Overexpression of Kruppel-like factor 7 regulates adipocytokine gene expressions in human adipocytes and inhibits glucose-induced insulin secretion in pancreatic beta-cell line

We have identified Kruppel-like factor 7 (KLF7) as a new candidate for conferring susceptibility to type 2 diabetes. To ascertain the possible involvement of KLF7 in the pathogenesis of type 2 diabetes, we examined the functional roles of KLF7 in various types of cells. In human adipocytes overexpressing KLF7, the expression of adiponectin and leptin was decreased compared with that in control cells, whereas expression of IL-6 was increased. In the insulin-secreting cell line (HIT-T15 cells), the expression and glucose-induced secretion of insulin were significantly suppressed in KLF7-overexpressed cells compared with control cells, accompanied by the reduction in the expression of glucose transporter 2, sulfonylurea receptor 1, Kir6.2, and pancreatic-duodenal homeobox factor 1. We also found that the overexpression of KLF7 resulted in the decrease of hexokinase 2 expression in smooth muscle cells, and of glucose transporter 2 expression in the HepG2 cells. These results suggest that KLF7 may contribute to the pathogenesis of type 2 diabetes through an impairment of insulin biosynthesis and secretion in pancreatic beta-cells and a reduction of insulin sensitivity in peripheral tissues. Therefore, we suggest that KLF7 plays an important role in the pathogenesis of type 2 diabetes, and may be a useful target for new drugs to aid in the prevention and treatment of this disease.     

Genetically modified rice seeds accumulating GLP-1 analogue stimulate insulin secretion from a mouse pancreatic beta-cell line

Glucagon-like peptide-1 (7-36) amide (GLP-1) is the most potent physiological insulinotropic hormone in humans. We produced large amounts of a GLP-1 analogue, [Ser8, Gln26, Asp34]-GLP-1, which is resistant to trypsin-digestion, as part of a chimeric rice seed storage protein, a 26 kDa globulin, in genetically modified rice seeds. Junction sites between GLP-1 analogue and globulin were replaced by tryptic cleavage sites. The highest level of GLP-1 analogue accumulation was approximately 20-50 microg per seed. We found that GLP-1 analogue derived from trypsin-digested genetically modified rice seeds stimulated insulin secretion from a mouse pancreatic beta-cell line, MIN6.     

Nitric oxide induces apoptosis via Ca2+-dependent processes in the pancreatic beta-cell line MIN6

An excessive production of nitric oxide (NO) in response to cytokines has been shown to be the major cause of the destruction of islet beta-cells associated with type 1 (insulin-dependent) diabetes mellitus. The NO-induced beta-cell death is the typical apoptosis. In the present study, we show evidence that supports a tight link between NO, Ca2+, protease and apoptosis in beta-cells. Three different NO donors, SNAP, NOR3 and NOC7, induced apoptosis in a beta-cell line, MIN6 cells, in a concentration-dependent manner. SNAP at 200 microM increased cytosolic Ca2+ concentration ([Ca2+]i) and induced apoptosis. The SNAP-induced apoptosis was blocked by a Ca2+ chelator, BAPTA-AM, and by an inhibitor of a Ca2+-dependent protease, calpain. In conclusion, an excessive NO production induces apoptosis, wherein an increase in [Ca2+]i and resultant activation of calpain play a key role.     

A role for transglutaminase in glucose-stimulated insulin release from the pancreatic beta-cell.

Preincubation of rat islets of Langerhans with the potent inhibitors of islet transglutaminase activity, monodansylcadaverine (30-100 microM) and N-(5-aminopentyl)-2-naphthalenesulphonamide (100-200 microM), led to significant inhibition of glucose-stimulated insulin release from islets. In contrast, the respective N'-dimethylated derivatives of these two compounds, which did not inhibit islet transglutaminase activity, were much less effective as inhibitors of glucose-stimulated insulin release. None of the compounds inhibited rat spleen protein kinase C activity at concentrations which gave rise to inhibition of glucose-stimulated insulin release. When tested for their effects on calmodulin-stimulated bovine heart phosphodiesterase activity, of the compounds that inhibited insulin release, only monodansylcadaverine did not act as an effective antagonist of calmodulin at concentrations (up to 50 microM) that gave rise to significant inhibition of glucose-stimulated insulin release. Furthermore, at 50 microM, monodansylcadaverine did not inhibit methylation of islet lipids. The inhibition of glucose-stimulated insulin release by monodansylcadaverine is therefore likely to be attributable to its interference with islet transglutaminase activity. The sensitivity of islet transglutaminase to activation by Ca2+ was investigated by using a modified assay incorporating dephosphorylated NN'-dimethylcasein as a substrate protein. The Km for Ca2+ obtained (approx. 3 microM) was an order of magnitude lower than previously reported for the islet enzyme [Bungay, Potter & Griffin (1984) Biochem. J. 219, 819-827]. Mg2+ (2 mM) was found to have little effect on the sensitivity of the enzyme to Ca2+. Investigation of the endogenous substrate proteins of islet transglutaminase by using the Ca2+-dependent incorporation of [14C]methylamine into proteins of islet homogenates demonstrated that most of the incorporated radiolabel was present in cross-linked polymeric aggregates which did not traverse 3% (w/v) acrylamide gels. The radiolabelled polymeric aggregates were present in 71 000 g-sedimented material of homogenates, and their formation was transglutaminase-mediated. These findings provide new evidence for the involvement of islet transglutaminase in the membrane-mediated events necessary for glucose-stimulated insulin release.     

Glucose promotes pancreatic islet beta-cell survival through a PI 3-kinase/Akt-signaling pathway

The concentration of glucose in plasma is an important determinant of pancreatic beta-cell mass, whereas the relative contributions of hypertrophy, proliferation, and cell survival to this process are unclear. Glucose results in depolarization and subsequent calcium influx into islet beta-cells. Because depolarization and calcium (Ca(2+)) influx promote survival of neuronal cells, we hypothesized that glucose might alter survival of islet beta-cells through a similar mechanism. In the present studies, cultured mouse islet beta-cells showed a threefold decrease in apoptosis under conditions of 15 mM glucose compared with 2 mM glucose (P < 0.05). MIN6 insulinoma cells incubated in 25 mM glucose for 24 h showed a threefold decrease in apoptosis compared with cells in 5 mM glucose (1.7 +/- 0.2 vs. 6.3 +/- 1%, respectively, P < 0.001). High glucose (25 mM) enhanced survival-required depolarization and Ca(2+) influx and was blocked by phosphatidylinositol (PI) 3-kinase inhibitors. Glucose activation of the protein kinase Akt was demonstrated in both insulinoma cells and cultured mouse islets by means of an antibody specific for Ser(473) phospho-Akt and by an in vitro Akt kinase assay. Akt phosphorylation was dependent on PI 3-kinase but not on MAPK. Transfection of insulinoma cells with an Akt kinase-dead plasmid (Akt-K179M) resulted in loss of glucose-mediated protection, whereas transfection with a constitutively active Akt enhanced survival in glucose-deprived insulinoma cells. The results of these studies defined a novel pathway for glucose-mediated activation of a PI 3-kinase/Akt survival-signaling pathway in islet beta-cells. This pathway may provide important targets for therapeutic intervention.     

Inhibition of calpain blocks pancreatic beta-cell spreading and insulin secretion

In addition to promoting insulin secretion, an increase in cytosolic Ca(2+) triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca(2+)-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca(2+)-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca(2+)]i. Finally, calpeptin was shown to affect the [Ca(2+)]i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic beta-cells. It is therefore suggested that calpain could be a mediator of Ca(2+)-induced-insulin secretion and beta-cell spreading.     

Phorbol ester stimulation of pancreatic beta-cell replication, polyamine content and insulin secretion.

Long-term effects of the protein kinase C activating phorbol ester, TPA, on pancreatic beta-cell proliferation and insulin production were investigated. It was found that beta-cell replication and long-term insulin secretion were enhanced in TPA-treated islets. This was not accompanied by a corresponding increase in (pro)insulin biosynthesis, presumably contributing to the lowered islet insulin content. TPA also increased islet polyamine content but when this increase was prevented by blocking polyamine synthesis, DNA replication and insulin secretion remained elevated. These findings indicate that TPA stimulates beta-cell replication and insulin secretion and suggest a stimulatory role for protein kinase C, but not for polyamines, in these processes.     

Calcium and pancreatic beta-cell function. I. Stimulatory effects of pentobarbital on insulin release.

Islets microdissected from ob/ob-mice were exposed to 3mM pentobarbital in media which were normal or deficient in Ca2+. This treatment resulted in marked decrease of the islet content of cyclic AMP recorded in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Pentobarbital had a dual effect on insulin release. In addition to being a potent inhibitor of glucose-stimulated insulin release in media containing 2.56 mM Ca2+ it increased the amounts of insulin released in high glucose media deficient in Ca2+. There was a transient stimulation with ordinary concentrations of Ca2+ and 3mM glucose whtn the media also contained 3-isobutyl-1-methylxanthine. The stimulatory effect of pentobarbital persisted after replacing part of the Ca2+ in the beta-cell membrane with lanthanum ions and it could not be mimicked by lowering the oxygen tension of the incubation medium. It is suggested that pentobarbital stimulation of insulin release is the result of a specific action of the drug on the distribution of Ca2+ within the pancreatic beta-cells.     

Glucose induces synchronous mitochondrial calcium oscillations in intact pancreatic islets

Mitochondria shape Ca(2+) signaling and exocytosis by taking up calcium during cell activation. In addition, mitochondrial Ca(2+) ([Ca(2+)](M)) stimulates respiration and ATP synthesis. Insulin secretion by pancreatic beta-cells is coded mainly by oscillations of cytosolic Ca(2+) ([Ca(2+)](C)), but mitochondria are also important in excitation-secretion coupling. Here, we have monitored [Ca(2+)](M) in single beta-cells within intact mouse islets by imaging bioluminescence of targeted aequorins. We find an increase of [Ca(2+)](M) in islet-cells in response to stimuli that induce either Ca(2+) entry, such as extracellular glucose, tolbutamide or high K(+), or Ca(2+) mobilization from the intracellular stores, such as ATP or carbamylcholine. Many cells responded to glucose with synchronous [Ca(2+)](M) oscillations, indicating that mitochondrial function is coordinated at the whole islet level. Mitochondrial Ca(2+) uptake in permeabilized beta-cells increased exponentially with increasing [Ca(2+)], and, particularly, it became much faster at [Ca(2+)](C)>2 microM. Since the bulk [Ca(2+)](C) signals during stimulation with glucose are smaller than 2 microM, mitochondrial Ca(2+) uptake could be not uniform, but to take place preferentially from high [Ca(2+)](C) microdomains formed near the mouth of the plasma membrane Ca(2+) channels. Measurements of mitochondrial NAD(P)H fluorescence in stimulated islets indicated that the [Ca(2+)](M) changes evidenced here activated mitochondrial dehydrogenases and therefore they may modulate the function of beta-cell mitochondria. Diazoxide, an activator of K(ATP), did not modify mitochondrial Ca(2+) uptake.