Effects of alcohol on the endocrine system.

Chronic consumption of a large amount of alcohol disrupts the communication between nervous, endocrine, and immune system and causes hormonal disturbances that lead to profound and serious consequences at physiologic and behavioral levels. These alcohol-induced hormonal dysregulations affect the entire body and can result in various disorders such as stress abnormalities, reproductive deficits, body growth defect, thyroid problems, immune dysfunction, cancers, bone disease, and psychological and behavioral disorders. This review summarizes the findings from human and animal studies that provide consistent evidence on the various effects of alcohol abuse on the endocrine system.

An improved measurement of isotopic ratios by high resolution mass spectrometry.

The study of protein kinetics requires an accurate measurement of isotopic ratios of peptides. Although Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometers yield accurate mass measurements of analytes, the isotopologue ratios are consistently lower than predicted. Recently, we demonstrated that the magnitude of the spectral error in the FT-ICR mass spectrometer is proportional to the scan duration of ions. Here, we present a novel isotopic ratio extrapolation (IRE) method for obtaining accurate isotopic ratio measurements. Accuracy is achieved by performing scans with different duration and extrapolation of the data to the initial moment of the ion rotation; IRE minimizes the absolute isotopic ratio error to ≤1%. We demonstrate the application of IRE in protein turnover studies using (2)H(2)O-metabolic labeling. Overall, this technique allows accurate measurements of the isotopic ratios of proteolytic peptides, a critical step for enabling routine studies of proteome dynamics.

Plasma proteome dynamics: analysis of lipoproteins and acute phase response proteins with 2H2O metabolic labeling.

Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a (2)H(2)O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis. (2)H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with (2)H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions.

Measuring protein synthesis using metabolic ²H labeling, high-resolution mass spectrometry, and an algorithm.

We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O.

Measuring proteome dynamics in vivo: as easy as adding water?

Proteomics investigations typically yield information regarding static gene expression profiles. The central issues that limit the study of proteome dynamics include how to (i) administer a labeled amino acid in vivo, (ii) measure the isotopic labeling of a protein(s) (which may be low), and (iii) reliably interpret the precursor/product labeling relationships. In this study, we demonstrate the potential of quantifying proteome dynamics by coupling the administration of stable isotopes with mass spectrometric assays. Although the direct administration of a labeled amino acid(s) is typically used to measure protein synthesis, we explain the application of labeled water, comparing (2)H(2)O versus H(2)(18)O for measuring albumin biosynthesis in vivo. This application emphasizes two distinct advantages of using labeled water over a labeled amino acid(s). First, in long term studies (e.g. days or weeks), it is not practical to continuously administer a labeled amino acid(s); however, in the presence of labeled water, organisms will generate labeled amino acids. Second, to calculate rates of protein synthesis in short term studies (e.g. hours), one must utilize a precursor/product labeling ratio; when using labeled water it is possible to reliably identify and easily measure the precursor labeling (i.e. water). We demonstrate that labeled water permits studies of protein synthesis (e.g. albumin synthesis in mice) during metabolic "steady-state" or "non-steady-state" conditions, i.e. integrating transitions between the fed and fasted state or during an acute perturbation (e.g. following a meal), respectively. We expect that the use of labeled water is applicable to wide scale investigations of proteome dynamics and can therein be used to obtain a functional image of gene expression in vivo.

Endothelin-1-stimulated glucose uptake is desensitized by tumor necrosis factor-alpha in 3T3-L1 adipocytes.

Tumor necrosis factor-alpha (TNF-alpha) is a potent inducer of insulin resistance, and increased TNF-alpha expression is associated with impaired glucose disposal. Although insulin is the primary regulator of glucose transport in adipose, endothelin-1, a vasoconstrictor peptide that signals through the heterotrimeric G proteins Galphaq/11, potently stimulates glucose uptake in 3T3-L1 adipocytes by a mechanism independent of phosphatidylinositol (PI) 3-kinase. Here, we report that exposure of 3T3-L1 adipocytes to TNF-alpha for 48 h dose-dependently decreased endothelin-1-stimulated glucose uptake and translocation of GLUT4 to the plasma membrane. TNF-alpha exposure had no effect on endothelin-1 receptor number at the cell surface. In contrast, TNF-alpha treatment reduced the quantity of Galphaq/11 and proline-rich tyrosine kinase 2 (PYK2) and decreased endothelin-1-stimulated PYK2-Tyr402 tyrosine phosphorylation. Taken together, these results suggest that TNF-alpha-induced desensitization of endothelin-1-stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes is due, at least in part, to a decreased expression of Galphaq/11, leading to a suppression in tyrosine phosphorylation of PYK2.