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Stephen F Previs

Publications and source records attributed to Stephen F Previs.

14 recordsLinked to original sources
PubMed2007

Exposure to azide markedly decreases the abundance of mRNAs encoding cholesterol synthetic enzymes and inhibits cholesterol synthesis.

This study was performed to identify genes that are regulated in the adaptive response to prolonged inhibition of oxidative phosphorylation. Gene microarray analysis in control Clone 9 cells and Clone 9 cells exposed to 5 mM azide for 24 h was carried out as a condition of "Chemical hypoxia." Among several hundred mRNAs whose abundances were either increased or decreased, we noted that the abundance of mRNAs encoding enzymes that catalyze the sequential steps of cholesterol synthesis was decreased; this finding was verified by real-time PCR. Exposure to azide for 24 h markedly inhibited the biosynthesis of cholesterol by approximately 90% and decreased the cellular content of cholesterol by 30%, similar results were observed in HepG2 cells. The abundance of sterol regulatory element binding protein (SREBP)-2 mRNA decreased to 0.37 and 0.25 that of controls after 2 and 24 h exposure, respectively. After 24 h of exposure to azide the precursor and nuclear forms of SREBP-2 protein decreased by approximately 80% and approximately 50%, respectively. Stimulation of AMP-activated protein kinase (AMPK) by AICAR in Clone 9 cells increased the abundance of mRNAs encoding cholesterol biosynthetic enzymes and that of SREBP-1c, and had no effect on SREBP-2 mRNA abundance. We conclude that the decrease in the abundance of multiple mRNAs encoding cholesterol biosynthetic enzymes may be mediated by decreased expression of SREBP-2 mRNA and protein and does not involve stimulation of AMPK. The decrease in SREBP-2 mRNA and protein abundance in the face of decreased cell cholesterol content raises the possibility of a novel regulatory pathway.

Animals↗
PubMed2007

Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta are protected against diet-induced obesity.

The CCAAT/enhancer-binding protein beta (C/EBPbeta) is required for adipocyte differentiation and maturation. We have studied the role of the transcription factor, C/EBPbeta, in the development of diet-induced obesity. Mice with a deletion in the gene for C/EBPbeta (C/EBPbeta(-/-)) and wild-type mice were fed a high-fat diet (60% fat) for 12 weeks. The C/EBPbeta(-/-) mice lost body fat, whereas the wild-type mice increased their total body fat on a high-fat diet. The C/EBPbeta(-/-) mice had lower levels of blood triglycerides, free fatty acids, cholesterol, and hepatic triglyceride accumulation compared with the wild-type mice, thus protecting them from diet-induced obesity and fatty liver on a high-fat diet. Deletion of C/EBPbeta gene resulted in greatly reducing hepatic lipogenic genes, acetyl CoA carboxylase, and fatty acid synthase and increasing the expression of beta-oxidation genes in the brown adipose tissue. CO(2) production was significantly higher in the C/EBPbeta(-/-) mice as was the level of uncoupling protein (UCP)-1 and UCP-3 in the muscle. In conclusion, the transcription factor C/EBPbeta is an important regulator in controlling lipid metabolism and in the development of diet-induced obesity.

Animals↗
PubMed2006

Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis.

Determining how the regulation of cellular processes is impacted in cystic fibrosis (CF) is fundamental to understanding disease pathology and to identifying new therapeutic targets. In this study, unesterified cholesterol accumulation is observed in lung and trachea sections obtained from CF patients compared with non-CF tissues, suggesting an inherent flaw in cholesterol processing. An alternate staining method utilizing a fluorescent cholesterol probe also indicates improper lysosomal storage of cholesterol in CF cells. Excess cholesterol is also manifested by a significant increase in plasma membrane cholesterol content in both cultured CF cells and in nasal tissue excised from cftr(-/-) mice. Impaired intracellular cholesterol movement is predicted to stimulate cholesterol synthesis, a hypothesis supported by the observation of increased de novo cholesterol synthesis in lung and liver of cftr(-/-) mice compared with controls. Furthermore, pharmacological inhibition of cholesterol transport is sufficient to cause CF-like elevation in cytokine production in wild-type cells in response to bacterial challenge but has no effect in CF cells. These data demonstrate via multiple methods in both cultured and in vivo models that cellular cholesterol homeostasis is inherently altered in CF. This perturbation of cholesterol homeostasis represents a potentially important process in CF pathogenesis.

4-Chloro-7-nitrobenzofurazan↗
PubMed2006

Reproducibility of gas chromatography-mass spectrometry measurements of 2H labeling of water: application for measuring body composition in mice.

Deuterium-labeled water (2H2O) has emerged as a novel isotope tracer. Following the administration of 2H2O, it is possible to study the dynamics of carbohydrate, protein, lipid, and DNA and to determine body composition. Those studies require reliable measurements of the 2H labeling of water. Although simple gas chromatography-mass spectrometry (GC-MS) methods have been developed for measuring the 2H enrichment of biological fluids, investigators have not reported on the intra- and/or interdaily variability of the measurements. We have experimentally examined the reproducibility of one GC-MS method for measuring the 2H labeling of water. Briefly, hydrogen (deuterium) atoms in water were exchanged with those bound to acetone, and the 2H labeling of acetone was then determined under electron impact ionization. We found that the coefficient of variation is generally less than 0.5% when water is labeled between 0 and 2.8 mole percentage excess 2H. We demonstrated that this highly reproducible result allows one to use 2H2O and the "acetone method" to measure physiological parameters such as body composition in mice.

Adipose Tissue↗
PubMed2006

Lipid-lowering actions of imidazoline antihypertensive agents in metabolic syndrome X.

Agonists active at I1-imidazoline receptors (I1R) not only lower blood pressure but also ameliorate glucose intolerance, insulin resistance, and hyperlipidemia with long-term treatment. We sought to determine the possible mechanism for the lipid-lowering actions of imidazolines in a model of metabolic Syndrome X, the spontaneously-hypertensive obese (SHROB) rat. The acute actions of moxonidine and rilmenidine, selective I1R agonists, were compared to a specific alpha2-adrenergic receptor agonist, guanabenz, with and without selective receptor blockers. Moxonidine and rilmenidine rapidly reduced plasma triglyceride (20+/-4% and 21+/-5%, respectively) and cholesterol (29+/-9% and 27+/-9%). In contrast, the specific alpha2-adrenergic receptor agonist guanabenz failed to reduce plasma lipids. Blocking experiments showed that moxonidine's actions were mediated by I1R and not alpha2-adrenergic receptors. To evaluate a hepatic site of action, radioligand binding studies with liver plasma membranes confirmed the presence of I1R. Intraportal moxonidine reduced plasma triglycerides by 23+/-3% within 10 min. Moxonidine inhibited hepatic triglyceride secretion by 75% compared to vehicle treatment. Tracer studies with 2H2O suggested that moxonidine inhibits de novo fatty acid synthesis. Thus, activation of I1R lowers plasma lipids, with the main site of action probably within the liver to reduce synthesis and secretion of triglycerides. More selective I1R agonists might provide monotherapy for hyperlipidemic hypertension.

Adrenergic alpha-2 Receptor Agonists↗
PubMed2005

Novel application of the "doubly labeled" water method: measuring CO2 production and the tissue-specific dynamics of lipid and protein in vivo.

The partitioning of whole body carbon flux between fat and lean compartments affects body composition. We hypothesized that it is possible to simultaneously determine whole body carbon (energy) balance and the dynamics of lipids and proteins in specific tissues in vivo. Growing C57BL/6J mice fed a high-fat low-carbohydrate diet were injected with a bolus of "doubly labeled" water (i.e., (2)H2O and H2(18)O). The rate of CO2 production was determined from the difference between the elimination rates of 2H and 18O from body water. The rates of synthesis and degradation of triglycerides extracted from epididymal fat pads and of proteins extracted from heart muscle were determined by mathematically modeling the 2H labeling of triglyceride-bound glycerol and protein-bound alanine, respectively. We found that mice were in positive carbon balance (approximately 20% retention per day) and accumulated lipid in epididymal fat pads (approximately 9 micromol triglyceride accumulated per day). This is consistent with the fact that mice were studied during a period of growth. Modeling the 2H labeling of triglycerides revealed a substantial rate of lipid breakdown during this anabolic state (equivalent to approximately 25% of the newly synthesized triglyceride). We found equal rates of protein synthesis and breakdown in heart muscle (approximately 10% of the pool per day), consistent with the fact that the heart muscle mass did not change. In total, these findings demonstrate a novel application of the doubly labeled water method. Utilization of this approach, especially in unique rodent models, should facilitate studies aimed at quantifying the efficacy of interventions that modulate whole body carbon balance and lipid flux while in parallel determining their impact on (cardiac) muscle protein turnover. Last, the simplicity of administering doubly labeled water and collecting samples allows this method to be used in virtually any laboratory setting.

Alanine↗
PubMed2005

Using 2H2O to study the influence of feeding on protein synthesis: effect of isotope equilibration in vivo vs. in cell culture.

We previously reported that 2H2O can be used to measure rates of protein synthesis during prolonged steady-state conditions (Previs SF, Fatica R, Chandramouli V, Alexander JC, Brunengraber H, and Landau BR. Am J Physiol Endocrinol Metab 286: E665-E672, 2004). The underlying premise of our method is that following the administration of 2H2O, 2H atoms in body water rapidly equilibrate with free alanine before it is incorporated into newly synthesized proteins. We have now directly examined whether 2H2O can be used to measure the influence of a single meal on protein synthesis. In addition, we have compared the use of 2H2O for measuring rates of protein synthesis in vivo vs. in cell culture. Using a rat model, we observed rapid equilibration between 2H in body water and free alanine; therefore we were able to study the response of protein synthesis to a single meal. We observed that approximately 50% of the plasma albumin that is synthesized over the course of 24 h is made within approximately 5 h after eating (in rats trained to eat a complete 24-h ration of food in a single meal). Contrary to what we observed in vivo, feeding (the replenishment of cell culture medium) does influence the use of 2H2O for in vitro studies. In particular, since there can be slow equilibration of 2H between water and alanine in the cell culture medium, special consideration must be made to avoid underestimating the rate of protein synthesis in vitro.

Alanine↗
PubMed2004

Using isotope tracers to study metabolism: application in mouse models.

The application of isotope tracers for investigating metabolism in mice is discussed. To familiarize the reader, some basic principles regarding the use of tracer methods are outlined. Emphasis is placed on showing how investigators are using isotope tracers to study the regulation of carbohydrate, fat and/or protein turnover in vivo. Finally, some of the advantages of using labeled water (i.e., 2H(2)O and/or H(2)18O) to trace the kinetics of biological processes are considered. The background provided in this report should assist engineers in designing studies that enhance our understanding of conditions in which metabolism is altered (e.g., diabetes, cancer cachexia, failure to thrive and travel at zero-gravity).

Animals↗
PubMed2003

Quantifying rates of protein synthesis in humans by use of 2H2O: application to patients with end-stage renal disease.

A method is introduced for quantitating protein synthetic rates in humans by use of (2)H(2)O. Its validity was tested in subjects with end-stage renal disease. Six clinically stable subjects, hemodialyzed three times weekly, ingested (2)H(2)O to a body water (2)H enrichment of approximately 0.4%. On dialysis, body water enrichment declined to approximately 0.1%. Enrichment of the alpha-hydrogen of plasma free alanine was also approximately 0.4% before and approximately 0.1% after dialysis. Beta-hydrogen enrichment was approximately 80-100% of alpha-hydrogen enrichment. (2)H(2)O was ingested to replace (2)H(2)O removed after each dialysis for 15-51 days, returning enrichment to approximately 0.4%. Enrichment of alanine from plasma albumin gradually increased, with again approximately 80-100% as much (2)H in beta- as in alpha-hydrogens. With continued dialyses, without (2)H(2)O replacement, alanine from albumin enrichment gradually declined, whereas free alanine and water enrichments were negligible. The fractional albumin synthesis rate, calculated from the increase in enrichment in alanine from albumin, was 4.0 +/- 0.5%/day, and from the decrease, 4.6 +/- 0.2%/day. Thus body water enrichment in a subject given (2)H(2)O can be maintained constant long term. A rapid exchange, essentially complete, occurs between the hydrogens of alanine and body water. An integrated measure over a long period of albumin's synthetic rate can be estimated from both the rise in enrichment of alanine from the protein during (2)H(2)O ingestion and fall on (2)H(2)O withdrawal, while the subject's living routine is uninterrupted. Estimates are in subjects with renal disease, but the method should be applicable to estimates of protein synthetic rates in normal subjects and in other pathological states.

Adult↗
PubMed2003

Influence of diet on the modeling of adipose tissue triglycerides during growth.

We have studied the accretion of lipids in growing mice. We measured the rates of synthesis and degradation of triglycerides in epididymal fat pads of mice maintained for 44 days on a low-fat, high-carbohydrate diet (I) or a high-fat, low-carbohydrate diet (II). 2H2O was added to the drinking water for 14 days. Rates of incorporation/washout of 2H to/from C1 of triglyceride-glycerol showed that triglyceride synthesis was greater than triglyceride degradation (net triglyceride balance was approximately 2.5 times greater in II than in I). The data also show that the contribution of de novo lipogenesis to triglyceride-bound palmitate was approximately 3 times greater in I than in II. This was consistent with a greater relative intake of carbohydrate in I vs. II. The rates of incorporation and washout of newly synthesized (2H-labeled) palmitate into and from triglycerides were also measured. Those data suggested a remodeling of triglyceride-bound fatty acids. On measuring the profile of triglyceride-bound fatty acids, we observed a decrease in the relative abundance of triglyceride-bound palmitate and stearate and an increase in triglyceride-bound oleate and linoleate. This was observed in I and II. In summary, diet substantially affects the deposition and modeling of triglycerides in adipose tissue during growth. 2H2O can be used to examine the mechanisms responsible for the accumulation of triglycerides, e.g., factors that affect 1) triglyceride synthesis and degradation and 2) the source of fatty acids that are used in esterification.

Adipose Tissue↗
PubMed2003

Measuring gluconeogenesis using a low dose of 2H2O: advantage of isotope fractionation during gas chromatography.

The contribution of gluconeogenesis to glucose production can be measured by enriching body water with (2)H(2)O to approximately 0.5% (2)H and determining the ratio of (2)H that is bound to carbon-5 vs. carbon-2 of blood glucose. This labeling ratio can be measured using gas chromatography-mass spectrometry after the corresponding glucose carbons are converted to formaldehyde and then to hexamethylenetetramine (HMT). We present a technique for integrating ion chromatograms that allows one to use only 0.05% (2)H in body water (i.e., 10 times less than the current dose). This technique takes advantage of the difference in gas chromatographic retention times of naturally labeled HMT and [(2)H]HMT. We discuss the advantage(s) of using a low dose of (2)H(2)O to quantify the contribution of gluconeogenesis.

Animals↗
PubMed2003

Measuring in vivo metabolism using heavy water.

PURPOSE OF REVIEW: Our goal is to explain how 2H(2)O, a stable isotope of water, can be used to quantify metabolic flux in vivo. Although 2H(2)O was originally used more than 65 years ago, recent reports have described new applications. Investigators have also refined the original work. We discuss those points, emphasizing factors that should be considered when interpreting data. As our goal is to discuss why 2H(2)O is a novel tracer for studying metabolism, we do not comment on the interpretation(s) of physiological data. RECENT FINDINGS: It is clear that 2H(2)O can now be used to address questions related to carbohydrate, lipid, protein and DNA synthesis. A particular advantage of using 2H(2)O is that investigators can study metabolism in free-living individuals. Using this novel tracer method, it is thus possible to elucidate new, highly relevant, knowledge regarding health and disease. SUMMARY: As the use of 2H(2)O requires few assumptions, the application of this tracer should yield sound information regarding the regulation of biochemical reactions in vivo.

Animals↗
PubMed2002

Gas chromatography-mass spectrometry assay of the (18)o enrichment of water as trimethyl phosphate.

We have developed an assay for determining the 18O enrichment of water in biological fluids. Urine, plasma, or whole blood is reacted with phosphorous pentachloride to yield phosphoric acid. Derivatization of phosphoric acid with diazomethane generates trimethyl phosphate. The enrichment of trimethyl phosphate is nearly four times that of water and is assayed using gas chromatography-mass spectrometry (electron impact ionization). Yang et al. (1998, Anal. Biochem. 258, 315-321) assayed the 2H enrichment of body water after exchange with acetone, by gas chromatography-mass spectrometry. The combination of our 18O method and the 2H method of Yang et al. allows one to measure energy expenditure via "doubly labeled" water (2H(2)O + H(2)18O), using small samples of body fluids. These techniques were used to measure energy expenditure in mice, in which the 18O enrichment of body water can be monitored down to 0.025%.

Animals↗