| 2. |
- Hahn, Max, 1993-
(författare)
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Characterizing the pancreatic "isletome" : 3D optical imaging to study diabetes
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
- Pardo, F N, et al.
(författare)
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The role of Raf-1 kinase inhibitor protein in the regulation of pancreatic beta cell proliferation in mice.
- 2012
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 0012-186X .- 1432-0428. ; 55:12, s. 3331-40
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Tidskriftsartikel (refereegranskat)abstract
- AIMS/HYPOTHESIS: Manoeuvres aimed at increasing beta cell mass have been proposed as regenerative medicine strategies for diabetes treatment. Raf-1 kinase inhibitor protein 1 (RKIP1) is a common regulatory node of the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways and therefore may be involved in regulation of beta cell homeostasis. The aim of this study was to investigate the involvement of RKIP1 in the control of beta cell mass and function.METHODS: Rkip1 (also known as Pebp1) knockout (Rkip1 (-/-)) mice were characterised in terms of pancreatic and glucose homeostasis, including morphological and functional analysis. Glucose tolerance and insulin sensitivity were examined, followed by assessment of glucose-induced insulin secretion in isolated islets and beta cell mass quantification through morphometry. Further characterisation included determination of endocrine and exocrine proliferation, apoptosis, MAPK activation and whole genome gene expression assays. Capacity to reverse a diabetic phenotype was assessed in adult Rkip1 (-/-) mice after streptozotocin treatment.RESULTS: Rkip1 (-/-) mice exhibit a moderately larger pancreas and increased beta cell mass and pancreatic insulin content, which correlate with an overall improvement in whole body glucose tolerance. This phenotype is established in young postnatal stages and involves enhanced cellular proliferation without significant alterations in cell death. Importantly, adult Rkip1 (-/-) mice exhibit rapid reversal of streptozotocin-induced diabetes compared with control mice.CONCLUSIONS/INTERPRETATION: These data implicate RKIP1 in the regulation of pancreatic growth and beta cell expansion, thus revealing RKIP1 as a potential pharmacological target to promote beta cell regeneration.
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| 4. |
- Quilichini, Evans, et al.
(författare)
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Insights into the etiology and physiopathology of MODY5/HNF1B pancreatic phenotype with a mouse model of the human disease
- 2021
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Ingår i: Journal of Pathology. - : John Wiley & Sons. - 0022-3417 .- 1096-9896. ; 254:1, s. 31-45
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Tidskriftsartikel (refereegranskat)abstract
- Maturity‐onset diabetes of the young type 5 (MODY5) is due to heterozygous mutations or deletion of HNF1B. No mouse models are currently available to recapitulate the human MODY5 disease. Here, we investigate the pancreatic phenotype of a unique MODY5 mouse model generated by heterozygous insertion of a human HNF1B splicing mutation at the intron‐2 splice donor site in the mouse genome. This Hnf1bsp2/+ model generated with targeted mutation of Hnf1b mimicking the c.544+1G>T (T) mutation identified in humans, results in alternative transcripts and a 38% decrease of native Hnf1b transcript levels. As a clinical feature of MODY5 patients, the hypomorphic mouse model Hnf1bsp2/+ displays glucose intolerance. Whereas Hnf1bsp2/+ isolated islets showed no altered insulin secretion, we found a 65% decrease in pancreatic insulin content associated with a 30% decrease in total large islet volume and a 20% decrease in total β‐cell volume. These defects were associated with a 30% decrease in expression of the pro‐endocrine gene Neurog3 that we previously identified as a direct target of Hnf1b, showing a developmental etiology. As another clinical feature of MODY5 patients, the Hnf1bsp2/+ pancreases display exocrine dysfunction with hypoplasia. We observed chronic pancreatitis with loss of acinar cells, acinar‐to‐ductal metaplasia, and lipomatosis, with upregulation of signaling pathways and impaired acinar cell regeneration. This was associated with ductal cell deficiency characterized by shortened primary cilia. Importantly, the Hnf1bsp2/+ mouse model reproduces the pancreatic features of the human MODY5/HNF1B disease, providing a unique in vivo tool for molecular studies of the endocrine and exocrine defects and to advance basic and translational research.
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