Serotonin regulates glucose-stimulated insulin secretion from pancreatic β cells during pregnancy

In preparation for the metabolic demands of pregnancy, β cells in the maternal pancreatic islets increase both in number and in glucose-stimulated insulin secretion (GSIS) per cell. Mechanisms have been proposed for the increased β cell mass, but not for the increased GSIS. Because serotonin production increases dramatically during pregnancy, we tested whether flux through the ionotropic 5-HT3 receptor (Htr3) affects GSIS during pregnancy. Pregnant Htr3a^−/− mice exhibited impaired glucose tolerance despite normally increased β cell mass, and their islets lacked the increase in GSIS seen in islets from pregnant wild-type mice. Electrophysiological studies showed that activation of Htr3 decreased the resting membrane potential in β cells, which increased Ca²⁺ uptake and insulin exocytosis in response to glucose. Thus, our data indicate that serotonin, acting in a paracrine/autocrine manner through Htr3, lowers the β cell threshold for glucose and plays an essential role in the increased GSIS of pregnancy.Pregnancy places unique demands on the metabolism of the mother. As the pregnancy progresses and the nutrient requirements of the fetus increase, rising levels of placental hormones reduce maternal insulin sensitivity, thereby maintaining the maternal/fetal gradient of glucose and the flow of nutrients to the fetus. The mother balances the resulting increase in insulin demand with structural and functional changes in the islets that generate increased and hyperdynamic insulin secretion. β cell numbers increase, the threshold for glucose decreases, and glucose-stimulated insulin secretion (GSIS) increases (1–3). Failure to reach this balance of insulin demand with insulin production results in gestational diabetes (4).However, the changes in the maternal islets are not simply a response to increased insulin demand, as they precede the development of insulin resistance. Instead, these changes correlate more closely with levels of circulating maternal lactogens (prolactin and placental lactogen) that signal through the prolactin receptor on the β cell (5–9). Downstream of the prolactin receptor, multiple pathway components have been identified that contribute to the maternal increase in β cell mass (10–16), but not the changes in GSIS.In response to the lactogen signaling during pregnancy, levels of both isoforms of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of serotonin (5-hydroxytryptamine; 5-HT), rise dramatically in the islet (13, 17, 18). Islet serotonin acts in an autocrine/paracrine manner through the Gα_q-coupled serotonin receptor 5-HT2b receptor (Htr2b) to increase β cell proliferation and mass at midgestation and through Gα_i-coupled 5-HT1d receptor (Htr1d) to reduce β cell mass at the end of gestation (13). These dynamic changes in 5-HT receptor (Htr) expression can explain the shifts in β cell proliferation during pregnancy.In addition to Htr2b and Htr1d, β cells also express Htr3a and Htr3b (13). Unlike the 12 other Htr genes in the mouse genome, which encode G-protein coupled serotonin receptors, Htr3a and Htr3b encode subunits of the serotonin-gated cation channel Htr3 (19, 20). Five identical Htr3a subunits or a mixture of Htr3a and Htr3b make up a functional Htr3 channel (21). The channel is predominantly Na⁺- and K⁺-selective, and its opening in response to serotonin actives an inward current and depolarizes the cell membrane (22, 23). Glucose also depolarizes β cells: Rising ATP from glucose catabolism depolarizes the cell by closing ATP-sensitive K⁺ channels, which causes Ca²⁺ to enter the cell through voltage-gated Ca²⁺ channels and trigger insulin granule exocytosis (24).Therefore, we tested the possibility that Htr3 may regulate β cell insulin secretion during pregnancy. We found that lactogen-induced serotonin in the pregnant islet acts through Htr3 to depolarize β cells, thereby lowering the threshold for glucose and enhancing GSIS during pregnancy.     

Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs

β cells produce, store, and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of β cell function is mostly derived from studies of ex vivo isolated islets in rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo–labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

Dysfunction of pancreatic islet β cells is a key contributor to type 2 diabetes mellitus (T2DM) (1, 2), starting in the early onset of the disease (3). Each β cell contains several thousand insulin secretory granules (SGs) (4, 5). However, only a small percentage of insulin SGs undergo exocytosis upon glucose stimulation (6). Insulin is secreted in two phases: a rapid first and a sustained second phase (7–9). On the level of insulin SGs, our understanding of insulin secretion has been shaped by two basic concepts: 1) the recruitment of SG pools defined by their spatial confinement in the cell and 2) the higher probability of young SGs for exocytosis. In model 1), the so-called, readily releasable pool consists of SGs that are already docked with the plasma membrane and are released immediately upon glucose stimulation, thereby creating the first rapid phase of insulin secretion (10). The second prolonged phase is then caused by the recruitment of SGs from the reserve pool, which is located deeper inside the β cell (6). The detailed properties of SGs of the different pools have been debated and refined recently (11). Additionally, data obtained by radio-labeling experiments suggest that young insulin SGs are preferentially secreted (12, 13). A method that allows for the visualization of age-defined pools of the desired protein is to fuse it with the SNAP-tag, a 20-kDa protein tag that reacts covalently in a bioorthogonal manner with fluorescent benzylguanine (BG)-fused substrates in living cells and organisms (14). By using a pulse–chase-labeling approach to track SGs containing an insulin-SNAP chimera, we could confirm the preferential exocytosis of young SGs and also show the preferential intracellular degradation of old SGs (15–17). Furthermore, we found that in insulinoma INS-1 cells a pool of young SGs travels fast on microtubules, while this property is lost for old SGs (18). Young insulin SGs additionally have a more acidic luminal pH compared to old ones (19). Addressing the heterogeneity of insulin SGs and their differential reaction to stimuli and pharmaceutical intervention poses possibilities for the treatment of T2DM. Genetically modified mouse models have been the method of choice to investigate intracellular signaling, as well as metabolism, in diabetes research. Recently, transgenic pigs have been made available that allow for conducting β cell research in a context even closer to humans (20).Here, we describe the generation and characterization of a transgenic pig model with the SNAP-tag fused to insulin, called the Study OF Insulin granule Aging (SOFIA) pig. We demonstrate the correct targeting and processing of insulin-SNAP to insulin SGs. Finally, we show successful in vivo labeling with one and two SNAP-substrates staining pancreatic islets and distinct insulin SG pools. In summary, our pig model is a valuable system enabling the imaging-based investigation of insulin SG turnover in a large living mammal.     

Human islet T cells are highly reactive to preproinsulin in type 1 diabetes

Cytotoxic CD8 T lymphocytes play a central role in the tissue destruction of many autoimmune disorders. In type 1 diabetes (T1D), insulin and its precursor preproinsulin are major self-antigens targeted by T cells. We comprehensively examined preproinsulin specificity of CD8 T cells obtained from pancreatic islets of organ donors with and without T1D and identified epitopes throughout the entire preproinsulin protein and defective ribosomal products derived from preproinsulin messenger RNA. The frequency of preproinsulin-reactive T cells was significantly higher in T1D donors than nondiabetic donors and also differed by individual T1D donor, ranging from 3 to over 40%, with higher frequencies in T1D organ donors with HLA-A*02:01. Only T cells reactive to preproinsulin-related peptides isolated from T1D donors demonstrated potent autoreactivity. Reactivity to similar regions of preproinsulin was also observed in peripheral blood of a separate cohort of new-onset T1D patients. These findings have important implications for designing antigen-specific immunotherapies and identifying individuals that may benefit from such interventions.

Type 1 diabetes (T1D) results from the chronic immune-mediated targeting of insulin-producing beta cells within pancreatic islets with T cells playing a central role in disease pathogenesis (1, 2). Tissue specificity and a strong genetic association with the human leukocyte antigen (HLA) locus suggest that antigen specificity is necessary for T cells to attack beta cells and induce T1D (3). Therefore, considerable efforts have been undertaken to identify antigens for disease-associated T cells in order to understand the disorder’s pathogenesis and develop therapies to prevent T1D (4–8). As such, many large well-controlled clinical trials evaluating antigen-specific immunotherapies have been conducted (e.g., oral, intranasal, subcutaneous, and DNA vaccines), especially with a focus on insulin- or preproinsulin-related epitopes. Unfortunately, none have achieved favorable clinical outcomes to date (9–14). However, several trials have identified subsets of responders who may potentially benefit from antigen-specific immunotherapy (13–18), thus suggesting the potential for heterogeneity of antigen specificity targeted by the adaptive immune system in individual T1D patients. Indeed, a recent report from the Environmental Determinants of Diabetes in the Young study demonstrated a trend of first appearing islet autoantibodies classified by HLA haplotypes (19), implicating that individual patients may have different antigen specificities that initiate and drive T1D development. To improve prevention efforts and direct tissue- and autoantigen-specific immunotherapy, an improved understanding of epitopes that activate T cells within inflamed pancreatic islets and subsequent reactivity in the peripheral blood is needed to optimally select patients for these therapies.While the contribution of both CD4 and CD8 T cells to T1D development is evident, several lines of evidence have highlighted the importance of CD8 T cells as mediators of disease pathogenesis. First, CD8 T cells predominate within the immune infiltrates of inflamed pancreatic islets from T1D organ donors (7, 20). Second, as HLA class I molecules present peptides to activate CD8 T cells, beta cells within inflamed T1D islets up-regulate HLA class I molecules, which has the potential to enhance CD8 T cell−beta cell–specific interactions (20, 21). Finally, islet-specific CD8 T cells have been measured in the peripheral blood of T1D patients using fluorescent peptide/major histocompatibility complex (MHC) multimers (22, 23), with some of these specificities correlating to the functional loss of insulin secretion following the clinical onset of T1D (24, 25).Fluorescent peptide/MHC multimer reagents have previously been used to identify self-reactive CD8 T cells within human T1D islets by in situ staining frozen pancreas sections (20, 26–28). These studies focused on islet peptides presented by the HLA-A2 variant, as it is most frequent in both T1D and the general population, with estimated frequencies of 50 to 70% and 20 to 40%, respectively (29). While these assays using peptide/MHC reagents efficiently detect antigen-specific T cells, they are biased toward limited epitopes and HLA usage and sometimes cannot discriminate T1D patients from nondiabetic controls, especially when staining T cells from the peripheral blood (26, 30–32). Thus, a compelling need exists to comprehensively examine CD8 T cell antigen specificity across various HLA class I molecules within human pancreatic islets. Therefore, in this study, we analyzed the antigen specificity from hundreds of CD8 T cells obtained from the islets of both T1D and nondiabetic organ donors without bias for HLA types and with minimal ex vivo amplification. Herein, we provide evidence that islet-infiltrating CD8 T cells reactive to a major self-antigen, insulin and its precursor preproinsulin, are abundant in three of seven T1D organ donors but not in nondiabetic donors. Preproinsulin-reactive CD8 T cell epitopes are presented by various HLA class I molecules, including epitopes spread throughout the entire preproinsulin protein with three hot spots, and elicit reactivity from the peripheral blood in a separate cohort of newly diagnosed T1D patients.     

Synaptotagmin-7 phosphorylation mediates GLP-1–dependent potentiation of insulin secretion from β-cells

Glucose stimulates insulin secretion from β-cells by increasing intracellular Ca²⁺. Ca²⁺ then binds to synaptotagmin-7 as a major Ca²⁺ sensor for exocytosis, triggering secretory granule fusion and insulin secretion. In type-2 diabetes, insulin secretion is impaired; this impairment is ameliorated by glucagon-like peptide-1 (GLP-1) or by GLP-1 receptor agonists, which improve glucose homeostasis. However, the mechanism by which GLP-1 receptor agonists boost insulin secretion remains unclear. Here, we report that GLP-1 stimulates protein kinase A (PKA)-dependent phosphorylation of synaptotagmin-7 at serine-103, which enhances glucose- and Ca²⁺-stimulated insulin secretion and accounts for the improvement of glucose homeostasis by GLP-1. A phospho-mimetic synaptotagmin-7 mutant enhances Ca²⁺-triggered exocytosis, whereas a phospho-inactive synaptotagmin-7 mutant disrupts GLP-1 potentiation of insulin secretion. Our findings thus suggest that synaptotagmin-7 is directly activated by GLP-1 signaling and may serve as a drug target for boosting insulin secretion. Moreover, our data reveal, to our knowledge, the first physiological modulation of Ca²⁺-triggered exocytosis by direct phosphorylation of a synaptotagmin.Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells follows a biphasic time course consisting of an initial, transient first phase lasting 5–10 min followed by a slowly developing, sustained second phase (1). Type 2 diabetes (T2D) is associated with partial or complete loss of the first insulin secretion phase and a reduction in the second insulin secretion phase (2, 3). Incretins, especially GLP-1, boost GSIS in T2D patients, thereby improving glucose homeostasis (4). GLP-1 exerts its action by activating GLP-1R, a G-protein–coupled receptor expressed on the surface of β-cells, which leads to an increase of adenylate cyclase activity and production of cAMP. Elevated cAMP levels in β-cells enhance GSIS through PKA-dependent and -independent (mediated by Epac2) mechanisms (5, 6). Mouse models with constitutively increased PKA activity have established PKA’s predominant role in the GLP-1–induced potentiation of β-cell GSIS (7, 8), but the downstream effectors remain unidentified.Insulin is secreted in response to glucose by regulated exocytosis of insulin-containing secretory granules. Electrical activity leads to opening of plasmalemmal voltage-gated Ca²⁺ channels (VGCCs) on the β-cell plasma membrane; the resulting increase in [Ca²⁺]_i then triggers Ca²⁺-dependent exocytosis (9). Insulin granule exocytosis is mediated by a multiprotein complex composed of soluble SNAP-receptor (SNARE) proteins (SNAP-25, Syntaxin, and synaptobrevin-2) and Sec1/Munc18-like (SM) proteins (Munc18-1) by a process that shares similarities with synaptic vesicle exocytosis in neurons (10). To date, numerous SNARE isoforms have been implicated in GSIS (11, 12), including Syntaxin-1, Syntaxin-4, SNAP-25 or SNAP-23, and synaptobrevin-2/3 (or VAMP2/3), whereas VAMP8, a nonessential SNARE for GSIS, may be involved in the regulation of GLP-1 potentiation of insulin secretion (13).In addition to SNARE and SM proteins, a Ca²⁺ sensor is required to initiate membrane fusion during exocytosis. Synaptotagmins, expressed mainly in neurons and endocrine cells, share a similar domain structure: a short N-terminal domain, followed by a transmembrane domain, a linker region with variable length, and two tandem Ca²⁺-binding C₂ domains (C₂A and C₂B) at the C terminus (14, 15). Some synaptotagmins bind to phospholipids in a Ca²⁺-dependent manner and have been identified as major Ca²⁺ sensors for regulated exocytosis (14, 16). Synaptotagmin-1, -2, -7, and -9 function as Ca²⁺ sensors for neurotransmitter release, whereas synaptotagmin-1, -7 (Syt7), and -10 regulate hormone secretion and neuropeptide release (9, 17, 18). Specifically, Syt7 regulates insulin granule exocytosis in insulin-secreting cell lines (19, 20). Syt7 is highly expressed in human pancreatic β-cells, and Syt7 KO mice exhibit reduced insulin secretion and consequently impaired glucose tolerance following glucose stimulation (21–23). Collectively, these studies demonstrate that Syt7 is a major Ca²⁺ sensor mediating GSIS in β-cells.Given that GLP-1 potentiates insulin secretion in a glucose-dependent manner, it is highly likely that its insulinotropic action is exerted distally to the initiation of electrical activity, possibly at the level of Ca²⁺ sensing and membrane fusion. Here we report that Syt7 is a stoichiometric substrate for PKA and functions as a downstream target of PKA activated by GLP-1 signaling. Compared with wild-type mice, Syt7 KO mice showed reduced insulin secretion ex vivo and in vivo in response to treatment with the GLP-1 analog exendin-4 in a manner that depended on Syt7 phosphorylation at serine-103. Our data not only provide a mechanism by which GLP-1 stimulates insulin secretion, but also report the physiological regulation of a synaptotagmin by phosphorylation.     

Molecular architecture of the αβ T cell receptor–CD3 complex

αβ T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3εγ, CD3εδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR–CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR–CD3 complex. Small-angle X-ray scattering of the soluble TCR–CD3εδ complex reveals the CD3εδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR–CD3 integral membrane complex, in which the CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR β-chain, respectively. Interestingly, the TCR–CD3 transmembrane complex bound to peptide–MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR–CD3 complex, and provides a conceptual framework for the TCR activation mechanism.T cells are key mediators of the adaptive immune response. Each αβ T cell contains a unique αβ T-cell receptor (TCR), which binds antigens (Ags) displayed by major histocompatibility complexes (MHCs) and MHC-like molecules (1). The TCR serves as a remarkably sensitive driver of cellular function: although TCR ligands typically bind quite weakly (1–200 μM), even a handful of TCR ligands are sufficient to fully activate a T cell (2, 3). The TCR does not possess intracellular signaling domains, uncoupling Ag recognition from T-cell signaling. The TCR is instead noncovalently associated with a multisubunit signaling apparatus, consisting of the CD3εγ and CD3εδ heterodimers and the CD3ζζ homodimer, which collectively form the TCR–CD3 complex (4, 5). The CD3γ/δ/ε subunits each consist of a single extracellular Ig domain and a single immunoreceptor tyrosine-based activation motif (ITAM), whereas CD3ζ has a short extracellular domain (ECD) and three ITAMs (6–11). The TCR–CD3 complex exists in 1:1:1:1 stoichiometry for the αβTCR:CD3εγ:CD3εδ:CD3ζζ dimers (12). Phosphorylation of the intracellular CD3 ITAMs and recruitment of the adaptor Nck lead to T-cell activation, proliferation, and survival (13, 14). Understanding the underlying principles of TCR–CD3 architecture and T-cell signaling is of therapeutic interest. For example, TCR–CD3 is the target of therapeutic antibodies such as the immunosuppressant OKT3 (15), and there is increasing interest in manipulating T cells in an Ag-dependent manner by using naturally occurring and engineered TCRs (16).Assembly of the TCR–CD3 complex is primarily driven by each protein’s transmembrane (TM) region, enforced through the interaction of evolutionarily conserved, charged, residues in each TM region (4, 5, 12). What, if any, role interactions between TCR and CD3 ECDs play in the assembly and function of the complex remains controversial (5): there are several plausible proposed models of activation, which are not necessarily mutually exclusive (5, 17–19). Although structures of TCR–peptide–MHC (pMHC) complexes (2), TCR–MHC-I–like complexes (1), and the CD3 dimers (6–10) have been separately determined, how the αβ TCR associates with the CD3 complex is largely unknown. Here, we use two independent structural approaches to gain an understanding of the TCR–CD3 complex organization and structure.     

Fetal endocannabinoids orchestrate the organization of pancreatic islet microarchitecture

Endocannabinoids are implicated in the control of glucose utilization and energy homeostasis by orchestrating pancreatic hormone release. Moreover, in some cell niches, endocannabinoids regulate cell proliferation, fate determination, and migration. Nevertheless, endocannabinoid contributions to the development of the endocrine pancreas remain unknown. Here, we show that α cells produce the endocannabinoid 2-arachidonoylglycerol (2-AG) in mouse fetuses and human pancreatic islets, which primes the recruitment of β cells by CB₁ cannabinoid receptor (CB₁R) engagement. Using subtractive pharmacology, we extend these findings to anandamide, a promiscuous endocannabinoid/endovanilloid ligand, which impacts both the determination of islet size by cell proliferation and α/β cell sorting by differential activation of transient receptor potential cation channel subfamily V member 1 (TRPV1) and CB₁Rs. Accordingly, genetic disruption of TRPV1 channels increases islet size whereas CB₁R knockout augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ω-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB₁R^−/− islet microstructure and improves coordinated hormone secretion. Overall, our data mechanistically link endocannabinoids to cell proliferation and sorting during pancreatic islet formation, as well as to life-long programming of hormonal determinants of glucose homeostasis.Anandamide (AEA) and 2-arachydonoylglycerol (2-AG), major endocannabinoids (eCBs), are involved in the regulation of energy homeostasis through coordinated actions in peripheral organs (adipose tissue, liver, and pancreas) and brain (hypothalamus, ventral striatum) (1). eCB signals are particularly significant to coordinate the regulated release of insulin and glucagon from mature pancreatic islets (2–6). Genetic evidence from CB₁ cannabinoid receptor^−/− (CB₁R^−/−) mice supports these findings because CB₁R^−/− mice are lean, resistant to high fat diet-induced obesity and diabetes (4, 7–9). Whether eCBs impact the formation of the endocrine pancreas and predispose it to long-lasting changes in hormone release postnatally remains unknown.Because eCBs broadly affect cell proliferation, fate, motility, and differentiation (e.g., in sperm, hematopoietic and T cells, and neurons) (10–13), it is likely that they play a role in the cellular organization of developing pancreatic islets, possibly by affecting the spatial segregation of α and β cells. A contribution of eCBs to cell diversification and positioning in the developing pancreas is supported by the temporal control of their levels in fetal tissues (14) and circulation (15). Moreover, α and β cells in mature pancreatic islets express the molecular machinery for eCB metabolism together with CB₁Rs and transient receptor potential cation channels, particularly subfamily V member 1 (TRPV1) (2, 16, 17). Understanding these developmental processes is also relevant to postnatal life because pancreatic α- and β-cell placement can be reconfigured upon metabolic demands in both rodents (18) and humans (19), altering the efficacy of endocrine responsiveness.Differential ligand and receptor recruitment within pleiotropic eCB signaling networks might facilitate a cellular context- and stage-dependent diversification of eCB signals. Thus, the coordinated availability of 2-AG and AEA and their varied action on CB₁Rs (20) and TRPV1 channels (21) are well-positioned to orchestrate progenitor proliferation and the survival, migration, and ability of hormone secretion from ensuing differentiated cell lineages. Here, we demonstrate that paracrine 2-AG signaling determines cell segregation via CB₁R-mediated adhesion signaling in the fetal mouse pancreas. In turn, chemical or genetic inactivation of TRPV1s on β cells increases cell proliferation both in vitro and in vivo, typically affecting the size of islets formed. Reducing eCB precursor bioavailability during pregnancy by ω-3 polyunsaturated fatty acid (PUFA) intake increases cellular heterogeneity and improves the temporal coordination of glucagon/insulin release, phenocopying CB₁R^−/− mice, as well as human islets (19). Cumulatively, our results outline a candidate mechanism for life-long cellular adaptation to metabolic challenges.     

A therapeutic convection–enhanced macroencapsulation device for enhancing β cell viability and insulin secretion

Islet transplantation for type 1 diabetes treatment has been limited by the need for lifelong immunosuppression regimens. This challenge has prompted the development of macroencapsulation devices (MEDs) to immunoprotect the transplanted islets. While promising, conventional MEDs are faced with insufficient transport of oxygen, glucose, and insulin because of the reliance on passive diffusion. Hence, these devices are constrained to two-dimensional, wafer-like geometries with limited loading capacity to maintain cells within a distance of passive diffusion. We hypothesized that convective nutrient transport could extend the loading capacity while also promoting cell viability, rapid glucose equilibration, and the physiological levels of insulin secretion. Here, we showed that convective transport improves nutrient delivery throughout the device and affords a three-dimensional capsule geometry that encapsulates 9.7-fold-more cells than conventional MEDs. Transplantation of a convection-enhanced MED (ceMED) containing insulin-secreting β cells into immunocompetent, hyperglycemic rats demonstrated a rapid, vascular-independent, and glucose-stimulated insulin response, resulting in early amelioration of hyperglycemia, improved glucose tolerance, and reduced fibrosis. Finally, to address potential translational barriers, we outlined future steps necessary to optimize the ceMED design for long-term efficacy and clinical utility.

Diabetes mellitus currently burdens over 387 million people worldwide, of which 5 to ∼10% are accounted by patients with type 1 diabetes (T1D) (1). T1D is characterized by the immune destruction of insulin-secreting β cells and the loss of glycemic regulation. Although intensive insulin injection regimens and the use of glucose monitors have been shown to effectively regulate blood glucose, patients are still unable to meet glycemic control targets. In particular, those with severe hypoglycemic events and glycemic lability cannot be effectively stabilized with these technologies (2). In 2000, the Edmonton protocol was developed as a procedure that directly infuses pancreatic islets, isolated from cadaveric donors, into the portal vein of T1D patients. This procedure led to insulin independence in patients for a short period postinfusion (3, 4). However, poor long-term graft survival due to alloimmune and autoimmune rejections and engraftment inefficiency prevents sustained, therapeutic effects (5–7). Although immunosuppressants are coadministered with the transplanted cells to prevent graft rejection, 56% of patients experience partial to complete graft loss after 1 y, and only 10% of patients remain insulin independent after 5 y (4, 8). The majority of patients also experience complications from immunosuppression, including elevated risk of opportunistic infections and cancer (4, 8, 9). In addition, islet transplantation is burdened by a major islet donor shortage, since often two or more human pancreases are needed to achieve a sufficient number of islets (10).The complications of immune rejection could be overcome with macroencapsulation devices (MEDs), in which glucose-sensing, insulin-secreting cell sources like pluripotent, stem cell–derived β clusters (SC-βCs) (11), or other islet sources, are transplanted within an immune-isolating vehicle to promote cell survival and function. In MEDs, islets are housed in a single compartment that selectively permits the exchange of nutrients while obstructing host immune effectors such as cells and antibodies. Over the past few decades, MEDs have successfully restored insulin independence and normoglycemia in T1D animal models (10). However, scaling these devices for human applications has been challenging. Currently, passive diffusion-based MEDs, including Encaptra, βAir Βio-Artificial Pancreas, Cell Pouch, and MAILPAN, are being explored in phase I/II clinical trials (12–14). Nevertheless, these diffusion-based devices still suffer from limitations in the transport of glucose, insulin, and other biomolecules to the core of these devices, which compromise the survival and function of encapsulated cells. Ultimately, these devices are restricted in geometry, thickness, and cell-loading capacity.More specifically, a significant portion of encapsulated cells become nonviable immediately after transplantation because of the lack of vascularization, which results in hypoxia and limited nutrient availability. Thus, during the initial prevascularization period, which lasts ∼14-d posttransplantation, solute exchange and insulin secretion cannot occur effectively using conventional MEDs (15–17). For this reason, many encapsulated cells prematurely lose their function and eventually die. Various strategies have been developed to expediate angiogenesis around the device, especially during the initial hypoxic period after device transplantation, to reduce cell loss. Examples include prevascularization of the device, infusion of vascular endothelial growth factor, and cotransplantation of mesenchymal stem cells (15, 18–21). In another instance, βAir Βio-Artificial Pancreas incorporated a daily refillable oxygen chamber in between two islet slabs to maintain adequate oxygen supply, but the chamber is 15- to 30-fold thicker than islet layers. Despite improvements in cell viability, these strategies still cannot guarantee adequate glucose sensing and insulin release kinetics of the islets, and they further limit the available space for cell packing (22).To supply cells with enough nutrients, it has been suggested that the islet density of the MEDs should be set to 5 to ∼10% of the volume fraction (23). Consequently, a limited mass of islets must be placed within a large device to ensure optimal nutrient distribution. Otherwise, devices exhibit extreme cell loss. For instance, TheraCyte, which packs 70 to ∼216 islet equivalent (IEQ) in 4.5 μL or 1,000 IEQ in 40 μL volume, exhibited poor cell survival (19). The remaining cells were neither capable of restoring euglycemia in rodents (1,000 to ∼2,000 IEQ required) nor sustaining a therapeutic dosage needed for humans (∼500,000 islets) in a reasonably sized device (13, 24–26).To overcome these nutrient delivery challenges and improve cell-loading capacity, we designed a convection-enhanced MED (ceMED) to perfuse the device continuously. We hypothesized that convective nutrient transport in ceMED would 1) deliver more nutrients compared with diffusion-based devices, 2) increase cell survival beyond the distance limit of diffusion, 3) support a three-dimensional (3D), expanded cell layer to increase the loading capacity, 4) improve glucose sensitivity and timely insulin secretion via faster biomolecule transport, and 5) show efficacy in vivo by reducing hyperglycemia before vascularization. Overall, we demonstrated that the convective motion promotes survival of insulin-secreting β cells encapsulated at high density. We also demonstrated that it effectively captures the dynamics of glucose concentrations in the transplantation site, resulting in more appropriate insulin secretion with faster on/off responses. Finally, the ceMED showed early, vascular-independent reduction in blood glucose levels in hyperglycemic rat models several days prior to the critical 14-d posttransplantation.     

Nanobodies and chemical cross-links advance the structural and functional analysis of PI3Kα

Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.

Class I phosphoinositol 3-kinases (PI3Ks) are a family of lipid kinases, each composed of a regulatory and a catalytic subunit. Class IA of PI3K consists of three isoforms of the catalytic subunit, p110α, p110β, and p110δ, that are bound to the regulatory subunit p85α or one of its isoforms, p55α, p50α, or p85β (1, 2). The dimer of p110α–p85α is the topic of this investigation and will be referred to as PI3Kα. Class IB contains a single isoform of the catalytic subunit, p110γ, which is associated with the regulatory subunit p101 or p84.PI3Ks control signaling events that are essential for cell growth and survival, and PI3K activity can be an important factor in tumor formation (2). Starting with the discovery of cancer-specific gain-of-function mutations, PI3Ks became prominent drug targets (3–5). Numerous PI3K inhibitors have been disclosed, but only five are currently approved for therapeutic use (6–10). The field of PI3K inhibitors has moved from so-called “panspecific” compounds toward greater target specificity. The Food and Drug Administration–approved PI3K inhibitors are isoform-specific or isoform-selective. However, since these inhibitors target the wild-type enzymes, toxicities are unavoidable and remain a major clinical problem (11–13). Another strategy is to develop inhibitors that are specific for the cancer-associated mutants of PI3K, and there are encouraging signs that this degree of specificity can be reached (14, 15). Novel chemical strategies that do not rely exclusively on ATP competition also appear promising (16–19). Mutant-specific immunotherapy is now also on the horizon (20).Detailed structural information could make an important contribution to these efforts. Crystallography, NMR, and hydrogen–deuterium exchange have provided valuable data on large parts but not all of the PI3Kα complex (6, 21–31). The efforts to achieve complete structural data of PI3Ks have recently been complemented by single-particle analysis in cryo–electron microscopy (cryo-EM) (32–34). For PI3Kα, conformational changes associated with inhibition and with activation have been defined (32). However, the positional flexibility of the regulatory subunit domains BH, SH3, and cSH2 prevented the identification of these domains and their positions in the cryo-EM analysis. We have therefore endeavored to use nanobodies and chemical cross-linking to achieve additional insights into the PI3Kα structure, notably the flexible domains. Nanobody binding and chemical cross-linking mass spectrometry (CXMS) provided the identity and docking data for the BH, SH3, and cSH2 domains of the regulatory subunit p85 and revealed information on mechanistic aspects of PI3K flexibility.     

CD11b regulates obesity-induced insulin resistance via limiting alternative activation and proliferation of adipose tissue macrophages

Obesity-associated inflammation is accompanied by the accumulation of adipose tissue macrophages (ATMs), which is believed to predispose obese individuals to insulin resistance. CD11b (integrin α_M) is highly expressed on monocytes and macrophages and is critical for their migration and function. We found here that high-fat diet–induced insulin resistance was significantly reduced in CD11b-deficient mice. Interestingly, the recruitment of monocytes to adipose tissue is impaired when CD11b is deficient, although the cellularity of ATMs in CD11b-deficient mice is higher than that in wild-type mice. We further found that the increase in ATMs is caused mainly by their vigorous proliferation in the absence of CD11b. Moreover, the proliferation and alternative activation of ATMs are regulated by the IL-4/STAT6 axis, which is inhibited by CD11b through the activity of phosphatase SHP-1. Thus, CD11b plays a critical role in obesity-induced insulin resistance by limiting the proliferation and alternative activation of ATMs.Obesity is associated with chronic inflammation characterized by progressive accumulation of immune cells in adipose tissue. Cytokines secreted by these immune cells, such as TNF-α, have been demonstrated to augment adipose tissue inflammation and consequentially induce insulin resistance (1, 2). As one of the major cell types that contribute to the proinflammatory response, macrophages are central players in obesity-related inflammation (3, 4). This heterogeneous cell population possesses broad plasticity that can be influenced by the local microenvironment. Changes in macrophage properties play distinct roles in regulating inflammation and also metabolic responses (5, 6). Classically activated macrophages (CAMs) secrete proinflammatory cytokines such as TNF-α, IL-6, and IL-1 (7). The accumulation of CAMs in adipose tissue exacerbates the development of obesity and subsequent tissue inflammation and insulin resistance. On the other hand, resident macrophages in adipose tissue of lean mice display a phenotype of alternatively activated macrophages (AAMs) with high expression of IL-10, Ym1/chitinase3-like3, and arginase1 (8). It has been reported that AAMs driven by IL-4 and IL-13 could improve insulin sensitivity (9, 10). Thus, differential accumulation of these two macrophage populations could result in distinct metabolic states and could influence the progression of insulin resistance.Traditionally, the accumulation of macrophages is considered as the migration of monocytes to inflammatory sites and subsequent differentiation into macrophages (5). One of the molecules that control monocytes immigration is integrin α_M (CD11b), which combines with integrin β₂ (CD18) to form MAC-1 (integrin α_Mβ₂). MAC-1 is well known for its role in regulating leukocyte transmigration through endothelial cells (11, 12). The function of CD11b is dependent on a cascade of inside–out and outside–in activation signaling processes. The activation of tyrosine kinases, especially members of the Src family, is indispensable for the outside–in signaling of different integrins (13). In addition to its role in regulating leukocyte adhesion and migration, CD11b has been reported to modulate various aspects of immune responses. Our previous studies showed that CD11b on antigen-presenting cells plays a critical role in promoting oral tolerance by inhibiting Th17 differentiation (14). Moreover, CD11b also has been shown to suppress Toll-like receptor–initiated signals in macrophages and thus to protect animals from endotoxic shock (15). Interestingly, activation of CD11b impedes the accumulation of lipid in macrophages and the formation of foam cells in the presence of IL-13 (16). Its negative regulation of the immune response also can be observed in B cells, because CD11b has been shown to inhibit the autoreactive B-cell response in systemic lupus erythematous (17). Importantly, CD11b is found to be involved in regulating body fat deposition (18); however, the effect of CD11b in obesity-induced insulin resistance has not been reported. In the present study, we show that CD11b is important for the influx of monocytes to adipose tissue during the development of obesity. Surprisingly, the accumulation of macrophages in the adipose tissue of CD11b-deficient mice is significantly increased compared with that in wild-type mice. We further demonstrated that CD11b deficiency promotes in situ proliferation of adipose tissue macrophages (ATMs), a process mediated by the IL-4/STAT6 signaling pathway. The infiltrated ATMs in CD11b-deficient mice phenotypically resemble AAMs. Depletion of these ATMs reversed the reduction of insulin resistance. Thus, our studies revealed a previously unidentified role of CD11b in regulating macrophage cellularity and function in adipose tissue and insulin resistance.     

Cross-talk between amyloidogenic proteins in type-2 diabetes and Parkinson’s disease

In type-2 diabetes (T2D) and Parkinson’s disease (PD), polypeptide assembly into amyloid fibers plays central roles: in PD, α-synuclein (aS) forms amyloids and in T2D, amylin [islet amyloid polypeptide (IAPP)] forms amyloids. Using a combination of biophysical methods in vitro we have investigated whether aS, IAPP, and unprocessed IAPP, pro-IAPP, polypeptides can cross-react. Whereas IAPP forms amyloids within minutes, aS takes many hours to assemble into amyloids and pro-IAPP aggregates even slower under the same conditions. We discovered that preformed amyloids of pro-IAPP inhibit, whereas IAPP amyloids promote, aS amyloid formation. Amyloids of aS promote pro-IAPP amyloid formation, whereas they inhibit IAPP amyloid formation. In contrast, mixing of IAPP and aS monomers results in coaggregation that is faster than either protein alone; moreover, pro-IAPP can incorporate aS monomers into its amyloid fibers. From this intricate network of cross-reactivity, it is clear that the presence of IAPP can accelerate aS amyloid formation. This observation may explain why T2D patients are susceptible to developing PD.Parkinson’s disease (PD) is the second most common neurological disorder and the most common movement disorder. It is characterized by widespread degeneration of subcortical structures of the brain, especially dopaminergic neurons in the substantia nigra. These changes are coupled with bradykinesia, rigidity, and tremor, resulting in difficulties in walking and abnormal gait in patients (1). The assembly process of the intrinsically unstructured 140-residue protein α-synuclein (aS) into amyloid fibers has been linked to the molecular basis of PD. aS is a major component of amyloid aggregates found in Lewy body inclusions, which are the pathological hallmark of PD, and duplications, triplications, and point mutations in the aS gene are related to familial PD cases (2, 3). The exact function of aS is unknown, but it is suggested to be involved in synaptic vesicle release and trafficking, regulation of enzymes and transporters, and control of the neuronal apoptotic response (4, 5). aS is present at presynaptic nerve terminals (6–8) and, intriguingly, also in many cells outside the brain (e.g., red blood cells and pancreatic β-cells). aS can assemble via oligomeric intermediates to amyloid fibrils under pathological conditions (9). Although soluble aS oligomers have been proposed to be toxic (10, 11), work with preformed aS fibrils has demonstrated that the amyloid fibrils themselves are toxic and can be transmitted from cell to cell and are also able to cross the blood–brain barrier (12–14).Type-2 diabetes (T2D) is another disease involving amyloid formation. Here, the primary pathological characteristic is islet amyloid of the hormone amylin, also known as islet amyloid polypeptide (IAPP), in pancreatic β-cells (15–18). The process of islet amyloid formation (19–21) leads to pancreatic β-cell dysfunction, cell death, and development of diabetes. IAPP (37 residues, natively unfolded) is cosecreted with insulin after enzymatic maturation of prohormones pro-IAPP (67 residues) and proinsulin in secretory granules. IAPP and insulin play roles in controlling gastric emptying, glucose homeostasis, and in the suppression of glucagon release. Although not understood on a mechanistic level, impairment of prohormone processing has been thought to play a role in initiation and progression of T2D (22, 23). Insulin and pro-IAPP (22, 24–26), but not proinsulin, can inhibit IAPP amyloid formation in vitro and in mice, suggesting that accumulation of unprocessed proinsulin may promote IAPP amyloid formation (22, 24). Insulin-degrading enzyme (IDE) is a conserved metallopeptidase that can degrade insulin and a variety of other small peptides including IAPP in the pancreas (27, 28). Genome-wide association studies have linked IDE to T2D (29, 30) and Ide mutant mice were found to have impaired glucose-stimulated insulin secretion as well as increased levels of IAPP, insulin, and, surprisingly, aS in pancreatic islets (31, 32). Here, aS may be associated with insulin biogenesis and exocytic release, as it was found to localize with insulin-secretory granules in pancreatic β-cells (33). We recently demonstrated in vitro that IDE readily inhibits aS amyloid formation via C-terminal binding and, in parallel, IDE activity toward insulin and other small substrates increases (34, 35).Together, the key role of aS in PD and the inverse correlation of impaired insulin secretion and increased aS levels in the pancreatic β-cells, imply that PD and T2D may be connected. In support, reports have suggested that patients with T2D are predisposed toward PD (36, 37). For Alzheimer’s disease (AD), a direct link with T2D was found (15, 38). Amyloid fiber seeds of the AD peptide, amyloid-β, were shown to efficiently accelerate amyloid formation of IAPP in vitro (39, 40) and IAPP was part of amyloid-β plaque found in mice brains (41). To address the unexplored question of cross-reactivity between the amyloidogenic peptides in PD and T2D, we here investigated cross-reactivity among aS, IAPP, and pro-IAPP using biophysical methods in vitro.     

Sodium/hydrogen exchanger NHA2 is critical for insulin secretion in β-cells

NHA2 is a sodium/hydrogen exchanger with unknown physiological function. Here we show that NHA2 is present in rodent and human β-cells, as well as β-cell lines. In vivo, two different strains of NHA2-deficient mice displayed a pathological glucose tolerance with impaired insulin secretion but normal peripheral insulin sensitivity. In vitro, islets of NHA2-deficient and heterozygous mice, NHA2-depleted Min6 cells, or islets treated with an NHA2 inhibitor exhibited reduced sulfonylurea- and secretagogue-induced insulin secretion. The secretory deficit could be rescued by overexpression of a wild-type, but not a functionally dead, NHA2 transporter. NHA2 deficiency did not affect insulin synthesis or maturation and had no impact on basal or glucose-induced intracellular Ca²⁺ homeostasis in islets. Subcellular fractionation and imaging studies demonstrated that NHA2 resides in transferrin-positive endosomes and synaptic-like microvesicles but not in insulin-containing large dense core vesicles in β-cells. Loss of NHA2 inhibited clathrin-dependent, but not clathrin-independent, endocytosis in Min6 and primary β-cells, suggesting defective endo–exocytosis coupling as the underlying mechanism for the secretory deficit. Collectively, our in vitro and in vivo studies reveal the sodium/proton exchanger NHA2 as a critical player for insulin secretion in the β-cell. In addition, our study sheds light on the biological function of a member of this recently cloned family of transporters.Sodium/hydrogen exchangers [NHEs; solute carrier 9 (Slc9) gene family] exchange monovalent cations such as Na⁺, Li⁺, or K⁺ with protons across lipid bilayers and are present in prokaryotes and eukaryotes (1). In mammals, 12 NHE isoforms are known so far (2). Recent additions to the large mammalian NHE family include NHA1 and NHA2, which possess higher homology to prokaryotic NHEs than the previously known NHEs 1–9 (2). Although NHA1 (also known as NHEDC1 or SLC9B1) is testis-specific, NHA2 (also known as NHEDC2 or SLC9B2) is widely expressed, including in the pancreas (3, 4). The physiological function of NHAs remains unknown. Based on chromosomal localization of the NHA2 gene, transport characteristics, and inhibitor sensitivity, NHA2 was proposed to be the long-sought sodium/lithium countertransporter (3). Sodium/lithium countertransporter activity is a highly heritable trait that was linked to the development of essential hypertension and diabetes in humans (5, 6). Given the reported expression of NHA2 in the pancreas and the epidemiological evidence linking NHA2 to the pathogenesis of diabetes mellitus in humans, we carried out in vitro and in vivo experiments to define the role of NHA2 in the endocrine pancreas.