Trafficking of dietary oleic, linolenic, and stearic acids in
fasted or fed lean rats
Daniel H.
Bessesen,
S. Holly
Vensor, and
Matthew R.
Jackman
Division of Endocrinology, Department of Medicine, Denver Health
Medical Center, Denver, Colorado 80204-4507
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ABSTRACT |
Increasing
evidence supports the notion that there are significant differences in
the health effects of diets enriched in saturated, as opposed to
monounsaturated or polyunsaturated fat. However, the current
understanding of how these types of fat differ in their handling by
relevant tissues is incomplete. To examine the effects of fat type and
nutritional status on the metabolic fate of dietary fat, we
administered 14C-labeled oleic, linolenic, or stearic acid
with a small liquid meal to male Sprague-Dawley rats previously fasted
for 15 h (fasted) or previously fed ad libitum (fed).
14CO2 production was measured for 8 h after
tracer administration. The 14C content of gastrointestinal
tract, serum, liver, skeletal muscle (soleus, lateral, and medial
gastrocnemius), and adipose tissue (omental, retroperitoneal, and
epididymal) was measured at six time points (2, 4, 8, 24, and 48 h and
10 days) after tracer administration. Plasma levels of glucose,
insulin, and triglyceride were also measured. Oxidation of stearic acid
was significantly less than that of either linolenic or oleic acid in
both the fed and fasted states. This reduction was in part explained by
a greater retention of stearic acid within skeletal muscle and liver.
Oxidation of oleate and stearate were significantly lower in the fed
state than in the fasted state. In the fasted state, liver and skeletal muscle were quantitatively more important than adipose tissue in the
uptake of dietary fat tracers during the immediate postprandial period.
In contrast, adipose tissue was quantitatively more important than
skeletal muscle or liver in the fed state. The movement of carbons
derived from dietary fat between tissues is a complex time-dependent
process, which varies in response to the type of fat ingested and the
metabolic state of the organism.
dietary fat; skeletal muscle; adipose tissue; fuel partitioning; triglyceride
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INTRODUCTION |
OVER THE COURSE OF HUMAN EVOLUTION, the nutrient
content of the diet has changed dramatically (16). Compared with
ancient times, the diet of modern societies is characterized by an
increase in the consumption of fat, a decrease in the ratio of
polyunsaturated fat to saturated fat, and an increase in the relative
consumption of -6 fatty acids (15). These changes in the patterns of
dietary fat intake have been associated with an increase in the
prevalence of diabetes, coronary artery disease, and obesity (7, 35, 37). A growing body of literature suggests that the metabolic effects
of diets high in saturated fat are quite different from those high in
either monounsaturated fats or -3 polyunsaturated fats (10, 18).
Diets high in saturated fat have been associated with reductions in
insulin sensitivity and increases in serum low-density lipoproteins and
body weight (14, 21-23, 28, 30, 38). One hypothesis that has been
offered is that, as different fatty acid types are incorporated into
cell membrane phospholipid, alterations in membrane fluidity, clotting,
and vascular reactivity are produced (12, 24, 29, 33). An alternative
hypothesis is that the differences in the health effects of these fats
come from differences in the metabolic handling of these different fatty acids by relevant tissues.
We have been interested in how abnormalities in the metabolism of
dietary fat might relate to the development of obesity. Our previous
studies examined the movement of 14C-labeled oleic acid
between the gastrointestinal (GI) tract, skeletal muscle, liver, and
adipose tissue in genetically obese Zucker rats. These studies
demonstrated a reduction in the oxidation and excessive storage of a
dietary fat tracer in obese rats relative to lean. More specifically,
the previous studies suggested a defect in the handling of dietary fat
by skeletal muscle in both obese and reduced obese rats. To more
completely define the time course of the handling of dietary fat, we
have also performed dietary fat tracer studies in lean Sprague-Dawley
rats (4). These studies suggested that the movement of carbons derived
from dietary fat between tissues is a complex and dynamic process over
time, perhaps better described by the term "trafficking" than by
the more widely used term "partitioning." They showed that, in
lean rats, skeletal muscle and liver are quantitatively more important
than adipose tissue in the early clearance of dietary fat. The
conclusions of these studies, however, are limited to the specific type
of dietary fat used (oleate) and the metabolic state studied (only previously fasted rats were studied). Would the results of these studies be the same if another fatty acid tracer had been used or if
the study had been conducted in fed rats? We wondered what the most
representative fatty acid tracer would be to use in studies of the
metabolism of dietary fat. A basic assumption in all tracer studies is
that the tracer will behave metabolically in an identical manner to the
tracee. Most metabolic tracer studies have used 14C- or
13C-labeled palmitic acid or, less commonly, oleic acid.
This has been done on the basis of studies in which the fractional
uptake of different fatty acids by skeletal muscle was found to be
similar by arteriovenous balance (19). It is less clear
that the handling of different fats by the GI tract, adipose tissue,
and liver is similar, or that the intracellular storage or oxidation of
the different fats is similar. In addition, many previous studies have
been conducted in the fasted state. We speculated that some of the
effects of diets varying in fatty acid composition were due to
differences in the postingestive movement of these different fatty
acids between tissues. In addition, we and others (2, 34) have
demonstrated that lipoprotein lipase in adipose tissue and muscle is
regulated in a manner that would predict that the tissue-specific
clearance of dietary fat might be quite different in the fed state than
in the previously fasted state.
To date, the trafficking of different dietary fats in the fasted and
fed states has not been systematically examined. In an effort to more
completely examine the effects that nutritional status and degree of
saturation have on the metabolic fate of dietary fat after ingestion,
we have performed tracer studies in both fasted and fed rats, using
three dietary fat tracers: stearic acid (18:0), oleic acid (18:1,
-9), and linolenic acid (18:3, -3). These studies
demonstrate significant effects of both fat type and nutritional state
on the trafficking of dietary fat.
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METHODS |
Animals.
Male Sprague-Dawley rats (n = 135) weighing 250-300 g were
obtained from Sasco or Harlan. The rats were housed in the Surgical Research Facility at the Denver Health Medical Center (DHMC) in a
temperature-controlled, 12:12-h light-dark cycle environment. Before
studies were performed, rats were fed a semisynthetic diet containing
21% protein, 56% carbohydrate, and 23% fat, with a polyunsaturated-to-saturated ratio of 2:1 (Research Diets no. D12449L)
for 7 days. Rats were observed and acclimatized to the facility for 7 days before surgery. Protocols for these experiments were approved by
the Animal Care and Use Committees at the University of Colorado Health
Sciences Center and DHMC.
Surgery.
Gastric feeding tubes were placed in a manner similar to that described
by Elizalde and Sclafani (17). Rats were fasted overnight before
surgery. After anesthesia was introduced (ketamine 80 mg/kg + xylazine
12 mg/kg), a midline abdominal incision was made and the stomach was
withdrawn from the abdominal cavity. A 3-mm incision was made along the
midportion of the greater curvature of the stomach, and a purse-string
suture was placed along the margin of the incision. A Silastic tube
(no. AST062095, Dow Corning, Konigsburg Instruments, Pasadena, CA) was
introduced into the lumen of the stomach through this incision. The
tube had been previously prepared with a drop of silicone rubber near
the tip. The purse-string suture was then tightened and tied to secure the feeding tube in the gastric lumen. A small piece of
marlex mesh (Davol, Cranston, RI) was then glued to the
tube and the outer surface of the stomach with methyl-methacrylate to
further secure the tube in place. The remaining length of the tube was brought through the abdominal wall, tunneled subcutaneously to an exit
site in the interscapular region, and trimmed to length, and a luer loc
hub from an intravenous catheter was glued to the tube. The hub was
then capped. Both surgical sites were closed with interrupted silk
sutures. Rats recovered from surgery for 24 h. After recovery, and on a
daily basis thereafter until studies were performed, a liquid meal
containing 3 kcal (16% protein, 64% carbohydrate, and 20% fat;
Ensure, Ross Laboratories) was introduced through the feeding tubes to
acclimate the rats to being fed in this manner.
14C feeding experiments.
Rats were allowed to recover from surgery for 7 days before tracer
studies were performed. During this time, rats were weighed each day.
Any rats that lost >10% of their presurgery body weight during the
postoperative period were removed from the study. 14C
feeding studies were conducted as previously described (3, 4). Before
tracer was administered, rats were either fasted for 15 h (fasted) or
allowed to eat ad libitum (fed). At 0830 on the morning of the study,
rats were given a tracer amount of linolenic, oleic, or stearic acid
labeled at the 1 position with 14C (8.3 × 106 dpm total dose, specific activity = 52-55
µCi/mmol; Amersham) in olive oil followed by a "chase" of cold
olive oil to ensure complete delivery of the tracer through the feeding
tube. This was immediately followed by an Ensure liquid meal, as
described above. The total nutrients delivered in this test meal then
included 4 kcal, with 48% fat, 35% carbohydrate, and 17% protein.
The fat content of the meal was 163 mg, with 65% monounsaturated, 23% polyunsaturated, and 12% saturated fat. Test meals had the same composition in all groups with the exception of the tracer quantity of
the labeled fatty acid being tested. After administration of the
tracer, rats were placed in an airtight respiratory chamber. Room air
was passed through barium hydroxide lime (Baralyme, Allied Healthcare
Products, St. Louis, MO) to remove CO2 and then passed through the chamber at a flow rate of 3.0 l/min. The effluent CO2 from the chamber was collected over 20-min intervals in
3.0-ml aliquots of a 2:1 mixture of methanol and methylbenzethonium
(hyamine) hydroxide. The 14C content of these samples was
then measured with a Beckman LS6500 scintillation counter. Background
14C activity, determined by counting a sample containing
only scintillation fluid and hyamine hydroxide, was subtracted from
experimental values. Exhaled CO2 was collected in this
manner for 8 h after tracer administration or until the time of tissue
collection for the 2- and 4-h time points. Rats representing time
points of >8 h were returned to their cages and given ad libitum
access to the baseline diet. These animals were returned to the
respiratory chamber, and CO2 was collected for 20 min in
the manner described above for 24 and 48 h and 10 days after tracer
administration. Therefore, rats in the fasted group were fasted for 8 h
after tracer administration but had ad libitum access to food before tissue collection at the later time points.
Determinations of 14C content in tissues.
At 2, 4, 8, 24, and 48 h and 10 days after the administration of
tracers, tissues were collected for determination of 14C
content. Studies were performed in a random order with regard to
nutritional state (fed vs. fasted), tracer (linolenate, oleate, or
stearate), and time point to minimize any effect of season or
systematic laboratory/procedural drift on the data. Rats were deeply
anesthetized with pentobarbital, and skeletal muscle samples including
lateral gastrocnemius (mixed fiber type), medial gastrocnemius (predominantly glycolytic), and soleus (predominantly oxidative) were
removed and cleaned of any visible fat and connective tissue. Samples
were frozen in liquid nitrogen and then stored at
80°C until
analyzed. A sample of blood was obtained from the vena cava, and rats
were then euthanized with an intracardiac injection of pentobarbital.
Blood was centrifuged, and the serum was stored at
20°C
until analyses of insulin, triglyceride, and glucose were performed.
The entire liver was removed and weighed. Samples of liver were then
frozen in liquid nitrogen and stored at
80°C. The GI tract
was removed, stripped of all omental fat, and weighed. Epididymal and
retroperitoneal fat pads were also removed and weighed.
The GI tract liver and muscle samples were homogenized in ice-cold
0.9% saline. Duplicate samples (0.5 ml) of tissue homogenate were then
digested with 1.0 ml of tissue solubilizer (Solvable; NEN) at 50°C
overnight. Samples were bleached with H2O2, and
14C content was determined by scintillation counting. The
14C content of serum samples was also determined in this manner.
The 14C content of adipose tissue samples was measured
after extraction of lipid with chloroform-methanol (2:1, vol/vol).
Phases were separated with the addition of
H2SO4 and centrifugation. The lower phase was
removed and allowed to dry overnight under nitrogen gas, and
14C content was measured by scintillation counting. The
lipid content of the remaining carcass was determined by lipid
extraction after homogenization.
Other assays.
Insulin levels were measured with a commercially available
radioimmunoassay (Linco catalog no. RI-13K). Serum triglyceride was
assessed by measuring glycerol released after acid hydrolysis of the
sample (Sigma kit no. 337-b). Serum glucose was measured with a Yellow
Springs Instruments model 1500 glucose analyzer.
Calculations and statistics.
The 14C content of each tissue was calculated from the
14C activity per gram of tissue multiplied by the total
weight of the tissue. Whole body skeletal muscle 14C
content was calculated by multiplying the average 14C
activity per gram in lateral and medial gastrocnemius by the body mass
times the percent skeletal muscle [38% of body weight as
estimated from the tissue data of Caster et al. (11)]. Similarly, whole body adipose tissue 14C content was calculated by
multiplying the average 14C activity per gram of epididymal
and retroperitoneal fat by percent body fat (measured by carcass
analysis). Serum 14C content was calculated as the measured
14C activity per milliliter of serum multiplied by 0.0385 (%body mass accounted for by serum) times body mass (11). All data are
presented in graphic form as the mean ± SE of 4-6 rats per time
point per tracer per metabolic state. The data were inspected for
apparent group differences, and one- (fasted vs. fed, for hormone and
substrate data) or two-way (fasted/fed and/or
linolenate/stearate/oleate) ANOVA was performed between groups (tissue,
time point) where appropriate (Sigma Stat, SPSS, Chicago,
IL). Trends within a group across time were tested
for with a pairwise multiple comparison procedure.
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RESULTS |
Rat weights and hormone and substrate concentrations.
Mean rat weights after the placement of gastric feeding tubes declined
by 7 g. This decline was not statistically significant. Weights
returned to baseline and were stable by postoperative day 5.
Serum levels of insulin, glucose, and triglyceride seen in fasted or
fed rats are given in Table 1. Because the
caloric content and macronutrient composition of the chronic diet and test meals were similar, these values were not different in the different fatty acid tracer groups. Therefore, data from the three tracer groups (stearate, oleate, and linolenate, either fasted or fed)
have been pooled. Serum insulin levels were significantly higher in fed
rats for 24 h after the tracer administration (P < 0.003).
Interestingly, at 48 h there was a trend for insulin levels to be
higher in fasted rats (P = 0.056). This is likely due to
rebound hyperphagia after the initial period of fasting. The rise in
insulin levels seen in fasted rats over time was significant (P < 0.05, 48 h vs. 2, 4, or 8 h). Serum glucose levels were measured over the time course of the experiment. There were no significant differences between groups at any time point. As might be expected, the
mean level of triglyceride was significantly higher in fed rats
compared with fasted rats at early time points (P = 0.039 between groups at 2 h, P < 0.001 at 4 h). The decline in
triglyceride levels seen in fed rats was statistically significant
(P < 0.05 at 2 vs. 8 h and at 4 vs. 8 h). The increase in
serum triglyceride levels seen in fasted rats was also significant
(P < 0.05 at 4 or 8 h vs. 48 h).
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Table 1.
Serum insulin, glucose, and triglyceride levels after
administration of dietary fat tracers with a small meal
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Tracer oxidation.
The production of 14C-labeled CO2 is shown in
Fig. 1. In all groups, tracer oxidation
appears to peak between 4 and 8 h. The striking finding is the reduced
production of 14CO2 in the animals that
received labeled stearate (P < 0.05). Oxidation of dietary
oleate or stearate over the 8 h after administration was found to be
significantly greater in fasted rats (P < 0.05), although
this effect was not seen in the linolenate group. Feeding effects were
most pronounced over the first 2 h after meal administration.

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Fig. 1.
Production of 14CO2 over time by rats after
administration of a meal containing 1-14C-labeled linolenic
(gray diamonds), oleic (open triangles), or stearic acids (closed
circles). A: data from fasted rats; B: data from rats
allowed to eat ad libitum overnight. Rats received 8.3 × 106 dpm at time 0 in a 3-kcal meal given through a
gastric feeding tube. Each time point represents
14CO2 (means ± SE) produced over that 20-min
interval (n = 25/group at time points <2 h, to n = 4 in each group at 10 days).
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Tracer content in serum and tissues.
Differences in tracer oxidation could be due to decreased GI absorption
between groups, decreased serum specific activity due to differences in
triglyceride concentration, or differences in the handling of tracer by
tissues. To better understand the mechanisms underlying the differences
in tracer oxidation seen, the 14C content of the GI tract,
serum, liver, skeletal muscle, and adipose tissue was measured and is
depicted graphically in Figs. 2-5. Data were evaluated by a
two-way ANOVA (fed/fasted, linolenic/oleic/stearic) at each time point.
The 14C content of the GI tract is depicted in Fig.
2. The individual values represent the
total 14C content of the entire GI tract (stomach to
rectum) at that time point. At the 2-h time point there was a
significant difference between fasted and fed groups (P = 0.017), with the fed state being associated with greater tracer
retention within the GI tract. This occurred at the same time that
oxidation of tracer was higher in fasted rats. There were no other
significant differences between groups.

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Fig. 2.
14C content of gastrointestinal (GI) tract. The entire GI
tract from the stomach to the rectum was removed at the time point
indicated after tracer administration. All visible omental adipose
tissue was stripped, and 14C content/g was determined. The
total 14C content was calculated as the content/g times the
total mass of the GI tract (n = 4-5/group at each time
point). Fasted (A) and fed (B) groups were
significantly different from each other at 2 h (P = 0.017).
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The 14C content of serum is depicted in Fig.
3. The values represent 14C
content within the total serum pool at each time point. 14C
activity within the serum pool could be associated with dietary fat
being absorbed, nonesterified fatty acids released into the circulation
after lipoprotein lipase-mediated hydrolysis, or 14C in
bicarbonate or dissolved CO2. The only significant
difference found was between tracer groups at the 2-h time point
(P = 0.02), with the difference being largely accounted for by
the difference between linolenic acid and stearic acid.

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Fig. 3.
14C content of serum from fasted (A) and fed
(B) rats. 14C content/ml of plasma was determined,
and the total body plasma content of 14C was estimated by
multiplying this value times body mass times 0.0385 (see text for
rationale). Two-way ANOVA identified significant differences among the
different fats at 2 h (P = 0.02).
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14C content of the liver is depicted in Fig.
4. The most striking finding is the
retention of tracer within the liver in the animals receiving labeled
stearic acid at 8 and 24 h. Also of note is the retention of labeled
linolenic acid in the livers of fasted rats relative to fed rats. A
number of significant differences between groups were identified. At
the 8-h time point, there is a significant effect of tracer type
(P = 0.005), with stearate being significantly higher than
either oleate or linolenate. At the 24-h time point, there was also a
significant effect of fat type (P < 0.001), with labeled
stearate content in the liver being significantly greater than either
oleate or linolenate. In addition, a significant difference was seen
between the fasted and fed linolenic acid groups at this time point
(P < 0.05). At the 48-h time point a significant effect of
fat type was seen, with stearic acid again being greater than oleic
acid (P = 0.01).

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Fig. 4.
14C content of liver from fasted (A) and fed
(B) rats. The total 14C content of the liver was
calculated as the content/g times the total mass of the liver
(n = 4-5/group at each time point). At 8, 24, and 48 h, there was a significant difference between fat types (P < 0.02). A significant difference between fasted and fed groups was
seen at 24 h (P < 0.05).
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14C content of skeletal muscle is depicted in Fig.
5. Although less dramatic than what was
seen in the liver, again the main finding is increased retention of the
stearate tracer. This difference was statistically significant at the
24-h time point (P < 0.001), with the stearic acid group
being greater than either the oleic or linolenic acid groups.

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Fig. 5.
14C content of whole body skeletal muscle in fasted
(A) and fed (B) rats. The 14C content of
whole body skeletal muscle was estimated as the average 14C
activity/g in lateral and medial gastrocnemius multiplied by body mass
times %skeletal muscle (n = 5/group at each time point). A
significant difference between fat types was seen at 24 h
(* P < 0.001).
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The 14C content of adipose tissue is depicted in Fig.
6. As was seen in our previous studies,
tracer content in adipose tissue continues to rise after it has already
peaked in liver and skeletal muscle. In fact, it doesn't peak until 48 h after ingestion in most groups, and it doesn't peak until 10 days in
the stearic acid groups. The other obvious finding in this tissue was
the marked feeding/fasting effects in the unsaturated fat groups. Differences between fed and fasted groups were significant (P < 0.002) at all time points, and differences between fat types (linolenic, oleic, and stearic) were significant at the 8-h, 24-h (P < 0.001), and 10-day (P = 0.007) time points. The
differences at the 8- and 24-h time points are the result of the
greater adipose tissue tracer content in the oleate- and linolenate-fed
groups. At the 10-day time point, the differences are due to the
greater adipose tissue tracer content in the fasted stearate groups.

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Fig. 6.
14C content of whole body adipose tissue in fasted
(A) and fed (B) rats. 14C content of whole
body adipose tissue was calculated as the average 14C
activity/g in epididymal and retroperitoneal adipose tissue times body
mass times %body fat measured by carcass analysis (n = 5/group
at each time point). Significant differences between fasted and fed
groups were seen at all time points (* P < 0.002), and
significant differences between fat types were seen at 8 h, 24 h, and
10 days (+ P < 0.01).
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DISCUSSION |
In this study, we have attempted to examine the effects of degree of
saturation and nutritional state on the postprandial movement of
dietary fat tracers between tissues. Both appear to have major effects
on fat trafficking. Specifically, the postprandial oxidation of the
saturated fat stearic acid was found to be considerably less than that
of either oleic or linolenic acids. One way of understanding this
reduction in the oxidation of stearic acid after ingestion is to follow
the content of tracer in several tissues over time. These relationships
for linolenic acid and stearic acid administered to fasted rats are
depicted in Fig. 7, which suggests that the
reduction in the oxidation of stearic relative to linolenic acid is not
due to a delay in GI absorption but rather to a retention of this fatty
acid within the liver and skeletal muscle pools. The movement of
stearic acid to the ultimate site of oxidation appears to be
restricted; its trafficking "shifted to the right" in
relationship to time. Most of the recent work examining the effects of
dietary fat type on insulin sensitivity has focused on the degree to
which the ingestion of these fats influences plasma membrane fluidity
(33). It is also possible that differences in fluidity between fatty
acids with differing degrees of saturation affect their
movement from the GI tract to the plasma compartment, into liver and
skeletal muscle, and ultimately into mitochondria where they are
oxidized. Saturated fat may be a less suitable substrate for
lipoprotein lipase, may bind less effectively to fatty acid-binding
proteins, or may be less able to move freely within cell membranes (9,
26). These alterations in fatty acid movement may underlie some of the
effects seen with diets enriched in saturated fat.

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Fig. 7.
Trafficking of different dietary fats between tissues in the fasted
state. Tissue 14C content of the liver (diamonds), whole
body muscle tissue (triangles), whole body adipose tissue (circles),
and the entire GI tract (squares) is shown for rats receiving
1-14C-labeled stearic acid (A) and linolenic acid
(B).
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In a previous study that systematically examined the effect of chain
length and degree of saturation on the oxidation of dietary fat, Leyton
et al. (25) found some of these same features. This study suggested
that fatty acid oxidation increased with higher degrees of unsaturation
but was inversely related to chain length. In addition, there was
increased retention of saturated fat in liver 24 h after tracer
administration compared with either mono- or polyunsaturated fats.
However, this study had a number of important limitations. First, the
rats used were quite young (21 days) and weighed 60-80 g. Second,
measurements did not take into account the different specific
activities of the different tracers used. This is to say that the
14C per nanomole of carbon varied between tracer groups as
a result of differing chain lengths. Third, the effect of nutritional
state was not studied. Fourth, in these studies relevant tissues,
including skeletal muscle, GI tract, and adipose tissue were not
sampled; as a result, it was not possible to speculate on the
mechanisms underlying the reduced oxidation of saturated fat. Finally,
serum, carcass, and liver distribution of tracer was examined only at a
single time point. Another study, by Bottino et al. (6), also found
lower oxidation of a saturated fat tracer compared with mono- or
polyunsaturated fat tracers. By sampling multiple tissues over a more
extensive time course, the current study extends the observations of
Leyton et al. and Bottino et al.
A second finding of the current study was that the oxidation of dietary
fat was in general lower in the fed compared with the fasted state.
This reduction in tracer oxidation seen in fed rats was associated with
an increase in the fraction of the dietary fat tracer stored in adipose
tissue. This is depicted in Fig. 8, which
compares the trafficking of oleic acid in the fed and fasted states.
These data are in accord with predictions made by Tan et al. (36) many
years ago on the basis of estimates of total body muscle and adipose
tissue lipoprotein lipase activities in the fasted or fed states.
Specifically, in fasted rats, skeletal muscle and liver play a
quantitatively more important role in the clearance of dietary fat than
in that of adipose tissue during the 8 h after ingestion, independent
of the type of fat. However, in the fed state, adipose tissue plays an
important role right from the start. It was surprising to see the
tracer content in adipose tissue rise between 24 and 48 h after
administration. This is in line with data obtained by Marin et al. (27)
in humans, which showed that a dietary fat tracer gradually accumulates
in adipose tissue for 1 mo after ingestion. This finding highlights the
importance of adipose tissue as a "storage site of last resort" for dietary fat. In contrast, it appears that skeletal muscle and liver
play important roles in the storage of dietary fat after periods of
negative caloric balance.

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Fig. 8.
Trafficking of dietary oleic acid between tissues in fasted rats
(A) vs. fed rats (B). 14C content of the
liver, whole body muscle tissue, whole body adipose tissue, and the
entire GI tract is shown.
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In this study, fat tracers were administered in the diet. This design
is a departure from the traditional approach used in classic studies of
chylomicron triglyceride (Tg) metabolism, in which the tracer is
delivered into the vascular compartment (1, 8, 20), but it has been
used in studies of both animals and humans by a number of investigators
(5, 31). The traditional experimental design that creates a steady
state allows quantitative measures to be calculated of the rate of
appearance or disappearance of the metabolite of interest to and from
the vascular compartment. A central issue in this experimental design,
then, is the specific activity (SA) of the tracer in the vascular
compartment. In the present study, the plasma SA is not constant over
the duration of the study. In particular, in the fed state the
plasma Tg concentration is increased relative to the fasted state.
This difference would produce a decreased SA of 14C/Tg
within the plasma compartment in the fed state, and a reduction in the
rate of 14CO2 evolution would be an expected
consequence. An alternative conceptual framework that we have employed
in the current study is to "trace the meal." Given this as the
starting point, the relevant SA is the SA of tracer in the meal. A
reduction in the production of 14CO2 as a
result of dilution of the tracer upon entering the plasma compartment
in fed rats does not mean that the assessment of the oxidation is
artificially low; it simply suggests an etiology for that reduction.
The present study attempts simply to follow the movement of dietary fat
tracers through the body. No attempt is made to quantitatively assess
hepatic Tg production or to comment on peripheral Tg clearance. Because
the tracer is placed directly into the relevant pool (the meal), the
behavior of the tracer should model the behavior of dietary fat quite
well. An SA for tracer in the dietary fat pool could have been
calculated; however, because the different tracers behave differently,
this method seems inappropriate. Alternatively, an SA could be
calculated as labeled saturated fat per total saturated fat in the
meal, labeled polyunsaturated fat per unlabeled polyunsaturated fat, and labeled monounsaturated fat per unlabeled monounsaturated fat in
each experimental condition. Because all meals contained the same dose
of tracer, yet the content of monounsaturated fat in the meal was high
and the saturated fat was quite low, this type of calculation would
make the reduced oxidation seen with stearic acid even more prominent.
The data are presented as disintegrations per minute, as this is the
most conservative way to present the data. A problem introduced by this
design is that the metabolites are not in steady state, and as a result
the quantitative analyses that can be performed on the data are
limited. However, we believe that this limitation is counterbalanced by
the physiological nature of the experiment and the comprehensive tissue
tracer content information obtained at multiple time points.
A second limitation is the nutritional context in which the studies
were performed. The results of this study describe the behavior of
these fatty acids in the setting of a small meal containing a
relatively high fat content, in particular a relatively high content of
monounsaturated fat, given to rats chronically consuming a diet
containing 23% fat. This baseline diet was chosen because it more
closely mimics the diet consumed by humans than the 10% fat content of
standard rat chow. The composition of the baseline diet, the caloric
content of the test meal, the fat content, and the type of fat in the
test meal likely play important roles in determining the overall
pattern of trafficking of dietary fat. The specific effect of varying
these parameters on trafficking, however, would need to be determined
experimentally. Third, the exact biochemical nature of the compounds
labeled with 14C is not known. It is possible that both the
chain length and degree of saturation may change after ingestion (13).
However, these possibilities do not alter the conclusion that ingested stearic acid is metabolized differently than ingested linolenic acid.
Finally, 14CO2 recovered may not quantitatively
reflect total rates of dietary fat oxidation because of dilution in the
bicarbonate pool and fixation of label within isotopic exchange
reactions (32). These differences may even be systematically different
between groups, in particular between the fed and fasted states.
However, because of the number of tissues tested, the length of the
time course examined, the consistent picture seen when the data are
taken as a whole, and the lack of accepted approaches to correcting tracer oxidation estimates in the fed state, it seems reasonable to
conclude that the present studies do suggest important differences in
the metabolic handling of different fatty acids that should be
considered in future studies.
In summary, the present study sought to examine the effects of
nutritional state and the type of fatty acid ingested on the trafficking of dietary fat. Each of these appears to have important effects. Most studies of fat metabolism have utilized palmitate or
oleate tracers administered to fasted animals or humans. It has been
assumed that the metabolic behavior of these tracers is similar to that
of other fats and that experimental results obtained in the fasting
state can be extrapolated to the fed state. The present study suggests
that these assumptions have limitations. In particular, the effect of
fat type may vary depending on the tissue and the time point examined.
Although stearate was excessively stored in liver and skeletal muscle
at intermediate time points, the content in adipose tissue was actually
reduced at these same time points relative to the unsaturated fats
studied. The effect of the degree of saturation on the oxidation of a
fatty acid ingested in the diet appeared to be dramatic. These
differences should be considered when a fat tracer is selected for
metabolic studies. Perhaps most importantly, these findings may have
relevance to understanding the effects of saturated fat on insulin
sensitivity and the health effects of diets high in saturated fat.
 |
ACKNOWLEDGEMENTS |
Support for this work was provided by National Institute of
Diabetes, Digestive, and Kidney Diseases (NIDDK) Grant R29 DK-47311. Assistance and technical support were also provided through the NIDDK-funded Colorado Clinical Nutrition Research Unit DK-48520.
 |
FOOTNOTES |
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.
Address for reprint requests and other correspondence: D. H. Bessesen, Mail Code 4000, Denver Health Medical Center, 777 Bannock St., Denver, CO 80204-4507 (E-mail: daniel.bessesen{at}uchsc.edu).
Received 16 August 1999; accepted in final form 18 January 2000.
 |
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