Effects of sequential treatment with lixisenatide, insulin glargine, or their combination on meal-related glycaemic excursions, insulin and glucagon secretion, and gastric emptying in patients with type 2 diabetes.

AIM: To examine the glucose-lowering mechanisms of the glucagon-like peptide-1 receptor agonist lixisenatide after two subsequent meals and in combination with basal insulin. MATERIALS AND METHODS: Twenty-eight metformin-treated patients with type 2 diabetes were randomly assigned to treatment sequences with either lixisenatide or insulin glargine alone for 4 weeks, and a combination of both treatments for 4 weeks. Metabolic examinations were performed before and after each treatment period following breakfast and a late lunch 8 hours later. RESULTS: Lixisenatide mainly reduced postprandial glycaemia, while insulin glargine mainly reduced fasting glucose after breakfast (P < 0.05). This was partially preserved after a late lunch (P < 0.05). After breakfast, lixisenatide reduced insulin secretion and glucagon levels significantly. These effects were lost after a late lunch. Insulin glargine did not significantly reduce glucagon or insulin secretion. Gastric emptying was slowed by lixisenatide, but not by insulin glargine after breakfast. After the late lunch, lixisenatide slightly accelerated gastric emptying. CONCLUSIONS: Lixisenatide decelerates gastric emptying after breakfast, thereby reducing glycaemic excursions, insulin secretion and glucagon levels. The glycaemic reduction persists until after a late lunch, despite accelerated gastric emptying. The combination with insulin glargine enhances the glucose-lowering effect because of complementary modes of action.

Selective chemical imaging of static actin in live cells.

We have characterized rationally designed and optimized analogues of the actin-stabilizing natural products jasplakinolide and chondramide C. Efficient actin staining was achieved in fixed permeabilized and non-permeabilized cells using different combinations of dye and linker length, thus highlighting the degree of molecular flexibility of the natural product scaffold. Investigations into synthetically accessible, non-toxic analogues have led to the characterization of a powerful cell-permeable probe to selectively image static, long-lived actin filaments against dynamic F-actin and monomeric G-actin populations in live cells, with negligible disruption of rapid actin dynamics.

HNF4alpha reduces proliferation of kidney cells and affects genes deregulated in renal cell carcinoma.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue-specific transcription factor known to regulate a large number of genes in hepatocytes and pancreatic beta cells. Although HNF4alpha is highly expressed in some sections of the kidney, little is known about its role in this organ and about HNF4alpha-regulated genes in the kidney cells. The abundance and activity of HNF4alpha are frequently reduced in renal cell carcinoma (RCC) indicating some tumor suppressing function of HNF4alpha in renal cells. To determine the potential role of HNF4alpha in RCC, we used Flp recombinase-mediated gene integration to generate human embryonic kidney cells (HEK293) that conditionally express wild-type or mutated HNF4alpha. Expression of wild-type HNF4alpha but not of the mutants led to reduction of proliferation and alterations of cell morphology. These effects were reversible and induced at physiological concentrations of HNF4alpha. Using gene expression profiling by microarrays, we determined genes regulated by HNF4alpha. Interestingly, many of the genes regulated by HNF4alpha have been shown to be deregulated in RCC microarray studies. These genes (ACY1, WT1, SELENBP1, COBL, EFHD1, AGXT2L1, ALDH5A1, THEM2, ABCB1, FLJ14146, CSPG2, TRIM9 and HEY1) are good candidates for genes whose activity is changed upon the decrease of HNF4alpha in RCC.

Conditional expression of hepatocyte nuclear factor-1beta, the maturity-onset diabetes of the young-5 gene product, influences the viability and functional competence of pancreatic beta-cells.

Mutations in the gene encoding hepatocyte nuclear factor (HNF)1beta result in maturity-onset diabetes of the young-(MODY)5, by impairing insulin secretory responses and, possibly, by reducing beta-cell mass. The functional role of HNF1beta in normal beta-cells is poorly understood; therefore, in the present study, wild-type (WT) HNF1beta, or one of two naturally occurring MODY5 mutations (an activating mutation, P328L329del, or a dominant-negative form, A263insGG) were conditionally expressed in the pancreatic beta-cell line, insulin-1 (INS-1), and the functional consequences examined. Surprisingly, overexpression of the dominant-negative mutant did not modify any of the functional properties of the cells studied (including insulin secretion, cell growth and viability). By contrast, expression of WT HNF1beta was associated with a time- and dose-dependent inhibition of INS-1 cell proliferation and a marked increase in apoptosis. Induction of WT HNF1beta also inhibited the insulin secretory response to nutrient stimuli, membrane depolarisation or activation of protein kinases A and C and this correlated with a significant decrease in pancrease-duodenum homeobox-1 protein levels. The attenuation of insulin secretion was, however, dissociated from the inhibition of proliferation and loss of viability, since expression of the P328L329del mutant led to a reduced rate of cell proliferation, but failed to induce apoptosis or to alter insulin secretion. Taken together, the present results suggest that mature rodent beta-cells are sensitive to increased expression of WT HNF1beta and they imply that the levels of this protein are tightly regulated to maintain secretory competence and cell viability.

Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4alpha and HNF1beta in a pancreatic beta-cell line.

Using the rat insulinoma cell line INS-1 we generated beta-cell clones that are most efficient for gene transfer, as they contain an FRT site for Flp recombinase-mediated, site-directed integration of a single copy transgene. Therefore, the gene-of-interest can be introduced by DNA transfection without the need to select individual cell clones. Additionally, the clones contain the tetracycline repressor allowing tetracycline induction of the transgene. By oligonucleotide microarray we define the beta-cell specific phenotype of the Flp-In T-REx cell clones. Using a clone expressing the HNF6, HNF4alpha and HNF1beta transcription factors at a limited level, we introduced the expression vectors encoding these factors. We show efficient tetracycline induction of these transcription factors by western blots and immunocytochemistry. Microarrays reveal that these three factors affect a similar number of genes with only few genes regulated in common. Statistical analysis reveals that the three transcription factors affect genes categorized to different biological processes. Furthermore, we document the usefulness of these Flp-In T-REx cells for the functional analysis of mutated HNF1beta transcription factors found in human MODY5 patients. We show that the expression of the mutant P328L329del and A263insGG affects only very few transcripts and these are predominantly distinct from those induced by wild-type HNF1beta.

Tissue-specific transcription factor HNF4alpha inhibits cell proliferation and induces apoptosis in the pancreatic INS-1 beta-cell line.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue-specific transcription factor expressed in many cell types, including pancreatic beta-cells. Mutations in the HNF4alpha gene in humans give rise to maturity-onset diabetes of the young (MODY1) characterized by defective insulin secretion by beta-cells. To elucidate the mechanism underlying this disease, we introduced the splice form HNF4alpha2 or HNF4alpha8 into the rat beta-cell line INS-1. Upon tetracycline-induced expression, both HNF4alpha isoforms caused distinct changes in cell morphology and a massive loss of cell numbers that was correlated with reduced proliferation and induced apoptosis. This differential activity was reflected in oligonucleotide microarray analysis that identified more genes affected by HNF4alpha2 compared to HNF4alpha8, and suggests that both isoforms regulate largely the same set of genes, with HNF4alpha2 being a stronger transactivator. We verified the induction of selected transcripts by real-time RT-PCR, including KAI1 and AIF, both known to have apoptotic potential. By establishing cell lines with inducible expression of these target genes, we deduce that both factors are insufficient to induce apoptosis. We propose that the anti-proliferative and apoptotic properties of HNF4alpha may be an essential feature impaired in MODY1 and possibly also in type 2 diabetes.

Murine Nr4a1 and Herpud1 are up-regulated by Wnt-1, but the homologous human genes are independent from beta-catenin activation.

The Wnt signal transduction pathway regulates morphogenesis and mitogenesis of cells in multicellular organisms. A major downstream consequence of Wnt-1 signalling is the activation of beta-catenin/T-cell factor (TCF)-mediated transcription. We compared Wnt-1-transformed murine mammary epithelial cells with control cells by subtractive hybridization. We found the two genes Nr4a1 and Herpud1 to be overexpressed in Wnt-1-transformed cells. Remarkably, the transcription levels of the two homologous human genes NR4A1 and HERPUD1 are neither activated in cells with activated beta-catenin/TCF-mediated transcription nor can be induced by beta-catenin transfection. These results indicate different regulation mechanisms of the two genes in murine and human cells.