RESUMEN

AIMS: To compare diagnosis characteristics, diabetes management and comorbidities in a population diagnosed with type 1 diabetes in childhood with those in a similar population diagnosed in adulthood to identify disease differences related to the age of diabetes onset. METHODS: This analysis was performed using the T1D Exchange Clinic Registry, a cross-sectional survivor cohort. Retrospectively collected characteristics were compared across the following age-at-diagnosis groups: <10, 10-17, 18-24, 25-39 and ≥40 years. RESULTS: The entire cohort included 20 660 participants [51% female, median (interquartile range) age 18 (14-36) years, 82% non-Hispanic white]. Diabetic ketoacidosis at diagnosis was more common among those with onset in childhood. Participants diagnosed as adults were more likely to be overweight/obese at diagnosis and to have used oral agents preceding type 1 diabetes diagnosis (57%). Current insulin pump use was less frequent in participants diagnosed at older ages. Current glycaemic control, measured by HbA1c , insulin requirements and use of a continuous glucose monitor were not different by age at diagnosis. Coeliac disease was the only comorbidity that was observed to have a different frequency by age at diagnosis, being more common in the participants diagnosed at a younger age. CONCLUSIONS: These results show differences and similarities between type 1 diabetes diagnosed in childhood vs adulthood; notably, there was a tendency for there was a higher frequency of diabetic ketoacidosis at onset in children and a higher frequency of use of oral antidiabetes agents in adults. The data indicate that there is little distinction between the clinical characteristics and outcomes of type 1 diabetes diagnosed in childhood vs adulthood. Optimizing glycaemic control remains a challenge in all age groups, with lower use of insulin pumps impacting those diagnosed as adults.

Asunto(s)

Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Adolescente , Adulto , Edad de Inicio , Automonitorización de la Glucosa Sanguínea , Niño , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Bombas de Infusión Implantables , Insulina/uso terapéutico , Sistemas de Infusión de Insulina , Masculino , Triglicéridos/sangre , Adulto Joven

RESUMEN

OBJECTIVE: Dopamine is a neurotransmitter that mediates the reward value of food. Methylphenidate (MPH) selectively binds and inhibits the dopamine transporter, thus increasing brain dopamine levels shortly after oral administration. This investigation studied whether a single dose of MPH decreases energy intake (EI) in obese teenagers compared to placebo (P). METHODS: This study used a single-blind, placebo-controlled, within subject design. Teenagers with body mass index (BMI) ≥95th percentile underwent two identical meal tests (P or MPH) after a 10 h fast in random order. Food was weighed before and after the meals, and EI was calculated as energy content/gram of consumed foods. Total and macronutrient EI (mean ± SD) were analyzed by Mann-Whitney U and Wilcoxon tests. RESULTS: Twenty-two subjects (15 females, 7 males) completed the study. Participants were 13.4 ± 2.2 years old and had BMI 34.9 ± 10.7 kg/m². EI from fat (167 vs. 203 kcal, P = 0.03) and carbohydrates (311 vs. 389 kcal, P = 0.04) was decreased for MPH compared to P meals, with a trend in decreased total EI (545 vs. 663 kcal, P = 0.06). CONCLUSION: A single dose of MPH decreases EI from fat and carbohydrates in obese adolescents. This effect underscores the importance of central dopamine signaling on eating behavior.

Asunto(s)

Carbohidratos de la Dieta/administración & dosificación , Grasas de la Dieta/administración & dosificación , Metilfenidato/administración & dosificación , Obesidad/tratamiento farmacológico , Adolescente , Negro o Afroamericano , Índice de Masa Corporal , Niño , Proteínas en la Dieta/administración & dosificación , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Relación Dosis-Respuesta a Droga , Ingestión de Energía , Femenino , Humanos , Insulina/sangre , Masculino , Comidas , Método Simple Ciego , Población Blanca

RESUMEN

The purpose of this study was to determine low-grade inflammation associated with obesity that is mediated partially by TNF-α, an adipocytokine which stimulates sphingomyelinase activity in adipocytes. Circulating ceramide (Cer) and sphingosine 1-phosphate (S1P) are elevated in genetically obese (ob/ob) mice. We aimed to determine whether serum sphingolipid concentrations correlate with measures of obesity, insulin resistance, and lipid profiles in overweight versus lean adolescents. This cross-sectional study recruited 30 healthy overweight (body mass index, BMI ≥ 85%) and 15 lean (BMI 10-84%) adolescents. Anthropometric measurements and fasting blood samples were collected at one clinic visit. Serum glucose, insulin, and fasting lipid profiles were measured. Serum adipocytokine concentrations were measured by ELISA or colorimetric assay and sphingolipids were measured by HPLC-mass spectrometry. Between group differences in serum sphingolipid concentrations were assessed. Correlations between sphingolipid concentrations and (i) body mass index, (ii) calculated homeostasis model assessment of insulin resistance (HOMA-IR), (iii) adipocytokines, and (iv) lipoproteins were determined. The results showed that significant differences in HOMA-IR (4.5 ± 3.2 vs. 1.2 ± 0.7), free fatty acids (0.8 ± 0.3 mmol/l vs. 0.4 ± 0.3 mmol/l), and adiponectin (6.4 ± 3.8 vs. 12.6 ± 9.9 µg/ml) were seen between groups (overweight vs. lean). There were significant correlations between Cer and TNF-α (r = 0.429), S1P and TNF-α (r = 0.288), Cer and adiponectin (r = 0.321), Cer:S1P and adiponectin (r = 0.324), Cer and HOMA-IR (r = 0.307), and Cer:S1P and LDL cholesterol (r = 0.453); these associations persisted after adjustment for BMI Z-score, sex, and Tanner stage. We concluded that elevated sphingolipid concentrations correlate with TNF-α, adiponectin, lipoprotein profiles, and HOMA-IR. Ceramide is associated with atherogenic lipid profiles and the development of insulin resistance in obese adolescents, similar to adults.

Asunto(s)

Adipoquinas/sangre , Mediadores de Inflamación/sangre , Síndrome Metabólico/etiología , Obesidad/sangre , Obesidad/fisiopatología , Esfingolípidos/sangre , Adiponectina/sangre , Adolescente , Índice de Masa Corporal , Ceramidas/sangre , LDL-Colesterol/sangre , Estudios Transversales , Ácidos Grasos no Esterificados/sangre , Femenino , Humanos , Resistencia a la Insulina , Lisofosfolípidos/sangre , Masculino , Síndrome Metabólico/epidemiología , New York/epidemiología , Obesidad/inmunología , Obesidad/metabolismo , Riesgo , Esfingosina/análogos & derivados , Esfingosina/sangre , Factor de Necrosis Tumoral alfa/sangre

RESUMEN

To evaluate the role of sphingosine kinase 1 (SphK1) in insulin secretion, we used stable transfection to knock down the expression of the Sphk1 gene in the rat insulinoma INS-1 832/13 cell line. Cell lines with lowered Sphk1 mRNA expression and SphK1 enzyme activity (SK11 and SK14) exhibited lowered glucose- and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine-stimulated insulin release and low insulin content associated with decreases in the mRNA of the insulin 1 gene. Overexpression of the rat or human Sphk1 cDNA restored insulin secretion and total insulin content in the SK11 cell line, but not in the SK14 cell line. The Sphk1 cDNA-transfected SK14 cell line expressed significantly less SphK1 activity than the Sphk1 cDNA-transfected SK11 cells suggesting that the shRNA targeting SK14 was more effective in silencing the exogenous rat Sphk1 mRNA. The results indicate that SphK1 activity is important for insulin synthesis and secretion.

Asunto(s)

Técnicas de Silenciamiento del Gen , Insulina/biosíntesis , Insulina/metabolismo , Insulinoma/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Animales , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Humanos , Secreción de Insulina , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ratas , Esfingosina/análogos & derivados , Esfingosina/farmacología

RESUMEN

Sphingosine kinase (SPHK) catalyzes sphingosine 1-phosphate production, promoting cell survival and reducing apoptosis in isolated rat pancreatic islets. Glucose, the primary islet beta-cell growth factor and insulin secretagogue, increased islet SPHK activity by 3- to 5-fold following acute (1 h) or prolonged (7 days) stimulation. Prolonged stimulation of islets with glucose induced SPHK1a and SPHK2 mRNA levels; there were no changes in SPHK protein expression. To isolate the metabolic effects of glucose on SPHK activation, islets were stimulated with glucose analogs or metabolites. 2-deoxy-D-glucose (2-DG), an analog phosphorylated by glucokinase but not an effective energy source, activated SPHK similarly to glucose. In contrast, 3-o-methylglucose (3-oMeG), which is transported but neither phosphorylated nor metabolized, did not increase islet SPHK activity. Glyceraldehyde and alpha-ketoisocaproic acid (KIC), metabolites that stimulate glycolysis and the citric acid cycle, respectively, did not activate islet SPHK. Moreover, inorganic phosphate blocked glucose-induced SPHK activation. A role for SPHK activity in beta-cell growth was confirmed when small interfering (si)SPHK2 RNA transfection reduced rat insulinoma INS-1e cell SPHK levels and activity and cell growth. Glucose induced an early and sustained increase in islet SPHK activity that was dependent on glucose phosphorylation, but independent of ATP generation or new protein biosynthesis. Glucose-supported beta-cell growth appears to be in part mediated by SPHK activity.

Asunto(s)

Adenosina Trifosfato , Glucosa/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , ARN Interferente Pequeño/genética , Ratas , Factores de Tiempo

RESUMEN

Polyubiquitin (Ub) chains linked through Lys-48-Gly-76 isopeptide bonds represent the principal signal by which substrates of the Ub-dependent protein degradation pathway are targeted to the 26 S proteasome, but the mechanism(s) whereby these chains are assembled on substrate proteins is poorly understood. Nor have assembly mechanisms or definitive functions been assigned to polyubiquitin chains linked through several other lysine residues of ubiquitin. We show that rabbit reticulocyte lysate harbors enzymatic components that catalyze the assembly of unanchored Lys-29-linked polyubiquitin chains. This reaction can be reconstituted using the ubiquitin-conjugating enzyme (E2) known as UbcH5A, a 120-kDa protein(s) that behaves as a ubiquitin-protein ligase (E3), and ubiquitin-activating enzyme (E1). The same partially purified E3 preparation also catalyzes the assembly of unanchored chains linked through Lys-48. Kinetic studies revealed a K(m) of approximately 9 microM for the acceptor ubiquitin in the synthesis of diubiquitin; this value is similar to the concentration of free ubiquitin in most cells. Similar kinetic behavior was observed for conjugation to Lys-48 versus Lys-29 and for conjugation to tetraubiquitin versus monoubiquitin. The properties of these enzymes suggest that there may be distinct pathways for ubiquitin-ubiquitin ligation versus substrate-ubiquitin ligation in vivo.

Asunto(s)

Biopolímeros/metabolismo , Ligasas/metabolismo , Lisina/metabolismo , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Enzimas Ubiquitina-Conjugadoras , Ubiquitinas/metabolismo , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida , Cinética , Poliubiquitina , Conformación Proteica , Conejos , Ubiquitina-Proteína Ligasas

RESUMEN

The mammalian ubiquitin conjugating enzyme known as E2-25K catalyzes the synthesis of polyubiquitin chains linked exclusively through K48-G76 isopeptide bonds. The properties of truncated and chimeric forms of E2-25K suggest that the polyubiquitin chain synthesis activity of this E2 depends on specific interactions between its conserved 150-residue core domain and its unique 50-residue tail domain [Haldeman, M. T., Xia, G., Kasperek, E. M., and Pickart, C. M. (1997) Biochemistry 36, 10526-10537]. In the present study, we provide strong support for this model by showing that a point mutation in the core domain (S86Y) mimics the effect of deleting the entire tail domain: the ability to form an E2 approximately ubiquitin thiol ester is intact, while conjugation activity is severely inhibited (>/=100-fold reduction in kcat/Km). The properties of E2-25K enzymes carrying the S86Y mutation indicate that this mutation strengthens the interaction between the core and tail domains: both free and ubiquitin-bound forms of S86Y-25K are completely resistant to tryptic cleavage at K164 in the tail domain, whereas wild-type enzyme is rapidly cleaved at this site. Other properties of S86Y-26K suggest that the active site of this mutant enzyme is more occluded than the active site of the wild-type enzyme. (1) Free S86Y-25K is alkylated by iodoacetamide 2-fold more slowly than the wild-type enzyme. (2) In assays of E2 approximately ubiquitin thiol ester formation, S86Y-25K shows a 4-fold reduced affinity for E1. (3) The ubiquitin thiol ester adduct of S86Y-25K undergoes (uncatalyzed) reaction with dithiothreitol 3-fold more slowly than the wild-type thiol ester adduct. One model to accommodate these findings postulates that an enhanced interaction between the core and tail domains, induced by the S86Y mutation, causes a steric blockade at the active site which prevents access of the incoming ubiquitin acceptor to the thiol ester bond. Consistent with this model, the S86Y mutation inhibits ubiquitin transfer to macromolecular acceptors (ubiquitin and polylysine) more strongly than transfer to small-molecule acceptors (free lysine and short peptides). These results suggest that unique residues proximal to E2 active sites may influence specific function by mediating intramolecular interactions.

Asunto(s)

Biopolímeros/antagonistas & inhibidores , Ligasas/antagonistas & inhibidores , Mutación Puntual , Serina/genética , Tirosina/genética , Enzimas Ubiquitina-Conjugadoras , Ubiquitinas/antagonistas & inhibidores , Alquilación , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión/genética , Biopolímeros/biosíntesis , Biopolímeros/genética , Catálisis , Bovinos , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Humanos , Hidrólisis , Ligasas/genética , Ligasas/metabolismo , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fenotipo , Poliubiquitina , Estructura Terciaria de Proteína , Conejos , Relación Estructura-Actividad , Tripsina , Ubiquitinas/biosíntesis , Ubiquitinas/genética

RESUMEN

A global cellular reorganization occurs during the reticulocyte stage of erythroid differentiation. This reorganization is accomplished partly through programmed protein degradation. The selection of proteins for degradation can be mediated by covalent attachment of ubiquitin. We have cloned cDNAs encoding two ubiquitin-conjugating (E2) enzymes, E2-20K and E2-230K, and found their genes to be strongly induced during the differentiation of erythroblasts into reticulocytes. Induction of the E2-20K and E2-230K genes is specific, as transcript levels for at least two other ubiquitinating enzymes fall during erythroblast differentiation. In contrast to most proteins induced in reticulocytes, E2-20K and E2-230K enzymes are present at strongly reduced levels in erythrocytes and thus decline in abundance as reticulocyte maturation is completed. This result suggests that both enzymes function during the reticulocyte stage, when enhanced protein degradation has been observed. These data implicate regulated components of the ubiquitin conjugation machinery in erythroid differentiation.

Asunto(s)

Eritroblastos/citología , Eritroblastos/enzimología , Ligasas/biosíntesis , Reticulocitos/citología , Reticulocitos/enzimología , Secuencia de Aminoácidos , Animales , Anticuerpos , Bovinos , Diferenciación Celular , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Inducción Enzimática , Eritropoyesis , Femenino , Immunoblotting , Isoenzimas/biosíntesis , Isoenzimas/aislamiento & purificación , Ligasas/sangre , Ligasas/aislamiento & purificación , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Especificidad de Órganos , Péptidos/síntesis química , Péptidos/inmunología , Fenilhidrazinas , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Transcripción Genética , Enzimas Ubiquitina-Conjugadoras , Ubiquitinas/metabolismo

RESUMEN

We propose the use of the information-theoretical entrophy, S = -sigman pi log2 pi, as a measure of variability at a given position in a set of aligned sequences. pi stands for the fraction of times the i-th type appears at a position. For protein sequences, the sum has up to 20 terms, for nucleotide sequences, up to 4 terms, and for codon sequences, up to 61 terms. We compare S and Vs, a related measure, in detail with Vk, the traditional measure of immunoglobulin sequence variability, both in the abstract and as applied to the immunoglobulins. We conclude that S has desirable mathematical properties that Vk lacks and has intuitive and statistical meanings that accord well with the notion of variability. We find that Vk and the S-based measures are highly correlated for the immunoglobulins. We show by analysis of sequence data and by means of a mathematical model that this correlation is due to a strong tendency for the frequency of occurrence of amino acid types at a given position to be log-linear. It is not known whether the immunoglobulins are typical or atypical of protein families in this regard, nor is the origin of the observed rank-frequency distribution obvious, although we discuss several possible etiologies.

Asunto(s)

Variación Genética , Inmunoglobulinas/genética , Alineación de Secuencia , Secuencia de Aminoácidos , Fenómenos Químicos , Química Física , Humanos , Teoría de la Información , Modelos Estadísticos , Datos de Secuencia Molecular