Pharmacological inhibition of Eph receptors enhances glucose-stimulated insulin secretion from mouse and human pancreatic islets.

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by impaired glucose-stimulated insulin secretion (GSIS) from pancreatic islets. Since erythropoietin-producing hepatoma (Eph)-ephrin bidirectional signalling fine-tunes GSIS from pancreatic beta cells, we investigated Eph receptor tyrosine kinases (RTK) as potential drug targets for selectively increasing GSIS. METHODS: Insulin secretion assays were carried out using mouse and human pancreatic islets as well as mouse insulinoma (MIN6) cells in the presence or absence of two Eph RTK inhibitors. Furthermore, the most potent inhibitor was injected into mice to evaluate its effects on glucose tolerance and plasma insulin levels. RESULTS: We showed that the Eph RTK inhibitors selectively increased GSIS from MIN6 cells as well as mouse and human islets. Our results also showed that the insulin secretory effects of these compounds required Eph-ephrin signalling. Finally, pharmacological inhibition of Eph receptor signalling improved glucose tolerance in mice. CONCLUSIONS/INTERPRETATION: We showed for the first time that Eph RTKs represent targets for small molecules to selectively increase GSIS and improve glucose tolerance.

Empagliflozin, a novel selective sodium glucose cotransporter-2 (SGLT-2) inhibitor: characterisation and comparison with other SGLT-2 inhibitors.

AIMS: Empagliflozin is a selective sodium glucose cotransporter-2 (SGLT-2) inhibitor in clinical development for the treatment of type 2 diabetes mellitus. This study assessed pharmacological properties of empagliflozin in vitro and pharmacokinetic properties in vivo and compared its potency and selectivity with other SGLT-2 inhibitors. METHODS: [(14)C]-alpha-methyl glucopyranoside (AMG) uptake experiments were performed with stable cell lines over-expressing human (h) SGLT-1, 2 and 4. Two new cell lines over-expressing hSGLT-5 and hSGLT-6 were established and [(14)C]-mannose and [(14)C]-myo-inositol uptake assays developed. Binding kinetics were analysed using a radioligand binding assay with [(3)H]-labelled empagliflozin and HEK293-hSGLT-2 cell membranes. Acute in vivo assessment of pharmacokinetics was performed with normoglycaemic beagle dogs and Zucker diabetic fatty (ZDF) rats. RESULTS: Empagliflozin has an IC(50) of 3.1 nM for hSGLT-2. Its binding to SGLT-2 is competitive with glucose (half-life approximately 1 h). Compared with other SGLT-2 inhibitors, empagliflozin has a high degree of selectivity over SGLT-1, 4, 5 and 6. Species differences in SGLT-1 selectivity were identified. Empagliflozin pharmacokinetics in ZDF rats were characterised by moderate total plasma clearance (CL) and bioavailability (BA), while in beagle dogs CL was low and BA was high. CONCLUSIONS: Empagliflozin is a potent and competitive SGLT-2 inhibitor with an excellent selectivity profile and the highest selectivity window of the tested SGLT-2 inhibitors over hSGLT-1. Empagliflozin represents an innovative therapeutic approach to treat diabetes.

Long-term treatment with empagliflozin, a novel, potent and selective SGLT-2 inhibitor, improves glycaemic control and features of metabolic syndrome in diabetic rats.

Empagliflozin is a potent, selective sodium glucose co-transporter-2 inhibitor that is in development for the treatment of type 2 diabetes. This series of studies was conducted to assess the in vivo pharmacological effects of single or multiple doses of empagliflozin in Zucker diabetic fatty rats. Single doses of empagliflozin resulted in dose-dependent increases in urinary glucose excretion and reductions in blood glucose levels. After multiple doses (5 weeks), fasting blood glucose levels were reduced by 26 and 39% with 1 and 3 mg/kg empagliflozin, respectively, relative to vehicle. After 5 weeks, HbA1c levels were reduced (from a baseline of 7.9%) by 0.3 and 1.1% with 1 and 3 mg/kg empagliflozin, respectively, versus an increase of 1.1% with vehicle. Hyperinsulinaemic-euglycaemic clamp indicated improved insulin sensitivity with empagliflozin after multiple doses versus vehicle. These findings support the development of empagliflozin for the treatment of type 2 diabetes.

Increased frequency of a null-allele for NAD(P)H: quinone oxidoreductase in patients with urological malignancies.

The NQO1 locus on chromosome 16q2.2 encodes NAD(P)H:quinone oxidoreductase, an enzyme implicated in detoxication and protection against redox cycling. Two alleles have been identified in the human population, the rarer one, termed the null-allele, coding for a nonfunctional enzyme. Since lack of NQOR activity has been suggested to increase susceptibility to certain cancers, the distribution of the two alleles was determined by polymerase chain reaction-restriction fragment length polymorphism analysis in patients with renal cell carcinoma (n = 131) and urothelial carcinoma (n = 99) compared with a normal population (n = 260). Allele distribution in the normal population followed a Hardy-Weinberg distribution with frequencies of 0.867 for the major allele and 0.133 for the null-allele. Increased frequencies of the null-allele were found in the tumour patient groups (0.191 and 0.182, respectively) due to an increased number of both homo- and heterozygotes. The odds ratios for homozygous null-allele vs. wild-type genotypes were 1.7 and 3.6 for renal cell carcinoma and urothelial carcinoma, respectively. These data are compatible with the assumption that diminished activity of NQOR in some individuals increases susceptibility to certain cancers.

Chemosensitivity of head and neck squamous carcinoma cell lines is not primarily correlated with glutathione level but is modified by glutathione depletion.

Glutathione has a variety of important physiological functions in cellular metabolism and defense, including protection from radicals, oxidative stress, and electrophilic compounds. On the basis of this interaction with both endogenous and synthetic substances, glutathione and the key enzyme for its conjugation, glutathione S-transferase, appear to be critical determinants in tumor cell resistance to several antineoplastic drugs, e.g. platinum analogs. In ten established head and neck cancer cell lines (UM-SCC 10A, 10B, 11B, 14A, 14B, 14C, and 22B, HLac79, 8029NA, and 8029DDP4) chemosensitivity to cisplatin, carboplatin, 5-fluorouracil, and bleomycin, as well as cellular glutathione content and activity of glutathione S-transferase were determined. The results revealed no correlation between the sensitivity of tumor cells to any of the drugs tested and the level of glutathione or the activity of glutathione S-transferase. However, the cisplatin-resistant subpopulation 8029DDP4 showed the highest glutathione level and marked cross-resistance to bleomycin. Glutathione depletion with buthionine sulfoximine led to moderately increased sensitivity towards cisplatin and carboplatin in all cell lines, but did not affect their response to 5-fluorouracil or bleomycin. These results suggest that the level of glutathione or the activity of glutathione S-transferase is not a suitable parameter for the assessment of chemosensitivity in head and neck squamous-cell carcinoma lines. However, response to platinum analogs is influenced by alterations of the initial intracellular glutathione concentration.

Turnover of glutathione S-transferase alpha mRNAs is accelerated by 12-O-tetradecanoyl phorbol-13-acetate in human hepatoma and colon carcinoma cell lines.

The phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), known to induce murine glutathione S-transferase (GST) Ya, was examined for its effect on the expression of human GST alpha. Unexpectedly, 24-h treatment of the human hepatoma cell line HepG2 with 100 nmol/l TPA caused a decrease of the GST alpha mRNA level to below 5% of controls, i.e. opposite to the known response in the mouse. The level of mRNA for GST Mu was also decreased, but the mRNAs of c-jun and jun-B were elevated after 2 h. The decrease of GST alpha mRNAs was inhibited by staurosporine, suggesting an involvement of protein kinase C. Inhibition of transcription and translation by actinomycin D and cycloheximide also partially inhibited the effect of TPA on the expression of GST alpha. In the presence of actinomycin D, GST alpha mRNA halflife was 14.5 h, compared to 3.5 h in the presence of TPA. The calcium ionophore A23187 caused a loss of GST alpha mRNAs to levels almost as low as those obtained with TPA. The effects of TPA and the calcium ionophore were also observed in CaCo2 colon carcinoma cells. As a consequence of the decrease of mRNA levels, GST alpha protein levels and total GST enzyme activity were also diminished. Also, the morphology of the cells was changed after 3 h exposure to TPA. These data suggest that human GST alpha expression can be regulated at the level of mRNA stability by a pathway involving protein kinase C.

Loss of heterozygosity at the NAD(P)H: quinone oxidoreductase locus associated with increased resistance against mitomycin C in a human bladder carcinoma cell line.

The human bladder carcinoma cell line RT112 and the mitomycin C-resistant cell line RT112MMC, derived from RT112 cells, were examined for their expression of NAD(P)H:quinone oxidoreductase (NQOR) and glutathione S-transferases (GSTs). RT112 cells were 40-fold more sensitive towards mitomycin C than RT112MMC cells. The NQOR mRNA level in RT112MMC cells was decreased to 15% as compared to RT112 cells. NQOR enzyme activity was 391 +/- 140 mU/mg protein in RT112 cells, whereas NQOR activity in RT112MMC cells was not measurable. As shown by a fast PCR-based assay and DNA-sequencing, the cell line RT112 is heterozygous, whereas RT112MMC is homozygous for a null allele of the NQOR gene without enzymatic activity. Accordingly, both wild-type and null allele mRNAs were present in RT112 cells, whereas only null allele mRNA was found in RT112MMC. The lack of NQOR enzyme activity in RT112MMC cells was thus associated with loss of heterozygosity at the NQOR locus. By a PCR-RFLP assay, three kidney carcinoma patients without measurable NQOR enzyme activity were shown to be homozygous for the null allele. The PCR assay described here is useful for examination of large numbers of samples. The relative amount of GST-Pi mRNA was decreased by 30% in RT112MMC as compared to RT112, contributing to a diminished level of GST enzyme activity, using CDNB as a substrate, from 95 +/- 62 mU/mg protein in RT112 to 26 +/- 6 mU/mg protein in RT112MMC.

Lowered amounts of the tissue-specific transcription factor LFB1 (HNF1) correlate with decreased levels of glutathione S-transferase alpha messenger RNA in human renal cell carcinoma.

Human renal cell carcinoma is characterized by the loss of differentiation markers such as glutathione S-transferase alpha (GST-alpha). In this paper we show that the promoter of a GST-alpha gene contains a functional binding site for the cell-specific transcription factor LFB1 (HNF1). To investigate the potential role of LFB1 in the down-regulation of GST-alpha expression, we have compared the amount and the binding activity of the LFB1 protein between normal kidney and tumor tissue. By Western analysis and gel retardation assay using a monoclonal antibody specific for LFB1 we show that in 11 of 14 carcinomas the amount of LFB1 is clearly reduced compared to the corresponding normal tissue and that in all 14 renal carcinomas LFB1 binding activity is diminished. As in the same samples the abundance of GST-alpha mRNA is lower than in the normal tissue, we postulate that the loss of LFB1 binding activity might be responsible for the decreased expression of the GST-alpha gene in renal cell carcinoma.

Expression of NAD(P)H:quinone oxidoreductase and glutathione S-transferases alpha and pi in human renal cell carcinoma and in kidney cancer-derived cell lines.

NAD(P)H:quinone oxidoreductase (NQOR) and glutathione S-transferases (GST) are enzymes of interest in cell defence and drug resistance. Relative levels of NQOR mRNA in renal cell carcinomas were 28 +/- 24% (n = 21) of those in non-neoplastic tissue and the enzyme activity decreased from 41 +/- 39 to 18 +/- 27 mU/mg protein (n = 23). In three of the cases, there was no measurable NQOR enzyme activity at all, indicating a polymorphism in the population for this gene. Relative GST-alpha mRNA levels in the tumours were on average 6 +/- 6% (n = 22) of the control value, whereas for GST-pi mRNA smaller decreases as well as increases were found in the tumours as compared to control tissue, but, on average, the level remained unchanged. Overall GST activity measured with CDNB as a substrate was 152 +/- 157 mU/mg protein in tumour tissue and 342 +/- 177 mU/mg protein in non-neoplastic tissue (n = 23). In all kidney tumour-derived cell lines NQOR mRNA was strongly expressed and on a per protein basis NQOR activity was about 10-fold higher than in the kidney tumour samples. GST-pi but not GST-alpha mRNA was also present. Total GST enzyme activities in these cell lines were similar to those in kidney tumour samples. HepG2 cells exhibited expression of NQOR and GST-alpha; GST-pi was not detectable. NQOR activity in HepG2 was about four-fold higher than in kidney-derived cell lines. Thus, NQOR and GST-alpha are both down-regulated in renal carcinoma, but their expression diverges in carcinoma cell lines.

Increase of beta-actin mRNA upon hypotonic perfusion of perfused rat liver.

beta-Actin mRNA levels in livers exposed to hypotonic perfusion (from 305 to 225 mosmol/l) for one hour are increased 2-fold relative to albumin mRNA. Like albumin, glyceraldehyde-3-phosphate dehydrogenase and tyrosine aminotransferase mRNAs remain at the levels observed under normotonic conditions. The increase in beta-actin mRNA is interpreted as a cytoskeletal response due to cell swelling.