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1.
Am J Physiol Endocrinol Metab ; 320(4): E702-E715, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33522396

RESUMEN

Elevated postprandial lipemia is an independent risk factor for cardiovascular disease, yet methods to quantitate postmeal handling of dietary lipids in humans are limited. This study tested a new method to track dietary lipid appearance using a stable isotope tracer (2H11-oleate) in liquid meals containing three levels of fat [low fat (LF), 15 g; moderate fat (MF), 30 g; high fat (HF), 60 g]. Meals were fed to 12 healthy men [means ± SD, age 31.3 ± 9.2 yr, body mass index (BMI) 24.5 ± 1.9 kg/m2] during four randomized study visits; the HF meal was administered twice for reproducibility. Blood was collected over 8 h postprandially, triglyceride (TG)-rich lipoproteins (TRL), and particles with a Svedberg flotation rate >400 (Sf > 400, n = 8) were isolated by ultracentrifugation, and labeling of two TG species (54:3 and 52:2) was quantified by LC-MS. Total plasma TRL-TG concentrations were threefold greater than Sf > 400-TG. Both Sf > 400- and TRL-TG 54:3 were present at higher concentrations than 52:2, and singly labeled TG concentrations were higher than doubly labeled. Furthermore, TG 54:3 and the singly labeled molecules demonstrated higher plasma absolute entry rates differing significantly across fat levels within a single TG species (P < 0.01). Calculation of fractional entry showed no significant differences in label handling supporting the utility of either TG species for appearance rate calculations. These data demonstrate the utility of labeling research meals with stable isotopes to investigate human postprandial lipemia while simultaneously highlighting the importance of examining individual responses. Meal type and timing, control of prestudy activities, and effects of sex on outcomes should match the research goals. The method, optimized here, will be beneficial to conduct basic science research in precision nutrition and clinical drug development.NEW & NOTEWORTHY A novel method to test human intestinal lipid handling using stable isotope labeling is presented and, for the first time, plasma appearance and lipid turnover were quantified in 12 healthy men following meals with varying amounts of fat. The method can be applied to studies in precision nutrition characterizing individual response to support basic science research or drug development. This report discusses key questions for consideration in precision nutrition that were highlighted by the data.


Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Hiperlipidemias/sangre , Lípidos/sangre , Periodo Posprandial , Espectrometría de Masas en Tándem/métodos , Adolescente , Adulto , Cromatografía Liquida/métodos , Estudios Cruzados , Grasas de la Dieta/administración & dosificación , Humanos , Hiperlipidemias/diagnóstico , Lípidos/análisis , Masculino , Comidas , Ciencias de la Nutrición/métodos , Ciencias de la Nutrición/tendencias , Medicina de Precisión/métodos , Medicina de Precisión/tendencias , Reproducibilidad de los Resultados , Adulto Joven
2.
J Nat Prod ; 84(3): 814-823, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33523676

RESUMEN

Natural product congeners serve a useful role in the understanding of natural product biosynthesis and structure-activity relationships. A minor congener with superior activity, selectivity, and modifiable functional groups could serve as a more effective lead structure and replace even the original lead molecule that was used for medicinal chemistry modifications. Currently, no effective method exists to discover targeted congeners rapidly, specifically, and selectively from producing sources. Herein, a new method based on liquid-chromatography tandem-mass spectrometry combination is evaluated for targeted discovery of congeners of platensimycin and platencin from the extracts of Streptomyces platensis. By utilizing a precursor-ion searching protocol, tandem mass spectrometry not only confirmed the presence of known congeners but also provided unambiguous detection of many previously unknown congeners of platensimycin and platencin. This high-throughput and quantitative method can be rapidly and broadly applied for dereplication and congener discovery from a variety of producing sources, even when the targeted compounds are obscured by the presence of unrelated natural products.


Asunto(s)
Adamantano/química , Aminobenzoatos/química , Aminofenoles/química , Anilidas/química , Ensayos Analíticos de Alto Rendimiento/métodos , Compuestos Policíclicos/química , Streptomyces/química , Adamantano/aislamiento & purificación , Aminobenzoatos/aislamiento & purificación , Aminofenoles/aislamiento & purificación , Anilidas/aislamiento & purificación , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Cromatografía Liquida , Estructura Molecular , Compuestos Policíclicos/aislamiento & purificación , Relación Estructura-Actividad , Espectrometría de Masas en Tándem
3.
Am J Physiol Endocrinol Metab ; 317(5): E852-E862, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31503515

RESUMEN

Altered lipid metabolism and inflammation are involved in the pathogenesis of both nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). Even though high-density lipoprotein (HDL), a CVD protective marker, is decreased, whether HDL metabolism and function are perturbed in NAFLD are currently unknown. We examined the effect of NAFLD and disease severity on HDL metabolism and function in patients with biopsy-proven simple steatosis (SS), nonalcoholic steatohepatitis (NASH), and healthy controls. HDL turnover and HDL protein dynamics in SS (n = 7), NASH (n = 8), and healthy controls (n = 9) were studied in vivo. HDL maturation and remodeling, antioxidant, cholesterol efflux properties, and activities of lecithin-cholesterol ester acyltransferase and cholesterol ester transfer protein (CETP) were quantified using in vitro assays. All patients with NAFLD had increased turnover of both HDL cholesterol (HDLc; 0.16 ± 0.09 vs. 0.34 ± 0.18 days, P < 0.05) and apolipoprotein A1 (ApoAI) (0.26 ± 0.04 vs. 0.34 ± 0.06 days, P < 0.005) compared with healthy controls. The fractional catabolic rates of other HDL proteins, including ApoAII (and ApoAIV) were higher (P < 0.05) in patients with NAFLD who also had higher CETP activity, ApoAI/HDLc ratio (P < 0.05). NAFLD-induced alterations were associated with lower antioxidant (114.2 ± 46.6 vs. 220.5 ± 48.2 nmol·mL-1·min-1) but higher total efflux properties of HDL (23.4 ± 1.3% vs. 25.5 ± 2.3%) (both P < 0.05), which was more pronounced in individuals with NASH. However, no differences were observed in either HDL turnover, antioxidant, and cholesterol efflux functions of HDL or HDL proteins' turnover between subjects with SS and subjects with NASH. Thus, HDL metabolism and function are altered in NAFLD without any significant differences between SS and NASH.


Asunto(s)
Lipoproteínas HDL/sangre , Enfermedad del Hígado Graso no Alcohólico/sangre , Adulto , Anciano , Antioxidantes/metabolismo , Apolipoproteína A-II/metabolismo , Biomarcadores/sangre , Colesterol/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Proteómica
4.
Am J Physiol Endocrinol Metab ; 316(6): E1105-E1117, 2019 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-30912961

RESUMEN

The regulation of nutrient homeostasis, i.e., the ability to transition between fasted and fed states, is fundamental in maintaining health. Since food is typically consumed over limited (anabolic) periods, dietary components must be processed and stored to counterbalance the catabolic stress that occurs between meals. Herein, we contrast tissue- and pathway-specific metabolic activity in fasted and fed states. We demonstrate that knowledge of biochemical kinetics that is obtained from opposite ends of the energetic spectrum can allow mechanism-based differentiation of healthy and disease phenotypes. Rat models of type 1 and type 2 diabetes serve as case studies for probing spatial and temporal patterns of metabolic activity via [2H]water labeling. Experimental designs that capture integrative whole body metabolism, including meal-induced substrate partitioning, can support an array of research surrounding metabolic disease; the relative simplicity of the approach that is discussed here should enable routine applications in preclinical models.


Asunto(s)
Aminoácidos/metabolismo , Glucemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Ayuno/metabolismo , Ácidos Grasos/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , Periodo Posprandial , Animales , Óxido de Deuterio , Modelos Animales de Enfermedad , Glucógeno/metabolismo , Cinética , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Redes y Vías Metabólicas , Metabolómica , Ratas , Ratas Wistar , Ratas Zucker , Análisis Espacio-Temporal
5.
Am J Physiol Endocrinol Metab ; 315(3): E416-E424, 2018 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-29509438

RESUMEN

Numerous studies have implicated dyslipidemia as a key factor in mediating insulin resistance. Ceramides have received special attention since their levels are inversely associated with normal insulin signaling and positively associated with factors that are involved in cardiometabolic disease. Despite the growing literature surrounding ceramide biology, there are limited data regarding the activity of ceramide synthesis and turnover in vivo. Herein, we demonstrate the ability to measure ceramide kinetics by coupling the administration of [2H]water with LC-MS/MS analyses. As a "proof-of-concept" we determined the effect of a diet-induced alteration on ceramide flux; studies also examined the effect of myriocin (a known inhibitor of serine palmitoyltransferase, the first step in sphingosine biosynthesis). Our data suggest that one can estimate ceramide synthesis and draw conclusions regarding the source of fatty acids; we discuss caveats in regards to method development in this area.


Asunto(s)
Ceramidas/farmacocinética , Animales , Cromatografía Líquida de Alta Presión , Óxido de Deuterio/farmacocinética , Dieta , Inhibidores Enzimáticos , Ácidos Grasos Monoinsaturados/farmacología , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Trazadores Radiactivos , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Espectrometría de Masas en Tándem
6.
Am J Physiol Endocrinol Metab ; 315(1): E63-E71, 2018 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-29351479

RESUMEN

An increased contribution of de novo lipogenesis (DNL) may play a role in cases of dyslipidemia and adipose accretion; this suggests that inhibition of fatty acid synthesis may affect clinical phenotypes. Since it is not clear whether modulation of one step in the lipogenic pathway is more important than another, the use of tracer methods can provide a deeper level of insight regarding the control of metabolic activity. Although [2H]water is generally considered a reliable tracer for quantifying DNL in vivo (it yields a homogenous and quantifiable precursor labeling), the relatively long half-life of body water is thought to limit the ability of performing repeat studies in the same subjects; this can create a bottleneck in the development and evaluation of novel therapeutics for inhibiting DNL. Herein, we demonstrate the ability to perform back-to-back studies of DNL using [2H]water. However, this work uncovered special circumstances that affect the data interpretation, i.e., it is possible to obtain seemingly negative values for DNL. Using a rodent model, we have identified a physiological mechanism that explains the data. We show that one can use [2H]water to test inhibitors of DNL by performing back-to-back studies in higher species [i.e., treat nonhuman primates with platensimycin, an inhibitor of fatty acid synthase]; studies also demonstrate the unsuitability of [13C]acetate.


Asunto(s)
Óxido de Deuterio/farmacología , Ácido Palmítico/sangre , Acetatos/sangre , Adipogénesis , Animales , Femenino , Semivida , Lipogénesis/efectos de los fármacos , Macaca mulatta , Masculino , Ratones Endogámicos C57BL
7.
Arterioscler Thromb Vasc Biol ; 37(9): 1770-1775, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28729361

RESUMEN

OBJECTIVE: Lp(a) [lipoprotein (a)] is composed of apoB (apolipoprotein B) and apo(a) [apolipoprotein (a)] and is an independent risk factor for cardiovascular disease and aortic stenosis. In clinical trials, anacetrapib, a CETP (cholesteryl ester transfer protein) inhibitor, causes significant reductions in plasma Lp(a) levels. We conducted an exploratory study to examine the mechanism for Lp(a) lowering by anacetrapib. APPROACH AND RESULTS: We enrolled 39 participants in a fixed-sequence, double-blind study of the effects of anacetrapib on the metabolism of apoB and high-density lipoproteins. Twenty-nine patients were randomized to atorvastatin 20 mg/d, plus placebo for 4 weeks, and then atorvastatin plus anacetrapib (100 mg/d) for 8 weeks. The other 10 subjects were randomized to double placebo for 4 weeks followed by placebo plus anacetrapib for 8 weeks. We examined the mechanisms of Lp(a) lowering in a subset of 12 subjects having both Lp(a) levels >20 nmol/L and more than a 15% reduction in Lp(a) by the end of anacetrapib treatment. We performed stable isotope kinetic studies using 2H3-leucine at the end of each treatment to measure apo(a) fractional catabolic rate and production rate. Median baseline Lp(a) levels were 21.5 nmol/L (interquartile range, 9.9-108.1 nmol/L) in the complete cohort (39 subjects) and 52.9 nmol/L (interquartile range, 38.4-121.3 nmol/L) in the subset selected for kinetic studies. Anacetrapib treatment lowered Lp(a) by 34.1% (P≤0.001) and 39.6% in the complete and subset cohort, respectively. The decreases in Lp(a) levels were because of a 41% reduction in the apo(a) production rate, with no effects on apo(a) fractional catabolic rate. CONCLUSIONS: Anacetrapib reduces Lp(a) levels by decreasing its production. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00990808.


Asunto(s)
Anticolesterolemiantes/uso terapéutico , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Hipercolesterolemia/tratamiento farmacológico , Lipoproteína(a)/sangre , Oxazolidinonas/uso terapéutico , Adulto , Anciano , Anticolesterolemiantes/efectos adversos , Biomarcadores/sangre , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Cromatografía Liquida , Método Doble Ciego , Regulación hacia Abajo , Femenino , Humanos , Hipercolesterolemia/sangre , Hipercolesterolemia/diagnóstico , Masculino , Persona de Mediana Edad , Ciudad de Nueva York , Oxazolidinonas/efectos adversos , Pennsylvania , Índice de Severidad de la Enfermedad , Espectrometría de Masas en Tándem , Factores de Tiempo , Resultado del Tratamiento
8.
J Lipid Res ; 58(8): 1561-1578, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28583918

RESUMEN

GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.


Asunto(s)
Tejido Adiposo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Lipólisis , Receptores Acoplados a Proteínas G/metabolismo , Tejido Adiposo/efectos de los fármacos , Animales , Células CHO , Cricetinae , Cricetulus , Diabetes Mellitus Experimental/patología , Regulación de la Expresión Génica/efectos de los fármacos , Resistencia a la Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/fisiopatología , Lipólisis/efectos de los fármacos , Masculino , Ratones , Ratas , Receptores Acoplados a Proteínas G/agonistas
9.
Am J Physiol Endocrinol Metab ; 312(4): E235-E243, 2017 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-28143858

RESUMEN

Insulin resistance and diabetes can develop spontaneously with obesity and aging in rhesus monkeys, highly similar to the natural history of obesity, insulin resistance, and progression to type 2 diabetes in humans. The current studies in obese rhesus were undertaken to assess hepatic and adipose contributions to systemic insulin resistance-currently, a gap in our knowledge-and to benchmark the responses to pioglitazone (PIO). A two-step hyperinsulinemic-euglycemic clamp, with tracer-based glucose flux estimates, was used to measure insulin resistance, and in an intervention study was repeated following 6 wk of PIO treatment (3 mg/kg). Compared with lean healthy rhesus, obese rhesus has a 60% reduction of glucose utilization during a high insulin infusion and markedly impaired suppression of lipolysis, which was evident at both low and high insulin infusion. However, obese dysmetabolic rhesus manifests only mild hepatic insulin resistance. Six-week PIO treatment significantly improved skeletal muscle and adipose insulin resistance (by ~50%). These studies strengthen the concept that insulin resistance in obese rhesus closely resembles human insulin resistance and indicate the value of obese rhesus for appraising new insulin-sensitizing therapeutics.


Asunto(s)
Tejido Adiposo/metabolismo , Hipoglucemiantes/farmacología , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Tiazolidinedionas/farmacología , Tejido Adiposo/efectos de los fármacos , Animales , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Técnica de Clampeo de la Glucosa , Hipoglucemiantes/uso terapéutico , Lipólisis/fisiología , Hígado/efectos de los fármacos , Macaca mulatta , Músculo Esquelético/efectos de los fármacos , Obesidad/tratamiento farmacológico , Pioglitazona , Tiazolidinedionas/uso terapéutico
10.
J Pharmacol Exp Ther ; 363(1): 80-91, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28724692

RESUMEN

Drug discovery and development efforts are largely based around a common expectation, namely, that direct or indirect action on a cellular process (e.g., statin-mediated enzyme inhibition or insulin-stimulated receptor activation) will have a beneficial impact on physiologic homeostasis. To expand on this, one could argue that virtually all pharmacologic interventions attempt to influence the flow of "traffic" in a biochemical network, irrespective of disease or modality. Since stable isotope tracer kinetic methods provide a measure of traffic flow (i.e., metabolic flux), their inclusion in study designs can yield novel information regarding pathway biology; the application of such methods requires the integration of knowledge in physiology, analytical chemistry, and mathematical modeling. Herein, we review the fundamental concepts that surround the use of tracer kinetics, define basic terms, and outline guiding principles via theoretical and experimental problems. Specifically, one needs to 1) recognize the types of biochemical events that change isotopic enrichments, 2) appreciate the distinction between fractional turnover and flux rate, and 3) be aware of the subtle differences between tracer kinetics and pharmacokinetics. We hope investigators can use the framework presented here to develop applications that address their specific questions surrounding biochemical flux, and thereby gain insight into the pathophysiology of disease states, and examine pharmacodynamic mechanisms.


Asunto(s)
Descubrimiento de Drogas/métodos , Análisis de Flujos Metabólicos/métodos , Animales , Humanos , Marcaje Isotópico , Isótopos/química , Agua/química , Agua/metabolismo
11.
J Lipid Res ; 57(12): 2150-2162, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27707816

RESUMEN

SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/genética , Lípidos/sangre , Proteínas de la Membrana/genética , Proproteína Convertasa 9/genética , ARN Interferente Pequeño/genética , Receptores de LDL/genética , Animales , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Hipolipemiantes/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipogénesis , Hígado/enzimología , Macaca mulatta , Masculino , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Proproteína Convertasa 9/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de LDL/metabolismo , Transducción de Señal , Simvastatina/farmacología , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo
12.
J Lipid Res ; 57(3): 398-409, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26658238

RESUMEN

Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([(2)H11] and [(13)C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is ∼ 13 mg · h(-1) · kg(-1) and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions.


Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Oxazolidinonas/farmacología , Triglicéridos/metabolismo , Animales , Lipoproteínas HDL/sangre , Macaca mulatta , Masculino , Modelos Biológicos , Triglicéridos/sangre
13.
J Proteome Res ; 15(7): 2115-22, 2016 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-27229456

RESUMEN

We describe a stochastic model to compute in vivo protein turnover rate constants from stable-isotope labeling and high-throughput liquid chromatography-mass spectrometry experiments. We show that the often-used one- and two-compartment nonstochastic models allow explicit solutions from the corresponding stochastic differential equations. The resulting stochastic process is a Gaussian processes with Ornstein-Uhlenbeck covariance matrix. We applied the stochastic model to a large-scale data set from (15)N labeling and compared its performance metrics with those of the nonstochastic curve fitting. The comparison showed that for more than 99% of proteins, the stochastic model produced better fits to the experimental data (based on residual sum of squares). The model was used for extracting protein-decay rate constants from mouse brain (slow turnover) and liver (fast turnover) samples. We found that the most affected (compared to two-exponent curve fitting) results were those for liver proteins. The ratio of the median of degradation rate constants of liver proteins to those of brain proteins increased 4-fold in stochastic modeling compared to the two-exponent fitting. Stochastic modeling predicted stronger differences of protein turnover processes between mouse liver and brain than previously estimated. The model is independent of the labeling isotope. To show this, we also applied the model to protein turnover studied in induced heart failure in rats, in which metabolic labeling was achieved by administering heavy water. No changes in the model were necessary for adapting to heavy-water labeling. The approach has been implemented in a freely available R code.


Asunto(s)
Química Encefálica , Hígado/química , Proteínas/metabolismo , Proteoma/metabolismo , Animales , Cromatografía Liquida , Interpretación Estadística de Datos , Marcaje Isotópico , Cinética , Espectrometría de Masas , Ratones , Distribución Normal , Proteómica/métodos , Procesos Estocásticos
14.
J Proteome Res ; 15(9): 3388-404, 2016 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-27439437

RESUMEN

Nonalcoholic fatty liver disease (NAFLD) is associated with an increased risk of cardiovascular disease. Because the liver is the major source of circulatory proteins, it is not surprising that hepatic disease could lead to alterations in the plasma proteome, which are therein implicated in atherosclerosis. The current study used low-density lipoprotein receptor-deficient (LDLR(-/-)) mice to examine the impact of Western diet (WD)-induced NAFLD on plasma proteome homeostasis. Using a (2)H2O-metabolic labeling method, we found that a WD led to a proinflammatory distribution of circulatory proteins analyzed in apoB-depleted plasma, which was attributed to an increased production. The fractional turnover rates of short-lived proteins that are implicated in stress-response, lipid metabolism, and transport functions were significantly increased with WD (P < 0.05). Pathway analyses revealed that alterations in plasma proteome dynamics were related to the suppression of hepatic PPARα, which was confirmed based on reduced gene and protein expression of PPARα in mice fed a WD. These changes were associated with ∼4-fold increase (P < 0.0001) in the proinflammatory property of apoB-depleted plasma. In conclusion, the proteome dynamics method reveals proinflammatory remodeling of the plasma proteome relevant to liver disease. The approach used herein may provide a useful metric of in vivo liver function and better enable studies of novel therapies surrounding NAFLD and other diseases.


Asunto(s)
Dieta Occidental , Enfermedad del Hígado Graso no Alcohólico/sangre , Proteoma/metabolismo , Animales , Modelos Animales de Enfermedad , Mediadores de Inflamación , Ratones , Ratones Noqueados , PPAR alfa/metabolismo , Plasma/química , Plasma/metabolismo , Proteoma/análisis , Receptores de LDL/deficiencia , Receptores de LDL/genética
15.
Am J Physiol Endocrinol Metab ; 311(6): E911-E921, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27651111

RESUMEN

Aberrant regulation of glucose production makes a critical contribution to the impaired glycemic control that is observed in type 2 diabetes. Although isotopic tracer methods have proven to be informative in quantifying the magnitude of such alterations, it is presumed that one must rely on venous access to administer glucose tracers which therein presents obstacles for the routine application of tracer methods in rodent models. Since intraperitoneal injections are readily used to deliver glucose challenges and/or dose potential therapeutics, we hypothesized that this route could also be used to administer a glucose tracer. The ability to then reliably estimate glucose flux would require attention toward setting a schedule for collecting samples and choosing a distribution volume. For example, glucose production can be calculated by multiplying the fractional turnover rate by the pool size. We have taken a step-wise approach to examine the potential of using an intraperitoneal tracer administration in rat and mouse models. First, we compared the kinetics of [U-13C]glucose following either an intravenous or an intraperitoneal injection. Second, we tested whether the intraperitoneal method could detect a pharmacological manipulation of glucose production. Finally, we contrasted a potential application of the intraperitoneal method against the glucose-insulin clamp. We conclude that it is possible to 1) quantify glucose production using an intraperitoneal injection of tracer and 2) derive a "glucose production index" by coupling estimates of basal glucose production with measurements of fasting insulin concentration; this yields a proxy for clamp-derived assessments of insulin sensitivity of endogenous production.


Asunto(s)
Glucemia/metabolismo , Indicadores y Reactivos , Animales , Glucemia/efectos de los fármacos , Isótopos de Carbono , Dieta Alta en Grasa , Femenino , Técnica de Clampeo de la Glucosa , Hipoglucemiantes/farmacología , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Resistencia a la Insulina , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Proyectos Piloto , Ratas , Ratas Sprague-Dawley , Ratas Zucker , Rosiglitazona , Tiazolidinedionas/farmacología
16.
Anal Bioanal Chem ; 408(1): 97-105, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26511226

RESUMEN

Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) that affect a broad range of physiological processes, including cell proliferation, inflammation, inflammation resolution, and vascular function. Moreover, oxylipins are readily detectable in plasma, and certain subsets of oxylipins have been detected in human atherosclerotic lesions. Taken together, we set out to produce a detailed quantitative assessment of plasma and plaque oxylipins in a widely used model of atherosclerosis, to identify potential biomarkers of disease progression. We administered regular chow or regular chow supplemented with 0.5% cholesterol (HC) to male New Zealand white rabbits for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our targeted lipidomic analyses of oxylipins on plaques isolated from rabbits fed the HC diet detected 34 oxylipins, 28 of which were in compliance with our previously established quality control acceptance criteria. The arachidonic acid (AA) metabolite derived from the COX pathway, 6-keto-PGF1α was the most abundant plaque oxylipin, followed by the linoleic acid (LA) metabolites 9-HODE, 13-HODE and 9,12,13-TriHOME and the arachidonic acid (AA)-derivatives 11-HETE and 12-HETE. We additionally found that the most abundant oxylipins in plasma were three of the five most abundant oxylipins in plaque, namely 11-HETE, 13-HODE, and 9-HODE. The studies reported here make the first step towards a comprehensive characterization of oxylipins as potentially translatable biomarkers of atherosclerosis.


Asunto(s)
Hipercolesterolemia/sangre , Oxilipinas/sangre , Placa Aterosclerótica/sangre , Animales , Cromatografía Líquida de Alta Presión , Ácidos Grasos Insaturados/metabolismo , Humanos , Hipercolesterolemia/metabolismo , Masculino , Espectrometría de Masas , Oxilipinas/metabolismo , Placa Aterosclerótica/metabolismo , Conejos
17.
J Biol Chem ; 289(52): 35770-80, 2014 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-25381252

RESUMEN

Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL(-/-) mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs.


Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Lipasa/metabolismo , Gotas Lipídicas/enzimología , Proteínas del Núcleo Viral/fisiología , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Humanos , Hidrólisis , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Triglicéridos/metabolismo
18.
Biochim Biophys Acta ; 1842(3): 402-13, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23707557

RESUMEN

Our ability to understand the pathogenesis of problems surrounding lipid accretion requires attention towards quantifying lipid kinetics. In addition, studies of metabolic flux should also help unravel mechanisms that lead to imbalances in inter-organ lipid trafficking which contribute to dyslipidemia and/or peripheral lipid accumulation (e.g. hepatic fat deposits). This review aims to outline the development and use of novel methods for studying lipid kinetics in vivo. Although our focus is directed towards some of the approaches that are currently reported in the literature, we include a discussion of the older literature in order to put "new" methods in better perspective and inform readers of valuable historical research. Presumably, future advances in understanding lipid dynamics will benefit from a careful consideration of the past efforts, where possible we have tried to identify seminal papers or those that provide clear data to emphasize essential points. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.


Asunto(s)
Tejido Adiposo/metabolismo , Metabolismo de los Lípidos , Lípidos/biosíntesis , Triglicéridos/metabolismo , Distribución de la Grasa Corporal , Colesterol/biosíntesis , Colesterol/metabolismo , Metabolismo Energético , Humanos , Cinética , Triglicéridos/química
19.
Am J Physiol Endocrinol Metab ; 309(8): E727-35, 2015 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-26330343

RESUMEN

Two groups recently used different tracer methods to quantify liver-specific flux rates. The studies had a similar goal, i.e., to characterize mitochondrial oxidative function. These efforts could have a direct impact on our ability to understand metabolic abnormalities that affect the pathophysiology of fatty liver and allow us to examine mechanisms surrounding potential therapeutic interventions. Briefly, one method couples the continuous infusion of [(13)C]acetate with direct real-time measurements of [(13)C]glutamate labeling in liver; the other method administers [(13)C]propionate, in combination with other tracers, and subsequently measures the (13)C labeling of plasma glucose and/or acetaminophen-glucuronide. It appears that a controversy has arisen, since the respective methods yielded different estimates of the anaplerotic/TCA flux ratio (VANA:VTCA) in "control" subjects, i.e., the [(13)C]acetate- and [(13)C]propionate-derived VANA:VTCA flux ratios appear to be ∼1.4 and ∼5, respectively. While the deep expertise in the respective groups makes it somewhat trivial for each to perform the tracer studies, the data interpretation is inherently difficult. The current perspective was undertaken to examine potential factors that could account for or contribute to the apparent differences. Attention was directed toward 1) matters of practicality, 2) issues surrounding stoichiometry, and 3) hidden assumptions. We believe that the [(13)C]acetate method has certain weaknesses that limit its utility; in contrast, the [(13)C]propionate method likely yields a more correct answer. We hope our discussion will help clarify the differences in the recent reports. Presumably this will be of interest to investigators who are considering tracer-based studies of liver metabolism.


Asunto(s)
Acetatos , Ciclo del Ácido Cítrico , Hígado Graso/metabolismo , Hígado/metabolismo , Análisis de Flujos Metabólicos/métodos , Mitocondrias Hepáticas/metabolismo , Oxidación-Reducción , Propionatos , Acetaminofén/análogos & derivados , Acetaminofén/metabolismo , Glucemia/metabolismo , Isótopos de Carbono , Ácido Glutámico/metabolismo , Humanos , Análisis de Flujos Metabólicos/normas , Mitocondrias/metabolismo , Trazadores Radiactivos
20.
Am J Physiol Endocrinol Metab ; 309(2): E115-21, 2015 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-26015435

RESUMEN

The synthesis of various molecules can be estimated by measuring the incorporation of a labeled precursor into a product of interest. Unfortunately, a central problem in many studies has been an inability to estimate the intracellular dilution of the precursor and therein correctly calculate the synthesis of the product; it is generally assumed that measuring the true product labeling is straightforward. We initiated a study to examine liver collagen synthesis and identified an apparent problem with assumptions regarding measurements of the product labeling. Since it is well known that collagen production is relatively slow, we relied on the use of [(2)H]H2O labeling (analogous to a primed infusion) and sampled animals over the course of 16 days. Although the water labeling (the precursor) remained stable and we observed the incorporation of labeled amino acids into collagen, the asymptotic protein labeling was considerably lower than what would be expected based on the precursor labeling. Although this observation is not necessarily surprising (i.e., one might expect that a substantial fraction of the collagen pool would appear "inert" or turn over at a very slow rate), its implications are of interest in certain areas. Herein, we discuss a novel situation in which tracers are used to quantify rates of flux under conditions where a product may not undergo complete replacement. We demonstrate how heterogeneity in the product pool can lead one to the wrong conclusions regarding estimates of flux, and we outline an approach that may help to minimize errors surrounding data interpretation.


Asunto(s)
Colágeno/metabolismo , Deuterio/farmacocinética , Marcaje Isotópico/métodos , Biosíntesis de Proteínas , Aminoácidos/metabolismo , Animales , Humanos , Indicadores y Reactivos/farmacocinética , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Transporte de Proteínas , Agua/metabolismo
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