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We previously showed that α cell function and gene expression in islets from donors with type 1 diabetes (T1D) was significantly compromised. The reason (s) for these intrinsic α cell changes are unknown but may include loss of α-to-β cell contact, chronic hyperglycemia, and/or repeated hypoglycemic events. To test these concurrent hypotheses, we modeled human T1D islets using α cell-enriched (“T1D-like") human pseudoislets. The synchronous acquisition of intracellular Ca2+ and hormone response showed impaired intracellular Ca2+ signaling and glucagon secretion in T1D-like pseudoislets compared to controls containing both α and β cells. For modeling of chronic hyperglycemia, we transplanted pseudoislets into Nod-SCID-IL2Rγnull; RIP-Diphtheria Toxin Receptor mice made diabetic by diphtheria toxin (DT) -induced β cell depletion. In DT-treated mice, α cells in T1D-like pseudoislets maintained expression of MAFB and ARX but began to express the β cell-enriched transcription factor NKX6.1 (control vs. T1D-like, 3.43±2.49 vs. 54.8±20.8% NKX6.1+ α cells; P=0.046, N=3-5 mice, 2 donors/group) . To mimic repeated hypoglycemic events, we intermittently exposed T1D-like pseudoislets to low glucose (1.7 mM) (3 events x 3hr each) in vitro, using those remaining in basal glucose (5 mM) as control. Following multiple low glucose (MLG) exposures, T1D-like pseudoislets had reduced glucagon secretion in the presence of both 1.7 mM (control vs. MLG, 1.51±0.18 vs. 0.79±0.13 % glucagon content; P=0.011, N=4 donors) and 16.7 mM glucose (control vs. MLG, 0.79±0.13 vs. 0.39±0.% glucagon content; P=0.023, N=4) . Native human islets exposed to MLG had similar changes in glucagon response (control vs. MLG, 1.7mM: 0.63±0.vs. 0.22±0.% glucagon content, P=0.007; 16.7mM: 0.18±0.vs. 0.05±0.% glucagon content, P=0.029; N=4 donors) . These data suggest that loss of α-to-β cell contact and repeated exposure to low glucose impair α cell function. Further, exposure to chronic hyperglycemia may lead to changes in α cell identity state after β cell loss. Disclosure Y.D.Pettway: None. C.Dai: None. T.M.Richardson: None. J.T.Walker: None. R.Aramandla: None. G.Poffenberger: None. A.Bradley: None. A.C.Powers: None. M.Brissova: None.

ATP release from β cell secretory vesicles during insulin exocytosis is postulated to modulate islet hormone secretion via purinergic signaling in an autocrine or paracrine manner. Ecto-enzymes, including a family of ectonucleotidases (NTPDase) , control the concentration of extracellular nucleotides. One member of this family, ectonucleotidase triphosphate diphosphohydrolase 3 (NTPDase3) , is specifically expressed in human β cells during postnatal maturation, and its enzymatic activity is present in isolated adult human islets. The role of NTPDase3 in islet hormone secretion is not understood. We hypothesized that NTPDase3 regulates islet hormone secretion by modulating local extracellular nucleotide levels that signal through purinergic receptors expressed by islet cells. To define the cellular distribution of purinergic ecto-enzymes and nucleotide receptors in human islets, we used single-cell RNA sequencing (scRNA-seq) data. In addition to β cell NTPDase3 expression, ecto-enzymes ENPP1, and ENPP2 were abundant in α and ε cells while ecto-5'-NT and ADA were expressed in endothelial and stellate cells. These ecto-enzymes can further hydrolyze β cell-derived ATP to ADP, AMP, and adenosine which all bind purinergic receptors. We found that the ATP receptor, P2RY1, was enriched in β cells, and an adenosine receptor, ADORA2A, was expressed in α cells by both bulk and scRNA-seq. To study the function of this purinergic niche, we further assessed extracellular ATP levels which increased concomitantly (4.2E-12 vs. 4.3E-13 moles/100 IEQ/hour; n=9; p=0.03) with insulin secretion (21 vs. 60 ng/100 IEQ/hour; n=9; p=0.02) upon stimulation with 16.7 mM glucose + IBMX. To directly test NTPDase3 loss of function, we genetically manipulated human pseudoislets using an shRNA knockdown approach, resulting in a 1.7-fold increase of insulin secretion (AUC=3 vs. 5 ng/100 IEQs; n=3) . Overall, these studies provide an insight into how extracellular nucleotides and purinergic signaling mechanisms fine-tune human islet hormone secretion. Disclosure T. M. Richardson: None. D. C. Saunders: None. K. C. Coate: None. S. C. Robson: Advisory Panel; eGenesis, Consultant; Puretech, Synlogic, Research Support; Tizona Inc, Stock/Shareholder; EPurines, Purinomia. A. C. Powers: None. M. Brissova: None. C. Dai: None. R. Aramandla: None. H. H. Durai: None. C. Reihsmann: None. C. Davis: None. S. Mei: None. D. Gibson: None. S. Shrestha: None.

A variety of islet autoantibodies (AAbs) can predict and possibly dictate eventual type 1 diabetes (T1D) diagnosis. Upwards of 75% of those with T1D are positive for AAbs against glutamic acid decarboxylase (GAD65), a producer of gamma-aminobutyric acid (GABA) in human pancreatic beta cells. Interestingly, bacterial populations within the human gut also express GAD65 and produce GABA. Evidence suggests that dysbiosis of the microbiome may correlate with T1D pathogenesis and physiology. Therefore, autoimmune linkages between the gut microbiome and islets susceptible to autoimmune attack need to be further elucidated. Utilizing silico analyses, we show here that 25 GAD sequences from different human gut bacterial sources show sequence and motif similarities to human beta cell GAD65. Our motif analyses determined that a majority of gut GAD sequences contain the pyroxical dependent decarboxylase domain of human GAD65 which is important for its enzymatic activity. Additionally, we showed overlap with known human GAD65 T-cell receptor epitopes which may implicate the immune destruction of beta cells. Thus, we propose a physiological hypothesis in which changes in the gut microbiome in those with T1D result in a release of bacterial GAD, thus causing miseducation of the host immune system. Due to the notable similarities, we found between humans and bacterial GAD, these deputized immune cells may then go on to target human beta cells leading to the development of T1D.

The nucleolus is a prominent nuclear condensate that plays a central role in ribosome biogenesis by facilitating the transcription and processing of nascent ribosomal RNA (rRNA). A number of studies have highlighted the active viscoelastic nature of the nucleolus, whose material properties and phase behavior are a consequence of underlying molecular interactions. However, the ways in which the material properties of the nucleolus impact its function in rRNA biogenesis are not understood. Here we utilize the Cry2olig optogenetic system to modulate the viscoelastic properties of the nucleolus. We show that above a threshold concentration of Cry2olig protein, the nucleolus can be gelled into a tightly linked, low mobility meshwork. Gelled nucleoli no longer coalesce and relax into spheres but nonetheless permit continued internal molecular mobility of small proteins. These changes in nucleolar material properties manifest in specific alterations in rRNA processing steps, including a buildup of larger rRNA precursors and a depletion of smaller rRNA precursors. We propose that the flux of processed rRNA may be actively tuned by the cell through modulating nucleolar material properties, which suggests the potential of materials-based approaches for therapeutic intervention in ribosomopathies., SCOPUS: ar.j, info:eu-repo/semantics/published

Endogenous β cell regeneration could alleviate diabetes, but proliferative stimuli within the islet microenvironment are incompletely understood. We previously found that β cell recovery following hypervascularization-induced β cell loss involves interactions with endothelial cells (ECs) and macrophages (MΦs). Here we show that proliferative ECs modulate MΦ infiltration and phenotype during β cell loss, and recruited MΦs are essential for β cell recovery. Furthermore, VEGFR2 inactivation in quiescent ECs accelerates islet vascular regression during β cell recovery and leads to increased β cell proliferation without changes in MΦ phenotype or number. Transcriptome analysis of β cells, ECs, and MΦs reveals that β cell proliferation coincides with elevated expression of extracellular matrix remodeling molecules and growth factors likely driving activation of proliferative signaling pathways in β cells. Collectively, these findings suggest a new β cell regeneration paradigm whereby coordinated interactions between intra-islet MΦs, ECs, and extracellular matrix mediate β cell self-renewal.

Endogenous β cell regeneration could alleviate diabetes, but proliferative stimuli within the islet microenvironment are incompletely understood. We previously found that β cell recovery following hypervascularization-induced β cell loss involves interactions with endothelial cells (ECs) and macrophages (MΦs). Here we show that proliferative ECs modulate MΦ infiltration and phenotype during β cell loss, and recruited MΦs are essential for β cell recovery. Furthermore, VEGFR2 inactivation in quiescent ECs accelerates islet vascular regression during β cell recovery and leads to increased β cell proliferation without changes in MΦ phenotype or number. Transcriptome analysis of β cells, ECs, and MΦs reveals that β cell proliferation coincides with elevated expression of extracellular matrix remodeling molecules and growth factors likely driving activation of proliferative signaling pathways in β cells. Collectively, these findings suggest a new β cell regeneration paradigm whereby coordinated interactions between intra-islet MΦs, ECs, and extracellular matrix mediate β cell self-renewal.

Impaired counterregulatory response of glucagon to hypoglycemia is a common complication of type 1 diabetes (T1D). One proposed contributor to abnormal glucagon secretion is decreased sympathetic innervation in T1D islets. To systematically assess the innervation of human pancreatic tissues, we examined samples from donors with recent-onset T1D (10 years, age 27-63 years, n = 3), and nondiabetic controls (age 10-52 years, n = 4). Islet and exocrine tissue innervation was visualized by pan-neuronal marker tubulin β-3 and analyzed by a 2-D morphometry and 3-D rendering. By quantifying the nerve fiber length (1.9±0.4 nm/μm2) and density (646±94 fibers/mm2), we found that innervation in control human islets was nearly 20-fold less than previously reported in mouse islets. In contrast to mouse, human exocrine tissue was far more innervated than islets with fiber length of 6.8±0.4 nm/μm2(p0.05), and so was the 3-D arrangement of nerve fibers in exocrine tissue and islets. Regardless of T1D duration, the islet fiber length (recent-onset: 1.5±0.2 nm/μm2; long-standing: 1.0±0.2 nm/μm2) and density (recent-onset: 623±61 fibers/mm2; longstanding: 486±49 fibers/mm2), as well as exocrine fiber length (recent-onset: 5.2±.5 nm/μm2; longstanding: 5.8±0.4 nm/μm2) and density (recent-onset: 1119±77 fibers/mm2; longstanding: 1537±72 fibers/mm2) did not differ from controls (p>0.05). These data indicate that neuronal patterning of human pancreas is significantly different from that of mouse and islet innervation does not appear to be altered in T1D. Disclosure T.M. Richardson: None. R. Haliyur: None. R. Aramandla: None. R. Bottino: Research Support; Self; Imagine Pharma. A.C. Powers: None. M. Brissova: None.

Exposure to chemotherapeutic agents has been linked to an increased risk of type 2 diabetes (T2D), a disease characterized by both the peripheral insulin resistance and impaired glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. Using the rat β-cell line INS-1 832/13 and isolated mouse pancreatic islets, we investigated the effect of the chemotherapeutic drug doxorubicin (Adriamycin) on pancreatic β-cell survival and function. Exposure of INS-1 832/13 cells to doxorubicin caused impairment of GSIS, cellular viability, an increase in cellular toxicity, as soon as 6 h post-exposure. Doxorubicin impaired plasma membrane electron transport (PMET), a pathway dependent on reduced equivalents NADH and NADPH, but failed to redox cycle in INS-1 832/13 cells and with their lysates. Although NADPH/NADP(+ )content was unaffected, NADH/NAD(+ )content decreased at 4 h post-exposure to doxorubicin, and was followed by a reduction in ATP content. Previous studies have demonstrated that doxorubicin functions as a topoisomerase II inhibitor via induction of DNA cross-linking, resulting in apoptosis. Doxorubicin induced the expression of mRNA for mdm2, cyclin G1, and fas whereas downregulating p53, and increased the melting temperature of genomic DNA, consistent with DNA damage and induction of apoptosis. Doxorubicin also induced caspase-3 and -7 activity in INS-1 832/13 cells and mouse islets; co-treatment with the pan-caspase inhibitor Z-VAD-FMK temporarily attenuated the doxorubicin-mediated loss of viability in INS-1 832/13 cells. Together, these data suggest that DNA damage, not H2O2 produced via redox cycling, is a major mechanism of doxorubicin toxicity in pancreatic β-cells.

The nucleolus and other ribonucleoprotein (RNP) bodies are membrane-less organelles that appear to assemble through phase separation of their molecular components. However, many such RNP bodies contain internal subcompartments, and the mechanism of their formation remains unclear. Here, we combine in vivo and in vitro studies, together with computational modeling, to show that subcompartments within the nucleolus represent distinct, coexisting liquid phases. Consistent with their in vivo immiscibility, purified nucleolar proteins phase separate into droplets containing distinct non-coalescing phases that are remarkably similar to nucleoli in vivo. This layered droplet organization is caused by differences in the biophysical properties of the phases-particularly droplet surface tension-which arises from sequence-encoded features of their macromolecular components. These results suggest that phase separation can give rise to multilayered liquids that may facilitate sequential RNA processing reactions in a variety of RNP bodies. PAPERCLIP.

T2D is characterized by progressive pancreatic islet dysfunction; however, dissecting causes of this dysfunction in humans is challenging due to disease heterogeneity and the difficulty of obtaining relevant pancreatic samples. To address these challenges, we used an integrated approach to functionally and molecularly study the native pancreas and isolated islets from the same human T2D cadaveric donor. Using associated donor clinical information, we focused on donors with short disease duration (n=10, age 47-66 years, 2-7-year duration, oral medications). Compared to age-matched control islets, T2D islets had dysfunctional insulin (decreased) and glucagon (increased) secretion despite similar insulin and glucagon content. Tissue-section analysis of T2D pancreata showed no difference in relative endocrine (α, β and δ) cell composition. To assess for changes in the islet microenvironment, we analyzed macrophages by staining with Iba1 and endothelial cells with Caveolin-1 and found a 2-fold increase in intraislet macrophages (T2D vs. control: 3.01% vs. 1.45% by area) but no change in intraislet endothelial cell area (T2D vs. control: 8.97% vs. 8.21% by area). Interestingly, costaining with Iba1 and thioflavin S demonstrated that T2D islets with amyloid did not have increased macrophage infiltration compared to T2D islets without amyloid although a close spatial association of macrophages and amyloid deposits was noted. RNA-sequencing analysis of FACS-purified α and β cells from T2D islets showed increased inflammatory signaling pathways in both cell types. Specifically, transcripts for cytokine receptors such as IL6R, IL1R1, and IFNGR1 as well as downstream regulators including NFKB1 and NFKB2 were elevated in T2D α and/or β cells. Together, these data suggest that early in the course of T2D, increased macrophages in the islet microenvironment may lead to elevated α and β cell inflammatory signaling and contribute to human α and β cell dysfunction. Disclosure J.T. Walker: None. S. Shrestha: None. N. Prasad: None. T. Richardson: None. R. Aramandla: None. G. Poffenberger: None. R. Bottino: None. M. Brissova: None. A.C. Powers: None.