Differential induction of apoptosis by type A and B trichothecenes in Jurkat T-lymphocytes.

Several studies have shown that the mycotoxins T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON) and nivalenol (NIV) affect lymphocyte functioning. However, the molecular mechanisms underlying the immunomodulatory effects of these trichothecenes are not defined yet. In this study, the potency of the type A trichothecenes T-2 toxin and DAS, and the type B trichothecenes DON (and its metabolite de-epoxy-deoxynivalenol; DOM-1) and NIV to reduce mitochondrial activity and to induce apoptosis of Jurkat T cells (human T lymphocytes) were examined. T-2 toxin and DAS are much more cytotoxic at low concentrations than DON and NIV as shown by the AlamarBlue cytotoxicity assay. In addition, the mechanism whereby DON and NIV induced cytotoxicity is mainly via apoptosis as we observed phosphatidylserine externalization, mitochondrial release of cytochrome c, procaspase-3 degradation and Bcl-2 degradation. In contrast, type A trichothecenes reduce the mitochondrial activity at approximately 1000-fold lower concentrations than the type B trichothecenes, resulting in necrosis. These data suggest that the mechanisms resulting in cytotoxic effects are different for type A and type B trichothecenes.

Adrenergic receptor stimulation attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes by inhibiting GLUT4 translocation.

Activation of the sympathetic nervous system inhibits insulin-stimulated glucose uptake. However, the underlying mechanisms are incompletely understood. Therefore, we studied the effects of catecholamines on insulin-stimulated glucose uptake and insulin-stimulated translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. We found that epinephrine (1 microM) nearly halved insulin-stimulated 2-deoxyglucose uptake. The beta-adrenoceptor antagonist propranolol (0.3 microM) completely antagonized the inhibitory effect of epinephrine on insulin-stimulated glucose uptake, whereas the alpha-adrenoceptor antagonist phentolamine (10 microM) had no effect. When norepinephrine was used instead of epinephrine, the results were identical. None of the individual selective beta-adrenoceptor antagonists (1 microM, beta(1): metoprolol, beta(2): ICI-118551, beta(3): SR-59230A) could counteract the inhibitory effect of epinephrine. Combination of ICI-118551 and SR-59230A, as well as combination of all three selective beta-adrenoceptor antagonists, abolished the effect of epinephrine on insulin-stimulated glucose uptake. After differential centrifugation, we measured the amount of GLUT1 and GLUT4 in the plasma membrane and in intracellular vesicles by means of Western blotting. Both epinephrine and norepinephrine reduced insulin-stimulated GLUT4 translocation to the plasma membrane. These results show that beta-adrenergic (but not alpha-adrenergic) stimulation inhibits insulin-induced glucose uptake in 3T3-L1 adipocytes, most likely via the beta(2)- and beta(3)-adrenoceptor by interfering with GLUT4 translocation from intracellular vesicles to the plasma membrane.

Regulation of GLUT1-mediated glucose uptake by PKClambda-PKCbeta(II) interactions in 3T3-L1 adipocytes.

Members of the PKC (protein kinase C) superfamily play key regulatory roles in glucose transport. How the different PKC isotypes are involved in the regulation of glucose transport is still poorly defined. PMA is a potent activator of conventional and novel PKCs and PMA increases the rate of glucose uptake in many different cell systems. In the present study, we show that PMA treatment increases glucose uptake in 3T3-L1 adipocytes by two mechanisms: a mitogen-activated protein kinase kinase-dependent increase in GLUT1 (glucose transporter 1) expression levels and a PKClambda-dependent translocation of GLUT1 towards the plasma membrane. Intriguingly, PKClambda co-immunoprecipitated with PKCbeta(II) and did not with PKCbeta(I). Previously, we have described that down-regulation of PKCbeta(II) protein levels or inhibiting PKCbeta(II) by means of the myristoylated PKCbetaC2-4 peptide inhibitor induced GLUT1 translocation towards the plasma membrane in 3T3-L1 adipocytes. Combined with the present findings, these results suggest that the liberation of PKClambda from PKCbeta(II) is an important factor in the regulation of GLUT1 distribution in 3T3-L1 adipocytes.

Hexosamines are unlikely to function as a nutrient-sensor in 3T3-L1 adipocytes: a comparison of UDP-hexosamine levels after increased glucose flux and glucosamine treatment.

Whether the hexosamine biosynthesis pathway acts as a nutrient-sensing pathway is still unclear. Glucose is directed into this pathway by GFAT. Because the activity of GFAT is tightly regulated, we examined whether UDP-hexosamine levels can increase significantly and dose-dependently in response to elevated glucose concentrations. In glucosamine-treated 3T3-L1 adipocytes, inhibition of insulin-stimulated glucose uptake was highly correlated with UDP-hexosamine levels (r = -0.992; p < 0.0001 for UDP-GlcNAc and r = -0.996; p < 0.0001 for UDP-GalNAc). Incubation of 3T3-L1 adipocytes with 0.1 microM insulin for 24 h in medium containing 1 and 5 mM glucose increased the rate of glucose uptake by 365% and 175% compared to untreated cells, respectively. This increase was not observed when the cells were incubated for 24 h with insulin in medium containing 10 or 25 mM glucose. However, treatment of cells with insulin and 1, 5, 10, or 25 mM glucose resulted in similar increases in levels of UDP-GlcNAc and UDP-GalNAc that always amounted to approx 30-40% above baseline values. This led us to conclude that despite exposure of adipocytes to conditions of extreme and prolonged glucose disposal, the increases in cellular UDP-hexosamines were minimal and not dependent on the extracellular glucose concentration. Taken together, our results are in line with the hypothesis that in glucosamine-treated adipocytes UDP-hexosamines influence insulin-stimulated glucose uptake. However, our observations in glucose-treated adipocytes argue against the possibility that UDP-hexosamines function as a nutrient-sensor, and question the role of the hexosamine biosynthesis pathway in the pathogenesis of insulin resistance.

Insulin inhibits amyloid beta-induced cell death in cultured human brain pericytes.

Amyloid-beta (Abeta) deposition in the cerebral arterial and capillary walls is one of the characteristics of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type. In vitro, Abeta1-40, carrying the "Dutch" mutation (DAbeta1-40), induced reproducible degeneration of cultured human brain pericytes (HBP), by forming fibrils at the cell surface. Thus, this culture system provides an useful model to study the vascular pathology seen in Alzheimer's disease. In this study, we used this model to investigate the effects of insulin on Abeta-induced degeneration of HBP, as it has been mentioned previously that insulin is able to protect neurons against Abeta-induced cell-death. The toxic effect of DAbeta1-40 on HBP was inhibited by insulin in a dose-dependent matter. Insulin interacted with Abeta and inhibited fibril formation of Abeta in a cell-free assay, as well as at the cell surface of HBP. Our data indicate that the formation of a fibril network is essential for Abeta-induced cell death in HBP. Additionally, insulin may be involved in the regulation of Abeta fibrillization in AD.

Molecular beacon reverse transcription-PCR of human chorionic gonadotropin-beta-3, -5, and -8 mRNAs has prognostic value in breast cancer.

BACKGROUND: The beta-subunit of human chorionic gonadotropin (hCG) is encoded by four genes, of which expression of the hCGbeta-3, -5, and -8 genes could have prognostic value in breast cancer. METHODS: Applying a new, modified Molecular Beacon reverse transcription-PCR assay, we investigated the prognostic value of the hCGbeta-3, -5, and -8 gene transcripts in 129 sporadic unilateral breast cancer samples from patients with a median follow-up of 62.3 months. RESULTS: Expression of hCGbeta-3, -5, -8 was significantly (P = 0.020) associated with relapse-free survival (RFS). In multivariate survival analysis, hCGbeta-3, -5, and -8 maintained prognostic value for RFS, with high expression predicting shorter RFS (P = 0.015; hazard ratio, 2.25; 95% confidence interval, 1.17-4.34). Only 1 of 24 (4%) node-negative patients with low hCGbeta-3, -5, -8 expression relapsed, in contrast to 7 of 26 (27%) patients with high expression (P = 0.046). CONCLUSIONS: Expression of hCGbeta-3, -5, -8, which differ by only one nucleotide from other hCGbeta genes, can be assessed by our modified Molecular Beacon assay in breast cancer tissues. Expression of hCGbeta-3, -5, -8 has independent, prognostic value for RFS in breast cancer and may help identify node-negative patients with poor prognosis.

Exploring levels of hexosamine biosynthesis pathway intermediates and protein kinase C isoforms in muscle and fat tissue of Zucker Diabetic Fatty rats.

Many studies suggest that insulin resistance develops and/or is maintained by an increased flux of glucose through the hexosamine biosynthesis pathway. This pathway may attenuate insulin-stimulated glucose uptake by activating protein kinase C (PKC). Therefore, we investigated whether the concentrations of the major hexosamine metabolites, uridine diphosphate- N-acetyl-glucosamine (UDP-GlcNAc) and uridine diphosphate- N-acetyl-galactosamine (UDP-GalNAc), and the expression levels of PKC isoforms were affected in Zucker Diabetic Fatty (ZDF) rats, an animal model widely used to study type 2 diabetes mellitus. At the age of 6 wk, control and ZDF rats were normoglycemic. Whereas control rats remained normoglycemic, the ZDF rats became hyperglycemic. The amount of UDP-GlcNAc and UDP-GalNAc in muscle tissue of ZDF rats was similar at 6, 12, 18, and 24 wk of age. Moreover, the concentration of both hexosamines did not differ among ZDF, phlorizin-treated ZDF, and control rats. Western blot analysis revealed that PKCalpha, delta, epsilon, andzeta, but not PKCbeta and gamma, were expressed in muscle and fat tissues from 6- and 24-wk-old control and ZDF rats. In addition, we did not observe changes in the expression levels of the PKC isoforms following prolonged hyperglycemia. Taken together, these findings indicate that the amounts of several metabolites from the hexosamine biosynthesis pathway and PKC isoforms, both hypothesized to be important in the development and/or maintenance of the insulin-resistant state of muscle and fat tissue, are not different in ZDF compared with nondiabetic rats.

Inhibition of protein kinase CbetaII increases glucose uptake in 3T3-L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane.

The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the rate of glucose uptake in many different cell systems. We attempted to investigate the mechanism via which PMA stimulates glucose transport in 3T3-L1 adipocytes in more detail. We observed a good correlation between the rate of disappearance of PKCbetaII during prolonged PMA treatment and the increase in glucose uptake. Moreover, inhibition of PKCbetaII with a specific myristoylated PKCbetaC2-4 peptide inhibitor significantly increased the rate of glucose transport. Western blot analysis demonstrated that both PMA treatment and incubation with the myristoylated PKCbetaC2-4 pseudosubstrate resulted in more glucose transporter (GLUT)-1 but not GLUT-4 at the plasma membrane. To our knowledge, we are the first to demonstrate that inactivation of PKC, most likely PKCbetaII, elevates glucose uptake in 3T3-L1 adipocytes. The observation that PKCbetaII influences the rate of glucose uptake through manipulation of GLUT-1 expression levels at the plasma membrane might reveal a yet unidentified regulatory mechanism involved in glucose homeostasis.

R-Ras alters Ca2+ homeostasis by increasing the Ca2+ leak across the endoplasmic reticular membrane.

Evidence in the literature implicating both Ras-like Ras (R-Ras) and intracellular Ca(2+) in programmed cell death and integrin-mediated adhesion prompted us to investigate the possibility that R-Ras alters cellular Ca(2+) handling. Chinese hamster ovary cells expressing the cholecystokinin (CCK)-A receptor were loaded with indo-1 to study the effects of constitutively active V38R-Ras and dominant negative N43R-Ras on the kinetics of the thapsigargin (Tg)- and CCK(8)-induced Ca(2+) rises using high speed confocal microscopy. In the absence of extracellular Ca(2+), both 1 microm Tg, a potent and selective inhibitor of the Ca(2+) pump of the intracellular Ca(2+) store, and 100 nm CCK(8) evoked a transient rise in Ca(2+), the size of which was decreased significantly after expression of V38R-Ras. At 0.1 nm, CCK(8) evoked periodic Ca(2+) rises. The frequency of these Ca(2+) oscillations was reduced significantly in V38R-Ras-expressing cells. In contrast to V38R-Ras, N43R-Ras did not alter the kinetics of the Tg- and CCK(8)-induced Ca(2+) rises. The present findings are compatible with the idea that V38R-Ras expression increases the passive leak of Ca(2+) of the store leading to a decrease in Ca(2+) content of this store, which, in turn, leads to a decrease in frequency of the CCK(8)-induced cytosolic Ca(2+) oscillations. The effect of V38R-Ras on the Ca(2+) content of the intracellular Ca(2+) store closely resembles that of the antiapoptotic protein Bcl-2 observed earlier. Together with reports on the role of dynamic Ca(2+) changes in integrin-mediated adhesion, this leads us to propose that the reduction in endoplasmic reticulum Ca(2+) content may underlie the antiapoptotic effect of R-Ras, whereas the decrease in frequency of stimulus-induced Ca(2+) oscillations may play a role in the inhibitory effect of R-Ras on stimulus-induced cell detachment and migration.

Expression of the transcription factor Ets-1 is an independent prognostic marker for relapse-free survival in breast cancer.

The transcription factor Ets-1 regulates the expression of several angiogenic and extracellular matrix remodeling factors, and might be implicated in disease progression of breast cancer. In the present study, the prognostic value of Ets-1 expression was assessed by quantitative real-time fluorescence RT-PCR in 123 sporadic primary breast cancer samples of patients with a median follow-up time of 62 months. Ets-1 expression levels correlated significantly with VEGF and PAI-1 in the same tissue. In univariate (P=0.0011) and multivariate (P=0.005) analyses, Ets-1 expression showed significant prognostic value for relapse-free survival. Ets-1 is a strong, independent predictor of poor prognosis in breast cancer. This seems - at least in part - to be attributable to its role in transcriptional regulation of factors involved in angiogenesis (VEGF), and extracellular matrix remodeling (PAI-1).