Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 87
Filtrar
Mais filtros

Base de dados
País/Região como assunto
Tipo de documento
Intervalo de ano de publicação
1.
Int J Obes (Lond) ; 44(2): 500-509, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-30705392

RESUMO

BACKGROUND: Obesity is associated with development of insulin resistance in adipose tissue (AT). Human obesity has been associated with increased glycogen deposition in adipocytes. Adipocytes synthesise glycogen prior to the formation of lipids. The present study examined adipose glycogen content in obese Zucker rats and the effect of fasting on glycogen-metabolising enzymes. We hypothesised that obesity imposes a blunted response to fasting through impaired activation of glycogen-metabolizing enzymes, which dampens glycogen mobilization in obese Zucker rats. METHODS: We investigated the effect of 24h fasting on AT glycogen metabolism in 12-week old obese Zucker rats. Epididymal fat pads were collected from rats fed ad-libitum and fasted for 24h. Glycogen content, glycogen synthase and phosphorylase enzyme activity, and PKA activity were analysed as well as total and phosphorylated protein content for glycogen-metabolizing enzymes glycogen synthase and phosphorylase, glucose transporter GLUT4, and cAMP-dependent response element binding protein levels. RESULTS: Twelve-week old obese Zucker rats showed increased AT glycogen content (adipose glycogen content [mean ± SD], lean: 3.95 ± 2.78 to 0.75 + 0.69 µg.mg-1; p < 0.005 fed vs fasted, and obese: 5.23 ± 3.38 to 5.019 ± 1.99 µg.mg-1; p = ns fed and fasted and p < 0.005 lean vs obese), and impaired fasting-induced glycogen mobilization following a 24h fast. These defects were associated with dysfunctional glycogen-metabolizing enzymes, characterized by: (1) blunted phosphorylation-mediated activation and downregulated protein expression of glycogen phosphorylase, and (2) an impaired phosphorylation-mediated inactivation of glycogen synthase. Furthermore, these defects were related to impaired fasting-induced protein kinase A (PKA) activation. CONCLUSION: This study provides evidence of a defective glycogen metabolism in the adipose associated with impaired fasting-induced activation of the upstream kinase protein kinase A, which render a converging point to obesity-related primary alterations in carbohydrate and lipid metabolism in the AT.


Assuntos
Tecido Adiposo/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Jejum/fisiologia , Glicogênio/metabolismo , Obesidade/metabolismo , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Feminino , Insulina/metabolismo , Masculino , Ratos , Ratos Zucker
2.
Chembiochem ; 19(3): 229-238, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29193585

RESUMO

The ß subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which exists as two isoforms (ß1 and ß2) in humans, has a carbohydrate-binding module (CBM) that interacts with glycogen. Although the ß1- and ß2-CBMs are structurally similar, with strictly conserved ligand-contact residues, they show different carbohydrate affinities. ß2-CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into ß2-CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why ß2-CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for ß2 and Thr for ß1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant ß2-Thr101Δ/Val134Thr differs from that of ß1-CBM, and mutant ß1-Thr101ins/Thr134Val differs from that of ß2-CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild-type ß-CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant ß1-CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of ß2-CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.


Assuntos
Proteínas Quinases Ativadas por AMP/química , Carboidratos/química , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Sítios de Ligação , Calorimetria , Espectroscopia de Ressonância Magnética , Mutagênese Sítio-Dirigida , Ratos , Termodinâmica
3.
J Occup Rehabil ; 28(4): 574-583, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29948473

RESUMO

Purpose Examine the potential for using information in short-term disability claims to identify workers at high risk of leaving the workforce and entering Social Security Disability Insurance (SSDI). Methods We analyze state-wide California data on claimants of State Disability Insurance (SDI) and Workers' Compensation (WC) and present statistics on: (1) characteristics (primary diagnosis, sex, age, geography, wage level) by claim duration (0-3, 4-6, 7-12, 12 + months); and (2) the ability of initial claim characteristics to predict duration of at least 12 months. All data are for claims with disability lasting more than 1 week. Results 22.2% of SDI claims last longer than 6 months and 12.5% last 12 months. More WC claims reach these durations: 33.7 and 18.6%, respectively. Long-term SDI and WC claimants are similar to SSDI awardees, nationwide, but differ in age distribution; they are typically younger. Conclusions Characteristics of SDI and WC claims can help predict claims likely to last 12 months, but more information is needed to effectively target early intervention services. Waiting longer to intervene improves targeting but risks missing opportunities where early intervention could be more effective. Collecting additional information at SDI or WC entry or soon thereafter could improve both the efficiency and timing of interventions.


Assuntos
Seguro por Deficiência/estatística & dados numéricos , Doenças Profissionais/reabilitação , Traumatismos Ocupacionais/reabilitação , Previdência Social/estatística & dados numéricos , Indenização aos Trabalhadores/estatística & dados numéricos , Adolescente , Adulto , California , Feminino , Previsões , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Fatores de Tempo , Adulto Jovem
4.
Am J Physiol Cell Physiol ; 311(1): C35-42, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27099349

RESUMO

The 5'-AMP-activated protein kinase (AMPK), a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, is activated in skeletal muscle in response to exercise and utilization of stored energy. The diffusibility properties of α- and ß-AMPK were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus and oxidative soleus muscles from which the surface membranes were removed by mechanical dissection. After the muscle segments were washed for 1 and 10 min, ∼60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. After in vitro stimulation of the muscle, which resulted in an ∼80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, as determined by addition of a wash step containing amylase. Stimulation of extensor digitorum longus muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target acetyl carboxylase-CoA. This, however, had no effect on the proportion of total ß2-AMPK that was phosphorylated in whole muscle homogenates measured by immunoprecipitation. These findings suggest that, in rat skeletal muscle, ß2-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate ß2-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of ß2-AMPK in skeletal muscle.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo Energético , Glicogênio/metabolismo , Contração Muscular , Fibras Musculares de Contração Rápida/enzimologia , Acetil-CoA Carboxilase/metabolismo , Animais , Estimulação Elétrica , Técnicas In Vitro , Masculino , Fosforilação , Ligação Proteica , Subunidades Proteicas , Ratos Sprague-Dawley , Treonina , Fatores de Tempo
5.
J Biol Chem ; 290(18): 11715-28, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25792737

RESUMO

The mammalian AMP-activated protein kinase (AMPK) is an obligatory αßγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the ß-subunit (AMPKß) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or ß1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKß is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glicogênio/metabolismo , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/genética , Ativação Enzimática , Células HEK293 , Células Hep G2 , Humanos , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Transporte Proteico , Treonina
6.
Plant J ; 82(2): 183-92, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25736509

RESUMO

SNF1-related protein kinase 1 (SnRK1) is the plant orthologue of the evolutionarily-conserved SNF1/AMPK/SnRK1 protein kinase family that contributes to cellular energy homeostasis. Functional as heterotrimers, family members comprise a catalytic α subunit and non-catalytic ß and γ subunits; multiple isoforms of each subunit type exist, giving rise to various isoenzymes. The Arabidopsis thaliana genome contains homologues of each subunit type, and, in addition, two atypical subunits, ß(3) and ßγ, with unique domain architecture, that are found only amongst plants, suggesting atypical heterotrimers. The AtSnRK1 subunit structure was determined using recombinant protein expression and endogenous co-immunoprecipitation, and six unique isoenzyme combinations were identified. Each heterotrimeric isoenzyme comprises a catalytic α subunit together with the unique ßγ subunit and one of three non-catalytic ß subunits: ß(1), ß(2) or the plant-specific ß(3) isoform. Thus, the AtSnRK1 heterotrimers contain the atypical ßγ subunit rather than a conventional γ subunit. Mammalian AMPK heterotrimers are phosphorylated on the T-loop (pThr175/176) within both catalytic a subunits. However, AtSnRK1 is insensitive to AMP and ADP, and is resistant to T-loop dephosphorylation by protein phosphatases, a process that inactivates other SNF1/AMPK family members. In addition, we show that SnRK1 is inhibited by a heat-labile, >30 kDa, soluble proteinaceous factor that is present in the lysate of young rosette leaves. Finally, none of the three SnRK1 carbohydrate-binding modules, located in the ß(1), ß(2) and ßγ subunits, associate with various carbohydrates, including starch, the plant analogue of glycogen to which AMPK binds in vitro. These data clearly demonstrate that AtSnRK1 is an atypical member of the SNF1/AMPK/SnRK1 family.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas Quinases Ativadas por AMP/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Imunoprecipitação , Isoenzimas/genética , Isoenzimas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
7.
Biochem J ; 468(2): 245-57, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25774984

RESUMO

AMP-activated protein kinase (AMPK) is an αßγ heterotrimer that is important in regulating energy metabolism in all eukaryotes. The ß-subunit exists in two isoforms (ß1 and ß2) and contains a carbohydrate-binding module (CBM) that interacts with glycogen. The two CBM isoforms (ß1- and ß2-CBM) are near identical in sequence and structure, yet show differences in carbohydrate-binding affinity. ß2-CBM binds linear carbohydrates with 4-fold greater affinity than ß1-CBM and binds single α1,6-branched carbohydrates up to 30-fold tighter. To understand these affinity differences, especially for branched carbohydrates, we determined the NMR solution structure of ß2-CBM in complex with the single α1,6-branched carbohydrate glucosyl-ß-cyclodextrin (gBCD) which supported the dynamic nature of the binding site, but resonance broadening prevented defining where the α1,6 branch bound. We therefore solved the X-ray crystal structures of ß1- and ß2-CBM, in complex with gBCD, to 1.7 and 2.0 Å (1 Å=0.1 nm) respectively. The additional threonine (Thr101) of ß2-CBM expands the size of the surrounding loop, creating a pocket that accommodates the α1,6 branch. Hydrogen bonds are formed between the α1,6 branch and the backbone of Trp99 and Lys102 side chain of ß2-CBM. In contrast, the α1,6 branch could not be observed in the ß1-CBM structure, suggesting that it does not form a specific interaction. The orientation of gBCD bound to ß1- and ß2-CBM is supported by thermodynamic and kinetic data obtained through isothermal titration calorimetry (ITC) and NMR. These results suggest that AMPK containing the muscle-specific ß2-isoform may have greater affinity for partially degraded glycogen.


Assuntos
Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Glicogênio/metabolismo , Oligossacarídeos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Isoenzimas , Cinética , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas , Especificidade por Substrato
8.
Clin Exp Pharmacol Physiol ; 42(4): 415-25, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25676548

RESUMO

Cardiac glycogen regulation involves a complex interplay between multiple signalling pathways, allosteric activation of enzymes, and sequestration for autophagic degradation. Signalling pathways appear to converge on glycogen regulatory enzymes via insulin (glycogen synthase kinase 3ß, protein phosphatase 1, allosteric action of glucose-6-phosphate), ß-adrenergic (phosphorylase kinase protein phosphatase 1 inhibitor), and 5' adenosine monophosphate-activated protein kinase (allosteric action of glucose-6-phosphate, direct glycogen binding, insulin receptor). While cytosolic glycogen synthesis and breakdown are relatively well understood, recent findings relating to phagic glycogen degradation highlight a new area of investigation in the heart. It has been recently demonstrated that a specific glycophagy pathway is operational in the myocardium. Proteins involved in recruiting glycogen to the forming phagosome have been identified. Starch-binding domain-containing protein 1 is involved in binding glycogen and mediating membrane anchorage via interaction with a homologue of the phagosomal protein light-chain 3. Specifically, it has been shown that starch-binding domain-containing protein 1 and light-chain 3 have discrete phagosomal immunolocalization patterns in cardiomyocytes, indicating that autophagic trafficking of glycogen and protein cargo in cardiomyocytes can occur via distinct pathways. There is strong evidence from glycogen storage diseases that phagic/lysosomal glycogen breakdown is important for maintaining normal cardiac glycogen levels and does not simply constitute a redundant 'alternative' breakdown route for glycogen. Advancing understanding of glycogen handling in the heart is an important priority with relevance not only to genetic glycogen storage diseases but also to cardiac metabolic stress disorders such as diabetes and ischaemia.


Assuntos
Doença de Depósito de Glicogênio/metabolismo , Glicogênio/metabolismo , Cardiopatias/metabolismo , Miocárdio/metabolismo , Animais , Metabolismo Energético , Doença de Depósito de Glicogênio/patologia , Doença de Depósito de Glicogênio/fisiopatologia , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Humanos , Cinética , Lisossomos/metabolismo , Miocárdio/patologia , Fagossomos/metabolismo , Transdução de Sinais
9.
J Bacteriol ; 196(11): 1941-9, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24610710

RESUMO

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-ß-cyclodextrin (Glc4-ß-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc4-ß-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-ß-cyclodextrin [(Glc4)2-ß-CD]; these were 6(1),6(3)- and 6(1),6(4)-dimaltotetraosyl-α-1,6-ß-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the kcat and Km values for transglycosylation were 1.78 × 10(3) s(-1) and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10(3) s(-1) and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the kcat/Km ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Glicogênio/metabolismo , Sulfolobus solfataricus/enzimologia , Sulfolobus solfataricus/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Sistema da Enzima Desramificadora do Glicogênio/genética , Glicosilação , Hidrólise , Especificidade por Substrato , Sulfolobus solfataricus/genética
10.
Am J Physiol Heart Circ Physiol ; 306(8): H1240-5, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24561860

RESUMO

Disturbed systemic glycemic and insulinemic status elicits cardiomyocyte metabolic stress and altered glucose handling. In diabetes, pathological myocardial glycogen accumulation occurs. Recently, evidence of a specific myocardial autophagic degradation pathway for glycogen ("glycophagy") has been reported, differentiated from the more well-characterized protein "macrophagy" pathway. The goal of this study was to identify potential mechanisms involved in cardiac glycogen accumulation, glycophagy, and macrophagy regulation using cultured neonatal rat ventricular myocytes (NRVMs). In NRVMs, insulin-induced Akt phosphorylation was evident with 5 mM-glucose conditions (∼2.3-fold increased). Under high-glucose (30 mM) conditions, insulin-augmented phosphorylation was not observed. Accumulation of glycogen was observed in response to insulin only in high-glucose conditions (∼2-fold increase). Increased expression of the glycophagy marker starch-binding domain-containing protein-1 (STBD1, 25% increase) was observed under high-glucose and insulin conditions. Expression levels of the macrophagy markers p62 and light chain protein 3BII:I were not increased by insulin at either glucose level. Preliminary results from hearts of streptozotocin-treated diabetic rats are supportive of the findings obtained in NRVMs, suggesting diabetes induced elevated expression of STBD1 and of an additional glycophagy marker GABA(A) receptor-associated protein-like 1. Confocal microscopy demonstrated that light chain protein 3B and STBD1 immunomarkers were not colocalized in NRVMs. These findings provide the first evidence that cardiomyocyte glycophagy induction occurs under the influence of insulin and is responsive to extracellular high glucose. This study suggests that the regulation of glycogen content and glycophagy induction in the cardiomyocyte may be linked, and it is speculated that glycogen pathology in diabetic cardiomyopathy has glycophagic involvement.


Assuntos
Glucose/farmacologia , Glicogênio/metabolismo , Insulina/fisiologia , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Autofagia , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Feminino , Glucose/metabolismo , Insulina/farmacologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
11.
Biomacromolecules ; 15(2): 660-5, 2014 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-24372590

RESUMO

Liver glycogen, a highly branched polymer of glucose, is important for maintaining blood-glucose homeostasis. It was recently shown that db/db mice, a model for Type 2 diabetes, are unable to form the large composite glycogen α particles present in normal, healthy mice. In this study, the structure of healthy mouse-liver glycogen over the diurnal cycle was characterized using size exclusion chromatography and transmission electron microscopy. Glycogen was found to be formed as smaller ß particles, and then only assembled into large α particles, with a broad size distribution, significantly after the time when glycogen content had reached a maximum. This pathway, missing in diabetic animals, is likely to give optimal blood-glucose control during the daily feeding cycle. Lack of this control may contribute to, or result from, diabetes. This discovery suggests novel approaches to diabetes management.


Assuntos
Glicemia/metabolismo , Ritmo Circadiano , Gorduras na Dieta/administração & dosagem , Glicogênio/química , Animais , Cromatografia em Gel , Glicogênio/isolamento & purificação , Glicogênio/metabolismo , Fígado/química , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Microscopia Eletrônica de Transmissão
12.
Proteome Sci ; 11(1): 11, 2013 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-23521774

RESUMO

BACKGROUND: Glycogen is a branched polysaccharide of glucose residues, consisting of α-1-4 glycosidic linkages with α-1-6 branches that together form multi-layered particles ranging in size from 30 nm to 300 nm. Glycogen spatial conformation and intracellular organization are highly regulated processes. Glycogen particles interact with their metabolizing enzymes and are associated with a variety of proteins that intervene in its biology, controlling its structure, particle size and sub-cellular distribution. The function of glycogen in adipose tissue is not well understood but appears to have a pivotal role as a regulatory mechanism informing the cells on substrate availability for triacylglycerol synthesis. To provide new molecular insights into the role of adipocyte glycogen we analyzed the glycogen-associated proteome from differentiated 3T3-L1-adipocytes. RESULTS: Glycogen particles from 3T3-L1-adipocytes were purified using a series of centrifugation steps followed by specific elution of glycogen bound proteins using α-1,4 glucose oligosaccharides, or maltodextrins, and tandem mass spectrometry. We identified regulatory proteins, 14-3-3 proteins, RACK1 and protein phosphatase 1 glycogen targeting subunit 3D. Evidence was also obtained for a regulated subcellular distribution of the glycogen particle: metabolic and mitochondrial proteins were abundant. Unlike the recently analyzed hepatic glycogen proteome, no endoplasmic proteins were detected, along with the recently described starch-binding domain protein 1. Other regulatory proteins which have previously been described as glycogen-associated proteins were not detected, including laforin, the AMPK beta-subunit and protein targeting to glycogen (PTG). CONCLUSIONS: These data provide new molecular insights into the regulation of glycogen-bound proteins that are associated with the maintenance, organization and localization of the adipocyte glycogen particle.

13.
Biophys J ; 102(2): 305-14, 2012 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-22339867

RESUMO

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the ß-subunit, for which there are two isoforms (ß(1) and ß(2)). Muscle-specific ß(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous ß(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of ß(2)-CBM is concurrent with an increase in flexibility in ß(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to ß(1)-CBM, unbound ß(2)-CBM showed microsecond-to-millisecond motion at the base of a ß-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this ß-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from ß(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the ß(2)-CBM mutant. Insertion of a threonine into the background of ß(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo dos Carboidratos , Modelos Moleculares , Movimento , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/genética , Sequência de Aminoácidos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato
14.
Am J Physiol Cell Physiol ; 303(11): C1146-55, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23015546

RESUMO

To understand how glycogen affects skeletal muscle physiology, we examined enzymes essential for muscle glycogen synthesis and degradation using single fibers from quiescent and stimulated rat skeletal muscle. Presenting a shift in paradigm, we show these proteins are differentially associated with glycogen granules. Protein diffusibility and/or abundance of glycogenin, glycogen branching enzyme (GBE), debranching enzyme (GDE), phosphorylase (GP), and synthase (GS) were examined in fibers isolated from rat fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscle. GDE and GP proteins were more abundant (~10- to 100-fold) in fibers from EDL compared with SOL muscle. GS and glycogenin proteins were similar between muscles while GBE had an approximately fourfold greater abundance in SOL muscle. Mechanically skinned fibers exposed to physiological buffer for 10 min showed ~70% total pools of GBE and GP were diffusible (nonbound), whereas GDE and GS were considerably less diffusible. Intense in vitro stimulation, sufficient to elicit a ~50% decrease in intracellular glycogen, increased diffusibility of GDE, GP, and GS (~15-60%) and decreased GBE diffusibility (~20%). Amylase treatment, which breaks α-1,4 linkages of glycogen, indicated differential diffusibilities and hence glycogen associations of GDE and GS. Membrane solubilization (1% Triton-X-100) allowed a small additional amount of GDE and GS to diffuse from fibers, suggesting the majority of nonglycogen-associated GDE/GS is associated with myofibrillar/contractile network of muscle rather than membranes. Given differences in enzymes required for glycogen metabolism, the current findings suggest glycogen particles have fiber-type-dependent structures. The greater catabolic potential of glycogen breakdown in fast-twitch fibers may account for different contraction induced rates of glycogen utilization.


Assuntos
Glicogênio/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Proteínas Musculares/metabolismo , Animais , Masculino , Ratos , Ratos Long-Evans
15.
J Biol Chem ; 286(48): 41331-41343, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-21976663

RESUMO

The binding by HIV-1 gp120 to CD4 and a chemokine receptor activates the membrane fusion glycoprotein, gp41. The fusion function of gp41 involves the refolding of its core into a 6-helix bundle, which apposes the lipophilic termini (the fusion peptide and transmembrane domain) and the associated cell and viral membranes, leading to their fusion. In this study, we examined the functional role of the polar segment and membrane proximal external region (MPER), which link the fusion peptide and transmembrane domain, respectively, to the core domain and interact to form a terminal clasp adjacent to the core. Limited proteolysis indicated that the terminal clasp is destabilized by simultaneous I535A/V539G mutations within the polar segment and mutations within the MPER. The destabilizing effects of I535A/V539G correlated with defective cell-cell fusion, viral entry, and viral replication. By using lipophilic and cytoplasmic fluorescent dye transfer assays, we found that terminal clasp destabilization is linked to a block in the lipid mixing/hemifusion phase of the membrane fusion cascade. Because the biosynthesis of the prefusion gp120-gp41 complex did not appear to be affected by I535A/V539G, we infer that the hemifusion block is due to a specific effect on the trimer of hairpins conformation of gp41. By contrast, the decreased fusion function of the MPER mutants correlated with a decrease in the interfacial hydropathy of the MPER sequence, suggesting that the prefusion Env complex had been adversely affected in these cases. These findings reveal a novel conserved functional target for the discovery of fusion inhibitors.


Assuntos
Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Dobramento de Proteína , Internalização do Vírus , Substituição de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Humanos , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
16.
Biomacromolecules ; 13(11): 3805-13, 2012 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-23004915

RESUMO

Glycogen, a hyperbranched complex glucose polymer, is an intracellular glucose store that provides energy for cellular functions, with liver glycogen involved in blood-glucose regulation. Liver glycogen comprises complex α particles made up of smaller ß particles. The recent discovery that these α particles are smaller and fewer in diabetic, compared with healthy, mice highlights the need to elucidate the nature of α-particle formation; this paper tests various possibilities for binding within α particles. Acid hydrolysis effects, examined using dynamic light scattering and size exclusion chromatography, showed that the binding is not simple α-(1→4) or α-(1→6) glycosidic linkages. There was no significant change in α particle size after the addition of various reagents, which disrupt disulfide, protein, and hydrogen bonds and hydrophobic interactions. The results are consistent with proteinaceous binding between ß particles in α particles, with the inability of protease to break apart particles being attributed to steric hindrance.


Assuntos
Glicogênio Hepático/química , Proteínas/metabolismo , Animais , Diabetes Mellitus , Dissulfetos/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Glicogênio Hepático/metabolismo , Camundongos , Ligação Proteica , Suínos
17.
Inquiry ; 49(2): 141-63, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22931021

RESUMO

This analysis used propensity score matching to construct a comparison sample that is observationally similar at baseline interview to older workers who later experience the onset of a medical condition that limits their ability to work. Using these matched onset and comparison samples, we studied trajectories in earnings and income around onset of the work limitation. Earnings two years after onset for the work-limitation group were 50% lower and poverty rates were nearly double. Income from unemployment insurance, workers' compensation, and retirement and disability benefits offset only a small amount of the earnings declines, resulting in decreased overall household income after onset of the work-limiting condition.


Assuntos
Envelhecimento , Pessoas com Deficiência/estatística & dados numéricos , Emprego/economia , Renda/estatística & dados numéricos , Assistência Pública/estatística & dados numéricos , Emprego/estatística & dados numéricos , Feminino , Nível de Saúde , Humanos , Cobertura do Seguro/estatística & dados numéricos , Seguro Saúde/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Indenização aos Trabalhadores/economia , Indenização aos Trabalhadores/estatística & dados numéricos
18.
Biomacromolecules ; 12(6): 1983-6, 2011 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-21591708

RESUMO

Glycogen is a highly branched glucose polymer functioning as a glucose buffer in animals. Multiple-detector size exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis were used to examine the structure of undegraded native liver glycogen (both whole and enzymatically debranched) as a function of molecular size, isolated from the livers of healthy and db/db mice (the latter a type 2 diabetic model). Both the fully branched and debranched levels of glycogen structure showed fundamental differences between glycogen from healthy and db/db mice. Healthy glycogen had a greater population of large particles, with more α particles (tightly linked assemblages of smaller ß particles) than glycogen from db/db mice. These structural differences suggest a new understanding of type 2 diabetes.


Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glicogênio Hepático , Animais , Glicemia/análise , Configuração de Carboidratos , Cromatografia em Gel , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/metabolismo , Eletroforese , Feminino , Humanos , Insulina/metabolismo , Fígado/química , Fígado/metabolismo , Glicogênio Hepático/química , Glicogênio Hepático/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Receptores para Leptina/genética , Receptores para Leptina/metabolismo
19.
Soc Secur Bull ; 71(3): 35-59, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21910298

RESUMO

We present longitudinal employment and work-incentive statistics for individuals who began receiving Social Security Disability Insurance (DI) benefits from 1996 through 2006. For the longest-observed cohort, 28 percent returned to work, 6.5 percent had their benefits suspended for work in at least 1 month, and 3.7 percent had their benefits terminated for work. The corresponding percentages are much higher for those who were younger than age 40 when they entered the DI program. Most first suspensions occurred within 5 years after entry. Cross-state variation in outcomes is high, and, to the extent observed, statistics for more recent cohorts are lower.


Assuntos
Pessoas com Deficiência/estatística & dados numéricos , Emprego/estatística & dados numéricos , Seguro por Deficiência/estatística & dados numéricos , Previdência Social/estatística & dados numéricos , Adolescente , Adulto , Estudos Transversais , Emprego/economia , Feminino , Humanos , Seguro por Deficiência/economia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Previdência Social/economia , Estados Unidos , United States Social Security Administration/economia , United States Social Security Administration/estatística & dados numéricos , Adulto Jovem
20.
Soc Secur Bull ; 71(3): 83-103, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21910300

RESUMO

We use a new variable in the Social Security Administration's Ticket Research File to produce statistics on the first month of suspension or termination for work (STW) for Social Security Disability Insurance (DI) and Supplemental Security Income (SSI)-only beneficiaries as well as on the number of months in nonpayment status following suspension or termination for work (NSTW) before their return to the rolls, attainment of the full retirement age, or death--in each year from 2002 through 2006. Less than 1 percent of beneficiaries experienced their first STW in each year, but more were in NSTW in at least 1 month. Ticket to Work (TTW) participants were more likely to have a first STW than nonparticipants, but most of those who had an STW were not TTW participants, reflecting low use of TTW. Employment networks often failed to file claims for outcome payments during months when their TTW clients were in NSTW.


Assuntos
Pessoas com Deficiência/estatística & dados numéricos , Readaptação ao Emprego/estatística & dados numéricos , Seguro por Deficiência/estatística & dados numéricos , Previdência Social/estatística & dados numéricos , Readaptação ao Emprego/economia , Humanos , Benefícios do Seguro/economia , Benefícios do Seguro/estatística & dados numéricos , Cobertura do Seguro/economia , Cobertura do Seguro/estatística & dados numéricos , Seguro por Deficiência/economia , Seguro por Deficiência/legislação & jurisprudência , Previdência Social/economia , Fatores de Tempo , Estados Unidos , United States Social Security Administration/economia , United States Social Security Administration/estatística & dados numéricos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA