Meenal Francis,Smitha Bhaskar,Saarwani Komanduri,Preethi Sheshadri,Jyothi Prasanna,Anujith Kumar
Deubiquitinase USP1 influences the dedifferentiation of mouse pancreatic β-cells
Molecular interaction,Diabetology,Specialized functions of cells
Summary Loss of insulin-secreting β-cells in diabetes may be either due to apoptosis or dedifferentiation of β-cell mass. The ubiquitin-proteasome system comprising E3 ligase and deubiquitinases (DUBs) controls several aspects of β-cell functions. In this study, screening for key DUBs identified USP1 to be specifically involved in dedifferentiation process. Inhibition of USP1 either by genetic intervention or small-molecule inhibitor ML323 restored epithelial phenotype of β-cells, but not with inhibition of other DUBs. In absence of dedifferentiation cues, overexpression of USP1 was sufficient to induce dedifferentiation in β-cells; mechanistic insight showed USP1 to mediate its effect via modulating the expression of inhibitor of differentiation (ID) 2. In an in vivo streptozotocin (STZ)-induced dedifferentiation mouse model system, administering ML323 alleviated hyperglycemic state. Overall, this study identifies USP1 to be involved in dedifferentiation of β-cells and its inhibition may have a therapeutic application of reducing β-cell loss during diabetes.
Deubiquitinase USP1 influences the dedifferentiation of mouse pancreatic β-cells

Loss of insulin-secreting β-cells in diabetes may be either due to apoptosis or dedifferentiation of β-cell mass. The ubiquitin-proteasome system comprising E3 ligase and deubiquitinases (DUBs) controls several aspects of β-cell functions. In this study, screening for key DUBs identified USP1 to be specifically involved in dedifferentiation process. Inhibition of USP1 either by genetic intervention or small-molecule inhibitor ML323 restored epithelial phenotype of β-cells, but not with inhibition of other DUBs. In absence of dedifferentiation cues, overexpression of USP1 was sufficient to induce dedifferentiation in β-cells; mechanistic insight showed USP1 to mediate its effect via modulating the expression of inhibitor of differentiation (ID) 2. In an in vivo streptozotocin (STZ)-induced dedifferentiation mouse model system, administering ML323 alleviated hyperglycemic state. Overall, this study identifies USP1 to be involved in dedifferentiation of β-cells and its inhibition may have a therapeutic application of reducing β-cell loss during diabetes.

Diabetes is a metabolic disease presented with a condition of hyperglycemia owing to the weaning of pancreatic β-cells. Autoimmune destruction of β-cells and β-cell stress due to systemic resistance to insulin are the major reasons for the loss of pancreatic β-cells., Recent theories divulge in accounting to loss of β-cells to multiple cellular attires such as transdifferentiation and dedifferentiation., The latest “Anna Karenina Model” suggests dedifferentiation to be a transitory fate change depending on the stage of the disease. Several cell-based lucrative avenues to restore β-cells have been postulated. Transplantation of cadaver donor islets or β-cells derived from differentiation of pluripotent stem cells are ideal cell-based strategies; however, they are fraught with shortage of donors and inefficient differentiation, respectively., These hurdles could be overcome by formulating a protocol that enhances in vitro differentiation or find an approach to enhance endogenous β-cell mass either by blocking transdifferentiation or dedifferentiation. The in vitro culture of islet β-cells is an extremely difficult task owing to the non-proliferative disposition of islet cells and unaccountable fibroblast-like cells emerging from the islet clusters.,, In a later study, serum-free cultures of β-cells indicated the ability of the cells to shuttle between functional epithelial and mesenchymal fates. There have been multiple experimentally proven works including lineage tracing studies showing the dedifferentiation process to be occurring in β-cells and proved to be one of the major reason in the manifestation of diabetes.,,, Dedifferentiation process involves epithelial β-cells transforming to a proliferative mesenchymal cell-like phenotype with non-functional progenitor state. Cellular signatures also transit from loss of mature β-cell-specific markers such as MAFA and PDX1 to transcribing forbidden genes such as NGN3 and ALDH1A3 and even pluripotent genes. Molecular detailing showed transcription factor forkhead box protein O1 (FOXO1) to translocate to the nucleus under metabolic stress conditions and inhibit the matured β-cells undergoing reprograming to the progenitor state. FOXO1 also rescues key parameters related to diabetes like decreased β-cell mass and function.,,, Injury models such as pancreatic ductal ligation or pancreatectomy conditions have shown to drive the dedifferentiation of β-cells and the mechanism has been attributed to the signaling of TGFβ secreted by the immune cells. Moreover, adult β-cells during this condition begin expressing epithelial-mesenchymal transition (EMT) gene Snail2. The homeostasis of transcription factors (TFs) regulating dedifferentiation is regulated either at transcriptional level or by targeting them to ubiquitin-proteasome system (UPS) or lysosomal autophagy pathway.,,,,,, More than 60% of cellular proteins are subjected to proteasome-mediated degradation. The UPS involves ligases (E1, E2, and E3) that facilitate various degrees of ubiquitination resulting in mono-, oligo-, or polyubiquitinated forms of proteins. In general, proteins ubiquitinated at K48 are targeted to proteasomal degradation., In contrary, deubiquitinases (DUBs) are set of enzymes which deubiquitinate targeted proteins and thereby salvage them from proteasomal degradation., Several events occurring during pancreatic development or dedifferentiation of β-cells are governed by the UPS. The TF PDX1 which is important for the development of pancreatic epithelial cells is targeted to proteasomal degradation by E3 ligase PDX1 C-terminus-interacting factor-1. Deletion of FBW7 ubiquitin ligase in adult pancreas drives ductal cells toward β-cells. E3 ligase Mind bomb1 targets Notch receptor Delta to proteasomal degradation and regulates the pancreatic proximal-distal axis patterning of pancreas.,,, The role of DUBs in the context of dedifferentiation of cancer cells is well documented. SNAIL1, a key regulator of dedifferentiation, is stabilized in esophageal squamous cell carcinoma, breast cancer cells, and lung carcinoma cells by set of DUBs including OTUB1, DUB3, and USP37, respectively.,, DUB3 has also been shown to deubiquitinate SLUG and TWIST, the other two essential regulators of dedifferentiation. Recent few reports provided information on direct impact of DUBs on β-cell physiology. CLEC16A-RNF41 and USP8 form a tripartite complex, wherein CLEC16A is an E3 ligase that ubiquitinates RNF41 and USP8 is the DUB that deubiquitinates RNF41. The fine-tuning of RNF41 protein level dictates the mitochondrial quality in pancreatic β-cells and facilitates insulin secretion. Accumulation of toxic misfolded IAPP (Islet amyloid polypeptide) oligomers is one of the reasons behind β-cell dysfunction and this is attributed majorly to failure in deubiquitination of IAPP by UCHL1 which further results in aggregation of polyubiquitinated forms. Similarly, OTUB2 has been shown to deubiquitinate TRAF6 which enhances the survival activity of NF-κB in β-cells. MCPIP1 is a unique protein which possesses both DUB and RNase-like activities and is responsible in shielding β-cells from cytokine toxicity. Despite these initial insights, comprehensive information about DUBs in dedifferentiation of β-cells largely remains elusive and identifying the key DUBs that regulate dedifferentiation beams to be a resilient proposition to elucidate a novel therapeutic approach. In the present study, we aimed to investigate and identify key DUBs involved in dedifferentiation of pancreatic β-cells. Screening for DUBs revealed USP1 to be the probable candidate and loss and gain of function further reiterated the important role of USP1 in dedifferentiation. Administration of small-molecule inhibitor of USP1 in a dedifferentiation diabetic mouse model reduced the blood glucose levels and maintained the glucose tolerance test (GTT) and islet architecture near to normalcy. Finally, experiments to examine the downstream effector of USP1 revealed inhibitor of differentiation 2 (ID2) to be the downstream protein responsible for the dedifferentiation process.

To identify the key DUB involved in dedifferentiation of β-cells, we generated in vitro dedifferentiation model system by subjecting MIN6 cells to TGFβ and proteasomal inhibitor (PI) treatment., The cells showed reduced epithelial marker E-Cadherin and a corresponding decrease in insulin and a moderate increase in mesenchymal marker Snail1 at the transcript level (Figure S1A). Flow cytometry and Western blot confirmed the observation by showing reduced levels of E-CADHERIN and PDX1 levels (Figure S1B and S1C), cumulatively exhibiting a dedifferentiated state. In this model system, the expression level of multiple DUBs was analyzed and identified an increased expression of Usp1 compared to other DUBs (Figures 1A and 1B). To confirm the observation, the transcript analysis was performed along with other DUB Usp30 and observed an enhanced expression of Usp1, but not the Usp30, in TGFβ +PI-treated condition compared to untreated cells (Figure 1B). The upregulation of USP1 was consistent at the protein level as observed by flow cytometry analysis (Figure 1C). Mimicking the hyperglycemic condition, MIN6 cells exposed to higher glucose concentration showed decreased insulin expression and in parallel increased expression of USP1 (Figures S1D and S1E). To validate the elevated expression of USP1 in a dedifferentiated condition, we isolated primary islets from the mice and authenticated the identity of insulin-producing cells by DTZ staining, immunofluorescence of C-peptide (Figures S2A, S2B and S2C), and transcript analysis of Insulin, Nkx6.1, and E-Cadherin (Figure S2D). The primary islets cultured on fibronectin-coated dish displayed the emanating fibroblast-like cells from the islets (Figure S3A) and transcript analysis showed downregulation of pancreatic matured β-cell-specific markers Insulin, Nkx6.1, Pdx1, and epithelial marker E-Cadherin and increased expression of mesenchymal markers Snail1, Snail2, and Twist compared to the isolated islets (Figures S3B and S3C). However, they lacked the expression of Usp1 (Data not shown). Subjecting the primary islets to TGFβ treatment showed upregulated expression of Usp1, but not the other DUBs, similar to that observed in MIN6 cells treated with TGFβ+PI (Figures 1D and 1E). In corroboration with the key feature of dedifferentiation process as to lose the expression of mature β-cell-specific markers and increase the transcription of forbidden genes, we observed enhanced expression of progenitor genes Ngn3 and Aldh1a3 and decreased expression of matured genes Pdx1 and Mafa (Figures 1F–1I). These results signify a strong correlation between USP1 and dedifferentiation process in β-cells.

In order to explore the role of USP1 in the dedifferentiation process, we knocked down the expression using USP1-specific short hairpin RNA (shRNA). The USP1 shRNA efficiently inhibited the expression of USP1 which was monitored at the transcript level (Figures S4A and S4B) as well at the protein level (Figure S4C). Treatment of MIN6 cells with TGFβ+PI and transduced with scrambled shRNA showed 70% reduction in the expression of E-Cadherin and 76% reduction in Insulin expression at transcript level as assayed by qPCR and E-CADHERIN expression at protein level as analyzed by the flow cytometry (Figures 2A and 2B). Interestingly, transduction of these cells with USP1-specific shRNA showed significant rescue in the expression of E-Cadherin and Insulin at transcript level and E-CADHERIN at protein level (Figures 2A and 2B). Supporting the observation, compared to TGFβ+PI-treated cells, there was 30% reduction in expression of mesenchymal gene VIMENTIN in cells transduced with USP1 shRNA (Figure S5). However, similar inhibition of USP30 using USP30 shRNA lacked the ability to rescue the expression of E-CADHERIN, denoting the specificity of USP1 in the dedifferentiation process (Figure S6). In parallel, similar rescue effect was observed in in vitro dedifferentiated mouse primary islets as represented by transcript analysis for the expression of E-Cadherin and Insulin under conditions of USP1shRNA-transduced cells (Figure S4D). Previous studies showed USP1 activity could be allosterically inhibited using small-molecule ML323. Inhibition of USP1 using ML323 in TGFβ+PI-treated dedifferentiated MIN6 cells displayed a rescue in the transcript of E-Cadherin and Insulin at transcript level (Figure 2C) and increased expression of E-CADHERIN and decreased expression of VIMENTIN at protein level (Figures 2D, and S7B). Similar treatment of ML323 in TGFβ-treated in vitro dedifferentiated mouse primary islets showed rescue in the expression of E-cadherin and insulin (Figure S7B). Previous studies had shown the mesenchymal cells emanating from the cultured mouse islets to have the ability to redifferentiate back into islet-like clusters when cultured in low adherent condition. To further understand the advantage of inhibiting USP1 in redifferentiation, MIN6 cells were subjected to sphere formation and observed decreased sphere size, sphere count, and E-Cadherin mRNA expression in presence of TGFβ+PI and all the said parameters were rescued upon inhibition of USP1 (Figures S8A–S8D). Compared to monolayer, there was increase in INSULIN-positive cells in MIN6 spheres, but upon inhibition of USP1 by ML323 there was 4-fold further increase in Insulin transcript and ∼2-fold increase in E-CADHERIN protein compared to spheres (Figures S8E–S8H). Overexpression of USP1 in spheres also decreased the expression of E-Cadherin transcript which was rescued by inhibiting USP1 by ML323 (Figure S8I). These results indicate the essential role of USP1 in the dedifferentiation of pancreatic β-cells.

To further confirm the role of USP1 in dedifferentiation of pancreatic β-cells, we analyzed the dedifferentiation process in a USP1-overexpressed state. For this, MIN6 cells were transduced with a doxycycline-inducible USP1 overexpression construct which upon addition of doxycycline showed enhanced expression of Usp1 (Figure S9A). Overexpression of USP1 was sufficient to reduce the expression of matured β-cell genes E-Cadherin, Insulin, Pdx1, and Mafa and concomitantly increased expression of progenitor genes Ngn3 and Aldh1a3 and mesenchymal gene Snail2 at transcript level (Figures 3A–3E and Figure S9B). Decreased expression of PDX1 and INSULIN and increased expression of ALDH1A3 was observed at protein level as assayed by flow cytometry (Figures 3F–3J) and immunofluorescence (Figure 3K). To confirm the endogenous expression of progenitor gene NGN3 in MIN6 cells during the course of dedifferentiation, we transduced the Ngn3::GFP construct, wherein the expression of GFP indicates the Ngn3 promoter activity. Compared to control, overexpression of USP1 showed increased GFP expression, indicating increased Ngn3 promoter activity (Figures 3L and 3M). To reiterate the specificity of the USP1’s contribution in the dedifferentiation process, USP1 was overexpressed in USP1 shRNA background and analyzed for the expression of epithelial genes. Compared to untreated control, TGFβ+PI-treated cells showed 2-fold decrease in the expression of E-Cadherin at transcript level and 13% decrease at protein level. Transduction of USP1 shRNA in this condition rescued the expression of E-Cadherin and showed 6-fold increase in expression at transcript level and 12% increased expression at protein level compared to TGFβ+PI-treated cells. Overexpression of USP1 in USP1 shRNA background significantly decreased the expression of E-CADHERIN both at transcript and protein level (Figures 4A, 4B, and 4C). To strengthen the observation, instead of shRNA we performed similar experiment in presence of ML323 and observed rescue in the expression of E-Cadherin at transcript level and protein levels in cells treated with TGFβ+PI and ML323 in comparison to cells treated with TGFβ+PI alone. Overexpression of USP1 in ML323-treated cells significantly reduced the expression of E-CADHERIN and maintained the expression similar to that of TGFβ+PI-treated condition (Figures 4D, 4E, and 4F). These results indicate that the increased dedifferentiation observed in β-cells is the consequence of specific upregulation in the expression of USP1 protein.

Extending the study to an in vivo β-cell dedifferentiation model, we followed the protocol designed by Sachs et al., 2020, wherein β-cell dedifferentiation was established by administering mice with intraperitoneal injections of streptozotocin (STZ) for 6 consecutive days. In the mice experiments, the following experimental set up was formulated: a) control mice N = 5, b) STZ mice N = 5, c) ML323 treatment prior to STZ injection N = 5 mice, and d) ML323 treatment after STZ injection N = 5 mice. Transcript analysis of pancreas in STZ mice showed increased expression of Usp1 and forbidden gene Aldh1a3 and decreased expression of glucose transporter isoform Glut2. The mice treated with USP1 inhibitor either prior or after STZ treatment showed decreased expression of Usp1 and Aldh1a3 and rescued the expression of Glut2 (Figure 5A,5B, and 5C). Blood glucose levels were monitored on alternate day points up to day 35. Blood glucose of mice treated with STZ showed an average of 400–500 mg/dl. Interestingly, the mice treated with ML323 either 3 days before or 3 days after STZ injection showed reduced blood glucose level with an average of 200–250 mg/dl (Figure 5D). An intraperitoneal GTT was performed after 30 days in mice that received ML323 and observed better clearance of glucose compared to STZ-treated and ML323-untreated mice (Figure 5E). Histology of the pancreas of diabetic mice treated with ML323, untreated diabetic, and normal animals was analyzed by H&E staining and immunohistochemical staining for c-peptide. H&E (Figures 5F–5I) and immunohistochemical staining (Figures 4J–4Q) studies showed rescue in islet architecture in pancreatic sections of mice treated with ML323 compared to STZ-treated mice. Similar rescue in β-cell mass, islet volume, and β-cell number/islet was observed in mice treated with ML323 (Figure 5R–5T). Estimation of serum insulin showed reduced insulin amount in STZ-treated mice compared to control whereas treatment with ML323 either prior or after STZ treatment showed ∼3-fold rescue in serum insulin amount compared to STZ mice (Figure 5U). Clustering the entire observation, we propose that diabetic animals treated with ML323 sustain functionality of β-cells in the prevailing diabetic conditions probably by inhibiting dedifferentiation.

In the prospects of understanding the underlying mechanism of USP1-mediated dedifferentiation of β-cells, we sought after the available information in the literature and found ID family proteins to be the substrates of USP1 in certain metastatic conditions., This intrigued us to investigate whether ID2 participates in the USP1-mediated dedifferentiation process in the β-cells. Analysis at the transcript level for the expression of ID family genes, ID1, ID2, and ID3, in islet cells treated with TGFβ compared to untreated controls revealed the upregulation of ID2 compared to ID1 and ID3 (Figure 6A). Furthermore, we looked into the expression of ID2 genes in a USP1-inhibited condition and found islets transduced with USP1sh RNA and treated with TGFβ showed ID2 to be highly downmodulated (Figure 6B). This indicated ID2 protein to be responding to the modulation in USP1 expression level. To analyze whether the stability of ID2 is determined by USP1, we overexpressed USP1 in MIN6 cells cultured in presence or absence of translational inhibitor cycloheximide and found ID2 protein to be stabilized in cells overexpressing USP1 compared to vector control (Figure 6C). The stability of ID2 was further corroborated by decreased expression of NeuroD transcript in cells overexpressing USP1 and the inhibition being reversed by inhibiting USP1 (Figures 6D and 6E). NeuroD has been shown to be the direct downstream target and being repressed by ID2 protein. To further reveal whether ID2 to be the downstream effector of USP1 in regulating the dedifferentiation process, we strategized to perform ID2 loss and gain of function in presence or absence of USP1 in MIN6 cells. Overexpression of ID2 in cells cultured in presence of TGFβ+PI reduced the expression of E-Cadherin by 2-fold at transcript level and by 12% at protein level compared to TGFβ+PI-treated cells (Figure 7 a-c). Inhibition of ID2 expression by ID2-specific shRNA reversed the TGFβ+PI-mediated reduced expression of E-Cadherin and dedifferentiation process. There was ∼3.2-fold increase in the expression of E-Cadherin at transcript level and ∼8% increase at protein level compared to TGFβ+PI-treated scrambled control (Figure 7 d-f). To correlate the relation between USP1 and ID2, we knocked down ID2 in presence of USP1 overexpression condition and analyzed for the expression of E-CADHERIN in MIN6 cells. In cells overexpressing USP1, ∼ 2-fold decrease in the expression of E-Cadherin at a transcript level and 22% at protein level compared to vector control was observed. Inhibition of ID2 in this USP1 overexpression condition by transducing ID2-specific shRNA reversed the expression of E-Cadherin by ∼4.5-fold at transcript level and 18% at protein level compared to USP1 overexpression and scrambled shRNA-transduced condition (Figure 7 g-i). In conclusion, these experiments reveal the activation of USP1 during dedifferentiation process to coincide with ID2 expression and indicate the likeliness of ID2 being a downstream effector of USP1 during β-cells dedifferentiation.

Dedifferentiation is a cardinal process playing a dogmatic role during pancreatic β-cell development as well in metabolic diseases like diabetes.,, Several experimental evidences showed the occurrence of β-cell dedifferentiation which could be one of the major reason for depleted mass in hyperglycemic state., Retaining in vivo β-cell mass by avoiding dedifferentiation is an ideal alternative strategy to combat β-cell loss. In the present study, we investigated the role of DUB USP1 and observed its role in in vitro and in vivo dedifferentiation of pancreatic β-cells. Inhibition of USP1 facilitated in retaining the epithelial phenotype and thereby properties of β-cell. Attempts to understand the mechanism shed light on ID2 to be the probable downstream substrate of USP1 during this process. The outcome of the present study helps in better understanding the process of dedifferentiation in β-cells and small-molecule-mediated inhibition of USP1 takes a step closer to sustain the β-cell mass. Several studies showed various signaling pathways to participate in the dedifferentiation process. Recent study by Xiao et al. showed the TGFβ secreted by M2 macrophages to be responsible for the dedifferentiation process of β-cells in a ductal ligated mouse model system. Due to lesser number of DUBs being encoded by mouse genome compared to E3 ligases, screening for DUBs in dedifferentiation process proved more feasible. Considering the evidence of TGFβ in dedifferentiation, we dedifferentiated MIN6 cells line by treating with TGFβ and PI and screened for key DUBs based on their expression level. We detected the transcript levels of Usp1 to be highly upregulated compared to other DUBs. Furthermore, enhanced expression of USP1 was also observed in primary β-cells treated with TGFβ. Several DUBs have been identified to enhance the downstream TGFβ activity by stabilizing the intermediates of TGFβ pathway. Interestingly, the deubiquitinase enzyme FAM/USP9X stabilizes Smurf1, the downstream of TGFβ cascade, and facilitates in enhancing the TGFβ activity. However, in our screening process, we failed to detect the enhanced expression of any other DUBs other than USP1. Inhibition of USP1 either by shRNA or by small-molecule ML323 showed similar effect on dedifferentiation process. Previous studies had shown ML323 to inhibit the enzymatic activity of USP1 allosterically., However, in our present study, we consistently observed the downregulation of USP1 expression at transcript and protein levels also when the cells were treated with ML323. This decrease in Usp1 transcript could be probably a secondary effect caused due to the induction of dedifferentiation process rather than the direct effect of ML323 on the transcription of Usp1 expression. There are a few recent reports which showed a direct correlation between USP1’s activity and manifestation of β-cell failure. Inhibition of USP1 was shown to protect β-cells from glucose toxicity-induced apoptosis and the mechanistic insights showed USP1 inhibition to enhance the DNA damage response and in turn to facilitate the β-cell survival. In our study, though we failed to find difference in β-cell survival upon overexpression or inhibition of USP1, our experimental results indicate inhibition of dedifferentiation in USP1 knockdown condition to be the other alternative mechanism involved in retaining β-cell function. To date, there has been an ongoing debate on whether β-cells per se undergo dedifferentiation or the mesenchymal cells exist from the pre-existing mesenchymal cells or is it the transdifferentiation of other islet cells. Though the ambiguity persists until date, several lineage tracing studies including the elegant study by Talchai et al. showed the β-cell-specific knockout of FOXO1 to result in dedifferentiation of β-cells that results in diabetes. Similarly, Mak et al. demonstrated β-cell dedifferentiation to be part of EMT and indicated miR-7 to be the key player in enhancing the expression of mesenchymal genes leading to EMT. All this collective information portrayed dedifferentiation process to exist in β-cells which triggers loss of β-cell mass and imply to be one of the major causatives of diabetes. Interestingly, in our both in vitro and in vivo model system, we observed the enhanced expression of forbidden genes and decreased expression of matured β-cell genes, authenticating the dedifferentiation process. Though we observed the occurrence of dedifferentiation in primary islet cells, we hesitate to rule out the contribution of other cells of the islets to participate in this process, as our analysis considered the entire pool of dedifferentiated cells emanating from the cultured islets and not from the fractionated homogeneous β-cells. Since larger part of islets is populated with β-cells (60%–70%), we still strongly consider the role of USP1 in the process of dedifferentiation of β-cells. To conclusively ascertain the role of USP1 in dedifferentiation, we adapted an in vivo diabetic dedifferentiation model system. Sachs et al. demonstrated that STZ-induced diabetes model could be utilized to study β-cell dedifferentiation in mice. In the present study, in STZ-treated pancreas, there was increased expression of the pancreatic progenitor gene Aldh1a3 along with Usp1. Administration of USP1 inhibitor ML323 in STZ-induced dedifferentiation model system reversed partially the hyperglycemic state and β-cell mass and number of islet-positive cells compared to STZ alone-treated mice. The islet architecture as well the insulin secretion in serum of diabetic mice treated with ML323 showed similar phenotype to that of non-diabetic controls but the untreated diabetic animals showed regressed islet clusters and reduced serum insulin. These results convincingly portrayed the significant role of USP1 in in vitro and in vivo dedifferentiation of β-cells. Mechanistically, dedifferentiation is controlled by several transcription factors. For instance, hypoxia-inducible factor-1α has been shown to facilitate dedifferentiation of β-cells. Conditional knockout of FOXO1 TF resulted in dedifferentiation of β-cells and has been shown to be the major cause of β-cell failure in type II diabetes. During unfavorable dedifferentiation inducing conditions, FOXO1 translocates to the nucleus and protects the β-cells by facilitating the expression of matured markers NEUROD1 and MAFA. The other important TFs family that regulates dedifferentiation process is ID family members. Elevated ID proteins are observed in several dedifferentiated primary human malignancies including pancreatic carcinoma and neuroblastomas., There are various DUBs which regulate the homeostasis of several proteins that dictate the degree of dedifferentiation process. For instance, homeostasis of NGN3, the TF that ear marks islet precursor cells, has been shown to be dependent on the presence of NOTCH signaling. DUB USP9x stabilizes the components of NOTCH and thereby increases NGN3 activity which in turn probably increases the dedifferentiation process. An independent study also showed the expression of SNAIL in NGN3-expressing precursor cells which inhibited the maturation to β-cells and in parallel facilitated the dedifferentiation process. Recent study in an independent cellular system showed USP1 to deubiquitinate SNAIL1 and enhance its stability. In the present study, with the goal of identifying the mechanism by which USP1 executes its role in dedifferentiation, we considered the previously available information from literature which showed the deubiquitination of ID proteins by USP1 to be essential for maintenance of mesenchymal phenotype in osteosarcoma cells (Williams et al., 2011). In the process of testing whether the similar mechanism holds true in dedifferentiation of β-cells, we observed enhanced expression of ID2 compared to other ID family members in dedifferentiated MIN6 cells. Overexpression of ID2 was sufficient to induce the dedifferentiation of MIN6 cells even in absence of USP1, and inhibition of ID2 even in USP1 overexpression condition was adequate to retain the epithelial gene expression. These results indicated ID2 to be the probable downstream candidate of USP1 and deubiquitination of which probably enhances dedifferentiation of β-cells. Endogenous β-cells undergoing dedifferentiation-mediated changes in in vitro culture has crippled the long-term expansion of β-cells. Therefore, it is prudent to have a better understanding of the dedifferentiation process in context of β-cells to retain functional cells in in vitro as well for in vivo therapeutic applications. Considering the outcome of the present study, altering USP1 activity could be envisaged as an alternative means to sustain in vitro β-cell culture as well to inhibit the in vivo β-cell loss in diseased state.

There are certain limitations in the study. Though we elegantly deciphered the role of USP1 in dedifferentiation using MIN6 cell line and primary β-cells, the role could be more affirmatively addressed by conditional knockout of USP1 in β-cells. In the in vivo diabetic model of dedifferentiation, we did demonstrate the reversal of hyperglycemia along with β-cell mass and serum insulin secretion. However, an in-depth in vivo characterization of the reversal of diabetes which includes the parameters like glucose-stimulated insulin secretion in the diabetic mice treated with USP1 inhibitor has not been addressed.



Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Anujith Kumar: anujith.kumar@manipal.edu.

No unique reagents were generated in this project.

All animal experiments were conducted post approval by the Animal Ethics Committee of Manipal Academy of Higher Education (MAHE) (IAEC/KMC/70/2020)). Animals were obtained from Central Animal House Facility, MAHE, Manipal. For in vivo experiments Swiss Albino male mice (10-12-week of age 30-35g of body weight) (N = 20) were used. To isolate endogenous islets Swiss Albino male mice (4-5-week of age 14-16g of body weight) (N = 20) were used. The animals were euthanized by cervical dislocation. All animals were handled in accordance with the national ethical norms, and they were maintained in a facility compliant to national regulations.

MIN6 (National Center for Cell Science, Cell repository, Pune, India) and HEK293T (Kind gift from Prof. Catherine Verfaillie, Leuven) cells were maintained in DMEM HG media (Gibco, 11965-092) containing 10% FBS (Gibco 10270-106), 2 mM Glutamax (Gibco, A12860), Penicillin 100units/ml and Streptomycin 100 μg/ml (Gibco, 15140-122). For dedifferentiation of MIN6 cells, the cells were trypsinized with 0.25% trypsin (Sigma Aldrich, T4049), counted and plated at the rate of 2 ×105 cells/35 mm dish and factors added the following day. The cells were cultured with and without 20 ng TGFβ (Sino Biologicals, 10804-HNAC) & 50 nM proteasome inhibitor (PI) MG132 (Sigma Aldrich, M7449), ML323 (Sigma Aldrich, SML1177) and maintained for 4 days and later harvested for analysis. Handpicked endogenous islets were plated on 10 μg/ml fibronectin (Sigma Aldrich, F1056) coated wells and maintained in RPMI media (Gibco, 11875-093) containing 10% FBS, 2 mM Glutamax, Penicillin 100units/ml and Streptomycin 100 μg/ml with or without 20 ng TGFβ for 4 days and later harvested for analysis.

Pancreas from 2 mice were isolated and digested for 20 min at 37°C in a shaker incubator in digesting enzyme liberase (Sigma Aldrich). The digest was neutralized with 1X HBSS (Sigma Aldrich) and the pellet was subjected to Dextran (Sigma Aldrich) based gradient centrifugation and the top most layer was separated and isolated islet clusters were handpicked using a Nikon SMZ1500 dissection microscope.

Mice was restrained in a tube with tail left out. With a lancet needle prick a droplet of blood was drawn and applied onto glucometer strips and blood glucose concentration was obtained through the glucometer reading (Accu-Chek) in mg/dl.

16 h starved mouse were injected intra-peritoneally with glucose (HiMedia) 2 g/kg body mass and blood glucose levels monitored at 0 min, 15th min, 30th min, 60th min and 120th min. The readings were graphically represented in line graph.

With single hand restrain mice starved for 6 h were administered intra-peritoneally with 50 mg/kg body weight of streptozotocin (Sigma Aldrich) for 6 consecutive days. Along with normal and STZ treated conditions, two conditions of ML323 (Sigma Aldrich) treatment were maintained, a) daily treatment of ML323 6 days post STZ treatment and b) treatment of ML323 beginning 3-day prior to STZ treatment. ML323 administered at the rate of 15 μg–30μg/dose.

HEK293T3 Cells were plated at the rate of 3 ×106 cells per 60 mm dish a day prior to transfection. Transfection agent X-tremeGENE (Merck) was added to optiMEM media (Gibco) at the rate of 1:4 ratio of plasmid concentration, vortexed and incubated for 10 min at room temperature. To the transfection mix, the defined concentration of viral packaging plasmids VSV-G and GAG-POL for overexpressing USP1 and ID2 and PMD2G and PsPAX2 packaging plasmid for expressing USP1 shRNA and ID2 shRNA, were added and incubated for 30 min at room temperature. Following which the mixture was added to the HEK293T3 cells and incubated at 37°C in CO2 incubator. The culture supernatant containing the viral particles were collected after 48hrs and 72 h and were concentrated by incubating in PEG with a final concentration of 50% on a rocker at 4°C overnight and centrifuged at 1600rpm for 1 h at 4°C. The viral palette was suspended in 200 μL media and added to respective cells for transduction. Post 24hrs 1 μg/ml of puromycin (HiMedia) was added to respective cultures for selection. Transduced cells were further used for respective experiments.

RNA was isolated from cultured cells using TriReagent (Sigma Aldrich) in accordance with manufacturer’s protocol. Briefly, monolayer cultures were homogenized with TRIReagent to the mixture. 200 μL/mL chloroform was added vortexed for 10 s and centrifuged at 12000g for 10 min at 4°C. The aqueous layer was collected and RNA was precipitated by adding 500 μL of isopropanol per mL TRIReagent and incubated at room temperature for 10min, then centrifuged at 12000g for 10 min at 4°C. The RNA pellet obtained was washed with 75% ethanol by centrifuging at 7500g for 5 min at 4°C. After which the supernatant was discarded and the pellet air-dried and suspended in DNAse/RNase free water. RNA was quantified using Naodrop and 1 μg RNA was used for cDNA synthesis using commercial cDNA preparatory kit (Takara 6110A). For gene analysis the cDNA was amplified using gene specific primers by qPCR with Takara 2X SYBR Green Mix (Takara RR420A) and analyzed in real-time PCR machine (7500 Applied Biosystems). Gene expression was normalized to the expression of housekeeping gene and the relative fold change was calculated using the 2 -ΔΔCt method. List of primers is provided in Table S1.

Cells to be analyzed were trypsinized and washed with 1X PBS and fixed by suspending in 4% Paraformaldehyde (PFA) and incubating at 4⁰C overnight. For flow cytometry analysis, the cells were counted and equal number of cells distributed into flow tubes, washed and permeabilized with 1X BD Permwash (BD). The cells were incubated with primary antibody overnight, followed by washes and incubation with secondary antibody for 1 h again washed and re-suspended in FACS buffer for analysis using BD LSR II or BD FACS Caliber.

Cells were fixed with 4% PFA and incubated at 4°C. For the experiment, the cells were washed with 1X PBS, then permeabilized with 0.2% Triton X-100 in PBS for 15 min at RT, blocked in blocking buffer containing 3% BSA in 1X PBS Solution at RT for 1h. Antibodies were diluted in blocking buffer and cells were incubated with the primary antibody at 4°C overnight. The cells were subsequently washed with 0.05% PBST and stained with secondary antibody for 1 h at RT and counterstained with DAPI (1:10000) (Life Technologies) for 1 min and observed under fluorescent microscope Nikon Eclipse TE2000U.

Cells were harvested and lysed with ice-cold RIPA lysis buffer with freshly added protease inhibitor cocktail (Sigma Aldrich, Bangalore). The lysate was spun down at 12000 rpm for 15min and the supernatant collected and stored in −20°C until further use. 100 μg of protein was denatured by heating at 95°C with protein loading buffer and resolved using 10% SDS-Polyacrylamide gel. The resolved proteins were transferred onto charged PVDF membrane (Millipore) using semi-dry blotting apparatus. The membrane was blocked using 3% skimmed milk in 1x TBST. The blots were probed with primary antibody and incubated overnight at 4°C on a rocker. Following day, the blots were washed trice with 1X TBST and incubated with HorseRadish Peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature on a rocker. The blots were then washed trice with 1X TBST. The blots were developed using Western Bright HRP substrate (Advansta) and imaged on LI-COR C digit blot scanner.

10 mg/ml DTZ (Sigma Aldrich) solution was added onto freshly handpicked mouse endogenous islets and incubated for 3–5 min. The stain removed and islets washed with 1X HBSS and phase contrast colored pictures of red stained islets were captured on Nikon Eclipse TE2000U.

The fixed tissues were embedded in paraffin and 5 μm sections were stained by H & E staining. The sections were deparaffinised with Xylene washes, rehydrated with gradient alcohol washes, the slides were stained with hematoxylin for 1 min and post a 70% alcohol wash stained with Eosin stain for 1 min. The slides were then processed through graded alcohols and xylene and mounted with DPX and subsequently observed using a phase contrast microscope Nikon Eclipse 80i.

Serum from mice of all experimental conditions were collected and insulin levels were measured following user guide (10-1249-01 Mercodia Ultrasensitive Mouse Insulin Elisa KIT). The OD were read on Perkin Elmer multi-mode spectrophotometer at 540 nm, values were plotted against standard curve, and concentration of serum insulin was calculated.

Whole pancreas from all conditions of 3-independent experiments were considered for each parameter. The average value from 15 random 5-microns histological sections of each pancreas into five consecutive sections of 200 μm apart were analyzed.

β-cell mass was calculated by measuring the ratio of C-PEPTIDE positive area and pancreatic tissue area and multiplied with the weight of the pancreas.

β-cell volume was calculated by multiplying total insulin positive area with the distance between the sections.

β-cell number in islets was calculated using ImageJ software using C-PEPTIDE positive cells. ITCN plugin was installed to serve the purpose of automated counting of cell nuclei.

Paraffin embedded sections were deparaffinized by placing the slides in a 65°C hot plate for 30 s to 1 min followed by xylene washes and rehydrated with gradient alcohols. The sections were subjected to antigen retrieval by 20min incubation in boiling in 10 mM citrate buffer pH 6.0. Blocking with 5% normal serum +1% BSA in 0.05M Tris Hcl buffer pH 7.5 for 1h. Washed in Tris Hcl buffer and added primary antibody and incubated overnight. Following day washed in Tris Hcl buffer twice and incubated with fluorescent secondary antibody overnight and counter stained with DAPI, mounted with mounting media and observed under Nikon TE80i.

Student’s t test was used to analyze the difference between the test cells and its respective controls. The values with p < 0.05 were considered statistically significant indicated as ∗ and those with p < 0.001 were considered highly significant indicate as ∗∗.