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Topologically selective islet vulnerability and self-sustained downregulation of markers for β-cell maturity in streptozotocin-induced diabetes
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).ORCID iD: 0000-0002-0712-8256
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).ORCID iD: 0000-0001-9401-6844
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
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2020 (English)In: Communications Biology, E-ISSN 2399-3642, Vol. 3, no 1, article id 541Article in journal (Refereed) Published
Abstract [en]

Mouse models of Streptozotocin (STZ) induced diabetes represent the most widely used preclinical diabetes research systems. We applied state of the art optical imaging schemes, spanning from single islet resolution to the whole organ, providing a first longitudinal, 3D-spatial and quantitative account of β-cell mass (BCM) dynamics and islet longevity in STZ-treated mice. We demonstrate that STZ-induced β-cell destruction predominantly affects large islets in the pancreatic core. Further, we show that hyperglycemic STZ-treated mice still harbor a large pool of remaining β-cells but display pancreas-wide downregulation of glucose transporter type 2 (GLUT2). Islet gene expression studies confirmed this downregulation and revealed impaired β-cell maturity. Reversing hyperglycemia by islet transplantation partially restored the expression of markers for islet function, but not BCM. Jointly our results indicate that STZ-induced hyperglycemia results from β-cell dysfunction rather than β-cell ablation and that hyperglycemia in itself sustains a negative feedback loop restraining islet function recovery.

Place, publisher, year, edition, pages
Nature Publishing Group, 2020. Vol. 3, no 1, article id 541
National Category
Endocrinology and Diabetes Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-176312DOI: 10.1038/s42003-020-01243-2ISI: 000576947100003PubMedID: 32999405Scopus ID: 2-s2.0-85091723089OAI: oai:DiVA.org:umu-176312DiVA, id: diva2:1484545
Available from: 2020-10-29 Created: 2020-10-29 Last updated: 2023-03-24Bibliographically approved
In thesis
1. Characterizing the pancreatic "isletome": 3D optical imaging to study diabetes
Open this publication in new window or tab >>Characterizing the pancreatic "isletome": 3D optical imaging to study diabetes
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Tredimensionella analyser av de Langerhanska öarna vid diabetes
Abstract [en]

The pancreas is a specialised multipurpose organ, that can be separated into two major compartments: endocrine and exocrine. The exocrine part makes up the majority of the organ volume and functions to secrete digestive enzymes into the small intestine. Notably, endocrine islets of Langerhans are embedded and scattered in vast numbers throughout the exocrine space. These miniature functional units are composed of different cell types that secrete hormones into the blood stream. The most abundant islet-cell is the insulin-producing β-cell. Highly coordinated, the endocrine cells are the primary regulators of energy homeostasis in the body. Together, the collective islet volume constitutes the pancreatic “isletome”, a synchronised, complex and size-equilibrated system that is able to respond to various metabolic conditions. Indeed, environmental and/or genetic conditions often lead to impaired islet function and/or β-cell destruction leading to elevated blood glucose levels over time and eventually diabetes. 

Diabetes mellitus is a disease that currently affects more than 400 million individuals worldwide. As such, understanding pancreatic disease-related mechanisms is pivotal to the development of new and more effective therapeutic, or even curative, regimens. The deep location of the pancreas in the abdomen and the relatively low resolution of current clinical imaging approaches, however, render the pancreatic islets difficult to study when visually assessing endocrine function. Although non-invasive imaging techniques have yet to reach their full potential, post-mortem studies of the pancreas and rodent disease models offer unique insights into the process of diabetes disease dynamics.

Diabetes induced by streptozotocin (STZ) is a widely used model system in pre-clinical research, where it is generally believed that the b-cells are depleted upon the administration of the drug. Yet, quantification of β-cell volume dynamics and underlying disease mechanisms have not been extensively described. Using optical projection tomography (OPT), light sheet fluorescence microscopy (LSFM) and advanced protocols for ex vivo whole organ three-dimensional (3D) imaging, this study demonstrated that STZ-induced β-cell depletion is modest, primarily affecting large islets, and is not the primary cause for the development of diabetes in STZ-diabetic mice. Combined with islet gene expression studies, the remaining β-cell volume in STZ-diabetic mice displayed a downregulation of glucose transporter type 2 (GLUT2), a transmembrane carrier vital for sensing blood glucose levels. Islet transplantation into the anterior chamber of the eye (ACE) reversed the STZ-induced hyperglycaemia and partially restored islet function, including GLUT2, but did not restore β-cell volume loss. Extensive 3D image datasets were generated as a resource to the research community. The combined results of this study indicated that STZ-induced hyperglycaemia is not caused by β-cell loss, but rather by dysfunctional β-cells and that recovery of islet function is restrained by continuous hyperglycaemia.

3D imaging using OPT has proven to be a reliable technique in quantifying cellular/anatomical features of the mouse pancreas. However, the technique has rarely been applied to patient-derived tissues. Here, a label-free and non-destructive method was developed to assess clinical biopsies within hours of collection. Specifically, this study showed that autofluorescence-based imaging can be used to delineate tumours of the pancreas (pancreatic ductal adenocarcinoma, PDAC) in 3D, which may aid in identifying tumour margins in conjunction with resective surgery. Importantly, the protocol included a reversal pipeline so that other histological workflows could be applied to the same specimen. Furthermore, this study demonstrated that natural fluorescent substances in the endocrine cells provide sufficient contrast when quantifying both the volume and number of islets of Langerhans in the healthy pancreas. Altogether, the developed technique may provide a novel tool for the rapid 3D analysis of pancreatic biopsies that may complement and improve traditional pathological assessments.

With the emergence of islet transplantation networks worldwide, access to fixed pancreatic tissues from diseased donors has dramatically improved. Hereby, the near instant autolysis of the pancreas post-mortem can generally be avoided, which provides the opportunity to quantitatively study the entire gland ex vivo within a conserved spatial context. Yet, mesoscopic 3D imaging of the pancreas (by OPT and/or LSFM) has been limited predominantly due to the obstacle of labelling larger tissue volumes. As such, a simple approach to antibody labelling and cellular imaging was developed in cubic centimetre-sized tissue cuboids that were mapped to the whole organ. By stitching the resultant datasets back into 3D space, this approach demonstrated how essentially any human organ may be analysed in full with high resolution. This technique was applied to pancreata from non-diabetic and type 2 diabetic (T2D) donors, analysing over 200 thousand islets, revealing features of the human pancreas that were not analysed in 3D previously, including high islet dense regions and intra-islet haemorrhaging. Crucially, this new technique may contribute to unveil a wealth of new insights into the complex pathophysiology of the “diabetic pancreas”.

By applying the above method to the entire volume of the human pancreas, the absolute distribution and volume of insulin-positive cells in a pancreas from a donor with longstanding type 1 diabetes (T1D) was demonstrated for the first time. By dividing the 19 cm long organ into smaller pieces, followed by insulin labelling, OPT imaging and reconstruction in 3D space, approximately 173,000 insulin-positive objects were identified. By utilising tissue autofluorescence, the entire organ was reconstructed in 3D, together with blood vessels and ducts. These data indicated several important regional differences in β-cell mass, such as the uncinate process showing the highest density, which potentially reflects key aspects of disease dynamics. Furthermore, regions with a “punctated distribution” of single β-cells in close proximity to each other were identified. Although the significance of these observations needs to be elucidated, we speculate that these regions could be associated with pancreatic regeneration, which might permit the development of new interventions for clinical regenerative processes in the future. Altogether, this study represents the first whole organ account of β-cell distribution at the current level of resolution in an entire organ. As such, it may serve as an important advancement towards detailed whole organ analyses of endocrine cell identity/function, via a wide range of markers, in the study of normal anatomy and pathophysiology of the human pancreas.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2022. p. 92
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2177
Keywords
3D imaging, fluorescence microscopy, 3D image analysis, technique development, diabetes, Islet of Langerhans, Insulin, β-cell mass, pancreas, anatomy
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biomedical Laboratory Science/Technology
Research subject
molecular medicine (medical sciences); Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-193479 (URN)978-91-7855-775-2 (ISBN)978-91-7855-776-9 (ISBN)
Public defence
2022-04-29, Aula Anatomica, BIO.A.206, Biology Building, Umeå University, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilInsamlingsstiftelsen Diabetes WellnessSwedish Child Diabetes FoundationDiabetesfondenNovo NordiskThe Kempe Foundations
Available from: 2022-04-08 Created: 2022-04-04 Last updated: 2023-04-29Bibliographically approved

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Hahn, MaxNord, ChristofferAlanentalo, TomasMorini, FedericoEriksson, MariaKostromina, ElenaAhlgren, Ulf

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