Loss of regulation of lipogenesis in the Zucker diabetic rat. II. Changes in stearate and oleate synthesis

Sara Bassilian, Syed Ahmed, Shu K. Lim, Laszlo G. Boros, Catherine S. Mao, and W.-N. Paul Lee

Department of Pediatrics, Research and Education Institute, Harbor-UCLA Medical Center, Torrance, California 90502


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

De novo lipogenesis and dietary fat uptake are two major sources of fatty acid deposits in fat of obese animals. To determine the relative contribution of fatty acids from these two sources in obesity, we have determined the distribution of c16 and c18 fatty acids of triglycerides in plasma, liver, and epididymal fat pad of Zucker diabetic fatty (ZDF) rats and their lean littermates (ZL) under two isocaloric dietary fat conditions. Lipogenesis was also determined using the deuterated water method. Conversion of palmitate to stearate and stearate to oleate was calculated from the deuterium incorporation by use of the tracer dilution principle. In the ZL rat, lipogenesis was suppressed from 70 to 24%, conversion of palmitate to stearate from 86 to 78%, and conversion of stearate to oleate from 56 to 7% in response to an increase in the dietary fat-to-carbohydrate ratio. The results suggest that suppression of fatty acid synthase and stearoyl-CoA desaturase activities is a normal adaptive mechanism to a high-fat diet. In contrast, de novo lipogenesis, chain elongation, and desaturation were not suppressed by dietary fat in the ZDF rat. The lack of ability to adapt to a high-fat diet resulted in a higher plasma triglyceride concentration and excessive fat accumulation from both diet and de novo synthesis in the ZDF rat.

chain elongation; stearoyl-coenzyme A desaturase; tissue fatty acid composition


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ABNORMALITIES IN UPTAKE AND TRANSPORT, as well as de novo synthesis, of fatty acids are characteristics found in many animal models of obesity. These abnormalities in fatty acid metabolism often lead to elevated plasma triglycerides and fatty acids, culminating in the phenomenon of "lipotoxicity," which results in tissue dysfunction and apoptosis (12, 25, 26). In the Zucker diabetic fatty (ZDF) rat, an animal model of obesity resulting from leptin resistance, the primary abnormality is in increased activation of the sterol-regulatory element-binding protein-1 (SREBP-1), a transcriptional factor for lipogenic enzyme gene expression, and lipogenesis (21). In contrast to the streptozotocin-induced diabetes model, in which hyperglycemia is accompanied by low serum insulin concentration and depressed liver SREBP expression, the ZDF rat has elevated insulin concentration and two to three times the level of SREBP in the liver (12, 23). The increased de novo lipogenesis and dietary fat intake both contribute to the excessive deposit of triglycerides in the liver and adipose tissues and ectopically in the islets (25).

The expression of lipogenic enzymes and their activities can be modulated by dietary fat intake through regulatory hormonal signals such that fatty acid composition in triglycerides remains relatively stable over a wide range of dietary intakes (2). Lipogenic regulation by diet has been shown to be defective in the ZDF rat (16). In other studies, the expression of fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD) is elevated in hepatic tissue in ZDF animals and remains elevated under high dietary fat conditions (10, 26). It has not been demonstrated how de novo synthesis interacts with dietary fatty acid uptake to result in the observed plasma fatty acid profile.

Palmitate, stearate, and oleate are three major fatty acids in dietary fat and in plasma triglycerides. They are also the products of the fatty acid synthesis pathways. The enzymes critical for producing these fatty acids to maintain homeostasis are FAS, elongase, and SCD. These enzymes link the c16 and c18 nonessential fatty acids in a network of pathways (Fig. 1). As a continuation of our previous report (16), this paper reports the contribution of chain elongation and desaturation relative to fatty acid synthesis to the accumulation of plasma and tissue triglycerides. Distribution of saturated and monounsaturated fatty acid (c16 and c18 fatty acids) was determined in plasma, liver, and epididymal fat pad triglycerides of ZL and ZDF rats of the previous study of lipogenic regulation by dietary fat. Differences between fatty acid profiles of the ZL and ZDF animals reflect the effect of leptin-receptor deficiency on substrate flux through the lipogenic pathways under the two dietary fat conditions.


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Fig. 1.   Pathways of lipogenesis and fatty acid interconversion. The pathways show a precursor-product relationship between stearate and palmitate and between oleate and stearate. The synthesis of palmitate is under the control of fatty acid synthase (FAS), and synthesis of oleate is under the control of stearoyl-CoA desaturase (SCD). TG, triglyceride; GPAT, glycerol-3-phosphate acyltransferase. Deuterium-labeled palmitate is the source for deuterated stearate and oleate. Thus the enrichment in palmitate is greater than that in stearate and oleate according to the precursor-product relationship. The possible participation of substrate in the regulation of flow through these pathways is indicated by the stimulatory (+) or inhibitory (-) signs.


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

Animals. Male ZL (72 days old) and ZDF rats (65 days old) from Genetic Models (Indianapolis, IN) were used in this study. Animal studies were conducted in accordance with the Institutional Laboratory Animal Care and Use Committee guidelines after approval by our Institutional Review Board. The physical and biochemical characteristics and dietary treatment protocol are as previously published (see Table 1 in Ref. 16). The experimental animals were fed a constant energy intake of ~90 cal/day of a control (ID 98246) diet, providing 10% of the energy intake as fat, or of a high-fat (ID 98247) diet, providing 30% energy intake as fat. These diets have identical contents in protein, polyunsaturated fatty acids, vitamins, and minerals and are formulated to provide 3.6 cal/g (16). Fatty acid composition of the two diets is shown in Table 1. The respective daily intake of each fatty acid is calculated on the basis of an intake of 25 g of food per day per animal. The high-fat diet provides excess amounts of palmitate, stearate, and oleate to both the lean and obese animals.

                              
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Table 1.   Gas-chromatography RT of fatty acids and the corresponding ranges of masses (m/z) monitored for quantitative analysis by mass spectrometry

After acclimatization until constant intake was established, animals were given an intraperitoneal injection of deuterated water and then maintained on water containing 6% deuterium oxide (2H2O) for the determination of de novo lipogenesis. At the end of the experimental period on day 14, rats were anesthetized and killed. Blood samples were collected, and plasma was separated from each of these samples. Livers and fat pads were removed and quickly frozen in liquid nitrogen for lipid analysis.

Gas chromatography-mass spectrometry analysis. Lipid extraction was performed using methods described by Lowenstein et al. (18). Gas chromatography-mass spectrometry (GC-MS) analysis was performed on a Hewlett-Packard model 5973 Mass Selective Detector connected to a model 6890 gas chromatograph using electron impact ionization. A glass capillary column BPX70 (from SGE, Austin, TX) measuring 30 m × 250 µm (ID) was used to separate fatty acid methyl esters. The GC conditions were: carrier gas (helium) flow rate, 1 ml/min; injector temperature, 250°C; and oven temperature, programmed from 120-220°C at 5°C/min. The retention times and mass-to-charge (m/z) ion clusters for selected ion monitoring of the fatty acids are summarized in Table 2.1 After determining mass isotopomer distribution from the respective mass spectra (15), we calculated the average number of deuterium incorporated per molecule, as well as fractional new synthesis (FNS) (3, 16).

                              
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Table 2.   Fatty acid composition in diets and approximate daily intake by rats

Analysis of fatty acid composition. Total ion chromatogram of fatty acids in various tissues allows the quantitation of the relative amounts of each fatty acid present in the tissue.2 The major peaks in the ion chromatogram are the long-chain fatty acids palmitate (c16:0) and stearate (c18:0) and the monounsaturated palmitelaidate (c16:1 trans), palmitoleate (c16:1 cis), oleate (C18:1 cis), and elaidate (C18:1 trans). The oleate peak is the largest among the monounsaturated fatty acids and was the most abundant besides palmitate and stearate. Other long-chain and polyunsaturated fatty acids were also detected in small amounts, representing <10% of the total fatty acids. The area under each peak was integrated using the ChemStation software. The long-chain and monounsaturated fatty acid distribution was calculated as a percentage of total c16 and c18 fatty acids, ignoring the contribution from minor peaks, and as a percentage of palmitate.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Contribution of de novo lipogenesis and dietary fat to liver fatty acids. Figure 2 shows the relative amounts of the saturated and unsaturated fatty acids relative to palmitate in liver of ZL and ZDF rats on low- and high-fat diets. Under low-fat conditions, palmitate and stearate are present in almost equal amounts in the liver extract of the lean animal, with the stearate fraction being slightly lower than the palmitate. Under high-fat conditions, the relative amounts of stearate and oleate increase in the ZL liver, suggesting an increase in the conversion of palmitate by chain elongation and desaturation giving rise to a distinctly different fatty acid profile. In the obese diabetic liver, stearate was rapidly converted to oleate. The relative amount of stearate is lower in the liver of the ZDF rat compared with that of the ZL rat. Palmitate and oleate are the major long-chain fatty acids in the obese diabetic liver. The fatty acid distribution in ZDF liver is not affected by the high dietary fat intake. The fatty acid profile of the ZL is distinctly different from that of the ZDF. The ZL rat differs from the ZDF animals in the ratio of saturated to monounsaturated c18 fatty acids regardless of dietary fat condition. Stearate is the major c18 fatty acid in the ZL animal, whereas oleate is the major c18 fatty acid in the ZDF animal.


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Fig. 2.   Fatty acid composition in liver tissue of Zucker lean (ZL; A) and Zucker diabetic fatty (ZDF; B) rats on low-fat (LF; hatched bars) and high-fat (HF; open bars) diet. Error bars, SD. aSignificant differences between ZL and ZDF samples by t-test (P < 0.005); bsignificant differences between values from HF and LF diets (P < 0.015); csignificant difference between liver and plasma fatty acid in the ZL rat (P < 0.02).

The contribution of de novo lipogenesis to the various fatty acids can be deduced from the individual fractional new synthesis (FNS) shown in Fig. 3. The FNS in palmitate is greater than that in stearate, which is greater than that of oleate. The order of FNS reflects the precursor-product relationship of these three fatty acids in the normal lean animal. De novo lipogenesis for palmitate, stearate, and oleate is greatly suppressed in the ZL by the high-fat diet. The suppression of FNS is less prominent in the ZDF liver because de novo synthesis continues at high rates. As pointed out in our previous article (16), the apparent suppression of de novo lipogenesis in the ZDF rat is probably due to dilution of deuterated fatty acid by dietary fat.


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Fig. 3.   Fractional new synthesis (FNS) of palmitate (c16), stearate (c18), and oleate (c18:1) in liver tissue of ZL (A) and ZDF rats (B) on LF (hatched bars) and HF (open bars) diets. Error bars, SD.

Contribution of de novo lipogenesis and dietary fat to plasma fatty acids. Figure 4 shows the amounts of each of these fatty acids relative to the amounts of palmitate in the plasma of ZL and ZDF animals. The composition of fatty acids in plasma of the ZL rat is different from that of the diets and has a lower level of palmitate and a higher level of stearate than those of the diets regardless of the level of fat intake. The fatty acid profile of plasma in the ZL animal resembles that of the liver. Both c18:0 and c18:1 fatty acid-to-palmitate ratios are higher in the plasma and liver triglycerides under high-fat intake. This discrepancy between plasma and liver fatty acid profiles of high- and low-fat diets is even more evident in the ZDF animal, suggesting preferential use of stearate and oleate for plasma triglyceride synthesis. The FNS of each fatty acid in the plasma relative to each other follows the same pattern as that in the liver, suggesting that the liver is the main source of plasma fatty acids (data reported in Ref. 16). The newly synthesized fraction of each fatty acid in the plasma is less than the FNS of the respective fatty acid in the liver.


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Fig. 4.   Fatty acid composition in plasma lipids of ZL (A) and ZDF rats (B) on LF (hatched bars) and HF (open bars) diets. Error bars, SD. bSignificant differences between values from HF and LF diets (P < 0.015); csignificant difference between liver and plasma fatty acid in the ZL rat (P < 0.02); dsignificant difference between ZL and ZDF on HF diet.

The contributions from dietary fat and de novo lipogenesis to plasma fatty acids in ZL and ZDF animals were calculated using the information of fatty acid distribution and FNS and shown in Table 3. The content of each fatty acid is first expressed as a percentage of the total. The plasma triglyceride concentration under low-fat conditions was similar to that observed under the high-fat conditions in the ZL rat. De novo synthesis of palmitate provides 70.7%, and dietary fat accounts for the remaining 29.3% (rightmost column of Table 3.) If one assumes that the deuterated c18:0 and c18:1 are derived from chain elongation of deuterated c16:0 and desaturation of c18:0, the contribution from dietary sources can be estimated by the decrease in FNS. The dietary contributions, including the conversion of dietary palmitate to plasma stearate and oleate, are 13.7 and 43.9%, respectively. Under high-fat conditions, dietary contributions to the three fatty acids increase to 76, 22, and 93%, with a concomitant decrease of de novo synthesis.

                              
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Table 3.   Relative contribution of new synthesis and dietary fat to plasma triglyceride fatty acids

The fatty acid composition in the plasma of the diabetic rats on the low-fat diet is also distinctly different from the fatty acid composition of the diet. The plasma palmitate, stearate, and oleate concentrations are higher in the ZDF than in the ZL rat. FNS in these rats is >80% in the period of the experiment, and the high levels of stearate and oleate come mostly from new synthesis. The dietary contributions to plasma stearate and oleate are much lower than those of the ZL rat because of the high de novo synthesis rates, being 9.5, 2.9, and 11%, respectively. The diabetic rats on the high-fat diet show slightly lower FNS rates. Under high-fat conditions, dietary contributions to the three fatty acids increase to 26, 22, and 25%.

If one assumes that synthesis of palmitate, stearate, and oleate follows a precursor-product relationship, such a relationship would predict that the enrichment in palmitate would be higher than that in stearate, which is higher than that in oleate. Furthermore, the steady-state ratio of deuterium enrichment of the product to that of the precursor represents the contribution of the precursor to the product. The values for the new fraction of c18:0 divided by the new fraction of c16:0 in Table 3 are used to estimate the chain elongase contribution to stearate, and the values for the new fraction of c18:1 over the new fraction of c18 are used to estimate the desaturase contribution to oleate. These estimates are summarized in Table 4. Elongation of hepatic palmitate contributes 86% of the plasma stearate under the low-fat diet. The contribution of elongation of palmitate to the stearate pool is 76% in the lean rat on the high-fat diet. The chain elongation in the liver of the ZDF rat provides 92% of the stearate on the low-fat diet and 78% on the high-fat diet. Desaturation of stearate provides only 56% of oleate in the lean rat, which is suppressed to 7.6% on the high-fat diet, suggesting a suppression of desaturase activity. In contrast to the desaturase activity in the ZL rat, desaturation of stearate to produce oleate contributes to >89% of the plasma oleate. In the diabetic rats, with both diet conditions this activity is higher than that in the lean rats on the low-fat diet.

                              
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Table 4.   Estimates of elongase and desaturase contribution in plasma stearate and oleate

Fatty acid uptake in adipose tissue. We have analyzed fatty acids extracted from epididymal fat pads of the ZL and ZDF animals. Figure 5 shows the relative amounts of the saturated and unsaturated fatty acids in epididymal fat tissue of ZL and ZDF rats on the low- and high-fat diets. The distributions of fatty acids of the ZL and ZDF rats are similar to each other but distinct from those of the plasma fatty acids. Oleate is the predominant fatty acid in adipose tissue, comprising 40% of total fatty acids. Stearate, the major c18 fatty acid in plasma, constitutes only 5% of the total in the fat pads. Figure 6 shows that lipogenesis in the fat tissue is much lower than that in the liver or the plasma. Therefore, plasma triglycerides are most likely transported from the liver and not from adipose tissues. The lower deuterium enrichment in fatty acids from adipose tissue is probably due to dilution of labeled molecules by the preexisting fat store. FNS is almost completely blocked in the ZL rat on the high-fat diet. The deuterium enrichment in these fatty acids in the ZDF rat, however, follows a different order from that in the plasma fatty acids.


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Fig. 5.   Fatty acid composition in epididymal fat tissue of ZL (A) and ZDF rats (B) on LF (hatched bars) and HF (open bars) diets. Error bars, SD. The ratio of c18:0 to c18:1 is significantly lower than the ratios of plasma or liver profiles (P < 0.005).



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Fig. 6.   FNS of palmitate, stearate, and oleate in epididymal fat tissue of ZL (A) and ZDF rats (B) on LF (hatched bars) and HF (open bars) diets. Error bars, SD.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasma triglyceride concentration and its fatty acid composition are the result of the balance between production and utilization of each individual fatty acid regulated by lipogenic enzymes and lipases. The regulation of lipogenic enzyme gene expression has been well studied. Under fasting-refeeding conditions, SREBP-1 is expressed, resulting in increased expression of acetyl-CoA carboxylase (ACC), FAS, and SCD genes in the liver (21). Diets rich in saturated fatty acids or cholesterol induce desaturase activity (6, 7), whereas polyunsaturated fatty acids diminish SCD activity in rat liver (14). Changes in the type and quantity of fat ingested further modulate the activity of enzymes through changes in substrate concentrations. The final triglyceride concentration and composition are the result of integrated interactions of these genomic and nongenomic factors. Previous studies of lipogenesis have mainly emphasized the role of mRNA expression of lipogenic enzymes as a surrogate marker of lipogenesis.

Role of substrate regulation of lipogenic enzymes and contribution of dietary fat to fat mass could not be assessed. The regulation of fatty acid synthesis and interconversion by specific fatty acids has been demonstrated in HepG2 cells in culture (17). Added amounts of stearate provided in culture medium have been shown to stimulate chain elongation to form arachidate (c20) and behenate (c22). It also participates in beta -oxidation to form palmitate, which acts to inhibit its own synthesis. Exogenous stearate also stimulates SCD, leading to the formation of oleate.

Our present study examines the effects of interaction of dietary fat and lipogenic enzyme expression on substrate flux through the lipogenic pathways. Results of our present study suggest a system of connected pathways of lipogenesis from palmitate to oleate, leading to triglyceride synthesis (Fig. 1). This system is regulated both by the levels of enzyme expression and by the availability of substrates, resulting in the observed pattern of fatty acid distribution in plasma triglycerides. The resultant fatty acid profile is clearly not predictable by the mRNA expression of these enzymes alone.

Under low-fat conditions, the activity of FAS produces 70% of the palmitate. Eighty-six percent of the stearate is derived from chain elongation of liver palmitate and 56% of the oleate from desaturation of liver stearate. The flow of substrates is in the direction of palmitate to stearate and oleate. The end product oleate probably stimulates its own removal in the synthesis of triglycerides. This order of events probably explains the increased proportion of stearate and oleate in plasma triglycerides over that of the liver in the ZL rat. The high-fat diet suppresses the activity of FAS. About 24% of liver palmitate is from de novo synthesis. Despite the lower FNS, a similar fraction of palmitate (78%) is converted to stearate. Isotope enrichment in stearate is only slightly diluted by dietary stearate, suggesting that stearate, an intermediate of oleate synthesis from palmitate, is in isotopic steady state. In normal adaptation, high dietary fat suppresses the expression of SCD. Only 7% of the oleate is from the conversion of stearate; the majority of the oleate is derived from the diet. These results support the role of substrate regulation of triglyceride synthesis in addition to the adaptive changes of FAS and SCD to maintain a relatively stable plasma profile of these energy-rich molecules over a wide range of dietary intakes. Fatty acids from both de novo synthesis and the diet are utilized for the synthesis of plasma triglyceride. Thus plasma triglyceride concentration and its fatty acid content are both regulated by gene expression as well as substrate availability through the diet.

The effects of lipogenic enzyme expression and substrate regulation can be seen in the fatty acid synthesis in the ZDF rat model. Hepatic lipogenic enzyme gene-expression is regulated by the opposing effects of insulin and leptin (11). In the absence of leptin/leptin-receptor regulation, the stimulatory effect of insulin on lipogenesis prevails. Enzymes necessary for triglyceride synthesis, such as ACC, FAS, SCD, and glycerol-3-phosphate acyltransferase (GPAT) genes, are highly expressed in the liver and other tissues (11). The expression of these genes in animal models with leptin/leptin-receptor malfunction can be modified by drugs such as thiazolidenedione and diazoxide (13, 24) but not by dietary changes (4, 5, 10). We (16) have previously reported the unsuppressed FNS of plasma fatty acids by high-fat diet. The increased FAS expression in ZDF results in almost a fourfold increase in palmitate synthesis regardless of dietary fat intake. The increased palmitate synthesis drives the chain elongation reaction, contributing to 80-98% of the liver stearate. These data are consistent with high mRNA expression of the desaturase in the ZDF rat. The increased SCD and GPAT expression has the effect of converting stearate to oleate and triglyceride synthesis. The overall effect is a higher plasma triglyceride concentration and a higher oleate-to-stearate ratio in the liver and plasma. Under high-fat conditions, dietary contributions to the three fatty acids increase slightly to 26, 22, and 25%, suggesting a dilution of the newly synthesized fatty acids by dietary fatty acids. Synthesis of palmitate, stearate, and oleate is at a maximum rate and is unaffected by dietary fat.

The significance of lipogenesis in adipose tissue remains controversial. In the study of the fasting-refeeding model, lipogenic enzymes are induced by refeeding in the liver but not in adipose tissue (1, 21). Deuterium enrichment in fatty acids from adipose tissue is less than one-half of that found in plasma fatty acids at the time of death (day 14). This lower enrichment argues against adipose tissue being the source of plasma triglycerides; it rather suggests that fatty acids in adipose tissue are derived from plasma triglycerides. The distinctive fatty acid distribution suggests preferential uptake or synthesis of stearate and oleate by chain elongation and desaturation over that of palmitate in adipose tissue of the ZDF rat. SCD activity is elevated in adipose tissue, and the activity is higher in obese than in lean animals (10). This increase in SCD activity has the effect of increasing stearate turnover in adipose tissue. Palmitate taken up by adipose tissue is quickly converted to stearate and oleate, which become the major c18 fatty acid. The increased turnover of stearate and oleate is exaggerated by the increased SCD activity in ZDF animals, resulting in the relatively higher deuterium enrichment of stearate and oleate over that of palmitate. The fatty acid profile with increased monounsaturated fatty acids is a typical fatty acid profile of triglycerides from adipose tissues (8, 20).

Excessive accumulation of fat is a dominant feature of obesity and "lipotoxicity" (25, 26). It is an important cause of steatohepatitis, islet cell injury, and diabetes (19). De novo lipogenesis and dietary fat are two major sources contributing to fat deposits in various tissues. The degree of fat deposition is influenced by the availability of substrate and the regulation of lipogenic enzyme expression induced by hormonal signals. In the normal triglyceridemic ZL rat, excess accumulation of fat is prevented by the suppression of lipogenesis. The loss of leptin receptor allows an unopposed insulin effect on the expression of lipogenic enzymes, resulting in excess production of fatty acids and triglycerides. The inability to adapt to high fat intake by reducing de novo lipogenesis leads to higher plasma triglyceride and excess fat deposits in liver and adipose tissues. The experimental approach with deuterated water allows a quantitative assessment of the relative contribution of de novo lipogenesis and dietary fat uptake in the normal and abnormal adaptive responses to dietary fat. Such understanding of the regulation of fatty acid interconversion and triglyceride synthesis in adaptation to high fat intake is important to our management of hypertriglyceridemia in diabetes and obesity.


    ACKNOWLEDGEMENTS

We thank Vy Ngo and Samuel Lee for performing some of the data reduction during their summer fellowship in 1999.


    FOOTNOTES

This work was supported by National Institutes of Health Grant DK-56090-A1 and by a grant from the American Diabetes Association. The GC-MS Facility is supported by Public Health Service Grants P01-CA-42710 to the UCLA Clinical Nutrition Research Unit and Stable Isotope Core, and M01-RR-00425 to the General Clinical Research Center.

1  This specific mass spectral analysis was designed to monitor saturated and monounsaturated fatty acids. Because of the different fragmentation patterns of c18:2 and c20:4, the resulting quantitation would not show correct amounts of linoleate (c18:2) and c20:4. The polyunsaturated fatty acids are not included in this study.

2  The precise method of quantitation of fatty acid is with the use of a flame ionization detector. Here, we assume equal ionization efficiency of the c16 and c18 fatty acid methyl esters under electron impact ionization. When consistently applied, the method provides results that can be used to compare relative changes of fatty acid concentration in different experiments.

Address for reprint requests and other correspondence: W.-N. Paul Lee, Harbor-UCLA Medical Center, RB1, 1124 W. Carson St., Torrance, California 90502 (E-mail: Lee{at}gcrc.rei.edu).

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. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00211.2001

Received 14 May 2001; accepted in final form 6 November 2001.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 282(3):E507-E513
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