Loss of regulation of lipogenesis in the Zucker diabetic (ZDF) rat

W.-N. Paul Lee, Sara Bassilian, Shu Lim, and Laszlo G. Boros

Research and Education Institute, Harbor-University of California Los Angeles Medical Center, Torrance, California 90502


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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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Table 1.   Physical and biochemical characteristics of Zucker rats in the two dietary groups


                              
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Table 2.   Nutrient composition of the two experimental (Harlan Teklad) diets

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
m<SUB>2</SUB>/m<SUB>1</SUB>=(N−1)/2×p<IT>/</IT>(<IT>1−</IT>p) (1)
We have previously shown that the number of hydrogen atoms (N) derived from body water per molecule of palmitate is 21 (12). Therefore, we can determine deuterium enrichment (p) from the m2/m1 mass isotopomer ratio
p<IT>=</IT>[<IT>m<SUB>2</SUB>/m<SUB>1</SUB>×2/</IT>(<IT>N−1</IT>)]<IT>/</IT>[<IT>1+m<SUB>2</SUB>/m<SUB>1</SUB>×2/</IT>(<IT>N−1</IT>)] (2)
For a given deuterium enrichment (specific activity) in water (p), the newly synthesized molecule contains on average p × N deuterium per molecule. If the observed isotope incorporation (deuterium atoms per molecule) is ME, the fraction of newly synthesized molecules (FNS) is given by
FNS<IT>=</IT>ME(palmitate)<IT>/</IT>(p<IT>×N</IT>) (3)
Assuming plasma fatty acids to be a mixture of the newly synthesized and dietary lipids, the steady-state level of FNS represents the lipogenic flux or the contribution of newly synthesized fatty acids entering the plasma pool per day. Lipogenic flux expressed as percent energy (%EN) is given by
FNS (<IT>%</IT>EN)<IT>=</IT>(<IT>%</IT>fat energy)<IT>×</IT>(FNS)<IT>/</IT>(<IT>1−</IT>FNS). (4)
For example, a steady-state FNS of 70% means that the dietary fatty acids contribute to 30% and newly synthesized fatty acids 70% of fatty acids entering the plasma pool per day. If dietary intake of fatty acids amounts to 10% energy per day, the newly synthesized fatty acids will amount to 23.3% energy by proportion. It should be noted that unlabeled fatty acids also come from lipolysis of preexisting fat. If such contribution is significant, lipogenic flux as calculated by the above equation generally underestimates the actual lipogenic flux.

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.

Fractional synthesis of stearate was calculated using Eq. 3. The number of possible deuterium substitutions (N) for stearate was previously determined to be 24. Oleate is either derived from the diet or synthesized from stearate by desaturation. Because of the deuterium isotope effect on the desaturase reaction, stearate molecules having a deuterium atom on either the C-9 or C-10 position may not be converted to oleate (7). To simplify the calculation, we assume that there is no loss of deuterium in the conversion. Fractional synthesis of oleate was approximated using N of 24 and Eq. 3.

Statistical analysis. Data are reported as means ± SD. The significance of difference between variables was calculated by Student's t-test for nonpaired groups.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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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Fig. 1.   Time course of deuterium enrichment in palmitate (A), stearate (B), and oleate (C) of the Zucker lean (ZL) rat expressed as molar enrichment (ME; deuterium atoms/molecule). , Enrichment of plasma fatty acid from ZL on low-fat-to-carbohydrate (Fat/Cho) diet; diamond , enrichment of plasma fatty acid from ZL on a high-Fat/Cho diet. Dashed lines in A-C represent the ME due to dilution by unlabeled fatty acids alone in the high-Fat/Cho group. The calculation of the expected enrichment is provided in the text. Deviation from this line represents increased or decreased lipogenesis.


                              
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Table 3.   De novo synthesis of palmitate, stearate, and oleate at steady state from rats fed either low- or high-fat/carbohydrate diets



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Fig. 2.   Time course of deuterium enrichment in palmitate (A), stearate (B), and oleate (C) of the Zucker diabetic fatty (ZDF) rat expressed as ME (deuterium atoms/molecule). , Enrichment of plasma fatty acid from ZDF on low-Fat/Cho diet; diamond , enrichment of plasma fatty acid from ZDF on high-Fat/Cho diet. Dashed lines in A-C represent the ME due to dilution by unlabeled fatty acids alone in the high-Fat/Cho group.



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Fig. 3.   Time course of deuterium enrichment in palmitate (A), stearate (B), and oleate (C) of the Zucker obese (ZO) rat expressed as ME (deuterium atoms/molecule). , Enrichment of plasma fatty acid from ZO on low-Fat/Cho diet; diamond , enrichment of plasma fatty acid from ZO on high-Fat/Cho diet. Dashed lines in A-C represent the ME due to dilution by unlabeled fatty acids alone in the high-Fat/Cho group.

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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Fig. 4.   The m2/m1 ratio of stearate is plotted against the m2/m1 ratio of palmitate for each animal. ×, ZDF and ZO on low-fat diet; diamond , ZDF and ZO on high-fat diet; diamond , ZL on low-fat diet; open circle , ZL on high-fat diet. In the absence of chain elongation on unlabeled palmitate, the m2/m1 ratio 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 low-fat diet fell along the theoretical line. Linear regression analysis of these points gives the equation y = (1.16 ± 0.0087)x with R2 = 0.812 and P < 0.0001. 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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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.


    APPENDIX
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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.

For the data presented in Figs. 1 and 2, the suppression of deuterium enrichment in the fatty acids in plasma of rats consuming the high-fat diet can be due to dilution by dietary fat or dietary fat and adipose fat. If the change in enrichment between the two diets is entirely due to a change in the influx of adipose fat, one has to conclude that fatty acid released from adipose tissue is increased in animals on the high-fat diet in the ZL but not the ZDF animals. This conclusion appears to be unlikely.

Moreover, in the calculation of the expected enrichment (indicated by the dashed lines in Figs. 1, 2, and 3), we assumed that fatty acids in plasma lipids are from either de novo lipogenesis or from dietary fat. The additional influx of fatty acids from adipose tissue will have the effect of increasing the expected enrichment (raising the dashed lines above those shown). The expected depression of enrichment from that of the low-fat diet would be less. If additional influx from adipose tissue is substantial, the difference between the observed and the expected enrichment in the case of the high-fat diet will be greater in the ZL rat than that shown in Fig. 1. This observation can only strengthen our conclusion of suppression of lipogenesis by the high-fat diet in the ZL rat.

It can be shown from the above equation [FNS + (1 - FNS) × (30 + x)/(10 + x)] that the effect of influx from adipose tissue can be estimated. We have determined FNS molecules in the epididymal fat pad to be ~30% of that of the plasma at the time of death on day 14. FNS molecules can be either from local synthesis or from an exchange with labeled plasma fatty acids. The contribution of adipose fat to the plasma fatty acids would be the greatest in the exchange process. The observed FNS suggests a maximal exchange to be ~2.1% (30/14) of the fat depot per day. Assuming 50% body fat, this amount of fat released from adipose tissue is <2.6 g (2.1% × 125 g) in a 250-g rat. This is 2.6 times that of the dietary fat intake of the low-fat diet per day. In the case of ZDF with FNS of 90%, the expected enrichment, if there is no contribution from adipose tissue, is 1/[(0.9 + 0.1 × 3)] or 0.83 of 90%. Assuming x to be equal to dietary fat of the low-fat diet, the expected enrichment is 1/[0.9 + 0.1 × (3 + 2.6)/(1 + 2.6)] or 0.95 of 90%. The difference is only 14% for this large contribution from adipose tissue. This difference does not change our conclusion regarding the nonsuppressibility of lipogenesis in the ZDF rat.

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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