Non-diabetic Donors Research Articles

Stevioside Potentiates Calcium Activity and Insulin Secretion in Human Pancreatic Islets Through Potentiation of TRPM5

TRPM5 is a calcium-activated monovalent cation channel that is expressed in human and murine islets. Ion channels of the TRP family are multimodal sensors and are activated by different stimuli such as voltage, temperature and ligands, including natural compounds. Previous work showed that mice lacking TRPM5 have increased postprandial blood glucose levels and an impaired insulin secretion. There are increased observations of TRPM5 mutations in patients suffering from diabetes or metabolic syndrome. Stevioside or stevia is a sweet-tasting organic compound isolated from the plant Stevia rebaudiana, and is widely used as a low-caloric sweetener. Despite the use of stevioside in consumer products, there is limited knowledge about its molecular interaction in the body. It was previously shown in mice that stevioside potentiates TRPM5, which leads to increased insulin secretion and ultimately better glycemic control in diabetic animal models. In this work, we translate these observations to freshly isolated human islets. We show that TRPM5 expression is limited to a subset of the insulin-expressing beta-cells in islets from non-diabetic donors. In islets isolated from diabetic donors, we also see distinct TRPM5 positive cells that are insulin negative. There are substantial differences observed in the microanatomy of the islets and the functional synchronization of calcium signaling in beta cells between mouse and man. In perifusion studies, there is a potentiation of the glucose-induced insulin secretion with the application of stevioside. These studies provide insight in the molecular mechanism of the action of stevioside in the human islets. Furthermore, we confirm the importance of TRPM5 in modulating the glucose-stimulated insulin secretion in human islets. Modulating TRPM5 with stevioside constitutes a potential novel

Islet sympathetic innervation and islet neuropathology in patients with type 1 diabetes

Dysregulation of glucagon secretion in type 1 diabetes (T1D) involves hypersecretion during postprandial states, but insufficient secretion during hypoglycemia. The sympathetic nervous system regulates glucagon secretion. To investigate islet sympathetic innervation in T1D, sympathetic tyrosine hydroxylase (TH) axons were analyzed in control non-diabetic organ donors, non-diabetic islet autoantibody-positive individuals (AAb), and age-matched persons with T1D. Islet TH axon numbers and density were significantly decreased in AAb compared to T1D with no significant differences observed in exocrine TH axon volume or lengths between groups. TH axons were in close approximation to islet α-cells in T1D individuals with long-standing diabetes. Islet RNA-sequencing and qRT-PCR analyses identified significant alterations in noradrenalin degradation, α-adrenergic signaling, cardiac β-adrenergic signaling, catecholamine biosynthesis, and additional neuropathology pathways. The close approximation of TH axons at islet α-cells supports a model for sympathetic efferent neurons directly regulating glucagon secretion. Sympathetic islet innervation and intrinsic adrenergic signaling pathways could be novel targets for improving glucagon secretion in T1D.

Pro-Inflammatory Cytokines Induce Insulin and Glucagon Double Positive Human Islet Cells That Are Resistant to Apoptosis.

The presence of islet cells double positive for insulin and glucagon (Ins+/Glu+) has been described in the pancreas from both type 2 (T2D) and type 1 (T1D) diabetic subjects. We studied the role of pro-inflammatory cytokines on the occurrence, trajectory, and characteristics of Ins+/Glu+ cells in human pancreatic islets. Pancreas samples, isolated islets, and dispersed islet cells from 3 T1D and 11 non-diabetic (ND) multi-organ donors were studied by immunofluorescence, confocal microscopy, and/or electron microscopy. ND islet cells were exposed to interleukin-1β and interferon-γ for up to 120 h. In T1D islets, we confirmed an increased prevalence of Ins+/Glu+ cells. Cytokine-exposed islets showed a progressive increase of Ins+/Glu+ cells that represented around 50% of endocrine cells after 120h. Concomitantly, cells expressing insulin granules only decreased significantly over time, whereas those containing only glucagon granules remained stable. Interestingly, Ins+/Glu+ cells were less prone to cytokine-induced apoptosis than cells containing only insulin. Cytokine-exposed islets showed down-regulation of β-cell identity genes. In conclusion, pro-inflammatory cytokines induce Ins+/Glu+ cells in human islets, possibly due to a switch from a β- to a β-/α-cell phenotype. These Ins+/Glu+ cells appear to be resistant to cytokine-induced apoptosis.

Insulitis in the pancreas of non-diabetic organ donors under age 25 years with multiple circulating autoantibodies against islet cell antigens

Autoantibodies against islet cell antigens are routinely used to identify subjects at increased risk of symptomatic type 1 diabetes, but their relation to the intra-islet pathogenetic process that leads to positivity for these markers is poorly understood. We screened 556 non-diabetic organ donors (3 months to 24 years) for five different autoantibodies and found positivity in 27 subjects, 25 single- and two double autoantibody-positive donors. Histopathological screening of pancreatic tissue samples showed lesion characteristic for recent-onset type 1 diabetes in the two organ donors with a high-risk profile, due to their positivity for multiple autoantibodies and HLA-inferred risk. Inflammatory infiltrates (insulitis) were found in a small fraction of islets (<5%) and consisted predominantly of CD3+CD8+ T-cells. Islets with insulitis were found in close proximity to islets devoid of insulin-positivity; such pseudo-atrophic islets were present in multiple small foci scattered throughout the pancreatic tissue or were found to be distributed with a lobular pattern. Relative beta cell area in both single and multiple autoantibody-positive donors was comparable to that in autoantibody-negative controls. In conclusion, in organ donors under age 25 years, insulitis and pseudo-atrophic islets were restricted to multiple autoantibody-positive individuals allegedly at high risk of developing symptomatic type 1 diabetes, in line with reports in older age groups. These observations may give further insight into the early pathogenetic events that may culminate in clinically overt disease.

Functional Characterization of the Human Islet Microvasculature Using Living Pancreas Slices.

Pancreatic islets are clusters of endocrine cells that secrete different hormones to regulate blood glucose levels. Efficient hormone secretion requires a close interaction of endocrine cells with their vascular system. Islets receive blood through feeding arteriole(s) that branch into capillaries made of endothelial cells covered by pericytes. While a lot is known about rodent islet blood vessels, the structure and function of the human islet microvasculature has been less investigated. In this study, we used living pancreas slices from non-diabetic human donors to examine the function of human islet blood vessels. Living human pancreas slices were incubated with a membrane permeant calcium indicator and pericytes/smooth muscle cells were visualized with a fluorescent antibody against the mural cell marker NG2 proteoglycan. By confocal microscopy, we simultaneously recorded changes in the diameter of lectin-labeled blood vessels and cytosolic calcium levels in mural cells in islets. We tested several stimuli with vasoactive properties, such as norepinephrine, endothelin-1 and adenosine and compared human vascular responses with those previously published for mouse islet blood vessels. Norepinephrine and endothelin-1 significantly constricted human islet feeding arterioles, while adenosine dilated them. Islet capillaries were less responsive and only 15–20% of the mouse and human islet capillary network showed vasomotion. Nevertheless, in these responsive regions, norepinephrine and endothelin-1 decreased both mouse and human islet capillary diameter. Changes in islet blood vessel diameter were coupled to changes in cytosolic calcium levels in adjacent mouse and human islet mural cells. Our study shows that mural cells in islets are the targets of different regulatory mechanisms of islet blood perfusion. Several alterations of the human islet microvasculature occur during diabetes progression. Elucidating their functional consequences in future studies will be critical for our understanding of disease pathogenesis.

The effect of liver donors' age, gender and metabolic state on pancreatic lineage activation.

Autologous cells replacement therapy by liver to pancreas transdifferentiation (TD)allows diabetic patients to be also the donors of their own therapeutic tissue. Aim: To analyze whether the efficiency of the process is affected by liver donors' heterogeneity with regard to age, gender and the metabolic state. Materials &methods:TDof liver cells derived from nondiabetic and diabetic donors at different ages was characterized at molecular and cellular levels, in vitro. Results: Neither liver cells proliferation nor the propagated cells TDefficiency directly correlate with the age (3-60years), gender or the metabolic state of the donors. Conclusion: Human liver cells derived from a wide array of ages and metabolic states can be used for autologous cells therapies for diabetics.

The relationship between systemic disorders and anatomical outcomes after Descemet membrane endothelial keratoplasty.

The aim of this study was to investigate the association of anatomical outcomes and medications of patients with systemic diseases who underwent Descemet membrane endothelial keratoplasty with donor factors. Sixty nondiabetic donors of endothelial grafts and 60 patients who underwent operation by a single surgeon were included in this retrospective study. The patients' data, including the presence of diabetes mellitus and hypertension, antidiabetic-antihypertensive medications, and intracameral tamponades and anatomical outcomes, were recorded. The donor data were obtained from eye bank records. Eighteen patients had type 2 diabetes mellitus (30%) and 34 had hypertension (56.6%). Among the patients with diabetes mellitus, 13 were receiving a single-agent antidiabetic drug, 4 were receiving dual oral antidiabetic therapy, and 1 was receiving insulin therapy. Among the hypertensive patients, 11 had monotherapy and 23 had dual antihypertensive therapy. Postoperatively, 35 patients (58.3%) had an endothelial attachment, 8 (13.3%) received reinjection, 7 (11.7%) required re-Descemet membrane endothelial keratoplasty, and 10 (16.7%) underwent penetrating keratoplasty. The mean donor age was 51.2 ± 14.1 years. The most common cause of donor death was cardiopulmonary arrest (36/60 cases; 60.0%). Regression analysis revealed that the presence of diabetes mellitus significantly disrupted graft attachment (p=0.034), while the presence of hypertension, antidiabetic and antihypertensive medication use, and the type of tamponade used in the patients, and the age, sex, cause of death, and specular endothelial cell count of donors were not statistically significantly associated with graft attachment (p>0.05). In this study, the anatomical outcomes of Descemet membrane endothelial keratoplasty surgery were affected by recipient and donor factors. The presence of diabetes mellitus in the recipient significantly negatively affected graft attachment.

Post-donation variations in various hematological parameters, serum ferritin and Hba1c level in diabetic and non-diabetic donors: A study report

Introduction: Whole blood donation is almost obsolete in developed country but still it’s the main stay of transfusion practice in developing countries. Iron deficiency is a complication of regular blood donation. Objective: To study the various hematological parameters along with serum ferritin & HBA1C level in non-diabetic and diabetic blood donor population. Materials and Methods: Complete blood count was performed using six part analyser, serum ferritin using ELISA and HBA1C using HPLC method. Results: In our study there was a decrease in level of some of the hematological parameters, serum ferritin and HBA1C level after donation. Conclusion: From our study we conclude that only hemoglobin estimation in blood donors is not sufficient for acceptance of donations and serum ferritin level should be measured in regular blood donors. For diabetic donors on oral medication at least HBA1C level should be repeated after two months of donation. Keywords: Iron deficiency, Blood donation, Hematological parameters, Serum Ferritin, HBA1C level.

Pancreatic Islet Composition Affects Hormone Secretion in Isolated Alpha and Beta cells

Background/Objective: &#x0D; The Integrated Islet Distribution Program (IIDP) distributes islets from five isolation Centers and serves as the main source of human islets for research in the U.S. In 2016, the IIDP initiated the Human Islet Phenotyping Program (HIPP), which provides standardized post-shipment assessment of islet hormone secretion and endocrine cell composition for each IIDP-supported islet isolation. To date, islets from 276 non-diabetic donors have been analyzed. We hypothesized that analysis of this unique resource will provide novel insights into how demographic and clinical features impact islet health. &#x0D; &#x0D; Methods: &#x0D; Relationships between insulin and glucagon secretion assessed by perifusion and islet composition (% of β- and α-cells) were analyzed using SAS Version 9.4. For each analysis, the isolation center was used as a covariate. &#x0D; &#x0D; Results: &#x0D; Of the 276 donors, 60% were male; 59% of donors were Caucasian, 28% were Hispanic, 9% were African-American; 4% were Asian; and 0.36% were American Indian. The % of β-cells was moderately correlated with insulin responses to 16.7 mM glucose (r=0.2785; p&lt;0.0001) and 20 mM KCL (r=0.3109, p&lt;0.0001). Similarly, the α-cell% was moderately correlated with total glucagon content (r=0.3362, p&lt;0.0001) and glucagon responses to 1.7 mM glucose + 1mM epinephrine (r=0.2015, p=0.0001). The % of β -cells was negatively correlated with glucagon total content (r=-.243,p=.0001), while the α-cell% was negatively correlated with insulin stimulation index to KCL (r=-0.2573, p&lt;0.0001). Notably, Asian donors exhibited a significantly higher β-cell% compared to other groups (p&lt;0.05). Consistent with this, glucose-stimulated insulin secretion was higher in Asian donors compared to responses observed in islets from African American donors (p&lt;0.01). &#x0D; &#x0D; Conclusion: &#x0D; These data indicate that islet cell composition influences insulin and glucagon secretory responses and suggests that race may impact islet composition and hormone secretion. Continued analysis of the HIPP dataset may aid in our understanding of risk factors for the development islet dysfunction in diabetes.

The impact of donor and recipient diabetes on renal transplant outcomes.

The use of diabetic kidneys is increasing worldwide with better outcome than being on waitlist and possible reversal of diabetic changes in transplanted kidneys. But particular caution is warranted in diabetic donor-recipient combination. Total 1223 deceased donor kidney transplants were performed at our center between 2008 and 2018. 689 from non-diabetic donor (NDD) to non-diabetic recipient, 400 from non-diabetic donor to diabetic recipient, 97 from diabetic to non-diabetic recipient, and 32 from diabetic donor (DD) to diabetic recipient. The DD was older than NDDs (median age 48 vs 39years, P<0.0001). DD had higher BMI (35.6 vs 26.9, P<0.0001), higher KDPI (74% vs 37%, P<0.0001), and higher terminal creatinine (1.10mg/dl vs 0.95mg/dl, p 0.0046) than the NDD. Diabetes recipients were comparatively older (57 vs 54, P<0.001). DD recipients had higher serum creatinine at 6months (1.70 vs 1.50mg/dl, p 0.00304) and 2years post-transplant (1.70 vs 1.50mg/dl P<0.0002). DD recipients had more favorable end CPRA than NDD recipients (77.5% at 0% vs 67.4% at 0, P=0.0074). Ten-year patient and graft survival was best in NDD-recipient pair and worse in DD-recipient pair. Diabetic donor kidneys to diabetic recipients have lower 1-, 3-, and 5-year graft survival.

Pancreatic Alpha-Cells Contribute Together With Beta-Cells to CXCL10 Expression in Type 1 Diabetes.

C-X-C Motif Chemokine Ligand 10 (CXCL10) is a pro-inflammatory chemokine specifically recognized by the ligand receptor CXCR3 which is mostly expressed in T-lymphocytes. Although CXCL10 expression and secretion have been widely associated to pancreatic islets both in non-obese diabetic (NOD) mice and in human type 1 diabetic (T1D) donors, the specific expression pattern among pancreatic endocrine cell subtypes has not been clarified yet. Therefore, the purpose of this study was to shed light on the pancreatic islet expression of CXCL10 in NOD, in C57Bl/6J and in NOD-SCID mice as well as in human T1D pancreata from new-onset T1D patients (DiViD study) compared to non-diabetic multiorgan donors from the INNODIA European Network for Pancreatic Organ Donors with Diabetes (EUnPOD). CXCL10 was expressed in pancreatic islets of normoglycaemic and new-onset diabetic NOD mice but not in C57Bl/6J and NOD-SCID mice. CXCL10 expression was increased in pancreatic islets of new-onset diabetic NOD mice compared to normoglycaemic NOD mice. In NOD mice, CXCL10 colocalized both with insulin and glucagon. Interestingly, CXCL10-glucagon colocalization rate was significantly increased in diabetic vs. normoglycaemic NOD mouse islets, indicating an increased expression of CXCL10 also in alpha-cells. CXCL10 was expressed in pancreatic islets of T1D patients but not in non-diabetic donors. The analysis of the expression pattern of CXCL10 in human T1D pancreata from DiViD study, revealed an increased colocalization rate with glucagon compared to insulin. Of note, CXCL10 was also expressed in alpha-cells residing in insulin-deficient islets (IDI), suggesting that CXCL10 expression in alpha cells is not driven by residual beta-cells and therefore may represent an independent phenomenon. In conclusion, we show that in T1D CXCL10 is expressed by alpha-cells both in NOD mice and in T1D patients, thus pointing to an additional novel role for alpha-cells in T1D pathogenesis and progression.

In-vitro and ex-situ regional mass spectral analysis of phospholipids and glucose in the vitreous humor from diabetic and non-diabetic human donors

The causes of vitreous humor (VH) liquefaction remain unclear. Diabetes accelerates this process and other ocular diseases. The weakening of the blood-retina barrier observed with diabetes could enhance the rate of transfer of relatively small molecules such as glucose (Glu) and phospholipids (PLs) from the retina to the VH. Glucose and PLs have been detected previously in VH but their regional distributions are not known. The mapping of Glu and PLs in VHs from subjects with and without diabetes could reveal the roles of these molecules in VH liquefaction. Diabetic and non-diabetic human eyes were acquired from the Kentucky Lions Eye Bank and frozen immediately. Each VH was removed and halved along the sagittal plane. One half was stamped on a matrix assisted laser desorption ionization (MALDI) plate. Either p-Nitroanaline (26 mg/mL MeOH:CHCl3) or 2,5-dihydroxybenzoic acid (20 mg/mL H2O:acetonitrile) was used as matrix. Glu and PLs were extracted from the remaining sections and analyzed. Data were acquired using a MALDI-mass spectrometer. The levels of Glu and PLs were significantly greater in VH from diabetics (VHd) compared with VH from non-diabetics (VHnd). VHds showed the highest relative levels of PLs in the posterior VH, followed by the anterior and central regions. Throughout the entire VH, the most abundant PLs were phosphatidylcholines followed by sphingomyelins. For Glu, the relative intensities were ~3 times higher in the posterior region of VHd (12 ± 1.3) compared with VHnd (6.5 ± 0.7) VHs. Regional studies showed that relative to the posterior VHd, the Glu levels were lower in the anterior (8.1 ± 1.0) and central (6.7 ± 0.8) regions. For the VHnds, the values for the central and anterior regions were 5.9 ± 1.2 and 4.7 ± 0.9, respectively. PLs and Glu are most abundant in the posterior region relative to the central and anterior zones of VHs. This trend was observed in VHd and VHnd, but PLs and Glu levels were significantly higher in VHds. These results support the possibility that higher levels of Glu and PLs accelerate VH liquefaction in diabetic patients. As liquefaction begins in the posterior region, the higher abundance of PLs and Glu in this zone also suggests that they may play a role in liquefaction. The specific molecular interactions affected by Glu and PLs in the collagen/hyaluronan/water network need to be examined.

Direct suppression of human islet dedifferentiation, progenitor genes, but not epithelial to mesenchymal transition by liraglutide

β-cell dedifferentiation has been accounted as one of the major mechanisms for β-cell failure; thus, is a cause to diabetes. We study direct impacts of liraglutide treatment on ex vivo human dedifferentiated islets, and its effects on genes important in endocrine function, progenitor states, and epithelial mesenchymal transition (EMT). Human islets from non-diabetic donors, were purified and incubated until day 1 and day 4, and were determined insulin contents, numbers of insulin (INS+) and glucagon (GCG+) cells. The islets from day 3 to day 7 were treated with diabetic drugs, the long acting GLP-1 receptor agonist, liraglutide. As observed in pancreatic islets of type 2 diabetic patients, ex vivo dedifferentiated islets showed more than 50% reduced insulin contents while number of glucagon increased from 10% to about 20%. β-cell specific genes: PDX1, MAFA, as well as β-cell functional markers: GLUT1 and SUR1, were significantly depleted more than 40%. Notably, we found increased levels of glucagon regulator, ARX and pre-glucagon transcripts, and remarkably upregulated progenitor expressions: NEUROG3 and ALDH1A identified as β-cell dysfunction markers in diabetic models. Hyperglucagonemia was often observed in type 2 patients that could lead to over production of gluconeogenesis by the liver. Liraglutide treatments resulted in decreased number of GCG+ cells, increased numbers of GLP-1 positive cells but did not alter elevated levels of EMT marker genes: ACTA2, CDH-2, SNAIL2, and VIM. These effects of liraglutide were blunted when FOXO1 transcripts were depleted. This work illustrates that ex vivo human isolated islets can be used as a tool to study different aspects of β-cell dedifferentiation. Our novel finding suggests a role of GLP-1 pathway in beta-cell maintenance in FOXO1-dependent manner. Importantly, dedifferentiated islets ex vivo is a useful model that can be utilized to verify the actions of potential drugs to diabetic β-cell failure.

Expression Profile of SARS-CoV-2 Host Receptors in Human Pancreatic Islets Revealed Upregulation of ACE2 in Diabetic Donors.

Cellular entry of SARS-CoV-2 is thought to occur through the binding of viral spike S1 protein to ACE2. The entry process involves priming of the S protein by TMPRSS2 and ADAM17, which collectively mediate the binding and promote ACE2 shedding. In this study, microarray and RNA-sequencing (RNA-seq) expression data were utilized to profile the expression pattern of ACE2, ADAM17, and TMPRSS2 in type 2 diabetic (T2D) and non-diabetic human pancreatic islets. Our data show that pancreatic islets express all three receptors irrespective of diabetes status. The expression of ACE2 was significantly increased in diabetic/hyperglycemic islets compared to non-diabetic/normoglycemic. Islets from female donors showed higher ACE2 expression compared to males; the expression of ADAM17 and TMPRSS2 was not affected by gender. The expression of the three receptors was statistically similar in young (≤40 years old) versus old (≥60 years old) donors. Obese (BMI > 30) donors have significantly higher expression levels of ADAM17 and TMPRSS2 relative to those from non-obese donors (BMI < 25). TMPRSS2 expression correlated positively with HbA1c and negatively with age, while ADAM17 and TMPRSS2 correlated positively with BMI. The expression of the three receptors was statistically similar in muscle and subcutaneous adipose tissues obtained from diabetic and nondiabetic donors. Lastly, ACE2 expression was higher in sorted pancreatic β-cell relative to other endocrine cells. In conclusion, ACE2 expression is increased in diabetic human islets. More studies are required to investigate whether variations of ACE2 expression could explain the severity of COVID-19 infection-related symptoms between diabetics and non-diabetic patients.

Adipose Tissue-Derived Stem Cells from Type 2 Diabetics Reveal Conservative Alterations in Multidimensional Characteristics.

Background and ObjectivesAdipose tissue-derived mesenchymal stem cells (ASCs) are recognized as an advantaged source for the prevention and treatment of diverse diseases including type 2 diabetes mellitus (T2DM). However, alterations in characteristics of ASCs from the aforementioned T2DM patients are still obscure, which also hinder the rigorous and systematic illumination of progression and pathogenesis.Methods and ResultsIn this study, we originally isolated peripancreatic adipose tissue-derived mesenchymal stem cells from both human type 2 diabetic and non-diabetic donors (T2DM-ASCs, ND-ASCs) with the parental consent, respectively. We noticed that T2DM-ASCs exhibited indistinguishable immunophenotype, cell vitality, chondrogenic differentiation and stemness as ND-ASCs. Simultaneously, there’s merely alterations in migration and immunoregulatory capacities in T2DM-ASCs. However, differing from ND-ASCs, T2DM-ASCs exhibited deficiency in adipogenic and osteogenic differentiation, and in particular, the delayed cell cycle and different cytokine expression spectrum.ConclusionsThe conservative alterations of T2DM-ASCs in multifaceted characteristics indicated the possibility of autologous application of ASCs for cell-based T2DM treatment in the future.

Differential Expression of Ormdl Genes in the Islets of Mice and Humans with Obesity

SummaryThe orosomucoid-like (Ormdl) proteins play a critical role in sphingolipid homeostasis, inflammation, and ER stress, all of which are associated with obesity and βcell dysfunction. However, their roles in β cells and obesity remain unknown. Here, we show that islets from overweight/obese human donors displayed marginally reduced ORMDL1-2 expression, whereas ORMDL3 expression was significantly downregulated compared with islets from lean donors. In contrast, Ormdl3 was substantially upregulated in the islets of leptin-deficient obese (ob/ob) mice compared with lean mice. Treatment of ob/ob mice and their islets with leptin markedly reduced islet Ormld3 expression. Ormdl3 knockdown in a β cell line induced expression of pro-apoptotic markers, which was rescued by ceramide synthase inhibitor fumonisin B1. Our results reveal differential expression of Ormdl3 in the islets of a mouse model and humans with obesity, highlight the potential effect of leptin in this differential regulation, and suggest a role for Ormdl3 in β cell apoptosis.

122-OR: Profile of Type 2 Diabetes (T2D) Donors in the Integrated Islet Distribution Program (IIDP)

The IIDP has been supplying isolated human islets and corresponding data from nondiabetic (ND) donors to diabetes researchers for &amp;gt; 15 years. Since 2010 IIDP also has offered islets from T2D donors, with up to 68% of IIDP researchers interested in receiving them. From 2010-19, this unique desirable resource comprised 7.3% of IIDP isolations (98/1,333). Analysis of ND vs. T2D donor demographics showed similar trends (p=0.94) by sex (M/F ratio 1.37 vs. 1.39, respectively). Donor race/ethnicity differed significantly (p&amp;lt;0.001), with a higher rate of Hispanic in T2D (White: ND 64.0%, T2D 43.9%; Hispanic: ND 20.5%, T2D 40.8%; Black: ND 11.1%, T2D 13.3%; Other [Asian, Hawaiian, Native American]: ND 4.4%, T2D 2.0%). This finding is in line with CDC data that Hispanics are more likely to develop T2D. Median donor age differed significantly (p&amp;lt;0.001), with a median of 46 yrs in ND (range: 4-73) vs. 53 yrs in T2D (range: 26-69). Body Mass Index (BMI) was significantly higher for T2D donors (p&amp;lt;0.001), with ND median of 29.2 (range: 11.8- 64.8) vs. 32.5 for T2D (range: 20.7-50.0). Cause of Death (COD) also differed significantly (p=0.003), with a higher rate of stroke for T2D (50.1% for ND vs. 68.4% in T2D), and higher head trauma in ND (31.2% ND vs. 13.3% T2D); other COD was 18% for both donor types. This difference is likely due to higher cardiovascular disease among T2D Americans. While median total islet equivalents (IEQ) yield was significantly higher (p=0.017) for ND donors (ND median IEQ: 278,792, range 35,000-1,015,455 vs. T2D median IEQ: 221,734, range 50,000-1,032,212), this difference was not as great as expected. A previous IIDP publication showed COD and BMI can affect islet yield. Evaluating the difference in yield between ND and T2D remained significant (p=0.02) after adjusting for these factors, with BMI category being a significant covariate, while COD was not significant in this relatively small sample. Perhaps as our sample of these unique donors grows additional associations with islet yield may emerge. Disclosure B.J. Olack: None. J. Kilburn: None. J.J. Chuang: None. J. Cravens: None. J.C. Niland: None. Funding 2UC4DK098085-02

2087-P: Functional Genomics of Human Islet Gluco/Lipotoxicity and Type 2 Diabetes

Gluco- and lipotoxicity impair β cell function and survival, potentially contributing to type 2 diabetes onset and progression. Here we profiled transcriptomes of human islets in gluco/lipotoxic and type 2 diabetes conditions. Human islets from 26 nondiabetic donors were exposed to 0.5 mM palmitate (P), 22.2 mM glucose (G), P+G, 1 mM palmitate+oleate, (1:2 ratio, P+O) and P+O+G for 2 days (n=2-5/condition), followed by 4 days of washout. Glucose-stimulated insulin secretion and RNA-seq transcriptomes were assessed. Data were compared to RNA-seq of islets from 34 type 2 diabetic and 62 nondiabetic donors by rank-rank hypergeometric overlap (RRHO), and to the Connectivity Map to identify therapeutic targets. PO did not impair insulin secretion, P or G induced transient dysfunction (resolved after washout), while PG or POG induced persistent β cell dysfunction. Among common upregulated pathways in lipo- and gluco/lipotoxicity were PPAR signaling, protein processing in the endoplasmic reticulum and aminoacyl tRNA biosynthesis (gene set enrichment analysis). The greatest overlap by RRHO between upregulated genes in gluco/lipotoxicity and type 2 diabetes was seen for washout of PG and POG (conditions of persistent β cell dysfunction), with gene set enrichment for inflammatory responses and extracellular matrix organization. Comparison of gluco/lipotoxic and type 2 diabetes gene signatures with the Connectivity Map identified endoplasmic reticulum stress signaling and knockdown of the cholesterol sensor SCAP as strongly positively correlated pathways. A negative correlation was found for JNK and DNA protein kinase inhibitors, pointing to compounds that could revert gluco/lipotoxic and type 2 diabetes gene expression signatures. In conclusion, human islet transcriptome signatures of transient or persistent gluco/lipotoxicity identify similarities to type 2 diabetes islet transcriptomes and point to novel therapeutic targets. Disclosure X. Yi: None. L. Marselli: None. A. Piron: None. M. Suleiman: None. D.L. Eizirik: None. P. Marchetti: Consultant; Self; Menarini Group. Research Support; Self; Janssen Pharmaceuticals, Inc. M. Cnop: None.

In vivo studies of glucagon secretion by human islets transplanted in mice.

Little is known about regulated glucagon secretion by human islet α cells compared to insulin secretion from β cells, despite conclusive evidence of dysfunction in both cell types in diabetes mellitus. Distinct insulins in humans and mice permit in vivo studies of human β cell regulation after human islet transplantation in immunocompromised mice, whereas identical glucagon sequences prevent analogous in vivo measures of glucagon output from human α cells. Here we use CRISPR/Cas9 editing to remove glucagon codons 2–29 in immunocompromised NSG mice, preserving production of other proglucagon-derived hormones. Glucagon knockout-NSG (GKO-NSG) mice have metabolic, liver and pancreatic phenotypes associated with glucagon signaling deficits that revert after transplantation of human islets from non-diabetic donors. Glucagon hypersecretion by transplanted islets from donors with type 2 diabetes revealed islet-intrinsic defects. We suggest that GKO-NSG mice provide an unprecedented resource to investigate human α cell regulation in vivo.

252-LB: Insulin Secretion Pathway Genes Correlate with Markers Associated with Viral Infections in Human Islets

Type 1 diabetes (T1D) is an autoimmune disease hypothesized, to be enhanced or triggered by viral infections. In this study we used organ donor pancreas to isolate islets that expressed known signs of viral infections, and subsequently investigated pathways that may be secondarily affected through analysis of gene expression profiles. Using samples provided by the Network for Pancreatic Organ Donors with Diabetes, (nPOD), islets were collected, by laser-capture, from nondiabetic donors (Control), autoantibody positive nondiabetic donors (AB+) and donors with T1D (T1D). Islets were categorized based on the presence of markers associated with viral infection (VIMs), HLA, Mx1, dsRNA, and PKR, identified via immunohistochemical staining. From each donor islets were pooled based on the number of VIMs (0 VIMs, 1 VIMs or ≥2VIMs). RNA was extracted, and microarray used to assess transcriptomes. GeneSpring software was used to generate a list of genes with differential expression between donors/VIMs. Using WebGestalt we identified pathways enriched by this list. A total of 383 genes with a fold change of ≥1.1 and p-value=0.001 had differential expression between islets with 0 VIMs and ≥1 VIMs. Pathway analysis showed strongest enrichment for genes in the insulin secretion pathway (p&amp;lt; 5.7E-5). As an example; CACNA1C and ABCC8 (important for the secretion of insulin), had lower expression in the islets with ≥1VIMs compared to 0 VIMs (p&amp;lt;0.03). CACNA1C also had lower expression in islets with 0 VIM vs. ≥1VIMs when only comparing samples from control donors (p=0.02). Islets selected for high expression for genes associated with viral infection had decreased expression of genes important for insulin secretion. This may suggest a pattern of dedifferentiation and/or functional impairment of beta cells in the setting of viral infection. Disclosure G. Nelson: None. N.I. Lenchik: None. T. Rodriguez-Calvo: None. D. Balcacean: None. I.C. Gerling: None. Funding JDRF; National Institute of Diabetes and Digestive and Kidney Diseases; Lebonheur Children’s Hospital