Steinunn Baekkeskov

Pancreatic beta cells synthesize and secrete the neurotransmitter GABA (γ-aminobutyric acid) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory-vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here, we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel, functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from patients with type 1 and type 2 diabetes, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.

Scientific Reports 7: Article number: 45961; published online: 12 April 2017; updated: 09 May 2017 This Article contains a typographical error in the Methods section under subheading ‘Data Availability. Associated Article’, “Protocol Exchange doi: 1038/Protex.2017.039 (2017)”. should read: “ProtocolExchange doi: 10.

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells.

The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D.

Pancreatic islet β-cells are particularly susceptible to endoplasmic reticulum (ER) stress, which is implicated in β-cell dysfunction and loss during the pathogenesis of type 1 diabetes (T1D). The peripheral membrane protein GAD65 is an autoantigen in human T1D. GAD65 synthesizes γ-aminobutyric acid, an important autocrine and paracrine signaling molecule and a survival factor in islets. We show that ER stress in primary β-cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in trans-Golgi membranes. The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen-presenting cells and T-cell stimulation compared with the nonpalmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human β-cells in pancreatic sections from GAD65 autoantibody-positive individuals who have not yet progressed to clinical onset of T1D and from patients with T1D with residual β-cell mass and ongoing T-cell infiltration of islets. We propose that aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system after release from stressed and/or damaged β-cells, triggering autoimmunity.

The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.

The inhibitory neurotransmitter γ-amino butyric acid (GABA) is synthesized by two isoforms of the enzyme glutamic acid decarboxylase (GAD): GAD65 and GAD67. Whereas GAD67 is constitutively active and produces >90% of GABA in the central nervous system, GAD65 is transiently activated and augments GABA levels for rapid modulation of inhibitory neurotransmission. Hydrophobic lipid modifications of the GAD65 protein target it to Golgi membranes and synaptic vesicles in neuroendocrine cells. In contrast, the GAD67 protein remains hydrophilic but has been shown to acquire membrane association by heterodimerization with GAD65. Here, we identify a second mechanism that mediates robust membrane anchoring, axonal targeting, and presynaptic clustering of GAD67 but that is independent of GAD65. This mechanism is abolished by a leucine-103 to proline mutation that changes the conformation of the N-terminal domain but does not affect the GAD65-dependent membrane anchoring of GAD67. Thus two distinct mechanisms target the constitutively active GAD67 to presynaptic clusters to facilitate accumulation of GABA for rapid delivery into synapses.