Yeon Jin Jang

In Vivo Dual Effects of Vitamin C on Paraquat-Induced Lung Damage: Dependence on Released Metals from the Damaged Tissue

Vitamin C, a potent antioxidant, can act as a pro-oxidant in the presence of free transition metal ions by accelerating the Fenton reaction. An in vivo pro-oxidant role of vitamin C has been suggested, but direct evidence for it is scant. Here, we report the dual role of vitamin C on paraquat-induced lung injury, which appears to depend on the metal ions released from damaged cells. Vitamin C (10 mg/kg) given at the time when the extensive tissue damage was in progress aggravated the oxidative damage, while it protected against the damage when given before the initiation of the damage. The extent of oxidative tissue damage was monitored by measuring the expiratory ethane, one of the hydrocarbons produced during lipid peroxidation. Deferoxamine, given intraperitoneally as a bolus dose of 50 mg/kg, completely blocked the aggravation of oxidative damage by vitamin C. Moreover, deferoxamine unmasked the antioxidant effect of vitamin C. The results show that vitamin C can either aggravate or alleviate the oxidative tissue damage depending on the presence of metal ions released from damaged cells.

Effects of weight reduction on serum vaspin concentrations in obese subjects: modification by insulin resistance.

Visceral adipose tissue‐derived serpin (vaspin) has been regarded as a novel adipokine with potential insulin sensitizing properties. We investigated the changes of serum vaspin concentration in response to weight reduction, and the associations between changes in serum vaspin concentrations and changes of anthropometric and metabolic variables in obese subjects after weight reduction. We performed a longitudinal clinical intervention study on 63 obese persons enrolled in a 12‐week weight reduction program that included lifestyle modification and adjuvant treatment with the antiobesity agent orlistat. Anthropometric variables, lipid profiles, fasting glucose, fasting insulin, and serum vaspin concentrations were measured. Statistical analyses were performed according to the homeostasis model assessment of insulin resistance (HOMAIR). Serum vaspin concentrations decreased significantly in responders (≥2% reduction in baseline weight), but not in nonresponders (<2% reduction in baseline weight). Changes in serum vaspin concentrations were significantly correlated with body weight, BMI, waist circumference, and hip circumference in the higher, but not in the lower, HOMAIR group. In multivariate linear regression analysis, change in serum vaspin concentrations in the higher, but not in the lower, HOMAIR group was positively correlated with change in BMI and negatively correlated with initial HOMAIR level. The associations between changes in serum vaspin concentrations and changes in anthropometric and metabolic parameters differed according to insulin resistance status in obese subjects. These relationships were more prominent in the higher HOMAIR group. Insulin resistance may influence the correlations between changes in serum vaspin concentration and related metabolic variables.

Association between low SIRT1 expression in visceral and subcutaneous adipose tissues and metabolic abnormalities in women with obesity and type 2 diabetes

AIMS To assess the importance of adipose tissue sirtuin 1 (SIRT1) in the regulation of whole-body metabolism in humans with obesity and type 2 diabetes. METHODS In total, 19 non-diabetic obese women, 19 type 2 diabetic women undergoing gastric bypass surgery, and 27 normal-weight women undergoing gynecological surgery (total 65 women) were enrolled. Their anthropometric variables, abdominal fat distribution and metabolic parameters, serum adiponectin concentrations, and SIRT1 mRNA and protein and adiponectin mRNA expressions in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were measured. RESULTS SIRT1 mRNA levels in VAT and SAT were similar and these levels were suppressed in obese and type 2 diabetic women compared to normal-weight subjects. These decreases in SIRT1 expression were observed in both adipocytes and non-fat cells. There was a strong association between adipose tissue SIRT1 mRNA and protein levels. Adipose SIRT1 expression correlated inversely with HOMA-IR and other insulin resistance-related parameters. Adipose SIRT1 and adiponectin mRNA expression correlated very strongly and positively. SIRT1 mRNA level in VAT correlated inversely with visceral obesity whereas its expression in SAT correlated negatively with body mass index. CONCLUSIONS Adipose tissue SIRT1 may play a key role in the regulation of whole body metabolic homeostasis in humans. Downregulation of SIRT1 in VAT may contribute to the metabolic abnormalities that are associated with visceral obesity.

IL-34 is associated with obesity, chronic inflammation, and insulin resistance.

OBJECTIVES IL-34 is a recently identified alternative ligand for colony-stimulating factor-1 (CSF-1) receptor. IL-34 and CSF-1 are regulators of differentiation, proliferation, and survival in mononuclear phagocytes. Here, we investigated the IL-34 serum concentration and expression in human adipose tissues and any associations with insulin resistance. METHODS We recruited 19 nondiabetic obese women, 9 type 2 diabetic women, and 27 normal-weight women. Metabolic parameters, abdominal fat distribution, serum IL-34 concentration, and IL-34 mRNA expression were measured in abdominal sc adipose tissue (SAT) and visceral adipose tissue (VAT). In addition, the expression/secretion and putative effects of IL-34 were assessed in human differentiated adipocytes. Serum IL-34 concentration was measured before and 5 to 9 months after laparoscopic Roux-en-Y gastric bypass surgery was performed on the 20 obese patients. RESULTS Regardless of diabetes status, obese patients demonstrated significantly higher serum IL-34 concentrations than controls. Serum IL-34 was significantly and positively correlated with insulin resistance-related metabolic parameters. IL-34 mRNA was significantly higher in VAT than SAT. IL-34 was expressed in adipocytes as well as nonadipocytes, and expression was significantly higher during adipogenesis. In differentiated adipocytes, the expression/secretion of IL-34 was enhanced by TNFα and IL-1β. In addition, IL-34 augmented fat accumulation and inhibited the stimulatory effects of insulin on glucose transport. Moreover, serum IL-34 was significantly decreased after Roux-en-Y gastric bypass-induced weight loss. CONCLUSION The present study demonstrates, for the first time, that IL-34 is expressed in human adipose tissues and the circulating concentration is significantly elevated in obese patients. This suggests that IL-34 is associated with insulin resistance.

Implication of phosphorylation of the myosin II regulatory light chain in insulin-stimulated GLUT4 translocation in 3T3-F442A adipocytes.

In adipocytes, insulin stimulates glucose transport primarily by promoting the translocation of GLUT4 to the plasma membrane. Requirements for Ca2+/ calmodulin during insulin-stimulated GLUT4 translocation have been demonstrated; however, the mechanism of action of Ca2+ in this process is unknown. Recently, myosin II, whose function in non-muscle cells is primarily regulated by phosphorylation of its regulatory light chain by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), was implicated in insulin-stimulated GLUT4 translocation. The present studies in 3T3- F442A adipocytes demonstrate the novel finding that insulin significantly increases phosphorylation of the myosin II RLC in a Ca2+-dependent manner. In addition, ML-7, a selective inhibitor of MLCK, as well as inhibitors of myosin II, such as blebbistatin and 2,3-butanedione monoxime, block insulin- stimulated GLUT4 translocation and subsequent glucose transport. Our studies suggest that MLCK may be a regulatory target of Ca2+/calmodulin and may play an important role in insulin-stimulated glucose transport in adipocytes.

Association between serum vaspin concentrations and visceral adipose tissue in Korean subjects.

Adipokines modulate multiple signaling pathways of insulin resistance via endocrine, paracrine, or autocrine mechanisms. Visceral adipose tissue (VAT)-derived serpin (vaspin) is a novel adipokine with potential insulin-sensitizing effects. We investigated the association between serum vaspin concentrations and abdominal adiposity. We recruited subjects (N = 150) aged 20 to 69 years who visited our hospital for regular health examinations. Abdominal VAT and subcutaneous adipose tissue areas were assessed by computed tomography. We measured serum vaspin concentrations by enzyme-linked immunosorbent assay. Statistical analysis was performed after stratification, using a homeostasis model for insulin resistance (HOMA-IR). Serum vaspin concentrations correlated positively with age (r = 0.196) when data from all subjects were analyzed. In the higher-HOMA-IR group, serum vaspin levels correlated more prominently with age (r = .344) and VAT area (r = .327) although these associations were not found in the lower-HOMA-IR group. In multivariate linear regression analysis, the VAT area was independently correlated with serum vaspin concentrations in the higher-HOMA-IR group. The association between serum vaspin concentrations and VAT differs according to insulin resistance. Insulin resistance might influence the correlation between serum vaspin concentration and VAT in human subjects.

Stretch-activated currents in cardiomyocytes isolated from rabbit pulmonary veins.

Evidence is growing of a relationship between atrial dilation and atrial fibrillation (AF), the most prevalent type of arrhythmia. Pulmonary veins, which are important ectopic foci for provoking AF, are of increasing interest in relation to the early development of AF. Here, using single cardiomyocytes isolated from rabbit pulmonary veins, we characterised the stretch-activated currents induced by swelling and axial mechanical stretching. Swelling induced both a stretch-activated nonselective cationic current (NSC) and a Cl(-) current. The swelling-induced Cl(-) current (I Cl,swell) was inhibited by DIDS, whereas the swelling-induced NSC (I NSC,swell) was inhibited by Gd3+. The cationic selectivity of the I NSC,swell was K+ >Cs+ >Na+ >Li+, whilst the PK/PNa, PCs/PNa, and PLi/PNa permeability ratios were 2.84, 1.86, and 0.85, respectively. Activation of the I NSC,swell was faster than that of the I Cl,swell. Given a high K+ concentration in the bath solution, the I NSC,swell showed limited amplitude (<-70 mV). Mechanical stretching induced an immediate Gd3+- and streptomycin-sensitive NSC (I NSC,stretch) that was permeable to Na+, K+, Cs+ and NMDG. Persistent stretching activated a DIDS-sensitive current (I Cl,stretch). The I NSC,stretch, but not the I NSC,swell, was completely blocked by 400 microM streptomycin; therefore, the two currents may not be associated with the same channel. In addition, the type of current induced may depend on the type of stretching. Thus, stretch-induced anionic and cationic currents are functionally present in the cardiomyocytes of the main pulmonary veins of rabbits, and they may have pathophysiological roles in the development of AF under stretched conditions.

Simulation of Ca2+-activated Cl- current of cardiomyocytes in rabbit pulmonary vein : implications of subsarcolemmal Ca2+ dynamics

In recent studies, we recorded transiently activated outward currents by the application of three-step voltage pulses to induce a reverse mode of Na+–Ca2+ exchange (NCX). We found that these currents were mediated by a Ca2+-activated Cl− current. Based on the recent reports describing the atrial Ca2+ transients, the Ca2+ transient at the subsarcolemmal space was initiated and then diffused into the cytosolic space. Because the myocardium in the pulmonary vein is an extension of the atrium, the Ca2+-activated Cl− current may reflect the subsarcolemmal Ca2+ dynamics. We tried to predict the subsarcolemmal Ca2+ dynamics by simulating these current traces. According to recent reports on the geometry of atrial myocytes, we assumed that there were three compartments of sarcoplasmic reticulum (SR): a network SR, a junctional SR and a central SR. Based on these structures, we also divided the cytosolic space into three compartments: the junctional, subsarcolemmal and cytosolic spaces. Geometry information and cellular capacitance suggested that there were essentially no T-tubules in these cells. The basic physical data, such as the compartmental volumes, the diffusion coefficients and the stability coefficients of the Ca2+ buffers, were obtained from the literature. In the simulation, we incorporated the NCX, the L-type Ca2+ channel, the rapid activating outward rectifier K+ channel, the Na+–K+ pump, the SR Ca2+-pump, the ryanodine receptor, the Ca2+-activated Cl− channel and the dynamics of Na+, K+, Ca2+ and Cl−. In these conditions, we could successfully reconstruct the Ca2+-activated Cl− currents. The simulation allowed estimation of the Ca2+ dynamics of each compartment and the distribution of the Ca2+-activated Cl− channel and the NCX in the sarcolemma on the junctional or subsarcolemmal space.

Fibronectin Gene Expression in Human Adipose Tissue and Its Associations with Obesity-Related Genes and Metabolic Parameters

BackgroundLimited data are available on the in vivo expression of fibronectin, one of the main extracellular matrix components. We investigated the expression of fibronectin in abdominal visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) and the associations of leptin, adiponectin, and vaspin gene expression with metabolic parameters in obese women.MethodsWe recruited female subjects undergoing bariatric surgery for obesity (n = 24) and patients undergoing benign gynecological surgery as the control group (n = 23). We measured anthropometric variables, abdominal fat distribution, metabolic parameters, serum concentrations of leptin, adiponectin, and vaspin, and depot-specific mRNA expression of fibronectin, leptin, adiponectin, and vaspin.ResultsFibronectin expression in both VAT and SAT was significantly lower in the obese group than in the control group. Fibronectin expression in both VAT and SAT were negatively correlated with body mass index or waist circumference, with higher prominence in VAT. In multiple regression analysis, fibronectin expression in both VAT and SAT was negatively correlated with serum leptin concentration. Fibronectin expression in VAT was negatively correlated with leptin expression in VAT. Additionally, fibronectin expression in SAT was negatively correlated with leptin expression in SAT and positively correlated with adiponectin expression in VAT and SAT.ConclusionsWe found significant negative associations between depot-specific fibronectin expression in human adipose tissue and obesity indices and obesity-related biomarkers. Our results suggest that fibronectin expression may contribute to obesity and metabolic dysregulation in humans.

Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein

Human fibroblast growth factor 21 (hFGF21) has been characterized as an important regulator of glucose and lipid metabolism homeostasis. Here, to produce hFGF21 efficiently in Escherichia coli, the expression and solubility of hFGF21 were tested and optimised by fusing the protein with one of eight tags: hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b′a′ domain of PDI (PDIb′a′). Each tag increased solubility of the protein when the expression temperature was 18°C. Unlike many other tags that were tested, MBP significantly enhanced the solubility of the protein also in the culture condition at 37°C. Thus, the MBP-hFGF21 construct was further pursued for optimisation of affinity chromatography purification. After tag removal, 8.1 mg of pure hFGF21 was obtained as a final product from 500 mL of starting culture. The protein was then characterised by mass spectroscopy and an in vitro functional assay using NIH-3T3 cells transfected with a β-klotho reporter gene. These characteristics are similar to those of commercial hFGF21. Thus, the MBP tag is useful for efficient prokaryotic production and purification of bioactive hFGF21.