation of C-terminal cysteine residues on isoprenylated proteins. The effect of Ado and Homo on TNF-alpha cytotoxicity was at least partly mimicked by S-farnesylthioacetic acid, a selective inhibitor of the isoprenylcysteine carboxyl MTase, suggesting involvement of methylations of prenylated proteins in TNF-alpha-mediated cytotoxicity. Blockage of methylation reactions was associated with an enhancement of the TNF-alpha-induced disruption of the mitochondrial membrane potential (delta psi(m)). In nude mice, a combination of Ado, Homo and TNF-alpha led to TNF-alpha-induced hemorrhagic necrosis and growth inhibition of TNF-sensitive L929 tumors, whereas little effect was observed with TNF-alpha alone. Even more important, the TNF-resistant L929 M1 tumors were rendered TNF-sensitive by the combined action of Ado and Homo. We conclude that Ado and Homo together enhance the effectiveness of TNF-alpha in vitro and in vivo, results that may have therapeutic implications.

Nitric-oxide-induced apoptosis in human leukemic lines requires mitochondrial lipid degradation and cytochrome C release.

We have previously shown that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through activation of caspases not only via CD95/CD95L interaction, but also independently of such death receptors. Here we investigated mitochondria-dependent mechanisms of NO-induced apoptosis in Jurkat leukemic cells. NO donor glycerol trinitrate (at the concentration, which induces apoptotic cell death) caused (1) a significant decrease in the concentration of cardiolipin, a major mitochondrial lipid; (2) a downregulation in respiratory chain complex activities; (3) a release of the mitochondrial protein cytochrome c into the cytosol; and (4) an activation of caspase-9 and caspase-3. These changes were accompanied by an increase in the number of cells with low mitochondrial transmembrane potential and with a high level of reactive oxygen species production. Higher resistance of the CD95-resistant Jurkat subclone (APO-R) cells to NO-mediated apoptosis correlated with the absence of cytochrome c release and with less alterations in other mitochondrial parameters. An inhibitor of lipid peroxidation, trolox, significantly suppressed NO-mediated apoptosis in APO-S Jurkat cells, whereas bongkrekic acid (BA), which blocks mitochondrial permeability transition, provided only a moderate antiapoptotic effect. Transfection of Jurkat cells with bcl-2 led to a complete block of apoptosis due to the prevention of changes in mitochondrial functions. We suggest that the mitochondrial damage (in particular, cardiolipin degradation and cytochrome c release) induced by NO in human leukemia cells plays a crucial role in the subsequent activation of caspase and apoptosis.

Tumor necrosis factor-alpha-induced cell killing and activation of transcription factor NF-kappaB are uncoupled in L929 cells.

The induction of transcription factor NF-kappaB has been shown to counteract tumor necrosis factor (TNF)-alpha-induced cell death in various cell types. In this study, we investigated the role of NF-kappaB for TNF-alpha-triggered cell death in the widely used mouse cell line L929 by various approaches. Inhibition of the mitochondrial permeability transition by bongkrekic acid impaired TNF-alpha-induced cell death without affecting the activity of NF-kappaB. The reduction of NF-kappaB-mediated gene expression by the synthetic steroid dexamethasone was associated with a decrease in TNF-alpha-mediated cell killing, suggesting that NF-kappaB does not protect L929 cells from TNF-alpha-induced cell death. This concept was reinforced by experiments employing L929 cell lines stably overexpressing a transdominant negative form of IkappaB-alpha. These cell lines were unable to activate NF-kappaB and to inducibly express the IL-6 gene, but they showed the same susceptibility toward TNF-alpha-mediated cell death as L929 wild-type cells.

Sesquiterpene lactones specifically inhibit activation of NF-kappa B by preventing the degradation of I kappa B-alpha and I kappa B-beta.

Extracts from certain Mexican Indian medicinal plants used in traditional indigenous medicine for the treatment of inflammations contain sequiterpene lactones (SLs), which specifically inhibit the transcription factor NF-kappa B (Bork, P. M., Schmitz, M. L., Kuhnt, M., Escher, C., and Heinrich, M. (1997) FEBS Lett. 402, 85-90). Here we show that SLs prevented the activation of NF-kappa B by different stimuli such as phorbol esters, tumor necrosis factor-alpha, ligation of the T-cell receptor, and hydrogen peroxide in various cell types. Treatment of cells with SLs prevented the induced degradation of I kappa B-alpha and I kappa B-beta by all these stimuli, suggesting that they interfere with a rather common step in the activation of NF-kappa B. SLs did neither interfere with DNA binding activity of activated NF-kappa B nor with the activity of the protein tyrosine kinases p59fyn and p60arc. Micromolar amounts of SLs prevented the induced expression of the NF-kappa B target gene intracellular adhesion molecule 1. Inhibition of NF-kappa B by SLs resulted in an enhanced cell killing of murine fibroblast cells by tumor necrosis factor-alpha. SLs lacking an exomethylene group in conjugation with the lactone function displayed no inhibitory activity on NF-kappa B. The analysis of the cellular redox state by fluorescence-activated cell sorter showed that the SLs had no direct or indirect anti-oxidant properties.

HIV type 1 glycoprotein 120 amplifies tumor necrosis factor-induced NF-kappa B activation in Jurkat cells.

This article demonstrates that human immunodeficiency virus type 1 (HIV-1) gp120 amplifies the activity of tumor necrosis factor alpha (TNF-alpha), a cytokine that stimulates HIV-1 replication through activation of NF-kappa B. In CD4-positive Jurkat cells, gp120 potentiates TNF-induced NF-kappa B activation. TNF-mediated activation of NF-kappa B is known to involve the intracellular formation of reactive oxygen intermediates (ROIs). Accordingly, we examined the influence of gp120 on the cellular redox state. We found that gp 120-modulated TNF-induced NK-kappa B activation was inhibited by the antioxidant butylated hydroxyanisole, indicating the involvement of redox-dependent mechanisms. In addition, we showed that gp120 induces intracellular formation of hydrogen peroxide, which is accompanied by a decrease in the ratio of glutathione to glutathione disulfide. In contrast, in the p56lck-deficient J.CaM1.6 T cell line, a derivative of the Jurkat cell line, gp120 was unable to stimulate hydrogen peroxide, to decrease the ratio of GSH to GSSG, and has no effect on TNF-induced NF-kappa B activation. This demonstrated that p56lck protein tyrosine kinase plays an active role in transmitting a signal that increases the oxidative state of the cell and as a consequence amplifies TNF-mediated NF-kappa B DNA binding. We have demonstrated that Tat protein decreased both the Mn-dependent superoxide dismutase (MnSOD) and the cellular glutathione content (GSH). Here we show that, in contrast to Tat, gp120 is unable to inhibit activity and expression of MnSOD and to decrease GSH content. Taken together, our data suggest that gp120 potentiates TNF-induced NF-kappa B activation by stimulating a signal pathway that involves p56lck and the increased formation of reactive oxygen intermediates such as H2O2. These findings may be relevant for the regulation of HIV-1 replication in T cells.

S-adenosylhomocysteine as a physiological modulator of Apo-1-mediated apoptosis.

APO-1/Fas (CD95) is a member of the tumor necrosis factor/nerve growth factor receptor superfamily and mediates apoptosis in various cell types. Here we show that L929 cells, expressing human APO-1 treated with agonistic antibodies (anti-APO-1), elicit an early and transient increase of S-adenosylhomocysteine (AdoHcy), a potent inhibitor of S-adenosylmethionine (AdoMet)-dependent methylation reactions. In contrast, anti-APO-1 did not induce an AdoHcy increase in L929-APO-1 Delta4 cells expressing a C-terminally truncated APO-1 lacking part of the 'death domain' known to be required for the transduction of apoptotic signals. Addition of adenosine and D, L-homocysteine also led to an increase of cellular AdoHcy thus enhancing anti-APO-1-induced killing of L929-APO-1 cells. Treatment with anti-APO-1 also induced release of arachidonic acid from phospholipids: this effect was augmented by elevated levels of AdoHcy. In contrast, AdoHcy had only a minor effect on anti-APO-1-mediated DNA fragmentation. These findings suggest that AdoHcy functions as a physiological modulator of APO-1-mediated cell death in L929 cells and enhances anti-APO-1-induced cell killing at least partially by acting via the phospholipase A2 pathway.

Adenosine and homocysteine together enhance TNF-mediated cytotoxicity but do not alter activation of nuclear factor-kappa B in L929 cells.

This paper shows that a combination of adenosine and homocysteine potentiates TNF-alpha-mediated cytotoxicity, but does not modulate activation of NF-kappa B transcription factor controlling the expression of various TNF-alpha-inducible genes. Adenosine and homocysteine at concentrations (1 mM each) that enhance TNF-alpha-induced cytotoxicity accumulate S-adenosyl-L-homocysteine (AdoHcy), a potent inhibitor of S-adenosyl-L-methionine-dependent methylation reactions. In addition, preloading L929 cells with AdoHcy resulted in enhanced responses to TNF-alpha, suggesting that AdoHcy potentiates TNF-alpha-induced cytotoxicity. Moreover, the combination of adenosine and homocysteine changed the dependency of the TNF-alpha-mediated cytolysis on reactive oxygen intermediates. In the absence of adenosine and homocysteine TNF-alpha-mediated cytotoxicity was inhibited by antioxidants such as butylated hydroxyanisole and pyrrolidine dithiocarbamate. In the presence of adenosine and homocysteine, however, TNF-alpha-mediated cytotoxicity is not inhibited by these antioxidants. A L929 subclone, defective in the respiratory chain, resisted the cytotoxic action of TNF-alpha, but was rendered TNF-alpha sensitive in the presence of adenosine and homocysteine. Unlike TNF-alpha-mediated cytotoxicity, the TNF-alpha-induced activation of NF-kappa B was inhibited by antioxidants regardless whether adenosine and homocysteine were present or absent in the culture medium. In conclusion, a combination of adenosine and homocysteine selectively modulates TNF-alpha-mediated cytotoxicity without changing the TNF-alpha-induced activation of NF-kappa B. Our results could facilitate the development of strategies that permit dissection of cytotoxic and gene-activating pathways.