Insulin secretion from pancreatic β-cells occurs in a biphasic manner. The loss of the early phase of insulin secretion, which occurs due to the inability of insulin granules to fuse with the plasma membrane (PM) via the formation of the SNARE complex, acts as a key determinant in impaired glucose tolerance and development of type 2 diabetes (T2D). Thus, it is critical to elucidate molecular mechanisms underlying early-phase insulin secretion. Tomosyn-2 binds to Stx1A, a key component of the SNARE complex, and functions as an endogenous inhibitor of insulin secretion. The mechanism by which Tomosyn-2 regulates the fusion of insulin granules is not completely understood. Our preliminary data show that Tomosyn-2 binds, colocalizes, and co-fractionates with synaptotagmin-9 (Syt9) at high glucose; Syt9 protein is present in the insulin granule membrane, and its role in insulin secretion remains poorly defined. Our data show that mice lacking Syt9 (Syt9-/-) have improved glucose clearance, increased in vivo early-phase insulin secretion at 5 min and 15 min post-glucose challenge with no change in insulin tolerance vs. the wild type control mice, suggesting that Syt9 regulates the early phase of insulin secretion from β-cells. Strikingly, Syt9-/- mouse islets have reduced Tomosyn-2 levels (∼50%) without altering the levels of other key SNARE proteins. The knockdown of Tomosyn in INS1 β-cells treated with KCL prevents the localization of Syt9 with Stx1A. These results show that Syt9 binding stabilizes Tomosyn-2 protein in a non-fusogenic complex by potentially blocking insulin granules’ access to the PM-Stx1A for membrane fusion and insulin secretion. In summary, our data reveal a novel molecular complex in Stx1A-Tomosyn-2-Syt-9 to regulate the early-phase insulin secretion. Disclosure A. Pathak: None. H. Alsharif: None. J. E. Trombley: None. M. Rahman: None. S. Bhatnagar: None. Funding National Institutes of Health (R01DK120684-01)

Effective energy expenditure is critical for maintaining body weight (BW). However, underlying mechanisms contributing to increased BW remain unknown. We characterized the role of brain angiogenesis inhibitor-3 (BAI3/ADGRB3), an adhesion G-protein coupled receptor (aGPCR), in regulating BW. A CRISPR/Cas9 gene editing approach was utilized to generate a whole-body deletion of the BAI3 gene (BAI3−/−). In both BAI3−/− male and female mice, a significant reduction in BW was observed compared to BAI3+/+ control mice. Quantitative magnetic imaging analysis showed that lean and fat masses were reduced in male and female mice with BAI3 deficiency. Total activity, food intake, energy expenditure (EE), and respiratory exchange ratio (RER) were assessed in mice housed at room temperature using a Comprehensive Lab Animal Monitoring System (CLAMS). While no differences were observed in the activity between the two genotypes in male or female mice, energy expenditure was increased in both sexes with BAI3 deficiency. However, at thermoneutrality (30 °C), no differences in energy expenditure were observed between the two genotypes for either sex, suggesting a role for BAI3 in adaptive thermogenesis. Notably, in male BAI3−/− mice, food intake was reduced, and RER was increased, but these attributes remained unchanged in the female mice upon BAI3 loss. Gene expression analysis showed increased mRNA abundance of thermogenic genes Ucp1, Pgc1α, Prdm16, and Elov3 in brown adipose tissue (BAT). These outcomes suggest that adaptive thermogenesis due to enhanced BAT activity contributes to increased energy expenditure and reduced BW with BAI3 deficiency. Additionally, sex-dependent differences were observed in food intake and RER. These studies identify BAI3 as a novel regulator of BW that can be potentially targeted to improve whole-body energy expenditure.

Secreted proteins are important metabolic regulators in the healthy and disease states. Using network biology approaches, our laboratory identified that complement-1q like-3 (C1ql3) secreted protein has a role in affecting pancreatic islet function. We demonstrated that the recombinant C1ql3 protein inhibits insulin secretion in response to stimulation by exendin-4 (an agonist for the stimulatory Gs-coupled GLP-1 receptor) from human and mouse islets. C1ql3 is expressed in β-cells but not in α- or δ-cells of mouse islets. Its expression is also conserved in human islets. Our goal is to investigate the role of β-cell C1ql3 in whole-body glucose homeostasis. We generated mice with β-cell-specific deletion of C1ql3 (βKO) by crossing C1ql3fl/fl and RIP-Cre+ mice. Preliminary data show that C1ql3 βKO mice have increased body weight with no change in fasting plasma glucose levels than the control mice. The glucose clearance was significantly improved after oral glucose challenge, whereas no difference in insulin action was observed in the C1ql3 βKO vs. control mice. Moreover, the plasma insulin levels were significantly increased at 15 min after the glucose challenge in the C1ql3 βKO vs. control mice. These results suggest that the loss of C1ql3 in β-cells increases insulin secretion to improve glucose tolerance. The expression-coupled secretion of C1ql3 from islets is increased with obesity. Interestingly, reduction in C1ql3 secretion is inversely correlated with increased insulin secretion from islets of C1ql3 βKO Leptinob/ob compared to Leptinob/ob control mice. These findings identify that C1ql3 in an autocrine manner inhibits insulin secretion from β-cells to regulate whole-body glucose homeostasis. Our work identifies a novel C1ql3-signaling pathway that may contribute to the observed β-cell dysfunction in obesity and type 2 diabetes. Disclosure M. Rahman: None. H. Alsharif: None. J. E. Trombley: None. A. Pathak: None. S. Bhatnagar: None.

Insulin secretion from beta-cells of pancreatic islets is essential for regulating whole-blood glucose homeostasis. Impaired insulin secretion is an early event in the development of obesity-linked type 2 diabetes (T2D). However, underlying molecular mechanisms that cause a reduction in insulin secretion are not completely understood. We recently identified that brain angiogenesis inhibitor-3 (BAI3) adhesion G-protein coupled receptor is expressed in beta-cells, and when activated by its native ligand, complement-1q like-3 (C1ql3), blunts insulin secretion in response to cyclic adenosine monophosphate (cAMP) in an autocrine/paracrine manner. The expression of BAI3 is conserved in human islets, yet the role of islet BAI3 in whole-body glucose homeostasis remains uncharacterized. We generated C57BL6/J mice that are homozygous for the loss of the BAI3 gene by using CRISPR technology. Our data show that the BAI3-/- mice have reduced body weight and impaired glucose tolerance compared to the wild-type control (WT) mice. These mice have elevated in vivo insulin secretion after 10 min of glucose challenge and an increased ex vivo islet insulin secretion in response to stimulation by 40 mM KCL and 11 mM glucose with no effect observed at 2.8 mM glucose. Concomitantly, the insulin action is decreased in the BAI3-/- mice. These outcomes show that increased insulin secretion from beta-cells due to the loss of the BAI3 gene contributes to reduced insulin sensitivity and glucose tolerance. The expression of BAI3 is increased by obesity, indicating that the activation of BAI3 signaling may contribute to reduced insulin secretion, causing beta-cell dysfunction in T2D. In sum, we have established an important role of an understudied BAI3 GPCR signaling in inhibiting insulin secretion, potentially identifying an attractive target for novel T2D therapeutics. Disclosure H. Alsharif: None. M. Rahman: None. A. Pathak: None. J. E. Trombley: None. S. Bhatnagar: None. Funding National Institutes of Health (R01DK120684-01)

Stimulus‐coupled insulin secretion from the pancreatic islet β‐cells involves the fusion of insulin granules to the plasma membrane (PM) via SNARE complex formation—a cellular process key for maintaining whole‐body glucose homeostasis. Less is known about the role of endogenous inhibitors of SNARE complexes in insulin secretion. We show that an insulin granule protein synaptotagmin‐9 (Syt9) deletion in mice increased glucose clearance and plasma insulin levels without affecting insulin action compared to the control mice. Upon glucose stimulation, increased biphasic and static insulin secretion were observed from ex vivo islets due to Syt9 loss. Syt9 colocalizes and binds with tomosyn‐1 and the PM syntaxin‐1A (Stx1A); Stx1A is required for forming SNARE complexes. Syt9 knockdown reduced tomosyn‐1 protein abundance via proteasomal degradation and binding of tomosyn‐1 to Stx1A. Furthermore, Stx1A‐SNARE complex formation was increased, implicating Syt9‐tomosyn‐1‐Stx1A complex is inhibitory in insulin secretion. Rescuing tomosyn‐1 blocked the Syt9‐knockdown‐mediated increases in insulin secretion. This shows that the inhibitory effects of Syt9 on insulin secretion are mediated by tomosyn‐1. We report a molecular mechanism by which β‐cells modulate their secretory capacity rendering insulin granules nonfusogenic by forming the Syt9‐tomosyn‐1‐Stx1A complex. Altogether, Syt9 loss in β‐cells decreases tomosyn‐1 protein abundance, increasing the formation of Stx1A‐SNARE complexes, insulin secretion, and glucose clearance. These outcomes differ from the previously published work that identified Syt9 has either a positive or no effect of Syt9 on insulin secretion. Future work using β‐cell‐specific deletion of Syt9 mice is key for establishing the role of Syt9 in insulin secretion.