Beta‐Cell Tipe1 Orchestrates Insulin Secretion and Cell Proliferation by Promoting Gαs/cAMP Signaling via USP5

Abstract Inadequate β‐cell mass and insulin secretion are essential for the development of type 2 diabetes (T2D). TNF‐α‐induced protein 8‐like 1 (Tipe1) plays a crucial role in multiple diseases, however, a specific role in T2D pathogenesis remains largely unexplored. Herein, Tipe1 as a key regulator in T2D, contributing to the maintenance of β cell homeostasis is identified. The results show that the β‐cell‐specific knockout of Tipe1 (termed Ins2‐Tipe1BKO) aggravated diabetic phenotypes in db/db mice or in mice with high‐fat diet‐induced diabetes. Notably, Tipe1 improves β cell mass and function, a process that depends on Gαs, the α subunit of the G‐stimulating protein. Mechanistically, Tipe1 inhibited the K48‐linked ubiquitination degradation of Gαs by recruiting the deubiquitinase USP5. Consequently, Gαs or cAMP agonists almost completely restored the dysfunction of β cells observed in Ins2‐Tipe1BKO mice. The findings characterize Tipe1 as a regulator of β cell function through the Gαs/cAMP pathway, suggesting that Tipe1 may emerge as a novel target for T2D intervention.


Supplementary Materials
Figure S1.Tipe1 is highly expressed in pancreatic islet β cells.A) Human pancreatic tissue microarray of patients with pancreatic cancer was used to perform IHC staining with anti-TIPE1 antibody and paracancerous tissues were analyzed.Scale bar, 50 μm.(n = 12).B) Representative images for multiplexed immunofluorescence staining (TIPE1, green; Insulin, red; Glucagon, orange; Somatostatin, pink) in paracancerous tissues from patients with pancreatic cancer.C,D) The median fluorescence intensity (MFI) and frequency for the expression of Tipe1 in β, α, and δ cells in human islet tissues of paracancerous tissues from patients with pancreatic cancer.(n = 16).E) Sections of pancreas tissues from 12-week-old WT mice were used to perform IHC staining with anti-TIPE1 antibody.Nuclei were stained with Hematoxylin (blue).Scale bar, 50 μm.F) Total RNAs isolated from β cells and non-β-cells of 8-12-week-old WT mice were subjected to qRT-PCR analysis for the indicated genes.mRNA levels were normalized to β-actin mRNA.(n =10).G) IF staining of β cells (insulin, red), α cells (glucagon, red) and δ cells (somatostatin, red) in islets of 3-month-old male WT mice.Nuclei were stained with DAPI (blue) and TIPE1-positive cells were stained in green.Scale bar, 50 μm.Data are presented as the mean ± SEM.Data were statistically analyzed by Student's t test.**P<0.01,***P<0.001,ns indicates no significant difference.

Figure S2 .
Figure S2.Tipe1 expression in pancreatic islet is reduced in fasting status, and restored after refeeding.A) Schematic description of a fasting-refeeding model.B) Total RNAs isolated from islets were subjected to qRT-PCR analysis for gene expression.Gene mRNA was normalized to β-actin mRNA.(n = 6 mice/group).C) Blood glucose levels of mice.(n = 3 mice/group).D) Serum insulin levels of mice.(n = 3 mice/group).E) IF staining of pancreatic sections of fasting and refeeding mice with anti-TIPE1 and anti-Insulin antibodies.Scale bar, 50 μm.F) GEPIA database was used to analyze the relationship between Tipe1 and proliferation or insulin regulation related genes in pancreas of healthy individuals.Data are presented as the mean ± SEM.Data were statistically analyzed by Student's t test.*P<0.05,**P<0.01,***P<0.001,ns indicates no significant difference.

Figure S3 .
Figure S3.Characterization of Ins2-Tipe1BKO mice.A) Strategy of islet β cell specific Tipe1-knockout mice.B) Schematic description of genotyping.C) Protein extracts isolated from hypothalamus and islet tissues of 8-12-week-old Ins2-Cre and Ins2-Tipe1BKO mice were subjected to Western blotting analysis for TIPE1 expression.D) Total RNAs isolated from the pancreas of 8-12-week-old Ins2-Cre and Ins2-Tipe1BKO mice were subjected to qRT-PCR analysis for Tipe1 expression.Tipe1 mRNA was normalized to β-actin mRNA.(n = 3 mice/group).E) Proteins isolated from different tissues of Ins2-Cre and Ins2-Tipe1BKO mice were subjected to Western blotting analysis for Tipe1 expression.F) Indicated tissues of 12-week-old mice were subjected to IHC staining for TIPE1 expression.G) IF staining of pancreatic sections of Ins2-Cre and Ins2-Tipe1BKO mice with anti-TIPE1 and anti-Insulin antibodies.Scale bar, 50 μm.Data are presented as the mean ± SEM.Data were statistically analyzed by Student's t test.**P<0.01,ns indicates no significant difference.

Figure S5 .
Figure S5.The HOMA-IR and improved insulin signaling in Ins2-Tipe1BKOdb/db mice.A) The HOMA-IR of Ins2-Cre-db/db and Ins2-Tipe1BKO-db/db mice (n = 7 mice/group).B) Proteins isolated from perirenal adipose tissue (PAT), epididymal adipose tissue (EAT) and skeletal muscles of Ins2-Cre-db/db and Ins2-Tipe1BKOdb/db mice were subjected to Western blotting analysis for p-AKT, AKT, p-IR and IR expression.Data are presented as the mean ± SEM.Data were statistically analyzed by Student's t test.**P<0.01,ns indicates no significant difference.

Figure S7 .
Figure S7.Tipe1 deficiency reduces β cell intracellular insulin and proliferation.A) Pancreatic islets from 16-week-old Ins2-Cre-db/db and Ins2-Tipe1BKO-db/db mice were detected intracellular insulin level.(n = 3 mice/group).B) IF staining of Ki67.Ki67-positive cells in islets were normalized to total insulin-positive cells in the same area.Scale bar, 50 μm.(n = 4 mice/group).C) MIN6 cells were infected with Ad-Tipe1 adenovirus for 48 h, and the protein level of TIPE1 and TIPE1-HA were detected by Western blotting.D) MIN6 cells were silenced Tipe1 for 24 h, then infected with either Ad-Con or Ad-Tipe1 for 48 h, were subjected to EdU incorporation assays by flow cytometry.Flow cytometry histograms showing the level of EdU in MIN6 cell line.(n = 5).E) Pancreatic islets from 8-week-old Ins2-Cre and Ins-Tipe1BKO mice were treated with either Ad-Con or Ad-Tipe1 for 48 h, subsequently treated with 2.8 or 16.7 mM glucose in an in vitro setting.Insulin levels in the culture supernatants were determined by ELISA.(n = 3 mice/group).F) Islets were treated as in E, and qRT-PCR was performed to detect the indicated gene expression.mRNA levels were normalized to β-actin mRNA (n = 5 mice/group).Data are presented as the mean ± SEM.Data were statistically analyzed by Student's t test.*P<0.05,**P<0.01.***P<0.001,ns indicates no significant difference.

Figure S8 .
Figure S8.Tipe1 regulates β cell function not through Gprasp1.A) Intracellular interaction between Tipe1 and GASP1, followed by IP with anti-Flag, probed with anti-GASP1.B) Gprasp1 expression in islets of Ins2-Cre and Tipe1BKO mice was measured by qRT-PCR.mRNA levels were normalized to β-actin mRNA.(n/ Ins2-Cre = 3; n/ Ins2-Tipe1BKO = 4).C,D) Gprasp1 expression of MIN6 cells infected with either Pultra-NC or Pultra-Tipe1 lentiviruses was measured by qRT-PCR and Western blotting.mRNA levels were normalized to β-actin mRNA.(n = 6).E) MIN6 cells were infected with either Pultra-NC or Pultra-Tipe1 lentiviruses and silenced Gprasp1 for 48 h, protein levels were detected by Western blotting.Data are presented as the mean ± SEM.Data were statistically analyzed by Student's t test.ns indicates no significant difference.

Figure S9 .
Figure S9.Tipe1 activates the cAMP/PKA/CREB pathway in MIN6 cells.A) MIN6 cells were infected with Ad-Gnas adenovirus for 48 h, and the protein level of Gαs was detected by Western blotting.B) The fluorescence was observed in islets infected with Ad-Gnas adenovirus.C) The protein level of Gαs in islets infected with Ad-Gnas adenovirus for 48 h was detected by Western blotting.D) MIN6 cells were silenced Tipe1 24 h after infected with Ad-Gnas adenovirus for 48 h, and the protein levels of PKA, p-PKA (Thr198), CREB and p-CREB (Ser133) were detected by Western blotting.E) MIN6 cells silenced Tipe1 for 48 h, then treated with cAMP agonist (Colforsin, 10 μM) for 48 h.The protein levels of p-AMPK and AMPK were detected by Western blotting.F,G) MIN6 cells silenced Tipe1 for 48 h, then treated with p-AMPK inhibitor (Comp-C, 20μM) for 48 h.PCNA expression was detected by qRT-PCR and Western blotting.mRNA levels were normalized to β-actin mRNA.H) Pancreatic islets from 8-week-old Ins2-Cre and Ins2-Tipe1BKO mice were treated with GLP-1 receptor agonist (exendin 4, 100 nM) for 48 h, subsequently treated with 2.8 or 16.7 mM glucose in an in vitro setting.Insulin levels in the culture supernatants were determined by ELISA.(n = 3 mice/group).Data are presented as the mean ± SEM.Data were statistically analyzed by Student's t test.*P<0.05,**P<0.01,***P<0.001,ns indicates no significant difference.

Figure S10 .
Figure S10.Both N and C-terminal of Tipe1 interacts with Gαs.A) HEK293T cells expressing mTipe1-Flag and mGnas-HA.Gnas-HA was immunoprecipitated from HEK293T cell lysates and immunoblotted with anti-Flag and anti-HA, respectively.B) HeLa cells were transfected with human Tipe1-HA and Gnas-Flag overexpression plasmid.Then, the cells were subjected to IF staining with antibodies against HA (red), Flag (green).Nuclei were stained with DAPI (blue).Confocal assay was performed.Scale bar, 10 μm.C) Islets from 12-week-old WT mice were subjected to IF staining with antibodies against TIPE1 (red), Gαs (green).Nuclei were stained with DAPI (blue).Confocal assay was performed.Scale bar, 20 μm.D,E) Flag-Tagged TIPE1, Gnas and their truncates were constructed and co-transfected into HEK293 cells, and cell lysates were precipitated with anti-Flag antibody and immunoblotted with anti-HA antibody as indicated (The orange arrow indicates the band).