Weight Change Alters the Small RNA Profile of Urinary Extracellular Vesicles in Obesity.

INTRODUCTION: Various kidney diseases reportedly show different urinary extracellular vesicle (EV) RNA profiles. Although obesity is one of the main causes of chronic kidney disease, the expression pattern of urinary EV RNA in obesity is uncertain. Our aim was to sequence the small RNA profiles of urinary EVs in obese patients before and after weight reduction and compare them to those of healthy volunteers (HVs). METHODS: We recruited age-sex-matched obese patients and HVs. The small RNA profiles of urinary EVs were analyzed using RNA sequencing. To evaluate the effect of weight reduction, small RNA profiles of urinary EVs 6 months after bariatric surgery were also analyzed. RESULTS: The proportion of urinary EVs transfer RNA and microRNA of obese patients differed from that of HVs. Obese patients showed differential expression of 1,343 small RNAs in urinary EVs compared to HVs (fold change ≥2 and p value <0.05). Among those, 61 small RNAs were upregulated in obese patients and downregulated after weight reduction, whereas 167 small RNAs were downregulated in obese patients and upregulated after weight reduction. RNA sequencing revealed the correlation between the specific urinary EV small RNAs and clinical parameters including body weight, low-density lipoprotein cholesterol, triglyceride, high-density lipoprotein cholesterol, serum glucose, estimated glomerular filtration rate, and albuminuria. CONCLUSION: Obese patients showed distinct urinary EV small RNA profiles compared to HVs. Weight reduction altered urinary EV small-RNA profiles in obese patients.

Metabolic Subtyping of Adrenal Tumors: Prospective Multi-Center Cohort Study in Korea.

BACKGROUND: Conventional diagnostic approaches for adrenal tumors require multi-step processes, including imaging studies and dynamic hormone tests. Therefore, this study aimed to discriminate adrenal tumors from a single blood sample based on the combination of liquid chromatography-mass spectrometry (LC-MS) and machine learning algorithms in serum profiling of adrenal steroids. METHODS: The LC-MS-based steroid profiling was applied to serum samples obtained from patients with nonfunctioning adenoma (NFA, n=73), Cushing's syndrome (CS, n=30), and primary aldosteronism (PA, n=40) in a prospective multicenter study of adrenal disease. The decision tree (DT), random forest (RF), and extreme gradient boost (XGBoost) were performed to categorize the subtypes of adrenal tumors. RESULTS: The CS group showed higher serum levels of 11-deoxycortisol than the NFA group, and increased levels of tetrahydrocortisone (THE), 20α-dihydrocortisol, and 6ß-hydroxycortisol were found in the PA group. However, the CS group showed lower levels of dehydroepiandrosterone (DHEA) and its sulfate derivative (DHEA-S) than both the NFA and PA groups. Patients with PA expressed higher serum 18-hydroxycortisol and DHEA but lower THE than NFA patients. The balanced accuracies of DT, RF, and XGBoost for classifying each type were 78%, 96%, and 97%, respectively. In receiver operating characteristics (ROC) analysis for CS, XGBoost, and RF showed a significantly greater diagnostic power than the DT. However, in ROC analysis for PA, only RF exhibited better diagnostic performance than DT. CONCLUSION: The combination of LC-MS-based steroid profiling with machine learning algorithms could be a promising one-step diagnostic approach for the classification of adrenal tumor subtypes.

Status of Diabetic Neuropathy in Korea: A National Health Insurance Service-National Sample Cohort Analysis (2006 to 2015).

This report presents the status of diabetic neuropathy (DN) in Korea as determined using a National Health Insurance ServiceNational Sample Cohort (NHIS-NSC). Annual prevalences of DN were estimated by age and gender using descriptive statistics. Pharmacological treatments for DN were also analyzed. The annual prevalence of DN increased from 24.9% in 2006 to 26.6% in 2007, and thereafter, gradually subsided to 20.8% in 2015. In most cases, pharmacological treatments involved a single drug, which accounted for 91.6% of total prescriptions in 2015. The most commonly used drugs (in decreasing order) were thioctic acid, an anti-convulsive agent, or a tricyclic antidepressant. In conclusion, the prevalence of DN decreased over the 10-year study period. Thioctic acid monotherapy was usually prescribed for DN. To reduce the socio-economic burden of DN, more attention should be paid to the diagnosis of this condition and to the appropriate management of patients.

Infliximab partially alleviates the bite force reduction in a mouse model of temporomandibular joint pain.

Temporomandibular joint (TMJ) disorder is clinically important because of its prevalence, chronicity, and therapy-refractoriness of the pain. In this study, we investigated the effect of infliximab in a mouse model of TMJ pain using a specially-engineered transducer for evaluating the changes in bite force (BF). The mice were randomly divided into three groups (7 mice per group): the control group, the complete Freund's adjuvant (CFA) group, and the infliximab group. BF was measured at day 0 (baseline BF). After measuring the baseline BF, CFA or incomplete Freund's adjuvant was injected into both TMJs and then the changes in BF were measured at days 1, 3, 5, 7, 9, and 13 after the TMJ injection. For measuring the BF, we used a custom-built BF transducer. Control, CFA, and infliximab groups showed similar baseline BF at day 0. From day 1, a significant reduction in BF was observed in the CFA group, and this reduction in BF was statistically significant compared to that in the control group (P < 0.05). This reduction in BF was maintained until day 7, and BF started to recover gradually from day 9. In the infliximab group also, the reduction in BF was observed on day 1, and this reduction was maintained until day 7. However, the degree of reduction in BF was less remarkable compared to that in the CFA group. The reduction in BF caused by injection of CFA into the TMJ could be partially alleviated by the injection of anti-tumor necrosis factor alpha, infliximab.

Melatonin prevents pancreatic ß-cell loss due to glucotoxicity: the relationship between oxidative stress and endoplasmic reticulum stress.

Prolonged hyperglycemia results in pancreatic ß-cell dysfunction and apoptosis, referred to as glucotoxicity. Although both oxidative and endoplasmic reticulum (ER) stresses have been implicated as major causative mechanisms of ß-cell glucotoxicity, the reciprocal importance between the two remains to be elucidated. The aim of this study was to evaluate the differential effect of oxidative stress and ER stress on ß-cell glucotoxicity, by employing melatonin which has free radical-scavenging and antioxidant properties. As expected, in ß-cells exposed to prolonged high glucose levels, cell viability and glucose-stimulated insulin secretion (GSIS) were significantly impaired. Melatonin treatment markedly attenuated cellular apoptosis by scavenging reactive oxygen species via its plasmalemmal receptor-independent increase in antioxidant enzyme activity. However, treatments with antioxidants alone were insufficient to recover the impaired GSIS. Interestingly, 4-phenylbutyric acid (4-PBA), a chemical chaperone that attenuate ER stress by stabilizing protein structure, alleviated the impaired GSIS, but not apoptosis, suggesting that glucotoxicity induces oxidative and ER stress independently. We found that cotreatment of glucotoxic ß-cells with melatonin and 4-PBA dramatically improved both their survival and insulin secretion. Taken together, these results suggest that ER stress may be the more critical mechanism for prolonged high-glucose-induced GSIS impairment, whereas oxidative stress appears to be more critical for the impaired ß-cell viability. Therefore, combinatorial therapy of melatonin with an ER stress modifier may help recover pancreatic ß-cells under glucotoxic conditions in type 2 diabetes.

TNF-α polymorphisms and coronary artery disease: association study in the Korean population.

Coronary artery disease (CAD) results from atherosclerosis, a chronic inflammatory disease mediated in part by proinflammatory cytokines, particularly tumor necrosis factor-α (TNF-α), which is expressed by atherosclerotic plaques. In this study, we investigated whether TNF-α gene promoter polymorphisms affect the incidence of CAD in Koreans by genotyping. 404 Control subjects and 197 patients who previously received a coronary artery stent for the G/A, C/T, and C/A polymorphisms at position -238, -857 and -863, respectively. The G/G, G/A and A/A genotypes at position -238 occurred in 85.8%, 14.2% and 0% CAD patients and 91.8%, 7.9% and 0.3% control subjects, respectively. The G/A polymorphisms at position -238 were significantly associated with CAD when assuming a dominant model of inheritance (OR = 1.87; 95% CI = 1.10-3.20; P = 0.02), and A allele carriers had a significantly increased risk of developing CAD relative to the G allele (OR = 1.74; 95% CI = 1.04-2.92; P = 0.03). However, the polymorphisms at positions -857 and -863 were not associated with CAD. Haplotype-based analysis revealed the CAD and control groups differed significantly in the frequencies of haplotype ACC at positions -238, -857 and -863 (OR = 1.77; 95% CI = 1.05-2.98; P = 0.03). This was confirmed by multivariate analysis after adjusting body mass index and the presence of diabetes and hypertension (OR = 2.06; 95% CI = 1.15-3.68; P = 0.015). Thus, the -238A allele of TNF-α is associated with an increased risk of CAD and could be used as predictor for CAD in Koreans. Further studies are needed to elucidate the clinical implications of these findings.

Reconstruction of defects after excision of facial skin cancer using a venous free flap.

After extensive excision of skin cancer on the face, or when skin cancer is located on the 3-dimensional structures of the face, reconstruction with a local flap can be impossible, or clinicians are reluctant to reconstruct defects with a skin graft because of postoperative contraction, hyperpigmentation, or other complication. Instead, an arterialized venous free flap can be used as an alternative method of reconstruction to prevent distortion and recurrence. Eight patients underwent surgery with an arterialized venous-free flap. We evaluated the cosmetic results using ordinary scale methods on the basis of 4 categories (color, contour, texture, and distortion of surrounding structures) and recurrence and metastases of skin cancer physically. The follow-up period ranged between 24 and 48 months, with an average of 33 months. All of the soft-tissue defects made by excising the tumor were reconstructed with good outcomes, except for 1 case. Regarding the cosmetic evaluation, the color was fair, the contour and texture were good, absence of distortion of surrounding structures was excellent, and the overall results in most all cases were good. There were no recurrences or metastases during the follow-up period. The arterialized venous free flap is an alternative plan among several reconstruction methods when skin cancer on the face is extensively excised.

A novel in vitro pancreatic carcinogenesis model.

Environmental factors (e.g., BaP) have been pointed out as one of the etiologies of pancreatic cancer. However, very limited experimental assays are available to identify pancreatic specific environmental mutagens or susceptibility genes. In this study, we have developed a simple in vitro cell culture model system that can be used to study the molecular and biochemical aspects of carcinogenesis in a near-normal immortalized pancreatic ductal epithelial cell lines. In order to demonstrate that xenobiotic stress response is intact in these cells, we employed standard molecular biology techniques. For examples, luciferase reporter and/or real-time quantitative PCR assays were used to determine stress-induced CYP1A1 and CYP1B1 gene expression. Western blotting and immunocytochemistry assays were used to demonstrate that TCDD or BaP could activate AhR signaling. For exploring the carcinogenesis mechanism, we incubated cells with [³H]BaP and determined BaP-DNA binding activity by measuring its radioactivity. BaP-DNA adduct formation was further confirmed by [³²P]-postlabeling assay. Finally, we demonstrated the effects of endogenous AhR or BRCA1 in BaP-DNA adduct accumulation in our cell system. As results, no apparent BaP-DNA adduct accumulation by [³²P]-postlabeling assay was found in either control-siRNA or AhR-siRNA pretreated cells. On the other hand, a significant increase of BaP-DNA adduct accumulation was found in BRCA1 knockdown cells. In conclusion, we suggest that this in vitro model may provide the feasibility for future studies on the molecular basis of pancreatic ductal cell carcinogenesis caused by dietary mutagens.

The Association between Serum GGT Concentration and Diabetic Peripheral Polyneuropathy in Type 2 Diabetic Patients.

BACKGROUND: Diabetic peripheral polyneuropathy (DPP) is one of the common complications of diabetes mellitus (DM) and can lead to foot ulcers or amputation. The pathophysiology of DPP includes several factors such as metabolic, vascular, autoimmune, oxidative stress and neurohormonal growth-factor deficiency and recent studies have suggested the use of serum gamma-glutamyl transferase (GGT) as an early marker of oxidative stress. Therefore, we investigated whether serum GGT may be useful in predicting DPP. METHODS: We assessed 90 patients with type 2 DM who were evaluated for the presence of DPP using clnical neurologic examinations including nerve conduction velocity studies. We evaluated the association between serum GGT and the presence of DPP. RESULTS: The prevalence of DPP was 40% (36 cases) according to clinical neurological examinations. The serum GGT concentration was significantly elevated in type 2 diabetic patients with DPP compared to patients without DPP (P < 0.01). There were other factors significantly associated with DPP including smoking (P = 0.019), retinopathy (P = 0.014), blood pressure (P < 0.05), aspartate aminotransferase (P = 0.022), C-reactive protein (P = 0.036) and urine microalbumin/creatinine ratio (P = 0.004). Serum GGT was independently related with DPP according to multiple logistic analysis (P < 0.01). CONCLUSION: This study shows that increased levels of serum GGT may have important clinical implications in the presence of DPP in patients with type 2 diabetes.

Glucagon-like peptide-1 protects NSC-34 motor neurons against glucosamine through Epac-mediated glucose uptake enhancement.

Bioenergetic deficits are considered a common cause of neurodegenerative diseases. Although creatine supplementation has been shown to be effective in certain neurodegenerative disorders, it is less effective in amyotrophic lateral sclerosis, a disease that primarily affects motor neurons. These neurons are particularly vulnerable to a cellular energy deficit. Using the ATP-depleting drug glucosamine, we evaluated whether the incretin hormone glucagon-like peptide (GLP)-1 protects motor neurons against glucosamine-induced cytotoxicity. Undifferentiated NSC-34 cells were differentiated into glutamate-sensitive motor neurons by a modified serum deprivation technique. Glucosamine inhibited the viability of differentiated NSC-34 cells in a time- and dose-dependent manner. Glucosamine also acutely reduced cellular glucose uptake, glucokinase activity and intracellular ATP levels. As a result, the activity of AMP-activated protein kinase as well as endoplasmic reticulum stress increased. Pretreatment with GLP-1 significantly alleviated glucosamine-mediated neurotoxicity by restoring cellular glucose uptake, glucokinase activity and intracellular ATP levels. The protective effect of GLP-1 was replicated by Exendin-4 but not Exendin-9, and not blocked by inhibitors of phosphoinositide-3 kinase, protein kinase A, cSrc, or epidermal growth factor receptor, but it was blocked by an adenylate cyclase inhibitor. A selective activator for exchange proteins directly activated by cAMP (Epac), but not a selective activator for protein kinase A, mimicked the GLP-1 effect. Therefore GLP-1 may exert its effect mainly through cAMP-dependent, Epac-mediated restoration of glucose uptake that is typically impaired by glucosamine. These findings indicate that GLP-1 could be employed therapeutically to protect motor neurons that are susceptible to bioenergetic deficits.

Carbamylated albumin stimulates microRNA-146, which is increased in human renal cell carcinoma.

Carbamylation is a post-translational modification, the pathophysiological consequences of which remain poorly understood. MicroRNAs (miRNAs) are endogenous non-coding small ribonucleic acids that have emerged as one of the central players in gene expression regulation. This study was designed to determine the effect of carbamylated albumin (cAlb) on the expression of miRNAs. Albumin was carbamylated, and the extent of carbamylation was monitored using trinitrobenzenesulphonic acid. Albumin or cAlb were added to rat mesangial cells (RMCs), and RNA was extracted. miRNA microarray analysis was performed. The expression of microRNA-146a (miR-146a) and microRNA-146b (miR-146b) was analyzed by real-time RT-PCR. Of 365 miRNAs analyzed, the expression of miR-146a/b was found to be markedly induced by cAlb (miR-146a, 12.75-fold increase; miR-146b, 5.88-fold increase). Real-time RT-PCR analysis confirmed the increased levels of miR-146a/b by cAlb (p<0.05). It was also found that expression levels of miR-146a/b were increased in renal cell carcinoma tumor tissues compared to corresponding non-tumor tissues (p<0.05). Our data suggest that cAlb stimulates miR-146a/b in RMCs, the levels of which are increased in renal cell carcinoma. Further studies on the function of cAlb may provide new insights into the pathophysiology of renal cell carcinoma.

Down-regulation of IL-6, IL-8, TNF-α and IL-1ß by glucosamine in HaCaT cells, but not in the presence of TNF-α

There is considerable evidence that glucosamine exerts an inhibitory effect on inflammatory cytokine expression in cells. Glucosamine has been recommended as a promising anti-inflammatory modulator, which has been applied in clinical trials for attenuation of the inflammatory process. However, it is unknown whether glucosamine reduces the expression of TNF-α-induced inflammatory cytokines in HaCaT cells. The anti-inflammatory effects of curcumin in HaCaT cells have been extensively investigated in several studies. Thus, in this study we investigated the expression of IL-6, IL-8, TNF-α and IL-1ß in glucosamine-treated HaCaT cells, and the effects of glucosamine were compared to those of curcumin-treated HaCaT cells. Our data showed that the expression of IL-6, IL-8, TNF-α and IL-1ß was decreased by glucosamine treatment in the HaCaT cells. In contrast, the expression of IL-6, IL-8, TNF-α and IL-1ß was not attenuated by glucosamine treatment in the TNF-α-treated HaCaT cells. Notably, curcumin induced an increased expression of IL-8 and IL-1ß in the HaCaT cells, but not that of IL-6 and TNF-α. On the other hand, curcumin attenuated the expression of IL-6 and IL-8 in the TNF-α-treated HaCaT cells. Our data indicated that glucosamine induced the down-regulation of IL-6, IL-8, TNF-α and IL-1ß expression in the HaCaT cells. However, the stimulation of TNF-α abolished the inhibitory effects of glucosamine on the expression of inflammatory cytokines in the HaCaT cells. Thus, even though glucosamine induces the down-regulation of inflammatory cytokines in HaCaT cells, the anti-inflammatory role of glucosamine in TNF-α-mediated inflammatory skin diseases should be investigated.

Forkhead transcription factor FoxO1 inhibits insulin- and transforming growth factor-beta-stimulated plasminogen activator inhibitor-1 expression.

Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are considered a risk factor for chronic liver disease in patients with hyperinsulinemia. Insulin increases the expression of PAI-1, and inactivates the forkhead box-containing protein FoxO1. We were interested in whether the inactivation of FoxO1 is involved in the activation of PAI-1 expression under conditions of insulin stimulation. Here, we examined whether adenoviral-mediated expression of a constitutively active form of FoxO1 (Ad-CA-FoxO1) inhibited insulin-stimulated PAI-1 expression in human HepG2 hepatocellular liver carcinoma cells and mouse AML12 hepatocytes. Treatment of cells with insulin increased PAI-1 gene expression, and this effect was abolished by Ad-CA-FoxO1. Insulin also increased the transforming growth factor (TGF)-beta-induced expression of PAI-1 mRNA, and Ad-CA-FoxO1 inhibited this effect. Transient transfection assays using a reporter gene under the control of the PAI-1 promoter revealed that CA-FoxO1 inhibits Smad3-stimulated PAI-1 promoter activity. Taken together, our results indicate that FoxO1 inhibits PAI-1 expression through the inhibition of TGF-beta/Smad-mediated signaling pathways. Our data also suggest that in the hyperinsulinemic state, FoxO1 is inactivated by increased levels of insulin, and does not function as an inhibitor of TGF-beta-induced PAI-1 expression.

Uncoupling by (--)-epigallocatechin-3-gallate of ATP-sensitive potassium channels from phosphatidylinositol polyphosphates and ATP.

Of green tea catechins, (--)-epigallocatechin-3-gallate (EGCG) and (--)-epicatechin-3-gallate (ECG), but not (--)-epicatechin and (--)-epigallocatechin, inhibit the activity of ATP-sensitive potassium (K(ATP)) channels at tens of micromolar concentrations, ECG being three times more effective than EGCG. Further, we found that by using cloned beta cell-type K(ATP) channels, only EGCG at 1 microM, a readily achievable plasma concentration by oral intake in humans, but not other epicatechins, significantly blocked channel reactivation after ATP wash-out, suggesting that interaction of phosphatidylinositol polyphosphates (PIP) with the channel was impaired by EGCG. In addition, a 10-fold higher concentration of EGCG reduced the channel sensitivity to ATP, but not AMP and ADP. This effect of EGCG was greater in the channel with the sulfonylurea receptor (SUR) than with the inwardly rectifying K(+) channel (Kir6.2) alone. Neomycin, a polycation, profoundly suppressed the effect of EGCG. Expectedly, glucose-stimulated cytosolic Ca(2+) elevation in rat pancreatic beta cells, and insulin secretory responses to high glucose loading in vivo were impaired by EGCG. In rabbit cardiac myocytes, dinitrophenol-induced opening of the channel was delayed by 1 microM EGCG. These results suggest that EGCG may interact with PIP-binding sites on the Kir6.2 subunit. SUR further endows EGCG with an ability to interfere with an interaction of the gamma-phosphate tail of ATP with Kir6.2. The specificity of EGCG possibly implies that 5'-OH of the B-ring on the pyrogallol moiety in the EGCG molecule may be critical for these actions of EGCG on the K(ATP) channel.