Cell Secretory Function Research Articles

Zhibai Dihuang Pill Alleviates Ureaplasma urealyticum-Induced Spermatogenic Failure and Testicular Dysfunction via MAPK Signaling Pathway.

The testicles and sperm are extremely susceptible to inflammation and oxidative stress. Although Zhibai Dihuang Pill (ZDP) has been reported to treat various infertilities including male infertility induced by Ureaplasma urealyticum (UU) infection, its mechanism is still poorly understood. This study is aimed at clarifying the underlying mechanism of ZDP to protect against UU-infected male infertility. We found that UU-infected infertile rats exhibited weight loss, reduced food intake, and decreased sperm count and vitality. The administration of ZDP improved the general state and sperm motility of rats. In addition, UU infection led to spermatogenesis disorders, impaired secretory function and blood-testis barrier (BTB) of Sertoli cells, and elevated inflammation and oxidative stress. As expected, ZDP suppressed inflammation and oxidative stress to alleviate spermatogenesis disorders. Our research showed that ZDP could improve spermatogenesis disorders and testicular function primarily through the mitogen-activated protein kinase (MAPK) signaling pathway. ZDP exerts its anti-inflammatory and antioxidant effects via the MAPK signaling pathway, thus playing an important role in ameliorating spermatogenesis failure and testicular dysfunction.

Bile acid- and ethanol-mediated activation of Orai1 damages pancreatic ductal secretion in acute pancreatitis.

Regardless of its aetiology, sustained intracellular Ca2+ overload is a well-known hallmark of acute pancreatitis (AP). Toxic Ca2+ elevation induces pancreatic ductal cell damage characterized by impaired ion and fluid secretion - essential to wash out the protein-rich fluid secreted by acinar cells while maintaining the alkaline intra-ductal pH under physiological conditions - and mitochondrial dysfunction. While prevention of ductal cell injury decreases the severity of AP, no specific drug target has yet been identified in the ductal cells. Although Orai1, a store-operated Ca2+ influx channel, is known to contribute to sustained Ca2+ overload in acinar cells, details concerning its expression and function in ductal cells are currently lacking. In this study, we demonstrate that functionally active Orai1 channels reside predominantly in the apical plasma membrane of pancreatic ductal cells. Selective CM5480-mediated Orai1 inhibition impairs Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. Furthermore, prevention of sustained extracellular Ca2+ influx protects ductal cell secretory function in vitro and decreases pancreatic ductal cell death. Finally, Orai1 inhibition partially restores and maintains proper exocrine pancreatic secretion in in vivo AP models. In conclusion, our results indicate that Orai1 inhibition prevents AP-related ductal cell function impairment and holds the potential of improving disease outcome. KEY POINTS: Sustained intracellular Ca2+ overload in pancreatic acinar and ductal cells is a hallmark of biliary and alcohol-induced acute pancreatitis, which leads to impaired ductal ion and fluid secretion. Orai1 is a plasma membrane Ca2+ channel that mediates extracellular Ca2+ influx upon endoplasmic reticulum Ca2+ depletion. Results showed that Orai1 is expressed on the luminal plasma membrane of the ductal cells and selective Orai1 inhibition impaired Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. The prevention of sustained extracellular Ca2+ influx protected ductal cell secretory functions in in vitro models and maintained exocrine pancreatic secretion in in vivo acute pancreatitis models. Orai1 inhibition prevents the bile acid- and alcohol-induced damage of the pancreatic ductal secretion and holds the potential of improving the outcome of acute pancreatitis.

Isolation, culture, and identification of ceruminous gland cells.

External auditory canal (EAC) stenosis or atresia usually requires a skin graft to repair, but due to the lack of a graft containing functional glands, postoperative complications such as infection and eczema are common. The aim of this study was to isolate and characterize seed cells for the construction of tissue engineered EAC skin containing ceruminous gland by isolating and cultivating cells of ceruminous gland. In this study, EAC skin samples were harvested from adult goats for ceruminous gland cell isolation. Cell morphology and proliferation rates, expression of CK7, CK8, CK18, and CK19 (glandular cell specific-markers), and secretion of β-defensin-1, lysozyme, and polysaccharides were evaluated at different passages to verify the presence of ceruminous gland cells and determine whether function and proliferation potential were maintained. Ceruminous glands were successfully isolated and extracted from goat EAC skin. Furthermore, the isolated glandular cells maintained robust proliferation potential, exhibited high expression of CK7, CK8, CK18, and CK19, and vigorously secreted β-defensin-1, lysozyme, and polysaccharides in this culture system. However, expression of glandular cell specific-markers and secretory function gradually declined with increasing passage number, indicating dedifferentiation of the subcultured ceruminous gland cells after five passages. In conclusion, ceruminous glands were successfully isolated, cultured, and expanded from goat EAC skin using the serumcontaining culture system. Importantly, the isolated glandular cells retained robust proliferation potential and maintained their phenotype and function in early passages (P1-P3), indicating the method's potential application for ceruminous gland regeneration.

Encapsulation of Primary Salivary Gland Acinar Cell Clusters and Intercalated Ducts (AIDUCs) within Matrix Metalloproteinase (MMP)-Degradable Hydrogels to Maintain Tissue Structure and Function.

Progress in the development of salivary gland regenerative strategies is limited by poor maintenance of the secretory function of salivary gland cells (SGCs) in vitro. To reduce the precipitous loss of secretory function, a modified approach to isolate intact acinar cell clusters and intercalated ducts (AIDUCs), rather than commonly used single cell suspension, is investigated. This isolation approach yields AIDUCs that maintain many of the cell-cell and cell-matrix interactions of intact glands. Encapsulation of AIDUCs in matrix metalloproteinase (MMP)-degradable PEG hydrogels promotes self-assembly into salivary gland mimetics (SGm) with acinar-like structure. Expression of Mist1, a transcription factor associated with secretory function, is detectable throughout the in vitro culture period up to 14 days. Immunohistochemistry also confirms expression of acinar cell markers (NKCC1, PIP and AQP5), duct cell markers (K7 and K5), and myoepithelial cell markers (SMA). Robust carbachol and ATP-stimulated calcium flux is observed within the SGm for up to 14 days after encapsulation, indicating that secretory function is maintained. Though some acinar-to-ductal metaplasia is observed within SGm, it is reduced compared to previous reports. In conclusion, cell-cell interactions maintained within AIDUCs together with the hydrogel microenvironment may be a promising platform for salivary gland regenerative strategies.

The role of inflammatory mediators in the development of endometrosis in mare

Equine endometrosis is a chronic degenerative condition with progressive fibrosis that forms in the endometrial stroma and around the endometrial glands. Equine endometrosis causes histological changes and alterations in the secretory function of endometrial cells. These alterations lead to changes in the endometrium and early pregnancy dysfunction. Thus, this condition is one of the main causes of subfertility in mares, resulting in enormous economic losses to the horse-breeding industry. In general, fibrosis is characterized by excessive deposition of extracellular matrix (ECM) components. It destroys normal tissue architecture and compromises tissue function. The cellular and molecular events associated with the development of tissue fibrosis are complex and poorly understood. In recent years, research has been carried out on the involvement of immune cells and their products in the pathogenesis of endometrosis in mares. These studies focused mainly on the role of cytokines, such as transforming growth factor (TGF)-β1, interleukin (IL) 6, IL-1β, prostaglandin (PG), and components of neutrophil extracellular traps (NET), such as myeloperoxidase, cathepsin G, and elastase, in the most important processes associated with the development of endometrosis. The results showed that TGF-β1, IL-1β, IL-6, and PGs act on myofibroblast differentiation, fibroblast proliferation, deposition of ECM protein, and expression of matrix metalloproteinases (MMPs), and their inhibitors. It suggests their involvement in processes associated with pathological endometrial remodeling. Moreover, the response of endometrial tissue to selected factors changes according to the stage of endometrosis. The action of inflammatory mediators on processes related to the development of fibrosis shows a connection between inflammation and endometrosis.

Reprogramming of Chinese hamster ovary cells towards enhanced protein secretion

The deficient secretory phenotype of Chinese hamster ovary (CHO) cells is a major limitation for high-level production of biopharmaceuticals, particularly for those with complex molecular architectures and post-translational modifications. To improve CHO cell secretory capacity, we recently engineered CHO cell hosts to overexpress BLIMP1 (CHOB), in a cell engineering strategy that transformed the cellular machinery and led to significantly higher product yields and cell-specific productivities for different rproteins. Here, as a follow-up to our previous study, we developed new CHO cell hosts that co-overexpress BLIMP1 and XBP1s ( CHOBX ), two transcription factors that together drive the professional secretory function of antibody-producing plasma cells. We found that the CHOBX cells presented an improved performance over that of CHOB cells, with better product yields and cell-specific productivities for a recombinant IgG1 and a ‘difficult-to-express’ EPO-Fc fusion protein. These improvements in the CHOBX-derived cell lines resulted from a series of physiological and metabolic changes due to the synergetic co-expression of BLIMP1 and XBP1s. Firstly, cells presented an inhibited cell growth and arrested cell cycle in G1/G0 phase, features that were directly linked to BLIMP1 expression levels. Secondly, cells increased protein translation (both overall and recombinant protein), expanded the endoplasmic reticulum and improved their capacity to secrete protein more effectively. Lastly, cells showed a metabolic profile favouring energy production, with a pronounced lactate switch and increased consumption of amino acids. This study highlights the value of transcription factors for reprogramming CHO cells towards a desired phenotype, offering the potential to engineer cells with new functionalities for enhanced manufacturing of recombinant therapeutic proteins.

FSH inhibits autophagy and lysosomal biogenesis to regulate protein degradation in cultured goat Sertoli cells

Although the follicle-stimulating hormone (FSH) plays a vital role in male reproduction, the molecular relationships among FSH, autophagy, and the secretory function of Sertoli cells remain largely undetermined. In this study, we sought to investigate the effects of FSH on dairy goat Sertoli cell autophagy and the role of autophagy in protein clearance. FSH treatment of primary Sertoli cells was found to enhance the expression level of LC3-II, reduce p62 degradation and the number of lysosomes, and downregulate the levels of LAMP2 protein and lysosomal gene mRNAs. Further analyses revealed that starvation-induced autophagy promotes the translocation of transcription factor EB (TFEB) from the cytoplasm to nucleus and its binding to the promoter region of LAMP2, whereas FSH suppresses the nuclear translocation of TFEB. Moreover, we found that the FSH-mediated inhibition of autophagy extends the biological half-lives of androgen-binding protein (ABP), glial-derived neurotrophic factor (GDNF), and stem cell factor (SCF) and promotes the secretion of these proteins. Collectively, these observations indicate that FSH inhibits autophagy by reducing lysosomal biogenesis, which is associated with the suppression of TFEB nuclear translocation via activation of the PI3K/Akt/mTOR pathway, thereby extending the biological half-lives and enhancing the expression of ABP, GDNF, and SCF in dairy goat Sertoli cells.

Next-Generation Sequencing to Assess the Impact of Low Dose Melphalan on Residual Disease before High-Dose Melphalan and Stem Cell Transplant in Multiple Myeloma

High-Dose Melphalan (HDM) followed by stem cell transplant (SCT) remains the standard-of-care for transplant-eligible patients newly-diagnosed with multiple myeloma (MM). However, ~1/3 of patients relapse <2 years after undergoing HDM-SCT, indicating that melphalan-sensitivity is limited to a subset of patients and is currently not predictable. Currently, models that predict melphalan-resistance before proceeding to transplant are lacking. Rather, transplant-eligibility is defined mostly based on adequate organ function and performance status. Therefore, there is an urgent and unmet clinical need to develop strategies that accurately predict melphalan sensitivity among MM patients prior to HDM-SCT and save melphalan-resistant patients from undergoing this highly morbid procedure, if no demonstrable benefit is expected from it.Traditional disease-measurement methods based on International Myeloma Working Group (IMWG) criteria rely on the secretory function of myeloma cells and measure monoclonal protein levels. Following induction therapy, pre-transplant monoclonal protein levels are usually very low, and further reduction in myeloma secretory function are not detectable. In addition, the long half-life of monoclonal proteins makes assessing short-term disease changes problematic. Methods to accurately detect minor changes in disease burden following a low dose of melphalan (LDM) as a marker of melphalan sensitivity are needed to better predict patient responses to LDM. Next-generation sequencing (NGS), is an alternative approach that may allow for the highly sensitive, rapid, real-time detection of minuscule changes in tumor volume that are not influenced by the long half-life of monoclonal proteins.Here, we propose to use NGS-based tumor assessment to evaluate changes in disease volume following LDM before proceeding to HDM-SCT. Evidence is lacking to determine whether a single LDM generates a decrease in myeloma burden that is measurable by NGS. Our central hypothesis is that NGS of bone marrow aspirates from newly-diagnosed, post-induction transplant-eligible MM patients will provide a method to precisely determine the effect of LDM on disease burden. ClonoSEQ assay is an FDA-cleared, highly sensitive, specific, and standardized method to detect and monitor MRD, in MM patients. clonoSEQ leverages the power of NGS and offers an accurate and reliable way to assess how disease burden changes over time in response to treatment. Therefore, we propose a proof-of-principle study to assess the validity of this strategy and to provide essential data for future trial design investigating individualized approaches based on NGS sequencing and low doses of therapeutic agents. We will test the central hypothesis that LDM, administered at 16 mg/m 2, generates a detectable reduction in tumor burden measured by NGS. A detectable reduction in tumor burden is defined as a ≥ 20% decrease in NGS clonal count in at least 30% of subjects. We will administer propylene glycol-free melphalan formulation (EVOMELA) due to greater stability upon reconstitution than AlKERAN formulation in order to diminish the variability in the effective administered dose. The primary and secondary objectives and endpoints of the study are listed in Table-1,2.Statistical Considerations: Clonoseq detects measurable residual disease at the level of a single cell given sufficient sample input. The specific hypothesis of this pilot trial is LDM produces a measurable disease reduction that is readily detectable by clonoSEQ with at least a 20% reduction in at least 30% of patients. Assuming a 100% yield for VJD clonal sequencing and calibration efficacy by clonoSEQ, the sample size required to test the null hypothesis of 5% patients with positive MRD test against alternative 30% patients with positive MRD test is 16 patients. The sample size estimation is using two-sided chi-square test with 80% power. The sample size estimation is n = 21, when power = 90% based on one sample Binomial distribution theory. We will assume 20% failure rate for VJD clonal sequencing and calibration efficacy by clonoSEQ. Therefore, by enrolling 20 patients, we expect that at least 16 patients will have MRD assessable by NGS method. [Display omitted] DisclosuresMalek: BMS: Honoraria, Research Funding; Amgen: Honoraria; Bluespark Inc.: Research Funding; Sanofi: Other: Advisory Board; Cumberland Inc.: Research Funding; Takeda: Honoraria; Janssen: Other: Advisory board ; Medpacto Inc.: Research Funding. Metheny: Incyte: Speakers Bureau; Pharmacosmos: Honoraria. de Lima: Miltenyi Biotec: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

2′-Fucosyllactose impacts the expression of mucus-related genes in goblet cells and maintains barrier function of gut epithelial cells

Cost-effective microbial biosynthesis of 2′-fucosyllactose (2′-FL) allows its application in infant formula. The specific effects of 2′-FL on the gastrointestinal immune barrier are still largely unknown. Here, we quantified and compared the effects of HMOs isolated from human milk, 2′-FL/lactose (Lac), and 2′-FL on the expression of the mucus associated genes MUC2, TFF3, RETLB and the Golgi sulfotransferases, CHST5, and GAL3ST5, in human goblet cells. We also determined whether these compounds have protective effects on A23187-induced barrier disruption of human T84 gut epithelial cells in vitro. The impact of isolated HMOs and 2′-FL/Lac on the mRNA expression of the mucus-related genes was minor while pure 2′-FL significantly induced GAL3ST2 and CHST5. Isolated HMOs, 2′-FL/Lac and 2′-FL all prevented A23187-induced barrier disruption in human T84 cells. Our findings indicate that 2′-FL modulates the secretory function of goblet cells and protects gut epithelial cells.

Zinc status, insulin resistance and glycoxidative stress in elderly subjects with type 2 diabetes mellitus.

Zinc deficiencies have been reported in numerous pathologies, such as diabetes mellitus, but also in the physiological process of ageing. Similarly, the end products of glycoxidation processes, advanced glycation end products (AGEs), are damaging compounds, a myriad of reports linking them to the development and progression of several age-associated chronic diseases. The aim of the present study was to analyze the relationships between zinc status, glycoxidative stress and insulin resistance (IR) in elderly subjects with type 2 diabetes mellitus (T2DM). A group of 52 non-smoking subjects (9 men and 43 women, aged 65-83 years) were enrolled in this cross-sectional study: 27 patients with T2DM, and 25 apparently healthy control subjects. Serum zinc (Zn) levels were assessed using a commercial kit based on an end-point colorimetric method, and serum AGEs were evaluated with a fluorimetric analytic procedure. The calculated glucose-to-zinc ratio (Gly/Zn), insulin-to-zinc ratio (Ins/Zn) and insulin-zinc resistance index (HOMA-IR/Zn) were further used to study the associations between serum Zn levels, secretory function of β-pancreatic cells and AGEs. T2DM patients presented significantly higher serum insulin and Zn levels, as compared to the controls. We found a significant inverse correlation between Zn and AGEs, and a strong positive correlation between AGEs and the Gly/Zn ratio, suggesting that both Zn and AGEs are biomarkers that could reflect the persistence of hyperglycemia. We identified new surrogate biomarkers useful for the assessment of glycemic control with great potential for the development of preventive and therapeutic strategies for elderly diabetics, based on the evaluation of serum Zn levels.

Admixture with indigenous people helps local adaptation: admixture-enabled selection in Polynesians

BackgroundHomo sapiens have experienced admixture many times in the last few thousand years. To examine how admixture affects local adaptation, we investigated genomes of modern Polynesians, who are shaped through admixture between Austronesian-speaking people from Southeast Asia (Asian-related ancestors) and indigenous people in Near Oceania (Papuan-related ancestors).MethodsIn this study local ancestry was estimated across the genome in Polynesians (23 Tongan subjects) to find the candidate regions of admixture-enabled selection contributed by Papuan-related ancestors.ResultsThe mean proportion of Papuan-related ancestry across the Polynesian genome was estimated as 24.6% (SD = 8.63%), and two genomic regions, the extended major histocompatibility complex (xMHC) region on chromosome 6 and the ATP-binding cassette transporter sub-family C member 11 (ABCC11) gene on chromosome 16, showed proportions of Papuan-related ancestry more than 5 SD greater than the mean (> 67.8%). The coalescent simulation under the assumption of selective neutrality suggested that such signals of Papuan-related ancestry enrichment were caused by positive selection after admixture (false discovery rate = 0.045). The ABCC11 harbors a nonsynonymous SNP, rs17822931, which affects apocrine secretory cell function. The approximate Bayesian computation indicated that, in Polynesian ancestors, a strong positive selection (s = 0.0217) acted on the ancestral allele of rs17822931 derived from Papuan-related ancestors.ConclusionsOur results suggest that admixture with Papuan-related ancestors contributed to the rapid local adaptation of Polynesian ancestors. Considering frequent admixture events in human evolution history, the acceleration of local adaptation through admixture should be a common event in humans.

Tetrahedral Framework Nucleic Acids Reestablish Immune Tolerance and Restore Saliva Secretion in a Sjögren's Syndrome Mouse Model.

As one of the most frequent autoimmune diseases, Sjogren's syndrome (SS) is characterized by overactive lymphocytic infiltration in the exocrine glands, with ensuing dry mouth and dry eyes. Unfortunately, so far, there are no appropriate therapies without causing overall immunosuppression. Tetrahedral framework nucleic acids (tFNAs) were regarded as promising nanoscale materials whose immunomodulatory capabilities have already been verified. Herein, we reveal, for the first time, that tFNAs were utilized to treat SS in female nonobese diabetic (NOD) mice, the animal model used for SS. We proved a 250 nM tFNA treatment was successful in suppressing inflammation and stimulating saliva secretion in NOD mice. Specialised proteins for the secretory function and structure of acinar cells in submandibular glands (SMGs) were restored. It has been the permanent goal for SS treatment to establish immune tolerance and stop disease development. Surprisingly, tFNA treatment guided T cells toward regulatory T cells (Tregs), while suppressing T helper (Th) cell responses. Th cells include Th1, Th17, and follicular helper T (Tfh) cells. Tregs are highly significant in immune tolerance. Inducing Tregs is a promising approach to reestablish immune tolerance. Comparable results were also observed in B cell responses. Reductions in the percentage of germinal center (GC) B cells and plasma cells were detected, and a marked increase in the percentage of regulatory B cells (Bregs) was also noticed. The mechanisms of inducing Tregs may associated with cytokine changes. Changes of T cell subsets, especially changes of Tfh, may influence the differentiation of B cells accordingly. Collectively, our results demonstrated the immunomodulatory capacities of tFNAs once again, which may provide a novel, safe, and effective option for the treatment of SS and other autoimmune diseases.

Effect of Different Titanium Dental Implant Surfaces on Human Adipose Mesenchymal Stem Cell Behavior. An In Vitro Comparative Study

Background: The aim of this research was to evaluate the effects of three different titanium (Ti) implant surfaces on the viability and secretory functions of mesenchymal stem cells isolated from a Bichat fat pad (BFP-MSCs). Methods: Four different Ti disks were used as substrate: (I) D1: smooth Ti, as control; (II) D2: chemically etched, resembling the Kontact S surface; (III) D3: sandblasted, resembling the Kontact surface; (IV) D4: blasted/etched, resembling the Kontact N surface. BFP-MSCs were plated on Ti disks for 72 h. Cell viability, adhesion on disks and release of a panel of cytokines, chemokines and growth factor were evaluated. Results: BFP-MSCs plated in wells with Ti surface showed a viability rate (~90%) and proliferative rate comparable to cells plated without disks and to cells plated on D1 disks. D2 and D4 showed the highest adhesive ability. All the Ti surfaces did not interfere with the release of cytokines, chemokines and growth factors by BFP-MSCs. However, BFP-MSCs cultured on D4 surface released a significantly higher amount of Granulocyte Colony-Stimulating Factor (G-CSF) compared either to cells plated without disks and to cells plated on D1 and D2. Conclusions: The implant surfaces examined do not impair the BFP-MSCs cell viability and preserve their secretion of cytokines and chemokines. Further in vitro and in vivo studies are necessary to define the implant surface parameters able to assure the chemokines’ optimal release for a real improvement of dental implant osseointegration.

Vitamin D status determines the impact of metformin on circulating prolactin levels in premenopausal women.

Metformin was found to normalize secretory function of overactive pituitary cells. Its effect on circulating thyrotropin levels was more pronounced in women receiving exogenous vitamin D. The aim of the current study was to investigate whether vitamin D status determines the impact of metformin on prolactin levels in premenopausal women with hyperprolactinaemia. The study population consisted of three groups of women with prediabetes and elevated prolactin levels: vitamin D-naïve women with vitamin D insufficiency (group 1; n=19), women receiving vitamin D preparations because of vitamin D deficiency (group 2 n=20), as well as vitamin D-naïve women with normal vitamin D status (group 3 n=23). All participants were then treated with metformin (2.55-3g daily). Circulating levels of glucose, insulin, prolactin, thyrotropin, free thyroid hormones, gonadotropins, estradiol, calcium and 25-hydroxyvitamin were determined at baseline and six months later. At baseline, prolactin levels were higher in group 1 than in the remaining groups of patients. Although metformin decreased glucose levels and improved insulin sensitivity in all treatment groups, this effect was more pronounced in groups 2 and 3. Only in subjects with 25-hydroxyvitamin D levels within the reference range, metformin reduced prolactin levels. The impact on prolactin levels correlated with 25-hydroxyvitamin D levels and with the improvement in insulin sensitivity. The drug produced a neutral effect on circulating levels of thyrotropin, free thyroid hormones, gonadotropins, estradiol, calcium and 25-hydroxyvitamin D. The results of the current study suggest that the impact of metformin on secretory function of overactive lactotropes depends on the vitamin D status of patients.

117-LB: Glucokinase Activator Dorzagliatin (HMS5552) Regulates GLP-1 Release in T2D Patients and Is Synergistic with Sitagliptin and Empagliflozin in Optimizing Beta-Cell Function

Glucokinase (GK) is a glucose sensor and plays a central role in glucose homeostasis through regulation of threshold of hormone release from α and β cell that controls blood glucose set point. Based on this concept, Dorzagliatin as a glucose sensitizer for the treatment of diabetes has completed pivotal registration trial in China with desirable outcome. GLP-1 is an important hormone involved in β cell secretory function and such plays an important role in glucose homeostasis. It has been debated over the last decade on whether GK is involved in GLP-1 secretion. The dominant reports support the GLP-1 secretory organ is intestine L cell and the secreted hormone traveled to pancreas regulating β cell secreting function. Others suggested β cell regulates GLP-1 secretion from α cell through paracrine and is mediated through GLP-1 receptor on β cell. Here we report that a glucokinase activator Dorzagliatin stimulates GLP-1 release in T2D patients and is synergistic with Sitagliptin, a DDP-IV inhibitor, to increase circulating active GLP-1 in T2D patients with AUEC0-4h of 94.9% over Dorzagliatin. Increase of total GLP-1 was observed in the Dorzagliatin treatment with AUEC0-4h increase of 103.6% and Cmax increase of 141% compared with Dorzagliatin in combination with Sitagliptin. The effect on β cell function is also seen in patients taking Dorzagliatin together with Sitagliptin and Empagliflozin, a SGLT-2 inhibitor, in the OGTT study with increased C-peptide secretion AUEC0-4h of 46.6% (p &amp;lt; 0.01) and 20.4%, Cmax of 52.5% (p &amp;lt; 0.01) and 22%, respectively. Furthermore, the C-peptide secretion increased in combination treatment with AUEC0-4h of 30.3% (p &amp;lt; 0.05) and Cmax of 31%(p &amp;lt; 0.01), compared with Empagliflozin alone. In both combination studies with Sitagliptin and Empagliflozin, reduction of glucose AUEC0-4h is 33.1% and 37.6% with reduction glucose Cmax of 13.9% and 27.8% over Sitagliptin and Empagliflozin alone. Disclosure L. Chen: Board Member; Self; Hua Medicine. J. Zhang: Employee; Self; Hua Medicine. R. Yang: Employee; Self; Hua Medicine. L. Feng: Employee; Self; Hua Medicine. Hua medicine: n/a.

163-OR: Evidence for Circadian Control of Glucose-Stimulated Insulin Secretion in Transplanted Human Stem-Cell-Derived Pancreatic ß-Cells

Cell replacement strategies utilizing stem cell-derived β-cells (SC-β cells) hold therapeutic potential for patients with diabetes. In order for cell therapy to be effective, transplanted SC-β cells must recapitulate physiological features of insulin secretion in humans. In this regard, circadian regulation of insulin release is critical for normal β cell physiology, where intrinsic β cell circadian clocks function to optimize glucose-stimulated insulin secretion (GSIS) during the active/feeding phase of the circadian cycle. Thus, to elucidate whether transplanted SC-β cells retain capacity for circadian control of insulin secretion, SC-β cells were generated using a six-step directed differentiation protocol and transplanted (n=13 independent transplants) under the kidney capsule into immunocompromised (SCID-beige) mice. Prior to transplantation, differentiated SC-β cells were confirmed to express core circadian clock genes (e.g.,BMAL1, CLOCK, PER1 and REV-ERBα). Provided this information, we next assessed in vivo glucose tolerance and corresponding insulin secretory response (using human insulin ELISA) in SCID-beige mice during the inactive/sleep and active/feeding circadian cycles. Both parameters demonstrated robust diurnal rhythmicity (p&amp;lt;.05, inactive vs. active) with markedly enhanced glucose tolerance and in vivo SC-β cell secretory function evident during active/feeding circadian phase in mice. Notably, among 13 independent transplantations, ~90% of mice demonstrated statistically significant GSIS in response to intraperitoneal glucose injection during active/feeding compared to ~30% during inactive/sleep circadian phase. Our results provide evidence that transplanted SC-β cells retain circadian control of insulin secretion which should be taken into consideration for assessment and design of future β cell replacement strategies. Disclosure S. K. Sen: None. S. Hassoun: None. Q. P. Peterson: Other Relationship; Self; Semma Therapeutics, Inc. A. Matveyenko: None. Funding National Institutes of Health (R01DK098468 to A.M.)

Pathological Hyperinsulinemia and Hyperglycemia in the Impaired Glucose Tolerance Stage Mediate Endothelial Dysfunction Through miR-21, PTEN/AKT/eNOS, and MARK/ET-1 Pathways.

BackgroundImpaired glucose tolerance (IGT) is an important prediabetic stage characterized by elevated concentrations of glucose and insulin in the blood. The pathological hyperglycemia and hyperinsulinemia in IGT may regulate the expression of microRNA-21 (miR-21) and affect the downstream insulin signaling pathways, leading to endothelial cell dysfunction and early renal damage.MethodsThe individual and combined effects of insulin and glucose were investigated using human glomerular endothelial cells (HGECs). The expression levels of miR-21, and PTEN/AKT/eNOS and MAPK/ET-1 pathway proteins in the treated cells were measured. The levels of nitric oxide (NO) and endothelin-1 (ET-1) secreted by the cells were also measured. The role of miR-21 in mediating the regulatory effects of insulin and glucose was assessed by overexpression/inhibition of this miRNA using mimics/inhibitor.ResultsHigh (>16.7 mmol/L) concentration of glucose upregulated the expression of miR-21, leading to the activation and inhibition of the PTEN/AKT/eNOS and MAPK/ET-1 pathways, and upregulation of NO and downregulation of ET-1 secretion, respectively. High (>25 ng/mL) concentration of insulin downregulated the expression of miR-21, and lead to the activation of the MAPK/ET-1 and inhibition of the PTEN/AKT/eNOS pathway, thereby upregulating the expression of ET-1 and downregulating the secretion of NO. MiR-21 was observed to play a key role by directly controlling the activation of the insulin signaling pathways when the cells were cotreated with different concentrations of insulin and glucose. The expression of miR-21 was found to be dependent on the relative concentration of insulin and glucose. Under simulated conditions of the IGT stage (8.3 mmol/L glucose + 50 ng/mL insulin), the inhibitory effect of high insulin concentration on miR-21 expression in the cells attenuated the activation by high glucose concentration, resulting in the downregulation of miR-21, upregulation of ET-1 and downregulation of NO secretion.ConclusionTaken together, these results indicate that high insulin and glucose concentrations regulate the secretory function of glomerular endothelial cells in opposite ways by regulating the expression of miRNA-21. Pathological concentrations of insulin and glucose in the IGT stage may lead to a decrease in miR-21 expression, thereby disordering the secretion of vasoactive factors, resulting in renal tubule ischemia.

Resistance exercise training improves glucose homeostasis by enhancing insulin secretion in C57BL/6 mice

Resistance exercise exerts beneficial effects on glycemic control, which could be mediated by exercise-induced humoral factors released in the bloodstream. Here, we used C57Bl/6 healthy mice, submitted to resistance exercise training for 10 weeks. Trained mice presented higher muscle weight and maximum voluntary carrying capacity, combined with reduced body weight gain and fat deposition. Resistance training improved glucose tolerance and reduced glycemia, with no alterations in insulin sensitivity. In addition, trained mice displayed higher insulinemia in fed state, associated with increased glucose-stimulated insulin secretion. Islets from trained mice showed reduced expression of genes related to endoplasmic reticulum (ER) stress, associated with increased expression of Ins2. INS-1E beta-cells incubated with serum from trained mice displayed similar pattern of insulin secretion and gene expression than isolated islets from trained mice. When exposed to CPA (an ER stress inducer), the serum from trained mice partially preserved the secretory function of INS-1E cells, and prevented CPA-induced apoptosis. These data suggest that resistance training, in healthy mice, improves glucose homeostasis by enhancing insulin secretion, which could be driven, at least in part, by humoral factors.

Attenuated lncRNA NKILA Enhances the Secretory Function of Airway Epithelial Cells Stimulated by Mycoplasma pneumoniae via NF-κB.

The secretory function of airway epithelial cells is important in the pathogenesis of Mycoplasma pneumoniae pneumonia (MPP). To investigate the regulatory function of NKILA (nuclear factor-κB (NF-κB) interacting long noncoding RNA (lncRNA)) in MPP, we first detected NKILA as well as the concentration of interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid of children with MPP. Then, NKILA was knocked down in epithelial cells to investigate its effect on their secretory function. The results suggested that NKILA was downregulated in children with MPP, while IL-8 and TNF-α levels increased. Knockdown of NKILA in vitro promoted the inflammatory effects of Mycoplasma pneumoniae (MP) in epithelial A549 and BEAS-2B cells. Knockdown of NKILA promoted inhibitor of κBα (IκBα) phosphorylation and degradation, and NF-κB p65 nuclear translocation. Furthermore, RNA immunoprecipitation showed that NKILA could physically bind to IκBα in MP-treated A549 cells. Collectively, our data demonstrated that attenuation of NKILA enhances the effects of MP-stimulated secretory functions of epithelial cells via regulation of NF-κB signaling.

ОЦЕНКА ВЛИЯНИЯ ТЕРАПИИ ИНКРЕТИНОМИМЕТИКАМИ И ГЛИФЛОЗИНАМИ НА ФУНКЦИЮ АЛЬФА И БЕТА-КЛЕТОК ПОДЖЕЛУДОЧНОЙ ЖЕЛЕЗЫ У ПАЦИЕНТОВ С СД 2 ТИПА

Defect in the function of pancreatic cells is one of the key pathophysiological factors in type 2 diabetes, in particular, an imbalance between the secretion of insulin and glucagon, as well as a decrease in the efficiency of converting proinsulin into insulin, often observed. The aim of the work was to compare the characteristics of the secretory function of pancreatic alpha and beta cells in 43 patients with decompensated type 2 diabetes mellitus when using new classes of anti-diabetic drugs: glucagon-like peptide receptors agonists (GLP1ra) and inhibitors of the sodium-glucose transporter-2 (SGLT2i). Long-term treatment with these drugs (for 12 months) contributed to the normalization of the HbA1c level and a decrease in the % of visceral fat, as well as a decrease in the glucagon level compared to the baseline level. In both groups of patients, subgroups with high and low proinsulin levels have been identified. Under the influence of GLP1ra therapy, in patients of the low proinsulin subgroup, serum concentration of proinsulin and C-peptide significantly decreased, indicating a decrease in insulin secretion and a possible reduce in adaptive release of proinsulin as a result of glycemia decrease. Among patients treated with SGLT-2i, high pre-treatment proinsulin levels associated with higher glucagon and C-peptide concentrations comparing the low proinsulin subgroup. After 12 months of treatment, no significant changes in the concentration of glucagon, C-peptide and proinsulin were observed in both subgroups of patients treated with SGLT2i.