RESUMO
BACKGROUND & AIMS: 3-Hydroxyisobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, which stimulates muscle fatty acid uptake, has been implicated in the pathogenesis of insulin resistance. We tested the hypothesis that circulating 3-HIB herald insulin resistance and that metabolic improvement with weight loss are related to changes in BCAAs and 3-HIB. METHODS AND RESULTS: We analyzed plasma and urine in 109 overweight to obese individuals before and after six months on hypocaloric diets reduced in either carbohydrates or fat. We calculated the homeostasis model assessment index (HOMA-IR) and whole body insulin sensitivity from oral glucose tolerance tests and measured intramyocellular fat by magnetic resonance spectroscopy. BCAAs and 3-HIB plasma concentrations were inversely related to insulin sensitivity but not to intramyocellular fat content at baseline. With 7.4 ± 4.5% weight loss mean BCAA and 3-HIB plasma concentrations did not change, irrespective of dietary macronutrient content. Individual changes in 3-HIB with 6-month diet but not BCAAs were correlated to the change in whole body insulin sensitivity and HOMA-IR independently of BMI changes. CONCLUSIONS: 3-HIB relates to insulin sensitivity but is not associated with intramyocellular fat content in overweight to obese individuals. Moreover, changes in 3-HIB rather than changes in BCAAs are associated with metabolic improvements with weight loss. Registration number for clinical trials: ClinicalTrials.gov Identifier: NCT00956566.
Assuntos
Aminoácidos de Cadeia Ramificada/sangue , Restrição Calórica , Dieta com Restrição de Carboidratos , Dieta com Restrição de Gorduras , Hidroxibutiratos/sangue , Resistência à Insulina , Obesidade/dietoterapia , Redução de Peso , Tecido Adiposo/metabolismo , Adulto , Biomarcadores/sangue , Glicemia/metabolismo , Feminino , Teste de Tolerância a Glucose , Humanos , Insulina/sangue , Espectroscopia de Ressonância Magnética , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Obesidade/sangue , Obesidade/diagnóstico , Estudos Prospectivos , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND & AIMS: Amino acids may interfere with insulin action, particularly in obese individuals. We hypothesized that increased circulating branched-chain and aromatic amino acids herald insulin resistance and ectopic fat storage, particularly hepatic fat accumulation. METHODS AND RESULTS: We measured fasting branched-chain and aromatic amino acids (tryptophan, tyrosine, and phenylalanine) by mass spectrometry in 111 overweight to obese subjects. We applied abdominal magnetic resonance imaging and spectroscopy to assess adipose tissue distribution and ectopic fat storage, respectively. Plasma branched-chain amino acids concentrations were related to insulin sensitivity and intrahepatic fat independent from adiposity, age and gender, but not to abdominal adipose tissue or intramyocellular fat. CONCLUSIONS: In weight stable overweight and obese individuals, branched-chain amino acid concentrations are specifically associated with hepatic fat storage and insulin resistance.
Assuntos
Adiposidade , Aminoácidos Aromáticos/sangue , Aminoácidos de Cadeia Ramificada/sangue , Proteínas Alimentares/sangue , Resistência à Insulina , Fígado/metabolismo , Obesidade/sangue , Adulto , Biomarcadores/sangue , Glicemia/metabolismo , Estudos Transversais , Feminino , Alemanha , Humanos , Insulina/sangue , Fígado/diagnóstico por imagem , Fígado/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Espectrometria de Massas , Metabolômica/métodos , Pessoa de Meia-Idade , Obesidade/diagnóstico por imagem , Obesidade/dietoterapia , Obesidade/fisiopatologiaRESUMO
Endurance training at an intensity eliciting maximal fat oxidation may have a beneficial effect on body weight and glucose metabolism in obese patients. However, the exercise intensity at which maximal fat oxidation occurs and the factors limiting fat oxidation are not well studied in this population. Obese, otherwise healthy men (n=38) and women (n=91) performed an incremental exercise test up to exhaustion on a cycle ergometer. Substrate oxidation was estimated using indirect calorimetry. Magnetic resonance tomography and spectroscopy were conducted to assess body fat distribution and intramyocellular fat content. We determined the exercise intensity at which maximal body fat oxidation occurs and assessed whether body composition, body fat distribution, intramyocellular fat content, or oxidative capacity predict exercise-induced fat oxidation. Maximal exercise-induced fat oxidation was 0.30+/-0.02 g/min in men and 0.23+/-0.01 g/min in women (p<0.05). Exercise intensity at the maximum fat oxidation was 42+/-2.2% VO (2 max) in men and 43+/-1.7% VO (2 max) in women. With multivariate analysis, exercise-induced fat oxidation was related to fat-free mass, percent fat mass, and oxidative capacity, but not to absolute fat mass, visceral fat, or intramyocellular fat content. We conclude that in obese subjects the capacity to oxidize fat during exercise appears to be limited by skeletal muscle mass and oxidative capacity rather than the availability of visceral or intramyocellular fat.
Assuntos
Exercício Físico/fisiologia , Metabolismo dos Lipídeos , Obesidade/metabolismo , Caracteres Sexuais , Área Sob a Curva , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Oxirredução , Oxigênio/metabolismo , Consumo de Oxigênio/fisiologia , Análise de RegressãoRESUMO
Mutations in the 2Cl(-)/1H(+)-exchanger ClC-7 impair osteoclast function and cause different types of osteoclast-rich osteopetrosis. However, it is unknown to what extent ClC-7 function has to be reduced to become rate-limiting for bone resorption. In osteoclasts from osteopetrosis patients expression of the mutated ClC-7 protein did not correlate with disease severity and resorption impairment. Therefore, a series of transgenic mice expressing ClC-7 in osteoclasts at different levels was generated. Crossing of these mice with Clcn7(-/-) mutants rescued the osteopetrotic phenotype to variable degrees. One resulting double transgenic line mimicked human autosomal dominant osteopetrosis. The trabecular bone of these mice showed a reduction of osteoblast numbers, osteoid, and osteoblast marker gene expression indicative of reduced osteoblast function. In osteoclasts from these mutants ClC-7 expression levels were 20 to 30% of wildtype levels. These reduced levels not only impaired resorptive activity, but also increased numbers, size and nucleus numbers of osteoclasts differentiated in vitro. Although ClC-7 was expressed in the stomach and PTH levels were high in Clcn7(-/-) mutants loss of ClC-7 did not entail a relevant elevation of gastric pH. In conclusion, we show that in our model a reduction of ClC-7 function by approximately 70% is sufficient to increase bone mass, but does not necessarily enhance bone formation. ClC-7 does not appear to be crucially involved in gastric acid secretion, which explains the absence of an osteopetrorickets phenotype in CLCN7-related osteopetrosis.
Assuntos
Remodelação Óssea , Canais de Cloreto/genética , Ácido Gástrico/metabolismo , Animais , Reabsorção Óssea/complicações , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Cálcio/metabolismo , Contagem de Células , Diferenciação Celular/genética , Fusão Celular , Canais de Cloreto/deficiência , Canais de Cloreto/metabolismo , Genes Dominantes , Genes Recessivos , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/genética , Osteopetrose/complicações , Osteopetrose/genética , Osteopetrose/patologia , Osteopetrose/fisiopatologia , FenótipoRESUMO
The brain-specific GDP/GTP exchange factor collybistin interacts with the receptor-anchoring protein gephyrin and activates the Rho-like GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous gephyrin aggregates which cluster with hetero-oligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind gephyrin upon coexpression in heterologous cells, in GST pull-down assays and in the yeast two-hybrid system. Site-directed mutagenesis revealed polar amino acid residues as essential determinants of gephyrin binding. Furthermore, in vitro gephyrin bound simultaneously to both collybistin and the GlyR beta-subunit binding motif. Our data are consistent with collybistin-gephyrin interactions occuring during inhibitory postsynaptic membrane formation.
Assuntos
Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Conformação Proteica , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Fatores de Troca de Nucleotídeo Guanina RhoRESUMO
gamma-Aminobutyric acid type A receptors (GABA(A)Rs) are ligand-gated chloride channels that exist in numerous distinct subunit combinations. At postsynaptic membrane specializations, different GABA(A)R isoforms colocalize with the tubulin-binding protein gephyrin. However, direct interactions of GABA(A)R subunits with gephyrin have not been reported. Recently, the GABA(A)R-associated protein GABARAP was found to bind to the gamma2 subunit of GABA(A)Rs. Here we show that GABARAP interacts with gephyrin in both biochemical assays and transfected cells. Confocal analysis of neurons derived from wild-type and gephyrin-knockout mice revealed that GABARAP is highly enriched in intracellular compartments, but not at gephyrin-positive postsynaptic membrane specializations. Our data indicate that GABARAP-gephyrin interactions are not important for postsynaptic GABA(A)R anchoring but may be implicated in receptor sorting and/or targeting mechanisms. Consistent with this idea, a close homolog of GABARAP, p16, has been found to function as a late-acting intra-Golgi transport factor.
Assuntos
Proteínas de Transporte/metabolismo , Canais de Cloreto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Transporte Biológico , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Humanos , Invertebrados/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Neurônios/metabolismo , Células PC12 , Ratos , Retina/metabolismo , Homologia de Sequência de Aminoácidos , Frações SubcelularesRESUMO
Cystathionine gamma-synthase, the enzyme catalysing the first reaction specific for methionine biosynthesis, has been cloned from Nicotiana tabacum, overexpressed in Escherichia coli and purified to homogeneity. The recombinant cystathionine gamma-synthase catalyses the pyridoxal 5'-phosphate dependent formation of L-cystathionine from L-homoserine phosphate and L-cysteine with apparent Km-values of 7.1+/-3.1 mM and of 0.23+/-0.07 mM, respectively. The enzyme was irreversibly inhibited by DL-propargylglycine (Ki = 18 microM, k(inact) = 0.56 min(-1)), while the homoserine phosphate analogues 3-(phosphonomethyl)pyridine-2-carboxylic acid, 4-(phosphonomethyl)pyridine-2-carboxylic acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competitive inhibitors with Ki values of 0.20, 0.30, 0.45, and 0.027 mM, respectively. In combination these results suggest a ping-pong mechanism for the cystathionine gamma-synthase reaction, with homoserine phosphate binding to the enzyme first. Large single crystals of cystathionine gamma-synthase diffracting to beyond 2.7 A resolution were obtained by the sitting drop vapour diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell constants a = 120.0 A, b = 129.5 A, c = 309.8 A, corresponding to two tetramers per asymmetric unit.