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1.
J Endocr Soc ; 5(12): bvab162, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34870058

RESUMO

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 ß-cell mass with some islets devoid of ß cells (29.3% of 311 islets) while other islets had many ß 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 ß-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.

2.
J Vis Exp ; (176)2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34747411

RESUMO

Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic ß 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.

3.
Cell Transplant ; 30: 9636897211057440, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34757864

RESUMO

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 (n = 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 (n = 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 (P=0.028). Glycolytic metabolism of islet preparations directly correlated with stimulated C-peptide secretion both before and after TPAIT (P=0.01, R 2=0.489 and P=0.03, R 2=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 (P<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.

4.
Pancreas ; 50(7): 1000-1006, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34629454

RESUMO

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.

5.
Am J Physiol Gastrointest Liver Physiol ; 321(5): G449-G460, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34523348

RESUMO

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.NEW & NOTEWORTHY 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.


Assuntos
Células Acinares/metabolismo , Perfilação da Expressão Gênica , Pâncreas/metabolismo , Pancreatite Crônica/genética , RNA-Seq , Análise de Célula Única , Transcriptoma , Células Acinares/efeitos dos fármacos , Células Acinares/patologia , Desdiferenciação Celular , Análise por Conglomerados , Estudos de Viabilidade , Humanos , Agonistas Muscarínicos/farmacologia , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pâncreas/cirurgia , Pancreatectomia , Pancreaticoduodenectomia , Pancreatite Crônica/metabolismo , Pancreatite Crônica/patologia , Pancreatite Crônica/cirurgia , Inibidores de Proteases/farmacologia
6.
Transplantation ; 105(9): 1980-1988, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34416751

RESUMO

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.


Assuntos
Bactérias/crescimento & desenvolvimento , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Microbioma Gastrointestinal , Controle Glicêmico , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Animais , Bactérias/classificação , Bactérias/genética , Biomarcadores/sangue , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/microbiologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/microbiologia , Disbiose , Fezes/microbiologia , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ribotipagem , Estreptozocina , Técnicas de Cultura de Tecidos
7.
JCI Insight ; 6(18)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34428183

RESUMO

Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and ß 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 ß cells based on combinatorial TF expression. Subpopulations of islet cells coexpressing ARX/MAFB (α cells) and MAFA/MAFB (ß 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 ß cells showed enhanced electrophysiological activity. Thus, these results indicate that combinatorial TF expression in islet α and ß cells predicts highly functional, mature subpopulations.

8.
Front Med (Lausanne) ; 8: 660877, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33937296

RESUMO

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 in vitro studies help to elucidate the mechanisms including reduction of cytokine stress by which the HMCs support islet graft protection in vivo. Derivation, stability, and scalability of the HMC source may offer unique advantages for clinical applications, including fewer islets needed for successful islet transplantation.

9.
Transl Psychiatry ; 11(1): 59, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33589583

RESUMO

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 ß-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 ß-cell adrenergic and dopaminergic receptors with different affinities to regulate glucagon and insulin release. Significantly, we show DA functions as a biased agonist at α2A-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 D2-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 ß-cell targets to produce metabolic disturbances.


Assuntos
Dopamina , Glucagon , Adrenérgicos , Glucagon/metabolismo , Insulina/metabolismo , Secreção de Insulina , Norepinefrina , Pâncreas/metabolismo
10.
Gastroenterology ; 160(4): 1330-1344.e11, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33212097

RESUMO

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.


Assuntos
Núcleo Celular/metabolismo , Pâncreas Exócrino/citologia , Adolescente , Adulto , Fatores Etários , Idoso , Animais , Fracionamento Celular , Criança , Pré-Escolar , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Pessoa de Meia-Idade , Modelos Animais , Pâncreas Exócrino/crescimento & desenvolvimento , Pâncreas Exócrino/metabolismo , RNA-Seq , Análise de Célula Única/métodos , Suínos , Adulto Jovem
11.
Cell Metab ; 32(6): 1028-1040.e4, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33207245

RESUMO

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 ß cells. This would require binding and entry of SARS-CoV-2 into ß 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 ß cells. In pancreatic sections, ACE2 and TMPRSS2 protein was not detected in ß 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 ß cells in vivo through ACE2 and TMPRSS2.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , Diabetes Mellitus/metabolismo , SARS-CoV-2/fisiologia , Serina Endopeptidases/metabolismo , Internalização do Vírus , Enzima de Conversão de Angiotensina 2/análise , Enzima de Conversão de Angiotensina 2/genética , Animais , COVID-19/complicações , COVID-19/genética , Células Cultivadas , Complicações do Diabetes/genética , Complicações do Diabetes/metabolismo , Diabetes Mellitus/genética , Expressão Gênica , Humanos , Células Secretoras de Insulina/metabolismo , Camundongos , Microvasos/metabolismo , Pâncreas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Serina Endopeptidases/análise , Serina Endopeptidases/genética
12.
bioRxiv ; 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-33106804

RESUMO

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 ß cells. This would require binding and entry of SARS-CoV-2 into host ß 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. ACE2 and TMPRSS2 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 ß 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 ß cells through these cell entry proteins.

13.
Xenotransplantation ; 27(5): e12623, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32596829

RESUMO

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.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/complicações , Xenoenxertos , Pneumonia Viral/complicações , Transplante Heterólogo , COVID-19 , Xenoenxertos/virologia , Humanos , Pandemias , SARS-CoV-2 , Doadores de Tecidos/estatística & dados numéricos , Obtenção de Tecidos e Órgãos/métodos , Transplante Heterólogo/ética , Estados Unidos
14.
JCI Insight ; 5(10)2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32352931

RESUMO

Pancreatic islets secrete insulin from ß 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 ß 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 ß and α cells.


Assuntos
Técnicas Biossensoriais , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Feminino , Células Secretoras de Glucagon/citologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/citologia , Masculino
15.
Endocrinology ; 161(8)2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32428240

RESUMO

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.


Assuntos
Compostos Benzidrílicos/farmacologia , Células Secretoras de Glucagon/efeitos dos fármacos , Glucosídeos/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Adolescente , Adulto , Animais , Células Cultivadas , Feminino , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Xenoenxertos , Humanos , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Especificidade da Espécie , Adulto Jovem
16.
Diabetologia ; 63(7): 1418-1423, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32388592

RESUMO

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 × 109 vs 121.6 ± 12.2 × 109 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.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Pâncreas Exócrino/metabolismo , Células Acinares/metabolismo , Animais , Feminino , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Pâncreas/metabolismo
17.
Methods Mol Biol ; 2110: 289-314, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32002916

RESUMO

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.


Assuntos
Xenoenxertos , Transplante das Ilhotas Pancreáticas/métodos , Transplante Heterólogo/métodos , Animais , Animais Recém-Nascidos , Biomarcadores , Separação Celular/métodos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Rejeição de Enxerto , Sobrevivência de Enxerto/imunologia , Humanos , Transplante das Ilhotas Pancreáticas/efeitos adversos , Macaca , Modelos Animais , Suínos , Tolerância ao Transplante , Transplante Heterólogo/efeitos adversos
18.
Development ; 147(6)2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32108026

RESUMO

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 ß-, 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 ß-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to ß-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.


Assuntos
Diferenciação Celular/genética , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Suínos , Animais , Animais Recém-Nascidos , Células Cultivadas , Embrião de Mamíferos , Feminino , Feto/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/fisiologia , Humanos , Ilhotas Pancreáticas/citologia , Camundongos , Organogênese/genética , Gravidez , Suínos/embriologia , Suínos/genética , Suínos/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
19.
JCI Insight ; 5(1)2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31941840

RESUMO

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 ß cell mass. Interestingly, these ß cell abnormalities reversed after withdrawal of drug treatment. Furthermore, cotreatment with a GLP-1 receptor agonist completely prevented TAC-induced ß cell dysfunction and partially prevented SIR-induced ß cell dysfunction. These results highlight the importance of both calcineurin and mTOR signaling in normal human ß cell function in vivo and suggest that modulation of these pathways may prevent or ameliorate PTDM.


Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Sirolimo/farmacologia , Tacrolimo/farmacologia , Animais , Calcineurina/metabolismo , Diabetes Mellitus , Rejeição de Enxerto , Humanos , Imunossupressores/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/efeitos dos fármacos
20.
Artigo em Inglês | MEDLINE | ID: mdl-31421261

RESUMO

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.


Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Pâncreas/patologia , Pancreatite/patologia , eIF-2 Quinase/metabolismo , Células Acinares/patologia , Animais , Asparaginase/administração & dosagem , Asparaginase/metabolismo , Asparagina/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Linhagem Celular , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Pâncreas/citologia , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima , eIF-2 Quinase/antagonistas & inibidores
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