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Glioblastoma multiforme (GBM) is the most lethal form of brain cancer in humans, with a grim prognosis and a median overall survival rate of less than 15 months. Recently, glioma stem cells (GSCs) have been identified as a crucial factor in both tumor initiation and therapy resistance in GBM. Analysis of datasets and direct differentiation experiments on GSCs have both confirmed that CREB5 is expressed more highly in GSCs than in differentiated GSCs. Furthermore, studies on loss of function have shown that CREB5 promotes the proliferation and self-renewal ability of GSCs, while also reducing their tumor-forming ability in vivo. RNA-sequencing, Luciferase reporter assay, and ChIP assay have demonstrated a direct association between CREB5 and OLIG2. These findings suggest that targeting CREB5 could be an effective approach to overcoming GSCs.

Exogenous polyamines are able to induce life span and improve glucose homeostasis and insulin sensitivity. However, the effects of exogenous polyamines on adipocyte differentiation and which polyamine transporters mediate them have not been elucidated yet. Here, we identified for the first time that exogenous polyamines can clearly stimulate adipocyte differentiation through polyamine transporters, solute carrier family 3 member A2 (SLC3A2) and SLC7A1. Exogenous polyamines markedly promote 3T3-L1 adipocyte differentiation by increasing the intracellular lipid accumulation and the expression of both adipogenic and lipogenic genes in a concentration-dependent manner. In particular, exogenous putrescine mainly regulates adipocyte differentiation in the early and intermediate stages. Moreover, we have assessed the expression of polyamine transporter genes in 3T3-L1 preadipocytes and adipocytes. Interestingly, the putrescine-induced adipocyte differentiation was found to be significantly suppressed in response to a treatment with a polyamine transporter inhibitor (AMXT-1501). Furthermore, knockdown experiments using siRNA that specifically targeted SLC3A2 or SLC7A2, revealed that both SLC3A2 and SLC7A2 act as important transporters in the cellular importing of exogenous putrescine. Thus, the exogenous putrescine entering the adipocytes via cellular transporters is involved in adipogenesis through a modulation of both the mitotic clonal expansion and the expression of master transcription factors. Taken together, these results suggest that exogenous polyamines (such as putrescine) entering the adipocytes through polyamine transporters, can stimulate adipogenesis.

Inflammation mediated by the innate immune system is the organism’s protective mechanism against infectious environmental risk factors. Uncontrolled acute inflammation can become chronic, contributing to various chronic inflammatory diseases such as arthritis, asthma, autoimmune diseases, and atherosclerosis. Although microalgae are increasingly receiving attention as a source of bioactive molecules with therapeutic potential for various human diseases, the underlying mechanisms are not yet well understood. In the present study, we investigated the molecular mechanisms underlying the anti-inflammatory and anti-aging activities of ethanol extracts of Antarctic freshwater microalga Micractinium simplicissimum. Using RAW 264.7 macrophages, microalgal extracts exerted anti-inflammatory activity by regulating the major inflammatory indicators including cyclooxy-genase (COX)-2, interleukin (IL)-6, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α and nitric oxide (NO). Besides, we observed the anti-aging activity of the microalgal extract by suppressing MMP-1 production in human dermal fibroblast. Taken together, these data suggest that anti-inflammatory and anti-aging activities of Antarctic freshwater microalga, Micractinium simplicissimum, can provide new clues to understanding the molecular link between inflammation and diseases, and be a potential anti-inflammatory agent.

Iris bungei Maxim. (IB), which is native to China and Mongolia, is used as a traditional medicine for conditions such as inflammation, cancer, and bacterial infections. However, the effects of Iris bungei Maxim. on adipocyte differentiation have not been studied. In the present study, we first demonstrated the molecular mechanisms underlying the adipogenic activity of the methanol extract of Mongolian I. bungei Maxim. (IB). IB significantly enhanced intracellular lipid accumulation and adipocyte differentiation in 3T3-L1 preadipocytes in a concentration-dependent manner. Moreover, IB markedly stimulated the expression of genes related to adipogenesis such as peroxisome proliferator-activated receptor γ, adiponectin, and aP2. In addition, we also observed that IB induces lipogenic genes such as fatty acid synthase, sterol regulatory element binding protein 1c, stearoyl-CoA desaturase, and acetyl-CoA carboxylase. Interestingly IB regulated adipocyte differentiation in both the early and middle stages. Taken together, these adipogenic and lipogenic effects of IB suggest its efficacy for the prevention and/or treatment of type 2 diabetes.

In obesity, increased mitochondrial metabolism with the accumulation of oxidative stress leads to mitochondrial damage and β-cell dysfunction. In particular, β-cells express antioxidant enzymes at relatively low levels and are highly vulnerable to oxidative stress. Early in the development of obesity, β-cells exhibit increased glucose-stimulated insulin secretion in order to compensate for insulin resistance. This increase in β-cell function under the condition of enhanced metabolic stress suggests that β-cells possess a defense mechanism against increased oxidative damage, which may become insufficient or decline at the onset of type 2 diabetes. Here, we show that metabolic stress induces β-cell hypoxia inducible factor 2α (HIF-2α), which stimulates antioxidant gene expression (e.g., Sod2 and Cat) and protects against mitochondrial reactive oxygen species (ROS) and subsequent mitochondrial damage. Knockdown of HIF-2α in Min6 cells exaggerated chronic high glucose–induced mitochondrial damage and β-cell dysfunction by increasing mitochondrial ROS levels. Moreover, inducible β-cell HIF-2α knockout mice developed more severe β-cell dysfunction and glucose intolerance on a high-fat diet, along with increased ROS levels and decreased islet mitochondrial mass. Our results provide a previously unknown mechanism through which β-cells defend against increased metabolic stress to promote β-cell compensation in obesity.

In obesity, increased mitochondrial metabolism with the accumulation of oxidative stress leads to mitochondrial damage and beta cell dysfunction. In particular, beta cells express anti-oxidant enzymes at relatively low levels and are highly vulnerable to oxidative stress. Early in the development of obesity, beta cells exhibit increased GSIS, in order to compensate for insulin resistance. This increase in beta cell function under the condition of enhanced metabolic stress suggests that beta cells possess a defense mechanism(s) against increased oxidative damage, which may become insufficient or decline at the onset of type 2 diabetes. Here, we show that metabolic stress induces beta cell HIF-2a, which stimulates anti-oxidant gene expression (e.g. Sod2 and Cat) and protects against mitochondrial ROS and subsequent mitochondrial damage. Knockdown of HIF-2a in Min6 cells exaggerated chronic high glucose-induced mitochondrial damage and beta cell dysfunction by increasing mitochondrial ROS levels. Moreover, inducible beta cell HIF-2a KO mice developed more severe beta cell dysfunction and glucose intolerance on high fat diet, along with increased ROS levels and decreased islet mitochondrial mass. Our results provide a previously unknown mechanism for how beta cell defends against increased metabolic stress to promote beta cell compensation in obesity.

Colorectal cancer (CRC) is a deadly disease regardless of sex, and a few therapeutic approaches have been fully developed at advanced stages, even if some strategies have durable clinical benefits, such as immunotherapy and chemotherapy.

The aim of this study is to analyse the determinants of women's vaginal dryness using machine learning. Data came from Korea University Anam Hospital in Seoul, Republic of Korea, with 3298 women, aged 40-80 years, who attended their general health check from January 2010 to December 2012. Five machine learning methods were applied and compared for the prediction of vaginal dryness, measured by a Menopause Rating Scale. Random forest variable importance, a performance gap between a complete model and a model excluding a certain variable, was adopted for identifying major determinants of vaginal dryness. In terms of the mean squared error, the random forest (1.0597) was much better than linear regression (17.9043) and artificial neural networks with one, two and three hidden layers (1.7452, 1.7148 and 1.7736, respectively). Based on random forest variable importance, the top-10 determinants of vaginal dryness were menopause age, age, menopause, height, thyroid stimulating hormone, neutrophils, years since menopause, lymphocytes, alkaline phosphatase and blood urea nitrogen. In addition, its top-20 determinants were peak expiratory flow rate, low-density lipoprotein cholesterol, white blood cells, monocytes, cancer antigen 19-9, creatinine, eosinophils, total cholesterol, triglyceride and amylase. Machine learning presents a great decision support system for the prediction of vaginal dryness. For preventing vaginal dryness, preventive measures would be needed regarding early menopause, the thyroid function and systematic inflammation.Impact Statement

β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein–coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P(2)). We found that β-arrestin also helped promote synthesis of PI(4,5)P(2) and up-regulated GPCR endocytosis. We studied these questions with the G(q)-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P(2), which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P(2,) could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P(2) could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P(2)-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P(2) generation that promotes recruitment of PI(4,5)P(2)-dependent endocytic machinery.

β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein-coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P2). We found that β-arrestin also helped promote synthesis of PI(4,5)P2 and up-regulated GPCR endocytosis. We studied these questions with the Gq-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P2, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P2 could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P2-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P2 generation that promotes recruitment of PI(4,5)P2-dependent endocytic machinery.

Background To analyze the factors associated with women's vasomotor symptoms (VMS) using machine learning. Methods Data on 3,298 women, aged 40–80 years, who attended their general health check-up from January 2010 to December 2012 were obtained from Korea University Anam Hospital in Seoul, Korea. Five machine learning methods were applied and compared for the prediction of VMS, measured by the Menopause Rating Scale. Variable importance, the effect of a variable on model performance, was used for identifying the major factors associated with VMS. Results In terms of the mean squared error, the random forest (0.9326) was much better than linear regression (12.4856) and artificial neural networks with one, two, and three hidden layers (1.5576, 1.5184, and 1.5833, respectively). Based on the variable importance from the random forest, the most important factors associated with VMS were age, menopause age, thyroid-stimulating hormone, and monocyte, triglyceride, gamma glutamyl transferase, blood urea nitrogen, cancer antigen 19-9, C-reactive protein, and low-density lipoprotein cholesterol levels. Indeed, the following variables were ranked within the top 20 in terms of variable importance: cancer antigen 125, total cholesterol, insulin, free thyroxine, forced vital capacity, alanine aminotransferase, forced expired volume in 1 second, height, homeostatic model assessment for insulin resistance, and carcinoembryonic antigen. Conclusion Machine learning provides an invaluable decision support system for the prediction of VMS. For managing VMS, comprehensive consideration is needed regarding thyroid function, lipid profile, liver function, inflammation markers, insulin resistance, monocyte count, cancer antigens, and lung function., Graphical Abstract

Inflammation mediated by the innate immune system is the organism's protective mechanism against infectious environmental risk factors. Uncontrolled acute inflammation can become chronic, contributing to various chronic inflammatory diseases such as arthritis, asthma, autoimmune diseases, and atherosclerosis. Although microalgae are increasingly receiving attention as a source of bioactive molecules with therapeutic potential for various human diseases, the underlying mechanisms are not yet well understood. In the present study, we investigated the molecular mechanisms underlying the anti-inflammatory and anti-aging activities of ethanol extracts of Antarctic freshwater microalga

Background To analyze the determinants of women’s vasomotor symptoms (VMS) using machine learning. Methods Data came from Korea University Anam Hospital in Seoul, Korea, with 3298 women, aged 40–80 years, who attended their general health check from January 2010 to December 2012. Five machine learning methods were applied and compared for the prediction of VMS, measured by a Menopause Rating Scale. Variable importance, the effect of a variable on model performance, was used for identifying major determinants of VMS. Results In terms of the mean squared error, the random forest (0.9326) was much better than linear regression (12.4856) and artificial neural networks with one, two and three hidden layers (1.5576, 1.5184 and 1.5833, respectively). Based on variable importance from the random forest, the most important determinants of VMS were age, menopause age, thyroid stimulating hormone, monocyte and triglyceride, as well as gamma glutamyl transferase, blood urea nitrogen, cancer antigen 19 − 9, C-reactive protein and low-density-lipoprotein cholesterol. Indeed, the following determinants ranked within the top 20 in terms of variable importance: cancer antigen 125, total cholesterol, insulin, free thyroxine, forced vital capacity, alanine aminotransferase, forced expired volume in one second, height, homeostatic model assessment for insulin resistance and carcinoembryonic antigen. Conclusions Machine learning provides an invaluable decision support system for the prediction of VMS. For preventing VMS, preventive measures would be needed regarding the thyroid function, the lipid profile, the liver function, inflammation markers, insulin resistance, the monocyte, cancer antigens and the lung function.

Decreased adipose tissue oxygen tension and increased HIF-1α expression can trigger adipose tissue inflammation and dysfunction in obesity. Our current understanding of obesity-associated decreased adipose tissue oxygen tension is mainly focused on changes in oxygen supply and angiogenesis. Here, we demonstrate that increased adipocyte O2 demand, mediated by ANT2 activity, is the dominant cause of adipocyte hypoxia. Deletion of adipocyte Ant2 improves obesity-induced intracellular adipocyte hypoxia by decreasing obesity-induced adipocyte oxygen demand, without effects on mitochondrial number or mass, or oligomycin-sensitive respiration. This led to decreased adipose tissue HIF-1α expression and inflammation with improved glucose tolerance and insulin resistance in both a preventative or therapeutic setting. Our results suggest that ANT2 may be a target for the development of insulin sensitizing drugs and that ANT2 inhibition might have clinical utility.

Summary Insulin resistance is a major factor in obesity-linked type 2 diabetes. PPARγ is a master regulator of adipogenesis, and small molecule agonists, termed thiazolidinediones, are potent therapeutic insulin sensitizers. Here, we studied the role of transcriptional co-activator with PDZ-binding motif (TAZ) as a transcriptional co-repressor of PPARγ. We found that adipocyte-specific TAZ knockout (TAZ AKO) mice demonstrate a constitutively active PPARγ state. Obese TAZ AKO mice show improved glucose tolerance and insulin sensitivity compared to littermate controls. PPARγ response genes are upregulated in adipose tissue from TAZ AKO mice and adipose tissue inflammation was also decreased. In vitro and in vivo mechanistic studies revealed that the TAZ-PPARγ interaction is partially dependent on ERK-mediated Ser112 PPARγ phosphorylation. As adipocyte PPARγ Ser112 phosphorylation is increased in obesity, repression of PPARγ activity by TAZ could contribute to insulin resistance. These results identify TAZ as a new factor in the development of obesity-induced insulin resistance.

Decreased adipose tissue oxygen tension and increased HIF-1α expression can trigger adipose tissue inflammation and dysfunction in obesity. Our current understanding of obesity-associated decreased adipose tissue oxygen tension is mainly focused on changes in oxygen supply and angiogenesis. Here, we demonstrate that increased adipocyte O2 demand, mediated by ANT2 activity, is the dominant cause of adipocyte hypoxia. Deletion of adipocyte Ant2 improves obesity-induced intracellular adipocyte hypoxia by decreasing obesity-induced adipocyte oxygen demand, without effects on mitochondrial number or mass, or oligomycin-sensitive respiration. This led to decreased adipose tissue HIF-1α expression and inflammation with improved glucose tolerance and insulin resistance in both a preventative or therapeutic setting. Our results suggest that ANT2 may be a target for the development of insulin sensitizing drugs and that ANT2 inhibition might have clinical utility.

The nature of obesity-associated islet inflammation and its impact on β cell abnormalities remains poorly defined. Here, we explore immune cell components of islet inflammation and define their roles in regulating β cell function and proliferation. Islet inflammation in obese mice is dominated by macrophages. We identify two islet-resident macrophage populations, characterized by their anatomical distributions, distinct phenotypes, and functional properties. Obesity induces the local expansion of resident intra-islet macrophages, independent of recruitment from circulating monocytes. Functionally, intra-islet macrophages impair β cell function in a cell-cell contact-dependent manner. Increased engulfment of βcell insulin secretory granules by intra-islet macrophages in obese mice may contribute to restricting insulin secretion. In contrast, both intra- and peri-islet macrophage populations from obese mice promote β cell proliferation in a PDGFR signaling-dependent manner. Together, these data define distinct roles and mechanisms for islet macrophages in the regulation of islet β cells.

Extracellular adenosine-5'-triphosphate (ATP) regulates cell death and survival of neighboring cells. The detailed effects are diverse depending on cell types and extracellular ATP concentration. We addressed the effect of ATP on ethanol-induced cytotoxicity in epithelial cells, the cell type that experiences the highest concentrations of alcohol. Using pancreatic duct epithelial cells (PDEC), we found that a micromolar range of ATP reverses all intracellular toxicity mechanisms triggered by exceptionally high doses of ethanol and, thus, improves cell viability dramatically. Out of the many purinergic receptors expressed in PDEC, the P2Y1 receptor was identified to mediate the protective effect, based on pharmacological and siRNA assays. Activation of P2Y1 receptors increased intracellular cyclic adenosine monophosphate (cAMP). The protective effect of ATP was mimicked by forskolin and 8-Br-cAMP but inhibited by a protein kinase A (PKA) inhibitor, H-89. Finally, ATP reverted leakiness of PDEC monolayers induced by ethanol and helped to maintain epithelial integrity. We suggest that purinergic receptors reduce extreme alcohol-induced cell damage via the cAMP signal pathway in PDEC and some other types of cells.

Hepatic TANK (TRAF family member associated NFκB activator)-binding kinase 1 (TBK1) activity is increased during obesity, and administration of a TBK1 inhibitor reduces fatty liver. Surprisingly, liver-specific TBK1 knockout in mice produces fatty liver by reducing fatty acid oxidation. TBK1 functions as a scaffolding protein to localize acyl-CoA synthetase long-chain family member 1 (ACSL1) to mitochondria, which generates acyl-CoAs that are channeled for β-oxidation. TBK1 is induced during fasting and maintained in the unphosphorylated, inactive state, enabling its high affinity binding to ACSL1 in mitochondria. In TBK1-deficient liver, ACSL1 is shifted to the endoplasmic reticulum to promote fatty acid re-esterification in lieu of oxidation in response to fasting, which accelerates hepatic lipid accumulation. The impaired fatty acid oxidation in TBK1-deficient hepatocytes is rescued by the expression of kinase-dead TBK1. Thus, TBK1 operates as a rheostat to direct the fate of fatty acids in hepatocytes, supporting oxidation when inactive during fasting and promoting re-esterification when activated during obesity.

We have previously reported that the fractalkine (FKN)/CX3CR1 system represents a novel regulatory mechanism for insulin secretion and β cell function. Here, we demonstrate that chronic administration of a long-acting form of FKN, FKN-Fc, can exert durable effects to improve glucose tolerance with increased glucose-stimulated insulin secretion and decreased β cell apoptosis in obese rodent models. Unexpectedly, chronic FKN-Fc administration also led to decreased α cell glucagon secretion. In islet cells, FKN inhibited ATP-sensitive potassium channel conductance by an ERK-dependent mechanism, which triggered β cell action potential (AP) firing and decreased α cell AP amplitude. This results in increased glucose-stimulated insulin secretion and decreased glucagon secretion. Beyond its islet effects, FKN-Fc also exerted peripheral effects to enhance hepatic insulin sensitivity due to inhibition of glucagon action. In hepatocytes, FKN treatment reduced glucagon-stimulated cAMP production and CREB phosphorylation in a pertussis toxin-sensitive manner. Together, these results raise the possibility of use of FKN-based therapy to improve type 2 diabetes by increasing both insulin secretion and insulin sensitivity.

The Ca(2+) sensor synaptotagmin-1 is thought to trigger membrane fusion by binding to acidic membrane lipids and SNARE proteins. Previous work has shown that binding is mediated by electrostatic interactions that are sensitive to the ionic environment. However, the influence of divalent or polyvalent ions, at physiological concentrations, on synaptotagmin's binding to membranes or SNAREs has not been explored. Here we show that binding of rat synaptotagmin-1 to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2) is regulated by charge shielding caused by the presence of divalent cations. Surprisingly, polyvalent ions such as ATP and Mg(2+) completely abrogate synaptotagmin-1 binding to SNAREs regardless of the presence of Ca(2+). Altogether, our data indicate that at physiological ion concentrations Ca(2+)-dependent synaptotagmin-1 binding is confined to PIP2-containing membrane patches in the plasma membrane, suggesting that membrane interaction of synaptotagmin-1 rather than SNARE binding triggers exocytosis of vesicles.

Our basic hypothesis is that mammalian pinealocytes have cycling electrical excitability and Ca2+ signalling that may contribute to the circadian rhythm of pineal melatonin secretion. This study asked whether the functional expression of voltage-gated Ca2+ channels (CaV channels) in rat pinealocytes is changed by culturing them in noradrenaline (NA) as a surrogate for the night signal. Channel activity was assayed as ionic currents under patch clamp and as optical signals from a Ca2+-sensitive dye. Channel mRNAs were assayed by quantitative polymerase chain reaction. Cultured without NA, pinealocytes showed only non-inactivating L-type dihydropyridine-sensitive Ca2+ current. After 24 h in NA, additional low-voltage activated transient Ca2+ current developed whose pharmacology and kinetics corresponded to a T-type CaV3.1 channel. This change was initiated by β-adrenergic receptors, cyclic AMP and protein kinase A as revealed by pharmacological experiments. mRNA for CaV3.1 T-type channels became significantly elevated, but mRNA for another T-type channel and for the major L-type channel did not change. After only 8 h of NA treatment, the CaV3.1 mRNA was already elevated, but the transient Ca2+ current was not. Even a 16 h wait without NA following the 8 h NA treatment induced little additional transient current. However, these cells were somehow primed to make transient current as a second NA exposure for only 60 min sufficed to induce large T-type currents. The NA-induced T-type channel mediated an increased Ca2+ entry during short depolarizations and supported modest transient electrical responses to depolarizing stimuli. Such experiments reveal the potential for circadian regulation of excitability.

Binding of agonists to G-protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M1 muscarinic acetylcholine receptors (M1Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M1R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane.

Store-operated Ca2+ channels (SOCs) are activated by depletion of intracellular Ca2+ stores following agonist-mediated Ca2+ release. Previously we demonstrated that Ca2+ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca2+ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca2+ influx as Ca2+ was released from intracellular stores. Direct measurement of [Ca2+] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP3 or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ~ 8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La3+ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca2+ entry through SOCs fills internal Ca2+ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca2+ such as salt and mucin secretion from the exocrine pancreatic ducts.

Pinealocytes secrete melatonin at night in response to norepinephrine released from sympathetic nerve terminals in the pineal gland. The gland also contains many other neurotransmitters whose cellular disposition, activity, and relevance to pineal function are not understood. Here, we clarify sources and demonstrate cellular actions of the neurotransmitter γ-aminobutyric acid (GABA) using Western blotting and immunohistochemistry of the gland and electrical recording from pinealocytes. GABAergic cells and nerve fibers, defined as containing GABA and the synthetic GAD67, were identified. The cells represent a subset of interstitial cells while the nerve fibers were distinct from the sympathetic innervation. The GABAA receptor subunit α1 was visualized in close proximity of both GABAergic and sympathetic nerve fibers as well as fine extensions among pinealocytes and blood vessels. The GABAB 1 receptor subunit was localized in the interstitial compartment but not in pinealocytes. Electrophysiology of isolated pinealocytes revealed that GABA and muscimol elicit strong inward chloride currents sensitive to bicuculline and picrotoxin, clear evidence for functional GABAA receptors on the surface membrane. Applications of elevated potassium solution or the neurotransmitter acetylcholine depolarized the pinealocyte membrane potential enough to open voltage-gated Ca2+ channels leading to intracellular calcium elevations. GABA repolarized the membrane and shut off such calcium rises. In 48–72-h cultured intact glands, GABA application neither triggered melatonin secretion by itself nor affected norepinephrine-induced secretion. Thus, strong elements of GABA signaling are present in pineal glands that make large electrical responses in pinealocytes, but physiological roles need to be found. Fil: Yu, Haijie. University of Washington; Estados Unidos Fil: Benitez, Sergio Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Jung, Seung-Ryoung. University of Washington; Estados Unidos Fil: Kruse, Martin. University of Washington; Estados Unidos Fil: Seo, Jong Bae. University of Washington; Estados Unidos Fil: Koh, Duk-Su. University of Washington; Estados Unidos Fil: Muñoz, Estela Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Hille, Bertil. University of Washington; Estados Unidos

In most eukaryotic cells, microtubules and filamentous actin (F-actin) provide tracks on which intracellular organelles move using molecular motors. Here we report that cytoplasmic movement of both mitochondria and lysosomes is slowed by F-actin meshwork formation in pancreatic duct epithelial cells (PDEC). Mitochondria and lysosomes were labeled with fluorescent Mitotracker Red CMXRos and Lysotracker Red DND-99, respectively, and their movements were monitored using epi-fluorescence and confocal microscopy. Mitochondria and lysosomes moving actively at rest stopped rapidly within several seconds after an intracellular Ca(2+) rise induced by activation of P2Y(2) purinergic receptors. The 'freezing' of the organelles was inhibited by blocking the Ca(2+) rise or by pretreatment with latrunculin B, an inhibitor of F-actin formation. Indeed, this freezing effect on the organelles was accompanied by the formation of F-actin in the whole cytoplasm as stained with Alexa 488-phalloidin in fixed PDEC. For real-time monitoring of F-actin formation in live cells, we expressed sGFP-fimbrin actin binding domain2 (fABD2) in PDEC. Rapid recruitment of the fluorescent probe near the nucleus and lysosomes suggested dense F-actin formation around intracellular structures. The development of F-actin paralleled that of organelle freezing. We conclude that rapid Ca(2+)-dependent F-actin formation physically restrains intracellular organelles and reduces their mobility non-selectively in PDEC.

The fruit of Terminalia chebula Retzius has been used as a panacea in India and Southeast Asia but its biological activities have not been fully elucidated. Here we report anti-arthritic and analgesic effect of NDI10218, a standardized ethanol extract of Terminalia chebula, on collagen-induced arthritis and acetic acid-induced writhing model, respectively. Arthritis was induced in DBA/1J mice by immunizing bovine type II collagen and mice were treated with NDI10218 daily for 5 weeks after the onset of the disease. NDI10218 reduced the arthritis index and blocked the synovial hyperplasia in a dose-dependent manner. The serum levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were significantly reduced in mice treated with NDI10218. Production of the inflammatory IL-17, but not immunosuppressive IL-10, was also inhibited in splenocytes isolated from NDI10218-treated arthritis mice. Administration of NDI10218 markedly decreased the number of T cell subpopulations in the regional lymph nodes of the arthritis mice. Finally, NDI10218 reduced the number of abdominal contractions in acetic acid-induced writhing model, suggesting an analgesic effect of this extract. Taken together, these results suggest that NDI10218 can be a new therapeutic candidate for the treatment of rheuma-toid arthritis.

We have previously reported anti-obesity effects of Lysimachia foenum-graecum in high-fat diet (HFD)-induced obesity model. Here we isolated a triterpene saponin foenumoside B as an active component of L. foenum-graecum. Foenumoside B blocked the differentiation of 3T3-L1 preadipocytes in a dose-dependent manner with an IC50 of 0.2 μg/ml in adipogenesis assay and suppressed the induction of PPARγ, the master regulator of adipogenesis. Foenumoside B induced the activation of AMP-activated protein kinase (AMPK), and modulated the expression of genes involved in lipid metabolism towards lipid breakdown in differentiated adipocytes. In mouse model, oral administration of foenumoside B (10mg/kg/day for 6 weeks) reduced HFD-induced body weight gain significantly without affecting food intake. Treatment of foenumoside B suppressed lipid accumulation in white adipose tissues and the liver, and lowered blood levels of glucose, triglycerides, ALT, and AST in HFD-induced obese mice. Consistent with the in vitro results, foenumoside B activated AMPK signaling, suppressed the expression of lipogenic genes, and enhanced the expression of lipolytic genes in vivo. Foenumoside B also blocked HFD-induced proinflammatory cytokine production in adipose tissue, suggesting its protective role against insulin resistance. Taken together, these findings demonstrate that foenumoside B represents the anti-obesity effects of L. foenum-graecum, and suggest therapeutic potential of foenumoside B in obesity and obesity-related metabolic diseases.

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically.

The mammalian pineal has a circadian rhythm of melatonin secretion at night triggered by norepinephrine (NE) released from sympathetic nerve terminals. We asked whether functional expression of voltage-gated calcium channels in rat pinealocytes is changed by culturing them in NE as a surrogate for the night signal. Channel activity was assayed as ionic currents under patch clamp. Cultured without NE, pinealocytes showed only non-inactivating L-type dihydropyridine-sensitive calcium current. After 24 h in NE, an additional low-voltage activated transient calcium current developed whose pharmacology and kinetics corresponded to a T-type channel. This change was initiated by β-adrenergic receptors, cyclic AMP, and protein kinase A as revealed by pharmacological experiments. Quantitative PCR experiments showed mRNA for Cav1.4 (L-type), Cav3.1 and Cav3.2 (both T-type). Cav3.1 T-type channel mRNA was significantly elevated by culture in NE, but those for Cav1.4 and Cav3.2 were not. After only 8 h of NE treatment, Cav3.1 mRNA was already elevated, but the transient calcium current was not. Even a 16 h wait without NE following the 8 h NE treatment induced little additional transient current. However, these cells were primed to make transient current after a second NE exposure. Induction of transient current was sensitive to the inhibitors of intracellular protein trafficking. The NE-induced T-type channel mediated an increased calcium entry during short depolarizations and supported modest transient electrical responses to depolarizing stimuli. Such experiments reveal a potential for circadian regulation of pinealocyte electrical excitability and calcium signaling. This work is supported by National Institutes of Health grants GM-83913, NS-08174 and DK-080840.

MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis.

The mammalian pineal gland is a neuroendocrine organ that responds to circadian and seasonal rhythms. Its major function is to secrete melatonin as a hormonal night signal in response to nocturnal delivery of noradrenaline from sympathetic neurons. Culturing rat pinealocytes in noradrenaline for 24 h induced a low-voltage activated transient Ca(2+) current whose pharmacology and kinetics corresponded to a CaV3.1 T-type channel. The upregulation of the T-type Ca(2+) current is initiated by β-adrenergic receptors, cyclic AMP and cyclic AMP-dependent protein kinase. Messenger RNA for CaV3.1 T-type channels is significantly elevated by noradrenaline at 8 h and 24 h. The noradrenaline-induced T-type channel mediated an increased Ca(2+) entry and supported modest transient electrical responses to depolarizing stimuli, revealing the potential for circadian regulation of pinealocyte electrical excitability and Ca(2+) signalling.Our basic hypothesis is that mammalian pinealocytes have cycling electrical excitability and Ca(2+) signalling that may contribute to the circadian rhythm of pineal melatonin secretion. This study asked whether the functional expression of voltage-gated Ca(2+) channels (CaV channels) in rat pinealocytes is changed by culturing them in noradrenaline (NA) as a surrogate for the night signal. Channel activity was assayed as ionic currents under patch clamp and as optical signals from a Ca(2+)-sensitive dye. Channel mRNAs were assayed by quantitative polymerase chain reaction. Cultured without NA, pinealocytes showed only non-inactivating L-type dihydropyridine-sensitive Ca(2+) current. After 24 h in NA, additional low-voltage activated transient Ca(2+) current developed whose pharmacology and kinetics corresponded to a T-type CaV3.1 channel. This change was initiated by β-adrenergic receptors, cyclic AMP and protein kinase A as revealed by pharmacological experiments. mRNA for CaV3.1 T-type channels became significantly elevated, but mRNA for another T-type channel and for the major L-type channel did not change. After only 8 h of NA treatment, the CaV3.1 mRNA was already elevated, but the transient Ca(2+) current was not. Even a 16 h wait without NA following the 8 h NA treatment induced little additional transient current. However, these cells were somehow primed to make transient current as a second NA exposure for only 60 min sufficed to induce large T-type currents. The NA-induced T-type channel mediated an increased Ca(2+) entry during short depolarizations and supported modest transient electrical responses to depolarizing stimuli. Such experiments reveal the potential for circadian regulation of excitability.

X-box binding protein 1 (XBP1), a transcription factor of the unfolded protein response, plays various roles in many biological processes. We examined its pro-adipogenic activity and target genes during adipogenic differentiation in wild-type and genetically modified 3T3-L1 cells. Signalling pathways that contribute to Xbp1 mRNA splicing, and the correlation of the transcriptionally active XBP1 isoform (XBP1s) level with body mass index and the level of peroxisome proliferator-activated receptor γ2 (PPARγ2) in human adipose tissues were also examined. The mRNA and nuclear protein expression levels of XBP1s increased immediately following hormonal induction of adipogenesis, reaching a peak at 6 h. Results from cDNA microarray and gene expression analyses using genetically modified cells indicated that PPARγ2 was a principal target of XBP1s. The XBP1s-specific binding motif, which is distinct from the CCAAT/enhancer-binding protein α binding site, was identified in the PPARγ2 promoter by site-directed mutagenesis. Fetal bovine serum, insulin, 3-isobutyl-1-methylxanthine and dexamethasone contributed independently to Xbp1 mRNA splicing. In human subcutaneous adipose tissues, the levels of both Xbp1s and Pparγ2 mRNA increased proportionally with body mass index, and there was a significant positive correlation between the two genes. These data suggest for the first time that positive regulation of PPARγ2 is a principal mechanism of XBP1s-mediated adipogenesis in 3T3-L1 cells.

Lysimachia foenum-graecum has been used as an oriental medicine with anti-inflammatory effect. The anti-obesity effect of L. foenum-graecum extract (LFE) was first discovered in our screening of natural product extract library against adipogenesis. To characterize its anti-obesity effects and to evaluate its potential as an anti-obesity drug, we performed various obesity-related experiments in vitro and in vivo. In adipogenesis assay, LFE blocked the differentiation of 3T3-L1 preadipocyte in a dose-dependent manner with an IC50 of 2.5 µg/ml. In addition, LFE suppressed the expression of lipogenic genes, while increasing the expression of lipolytic genes in vitro at 10 µg/ml and in vivo at 100 mg/kg/day. The anti-adipogenic and anti-lipogenic effect of LFE seems to be mediated by the inhibition of PPARγ and C/EBPα expression as shown in in vitro and in vivo, and the suppression of PPARγ activity in vitro. Moreover, LFE stimulated fatty acid oxidation in an AMPK-dependent manner. In high-fat diet (HFD)-induced obese mice (n = 8/group), oral administration of LFE at 30, 100, and 300 mg/kg/day decreased total body weight gain significantly in all doses tested. No difference in food intake was observed between vehicle- and LFE-treated HFD mice. The weight of white adipose tissues including abdominal subcutaneous, epididymal, and perirenal adipose tissue was reduced markedly in LFE-treated HFD mice in a dose-dependent manner. Treatment of LFE also greatly improved serum levels of obesity-related biomarkers such as glucose, triglycerides, and adipocytokines leptin, adiponectin, and resistin. All together, these results showed anti-obesity effects of LFE on adipogenesis and lipid metabolism in vitro and in vivo and raised a possibility of developing LFE as anti-obesity therapeutics.

Systematic imaging studies and modeling reveal new details of the regulation of the Gq-coupled GPCR, protease-activated receptor 2, by phosphorylation and β-arrestin., Activated Gq protein–coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein–tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein–coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary depletion of PIP2. Indeed, artificial recruitment of PIP5K removed the secondary loss of PIP2 completely. Altogether, our experimental and theoretical approaches demonstrate roles and dynamics of the protein kinases, β-arrestin, and PIP5K in the desensitization of PAR2.

Liver X receptors (LXRs) are nuclear hormone receptors that regulate cholesterol and fatty acid metabolism in liver tissue and in macrophages. Although LXR activation enhances lipogenesis, it is not well understood whether LXRs are involved in adipocyte differentiation. Here, we show that LXR activation stimulated the execution of adipogenesis, as determined by lipid droplet accumulation and adipocyte-specific gene expression in vivo and in vitro. In adipocytes, LXR activation with T0901317 primarily enhanced the expression of lipogenic genes such as the ADD1/SREBP1c and FAS genes and substantially increased the expression of the adipocyte-specific genes encoding PPARgamma (peroxisome proliferator-activated receptor gamma) and aP2. Administration of the LXR agonist T0901317 to lean mice promoted the expression of most lipogenic and adipogenic genes in fat and liver tissues. It is of interest that the PPARgamma gene is a novel target gene of LXR, since the PPARgamma promoter contains the conserved binding site of LXR and was transactivated by the expression of LXRalpha. Moreover, activated LXRalpha exhibited an increase of DNA binding to its target gene promoters, such as ADD1/SREBP1c and PPARgamma, which appeared to be closely associated with hyperacetylation of histone H3 in the promoter regions of those genes. Furthermore, the suppression of LXRalpha by small interfering RNA attenuated adipocyte differentiation. Taken together, these results suggest that LXR plays a role in the execution of adipocyte differentiation by regulation of lipogenesis and adipocyte-specific gene expression.

Adiponectin is exclusively expressed in differentiated adipocytes and plays an important role in regulating energy homeostasis, including the glucose and lipid metabolism associated with increased insulin sensitivity. However, the control of adiponectin gene expression in adipocytes is poorly understood. We show here that levels of adiponectin mRNA and protein are reduced in the white adipose tissue of ob/ob and db/db mice and that there is a concomitant reduction of the adipocyte determination- and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein 1c (SREBP1c) transcription factor. To determine whether ADD1/SREBP1c regulates adiponectin gene expression, we isolated and characterized the mouse adiponectin promoter. Analysis of the adiponectin promoter revealed putative binding sites for the adipogenic transcription factors ADD1/SREBP1c, peroxisomal proliferator-activated receptor gamma and CCAAT enhancer-binding proteins. DNase I footprinting and chromatin immunoprecipitation analyses revealed that ADD1/SREBP1c binds in vitro and in vivo to the proximal promoter containing sterol regulatory element (SRE) motifs. A luciferase reporter containing the promoter was transactivated by ADD1/SREBP1c, and introduction of SRE mutations into the construct abolished transactivation. Adenoviral overexpression of ADD1/SREBP1c also elevated adiponectin mRNA and protein levels in 3T3-L1 adipocytes. Furthermore, insulin stimulated adiponectin mRNA expression in adipocytes and augmented transactivation of the adiponectin promoter by ADD1/SREBP1c. Taken together, these data indicate that ADD1/SREBP1c controls adiponectin gene expression in differentiated adipocytes.

Recent studies with murine models propose that resistin would be a possible mediator to link between obesity and insulin resistance. Although it has been reported that resistin is highly expressed and secreted by adipocytes, transcription factors that are involved in resistin gene expression have not been well characterized. To investigate the molecular mechanisms of resistin gene expression, we cloned and characterized the human resistin promoter. Sequence analysis of the resistin promoter revealed several putative binding sites for adipogenic transcription factors including adipocyte determination- and differentiation-dependent factor 1 (ADD1)/sterol regulatory element binding protein 1c (SREBP1c) and CCAAT enhancer binding protein-alpha (C/EBP alpha). EMSA and chromatin immunoprecipitation assays demonstrated that ADD1/SREBP1c binds to the human resistin promoter in vitro and in vivo. Expression of ADD1/SREBP1c transactivated the luciferase reporter gene activity, the promoter region of which contains a human resistin promoter in a sterol regulatory element (SRE)-dependent manner. Furthermore, ectopic expression of ADD1/SREBP1c by adenovirus significantly increased the expression of resistin mRNA in adipocytes. Human resistin promoter was also activated by C/EBP alpha expression, although ectopic expression of both transcription factors did not show any synergistic effects on the activation of resistin promoter. Together, these data suggest that ADD1/SREBP1c and C/EBP alpha may play discrete roles in the regulation of the resistin gene expression.