Moreover, most minor attempts were made to understand the possible mode of action of PARP1 in β cell regeneration or stem cell-derived differentiation. Islet neogenesis is one such pathway for new islet cell development and is highly dependent on the spatial—temporal expression of key transcription factors.

This has been very recently established that PARP activity and DNA-PARP Protein interaction conversely modulate a key transcriptional factor involved in islet differentiation pathways such as Sox2, Ngn-3, PDX-1, MafA, Reg-1 etc 6 , As the concept is pretty new and relatively unexplored, in the present study, we aim to investigate the role of the PARP1 enzyme in human islet differentiation using a pancreatic progenitor cell line model- PANC-1 to mimic the embryonic islet developmental pathway.

Our ultimate aim was to delineate the mechanistic action of PARP1 regulation in new islet formation either by its catalytic activity or independently due to the presence of unmodified protein per se. We performed routine islet differentiation using human progenitor cell line, PANC-1 and activin-A as established differentiating agents by employing catalytic inactivation of PARP using potent pharmacological PARP inhibitor- PJ or attaining absolute protein loss using DNA vector-based RNAi technology.

Our intention in abrogating all PARP enzyme activity using PJ was to answer if PARP1 protein presence is indispensable to facilitate islet neogenesis with activin-A. Additionally, using stable knockdown of PARP1 protein with shRNA, we tested the effect of PARP-1 absence on the islet cell differentiation pathway.

Additionally, pharmacological PARP-inhibitor- PJ is a potent nonspecific inhibitor that blocks PARP1 and PARP2 while all other isoforms of PARPs are allowed to carry out DNA repair function, thereby allowing cells to have free access to unmodified PARP protein for molecular interaction with targeted transcription factor proteins in modulating endocrine differentiation other than DNA damage induced repair mechanism 8 , 10 , 34 , 35 , In our study, PJ alone does not impact the islet neogenesis pathway.

Still, in combination with activin-A, the PARPi effect was highly pronounced to show accelerated islet clusters differentiation and maturation that was translated to hormone insulin and glucagon production.

This is evident since the SFM control medium is not rich in growth factors and quickly causes terminal mild-to-severe DNA damage due to starvation, leading to PARP over-activation and catalytically activating the auto modification domain.

On another side, PARPi with PJ facilitates free but unmodified PARP protein and allows for direct interaction and recruitment for essential endocrine transcription factor machinery for improved islet differentiation.

Because of the catalytic inactivation and its unmodified structure, free PARP1 interacts with regulators including Pdx-1, Ngn-3 and p38 MAPK upon phosphorylation.

It became interesting to record the mechanism for the complementary effect of PARP-1 action since this remained largely unstudied, despite a promise for PARPi in clinical trials In parallel, separately to PJ inhibition that highlighted the role of PARP1 protein in stimulating islet cell formation, we adopted a reverse strategy to completely abrogate PARP1 levels in the differentiated cells using the gene knockdown approach.

The idea was to test whether the absence of PARP1 protein is required for islet formation. To investigate this, we used the DNA vector-based RNAi approach, previously established by Shah GM et.

We observed that PARP-depleted SiP cells lose the capacity to form insulin-producing cells when treated with activin-A. In contrast, PARP1-control U6 cells could still form routine islet-like clusters that were intensely positive for insulin and glucagon hormones, as measured by DTZ staining and immunocytochemistry.

Strikingly, PARP1-depleted SiP-differentiated cells displayed reduced cluster size and retarded islet yield despite activin-A exposure. A mechanistic blueprint from key transcription factors captured after immunoblotting Sip and U6-derived cells clearly reflected the involvement of PARP1 protein in endocrine cell differentiation, confirming the indispensable presence of PARP1 for islet cell differentiation and maturation for 10 days differentiation protocol.

The study also shows an elevated expression of progenitor markers like Nestin and E-cadherin in PARP1 depleted cells, indicating that they failed to differentiate, unlike PARP-control U6 cells.

Interestingly, activin-A treatment upregulated phospho-p38 in SiP cells, but this was insufficient to translate the strong up-regulation of Ngn-3 and insulin expression.

In Contrast to PARP1 KO animals where Ngn3 expression was observed without nestin staining, RNAi-induced PARP1 SiP cells showed basal nestin trigger without stimulating Ngn3 activation. We believe that Nestin and Ngn3 arm for human islet cell differentiation and maturation might progress distinctly than in mice pancreas development, such as in PARP1-KO mice.

Our data from this study concludes that the absence of PARP1 in KO mice defines immature islets with increased but small size suggesting halted progenitors during morphogenesis. This was not observed when mimicked with RNAi-mediated PARP1-depleted human PANCislets. Based on these substantial datasets using PARP1 KO mice and PANC-1 line differentiated islets with pharmacological and gene manipulation approach, we hypothesize that the presence of PARP1 protein, but not its catalytic activation, is essential for forming functional islet clusters with activin-A summarized data shown in Table 1.

We also suggest that PARP-activation may negatively influence this event Fig. Our results reflected the two earlier observations in a simpler model of only β-cell lines that PARP-inhibitor could promote transcription of Reg-1 6 and MafA 29 genes, which are implicated in β-cell proliferation or insulin gene expression, respectively.

Unlike these studies that involved only mature β cells, our model requires differentiation of precursor cells to islet-like clusters for insulin and glucagon-producing cell generation, thereby mimicking the embryonic pancreatic ontology for islet development artificially.

Conceptual illustration describing the mode of action of PARP-1 function in enhancing and activation of islet cell differentiation, maturation and improved β cell production with PJ inhibition while completed abrogation of the process with RNAi mediated PARP1 silencing.

Poly ADP-Ribose polymerase-1 show necessary regulatory action in controlling islet differentiation and is inevitable for new β cell formation. To further understand the role of PARP1 catalytic activity or unmodified protein in controlling the translational processing of essential endocrine regulators-Pdx-1, Ngn-3, NeuroD1, MafA and Reg-1, further molecular studies should be conducted using gain and loss-of-PARP-1 function.

Our study suggests that PARP1 is required for the proper development and differentiation of human islets. Future exploration to harness the beneficial effects of PARP1 and PARPi in clinical settings is needed.

Human PANC-1 cells were cultured Originally procured from ATCC and given as a kind gift from Dr. After differentiation, islet-like clusters were tested for the presence of insulin with Dithizone DTZ staining.

The study is performed and reported in accordance with ARRIVE guidelines. Four young 6—8 week male PARP1 gene-deficient and wild-type SV mice 37 were procured from Jackson laboratories and used for harvesting pancreases for performing a histological examination of islet morphometry and islet distribution census.

The animals were not treated in any manner and were kept in 12 h light cycle with regular chaw and water supply. All animal experiments were performed in accordance with animal ethical guidelines and after obtaining institutional committee approval from Université Laval, Quebec, Canada.

PREP cells were isolated and cultured using the established protocol described in previously published report PARP1 gene was knocked down using specific shRNA against the PARP1 gene.

DNA vector-based RNAi approach, established in Dr. Girish M Shah's lab 8 was used to stably knockdown PARP1 in human PANC-1 cells SiP along with empty vector control PARP1-shRNA control PANCU6 clone , without shRNA.

The vector map and shRNA sequence chosen are shown in Fig. Transfection was done using lipofectamine with a DNA to lipofection ratio of Clones were purified from isolated colonies using clonal disc selection and further scaled up in 10 cm dishes.

An absolute knockdown for PARP1 in the isolated clones was confirmed using Immunoblotting using an anti-PARP-1 antibody. In order to better comprehend the differentiation procedure of islet neogenesis, differentiation of PANC-1, in presence and absence of PJ Inhibitor of PARP activity at a concentration of 10 μM.

Islet-like clusters from all groups were purified and counted for yield assessment and stained for DTZ and insulin immunostaining on the 10th day. Islet clusters from each plate were then processed for total protein isolation and immunoblotting of key transcription factors.

WE harvested four mice pancreas, fixed and sliced from each genotype. For morphometry and quantification, we used a minimum of 3 slides per animal per genotype. Each slide was stained with hematoxylin and eosin for at least one whole pancreas section and imaged to count for islet structures.

For understanding the role of PARP1 in islet differentiation, immunohistochemistry for insulin and glucagon and key islet differentiation markers were performed. Clusters obtained post differentiation of PANC-1 cells with and with PJ treatment and after knockdown of PARP1 using Sip , and U6 plasmids were stained for insulin and glucagon.

Briefly, clusters were collected on the 10th day and allowed to adhere to serum FBS coated glass coverslips for 4 h.

Once adhered, the clusters were fixed with ice-chilled methanol for 10 min at 4 °C. For mice tissue histology, paraffin fixed and microtome pancreatic tissue sections were first deparaffinized and rehydrated as described previously 31 followed by blocking and staining for primary antibodies.

Both tissue slides and clusters were then permeabilized with 0. Primary antibodies, as described in Table 2 , were used to stain tissue slides or clusters for 18 h at 4 °C in a humidified chamber.

Labelled conjugated secondary antibodies were then used to counterstain primary antibodies, as described in Table 2. Nuclear stain- DAPI was used at nM concentration, and samples were finally mounted with vecta-shield mounting media. Fluorescent images were captured on a Zeiss Axio-Vision Fluorescent microscope Carl Zeiss, Germany using Axiovision Zen 10 software.

TO confirm the regulatory role of PARP1 in islet neogenesis, clusters from each group were harvested, and total protein was isolated. After protein separation on poly acrylamide gels, migrated proteins were transferred and immunoblotted onto nitrocellulose membrane before probing for essential endocrine pathway proteins as specific antibodies, as described in Table 2.

Transcription factors like Nestin, E-Cadherin, Pdx-1, Ngn-3, P-p38, pMAPK, N-cadherin and PARP1 were probed as described earlier Extended uncropped image source data of immune blots for RNAi and PARPi protein profiling experiments along with corresponding ponceau blots to verify loading controls are submitted as supplementary data files please refer to Supplementary Fig.

The presence of islet markers to confirm islet differentiation was measured using the semiquantitative reverse transcriptase PCR method and normalized to the human beta-actin gene.

Total RNA was isolated and extracted using TRIZOL Sigma. Following RT, semi-quantitative gene expression was amplified and visualized using gene-specific primers under optimal PCR conditions for human insulin, glucagon and beta-actin genes.

Primer sequences and PCR conditions are shown in Table 3. Amplified PCR products were run on 1. This research finding has been approved by the institutional ethics committee of the MS University of Baroda, India and the University of Laval, Quebec, Canada. All relevant data are presented within the manuscript and its supplementary information files.

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The activity was expressed as micromoles of p-nitrophenol formed per minute per milligram of PARP-1 protein. Pancreatic islets were isolated as previously described 7. Protein expression was assayed with islet lysates with equal amounts of protein.

Lysates were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes for immunoblotting. The membranes were incubated with specific antibodies against β-actin, cleaved PARP-1, RegI, Reg II and Reg IV. Each value was the mean of three independent experiments. All data are presented as mean ± standard deviation SD from at least triplicate experiments.

Groups were evaluated by Student t test SPSS Insulin secretion by determining blood insulin levels in the glucose tolerance test before E and after G STZ. We used low-dose STZ to further test whether glucose homeostasis restricted with advanced age is associated with PARP After low-dose STZ, basal blood glucose levels were increased and blood insulin levels were decreased in all groups compared with before STZ Figures 1D , H.

Recent studies demonstrated that physiological and pathological states can affect islet sizes and architecture. In contrast, low-dose STZ treatment significantly decreased islet sizes of old WT mice. Results are representative of 5—6 slides spanning the whole pancreas of each mouse for at least 4 mice per group.

Pancreatic islets showed no change in structure, as shown by antiinsulin and antiglucagon antibody staining in all groups before and after low-dose STZ Figure 3A. Pancreatic weight did not differ before or after low-dose STZ in all groups Figure 3C.

Adaptive expansion of β-cell mass in young and old mice. A Immunofluorescence analysis of representative images of pancreatic sections from young and old mice before and after low-dose STZ treatment and stained with antiinsulin green and antiglucagon red antibodies.

β -Cell proliferation was assessed by BrdU antibody staining Figures 4A , B. However, the proportion of BrdU-positive nuclei was higher in old PARP-mice than in old WT mice 0. Similar results for β-cell proliferation were obtained after STZ treatment Figures 4B , C.

C Proportion of BrdU-positive β cells of young and old mice before and after STZ injection. Values were averaged from 5 slides for each mouse, with 4—5 mice in each group.

The islets of old WT mice showed higher levels of cleaved PARP-1 protein and PARP-1 activity than those of young WT mice, which was detected by Western blot Figures 5A , B and the universal colorimetric PARP-1 assay kit Figure 5C , respectively.

Furthermore, similar results were observed after low-dose STZ Figures 5A — C. Expression of cleaved PARP-1 and PARP-1 activity in the pancreas of WT mice before and after low-dose STZ treatment. A Levels of cleaved PARP-1 protein expression in the islets isolated from young and old mice by Western blot analysis.

B Analysis of immunoblot of cleaved PARP-1 protein normalized to β-actin. Values were representative of islets from 4—5 mice per group. C The PARP-1 activity of pancreatic islets was determined with the universal colorimetric PARP-1 assay kit, and results were expressed as micromoles of p-nitrophenol formed per minute per milligram of PARP-1 protein.

After STZ treatment, the level of pancreatic RegI was increased 3. The levels of RegII were slightly increased after STZ treatment in each group except for the old WT mice group Figure 6D.

However, RegII expression was significantly higher for old PARP-mice than WT mice Similar results were observed with the use of Western blot analysis Figures 7A — D. Expression of RegI, RegII and Reg IV protein in the pancreas before and after low-dose STZ treatment.

Levels of RegI A , RegII C and Reg IV E expression in the islets isolated from young and old mice by Western blot analysis. Analysis of immunoblot of RegI B , RegII D and Reg IV F normalized to β-actin.

Double staining for the islet hormones insulin and RegIV showed that RegIV gave a strong signal in the islets in each group Figure 8A. RegIV expression was found in almost all β cells; however, only a small number of insulin-negative islet cells were stained for RegIV.

RegIV expression was similar in young and old mouse groups regardless of STZ treatment Figure 8B. Furthermore, islets from each group did not show significant differences in expression of RegIV before or after STZ treatment, which was detected by Western blot analysis Figures 7E , F.

PARP-1 regulates pancreatic β-cell death, regeneration and insulin secretion. Many experiments have evaluated whether age plays a role in the proliferative capacity of pancreatic β cells by using distinct models of endogenous β -cell regeneration: low-dose STZ administration, high-fat diet, treatment with the glucagon-like peptide 1 analog exendin-4 and partial pancreatectomy 5 — 7.

In all models 5 — 7 , young mice could regenerate β cells, whereas old mice did not show a similar plasticity, which is similar to rats 19 , In recent years, the prevalence of type 2 diabetes among elderly people has increased worldwide 8 , 21 , Therefore, old human patients could have poor capacity to regenerate β-cell mass to fulfill the need.

PARP-1 is involved in the pathophysiological features of many age-related diseases, such as chronic obstructive pulmonary disease 23 , atherosclerotic lesions 24 and diabetes 25 , 26 , by regulating DNA integrity, transcription, inflammation, cell regeneration and cell death.

Low-dose STZ is a robust stimulus of β-cell regeneration that is well tolerated and does not cause diabetes 7. BrdU labeling revealed a similar result. Therefore, loss of PARP-1 resulted in resistance to age-dependent decreases in β-cell proliferation in mice.

The first Reg gene was isolated from a regenerating islet-derived cDNA library in rats Reg family proteins act on β cells and other cells as autocrine or paracrine growth factors 16 , 28 — In mice, RegI and RegII proteins expressed in regenerating islets and pancreatic exocrine tissues 15 , 34 , RegIIIδ islet neogenesis-associated protein , expressed predominantly in exocrine pancreas, enhances the secretion of insulin and the transcription of many islet genes in controlling β-cell neogenesis and islet metabolism 36 , RegIIIα, RegIIIβ and RegIIIγ are expressed weakly in pancreas and strongly in the intestinal tract; however, in a recent study, RegIIIα and RegIIIβ were activated in pancreatic-specific insulin-like growth factor I IGF-I gene-deficient and diabetic mice and participated in islet regeneration after β-cell damage RegIV, the latest member of the Reg family to be identified, plays an important role in the progression of colo-rectal cancer Moreover, PARP inhibitors may enhance and stabilize the DNA-protein complex for Reg gene transcription in β cells by inhibiting autopoly ADP-ribosyl ation of PARP 14 , However, levels of RegIV were not changed further in each group after STZ treatment.

These findings might therefore represent a potential target in efforts to improve β-cell regeneration in treating diabetes in elderly patients. The authors declare that they have no competing interests as defined by Molecular Medicine , or other interests that might be perceived to influence the results and discussion reported in this paper.

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Pieper AA, et al. Masutani M, et al. Yonemura Y, et al. Evidence of islet B-cell regeneration. Akiyama T, et al. Evidence in the second part of the paper suggested an alternative function of poly ADP-ribose synthetase inhibitors in the improvement of surgical diabetes. In this case, poly ADP-ribose synthetase inhibitors may relieve restriction of DNA replication and so cause B-cell regeneration.

Since a low capacity for B-cell regeneration has been suggested as a predisposition for the development of human diabetes, the present study may provide a novel clue for the prevention and treatment of human diabetes.

This output contributes to the following UN Sustainable Development Goals SDGs. T1 - The role of poly ADP-ribose synthetase in the development of insulin-dependent diabetes and islet B-cell regeneration.

N2 - It is reasonable to assume that poly ADP-ribose synthetase inhibitors induce pancreatic B-cell regeneration, thereby improving diabetes mellitus caused by partial pancreatectomy.

AB - It is reasonable to assume that poly ADP-ribose synthetase inhibitors induce pancreatic B-cell regeneration, thereby improving diabetes mellitus caused by partial pancreatectomy. The role of poly ADP-ribose synthetase in the development of insulin-dependent diabetes and islet B-cell regeneration.

HOSP - Surgery. Overview Fingerprint. Abstract It is reasonable to assume that poly ADP-ribose synthetase inhibitors induce pancreatic B-cell regeneration, thereby improving diabetes mellitus caused by partial pancreatectomy.