Fig 2: Human β cells expressing MAFAS64F demonstrate accelerated senescence and a species-specific SASP signature(A) SA-β-gal staining on EndoC-βH2 cells transduced to express WT MAFA or MAFAS64F and stained for MAFA (red). Scale bar, 100 μm.(B) Inset areas outlined in boxes in (A) are magnified in the left panel. Scale bar, 25 μm. β-gal+ area was significantly increased in MAFAS64F cells (right panel).(C) Human expression of β cell-enriched proteins and senescence associated proteins identified in male MafAS64F/+ mice. Two-tailed Student t test; *p < 0.05; **p < 0.01.(D) Immunostaining for senescence markers p21 (green) was increased and LaminB1 (red) decreased in MAFAS64F-expressing EndoC-βH2 cells.(E) CM from WT MAFA or MAFAS64F-expressing EndoC-βH2 cells were collected, purified and added to EndoC-βH2 cells cultured for 72 h. SASP genes upregulated in male MafAS64F/+ mice were not identified in EndoC-βH2 cells. However, novel, species-specific secretory senescence-associated factors were identified in this context. N.D., not detectable. One-way ANOVA; *p < 0.05; **p < 0.01; ****p < 0.0001.(F) Schematic of temporal senescence and aging responses in MafAS64F-expressing male β cells. In male MafAS64F/+, transiently high MafA protein triggers molecular insults to induce premature senescence marked by cell-cycle arrest, senescence staining (blue cytoplasmic shade), and initiation of a senescence-associated secretory phenotype (SASP, pink secretory factors), resulting in impaired GSIS. Late (progressive) senescence propagates this phenotype with SASP amplification and diversification.

Fig 3: Non–β cell hormone expression is increased in MafA∆β islets.(A and B) Analysis of MafA, Insulin 1 (Ins1), and non–β cell hormone mRNA levels in MafAWT and MafA∆β islets from 3-month-old adult male (A) and female (B) mice. Data are shown as mean ± SEM. n = 3–4 animals/group. *P < 0.05 by Student’s t test; n.d., not detectable. (C) Gast mRNA was induced more in male than in female MafA∆β islets. Data are shown as mean ± SEM. n = 3–4 animals/group. *P < 0.05 by Student’s t test. (D) Top panel: Stomach G cells from GASTWT and GASTKO mice served as positive and negative control for Gast antibody staining. n = 2–3 animals/group. Middle panels: Representative images for Gast (red) and Insulin (white) immunostaining in male MafAWT and MafA∆β islets. Insets for Gast staining are shown in right panels. Bottom left panel: Gastrin signal (A.U.) per mouse islet cell. Bottom right panel: Gast+ cells per islet (% of islet cells). Four to 6 islets per mouse. n = 3–4 animals/group were quantified using ImageJ. Scale bar, 50 μm. Data are shown as mean ± SEM. *P < 0.05 by Student’s t test.

Fig 4: MAFBKD, but not MAFAKD, induces non–β cell hormone expression in human β cells.(A–D) Analysis of MAFA/B protein (A and C) and hormone-related mRNA levels (B and D) in shControl (scrambled construct), shMAFA-, or shMAFB-treated EndoC-βH2 (i.e., only MAFB+) (A) and EndoC-βH1 (i.e., MAFA+MAFB+) cells (C). β-Actin served as the internal control and expression was normalized to shControl levels. MAFA knockdown in shMAFA-treated cells was reduced by ~55% of the control level, and shMAFB-treated cells decreased by ~71%. Data are shown as mean ± SEM. *P < 0.05, **P < 0.005 by Student’s t test in B. *P < 0.05 by 2-way ANOVA in D. n = 2–3 replicates/experiment, and experiments were repeated 4 times. (E) Immunostaining for SST (white) and GAST (green) in MAFBKD EndoC-βH2 cells. These proteins were undetectable in shControl-treated cells. Magnification, 20×. Left: Green arrows denote GAST+ SST– cells, while red arrows denote GAST+ SST+ cells. Right: Quantification of hormone+ cells shown in MAFBKD EndoC-βH2 cells relative to the total β cell number compared with shControl. Data are shown as mean ± SEM. n = 2–3 replicates/experiment, and experiments were repeated 4 times.

Fig 5: Islet Gast+ cells produced in insulin-resistant mice share molecular signatures with the broader MafA∆β islet population.(A) Administration of the S961 insulin receptor antagonist for 4 days elevated random blood glucose levels in relation to PBS-treated control mice. Data are shown as mean ± SEM. n = 2–3 animals/group. *P < 0.05 by Student’s t test. (B) MafA (red) protein levels were markedly reduced in S961-treated mice. Insulin (white). n = 2–4 animals/group. Scale bar, 50 μm. (C) UMAP analyses of the single-cell RNA-Seq data in male S961-treated islets. Left panel: Cluster annotation shows that ~80% of islet cells were Insulin+ β cells (left, circled in red). Right panels: Gast+ cells (blue dots) were enriched in the β cell population. (D) Heatmap showing the 20 upregulated genes found in S961 Gast+ cells by single-cell RNA sequencing (Supplemental Table 1) were also elevated in male MafA∆β islets. MafA∆β β cell RNA-Seq were was used in this analysis. n = 3 animals/experimental group. (E) Heatmap showing limited overlap between the upregulated genes found in S961 Gast+ cells and other exocrine and islet cell types.