Competitive inhibition of SGLT2 by tofogliflozin or phlorizin induces urinary glucose excretion through extending splay in cynomolgus monkeys.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors showed a glucose lowering effect in type 2 diabetes patients through inducing renal glucose excretion. Detailed analysis of the mechanism of the glucosuric effect of SGLT2 inhibition, however, has been hampered by limitations of clinical study. Here, we investigated the mechanism of urinary glucose excretion using nonhuman primates with SGLT inhibitors tofogliflozin and phlorizin, both in vitro and in vivo. In cells overexpressing cynomolgus monkey SGLT2 (cSGLT2), both tofogliflozin and phlorizin competitively inhibited uptake of the substrate (α-methyl-d-glucopyranoside; AMG). Tofogliflozin was found to be a selective cSGLT2 inhibitor, inhibiting cSGLT2 more strongly than did phlorizin, with selectivity toward cSGLT2 1,000 times that toward cSGLT1; phlorizin was found to be a nonselective cSGLT1/2 inhibitor. In a glucose titration study in cynomolgus monkeys under conditions of controlled plasma drug concentration, both tofogliflozin and phlorizin increased fractional excretion of glucose (FEG) by up to 50% under hyperglycemic conditions. By fitting the titration curve using a newly introduced method that avoids variability in estimating the threshold of renal glucose excretion, we found that tofogliflozin and phlorizin lowered the threshold and extended the splay in a dose-dependent manner without significantly affecting the tubular transport maximum for glucose (TmG). Our results demonstrate the contribution of SGLT2 to renal glucose reabsorption (RGR) in cynomolgus monkeys and demonstrate that competitive inhibition of cSGLT2 exerts a glucosuric effect by mainly extending splay and lowering threshold without affecting TmG.

A glaucoma-associated mutant of optineurin selectively induces death of retinal ganglion cells which is inhibited by antioxidants.

PURPOSE: Mutations in the coding region of the OPTN gene are associated with certain glaucomas. Although the function of the optineurin protein is yet to be elucidated, the most common mutation, E50K, is associated with a severe phenotype. This study explores some functional features of optineurin and its mutants. METHODS: Plasmids expressing normal or wild-type (WT) and E50K, R545Q, H26D, and H486R mutant optineurin were transfected into HeLa, Cos-1, IMR32, and the rat retinal ganglion cell (RGC) line RGC-5, and their effects on cell survival monitored by morphologic observation of cells were studied. Expression of optineurin and its mutants was monitored by immunofluorescence staining of cells and by Western blotting. RESULTS: The E50K mutant of optineurin selectively induced the death of retinal ganglion cells but not of the other cell lines tested. Although the expression of optineurin and E50K mutant suppressed cell death induced by tumor necrosis factor-alpha in HeLa cells, they potentiated this cell death in retinal ganglion cells. Cell death induced by the optineurin mutant in retinal ganglion cells was inhibited by the antioxidants N-acetylcysteine and Trolox. Reactive oxygen species (ROS) were produced upon expression of E50K, which were reduced by antioxidants. Coexpression of manganese superoxide dismutase with the E50K mutant abolished ROS production and inhibited cell death. CONCLUSIONS: The E50K mutation of optineurin acquired the ability to induce cell death selectively in retinal ganglion cells. This cell death was mediated by oxidative stress. The present findings raise the possibility of antioxidant use for delaying or controlling some forms of glaucoma.

Truncated variants of hyaluronan-binding protein 1 bind hyaluronan and induce identical morphological aberrations in COS-1 cells.

Hyaluronan (HA)-binding protein 1 (HABP1) is multifunctional in nature and exists as a trimer through coiled-coil interaction between alpha-helices at its N- and C-termini. To investigate the importance of trimeric assemblage and HA-binding ability of HABP1, we generated and overexpressed variants of HABP1 by truncating the alpha-helices at its termini. Subsequently, these variants were transiently expressed in COS-1 cells to examine the influence of these structural variations on normal cell morphology, as compared with those imparted by HABP1. Substantiating the centrality of coiled-coil interaction for maintaining the trimeric assembly of HABP1, we demonstrate that disruption of trimerization does not alter the affinity of variants towards its ligand HA. Transient expression of HABP1 altered the morphology of COS-1 cells by generating numerous cytoplasmic vacuoles along with disruption of the f-actin network. Interestingly, the truncated variants also imparted identical morphological changes. Characterization of the cytoplasmic vacuoles revealed that most of these vacuoles were autophagic in nature, resembling those generated under stress conditions. The identical morphological changes manifested in COS-1 cells on transient expression of HABP1 or its variants is attributed to their comparable HA-binding ability, which in concert with endogenous HABP1, may deplete the cellular HA pool. Such quenching of HA below a threshold level in the cellular milieu could generate a stress condition, manifested through cytoplasmic vacuoles and a disassembly of the f-actin network.

Ochratoxin A affects COS cell adhesion and signaling.

Ochratoxin A (OTA), a metabolite produced by strains of Aspergillus and Penicillium, has nephritogenic, carcinogenic, and teratogenic activity in animals and humans. Nanomolar concentrations of OTA promote apoptosis in a cell-type specific fashion. In this study, we have analyzed the molecular mechanism by which OTA affects COS cell adhesion and signaling resulting in an apoptotic response. OTA, at noncytotoxic doses, was able to detach collagen- and fibronectin-adherent cells from immobilized substratum. However, prior to inducing detachment of adherent cells, OTA caused apoptosis as measured by caspase-3 activation. The treatment of adherent cells by OTA caused a reduction of tyrosine phosphorylation levels of FAK and of the adapter protein paxillin. The down-regulation of FAK preceded apoptosis and cell detachment induced by OTA. The mycotoxin was also able to cause a decrease of the phosphorylation levels of the two Shc isoforms, P66 and P52, in adherent cells. Since these Shc isoforms have been implicated in the activation of protein kinase c-Src, which is required for FAK tyrosine phosphorylation, the observed dephosphorylation of FAK and of the FAK substrate paxillin by OTA could be ascribed to the early down-regulation of Shc isoforms. However, whether FAK and Shc phosphorylation contribute both to the same pathway leading to the induction of apoptosis by OTA or are involved in two parallel signaling pathways remains to be investigated.

Overexpression of BAD preferentially augments anoikis.

BAD is a BH3-only protein, and its proapoptotic activity is negatively regulated by serine phosphorylation. Here, we show that overexpression of BAD preferentially augments anchorage loss-induced apoptosis (anoikis). Gene transfer-mediated BAD overexpression alone did not induce apoptosis in attached MDCK cells but strongly augmented apoptosis when cells were cultured in suspension. In contrast, overexpression of another BH3-only protein, BID, displayed much lower augmentation of anoikis, suggesting a preferential contribution of BAD to anoikis. During suspension culture, unphosphorylated BAD was gradually increased and targeted to the mitochondria. Cotransfection of BAD with constitutively active Akt cDNA strongly inhibited this change. In contrast, the increase of unphosphorylated BAD was not significantly inhibited by several phosphatase inhibitors or cotransfection with a dominant negative calcineurin cDNA, implying that the increase may be mainly due to a decrease of serine kinase activity, such as that of Akt. Similar results were observed in COS-7 cells, suggesting that BAD overexpression can increase sensitivity of anchorage-dependent cancer cells to anoikis. Thus, we propose that BAD can serve as a valuable gene therapeutic molecule to inhibit carcinoma progression.

Fluorescence imaging of mobility shifts: an expression cloning method for identification of cell signaling targets.

There is a need for a simple global approach to identify signaling targets that are posttranslationally modified in response to physiologic or pathologic stimuli within living cells. Reported here is a simple method, fluorescence imaging of mobility shifts (FIMS), which relies on in-gel detection of cell-expressed green fluorescent protein fusion proteins undergoing electrophoretic mobility shifts. This detection method is applied to a small pool cDNA library screening protocol. The readout is essentially a differential display of posttranslational modifications. Unlike biochemical approaches to identifying signaling targets, the screen is performed in living cells using standard methods for transient transfection. This enables detection of intracellular targets modified in response to either molecularly defined stimuli, such as growth factors or drugs, or complex pathologic stimuli, such as oxidative stress or hypoglycemia. FIMS is rapid, sensitive, inexpensive, and nonradioactive and easily adapted to automated high throughput methods, including capillary electrophoresis. The technique is sufficiently sensitive to easily detect fluorescent proteins expressed in a single well in 384-well format. FIMS is applicable to traditional cDNA library screening, but the method will be especially attractive for screening preselected collections of autofluorescent fusion proteins. A bonus of the technique is that examination of transfected cells by fluorescence microscopy provides immediate information about intracellular localization and stimulus-induced translocation of putative targets. We illustrate the utility of the technique with pilot screens for apoptotic and mitogenic targets modified by staurosporine and serum stimulation, respectively.

Geldanamycin inhibits trichostatin A-induced cell death and histone H4 hyperacetylation in COS-7 cells.

As widely believed treating cells with trichostatin A (TSA), an inhibitor of histone deacetylase, results in histone H4 hyperacetylation and cell cycle arrest. This compound is often compared with other potential anticancer drugs in cell cycle, proliferation and differentiation research. Furthermore, geldanamycin (GA), a 90-kDa heat shock protein (HSP90) specific inhibitor, is a well-known potential anticancer agent. This study examines whether GA can affect the cellular functions induced by TSA. When using TSA treatment, although caused COS-7 cell death, pretreatment of 0.5 microg/ml GA for 30 min and an addition of 50 ng/ml TSA (GA + TSA) apparently averted cell death. Our results indicated that the cell survival rate was only approximately 20% when prolonged treatment was undertaken with 50 ng/ml TSA (TSA) alone for 24 h. In contrast, the cell survival rate was enhanced by two folds when treating with GA + TSA. Furthermore, DNA fragmentation assay revealed that fragmented DNA was produced 8 h after prolonged treatment with TSA alone. Within 16 h, the apoptotic percentages of TSA-treated cells were between 15-25%. In contrast, the other treatments did not exceed 6%. Furthermore, GA inhibited TSA-induced histone H4 hyperacetylation. Western blotting analysis further demonstrated that the HSP70 levels did not significantly increase in TSA-treated cells. However, the accumulated 70-kDa heat shock protein (HSP70) markedly increased up to 2 to 3 folds at 8 h in GA- and GA + TSA-treated cells, and the maximum amount up to 5 to 7 folds at 20 h. Conversely, HSP90 did not markedly increase in all treatments. Based on the results in this study, we suggest that apoptosis induced by TSA can be prevented by GA-induced increment of heat shock proteins, particularly HSP70.