In Vivo Response to α1‐Adrenoreceptor Stimulation in Human White Adipose Tissue

Objective: Recent studies in rats suggest an important effect of α₁‐adrenoreceptor stimulation on glucose uptake in white adipocytes. It is not known if α₁‐adrenoreceptor stimulation elicits similar metabolic effects in humans. Research Methods and Procedures: Three microdialysis catheters in abdominal subcutaneous adipose tissue were perfused with 0.00, 0.01, 0.10, 1.00, and 10.00 μM isoproterenol, phenylephrine, or phenylephrine plus 100 μM propranolol. Dialysate concentrations of ethanol, glycerol, glucose, and lactate were measured for estimating blood flow (ethanol‐dilution technique), lipolysis, and glycolysis, respectively. Results: Phenylephrine, with or without propranolol, did not elicit a change in ethanol ratio. In contrast, the ethanol ratio decreased markedly with isoproterenol. Dialysate glucose concentration decreased with phenylephrine with and without propranolol and increased with isoproterenol. Phenylephrine caused a dose‐dependent increase in dialysate glycerol concentration, with a maximal effect similar to that of isoproterenol. The effect was attenuated with propranolol. Discussion: Our findings suggest that α₁‐adrenoreceptor stimulation by phenylephrine increases glucose uptake and metabolism in human abdominal adipose tissue. Furthermore, phenylephrine elicits a marked increase in lipolytic activity in white adipose tissue through β‐adrenoreceptor activation.     

α1‐adrenergic stimulation mediates Ca2+‐dependent inositol phosphate formation through the α1B‐like adrenoceptor subtype in adult rat cardiac myocytes

We studied the effects of increased Ca²⁺ influx on α₁‐adrenoceptor‐stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific α₁‐adrenoceptor subtype. [³H]InsP responses to adrenaline were dependent on extracellular Ca²⁺ concentration, from 0.1 μM to 2 mM, and were completely blocked by Ca²⁺ removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca²⁺ concentrations higher than 1 μM had no effect on adrenaline‐stimulated [³H]InsP formation. Taken together these results suggest that [³H]InsP formation induced by α₁‐adrenergic stimulation is in part mediated by increased Ca²⁺ influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [³H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the α_1B‐adrenoceptor alkylating agent, CEC, [³H]InsP formation remained unaffected by increased Ca²⁺ concentrations, a pattern similar to that observed when intracellular Ca²⁺ was chelated with BAPTA. In contrast, addition of the α_1A‐subtype antagonist, 5′‐methyl urapidil, did not affect the Ca²⁺ dependence of [³H]InsP formation. Neither nifedipine, a voltage‐dependent Ca²⁺ channel blocker nor the inorganic Ca²⁺ channel blockers, Ni²⁺ and Co²⁺, had any effect on adrenaline stimulated [³H]InsP, at concentrations that inhibit Ca²⁺ channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein‐mediated response, α₁‐adrenergic‐stimulated [³H]InsP formation is activated by increased Ca²⁺ influx mediated by the α_1B‐subtype. J. Cell. Biochem. 84: 201–210, 2002. © 2001 Wiley‐Liss, Inc.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Norepinephrine Induces Apoptosis in Skin Melanophores by Attenuating cAMP‐PKA Signals Via α2‐Adrenoceptors in the Medaka,Oryzias Latipes

The density of skin melanophores in many teleost fish decreases during long‐term adaptation to a white background. Using the medaka, Oryzias latipes, we previously reported that apoptosis is responsible for the decrease in melanophores, and that a sympathetic neurotransmitter, norepinephrine (NE), induces their apoptosis in skin tissue cultures. In this study, we show that NE‐induced apoptosis of melanophores is mediated by the activation of α2‐adrenoceptors. Clonidine, an α2‐adrenoceptor agonist, induced apoptotic melanophore death in skin organ culture, while phenylephrine, an α1‐adrenoceptor agonist, had no effect. NE‐induced apoptosis was diminished by an α2‐adrenoceptor antagonist, yohimbine, but an α1‐adrenoceptor antagonist, prazosin, did not abrogate the effect of NE. Furthermore, forskolin inhibited NE‐induced apoptosis, while an inhibitor of PKA, H‐89, mimicked the effect of NE. These results suggest that NE induces apoptosis in melanophores by attenuating cAMP‐PKA signaling via α2‐adrenoceptors.     

Hyperphagia and Obesity in Na,K‐ATPase α2 Subunit‐Defective Mice

Objective: The Na,K‐ATPase α2 subunit gene (Atp1a2) is expressed in the brain, skeletal muscles, heart, and adipocytes. Specific function of the α2 subunit, such as involvement in differentiation and function of adipocytes, has not been addressed. The aim of this study was to examine whether Atp1a2‐defective heterozygous mice show obesity and reveal the mechanisms underlying the obesity. Research Methods and Procedures: We measured the differentiation and glucose uptake function of in vitro‐differentiated adipocytes derived from embryonic fibroblasts of Atp1a2‐defective mice. Food intake, body temperature, metabolic rate, and spontaneous activity and mRNA levels of neuropeptide genes were compared between the heterozygous and wild‐type adult mice. Results: Atp1a2 heterozygous female mice developed obesity after middle age. The time course of in vitro adipocyte differentiation of embryonic fibroblasts isolated from wild type, heterozygous, and homozygous mice was not different, glucose and Rb uptake activities of the in vitro‐differentiated adipocytes were not altered, and the effects of insulin on glucose uptake and those of monensin and ouabain on Rb uptake were similar among the genotypes. However, food intake in the light phase was significantly greater in the heterozygous mice than the wild type in the 24‐hour dark‐light cycle, whereas it was similar under constant‐light condition. Body temperature, metabolic rate at rest, and spontaneous motor activity of the heterozygous mice were similar to those of the wild type. Orexin mRNA level was lower in heterozygous than wild‐type mice. Discussion: The Na,K‐ATPase α2 subunit is not involved in the differentiation or in glucose and Rb uptake function of in vitro‐differentiated adipocytes. Hyperphagia is the likely primary cause of obesity in Atp1a2 heterozygous mice.     

Studies of phosphodiesterase effects on adipose tissue metabolism in obese subjects by the microdialysis technique.

The effect of non-selective (theophylline) inhibition of cyclic AMP breakdown on norepinephrine stimulated lipolysis rate was investigated in subcutaneous adipose tissue of obese subjects. In addition, changes in interstitial glucose and lactate concentration were assessed by means of the microdialysis technique. The interaction of endogenous released insulin and theophylline on adipocyte metabolism was determined. Theophylline and norepinephrine alone increased glycerol outflow significantly. When both agents were perfused in combination, interstitial glycerol concentration increased further. The enhanced glycerol level due to theophylline application was slightly decreased by insulin. In the presence of theophylline, extracellular glucose concentration increased, in contrast to the catecholamine. Norepinephrine decreased interstitial glucose level. When both drugs were added in combination, the level of interstitial glucose increased to about 1 mM, greater than with theophylline alone. With each intervention, lactate was synthesized. Local adipose tissue blood flow was increased by theophylline and theophylline plus norepinephrine. In conclusion, post-receptor mechanisms increased norepinephrine maximal stimulated lipolysis rate in subcutaneous adipose tissue. Glucose uptake was inhibited by the non-specific inhibitor of phosphodiesterase. The effect of insulin on inhibition of lipolysis was modest but sustained in the presence of high theophylline (10(-4) M) concentration. Phosphodiesterase activity may be relatively low in obese subjects in comparison with lean subjects. In lean subjects theophylline caused a transient reversal of the antilipolytic effect of insulin.     

Insulin‐ and Exercise‐Stimulated Skeletal Muscle Blood Flow and Glucose Uptake in Obese Men

Objective: Insulin resistance in obese subjects results in the impaired use of glucose by insulin‐sensitive tissues, e.g., skeletal muscle. In the present study, we determined whether insulin resistance in obesity is associated with an impaired ability of exercise to stimulate muscle blood flow, oxygen delivery, or glucose uptake. Research Methods and Procedures: Nine obese (body mass index = 36 ± 2 kg/m²) and 11 age‐matched nonobese men (body mass index = 22 ± 1 kg/m²) performed one‐legged isometric exercise during hyperinsulinemia. Rates of femoral muscle blood flow, oxygen consumption, and glucose uptake were measured simultaneously in both legs using [¹⁵O]H₂O, [¹⁵O]O₂, [¹⁸F]fluoro‐deoxy‐glucose, and positron emission tomography. Results: The obese subjects exhibited resistance to insulin stimulation of glucose uptake in resting muscle, regardless of whether glucose uptake was expressed per kilogram of femoral muscle mass (p = 0.001) or per the total mass of quadriceps femoris muscle. At similar workloads, oxygen consumption, blood flow, and glucose uptake were lower in the obese than the nonobese subjects when expressed per kilogram of muscle, but similar when expressed per quadriceps femoris muscle mass. Discussion: We conclude that obesity is characterized by insulin resistance of glucose uptake in resting skeletal muscle regardless of how glucose uptake is expressed. When compared with nonobese individuals at similar absolute workloads and under identical hyperinsulinemic conditions, the ability of exercise to increase muscle oxygen uptake, blood flow, and glucose uptake per muscle mass is blunted in obese insulin‐resistant subjects. However, these defects are compensated for by an increase in muscle mass.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Impaired integrin α5/β1‐mediated hepatocyte growth factor release by stellate cells of the aged liver

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α₅/β₁ is an important mechanosensor in HSC involved in shear stress‐induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α₅ and β₁ decreases in liver and HSC from aged rats. CRISPR/Cas9‐mediated integrin α₅ and β₁ knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α₅ and laminin gene expression whereas integrin β₁ remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin‐521 are lowered. The integrin α₅ knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin‐521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence‐associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α₅/β₁ in HSC contributes to age‐related reduction of ECM and HGF release that could affect liver regeneration.     

Expression of α2A adrenoceptors during rat neocortical development

Norepinephrine has been suggested to play a neurotrophic role during development and is present in the brain as early as embryonic day (E) 12. We have recently demonstrated that the α_2A adrenoceptor subtype is widely expressed during times of neuronal migration and differentiation throughout the developing brain. Here, we report the temporal and spatial expression pattern of α_2A adrenoceptors in neocortex during late embryonic and early postnatal development using in situ hybridization and receptor autoradiography. Functional α₂ receptors in embryonic rat cortex were also detected using agonist stimulated [³⁵S]GTPγS autoradiography. Both α_2A mRNA and protein expression were strongly increased by E19 and E20, respectively. The increased expression was in the cortical plate and intermediate and subventricular zones, corresponding to tiers of migrating and differentiating neurons. This transient up‐regulation of α_2A adrenoceptors was restricted to the lateral neocortex. At E20, functional α₂ adrenoceptors were also detected in deep layers of lateral neocortex. During the first week of postnatal development, the expression of α_2A mRNA and protein changed markedly, giving rise to a more mature pattern of anatomical distribution. The temporal and spatial distribution of α_2A adrenoceptors in developing neocortex is consistent with expression of functional proteins on migrating and differentiating layer IV to II neurons. These findings suggest that α_2A receptors may mediate a neurotrophic effect of norepinephrine during fetal cortical development. The early delineation of the lateral neocortex, which will develop into somatosensory and auditory cortices, suggests an intrinsic regulation of α_2A mRNA expression. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 259–269, 1999     

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

RalA GTPase Tethers Insulin Granules to L‐ and R‐Type Calcium Channels Through Binding α2δ‐1 Subunit

RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage‐gated calcium (Ca_v) channels. RalA knockdown (KD) in INS‐1 cells and primary rat β‐cells resulted in a reduction in Ca²⁺ currents arising specifically from L‐(Ca_v1.2 and Ca_v1.3) and R‐type (Ca_v2.3) Ca²⁺ channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca²⁺ currents. RalA co‐immunoprecipitated with the Ca_vα₂δ‐1 auxiliary subunit known to bind the three Ca_vs. Moreover, the functional molecular interactions between Ca_vα₂δ‐1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of α₂δ‐1 to insulin granules without affecting the localization of the other Ca_v subunits. Furthermore, we confirmed that RalA and α₂δ‐1 functionally interact since RalA KD‐induced inhibition of Ca_v currents could not be recovered by RalA when α₂δ‐1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α₂δ‐1 on insulin granules to tether these granules to PM Ca²⁺ channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.     

Epitope analysis of the multiphosphorylated peptide αs1‐casein(59–79)

The multiphosphorylated tryptic peptide α_s1‐casein(59–79) has been shown to be antigenic with anti‐casein antibodies. In an approach to determine the amino acyl residues critical for antibody binding we undertook an epitope analysis of the peptide using overlapping synthetic peptides. With α_s1‐casein(59–79) as the adsorbed antigen in a competitive ELISA only two of five overlapping synthetic peptides at 1 mM significantly inhibited binding of the anti‐casein antibodies. Peptides Glu‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu and Ile‐Val‐Pro‐Asn‐Ser(P)‐Val‐Glu‐Glu inhibited antibody binding by 20.0±3.6% and 60.3±7.9%, respectively. The epitope of Glu⁶³‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu⁷⁰ was further localised to the phosphoseryl cluster as the peptide Ser(P)‐Ser(P)‐Ser(P) significantly inhibited binding of the anti‐casein antibodies to α_s1‐casein(59–79) by 29.5±7.4%. Substitution of Ser(P)⁷⁵ with Ser⁷⁵ in the second inhibitory peptide Ile‐Val‐Pro‐Asn‐Ser(P)⁷⁵‐Val‐Glu‐Glu also abolished inhibition of antibody binding to α_s1‐casein (59–79) demonstrating that Ser(P)⁷⁵ is also a critical residue for recognition by the antibodies. These data show that the phosphorylated residues in the cluster sequence ‐Ser(P)⁶⁶‐Ser(P)‐Ser(P)⁶⁸ and in the sequence ‐Pro⁷³‐Asn‐Ser(P)‐Val‐Glu⁷⁷‐ are critical for antibody binding to α_s1‐casein(59–79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Heat shock factor 1 deficiency via its downstream target gene αB‐crystallin (Hspb5) impairs p53 degradation

Heat shock factor Hsf1 regulates the stress‐inducibility of heat shock proteins (Hsps) or molecular chaperones. One of the functions attributed to Hsps is their participation in folding and degradation of proteins. We recently showed that hsf1^?/? cells accumulate ubiquitinated proteins. However, a direct role for Hsf1 in stability of specific proteins such as p53 has not been elucidated. We present evidence that cells deficient in hsf1 accumulate wild‐type p53 protein. We further show that hsf1^?/? cells express lower levels of αB‐crystallin and cells deficient in αB‐crystallin also accumulate p53 protein. Reports indicate that αB‐crystallin binds to Fbx4 ubiquitin ligase, and they target cyclin D1 for degradation through a pathway involving the SCF (Skp1‐Cul1‐F‐box) complex. Towards determining a mechanism for p53 degradation involving αB‐crystallin and Hsf1, we have found that ectopic expression of Fbx4 in wild‐type mouse embryo fibroblasts (MEFs) expressing mutant p53 (p53R175H) leads to increase in its degradation, while MEFs deficient in hsf1 or αBcry are defective in degradation of this p53 protein. In addition, immunoprecipitated p53R175H from wild‐type MEFs is able to pull‐down both αB‐crystallin and Fbx4. Finally, immunoprecipitated wild‐type p53 from doxorubicin treated U2OS cells can pull‐down endogenous αB‐crystallin and Fbx4. These results indicate that hsf1‐ and αBcry‐deficient cells accumulate p53 due to reduced levels of αB‐crystallin in these cells. Elevated levels of p53 in hsf1‐ and αBcry‐deficient cells lead to their increased sensitivity to DNA damaging agents. These data reveal a novel mechanism for protein degradation through Hsf1 and αB‐crystallin. J. Cell. Biochem. 107: 504–515, 2009. © 2009 Wiley‐Liss, Inc.     

Potential of the bean α‐amylase inhibitor αAI‐1 to inhibit α‐amylase activity in true bugs (Hemiptera)

True bugs (Hemiptera) are an important pest complex not controlled by Bt‐transgenic crops. An alternative source of resistance includes inhibitors of digestive enzymes, such as protease or amylase inhibitors. αAI‐1, an α‐amylase inhibitor from the common bean, inhibits gut‐associated α‐amylases of bruchid pests of grain legumes. Here we quantify the in vitro activity of α‐amylases of 12 hemipteran species from different taxonomic and functional groups and the in vitro inhibition of those α‐amylases by αAI‐1. α‐Amylase activity was detected in all species tested. However, susceptibility to αAI‐1 varied among the different groups. α‐Amylases of species in the Lygaeidae, Miridae and Nabidae were highly susceptible, whereas those in the Auchenorrhyncha (Cicadellidae, Membracidae) had a moderate susceptibility, and those in the Pentatomidae seemed to be tolerant to αAI‐1. The species with αAI‐1 susceptible α‐amylases represented families which include both important pest species but also predatory species. These findings suggest that αAI‐1‐expressing crops have potential to control true bugs in vivo.     

Rap1 controls activation of the αMβ2 integrin in a talin‐dependent manner

The small GTPase Rap1 and the cytoskeletal protein talin regulate binding of C3bi‐opsonised red blood cells (RBC) to integrin α_Mβ₂ in phagocytic cells, although the mechanism has not been investigated. Using COS‐7 cells transfected with α_Mβ₂, we show that Rap1 acts on the β₂ and not the α_M chain, and that residues 732–761 of the β₂ subunit are essential for Rap1‐induced RBC binding. Activation of α_Mβ₂ by Rap1 was dependent on W747 and F754 in the β₂ tails, which are required for talin head binding, suggesting a link between Rap1 and talin in this process. Using talin1 knock‐out cells or siRNA‐mediated talin1 knockdown in the THP‐1 monocytic cell line, we show that Rap1 acts upstream of talin but surprisingly, RIAM knockdown had little effect on integrin‐mediated RBC binding or cell spreading. Interestingly, Rap1 and talin influence each other's localisation at phagocytic cups, and co‐immunoprecipitation experiments suggest that they interact together. These results show that Rap1‐mediated activation of α_Mβ₂ in macrophages shares both common and distinct features from Rap1 activation of α_IIbβ₃ expressed in CHO cells. J. Cell. Biochem. 111: 999–1009, 2010. © 2010 Wiley‐Liss, Inc.     

Engineering the serpin α1‐antitrypsin: A diversity of goals and techniques

α₁‐Antitrypsin (α₁‐AT) serves as an archetypal example for the serine proteinase inhibitor (serpin) protein family and has been used as a scaffold for protein engineering for >35 years. Techniques used to engineer α₁‐AT include targeted mutagenesis, protein fusions, phage display, glycoengineering, and consensus protein design. The goals of engineering have also been diverse, ranging from understanding serpin structure–function relationships, to the design of more potent or more specific proteinase inhibitors with potential therapeutic relevance. Here we summarize the history of these protein engineering efforts, describing the techniques applied to engineer α₁‐AT, specific mutants of interest, and providing an appended catalog of the >200 α₁‐AT mutants published to date.     

Endogenous CSE/H2S system mediates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes

Tumour necrosis factor‐α (TNF‐ α)is a major contributor to the pathogenesis of insulin resistance associated with obesity and type 2 diabetes. It has been found that endogenous hydrogen sulfide (H₂S) contributes to the pathogenesis of diabetes. We have hypothesized that TNF‐α‐induced insulin resistance is involved in endogenous H₂S generation. The aim of the present study is to investigate the role of endogenous H₂S in TNF‐α‐induced insulin resistance by studying 3T3‐L1 adipocytes. We found that treatment of 3T3‐L1 adipocytes with TNF‐α leads to deficiency in insulin‐stimulated glucose consumption and uptake and increase in endogenous H₂S generation. We show that cystathionine γ‐lyase (CSE) is catalysed in 3T3‐L1 adipocytes to generate H₂S and that CSE expression and activity are upregulated by TNF‐α treatment. Inhibited CSE by its potent inhibitors significantly attenuates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes, whereas H₂S treatment of 3T3‐L1 adipocytes impairs insulin‐stimulated glucose consumption and uptake. These data indicate that endogenous CSE/H₂S system contributes to TNF‐α‐caused insulin resistance in 3T3‐L1 adipocytes. Our findings suggest that modulation of CSE/H₂S system is a potential therapeutic avenue for insulin resistance. Copyright © 2012 John Wiley & Sons, Ltd.     

XOMA 052, an anti‐IL‐1β monoclonal antibody,prevents IL‐1β‐mediated insulin resistance in 3T3‐L1 adipocytes

Objective:

Interleukin‐1β (IL‐1β) has recently been implicated as a major cytokine that is involved in the pancreatic islet inflammation of type 2 diabetes mellitus. This inflammation impairs insulin secretion by inducing beta‐cell apoptosis. Recent evidence has suggested that in obesity‐induced inflammation, IL‐1β plays a key role in causing insulin resistance in peripheral tissues.

Design and Methods:

To further investigate the pathophysiological role of IL‐1β in causing insulin resistance, the inhibitory effects of IL‐1β on several insulin‐dependent metabolic processes in vitro has been neutralized by XOMA 052. The role IL‐1β plays in insulin resistance in adipose tissue was assessed using differentiated 3T3‐L1 adipocytes and several parameters involved in insulin signaling and lipid metabolism were examined.

Results and Conclusion:

IL‐1β inhibited insulin‐induced activation of Akt phosphorylation, glucose transport, and fatty acid uptake. IL‐1β also blocked insulin‐mediated downregulation of suppressor of cytokine signaling‐3 expression. Co‐preincubation of IL‐1β with XOMA 052 neutralized nearly all of these inhibitory effects in 3T3‐L1 adipocytes. These studies provide evidence, therefore, that IL‐1β is a key proinflammatory cytokine that is involved in inducing insulin resistance. These studies also suggest that the monoclonal antibody XOMA 052 may be a possible therapeutic to effectively neutralize cytokine‐mediated insulin resistance in adipose tissue.