Thomas M. Reinbothe

Insulin is secreted from the pancreatic β-cells in response to elevated glucose. In intact islets the capacity for insulin release is determined by a complex interplay between different cell types. This has made it difficult to specifically assess the role of β-cell defects to the insulin secretory impairment in type 2 diabetes. Here we describe a new approach, based on optogenetics, that enables specific investigation of β-cells in intact islets. We used transgenic mice expressing the light-sensitive cation channel Channelrhodopsin-2 (ChR2) under control of the insulin promoter. Glucose tolerance in vivo was assessed using intraperitoneal glucose tolerance tests, and glucose-induced insulin release was measured from static batch incubations. ChR2 localization was determined by fluorescence confocal microscopy. The effect of ChR2 stimulation with blue LED light was assessed using Ca2+ imaging and static islet incubations. Light stimulation of islets from transgenic ChR2 mice triggered prompt increases in ...

Inappropriate surface expression of voltage-gated Ca 2+ channels (Ca V ) in pancreatic ß-cells may contribute to the development of type 2 diabetes. First, failure to increase intracellular Ca 2+ concentrations at the sites of exocytosis impedes insulin release. Furthermore, excessive Ca 2+ influx may trigger cytotoxic effects. The regulation of surface expression of Ca V channels in the pancreatic β-cells remains unknown. Here, we used real-time 3D confocal and TIRFM imaging, immunocytochemistry, cellular fractionation, immunoprecipitation and electrophysiology to study trafficking of L-type Ca V 1.2 channels upon β-cell stimulation. We found decreased surface expression of Ca V 1.2 and a corresponding reduction in L-type whole-cell Ca 2+ currents in insulin-secreting INS-1 832/13 cells upon protracted (15–30 min) stimulation. This internalization occurs by clathrin-dependent endocytosis and could be prevented by microtubule or dynamin inhibitors. eIF3e (Eukaryotic translation initiation factor 3 subunit E) is part of the protein translation initiation complex, but its effect on translation are modest and effects in ion channel trafficking have been suggested. The factor interacted with Ca V 1.2 and regulated Ca V 1.2 traffic bidirectionally. eIF3e silencing impaired Ca V 1.2 internalization, which resulted in an increased intracellular Ca 2+ load upon stimulation. These findings provide a mechanism for regulation of L-type Ca V channel surface expression with consequences for β-cell calcium homeostasis, which will affect pancreatic β-cell function and insulin production.

Increasing evidence shows that hearing loss is a risk factor for tinnitus and hyperacusis. Although both often coincide, a causal relationship between tinnitus and hyperacusis has not been shown. Currently, tinnitus and hyperacusis are assumed to be caused by elevated responsiveness in subcortical circuits. We examined both the impact of different degrees of cochlear damage and the influence of stress priming on tinnitus induction. We used (1) a behavioral animal model for tinnitus designed to minimize stress, (2) ribbon synapses in inner hair cells (IHCs) as a measure for deafferentation, (3) the integrity of auditory brainstem responses (ABR) to detect differences in stimulus-evoked neuronal activity, (4) the expression of the activity-regulated cytoskeletal protein, Arc, to identify long-lasting changes in network activity within the basolateral amygdala (BLA), hippocampal CA1, and auditory cortex (AC), and (5) stress priming to investigate the influence of corticosteroid on trauma-induced brain responses. We observed that IHC ribbon loss (deafferentation) leads to tinnitus when ABR functions remain reduced and Arc is not mobilized in the hippocampal CA1 and AC. If, however, ABR waves are functionally restored and Arc is mobilized, tinnitus does not occur. Both central response patterns were found to be independent of a profound threshold loss and could be shifted by the corticosterone level at the time of trauma. We, therefore, discuss the findings in the context of a history of stress that can trigger either an adaptive or nonadaptive brain response following injury.

Voltage-gated calcium channels of the L-type have been shown to be essential for rodent pancreatic beta cell function, but data about their presence and regulation in humans are incomplete. We therefore sought to elucidate which L-type channel isoform is functionally important and its association with inherited diabetes-related phenotypes.

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.

Ratting Out a Diabetes Gene: Inbred animals with inherited susceptibility to disease can be especially informative regarding pathogenetic mechanisms because they carry naturally occurring genetic variants of the same type that cause disease in humans. This principle is illustrated byRosengrenet al.(p.217; published online 19 November), whose analysis of an inbred strain of rats prone to develop type 2 diabetes led to the discovery of a gene whose aberrant overexpression suppresses pancreatic insulin secretion in both rats and humans. The culprit gene, ADRA2A, encodes the alpha2A adrenergic receptor and is potentially a valuable lead for diabetes therapy because it can be targeted pharmacologically.

Nicotinamide adenine dinucleotide phosphate (NADPH) enhances Ca²⁺-induced exocytosis in pancreatic β-cells, an effect suggested to involve the cytosolic redox protein glutaredoxin-1 (GRX-1). We here detail the role of GRX-1 in NADPH-stimulated β-cell exocytosis and glucose-stimulated insulin secretion. Silencing of GRX-1 by RNA interference reduced glucose-stimulated insulin secretion in both clonal INS-1 832/13 cells and primary rat islets. GRX-1 silencing did not affect cell viability or the intracellular redox environment, suggesting that GRX-1 regulates the exocytotic machinery by a local action. By contrast, knockdown of the related protein thioredoxin-1 (TRX-1) was ineffective. Confocal immunocytochemistry revealed that GRX-1 locates to the cell periphery, whereas TRX-1 expression is uniform. These data suggest that the distinct subcellular localizations of TRX-1 and GRX-1 result in differences in substrate specificities and actions on insulin secretion. Single-cell exocytosis was likewise suppressed by GRX-1 knockdown in both rat β-cells and clonal 832/13 cells, whereas after overexpression exocytosis increased by approximately 40%. Intracellular addition of NADPH (0.1 mm) stimulated Ca²⁺-evoked exocytosis in both cell types. Interestingly, the stimulatory action of NADPH on the exocytotic machinery coincided with an approximately 30% inhibition in whole-cell Ca²⁺ currents. After GRX-1 silencing, NADPH failed to amplify insulin release but still inhibited Ca²⁺ currents in 832/13 cells. In conclusion, NADPH stimulates the exocytotic machinery in pancreatic β-cells. This effect is mediated by the NADPH acceptor protein GRX-1 by a local redox reaction that accelerates β-cell exocytosis and, in turn, insulin secretion.