Roles of unconventional ion channels and insulin granule structure in the pathogenesis of type-2 diabetes

T2D is the most widespread endocrine disease. In conventional stimulus secretion coupling increased blood glucose is metabolized causing an increased intracellular level of ATP and closure of the KATP channels, and this, in turn, depolarizes the cell membrane leading to the opening of voltage-gated Ca2+ channels, the influx of Ca2+ and exocytosis of insulin granules. This model has become almost an axiom in the diabetes research area. However, there are clear weak points that so far remain unclarified. More ion channels than potassium channels are needed to depolarize the membrane. Voltage-gated Ca2+ (Cav) channels have an essential role in beta cell function. The role of high voltage-activated Cav channels is well studied while the role of low voltage-activated T-type channels remain elusive. Moreover, glucose-stimulated insulin secretion (GSIS) causes beta cell swelling, which promotes us to explore mechanotransduction signalling pathways in beta cells represented by recently discovered Piezo1 mechanosensitive channel and aquaporins channels. Moreover, we also employed a dSTORM microscope combined with a single domain (SD) antibody to study the insulin granule cores (IGCs) at nano levels. Results: We find that the T-type Cav3.2 channel is abundantly expressed in human islets, and the gene expression is negatively correlated with HbA1c and strongly positively correlated with the expression of islet-predominantly expressed L-type subunits. CaV3.2 plays a fundamental role in maintaining normal insulin secretion by controlling Ca2+ signalling. Meanwhile, we also show that Piezo1 is expressed in pancreatic alpha and beta cells with heterogeneous distribution and is upregulated in T2D donors. Chronic hyperglycemia induces translocation of Piezo1 into the nucleus. In addition, silencing or inhibiting Piezo1 reduces Ca2+ signalling, membrane depolarization, and GSIS. Interestingly, beta- cell-specific Piezo1-knockout mice show impaired glucose tolerance in vivo and reduced electrical activity in islets. Subsequently, we identify that AQP1 gene expression is downregulated in islets from T2D individuals and silencing AQP1 decreased insulin secretion and insulin content. Whereas AQP1 overexpression significantly increased GSIS, AQP1 silencing elevated Ca2+ signalling due to elevated expression of CaV1.2 and CaV1.3 channels. Moreover, we demonstrate that AqB011, a selective AQP1 inhibitor blocking ion transport, substantially increases insulin secretion. Finally, nanoscale imaging for insulin granule cores exhibit that larger size located in exocytotic granules, the size and shape can be regulated by granule proteins cargo and the size is decreased after glucose stimulation, due to release of the readily releasable pool (RRP) part of insulin cores through incomplete granule fusion. Intriguingly, IGCs size was significantly decreased in pancreatic beta cells from human T2D donors and indicating that the lack of the RRP of the insulin core in the diabetic beta cells may be a primary cause for the impaired exocytosis. Conclusion: In this thesis, we have challenged the a consensus model and explored the importance of insulin granule structure in order to gain knowledge in the field around T2D pathophysiological progression and to facilitate finding new drug targets for the disease

Direct observation of secondary nucleation along the fibril surface of the amyloid β 42 peptide

Alzheimer's disease is a neurodegenerative condition which involves heavy neuronal cell death linked to oligomers formed during the aggregation process of the amyloid β peptide 42 (Aβ42). The aggregation of Aβ42 involves both primary and secondary nucleation. Secondary nucleation dominates the generation of oligomers and involves the formation of new aggregates from monomers on catalytic fibril surfaces. Understanding the molecular mechanism of secondary nucleation may be crucial in developing a targeted cure. Here, the self-seeded aggregation of WT Aβ42 is studied using direct stochastic optical reconstruction microscopy (dSTORM) with separate fluorophores in seed fibrils and monomers. Seeded aggregation proceeds faster than nonseeded reactions because the fibrils act as catalysts. The dSTORM experiments show that monomers grow into relatively large aggregates on fibril surfaces along the length of fibrils before detaching, thus providing a direct observation of secondary nucleation and growth along the sides of fibrils. The experiments were repeated for cross-seeded reactions of the WT Aβ42 monomer with mutant Aβ42 fibrils that do not catalyze the nucleation of WT monomers. While the monomers are observed by dSTORM to interact with noncognate fibril surfaces, we fail to notice any growth along such fibril surfaces. This implies that the failure to nucleate on the cognate seeds is not a lack of monomer association but more likely a lack of structural conversion. Our findings support a templating role for secondary nucleation, which can only take place if the monomers can copy the underlying parent structure without steric clashes or other repulsive interactions between nucleating monomers.ISSN:0027-8424ISSN:1091-649

A Palette of Fluorescent Aβ42 Peptides Labelled at a Range of Surface-Exposed Sites

Fluorescence-based single molecule techniques provide important tools towards understanding the molecular mechanism of complex neurodegenerative diseases. This requires efficient covalent attachment of fluorophores. Here we create a series of cysteine mutants (S8C, Y10C, S26C, V40C, and A42C) of Aβ42, involved in Alzheimer’s disease, based on exposed positions in the fibril structure and label them with the Alexa-fluorophores using maleimide chemistry. Direct stochastic optical reconstruction microscopy imaging shows that all the labelled mutants form fibrils that can be detected by virtue of Alexa fluorescence. Aggregation assays and cryo-electron micrographs establish that the careful choice of labelling position minimizes the perturbation of the aggregation process and fibril structure. Peptides labelled at the N-terminal region, S8C and Y10C, form fibrils independently and with wild-type. Peptides labelled at the fibril core surface, S26C, V40C and A42C, form fibrils only in mixture with wild-type peptide. This can be understood on the basis of a recent fibril model, in which S26, V40 and A42 are surface exposed in two out of four monomers per fibril plane. We provide a palette of fluorescently labelled Aβ42 peptides that can be used to gain understanding of the complex mechanisms of Aβ42 self-assembly and help to develop a more targeted approach to cure the disease.ISSN:1422-006

The calcium channel subunit gamma-4 is regulated by MafA and necessary for pancreatic beta-cell specification

Cheng Luan et al. report that the voltage-gated calcium channel CaVγ4 is necessary for maintaining pancreatic beta-cell function. They find that MafA, a transcription factor required for beta-cell maturation, directly regulates the gene encoding CaVγ4 and suggest that restoration of CaVγ4 may be a potential treatment for type 2 diabetes

Intracellular Reverse Transcription of Pfizer BioNTech COVID-19 mRNA Vaccine BNT162b2 In Vitro in Human Liver Cell Line

Preclinical studies of COVID-19 mRNA vaccine BNT162b2, developed by Pfizer and BioNTech, showed reversible hepatic effects in animals that received the BNT162b2 injection. Furthermore, a recent study showed that SARS-CoV-2 RNA can be reverse-transcribed and in-tegrated into the genome of human cells. In this study, we investigated the effect of BNT162b2 on the human liver cell line Huh7 in vitro. Huh7 cells were exposed to BNT162b2, and quantitative PCR was performed on RNA extracted from the cells. We detected high levels of BNT162b2 in Huh7 cells and changes in gene expression of long interspersed nuclear element-1 (LINE-1), which is an endogenous reverse transcriptase. Immunohistochemistry using antibody binding to LINE-1 open reading frame-1 RNA-binding protein (ORFp1) on Huh7 cells treated with BNT162b2 indicated increased nucleus distribution of LINE-1. PCR on genomic DNA of Huh7 cells exposed to BNT162b2 amplified the DNA sequence unique to BNT162b2. Our results indicate a fast up-take of BNT162b2 into human liver cell line Huh7, leading to changes in LINE-1 expression and distribution. We also show that BNT162b2 mRNA is reverse transcribed intracellularly into DNA in as fast as 6 h upon BNT162b2 exposure

The T-type calcium channel CaV3.2 regulates insulin secretion in the pancreatic β-cell

Voltage-gated Ca2+ (CaV) channel dysfunction leads to impaired glucose-stimulated insulin secretion in pancreatic β-cells and contributes to the development of type-2 diabetes (T2D). The role of the low-voltage gated T-type CaV channels in β-cells remains obscure. Here we have measured the global expression of T-type CaV3.2 channels in human islets and found that gene expression of CACNA1H, encoding CaV3.2, is negatively correlated with HbA1c in human donors, and positively correlated with islet insulin gene expression as well as secretion capacity in isolated human islets. Silencing or pharmacological blockade of CaV3.2 attenuates glucose-stimulated cytosolic Ca2+ signaling, membrane potential, and insulin release. Moreover, the endoplasmic reticulum (ER) Ca2+ store depletion is also impaired in CaV3.2-silenced β-cells. The linkage between T-type (CaV3.2) and L-type CaV channels is further identified by the finding that the intracellular Ca2+ signaling conducted by CaV3.2 is highly dependent on the activation of L-type CaV channels. In addition, CACNA1H expression is significantly associated with the islet predominant L-type CACNA1C (CaV1.2) and CACNA1D (CaV1.3) genes in human pancreatic islets. In conclusion, our data suggest the essential functions of the T-type CaV3.2 subunit as a mediator of β-cell Ca2+ signaling and membrane potential needed for insulin secretion, and in connection with L-type CaV channels

The TCF7L2-dependent high-voltage activated calcium channel subunit α2δ-1 controls calcium signaling in rodent pancreatic beta-cells

The transcription factor TCF7L2 remains the most important diabetes gene identified to date and genetic risk carriers exhibit lower insulin secretion. We show that Tcf7l2 regulates the auxiliary subunit of voltage-gated Ca2+ channels, Cacna2d1 gene/α2δ-1 protein levels. Furthermore, suppression of α2δ-1 decreased voltage-gated Ca2+ currents and high glucose/depolarization-evoked Ca2+ signaling which mimicked the effect of silencing of Tcf7l2. This appears to be the result of impaired voltage-gated Ca2+ channel trafficking to the plasma membrane, as Cav1.2 channels accumulated in the recycling endosomes after α2δ-1 suppression, in clonal as well as primary rodent beta-cells. This impaired the capacity for glucose-induced insulin secretion in Cacna2d1-silenced cells. Overexpression of α2δ-1 increased high-glucose/K+-stimulated insulin secretion. Furthermore, overexpression of α2δ-1 in Tcf7l2-silenced cells rescued the Tcf7l2-dependent impairment of Ca2+ signaling, but not the reduced insulin secretion. Taken together, these data clarify the connection between Tcf7l2, α2δ-1 in Ca2+-dependent insulin secretion

The highly expressed calcium-insensitive synaptotagmin-11 and synaptotagmin-13 modulate insulin secretion

AIM: SYT11 and SYT13, two calcium-insensitive synaptotagmins, are downregulated in islets from type-2 diabetic donors, but their function in insulin secretion is unknown. To address this, we investigated the physiological role of these two synaptotagmins in insulin secreting cells.METHODS: Correlations between gene expression levels were performed using previously described RNA-seq data on islets from 188 human donors. SiRNA knockdown was performed in EndoC-βH1 and INS-1 832/13 cells. Insulin secretion was measured with ELISA. Patch clamp was used for single cell electrophysiology. Confocal microscopy was used to determine intra-cellular localization.RESULTS: Human islet expression of the transcription factor PDX-1 was positively correlated with SYT11 (p = 2.4e -10 ) and SYT13 (p<2.2 e -16 ). Syt11 and Syt13 both co-localized with insulin, indicating their localization in insulin granules. Downregulation of Syt11 in INS-1 832/13 cells (siSYT11) resulted in increased basal and glucose-induced insulin secretion. Downregulation of Syt13 (siSYT13) decreased insulin secretion induced by glucose and K + .Interestingly, the cAMP raising agent forskolin was unable to enhance insulin secretion in siSYT13 cells. There was no difference in insulin content, exocytosis, or voltage-gated Ca 2+ currents in the two models. Double knockdown of Syt11 and Syt13 (DKD) resembled the results in siSYT13 cells. CONCLUSION: SYT11 and SYT13 have similar localization and transcriptional regulation but they regulate insulin secretion differentially. While downregulation of SYT11 might be a compensatory mechanism in type-2 diabetes, downregulation of SYT13 reduces the insulin secretory response and overrules the compensatory regulation of SYT11 in a way that could aggravate the disease

The structure of insulin granule core determines secretory capacity being reduced in type-2 diabetes

Exocytosis in excitable cells is essential for their physiological functions. Although the exocytotic machinery controlling cellular secretion has been well investigated, the function of the vesicular cargo, i.e. secretory granular content remains obscure. Here we combine dSTORM imaging and single-domain insulin antibody, to dissect the in situ structure of insulin granule cores (IGCs) at nano level. We demonstrate that the size and shape of the IGCs can be regulated by the juxta-granular molecules Nucleobindin-2 and Enolase-1, that further contribute to the stimulated insulin secretion. IGCs located at the plasma membrane are larger than those in the cytosol. The IGCs size is decreased by ∼20% after glucose stimulation due to the release of the peripheral part of IGCs through incomplete granule fusion. Importantly, the reduction of the IGCs size is also observed in non-stimulatory pancreatic β-cells from diabetic db/db mice, Akita (Ins2+/-) mice and human Type-2 diabetic donors, in accordance with impaired secretion. These findings overall highlight the structure of exocytotic insulin cores as a novel modality amenable to targeting in the stimulated exocytosis in β-cells with impaired insulin secretion.Competing Interest StatementThe authors have declared no competing interest

A critical role of the mechanosensor PIEZO1 in glucose-induced insulin secretion in pancreatic β-cells

Insulin secretion depends on action potential firing in pancreatic islet beta-cells, but the underlying mechanism is unclear. Here, the authors show that activation of the mechanosensor ion channel PIEZO1 plays a central role in beta-cell electrical activity and insulin release