RESUMEN
Labeling with deuterium oxide (D2O) has emerged as one of the preferred approaches for measuring the synthesis of individual proteins in vivo. In these experiments, the synthesis rates of proteins are determined by modeling mass shifts in peptides during the labeling period. This modeling depends on a theoretical maximum enrichment determined by the number of labeling sites (NEH) of each amino acid in the peptide sequence. Currently, NEH is determined from one set of published values. However, it has been demonstrated that NEH can differ between species and potentially tissues. The goal of this work was to determine the number of NEH for each amino acid within a given experiment to capture the conditions unique to that experiment. We used four methods to compute the NEH values. To test these approaches, we used two publicly available data sets. In a de novo approach, we compute NEH values and the label enrichment from the abundances of three mass isotopomers. The other three methods use the complete isotope profiles and body water enrichment in deuterium as an input parameter. They determine the NEH values by (1) minimizing the residual sum of squares, (2) from the mole percent excess of labeling, and (3) the time course profile of the depletion of the relative isotope abundance of monoisotope. In the test samples, the method using residual sum of squares performed the best. The methods are implemented in a tool for determining the NEH for each amino acid within a given experiment to use in the determination of protein synthesis rates using D2O.
Asunto(s)
Cromatografía Líquida con Espectrometría de Masas , Animales , Aminoácidos/química , Aminoácidos/análisis , Aminoácidos/metabolismo , Óxido de Deuterio , Cromatografía Líquida con Espectrometría de Masas/métodos , Péptidos/química , Péptidos/análisis , Proteínas/química , Proteínas/análisis , Proteínas/metabolismoRESUMEN
The rationale for the use of metformin as a treatment to slow aging was largely based on data collected from metabolically unhealthy individuals. For healthspan extension metformin will also be used in periods of good health. To understand the potential context specificity of metformin treatment on skeletal muscle, we used a rat model (high-capacity runner/low-capacity runner [HCR/LCR]) with a divide in intrinsic aerobic capacity. Outcomes of metformin treatment differed based on baseline intrinsic mitochondrial function, oxidative capacity of the muscle (gastroc vs soleus), and the mitochondrial population (intermyofibrillar vs. subsarcolemmal). Metformin caused lower ADP-stimulated respiration in LCRs, with less of a change in HCRs. However, a washout of metformin resulted in an unexpected doubling of respiratory capacity in HCRs. These improvements in respiratory capacity were accompanied by mitochondrial remodeling that included increases in protein synthesis and changes in morphology. Our findings raise questions about whether the positive findings of metformin treatment are broadly applicable.
RESUMEN
The rationale for the use of metformin as a treatment to slow aging was largely based on data collected from metabolically unhealthy individuals. For healthspan extension metformin will also be used in periods of good health. To understand potential context specificity of metformin treatment on skeletal muscle, we used a rat model (HCR/LCR) with a divide in intrinsic aerobic capacity. Outcomes of metformin treatment differed based on baseline intrinsic mitochondrial function, oxidative capacity of the muscle (gastroc vs soleus), and the mitochondrial population (IMF vs SS). Metformin caused lower ADP-stimulated respiration in LCRs, with less of a change in HCRs. However, a washout of metformin resulted in an unexpected doubling of respiratory capacity in HCRs. These improvements in respiratory capacity were accompanied by mitochondrial remodeling that included increases in protein synthesis and changes in morphology. Our findings raise questions about whether the positive findings of metformin treatment are broadly applicable.
RESUMEN
Chronic unpredictable mild stress (CMS), an animal model of depression, downregulates hippocampal CB1 receptors in adult male rats. Given that endocannabinoids are implicated in modulating stress and anxiety and that women are vulnerable to stress-related disorders, we tested the effects of CMS on both female and male rats. Gonadectomized (gndx) and gonadally intact male and female rats were exposed to a three-week chronic stress protocol. Following CMS, CB1 receptor and fatty-acid-amide-hydrolase (FAAH) expression levels in the hippocampus were assessed by western blot analysis. CMS reliably produced a downregulation of CB1 receptors ( approximately 50%) in the hippocampus of both gndx and intact males. This effect was more robust in the dorsal than in the ventral hippocampus. Conversely, CMS produced an upregulation of CB1 receptors ( approximately 150%) in the hippocampus of both gndx and intact females. This upregulation was only observed in the dorsal hippocampus of female animals. CMS produced an upregulation of FAAH levels in both male and female animals. In non-stress control animals, males were observed to have higher CB1 levels than females, but no differences in FAAH were found. These findings suggest that the endocannabinoid (eCB) system is preferentially organized in male and female animals to respond differentially to chronic stress. These sex differences in the eCB system may help development of novel treatments for stress and depression that are designed specifically for women and men.
Asunto(s)
Hipocampo/metabolismo , Hipocampo/fisiopatología , Receptor Cannabinoide CB1/metabolismo , Estrés Psicológico/fisiopatología , Amidohidrolasas/metabolismo , Animales , Western Blotting , Corticosterona/sangre , Regulación hacia Abajo , Femenino , Hormonas Esteroides Gonadales/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Estrés Psicológico/metabolismo , Regulación hacia ArribaRESUMEN
Steroid-mediated sexual differentiation of the brain is a developmental process that permanently organizes the brain into a male or female phenotype. Previous studies in the rodent have examined the steroid-mediated mechanisms of male brain development. In an effort to identify molecules involved in female brain development, a high-throughput proteomics approach called PowerBlot was used to identify signaling proteins differentially regulated in the neonatal male and female rat hypothalamus during the critical period for brain sexual differentiation. Focal adhesion kinase (FAK) and paxillin, both members of the focal adhesion complex family of proteins, were significantly elevated in the newborn female compared with the male hypothalamus. Sex differences in these proteins were not detected in brain regions that are not subject to substantial organizational effects of steroids. Estrogens, the aromatized products of testosterone in the male, can both masculinize and defeminize the male brain. Daily estradiol administration to neonatal females significantly reduced FAK and paxillin in the hypothalamus, and aromatase inhibition increased paxillin in males to levels comparable with females. Androgens also appear to modulate paxillin levels in combination with estrogen action. Across development, hypothalamic levels of FAK were significantly elevated in females compared with males on postnatal d 6. Synaptic circuits in the hypothalamus develop sex differences perinatally. Estradiol treatment of cultured hypothalamic neurons significantly enhanced axon branching (P<0.01), consistent with the phenotype of FAK-deficient neurons. Together, these data implicate FAK and paxillin as regulators of sex differences in neuronal morphology.