A Small Molecule That Promotes Cellular Senescence Prevents Fibrogenesis and Tumorigenesis.

Uncontrolled proliferative diseases, such as fibrosis or cancer, can be fatal. We previously found that a compound containing the chromone scaffold (CS), ONG41008, had potent antifibrogenic effects associated with EMT or cell-cycle control resembling tumorigenesis. We investigated the effects of ONG41008 on tumor cells and compared these effects with those in pathogenic myofibroblasts. Stimulation of A549 (lung carcinoma epithelial cells) or PANC1 (pancreatic ductal carcinoma cells) with ONG41008 resulted in robust cellular senescence, indicating that dysregulated cell proliferation is common to fibrotic cells and tumor cells. The senescence was followed by multinucleation, a manifestation of mitotic slippage. There was significant upregulation of expression and rapid nuclear translocation of p-TP53 and p16 in the treated cancer cells, which thereafter died after 72 h confirmed by 6 day live imaging. ONG41008 exhibited a comparable senogenic potential to that of dasatinib. Interestingly, ONG41008 was only able to activate caspase-3, 7 in comparison with quercetin and fisetin, also containing CS in PANC1. ONG41008 did not seem to be essentially toxic to normal human lung fibroblasts or primary prostate epithelial cells, suggesting ONG41008 can distinguish the intracellular microenvironment between normal cells and aged or diseased cells. This effect might occur as a result of the increased NAD/NADH ratio, because ONG41008 restored this important metabolic ratio in cancer cells. Taken together, this is the first study to demonstrate that a small molecule can arrest uncontrolled proliferation during fibrogenesis or tumorigenesis via both senogenic and senolytic potential. ONG41008 could be a potential drug for a broad range of fibrotic or tumorigenic diseases.

Eupatilin Promotes Cell Death by Calcium Influx through ER-Mitochondria Axis with SERPINB11 Inhibition in Epithelial Ovarian Cancer.

Ovarian cancer is the leading cause of gynecological cancer-related mortality. The anticancer effect of eupatilin, a family of flavonoids, is known in many cancer types, but it is unclear what mechanism it plays in ovarian cancer. In this study, eupatilin promoted cell death of ovarian cancer cells by activating caspases, cell cycle arrest, reactive oxygen species (ROS) generation, calcium influx, disruption of the endoplasmic reticulum (ER)-mitochondria axis with SERPINB11 inhibition, and downregulation of phosphoinositide 3-kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways. Additionally, eupatilin-reduced SERPINB11 expression enhanced the effect of conventional chemotherapeutic agents against ovarian cancer cell progression. Cotreatment with siSERPINB11 and eupatilin increased calcium-ion-dependent apoptotic activity in ovarian cancer cells. Although there were no significant toxic effects of eupatilin on embryos, eupatilin completely inhibited tumorigenesis in a zebrafish xenograft model. In addition, eupatilin suppressed angiogenesis in zebrafish transgenic models. Collectively, downregulating SERPINB11 with eupatilin against cancer progression may improve therapeutic activity.

Reversion of in vivo fibrogenesis by novel chromone scaffolds.

BACKGROUND: Myofibroblasts are known to play a key role in the development of idiopathic pulmonary fibrosis (IPF). Two drugs, pirfenidone and nintedanib, are the only approved therapeutic options for IPF, but their applications are limited due to their side effects. Thus, curative IPF drugs represent a huge unmet medical need. METHODS: A mouse hepatic stellate cell (HSC) line was established that could robustly differentiate into myofibroblasts upon treatment with TGF-ß. Eupatilin was assessed in diseased human lung fibroblasts from IPF patients (DHLFs) as well as in human lung epithelial cells (HLECs). The drug's performance was extensively tested in a bleomycin-induced lung fibrosis model (BLM). Global gene expression studies and proteome analysis were performed. FINDINGS: Eupatilin attenuated disease severity of BLM in both preventative and therapeutic studies. The drug inhibited the in vitro transdifferantiation of DHLFs to myofibroblasts upon stimulation with TGF-ß. No such induction of the in vitro transdifferantiation was observed in TGF-ß treated HLECs. Specific carbons of eupatilin were essential for its anti-fibrotic activity. Eupatilin was capable of dismantling latent TGF-ß complex, specifically by eliminating expression of the latent TGF-ß binding protein 1 (LTBP1), in ECM upon actin depolymerization. Unlike eupatilin, pirfenidone was unable to block fibrosis of DHLFs or HSCs stimulated with TGF-ß. Eupatilin attenuated phosphorylation of Smad3 by TGF-ß. Eupatilin induced myofibroblasts to dedifferentiate into intermediate HCS-like cells. INTERPRETATION: Eupatilin may act directly on pathogenic myofibroblasts, disarming them, whereas the anti-fibrotic effect of pirfenidone may be indirect. Eupatilin could increase the efficacy of IPF treatment to curative levels.

Role of Angptl4/Fiaf in exercise-induced skeletal muscle AMPK activation.

Angiopoietin-like protein 4 (Angptl4)/fasting-induced adipose factor (Fiaf) expression levels are increased by exercise in skeletal muscle. We have previously shown that Angptl4 regulates food intake and energy expenditure via modulation of hypothalamic AMP-activated protein kinase (AMPK) activity. AMPK is an important signaling molecule that integrates skeletal muscle metabolism during exercise. Therefore, we investigated the involvement of Angptl4 in exercise-induced AMPK activation in skeletal muscle. Angptl4 protein and mRNA expression levels were significantly increased in the gastrocnemius and soleus muscles of mice following a 50-min running bout. Treatment of C2C12 myotubes with Angptl4 increased phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), which were markers of AMPK activation, and the mitochondrial maximum respiratory capacity. Treadmill exercise increased AMPK and ACC phosphorylation in the gastrocnemius of normal mice; this phosphorylation increase was attenuated in mice lacking Angptl4. Endurance to swimming and hanging was also reduced in Angptl4 knockout mice. Taken together, our current data demonstrate that exercise-induced upregulation of skeletal muscle Angptl4 is critical for AMPK activation and exercise tolerance. These findings unveil a new role for skeletal muscle Angptl4 in exercise physiology. NEW & NOTEWORTHY 1) Angiopoietin-like protein 4 (Angptl4) treatment activates AMP-activated protein kinase (AMPK) signaling in skeletal muscle cells. 2) Angptl4 increases the maximum mitochondrial oxidative capacity through AMPK activation in skeletal muscle cells. 3) Lack of Angptl4 mitigates exercise-induced skeletal muscle AMPK activation. 4) Angptl4-deficient mice show a lower endurance to exercise.

Development of a mouse IgA monoclonal antibody-based enzyme-linked immunosorbent sandwich assay for the analyses of RBP4.

Elevated circulating Retinol-binding protein 4 (RBP4) has been associated with insulin resistance, dyslipidemia, and hypertension. However, many commonly used RBP4 ELISAs have limited dynamic range. We therefore developed an enzyme-linked immunosorbent sandwich assay (ELISA) employing a novel immunoglobulin A (IgA)-type capture mAb called AG102 instead of IgG subtypes, which was selected for its stability, capture efficiency, and specificity for human RBP 4. These features of RBP4 have hampered the development of quantitative immunological assays. Molecular analysis of AG102 revealed IgA heavy and light chains and a J chain, as expected. AG102 demonstrated notable detection of both bacterial- and HEK293-expressed RBP4 in Western blots. Serial and internal deletion experiments suggested that a putative epitope may be located in the first 35 amino acids of the mature RBP4. Compared with commercial ELISAs, the AG102-based system exhibited more significant recovery of RBP4 from serum or urine at any given dilution factor. To substantiate its quantitation capacity, comparison between RBP4 measurements from quantitative western blots and the AG102-based ELISA demonstrated a significant correlation (R2 = 0.859). After measurement for those analytes, our data suggested that IgA-based ELISA could be adapted for quantitative measurement of those analytes existing as major serum proteins or as multi-protein complexes like RBP4.

Angiopoietin-like peptide 4 regulates insulin secretion and islet morphology.

Insulin secretion from pancreatic islet ß-cells is primarily regulated by the blood glucose level, and also modulated by a number of biological factors produced inside the islets or released from remote organs. Previous studies have shown that angiopoietin-like protein 4 (Angptl4) controls glucose and lipid metabolism through its actions in the liver, adipose tissue, and skeletal muscles. In this present study, we investigated the possible role of Angptl4 in the regulation of insulin secretion from pancreatic islets. Angptl4 was found to be highly expressed in the α-cells but not ß-cells of rodent islets. Moreover, treatment of rodent islets with Angptl4 peptide potentiated glucose-stimulated insulin secretion through a protein kinase A-dependent mechanism. Consistently, Angptl4 knockout mice showed impaired glucose tolerance. In the cultured islets from Angptl4 knockout mice, glucose-stimulated insulin secretion was significantly lower than in islets from wild type mice. Angptl4 peptide replacement partially reversed this reduction. Moreover, Angptl4 knockout mice had dysmorphic islets with abnormally distributed α-cells. In contrast, the ß-cell mass and distribution were not significantly altered in these knockout mice. Our current data collectively suggest that Angptl4 may play a critical role in the regulation of insulin secretion and islet morphogenesis.

Preadipocyte factor 1 induces pancreatic ductal cell differentiation into insulin-producing cells.

The preadipocyte factor 1 (Pref-1) is involved in the proliferation and differentiation of various precursor cells. However, the intracellular signaling pathways that control these processes and the role of Pref-1 in the pancreas remain poorly understood. Here, we showed that Pref-1 induces insulin synthesis and secretion via two independent pathways. The overexpression of Pref-1 activated MAPK signaling, which induced nucleocytoplasmic translocation of FOXO1 and PDX1 and led to the differentiation of human pancreatic ductal cells into ß-like cells and an increase in insulin synthesis. Concurrently, Pref-1 activated Akt signaling and facilitated insulin secretion. A proteomics analysis identified the Rab43 GTPase-activating protein as a downstream target of Akt. A serial activation of both proteins induced various granular protein syntheses which led to enhanced glucose-stimulated insulin secretion. In a pancreatectomised diabetic animal model, exogenous Pref-1 improved glucose homeostasis by accelerating pancreatic ductal and ß-cell regeneration after injury. These data establish a novel role for Pref-1, opening the possibility of applying this molecule to the treatment of diabetes.

The ratio of skeletal muscle mass to visceral fat area is a main determinant linking circulating irisin to metabolic phenotype.

BACKGROUND: The aims of this study were to investigate whether circulating irisin is associated with favorable metabolic parameters and how the association differs according to body composition in humans. METHODS: A total of 424 subjects (233 men and 191 women), aged 23-73 years (mean age 47.1 years), were enrolled from the Seoul Metro City Diabetes Prevention Program. Body composition was determined using an impedance body composition analyzer, and serum irisin level was measured using a commercial kit. RESULTS: Serum irisin was correlated with favorable metabolic parameters including less obese, lower blood pressure and glucose levels and healthy lipid parameters. The skeletal muscle mass to visceral fat area ratio (SVR) was positively correlated with the serum irisin concentration (r = 0.10, P = 0.04). When the study subjects were divided into tertiles according to their SVR, serum irisin was correlated with favorable metabolic phenotypes in those subjects in the upper tertile. However, there were no such correlations in the lower tertile. In addition, serum irisin was inversely related to pre-diabetes/type 2 diabetes (T2D) independent of other risk factor for T2D and insulin resistance [OR (95 % CI); 0.66 (0.49-0.90), P = 0.009]. CONCLUSIONS: The compositions of skeletal muscle and visceral fat play key roles in the association between circulating irisin and a patient's metabolic phenotype.

Extracellular Vesicles as Drug Delivery Vehicles for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic, systemic and progressive autoimmune disease of connective tissues common in middle age. Dysregulation of the tissue homeostasis involving inflammation is the hallmark of disease pathogenesis, inducing autoimmune insults that frequently lead to permanent disability. Although the advent of immunosuppressive and anti-inflammatory drugs and, more recently, pathogenic TNF-TNF-R axis-targeting biologics significantly delayed progressive joint destruction with significant reduction of disability and physical improvement, a large proportion of RA patients failed to respond to the treatment. In this regard, mesenchymal stem/stromal cells (MSC) are particularly attractive to the refractory patients to the pharmacologic intervention for their immunosuppressive/anti-inflammatory capacity as well as tissue reparative and/or regenerative potential. Local or systemic delivery of MSCs led to promising results in preclinical as well as in clinical studies of RA and thus proposing that these cells can be further exploited for their therapeutic application in RA and other degenerative connective tissue diseases. Mechanistically, paracrine factors appear to be the main contributors of MSC-mediated tissue regeneration in a number of preclinical and clinical studies rather than direct tissue cell replacement. More recently, extracellular vesicles (EVs) released from MSCs emerged as key paracrine messengers that can also participate in the healing process through influencing the local microenvironment with anti-inflammatory effects. It is highly likely that the use of these EVs becomes beneficial in the treatment of RA. Yet, identification of key components involved in the regenerative process needs to be assessed for developing efficient MSC-based strategy of RA treatment.

Regulation of energy balance by the hypothalamic lipoprotein lipase regulator Angptl3.

Hypothalamic lipid sensing is important for the maintenance of energy balance. Angiopoietin-like protein 3 (Angptl3) critically regulates the clearance of circulating lipids by inhibiting lipoprotein lipase (LPL). The current study demonstrated that Angptl3 is highly expressed in the neurons of the mediobasal hypothalamus, an important area in brain lipid sensing. Suppression of hypothalamic Angptl3 increased food intake but reduced energy expenditure and fat oxidation, thereby promoting weight gain. Consistently, intracerebroventricular (ICV) administration of Angptl3 caused the opposite metabolic changes, supporting an important role for hypothalamic Angptl3 in the control of energy balance. Notably, ICV Angptl3 significantly stimulated hypothalamic LPL activity. Moreover, coadministration of the LPL inhibitor apolipoprotein C3 antagonized the effects of Angptl3 on energy metabolism, indicating that LPL activation is critical for the central metabolic actions of Angptl3. Increased LPL activity is expected to promote lipid uptake by hypothalamic neurons, leading to enhanced brain lipid sensing. Indeed, ICV injection of Angptl3 increased long-chain fatty acid (LCFA) and LCFA-CoA levels in the hypothalamus. Furthermore, inhibitors of hypothalamic lipid-sensing pathways prevented Angptl3-induced anorexia and weight loss. These findings identify Angptl3 as a novel regulator of the hypothalamic lipid-sensing pathway.

Progranulin and a five transmembrane domain-containing receptor-like gene are the key components in receptor activator of nuclear factor κB (RANK)-dependent formation of multinucleated osteoclasts.

Homeostatic bone remodeling is vital to maintain healthy bone tissue. Although the receptor activator of nuclear factor κB ligand (RANKL)/RANK axis is considered the master regulator of osteoclastogenesis, the underlying mechanisms including cell fusion remain incompletely defined. Here, we introduce a new axis in the formation of multinucleated cells via RANK signaling: the progranulin (PGRN)/PIRO (PGRN-induced receptor-like gene during osteoclastogenesis) axis. When mouse bone marrow-derived macrophages were stimulated with PGRN in the presence of RANKL, explosive OC formation was observed. PGRN knockdown experiments suggested that endogenous PGRN is an essential component of the RANKL/RANK axis. Our efforts for identifying genes that are induced by PGRN unveiled a remarkably induced (20-fold) gene named PIRO. Substantial PGRN and PIRO expression was detected after 2 and 3 days, respectively, suggesting that their sequential induction. PIRO was predicted to be a five transmembrane domain-containing receptor-like molecule. The tissue distribution of PGRN and PIRO mRNA expression suggested that bone marrow cells are the most suitable niche. Mouse and human PIRO are part of a multigene family. Knockdown experiments suggested that PIRO is a direct target for the formation of multinucleated cells by PGRN. PGRN levels were also substantially higher in ovariectomized mice than in sham control mice. These observations suggest that PGRN and PIRO form a new regulatory axis in osteoclastogenesis that is included in RANK signaling in cell fusion and OC resorption of osteoclastogenesis, which may offer a novel therapeutic modality for osteoporosis and other bone-associated diseases.

Massive elimination of multinucleated osteoclasts by eupatilin is due to dual inhibition of transcription and cytoskeletal rearrangement.

Osteoporosis is an aging-associated disease requiring better therapeutic modality. Eupatilin is a major flavonoid from Artemisia plants such as Artemisia princeps and Artemisia argyi which has been reported to possess various beneficial biological effects including anti-inflammation, anti-tumor, anti-cancer, anti-allergy, and anti-oxidation activity. Complete blockade of RANK-dependent osteoclastogenesis was accomplished upon stimulation prior to the receptor activator of nuclear factor κB (RANK)-ligand (RANKL) treatment or post-stimulation of bone marrow macrophages (BMCs) in the presence of RANKL with eupatilin. This blockade was accompanied by inhibition of rapid phosphorylation of Akt, GSK3ß, ERK and IκB as well as downregulation of c-Fos and NFATc1 at protein, suggesting that transcriptional suppression is a key mechanism for anti-osteoclastogenesis. Transient reporter assays or gain of function assays confirmed that eupatilin was a potent transcriptional inhibitor in osteoclasts (OC). Surprisingly, when mature osteoclasts were cultured on bone scaffolds in the presence of eupatilin, bone resorption activity was also completely blocked by dismantling the actin rings, suggesting that another major acting site of eupatilin is cytoskeletal rearrangement. The eupatilin-treated mature osteoclasts revealed a shrunken cytoplasm and accumulation of multi-nuclei, eventually becoming fibroblast-like cells. No apoptosis occurred. Inhibition of phosphorylation of cofilin by eupatilin suggests that actin may play an important role in the morphological change of multinucleated cells (MNCs). Human OC similarly responded to eupatilin. However, eupatilin has no effects on osteoblast differentiation and shows cytotoxicity on osteoblast in the concentration of 50 µM. When eupatilin was administered to LPS-induced osteoporotic mice after manifestation of osteoporosis, it prevented bone loss. Ovariectomized (OVX) mice remarkably exhibited bone protection effects. Taken together, eupatilin is an effective versatile therapeutic intervention for osteoporosis via; 1) transcriptional suppression of c-Fos and NFATc1 of differentiating OC and 2) inhibition of actin rearrangement of pathogenic MNCs.

Association of urinary RBP4 with insulin resistance, inflammation, and microalbuminuria.

OBJECTIVE: Serum concentrations of retinol-binding protein 4 (RBP4) are elevated in type 2 diabetes and associated with the severity of insulin resistance; however, there are few data about the relationship between urinary RBP4 levels and metabolic parameters. We assessed urinary RBP4 as a new biomarker by establishing its relationship with clinical parameters associated with insulin resistance and urinary albumin excretion. DESIGN AND METHODS: We measured RBP4 in the serum and urine of 689 subjects with diverse glucose tolerance status. We also evaluated the relationship between urinary RBP4 and cardiometabolic risk factors, including insulin resistance, high-sensitivity C-reactive protein (hsCRP), arterial stiffness, and microalbuminuria. RESULTS: Urinary RBP4 levels were higher in insulin-resistant subjects with prediabetes or type 2 diabetes than in subjects with normal glucose tolerance (NGT) (type 2 diabetes>prediabetes>NGT; all P<0.001). Urinary RBP4 correlated strongly with homeostasis model assessments of insulin resistance (HOMA-IR), fasting glucose, triglycerides, blood pressure, hsCRP, arterial stiffness, estimated glomerular filtration rate, and urinary albumin-to-creatinine ratio (all P<0.01). HOMA-IR and arterial stiffness were found to be independent determinants of urinary RBP4 concentration. Furthermore, urinary RBP4 was highly predictive of microalbuminuria (odds ratio 2.6, 95% CI 1.6-4.2), even after adjustment for other metabolic parameters. The area under the ROC curve for urinary RBP4 to detect the presence of microalbuminuria was 0.80±0.02 (95% CI 0.76-0.84) and the cut-off value was 157.01 µg/gCr. CONCLUSIONS: Urinary RBP4 concentrations were elevated in patients with dysregulation of glucose metabolism and were related to various cardiometabolic risk factors including insulin resistance, inflammation, and microalbuminuria.

Plasma clusterin (ApoJ) levels are associated with adiposity and systemic inflammation.

Obesity and insulin resistance are hallmarks of the metabolic syndrome, which is associated with low-grade chronic inflammation. Clusterin/apolipoprotein J is an abundant plasma chaperone protein that has recently been suggested as a potential biomarker that reflects the inflammatory process in Alzheimer's disease. In the present study, we investigated anthropometric and clinical factors affecting the plasma levels of clusterin in healthy Korean subjects. We measured fasting plasma clusterin levels in healthy Korean adults (111 men and 93 women) using ELISA kit. We analyzed the relationship between plasma clusterin concentrations and anthropometric and clinical parameters. Fasting plasma clusterin concentrations were higher in overweight and obese subjects than in lean subjects. Correlation analysis revealed that the plasma clusterin levels were positively associated with indices of obesity such as body mass index (BMI), waist circumference and waist-hip ratio and markers of systemic inflammation such as high sensitivity C-reactive protein (hsCRP), uric acid, ferritin and retinol binding protein-4. Multiple linear regression analysis showed that sex, BMI and hsCRP were independent determinants of plasma clusterin levels. Furthermore, plasma clusterin levels showed an upward trend with increasing numbers of metabolic syndrome components. These findings suggest that fasting plasma clusterin levels correlate with the parameters of adiposity and systemic inflammation in healthy adults. Therefore, the circulating clusterin level may be a surrogate marker for obesity-associated systemic inflammation.

Identification of adipokine clusters related to parameters of fat mass, insulin sensitivity and inflammation.

In obesity, elevated fat mass and ectopic fat accumulation are associated with changes in adipokine secretion, which may link obesity to inflammation and the development of insulin resistance. However, relationships among individual adipokines and between adipokines and parameters of obesity, glucose metabolism or inflammation are largely unknown. Serum concentrations of 20 adipokines were measured in 141 Caucasian obese men (n = 67) and women (n = 74) with a wide range of body weight, glycemia and insulin sensitivity. Unbiased, distance-based hierarchical cluster analyses were performed to recognize patterns among adipokines and their relationship with parameters of obesity, glucose metabolism, insulin sensitivity and inflammation. We identified two major adipokine clusters related to either (1) body fat mass and inflammation (leptin, ANGPTL3, DLL1, chemerin, Nampt, resistin) or insulin sensitivity/hyperglycemia, and lipid metabolism (vaspin, clusterin, glypican 4, progranulin, ANGPTL6, GPX3, RBP4, DLK1, SFRP5, BMP7, adiponectin, CTRP3 and 5, omentin). In addition, we found distinct adipokine clusters in subgroups of patients with or without type 2 diabetes (T2D). Logistic regression analyses revealed ANGPTL6, DLK1, Nampt and progranulin as strongest adipokine correlates of T2D in obese individuals. The panel of 20 adipokines predicted T2D compared to a combination of HbA1c, HOMA-IR and fasting plasma glucose with lower sensitivity (78% versus 91%) and specificity (76% versus 94%). Therefore, adipokine patterns may currently not be clinically useful for the diagnosis of metabolic diseases. Whether adipokine patterns are relevant for the predictive assessment of intervention outcomes needs to be further investigated.

Implication of circulating irisin levels with brown adipose tissue and sarcopenia in humans.

CONTEXT: Irisin is an exercise-induced novel myokine that drives brown-fat-like conversion of white adipose tissue and has been suggested to be a promising target for the treatment of obesity-related metabolic disorders. OBJECTIVE: To assess the association of circulating irisin concentrations with brown adipose tissue (BAT) and/or sarcopenia in humans. SETTING AND DESIGN: We examined irisin levels in 40 BAT-positive and 40 BAT-negative women detected by (18)F-fluorodeoxyglucose positron emission tomography ((18)FDG-PET). In a separate study, we also examined 401 subjects with or without sarcopenia defined by skeletal muscle mass index (SMMI) and appendicular skeletal muscle (ASM)/height(2) using dual-energy x-ray absorptiometry. RESULTS: Among 6877 consecutive (18)FDG-PET scans in 4736 subjects, 146 subjects (3.1%) had positive BAT scans. The BAT-detectable group and the matched BAT-undetectable group did not differ in circulating irisin levels measured using two different ELISA kits (P = .747 and P = .160, respectively). Serum irisin levels were not different between individuals with sarcopenia and those without sarcopenia using either kit (P = .305 and P = .569, respectively). Also, serum irisin levels were not different between groups defined by ASM/height(2) using either kit (P = .352 and P = .134, respectively). Although visceral fat area and skeletal muscle mass showed significant difference according to tertiles of SMMI levels, irisin concentrations did not differ. CONCLUSIONS: Circulating irisin levels were not different in individuals with detectable BAT or those with sarcopenia compared with control subjects and were not correlated with SMMI.

Clusterin/ApoJ enhances central leptin signaling through Lrp2-mediated endocytosis.

Hypothalamic leptin signaling plays a central role in maintaining body weight homeostasis. Here, we show that clusterin/ApoJ, recently identified as an anorexigenic neuropeptide, is an important regulator in the hypothalamic leptin signaling pathway. Coadministration of clusterin potentiates the anorexigenic effect of leptin and boosts leptin-induced hypothalamic Stat3 activation. In cultured neurons, clusterin enhances receptor binding and subsequent endocytosis of leptin. These effects are mainly mediated through the LDL receptor-related protein-2 (Lrp2). Notably, inhibition of hypothalamic clusterin, Lrp2 or endocytosis abrogates anorexia and hypothalamic Stat3 activation caused by leptin. These findings propose a novel regulatory mechanism in central leptin signaling pathways.

Implications of C1q/TNF-related protein-3 (CTRP-3) and progranulin in patients with acute coronary syndrome and stable angina pectoris.

BACKGROUND: C1q/TNF-related protein-3 (CTRP-3), an adiponectin paralog, and progranulin were recently identified as novel adipokines which may link obesity with glucose dysregulation and subclinical inflammation. We analyzed the relationship between CTRP-3, progranulin and coronary artery disease (CAD) in Korean men and women. METHODS: Circulating CTRP-3 and progranulin levels were examined in 362 Korean adults with acute coronary syndrome (ACS, n = 69), stable angina pectoris (SAP, n = 85), and control subjects (n = 208) along with various kinds of cardiometabolic risk factors. RESULTS: CTRP-3 concentrations were significantly decreased in patients with ACS or SAP compared to control subjects (P <0.001, respectively), whereas progranulin and adiponectin levels were similar. Correlation analysis adjusted for age and gender exhibited that CTRP-3 levels showed significant negative relationship with glucose (r = -0.110, P = 0.041) and high sensitive C-reactive protein (hsCRP) levels (r = -0.159, P = 0.005), and positive relationship with HDL-cholesterol (r = 0.122, P = 0.025) and adiponectin levels (r = 0.194, P <0.001). In a multivariate logistic regression analysis, the odds ratio for CAD risk was 5.14 (95% CI, 1.83-14.42) in the second, and 9.04 (95% CI, 2.81-29.14) in the first tertile of CTRP-3 levels compared to third tertile after adjusting for other cardiometabolic risk variables. CONCLUSIONS: Patients with ACS or SAP had significantly lower circulating CTRP-3 concentrations compared to control subjects, although progranulin levels were not different. These results suggest the possibility that CTRP-3 might be useful for assessing the risk of CAD. TRIAL REGISTRATION: (Clinical trials No.: NCT01594710).

Urinary adiponectin concentration is positively associated with micro- and macro-vascular complications.

BACKGROUND: A relationship between plasma adiponectin level and a number of metabolic conditions, including insulin resistance, obesity, and type 2 diabetes, has been reported. This study aimed to assess whether urinary adiponectin concentration is correlated with vascular complications. METHODS: The study comprised 708 subjects who enrolled in the Seoul Metro City Diabetes Prevention Program and were carefully monitored from September 2008 to December 2008. Levels of urinary adiponectin were measured using an enzyme linked immunosorbent assay (ELISA) kit (AdipoGen, Korea). Urinary albumin excretion was assessed by the ratio of urinary albumin to creatinine (A/C ratio). Participants were divided into three groups based on tertiles of urinary adiponectin concentration, and we investigated whether urinary adiponectin levels are associated with microalbuminuria and pulse wave velocity. RESULTS: Urinary adiponectin concentrations were significantly higher in subjects with microalbuminuria than subjects with normoalbuminuria (P < 0.001). Urinary adiponectin concentration was positively correlated with age, fasting plasma glucose level, HbA1C level, triglyceride level, HOMA-IR, systolic/diastolic blood pressure, and urinary A/C ratio (all P < 0.05). Subjects in the highest tertile of urinary adiponectin concentration had an increased likelihood of microalbuminuria than those in the lowest tertile (Odds ratio (OR), 6.437; 95% confidence interval (CI), 4.202 to 9.862; P < 0.001). After adjusting for age, sex, and estimated creatinine clearance rate (eCcr), the OR remained significant (OR, 5.607; 95% CI, 3.562 to 8.828; P < 0.001). Backward multiple linear regression analysis revealed urinary adiponectin concentration to be a significant determinant of mean brachial-ankle pulse wave velocity (baPWV). CONCLUSIONS: An increased urinary adiponectin concentration is significantly associated with microalbuminuria and increased mean baPWV. These results suggest that urinary adiponectin may play an important role as a biomarker for vascular dysfunction.

Salsalate and adiponectin ameliorate hepatic steatosis by inhibition of the hepatokine fetuin-A.

Fetuin-A was recently identified as a novel hepatokine which is associated with obesity, insulin resistance and non-alcoholic fatty liver disease. Salsalate, a prodrug of salicylate with an anti-inflammatory effect and lower side effect profile, significantly lowers glucose and triglyceride levels, and increased adiponectin concentrations in randomized clinical trials. In this study, we examined the effects and regulatory mechanisms of salsalate and full length-adiponectin (fAd) on fetuin-A expression, steatosis and lipid metabolism in palmitate-treated HepG2 cells. Incubation of hepatocytes with palmitate significantly increased fetuin-A and SREBP-1c expression which lead to steatosis and knock-down of fetuin-A by siRNA restored these changes. Salsalate significantly down-regulated palmitate-induced fetuin-A mRNA expression and secretion in a dose- and time-dependent manner. Inhibition of palmitate-induced fetuin-A by salsalate was mediated by AMPK-mediated reduction of NFκB activity, which was blocked by AMPK siRNA or an inhibitor of AMPK. Salsalate attenuated the excessive steatosis by palmitate through SREBP-1c regulation in hepatocytes. Furthermore, fAd also showed suppression of palmitate-induced fetuin-A through the AMPK pathway and improvement of steatosis accompanied by restoration of SREBP-1c, PAPR-α and CD36. In preliminary in vivo experiments, salsalate treatment inhibited high fat diet (HFD)-induced steatosis as well as fetuin-A mRNA and protein expression in SD rats. In conclusion, salsalate and fAd improved palmitate-induced steatosis and impairment of lipid metabolism in hepatocytes via fetuin-A inhibition through the AMPK-NFκB pathway.