The type 2 diabetes gene product STARD10 is a phosphoinositide-binding protein that controls insulin secretory granule biogenesis

Abstract

Objective: Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the β-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates β-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the β-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation.

Methods: We used isolated islets from mice deleted selectively in the β-cell for Stard10 (βStard10KO) and performed electron microscopy, pulse-chase, RNA sequencing, and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in the INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay was performed on purified STARD10 protein.

Results: βStard10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of "rod-like" dense cores. Correspondingly, basal secretion of proinsulin was increased versus wild-type islets. The solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides, and STARD10 was shown to bind to inositides phosphorylated at the 3' position. Lipidomic analysis of βStard10KO islets demonstrated changes in phosphatidylinositol levels, and the inositol lipid kinase PIP4K2C was identified as a STARD10 binding partner. Also consistent with roles for STARD10 in phosphoinositide signalling, the phosphoinositide-binding proteins Pirt and Synaptotagmin 1 were amongst the differentially expressed genes in βStard10KO islets.

Conclusion: Our data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis, and insulin processing.

Keywords: Insulin granule biogenesis; Lipid transporter; Pancreatic β-cell; Phosphoinositides; Type 2 diabetes.

Figure 1

βStard10KO β-cells display altered granule morphology. A, Representative Transmission Electron Microscopy images of control (Ctl) and βStard10KO β-cells. Red arrowhead: granules with a “rod-shaped” core. Scale bar = 1 μm. B, “Rod-shaped” core granule numbers are increased in the βStard10KO β-cells (n = 3 animals, 6 images per animal; P < 0.001, Student's t-test). C, β-cell granule diameter (nm) (n = 42 images from 3 animals, P < 0.001, Student's t-test). D, β-cell granule “circularity” (n = 42 images from 3 animals, P < 0.05, Welch's t-test). E, β-cell granule density (n = 19 images from 3 animals, ns, Mann–Whitney test). F, β-cell morphologically docked granules (per μm plasma membrane) (n = 18 images from 3 animals, ns, Mann–Whitney test). G, Representative trace for β-cell expressing the cytosolic eCALWY4 Zn²⁺ sensor. Steady-state fluorescence intensity ratio (citrine/cerulean) (1, R) was first measured before the maximum ratio (2, Rmax) was obtained under perfusion with buffer containing 50 μM TPEN (zinc-free condition). Finally, the minimum ratio (3, Rmin) was obtained under perfusion with buffer containing 5 μM pyrithione and 100 μM Zn²⁺ (zinc-saturated condition). Cytosolic free Zn²⁺ concentrations were calculated using the following formula: (R-Rmin)/(Rmax-Rmin). H, Cytosolic Zn²⁺ concentrations measured by eCALWY4 in Ctl and βStard10KO β-cells (n = 33–65 cells per genotype, ns, Mann–Whitney test). I, Total islet zinc measured by inductively coupled plasma mass spectrometry (ICP/MS) in Ctl and βStard10KO animals (n = 4 animals/genotype, ∗P < 0.05, unpaired t-test).

Figure 2

Deletion of Stard10 increased basal secretion of newly synthesised proinsulin but did not affect total secreted proinsulin:insulin ratio. A, Representative phosphorimages from reducing gels representing ³⁵S-labelled proinsulin and insulin (B chain) originating from cell lysate (C) or secreting medium (M) samples after 1.5 or 4 h of a chase in 5.5 mM glucose medium. B, Secreted proinsulin after 1.5 or 4 h of a chase in 5.5 mM glucose medium expressed as a percentage of total labelled proinsulin (n = 4–5 animals; ∗P < 0.05, Mann–Whitney test). C, Secreted proinsulin:insulin ratio after 1.5 or 4 h of a chase in 5.5 mM glucose medium (n = 4–5 animals; ∗P < 0.05, Mann–Whitney test). D, Fraction of stored processed insulin remaining inside the cells after 1.5 or 4 h of a chase in 5.5 mM glucose medium (n = 4–5 animals; ns, Mann–Whitney test). E, Cellular proinsulin:insulin ratio after 1.5 or 4 h of a chase in 5.5 mM glucose medium (n = 4–5 animals; ns, Mann–Whitney test). F, Representative phosphorimages from reducing gels representing ³⁵S-labelled proinsulin and insulin (B chain) originating from cell lysate (C) or secreting medium (M) samples after 30 min of a chase in 20 mM glucose medium. G, Secreted proinsulin after 30 min of a chase in 20 mM glucose medium expressed as a percentage of total labelled proinsulin (n = 4–5 animals; ns, Mann–Whitney test). H, Secreted proinsulin:insulin ratio after 30 min of a chase in 20 mM glucose medium (n = 4–5 animals; ns, Mann–Whitney test). Quantification for B, C, D, E, G, and H was done on the phosphorimages obtained from reducing gels. I, Secreted total proinsulin:insulin ratio after 30 min secretion by isolated islets in 3 or 17 mM glucose Krebs-HEPES-bicarbonate buffer.

Figure 3

Structure of H. sapiens STARD10. A, Three-dimensional views at 2.3 Å of the crystal structure of unliganded human STARD10: ribbon diagram coloured from the N-terminus (blue) to the C-terminus (red). B, Docking of phosphatidyl-inositol 3 phosphate (PI(3)P) to the human STARD10 structure. STARD10 cavity is larger than the phosphatidylcholine transporter protein STARD2 and, contrary to the latter, readily accommodates phosphatidylinositols. The three projections shown in A and B are rotated by 120° with respect to each other. C, Comparison of unliganded STARD10 (green) and its close family relative STARD2 (grey) bound to phosphatidylcholine.

Figure 4

Phosphoinositide binding to STARD10. A, Coomassie blue staining of 6His-MBP-STARD10 (75 kDa) purified by Immobilised Metal Affinity Column (IMAC): FT: flow-through, W1: first column wash, W5: last column wash, E: elution. B, Lipid overlay assay. LPA, lysophosphatidic acid; S1P, sphingosine-1-phosphate; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; PI(3)P, PI-(3)-phosphate; PI(4)P, PI-(4)-phosphate; PI(5)P, PI-(5)-phosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI(3,4)P₂, PI-(3,4)-bisphosphate; PI(3,5)P₂, PI-(3,5)-bisphosphate; PI(4,5)P₂, PI-(4,5)-bisphosphate; PIP(3,4,5)P₃, PI-(3,4,5)-trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine. Immunodetection of bound protein was performed using a primary anti-STARD10 antibody (Santa Cruz) and a secondary anti-goat-HRP antibody (Santa Cruz). STARD10 was bound to all PIP species.

Figure 5

Altered lipidomic profile in βStard10KO islets. The deletion of Stard10 in mouse pancreatic β-cells significantly increased the total cholesteryl esters, phosphatidylinositols, and the phosphatidylethanolamine species 34:0 in islets (n = 5 animals; ∗P < 0.05, ∗∗P < 0.01, paired t-test). See Supplemental Table 3 for a complete list of all the lipids measured.

Figure 6

Subcellular localisation of STARD10-GFP (left panel) and GFP-STARD10 (right panel) in EndoC-βH1 cells. Scale bar = 10 μm.