G Proteins in Reverse Mode: RECEPTOR-MEDIATED GTP RELEASE INHIBITS G PROTEIN AND EFFECTOR FUNCTION*

Active G protein-coupled receptors activate heterotrimeric Gαβγ proteins by catalyzing the exchange of GDP by GTP at the Gα subunit. A paradoxical attenuation of G protein-activated inwardly rectifying potassium channels (GIRK) upon stimulation of native cells with high concentrations of agonist is known. However, a deactivation of activated G proteins by active receptors has not been experimentally studied in intact cells. We monitored GIRK currents and G_o protein activation by means of fluorescence resonance energy transfer (FRET) in parallel. The results suggested that GIRK currents were paradoxically attenuated due to an inactivation of G_o proteins by active α_2A-adrenergic receptors. To study the mechanisms, G protein activation and receptor-G protein interactions were analyzed as a function of nucleotide type and nucleotide concentrations by means of FRET, while controlling intracellular nucleotides upon permeabilization of the cell membrane. Results suggested a receptor-catalyzed dissociation of GTP from activated heterotrimeric Gαβγ. Consequently, nucleotide-free G proteins were sequestrated in heterotrimeric conformation at the active receptor, thus attenuating downstream signaling in an agonist-dependent manner.     

A Kinetic Approach to Characterize the Electrostatic Environments of Thiol Groups in Proteins

In this study, we synthesized a zwitterionic DTNB derivative, 5-(2-aminoethyl)-dithio-2-nitrobenzoate (ADNB), and characterized its reactions with several cationic, anionic, and neutral thiols. Reactions with ADNB, unlike those with DTNB, are relatively insensitive to electrostatic environments and ionic strengths. At relatively low ionic strength, rate ratios,k_ADNB/k_DTNB, varied from 0.22 for reactions with low-molecular-weight cationic thiols to 3.0 for those with low-molecular-weight anionic thiols. Ak_ADNB/k_DTNBratio of 200 for Cys-34 of BSA appears to reflect a very anionic environment.k_ADNB/k_DTNBratios of 6 and 1, respectively, for canine and equine serum albumins, which have Glu-82 → Asp and Glu-82 → Ala substitutions suggest Glu-82 is the most important anionic residues affecting the reactivity of Cys-34 in BSA.k_ADNB/k_DTNBratios appear to be useful for characterizing electrostatic environments of thiol groups in proteins.     

Defect in Glucose Utilization in Adipose Tissue of Patients with Maturity-onset Diabetes

The ability of glucose, glucose-6-phosphate, and glycerol phosphate to support incorporation of ¹⁴C-palmitate into neutral lipid of adipose tissue has been studied in eight patients with maturity-onset diabetes. After two hours'' incubation glucose and glucose-6-phosphate supported incorporation rates relative to glycerol phosphate of 30% and 44% respectively in diabetic tissue, whereas the corresponding rates in paired non-diabetic controls were 93% and 95%. This failure of adipose tissue to metabolize glucose in maturity-onset diabetes might be responsible for a delay in the clearance of glucose from the blood stream. Alternatively, a defect in glucose utilization might be due to tissue changes associated with maturity-onset diabetes.     

Endoplasmic Reticulum Stress Is Increased in Adipose Tissue of Women with Gestational Diabetes

Maternal obesity and gestational diabetes mellitus (GDM) are two increasingly common and important obstetric complications that are associated with severe long-term health risks to mothers and babies. IL-1β, which is increased in obese and GDM pregnancies, plays an important role in the pathophysiology of these two pregnancy complications. In non-pregnant tissues, endoplasmic (ER) stress is increased in diabetes and can induce IL-1β via inflammasome activation. The aim of this study was to determine whether ER stress is increased in omental adipose tissue of women with GDM, and if ER stress can also upregulate inflammasome-dependent secretion of IL-1β. ER stress markers IRE1α, GRP78 and XBP-1s were significantly increased in adipose tissue of obese compared to lean pregnant women. ER stress was also increased in adipose tissue of women with GDM compared to BMI-matched normal glucose tolerant (NGT) women. Thapsigargin, an ER stress activator, induced upregulated secretion of mature IL-1α and IL-1β in human omental adipose tissue explants primed with bacterial endotoxin LPS, the viral dsRNA analogue poly(I:C) or the pro-inflammatory cytokine TNF-α. Inhibition of capase-1 with Ac-YVAD-CHO resulted in decreased IL-1α and IL-1β secretion, whereas inhibition of pannexin-1 with carbenoxolone suppressed IL-1β secretion only. Treatment with anti-diabetic drugs metformin and glibenclamide also reduced IL-1α and IL-1β secretion in infection and cytokine-primed adipose tissue. In conclusion, this study has demonstrated ER stress to activate the inflammasome in pregnant adipose tissue. Therefore, increased ER stress may contribute towards the pathophysiology of obesity in pregnancy and GDM.     

Elevated Soluble CD163 in Gestational Diabetes Mellitus: Secretion from Human Placenta and Adipose Tissue

Recently soluble CD163 (sCD163), a cleaved form of the macrophage receptor CD163, was identified as a macrophage-specific risk-predictor for developing Type 2 Diabetes. Here, we investigate circulating levels of sCD163 in gestational diabetes mellitus (GDM). Furthermore, given the role of the placenta in the pathogenesis of GDM, we assessed placental contribution to sCD163 secretion. Paired maternal (venous) and umbilical vein blood samples from GDM (n = 18) and Body Mass Index (BMI) matched control women (n = 20) delivered by caesarean section at 39–40 week gestation were assessed for circulating levels of sCD163, Tumour necrosis factor alpha (TNF-α) and Interleukin 6 (IL-6). Media from explant culture of maternal subcutaneous fat and corresponding placental tissues were assayed for these same molecules. CD163 positive cell numbers were determined in placental and adipose tissues of GDM and control women. We found significantly elevated circulating sCD163 levels in GDM mothers (688.4±46.9 ng/ml vs. 505.6±38.6 ng/ml) and their offspring (418.2±26.6 ng/ml vs. 336.3±24.4 ng/ml [p<0.05 for both]) as compared to controls, together with elevated circulating TNF-α and IL-6 levels. Moreover, both GDM placentae (268.1±10.8 ng/ml/mg vs. 187.6±20.6 ng/ml/mg) and adipose explants (41.1±2.7 ng/ml/mg vs. 26.6±2.4 ng/ml/mg) released significantly more sCD163 than controls. Lastly, significantly more CD163 positive cells were observed in GDM placentae (25.7±1.1 vs. 22.1±1.2) and adipose tissue (19.1±1.1 vs 12.7±0.9) compared to controls. We describe elevated sCD163 levels in GDM and identify human placenta as a novel source of sCD163 suggesting that placental tissues might contribute to the increased levels of circulating sCD163 in GDM pregnancies.     

Structural Mapping of Post-translational Modifications in Human Interleukin-24: ROLE OF N-LINKED GLYCOSYLATION AND DISULFIDE BONDS IN SECRETION AND ACTIVITY*

Human interleukin-24 (IL-24) is unique among the IL-10 superfamily as there is considerable evidence that it possesses multiple anti-cancer properties, including direct tumor cell cytotoxicity, helper T cell (TH1) immune stimulation, and anti-angiogenic activities. The primary sequence of human IL-24 differs from homologous cytokines, because it possesses three consensus N-linked glycosylation sites and the potential for a single disulfide bond. To address the significance of these modifications in human IL-24, we analyzed the relationship between post-translational modifications and the cytokine activity of the human IL-24 protein. In contrast to related interleukins, we identified a relationship between net glycosylation, protein solubility, and cytokine activity. In addition, abrogation of the two cysteine residues by mutagenesis dramatically altered the ability of IL-24 to secrete from host cells and resulted in the concomitant loss of IL-24 activity. We conclude that, unlike other IL-10 family members, human IL-24 must be glycosylated to maintain solubility and bioavailability. Further, a single, unique disulfide bond is required for secretion and activity. These structure-function relationships show that, although IL-24 is a member of the IL-19 subfamily of IL-10-like cytokines by sequence similarity, its surface properties and its distinctive disulfide arrangement make it unique. These observations could explain the novel biological activities measured of this cytokine. Understanding the structural basis of IL-24 activity will be important in the interpretation of the function of this cytokine and in the development of scale-up strategies for biophysical and clinical applications.Melanoma differentiation-associated gene 7 was identified as a novel tumor suppressor gene in human melanoma cells (1). Because of its physical location within the IL-10⁴ gene cluster, its homology with the IL-10 protein and its cytokine activity, melanoma differentiation-associated gene 7 was reclassified as interleukin-24 (IL-24) (2). Gene delivery of IL-24 using plasmid or adenoviral vectors has demonstrated that the product of the human IL-24 gene exhibits powerful tumor specific pro-apoptotic, growth inhibitory, and anti-angiogenic activities (3). In primary human endothelial cells, IL-24 interacts with a unique set of cell surface receptors, IL-22R1/IL-20R2 (4, 5), resulting in potent anti-angiogenic activity (3). In tumor cells that express these receptors, IL-24 induces apoptosis; however, no cytotoxicity is observed in normal cells that also express the IL-24 receptors (6). Given these properties, IL-24 is now being considered as a promising new bio-therapeutic agent in the treatment of various cancers (7, 8).IL-24 has been categorized as a member of the IL-19 subfamily of IL-10-like cytokines (9). This subfamily includes IL-19, IL-20, IL-22, and IL-24. The three-dimensional structures of two of the four IL-19 subfamily members, IL-19 and IL-22, have been determined to high resolution (10–12). Like IL-10, both IL-19 and IL-22 are composed of a bundle of 6 α-helices; however, unlike IL-10, these cytokines are active as monomers. Each of these cytokines possesses a set of highly conserved disulfide bonds, which are characteristic of each cytokine. Presumably, these disulfide bridges stabilize the α-helical structure for efficient receptor engagement in the extracellular environment (13). IL-24 is the lone exception, because it is missing two complementary cysteine residues that form consensus disulfide bonds in the other members of this family.In many of the known IL-10-like cytokines (IL-10, IL-20, IL-22, and others), glycosylation is not required for activity. Either the proteins are not glycosylated, as is the case for IL-10 and IL-20, or the cytokine is glycosylated, but the glycan can be removed either enzymatically or by mutagenesis without compromising activity, as is the case for IL-22. Refolding IL-22 from bacterially expressed protein has no effect on the cytokine activity or the overall three-dimensional structure (11, 14). Although similar claims have been reported for IL-24 (15–17), these analyses relied on very small amounts of protein, so it was difficult to accurately associate protein structure with function. It is also clear that IL-24 is not as robust as other related cytokines, because several groups have shown varied responses of this cytokine in different biological assays (18, 19). One possibility to explain the discrepancy between IL-24 and the other IL-19-like cytokines is that the IL-24 protein possesses unique structural features that contribute both to its distinctive biology and to the characteristic properties of the protein.In this study, we utilized cytokine activity assays in tandem with genomic and biophysical analyses to assess the role of post-translational modifications in IL-24. We found that, unlike the other members of the IL-19 subfamily, human IL-24 requires at least two contiguous glycosylated sites for efficient secretion and activity. These neighboring glycan groups likely mask a non-polar region on the surface of IL-24 located near the putative receptor binding site on helix B. Further, the IL-24 protein possesses a novel pairing of disulfide bonds that has not been identified in related interleukins. Understanding the structural characteristics of human IL-24 will be essential for its development as a therapeutic cytokine.     

Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION*

The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075–11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis.     

免疫细胞在棕色脂肪产热及白色脂肪棕色化过程中的功能研究进展

肥胖已经成为威胁人类健康的全球性问题,棕色脂肪(Brown adipose tissue,BAT)及米色脂肪因其能够通过产热作用增加能量消耗这一特性,已成为一种备受关注的潜在肥胖治疗方法。近年来的研究发现M2型巨噬细胞(Alternatively activated macrophages,M2 type)能够促进BAT产热和白色脂肪(White adipose tissue,WAT)的棕色化(即米色脂肪的形成过程),但随后的一些研究却得到了相反的结论。到目前为止,M2型巨噬细胞是否参与促进WAT的棕色化过程仍是一个备受争议的话题。主要对M2型巨噬细胞、II型固有淋巴细胞(Type 2 Innate Lymphoid Cells,ILC2s)和嗜酸性粒细胞(Eosinophils)对BAT产热和WAT的棕色化的促进作用,以及M2型巨噬细胞不参与/抑制WAT棕色化这两个方面的研究状况做一综述。     

Proteome of Acidic Phospholipid-binding Proteins: SPATIAL AND TEMPORAL REGULATION OF CORONIN 1A BY PHOSPHOINOSITIDES*

Reversible interactions between acidic phospholipids in the cellular membrane and proteins in the cytosol play fundamental roles in a wide variety of physiological events. Here, we present a novel approach to the identification of acidic phospholipid-binding proteins using nano-liquid chromatography-tandem mass spectrometry. We found more than 400 proteins, including proteins with previously known acidic phospholipid-binding properties, and confirmed that several candidates, such as Coronin 1A, mDia1 (Diaphanous-related formin-1), PIR121/CYFIP2, EB2 (end plus binding protein-2), KIF21A (kinesin family member 21A), eEF1A1 (translation elongation factor 1α1), and TRIM2, directly bind to acidic phospholipids. Among such novel proteins, we provide evidence that Coronin 1A activity, which disassembles Arp2/3-containing actin filament branches, is spatially and temporally regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Whereas Coronin 1A co-localizes with PI(4,5)P₂ at the plasma membrane in resting cells, it is dissociated from the plasma membrane during lamellipodia formation where the PI(4,5)P₂ signal is significantly reduced. Our in vitro experiments show that Coronin 1A preferentially binds to PI(4,5)P₂-containing liposomes and that PI(4,5)P₂ antagonizes the ability of Coronin 1A to disassemble actin filament branches, indicating a spatiotemporal regulation of Coronin 1A via a direct interaction with the plasma membrane lipid. Collectively, our proteomics data provide a list of potential acidic phospholipid-binding protein candidates ranging from the actin regulatory proteins to translational regulators.     

Characteristic Expression of Extracellular Matrix in Subcutaneous Adipose Tissue Development and Adipogenesis; Comparison with Visceral Adipose Tissue

Adipose tissue is a connective tissue specified for energy metabolism and endocrines, but functional differences between subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) have not been fully elucidated. To reveal the physiological role of SAT, we characterized in vivo tissue development and in vitro adipocyte differentiation. In a DNA microarray analysis of SAT and VAT in Wistar rats, functional annotation clusters of extracellular matrix (ECM)-related genes were found in SAT, and major ECM molecules expressed in adipose tissues were profiled. In a histological analysis and quantitative expression analysis, ECM expression patterns could be classified into two types: (i) a histogenesis-correlated type such as type IV and XV collagen, and laminin subunits, (ii) a high-SAT expression type such as type I, III, and V collagen and minor characteristic collagens. Type (i) was related to basal membrane and up-regulated in differentiated 3T3-L1 cells and in histogenesis at depot-specific timings. In contrast, type (ii) was related to fibrous forming and highly expressed in 3T3-L1 preadipocytes. Exceptionally, fibronectin was abundant in developed adipose tissue, although it was highly expressed in 3T3-L1 preadipocytes. The present study showed that adipose tissues site-specifically regulate molecular type and timing of ECM expression, and suggests that these characteristic ECM molecules provide a critical microenvironment, which may affect bioactivity of adipocyte itself and interacts with other tissues. It must be important to consider the depot-specific property for the treatment of obesity-related disorders, dermal dysfunction and for the tissue regeneration.     

外源性褪黑激素对黄鳝性腺发育及性腺激素分泌的影响

通过注射实验研究了外源性褪黑激素对黄鲜性腺发育及性腺激素分泌的影响。结果表明,处于不同性腺发育时期的黄鲜对餐源褪黑激素的反应存在季节性差异。上述结果暗示,黑激素影响黄鲜性腺发育的关键时间可能是在性腺静止期,亦即Ⅳ期或／和Ⅳ期之前。     

STIGMA OF SOLANUM TUBEROSUM CV SHEPODY: MORPHOLOGY,ULTRASTRUCTURE,AND SECRETION

The stigma of Solarium tuberosum L. cv Shepody has a bilobed papillate surface covered with a viscous secretion at anthesis. The secretion originates as osmiophilic droplets in the cytoplasm, accumulates in the intercellular space, and fills the base of the papillae, after lifting and rupturing the cuticular layer covering the stigma surface. Cytochemical evidence shows that the stigmatic secretion is lipidic in nature; it did not stain with the periodic acid-Schiffs reaction for carbohydrate or Coomassie Brilliant Blue R-250 for proteins, but did stain with Sudan black or Nile red, a fluorescence probe for lipids. Sodium dodecylsulfate-polyacrylamide gel electrophoresis has revealed that the secretion contained three polypeptides that appeared to be 'indigenous' to the stigmatic secretion, and not contaminants. Comparative analyses of this and other proteins found in stigma secretions may provide clues to their possible roles in pollen grain adhesion and germination.     

Metabolic Dysregulation and Adipose Tissue Fibrosis: Role of Collagen VI

Adipocytes are embedded in a unique extracellular matrix whose main function is to provide mechanical support, in addition to participating in a variety of signaling events. During adipose tissue expansion, the extracellular matrix requires remodeling to accommodate adipocyte growth. Here, we demonstrate a general upregulation of several extracellular matrix components in adipose tissue in the diabetic state, therefore implicating “adipose tissue fibrosis” as a hallmark of metabolically challenged adipocytes. Collagen VI is a highly enriched extracellular matrix component of adipose tissue. The absence of collagen VI results in the uninhibited expansion of individual adipocytes and is paradoxically associated with substantial improvements in whole-body energy homeostasis, both with high-fat diet exposure and in the ob/ob background. Collectively, our data suggest that weakening the extracellular scaffold of adipocytes enables their stress-free expansion during states of positive energy balance, which is consequently associated with an improved inflammatory profile. Therefore, the disproportionate accumulation of extracellular matrix components in adipose tissue may not be merely an epiphenomenon of metabolically challenging conditions but may also directly contribute to a failure to expand adipose tissue mass during states of excess caloric intake.Adipose tissue is a key regulator of systemic energy homeostasis. The physiological state of adipose tissue is driven by cell-autonomous processes within the adipocyte. In addition to this, the adipocyte itself is subject to major modifications by other cell types that infiltrate adipose tissue, such as macrophages and vascular cells; moreover, adipocytes can be markedly influenced by several hormones and cytokines that circulate systemically.Although all these cellular interactions have been the subject of extensive studies in numerous laboratories, the extracellular matrix of adipose tissue has received limited attention to date, despite evidence suggesting that it is a functionally relevant constituent of adipose tissue physiology.It is currently unknown what consequential effects metabolic stress exerts on the extracellular matrix and vice versa. In other words, what is the impact of dysregulation of the extracellular constituents of adipose tissue on the systemic metabolic state? Here, we approach this subject from two different perspectives. We first assessed the overall level of extracellular matrix components under different metabolic conditions and established that the extracellular constituents are globally upregulated during metabolically challenging conditions. We then selected a specific member of the collagen family, collagen VI (exhibiting predominant expression in adipose tissue), and utilized a genetic model of collagen VI disruption to investigate the effects of disruption of the extracellular matrix of adipose tissue. Remarkably, our studies demonstrated that such weakening of adipose tissue extracellular matrix results in considerable improvement of the metabolic phenotype in the context of both a high-fat diet and a challenge with the ob/ob mutation.Our observations highlight the extracellular matrix of adipose tissue as an important and novel site of modulation of systemic metabolism. Obese adipose tissue displays hallmarks similar to other fibrotic tissues, such as the liver; this suggests that specific constituents of this normally rather rigid extracellular matrix environment may provide possible targets for pharmacological intervention for the treatment of metabolic disorders.     

Insulin Signaling in Type 2 Diabetes: EXPERIMENTAL AND MODELING ANALYSES REVEAL MECHANISMS OF INSULIN RESISTANCE IN HUMAN ADIPOCYTES*

Type 2 diabetes originates in an expanding adipose tissue that for unknown reasons becomes insulin resistant. Insulin resistance reflects impairments in insulin signaling, but mechanisms involved are unclear because current research is fragmented. We report a systems level mechanistic understanding of insulin resistance, using systems wide and internally consistent data from human adipocytes. Based on quantitative steady-state and dynamic time course data on signaling intermediaries, normally and in diabetes, we developed a dynamic mathematical model of insulin signaling. The model structure and parameters are identical in the normal and diabetic states of the model, except for three parameters that change in diabetes: (i) reduced concentration of insulin receptor, (ii) reduced concentration of insulin-regulated glucose transporter GLUT4, and (iii) changed feedback from mammalian target of rapamycin in complex with raptor (mTORC1). Modeling reveals that at the core of insulin resistance in human adipocytes is attenuation of a positive feedback from mTORC1 to the insulin receptor substrate-1, which explains reduced sensitivity and signal strength throughout the signaling network. Model simulations with inhibition of mTORC1 are comparable with experimental data on inhibition of mTORC1 using rapamycin in human adipocytes. We demonstrate the potential of the model for identification of drug targets, e.g. increasing the feedback restores insulin signaling, both at the cellular level and, using a multilevel model, at the whole body level. Our findings suggest that insulin resistance in an expanded adipose tissue results from cell growth restriction to prevent cell necrosis.     

Changes in Intracellular Distribution of Thiol Groups During the Development of Acetabularia mediterranea

A cytochemical study of intracellular thiol distribution inAcetabularia mediterranea intact cells was performed using thefluorescent thiol-labelling agent monobromobimane (mBBr). Differentdevelopmental stages were examined during the vegetative phaseand generative phase of the algal life cycle up to cyst maturation.Important changes in thiol localization have been found to coincidewith turning-points ofAcetabularia development. During the rapid growth phase, overall thiol content steadilyincreased along the stalk, being maximal shortly before capdifferentiation. At this stage, the thiol distribution patternparalleled that of cap morphogenesis essential processes: thiolsbeing accumulated at the apex where morphogenesis is going tobe expressed. High thiol contents were also present in the rhizoidal partof the alga throughout the vegetative phase. At the onset ofthe generative phase, important alterations in rhizoid thioldistribution coincided with the presumptive time of nucleardivision. Overall thiol content strongly decreased and thiolsbecame highly concentrated in definite zones localized in thecentral area of the rhizoid. Later on, during the sequence of morphogenetic events leadingto cyst differentiation, changes in thiol localization and relativecontent were observed in the cap rays. Positioning of secondarynuclei into the cap coincided with a high increase in thiolcontent in the entire cap. During the process of cyst formation,thiol content slightly decreased and thiols were localized incyst domains. Thiol distribution was also studied during regenerative processesafter merotomy. A spatio-temporal coincidence was shown withcell wall regeneration. Key words: Acetabularia, thiols, development, bromobimanes