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3 Metabolism Unit, Shriners Hospital for Children, and Departments of 4 Surgery and 5 Anesthesiology, University of Texas Medical Branch, Galveston, TX 77550
* To whom correspondence should be addressed. E-mail: xzhang{at}utmb.edu.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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This study was designed to investigate alterations in lipid metabolism in diet-induced obese rabbits using stable isotope techniques. We hypothesized that obese rabbits are characterized by increased lipid turnover similar to obese humans and therefore would be a suitable model for the study of obesity. On the other hand, the rabbit is known to be naturally deficient in hepatic lipase (1), so its effect on hepatic lipid metabolism needs to be recognized.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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3.5 kg, were used for this study. The rabbits were housed in individual cages and were given 100–120 g/d of unpurified diet (Lab Rabbit Chow 5326, Purina Mills) for 1 wk for acclimation. This diet provided 203–244 kcal/d (850–1022 kJ/d) with 23% from protein, 7.9% from fat, and 69% from carbohydrate. They were then randomly assigned to 2 groups: lean and obese. This protocol complied with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, incorporated in the Institute for Laboratory Animal Research Guide for Care and Use of Laboratory Animals, and was approved by the Animal Care and Use Committee of The University of Texas Medical Branch at Galveston. Experimental design. The rabbits in the lean group were given the same diet as in the acclimation phase. The rabbits in the obese group consumed a high-fat diet ad libitum consisting of the same unpurified solid grain diet mixed with additional 10% corn oil and 8% lard; the energy sources consisted 13% from protein, 49% from fat, and 38% from carbohydrate. After 10 wk of feeding, the rabbits were food-deprived for 24 h before tracer infusion to ensure that there was no exogenous fat entry.
Isotope infusion protocol. Surgical procedures were performed to insert catheters into the carotid artery and jugular vein under general anesthesia (8). The arterial line was used for drawing blood and monitoring arterial blood pressure and heart rate; the venous line was used for infusing a stable isotope tracer and anesthetics. Tracheotomy was performed for placement of a tracheal tube, which was connected to a hood filled with oxygen-enriched room air. U-13C16-palmitate (99% enriched; Cambridge Isotope Laboratories), bound to albumin in a 5% solution, was infused at 0.11 µmol·kg–1·min–1 for 8 h. Blood samples were taken before the start of the tracer infusion and every hour during the tracer infusion. The blood samples were collected in prechilled tubes containing EDTA; plasma was separated by centrifugation and stored at –20°C. During the tracer infusion, heart rate, mean arterial blood pressure, and rectal temperature were maintained stable by adjusting the infusion rates of anesthetics and physiological saline and by using heating lamps. At the end of tracer infusion, the rabbits were killed by i.v. injection of 5 mL saturated KCl under general anesthesia.
Sample analysis. Blood glucose concentration was measured with a glucose analyzer (model 2300; Yellow Springs Instruments). To measure concentrations in plasma of nonesterified fatty acids (NEFA)7 and triglycerides (TG), heptadecanoic acid and triheptadecanoin were added to the plasma as internal standards. NEFA and TG fractions were isolated by TLC (9). Seven fatty acids (FA) in plasma NEFA were measured using a GC system with flame identification detection (model 6890, Aligent); their individual contributions to the total 7 FA were expressed as percents. The 7 FA were myristiate (14:0), palmitate (16:0), palmitoleate [16:1(n-1)], stearate (18:0), oleate [C18:1(n-9)], linoleate [18:2(n-6)], and linolenate [(18:3(n-3)]. Palmitate enrichment in the NEFA and TG fractions were measured using a GC-MS (MSD system, Aligent). Ions were selectively monitored at mass-to-charge ratios of 270, 285, and 286 for palmitate.
Calculations.
The rate of appearance (Ra) of plasma palmitate was calculated by tracer dilution. The equation is
 | (Eq. 1) |
where F is U-13C16-palmitate infusion rate in µmol·kg–1·min–1 and E is plasma nonesterified palmitate enrichment at plateau.
Plasma TG-bound palmitate enrichment was calculated by the following equation:
 | (Eq. 2) |
where enrichment (t) is plasma TG-bound palmitate enrichment at time t, A is the plateau enrichment, B is the increase in enrichment from baseline to plateau, and l is the initial slope.
The equation used to calculate percent of secretory TG synthesis from plasma NEFA is
 | (Eq. 3) |
where ETG is plateau enrichment of plasma TG and Epalmitate is plateau enrichment of plasma nonesterified palmitate.
The fractional secretion rate (FSR) of hepatic secretory TG from plasma NEFA was calculated from the following equation:
 | (Eq. 4) |
where (Et2 – Et1) is the increment of plasma TG-bound palmitate from time 1 to time 2; EP(t2 – t1) is the mean plasma nonesterified palmitate enrichment from time 1 to time 2.
Total FSR of plasma TG was calculated from the equation:
 | (Eq. 5) |
where FSRplasma is calculated from Eq. 4 and % TGpalmitate is calculated from Eq. 3.
Values are expressed as means ± SEM. Differences comparing 2 parameters were tested by t test either paired (within 1 group) or nonpaired (between 2 groups). Differences dealing with multiple time points within 1 group (comparisons to time 0) or between 2 groups (comparison between each pair of corresponding time points) were evaluated using 2-way repeated measures ANOVA followed by Bonferroni's multiple comparison test as appropriate. A P-value <0.05 was considered significant.
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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During the tracer infusion, the rectal temperature was similar in the lean and obese rabbits (Table 1). Heart rate and mean arterial pressure were higher in the obese than in the lean rabbits (Table 1). Plasma concentrations of glucose (Fig. 1A), NEFA (Fig. 1B), and TG (Fig. 1C) were higher (P < 0.05) in the obese than in the lean rabbits (Table 1). Palmitate accounted for 32.4 ± 0.2 and 24.8 ± 0.3% of the total 7 NEFA in the lean and obese groups, respectively (P = 0.05). These values were used to convert plasma nonesterified palmitate concentration to total NEFA concentration.
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Hyperlipidemia and elevated NEFA turnover are the primary alterations of systemic lipid metabolism in obesity. This reflects increased lipolysis in conjunction with relatively insufficient plasma clearance, leading to fat accumulation. In human obesity, the rate of lipolysis may increase by 20–30%, when expressed as per unit of fat-free mass compared with that of lean counterparts (11,13). In obese animals, plasma NEFA and TG concentrations are reported to be elevated (2,15,16); however, the lipid kinetics have not been adequately measured. We found that the Ra of NEFA (reflecting the rate of lipolysis) in the obese rabbits was 30 or 40% greater than that in lean rabbits during the total 8 h or the first 4 h of tracer infusion. These results are consistent with the previous report of insulin resistance in obese rabbits (2), because insulin would normally be expected to limit lipolysis and thus Ra NEFA.
The difference in FA sources used for hepatic secretory TG synthesis between the groups was striking. In the lean rabbits, 100% of the hepatic secretory TG was synthesized from plasma NEFA. In contrast, only 59% was synthesized from plasma NEFA in the obese rabbits. Hepatic secretory TG synthesis may use FA from plasma NEFA, de novo synthesized FA in the liver, FA released from hepatic TG degradation, and portal vein delivery of chylomicra and omental FA. Because the rabbits in this study were food deprived for 24 h before the tracer infusion, the contributions from hepatic de novo synthesis and from portal vein delivery of chylomicra can be excluded. Therefore, in the obese rabbits, the balance of hepatic TG synthesis (41%) was likely due to either hepatic TG degradation and/or portal vein delivery of omental FA.
Hepatic TG degradation consists of 2 potential components: plasma lipoprotein TG hydrolysis catalyzed by hepatic lipase in the sinusoids and hydrolysis of hepatic TG stored in the cytosol of hepatocytes. The latter is referred to as the delayed secretory pathway (17). The rabbit lacks hepatic lipase (18), which in other species binds to the sinusoidal surface of hepatic endothelial cells and plays a fundamental role in hydrolyzing the TG in lipoproteins and chylomicron remnants (19–21). Therefore, plasma lipoprotein TG hydrolysis can be excluded, meaning that hepatic TG degradation is exclusively attributable to the hydrolysis of storage TG. This is a unique characteristic of rabbits, because in lean fasted humans, plasma NEFA accounts for 90–94% of FA in VLDL-TG (22,23). Considering the complex nature of hepatic lipid metabolism, the deficiency in hepatic lipase in rabbits could be advantageous in quantification of hepatic lipid kinetics in future experiments because of a simplification of the necessary calculations.
In summary, the diet-induced obese rabbits had high plasma NEFA and TG concentrations and increased plasma NEFA turnover rate. These alterations in lipid kinetics are similar to those observed in obese humans. The deficiency of hepatic lipase in the rabbit provides a potential advantage in quantification of hepatic lipid metabolism, because it excludes hydrolysis of lipoprotein TG as a source of FA for synthesizing secretory TG.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosure: X-J. Zhang, D. L. Chinkes, A. Aarsland, D. N. Herndon, and R. R. Wolfe, no conflicts of interest.
6 Current address: University of Arkansas Medical School, 4301 Markham Slot 806, Little Rock, AR 72205. Tel: +15015265708, Fax: +15015265710. E-mail: rwolfe2{at}uams.edu.
7 Abbreviations used: FA, fatty acid; FSR, fractional secretion rate; NEFA; nonesterified fatty acid; Ra, rate of appearance; TG, triglyceride.
Manuscript received 10 September 2007. Initial review completed 10 October 2007. Revision accepted 18 December 2007.
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LITERATURE CITED |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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1. Taylor JM, Fan J. Transgenic rabbit models for the study of atherosclerosis. Front Biosci. 1997;2:298–308.
2. Carroll JF, Dwyer TM, Grady AW, Reinhart GA, Montani J-P, Cockrell K, Meydrech EF, Mizelle HL. Hypertension, cardiac hypertrophy, and neurohumoral activity in a new animal model of obesity. Am J Physiol. 1996;271:H373–8.[Medline]
3. Antic V, Belforti K-B, Tempini A, Van Vliet BN, Montani J-P. Role of the sympathetic nervous system during the development of obesity-induced hypertension in rabbits. Am J Hypertens. 2000;13:556–9.[Medline]
4. Carroll JF, King JW, Cohen JS. Hydralazine treatment alters body composition in the rabbit model of obesity. Acta Physiol Scand. 2004;181:183–91.[Medline]
5. Rong JX, Shen L, Chang YH, Richters A, Hodis HN, Sevanian A. Cholesterol oxidation products induce vascular foam cell lesion formation in hypercholoesterolemic New Zealand White rabbits. Arterioscler Thromb Vasc Biol. 1999;19:2179–88.
6. Dwyer TM, Mizelle HL, Cockrell K, Diveki K, Buhner P. Renal sinus lipomatosis and body composition in hypertensive, obese rabbits. Int J Obes. 1995;19:869–74.[Medline]
7. Carroll JF, Huang M, Hester RL, Cockrell HK, Mizelle HL. Hemodynamic alternations in hypertensive obese rabbits. Hypertension. 1995;26:465–70.
8. Zhang X-J, Sakurai Y, Wolfe RR. An animal model for measurement of protein metabolism in the skin. Surgery. 1996;119:326–32.[Medline]
9. Wolfe RR. Radioactive and stable isotope tracers in biomedicine. New York: Wiley-Liss; 1992.
10. Craig BW, Carthwaite SM, Holloszy JO. Adipocyte insulin resistance: effects of aging, obesity, exercise, and food restriction. J Appl Physiol. 1987;62:95–100.
11. Campbell PJ, Carlson MG, Nurjhan N. Fat metabolism in human obesity. Am J Physiol. 1994;266:E600–5.[Medline]
12. Ranneries C, Buülow J, Buemann B, Christensen NJ, Madsen J, Astrup A. Fat metabolism in formerly obese women. Am J Physiol. 1998;274:E155–61.[Medline]
13. Horowitz J, Klein S. Whole body and abdominal lipolytic sensitivity to epinephrine is suppressed in upper body obese women. Am J Physiol. 2000;278:E1144–52.
14. Racette SB, Horowitz JF, Mittendorfer B, Klein S. Racial differences in lipid metabolism in women with abdominal obesity. Am J Physiol. 2000;279:R944–50.
15. Buettner R, Parhofer KG, Woenckhaus M, Wrede CE, Kunz-Schughart LA, Schölmerich J, Bollheimer LC. Defining high-fat-diet rat models: metabolic and molecular effects of different fat types. J Mol Endocrinol. 2006;36:485–501.
16. Gauthier MS, Favier R, Lovoie JM. Time course of the development of non- alcoholic hepatic steatosis in response to high-fat diet-induced obesity in rats. Br J Nutr. 2006;95:273–81.[Medline]
17. Vedala A, Wang W, Neese RA, Christiansen MP, Hellerstein MC. Delayed secretory pathway contributions to VLDL-triglycerides from plasma NEFA, diet, and de novo lipogenesis in humans. J Lipid Res. 2006;47:2563–74.
18. Clay MA, Hopkins GJ, Ehnholm CP, Barter PJ. The rabbit as an animal model for hepatic lipase deficiency. Biochim Biophys Acta. 1989;1002:173–81.[Medline]
19. Jackson RL. Lipoprotein lipase and hepatic lipase. In: Boyer PD, editor. The enzymes. New York: Academic Press; 1983. p. 141–81.
20. Sultan F, Lagrange D, Jansen H, Grigli S. Inhibition of hepatic lipase activity impairs chylomicron-remnant removal. Biochim Biophys Acta. 1989;1042:150–2.
21. Rubinstein A, Gibson JC, Paterniti JR, Kakis G, Little A, Ginsberg HN, Brown WV. Effect of heparin-induced lipolysis on the distribution of apolipoprotein E in hepatic triglyceride lipase. J Clin Invest. 1985;75:710–21.[Medline]
22. Parks EJ, Krauss RM, Christiansen MP, Neese RA. Effects of a low-fat, high-carbohydrate diet on VLDL-triglyceride assembly, production, and clearance. J Clin Invest. 1999;104:1087–96.[Medline]
23. Sidossis LS, Mittendorfer B, Walser E, Chinkes D, Wolfe RR. Hyperglycemia-induced inhibition of splanchnic fatty acid oxidation increases hepatic triacylglycerol secretion. Am J Physiol. 1998;275:E798–805.[Medline]
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Different from 0 h within a group, P < 0.05.