surgical-injury-to-mouse-pancreas-through-ligation-pancreatic-duct-as

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

Vrije Universiteit Brussel

Generating functional beta-cells by inducing their proliferation may provide new perspectives for cell therapy in diabetes. Transcription factor E2F1 controls G(1)- to S-phase transition during the cycling of many cell types and is required for pancreatic beta-cell growth and function. However, the consequences of overexpression of E2F1 in beta-cells are unknown.

Ectopic expression of E2F1 stimulates beta-cell proliferation and function.

It was recently demonstrated that embryonic glucagon-producing cells in the pancreas can regenerate and convert into insulin-producing β-like cells through the constitutive/ectopic expression of the Pax4 gene. However, whether α cells in adult mice display the same plasticity is unknown. Similarly, the mechanisms underlying such reprogramming remain unclear. We now demonstrate that the misexpression of Pax4 in glucagon(+) cells age-independently induces their conversion into β-like cells and their glucagon shortage-mediated replacement, resulting in islet hypertrophy and in an unexpected islet neogenesis. Combining several lineage-tracing approaches, we show that, upon Pax4-mediated α-to-β-like cell conversion, pancreatic duct-lining precursor cells are continuously mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon(+) and a β-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition. Importantly, these processes can repeatedly regenerate the whole β cell mass and thereby reverse several rounds of toxin-induced diabetes, providing perspectives to design therapeutic regenerative strategies.

Adult duct-lining cells can reprogram into β-like cells able to counter repeated cycles of toxin-induced diabetes.

Long-term labelling of mice with halogenated thymidine analogues is an established method for quantifying the contribution of beta cell proliferation to in vivo beta cell mass expansion in (re)generation models. The method is believed to give accurate information on the accrued number of cycling beta cells over a period of time. Multiple thymidine analogue labelling is applied for evaluating the duration of postmitotic quiescence in beta cells. We hypothesise, however, that long-term labelling by thymidine analogues hampers beta cell proliferation.

Mouse beta cell proliferation is inhibited by thymidine analogue labelling.

Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon(+) cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.

The inactivation of Arx in pancreatic α-cells triggers their neogenesis and conversion into functional β-like cells.

We recently reported that human blood outgrowth endothelial cells (BOEC) are supportive to reverse hyperglycemia in marginal islet mass-transplanted diabetic mice. In this report, we investigated whether the observed effect was evoked by islet packing in a blood clot prior to transplantation or could be mimicked by another method of islet/cell delivery. A marginal islet mass with or without BOEC was grafted underneath the kidney capsule of diabetic recipient mice via a (blood clot-independent) tubing system and compared with previous islet packing in a blood clot. The effect on metabolic outcome of both delivery techniques as well as the additive effect of BOEC was subsequently evaluated. Marginal islet mass transplantation via a tubing system required more islets per recipient than via a blood clot. Using the tubing method, transplantation of a marginal islet mass combined with 5x10 (5) BOEC resulted in reversal of hyperglycemia, improved glucose tolerance and increased kidney insulin content. The present study provides evidence that (1) previous packing in a blood clot results in more effective islet delivery compared with tubing; (2) BOEC exert a beneficial effect on marginal islet transplantation, independent of grafting technique and potential blood clot-induced processes. These data further support the use of BOEC in (pre-) clinical studies that aim to improve current islet transplantation protocols.

Reversal of hyperglycemia in diabetic mice by a marginal islet mass together with human blood outgrowth endothelial cells is independent of the delivery technique and blood clot-induced processes.

Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.

Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice.

IL-6 was recently shown to control alpha cell expansion. As beta cells expand following partial pancreatic-duct ligation (PDL) in adult mice, we investigated whether PDL also causes alpha cells to expand and whether IL-6 signalling is involved. As alpha cells can reprogramme to beta cells in a number of beta cell (re)generation models, we examined whether this phenomenon also exists in PDL pancreas.

IL-6-dependent proliferation of alpha cells in mice with partial pancreatic-duct ligation.

Partial duct ligation: β-cell proliferation and beyond.

Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing β-cell neogenesis and proliferation. Macrophages (MΦs) were recently shown to promote β-cell proliferation after PDL, but they remain poorly characterized. We assessed myeloid cell diversity and the factors driving myeloid cell dynamics following acute pancreas injury by PDL. In naive and sham-operated pancreas, the myeloid cell compartment consisted mainly of two distinct tissue-resident MΦ types, designated MHC-II(lo) and MHC-II(hi) MΦs, the latter being predominant. MHC-II(lo) and MHC-II(hi) pancreas MΦs differed at the molecular level, with MHC-II(lo) MΦs being more M2-activated. After PDL, there was an early surge of Ly6C(hi) monocyte infiltration in the pancreas, followed by a transient MHC-II(lo) MΦ peak and ultimately a restoration of the MHC-II(hi) MΦ-dominated steady-state equilibrium. These intricate MΦ dynamics in PDL pancreas depended on monocyte recruitment by C-C chemokine receptor 2 and macrophage-colony stimulating factor receptor as well as on macrophage-colony stimulating factor receptor-dependent local MΦ proliferation. Functionally, MHC-II(lo) MΦs were more angiogenic. We further demonstrated that, at least in C-C chemokine receptor 2-KO mice, tissue MΦs, rather than Ly6C(hi) monocyte-derived MΦs, contributed to β-cell proliferation. Together, our study fully characterizes the MΦ subsets in the pancreas and clarifies the complex dynamics of MΦs after PDL injury.

Macrophage dynamics are regulated by local macrophage proliferation and monocyte recruitment in injured pancreas.

Macrophages are classically considered detrimental for pancreatic β-cell survival and function, thereby contributing to β-cell failure in both type 1 (T1D) and 2 (T2D) diabetes mellitus. In addition, adipose tissue macrophages negatively influence peripheral insulin signaling and promote obesity-induced insulin resistance in T2D. In contrast, recent data unexpectedly uncovered that macrophages are not only able to protect β cells during pancreatitis but also to orchestrate β-cell proliferation and regeneration after β-cell injury. Moreover, by altering their activation state, macrophages are able to improve insulin resistance in murine models of T2D. This review will elaborate on current insights in macrophage heterogeneity and on the evolving role of pancreas macrophages during organogenesis, tissue injury, and repair. Additional identification of macrophage subtypes and of their secreted factors might ultimately translate into novel therapeutic strategies for both T1D and T2D.

Concise Review: Macrophages: Versatile Gatekeepers During Pancreatic β-Cell Development, Injury, and Regeneration.

Identifying pathways for beta cell generation is essential for cell therapy in diabetes. Here we investigate the potential of 17β-estradiol (E2) and estrogen receptor (ER) signaling for stimulating beta cell generation during embryonic development and in severely injured adult pancreas. Estradiol concentration, ER activity and number of ERα transcripts were enhanced in pancreas injured by partial duct ligation (PDL), along with nuclear localization of ERα in beta cells. PDL-induced proliferation of beta cells depended on aromatase activity. The activation of Neurogenin3 (Ngn3) gene expression and beta cell growth in PDL pancreas were impaired when ERα was turned off chemically or genetically (ERα(-/-)), while in situ delivery of E2 promoted beta cell formation. In embryonic pancreas, beta cell replication, number of Ngn3(+) progenitor cells and expression of key transcription factors of the endocrine lineage were decreased by ERα inactivation. The present study reveals that E2 and ERα signaling can drive beta cell replication and formation in mouse pancreas.

Article	Year
Ectopic expression of E2F1 stimulates beta-cell proliferation and function. Diabetes\| PubMed ID: 20299467	2010
Adult duct-lining cells can reprogram into β-like cells able to counter repeated cycles of toxin-induced diabetes. Developmental cell\| PubMed ID: 23810513	2013
Mouse beta cell proliferation is inhibited by thymidine analogue labelling. Diabetologia\| PubMed ID: 24026213	2013
The inactivation of Arx in pancreatic α-cells triggers their neogenesis and conversion into functional β-like cells. PLoS genetics\| PubMed ID: 24204325	2013
Reversal of hyperglycemia in diabetic mice by a marginal islet mass together with human blood outgrowth endothelial cells is independent of the delivery technique and blood clot-induced proc... Islets\| PubMed ID: 24213480	2013
Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice. Nature biotechnology\| PubMed ID: 24240391	2014
IL-6-dependent proliferation of alpha cells in mice with partial pancreatic-duct ligation. Diabetologia\| PubMed ID: 24759958	2014
Partial duct ligation: β-cell proliferation and beyond. Diabetes\| PubMed ID: 25060885	2014
Macrophage dynamics are regulated by local macrophage proliferation and monocyte recruitment in injured pancreas. European journal of immunology\| PubMed ID: 25645754	2015
Concise Review: Macrophages: Versatile Gatekeepers During Pancreatic β-Cell Development, Injury, and Regeneration. Stem cells translational medicine\| PubMed ID: 25848123	2015
Estrogen Receptor α Regulates Beta Cell Formation During Pancreas Development and Following Injury. Diabetes\| PubMed ID: 26015547	2015

Gunter Leuckx

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