Tilo Moede

Selective Insulin Signaling through A and B Insulin Receptors Regulates Transcription of Insulin and Glucokinase Genes in Pancreatic β Cells

Insulin signaling is mediated by a complex network of diverging and converging pathways, with alternative proteins and isoforms at almost every step in the process. We show here that insulin activates the transcription of its own gene and that of the beta cell glucokinase gene (betaGK) by different mechanisms. Whereas insulin gene transcription is promoted by signaling through insulin receptor A type (Ex11-), PI3K class Ia, and p70s6k, insulin stimulates the betaGK gene by signaling via insulin receptor B type (Ex11+), PI3K class II-like activity, and PKB (c-Akt). Our data provide evidence for selectivity in insulin action via the two isoforms of the insulin receptor, the molecular basis being preferential signaling through different PI3K and protein kinases.

Noninvasive in vivo imaging of pancreatic islet cell biology

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions.

Adipsin Is an Adipokine that Improves β Cell Function in Diabetes

A hallmark of type 2 diabetes mellitus (T2DM) is the development of pancreatic β cell failure, which results in insulinopenia and hyperglycemia. We show that the adipokine adipsin has a beneficial role in maintaining β cell function. Animals genetically lacking adipsin have glucose intolerance due to insulinopenia; isolated islets from these mice have reduced glucose-stimulated insulin secretion. Replenishment of adipsin to diabetic mice treated hyperglycemia by boosting insulin secretion. We identify C3a, a peptide generated by adipsin, as a potent insulin secretagogue and show that the C3a receptor is required for these beneficial effects of adipsin. C3a acts on islets by augmenting ATP levels, respiration, and cytosolic free Ca(2+). Finally, we demonstrate that T2DM patients with β cell failure are deficient in adipsin. These findings indicate that the adipsin/C3a pathway connects adipocyte function to β cell physiology, and manipulation of this molecular switch may serve as a therapy in T2DM.

Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

Cysteine string proteins (CSPs) are novel synaptic vesicle‐associated protein components characterized by an N‐terminal J‐domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT–PCR analysis demonstrated CSP mRNA expression in insulin‐producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin‐secreting cell lines. Punctate CSP‐like immunoreactivity (CSP‐LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP‐LI in close association with membranes of secretory granules of cells in the endo‐ and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS‐1 cells or intracellular administration of CSP antibodies into mouse ob/ob β‐cells did not affect voltage‐dependent Ca2+‐channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS‐1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage‐gated Ca2+‐channels.

Identification of a nuclear localization signal, RRMKWKK, in the homeodomain transcription factor PDX-1

Pancreatic duodenal homeobox‐containing transcription factor 1 (PDX‐1) plays a crucial role in pancreas development and β‐cell gene regulation. Absence of PDX‐1 leads to pancreas agenesis and its malfunction causes MODY4 diabetes mellitus. PDX‐1 has been suggested to be involved in the glucose‐dependent regulation of insulin gene transcription. Whereas DNA‐binding and transactivation domains of PDX‐1 are in the process of being characterized, protein sequences responsible for its nuclear translocation remain unknown. By combining site‐directed mutagenesis of putative phosphorylation sites and nuclear localization signal (NLS) motifs with on‐line monitoring of GFP‐tagged PDX‐1 translocation, we demonstrate that the NLS motif RRMKWKK is necessary and in conjunction with the integrity of the ‘helix 3’ domain of the PDX‐1 homeodomain is sufficient for the nuclear import of PDX‐1. Furthermore, we show that there is no glucose‐dependent cytoplasmic‐nuclear cycling of PDX‐1.

Isoform-specific insulin receptor signaling involves different plasma membrane domains

In pancreatic β-cells, insulin selectively up-regulates the transcription of its own gene and that of the glucokinase gene by signaling through the two isoforms of the insulin receptor, i.e., A-type (Ex11−) and B-type (Ex11+), using different signaling pathways. However, the molecular mechanism(s) that allows the discrete activation of signaling cascades via the two receptor isoforms remains unclear. Here we show that activation of the insulin promoter via A-type and of the glucokinase promoter via B-type insulin receptor is not dependent on receptor isoform–specific differences in internalization but on the different localization of the receptor types in the plasma membrane. Our data demonstrate that localization and function of the two receptor types depend on the 12–amino acid string encoded by exon 11, which acts as a sorting signal rather than as a physical spacer. Moreover, our data suggest that selective activation of the insulin and glucokinase promoters occurs by signaling from noncaveolae lipid rafts that are differently sensitive toward cholesterol depletion.

Insulin-feedback via PI3K-C2α activated PKBα/Akt1 is required for glucose-stimulated insulin secretion

Phosphatidylinositide 3‐kinases (PI3Ks) play central roles in insulin signal transduction. While the contribution of class Ia PI3K members has been extensively studied, the role of class II members remains poorly understood. The diverse actions of class II PI3K‐C2α have been attributed to its lipid product PI(3)P. By applying pharmacological inhibitors, transient overexpression and small‐interfering RNA‐based knockdown of PI3K and PKB/Akt isoforms, together with PI‐lipid profiling and live‐cell confocal and total internal reflection fluorescence microscopy, we now demonstrate that in response to insulin, PI3K‐C2α generates PI(3, 4)P2, which allows the selective activation of PKBα/Akt1. Knockdown of PI3K‐C2α expression and subsequent reduction of PKBα/Akt1 activity in the pancreatic β‐cell impaired glucose‐stimulated insulin release, at least in part, due to reduced glucokinase expression and increased AS160 activity. Hence, our results identify signal transduction via PI3K‐C2α as a novel pathway whereby insulin activates PKB/Akt and thus discloses PI3K‐C2α as a potential drugable target in type 2 diabetes. The high degree of codistribution of PI3K‐C2α and PKBα/Akt1 with insulin receptor B type, but not A type, in the same plasma membrane microdomains lends further support to the concept that selectivity in insulin signaling is achieved by the spatial segregation of signaling events.—Leibiger, B., Moede, T., Uhles, S., Barker, C. J., Creveaux, M., Domin, J., Berggren, P.‐O., Leibiger, I. B. Insulin‐feedback via PI3K‐C2α activated PKBα/Akt1 is required for glucose‐stimulated insulin secretion. FASEB J. 24, 1824–1837 (2010). www.fasebj.org

Ionizing radiation modulates cell surface integrin expression and adhesion of COLO-320 cells to collagen and fibronectin in vitro.

Purpose: Adhesion of tumor cells to endothelial cells and to the extracellular matrix is a key step in the initial phase of metastasis. Since radiotherapy of tumors can induce alterations of the cell surface, we investigated the effect of ionizing radiation on the expression of integrins in the colorectal tumor cell line COLO-320 and the modulation of adhesion capacity of irradiated cells to collagen and fibronectin. Material and Methods: The cell surface expression of a broad range of integrins on COLO-320 cells was determined by flow cytometry during 144 hours after X-irradiation. The functional significance of increased adhesion molecule expression was assessed by cell-matrix adhesion and receptor blocking experiments. Results: Cell surface expression of the following integrin α and β subunits was quantified: β1 (CD29), α2 (CD49b), α5 (CD49e) and α6 (CD49f). The expression of α1, α2, α5, and α6 changes as a function of time after irradiation (5 Gy). For β1 even a function of dose (1–5 Gy) could be shown. Adhesion experiments confirmed a time dependent increase in adhesion to both collagen and fibronectin. Radiation-induced increase in adhesion was inhibited significantly by using a CD29 antibody. Conclusions: Ionizing radiation modulates cell surface expression of integrins and cell-matrix interactions. The β1-integrin subunit plays an important role in radiation-induced adhesion to both collagen and fibronectin. Possible consequences of these in-vitro results for radiotherapy of colorectal tumors in vivo are discussed.Hintergrund: Die Adhäsion zwischen Tumorzellen und Endothelzellen sowie der extrazellulären Matrix ist ein entscheidender Schritt in der Initialphase einer Metastasierung. Da eine Bestrahlung von Tumoren mittels Radiotherapie Änderungen der Zelloberfläche bewirken kann, wurde der Effekt von ionisierender Strahlung auf die Expression von Integrinen in der kolorektalen Tumorzelllinie COLO-320 sowie die Modulationder Adhäsionsfähigkeit der bestrahlten Zellen an Kollagen und Fibronektin untersucht. Material und Methode: Die Zelloberflächenexpression einer Reihe von Integrinen wurde durchflusszytometrisch an COLO-320-Zellen bis zu 144 Stunden nach Bestrahlung bestimmt. Die funktionelle Bedeutung einer gesteigerten Adhäsionsmolekülexpression wurde mittels Zell-Matrix-Adhäsionsversuchen und Rezeptorblockaden untersucht. Ergebnisse: Die Zelloberflächenexpression der folgenden α- und β-Integrinuntereinheiten wurde quantifiziert: β1 (CD29), α2 (CD49b), α5 (CD49e) und α6 (CD49f). Die Expression von α1, α2, α5 und α6 änderte sich zeitabhängig nach Bestrahlung (5 Gy). Für β1 konnte auch eine Dosisunabhängigkeit zwischen 1 und 5 Gy gezeigt werden. Adhäsionsversuche bestätigten einen zeitabhängigen Anstieg sowohl der Adhäsion zu Kollagen als auch zu Fibronektin. Der strahleninduzierte Anstieg der Adhäsion wurde signifiaktn durch einen CD29-Antikörper inhibiert. Schlussfolgerungen: Ionisierende Strahlung moduliert die Zelloberflächenexpression von Integrinen und Zell-Matrix-Interaktionen. Die β1-Integrinuntereinheit spielt eine wichtige Rolle bei der strahleninduzierten Adhäsion sowohl an Kollagen als auch an Fibronektin. Mögliche Konsequenzen dieser In-vitro-Ergebnisse für eine Radiotherapie kolorektaler Tumoren in vivo werden diskutiert.

Selective gene activation by spatial segregation of insulin receptor B signaling

Insulin exerts pleiotropic effects at the cellular level. Signaling via the two isoforms of the insulin receptor (IR) may explain the activation of different signaling cascades, while it remains to be explored how selectivity is achieved when utilizing the same IR isoform. We now demonstrate that insulin‐stimulated transcription of c‐fos and glucokinase genes is activated simultaneously in the insulin‐producing β‐cell via IR‐B localized in different cellular compartments. Insulin activates the glucokinase gene from plasma membrane‐standing IR‐B, while c‐fos gene activation is dependent on clathrin‐mediated IR‐B‐endocytosis and signaling from early endosomes. Moreover, glucokinase gene up‐regulation requires the integrity of the jux‐tamembrane IR‐B NPEY‐motif and signaling via PI3K‐C2α‐like/PDK1/PKB, while c‐fos gene activation requires the intact C‐terminal YTHM‐motif and signaling via PI3K Ia/Shc/MEK1/ERK. By using IR‐B as an example it is thus possible to demonstrate how spatial segregation allows simultaneous and selective signaling via the same receptor isoform in the same cell.—Uhles S., Moede T., Leibiger B., Berggren P.‐O., and Leibiger I. B. Selective gene activation by spatial segregation of insulin receptor B signaling. FASEB J. 21, 1609–1621 (2007)

Ciliary dysfunction impairs beta-cell insulin secretion and promotes development of type 2 diabetes in rodents

Type 2 diabetes mellitus is affecting more than 382 million people worldwide. Although much progress has been made, a comprehensive understanding of the underlying disease mechanism is still lacking. Here we report a role for the β-cell primary cilium in type 2 diabetes susceptibility. We find impaired glucose handling in young Bbs4(-/-) mice before the onset of obesity. Basal body/ciliary perturbation in murine pancreatic islets leads to impaired first phase insulin release ex and in vivo. Insulin receptor is recruited to the cilium of stimulated β-cells and ciliary/basal body integrity is required for activation of downstream targets of insulin signalling. We also observe a reduction in the number of ciliated β-cells along with misregulated ciliary/basal body gene expression in pancreatic islets in a diabetic rat model. We suggest that ciliary function is implicated in insulin secretion and insulin signalling in the β-cell and that ciliary dysfunction could contribute to type 2 diabetes susceptibility.