PurposeGlucagon-like peptide-1 (GLP-1) secretagogues offer therapeutic potential for type 2 diabetes. Bioactive compounds in citrus were supposed to be effective in treating diabetes, while exact compounds activating enteroendocrine L-cell pathways remain underexplored. This study aimed to identify citrus-derived compounds that stimulate GLP-1 secretion from L cells by screening a traditional Chinese medicine library and to elucidate the underlying cellular mechanisms.MethodsA reporter GLUTag cell line (murine L cell line), designed to introduce a luciferase into the proglucagon sequence, was utilized to screen a traditional Chinese medicine library. The effects of selected compounds on GLP-1 secretion were evaluated in both GLUTag cells and primary mouse ileac epithelial cells. Intracellular cAMP levels and calcium flux were measured to elucidate their underlying mechanism, and a dual-luciferase reporter assay was used to confirm their activation on TGR5 target. In vivo effect was confirmed in high-fat-diet-induced diabetic mice by oral administration.ResultsHesperetin and Naringenin significantly promoted GLP-1 secretion from L cells. Both compounds increased cAMP accumulation and intracellular calcium influx in L cells, effects that were largely absent in TGR5-deficient L cells. Subsequently, dual-luciferase reporter assays identified them as TGR5 activators. Furthermore, cellular thermal shift assay (CETSA) provided evidence that each flavonoid forms a stable complex with TGR5. Finally, in high-fat-diet-induced diabetic mice, oral administration of either compound enhanced glucose-stimulated serum GLP-1 levels and ameliorated obesity and hyperglycemia under obese diabetic conditions.ConclusionThis study demonstrated that Hesperetin and Naringenin promoted GLP-1 secretion from L-cells through TGR5 activation and alleviated type 2 diabetes.

Mitochondrial glucose metabolism is critical for glucose-stimulated insulin secretion and glucose homeostasis in pancreatic β cells. We previously showed that KCNH6, a voltage-dependent potassium (Kv) channel, participated regulation of insulin secretion in pancreatic β cells, however, its role in mitochondrial metabolism remains unclear. Since we recently found that KCNH6 distributed in mitochondria, in this study, we investigated the role of KCNH6 in regulating mitochondrial function in pancreatic β cells by using a β cell-specific knockout (KCNH6-βKO) mouse model. Proteomics analysis of islets indicated that multiple proteins involved in mitochondrial metabolism were dysregulated in islets of KCNH6-βKO mice. Additionally, KCNH6-deficient β cells exhibited damaged mitochondria morphology and oxidative respiration dysfunction, which manifested as decreased glucose-induced ATP production, elevated NADH/NAD+ ratio and ROS levels. Impaired mitochondrial metabolism in βKO islets were significantly alleviated after the re-expression of KCNH6. Mechanistically, a physical interaction between KCNH6 and complex I assembly subunit Ndufa13 was detected, providing direct evidence of KCNH6's ability to regulate mitochondrial function. These results suggested that KCNH6 could be a promising therapeutic target for improving energy metabolism in β cells.

The Coptis chinensis (Franch) is widely used in diabetes therapies in traditional folk medicine from China, and we previously reported that its main active component, berberine (BBR), acted as an insulin secretagogue through blocking the KCNH6 potassium channel. However, the specific actions of BBR on insulin secretory granule (ISG) dynamics are largely unknown. Here, we analyzed the docking and fusion of ISGs from β-cells exposed to either short-term or long-term treatment with BBR. Under short-term treatment, at 8.3 mmol/L glucose, BBR slightly induced insulin secretion with a gradually increasing second phase only, showing an increased number of ISGs fused without stable docking to the plasma membrane. However, in the presence of 16.7 mmol/L glucose, biphasic insulin secretion by BBR was augmented significantly. The intracellular Ca2+ level increased during the second phase by BBR, suggesting that the Ca2+ dynamics contribute to the dynamics of insulin exocytosis. Under long-term BBR treatment for db/db mice, BBR restored impaired biphasic phases of insulin secretion by recovering the number of ISGs fused with or without predocking to the plasma membrane. In addition, BBR enhanced the docking capacity and increased biphasic Ca2+ concentration after glucose stimulation. Further research revealed that the short-term treatment with BBR primarily promoted the fusion of ISGs through blocking KCNH6 channels, whereas the long-term treatment with BBR improved the docking and fusion of ISGs by an additional effect on activating the cAMP-PKA-CREB pathway. Hence, our study indicated that short-term and long-term treatment with BBR promoted insulin exocytosis through different mechanisms in pancreatic β cells. BBR could be a dual-action antidiabetic agent, acutely enhancing insulin secretion in response to glucose and chronically improving β-cell function in T2D.

Derived from enteroendocrine cells (EECs), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are pivotal incretin hormones crucial for blood glucose regulation. Medications of GLP-1 analogs and GLP-1 receptor activators are extensively used in the treatment of type 2 diabetes (T2D) and obesity. However, there are currently no agents to stimulate endogenous incretin secretion. Here, we find the pivotal role of KCNH2 potassium channels in the regulation of incretin secretion. Co-localization of KCNH2 with incretin-secreting EECs in the intestinal epithelium of rodents highlights its significance. Gut epithelial cell-specific KCNH2 knockout in mice improves glucose tolerance and increases oral glucose-triggered GLP-1 and GIP secretion, particularly GIP. Furthermore, KCNH2-deficient primary intestinal epithelial cells exhibit heightened incretin, especially GIP secretion upon nutrient stimulation. Mechanistically, KCNH2 knockdown in EECs leads to reduced K+ currents, prolonged action potential duration, and elevated intracellular calcium levels. Finally, we found that dofetilide, a KCNH2-specific inhibitor, could promote incretin secretion in enteroendocrine STC-1 cells in vitro and in hyperglycemic mice in vivo. These findings elucidate, for the first time, the mechanism and application of KCNH2 in regulating incretin secretion by EECs. Given the therapeutic promise of GLP-1 and GIP in diabetes and obesity management, this study advances our understanding of incretin regulation, paving the way for potential incretin secretagogue therapies in the treatment of diabetes and obesity.

Atrial fibrillation (AF) is associated with increased risk of stroke and mortality. It has been reported that the process of atrial fibrosis was regulated by β-catenin in rats with AF. However, pathophysiological mechanisms of this process in human with AF remain unclear. This study aims to investigate the possible mechanisms of β-catenin in participating in the atrial fibrosis using human right atrial appendage (hRAA) tissues . We compared the difference of β-catenin expression in hRAA tissues between the patients with AF and sinus rhythm (SR). The possible function of β-catenin in the development of AF was also explored in mice and primary cells. Firstly, the space between the membrane of the gap junctions of cardiomyocytes was wider in the AF group. Secondly, the expression of the gap junction function related proteins, Connexin40 and Connexin43, was decreased, while the expression of β-catenin and its binding partner E-cadherin was increased in hRAA and cardiomyocytes of the AF group. Thirdly, β-catenin colocalized with E-cadherin on the plasma membrane of cardiomyocytes in the SR group, while they were dissociated and accumulated intracellularly in the AF group. Furthermore, the expression of glycogen synthase kinase 3β (GSK-3β) and Adenomatous Polyposis Coli (APC), which participated in the degradation of β-catenin, was decreased in hRAA tissues and cardiomyocytes of the AF group. Finally, the development of atrial fibrosis and AF were proved to be prevented after inhibiting β-catenin expression in the AF model mice. Based on human atrial pathological and molecular analyses, our findings provided evidence that β-catenin was associated with atrial fibrosis and AF progression.

Cathepsin L (CTSL) is a promising therapeutic target for metabolic disorders. Current pharmacological interventions targeting CTSL have demonstrated potential in reducing body weight gain, serum insulin levels, and improving glucose tolerance. However, the clinical application of CTSL inhibitors remains limited. In this study, we used a combination of artificial intelligence and experimental methods to identify new CTSL inhibitors from natural products. Through a robust deep learning model and molecular docking, we screened 150 molecules from natural products for experimental validation. At a concentration of 100µM, we found that 36 of them exhibited more than 50% inhibition of CTSL. Notably, 13 molecules displayed over 90% inhibition and exhibiting concentration-dependent effects. The molecular dynamics simulation on the two most potent inhibitors, Plumbagin and Beta-Lapachone, demonstrated stable interaction at the CTSL active site. Enzyme kinetics studies have shown that these inhibitors exert an uncompetitive inhibitory effect on CTSL. In conclusion, our research identifies Plumbagin and Beta-Lapachone as potential CTSL inhibitors, offering promising candidates for the treatment of metabolic disorders and illustrating the effectiveness of artificial intelligence in drug discovery.

Activin receptor-like kinase 7 (ALK7) is a type I receptor in the TGF-β 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-1β release from adipose tissue macrophages. These findings support the feasibility of potential therapeutics targeting ALK7 as a treatment for obesity and diabetes.

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.

The hERG protein is a member of voltage-gated potassium (Kv) channels. Previous reports showed that patients with long QT syndrome due to mutations in hERG channel had higher responsive glucagon-like peptide-1 (GLP-1) secretion. However, the role of hERG in GLP-1 secretion remains uncertain. Here we report that hERG is co-expressed in GLP-1-expressing L cells in rodent intestinal epithelium (Fig. 1). In a murine L-cell model, GLUTag cell line, downregulation of hERG significantly prolonged action potential duration, increased intracellular calcium concentration ([Ca2+]i), and promoted GLP-1 secretion (Fig. 2) after stimulation of nutrients. These results reveal a direct role of hERG in GLP-1 secretion in murine L-cells, suggesting intestinal hERG is a potential target for the treatment of diabetes. Disclosure J.Yang: None. C.Liu: None. H.Wang: None. Y.Yuan: None. Funding National Natural Science Foundation of China (82170809, 81930019)

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important target for vaccine and drug development. However, the rapid emergence of variant strains with mutated S proteins has rendered many treatments ineffective. Cleavage of the S protein by host proteases is essential for viral infection. Here, we discovered that the S protein contains two previously unidentified Cathepsin L (CTSL) cleavage sites (CS-1 and CS-2). Both sites are highly conserved among all known SARS-CoV-2 variants. Our structural studies revealed that CTSL cleavage promoted S to adopt receptor-binding domain (RBD) “up” activated conformations, facilitating receptor-binding and membrane fusion. We confirmed that CTSL cleavage is essential during infection of all emerged SARS-CoV-2 variants (including the recently emerged Omicron variant) by pseudovirus (PsV) infection experiment. Furthermore, we found CTSL-specific inhibitors not only blocked infection of PsV/live virus in cells but also reduced live virus infection of ex vivo lung tissues of both human donors and human ACE2- transgenic mice. Finally, we showed that two CTSL-specific inhibitors exhibited excellent In vivo effects to prevent live virus infection in human ACE2- transgenic mice. Our work demonstrated that inhibition of CTSL cleavage of SARS-CoV-2 S protein is a promising approach for the development of future mutation-resistant therapy.

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 β-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.

A common variant in the RAB27A gene in adults was recently found to be associated with the fractional exhaled nitric oxide level, a marker of eosinophilic airway inflammation. The small GTPase Rab27 is known to regulate intracellular vesicle traffic, although its role in allergic responses is unclear. We demonstrated that exophilin-5, a Rab27-binding protein, was predominantly expressed in both of the major IL-33 producers, lung epithelial cells, and the specialized IL-5 and IL-13 producers in the CD44hiCD62LloCXCR3lo pathogenic Th2 cell population in mice. Exophilin-5 deficiency increased stimulant-dependent damage and IL-33 secretion by lung epithelial cells. Moreover, it enhanced IL-5 and IL-13 production in response to TCR and IL-33 stimulation from a specific subset of pathogenic Th2 cells that expresses a high level of IL-33 receptor, which exacerbated allergic airway inflammation in a mouse model of asthma. Mechanistically, exophilin-5 regulates extracellular superoxide release, intracellular ROS production, and phosphoinositide 3-kinase activity by controlling intracellular trafficking of Nox2-containing vesicles, which seems to prevent the overactivation of pathogenic Th2 cells mediated by IL-33. This is the first report to our knowledge to establish the significance of the Rab27-related protein exophilin-5 in the development of allergic airway inflammation, and provides insights into the pathophysiology of asthma.

Genetic variation in the ACVR1C gene has been reported to be associated with decreased waist-to-hip ratio and risk of type 2 diabetes in humans. However, the effect of this naturally occurring variation on ALK7 function is unclear. Here we show that the human ACVR1C variant I195T, which displays the greatest effect on waist-to-hip ratio adjusted for body mass index, fails to signal in response to ALK7 ligands in a cell-based assay. This is consistent with observations in mice that impaired ALK7 signaling increases lipolysis and decreases adiposity. As further validation of ALK7 as an obesity target, we present data showing in the naturally occurring polygenic obese TSOD mouse a specific high affinity neutralizing monoclonal anti-ALK7 antibody (ALK7 mAb) dramatically decreases leptin levels, adipose macrophages and adiposity. The specificity of the ALK7 mAb is demonstrated by its lack of effect in genetically deficient ALK7 mice. These data along with the knowledge that normal Balb/c mice harbor a homozygous ALK7 nonsense mutation suggests that targeting ALK7 in humans should decrease obesity, susceptibility to diabetes and have minimal on-target negative consequences. Disclosure Y. Bu: None. M. Zhao: None. M. Fredericks: None. M. Cannell: None. Y. Dagon: None. C.A. Emdin: None. K. Okunishi: None. H. Wang: None. D. Sako: None. R. Castonguay: Employee; Self; Acceleron Pharma. R.N.V. Suragani: None. S. Kathiresan: Advisory Panel; Self; Regeneron Genetics Center. Consultant; Self; Color Genomics, Corvia Medical, Inc., Medgenome, Pfizer Inc. Stock/Shareholder; Self; Catabasis Pharmaceuticals, Maze Therapeutics, San THerapeutics, Verve Therapeutics. A. Grinberg: None. J. Knopf: Stock/Shareholder; Self; Acceleron Phama. R.S. Pearsall: Employee; Self; Acceleron Pharma, Inc. Stock/Shareholder; Self; Acceleron Pharma, Inc. R. Kumar: Employee; Self; Acceleron Pharma. T. Izumi: Research Support; Self; Acceleron Pharma.

Direct observation of fluorescence-labeledsecretory granule exocytosis in living pancreatic β cells has revealedheterogeneous prefusion behaviors: some granules dwell beneath the plasmamembrane before fusion, while others fuse immediately once they are recruitedto the plasma membrane. Although the former mode seems to follow sequentialdocking-priming-fusion steps as found in synaptic vesicle exocytosis, thelatter mode, which is unique to secretory granule exocytosis, has not beenexplored well. Here, we show that melanophilin, one of the effectors of themonomeric GTPase Rab27 on the granule membrane, is involved in such anaccelerated mode of exocytosis. Both melanophilin-mutated leaden mouse and melanophilin-downregulated human pancreatic βcells exhibit impaired glucose-stimulated insulin secretion, with a specificreduction in fusion events that bypass stable docking to the plasma membrane. Uponstimulus-induced [Ca2+]irise, melanophilin mediates this type of fusion by dissociating granules frommyosin-Va and actin in the actin cortex and by associating them with a fusion-competent,open form of syntaxin-4 on the plasma membrane. These findings provide thehitherto unknown mechanism to support sustainable exocytosis by which granulesare recruited from the cell interior and fuse promptly without stablepredocking to the plasma membrane.

Recently, we reported the role of coixol (6-methoxy-2(3H)-benzoxazolone), an alkaloid from Scoparia dulcis, in glucose-dependent insulin secretion; however, its insulin secretory mechanism(s) remained unknown. Here, we explored the insulinotropic mechanism(s) of coixol in vitro and in vivo. Mice islets were batch incubated, perifused with coixol in the presence of agonists/antagonists, and insulin secretion was measured by ELISA. Intracellular cAMP levels were measured using enzyme immunoassay. K+- and Ca2+-currents were recorded in MIN6 cells using whole-cell patch-clamp technique. The in vivo glucose tolerance and the insulinogenic index were evaluated in diabetic rats treated with coixol at 25 and 50 mg/kg, respectively. Coixol, unlike sulfonylurea, enhanced insulin secretion in batch incubated and perifused islets at high glucose, with no effect at basal glucose concentrations. Coixol showed no pronounced effect on the inward rectifying K+- and Ca2+-currents in whole-cell patch recordings. Moreover, coixol-induced insulin secretion was further amplified in the depolarized islets. Coixol showed an additive effect with forskolin (10 μM)-induced cAMP level, and in insulin secretion; however, no additive effect was observed with isobutylmethylxanthine (IBMX, 100 μM)-induced cAMP level, nor in insulin secretion. The PKA inhibitor H-89 (50 μM), and Epac2 inhibitor MAY0132 (50 μM) significantly inhibited the coixol-induced insulin secretion (P < 0.01). Furthermore, insulin secretory kinetics revealed that coixol potentiates insulin secretion in both early and late phases of insulin secretion. In diabetic animals, coixol showed significant improvement in glucose tolerance and on fasting blood glucose levels. These data suggest that coixol amplifies glucose-stimulated insulin secretion by cAMP-mediated signaling pathways.

Exophilin-8 has been reported to play a role in anchoring secretory granules within the actin cortex, due to its direct binding activities to Rab27 on the granule membrane and to F-actin and its motor protein, myosin-Va. Here, we show that exophilin-8 accumulates granules in the cortical F-actin network not by direct interaction with myosin-Va, but by indirect interaction with a specific form of myosin-VIIa through its previously unknown binding partner, RIM-BP2. RIM-BP2 also associates with exocytic machinery, Cav1.3, RIM, and Munc13-1. Disruption of the exophilin-8-RIM-BP2-myosin-VIIa complex by ablation or knockdown of each component markedly decreases both the peripheral accumulation and exocytosis of granules. Furthermore, exophilin-8-null mouse pancreatic islets lose polarized granule localization at the β-cell periphery and exhibit impaired insulin secretion. This newly identified complex acts as a physical and functional scaffold and provides a mechanism supporting a releasable pool of granules within the F-actin network beneath the plasma membrane.

Granuphilin, an effector of the small GTPase Rab27a, mediates the stable attachment (docking) of insulin granules to the plasma membrane and inhibits subsequent fusion of docked granules, possibly through interaction with a fusion-inhibitory Munc18-1/syntaxin complex. However, phenotypes of insulin exocytosis differ considerably between Rab27a- and granuphilin-deficient pancreatic β cells, suggesting that other Rab27a effectors function in those cells. We found that one of the putative Rab27a effector family proteins, exophilin7/JFC1/Slp1, is expressed in β cells; however, unlike granuphilin, exophilin7 overexpressed in the β-cell line MIN6 failed to show granule-docking or fusion-inhibitory activity. Furthermore, exophilin7 has no affinities to either Munc18-1 or Munc18-1-interacting syntaxin-1a, in contrast to granuphilin. Although β cells of exophilin7-knockout mice show no apparent abnormalities in intracellular distribution or in ordinary glucose-induced exocytosis of insulin granules, they do show impaired fusion in response to some stronger stimuli, specifically from granules that have not been docked to the plasma membrane. Exophilin7 appears to mediate the fusion of undocked granules through the affinity of its C2A domain toward the plasma membrane phospholipids. These findings indicate that the two Rab27a effectors, granuphilin and exophilin7, differentially regulate the exocytosis of either stably or minimally docked granules, respectively.

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 β 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.