GLP-1 Treatment Improves Diabetic Retinopathy by Alleviating Autophagy through GLP-1R-ERK1/2-HDAC6 Signaling Pathway.

Objective: Apoptosis and autophagy of retinal cells, which may be induced by oxidative stress, are tightly associated with the pathogenesis of diabetic retinopathy (DR). The autophagy induced by oxidative stress is considered as excessively stimulated autophagy, which accelerates the progression of DR. This study aims to investigate the protective effect of GLP-1 treatment on alleviating apoptosis and autophagy of retinal cells in type 2 diabetic rats and reveals its possible mechanism. Methods: Type 2 diabetic rats were induced by fed with high sugar, high fat diet and followed with streptozotocin injection. GLP-1 was applied to treat the diabetic rats for one week after the onset of diabetes. The expressions of oxidative stress-related enzymes, retinal GLP-1R, mitochondria-dependent apoptosis- related genes, autophagy markers, and autophagy-associated pathway genes were studied by Western blotting or immunohistochemistry analysis. Results: GLP-1treatment reduced the levels of NOX3 and SOD2 in DR. The expression of BCL2 was increased, while the levels of caspase3 and LC3B were reduced through GLP-1 treatment in DR. GLP-1 treatment restored the GLP-1R expression and decreased the levels of phosphorylated AKT and phosphorylated ERK1/2, which was accompanied with the reduction of the HDAC6 levels in DR. Conclusions: GLP-1 treatment can alleviate autophagy which may be induced by oxidative stress; this protective effect is likely through GLP-1R-ERK1/2-HDAC6 signaling pathway.

A Prediction Model with a Combination of Variables for Diagnosis of Lung Cancer.

BACKGROUND Multivariate models with a combination of variables can predict disease more accurately than a single variable employed alone. We developed a logistic regression model with a combination of variables and evaluated its ability to predict lung cancer. MATERIAL AND METHODS The exhaled breath from 57 patients with lung cancer and 72 healthy controls without cancer was collected. The VOCs of exhaled breath were examined qualitatively and quantitatively by a novel electronic nose (Z-nose4200 equipment). The VOCs in the 2 groups were compared using the Mann-Whitney U test, and the baseline data were compared between the 2 groups using the chi-square test or ANOVA. Variables from VOCs and baseline data were selected by stepwise logistic regression and subjected to a prediction model for the diagnosis of lung cancer as combined factors. The receiver operating characteristic (ROC) curve was used to evaluate the predictive ability of this prediction model. RESULTS Nine VOCs in exhaled breath of lung cancer patients differed significantly from those of healthy controls. Four variables - age, hexane, 2,2,4,6,6-pentamethylheptane, and 1,2,6-trimethylnaphthalene - were entered into the prediction model, which could effectively separate the lung cancer samples from the control samples with an accuracy of 82.8%, a sensitivity of 76.0%, and a specificity of 94.0%. CONCLUSIONS The profile of VOCs in exhaled breath contained distinguishable biomarkers in the patients with lung cancers. The prediction model with 4 variables appears to provide a new technique for lung cancer detection.

Glucagon-Like Peptide-1 (GLP-1) Treatment Ameliorates Cognitive Impairment by Attenuating Arc Expression in Type 2 Diabetic Rats.

BACKGROUND Glucagon-like peptide-1 (GLP-1) has been reported to exert some beneficial effects on the central nervous system (CNS). However, the effect of GLP-1 on cognitive impairment associated with type 2 diabetes is not well known. This study investigated the effect of GLP-1 on ameliorating memory deficits in type 2 diabetic rats. MATERIAL AND METHODS Type 2 diabetic rats were induced by a high-sugar, high-fat diet, followed by streptozotocin (STZ) injection and then tested in the Morris Water Maze (MWM) 1 week after the induction of diabetes. The mRNA expression of Arc, APP, BACE1, and PS1 were determined by real-time quantitative PCR, and the Arc protein was analyzed by immunoblotting and immunohistochemistry. RESULTS Type 2 diabetic rats exhibited a significant decline in learning and memory in the MWM tests, but GLP-1 treatment was able to protect this decline and significantly improved learning ability and memory. The mRNA expression assays showed that GLP-1 treatment markedly reduced Arc, APP, BACE1, and PS1 expressions, which were elevated in the diabetic rats. Immunoblotting and immunohistochemistry results also confirmed that Arc protein increased in the hippocampus of diabetic rats, but was reduced after GLP-1 treatment. CONCLUSIONS Our findings suggest that GLP-1 treatment improves learning and memory deficits in type 2 diabetic rats, and this effect is likely through the reduction of Arc expression in the hippocampus.

A bitter pill for type 2 diabetes? The activation of bitter taste receptor TAS2R38 can stimulate GLP-1 release from enteroendocrine L-cells.

The bitter taste receptor TAS2R38 is a G protein coupled receptor (GPCR) that has been found in many extra-oral locations like the gastrointestinal (GI) system, respiratory system, and brain, though its function at these locations is only beginning to be understood. To probe the receptor's potential metabolic role, immunohistochemistry of human ileum tissues was performed, which showed that the receptor was co-localized with glucagon-like peptide 1 (GLP-1) in L-cells. In a previous study, we had modeled the structure of this receptor for its many taste-variant haplotypes (Tan et al. 2011), including the taster haplotype PAV. The structure of this haplotype was then used in a virtual ligand screening pipeline using a collection of ∼2.5 million purchasable molecules from the ZINC database. Three compounds (Z7, Z3, Z1) were purchased from the top hits and tested along with PTU (known TAS2R38 agonist) in in vitro and in vivo assays. The dose-response study of the effect of PTU and Z7 on GLP-1 release using wild-type and TAS2R38 knockout HuTu-80 cells showed that the receptor TAS2R38 plays a major role in GLP-1 release due to these molecules. In vivo studies of PTU and the three compounds showed that they each increase GLP-1 release. PTU was also chemical linked to cellulose to slow its absorption and when tested in vivo, it showed an enhanced and prolonged GLP-1 release. These results suggest that the GI lumen location of TAS2R38 on the L-cell makes it a relatively safe drug target as systemic absorption is not needed for a TAS2R38 agonist drug to effect GLP-1 release.

Ellagic acid and embelin affect key cellular components of pancreatic adenocarcinoma, cancer, and stellate cells.

Ellagic acid is a polyphenolic phytochemical present in many fruits and nuts with anticancer properties demonstrated in experimental tumor studies. Embelin is a benzoquinone phytochemical isolated from the Japanese herb Ardisiae Japonicae and has been shown to induce apoptosis in cancer cells. We found that ellagic acid and embelin each dose-dependently increased apoptosis and inhibited proliferation in human pancreatic cancer cells, MIA PaCa-2 and HPAF-II cells, and in pancreatic stellate cells, which are progenitors of pancreatic cancer desmoplasia. In each of these cell types, combinations of ellagic acid and embelin at low micromolar concentrations (0.5-3 µM) induced synergistic increases in apoptosis and decreases in proliferation. Ellagic acid decreased NF-κB transcriptional activity, whereas embelin decreased STAT-3 phosphorylation and protein expression of its downstream target survivin in cancer cells. In vivo dietary ellagic acid alone or in combination with embelin decreased tumor size and tumor cellularity in a subcutaneous xenograft mouse model of pancreatic cancer. These results show that ellagic acid and embelin interact with divergent intracellular signaling pathways resulting in augmentation of apoptosis and inhibition of proliferation at low micromolar concentrations for the key cellular components of pancreatic adenocarcinoma.

The alpha1-adrenergic receptor antagonist doxazosin inhibits EGFR and NF-kappaB signalling to induce breast cancer cell apoptosis.

The selective alpha(1)-adrenergic receptor antagonist doxazosin (dox) has been reported to inhibit prostate cancer proliferation. We now demonstrate that dox-treatment inhibits proliferation and induces apoptosis in breast cancer cells in vitro by mechanisms that do not wholly involve the alpha1-adrenergic receptor. Intriguingly, dox-treatment reduced phosphorylated EGFR expression, decreased pERK1/2 levels and decreased NF-kappaB, AP-1, SRE, E2F and CRE-mediated transcriptional activity. EGF- and TNFalpha treatment alone failed to block dox-mediated breast cancer apoptotic effects, but combination of EGF and TNFalpha treatments completely abrogated dox-induced breast cancer cell apoptosis, indicating doxazosin inhibits both EGFR and NF-kappaB signalling pathways to induce breast cancer cell apoptosis. Doxazosin is proposed as a possible novel medical therapy for breast cancer.

Adenovirus-mediated XIAP gene transfer reverses the negative effects of immunosuppressive drugs on insulin secretion and cell viability of isolated human islets.

Immunosuppressive drugs are routinely used to provide tolerance after whole pancreas and islet cell transplantations. While they are essential in inhibiting graft rejection, little is known about their effect on islet function and beta-cell viability. In this study, we report that tacrolimus, sirolimus, and mycophenolic acid, when added to cultures of freshly isolated human islets, induce a downregulation of the synthesis and secretion of insulin. These functional changes are associated with decreased islet cell viability. All three agents induce a decrease of intracellular levels of Bcl-2 and Bcl-xL, with an increased level of Smac, indicating that they are capable of promoting a downregulation of anti-apoptotic factors and an accumulation of pro-apoptotic mediators. Transduction of islet cells with the anti-apoptotic gene XIAP prevents the negative effects of these drugs on the function and viability of islets. XIAP-infected cells show a higher expression of phospho-CREB (cAMP-responsive element binding protein) and a reduced level of Smac, resulting in a significant reduction of apoptotic cells and a preservation of the glucose-dependent secretion of insulin. In conclusion, the present study demonstrates that genetically modified human islets expressing XIAP are resistant to the negative effects of immunosuppressive drugs on insulin secretion and cell viability.

Fatty acid translocase (FAT/CD36) is localized on insulin-containing granules in human pancreatic beta-cells and mediates fatty acid effects on insulin secretion.

The membrane receptor FAT/CD36 facilitates the major fraction of long-chain fatty acid (FA) uptake by muscle and adipose tissues. In line with the well-known effects of FA metabolism on carbohydrate utilization and insulin responsiveness, altered expression of CD36 has been linked to phenotypic features of the metabolic syndrome including insulin resistance and dyslipidemia. FA metabolism is also known to significantly affect insulin secretion. However, the role of CD36 in this process remains unknown, since its expression levels and function in the pancreas have not been explored. In the present study, freshly isolated human islets and a mouse-derived beta-cell line (MIN6) were shown positive for CD36 expression by RT-PCR, Western blot, and immunofluorescence. The identity of the PCR product was confirmed by microsequencing. The identified transcript was translated and the protein was expressed and subjected to the known posttranslational glycosylation. Fluorescence resonance energy transfer analysis and subcellular protein fractionation indicated that insulin and CD36 are colocalized in the secretory granules of beta-cells. Islet CD36 functioned in FA uptake because this process was blocked by the irreversible CD36 inhibitor sulfosuccinimidyl-oleate. More importantly, sulfosuccinimidyl-oleate reversed enhancing and inhibiting effects, respectively, of acute and long-term palmitate incubations on glucose-dependent insulin secretion. In conclusion, our study demonstrates that human islets express CD36 in the plasma membrane as well as in the insulin secretory granules. CD36 activity appears important for uptake of FA into beta-cells as well as for mediating their modulatory effects on insulin secretion.

Murine pituitary tumor-transforming gene functions as a securin protein in insulin-secreting cells.

Human pituitary tumor-transforming gene 1 (PTTG1) encodes a securin protein critically important in regulating chromosome separation. Murine PTTG (mPTTG) is 66% homologous to human PTTG1 and PTTG-null (PTTG-/-) mice exhibit pancreatic beta-cell hypoplasia and abnormal nuclear morphology with resultant diabetes. As we show that ductal beta-cell neogenesis is intact in PTTG-/- mice, we explored mechanism for defective beta-cell replication. We tested whether mPTTG exhibits securin properties in mouse insulin-secreting insulinoma MIN6 cells, using a live-cell system to monitor mitosis in cells transfected with an enhanced green fluorescent protein (EGFP)-tagged mPTTG conjugate (mPTTG-EGFP). To fulfill the criteria for securin properties, the protein should undergo degradation immediately before the metaphase-to-anaphase transition when expression levels are low, and should inhibit metaphase-to-anaphase transition when expression levels are high. EGFP itself did not undergo degradation throughout mitosis and high levels of EGFP per se did not affect normal mitosis progression (n=25). However, mPTTG-EGFP was degraded 2 min before the metaphase-to-anaphase transition when expression levels were low (n=19), and high mPTTG-EGFP levels blocked metaphase-to-anaphase transition in 13 cells. mPTTG-EGFP inhibited MIN6 cell proliferation and caused apoptosis. Immunocoprecipitation demonstrated binding of mPTTG-EGFP and separase. These results show that mPTTG exhibits properties consistent with a murine securin in insulin-secreting mouse cells and mPTTG overexpression inhibits cell proliferation, suggesting that defective beta-cell proliferation observed in PTTG-/- mice is likely due to abnormal cell-cycle progression.

[Glucagon-like peptide 1 improves learning and memory abilities of rats with type 2 diabetes].

OBJECTIVE: To investigate the effect of glucagon-like peptide 1 (GLP-1) on cognitive dysfunction in diabetic rats. METHODS: Male SD rats were randomly divided into normal control group, diabetes mellitus (DM) group, and GLP-1 treatment group. Rat models of type 2 diabetes were established by high-sugar and high-fat feeding and streptozotocin (STZ) injection, and 25 days after the onset of diabetes, GLP-1 was infused in GLP-1 treatment group at the rate of 30 pmol·kg-1·min-1 via a subcutaneous osmotic pump for 7 days. The learning and cognitive ability of the rats was assessed with Morris water maze test, and the expression of cognition-related genes in the hippocampus tissue was detected with real-time PCR, Western blotting and immunohistochemical staining. RESULTS: Compared with the normal control group, the diabetic rats showed significantly decreased learning and memory abilities (P<0.05) with increased hippocampal expressions of APP, BACE1, Arc, ERK1/2, PKA, and PKC mRNAs (P<0.05) and Arc protein. Compared with diabetic rats, GLP-1-treated rats showed significantly improvements in the learning and memory function (P<0.05) with decreased expressions of APP, BACE1, Arc, ERK1/2, and PKA mRNAs (P<0.05) and Arc protein. CONCLUSION: GLP-1 can improve cognitive dysfunctions in diabetic rats possibly by regulating the PKC, PKA, and ERK1/2 pathways and inhibiting Arc expression in the hippocampus.

Pancreatic beta-cells expressing GLP-1 are resistant to the toxic effects of immunosuppressive drugs.

Glucose intolerance is often observed after pancreatic islet cell transplantation. The administration of immunosuppressive agents (ISD), necessary to avoid tissue rejection, is in part responsible for hyperglycemia. To investigate whether mouse insulinoma (MIN6) cells transfected with the glucagon like peptide-1 (GLP-1) fragment of the proglucagon gene (RIP/GLP-1 MIN6 cells) are resistant to the toxicity derived from the administration of ISD. RIP/GLP-1 MIN6 cells, as well as parental MIN6 cells, were exposed to a cocktail of ISD. The secretion of insulin and the expression of apoptosis-related proteins were investigated by RIA and western blot analysis. Cell apoptosis was quantified by FACS analysis. Finally, to study whether the antiapoptotic action of GLP-1 was a function of its effect on insulin secretion, or rather it was a direct effect of GLP-1, cells were cultured with or without diazoxide or exendin-9. GLP-1 improved the functional activity and the viability of cells exposed to ISD. The insulin secretion of RIP/GLP-1 MIN6 cells after exposure to ISD was preserved. The expression of GLP-1 by beta-cells reduced the number of apoptotic cells and increased the expression of the antiapoptotic protein Bcl-2. GLP-1 also decreased the abundance of the proapoptotic markers PARP-p85 and Smac/Diablo. Treatment of cells with the diazoxide did not abolish the protective advantage that cells transfected with GLP-1 had; conversely the exposure of cells to exendin-9 was associated with a restored susceptibility to apoptosis. This report demonstrates that GLP-1 is capable of preserving beta-cell function and protecting cells from apoptotic cell death.

Intrasplenic transplantation of encapsulated genetically engineered mouse insulinoma cells reverses streptozotocin-induced diabetes in rats.

Pancreatic islet transplantation is limited by shortage of donor organs. Although beta-cell lines could be used, their secretion of insulin is characteristically glucose independent and immunoisolation is required. Here we show that intrasplenic transplantation of encapsulated glucose-responsive mouse insulinoma cells reversed streptozotocin (STZ)-induced diabetes in rats. MIN-6 cells derived from a transgenic mouse expressing SV 40 large T antigen in pancreatic beta-cells were transfected with minigene encoding for human glucagon-like-peptide-1 under the control of rat insulin promoter. The cells were encapsulated in alginate/poly-L-lysine and used for cell transplantation in STZ-diabetic rats. Rats with nonfasting blood glucose (n-FBG) greater than 350 mg/dl were used. In group I rats (n=6) 20 million encapsulated cells were injected into the spleen. Group II rats (n=6) received empty capsules. n-FBG was measured biweekly. After 4 and 8 weeks, an intraperitoneal glucose tolerance test (IPGTT) was performed in group I; normal rats served as controls. Plasma insulin level was measured every other week (RIA). After 8 weeks, spleens were removed 1 day before sacrifice. In rats transplanted with cells the n-FBG was 100-150 mg/dl until the end of the study. After splenectomy, all cell recipients became diabetic (glucose 400 +/- 20 mg/dl). Transplanted rats showed increase in body weight and insulin production (3.3 +/- 1.0 ng/ml versus 0.92 +/- 0.3 ng/ml; p < 0.01) and had normal IPGTT. Spleens contained capsules with insulin-positive cells. Overall, data from this work indicate that intrasplenic transplantation of xenogeneic encapsulated insulin-producing cells without immunosuppression reversed diabetes in rats. Excellent survival and function of the transplanted cells was due to the fact that the cells were separated from the bloodstream by the immunoisolatory membrane only and insulin was delivered directly to the liver (i.e., in a physiological manner).

GLP-1 inhibition of pancreatic islet cell apoptosis.

Apoptosis plays an important role in the normal physiology of the pancreas, the pathogenesis of diabetes mellitus (DM) and the success rate of islet transplantation. Glucagon-like peptide-1 (GLP-1), an incretin hormone with multiple effects on glucose metabolism and pancreatic gene expression, has recently been found to have antiapoptotic properties. This new property of GLP-1 has clinical relevance for the treatment of patients with overt DM, possible prevention of DM during the stage of impaired glucose tolerance and improvement in the outcome of islet transplantation. The pleiotropic effects of GLP-1 have fostered considerable interest in evaluating the efficacy of GLP-1, and might lead in the near future to its use in the prevention and/or treatment of DM.

Transfection of pancreatic-derived beta-cells with a minigene encoding for human glucagon-like peptide-1 regulates glucose-dependent insulin synthesis and secretion.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from the proglucagon gene, capable of regulating the transcription of the three major genes that determine the pancreatic beta-cell-specific phenotype: insulin, GLUT-2, and glucokinase. The aim of this study was to investigate the potential role of GLP-1 for the gene therapy of glucose-insensitive pancreatic beta-cells. We transfected mouse insulinoma cells with a DNA fragment of the human proglucagon gene containing the nucleotide sequence encoding for human GLP-1 but lacking the coding region for glucagon. Two constructs were generated: In one, the expression of GLP-1 was under the control of the cytomegalovirus (CMV) promoter (CMV/GLP-1), and the second was regulated by the rat insulin II promoter (RIP)/GLP-1). Northern blot, HPLC, and RIA analyses confirmed that the minigene was transcribed and the protein appropriately translated, processed, and secreted in the extracellular environment. Gene expression studies revealed that although CMV/GLP-1 cells did not gain a greater glucose sensitivity as a result of the transfection with GLP-1, compared with cells transfected with the plasmid alone, RIP/GLP-1 was capable of regulating the gene expression of insulin and GLP-1 based on the concentration of glucose in the culture medium. Detection of the counterpart proteins (insulin and GLP-1) in the culture medium paralleled the observation derived from the Northern blot analysis. GLP-1 action was mediated by an IDX-1 (islet/duodenum homeobox-1) dependent transactivation of the endogenous insulin promoter, as demonstrated by gel shift analysis. This was further suggested by a significant increase of the glucose-dependent binding of IDX-1 to the insulin promoter in RIP/GLP-1 cells but not in CMV/GLP-1 cells or control cells. Finally, we observed that although the GLP-1-dependent secretion of insulin was mediated by an increase in cAMP levels, the transcription of the insulin gene, in response to GLP-1, was in large part cAMP independent. The present study lays the research foundation to investigate the potential use of GLP-1 for the gene or cell therapy of diabetes.

Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5'-adenosine monophosphate-dependent protein kinase A- and a phosphatidylinositol 3-kinase-dependent pathway.

The activation of the glucagon-like peptide-1 (GLP-1) receptor has been shown to have an important role in the functional activity of islet beta-cells and in the expansion of the islet cell mass. Constant remodeling of islet cell mass is mediated in vivo by proliferative and apoptotic stimuli to ensure a dynamic response to a changing demand for insulin. The present study was undertaken to investigate the biological activity of GLP-1 when cells were challenged by a proapoptotic stimulus. We have shown that activation of the GLP-1 receptor inhibits H(2)O(2)-induced apoptosis in a cultured mouse insulinoma cell line, termed MIN6. GLP-1 reduced DNA fragmentation and improved cell survival. This was mediated by an increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. GLP-1 also prevented the H(2)O(2)-dependent cleavage of poly-(ADP-ribose)-polymerase. Inhibition of the GLP-1-dependent increase of cAMP by Rp-cAMP blocked the antiapoptotic action of GLP-1, as determined by DNA fragmentation and poly-(ADP-ribose)-polymerase assays and by detection of Bcl-2 and Bcl-xL protein levels. Investigation of the role of the protein kinases, PI-3 kinase (PI3K) and MAPK, by use of the inhibitors PD098059 and LY294002 demonstrated that the activation of PI3K, but not MAPK, was required to prevent proapoptotic events in cells exposed to H(2)O(2). The present study provides evidence that GLP-1 has an antiapoptotic action mediated by a cAMP- and PI3K-dependent signaling pathway.

Glucagon-like peptide-1 promotes islet cell growth and inhibits apoptosis in Zucker diabetic rats.

A constant remodeling of islet cell mass mediated by proliferative and apoptotic stimuli ensures a dynamic response to a changing demand for insulin. In this study, we investigated the effect of glucagon-like peptide-1 (GLP-1) in Zucker diabetic rats, an animal model in which the onset of diabetes occurs when the proliferative potential and the rate of beta-cell apoptosis no longer compensate for the increased demand for insulin. We subjected diabetic rats to a 2-d infusion of GLP-1 and tested their response to an ip glucose tolerance test. GLP-1 produced a significant increase of insulin secretion, which was paralleled by a decrease in plasma glucose levels (P < 0.001 and P < 0.01, respectively). Four days after the removal of the infusion pumps, rats were killed and the pancreas harvested to study the mechanism by which GLP-1 ameliorated glucose tolerance. Ex vivo immunostaining with the marker of cell proliferation, Ki-67, showed that the metabolic changes observed in rats treated with GLP-1 were associated with an increase in cell proliferation of the endocrine and exocrine component of the pancreas. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining, a marker of cellular apoptosis, indicated a reduction of apoptotic cells within the islet as well in the exocrine pancreas in GLP-1-treated rats. Double immunostaining for the apoptotic marker caspase-3 and for insulin showed a significant reduction of caspase-3 expression and an increase in insulin content in GLP-1-treated animals. Finally, staining of pancreatic sections with the nuclear dye 4,6-Diaminidino-2-phenyl-dihydrochloride demonstrated a marked reduction of fragmented nuclei in the islet cells of rats treated with GLP-1. Our findings provide evidence that the beneficial effects of GLP-1 in Zucker diabetic rats is mediated by an increase in islet cell proliferation and a decrease of cellular apoptosis.

Pancreas duodenum homeobox-1 regulates pancreas development during embryogenesis and islet cell function in adulthood.

Pancreas duodenum homeobox-1 (PDX-1) (also known as insulin promoter factor-1, islet/duodenum homeobox-1, somatostatin transactivating factor-1, insulin upstream factor-1 and glucose-sensitive factor) is a transcription factor encoded by a Hox-like homeodomain gene. In humans and other animal species, the embryonic development of the pancreas requires PDX-1, as demonstrated by the identification of an individual with pancreatic agenesis resulting from a mutation that impaired the transcription of a functionally active PDX-1 protein. In adult subjects, PDX-1 is essential for normal pancreatic islet function as suggested by its regulatory action on the expression of a number of pancreatic genes, including insulin, somatostatin, islet amyloid polypeptide, the glucose transporter type 2 and glucokinase. Furthermore, heterozygous mutations of PDX-1 have been linked to a type of autosomal dominant form of diabetes mellitus known as maturity onset diabetes of the young type 4. The dual action of PDX-1, as a differentiation factor during embryogenesis and as a regulator of islet cell physiology in mature islet cells, underscores the unique role of PDX-1 in health and disease of the human endocrine pancreas.

The short half-life of glucagon-like peptide-1 in plasma does not reflect its long-lasting beneficial effects.

The incretin hormone glucagon-like peptide-1 (GLP-1) is capable of ameliorating glucose-dependent insulin secretion in subjects with diabetes. However, its very short half-life (1.5-5 min) in plasma represents a major limitation for its use in the clinical setting. The present study was designed to characterize the duration of the effect of GLP-1 in the Zucker diabetic fatty (ZDF) rat. ZDF rats were subjected to a 48 h infusion of human GLP-1 (30 pmol/kg per min), followed by an i.p. glucose tolerance test (IPGTT) (1 g/kg body weight), 2 h after removing the infusion pump. At 15 min from the beginning of the test, GLP-1-treated animals had lower plasma glucose levels (442+/-38 mg/dl) than saline-infused controls (583+/-63 mg/dl, P<0.01). This was reflected in the higher insulin levels attained in the GLP-1-treated animals (1999+/-163 vs 1250+/-51 pmol/l, GLP-1 vs saline respectively, P<0.01). Repetition of the IPGTT on day 3, 9 and 16 from the removal of the infusion pump revealed a surprising lasting 'memory' of the exposure to GLP-1. Indeed, the best insulin secretory response was observed approximately 1 week after discontinuation of the GLP-1 infusion, and lasted up to 3 weeks from the early exposure to GLP-1. Detection of fasting plasma levels of GLP-1 during the 3 weeks of the experiment showed a very rapid decline, consistent with the data reported by others. Our findings provide evidence for a long-lasting beneficial effect of GLP-1 that persists for weeks even when the circulating levels of GLP-1 are back to normal.

Gene expression profiling of cultured human islet preparations.

The expression of functional and regulatory genes by islet cells is a key determinant for the success of islet transplantation. The aim of this study is twofold: first, to characterize the cluster of genes expressed in human islet isolations; and second, to validate the capability of gene array technology to assess with accuracy the expression of various transcripts. RNA from isolated islet preparations obtained from three independent donors was converted to cDNA and then transcribed to cRNA. Individual cRNA preparations were then hybridized to U133A microarrays carrying approximately 23,000 genes, and analyzed using GeneSpring (SiliconGenetics, Redwood City, CA) software. Real-time reverse transcription-polymerase chain reaction was performed to validate results obtained by microarray analysis. Microarray analysis identified the expression of about 7,000 genes transcribed in cultured human islet preparations. Enzymes represented the most abundant class of genes identified, followed by nuclear binding proteins, signal transduction molecules, transport proteins, and growth factor receptors and their ligands. Real-time polymerase chain reaction confirmed the identification of various islet-specific genes detected by microarray analysis, but also showed that such genes as pancreatic duodenal homeobox 1 protein and glucagon-like peptide 1 receptor, which were not detected by gene array, can be readily identified and quantified. In addition, gene array produced a suboptimal quantification of genes expressed in large amounts by islet cells. Indeed, the abundance of mRNA for insulin when compared with the level of somatostatin mRNA was not as different as one would have predicated based on the classic knowledge of islet physiology. Gene array analysis appears to be a valuable tool to obtain preliminary information of genes expressed by a given tissue. The expression levels of transcripts expressed in very low or very high quantities need to be confirmed by an independent technique.

Upregulation of bax Gene Expression Promotes Paclitaxel-induced Apoptosis in Esophageal Carcinoma Cells.

An inducible mammalian expression vector of bax gene was constructed and the control ability of metallothionein II promoter in esophageal carcinoma cell line was systematically identified with luciferase report gene. After the transfection of it into human esophageal carcinoma cell line Eca109, Bax protein expression was analyzed by immunolcytochemical method. Paclitaxel-induced apoptosis was determined by TUNEL assay, DNA ladder assay and flow cytometry. Results showed that 140 &mgr;mol/L ZnSO(4) for 12 h is optimal for induction of bax gene expression. Under these conditions, a clonal transfectant X1097(#), expressing bax gene effectively, was obtained. It was found that, X1097(#) had higher apoptotic rate and was more sensible than Eca109. These results implied that Bax protein may play an important role in paclitaxel-induced apoptosis. Therefore, bax protein may be promising as an helping drug to improve therapeutic effects of paclitaxel.