Defective glucose metabolism in ICA69 KO mice.

<p>(A) Body weight (<i>n</i> = 11, **<i>p</i><0.01). (B) Daily food intake (<i>n</i> = 8, **<i>p</i><0.01). (C) Daily water intake (<i>n</i> = 8, **<i>p</i><0.01). (D) Feeding basal glucose (<i>n</i> = 6, <i>p</i> = 0.17). (E) Fasting basal glucose (<i>n</i> = 11, <i>p</i> = 0.36). (F) Fasting basal insulin (<i>n</i> = 8, <i>p</i> = 0.21). (G) Intraperitoneal glucose tolerance test (IGTT) (<i>n</i> = 11, *<i>p</i><0.05). (H) Insulin tolerance test (ITT). The basal glucose level at 0 min was normalized as 100 (<i>n</i> = 7). (I) Glucose-stimulated insulin secretion (<i>n</i> = 8, *<i>p</i><0.05). (J) Total insulin level in isolated islets (<i>n</i> = 5, *<i>p</i><0.05). (K) Total proinsulin level in isolated islets (<i>n</i> = 12, *<i>p</i><0.05). (L) Proinsulin/insulin ratio in isolated islets (<i>n</i> = 6, *<i>p</i><0.05). (M) Immunoblotting of proinsulin and insulin in ICA69 group islets. GAPDH served as a loading control. (N) Quantification of proinsulin/insulin ratio in (M) (<i>n</i> = 4, *<i>p</i><0.05). (A–N) Data are represented as mean ± SEM.</p

Decreased insulin secretion and total content in PICK1 KO mice.

<p>(A) Glucose-stimulated insulin secretion from isolated islets (<i>n</i> = 10, *<i>p</i><0.05). (B) KCl-stimulated insulin secretion from isolated islets (<i>n</i> = 9, *<i>p</i><0.05). (C) Time-course of glucose-stimulated insulin secretion from isolated islets and its AUC analysis (<i>n</i> = 10, *<i>p</i><0.05, **<i>p</i><0.01). (D) Total insulin content in isolated islets (<i>n</i> = 16, *<i>p</i><0.05). (E) Normalized glucose-stimulated insulin secretion from isolated islets (<i>n</i> = 8). (A–E) Data are represented as mean ± SEM.</p

Normal islet structure in PICK1 KO mice.

<p>(A) Representative images of H&E staining from WT and PICK1 KO mice pancreas. Scale bar, 100 nm. (B) Quantification of the islet number (<i>n</i> = 3, <i>p</i> = 0.74). (C) Quantification of islet/pancreas area (<i>n</i> = 3, <i>p</i> = 0.05). (D) Quantification of islet size (<i>n</i> = 3, *<i>p</i><0.05). (E) Quantification of islet size distribution (<i>n</i> = 3). (A–E) Data are represented as mean ± SEM.</p

Increased proinsulin secretion in PICK1 KO islets is due to impaired insulin maturation.

<p>(A) Glucose-stimulated proinsulin secretion from isolated islets (<i>n</i> = 10, *<i>p</i><0.05, ***<i>p</i><0.001). (B) Total proinsulin level in isolated islets (<i>n</i> = 16, *<i>p</i><0.05). (C) Proinsulin–insulin ratio in isolated islets (<i>n</i> = 16, **<i>p</i><0.01). (D) Normalized glucose-stimulated proinsulin secretion from isolated islets (<i>n</i> = 5). (E) Immunoblotting of proinsulin and insulin from PICK1 KO and wild-type islets. GAPDH served as a loading control. (F) Quantification of proinsulin–insulin ratio in (E) (<i>n</i> = 4, *<i>p</i><0.05). (G) Ultrastructure of PICK1 KO beta cells. Arrows, immature granules; arrowheads, mature granules. Scale bar, 1 µm. (H) Immature/mature secretory granule (SG) ratio in WT and PICK1 KO beta cells (<i>n</i> = 37, 48, ***<i>p</i><0.001). (A–H) Data are represented as mean ± SEM.</p

PICK1 is expressed in pancreatic beta cells and partially co-localizes with insulin granules.

<p>(A) Upper panel: double staining of PICK1 (red) and insulin (green) on pancreatic cryosection. Scale bar, 100 µm. The lower panel is the magnification of upper panel. PICK1 partially co-localized with insulin positive granules in beta cells. Scale bar, 10 µm. (B) Double staining of PICK1 (red) and insulin (green) on INS-1E cells (upper panel) and MIN6 cells (lower panel). Scale bar, 10 µm. (C) Subcellular fractionation of INS-1E cells. PNS, post-nuclear supernatant; SGs, secretory granules. (D) Immuno-gold labeling of PICK1 on mouse islet beta cell. Arrowheads, ISG (immature secretory granules); arrows, MSG (mature secretory granules); N, nucleus. Scale bar, 1 µm.</p

PICK1 and ICA69 form tight complexes in the pancreas.

<p>(A) In vivo co-IP from islet extracts using anti-PICK1 antibody. (B) In vivo co-IP using anti-ICA69 antibody. (C) Western blotting of homogenates from WT and PICK1 KO islets. GAPDH served as a loading control. (D) Western blotting of homogenates from WT and ICA69 KO islets. GAPDH served as a loading control. (E) RT-PCR analysis in WT and PICK1 KO pancreas, using β-actin as an internal control. (F) Primary cultured PICK1 KO islet cells were transfected with GFP-PICK1 (green) and stained for ICA69 (red). Scale bar, 10 µm.</p

A model illustrating the roles of PICK1 and ICA69 in insulin granule trafficking.

<p>In this proposed model, PICK1 and ICA69 form heteromeric BAR domain complexes that are capable of sensing membrane curvatures and phospholipids on the membranes of the TGN. In addition, the PDZ domain of PICK1 may bind to membrane proteins on the insulin granules. This aids the formation of proinsulin immature secretory granules (ISGs) from the TGN with both PICK1 and ICA69 (P-I granules). The P-I granules gradually lose ICA69 as they mature and become PICK1 only granules (P-P granules). With more PDZ domains, the PICK1 homomeric complexes can bind to more membrane proteins and enrich secretory cargos in the mature insulin granules.</p

PICK1 and ICA69 associate with insulin granules at three maturation stages.

<p>(A) Triple staining of insulin (green), PICK1 (red), and ICA69 (blue) on INS-1E cells. Arrowheads, PICK1-insulin co-clusters without ICA69; arrows, PICK1-ICA69 co-clusters; asterisks, insulin granules without PICK1 and ICA69. Scale bar, 10 µm. (B) Triple staining as in (A) after glucose/KCl-stimulation. Arrows, PICK1-ICA69 co-clusters; arrowheads, PICK1-insulin co-clusters. Scale bar, 10 µm. (C) Co-localization quantification of PICK1 with insulin under different conditions from (A) and (B). Data are represented as mean ± SD; <i>n</i> = 31 cells from four independent experiments. **<i>p</i><0.01. NS, non-significant. (D) Triple staining of proinsulin (green), PICK1 (red), and ICA69 (blue) on INS-1E cells. Empty arrow heads, PICK1-ICA69-proinsulin co-clusters. (E) Triple staining of PICK1 (red), ICA69 (blue), and TGN38 (green) before (0′) and after BFA treatment (5 and 10′). Scale bar, 10 µm. (F) Co-localization quantification of PICK1 or ICA69 with TGN38 from (E). Data are represented as mean ± SD; <i>n</i> = 40 cells from three independent experiments. ***<i>p</i><0.001. (G) Triple staining of PICK1 (red), TGN38 (green), and GM130 (blue) on INS-1E cells without BFA treatment (0′) or with BFA treatment (5′). Scale bar, 10 µm.</p

PICK1 KO mice are diabetic due to insulin deficiency.

<p>(A) Body weight (<i>n</i> = 34, *<i>p</i><0.05). (B) Daily food intake (<i>n</i> = 14, *<i>p</i><0.05). (C) Daily water intake (<i>n</i> = 16, * <i>p</i><0.05). (D) Feeding basal glucose (<i>n</i> = 14, <i>p</i> = 0.6). (E) Fasting basal glucose (<i>n</i> = 16, <i>p</i> = 0.17). (F) Intraperitoneal glucose tolerance test (IGTT) and its area under curve (AUC) analysis (<i>n</i> = 16, *<i>p</i><0.05, **<i>p</i><0.01). (G) Fasting basal insulin (<i>n</i> = 20, <i>p</i> = 0.47). (H) Insulin tolerance test (ITT) and its AUC analysis. The basal glucose level at 0 min was normalized as 100 (<i>n</i> = 20). (I) Glucose-stimulated insulin secretion (GSIS) and its AUC analysis (<i>n</i> = 8, *<i>p</i><0.05, **<i>p</i><0.01). (A–I) Data are represented as mean ± SEM.</p

Supernate soluble TM in endotoxin induction group (A) and optical density in destained oil red O staining in steroid induction group (B) was significantly higher than the corresponding control group, respectively, whereas Icaritin dose-dependently lowered supernate soluble TM (A) and optical density in destained oil red O staining (B) when compared to the corresponding induction group, respectively.

<p>However, no differences were found between the seven parent flavonoids in EF at the concentration of 10⁻¹⁴ M and corresponding induction group both in supernate soluble TM (C) and optical density in destained oil red O staining (D). Note: * P<0.05 for comparison with the induction group. # P<0.05 for comparison with the low dose group.</p