Novel application of the “doubly labeled” water method: measuring CO₂production and the tissue-specific dynamics of lipid and protein in vivo

Abstract

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 2 H and 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 2 H labeling of triglyceride-bound glycerol and proteinbound alanine, respectively. We found that mice were in positive carbon balance (ϳ20% retention per day) and accumulated lipid in epididymal fat pads (ϳ9 mol triglyceride accumulated per day). This is consistent with the fact that mice were studied during a period of growth. Modeling the 2 H labeling of triglycerides revealed a substantial rate of lipid breakdown during this anabolic state (equivalent to ϳ25% of the newly synthesized triglyceride). We found equal rates of protein synthesis and breakdown in heart muscle (ϳ10% 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 OBESITY PRESENTS A GLOBAL HEALTH CHALLENGE (20,25), and many attempts are being made to determine the influence of environmental vs. genetic factors on the modeling of body composition. The application of isotope tracers in novel animal models offers a means of dissecting the importance of specific reactions on the pathogenesis and treatment of disease (12,15).We recently demonstrated the use of 2 H 2 O for measuring the turnover rates of triglycerides and proteins in vivo (3,10,24). Briefly, following the administration of 2 H 2 O, 2 H in body water equilibrates with the carbon-bound hydrogens of glycerol 3-phosphate and alanine. The rates of triglyceride and protein syntheses are determined by measuring the incorporation of the respective 2 H-labeled precursors into the appropriate end products. This simple and powerful method can be used in free-living subjects and complements other tracer methods for studying intermediary metabolism (12,15,18,22,36).In our previous studies (3, 10, 24), rates of biochemical flux were ...

Triglyceride Synthesis in Epididymal Adipose Tissue

“…Turning our attention toward triglyceride glycerol dynamics in adipose tissue, we observed reasonable rates of triglyceride turnover in mice fed the HC diet as compared with data in our previous studies (5,6). However, we were surprised by the substantial rates of triglyceride turnover in mice fed the CF diet.…”

Chronic Ethanol and Triglyceride Turnover in White Adipose Tissue in Rats

“…The 2 H-Labeling of Triglyceride-bound Glycerol-Frozen epididymal adipose tissue was hydrolyzed with 1 N KOH in 90% ethanol at 70°C for 2 h. After evaporation of ethanol, free glycerol was recovered as previously described (12,13). H 2 O (3 ml) was added, and the solution was acidified to ϳpH 1.…”

“…1B), and triglyceride-bound glycerol (Fig. 1C) using a procedure that was modified from previous reports (12,13). Briefly, the change in adipose mass over time was first modeled.…”

“…Triglyceride Turnover RatesRates of in vivo triglyceride synthesis and degradation in epididymal adipose tissue were determined during the last two weeks of ethanol feeding using 2 H 2 O as described under "Experimental Procedures" (12,13). Epididymal fat weight increased over time in both pairand ethanol-fed rats; the rate of increase was lower during chronic ethanol feeding (Fig.…”

“…After the administration of 2 H 2 O, 2 H in body water equilibrates with the carbon-bound hydrogens of glycerol 3-phosphate, and the rates of triglyceride synthesis and degradation are determined by measuring the incorporation/ washout of 2 H to/from carbon 1 of triglyceride-bound glycerol (13). The rate of triglyceride degradation during 2 weeks of ethanol feeding was increased by 2.3-fold.…”