3.1. ScRNA-seq showed Ube2c highly expressed in proliferating β-cells in mouse and human islets
To explore the pivotal mechanism promoting β-cell proliferation, scRNA-seq was performed on islets of C57BL/6J mice fed a HFD for 8 weeks which was confirmed by the compensatory proliferation period[21]. A total of 14108 β-cells from HFD- and CD-fed mice were annotated based on predominant hormone gene expression (Ins1) and then clustered into nine clusters using UMAP according to our previous study [28] (Fig. 1A). The cell cycle distribution of β-cells was further analysed, showing that Cluster-7 β-cells were in the G2M phase, whereas the other subpopulations were in the G1 and S phases (Fig. 1B). We then analysed the marker genes of cluster-7: Ube2c showed the Top3 highest expression in this cluster and was barely expressed in the remaining subpopulations (Fig. 1C). Therefore, we defined cluster-7 as Ube2chigh β-cells, and the remainder were defined as Ube2clow β-cells (Fig. 1D). Gene set enrichment analysis (GSEA) revealed that Ube2chigh β-cells were enriched significantly in “DNA replication”, “cell cycle”, and “ubiquitin-mediated proteolysis pathways” (Fig. 1F). Functional enrichment analyses using Gene Ontology (GO) revealed biological processes to be associated with “mitosis” and “chromosome segregation”, and molecular functions to be associated with “cell cycle-dependent serine/threonine kinase activity” (Fig. 1G).
Compared to the Ube2clow subpopulation, genes related to cell proliferation and cell cycle, such as Pcna, Mki67, Ccnb2, and Cdk1, were highly expressed in Ube2chigh subpopulation. However, the expression of genes related to β-cell maturation, such as Slc2a2, G6pc2, Ucn3, Chga, and Ero1lb, was slightly reduced. However, no difference was observed in the expression of Ins1 and Ins2 (Fig. 1E and H). Other DEGs are presented in Supplementary Table 5. Immunostaining confirmed the scRNA-seq findings, as Ki67, UBE2C, and INS were co-stained in pancreatic sections, indicating that high expression of Ube2c is a strong sign of proliferative β-cells (Fig. 2A).
To determine UBE2C expression levels in human islets, we used a recently reported dataset from non-diabetic donors [29]. Based on their annotated beta cells, UBE2C was specifically expressed in a very small population of cells (cluster 48). We defined cluster-48 as UBE2Chigh β-cells and the remainder as UBE2Clow β-cells (Supplementary Fig. 1A–C). Both GO and KEGG analyses suggested that UBE2Chigh β-cells were highly enriched in cell cycle and biological processes related to ubiquitination and DNA replication (Supplementary Fig. 1D–E). The cell proliferation-related genes MKI67, CCNB2, CDK1, PCNA, and PBK, and E3 ubiquitin ligase CUL1 were highly expressed in UBE2Chigh β-cells (Supplementary Fig. 1F). Other DEGs are presented in Supplementary Table 6. Moreover, a higher proportion of UBE2C positive β-cells was observed in human islets from an obese donor (BMI = 29.7) than in lean donors (BMI < 24) (Supplementary Fig. 1G). These results suggest that, similar to mice, UBE2C is also highly expressed in proliferating human β-cells.
3.2. Ube2c is highly expressed in islets of weaning and HFD-fed mice, and promotes β-cell proliferation
As previously reported, β-cells proliferate significantly physiologically during weaning and pathologically during the overnutrition-induced compensatory proliferation stage. The protein and RNA levels of islets were further investigated in CD and HFD-fed mice of various ages. In accordance with the RNA levels, WB analyses showed that UBE2C was highly expressed in juvenile mouse islets and increased when HFD was administered (Fig. 2B–D). This phenomenon was confirmed using immunohistochemical staining (Fig. 2E). However, the expression of UBE2C decreased with age. Importantly, these data were consistent with the decreased Ki67-positive β-cell ratio in mice fed an HFD in our previous study [21].
To determine whether Ube2c is the pivotal gene that regulates β-cell proliferation, gain- and loss-of-function were investigated in the mouse β-cell lines MIN6 and βTC-6. Increased cell viability and proliferation ratio were observed in the UBE2C over-expressing cell line; however, Ube2c knockdown diminished β-cell proliferation (Fig. 2F–M). As self-replication and expansion contribute to proliferation, PI staining combined with flow cytometry analysis was performed. We found a reduced proportion of cells in the G1 phase and an increased proportion of cells in the G2M phase in Ube2c knockdown βTC-6 cells compared to the control (Fig. 2N–O). Collectively, these data indicate that UBE2C promotes β-cell proliferation through self-replication based on cell cycle renewal.
3.3. β-cell specific Ube2c knockout impairs insulin secretion and glucose tolerance in vivo
To clarify the role of UBE2C in β-cells in vivo, βUbe2cKO mice were constructed (Supplementary Fig. 2A). Compared to wild-type (WT) mice, no significant difference in UBE2C expression was found in metabolic tissues of βUbe2cKO mice, such as liver, adipose tissue, and skeletal muscle, but expression was diminished in islets (Supplementary Fig. 2B–C). Physiologically, Ube2c knockout did not alter body weight (Fig. 3A) or fasting blood glucose levels but increased 2-h postprandial blood glucose levels in juvenile mice during 4-week-old weaning (Fig. 3B). Compared with control mice, βUbe2cKO mice developed glucose intolerance at 4 weeks of age and presented significantly higher plasma glucose levels during the glucose tolerance test (Fig. 3D). We further divided 4-week-old βUbe2cKO mice and their littermates randomly into HFD and CD groups to investigate the function of UBE2C during diabetic pathophysiology. Continuous monitoring of body weight showed increased body weight in HFD-fed mice compared with CD-fed mice; however, no significant difference was observed between βUbe2cKO and control mice (Fig. 3F). Mouse sex was also considered as a contributing factor for the metabolic phenotype, and no significant difference was found in body weight or blood glucose levels between βUbe2cKO and control female mice (data not shown); therefore, we selected male mice for subsequent studies. We found that the glucose clearance capacity of βUbe2cKO mice decreased significantly with prolonged HFD feeding compared to that of control mice (Fig. 3G–J).
To determine the main cause of decreased glucose clearance capacity, insulin and glucagon secretion, and insulin sensitivity of metabolic tissues were analysed. Compared to the control mice, βUbe2cKO mice showed impaired insulin secretion at 4 weeks old both in the fasting state or postprandially (Fig. 3C), and this phenomenon persisted in HFD feeding mice (Fig. 3L–M). However, glucagon secretion did not differ between βUbe2cKO and the control mice (Supplementary Fig. 4A–C). In addition, glucose-challenged insulin secretion was impaired in βUbe2cKO mice fed both CD and HFD (Fig. 3E and K). The ITT was used to estimate whole-body insulin sensitivity, and no difference was found between the control and βUbe2cKO mice (Supplementary Fig. 3A–C). We also assessed insulin sensitivity in the major insulin target organs; however, no significant change in the phosphorylated protein kinase B (p-AKT)/AKT ratio was found in βUbe2cKO mice compared to that in the control (Supplementary Fig. 3D–F). Collectively, these data suggest that Ube2c specific deletion in islet β-cells impairs insulin secretion, which contributes to hyperglycaemia in βUbe2cKO mice.
3.4. Decreased proliferation rate and secretory capacity of β-cells in β Ube2c KO mice result in impaired insulin secretion
To investigate the reasons for impaired insulin secretion in βUbe2cKO mice, pancreatic sections were used to analyse islet morphology. Based on the statistical analysis of hematoxylin and eosin (H&E) staining, a significant reduction in islet mass of βUbe2cKO mice compared to control mice had occurred by 4-week-old weaning (Fig. 4A). Moreover, islet size distribution analysis showed that the proportion of large islets decreased, but the proportion of small islets increased (Fig. 4B) in βUbe2cKO mice compared with control mice during CD feeding. The same tendency, but a more pronounced decrease in islet mass, was found after 4 weeks of HFD feeding in βUbe2cKO mice. Co-staining of insulin and glucagon was further performed to analyse whether β- or α-cell loss contributes to islet mass deficiency. Compared to the control mice, a smaller proportion of β-cells; however, a larger proportion of α cells in βUbe2cKO mice were noted (Fig. 4D). However, the comparatively high plasma glucagon levels in both fasting and postprandial states indicated that the relative increase in α-cell area was caused by β-cell reduction (Supplementary Fig. 4A–C). As described above, Ube2c was associated with β-cell replication, the proliferation indicators Ki67 and insulin were further co-stained, and a significantly decreased Ki67-positivite β-cell ratio was detected in βUbe2cKO mice both during off-lactation and in HFD feeding conditions (Fig. 4C and E). These data revealed that Ube2c knockout diminished the proliferative capacity of islet β-cells, resulting in inadequate amplification under metabolic stress, including nutrient changes from breastfeeding to carbohydrate or overnutrition.
Islet perfusion was performed on 4-week-old mice, and compared to the control mice, a significant decrease in glucose-stimulated second-phase insulin secretion was observed when corrected according to the DNA mass in βUbe2cKO mice (Fig. 4F). However, no difference was observed in the first phase of insulin secretion and insulin content. After 4 weeks of HFD feeding, the islet perfusion results of βUbe2cKO mice were similar to those of weaned mice, despite no statistical differences (Fig. 4G). In vitro GSIS experiments also showed that βUbe2cKO mice had significantly decreased insulin secretion in both CD and HFD feeding (Supplementary Fig. 4D and G). We further detected glucose-stimulated glucagon and somatostatin secretion, and no significant difference was found between βUbe2cKO and control mice in either CD or HFD feeding (Supplementary Fig. 4D–I). Moreover, Ube2c overexpression in primary islets and βTC-6 cells improved GSIS when corrected based on protein levels (Supplementary Fig. 4J–L), whereas knockdown of UBE2C in βTC-6 cells decreased GSIS. These data indicated that UBE2C positively regulates insulin secretion, except for its ability to promote β-cell proliferation.
Considering the dampened glucose-stimulated insulin secretion of the Ube2c knockout, TEM was used to investigate the ultrastructure of β-cells, especially insulin granules. The total number of insulin granules decreased in βUbe2cKO mice at 4-week-old or after 4 weeks of HFD feeding (Fig. 4H). The reduction in mature insulin granules was the primary reason for total granule reduction, but not for immature or docked granules (Fig. 4I and J). Collectively, these data suggest that reduced β-cell mass contributes to the basal decrease in plasma insulin levels, whereas reduced mature granules contribute to glucose-stimulated insulin secretion in vivo, which finally promotes diabetes onset.
3.5. UBE2C promotes β-cell proliferation by ubiquitinating of PER1
Usually, UBE2C interacts with substrates and E3 ubiquitin ligases to ubiquitinate target proteins and degrade them through the proteasome pathway. The Cys114 site of human UBE2C protein is used to binds ubiquitin molecules. We found that human and mouse UBE2C were highly conserved; therefore, we constructed a mouse plasmid with the Cys mutation at the UBE2C114 site (UBE2CC114S). The mouse cell line βTC-6 was transfected with WT, mutant (MT), and vector plasmids, and WB experiments revealed that UBE2CC114S did not affect UBE2C expression (Fig. 5A). Moreover, the CCK-8 and EdU assays showed that UBE2CC114S lost the capacity to promote β-cell proliferation compared to the WT plasmid (Fig. 5B–D), suggesting that the ubiquitin-binding site of UBE2C contributes to its molecular function.
To identify the possible substrate proteins and E3 binding to UBE2C, immune-precipitation and MS using the UBE2C antibody were performed. Using the immunoglobulin (Ig)G control, background proteins were removed, and 296 and 317 intercalating proteins were found in LV-GFP- (physiological condition) or LV-UBE2C- (proliferative condition) infected βTC-6 cells, respectively. In the physiological state, βTC-6 itself proliferates actively with high expression of UBE2C, and the high number of identified proteins (296) derived from the LV-GFP group reflects this condition. More interacting proteins (317) were identified after UBE2C overexpression, suggesting that the substrates of UBE2C in β-cells under physiological and proliferative conditions may differ. Among them, several core proteins in E3 ubiquitin ligase, including CUL1 (a member of the SCF complex), HERC2, CBLB, MID1, and members of the E1 ubiquitin activator UBA6, were detected (Fig. 5E). Quantification TMT proteomics of βTC-6 cell lines was also performed to identify possible target proteins that were expressed at lower levels after UBE2C overexpression (Fig. 5F). Venn diagrams showed that PER1 was the only protein detected in both the control and UBE2C-overexpression groups using the co-immunoprecipitation technique, and PER1 was decreased significantly after UBE2C overexpression (Fig. 5G). As ever reported that PER1 is an important negative regulator of “biological clock” that negatively regulates tumour proliferation, and the SCF complex can degrade PER1 through ubiquitination [30, 31]. Besides, RNA-seq performed on HFD-fed control and βUbe2cKO mouse islets revealed that significant expressed genes enriched in “circadian entrainment pathway” (Fig. 5H–J), which corresponds to the function of the biological-clock gene Per1. Together, these data provide evidence that the UBE2C-CUL1-PER1 complex participates in β-cell proliferation.
We further proved that PER1 and CUL1 could co-precipitate using the UBE2C antibody (Fig. 6A), indicating that UBE2C interacts with PER1 and CUL1. The colocalisation of UBE2C and PER1 in islet β-cells was further verified by immunofluorescence (Fig. 6E). The transcriptional level of Per1 was detected using PCR, and we found that neither Ube2c overexpression nor knockout in primary islets influenced Per1 expression (Fig. 6F). However, WB showed that PER1 expression was reduced upon UBE2C overexpression, but increased in βUbe2cKO mice (Fig. 6B). We questioned whether UBE2C regulates PER1 expression through ubiquitination. Thus, we transfected the UBE2C WT and UBE2CC114S plasmids into βTC-6 cells and found that the UBE2CC114S plasmid did not cause a change in PER1 expression (Fig. 6C). The protein synthesis inhibitor cycloheximide (CHX) and proteasome inhibitor MG132 were then applied, and PER1 degradation was found to be significantly inhibited by MG132 (Fig. 6D), similar to the effect of Ube2c knockdown (Fig. 6G). The ubiquitination of PER1 was then detected in UBE2C WT and UBE2CC114S plasmid overexpressing cells, and UBE2C WT, but not UBE2CC114S, mediated the notable increase in PER1 ubiquitination (Fig. 6H). Co-immunoprecipitation also showed that UBE2C knockdown significantly reduced PER1 ubiquitination (Fig. 6I). The regulatory role of UBE2C to PER1 in β-cell proliferation and insulin secretion was further confirmed. PER1 inhibition significantly rescued UBE2C knockout-induced growth inhibition, and insulin secretion was observed in βTC-6 cells (Fig. 6J–L). Collectively, these results indicate that UBE2C reduced PER1 expression through ubiquitin-mediated proteasomal degradation, which contributes to the proliferation and glucose-stimulation of β-cells.
3.6. Restored UBE2C expression promotes β-cell regeneration in streptozotocin (STZ)-induced diabetic mice
As UBE2C promotes β-cell proliferation, we treated STZ-induced diabetic mice with massive β-cell loss (Fig. 7A). Low doses of STZ were injected into 4-week-old C57BL/6J mice for 5 d, and hyperglycaemia was monitored 1 month before virus injection (Fig. 7E–F). Ube2c-overexpressing and control lentiviruses were injected into the pancreatic duct at 8-week-old. Two weeks post injection, the UBE2C restored mice began to show a decrease in blood glucose levels and an increase in insulin levels (Fig. 7G–J).
With decreasing glucose levels, the body weight of Ube2c restored mice increase gradually compared to that of the control mice (Fig. 7B). Glucose tolerance was also improved in Ube2c restored mice owing to the increased glucose-stimulated insulin secretion in vivo (Fig. 7C and D). However, glucagon levels were not different between the groups (Fig. 7K), suggesting a specific effect of UBE2C on β-cells.
The co-staining of GFP and insulin showed widely expressed lentiviruses in the pancreas and more pronounced enrichment in islets (Fig. 7L). Interestingly, co-staining of Ki67 and insulin showed an increased proliferation rate of β-cell from Ube2c restored mice (Fig. 7M), which improved the proportion of β-cells to α-cells (Fig. 7N), and recovered islet mass terminally (Fig. 7O). These data indicate the molecular function of Ube2c in β-cell proliferation and show its application prospects in diabetic patients with β-cell loss.