Imeglimin attenuates NLRP3 inflammasome activation by restoring mitochondrial functions in macrophages.

Imeglimin is a novel oral antidiabetic drug for treating type 2 diabetes. However, the effect of imeglimin on NLRP3 inflammasome activation has not been investigated yet. Here, we aimed to investigate whether imeglimin reduces LPS-induced NLRP3 inflammasome activation in THP-1 macrophages and examine the associated underlying mechanisms. We analyzed the mRNA and protein expression levels of NLRP3 inflammasome components and IL-1ß secretion. Additionally, reactive oxygen species (ROS) generation, mitochondrial membrane potential, and mitochondrial permeability transition pore (mPTP) opening were measured by flow cytometry. Imeglimin inhibited NLRP3 inflammasome-mediated IL-1ß production in LPS-stimulated THP-1-derived macrophages. In addition, imeglimin reduced LPS-induced mitochondrial ROS production and mitogen-activated protein kinase phosphorylation. Furthermore, imeglimin restored the mitochondrial function by modulating mitochondrial membrane depolarization and mPTP opening. We demonstrated for the first time that imeglimin reduces LPS-induced NLRP3 inflammasome activation by inhibiting mPTP opening in THP-1 macrophages. These results suggest that imeglimin could be a promising new anti-inflammatory agent for treating diabetic complications.

Empagliflozin is associated with lower cardiovascular risk compared with dipeptidyl peptidase-4 inhibitors in adults with and without cardiovascular disease: EMPagliflozin compaRative effectIveness and SafEty (EMPRISE) study results from Europe and Asia.

BACKGROUND: Studies that have reported lower risk for cardiovascular outcomes in users of Sodium-Glucose Cotransporter-2 Inhibitors (SGLT-2i) are limited by residual cofounding and lack of information on prior cardiovascular disease (CVD). This study compared risk of cardiovascular events in patients within routine care settings in Europe and Asia with type 2 diabetes (T2D) initiating empagliflozin compared to dipeptidyl peptidase-4 inhibitors (DPP-4i) stratified by pre-existing CVD and history of heart failure (HF). METHODS AND RESULTS: Adults initiating empagliflozin and DPP-4i in 2014-2018/19 from 11 countries in Europe and Asia were compared using propensity score matching and Cox proportional hazards regression to assess differences in rates of primary outcomes: hospitalisation for heart failure (HHF), myocardial infarction (MI), stroke; and secondary outcomes: cardiovascular mortality (CVM), coronary revascularisation procedure, composite outcome including HHF or CVM, and 3-point major adverse cardiovascular events (MACE: MI, stroke and CVM). Country-specific results were meta-analysed and pooled hazard ratios (HR) with 95% confidence intervals (CI) from random-effects models are presented. In total, 85,244 empagliflozin/DPP4i PS-matched patient pairs were included with overall mean follow-up of 0.7 years. Among those with pre-existing CVD, lower risk was observed for HHF (HR 0.74; 95% CI 0.64-0.86), CVM (HR 0.55; 95% CI 0.38-0.80), HHF or CVM (HR 0.57; 95% CI 0.48-0.67) and stroke (HR 0.79; 95% CI 0.67-0.94) in patients initiating empagliflozin vs DPP-4i. Similar patterns were observed among patients without pre-existing CVD and those with and without pre-existing HF. CONCLUSION: These results from diverse patient populations in routine care settings across Europe and Asia demonstrate that initiation of empagliflozin compared to DPP-4i results in favourable cardioprotective effects regardless of pre-existing CVD or HF status.

Amphiregulin Induces iNOS and COX-2 Expression through NF-κB and MAPK Signaling in Hepatic Inflammation.

Background: Inflammation is a major cause of hepatic tissue damage and accelerates the progression of nonalcoholic fatty liver disease (NAFLD). Amphiregulin (AREG), an epidermal growth factor receptor ligand, is associated with human liver cirrhosis and hepatocellular carcinoma. We aimed to investigate the effects of AREG on hepatic inflammation during NAFLD progression, in vivo and in vitro. Methods: AREG gene expression was measured in the liver of mice fed a methionine choline-deficient (MCD) diet for 2 weeks. We evaluated inflammatory mediators and signaling pathways in HepG2 cells after stimulation with AREG. Nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were analyzed using an enzyme-linked immunosorbent assay and western blotting. Nuclear transcription factor kappa-B (NF-κB) and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, were analyzed using western blotting. Results: Proinflammatory cytokines (interleukin (IL)-6, IL-1ß, and IL-8) and immune cell recruitment (as indicated by L3T4, F4/80, and ly6G mRNA expression) increased, and expression of AREG increased in the liver of mice fed the MCD diet. AREG significantly increased the expression of IL-6 and IL-1ß and the production of NO, PGE2, and IL-8 in HepG2 cells. It also activated the protein expression of iNOS and COX-2. AREG-activated NF-κB and MAPKs signaling, and together with NF-κB and MAPKs inhibitors, AREG significantly reduced the protein expression of iNOS and COX-2. Conclusion: AREG plays a role in hepatic inflammation by increasing iNOS and COX-2 expression via NF-κB and MAPKs signaling.

Specific Signal Transduction of Constitutively Activating (D576G) and Inactivating (R476H) Mutants of Agonist-Stimulated Luteinizing Hormone Receptor in Eel.

We investigated the mechanism of signal transduction using inactivating (R476H) and activating (D576G) mutants of luteinizing hormone receptor (LHR) of eel at the conserved regions of intracellular loops II and III, respectively, naturally occurring in mammalian LHR. The expression of D576G and R476H mutants was approximately 58% and 59%, respectively, on the cell surface compared to those of eel LHR-wild type (wt). In eel LHR-wt, cAMP production increased upon agonist stimulation. Cells expressing eel LHR-D576G, a highly conserved aspartic acid residue, exhibited a 5.8-fold increase in basal cAMP response; however, the maximal cAMP response by high-agonist stimulation was approximately 0.62-fold. Mutation of a highly conserved arginine residue in the second intracellular loop of eel LHR (LHR-R476H) completely impaired the cAMP response. The rate of loss in cell-surface expression of eel LHR-wt and D576G mutant was similar to the agonist recombinant (rec)-eel LH after 30 min. However, the mutants presented rates of loss higher than eel LHR-wt did upon rec-eCG treatment. Therefore, the activating mutant constitutively induced cAMP signaling. The inactivating mutation resulted in the loss of LHR expression on the cell surface and no cAMP signaling. These data provide valuable information regarding the structure-function relationship of LHR-LH complexes.

Hepatoprotective effects of gemigliptin and empagliflozin in a murine model of diet-induced non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) includes a broad spectrum of liver diseases characterized by steatosis, inflammation, and fibrosis. This study aimed to investigate the potential of dipeptidyl peptidase-4 inhibitors and sodium-glucose cotransporter 2 inhibitors in alleviating the progression of NAFLD. The NAFLD model was generated by feeding male C57BL/6J mice a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 7 weeks. After 2 weeks of CDAHFD feeding, the NAFLD model mice were assigned to four groups, namely (ⅰ) VEHICLE, (ⅱ) gemigliptin (GEMI), (ⅲ) empagliflozin (EMPA), and (ⅳ) GEMI + EMPA. For the next 5 weeks, mice received the vehicle or the drug based upon the group to which they belonged. Thereafter, the triglyceride concentration, extent of fibrosis, and the expression of genes encoding inflammatory cytokines, chemokines, and antioxidant enzymes were analyzed in the livers of mice. The NAFLD activity score and hepatic fibrosis grade were assessed via hematoxylin and eosin and Sirius Red staining of the liver tissue samples. All mice belonging to the GEMI, EMPA, and GEMI + EMPA groups showed improvements in the accumulation of liver triglycerides and the expression of inflammatory cytokines and chemokines. Additionally, the oxidative stress was reduced due to inhibition of the c-Jun N-terminal kinase pathway and upregulation of the antioxidant enzymes. Furthermore, in these three groups, the galectin-3 and interleukin 33-induced activity of tumor necrosis factor-α was inhibited, thereby preventing the progression of liver fibrosis. These findings suggest that the GEMI, EMPA, and GEMI + EMPA treatments ameliorate hepatic steatosis, inflammation, oxidative stress, and fibrosis in CDAHFD-induced NAFLD mouse models.

Insight into the molecular structure and function of peptidoglycan recognition protein SC2 (PGRP-SC2) from Amphiprion clarkii: Investigating the role in innate immunity.

Peptidoglycan recognition proteins (PGRPs) belong to the pattern recognition receptor (PRR) family and are conserved from insects to mammals. PGRPs show specific binding abilities to peptidoglycans (PGNs) in various microbes. In this study, molecular and functional analyses of PGRP-SC2 from Amphiprion clarkii (AcPGRP-SC2) were conducted. The 492 bp ORF of AcPGRP-SC2 encoded a protein of 164 amino acids with a molecular weight of 17.58 kDa and pI of 8.9. The PGRP superfamily domain was identified from the protein sequence of AcPGRP-SC2 and sequence similarities were observed with homologous proteins. Quantitative polymerase chain reaction (qPCR) analysis revealed that AcPGRP-SC2 transcripts were ubiquitously expressed in all tested tissues, with high levels in the skin, and transcript expression was significantly modulated by immune stimulation with lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (poly I:C), and Vibrio harveyi post-immune challenge. Recombinant AcPGRP-SC2 with the maltose-binding protein fusion (rAcPGRP-SC2) was used to evaluate LPS-, PGN-, and bacterial-binding activities and to conduct bacterial agglutination assays, and the results demonstrated that AcPGRP-SC2 exhibited bacterial recognition, binding, and colonization abilities to a range of Gram-positive and Gram-negative bacterial strains. Moreover, rAcPGRP-SC2-pre-treated Fat Head Minnow (FHM) cells exhibited significant upregulation in NF-ĸB1, NF-ĸB2, and stat3 expression upon treatment with killed bacteria. Taken together, our findings suggest that AcPGRP-SC2 plays an important role in the immune response against microbial pathogens in A. clarkii.

Recommendation on an updated standardization of serum magnesium reference ranges.

PURPOSE: Serum magnesium is the most frequently used laboratory test for evaluating clinical magnesium status. Hypomagnesemia (low magnesium status), which is associated with many chronic diseases, is diagnosed using the serum magnesium reference range. Currently, no international consensus for a magnesemia normal range exists. Two independent groups designated 0.85 mmol/L (2.07 mg/dL; 1.7 mEq/L) as the low cut-off point defining hypomagnesemia. MaGNet discussions revealed differences in serum magnesium reference ranges used by members' hospitals and laboratories, presenting an urgent need for standardization. METHODS: We gathered and compared serum magnesium reference range values from our institutions, hospitals, and colleagues worldwide. RESULTS: Serum magnesium levels designating "hypomagnesemia" differ widely. Of 43 collected values, only 2 met 0.85 mmol/L as the low cut-off point to define hypomagnesemia. The remainder had lower cut-off values, which may underestimate hypomagnesemia diagnosis in hospital, clinical, and research assessments. Current serum magnesium reference ranges stem from "normal" populations, which unknowingly include persons with chronic latent magnesium deficit (CLMD). Serum magnesium levels of patients with CLMD fall within widely used "normal" ranges, but their magnesium status is too low for long-term health. The lower serum magnesium reference (0.85 mmol/L) proposed specifically prevents the inclusion of patients with CLMD. CONCLUSIONS: Widely varying serum magnesium reference ranges render our use of this important medical tool imprecise, minimizing impacts of low magnesium status or hypomagnesemia as a marker of disease risk. To appropriately diagnose, increase awareness of, and manage magnesium status, it is critical to standardize lower reference values for serum magnesium at 0.85 mmol/L (2.07 mg/dL; 1.7 mEq/L).

Association of prediabetes with death and diabetic complications in older adults: the pros and cons of active screening for prediabetes.

BACKGROUND: active screening can improve health outcomes for people with prediabetes. However, its efficacy in older adults remains uncertain. OBJECTIVE: the study aimed to analyse the progression from prediabetes to diabetes in older adults, including associated complications and mortality rates, to determine the benefits of active screening. DESIGN: retrospective cohort study. SETTING: Korean National Health Insurance Service claims database. SUBJECTS: a total of 36,946 adults aged ≥65 years who underwent national health examinations from 2006 to 2008. METHODS: follow-up was until 31 December 2015. Cox's proportional hazards models estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for death and diabetic complications. RESULTS: over a median follow-up of 8.2 years, 19.3% of older adults with prediabetes progressed to diabetes. Compared with normoglycaemic adults, the multivariable-adjusted HRs (95% CI) for major adverse cardiovascular events were 1.01 (0.95-1.07), 1.08 (0.95-1.23), 1.14 (1.05-1.23) and 1.50 (1.35-1.68) in adults with prediabetes, newly diagnosed diabetes, diabetes not on insulin and diabetes on insulin, respectively. The corresponding HRs (95% CI) for diabetic retinopathy risk were 1.28 (1.15-1.43), 3.16 (2.71-3.69), 6.58 (6.02-7.19) and 9.17 (8.21-10.24). Diabetic nephropathy risk also significantly increased. CONCLUSIONS: progression from prediabetes to diabetes is an important concern among older adults. Prediabetes is associated with the risk of microvascular complications, but not cardiovascular complications and mortality. Therefore, active regular screening for prediabetes is necessary to prevent microvascular complications.

Comparative toxicity study of waterborne two booster biocides (CuPT and ZnPT) on embryonic flounder (Paralichthys olivaceus).

A new generation of booster biocides that include metal pyrithiones (PTs) such as copper pyrithione (CuPT) and zinc pyrithione (ZnPT) are being used as tributyltin alternatives. In the marine environment, ZnPT can easily transchelate Cu to form CuPT, and the environmental fate and persistence of these two metal pyrithiones are closely related. Although some data on the toxicity of biocides on marine fish are available, little is known about their toxicity and toxic pathway. We thus compared the toxic effects of CuPT and ZnPT on embryonic olive flounder (Paralichthys olivaceus) by investigating their adverse effects based on developmental morphogenesis and transcriptional variation. In our study, the toxic potency of CuPT was greater with respect to developmental malformation and mortality than ZnPT. Consistent with the developmental effects, the expression of genes related to tail fin malformation (including plod2, furin, and wnt3a) was higher in embryonic flounder exposed to CuPT than in those exposed to ZnPT. Genes related to muscle and nervous system development exhibited significant changes on differential gene expression profiles using RNA sequencing (cutoff value P < 0.05). Gene ontology analysis of embryos exposed to CuPT revealed affected cellular respiration and kidney development, whereas genes associated with cell development, nervous system development and heart development showed significant variation in embryonic flounder exposed to ZnPT. Overall, our study clarifies the common and unique developmental toxic effects of CuPT and ZnPT through transcriptomic analyses in embryonic flounder.

The N-Linked Glycosylation Site N191 Is Necessary for PKA Signal Transduction in Eel Follicle-Stimulating Hormone Receptor.

The follicle-stimulating hormone receptor (FSHR) contains several N-linked glycosylation sites in its extracellular region. We conducted the present study to determine whether conserved glycosylated sites in eel FSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction. We used site-directed mutagenesis to induce four mutations (N120Q, N191Q, N272Q, and N288Q) in the N-linked glycosylation sites of eel FSHR. In the eel FSHR wild-type (wt), the cAMP response was gradually increased in a dose-dependent manner (0.01-1500 ng/mL), displaying a high response (approximately 57.5 nM/104 cells) at the Rmax level. Three mutants (N120Q, N272Q, and N288Q) showed a considerably decreased signal transduction as a result of high-ligand treatment, whereas one mutant (N191Q) exhibited a completely impaired signal transduction. The expression level of the N191Q mutant was only 9.2% relative to that of the eel FSHR-wt, indicating a negligible expression level. The expression levels of the N120Q and N272Q mutants were approximately 35.9% and 24% of the FSHG-wt, respectively. The N288Q mutant had an expression level similar to that of the eel FSHR-wt, despite the mostly impaired cAMP responsiveness. The loss of the cell surface agonist-receptor complexes was very rapid in the cells expressing eel FSHR-wt and FSHR-N288Q mutants. Specifically, the N191Q mutant was completely impaired by the loss of cell surface receptors, despite treatment with a high concentration of the agonist. Therefore, we suggest that the N191 site is necessary for cAMP signal transduction. This finding implies that the cAMP response, mediated by G proteins, is directly related to the loss of cell surface receptors as a result of high-agonist treatment.

Hyperprogressive disease during PD-1 blockade in patients with advanced hepatocellular carcinoma.

BACKGROUND & AIMS: Programmed cell death-1 (PD-1) inhibitor treatment can cause hyperprogressive disease (HPD), but the incidence, outcome, and predictive factors of HPD are unknown in patients with hepatocellular carcinoma (HCC). Herein, we assessed the existence and factors predictive of HPD in patients with advanced HCC treated with nivolumab. METHODS: We enrolled 189 patients with advanced HCC treated with nivolumab. Occurrence of HPD was investigated using tumour growth dynamics based on tumour growth kinetics (TGK) and tumour growth rate (TGR) before and after treatment, or time to treatment failure. We additionally analysed patients treated with regorafenib (n = 95) or best supportive care (BSC)/placebo (n = 103) after progression on sorafenib to compare tumour growth dynamics. RESULTS: Flare-up of tumour growth was observed in a fraction of patients upon PD-1 blockade, indicating the occurrence of HPD. Based on distinct patterns of disease progression exclusively observed in the nivolumab-treated cohort, but not in the regorafenib- or BSC/placebo-treated cohorts, 4-fold increases in TGK and TGR ratios as well as a 40% increase in TGR were the cut-off values used to define HPD; 12.7% of the patients (24/189) treated with nivolumab met all these criteria. Patients with HPD had worse progression-free survival (hazard ratio [HR] 2.194; 95% CI 1.214-3.964) and overall survival (HR 2.238; 95% CI 1.233-4.062) compared to patients with progressive disease without HPD. More than 90% of patients with HPD missed the opportunity for subsequent treatment because of rapid clinical deterioration. An elevated neutrophil-to-lymphocyte ratio (>4.125) was associated with HPD and an inferior survival rate. CONCLUSIONS: HPD occurs in a fraction of patients with HCC who receive PD-1 inhibitor treatment. Analyses of the baseline immune profile and on-treatment tumour growth dynamics could enable optimal patient selection and earlier identification of HPD. LAY SUMMARY: Hyperprogressive disease is an unexpected response pattern observed in patients treated with an immune checkpoint inhibitor. This study revealed that hyperprogressive disease occurs in a fraction of patients with advanced hepatocellular carcinoma treated with an anti-PD-1 antibody, providing evidence to encourage careful monitoring of patients to prevent clinical deterioration induced by PD-1 blockade.

Cardiovascular outcomes with sodium-glucose cotransporter-2 inhibitors vs other glucose-lowering drugs in 13 countries across three continents: analysis of CVD-REAL data.

BACKGROUND: Randomized, controlled cardiovascular outcome trials may not be fully representative of the management of patients with type 2 diabetes across different geographic regions. We conducted analyses of data from the multinational CVD-REAL consortium to determine the association between initiation of sodium-glucose cotransporter-2 inhibitors (SGLT-2i) and cardiovascular outcomes, including subgroup analyses based on patient characteristics. METHODS: De-identified health records from 13 countries across three continents were used to identify patients newly-initiated on SGLT-2i or other glucose-lowering drugs (oGLDs). Propensity scores for SGLT-2i initiation were developed in each country, with 1:1 matching for oGLD initiation. In the matched groups hazard ratios (HRs) for hospitalization for heart failure (HHF), all-cause death (ACD), the composite of HHF or ACD, myocardial infarction (MI) and stroke were estimated by country, and pooled using a weighted meta-analysis. Multiple subgroup analyses were conducted across patient demographic and clinical characteristics to examine any heterogeneity in treatment effects. RESULTS: Following matching, 440,599 new users of SGLT-2i and oGLDs were included in each group. Mean follow-up time was 396 days for SGLT-2i initiation and 406 days for oGLDs initiation. SGLT-2i initiation was associated with a lower risk of HHF (HR: 0.66, 95%CI 0.58-0.75; p < 0.001), ACD (HR: 0.52, 95%CI 0.45-0.60; p < 0.001), the composite of HHF or ACD (HR: 0.60, 95%CI 0.53-0.68; p < 0.001), MI (HR: 0.85, 95%CI 0.78-0.92; p < 0.001), and stroke (HR: 0.78, 95%CI 0.72-0.85; p < 0.001); regardless of patient characteristics, including established cardiovascular disease, or geographic region. CONCLUSIONS: This CVD-REAL study extends the findings from the SGLT-2i clinical trials to the broader setting of an ethnically and geographically diverse population, and across multiple subgroups. Trial registration NCT02993614.

Effectiveness and safety of sodium-glucose co-transporter-2 inhibitors compared with dipeptidyl peptidase-4 inhibitors in older adults with type 2 diabetes: A nationwide population-based study.

AIM: To examine the real-world cardiovascular effectiveness and safety associated with sodium-glucose co-transporter-2 (SGLT2) inhibitor compared with dipeptidyl peptidase-4 (DPP-4) inhibitor treatment in older adults with type 2 diabetes. MATERIALS AND METHODS: In this retrospective cohort study, older adults with type 2 diabetes (aged ≥65 years) were identified in the Korean National Health Insurance Service database from September 2014 to December 2016. In total, 408 506 new users of an SGLT2 inhibitor or DPP-4 inhibitor were propensity score matched. Cox regression was used to estimate the hazard ratios (HR) and 95% confidence interval (CI) for outcomes of interest: hospitalization for heart failure (HHF), all-cause death, myocardial infarction, stroke, diabetic ketoacidosis (DKA), bone fracture, severe hypoglycaemia, genital infection and urinary tract infection (UTI). RESULTS: Compared with DPP-4 inhibitors, new users of SGLT2 inhibitors had a lower risk of HHF (HR 0.86; 95% CI 0.76-0.97), all-cause death (HR 0.85; 95% CI 0.75-0.98) and stroke (HR 0.86; 95% CI 0.77-0.97), but a similar risk of myocardial infarction (HR 0.95; 95% CI 0.77-1.19). The risks of DKA, bone fracture and severe hypoglycaemia were similar between both groups, although genital infection (HR 2.44; 95% CI 2.22-2.67) and UTI (HR 1.05; 95% CI 1.00-21.11) were more frequent among new users of SGLT2 inhibitors compared with DPP-4 inhibitors. CONCLUSION: Our findings suggest that initiation of SGLT2 inhibitors offers cardiovascular disease protection and can be used safely in older adults with type 2 diabetes.

Lower cardiorenal risk with sodium-glucose cotransporter-2 inhibitors versus dipeptidyl peptidase-4 inhibitors in patients with type 2 diabetes without cardiovascular and renal diseases: A large multinational observational study.

AIMS: We compared the new use of sodium-glucose cotransporter-2 inhibitor (SGLT2i) versus dipeptidyl peptidase-4 inhibitor (DPP4i) and the risk of cardiorenal disease, heart failure (HF) or chronic kidney disease (CKD), in patients with type 2 diabetes without a history of prevalent cardiovascular and renal disease, defined as cardiovascular and renal disease (CVRD) free, managed in routine clinical practice. MATERIALS AND METHODS: In this observational cohort study, patients were identified from electronic health records from England, Germany, Japan, Norway, South Korea and Sweden, during 2012-2018. In total, 1 006 577 CVRD-free new users of SGLT2i or DPP4i were propensity score matched 1:1. Unadjusted Cox regression was used to estimate hazard ratios (HRs) for outcomes: cardiorenal disease, HF, CKD, stroke, myocardial infarction (MI), cardiovascular and all-cause mortality. RESULTS: Baseline characteristics were well balanced between the treatment groups (n = 105 130 in each group) with total follow-up of 187 955 patient years. Patients had a mean age of 56 years, 43% were women and they were indexed between 2013 and 2018. The most commonly used agents were dapagliflozin (91.7% of exposure time) and sitagliptin/linagliptin (55.0%), in the SGLT2i and DPP4i, groups, respectively. SGLT2i was associated with lower risk of cardiorenal disease, HF, CKD, all-cause and cardiovascular mortality; HR (95% confidence interval), 0.56 (0.42-0.74), 0.71 (0.59-0.86), 0.44 (0.28-0.69), 0.67 (0.59-0.77), and 0.61 (0.44-0.85), respectively. No differences were observed for stroke [0.87 (0.69-1.09)] and MI [0.94 (0.80-1.11)]. CONCLUSION: In this multinational observational study, SGLT2i was associated with a lower risk of HF and CKD versus DPP4i in patients with type 2 diabetes otherwise free from both cardiovascular and renal disease.

Visfatin exacerbates hepatic inflammation and fibrosis in a methionine-choline-deficient diet mouse model.

BACKGROUND AND AIM: Non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to non-alcoholic steatohepatitis, which is characterized by hepatic inflammation that can progress to fibrosis, cirrhosis, and hepatocellular carcinoma. Visfatin, an adipocytokine, was reported to induce pro-inflammatory cytokines and can be associated with liver fibrosis. We investigated the role of visfatin on hepatic inflammation and fibrosis in a methionine-choline-deficient (MCD)-diet-induced steatohepatitis mouse model. METHODS: Eight-week-old male C57BL/6 J mice were randomly assigned into one of three groups: (1) saline-injected control diet group; (2) saline-injected MCD diet group; and (3) visfatin-injected MCD diet group (n = 8 per group). Mice were administered intravenous saline or 10 µg/kg of recombinant murine visfatin for 2 weeks. Histologic assessment of liver and biochemical and molecular measurements of endoplasmic reticulum (ER) stress, reactive oxidative stress (ROS), inflammation, and fibrosis were performed in livers from these animals. RESULTS: Visfatin injection aggravated hepatic steatosis and increased plasma alanine aminotransferase and aspartate aminotransferase concentrations. Visfatin increased inflammatory cell infiltration (as indicated by F4/80, CD68, ly6G, and CD3 mRNA expression) and expression of chemokines in the liver. Visfatin also increased the expression of pro-inflammatory cytokines (IL-1ß, TNF-α, and IL-6) and activated fibrosis markers (CTGF, TIMP1, collagen 1α2, collagen 3α2, αSMA, fibronectin, and vimentin) in liver. Livers of visfatin-injected mice showed upregulation of ER stress and ROS and activation of JNK signaling. CONCLUSIONS: These results suggest that visfatin aggravates hepatic inflammation together with induction of ER and oxidative stress and exacerbates fibrosis in an MCD-diet-fed mouse model of NAFLD.

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro.

The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

Acute Glucose Shift Induces the Activation of the NLRP3 Inflammasome in THP-1 Cells.

We aimed to investigate the effect of acute glucose shift on the activation of the NLRP3 inflammasome, IL-1ß secretion, and underlying signaling pathways in THP-1 cells. THP-1 cells were divided into four groups and exposed to the following glucose concentrations for 24 h: constant normal glucose (NG, 5.5 mM), constant high glucose (HG, 25 mM), normal to high glucose shift (NG-to-HG, 5.5 to 25 mM), and high to normal glucose shift (HG-to-NG, 25 to 5.5 mM). Cell viability, oxidative stress, and the levels of NLRP3 inflammasome components were assessed. Both directions of the acute glucose shift increased the activation of the NLRP3 inflammasome, generation of reactive oxygen species (ROS), and expression of phosphorylated p38 MAPK, JNK, and NF-κB compared with either constant NG or HG. Treatment with N-acetylcysteine, a pharmacological antioxidant, inhibited the acute glucose shift-induced generation of ROS, activation of NLRP3 inflammasome, and upregulation of MAPK-NF-κB. Further analysis using inhibitors of p38 MAPK, JNK, and NF-κB indicated that acute glucose shifts promoted IL-1ß secretion by activating the signaling pathway in a ROS-MAPK-NF-κB-NLRP3 inflammasome in THP-1 cells. These findings suggested that acute changes in glucose concentration might cause monocyte inflammation, which is associated with diabetic complications.

Body mass index and cardiovascular outcomes in patients with acute coronary syndrome by diabetes status: the obesity paradox in a Korean national cohort study.

BACKGROUND: The "obesity paradox" has not been elucidated in the long-term outcomes of acute coronary syndrome (ACS). We investigated the association between obesity and cardiovascular (CV) outcomes in ACS patients with and without diabetes. METHODS: We identified 6978 patients with ACS aged 40-79 years from the Korean National Health Insurance Service-Health Screening Cohort between 2002 and 2015. Baseline body mass index (BMI) was categorized as underweight (< 18.5 kg/m2), normal weight (18.5-22.9 kg/m2), overweight (23.0-24.9 kg/m2), obese class I (25.0-29.9 kg/m2), and obese class II (≥ 30.0 kg/m2). The primary outcome was major adverse CV events (MACE)-CV death, myocardial infarction (MI), and stroke. The secondary outcomes were the individual components of MACE, hospitalization for heart failure (HHF), and all-cause death. RESULTS: After adjustment for confounding variables, compared to normal-weight patients without diabetes (reference group), obese class I patients with and without diabetes had a lower risk of MACE, but only significant in patients without diabetes (with diabetes: hazard ratio [HR] 0.95, 95% confidence interval [CI] 0.78-1.14; without diabetes: HR 0.78, 95% CI 0.62-0.97). Obese class II patient with diabetes had a higher risk of MACE with no statistical significance (HR 1.14, 95% CI 0.82-1.59). Underweight patients with and without diabetes had a higher risk of MACE, but only significant in patients with diabetes (with diabetes: HR 1.79, 95% CI 1.24-2.58; without diabetes: HR 1.23, 95% CI 0.77-1.97). CONCLUSION: In ACS patients, obesity had a protective effect on CV outcomes, especially in patients without diabetes.

Time-varying and dose-dependent effect of long-term statin use on risk of type 2 diabetes: a retrospective cohort study.

BACKGROUND: We evaluated the effect of statin use on new-onset type 2 diabetes among individuals without atherosclerotic cardiovascular disease (ASCVD) using nationally representative South Korean claims data (2002-2013, N = 1,016,820). METHODS: A total of 13,698 patients (statin users 5273, non-statin users 5273) aged 40-74 years, newly diagnosed with dyslipidemia but without any history of diabetes or ASCVD, were selected in 2005. We followed up the final sample until 2013 and evaluated the cumulative incidence of type 2 diabetes. We used extended Cox regression models to estimate the time-varying adjusted hazard ratios of statin use on new-onset type 2 diabetes. We performed further analyses based on the cumulative defined daily dose of statin received per year to evaluate the degree of risk compared to non-statin users. RESULTS: Over the mean follow-up period of 7.1 years, 3034 patients developed type 2 diabetes; the number of statin users exceeded that of non-users, demonstrating that statin use significantly increased the risk of new-onset type 2 diabetes. The risk of new-onset type 2 diabetes differed among statin users according to cDDD per year (adjusted HR = 1.31 [95% CI 1.18-1.46] for less than 30 cDDD per year; 1.58 [1.43-1.75] for 30-120 cDDD per year; 1.83 [1.62-2.08] for 120-180 cDDD per year; and 2.83 [2.51-3.19] for more than 180 cDDD per year). The diabetogenic effect of pitavastatin was not statistically significant, but the risk was the largest for atorvastatin. Long-term exposure (≥ 5 years) to statins was associated with a statistically significant increase in the risk of new onset type 2 diabetes in all statin subtypes explored, with the highest magnitude for simvastatin (HR = 1.916, 95% CI 1.647-2.228) followed by atorvastatin (HR = 1.830, 95% CI 1.487-2.252). CONCLUSIONS: Statin use was significantly associated with an increased risk of new-onset type 2 diabetes. We also found a dose-response relationship in terms of statin use duration and dose maintenance. Periodic screening and monitoring for incident type 2 diabetes may be warranted in long-term statin users.

CCL20 induced by visfatin in macrophages via the NF-κB and MKK3/6-p38 signaling pathways contributes to hepatic stellate cell activation.

Chemokines interact with hepatic resident cells during inflammation and fibrosis. CC chemokine ligand (CCL) 20 has been reported to be important in inflammation and fibrosis in the liver. We hypothesized that visfatin, an adipocytokine, could play a role in hepatic fibrosis via CCL20. We investigated the effect of visfatin on CCL20 in THP-1 human promonocytic cells and examined the molecular mechanisms involved. Following treatment of THP-1 cells with visfatin, CCL20 expression and secretion were assessed. We assessed the intracellular signaling molecules IKK/NF-κB, JAK2/STAT3, MAPKs, and MKK3/6 by western blotting. We treated THP-1 cells with visfatin and signaling inhibitors, and examined CCL20 mRNA and protein levels. To investigate the effect of visfatin-induced CCL20 expression in hepatic stellate cells (HSCs), LX-2 cells were co-cultured with the culture supernatant of THP-1 cells with or without anti-CCL20 neutralizing antibodies, and fibrosis markers were examined by RT-PCR and immunoblotting. In THP-1 cells, visfatin increased the CCL20 mRNA and protein levels. visfatin increased the activities of the NF-κB, p38, and MLK3/6 signaling pathways but not those of the JAK2/STAT3 and ERK pathways. Visfatin treatment together with an NF-κB, p38, or MLK3 inhibitor reduced the mRNA and protein levels of CCL20. The visfatin-induced CCL20 increased the expression of fibrosis markers and CCR6 in HSCs. Following neutralization of CCL20, the levels of fibrosis markers and CCR6 were decreased. Visfatin increases the expression of CCL20 via the NF-κB and MKK3/6-p38 signaling pathways in macrophages, and visfatin-induced CCL20 expression promotes the fibrosis markers in HSCs.