Fig 2: Altering the actin cytoskeleton triggers ER stress and affects β-cell identity and function. (A) Islet integrity disruption is further studied by altering the actin cytoskeleton using jasplakinolide (JP). Affecting the actin cytoskeleton in human islets with 0.1 μM JP for 24 h leads to decreased glucose-stimulated insulin secretion in two of three donors. GSIS was performed immediately after 24 h JP treatment. (B) Treatment of isolated human islets with 0.1 μM JP for 24 h leads to increased mRNA expression levels of the ER stress marker genes ATF3 and CHOP. (C) Treatment of isolated human islets with 0.1 μM JP for 24 h leads to decreased mRNA expression levels of the β-cell-specific gene MAFA but not PDX1, NKX6.1 and insulin as measured by qPCR. Data are presented as means ± SEM of fold change over untreated control islets. n = 5 donors; each data point represents one donor. * p < 0.05, ** p < 0.01 vs. untreated control islets.

Fig 3: Oxidative stress leads to loss of β-cell maturity markers and a concomitant increase in progenitor marker expression. To evaluate the effect of oxidative stress on β-cell maturity markers and progenitor markers, primary human islets (blue graphs) or EndoC-βH1 cells (red graphs) were treated with hydrogen peroxide (H2O2). (A) Oxidative stress induction in human islets by treatment with 200 μM H2O2 for 90 minutes leads to decreased mRNA expression levels of the β-cell-specific genes MAFA, PDX1, KIR6.2, MAFB, FOXA2, PAX6, NKX2.2, NEUROD1 and GLUT1 as measured by qPCR. (B) The level of β-cell-specific protein MAFA is decreased in human islets upon treatment with 200 μM H2O2 for 90 minutes as measured by Western blot. (C) Oxidative stress induction in EndoC-βH1 cells by treatment with 50 μM H2O2 for 24 hours and 200 μM H2O2 for 90 minutes leads to decreased mRNA expression levels of the β-cell-specific genes MAFA, PDX1, PAX4, NKX6.1, insulin, KIR6.2, MAFB, FOXA2, PAX6, NKX2.2, NEUROD1 and GLUT1 as measured by qPCR. (D) The level of β-cell-specific proteins MAFA and PDX1 is decreased in EndoC-βH1 cells upon treatment with 200 μM H2O2 for 90 minutes as measured by Western blot. (E) Oxidative stress induction in EndoC-βH1 cells by treatment with 50 μM H2O2 for 24 hours and 200 μM H2O2 for 90 minutes leads to increased mRNA expression levels of the progenitor cell-specific genes SOX9 and HES1 as measured by qPCR. (F) The level of progenitor cell-specific protein SOX9 is increased in human islets upon treatment with 200 μM H2O2 for 90 minutes as measured by Western blot. Data are presented as means ± SEM of fold change over untreated control islets (blue graphs) or EndoC-βH1 cells (red graphs). n=1-10 donors/batches; each data point represents one donor/batch. *p < 0.05, **p < 0.01, ***p < 0.0005, ****p < 0.0001 vs. untreated control islets/EndoC-βH1 cells as determined by a paired Student’s t test on the dCT values. Black circles = untreated control islets/EndoC-βH1 cells, upward-pointing triangles = H2O2-treated islets/EndoC-βH1 cells (50 μM 24h), downward-pointing triangles = H2O2-treated islets/EndoC-βH1 cells (200 μM 90min). h = hours, min = minutes, H2O2 = hydrogen peroxide.

Fig 4: TUDCA partially prevents oxidative stress-induced detrimental effects. To evaluate the effect of tauroursodeoxycholic acid (TUDCA) on oxidative stress-induced detrimental effects, primary human islets were pre-incubated overnight with TUDCA prior to the H2O2 treatments as shown in Supplementary Figure 1A , as well as co-incubated together with H2O2 for the duration of the treatments. (A) TUDCA partially or completely recovers the oxidative stress-induced decreased mRNA expression levels of the genes MAFA, MAFB, KIR6.2, PAX6, NKX2.2, NEUROD1 and GLUT1 in human islets as measured by qPCR. (B) The oxidative stress-induced decreased level of the β-cell-specific protein MAFA is recovered by TUDCA in human islets as measured by Western blot. Data are presented as means of technical triplicates as fold change over untreated control islets (blue graphs). n = 1 donor; Black circles = untreated control islets, upward-pointing triangles = H2O2-treated islets (50 μM 24h), downward-pointing triangles = H2O2-treated islets (200 μM 90min), green squares = H2O2 (50 μM 24h or 200 μM 90min) and TUDCA (1 mM) pre- and cotreated islets. h = hours, min = minutes, H2O2 = hydrogen peroxide, TUDCA = tauroursodeoxycholic acid.