α-Synuclein promotes IAPP fibril formation in vitro and ß-cell amyloid formation in vivo in mice.

Type 2 diabetes (T2D), alike Parkinson's disease (PD), belongs to the group of protein misfolding diseases (PMDs), which share aggregation of misfolded proteins as a hallmark. Although the major aggregating peptide in ß-cells of T2D patients is Islet Amyloid Polypeptide (IAPP), alpha-synuclein (αSyn), the aggregating peptide in substantia nigra neurons of PD patients, is expressed also in ß-cells. Here we show that αSyn, encoded by Snca, is a component of amyloid extracted from pancreas of transgenic mice overexpressing human IAPP (denoted hIAPPtg mice) and from islets of T2D individuals. Notably, αSyn dose-dependently promoted IAPP fibril formation in vitro and tail-vein injection of αSyn in hIAPPtg mice enhanced ß-cell amyloid formation in vivo whereas ß-cell amyloid formation was reduced in hIAPPtg mice on a Snca -/- background. Taken together, our findings provide evidence that αSyn and IAPP co-aggregate both in vitro and in vivo, suggesting a role for αSyn in ß-cell amyloid formation.

PAN-AMPK activator O304 improves glucose homeostasis and microvascular perfusion in mice and type 2 diabetes patients.

AMPK activated protein kinase (AMPK), a master regulator of energy homeostasis, is activated in response to an energy shortage imposed by physical activity and caloric restriction. We here report on the identification of PAN-AMPK activator O304, which - in diet-induced obese mice - increased glucose uptake in skeletal muscle, reduced ß cell stress, and promoted ß cell rest. Accordingly, O304 reduced fasting plasma glucose levels and homeostasis model assessment of insulin resistance (HOMA-IR) in a proof-of-concept phase IIa clinical trial in type 2 diabetes (T2D) patients on Metformin. T2D is associated with devastating micro- and macrovascular complications, and O304 improved peripheral microvascular perfusion and reduced blood pressure both in animals and T2D patients. Moreover, like exercise, O304 activated AMPK in the heart, increased cardiac glucose uptake, reduced cardiac glycogen levels, and improved left ventricular stroke volume in mice, but it did not increase heart weight in mice or rats. Thus, O304 exhibits a great potential as a novel drug to treat T2D and associated cardiovascular complications.

Imaging dynamics of CD11c⁺ cells and Foxp3⁺ cells in progressive autoimmune insulitis in the NOD mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: The aim of this study was to visualise the dynamics and interactions of the cells involved in autoimmune-driven inflammation in type 1 diabetes. METHODS: We adopted the anterior chamber of the eye (ACE) transplantation model to perform non-invasive imaging of leucocytes infiltrating the endocrine pancreas during initiation and progression of insulitis in the NOD mouse. Individual, ACE-transplanted islets of Langerhans were longitudinally and repetitively imaged by stereomicroscopy and two-photon microscopy to follow fluorescently labelled leucocyte subsets. RESULTS: We demonstrate that, in spite of the immune privileged status of the eye, the ACE-transplanted islets develop infiltration and beta cell destruction, recapitulating the autoimmune insulitis of the pancreas, and exemplify this by analysing reporter cell populations expressing green fluorescent protein under the Cd11c or Foxp3 promoters. We also provide evidence that differences in morphological appearance of subpopulations of infiltrating leucocytes can be correlated to their distinct dynamic behaviour. CONCLUSIONS/INTERPRETATION: Together, these findings demonstrate that the kinetics and dynamics of these key cellular components of autoimmune diabetes can be elucidated using this imaging platform for single cell resolution, non-invasive and repetitive monitoring of the individual islets of Langerhans during the natural development of autoimmune diabetes.

Retinoic acid promotes the generation of pancreatic endocrine progenitor cells and their further differentiation into beta-cells.

The identification of secreted factors that can selectively stimulate the generation of insulin producing beta-cells from stem and/or progenitor cells represent a significant step in the development of stem cell-based beta-cell replacement therapy. By elucidating the molecular mechanisms that regulate the generation of beta-cells during normal pancreatic development such putative factors may be identified. In the mouse, beta-cells increase markedly in numbers from embryonic day (e) 14.5 and onwards, but the extra-cellular signal(s) that promotes the selective generation of beta-cells at these stages remains to be identified. Here we show that the retinoic acid (RA) synthesizing enzyme Raldh1 is expressed in developing mouse and human pancreas at stages when beta-cells are generated. We also provide evidence that RA induces the generation of Ngn3(+) endocrine progenitor cells and stimulates their further differentiation into beta-cells by activating a program of cell differentiation that recapitulates the normal temporal program of beta-cell differentiation.

BMP4-BMPR1A signaling in beta cells is required for and augments glucose-stimulated insulin secretion.

Impaired glucose-stimulated insulin secretion (GSIS) and perturbed proinsulin processing are hallmarks of beta cell dysfunction in type 2 diabetes. Signals that can preserve and/or enhance beta cell function are therefore of great therapeutic interest. Here we show that bone morphogenetic protein 4 (Bmp4) and its high-affinity receptor, Bmpr1a, are expressed in beta cells. Mice with attenuated BMPR1A signaling in beta cells show decreased expression of key genes involved in insulin gene expression, proinsulin processing, glucose sensing, secretion stimulus coupling, incretin signaling, and insulin exocytosis and develop diabetes due to impaired insulin secretion. We also show that transgenic expression of Bmp4 in beta cells enhances GSIS and glucose clearance and that systemic administration of BMP4 protein to adult mice significantly stimulates GSIS and ameliorates glucose tolerance in a mouse model of glucose intolerance. Thus, BMP4-BMPR1A signaling in beta cells plays a key role in GSIS.

Dendritic cells in human thymus and periphery display a proinsulin epitope in a transcription-dependent, capture-independent fashion.

The natural expression of tissue-specific genes in the thymus, e.g., insulin, is critical for self-tolerance. The transcription of tissue-specific genes is ascribed to peripheral Ag-expressing (PAE) cells, which discordant studies identified as thymic epithelial cells (TEC) or CD11c+ dendritic cells (DC). We hypothesized that, consistent with APC function, PAE-DC should constitutively display multiple self-epitopes on their surface. If recognized by Abs, such epitopes could help identify PAE cells to further define their distribution, nature, and function. We report that selected Abs reacted with self-epitopes, including a proinsulin epitope, on the surface of CD11c+ cells. We find that Proins+ CD11c+ PAE cells exist in human thymus, spleen, and also circulate in blood. Human thymic Proins+ cells appear as mature DC but express CD8alpha, CD20, CD123, and CD14; peripheral Proins+ cells appear as immature DC. However, DC derived in vitro from human peripheral blood monocytes include Proins+ cells that uniquely differentiate and mature into thymic-like PAE-DC. Critically, we demonstrate that human Proins+ CD11c+ cells transcribe the insulin gene in thymus, spleen, and blood. Likewise, we show that mouse thymic and peripheral CD11c+ cells transcribe the insulin gene and display the proinsulin epitope; moreover, by using knockout mice, we show that the display of this epitope depends upon insulin gene transcription and is independent of Ag capturing. Thus, we propose that PAE cells include functionally distinct DC displaying self-epitopes through a novel, transcription-dependent mechanism. These cells might play a role in promoting self-tolerance, not only in the thymus but also in the periphery.

Derivation, characterization, and differentiation of human embryonic stem cells.

The derivation of human embryonic stem (hES) cells establishes a new avenue to approach many issues in human biology and medicine for the first time. To meet the increased demand for characterized hES cell lines, we present the derivation and characterization of six hES cell lines. In addition to the previously described immunosurgery procedure, we were able to propagate the inner cell mass and establish hES cell lines from pronase-treated and hatched blastocysts. The cell lines were extensively characterized by expression analysis of markers characteristic for undifferentiated and differentiated hES cells, karyotyping, telomerase activity measurement, and pluripotency assays in vitro and in vivo. Whereas three of the cell lines expressed all the characteristics of undifferentiated pluripotent hES cells, one cell line carried a chromosome 13 trisomy while maintaining an undifferentiated pluripotent state, and two cell lines, one of which carried a triploid karyotype, exhibited limited pluripotency in vivo. Furthermore, we clonally derived one cell line, which could be propagated in an undifferentiated pluripotent state.

The impact of smoking on inhaled insulin.

OBJECTIVE: This study, one of the first to address issues of pulmonary insulin delivery in smokers, compared pharmacokinetics of inhaled insulin delivered via the AERx insulin Diabetes Management System (iDMS) in nondiabetic cigarette smokers and nonsmokers. RESEARCH DESIGN AND METHODS: In this randomized two-period crossover efficacy and safety trial in 27 nondiabetic smokers and 16 nonsmokers (18 men/25 women, mean age 28 years, mean BMI 23.0 kg/m(2)), subjects received single doses of inhaled insulin (33.8 IU) following overnight fasting on consecutive dosing days. On one dosing day, smokers smoked three cigarettes immediately before insulin administration ("acute smoking"); on the other dosing day, smokers had not smoked since midnight ("nonacute smoking"). After inhalation, 6-h serum insulin and serum glucose profiles were determined. RESULTS: Pharmacokinetic results for evaluable subjects were derived from serum insulin profiles. The amount of insulin absorbed during the first 6 h after dosing (area under the exogenous serum insulin curve from 0 to 6 h [AUC((0-6 h))]) was significantly greater in smokers (63.2 vs. 40.0 mU l(-1) x h(-1), P = 0.0017); peak concentration was both higher and earlier in the smokers (maximal serum concentration of insulin [C(max)] 42.0 vs. 13.9 mU/l, P < 0.0001; time to maximal serum concentration of insulin [t(max)] 31.5 vs. 53.9 min, P = 0.0003). The estimated intrasubject variability of AUC((0-6 h)) was 13.7 and 16.5% for nonsmokers and smokers, respectively. No safety issues arose. CONCLUSIONS: Absorption of inhaled insulin via the AERx iDMS was significantly greater in smokers, with a higher AUC((0-6 h)) and C(max) and a shorter t(max). Intrasubject variability of AUC((0-6 h)) was low and similar in nonsmokers and smokers. These data prompt more extensive investigation of inhaled insulin in diabetic smokers.

Genetic dissection of cadherin function during nephrogenesis.

The distinct expression of R-cadherin in the induced aggregating metanephric mesenchyme suggests that it may regulate the mesenchymal-epithelial transition during kidney development. To address whether R-cadherin is required for kidney ontogeny, R-cadherin-deficient mice were generated. These mice appeared to be healthy and were fertile, demonstrating that R-cadherin is not essential for embryogenesis. The only kidney phenotype of adult mutant animals was the appearance of dilated proximal tubules, which was associated with an accumulation of large intracellular vacuoles. Morphological analysis of nephrogenesis in R-cadherin(-/-) mice in vivo and in vitro revealed defects in the development of both ureteric bud-derived cells and metanephric mesenchyme-derived cells. First, the morphology and organization of the proximal parts of the ureteric bud epithelium were altered. Interestingly, these morphological changes correlated with an increased rate of apoptosis and were further supported by perturbed branching and patterning of the ureteric bud epithelium during in vitro differentiation. Second, during in vitro studies of mesenchymal-epithelial conversion, significantly fewer epithelial structures developed from R-cadherin(-/-) kidneys than from wild-type kidneys. These data suggest that R-cadherin is functionally involved in the differentiation of both mesenchymal and epithelial components during metanephric kidney development. Finally, to investigate whether the redundant expression of other classic cadherins expressed in the kidney could explain the rather mild kidney defects in R-cadherin-deficient mice, we intercrossed R-cadherin(-/-) mice with cadherin-6(-/-), P-cadherin(-/-), and N-cadherin(+/-) mice. Surprisingly, however, in none of the compound knockout strains was kidney development affected to a greater extent than within the individual cadherin knockout strains.