Modular genetic control of sexually dimorphic behaviors.

Sex hormones such as estrogen and testosterone are essential for sexually dimorphic behaviors in vertebrates. However, the hormone-activated molecular mechanisms that control the development and function of the underlying neural circuits remain poorly defined. We have identified numerous sexually dimorphic gene expression patterns in the adult mouse hypothalamus and amygdala. We find that adult sex hormones regulate these expression patterns in a sex-specific, regionally restricted manner, suggesting that these genes regulate sex typical behaviors. Indeed, we find that mice with targeted disruptions of each of four of these genes (Brs3, Cckar, Irs4, Sytl4) exhibit extremely specific deficits in sex specific behaviors, with single genes controlling the pattern or extent of male sexual behavior, male aggression, maternal behavior, or female sexual behavior. Taken together, our findings demonstrate that various components of sexually dimorphic behaviors are governed by separable genetic programs.

The signaling protein GIV/Girdin mediates the Nephrin-dependent insulin secretion of pancreatic islet ß cells in response to high glucose.

Glucose-stimulated insulin secretion of pancreatic ß cells is essential in maintaining glucose homeostasis. Recent evidence suggests that the Nephrin-mediated intercellular junction between ß cells is implicated in the regulation of insulin secretion. However, the underlying mechanisms are only partially characterized. Herein we report that GIV is a signaling mediator coordinating glucose-stimulated Nephrin phosphorylation and endocytosis with insulin secretion. We demonstrate that GIV is expressed in mouse islets and cultured ß cells. The loss of function study suggests that GIV is essential for the second phase of glucose-stimulated insulin secretion. Next, we demonstrate that GIV mediates the high glucose-stimulated tyrosine phosphorylation of GIV and Nephrin by recruiting Src kinase, which leads to the endocytosis of Nephrin. Subsequently, the glucose-induced GIV/Nephrin/Src signaling events trigger downstream Akt phosphorylation, which activates Rac1-mediated cytoskeleton reorganization, allowing insulin secretory granules to access the plasma membrane for the second-phase secretion. Finally, we found that GIV is downregulated in the islets isolated from diabetic mice, and rescue of GIV ameliorates the ß-cell dysfunction to restore the glucose-stimulated insulin secretion. We conclude that the GIV/Nephrin/Akt signaling axis is vital to regulate glucose-stimulated insulin secretion. This mechanism might be further targeted for therapeutic intervention of diabetic mellitus.

Multiple pathways and independent functional pools in insulin granule exocytosis.

In contrast to synaptic vesicle exocytosis, secretory granule exocytosis follows a much longer time course, and thus allows for different prefusion states prior to stimulation. Indeed, total internal reflection fluorescence microscopy in living pancreatic ß cells reveals that, prior to stimulation, either visible or invisible granules fuse in parallel during both early (first) and late (second) phases after glucose stimulation. Therefore, fusion occurs not only from granules predocked to the plasma membrane but also from those translocated from the cell interior during ongoing stimulation. Recent findings suggest that such heterogeneous exocytosis is conducted by a specific set of multiple Rab27 effectors that appear to operate on the same granule; namely, exophilin-8, granuphilin, and melanophilin play differential roles in distinct secretory pathways to final fusion. Furthermore, the exocyst, which is known to tether secretory vesicles to the plasma membrane in constitutive exocytosis, cooperatively functions with these Rab27 effectors in regulated exocytosis. In this review, the basic nature of insulin granule exocytosis will be described as a representative example of secretory granule exocytosis, followed by a discussion of the means by which different Rab27 effectors and the exocyst coordinate to regulate the entire exocytic processes in ß cells.

The GDF3-ALK7 signaling axis in adipose tissue: a possible therapeutic target for obesity and associated diabetes?

ALK7, a type I receptor for the transforming growth factor-ß superfamily, is known to be predominantly expressed in adipocytes in both mice and humans. The present review describes recent findings suggesting that ALK7 plays a major role in regulating lipid metabolism and fat mass. Furthermore, the ligands and upstream regulators that activate ALK7 signaling are discussed. The focus is on findings in mice and their derivative tissues and cells that harbor the mutations of ALK7 and related molecules. Particular attention is paid to the contradictory nature of the current literature about the loss-of-function phenotypes and the relationship with insulin secretion and sensitivity. Additional attention is paid to the ALK7 gene variants found in humans and their associated traits. The goal is to seek a parsimonious, and preferably singular and unified, description of the underlying mechanism. This review also introduces recent promising findings about ALK7 neutralizing treatment to obese mice.

Munc13b stimulus-dependently accumulates on granuphilin-mediated, docked granules prior to fusion.

The Rab27 effector granuphilin plays an indispensable role in stable docking of secretory granules to the plasma membrane by interacting with the complex of Munc18-1 and the fusion-incompetent, closed form of syntaxins-1~3. Although this process prevents spontaneous granule exocytosis, those docked granules actively fuse in parallel with other undocked granules after stimulation. Therefore, it is postulated that the closed form of syntaxins must be converted into the fusion-competent open form in a stimulus-dependent manner. Although Munc13 family proteins are generally thought to prime docked vesicles by facilitating conformational change in syntaxins, it is unknown which isoform acts in granuphilin-mediated, docked granule exocytosis. In the present study, we show that, although both Munc13a and Munc13b are expressed in mouse pancreatic islets and their beta-cell line MIN6, the silencing of Munc13b, but not that of Munc13a, severely affects glucose-induced insulin secretion. Furthermore, Munc13b accumulates on a subset of granules beneath the plasma membrane just prior to fusion during stimulation, whereas Munc13a is translocated to the plasma membrane where granules do not exist. When fluorescently labeled granuphilin was introduced to discriminate between molecularly docked granules and other undocked granules in living cells, Munc13b downregulation was observed to preferentially decrease the fusion of granuphilin-positive granules immobilized to the plasma membrane. These findings suggest that Munc13b promotes insulin exocytosis by clustering on molecularly docked granules in a stimulus-dependent manner.Key words: docking, insulin, live cell imaging, priming, TIRF microscopy.

In vivo Roles of Rab27 and Its Effectors in Exocytosis.

The monomeric GTPase Rab27 regulates exocytosis of a broad range of vesicles in multicellular organisms. Several effectors bind GTP-bound Rab27a and/or Rab27b on secretory vesicles to execute a series of exocytic steps, such as vesicle maturation, movement along microtubules, anchoring within the peripheral F-actin network, and tethering to the plasma membrane, via interactions with specific proteins and membrane lipids in a local milieu. Although Rab27 effectors generally promote exocytosis, they can also temporarily restrict it when they are involved in the rate-limiting step. Genetic alterations in Rab27-related molecules cause discrete diseases manifesting pigment dilution and immunodeficiency, and can also affect common diseases such as diabetes and cancer in complex ways. Although the function and mechanism of action of these effectors have been explored, it is unclear how multiple effectors act in coordination within a cell to regulate the secretory process as a whole. It seems that Rab27 and various effectors constitutively reside on individual vesicles to perform consecutive exocytic steps. The present review describes the unique properties and in vivo roles of the Rab27 system, and the functional relationship among different effectors coexpressed in single cells, with pancreatic beta cells used as an example.Key words: membrane trafficking, regulated exocytosis, insulin granules, pancreatic beta cells.

Rab2a and Rab27a cooperatively regulate the transition from granule maturation to exocytosis through the dual effector Noc2.

Exocytosis of secretory granules entails budding from the trans-Golgi network, sorting and maturation of cargo proteins, and trafficking and fusion to the plasma membrane. Rab27a regulates the late steps in this process, such as granule recruitment to the fusion site, whereas Rab2a functions in the early steps, such as granule biogenesis and maturation. Here, we demonstrate that these two small GTPases simultaneously bind to Noc2 (also known as RPH3AL) in a GTP-dependent manner, although Rab2a binds only after Rab27a has bound. In pancreatic ß-cells, the ternary Rab2a-Noc2-Rab27a complex specifically localizes on perinuclear immature granules, whereas the binary Noc2-Rab27a complex localizes on peripheral mature granules. In contrast to the wild type, Noc2 mutants defective in binding to Rab2a or Rab27a fail to promote glucose-stimulated insulin secretion. Although knockdown of any component of the ternary complex markedly inhibits insulin secretion, only knockdown of Rab2a or Noc2, and not that of Rab27a, impairs cargo processing from proinsulin to insulin. These results suggest that the dual effector, Noc2, regulates the transition from Rab2a-mediated granule biogenesis to Rab27a-mediated granule exocytosis.

PI3K regulates endocytosis after insulin secretion by mediating signaling crosstalk between Arf6 and Rab27a.

In secretory cells, endocytosis is coupled to exocytosis to enable proper secretion. Although endocytosis is crucial to maintain cellular homeostasis before and after secretion, knowledge about secretagogue-induced endocytosis in secretory cells is still limited. Here, we searched for proteins that interacted with the Rab27a GTPase-activating protein (GAP) EPI64 (also known as TBC1D10A) and identified the Arf6 guanine-nucleotide-exchange factor (GEF) ARNO (also known as CYTH2) in pancreatic ß-cells. We found that the insulin secretagogue glucose promotes phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generation through phosphoinositide 3-kinase (PI3K), thereby recruiting ARNO to the intracellular side of the plasma membrane. Peripheral ARNO promotes clathrin assembly through its GEF activity for Arf6 and regulates the early stage of endocytosis. We also found that peripheral ARNO recruits EPI64 to the same area and that the interaction requires glucose-induced endocytosis in pancreatic ß-cells. Given that GTP- and GDP-bound Rab27a regulate exocytosis and the late stage of endocytosis, our results indicate that the glucose-induced activation of PI3K plays a pivotal role in exocytosis-endocytosis coupling, and that ARNO and EPI64 regulate endocytosis at distinct stages.

In vivo gene delivery by cationic tetraamino fullerene.

Application of nanotechnology to medical biology has brought remarkable success. Water-soluble fullerenes are molecules with great potential for biological use because they can endow unique characteristics of amphipathic property and form a self-assembled structure by chemical modification. Effective gene delivery in vitro with tetra(piperazino)fullerene epoxide (TPFE) and its superiority to Lipofectin have been described in a previous report. For this study, we evaluated the efficacy of in vivo gene delivery by TPFE. Delivery of enhanced green fluorescent protein gene (EGFP) by TPFE on pregnant female ICR mice showed distinct organ selectivity compared with Lipofectin; moreover, higher gene expression by TPFE was found in liver and spleen, but not in the lung. No acute toxicity of TPFE was found for the liver and kidney, although Lipofectin significantly increased liver enzymes and blood urea nitrogen. In fetal tissues, neither TPFE nor Lipofectin induced EGFP gene expression. Delivery of insulin 2 gene to female C57/BL6 mice increased plasma insulin levels and reduced blood glucose concentrations, indicating the potential of TPFE-based gene delivery for clinical application. In conclusion, this study demonstrated effective gene delivery in vivo for the first time using a water-soluble fullerene.

Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis.

The Rab27 effectors are known to play versatile roles in regulated exocytosis. In pancreatic beta cells, exophilin-8 anchors granules in the peripheral actin cortex, whereas granuphilin and melanophilin mediate granule fusion with and without stable docking to the plasma membrane, respectively. However, it is unknown whether these coexisting effectors function in parallel or in sequence to support the whole insulin secretory process. Here, we investigate their functional relationships by comparing the exocytic phenotypes in mouse beta cells simultaneously lacking two effectors with those lacking just one of them. Analyses of prefusion profiles by total internal reflection fluorescence microscopy suggest that melanophilin exclusively functions downstream of exophilin-8 to mobilize granules for fusion from the actin network to the plasma membrane after stimulation. The two effectors are physically linked via the exocyst complex. Downregulation of the exocyst component affects granule exocytosis only in the presence of exophilin-8. The exocyst and exophilin-8 also promote fusion of granules residing beneath the plasma membrane prior to stimulation, although they differentially act on freely diffusible granules and those stably docked to the plasma membrane by granuphilin, respectively. This is the first study to diagram the multiple intracellular pathways of granule exocytosis and the functional hierarchy among different Rab27 effectors within the same cell.

Targeting activin receptor-like kinase 7 ameliorates adiposity and associated metabolic disorders.

Activin receptor-like kinase 7 (ALK7) is a type I receptor in the TGF-ß superfamily preferentially expressed in adipose tissue and associated with lipid metabolism. Inactivation of ALK7 signaling in mice results in increased lipolysis and resistance to both genetic and diet-induced obesity. Human genetic studies have recently revealed an association between ALK7 variants and both reduced waist to hip ratios and resistance to development of diabetes. In the present study, treatment with a neutralizing mAb against ALK7 caused a substantial loss of adipose mass and improved glucose intolerance and insulin resistance in both genetic and diet-induced mouse obesity models. The enhanced lipolysis increased fatty acid supply from adipocytes to promote fatty acid oxidation in muscle and oxygen consumption at the whole-body level. The treatment temporarily increased hepatic triglyceride levels, which resolved with long-term Ab treatment. Blocking of ALK7 signals also decreased production of its ligand, growth differentiation factor 3, by downregulating S100A8/A9 release from adipocytes and, subsequently, IL-1ß release from adipose tissue macrophages. These findings support the feasibility of potential therapeutics targeting ALK7 as a treatment for obesity and diabetes.

Loss of granuphilin and loss of syntaxin-1A cause differential effects on insulin granule docking and fusion.

The Rab27 effector granuphilin/Slp4 is essential for the stable attachment (docking) of secretory granules to the plasma membrane, and it also inhibits subsequent fusion. Granuphilin is thought to mediate these processes through interactions with Rab27 on the granule membrane and with syntaxin-1a on the plasma membrane and its binding partner Munc18-1. Consistent with this hypothesis, both syntaxin-1a- and Munc18-1-deficient secretory cells, as well as granuphilin null cells, have been observed to have a deficit of docked granules. However, to date there has been no direct comparative analysis of the docking defects in those mutant cells. In this study, we morphometrically compared granule-docking states between granuphilin null and syntaxin-1a null pancreatic ß cells derived from mice having the same genetic background. We found that loss of syntaxin-1a does not cause a significant granule-docking defect, in contrast to granuphilin deficiency. Furthermore, we newly generated granuphilin/syntaxin-1a double knock-out mice, characterized their phenotypes, and found that the double mutant mice represent a phenocopy of granuphilin null mice and do not represent phenotypes of syntaxin-1a null mice, including their granule-docking behavior. Because granuphilin binds to syntaxin-2 and syntaxin-3 as well as syntaxin-1a, it likely mediates granule docking through interactions with those multiple syntaxins on the plasma membrane.

Cargo receptor Surf4 regulates endoplasmic reticulum export of proinsulin in pancreatic ß-cells.

Insulin is an essential peptide hormone that maintains blood glucose levels. Although the mechanisms underlying insulin exocytosis have been investigated, the mechanism of proinsulin export from the endoplasmic reticulum (ER) remains unclear. Here, we demonstrated that Surf4, a cargo receptor homolog, regulates the ER export of proinsulin via its recruitment to ER exit sites (ERES). Under high-glucose conditions, Surf4 expression was upregulated, and Surf4 proteins mainly localized to the ER at a steady state and accumulated in the ERES, along with proinsulin in rat insulinoma INS-1 cells. Surf4-knockdown resulted in proinsulin retention in the ER and decreased the levels of mature insulin in secretory granules, thereby significantly reducing insulin secretion. Surf4 forms an oligomer and can physically interact with proinsulin and Sec12, essential for COPII vesicle formation. Our findings suggest that Surf4 interacts with proinsulin and delivers it into COPII vesicles for ER export in co-operation with Sec12 and COPII.

Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies.

Many members of the tripartite motif (TRIM) family of ubiquitin ligases localize in spherical, membrane-free structures collectively referred to as cytoplasmic bodies (CBs) in a concentration-dependent manner. These CBs may function as aggresome precursors or storage compartments that segregate potentially harmful excess TRIM molecules from the cytosolic milieu. However, the manner in which TRIM proteins accumulate into CBs is unclear. In the present study, using TRIM32, TRIM5α and TRIM63 as examples, we demonstrated that CBs are in a liquid droplet state, resulting from liquid-liquid phase separation (LLPS). This finding is based on criteria that defines phase-separated structures, such as recovery after photobleaching, sensitivity to hexanediol, and the ability to undergo fusion. CB droplets, which contain cyan fluorescent protein (CFP)-fused TRIM32, were purified from HEK293 cells using a fluorescence-activated cell sorter and analyzed by LC-MS/MS. We found that in addition to TRIM32, these droplets contain a variety of endogenous proteins and enzymes including ubiquitin. Localization of ubiquitin within CBs was further verified by fluorescence microscopy. We also found that the activation of the intracellular ubiquitination cascade promotes the assembly of TRIM32 molecules into CBs, whereas inhibition causes suppression. Regulation is dependent on the intrinsic E3 ligase activity of TRIM32. Similar regulation by ubiquitination on the TRIM assembly was also observed with TRIM5α and TRIM63. Our findings provide a novel mechanical basis for the organization of CBs that couples compartmentalization through LLPS with ubiquitination.

Involvement of vps33a in the fusion of uroplakin-degrading multivesicular bodies with lysosomes.

The apical surface of the terminally differentiated mouse bladder urothelium is largely covered by urothelial plaques, consisting of hexagonally packed 16-nm uroplakin particles. These plaques are delivered to the cell surface by fusiform vesicles (FVs) that are the most abundant cytoplasmic organelles. We have analyzed the functional involvement of several proteins in the apical delivery and endocytic degradation of uroplakin proteins. Although FVs have an acidified lumen and Rab27b, which localizes to these organelles, is known to be involved in the targeting of lysosome-related organelles (LROs), FVs are CD63 negative and are therefore not typical LROs. Vps33a is a Sec1-related protein that plays a role in vesicular transport to the lysosomal compartment. A point mutation in mouse Vps33a (Buff mouse) causes albinism and bleeding (Hermansky-Pudlak syndrome) because of abnormalities in the trafficking of melanosomes and platelets. These Buff mice showed a novel phenotype observed in urothelial umbrella cells, where the uroplakin-delivering FVs were almost completely replaced by Rab27b-negative multivesicular bodies (MVBs) involved in uroplakin degradation. MVB accumulation leads to an increase in the amounts of uroplakins, Lysosomal-associated membrane protein (LAMP)-1/2, and the activities of beta-hexosaminidase and beta-glucocerebrosidase. These results suggest that FVs can be regarded as specialized secretory granules that deliver crystalline arrays of uroplakins to the cell surface, and that the Vps33a mutation interferes with the fusion of MVBs with mature lysosomes thus blocking uroplakin degradation.

Granuphilin is activated by SREBP-1c and involved in impaired insulin secretion in diabetic mice.

Granuphilin is a crucial component of the docking machinery of insulin-containing vesicles to the plasma membrane. Here, we show that the granuphilin promoter is a target of SREBP-1c, a transcription factor that controls fatty acid synthesis, and MafA, a beta cell differentiation factor. Potassium-stimulated insulin secretion (KSIS) was suppressed in islets with adenoviral-mediated overexpression of granuphilin and enhanced in islets with knockdown of granuphilin (in which granuphilin had been knocked down). SREBP-1c and granuphilin were activated in islets from beta cell-specific SREBP-1c transgenic mice, as well as in several diabetic mouse models and normal islets treated with palmitate, accompanied by a corresponding reduction in insulin secretion. Knockdown- or knockout-mediated ablation of granuphilin or SREBP-1c restored KSIS in these islets. Collectively, our data provide evidence that activation of the SREBP-1c/granuphilin pathway is a potential mechanism for impaired insulin secretion in diabetes, contributing to beta cell lipotoxicity.

Berberine is an insulin secretagogue targeting the KCNH6 potassium channel.

Coptis chinensis is an ancient Chinese herb treating diabetes in China for thousands of years. However, its underlying mechanism remains poorly understood. Here, we report the effects of its main active component, berberine (BBR), on stimulating insulin secretion. In mice with hyperglycemia induced by a high-fat diet, BBR significantly increases insulin secretion and reduced blood glucose levels. However, in mice with hyperglycemia induced by global or pancreatic islet ß-cell-specific Kcnh6 knockout, BBR does not exert beneficial effects. BBR directly binds KCNH6 potassium channels, significantly accelerates channel closure, and subsequently reduces KCNH6 currents. Consequently, blocking KCNH6 currents prolongs high glucose-dependent cell membrane depolarization and increases insulin secretion. Finally, to assess the effect of BBR on insulin secretion in humans, a randomized, double-blind, placebo-controlled, two-period crossover, single-dose, phase 1 clinical trial (NCT03972215) including 15 healthy men receiving a 160-min hyperglycemic clamp experiment is performed. The pre-specified primary outcomes are assessment of the differences of serum insulin and C-peptide levels between BBR and placebo treatment groups during the hyperglycemic clamp study. BBR significantly promotes insulin secretion under hyperglycemic state comparing with placebo treatment, while does not affect basal insulin secretion in humans. All subjects tolerate BBR well, and we observe no side effects in the 14-day follow up period. In this study, we identify BBR as a glucose-dependent insulin secretagogue for treating diabetes without causing hypoglycemia that targets KCNH6 channels.

Docking is not a prerequisite but a temporal constraint for fusion of secretory granules.

We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic beta cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.

Granuphilin molecularly docks insulin granules to the fusion machinery.

The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin-null mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin-null beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.