Factors controlling massive green tide blooms on the coasts of Jeju Island, Korea.

To determine the driving mechanisms between submarine groundwater discharge (SGD) and massive blooms of the green alga Ulva, we examined the magnitude of SGD and SGD-derived nutrient fluxes from November 2019 to July 2020 in Ihotewoo (north) and Bangdu (east) bays, Jeju Island, Korea. The variability of SGD flux at both the sites, where the hydraulic gradient is low, closely followed the daily variability of the tidal range, indicating that SGD flux is primarily driven by tidal pumping. Although the average annual SGD-driven nutrient fluxes were 24-37 % lower in Bangdu Bay than in Ihotewoo Bay, massive Ulva blooms only occurred in Bangdu Bay. A longer residence time (poor water exchange) and continuous SGD input with high dissolved inorganic nitrogen play a significant role in the growth and extentsion of Ulva blooms.

Origins and characteristics of dissolved organic matter fueling harmful dinoflagellate blooms revealed by δ13C and D/L-Amino acid compositions.

We measured the concentrations of dissolved inorganic and organic nutrients, dissolved organic carbon (DOC), total hydrolyzable amino acids (THAA), fluorescent dissolved organic matter (FDOM), phytoplankton pigments, and δ13C-DOC during the summer of 2019 in the harmful dinoflagellate bloom regions of the southern coast of Korea. In the harmful dinoflagellate bloom region, the concentrations of inorganic nitrogen were depleted, inhibiting the growth of diatoms, while the concentrations of dissolved organic components (nutrients, DOC, FDOM, and amino acids) which fuel dinoflagellates were unusually high. Thus, we attempted to investigate the origins and characteristics of DOM which fuels the harmful dinoflagellate blooms. The δ13C-DOC values (- 22.2‰ to - 18.2‰) indicate that the elevated DOC concentrations result from in-situ biological production rather than terrestrial inputs. The enantiomeric (D/L) ratios of THAA indicate that dissolved organic nitrogen was more labile in the early stage of harmful dinoflagellate bloom and became more refractory in the final stage. Our results suggest that the marine production of bioavailable DOM plays an important role in initiating and sustaining harmful dinoflagellate blooms.

Unique microbial module regulates the harmful algal bloom (Cochlodinium polykrikoides) and shifts the microbial community along the Southern Coast of Korea.

Harmful algal blooms (HABs) of Cochlodinium (aka Margalefidinium) polykrikoides cause huge economic and ecological damages and thus are considered environmental problems. Previous studies uncovered that the formation and collapse of phytoplankton blooms could be closely related to their associated microbes although their roles in C. polykrikoides bloom have not been elucidated yet. To explore the potential interactions between C. polykrikoides and other microbes (archaea, bacteria, and phytoplankton), we collected water samples in the free-living (FL) (0.22 to 3 µm), nanoparticle-associated (NP) (3 to 20 µm), and microparticle-associated (MP) (>20 µm) fractions when C. polykrikoides blooms occurred from July to August in 2016, 2017, and 2018 in the South Sea of Korea. The microbial composition of the C. polykrikoides-associated microbial cluster (Module I) significantly differed from those of other modules associated with Alexandrium, Chaetoceros or Chattonella. Over half of the interspecies interactions in Module I occurred within the module. That is, specific microbial clusters were associated with the C. polykrikoides bloom. Structural equation modeling (SEM) further confirmed the stronger effects of Module I on C. polykrikoides blooms compared to environmental factors. Among the operational taxonomic units (OTUs) directly correlated with C. polykrikoides, Marine Group I was presumed to supply vitamin B12, the essential element for C. polykrikoides growth, while the potential fish pathogens (Micrococcaceae and Piscirickettsiaceae) could contribute to the massive fish death together with C. polykrikoides itself. In addition, the zoospores of Syndiniales, a parasitoid to dinoflagellates, might be related to the sudden collapse of C. polykrikoides blooms. These microbial groups also contributed to significant alterations of the local microbial community structures. Collectively, network analysis and SEM revealed that the C. polykrikoides bloom is concomitant with distinct microbial communities, contributing to the rise and fall of the bloom, and finally determining the local microbial community structures.

Can algicide (the thiazolidinedione derivative TD49) truly contribute to the restoration of microbial communities?

Harmful algal blooms (HABs) are becoming a more serious ecological threat to marine environments; they not only produce toxins, resulting in the death of marine organisms, but they also adversely affect biodiversity, which is an indicator of the health of an ecosystem. Thus, to mitigate HABs, numerous studies have been conducted to develop an effective algicide, but few studies have elucidated the effect of algicides on marine environmental health. In this study, thiazolidinedione derivative 49 (TD49), which has been developed as an algicide for the dinoflagellate Heterocapsa circularisquama, was used, and we investigated changes in phytoplankton biomass (abundance, chlorophyll a, and carbon biomass) and biodiversity (diversity, evenness, and richness) following the application of TD49. To gain deeper understanding, a large-scale mesocosm (1300 L) experiment containing control and treatment with four different concentrations (0.2, 0.4, 0.6 and 1 µM) was conducted for 10 days. Based on a previous study, TD49 shows algicidal activity against H. circularisquama depending on its concentration. The phytoplankton biomass in the TD49 treatments was generally lower than that in the control due to the algicidal effect of TD49 on H. circularisquama. The biodiversity indices (e.g., the Shannon-Weaver index) in the treatments were consistently higher than those in the control before depletion of nitrite + nitrate. Interestingly, the 0.6 µM TD49 treatment had higher biodiversity indices than the high-concentration treatment (1 µM), which appeared to show a better algicidal effect. These findings suggest that mitigation of H. circularisquama blooms with TD49 treatment may enhance phytoplankton biodiversity, but treatment with excessively high concentrations can lead to harmful effects. During the study period, regardless of the control and TD49 treatments, the total biomass of phytoplankton gradually decreased from the midpoint of the experiment to the end of the experiment. This was more likely caused by the depletion of nutrients than by the toxicity of TD49.

Prevention and treatment effect of evogliptin on hepatic steatosis in high-fat-fed animal models.

Dipeptidyl peptidase 4 (DPP4) is an adipokine that interrupts insulin signaling. The resulting insulin resistance exacerbates hepatic steatosis. We previously reported that the novel DPP4 inhibitor evogliptin improves insulin resistance. This study aimed to verify the therapeutic potential of evogliptin for fatty liver. Evogliptin treatment was initiated simultaneously with a high-fat diet (HFD) feeding in normal mice and in a post-24 week HFD-fed rats. In a prevention study, insulin sensitivity was preserved in evogliptin-treated mice after a 16-week treatment. Overall plasma lipid levels stayed lower and hepatic lipid accumulation was drastically suppressed by evogliptin treatment. Evogliptin reduced hepatic expression of Srebf1, a key transcriptional factor for lipogenesis. Additionally, DPP4 inhibitor-treated mice showed less weight gain. In a treatment study, after evogliptin treatment for 14 weeks in pre-established HFD-fed obese rats, weight loss was marginal, while hepatic lipid accumulation and liver damage assessed by measuring plasma aminotransferase levels were completely resolved, suggesting weight loss-independent beneficial effects on fatty liver. Moreover, reduction in plasma non-esterified fatty acids supported the improvement of insulin resistance by evogliptin treatment. Conclusively, our findings suggest that evogliptin treatment ameliorates fatty liver by increasing insulin sensitivity and suppressing lipogenesis.

Hepatic role in an early glucose-lowering effect by a novel dipeptidyl peptidase 4 inhibitor, evogliptin, in a rodent model of type 2 diabetes.

Although multiple dipeptidyl peptidase 4 (DPP4) inhibitors have shown glucose-lowering effects by preserving pancreatic cells in high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice, the hepatic role in regulation of glucose homeostasis by DPP4 inhibitors in HFD/STZ mice remains elusive. In herein study, parallel comparison of effects on the liver (expression of gluconeogenic genes and the linked signaling molecules) and pancreas (islet morphology and relative area of alpha or beta cells) in combination with glucose-lowering effects were made at the end of 2- and 10-week of evogliptin treatment in HFD/STZ mice. Significant control of hyperglycemia was observed from the second week and persisted during 10-week treatment of 0.3% evogliptin in HFD/STZ mice. This effect was accompanied by increased level of plasma glucagon-like peptide-1 and preserved pancreas islet structure. Furthermore, the hepatic increases in gluconeogenic gene expression in HFD/STZ mice was significantly reduced by evogliptin treatment, which was accompanied by the suppression of cAMP response element-binding protein (CREB) phosphorylation and expression of transducer of regulated CREB protein 2. This hepatic effect of evogliptin treatment was reproduced in 2-week study, however, pancreatic beta-cell area was not altered yet although the expression of pancreatic and duodenal homeobox protein 1 was increased. We conclude that the suppression of hepatic gluconeogenesis by evogliptin is followed by preservation of pancreatic islet, leading to remarkable and persistent glucose-lowering effect in HFD/STZ mice. Our findings provide further insight for the hepatic role in DPP4 inhibitor-mediated glucose control in diabetes.

Beneficial Effects of Evogliptin, a Novel Dipeptidyl Peptidase 4 Inhibitor, on Adiposity with Increased Ppargc1a in White Adipose Tissue in Obese Mice.

Although dipeptidyl peptidase 4 (DPP4) is an adipokine known to positively correlate with adiposity, the effects of pharmacological DPP4 inhibition on body composition have not been fully understood. This study was aimed to assess the effects of DPP4 inhibitors on adiposity for the first time in the established obese mice model. The weight loss effects of multiple DPP4 inhibitors were compared after a 4 week treatment in diet-induced obese mice. In addition, a 2 week study was performed to explore and compare the acute effects of evogliptin, a novel DPP4 inhibitor, and exenatide, a glucagon-like peptide-1 (GLP-1) analogue, on whole body composition, energy consumption, various plasma adipokines and gene expression in white adipose tissue (WAT). After the 4 week treatment, weight loss and blood glucose reductions were consistently observed with multiple DPP4 inhibitors. Moreover, after 2-week treatment, evogliptin dose-dependently reduced whole body fat mass while increasing the proportion of smaller adipocytes. However, insulin sensitivity or plasma lipid levels were not significantly altered. In addition to increased active GLP-1 levels by plasma DPP4 inhibition, evogliptin also enhanced basal metabolic rate without reduction in caloric intake, in contrast to exenatide; this finding suggested evogliptin's effects may be mediated by pathways other than via GLP-1. Evogliptin treatment also differentially increased Ppargc1a expression, a key metabolic regulator, in WAT, but not in skeletal muscle and brown adipose tissue. The increased expression of the downstream mitochondrial gene, Cox4i1, was also suggestive of the potential metabolic alteration in WAT by DPP4 inhibitors. We are the first to demonstrate that pharmacological DPP4 inhibition by evogliptin directly causes fat loss in established obese mice. In contradistinction to exenatide, the fat-loss effect of DPP4 inhibitor is partly attributed to enhanced energy expenditure along with metabolic changes in WAT. These results provide insight into the regulation of energy storage in WAT caused by DPP4 inhibition.

Effects of the algicide, thiazolidinedione derivative TD49, on microbial communities in a mesocosm experiment.

We investigated the effects of the algicide thiazolidinedione derivative TD49 on microbial community in mesocosm experiments. The TD49 concentration exponentially decreased over time, with half-life of 3.5 h, following addition in the seawater (R2=0.98, P<0.001). Among microbial communities, heterotrophic bacteria and heterotrophic nanoflagellates (HNFs) grew well in all treatments following the addition of TD49. The abundance of HNFs lagged behind the increase in heterotrophic bacteria by 24 h in the 0.2 and 0.4 µM TD49 concentrations (R2=0.28, P<0.05), and by 48 h in the 0.6 and 1.0 µM TD49 concentrations (R2=0.30, P<0.05). This implies a strong concentration-dependent top-down effect of TD49 on microbial communities, with indications that the degradation of planktonic organisms, including the target alga, led to high heterotrophic bacteria concentrations, which in turn stimulated the population growth of predatory HNF. However, total ciliate numbers remained relatively low in the TD49 treatments relative to the control and blank groups, suggesting limited carbon flow from bacteria to these grazers even though the abundance of aloricate ciliates gradually increased toward the end of the experimental period, particularly at the high TD49 concentrations. TD49 appears to provide an environmentally safe approach to the control of harmful algal blooms (HABs) in aquatic ecosystems.

Proteomic analysis of INS-1 rat insulinoma cells: ER stress effects and the protective role of exenatide, a GLP-1 receptor agonist.

Beta cell death caused by endoplasmic reticulum (ER) stress is a key factor aggravating type 2 diabetes. Exenatide, a glucagon-like peptide (GLP)-1 receptor agonist, prevents beta cell death induced by thapsigargin, a selective inhibitor of ER calcium storage. Here, we report on our proteomic studies designed to elucidate the underlying mechanisms. We conducted comparative proteomic analyses of cellular protein profiles during thapsigargin-induced cell death in the absence and presence of exenatide in INS-1 rat insulinoma cells. Thapsigargin altered cellular proteins involved in metabolic processes and protein folding, whose alterations were variably modified by exenatide treatment. We categorized the proteins with thapsigargin initiated alterations into three groups: those whose alterations were 1) reversed by exenatide, 2) exaggerated by exenatide, and 3) unchanged by exenatide. The most significant effect of thapsigargin on INS-1 cells relevant to their apoptosis was the appearance of newly modified spots of heat shock proteins, thimet oligopeptidase and 14-3-3ß, ε, and θ, and the prevention of their appearance by exenatide, suggesting that these proteins play major roles. We also found that various modifications in 14-3-3 isoforms, which precede their appearance and promote INS-1 cell death. This study provides insights into the mechanisms in ER stress-caused INS-1 cell death and its prevention by exenatide.

Differential protective effects of exenatide, an agonist of GLP-1 receptor and Piragliatin, a glucokinase activator in beta cell response to streptozotocin-induced and endoplasmic reticulum stresses.

BACKGROUND: Agonists of glucagon-like peptide-1 receptor (GLP-1R) and glucokinase activators (GKA) act as antidiabetic agents by their ability protect beta cells, and stimulate insulin secretion. Oxidative and endoplasmic reticulum (ER) stresses aggravate type 2 diabetes by causing beta cell loss. It was shown that GLP-1R agonists protect beta cells from oxidative and ER stresses. On the other hand, little is known regarding how GKAs protect beta cells. We hypothesized that GKAs protect beta cells by mechanisms distinct from those underlying GLP-1R agonist and tested our hypothesis by comparing the molecular effects of exenatide, a GLP-1R agonist, and piragliatin, a GKA, on INS-1 cells under oxidative and ER-induced stresses. METHODS: BETA CELLS WERE TREATED WITH STREPTOZOTOCIN (STZ) TO INDUCE OXIDATIVE STRESS AND WITH PALMITATE OR THAPSIGARGIN (TG) TO INDUCE ER STRESS RESPECTIVELY, AND THE EFFECTS OF EXENATIDE AND PIRAGLIATIN ON THESE CELLS WERE INVESTIGATED BY: a) characterizing the kinases involved employing specific kinase inhibitors, and b) by identifying the differentially regulated proteins in response to stresses with proteomic analysis. RESULTS: Exenatide protected INS-1 cells from both ER and STZ-induced death. In contrast, piragliatin rescued the cells only from STZ-induced stress. Akt activation by exenatide appeared to contribute to its protective effects of beta cells while enhanced glucose utilization was the contributing factor in the case of piragliatin. Also, exenatide, not piragliatin, blocked changes in proteins 14-3-3ß, ε and θ, and preserved the 14-3-3θ levels under the ER stress. Isoform-specific modifications of 14-3-3, and the reduction of 14-3-3θ, commonly associated with beta cell death were assessed. CONCLUSIONS: Exenatide and piragliatin exert distinct effects on beta cell survival and thus on type 2 diabetes. This study which confirmed our hypothesis is also the first to observe specific modulation of 14-3-3 isoform in stress-induced beta cell death associated with progressive deterioration of type 2 diabetes.

Effects of chemically enhanced water-accommodated fraction of Iranian heavy crude oil on periphytic microbial communities in microcosm experiment.

Exposure experiments were conducted to determine the effects of an oil spill on a periphytic microbial community with the chemically enhanced water-accommodated fraction (CEWAF) of Iranian Heavy Crude oil in a microcosm containing artificial benthic substrates. Bacteria and heterotrophic nano-flagellates (HNF) grew well in all the treatments, except for the 100% CEWAF exposure. However, periphyton did not adapt to concentrations of CEWAF ≥ 20%. Among the periphyton, Cylindrotheca spp. dominated under treatment conditions, and the response of Cylindrotheca spp. to CEWAF (i.e., particularly 10%) closely followed the changes in chlorophyll a concentration. The concentrations of petroleum hydrocarbons from 10% and 20% CEWAF seemed to have a negative effect on periphyton and a growth-promoting effect on bacteria and HNF, respectively.

Two small molecule agonists of glucagon-like peptide-1 receptor modulate the receptor activation response differently.

The glucagon-like peptide-1 receptor (GLP-1R) is a target for type 2 diabetes treatment. Due to the inconvenience of peptide therapeutics, small-molecule GLP-1R agonists have been studied. Compound 2 (6,7-dichloro-2-methylsulfonyl-2-N-tert-butylaminoquinoxaline) and compound B (4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine) have been described as small molecule, ago-allosteric modulators of GLP-1R. However, their modes of action at the GLP-1R have not been elucidated. Thus, in this study, we compared the mechanisms of action between these two compounds. When compound 2 was treated with endogenous or exogenous peptide agonists (GLP-1 and exenatide) or fragments of peptide agonists (GLP-1(9-36), Ex3, Ex4, and Ex5), the response curve of these peptide agonists shifted left without a change in maximum efficacy. In contrast, compound B potentiated the response and increased maximum efficacy. However, N-terminal truncated orthosteric antagonists including Ex7, Ex9, and Ex10, augmented the response of compound 2 at the GLP-1R but did not alter compound B activity. Intriguingly, when we co-treated compound 2 with compound B in CHO cells expressing full-length hGLP-1R or N-terminal extracellular domain-truncated GLP-1R, the activation of both types of receptors increased additively, implying that the N-terminus of the receptor is not involved in the modulation by compound agonists. We confirmed that these two compounds increased calcium influx by different patterns in CHO cells expressing GLP-1R. Taken together, our findings suggest that compounds 2 and B have different modes of action to activate GLP-1R. Further study to identify the putative binding sites will help in the discovery of orally available GLP-1R agonists.

DA-1229, a novel and potent DPP4 inhibitor, improves insulin resistance and delays the onset of diabetes.

AIM: To characterize the pharmacodynamic profile of DA-1229, a novel dipeptidyl peptidase (DPP) 4 inhibitor. MAIN METHODS: Enzyme inhibition assays against DPP4, DPP8 and DPP9. Antidiabetic effects of DA-1229 in HF-DIO mice and young db/db mice. KEY FINDINGS: DA-1229 was shown to potently inhibit the DPP4 enzyme in human and murine soluble forms and the human membrane-bound form with IC(50) values of 0.98, 3.59 and 1.26 nM, respectively. As a reversible and competitive inhibitor, DA-1229 was more selective to human DPP4 (6000-fold) than to human DPP8 and DPP9. DA-1229 (0.1-3mg/kg) dose-dependently inhibited plasma DPP4 activity, leading to increased levels of plasma GLP-1 and insulin, and thereby lowering blood glucose levels in mice. In high fat diet-fed (HF) mice, a single oral dose of 100mg/kg of DA-1229 reduced plasma DPP4 activity by over 80% during a 24h period. Long-term treatment with DA-1229 for 8 weeks revealed significant improvements in glucose intolerance and insulin resistance, accompanied by significant body weight reduction. However, it remains unclear whether there is a direct causal relationship between DPP4 inhibition and body weight reduction. In young db/db mice, the DA-1229 treatment significantly reduced blood glucose excursions for the first 2 weeks, resulting in significantly lower levels of HbA1c at the end of the study. Furthermore, the pancreatic insulin content of the treatment group was significantly higher than that of the db/db control. SIGNIFICANCE: DA-1229 as a novel and selective DPP4 inhibitor improves the insulin sensitivity in HF mice and delays the onset of diabetes in young db/db mice.

Glucose exposure pattern determines glucagon-like peptide 1 receptor expression and signaling through endoplasmic reticulum stress in rat insulinoma cells.

Repeated fluctuation in plasma glucose levels, as well as chronic hyperglycemia, is an important phenomenon frequently observed in diabetic patients. Recently, several studies have reported that glucose fluctuation, compared to chronic hyperglycemia, mediates more adverse effects due to induced oxidative and/or endoplasmic reticulum (ER) stress. In type 2 diabetes, stimulation of insulin secretion by glucagon-like peptide-1 (GLP-1) has been found to be reduced, and the results of recent studies have shown that the expression of the GLP-1 receptor (GLP-1R) is reduced by chronic hyperglycemia. However, GLP-1R signaling in glucose fluctuation has not been elucidated clearly. In this study, we hypothesized that intermittent high glucose (IHG) conditions also reduced GLP-1-mediated cellular signaling via reduction in GLP-1R expression. To evaluate this hypothesis, rat insulinoma cells (INS-1) were exposed for 72 h to either sustained high glucose (SHG) conditions (30 mM glucose) or IHG conditions (11 and 30 mM glucose, alternating every 12h). In comparison to both the SHG and control groups, IHG conditions induced a more significant impairment of insulin release and calcium influx in response to 1nM GLP-1 treatment. In addition, the activity of caspase 3/7 as well as the gene expression of binding protein (Bip) and C/EBP homologous protein (CHOP), molecular markers of ER stress, was significantly higher in IHG-treated cells than in SHG-treated cells. Interestingly, the expression level of GLP-1R was significantly lower under IHG conditions than under SHG conditions. Collectively, these findings indicated that glucose fluctuation reduces GLP-1R expression through ER stress more profoundly than sustained hyperglycemia, which may contribute to the diminished response of GLP-1.

The development of non-peptide glucagon-like peptide-1 receptor agonist for the treatment of type 2 diabetes.

Glucagon-like peptide-1 (GLP-1) is the main member of the incretin family and stimulates insulin secretion by binding with its specific receptor on pancreatic ß-cells. In addition, GLP-1 exerts broad beneficial effects on the glucose regulation by suppressing food intake and delaying stomach emptying. Now, long acting GLP-1 analogs including exenatide and liraglutide have been approved for the treatment of diabetes mellitus type 2, however long-term injection can limit their use for these chronic patients. In this report, the authors provide a review on the development of non-peptide GLP-1 receptor agonists and introduce a novel agonist DA-15864.

PAM-1616, a selective peroxisome proliferator-activated receptor γ modulator with preserved anti-diabetic efficacy and reduced adverse effects.

Peroxisome proliferator-activated receptor (PPAR) γ is known to be a key regulator of insulin resistance. PAM-1616 is a novel, non-thiazolidinedione small molecule compound synthesized in Dong-A Research Center. In this study, we characterized the pharmacological and safety profiles of PAM-1616 as a selective PPARγ modulator. PAM-1616 selectively binds to human PPARγ (IC(50), 24.1±5.6 nM) and is a partial agonist for human PPARγ with an EC(50) of 83.6±43.7 nM and a maximal response of 24.9±7.1% relative to the full agonist, rosiglitazone. PAM-1616 was selective for human PPARγ than for human PPARα (EC(50), 2658±828 nM) without activating human PPARδ, which makes it a selective modulator of PPARγ. Treatment of high fat diet-induced obese C57BL/6J mice with PAM-1616 for 21 days improved HOMA-IR. Furthermore, PAM-1616 significantly improved hyperglycemia in db/db mice with little side effect when orally administered at a dose of 1 mg/kg/day for 28 days. Intriguingly, PAM-1616 was seen to increase the gene expression of inducible glucose transporter (GLUT4), while it partially induced that of a fatty acid carrier, aP2 in 3T3-L1 adipocytes, and it also showed partial recruitment of an adipogenic cofactor, TRAP220 as compared to rosiglitazone. PAM-1616 did not cause a significant increase in plasma volume of ICR mice when orally administered at a dose of 10 mg/kg/day for 9 days. PAM-1616 increased the expression of fluid retention-inducing genes such as serum/glucocorticoid-regulated kinase (SGK)-1 to a lesser extent as compared to rosiglitazone in human renal epithelial cells. These results suggest that PAM-1616 acts as a selective modulator of PPARγ with excellent antihyperglycemic property. The differential modulation of target gene by PAM-1616 might contribute to the improved side effect profiles.