Dependent Secretion Research Articles

Monoclonal therapy against calcitonin gene-related peptide lowers hyperglycemia and adiposity in type 2 diabetes mouse models

ObjectiveCalcitonin Gene-Related Peptide α (CGRPα) is a multifunctional neuropeptide found in the central and peripheral nervous system with cardiovascular, nociceptive, and gastrointestinal activities. CGRPα has been linked to obesity and insulin secretion but the role of this circulating peptide in energy metabolism remains unclear. Here, we thought to utilize a monoclonal antibody against circulating CGRPα to assess its ability to improve glucose homeostasis in mouse models of hyperglycemia and diabetes. MethodsWe examined the outcome of anti-CGRPα treatment in mouse models of diabetes and diet-induced obesity, using db/db mice, Streptozotocin (STZ) treatment to eliminate pancreatic islets, and high fat diet-fed mice. We also correlated these data with application of recombinant CGRPα peptide on cultured mature adipocytes to measure its impact on mitochondrial bioenergetics and fatty acid oxidation. Furthermore, we applied recombinant CGRPα to primary islets to measure glucose-stimulated insulin secretion (GSIS) and gene expression. ResultsBL6-db diabetic mice receiving anti-CGRPα treatment manifested weight loss, reduced adiposity, improved glucose tolerance, insulin sensitivity, GSIS and reduced pathology in adipose tissue and liver. Anti-CGRPα failed to modulate weight or glucose homeostasis in STZ-treated animals. High fat diet-fed mice showed reduced adiposity but no benefit on glucose homeostasis. Considering these findings, we postulated that CGRPα may have dual effects on adipocytes to promote lipid utilization while acting on pancreatic β-cells to modulate insulin secretion. Analysis of CGRPα in the pancreas showed that the peptide localized to insulin-positive cells and perivascular nerves surrounding islets. Ex-vivo analysis of pancreatic islets determined that CGRPα blocked GSIS and reduced insulin-2 gene expression. Mechanistical analysis revealed that recombinant CGRPα was able to reduce glycolytic capacity as well as fatty acid oxidation in primary white adipocytes. ConclusionsThese results establish a multifaceted role in energy metabolism for circulating CGRPα, with the ability to modulate thermogenic pathways in adipose tissue, as well as pancreatic β-cell dependent insulin secretion. Reducing circulating CGRPα levels with monoclonal therapy presents therapeutic potential for type 2 diabetes as shown in BL6-db/db mice but has reduced potential for models of hyperglycemia resulting from loss of β-cells (STZ treatment).

WHY SOME YARROWIA LIPOLYTICA STRAINS SECRET CITRIC ACID TO THE MEDIUM?

Despite many studies, the “check points” of metabolic regulation of citric acid (CA) secretion by the yeasts Y.lipolytica still remain unknown. In this manuscript, some possible aspects of strain dependent secretion as well as CA metabolism regulation were discussed. Keys enzymes’ activities, substrate concentration, affinity of the uptake systems, intracellular CA concentration and strains abilities were the main points taken into consideration. The direction for the future studies emerged from this review, mainly connected to cellular and mitochondrial citrate transport systems and cellular substrates transporters (glucose, fructose, glycerol, ethanol and acetate), give promising starting point for future efficient strain development.

Assessment of Risks and Benefits of Beta Cell Replacement Versus Automated Insulin Delivery Systems for Type 1 Diabetes.

Current approaches to insulin replacement in type 1 diabetes are unable to achieve optimal levels of glycemic control without substantial risk of hypoglycemia and substantial burden of self-management. Advances in biology and technology present beta cell replacement and automated insulin delivery as two alternative approaches. Here we discuss current and future prospects for the relative risks and benefits for biological and psychosocial outcomes from the perspective of researchers, clinicians, and persons living with diabetes. Beta cell replacement using pancreas or islet transplant can achieve insulin independence but requires immunosuppression. Although insulin independence may not be sustained, time in range of 80-90%, minimal glycemic variability and abolition of hypoglycemia is routine after islet transplantation. Clinical trials of potentially unlimited supply of stem cell-derived beta cells are showing promise. Automated insulin delivery (AID) systems can achieve 70-75% time in range, with reduced glycemic variability. Impatient with the pace of commercially available AID, users have developed their own algorithms which appear to be at least equivalent to systems developed within conventional regulatory frameworks. The importance of psychosocial factors and the preferences and values of persons living with diabetes are emerging as key elements on which therapies should be evaluated beyond their impact of biological outcomes. Biology or technology to deliver glucose dependent insulin secretion is associated with substantial improvements in glycemia and prevention of hypoglycemia while relieving much of the substantial burden of diabetes. Automated insulin delivery, currently, represents a more accessible bridge to a biologic cure that we expect future cellular therapies to deliver.

Enhanced cellular uptake of CpG DNA by α-helical antimicrobial peptide Kn2-7: Effects on macrophage responsiveness to CpG DNA

DNA containing unmethylated cytosine-guanine motifs (CpG DNA) initiates innate immune responses, including the secretion of cytokines from macrophages. Some antimicrobial peptides modulate the responses to CpG DNA, although the molecular mechanisms of this process remain unclear. This study examined the effects of four α-helical antimicrobial peptides on the immune responses induced by CpG DNA. The antimicrobial peptide FIKRIARLLRKIF, known as Kn2-7, increased the CpG DNA–dependent secretion of interleukin-10 (IL-10) and tumor necrosis factor-α from mouse macrophage-like RAW264.7 cells. Kn2-7 enhanced the cellular uptake of CpG DNA; this effect was decreased by the substitution of arginine residues with alanine residues, and increased by the substitution of lysine residues with arginine residues. The degree to which these peptides enhanced the cellular uptake of CpG DNA correlated well with their ability to increase CpG DNA–dependent IL-10 secretion. In contrast, Kn2-7 synthesized with d-amino acids did not increase CpG DNA–dependent IL-10 secretion, although the ability of the D-form of Kn2-7 to enhance the cellular uptake of CpG DNA was not diminished relative to that of Kn2-7. These results indicate that enhanced cellular uptake of CpG DNA is necessary but insufficient to augment CpG DNA–dependent immune responses.

Bax inhibitor-1 deficiency leads to obesity by increasing Ca2+-dependent insulin secretion

Transmembrane BAX inhibitor motif containing 6 (TMBIM6), also known as Bax inhibitor-1, is an evolutionarily conserved protein involved in endoplasmic reticulum (ER) function. TMBIM6 is an ER Ca2+ leak channel and its deficiency enhances susceptibility to ER stress due to inhibition of the ER stress sensor IRE1α. It was previously shown that TMBIM6 overexpression improves glucose metabolism and that TMBIM6 knockout mice develop obesity. We here examined the metabolic alterations underlying the obese phenotype and subjected TMBIM6 knockout mice to indirect calorimetry and euglycemic-hyperinsulinemic tests with stable isotope dilution to gauge tissue-specific insulin sensitivity. This demonstrated no changes in heat production, food intake, activity or hepatic and peripheral insulin sensitivity. TMBIM6 knockout mice, however, featured a higher glucose-stimulated insulin secretion in vivo as assessed by the hyperglycemic clamp test and hepatic steatosis. This coincided with profound changes in glucose-mediated Ca2+ regulation in isolated pancreatic β cells and increased levels of IRE1α levels but no differences in downstream effects of IRE1α like increased Xbp1 mRNA splicing or Ire1-dependent decay of insulin mRNA in the pancreas. We therefore conclude that lack of TMBIM6 does not affect insulin sensitivity but leads to hyperinsulinemia, which serves to explain the weight gain. TMBIM6-mediated metabolic alterations are mainly caused by its role as a Ca2+ release channel in the ER.Key messagesTMBIM6−/− leads to obesity and hepatic steatosis.Food intake and energy expenditure are not changed in TMBIM6−/− mice.No changes in insulin resistance in TMBIM6−/− mice.Increased insulin secretion caused by altered calcium dynamics in β cells.

Secretion of a low and high molecular weight \u03b2-glycosidase by Yarrowia lipolytica

BackgroundThe secretory production of recombinant proteins in yeast simplifies isolation and purification but also faces possible complications due to the complexity of the secretory pathway. Therefore, correct folding, maturation and intracellular transport of the recombinant proteins are important processing steps with a higher effort needed for complex and large proteins. The aim of this study was to elucidate the secretion potential of Yarrowia lipolytica for low and high molecular weight β-glycosidases in a comparative cultivation approach.ResultsA low sized β-glucosidase from Pyrococcus furiosus (CelB; 55 kDa) and a large sized β-galactosidase isolated from the metagenome (M1; 120 kDa) were integrated into the acid extracellular protease locus using the CRISPR–Cas9 system to investigate the size dependent secretion of heterologous proteins in Y. lipolytica PO1f. The recombinant strains were cultivated in the bioreactor for 78 h and the extra- and intracellular enzyme activities were determined. The secretion of CelB resulted in an extracellular volumetric activity of 187.5 µkatoNPGal/Lmedium, while a volumetric activity of 2.98 µkatoNPGal/Lmedium was measured during the M1 production. However, when the amount of functional intra- and extracellular enzyme was investigated, the high molecular weight M1 (85%) was secreted more efficiently than CelB (27%). Real-time PCR experiments showed a linear correlation between the transcript level and extracellular activity for CelB, while a disproportional high mRNA level was observed regarding M1. Interestingly, mass spectrometry data revealed the unexpected secretion of two endogenous intracellular glycolytic enzymes, which is reported for the first time for Y. lipolytica.ConclusionThe results of this study provide deeper insights into the secretion potential of Y. lipolytica. A secretion limitation for the low-size CelB was observed, while the large size M1 enzyme was produced in lower amounts but was secreted efficiently. It was shown for the first time that Y. lipolytica is a promising host for the secretion of heterologous high molecular weight proteins (> 100 kDa), although the total secreted amount has to be increased further.

NFkB mediated sustained inflammation promotes hepatobiliary injury in Sickle Cell Disease

Abstract Hepatic crisis is an emergent complication affecting sickle cell disease (SCD) patients, however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepatopathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, progressive inflammation, hyperbilirubinemia, and increased ductular reaction under basal condition. NFkB activation in the SCD mice liver inhibited FXR signaling and its downstream targets, leading to loss of canallicular bile transport, sustained inflammation and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canallicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mice liver. These findings are the first to identify that NFkB-FXR dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury and liver inflammation in SCD. Improved understanding of these processes could potentially benefit the development of new therapies to treat sickle cell hepatic crisis.

The P2X7 receptor mediates NLRP3-dependent IL-1β secretion and promotes phagocytosis in the macrophage response to Treponema pallidum

It is unclear whether P2X7 receptor (P2X7R) mediates NOD-like receptor family protein 3 (NLRP3)-dependent IL-1β secretion and spirochete phagocytosis in syphilis. This study was conducted to investigate the role of P2X7R in modifying NLRP3-dependent IL-1β secretion and regulating phagocytosis by Treponema pallidum (T. pallidum)-induced macrophages. Macrophages derived from a human acute monocytic leukemia cell line were cultured with T. pallidum. The activation of P2X7R in T. pallidum-treated macrophages occurred in a dose- and time-dependent manner. The P2X7R silencing group showed significantly decreased NLRP3 mRNA and protein levels (vs. the Tp group, P < 0.001). Similar results were observed for IL-1β secretion using ELISA (vs. the Tp group, P < 0.001). Furthermore, P2X7R siRNA transfection significantly decreased the percentage of spirochete-positive macrophages (29.73% vs. 70.83%, P < 0.001) and spirochete internalization (mean fluorescence intensity (MFI), 9.20 vs. 19.39, P < 0.001). This finding revealed that P2X7R played a role in the induction of NLRP3-dependent IL-1β secretion by T. pallidum-induced macrophages. Furthermore, we found that P2X7R plays an important role in IL-1β secretion and in the promotion of T. pallidum phagocytosis by macrophages. These results may not only contribute to our understanding of the immune mechanism that is active during T. pallidum infection but may also lay the groundwork for strategies to combat syphilis.

Abstract B53: Development of SB 11 and SB 12, structurally unique linkage analogs of SB 11285 as STING agonists for Immuno-oncology

Abstract Background: Agonists of the Stimulator of Interferon Genes (STING) pathway have great potential in cancer immunotherapy. Activation of STING in tumor cells and/or antige- presenting cells (APCs) can induce type I Interferon production, leading to the induction of innate and adaptive immune response. We recently reported the discovery of the cyclic dinucleotide SB 11285 as a potent, first-in-class, STING agonist. Herein, we report SB 11 and SB 12 as unique linkage analogs of SB 11285. Methods: (a) Synthesis: Several linkage analogs of SB 11285 were synthesized by standard solution-phase phosphoramidite chemistry and screened for STING agonistic activity using the previously described SZ14 reporter cell lines, and SB 11 and SB 12 were identified as lead compounds., (b) Binding affinity: Differential scanning fluorimetry was used to determine binding affinity of SB 11 and 12 to wtSTING, with 2´,3´-cGAMP being used as a positive control. (c) Induction of IRF3 and NF-KB: wtTHP-1 or R232-THP1 or RAW cells carrying the respective reporter constructs were treated with SB 11 and 12 or controls for 22hrs and induction of IRF3 and NF-KB was determined as % fold-change in luminescence compared to vehicle-treated cells. (d) Induction of cytokines: PBMCs and mBMDCs were treated with SB 11 and 12 at different doses, and cell supernatants were analyzed for IFN-β, TNF-α, and RANTES by ELISA or multiplexing Luminex assays. (e) In vivo studies: Both SB 11 and 12 were tested for antitumor efficacy in CT26 syngeneic mouse tumor model after intravenous (i.v.) administration at 3mg/kg on days 1,5,9,14. Results: (i) SB 11 and 12 demonstrated high binding affinity to human wild-type and mouse STING that is evident by 16° thermal shift. (ii) SB 11 and 12 showed potent induction of (a) STING-dependent IRF3 (EC50: 0.8 and 39.7 nM) and NF-κB (EC50: 9 and 5265 nM) signaling induction in wtTHP-1 cells; (b) IRF3 (EC50: 2.6 and 317 nM) and NF-κB (EC50: 83 and 3783 nM) induction in R232-THP-1 cells; (c) IRF3 (EC50: 67 and 46 nM) in RAW macrophages. (iii) Both SB 11 and SB 12 induced STING dependent secretion of IFN-β (~10 ng/ml) in mBMDCs. (iv) In the CT26 colon carcinoma syngeneic mouse models, both SB 11 and 12 showed potent antitumor activity when administered by i.v. route with 98 and 97% TGI and 91 and 79% TGD, respectively. Conclusion: SB 11 and SB 12 showed excellent safety and antitumor activity in syngeneic mouse model when administered by i.v. route. SB 11 and SB 12 were shown to cause STING-dependent activation of IRF3 and NF-κB signaling, as well as the induction of type I IFN signature and ISGs. Further preclinical studies of systemically administered analogs SB 11 and SB 12 are in progress. Citation Format: Shenghua Zhou, Sreerupa Challa, Diane Shmidt, Leena Suppiah, Vishal Nair, Anjaneyulu Sheri, Geeta Meher, Rayomand Gimi, Seetharamaiyer Padmanabhan, Dillon Cleary, Radhakrishnan Iyer. Development of SB 11 and SB 12, structurally unique linkage analogs of SB 11285 as STING agonists for Immuno-oncology [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B53.

Discovery of N-Cyano-sulfoximineurea Derivatives as Potent and Orally Bioavailable NLRP3 Inflammasome Inhibitors.

NLRP3 inflammasome mediated release of interleukin-1β (IL-1β) has been implicated in various diseases. In this study, rationally designed mimics of sulfonylurea moiety were investigated as NLRP3 inhibitors. Our results culminated into discovery of series of unprecedented N-cyano sulfoximineurea derivatives as potent NLRP3 inflammasome inhibitors. Compound 15 (IC50 = 7 nM) and analogues were found to be highly potent and selective NLRP3 inflammasome inhibitor with good pharmacokinetic profile. These effects translate in vivo, as 15, 29, and 34 significantly inhibit NLRP3 dependent IL-1β secretion in mice.

Abstract P3-10-02: Neutrophil elastase as a therapeutic target to inhibit metastasis in breast cancer

Abstract Background: Chronic inflammation, a hallmark of cancer, is associated with poor prognosis in human various malignancies. Predominantly, tumor associated neutrophils (TANs) and myeloid-derived suppressor cells (MDSCs) are immune cells well characterized to promote inflammation, support tumor growth and metastasis, by secreting chemokines, serine proteases and reactive oxygen species. Increased TANs and MDSCs are predictive markers of both tumor progression and metastasis, suggesting that their inhibition may provide viable anti-tumor strategies. Neutrophil elastase (NE), a serine protease exclusively secreted by TANs and MDSCs, is a crucial mediator of inflammation and tumor progression. Recent preliminary studies from our group suggests that genetic deletion of Elane (encoding NE) inhibits lung metastasis in in vivo models of breast cancer. Yet the precise mechanism(s) by which NE promotes tumorigenesis and metastasis of breast cancer remains to be elucidated. To address this gap in knowledge, the current focus of our work is to identify the tumor-intrinsic and -extrinsic mechanisms by which NE promotes metastasis. Methods: To determine the prognostic significance of NE, we analyzed 192 breast cancer patients (58% ER/PR+ve, 20%-HER-2 +ve and 22% TNBC) for infiltration of TANs. To examine the role of NE in breast cancer metastasis, we have generated Elane+/+ and Elane-/- mice in BALB/c and FVB/N genetic backgrounds, bearing 4T1 and PyMT tumors (orthotopic and spontaneous) respectively. Each of these mouse models develop lung metastasis in 80-90% in tumor-bearing mice within 1-3 months of primary tumor initiation. Results: Immunohistochemical staining of NE, a marker of TANs shows that higher infiltration of NE-positive TANs is associated with recurrence free survival with a hazard ratio of 3.4 (95% CI, 1.1-5.5). Genetic ablation of NE (Elane −/−) in the PyMT and 4T1 models strongly inhibits lung metastasis. Specifically, the number of lung foci was reduced by ~90% in PyMT Elane−/− mice compared to controls (21.8 vs. 2.7, respectively; p=0.0044). Similarly, ~50% decrease in the number of lung foci was observed in the 4T1 Elane−/− mice compared to control (7.8 vs. 3.4, respectively; p=0.0281). Comparison of inflammatory factors in the tumor microenvironment revealed that Csf3 (G-CSF), Cxcr2 (CXCR2) and Cxcl1 (CXCL1/KC) were significantly elevated in tumors from PyMT/4T1 Elane+/+ mice vs. PyMT/4T1 Elane-/- animals. Mechanistically, our result implies NE-dependent recruitment of immunosuppressive subsets (CXCL1 and related chemokines), thus sustaining inflammation at the primary tumor site. NE- dependent secretion of CXCL1 can further enrich for cancer stem-like cells (CSCs) via CXCR2 signaling, thus aiding metastasis. Lastly, we interrogated the efficacy reversible NE inhibitor AZD9668 in Elane+/+ mouse models. 100mg/kg daily treatment of AZD9668 reduced lung metastasis in PyMT mice by 94% compared to vehicle-treated mice (0.49+/- 0.21% vs. 0.03+/-0.01%; p=0.05). Conclusions: Collectively, our studies suggest that genetic and pharmacological ablation of NE reduces metastasis in in vivo models of breast cancer. NE inhibitors thus far have only been clinically tested in COPD patients and their efficacy in breast cancer remains to be evaluated. Our preclinical studies presented here are likely to provide the much needed rationale for the use of this class of NE inhibitors as a viable treatment strategy for the metastatic breast cancer. Citation Format: Amriti Rajender Lulla, Said Akli, Taylor T Chrisikos, Marc A. Pina, Yifan Zhou, Haiyan Li, Stephanie S Watowich, Khandan Keyomarsi. Neutrophil elastase as a therapeutic target to inhibit metastasis in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-02.

Enhanced Structure and Function of Stem Cell-Derived Beta-Like Cells Cultured on Extracellular Matrix

Differentiation of stem cells into insulin secreting beta-like cells holds great promise to treat diabetes. Current protocols drive the cells through stages of maturation and produce cells that secrete insulin. However, these cells still lack many of the characteristics of native beta cells. One important influence on native cell responses is the capillary basement membrane. Whether the stem cell-derived spheroids contain basement membrane and whether it plays any role in the cell responses is unknown. Here we show the spheroids do contain basement membrane proteins as a diffuse web-like structure. We demonstrate that the beta-like cells within the spheroid do not respond to this basement membrane and show no evidence for the cell polarization that characterizes native cells. We then isolated cells from the mature spheroids and show that culture on basement membrane protein coated dishes does impose cell polarity and favourably alters glucose dependent insulin secretion. We also show that cells cultured on the inner surface of basement membrane coated polymer devices survive implantation in a mouse and retain their identity. We conclude that the introduction of basement membrane and controlling the environment around stem cell-derived beta cells is a valuable route towards better phenocopying the native responses.

Joint Random Subcarrier Selection and Channel-Based Artificial Signal Design Aided PLS

A novel method for providing physical layer security (PLS) depending on the randomness of wireless channel is proposed. Specifically, a channel-based joint random subcarrier selection and artificial signal design are introduced to protect the communication in the presence of a strong passive eavesdropper. Our analysis assumes a window-based subcarrier selection method in which the strongest subcarriers in each window are selected. Chosen subcarriers are considered for secret sequence extraction. In addition the generated channel dependent secret sequence is used for both random subcarrier selection and artificial signal design. We evaluate the efficiency of the proposed method through some representative metrics, such as secret sequence disagreement rate (SSDR), throughput and bit error rate (BER), in both perfect and imperfect channel estimation cases. Simulation results are presented and insightful discussions are drawn.

The TCF7L2-dependent high-voltage activated calcium channel subunit α2δ-1 controls calcium signaling in rodent pancreatic beta-cells

The transcription factor TCF7L2 remains the most important diabetes gene identified to date and genetic risk carriers exhibit lower insulin secretion. We show that Tcf7l2 regulates the auxiliary subunit of voltage-gated Ca2+ channels, Cacna2d1 gene/α2δ-1 protein levels. Furthermore, suppression of α2δ-1 decreased voltage-gated Ca2+ currents and high glucose/depolarization-evoked Ca2+ signaling which mimicked the effect of silencing of Tcf7l2. This appears to be the result of impaired voltage-gated Ca2+ channel trafficking to the plasma membrane, as Cav1.2 channels accumulated in the recycling endosomes after α2δ-1 suppression, in clonal as well as primary rodent beta-cells. This impaired the capacity for glucose-induced insulin secretion in Cacna2d1-silenced cells. Overexpression of α2δ-1 increased high-glucose/K+-stimulated insulin secretion. Furthermore, overexpression of α2δ-1 in Tcf7l2-silenced cells rescued the Tcf7l2-dependent impairment of Ca2+ signaling, but not the reduced insulin secretion. Taken together, these data clarify the connection between Tcf7l2, α2δ-1 in Ca2+-dependent insulin secretion.

SPION-Decorated Exosome Delivered BAY55-9837 Targeting the Pancreas through Magnetism to Improve the Blood GLC Response.

BAY55-9837, a potential therapeutic peptide in the treatment of type 2 diabetes mellitus (T2DM), is capable of inducing glucose (GLC)-dependent insulin secretion. However, the therapeutic benefit of BAY55-9837 is limited by its short half-life, lack of targeting ability, and poor blood GLC response. How to improve the blood GLC response of BAY55-9837 is an existing problem that needs to be solved. In this study, a method for preparing BAY55-9837-loaded exosomes coupled with superparamagnetic iron oxide nanoparticle (SPIONs) with pancreas islet targeting activity and an enhanced blood GLC response with the help of an external magnetic force (MF) is demonstrated. The plasma half-life of BAY55-9837 loaded in exosome-SPION is 27-fold longer than that of BAY55-9837. The active targeting property of SIPONs enables BAY-exosomes to gain a favorable targeting property, which improves the BAY55-9837 blood GLC response capacity with the help of an external MF. In vivo studies show that BAY-loaded exosome-based vehicle delivery enhances pancreas islet targeting under an external MF and markedly increases insulin secretion, thereby leading to the alleviation of hyperglycemia. The chronic administration of BAY-exosome-SPION/MF significantly improves glycosylated hemoglobin and lipid profiles. BAY-exosome-SPION/MF maybe a promising candidate for a peptide drug carrier for T2DM with a better blood GLC response.

Mechanosensor transient receptor potential vanilloid member 4 (TRPV4) regulates mouse cholangiocyte secretion and bile formation.

Mechanosensitive signaling has emerged as a mechanism for the regulation of cholangiocyte transport and bile formation. The mechanical effect of fluid-flow, or shear, at the apical membrane of cholangiocytes regulates secretion through a process involving increases in [Ca2+]i and activation of Ca2+-activated Cl- channels. However, the initiating steps translating shear force to increases in intracellular calcium concentration ([Ca2+]i) are unknown. Transient receptor potential vanilloid member 4 (TRPV4), a nonselective cation channel present in the apical membrane of cholangiocytes, has been proposed as a potential mechanosensor. The aim of the present studies was to determine the potential role of TRPV4 in initiating mechanosensitive signaling in response to fluid-flow in cholangiocytes. TRPV4 expression was confirmed in both small and large mouse cholangiocytes. Exposure of cells to either fluid flow or specific TRPV4 pharmacological agonists rapidly increased both [Ca2+]i and membrane cation currents. Both flow- and agonist-stimulated currents displayed identical biophysical properties and were inhibited in the presence of TRPV4 antagonists or in cells after transfection with TRPV4 small interfering RNA. Transfection of mouse cholangiocytes with a TRPV4-enhanced green fluorescent protein construct increased the expression of TRPV4 and the magnitude of flow-stimulated currents. A specific TRPV4 agonist significantly increased the biliary concentration of ATP and bile flow in live mice when administered intravenously and increased ATP release from cholangiocyte monolayers when applied exogenously. The findings are consistent with a model in which activation of cholangiocyte TRPV4 translates shear force into an acute rise in membrane cation permeability, [Ca2+]i, ATP release, and bile flow. Understanding the role of mechanosensitive transport pathways may provide novel insights to modulate bile flow for the treatment of cholestatic liver disorders.NEW & NOTEWORTHY These studies functionally characterize TRPV4 as a mechanosensitive channel in mouse cholangiocytes. By mediating a rapid rise in intracellular Ca2+, necessary for Ca2+-dependent secretion, TRPV4 represents a mechanosensor responsible for translating fluid flow into intracellular signaling and biliary secretion. Furthermore, intravenous infusion of a specific TRPV4 agonist increases bile flow in live mice. Understanding the role of TRPV4 in mechanosensitive transport pathways may provide novel insights to modulate bile flow during cholestasis.

Control of Ion Transport by Tmem16a Expressed in Murine Intestine.

Cl– secretion by the human and murine intestinal epithelium occurs through the cystic fibrosis transmembrane conductance regulator (cftr). However, the Ca2+ activated Cl– channel Tmem16a was shown to contribute to Cl– secretion, mainly, but not exclusively, as a basolaterally located Cl– channel that controls basolateral Ca2+ signaling, and thus activation of basolateral Ca2+ dependent Sk4 K+ channels. In intestinal goblet cells, Tmem16a was shown to regulated Ca2+ signals required for exocytosis of mucus. Because a recent report denied the existence and functional role of Tmem16a in murine intestine, we reexamined in detail expression of mRNA and protein for Tmem16a in mouse colon. In experiments using short-circuited Ussing chamber and whole cell patch-clamp techniques, we further compared ion transport in wild type (WT) colon with that in mice with intestinal epithelial specific knockout of Tmem16a. As reported earlier we fully confirm expression of Tmem16a in colonic epithelial cells and the role of Tmem16a for both Ca2+-dependent and cAMP-regulated ion secretion.

Slc26a6 is an apical membrane anion exchanger that drives HCO3--dependent fluid secretion in murine pancreatic acinar cells.

The nonselective anion exchanger Slc26a6, also known as putative anion transporter 1 and chloride/formate exchanger, is thought to play a major role in transport in exocrine glands. In this study, Slc26a6 null mice were used to explore the function of Slc26a6 in the exocrine pancreas. Slc26a6 primarily localized to the apical membrane of pancreatic exocrine acinar cells. The volume of stimulated juice secretion by the ex vivo pancreas was significantly reduced ~35% in Slc26a6-/- mice, but no changes occurred in the gross structure or gland weights of Slc26a6 null mice. The secretion of pancreatic juice by Slc26a6+/+ mice was dependent on while, in contrast, fluid secretion by Slc26a6-/- mice was independent of , suggesting that Slc26a6 mediates the -dependent component of fluid secretion. Consistent with these observations, disruption of Slc26a6 also significantly reduced secretion by the pancreas ~35%. Taken together, these results demonstrate that the apical Slc26a6 anion exchanger in acinar cells is involved in -dependent fluid secretion but that another major -independent pathway is the primary driver of the fluid secretion process in the mouse pancreas.

Ca2+-independent but voltage-dependent quantal catecholamine secretion (CiVDS) in the mammalian sympathetic nervous system

Action potential-induced vesicular exocytosis is considered exclusively Ca2+ dependent in Katz's Ca2+ hypothesis on synaptic transmission. This long-standing concept gets an exception following the discovery of Ca2+-independent but voltage-dependent secretion (CiVDS) and its molecular mechanisms in dorsal root ganglion sensory neurons. However, whether CiVDS presents only in sensory cells remains elusive. Here, by combining multiple independent recordings, we report that [1] CiVDS robustly presents in the sympathetic nervous system, including sympathetic superior cervical ganglion neurons and slice adrenal chromaffin cells, [2] uses voltage sensors of Ca2+ channels (N-type and novel L-type), and [3] contributes to catecholamine release in both homeostatic and fight-or-flight like states; [4] CiVDS-mediated catecholamine release is faster than that of Ca2+-dependent secretion at the quantal level and [5] increases Ca2+ currents and contractility of cardiac myocytes. Together, CiVDS presents in the sympathetic nervous system with potential physiological functions, including cardiac muscle contractility.

The habenular Ca2+-dependent secretion activator 2 knock-down leads to despair-like behavior

The habenular Ca2+-dependent secretion activator 2 knock-down leads to despair-like behavior