BIG3 inhibits insulin granule biogenesis and insulin secretion

Abstract

While molecular regulation of insulin granule exocytosis is relatively well understood, insulin granule biogenesis and maturation and its influence on glucose homeostasis are relatively unclear. Here, we identify a novel protein highly expressed in insulin-secreting cells and name it BIG3 due to its similarity to BIG/GBF of the Arf-GTP exchange factor (GEF) family. BIG3 is predominantly localized to insulin- and clathrin-positive trans-Golgi network (TGN) compartments. BIG3-deficient insulin-secreting cells display increased insulin content and granule number and elevated insulin secretion upon stimulation. Moreover, BIG3 deficiency results in faster processing of proinsulin to insulin and chromogranin A to β-granin in β-cells. BIG3-knockout mice exhibit postprandial hyperinsulinemia, hyperglycemia, impaired glucose tolerance, and insulin resistance. Collectively, these results demonstrate that BIG3 negatively modulates insulin granule biogenesis and insulin secretion and participates in the regulation of systemic glucose homeostasis.

Keywords: insulin granule biogenesis; insulin resistance; insulin secretion; metabolism; proinsulin processing.

Figure 1. BIG3 associates with insulin granules and negatively regulates insulin secretion

A BIG3 protein expression profile in adult mouse tissues. B Immunostaining of mouse pancreas section showing BIG3 presence in insulin-positive cells. C Immunostaining of isolated mouse islet showing co-localization of BIG3 and insulin. D Representative electron microscopy (EM) image of mouse pancreatic β-cells. 87 ± 3% of BIG3 antibody-labeled gold particles (arrow heads) localized to insulin granules. N = 20 randomly selected images of β-cells. E Immunoblot for BIG3 and other proteins in BIG3-knockdown (BKD) and control (Ctrl) cells. F, G Insulin secretion time course (F) and quantification (area under curve, 0–60 min; G) in BKD and control cells under basal (2.8 mM glucose) and stimulated (16.7 mM glucose) condition in the absence or presence of 50 mM KCl (indicated as K). Data are presented as mean ± s.e.m. N = 6, *P < 0.05, **P < 0.01, t-test. H, I Representative membrane capacitance recordings (H) and calculated exocytosis events (I) from capacitance increases in BKD and control cells. Data are presented as mean ± s.e.m. N = 10–12, **P < 0.01, t-test. J Capacitance measurements of hBIG3 overexpression in BKD and control cells. Data are presented as mean ± s.e.m. N = 12–19, **P < 0.01, t-test.

Figure 2. Increased insulin secretion and granule number in BIG3-knockout (BKO) islets

A BKO vector: a Neo cassette was introduced into exon 12 to disrupt BIG3 gene expression. B PCR genotyping of wild-type and mutant allele. C, D Immunoblot of lysates of brain (C) and islets (D) from adult BKO and control mice. E Body weights of BKO and littermate control mice. Data are presented as mean ± s.e.m. N = 11 per group, t-test. F–I Metabolic parameters for food intake (F), water intake (G), respiratory exchange ratio (RER, H), and total energy expenditure (I). Data are presented as mean ± s.e.m. N = 6 per group, t-test. J, K Perifusion analysis of insulin secretion time course (J) and quantification (AUC, K) of first phase and total insulin secretion in isolated islets from BKO and control mice. Data are presented as mean ± s.e.m. N = 8 from three independent islet isolations, *P < 0.05, **P < 0.01, t-test. L, M Representative membrane capacitance recordings (L) and calculated exocytosis events (M) in BKO and control β-cells excited by five 50-ms (for measuring immediately releasable pool or IRP), and 8- to 500-ms depolarization pulses from −70 to 0 mV. RP, releasable pool. Data are presented as mean ± s.e.m. N = 16 from three independent islet isolations, **P < 0.01, t-test. N Representative EM images of islet β-cells. O Quantification of insulin granule density in the cytoplasm in β-cells. Data are presented as mean ± s.e.m. of 80 EM images of four mice per group, **P < 0.01, t-test. P Size of insulin granule dense core. Data are presented as mean ± s.e.m. of 12 randomly selected EM images per group, t-test. Q, R Insulin (Q) and (R) proinsulin content, normalized to total protein. Data are presented as mean ± s.e.m. N = 8 from three independent islet isolations, *P < 0.05, t-test. S Representative images of H&E-stained pancreatic sections from BKO and control mice. Scale bars = 1 mm. T–V Cumulative islet area (T), number (U), and size (V) were analyzed from 120 randomly selected pancreatic sections from four mice per group. Data are presented as mean ± s.e.m., t-test.

Figure 3. Hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance in BIG3-knockout (BKO) mice

A, B Postprandial glycemia (A) and insulinemia (B) in BKO and control mice. Data are presented as mean ± s.e.m. N = 14 per group, *P < 0.05, **P < 0.01, t-test. C, D Serum proinsulin (C) and proinsulin-to-insulin ratio (D) at rest, fast, and refed conditions. Data are presented as mean ± s.e.m. N = 8 per group, t-test. E  Glucose levels of oral glucose tolerance tests (OGTT) for BKO and control mice. Data are presented as mean ± s.e.m. N = 12 per group, *P < 0.05, t-test. F Insulin response during OGTT for BKO and control mice. Data are presented as mean ± s.e.m. N = 10 per group, *P < 0.05, t-test. G Postprandial glycemia time course after fasting-refeeding. Data are presented as mean ± s.e.m. N = 10 per group, *P < 0.05, **P < 0.01, t-test. H–K Hyperinsulinemic-euglycemic clamp measurements of glucose infusion rate (H), hepatic glucose production (HGP, I), overall glucose uptake (J), and muscle/adipose glucose uptake (K). BAT: brown adipose tissue; WAT: white adipose tissue; SOL: soleus muscle; EDL: extensor digitorum longus muscle. Data are presented as mean ± s.e.m. N = 3 per group, *P < 0.05, **P < 0.01, t-test.

Figure 4. BIG3 dynamically localizes to trans-Golgi network (TGN)-immature secretory granule (ISG) compartment

A–C Confocal images of MIN6 cells showing (A) TGN-granule localization of BIG3 under normal growth conditions, (B) perinuclear relocalization of BIG3 and chromogranin A (CGA) after high K⁺ and CHX treatments, and (C) intact TGN compartments during high K⁺ and CHX treatments. Scale bars = 10 μm.

Figure 5. BIG3 negatively regulates insulin granule biogenesis

A, B Cellular content of insulin (A) and proinsulin (B), normalized to total protein in BIG3-knockdown (BKD) and control cells. Data are presented as mean ± s.e.m. N = 6 per group, **P < 0.01, t-test. C, D Cellular content of β-granin and chromogranin A (CGA) by immunoblotting (C) and normalized to actin (D) in BKD and control cells. Data are presented as mean ± s.e.m. N = 4 per group, *P < 0.05, **P < 0.01, t-test. E Proinsulin processing rate, normalized to total protein and Time 0 after CHX treatment. Data are presented as mean ± s.e.m. N = 7 per group, **P < 0.01, t-test. F Cellular CGA and β-granin processing was assessed by pulse-chase experiments, and lysates were immunoprecipitated from BKD and control cells. Representative blots from three independent experiments are shown.