Vol. 282, Issue 3, E507-E513, March 2002
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
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ABSTRACT |
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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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
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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).
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.
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RESULTS |
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).
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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.
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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.
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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.
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.
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.
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DISCUSSION |
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 
-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.
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ACKNOWLEDGEMENTS |
We thank Vy Ngo and Samuel Lee for performing some of the data
reduction during their summer fellowship in 1999.
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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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REFERENCES |
1.
Berk, PD,
Zhou SL,
Kiang CL,
Stump D,
Bradbury M,
and
Isola LM.
Uptake of long chain free fatty acids is selectively up-regulated in adipocytes of Zucker rats with genetic obesity and non-insulin-dependent diabetes mellitus.
J Biol Chem
272:
8830-8835,
1997.
2.
Bessesen, DH,
Vensor SH,
and
Jackman MR.
Trafficking of dietary oleic, linolenic, and stearic acids in fasted or fed lean rats.
Am J Physiol Endocrinol Metab
278:
E1124-E1132,
2000.
3.
Blom, K,
Dybowsky C,
and
Munson B.
Mass spectral analysis of isotopically labeled compounds: average mass approach.
Anal Chem
59:
1372-1374,
1987.
4.
Clandinin, MT,
Cheema S,
Pehowich D,
and
Field CJ.
Effect of polyunsaturated fatty acids in obese mice.
Lipids
31, Suppl:
S13-S22,
1996.
5.
Enser, M.
Desaturation of stearic acid by liver and adipose tissue from obese-hyperglycemic mice (ob/ob).
Biochem J
148:
551-555,
1975.
6.
Garg, ML,
Snoswell AM,
and
Sabine JR.
Influences of dietary cholesterol on desaturase enzymes of rat liver microsomes.
Prog Lipid Res
25:
639-644,
1986.
7.
Garg, ML,
Wierzbicki AA,
Thomson ABR,
and
Clandinin MT.
Dietary cholesterol and/or n-3 fatty acid modulate D-9 desaturase activity in rat liver microsomes.
Biochim Biophys Acta
962:
330-336,
1988.
8.
Giacobino, JP,
and
Chmelar M.
The role of chain elongation systems in the supplying of fatty acids to the adipocyte membrane lipids.
Biochim Biophys Acta
487:
269-276,
1977.
9.
Horton, JD,
Bashmakov Y,
Shimomura I,
and
Shimano H.
Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice.
Proc Natl Acad Sci USA
95:
5987-5992,
1998.
10.
Jones, B,
Maher MA,
Banz WJ,
Zemel MB,
Whelan J,
Smith P,
and
Moustaid N.
Adipose tissue stearoyl-CoA desaturase mRNA is increased by obesity and decreased by polyunsaturated fatty acids.
Am J Physiol Endocrinol Metab
270:
E44-E49,
1996.
11.
Jump, DB,
Clarke SD,
Thelen A,
Liimatta M,
Ren B,
and
Badin MV.
Dietary fat, genes, and human health.
Adv Exp Med Biol
422:
167-176,
1997.
12.
Kakuma, T,
Lee Y,
Higa M,
Wang ZW,
Pan W,
Shimomura I,
and
Unger RH.
Leptin, troglitazone, and the expression of sterol regulatory element binding proteins in liver and pancreatic islets.
Proc Natl Acad Sci USA
97:
8536-8541,
2000.
13.
Komers, R,
and
Vrana A.
Thiazolidinediones
tools for the research of metabolic syndrome X.
Physiol Res
47:
215-225,
1998.
14.
Landau, JM,
Skowski A,
and
Hamm MW.
Dietary cholesterol and activity of stearoyl-CoA desaturase in rats: evidence for an indirect regulatory effect.
Biochim Biophys Acta
1345:
349-357,
1997.
15.
Lee, W-NP.
Stable isotope and mass isotopomer study of fatty acid and cholesterol synthesis: a review of the MIDA approach.
In: Dietary Fats, Lipids, Hormones and Tumorogenesis: New Horizons in Basic Research, edited by Heber D,
and Kritchevsky D. New York: Plenum, 1996, p. 95-114.
16.
Lee, W-NP,
Bassilian S,
Lim S,
and
Boros LG.
Loss of regulation of lipogenesis in the Zucker diabetic (ZDF) rat.
Am J Physiol Endocrinol Metab
279:
E425-E432,
2000.
17.
Lee, W-NP,
Lim S,
Bassilian S,
Bergner EA,
and
Edmond J.
Fatty acid cycling in human hepatoma cells and the effects of troglitazone.
J Biol Chem
273:
20929-20934,
1998.
18.
Lowenstein, JM,
Brunengraber H,
and
Wadke M.
Measurement of rates of lipogenesis with deuterated and tritiated water.
Methods Enzymol
34:
279-287,
1975.
19.
Marchesini, G,
Brizi M,
Bianchi G,
Tomassetti S,
Bugianesi E,
Lenzi M,
McCullough AJ,
Natale S,
Forlani G,
and
Melchionda N.
Nonalcoholic fatty liver disease: a feature of the metabolic syndrome.
Diabetes
50:
1844-1850,
2001.
20.
Ntambi, JM.
Regulation of stearoyl-CoA desaturase by polyunsaturated fatty acids and cholesterol.
J Lipid Res
40:
1549-1558,
1999.
21.
Shimano, H,
Yahagi N,
Amemiya-Kudo M,
Hasty AH,
Osuga J,
Tamura Y,
Shionoiri F,
Iizuka Y,
Ohashi K,
Harada K,
Gotoda T,
Ishibashi S,
and
Yamada N.
Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes.
J Biol Chem
274:
35832-35839,
1999.
22.
Shimomura, I,
Bashmakov Y,
and
Horton JD.
Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus.
J Biol Chem
274:
30028-30032,
1999.
23.
Shimomura, I,
Bashmakov Y,
Ikemoto S,
Horton JD,
Brown MS,
and
Goldstein JL.
Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes.
Proc Natl Acad Sci USA
96:
13656-13661,
1999.
24.
Standridge, M,
Alemzadeh R,
Zemel M,
Koontz J,
and
Moustaid-Moussa N.
Diazoxide down-regulates leptin and lipid metabolizing enzymes in adipose tissue of Zucker rats.
FASEB J
14:
455-460,
2000.
25.
Unger, RH,
and
Zhou YT.
Lipotoxicity of beta-cells in obesity and in other causes of fatty acid spillover.
Diabetes
50, Suppl1:
S118-S121,
2001.
26.
Zhou, YT,
Shimabukuro M,
Lee Y,
Koyama K,
Higa M,
Ferguson T,
and
Unger RH.
Enhanced de novo lipogenesis in the leptin-unresponsive pancreatic islets of prediabetic Zucker diabetic fatty rats: role in the pathogenesis of lipotoxic diabetes.
Diabetes
47:
1904-1908,
1998.
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