Research and Education Institute, Harbor-University of California Los Angeles Medical Center, Torrance, California 90502
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ABSTRACT |
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We present here a study on the role of leptin in the regulation of lipogenesis by examining the effect of dietary macronutrient composition on lipogenesis in the leptin receptor-defective Zucker diabetic fatty rat (ZDF) and its lean litter mate (ZL). Animals were pair fed two isocaloric diets differing in their fat-to-carbohydrate ratio providing 10 and 30% energy as fat. Lipogenesis was measured in the rats using deuterated water and isotopomer analysis. From the deuterium incorporation into plasma palmitate, stearate, and oleate, we determined de novo synthesis of palmitate and synthesis of stearate by chain elongation and of oleate by desaturation. Because the macronutrient composition and the caloric density were controlled, changes in de novo lipogenesis under these dietary conditions represent adaptation to changes in the fat-to-carbohydrate ratio of the diet. De novo lipogenesis was normally suppressed in response to the high-fat diet in the ZL rat to maintain a relatively constant amount of lipids transported. The ZDF rat had a higher rate of lipogenesis, which was not suppressed by the high-fat diet. The results suggest an important hormonal role of leptin in the feedback regulation of lipogenesis.
leptin receptor; animal model of diabetes; deuterated water; dietary fat
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INTRODUCTION |
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SINCE THE DISCOVERY OF LEPTIN and its receptor, experimental evidence has suggested both a central and a peripheral mechanism of action of leptin. Centrally, leptin, acting on its hypothalamic receptors, regulates food intake and adiposity (total body fat; see Refs. 8, 18, 19). On the other hand, chronic administration of leptin severely depletes triglyceride content in adipose tissue, the liver, skeletal muscles, and the pancreas in excess of the loss expected from reduced food intake (25, 26). Leptin also suppresses the expression of many genes for lipogenesis such as acetyl-CoA carboxylase and fatty acid synthetase under in vivo and in vitro conditions (10, 22, 26). The relative contribution of each of these regulatory mechanisms in maintaining energy balance and body composition is poorly understood.
We present here a study on the role of leptin in the regulation of lipogenesis by examining the effect of dietary macronutrient composition on lipogenesis in the leptin receptor-defective Zucker diabetic fatty (ZDF) rat and its lean litter mate (ZL). Animals were fed a constant energy intake of two isocaloric diets differing in their fat-to-carbohydrate (Fat/Cho) ratio. Lipogenesis was measured in the rats using deuterated water and isotopomer analysis. From the deuterium incorporation into plasma palmitate, stearate, and oleate, we determined de novo synthesis of palmitate and synthesis of stearate by chain elongation and of oleate by desaturation. Because the macronutrient composition and the caloric density were controlled, changes in de novo lipogenesis under these dietary conditions represent adaptation to changes in Fat/Cho of the diet. Any difference in lipogenesis in response to changes in Fat/Cho of the diet between the ZDF and the ZL animals reflects the effect of the loss of peripheral action of leptin in the ZDF animals.
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MATERIALS AND METHODS |
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Animals.
Male ZDF and Zucker lean (ZL) rats were purchased from Genetic models
(Indianapolis, IN). Male Zucker obese (ZO) rats were purchased from
Charles River (Modesto, CA) and were used as an additional group of
leptin receptor-defective rats. The age and weight of the animals at
the time of study and their biochemical characteristics are provided in
Table 1. Animals were housed in
individual cages in a vivarium, which maintains a constant temperature
and an artificial 12:12-h light-dark cycle. The experimental animals
were fed a constant energy intake either of a control (ID 98246) or of
a high-fat (ID 98247) diet purchased from Harlan Teklad (Madison, WI).
The energy intake was set (pair) to that consumed by the ZL rat when
given free access to the control diet. The control diet provides 10%
of the energy intake as fat in the form of safflower oil. The high-fat
diet provides 30% energy intake as fat. This is accomplished by
substituting part of the cornstarch with an amount of lard of equal
energy. These diets have identical contents in protein, polyunsaturated
fatty acids, vitamins, and minerals and are formulated to provide 3.6 kcal/g (Table 2). After acclimatization
for 2-3 days until constant intake was established, animals were
given an intraperitoneal injection of deuterated water equal to 4% of
lean body weight as saline to begin the experiment at 10:00 AM. Animals
were then maintained on water containing 6% deuterium oxide
(D2O). This procedure is designed to maintain deuterium
enrichment in body water at ~3% throughout the study (12).1 Blood
samples were obtained from the tail vein at 6-8 h and at 10:00 AM
on days 1, 2, 4, 8, and
14 of the study, and plasma was separated from these samples
for total lipid analyses. Rats were killed on day 14, and
blood was collected from the hepatic vein. Plasma glucose, lactate,
free fatty acids (FFA), cholesterol, and triglyceride of samples
collected at the time of death were analyzed using colorimetric methods
and insulin by RIA using rat insulin standards.
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Gas Chromatography-Mass Spectometry Analysis
Plasma total lipid extraction was performed using methods described by Lowenstein et al. (15). This procedure extracts fatty acids from triacylglycerol, cholesteryl esters, and phopsholipids in addition to FFA. Gas chromatography-mass spectometry (GC-MS) analysis was performed on a Hewlett Packard model 5973 Selective Mass Detector connected to a model 6890 gas chromatograph (GC) using electron impact ionization. A glass capillary column bpx 70 (SGE, Austin, TX) measuring 30 m × 250 µm (ID) was used to separate fatty acid methyl esters. The GC carrier gas (helium) flow rate was 1 ml/min; injector temperature was 250°C; and oven temperature was programmed from 120 to 220°C at 5°C/min. Respective retention times for palmitate, stearate, and oleate methyl esters were 9.1, 12.3, and 14.4 min. Selected ion monitoring was used to follow specific ions bracketing the molecular ions of methyl ester of palmitate at mass-to-charge ratio (m/z) 270, stearate at m/z 298, and oleate at m/z 264. The ion clusters monitored were m/z 269-276 for palmitate, m/z 297-304 for stearate, and m/z 263-270 for oleate. Mass isotopomer (m) distribution was determined using the method of Lee et al. (14), which corrects for the contribution of derivatizing agent and 13C natural abundance to the mass isotopomer distribution of the compound of interest. The calculated mass isotopomer distribution is expressed as molar fractions (m0, m1, m2, m3, etc.), which are the fractions of molecules containing 0, 1, 2, 3, etc., deuterium substitutions, respectively. Enrichment is expressed as a fraction of the total number of molecules. Thus the sum of all m[infi]i[r] values equals one. The average number of deuterium atoms incorporated per molecule (ME) is calculated from the mass isotopomer distribution using the following relationship: ME = 1 × m1 + 2 × m2 + 3 × m3 + ... or ME = E i×m[infi]i[r] (i = 1, 2, ..., N).ME calculated by this method is the same as the incremental change in the average mass using the average mass approach (2).
Calculation of de novo synthesis.
The method for calculating de novo lipogenesis of palmitate and
stearate has been described previously (11,
13). The deuterium enrichment in body water (p) was first
determined from the consecutive mass isotopomer ratio
m2/m1 using the
relationship
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(1) |
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(2) |
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(3) |
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(4) |
Estimation of chain elongation to stearate and of desaturation to
oleate.
In the synthesis of stearate in the presence of D2O, three
deuterium atoms are added to palmitate in chain elongation
(1).2 Because
circulating palmitate molecules in the form of esterified or
nonesterified fatty acids are either labeled or unlabeled, stearate
molecules derived from chain elongation also consist of two pools, one
having only three deuterium atoms and another having 24 deuterium atoms
per molecule. From Eq. 1 above, it is clear that the
consecutive mass isotopomer ratio
(m2/m1) of stearate is
going to follow the constraint (24 1)/2 × p/q
m2/m1
p/q. If every
molecule of stearate is synthesized from preexisting unlabeled
palmitate, m2/m1 of
stearate equals p/q. If stearate is completely synthesized from
acetyl-CoA, then m2/m1
equals (24
1)/2 × p/q or 11.5 × p/q. Thus the
amount of stearate synthesized by chain elongation of unlabeled
palmitate can be evaluated by comparing the
m2/m1 of stearate with
that of palmitate.
Statistical analysis. Data are reported as means ± SD. The significance of difference between variables was calculated by Student's t-test for nonpaired groups.
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RESULTS |
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The physical and biochemical characteristics of the ZL, ZDF, and ZO rats in the low-Fat/Cho and high-Fat/Cho groups are shown in Table 1. Characteristics of the weight-matched and age-matched ZDF rats are presented as separate groups. The rats each consumed 90 kcal of food/day. The ZL rats gained on average ~30 g over the 14-day period. In contrast, the young weight-matched ZDF rats gained ~94 g, the age-matched rats gained 60 g, and the ZO rats gained 70 g during the same period. The differences in weight gain between ZL and the leptin receptor-defective animals, ZDF (both age matched and weight matched) and ZO, were significant (P < 0.01). The ZDF rats had elevated random blood glucose (P < 0.02), insulin (P < 0.02), FFA (P < 0.005), cholesterol, and triglyceride (P < 0.05) values compared with their lean littermates. The ZO rats had elevated random blood glucose (P < 0.02) and insulin (P < 0.02) compared with the ZL rats.3 However, FFA, cholesterol, and triglyceride values were comparable to those of the ZL rats. Concentrations of blood glucose, insulin, lactate, FFA, cholesterol, and triglyceride within each group of animals were not affected by the high-Fat/Cho diet treatment.
Figure 1 shows the time course of
deuterium enrichment in palmitate, stearate, and oleate of the ZL rats
expressed as molar enrichment (ME; deuterium atoms/molecule). Deuterium
enrichment in plasma fatty acids appeared to follow a single
exponential kinetic and achieved a steady state within 2 wk, as
determined by curve fitting using the CONSAAM program. The steady-state
ME is a function of the rate of lipogenesis and the influx of unlabeled fatty acid from the diet. Therefore, changes in unlabeled fatty acid
influx and lipogenesis determine the changes in ME between the two
dietary treatment groups. A higher influx of unlabeled fatty acids from
the high-Fat/CHO diet alone can depress steady-state ME. The depression
of ME of fatty acid of animals by the increase in dietary fat alone can
be predicted. Because dietary fat content was changed from 10% energy
to 30% energy, the total flux of a fatty acid was higher in the
high-Fat/CHO-treated animals by a factor [(1 FNS*) × 3 + FNS*] compared with that of the low-Fat/CHO group if
lipogenesis is unchanged. FNS* is the FNS at plateau for the respective
fatty acid under low dietary fat conditions. Thus predicted ME of the
fatty acid from the high-Fat/Cho group is related to the ME of the
low-fat group by the equation ME* (high fat) = ME (low fat) × {1/[(1
FNS*) × 3 + FNS*]}, where ME (high
fat) and ME (low fat) are the predicted ME of that fatty acid for
animals on a high-fat and low-fat diet, respectively, assuming no
change in lipogenesis.4 This
expected ME due to high fat intake was calculated using respective FNS
values of animals on the low-fat diet in Table 3 and was indicated by the dashed lines
for each fatty acid in Figs. 1, 2, and
3. Adaptive changes in lipogenesis cause
the steady-state ME to deviate from this line in response to high
dietary fat intake. In the ZL animals, the plateau value of ME of
plasma fatty acids from animal fed a high-fat diet was significantly
less than that from animals fed a low-fat diet. The consumption of a
high-Fat/Cho diet depressed ME of palmitate, stearate, and oleate
beyond that expected from dilution alone. The curves representing ME of
the fatty acids in the high dietary fat-treated animals fall below the
dashed lines (P < 0.01), suggesting reduced de novo
lipogenesis in adaptation to the high-fat diet.
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The results of deuterium incorporation in fatty acids in plasma lipids of the ZDF rat are shown in Fig. 2, and those of the ZO rat are shown in Fig. 3. Because deuterium incorporation into lipids of both (age- and weight-matched) groups of ZDF rats was almost identical, the results of the two ZDF groups were combined. Fewer deuterium atoms were incorporated per fatty acid molecule in plasma of ZDF rats fed a high-fat diet than in plasma of animals fed a low-fat diet. However, the new ME matched the enrichment predicted based on a change in the influx of dietary fat alone, and de novo lipogenesis was essentially unchanged. There was an apparent lack of adaptation in the synthesis of palmitate, stearate, and oleate to high dietary fat intake. Similar lack of responsiveness was observed in the deuterium incorporation in plasma fatty acids in the ZO rats (Fig. 3).
The source of palmitate available for chain elongation in the synthesis
of stearate in these animals can be determined from m2/m1, which is plotted
in Fig. 4. In the absence of chain
elongation on unlabeled palmitate,
m2/m1 of stearate as a
function of that in palmitate is shown by the theoretical line with a
slope of 1.15. The points
m2/m1 of the ZDF and ZO
animals and those of the ZL on a low-fat diet fell along the
theoretical line. In the ZL animals fed a high-fat diet, the
m2/m1 fell below the
theoretical line, indicating the use of unlabeled (dietary) palmitate
for stearate synthesis.
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The isotope data and the calculated fractional synthesis of palmitate, stearate, and oleate at steady state are presented in Table 3. From the m2/m1, the deuterium enrichment in body water (p) was calculated to be 2.7-3.5%. More deuterium was incorporated in plasma palmitate of ZL animals fed a low-Fat/Cho diet than in palmitate of animals fed a high-Fat/Cho diet. After taking into account the deuterium enrichment in water, 70% of palmitate, 61% of stearate, and 34% of oleate of the lean animals were newly synthesized. These were suppressed to 24, 18, and 1.4%, respectively, by the consumption of the high-fat diet. The suppression of FNS was more than that predicted by dilution due to high influx of unlabeled dietary fatty acids (see Fig. 1). Deuterium incorporation into palmitate was not suppressed by the change in dietary fat in the age- or weight-matched ZDF animals and in ZO animals. A much smaller diminution of fractional synthesis of fatty acids was observed in the two groups of ZDF and ZO rats in response to the high-fat diet. This diminution was explained entirely by dilution due to high influx of unlabeled dietary fatty acids (see Figs. 2 and 3). In either dietary treatment, de novo lipogenesis of the ZDF and ZO animals was statistically greater than that in the ZL rat. These data demonstrated that, in adaptation to the consumption of a high-Fat/Cho diet, ZL rats were able to reduce their activities of fatty acid synthetase, elongase, and desaturase, leading to reduced lipogenesis. The suppression in these enzyme activities in adaptation to high dietary fat was lost in both the ZDF and ZO rats.
If we assume the transport of lipids in the circulatory system to be at steady state, as suggested by our dynamic data, the fractional enrichment in lipids is the result of the mixing of unlabeled fatty acids from the diet and deuterium-labeled fatty acids from lipogenesis. Because some of the unlabeled fatty acids also come from lipolysis of preexisting fat, lipogenic flux as calculated by Eq. 4 underestimates the actual lipogenic flux. A discussion of the effect of influx of unlabeled fatty acids from adipose tissues on our calculation is presented in the APPENDIX. Under the condition of low fat intake (dietary fat contributing to 10% energy), 70% of plasma fatty acid (represented by palmitate) in the lean animals was labeled with deuterium. Assuming proportionality between quantity of fatty acids and percent energy, the rate of de novo lipogenesis had to be about two times the dietary fat intake and amounted to 23% of the energy intake. Therefore, the total energy delivered by plasma triglycerides was ~10% plus 23.3% or 33% of the energy intake. By similar reasoning, de novo lipogenesis contributed only 24% to plasma fatty acids or 9.5% of the energy intake in rats fed a high-Fat/Cho diet. Thus 39.5% of the energy intake was transported as plasma triglycerides under the high-fat condition. De novo lipogenesis was downregulated to maintain the same percent energy transported as plasma lipids. The corresponding values of de novo lipogenesis as percent of total energy intake calculated for the ZDF animals were 90% under low-fat and 85% under high-fat intake. These values were very much higher than those of the ZL animals. There was no adaptation to high fat intake.
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DISCUSSION |
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The existence of a humoral factor that regulates food intake and energy metabolism has long been suspected since the parabiosis experiments with db/db, ob/ob, and wild-type mice (5, 6). This humoral factor was recently identified to be leptin (24). Leptin is produced principally in adipose tissue. Centrally, leptin decreases food intake and causes rapid weight loss. In peripheral tissues, leptin inhibits lipogenesis and stimulates fatty acid utilization. These endocrine and paracrine functions of leptin suggest a feedback control relationship between leptin and fuel energy metabolism. Leptin is secreted to regulate food intake, fatty acid synthesis, and utilization to achieve some targeted endpoint, which is believed to be total body fat. However, recent evidence suggests that fatty acid availability rather than adiposity may be the set point of the feedback regulation by leptin (3, 22). In the rat, high-fat feeding for 3 days resulted in a 50% decrease in the induction of skeletal muscle ob expression by leptin (23). In this study, we found that the consumption of an isocaloric diet, in which dietary fat was increased from 10 to 30% of energy intake, suppressed de novo lipogenesis, chain elongation, and desaturation in the ZL rats with an intact leptin feedback mechanism. The suppression of lipogenesis by the high-Fat/Cho diet was not observed in the leptin receptor-defective ZDF rats. Furthermore, de novo synthesis of long-chain fatty acids was higher in the ZDF compared with the ZL animals regardless of diet, age, or body weight. The observation of unregulated and increased lipogenesis has also been reported in the leptin-deficient ob/ob mouse that also has a defective leptin feedback loop. In the ob/ob mouse, lipogenesis was not suppressed in response to fast, but was corrected with leptin treatment (21). The observations in the ZDF rat and the ob/ob mouse strongly support our hypothesis that the leptin/leptin receptor system is part of the hormonal feedback loop in the regulation of lipogenesis. Because a change in the Fat/Cho ratio in the diet induced an adaptive change in FNS in the ZL but not in the ZDF rat, it appears that fatty acid availability rather than adiposity is the set point of the feedback regulation by leptin. The relation between leptin and fat metabolism is analogous to that between insulin and glucose metabolism. In glucose homeostasis, insulin is part of the feedback loop for the control of glucose production. When there is a loss of insulin/insulin receptor action, glucose production is unregulated, leading to hyperglycemia. Leptin is the counterpart of insulin in lipid homeostasis. When the leptin/leptin receptor system is defective, as in the ZDF rat, lipogenesis is unregulated, leading to hyperlipidemia and obesity in the affected animal.
Chain elongation and desaturation of nonessential fatty acids are responsible for the synthesis of long-chain fatty acids such as stearate and monounsaturated fatty acids such as oleate. These reactions potentially determine intracellular acetyl-CoA and acyl-CoA concentrations and the availability of reducing equivalents. The recycling by chain elongation/shortening and desaturation/hydrogenation of long-chain fatty acids has been shown to be affected by dietary polyunsaturated fatty acid intake (4). In our study, synthesis of stearate and oleate was depressed in animals fed a high-Fat/Cho diet, which is consistent with previous observations of changes in the expression of these enzymes by metabolic signals of the fed and fasted states (4). In the leptin receptor-defective ZDF rats, the regulation of these enzymes by the normal metabolic signals is lost. Therefore, we infer that leptin is part of the regulatory system influencing chain elongation and desaturation of nonessential fatty acids in response to a change in the Fat/Cho ratio in the diet.
The partitioning of metabolic fuels among tissues has important implications for health and disease (9). Leptin has been shown to affect lipid oxidation and fuel partitioning in skeletal muscles (16, 17, 20, 25). The observation that leptin treatment can normalize the lipid abnormalities in the ob/ob mouse suggests that leptin also regulates de novo lipogenesis by the liver and fatty acid utilization by the peripheral tissues. Leptin therefore may serve as the effector of a negative feedback loop between dietary fatty acid availability and lipogenesis. This regulation appears to be independent of serum lipid levels, insulin, energy intake, or body weight. In the ZDF and ZO animals, this regulation is lost, lipogenesis probably exceeds the animal's energy need, and the extra fat is stored in the adipose tissues, leading to obesity. Because we did not use C-13 as tracer, the source of acetyl units for fatty acid synthesis remains unknown. Finally, the loss of regulation of lipogenesis was associated with hyperglycemia in both ZDF and ZO rats. The possibility that the continued lipogenesis on the high-fat diet was driven by the hyperglycemia secondary to reduced glucose uptake by the muscle has not been ruled out. How this abnormality in lipid metabolism is connected to the regulation of carbohydrate utilization and glucose homeostasis remains to be explored.
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APPENDIX |
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Effect of unlabeled fatty acids from adipose tissues on the
predicted FNS.
The rate of lipogenesis relative to the influx of unlabeled fatty acid
is given by the FNS to the fraction of unlabeled molecules or
FNS/(1 FNS). The total flux is FNS + (1
FNS) or
one. When the influx of unlabeled fatty acid is increased from 10%
energy to 30% energy, the new total flux is FNS + (1
FNS) × 30/10, assuming FNS to be unchanged. Thus the total flux
relative to that of the low-fat diet is [FNS + (1
FNS) × 3] to one. When the contribution of unlabeled fatty acids
from other fat stores is significant, the relationship changes to
FNS + (1
FNS) × (30 + x)/(10 + x) where x is the contribution from other fat
stores. Because x is a positive number, the increase in
total flux relative to that in the low dietary fat condition will be
less, and the predicted FNS (dashed line) for the high-fat diet will be
greater than that provided in Figs. 1-4. In other words, the
greater the contribution from adipose fat, the smaller the effect of
diet on the plateau enrichment.
Effect of unlabeled fatty acids from adipose tissues on the lipogenic flux. The effect of influx of fatty acids from adipose tissue on the estimation of lipogenic flux of FNS (%energy) is substantial. Assuming the same estimation of 2.6 g/day or 2.6 times that of the low-fat intake, the FNS (%energy) of ZL on the low-fat diet is changed from 10% energy × (70/30) or 23.3% energy to 3.6 × 10% energy × (70/30) or 83.9% energy. The FNS (% energy) of ZL on a high-fat diet is changed from 30% energy × (24/76) or 9.5% energy to 5.6 × 30% energy × (24/76) or 53.0% energy. With the use of the same assumption of influx of 2.6 g of adipose fat, the FNS (%energy) of ZDF on a low-fat diet is changed from 10% energy × (91/9) or 101% energy to 3.6 × 10% energy × (91/9) or 364% energy. The FNS (%energy) of ZDF on a high-fat diet is changed from 30% energy × (74/26) or 85% energy to 5.6 × 30% energy × (74/26) or 478% energy. Because the calculated FNS (%energy) are so extraordinarily high for the ZDF rat, we believe that the contribution of adipose fat to the plasma total fatty acids is small.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the American Diabetes Association. The GC-MS Facility is supported by National Institutes of Health Grants P01-CA-42710 to the University of California at Los Angeles Clinical Nutrition Research Unit, Stable Isotope Core and M01-RR-00425 to the General Clinical Research Center.
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FOOTNOTES |
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Address for reprint requests and other correspondence: W.-N. P. Lee, Research and Education Institute, Harbor-UCLA Medical Center, RB1, 1124 W. Carson St., Torrance, CA 90502 (E-mail: Lee{at}GCRC.HUMC.EDU).
1 The final enrichment in plasma is always lower than that of the drinking water, depending on the relative contribution of metabolic water and moisture content of the air (see Ref. 12).
2 The number of hydrogen atoms derived from body water (N) for chain elongation was determined to be 3. Thus, in the presence of 100% D2O, three deuterium atoms are added to palmitate in chain elongation. By the same reasoning, the newly synthesized stearate has 24 deuterium atoms per molecule. When deuterium enrichment is <100%, a different degree of deuterium incorporation is possible, leading to the binomial distribution of mass isotopomers of the fatty acid.
3 The elevated blood glucose concentrations may represent development of diabetes in the ZO rat on the given diets.
4
The rate of lipogenesis relative to the influx of
unlabeled fatty acid is given by the FNS of unlabeled molecules or
FNS/(1 FNS). The total flux is FNS + (1
FNS) or
one. When the influx of unlabeled fatty acid is increased from 10%
energy to 30% energy, the total flux is FNS + (1
FNS) × 30/10. Thus the total flux relative to that of the low-fat
diet is [FNS + (1
FNS) × 3] to 1.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 14 July 1999; accepted in final form 13 March 2000.
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