Publications by authors named "Rita Bottino"

124 Publications

Autologous Islet Transplantation After Total Pancreatectomy in a Patient Recovered from SARS-CoV-2: A Case Report.

Am J Case Rep 2022 Feb 12;23:e935142. Epub 2022 Feb 12.

Department of Surgery, Division of Transplant Medicine, Allegheny Health Network, Pittsburgh, PA, USA.

BACKGROUND SARS-CoV-2 infection or COVID-19 disease has been linked to the onset of diabetes and metabolic dysregulation because it has been suggested that viral entry proteins, specifically ACE2 and TMPRSS2, are expressed in the exocrine cells and ductal epithelium of the pancreas. Because of the unknown effect this can have on islet function, there can be doubt that patients with previous SARS-CoV-2 infections are good candidates for autologous islet transplantation after total pancreatectomy (TPAIT). CASE REPORT A patient with a history of chronic pancreatitis and previous non-surgical interventions was presented as a viable candidate for TPAIT at our institution. Approximately 1 month later, the patient contracted a SARS-CoV-2 infection, resulting in a mild case of COVID-19. The infection resolved without the need for hospitalization. At the time of this occurrence, COVID-19 was primarily considered a respiratory ailment, and little was known of the potential association between metabolic dysfunction and SARS-CoV-2. Islet isolation and surgery proceeded in a textbook manner with no surgical complications. The patient was weaned off exogenous insulin within 3 months after transplantation. CONCLUSIONS Favorable outcomes after surgery included pain reduction, islet function, and improved quality of life for the patient in the first 6 months after the procedure. These successful results demonstrate that SARS-CoV-2 infection did not prevent the patient from achieving good glucose regulation after auto-islet transplantation. This outcome suggests that, at least in this instance of mild infection, there were no long-lasting negative COVID-19-associated effects on the transplanted islets that might impact islet function.
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http://dx.doi.org/10.12659/AJCR.935142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8848141PMC
February 2022

Integrated Analysis of the Pancreas and Islets Reveals Unexpected Findings in Human Male With Type 1 Diabetes.

J Endocr Soc 2021 Dec 29;5(12):bvab162. Epub 2021 Oct 29.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA.

Clinical and pathologic heterogeneity in type 1 diabetes is increasingly being recognized. Findings in the islets and pancreas of a 22-year-old male with 8 years of type 1 diabetes were discordant with expected results and clinical history (islet autoantibodies negative, hemoglobin A1c 11.9%) and led to comprehensive investigation to define the functional, molecular, genetic, and architectural features of the islets and pancreas to understand the cause of the donor's diabetes. Examination of the donor's pancreatic tissue found substantial but reduced β-cell mass with some islets devoid of β cells (29.3% of 311 islets) while other islets had many β cells. Surprisingly, isolated islets from the donor pancreas had substantial insulin secretion, which is uncommon for type 1 diabetes of this duration. Targeted and whole-genome sequencing and analysis did not uncover monogenic causes of diabetes but did identify high-risk human leukocyte antigen haplotypes and a genetic risk score suggestive of type 1 diabetes. Further review of pancreatic tissue found islet inflammation and some previously described α-cell molecular features seen in type 1 diabetes. By integrating analysis of isolated islets, histological evaluation of the pancreas, and genetic information, we concluded that the donor's clinical insulin deficiency was most likely the result autoimmune-mediated β-cell loss but that the constellation of findings was not typical for type 1 diabetes. This report highlights the pathologic and functional heterogeneity that can be present in type 1 diabetes.
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http://dx.doi.org/10.1210/jendso/bvab162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8633619PMC
December 2021

Bioenergetics of Islet Preparations in a Pilot Clinical Trial of Peri-Transplant Hydroxychloroquine for Autologous Islet Transplantation.

Cell Transplant 2021 Jan-Dec;30:9636897211057440

Endocrinology and Metabolism Institute, Cleveland Clinic, Cleveland, OH, USA.

The inflammatory response is an obstacle to success in both allogeneic and autologous islet transplantation. In autologous islet transplantation (AIT), however, the recipient is also the donor, permitting pretreatment of donor/recipient for a controlled duration prior to transplantation. We sought to exploit this feature of (AIT) by pretreating donor/recipients with chronic pancreatitis undergoing total pancreatectomy and autologous islet transplantation (TPAIT) to test the hypothesis that peri-transplant treatment with the FDA-approved anti-inflammatory hydroxychloroquine (HCQ) improves graft function. In this randomized placebo-controlled pilot clinical study, patients ( = 6) were treated with oral HCQ for 30 days prior to and 90 days after TPAIT. In vivo islet function was assessed via Mixed Meal Tolerance Testing before HCQ treatment, 6- and 12-months after surgery. In vitro islet bioenergetics were assessed at the time of transplantation via extracellular flux analysis of islet preparation samples from the clinical trial cohort and six additional patients ( = 12). Our study shows that HCQ did not alter clinical endpoints, but HCQ-treated patients showed greater spare respiratory capacity (SRC) compared to samples from control patients (=0.028). Glycolytic metabolism of islet preparations directly correlated with stimulated C-peptide secretion both before and after TPAIT (=0.01, =0.489 and =0.03, =0.674, respectively), and predicted in vivo islet function better than mitochondrial metabolism of islet preps or islet equivalents infused. Overnight culture of islet preparations altered bioenergetic function, significantly decreasing SRC and maximal respiration (<0.001). In conclusion, while HCQ did not alter clinical outcomes, it was associated with significantly increased SRC in islet preparations. Bioenergetic analyses of islet preparations suggests that culture should be avoided and that glycolysis may be a more sensitive indicator of in vivo islet function than current metrics, including islet oxygen consumption and islet equivalents infused.
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http://dx.doi.org/10.1177/09636897211057440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8586172PMC
February 2022

Adult Pig Islet Isolation.

J Vis Exp 2021 10 21(176). Epub 2021 Oct 21.

Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Institute of Translational Medicine, First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital;

Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic β cells, which results in little or no insulin production. Islet transplantation plays an important role in the treatment of T1DM, with the improved glycometabolic control, the reduced progression of complications, the reduction of hypoglycemic episodes when compared with traditional insulin therapy. The results of phase III clinical trial also demonstrated the safety and efficacy of islet allotransplantation in T1DM. However, the shortage of pancreas donors limits its widespread use. Animals as a source of islets such as the pig offer an alternative choice. Because the architecture of the pig pancreas is different from the islets of mice or humans, the pig islet isolation procedure is still challenging. Since the translation of alternative porcine islet sources (xenogeneic) to the clinical setting for treating T1DM through cellular transplantation is of great importance, a cost-effective, standardized, and reproducible protocol for isolating porcine islets is urgently needed. This manuscript describes a simplified and cost-effective method to isolate and purify adult porcine islets based on the previous protocols that have successfully transplanted porcine islets to non-human primates. This will be a beginners guide without the use of specialized equipment such as a COBE 2991 Cell Processor.
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http://dx.doi.org/10.3791/63017DOI Listing
October 2021

Clinical Significance of Postoperative Antibiotic Treatment for Positive Islet Cultures After Total Pancreatectomy With Islet Autotransplantation.

Pancreas 2021 08;50(7):1000-1006

From the Department of General Surgery, Digestive Disease and Surgery Institute, Cleveland Clinic, Cleveland, OH.

Objectives: Islet cultures are routinely performed in total pancreatectomy with islet autotransplantation (TPIAT), and the need for empiric antibiotic treatment based on culture results is unknown. We evaluated the effect of postoperative antibiotic treatment for positive islet cultures on clinical infection.

Methods: Seventy-nine patients undergoing TPIAT were reviewed. Prophylactic perioperative ceftriaxone and metronidazole were administered, and transplanted islet preparations included ciprofloxacin. Postoperative antibiotics were not routinely given for positive cultures unless a clinical infection was suspected. The primary end point was 30-day infectious complications.

Results: Fifty-one patients (65%) had a positive culture. Overall, 39 patients (87%) had organisms susceptible to our perioperative antibiotic regimen. There was no difference in the infectious complication rate between those with positive compared with negative cultures (16% vs 29%, P = 0.17). Patients with a positive culture had similar 30-day postoperative infectious complication rates whether receiving postoperative antibiotics (n = 7) or not (14% vs 16%, P = 0.91). Only 1 patient had a correlation of clinical and islet cultures.

Conclusions: Beyond prophylactic antibiotics, empiric antibiotic treatment for a positive culture is not warranted and provides a rationale for the abandonment of routine cultures in TPIAT.
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http://dx.doi.org/10.1097/MPA.0000000000001874DOI Listing
August 2021

Single-cell analyses of human pancreas: characteristics of two populations of acinar cells in chronic pancreatitis.

Am J Physiol Gastrointest Liver Physiol 2021 11 15;321(5):G449-G460. Epub 2021 Sep 15.

Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.

Chronic pancreatitis (CP) is a complex inflammatory disorder with numerous associated genetic and environmental risk factors. The most distressing characteristic of CP is recalcitrant pain, often requiring surgical resection including total pancreatectomy with islet autotransplantation (TPIAT). We studied five consented subjects undergoing pancreatic resection and processed isolated cells for single-cell RNA sequencing (scRNA-Seq). Using high-dimensional transcriptomic cluster analysis, we identified 11 unique cell clusters in the pancreas tissue. These cell clusters include a cluster of undifferentiated/dedifferentiated cells and two unique clusters of acinar cells, one of which appears to be in a transitional stage. To determine the cellular response to protease inhibitor and stimulation, we treated aliquots of cells from one subject with a protease inhibitor cocktail with and without bethanechol (a muscarinic receptor agonist) at 100 and 400 µM and compared gene expression profiles. The protease inhibitors appeared to reduce cell stress. Pancreatic digestive enzymes and islet hormones were upregulated in both doses of bethanechol-treated cells compared with naïve cells. High-dose bethanechol appeared to be toxic and consistent with hyperstimulation. These studies demonstrate the feasibility of investigating human acinar cell physiology at the single-cell level and initial evidence that these cells retain responsiveness to agonist stimulation with predicted second messenger and transcriptomic responses. We conducted single cell RNA sequencing on pancreas tissue from five individuals. We identified eleven unique cell clusters including a large population of dedifferentiated cells as well as two unique clusters of acinar cells, one of which appears to exist in a transitional state. We also examined the cellular response of pancreas tissue to stimulation and identified affected genes and pathways, including pancreatic digestive enzymes.
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http://dx.doi.org/10.1152/ajpgi.00482.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8616588PMC
November 2021

Combinatorial transcription factor profiles predict mature and functional human islet α and β cells.

JCI Insight 2021 09 22;6(18). Epub 2021 Sep 22.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells, and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled more than 40,000 cells from normal human islets by single-cell RNA-Seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells coexpressing ARX/MAFB (α cells) and MAFA/MAFB (β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-Seq, MAFA/MAFB-coexpressing β cells showed enhanced electrophysiological activity. Thus, these results indicate that combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.
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http://dx.doi.org/10.1172/jci.insight.151621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8492318PMC
September 2021

Controls of Hyperglycemia Improves Dysregulated Microbiota in Diabetic Mice.

Transplantation 2021 09;105(9):1980-1988

Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen University Health Science Center, Shenzhen University School of Medicine, First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, Guangdong, China.

Background: Type 1 diabetes (T1DM) is a chronic autoimmune disease characterized by T-cell-mediated destruction of insulin-producing beta cells. Evidence shows that patients with T1DM and mice used in specific diabetic models both exhibit changes in their intestinal microbiota and dysregulated microbiota contributes to the pathogenesis of T1DM. Islet transplantation (Tx) is poised to play an important role in the treatment of T1DM. However, whether treatment of T1DM with islet Tx can rescue dysregulated microbiota remains unclear.

Methods: In this study, we induced diabetic C57BL/6 mice with streptozotocin. Then treatment with either insulin administration, or homogenic or allogenic islet Tx was performed to the diabetic mice. Total DNA was isolated from fecal pellets and high-throughput 16S rRNA sequencing was used to investigate intestinal microbiota composition.

Results: The overall microbial diversity was comparable between control (nonstreptozotocin treated) and diabetic mice. Our results showed the ratio of the Bacteroidetes: Firmicutes between nondiabetic and diabetic mice was significant different. Treatment with islet Tx or insulin partially corrects the dysregulated bacterial composition. At the genus level, Bacteroides, Odoribacter, and Alistipes were associated with the progression and treatment efficacy of the disease, which may be used as a biomarker to predict curative effect of treatment for patients with T1DM.

Conclusions: Collectively, our results indicate that diabetic mice show changed microbiota composition and that treatment with insulin and islet Tx can partially correct the dysregulated microbiota.
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http://dx.doi.org/10.1097/TP.0000000000003603DOI Listing
September 2021

Human Hemangioblast-Derived Mesenchymal Stem Cells Promote Islet Engraftment in a Minimal Islet Mass Transplantation Model in Mice.

Front Med (Lausanne) 2021 15;8:660877. Epub 2021 Apr 15.

Institute of Cellular Therapeutics, Allegheny-Singer Research Institute, Allegheny Health Network, Pittsburgh, PA, United States.

Islet transplantation can restore glycemic control in patients with type 1 diabetes. Using this procedure, the early stages of engraftment are often crucial to long-term islet function, and outcomes are not always successful. Numerous studies have shown that mesenchymal stem cells (MSCs) facilitate islet graft function. However, experimental data can be inconsistent due to variables associated with MSC generation (including donor characteristics and tissue source), thus, demonstrating the need for a well-characterized and uniform cell product before translation to the clinic. Unlike bone marrow- or adipose tissue-derived MSCs, human embryonic stem cell-derived-MSCs (hESC-MSCs) offer an unlimited source of stable and highly-characterized cells that are easily scalable. Here, we studied the effects of human hemangioblast-derived mesenchymal cells (HMCs), (i.e., MSCs differentiated from hESCs using a hemangioblast intermediate), on islet cell transplantation using a minimal islet mass model. The co-transplantation of the HMCs allowed a mass of islets that was insufficient to correct diabetes on its own to restore glycemic control in all recipients. Our studies help to elucidate the mechanisms including reduction of cytokine stress by which the HMCs support islet graft protection . Derivation, stability, and scalability of the HMC source may offer unique advantages for clinical applications, including fewer islets needed for successful islet transplantation.
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http://dx.doi.org/10.3389/fmed.2021.660877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8081894PMC
April 2021

Dopamine regulates pancreatic glucagon and insulin secretion via adrenergic and dopaminergic receptors.

Transl Psychiatry 2021 02 16;11(1):59. Epub 2021 Feb 16.

Translational Neuroscience Program, Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA, USA.

Dopamine (DA) and norepinephrine (NE) are catecholamines primarily studied in the central nervous system that also act in the pancreas as peripheral regulators of metabolism. Pancreatic catecholamine signaling has also been increasingly implicated as a mechanism responsible for the metabolic disturbances produced by antipsychotic drugs (APDs). Critically, however, the mechanisms by which catecholamines modulate pancreatic hormone release are not completely understood. We show that human and mouse pancreatic α- and β-cells express the catecholamine biosynthetic and signaling machinery, and that α-cells synthesize DA de novo. This locally-produced pancreatic DA signals via both α- and β-cell adrenergic and dopaminergic receptors with different affinities to regulate glucagon and insulin release. Significantly, we show DA functions as a biased agonist at α-adrenergic receptors, preferentially signaling via the canonical G protein-mediated pathway. Our findings highlight the interplay between DA and NE signaling as a novel form of regulation to modulate pancreatic hormone release. Lastly, pharmacological blockade of DA D-like receptors in human islets with APDs significantly raises insulin and glucagon release. This offers a new mechanism where APDs act directly on islet α- and β-cell targets to produce metabolic disturbances.
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http://dx.doi.org/10.1038/s41398-020-01171-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884786PMC
February 2021

Single-Nucleus and In Situ RNA-Sequencing Reveal Cell Topographies in the Human Pancreas.

Gastroenterology 2021 03 16;160(4):1330-1344.e11. Epub 2020 Nov 16.

Center for Digital Health, Berlin Institute of Health and Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany. Electronic address:

Background & Aims: Molecular evidence of cellular heterogeneity in the human exocrine pancreas has not been yet established because of the local concentration and cascade of hydrolytic enzymes that can rapidly degrade cells and RNA upon pancreatic resection. We sought to better understand the heterogeneity and cellular composition of the pancreas in neonates and adults in healthy and diseased conditions using single-cell sequencing approaches.

Methods: We innovated single-nucleus RNA-sequencing protocols and profiled more than 120,000 cells from pancreata of adult and neonatal human donors. We validated the single-nucleus findings using RNA fluorescence in situ hybridization, in situ sequencing, and computational approaches.

Results: We created the first comprehensive atlas of human pancreas cells including epithelial and nonepithelial constituents, and uncovered 3 distinct acinar cell types, with possible implications for homeostatic and inflammatory processes of the pancreas. The comparison with neonatal single-nucleus sequencing data showed a different cellular composition of the endocrine tissue, highlighting the tissue dynamics occurring during development. By applying spatial cartography, involving cell proximity mapping through in situ sequencing, we found evidence of specific cell type neighborhoods, dynamic topographies in the endocrine and exocrine pancreas, and principles of morphologic organization of the organ. Furthermore, similar analyses in chronic pancreatitis biopsy samples showed the presence of acinar-REG cells, a reciprocal association between macrophages and activated stellate cells, and a new potential role of tuft cells in this disease.

Conclusions: Our human pancreas cell atlas can be interrogated to understand pancreatic cell biology and provides a crucial reference set for comparisons with diseased tissue samples to map the cellular foundations of pancreatic diseases.
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http://dx.doi.org/10.1053/j.gastro.2020.11.010DOI Listing
March 2021

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 Are Expressed in the Microvasculature and Ducts of Human Pancreas but Are Not Enriched in β Cells.

Cell Metab 2020 12 13;32(6):1028-1040.e4. Epub 2020 Nov 13.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA; VA Tennessee Valley Healthcare System, Nashville, TN 37212, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address:

Isolated reports of new-onset diabetes in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2 is directly cytotoxic to pancreatic islet β cells. This would require binding and entry of SARS-CoV-2 into β cells via co-expression of its canonical cell entry factors, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2); however, their expression in human pancreas has not been clearly defined. We analyzed six transcriptional datasets of primary human islet cells and found that ACE2 and TMPRSS2 were not co-expressed in single β cells. In pancreatic sections, ACE2 and TMPRSS2 protein was not detected in β cells from donors with and without diabetes. Instead, ACE2 protein was expressed in islet and exocrine tissue microvasculature and in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. These findings reduce the likelihood that SARS-CoV-2 directly infects β cells in vivo through ACE2 and TMPRSS2.
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http://dx.doi.org/10.1016/j.cmet.2020.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664344PMC
December 2020

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 are Expressed in the Pancreas but are Not Enriched in Islet Endocrine Cells.

bioRxiv 2020 Oct 20. Epub 2020 Oct 20.

Reports of new-onset diabetes and diabetic ketoacidosis in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2, the virus that causes COVID-19, is directly cytotoxic to pancreatic islet β cells. This would require binding and entry of SARS-CoV-2 into host β cells via cell surface co-expression of ACE2 and TMPRSS2, the putative receptor and effector protease, respectively. To define ACE2 and TMPRSS2 expression in the human pancreas, we examined six transcriptional datasets from primary human islet cells and assessed protein expression by immunofluorescence in pancreata from donors with and without diabetes. and transcripts were low or undetectable in pancreatic islet endocrine cells as determined by bulk or single cell RNA sequencing, and neither protein was detected in α or β cells from these donors. Instead, ACE2 protein was expressed in the islet and exocrine tissue microvasculature and also found in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. The absence of significant ACE2 and TMPRSS2 co-expression in islet endocrine cells reduces the likelihood that SARS-CoV-2 directly infects pancreatic islet β cells through these cell entry proteins.
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http://dx.doi.org/10.1101/2020.08.31.275719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587777PMC
October 2020

How the COVID-19 pandemic may impact public support for clinical xenotransplantation in the United States?

Xenotransplantation 2020 09 28;27(5):e12623. Epub 2020 Jun 28.

Institute of Cellular Therapeutics, Allegheny Health Network, Pittsburgh, PA, USA.

Many patients who would undergo organ transplantation cannot proceed due to the inability of human organ donation to satisfy medical needs. Xenotransplantation has the potential to offer unlimited availability of pig organs for transplantation, and pig-to-non-human primate models have demonstrated outcomes that may soon justify clinical trials. However, one of the unique ethical challenges faced by xenotransplantation is that the risk of introducing potential zoonotic disease into the community must be weighed along with the benefit to the patient. While most experts believe that zoonosis is manageable, apprehension over disease transmission from animal donors to human recipients remains a frequent concern of many who are undecided or opposed to clinical xenotransplantation. The COVID-19 pandemic represents a scenario (rapid worldwide spread of a highly contagious novel zoonotic disease with no natural defense in humans) that would seem to justify apprehension, especially in the United States, which has largely avoided previous pandemic outbreaks. However, there are many differences between zoonosis found in the wild or after xenotransplantation that favor the safety of the latter. Still, these differences, as well as the benefits of xenotransplantation, are not widely understood outside of the field. We must therefore ask what impact the COVID-19 pandemic will have on attitudes toward xenotransplantation.
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http://dx.doi.org/10.1111/xen.12623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361153PMC
September 2020

Dapagliflozin Does Not Directly Affect Human α or β Cells.

Endocrinology 2020 08;161(8)

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee.

Selective inhibitors of sodium glucose cotransporter-2 (SGLT2) are widely used for the treatment of type 2 diabetes and act primarily to lower blood glucose by preventing glucose reabsorption in the kidney. However, it is controversial whether these agents also act on the pancreatic islet, specifically the α cell, to increase glucagon secretion. To determine the effects of SGLT2 on human islets, we analyzed SGLT2 expression and hormone secretion by human islets treated with the SGLT2 inhibitor dapagliflozin (DAPA) in vitro and in vivo. Compared to the human kidney, SLC5A2 transcript expression was 1600-fold lower in human islets and SGLT2 protein was not detected. In vitro, DAPA treatment had no effect on glucagon or insulin secretion by human islets at either high or low glucose concentrations. In mice bearing transplanted human islets, 1 and 4 weeks of DAPA treatment did not alter fasting blood glucose, human insulin, and total glucagon levels. Upon glucose stimulation, DAPA treatment led to lower blood glucose levels and proportionally lower human insulin levels, irrespective of treatment duration. In contrast, after glucose stimulation, total glucagon was increased after 1 week of DAPA treatment but normalized after 4 weeks of treatment. Furthermore, the human islet grafts showed no effects of DAPA treatment on hormone content, endocrine cell proliferation or apoptosis, or amyloid deposition. These data indicate that DAPA does not directly affect the human pancreatic islet, but rather suggest an indirect effect where lower blood glucose leads to reduced insulin secretion and a transient increase in glucagon secretion.
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http://dx.doi.org/10.1210/endocr/bqaa080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375801PMC
August 2020

Decreased pancreatic acinar cell number in type 1 diabetes.

Diabetologia 2020 07 9;63(7):1418-1423. Epub 2020 May 9.

Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, 7465 Medical Research Bldg IV, 2215 Garland Avenue, Nashville, TN, 37232-0475, USA.

Aims/hypothesis: Individuals with longstanding and recent-onset type 1 diabetes have a smaller pancreas. Since beta cells represent a very small portion of the pancreas, the loss of pancreas volume in diabetes is primarily due to the loss of pancreatic exocrine mass. However, the structural changes in the exocrine pancreas in diabetes are not well understood.

Methods: To characterise the pancreatic endocrine and exocrine compartments in diabetes, we studied pancreases from adult donors with type 1 diabetes compared with similarly aged donors without diabetes. Islet cell mass, islet morphometry, exocrine mass, acinar cell size and number and pancreas fibrosis were assessed by immunohistochemical staining. To better understand possible mechanisms of altered pancreas size, we measured pancreas size in three mouse models of insulin deficiency.

Results: Pancreases from donors with type 1 diabetes were approximately 45% smaller than those from donors without diabetes (47.4 ± 2.6 vs 85.7 ± 3.7 g), independent of diabetes duration or age of onset. Diabetic donor pancreases had decreased beta cell mass (0.061 ± 0.025 vs 0.94 ± 0.21 g) and reduced total exocrine mass (42.0 ± 4.9 vs 96.1 ± 6.5 g). Diabetic acinar cells were similar in size but fewer in number compared with those in pancreases from non-diabetic donors (63.7 ± 8.1 × 10 vs 121.6 ± 12.2 × 10 cells/pancreas), likely accounting for the difference in pancreas size. Within the type 1 diabetes exocrine tissue, there was a greater degree of fibrosis. The pancreases in three mouse models of insulin deficiency were similar in size to those in control mice.

Conclusions/interpretation: Pancreases from donors with type 1 diabetes are smaller than normal donor pancreases because exocrine cells are fewer in number rather than smaller in size; these changes occur early in the disease process. Our mouse data suggest that decreased pancreas size in type 1 diabetes is not directly caused by insulin deficiency, but the precise mechanism responsible remains unclear.
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http://dx.doi.org/10.1007/s00125-020-05155-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403487PMC
July 2020

Integrated human pseudoislet system and microfluidic platform demonstrate differences in GPCR signaling in islet cells.

JCI Insight 2020 05 21;5(10). Epub 2020 May 21.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Pancreatic islets secrete insulin from β cells and glucagon from α cells, and dysregulated secretion of these hormones is a central component of diabetes. Thus, an improved understanding of the pathways governing coordinated β and α cell hormone secretion will provide insight into islet dysfunction in diabetes. However, the 3D multicellular islet architecture, essential for coordinated islet function, presents experimental challenges for mechanistic studies of intracellular signaling pathways in primary islet cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles. We demonstrate the utility of this experimental approach by studying the effects of Gi and Gq GPCR pathways on insulin and glucagon secretion by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of Gi signaling reduced insulin and glucagon secretion, while activation of Gq signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion, which occur through changes in intracellular Ca2+. The experimental approach of combining pseudoislets with a microfluidic system allowed the coregistration of intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human β and α cells.
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http://dx.doi.org/10.1172/jci.insight.137017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259531PMC
May 2020

Molecular and genetic regulation of pig pancreatic islet cell development.

Development 2020 03 30;147(6). Epub 2020 Mar 30.

Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA

Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases such as diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet β-, α- and δ-cells followed by transcriptome analysis (RNA-seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features that are not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet β-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to β-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.
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http://dx.doi.org/10.1242/dev.186213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132804PMC
March 2020

Pig-to-Macaque Islet Xenotransplantation.

Methods Mol Biol 2020 ;2110:289-314

Institute of Cellular Therapeutics, Allegheny Health Network Research Institute, Allegheny Health Network, Pittsburgh, PA, USA.

The advancement toward a clinical application for porcine islets to cure diabetes in humans must include reproducible long-term successes in non-human primate (NHP) models. Many dedicated researchers around the world are continuing to work toward this goal. In this chapter, we describe procedures for islet isolation of pancreatic islets from adult and neonatal/fetal pigs. We further include procedures for the induction of diabetes in non-human primates and subsequent insulin therapy, islet transplantation, immunosuppression, and also the daily maintenance of xenotransplanted NHPs. The procedures that we outline in this chapter are ones that we have successfully utilized in pig-to-NHP islet transplantation models. However, where appropriate, alternative methods will also be identified.
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http://dx.doi.org/10.1007/978-1-0716-0255-3_19DOI Listing
January 2021

Tacrolimus- and sirolimus-induced human β cell dysfunction is reversible and preventable.

JCI Insight 2020 01 16;5(1). Epub 2020 Jan 16.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, and.

Posttransplantation diabetes mellitus (PTDM) is a common and significant complication related to immunosuppressive agents required to prevent organ or cell transplant rejection. To elucidate the effects of 2 commonly used agents, the calcineurin inhibitor tacrolimus (TAC) and the mTOR inhibitor sirolimus (SIR), on islet function and test whether these effects could be reversed or prevented, we investigated human islets transplanted into immunodeficient mice treated with TAC or SIR at clinically relevant levels. Both TAC and SIR impaired insulin secretion in fasted and/or stimulated conditions. Treatment with TAC or SIR increased amyloid deposition and islet macrophages, disrupted insulin granule formation, and induced broad transcriptional dysregulation related to peptide processing, ion/calcium flux, and the extracellular matrix; however, it did not affect regulation of β cell mass. Interestingly, these β cell abnormalities reversed after withdrawal of drug treatment. Furthermore, cotreatment with a GLP-1 receptor agonist completely prevented TAC-induced β cell dysfunction and partially prevented SIR-induced β cell dysfunction. These results highlight the importance of both calcineurin and mTOR signaling in normal human β cell function in vivo and suggest that modulation of these pathways may prevent or ameliorate PTDM.
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http://dx.doi.org/10.1172/jci.insight.130770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7030815PMC
January 2020

Asparagine Synthetase Is Highly Expressed at Baseline in the Pancreas Through Heightened PERK Signaling.

Cell Mol Gastroenterol Hepatol 2020 14;9(1):1-13. Epub 2019 Aug 14.

Department of Pediatrics, Stanford University, Palo Alto, California. Electronic address:

Asparaginase (ASNase) causes pancreatitis in approximately 10% of leukemia patients, and the mechanisms underlying this painful complication are not known. ASNase primarily depletes circulating asparagine, and the endogenously expressed enzyme, asparagine synthetase (ASNS), replenishes asparagine. ASNS was suggested previously to be highly expressed in the pancreas. In this study, we determined the expression pattern of ASNS in the pancreas and the mechanism for increased pancreatic ASNS abundance. Compared with other organs, ASNS was highly expressed in both the human and mouse pancreas, and, within the pancreas, ASNS was present primarily in the acinar cells. The high baseline pancreatic ASNS was associated with higher baseline activation of protein kinase R-like endoplasmic reticulum kinase (PERK) signaling in the pancreas, and inhibition of PERK in acinar cells lessened ASNS expression. ASNase exposure, but not the common pancreatitis triggers, uniquely up-regulated ASNS expression, indicating that the increase is mediated by nutrient stress. The up-regulation of acinar ASNS with ASNase exposure was owing to increased transcriptional rather than delayed degradation. Knockdown of ASNS in the 266-6 acinar cells provoked acinar cell injury and worsened ASNase-induced injury, whereas ASNS overexpression protected against ASNase-induced injury. In summary, ASNS is highly expressed in the pancreatic acinar cells through heightened basal activation of PERK, and ASNS appears to be crucial to maintaining acinar cell integrity. The implications are that ASNS is especially hardwired in the pancreas to protect against both baseline perturbations and nutrient deprivation stressors, such as during ASNase exposure.
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http://dx.doi.org/10.1016/j.jcmgh.2019.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881672PMC
May 2021

Is the renal subcapsular space the preferred site for clinical porcine islet xenotransplantation? Review article.

Int J Surg 2019 Sep 30;69:100-107. Epub 2019 Jul 30.

Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA. Electronic address:

It can reasonably be anticipated that, within 5-10 years, islet allotransplantation or pig islet xenotransplantation may be the preferred options for β-cell replacement therapy. The portal vein/liver is currently the preferred clinical site for free islet transplantation, constituting 90% of clinical islet transplants. Despite being the site of choice for rodent and some large animal studies, the renal subcapsular space is rarely used clinically, even though the introduction of islets intraportally is not entirely satisfactory (particularly for pig islet xenotransplantation). We questioned why this might be so. Is it perhaps based on prior clinical evidence, or from experience in nonhuman primates? When we have questioned experts in the field, no definitive answers have been forthcoming. We have therefore reviewed the relevant literature, and still cannot find a convincing reason why the renal subcapsular space has been so relatively abandoned as a site for clinical islet transplantation. Owing to its sequestered environment, subcapsular transplantation might avoid some of the remaining challenges of intraportal transplantation. This may be particularly true when using pig islets for xenotransplantation, which are exceptionally pure in comparison to human islets used in auto- or allo-transplantation. With evidence from the literature, we question the notion that the subcapsular space is inhospitable to islet transplantation and suggest that, when porcine islet transplantation is introduced, this site should perhaps be reconsidered.
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http://dx.doi.org/10.1016/j.ijsu.2019.07.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745254PMC
September 2019

Change in Functional Beta Cell Capacity With Time Following Autologous Islet Transplantation.

Pancreas 2019 May/Jun;48(5):656-661

From the Endocrinology and Metabolism Institute and.

Objectives: Autologous islet transplantation (AIT) is performed to preserve insulin secretory function in chronic pancreatitis patients undergoing total pancreatectomy (TP). No data exist on the effect of time lapse on beta cell function post TP-AIT. We aimed to investigate the factor of time lapse on beta cell function following TP-AIT.

Methods: Retrospectively, we identified 31 adult patients with chronic pancreatitis who underwent TP-AIT between 2008 and 2016. Changes in beta cell function were assessed using (1) BETA-2 scores and (2) analysis of posttransplant mixed-meal tolerance testing.

Results: Significant decrease in functional beta cell capacity expressed by BETA-2 scores was seen in the first 2 years following TP-AIT, with an annual decrease of 6.3 points in median BETA-2 score (interquartile range, 4.6-11.6; P = 0.002). In the mixed-meal tolerance testing analysis, nonsignificant trends toward higher glucose, lower insulin, and lower C-peptide were seen with time lapse. Additionally, higher hemoglobin A1c values (P = 0.033) and higher insulin requirements (P = 0.04) were seen with longer follow-up after AIT.

Conclusions: A steady drop in functional beta cell capacity was observed in the 2 years following TP and AIT. To our knowledge, to date this is the first report of the BETA-2 score applicability in the AIT setting.
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http://dx.doi.org/10.1097/MPA.0000000000001315DOI Listing
February 2020

Mesenteric lymph nodes as alternative site for pancreatic islet transplantation in a diabetic rat model.

BMC Surg 2019 Apr 24;18(Suppl 1):126. Epub 2019 Apr 24.

Department of Drug Science, Biochemistry Section, University of Catania, Catania, Italy.

Background: Islet transplantation has progressively become a safe alternative to pancreas transplantation for the treatment of type 1 diabetes. However, the long-term results of islet transplantation could be significantly increased by improving the quality of the islet isolation technique even exploring alternative islet transplantation sites to reduce the number of islets required to mitigate hyperglycemia. The goal of the study was to test the lymph node as a suitable anatomical location for islet engraftment in a rodent model.

Methods: Forty Lewis rats, 6-8 weeks old, body weight 250-300 g, have been used as islet donors and recipients in syngeneic islet transplantation experiments. Ten rats were rendered diabetic by one injection of 65 mg/Kg of streptozotocin. After pancreas retrieval from non diabetic donors, islet were isolated and transplanted in the mesenteric lymph nodes of 7 diabetic rats. Rats were followed for 30 days after islet transplantation.

Results: A total of 7 islet transplantations in mesenteric lymph nodes have been performed. Two rats died 24 and 36 h after transplantation due to complications. No transplanted rat acquired normal glucose blood levels and insulin independence after the transplantation. However, the mean blood levels of glycemia were significantly lower in transplanted rats compared with diabetic rats (470.4 mg/dl vs 605 mg/dl, p 0.04). Interestingly, transplanted rats have a significant weight increase after transplantation compared to diabetic rats (mean value 295 g in transplanted rats vs 245 g in diabetic rats, p < 0.05), with an overall improvement of social activities and health. Immunohistochemical analysis of the 5 mesenteric lymph nodes of transplanted rats demonstrated the presence of living islets in one lymph node.

Conclusions: Although islet engraftment in lymph nodes is possible, islet transplantation in lymph nodes in rats resulted in few improvements of glucose parameters.
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http://dx.doi.org/10.1186/s12893-018-0452-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402566PMC
April 2019

Immunological aspects of allogeneic pancreatic islet transplantation: a comparison between mouse and human.

Transpl Int 2019 09 15;32(9):903-912. Epub 2019 May 15.

Department of Surgery, Geneva University Hospitals and Medical Faculty, Geneva, Switzerland.

Pancreatic islet allotransplantation is a treatment for patients with severe forms of type 1 diabetes. As long-term graft function and survival are not yet optimal, additional studies are warranted in order to continue improving transplant outcomes. The mechanisms of islet graft loss and tolerance induction are often studied in murine diabetes models. Despite numerous islet transplantation studies successfully performed over recent years, translation from experimental mouse models to human clinical application remains elusive. This review aims at critically discussing the strengths and limitations of current mouse models of diabetes and experimental islet transplantation. In particular, we will analyze the causes leading to diabetes and compare the immunological mechanisms responsible for rejection between mouse and human. A better understanding of the experimental mouse models should facilitate translation to human clinical application.
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http://dx.doi.org/10.1111/tri.13445DOI Listing
September 2019

Xenotransplantation literature update, January/February 2019.

Xenotransplantation 2019 03 11;26(2):e12518. Epub 2019 Apr 11.

Department of Surgery, Schulze Diabetes Institute, University of Minnesota, Minneapolis, Minnesota.

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http://dx.doi.org/10.1111/xen.12518DOI Listing
March 2019

HLA Class II Antigen Processing and Presentation Pathway Components Demonstrated by Transcriptome and Protein Analyses of Islet β-Cells From Donors With Type 1 Diabetes.

Diabetes 2019 05 4;68(5):988-1001. Epub 2019 Mar 4.

Division of Diabetes, Department of Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA

Type 1 diabetes studies consistently generate data showing islet β-cell dysfunction and T cell-mediated anti-β-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the β-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single β-cells. Consistent with immunohistological studies, β-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These β-cells also expressed mRNA for Class II and Class II antigen presentation pathway components, but lacked the macrophage marker CD68. Immunohistological study of three independent cohorts of donors with recent-onset type 1 diabetes showed Class II protein and its transcriptional regulator Class II MHC -activator protein expressed by a subset of insulinCD68 β-cells, specifically found in islets with lymphocytic infiltrates. β-Cell surface expression of HLA Class II was detected on a portion of CD45insulin β-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic β-cells from donors with type 1 diabetes express Class II molecules on selected cells with other key genes in those pathways and inflammation-associated genes. β-Cell expression of Class II molecules suggests that β-cells may interact directly with islet-infiltrating CD4 T cells and may play an immunopathogenic role.
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http://dx.doi.org/10.2337/db18-0686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477908PMC
May 2019

A Method for Islet Transplantation to the Omentum in Mouse.

J Vis Exp 2019 01 7(143). Epub 2019 Jan 7.

Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen University Health Science Center;

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.
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http://dx.doi.org/10.3791/57160DOI Listing
January 2019

Human islets expressing HNF1A variant have defective β cell transcriptional regulatory networks.

J Clin Invest 2019 01 3;129(1):246-251. Epub 2018 Dec 3.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA.

Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained β cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1α (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment.
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http://dx.doi.org/10.1172/JCI121994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307934PMC
January 2019
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