Fig 2: PITPNA expression is decreased in isolated islets of T2D human subjects.a Immunostaining of endogenous INSULIN (cyan), PITPNA (green), and protein markers (magenta) for the Golgi (GIANTIN), ER (CALR), and intracellular granules (CD63) in pancreatic islets isolated from a non-diabetic human subject. White arrows denote the colocalization of protein markers with PITPNA. b UMAP projection and graph-based clustering of scRNA-Seq analysis performed on isolated human pancreatic islet cell types. c Relative abundance of PITPNA in islet cell clusters from human donors. d Comparison of PITPNA expression in islet endocrine cell types from T2D (green) and non-diabetic donors (red). e Normalized PITPNA expression from bulk RNA sequencing of isolated human islets across non-diabetic (n = 51), pre-diabetic (n = 27), and diabetic human subjects (n = 11). Normalized expression values are shown in reads per million (RPM). PNon vs Pre-diabetes = 0.7248, PNon vs Diabetes = 0.025, PDiabetes vs Pre-diabetes = 0.1278, f Correlation analysis between normalized islet PITPNA expression and HbA1c of human subjects (n = 77). The R2 value indicates the correlation coefficient. g Correlation analysis between normalized islet PITPNA expression and body mass index (BMI) of human subjects (n = 89). The R2 value indicates the correlation coefficient. h qRT-PCR analysis of PITPNA mRNA expression in pancreatic islets isolated from non-diabetic (n = 15) and T2D (n = 5) human donors. P = 0.0006. Error bar: ND = 1.00 ± 0.02, T2D = 0.43 ± 0.04. i Western blot analysis of PITPNA and AGO2 expression in isolated islets of non-diabetic human donors (ND) and T2D donors (T2D). Data are presented as mean values ± SEM for (e), (h). *P < 0.05, ***P < 0.001. Ordinary one-way ANOVA with Turkey’s multiple comparisons test was used for (e). Linear regression was used for (f), (g). Two-tailed unpaired Student t-test were used for (h). All primary source data are reported in the Source data file.

Fig 3: PITPNA regulates insulin secretion in human pancreatic beta-cells.a Western blot analysis of PITPNA in isolated islets from non-diabetic human donors after treatment with lentiviruses encoding either an shRNA targeting PITPNA (sh-PITPNA), cDNA of human PITPNA (OE-PITPNA), or empty control vector (sh-Ctrl). b Quantification of insulin release in response to 2.8 mM and 25 mM glucose concentrations from isolated islets from non-diabetic human subjects after lentiviral-mediated over-expression of PITPNA (OE-PITPNA) or inhibition of PITPNA (sh-PITPNA) in comparison to treatment with control lentivirus (sh-Ctrl) (n = 4). Statistical Test: Ordinary one-way ANOVA with Turkey’s multiple comparisons test. Values: For 2.8 mM glucose, Summary: F = 3.937, P = 0.0591; Multiple comparison: sh-Ctrl vs sh-PITPNA: P = 0.7839, sh-Ctrl vs OE-PITPNA: P = 0.1625, sh-PITPNA vs OE-PITPNA: P = 0.0581. For 25 mM glucose, Summary: F = 137, P < 0.0001; Multiple comparisons: sh-Ctrl vs sh-PITPNA: P < 0.0001, sh-Ctrl vs OE-PITPNA: P = 0.0008, sh-PITPNA vs OE-PITPNA: P < 0.0001. c Quantification of intracellular Ca2+ concentration in isolated human islets after lentiviral-mediated inhibition of PITPNA (sh-PITPNA, n = 7) in comparison to control lentivirus (sh-Ctrl, n = 5). d, e Representative TIRF images of human beta-cells expressing the granule marker NPY-tdmOrange2 after treatment with lentiviruses encoding GFP control (OE-Ctrl), cDNA of human PITPNA (OE-PITPNA) in panel (d), as well as an shRNA pool targeting PITPNA (sh-PITPNA) or shRNA control (sh-Ctrl) in panel (e); Scale bar, 4 µm. f, g Cumulative time course of high K+-evoked exocytosis events normalized to cell area, for conditions as in (d), (e). Bars at individual time points indicate SEM., K+ was elevated to 75 mM during t = 10–50 s. h Total exocytosis measured during TIRF analysis of human beta-cells expressing the granule marker NPY-tdmOrange2 after lentivirus treatments represented in panels (d) and (e). Statistical Test: Two-way ANOVA with Tukey’s multiple comparisons test. Values: Summary: F = 11.67, P < 0.0001; Multiple comparison: sh-Ctrl vs sh-PITPNA: P = 0.0003, Ctrl vs OE-PITPNA: P = 0.001. The data set was generated from 3 unique human donors; dots and their color/symbol indicate individual cells and donors, respectively. Data presented as mean ± SEM, unless otherwise indicated. ***P < 0.001, and n.s. denotes not significant. All primary source data are reported in the Source data file.

Fig 4: PITPNA regulates insulin granule maturation and proinsulin processing in human pancreatic beta-cells.a Representative transmission electron micrographs of pancreatic beta-cells from non-diabetic human donors after lentiviral-mediated over-expression of PITPNA (OE-PITPNA) or inhibition of PITPNA (sh-PITPNA) in comparison to respective control lentiviruses (OE-Ctrl or sh-Ctrl). The second raw image represents a zoom-in view of the white box from the first raw image. Granule profile: immature secretory granules (blue box), mature secretory granules (red box), crystal-containing granules (yellow box), and empty secretory granules (orange box). b Quantification of granule density in beta-cells of lentiviral-treated human islets shown in panel (a) (n = 9). Psh-Ctrl vs sh-PITPNA = 0.0199, POE-Ctrl vs OE-PITPNA < 0.0001. c Quantification of docked vesicles in beta-cells of lentiviral-treated human islets shown in panel (a) (n = 9). Psh-Ctrl vs sh-PITPNA = 0.0004, POE-Ctrl vs OE-PITPNA = 0.00421. d Quantification of immature secretory granules (ISG, n = 7, 8, 5, 6 for sh-Ctrl, sh-PITPNA, OE-Ctrl, OE-PITPNA, respectively), mature secretory granules (MSG, n = 8, 8, 8, 6 for sh-Ctrl, sh-PITPNA, OE-Ctrl, OE-PITPNA, respectively), crystal-containing granules (CCG, n = 6, 8, 8, 8 for sh-Ctrl, sh-PITPNA, OE-Ctrl, OE-PITPNA, respectively), and empty secretory granules (ESG, n = 7, 7, 7, 7 for sh-Ctrl, sh-PITPNA, OE-Ctrl, OE-PITPNA, respectively) in beta-cells of isolated human islets after lentiviral treatments shown in panel (a). (Psh-Ctrl vs sh-PITPNA < 0.0001, POE-Ctrl vs OE-PITPNA = 0.0128 for ISG), (Psh-Ctrl vs sh-PITPNA < 0.0001, POE-Ctrl vs OE-PITPNA = 0.0008 for MSG), (Psh-Ctrl vs sh-PITPNA = 0.0068, POE-Ctrl vs OE-PITPNA = 0.6874 for CCG), (Psh-Ctrl vs sh-PITPNA = 0.0207, POE-Ctrl vs OE-PITPNA = 0.4262 for ESG). e Proinsulin expression in isolated human islets after lentiviral-mediated over-expression of PITPNA (OE-PITPNA, n = 5 biologically independent samples), knockdown of PITPNA (sh-PITPNA, n = 7 biologically independent samples) or control lentivirus (sh-Ctrl, n = 7 biologically independent samples). f Quantification of proinsulin in isolated human islets after densitometric analysis of western blots shown in panel (f). g Western blot analysis of PITPNA, and ER stress/unfolded protein response (UPR) proteins IRE1a, ERO1, PDI, and CHOP in human islets after lentiviral-mediated over-expression of PITPNA (OE-PITPNA), knockdown of PITPNA (sh-PITPNA) or control lentivirus (sh-Ctrl). Data are presented as mean values ± SEM for (b), (c), (d), e). *P < 0.05, **P < 0.01, ***P < 0.001. Ordinary one-way ANOVA with Turkey’s multiple comparisons test was used for (b), (c), (d), (e). All primary source data are reported in the Source data file.

Fig 5: Loss of Pitpna increases beta-cell apoptosis and expression of endoplasmic reticulum (ER) stress markers.a Immunostaining in paraffin-embedded pancreata from 8-week-old female Ins-Cre, Pitpnaflox/flox, and littermate control wild-type (WT) mice was performed to assess: insulin (magenta), glucagon (Gcg, green) and apoptotic marker TUNEL (cyan). White arrowheads point to TUNEL-positive and insulin-positive cells in (b), TUNEL-positive beta cell number (n = 6), P = 0.0007. c TUNEL-positive alpha cell number (n = 6). d Western blot analysis of Pitpna, BiP/GRP78, and CHOP after treatment of hydrogen peroxide (H2O2) in isolated islets of 8-week-old male Ins-Cre, Pitpnaflox/flox, and WT mice. e qRT-PCR analysis of Pitpna, PC1/3, PC2, CPE, CGA, and CGB mRNA expression in islets of male WT and Ins-Cre, Pitpnaflox/flox mice at age 10 weeks (n = 5), PPitpna < 0.0001, PPC1/3 < 0.0001, PPC2 = 0.0441; PCPE = 0.0488, PCGA = 0.1313, PCGB = 0.0585. Data are presented as mean values ± SEM for (b), (c), (e). *P < 0.05, ***P < 0.001 and n.s. denotes not significant. Two-tailed unpaired Student t-test were used in for (b), (c), (e). All primary source data are reported in the Source data file.