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, lowcarbohydrate diet (II).2 H2O was added to the drinking water for 14 days. Rates of incorporation/washout of 2 H to/from C1 of triglyceride-glycerol showed that triglyceride synthesis was greater than triglyceride degradation (net triglyceride balance was ϳ2.5 times greater in II than in I). The data also show that the contribution of de novo lipogenesis to triglyceride-bound palmitate was ϳ3 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 ( 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. 2 H2O 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 THERE IS AN ALARMING INCREASE in the number of reported cases of pediatric obesity (26, 32). Like obese adults, obese children present with metabolic abnormalities (e.g., impaired glucose tolerance, dyslipidemia) that are associated with an increased risk of developing a chronic disease (e.g., diabetes, cardiovascular disease) (8,9,21,24,32). A special concern regarding obese children is that many will remain obese as adults, which further increases the likelihood of developing a chronic disease. Presumably, the development of effective treatments for pediatric obesity will benefit from knowledge of how specific biochemical reactions drive lipid accumulation during growth.We hypothesized that, during growth, the nature of one's caloric intake will affect the rate of, and the mechanism of, fat accumulation. For example, as the ratio of dietary fat to carbohydrate increases, the rate of triglyceride accretion will increase. Also, the relative amount of carbohydrate in the diet will affect the relative contribution of de novo lipogenesis to the pool of triglyceride-bound fatty acids. We developed the use of 2 H 2 O to measure the rates of the reactions involved in triglyceride accumulation (e.g., triglyceride synthesis and triglyceride degradation and the contribution of de novo lipogenesis to the pool of triglyceride-bound fatty acids). This approach also allows us to determine the rate of remodeling of fatty acids that are bo...

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.

A high rate of cardiac work increases citric acid cycle (CAC) turnover and flux through pyruvate dehydrogenase (PDH); however, the mechanisms for these effects are poorly understood. We tested the hypotheses that an increase in cardiac energy expenditure: (1) activates PDH and reduces the product/substrate ratios ([NADH]/[NAD(+)] and [acetyl-CoA]/[CoA-SH]); and (2) increases the content of CAC intermediates. Measurements were made in anaesthetized pigs under control conditions and during 15 min of a high cardiac workload induced by dobutamine (Dob). A third group was made hyperglycaemic (14 mm) to stimulate flux through PDH during the high work state (Dob + Glu). Glucose and fatty acid oxidation were measured with (14)C-glucose and (3)H-oleate. Compared with control, the high workload groups had a similar increase in myocardial oxygen consumption ( and cardiac power. Dob increased PDH activity and glucose oxidation above control, but did not reduce the [NADH]/[NAD(+)] and [acetyl-CoA]/[CoA-SH] ratios, and there were no differences between the Dob and Dob + Glu groups. An additional group was treated with Dob + Glu and oxfenicine (Oxf) to inhibit fatty acid oxidation: this increased [CoA-SH] and glucose oxidation compared with Dob; however, there was no further activation of PDH or decrease in the [NADH]/[NAD(+)] ratio. Content of the 4-carbon CAC intermediates succinate, fumarate and malate increased 3-fold with Dob, but there was no change in citrate content, and the Dob + Glu and Dob + Glu + Oxf groups were not different from Dob. In conclusion, compared with normal conditions, at high myocardial energy expenditure (1) the increase in flux through PDH is regulated by activation of the enzyme complex and continues to be partially controlled through inhibition by fatty acid oxidation, and (2) there is expansion of the CAC pool size at the level of 4-carbon intermediates that is largely independent of myocardial fatty acid oxidation.

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The contribution of gluconeogenesis to glucose production can be measured by enriching body water with2H2O to ∼0.5% 2H and determining the ratio of 2H 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% 2H 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 [2H]HMT. We discuss the advantage(s) of using a low dose of 2H2O to quantify the contribution of gluconeogenesis.