Barbara Leibiger

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.

Insulin Signaling in the Pancreatic β-Cell

The appropriate function of insulin-producing pancreatic beta-cells is crucial for the regulation of glucose homeostasis, and its impairment leads to diabetes mellitus, the most common metabolic disorder in man. In addition to glucose, the major nutrient factor, inputs from the nervous system, humoral components, and cell-cell communication within the islet of Langerhans act together to guarantee the release of appropriate amounts of insulin in response to changes in blood glucose levels. Data obtained within the past decade in several laboratories have revitalized controversy over the autocrine feedback action of secreted insulin on beta-cell function. Although insulin historically has been suggested to exert a negative effect on beta-cells, recent data provide evidence for a positive role of insulin in transcription, translation, ion flux, insulin secretion, proliferation, and beta-cell survival. Current insights on the role of insulin on pancreatic beta-cell function are discussed.

Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic β cells

Inositol pyrophosphates are recognized components of cellular processes that regulate vesicle trafficking, telomere length, and apoptosis. We observed that pancreatic β cells maintain high basal concentrations of the pyrophosphate diphosphoinositol pentakisphosphate (InsP7 or IP7). Inositol hexakisphosphate kinases (IP6Ks) that can generate IP7 were overexpressed. This overexpression stimulated exocytosis of insulin-containing granules from the readily releasable pool. Exogenously applied IP7 dose-dependently enhanced exocytosis at physiological concentrations. We determined that IP6K1 and IP6K2 were present in β cells. RNA silencing of IP6K1, but not IP6K2, inhibited exocytosis, which suggests that IP6K1 is the critical endogenous kinase. Maintenance of high concentrations of IP7 in the pancreatic β cell may enhance the immediate exocytotic capacity and consequently allow rapid adjustment of insulin secretion in response to increased demand.

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.

Insulin feedback action on pancreatic β-cell function

Pancreatic β‐cell function is essential for the regulation of glucose homeostasis and its impairment leads to diabetes mellitus. Besides glucose, the major nutrient factor, inputs from neural and humoral components and intraislet cell–cell communication act together to guarantee an appropriate pancreatic β‐cell function. Data obtained over the last 5 years in several laboratories have revitalized a controversial concept, namely the autocrine feedback action of secreted insulin on β‐cell function. While, historically, insulin was suggested to exert a negative effect on β‐cells, recent data provide evidence for a positive role of insulin in transcription, translation, ion flux, insulin secretion and β‐cell survival.

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.

Removal of Ca2+ Channel β3 Subunit Enhances Ca2+ Oscillation Frequency and Insulin Exocytosis

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.

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.