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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.

The primary objective of this investigation was to determine whether [sup.2][H.sub.2]O and phenylalanine (Phe) flooding dose methods yield comparable fractional rates of protein synthesis (FSR) in skeletal muscle following a single bout of high-intensity resistance exercise (RE). Sprague-Dawley rats were assigned by body mass to either 4-h control (CON 4 h; n = 6), 4-h resistance exercise (RE 4 h; n = 6), 24-h control (CON 24 h; n = 6), or 24-h resistance exercise (RE 24 h; n = 6). The RE groups were operantly conditioned to engage in a single bout of high-intensity, 'squat-like' RE. All rats were given an intraperitoneal injection of 99.9% [sup.2][H.sub.2]O and provided 4.0% [sup.2][H.sub.2]O drinking water for either 24 (n = 12) or 4 h (n = 12) prior to receiving a flooding dose of L-[2,3,4,5,6-[sup.3]H]Phe 16 h post-RE. Neither method detected an effect of RE on FSR in the mixed gastrocnemius, plantaris, or soleus muscle. Aside from the qualitative similarities between methods, the 4-h [sup.2][H.sub.2]O FSR measurements, when expressed in percent per hour, were quantitatively greater than the 24-h [sup.2][H.sub.2]O and Phe flooding in all muscles (P < 0.001), and the 24-h [sup.2][H.sub.2]O was greater than the Phe flooding dose in the mixed gastrocnemius and plantaris (P < 0.05). In contrast, the actual percentage of newly synthesized protein was significantly higher in the 24- vs. 4-h [sup.2][H.sub.2]O and Phe flooding dose groups (P < 0.001). These results suggest that the methodologies provide 'qualitatively' similar results when a perturbation such as RE is studied. However, due to potential quantitative differences between methods, the experimental question should determine what approach should be used. fractional rates of protein synthesis; skeletal muscle

Whole body protein synthesis is reduced during the fed-to-fasted transition and in cases of chronic dietary restriction; however, less is known about tissue-specific alterations. We have assessed the extent to which protein synthesis in cardiac muscle responds to dietary perturbations compared with liver and skeletal muscle by applying a novel [sup.2][H.sub.2]O tracer method to quantify tissue-specific responses of protein synthesis in vivo. We hypothesized that protein synthesis in cardiac muscle would be unaffected by acute fasting or food restriction, whereas protein synthesis in the liver and gastrocnemius muscle would be reduced when there is a protein-energy deficit. We found that, although protein synthesis in liver and gastrocnemius muscle was significantly reduced by acute fasting, there were no changes in protein synthesis in the left ventricle of the heart for either the total protein pool or in isolated mitochondrial or cytosolic compartments. Likewise, a chronic reduction in calorie intake, induced by food restriction, did not affect protein synthesis in the heart, whereas protein synthesis in skeletal muscle and liver was decreased. The later observations are supported by changes in the phosphorylation state of two critical mediators of protein synthesis (4E-BP1 and eIF2[alpha]) in the respective tissues. We conclude that cardiac protein synthesis is maintained in cases of nutritional perturbations, in strong contrast to liver and gastrocnemius muscle, where protein synthesis is decreased by acute fasting or chronic food restriction. protein turnover; nitrogen homeostasis; left ventricular hypertrophy; calorie restriction; stable isotopes

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.sup.-/-] 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.sup.-/-] 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.

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., [sup.2][H.sub.2]O and [H.sub.2][sup.18]O). The rate of C[O.sub.2] production was determined from the difference between the elimination rates of [sup.2]H and [sup.18]O 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 [sup.2]H labeling of triglyceride-bound glycerol and proteinbound alanine, respectively. We found that mice were in positive carbon balance (~20% retention per day) and accumulated lipid in epididymal fat pads (~9 [micro]mol triglyceride accumulated per day). This is consistent with the fact that mice were studied during a period of growth. Modeling the [sup.2]H labeling of triglycerides revealed a substantial rate of lipid breakdown during this anabolic state (equivalent to ~25% of the newly synthesized triglyceride). We found equal rates of protein synthesis and breakdown in heart muscle (~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. metabolic regulation; carbon-energy balance; triglyceride turnover; protein turnover; stable isotope tracer kinetics

We previously reported that [sup.2][H.sub.2]O 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 [sup.2][H.sub.2]O, [sup.2]H atoms in body water rapidly equilibrate with free alanine before it is incorporated into newly synthesized proteins. We have now directly examined whether [sup.2][H.sub.2]O can be used to measure the influence of a single meal on protein synthesis. In addition, we have compared the use of [sup.2][H.sub.2]O for measuring rates of protein synthesis in vivo vs. in cell culture. Using a rat model, we observed rapid equilibration between [sup.2]H in body water and free alanine; therefore we were able to study the response of protein synthesis to a single meal. We observed that ~50% of the plasma albumin that is synthesized over the course of 24 h is made within ~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 [sup.2][H.sub.2]O for in vitro studies. In particular, since there can be slow equilibration of [sup.2]H between water and alanine in the cell culture medium, special consideration must be made to avoid underestimating the rate of protein synthesis in vitro. albumin turnover; nutritional status; stable isotopes; gas chromatography-mass spectrometry

Previs, Stephen F., Richard Fatica, Visvanathan Chandramouli, James C. Alexander, Henri Brunengraber, and Bernard R. Landau. Quantifying rates of protein synthesis in humans by use of [sup.2.H.sub.2.0]: application to patients with end-stage renal disease. Am J Physiol Endocrinol Metab 286: E665-E672, 2004. First published December 23, 2003; 10.1152/ajpendo.00271.2003.--A method is introduced for quantitating protein synthetic rates in humans by use of [sup.2.H.sub.2.O]. Its validity was tested in subjects with end-stage renal disease. Six clinically stable subjects, hemodialyzed three times weekly, ingested [sup.2.H.sub.2.0] to a body water [sup.2.H] enrichment of ~0.4%. On dialysis, body water enrichment declined to ~0.1%. Enrichment of the [alpha]-hydrogen of plasma free alanine was also ~0.4% before and ~0.1% after dialysis. [3-Hydrogen enrichment was ~80-100% of [alpha]-hydrogen enrichment. [sup.2.H.sub.2.0] was ingested to replace [sup.2.H.sub.2.0] removed after each dialysis for 15-51 days, returning enrichment to ~0.4%. Enrichment of alanine from plasma albumin gradually increased, with again ~80-100% as much [sup.2.H] in [beta]- as in [alpha]-hydrogens. With continued dialyses, without [sup.2.H.sub.2.0] 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 [+ or -] 0.5%/day, and from the decrease, 4.6 [+ or -] 0.2%/day. Thus body water enrichment in a subject given [sup.2.H.sub.2.0] 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 [sup.2.H.sub.2.0] ingestion and fall on [sup.2][H.sub.2O] 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. protein; albumin; alanine; deuterium oxide; hemodialysis

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 (H). [sup.2][H.sub.2]O was added to the drinking water for 14 days. Rates of incorporation/washout of [sup.2]H to/from C1 of triglyceride-glycerol showed that triglyceride synthesis was greater than triglyceride degradation (net triglyceride balance was ~2.5 times greater in H than in I). The data also show that the contribution of de novo lipogenesis to triglyceride-bound palmitate was ~3 times greater in I than in H. This was consistent with a greater relative intake of carbohydrate in I vs. H. The rates of incorporation and washout of newly synthesized ([sup.2]H-labeled) palmitate into and from triglycerides were also measured. Those data suggested a remodeling of triglyceridebound fatty acids. On measuring the profile of triglyceridebound 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. [sup.2][H.sub.2]0 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. deuterium; stable isotopes; lipid metabolism; pediatric obesity; insulin resistance

The contribution of gluconeogenesis to glucose production can be measured by enriching body water with [sup.2][H.sub.2]O to ~0.5% [sup.2]H and determining the ratio of [sup.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% [sup.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 [[sup.2]H]HMT. We discuss the advantage(s) of using a low dose of [sup.2][H.sub.2]O to quantify the contribution of gluconeogenesis. diabetes; stable isotopes; gas chromatography-mass spectrometry

The substrate-induced effects on rate of appearance of glycerol and glucose and total glucose production (f) during (2-13C)glycerol infusion in vivo in mice have been studied by using the mass isotopomer distribution analysis (MIDA). Five groups of mice were fasted for 30 hours and were then administered with (2-13C)glycerol at various rates and 13C enrichments. Results indicated that MIDA gave erroneous estimates of f with low infusion rates of (2-13C)glycerol.

Previous studies proposed the calculations of gluconeogenesis's contributions to glucose production and Cori cycling from mass isotopomer distributions in blood lactate and glucose during (U-(super 13)C(sub 6))glucose infusion. It is, however, hypothesized that there are limitations in the proposal including the inadequate differentiation of isotopic exchange from the dilution. Results demonstrate an underestimation of the contribution of gluconeogenesis which may be attributable to various reasons.

In vivo preparations involving fasted anesthesized dogs were utilized to determine the role of the liver and kidneys in gluconeogenesis. The presence of high insulin and glucose concentrations in the CHO-fasted dogs reduced the ratio of the rate of appearance (Ra) of glycerol to the Ra of free fatty acids from three to 2.4. The release of insulin from the liver was also inhibited after the infusion of glucose indicating the role of the kidneys and liver in mediating glycerol uptake.

The relationship between splanchnic and renal glycerol balance and the rates of appearance (Ra) of glycerol in the circulation was analyzed in normal human subjects after fasting for 60 hours. The subjects exhibited 3.5-fold increase in the Ra of glycerol and enhanced glucose uptake in the splanchnic bed. However, glycerol uptake in the liver was not detected in the livers of the 60-hour fasted human subjects while fatty acid reesterification was detected only in nonsplanchnic tissues.

A new technique that uses the phenylacetylglutamine (PAGN) probe to determine the 14C-labeling pattern of liver citric acid cycle is presented. The PAGN probe assumes that the labeling pattern of the glutamine moiety of PAGN is similar with liver alpha-ketoglutarate. Various stable isotopic tracers can be used with the probe. The labeling pattern of liver citric acid cycle is determined by gas chromatography-mass spectrometry. This new technique has been tested in primate liver and the results are discussed.

Controlled starvation of poorly differentiated (anaplastic) fast-growing tumour cells, but not host cells is possible in vivo, and this finding is useful for selective starving of the cancer cells that are unable to use fat-derived fuels, but can preserve nutrient supply to vital organs. An animal model is developed that requires all the cells to oxidize fatty acids, ketoacids, and 1,3-butane-diol to satisfy the energy needs. The tracer kinetic measurements show no change in the rate of glutamine appearance as predicted in the model.