From the Gifford Laboratories, Touchstone Center for
Diabetes Research, Department of Internal Medicine, University of Texas
Southwestern Medical Center, Dallas, Texas 75390-8854, the
¶ Department of Radiology, University of Texas Southwestern
Medical Center, Dallas, Texas 75390-9085, and the
Veterans
Affairs Medical Center, Dallas, Texas 75216
Received for publication, September 19, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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To test the hypothesis that the physiologic
liporegulatory role of hyperleptinemia is to prevent steatosis during
caloric excess, we induced obesity by feeding normal Harlan
Sprague-Dawley rats a 60% fat diet. Hyperleptinemia began within
24 h and increased progressively to 26 ng/ml after 10 weeks,
correlating with an ~150-fold increase in body fat
(r = 0.91, p < 0.0001). During this
time, the triacylglycerol (TG) content of nonadipose tissues rose only
1-2.7-fold implying antisteatotic activity. In rodents without leptin
action (fa/fa rats and ob/ob and
db/db mice) receiving a 6% fat diet, nonadipose tissue TG
was 4-100 times normal. In normal rats on a 60% fat diet, peroxisome
proliferator-activated receptor Compelling theoretical considerations coupled with corroborating
experimental evidence argue against the conventional view that the
physiologic role of leptin is to prevent obesity. First, plasma leptin
levels of rodents and humans are low in the lean and high in the obese
(1), hardly the credentials of an antiobesity hormone. Second,
diet-induced obesity is not prevented in hypoleptinemic mice by
restoring their plasma leptin levels to normal with recombinant leptin
(2). Third, there is no evidence that overnutrition and obesity have
ever posed a serious survival threat in evolution. On the contrary, the
principal survival threat throughout evolution has been famine, against
which obesity provides a measure of protection as the "thrifty
gene" hypothesis maintains (3). Finally, it seems implausible to
suggest that hormones evolve for the purpose of preventing the clinical
consequences of their own deficiency. Just as insulin evolved to confer
advantages in nutrient metabolism rather than to prevent diabetic
ketoacidosis, leptin must have evolved, not to prevent its deficiency
syndrome, obesity (4), but rather to confer a metabolic advantage that
has not as yet been identified.
We previously had suggested that the metabolic advantage conferred by
the hyperleptinemia of obesity might be the prevention of
overaccumulation of triacylglycerols
(TG)1 in nonadipose tissues
(5). Clearly, leptin does have powerful antilipogenic activity in some
such tissues (6). In the absence of leptin action, lipogenesis is
increased and fatty-acid (FA) oxidation is reduced (7), accounting for
the steatosis and lipotoxicity that occur in such circumstances (7-9).
For example, in Zucker Diabetic Fatty (ZDF) rats with a
loss-of-function mutation in the leptin receptors (10, 11), tissue TG
ranges from 10 to 50 times the normal content (8) and is associated
with functional impairment of pancreatic If the foregoing abnormalities develop in the absence of leptin action,
it follows that leptin must be able to prevent them. Certainly
hyperleptinemia induced by adenoviral transfer of the leptin gene has
remarkable lipopenic and antilipogenic activity in tissues of normal
rats, down-regulating the expression of genes involved in lipogenesis
while up-regulating those genes involved in Animals without Leptin Action--
Three groups of rodents were
employed. Obese homozygous (fa/fa) ZDF-Drt rats,
which are unresponsive to leptin because of a loss-of-function mutation
in their leptin receptor (10, 11), and lean wild-type (+/+) ZDF
controls were bred in our laboratory from ZDF/Drt-fa (F10) rats
purchased from Dr. R. Peterson (University of Indiana School of
Medicine, Indianapolis, IN). Two groups of mice,
C57BL/6J-ob/ob and C57BL/KS-J-db/db, and their
wild-type controls, C57BL/6J +/+ and C57BL/KS-J +/+ mice, were
purchased from the Jackson Laboratory (Bar Harbor, ME).
Animals with Leptin Action--
To achieve diet-induced
obesity in normal rats, Harlan Sprague-Dawley rats, purchased from
Charles River Laboratories (Raleigh, NC) were employed. They were
housed in individual metabolic cages (Nalgene, Rochester, NY) with a
constant temperature and 12 h of light altering with
12 h of darkness. Body weight and food intake were measured
weekly. Initially, all rats were fed standard chow (Teklad 4%
mouse/rat diet, Teklad Madison, WI) ad libitum and
had free access to water. At 4 weeks of age they either continued on
this diet, which contains 24.8% protein, 4% fat, and 3.94 Kcal/g, or
they were switched to a high fat diet (Purina Test Diet, Purina Mills,
Inc., Richmond, IN) containing 60% fat, 7.5% carbohydrate, 24.5%
protein, and 6.7 Kcal/g to produce diet-induced obesity.
Adenovirus Transfer of OB-Rb cDNA to Liver of fa/fa ZDF
Rats--
In in vivo experiments containing a total of
1 × 1012 plaque-forming units of recombinant
adenovirus containing the cDNA of the leptin receptor OB-Rb
(AdCMV-OB-Rb) or as a control Expression of Wild-type and Mutated OB-Rb in Liver and
Hypothalamus of fa/fa Rats--
To compare the expression of wild-type
OB-Rb in fa/fa rats with mutated OB-Rb, total RNA of rat
liver and hypothalamus were extracted using TRIzol reagent (Life
Technologies, Inc.). Reverse transcription of total RNA was carried out
after treating RNA samples with RNase-free DNase I. The first strand
cDNA was then used to PCR-amplify an OB-RbcDNA fragment
with OB-Rb-specific primers encompassing the region with the
fa/fa mutation as described previously (11). The conditions
of the PCR were as follows: denaturation for 45 s at 92 °C,
annealing for 45 s at 55 °C, and elongation for 1 min at
72 °C. The amplified PCR products were digested with MspI
at 37 °C 1 h and then run on a 1.2% agarose gel.
Northern Blot Analysis--
Total RNA was extracted by the
TRIzol isolation method, and Northern blot analysis was carried out as
described previously (21). cDNA probes for the oxidative enzymes,
acyl-CoA oxidase (ACO) and liver-carnitine palmitoyl- transferase-1
(LIVER-CPT-1), were prepared by reverse
transciptase-PCR using the following primers: ACO-sense (amino acids
2891-2910), 5'-GCCCTCAGCTATGGTATTAC-3' and ACO-antisense (amino acids
3505-3524), 5'-AGGAACTGCTCTCACAATGC-3' (GenBankTM
accession number J02752); and LIVER-CPT-1-sense (amino
acids 3094-3113), 5'-TATGTGAGGATGCTGCTTCC-3' and
LIVER-CPT-1-antisense (amino acids 3703-3722),
5'-CTCGGAGAGCTAAGCTTGTC-3' (GenBankTM accession number
L07736). The DNA fragment excised after digesting pAC CMV-OB-Rb (13)
with KpnI/HindIII restriction enzymes
hybridizes only the intracellular domain of OB-Rb was also used
as a probe of OB-Rb. The hybridization signals were analyzed by
Molecular Imager GS-363 (Bio-Rad). Values were normalized to the signal generated with an 18 S ribosomal RNA (rRNA) gene probe.
Multiplex Reverse Transcriptase PCR--
The procedure used was
based on methods described by Jensen et al. (22) and
O'Doherty et al. (23). Total RNA (1 µg) was treated with
RNase-free DNase (Promega), and first-strand cDNA was generated
with the oligo(dT) primer in the first-strand cDNA synthesis kit
(CLONTECH). Multiplex reverse transcriptase-PCR was
carried out in 25-µl reactions with 1.5 µl of the diluted cDNA reaction as template mixed with 23.5 µl of PCR mix
containing 1.25 units of Taq polymerase and buffer (Roche
Molecular Biochemicals) containing 25 µM dATP, dTTP, and
dGTP, 2.5 µCi of 2500 Ci/mmol [
Reaction products were separated on 7 M urea, 1× TBE (0.1 M Tris base, 83 mM boric acid, 1 mM
EDTA) and 6% polyacrylamide gels. Dried and PhosphorImager screens
were scanned by a Molecular Imager System (GS-363). TATA box-binding
protein mRNA was coamplified as an internal control, and data were
expressed as ratios to its signal.
To avoid biased results caused by potential interference between
individual amplicons, we analyzed the amplification kinetics of
individual amplicons in reactions where several products were coamplified. Representative experiments, in which mRNA encoding lipogenic enzymes and TATA box-binding protein in pancreatic islets were simultaneously amplified, show the noncompetitive amplification of
individual products and their almost identical rate of amplification as
indicated by the slopes within the exponential phase observed from a
linear regression analysis.
PPAR Magnetic Nuclear Resonance Spectroscopy (MRS) and Magnetic
Resonance Imaging--
Using the method of Stein et al.
(24), proton MRS and magnetic resonance imaging data were obtained with
a 4.7-T 40-cm-bore system (Omega chemical shift imaging model, Bruker
Instruments, Fremont, CA) with a 6-inch-diameter birdcage coil.
Anesthetized rats were placed supine within the coil and positioned in
the center of the magnet. Proton spectra of the rat were resolved into
water and fat resonances, the areas of which were quantified using the
nuclear magnetic resonance software program NRM-1 (Tripos Associates,
St. Louis, MO) assuming equal line widths for both resonances. Proton
images were obtained from the abdominal region of each rat. Spin-echo
transaxial images were acquired with the following parameters: two
transients, recycle time = 500 ms, echo time = 16 ms, 2-mm
slice thickness, 2-mm interslice gap, eight slices, a 140-mm field of
vision, and a 128 × 256 matrix. Images were analyzed using
NIH Image software (National Institute of Mental Health, Bethesda, MD).
TG Content of Tissues--
Animals were sacrificed under sodium
pentobarbital anesthesia. Tissues were dissected and placed in liquid
nitrogen. Total lipids were extracted from about 100 mg of tissue by
the method of Folch et al. (25) and dried under
N2 gas. TG was assayed by the method of Danno et
al. (26).
Plasma Measurements--
Tail vein blood was collected in
capillary tubes coated with EDTA. Plasma was stored at [3H]Palmitate Oxidation in Pancreatic
Islets--
Oxidation of [3H]palmitate by islets was
determined as described previously. Groups of 100-200 islets were
incubated in duplicate with 1 mM
9,10-[3H]palmitate for 3 days. Palmitate oxidation was
assessed by measuring tritiated water in the medium. Excess
[3H]palmitate was removed by precipitating twice with an
equal volume of 10% trichloroacetic acid and 2% bovine serum albumin.
Supernatants in a microcentrifuge tube were placed in a scintillation
vial containing unlabeled water and incubated at 50 °C for 18 h. Tritiated water was measured as described for use of
[3H]glucose (27).
[U-14C]Glucose Incorporation into Lipids in
Islets--
Incorporation of [U-14C]glucose (14.6 mmol/liter, PerkinElmer Life Sciences) into lipids was measured
in islets as described previously in detail (28). About 200 islets were
cultured for 3 days in medium containing 8 mmol/liter of glucose. After
3 days in culture, lipids were extracted from the islets according to the method of Bligh and Dyer (29), and counts incorporated into total
lipid were determined.
Statistical Analyses--
All values shown are expressed as
mean ± S.E. Statistical analysis was performed by two-tailed
unpaired Student's t test by one-way analysis of variance.
Response of Leptin Levels to Caloric Excess--
If the function
of leptin during caloric excess is to minimize the accumulation of
lipids in nonadipose tissues, hyperleptinemia should begin promptly at
the start of overnutrition and increase progressively as the
overnutrition continues. To test this theory, a group of 10 normal male Harlan Sprague-Dawley rats was fed a diet in which 60% of
the calories were derived from fat. Age-matched control rats received a
4% fat diet. Both groups were observed for 70 days. Plasma leptin
levels in control rats were relatively unchanged, rising by only
0.04 ± 0.002 ng/ml/day to a level of only 2.80 ± 0.77 ng/ml
on the final day of the 70-day study. By contrast, in rats on a 60%
fat diet, plasma leptin rose to 4.3 ± 0.2 ng/ml
(p < 0.001) within 24 h and increased
progressively thereafter by 0.37 ± 0.07 ng/ml/day to a level of
26 ng/ml at 70 days (Fig. 1A).
In this group the rise in plasma leptin levels paralleled the expansion
in body fat mass quantified by MRS (Fig. 1B); there was a
highly significant correlation between body fat and the plasma leptin
level (r = 0.91, p < 0.0001) (Fig.
1C). Thus, leptin levels appear to respond promptly to a
caloric excess, and they increase in proportion to enlargement of the
adipose mass, which is consistent with the postulated role.
TG Deposition in Nonadipose Tissues in the Presence of Leptin
Action--
If the hyperleptinemia induced by high fat feeding does in
fact protect nonadipose tissues of normal rats from overaccumulation of
lipids, their tissue TG content should remain low during the development of obesity despite the expansion of the adipose tissue mass
and the concomitant rise in plasma lipid levels. To test this
theory, we measured tissue TG content of nonadipose tissues 70 days after the start of the high fat diet at which point the total body
fat measured by MRS had increased an ~150-fold above the pre-diet
base line (Fig. 1B), and plasma TG and free fatty-acid levels were significantly higher (Fig.
2A). However, TG content in
nonadipose tissues increased only 1-2.7-fold above the base line (Fig.
2B). Thus, nonadipose tissues of leptin-responsive hyperleptinemic rats accumulated only a small fraction of the total
increase in body fat acquired over 70 days of excessive fat intake,
during which time the animals had became grossly obese (Fig.
1B).
Mechanism of Antisteatotic Protection in Liver--
In the liver,
protection against steatosis might involve not only increased secretion
of very low density liprotein but also enhanced FA oxidation. In
the latter case, an increase in the expression of PPAR Mechanism of the Antisteatotic Protection in Islets--
Unlike
liver, islets cannot export excess FA, which may account for their
vulnerability in obesity. To determine the mechanism of the protection
against lipid overaccumulation that prevails early in the course of
obesity, we measured the rate of oxidation of
[3H]palmitate in isolated islets of Harlan Sprague-Dawley
rats receiving either a 4 or 60% fat diet. Oxidation was 30% greater
in pancreatic islets of rats on the 60% fat diet than in controls that
were on the 4% fat diet (Fig.
4A). However, unlike in liver,
no change in ACO or liver-CPT-1 could be detected by
multiplex-PCR (data not shown). These findings suggest that the
preexisting oxidative machinery of the islets was able to accommodate
this increase in oxidation without an increase in expression of genes
encoding the enzymes.
We had previously reported that in the absence of leptin activity as in
fa/fa ZDF rats, increased lipogenesis was the most important
single factor in the ectopic overaccumulation of lipids in islets (7,
31). Accordingly, in normal rats the high fat diet should not induce
the increase in lipogenesis and lipogenic enzymes that had been
observed in fat-laden islets of the leptin-insensitive fa/fa
rats. As shown in Fig. 4, B and C, there was no
increase in the incorporation of [14C]glucose to lipids
or in the expression of lipogenic enzymes. In fact, a small but
significant decrease in lipogenesis and in fatty acid synthase
mRNA was evident (Fig. 4, B and C). This was in sharp contrast to the ZDF fa/fa rats in which lipogenesis
was 2.5 times greater.
Ectopic TG Deposition in the Absence of Leptin Action--
If the
antilipogenic protection observed in normal rats during caloric excess
did in fact require the action of the accompanying hyperleptinemia,
rodent models with either a leptin deficiency (ob/ob mice)
or a loss-of-function mutation in their leptin receptors (db/db mice and ZDF fa/fa rats) would be
unprotected from lipid overaccumulation. We, therefore, measured the
plasma leptin levels (Fig. 5A)
and the TG content of islets, skeletal muscle, heart, and liver of
these "unleptinized" rodents (Fig. 5B). Although their
diet contained only 6% fat, the TG content of their nonadipose tissues
ranged from ~4 to ~100-fold above normal controls on the same diet.
Thus, when leptin action is lacking, protection from lipid
overaccumulation in nonadipocytes is also lacking, even when the
dietary fat intake is normal.
Overexpression of Wild-type OB-Rb in Livers of ZDF fa/fa Rats
Prevents Steatosis--
If the marked hepatic steatosis and
hypertriglyceridemia of obese ZDF fa/fa rats are the result
of a lack of direct leptin action on the liver, transgenic
overexpression of the wild-type leptin receptor in the liver of these
leptin receptor-defective animals should protect them. Therefore, we
infused into 9-week-old ZDF fa/fa rats 1012
plaque-forming units of recombinant adenovirus containing the cDNA
of wild-type OB-Rb, the full-length isoform of the leptin receptor
(AdCMV-OB-Rb). AdCMV-
One week after treatment with AdCMV-OB-Rb plasma, TG levels of ZDF
fa/fa rats declined slightly below pretreatment levels and
remained significantly below the controls for 3 weeks after AdCMV-OB-Rb
treatment (Fig. 5B). Liver TG content was significantly less
than that of These findings suggest that a physiologic role of leptin during
overnutrition is to protect nonadipocytes from the adverse consequences
of lipid overaccumulation. This protection begins promptly at
the start of overfeeding as the result of progressively increasing
hyperleptinemia that continues to rise for the duration of
hypernutrition. This appeared to minimize overaccumulation of lipids
both by preventing the increase in lipogenesis that occurs in the
absence of leptin action (31) and through the up-regulation of
In islets the antilipogenic action of hyperleptinemia appears to be at
least as important as the increase in FA oxidation in protecting islets
from the lipid overload. When leptin action is lacking as in
hyperphagic fa/fa ZDF rats, the fat-laden islets have a high
rate of [14C]glucose incorporation into lipids in
association with increased PPAR The most compelling evidence in support of the antisteatotic role for
leptin was the in vivo demonstration in leptin-unresponsive fa/fa ZDF rats that transgenic overexpression of the
wild-type receptor in their livers prevented the severe hepatic
steatosis and hypertriglyceridemia that otherwise occurred. These
findings are congruent with earlier evidence of the antisteatotic
action of recombinant leptin (33) and of transplanted fat tissue in "fatless" mice with congenital lipodystrophy (34). Furthermore, in
our experiments the wild-type leptin receptors were expressed only in
the liver and not in the hypothalamus or anywhere else. Therefore, it
follows that the endogenous hyperleptinemia of those obese
fa/fa rats must have acted directly via the transgenic OB-Rb to prevent the lipid overaccumulation.
The prompt rise of plasma leptin levels on the very first day of the
high fat diet and their high degree of correlation with the expanding
body fat are all consistent with the response of an antilipogenic
hormone with a physiologic liporegulatory mission, namely to maintain
FA homeostasis in nonadipocytes during overnutrition. This protection
may account for the fact that in hyperleptinemic rats and humans the
lipotoxic complications of diet-induced obesity do not appear until
late in life when leptin effectiveness wanes (35, 36). When leptin is
absent as in congenital generalized lipodystrophy (33) or when leptin
receptors are congenitally defective as in ZDF rats, these
complications appear in severe form early in life.
It should be emphasized that we do not suggest that the direct
antisteatotic activity ascribed to the endogenous hyperleptinemia of
obesity occurs in normal lean animals. It appears to be a factor only
during overnutrition when plasma leptin levels approach or exceed the
threshold for transport across the blood-brain barrier, which is
probably in the vicinity of 10 ng/ml (37). In the absence of
overnutrition, plasma levels are below 5 ng/ml, and leptin action is
presumed to be largely on the hypothalamic centers for control of food
intake and thermoregulation (38).
protein and liver-carnitine
palmitoyltransferase-1 (L-CPT-1) mRNA increased in
liver. In their pancreatic islets, fatty-acid oxidation increased 30%
without detectable increase in the expression of peroxisome
proliferator-activated receptor-
or oxidative enzymes, whereas
lipogenesis from [14C]glucose was slightly below that of
the 4% fat-fed rats (p < 0.05). Tissue-specific
overexpression of wild-type leptin receptors in the livers of
fa/fa rats, in which marked steatosis is uniformly present,
reduced TG accumulation in liver but nowhere else. We conclude that a
physiologic role of the hyperleptinemia of caloric excess is to protect
nonadipocytes from steatosis and lipotoxicity by preventing the
up-regulation of lipogenesis and increasing fatty-acid oxidation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cells (12, 13) and
myocardium (9) and insulin resistance (14). Ultimately, the progressive
overaccumulation of lipids causes death of cells in pancreatic islets
and myocardium, resulting in diabetes and myocardial failure, which are
the most serious complications of obesity. It has been proposed that
the lipid overaccumulation enlarges the intracellular pool of fatty acyl-CoA beyond the oxidative requirements of the cell (15), thereby
providing substrate for potentially destructive nonoxidative pathways,
such as de novo ceramide formation (16) and lipid peroxidation (17, 18).
-oxidation and
thermogenesis (19). Although they are consistent with putative
antisteatotic activity of hyperleptinemia, such studies do not prove
that the actual physiologic role of adipocyte-derived hyperleptinemia
in obesity is to prevent the ectopic accumulation of TG in nonadipose
tissues. This study was designed to test this premise.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase (AdCMV-
-galactosidase), prepared as described previously (20), were
infused into conscious animals over a 10-min period through polyethylene tubing (PE-50, Becton Dickinson) previously anchored in
the left jugular vein of 9-week-old ZDF fa/fa rats under
sodium pentobarbital anesthesia (20).
-33P]dCTP (1 CI = 37 GBq) (Amersham Pharmacia Biotech), and 5 pmol of each
primer (Table I). The standard thermal
cycle profile was as follows for lipogenic enzyme mRNA (FAS, ACC,
and GPAT) and
-oxidative enzyme gene mRNA (liver-CPT-1
and ACO): denaturation of 94 °C for 1 min, annealing at 55 °C for
1 min, and extension at 72 °C for 1 min for 24 cycles in liver and
for 26 cycles in islets.
Primers employed for multiplex reverse transcriptase-PCR
Immunoprecipitation--
50 mg of liver from the rats
were homogenized in 2 ml of lysate buffer with proteinase inhibitors. A
total of 100 µg of protein in 0.5 ml of buffer were used for
precipitation with 1:500 goat anti-PPAR
(C-20, Santa Cruz
Biotechnology, Inc. Santa Cruz, CA). Protein A beads from Amersham
Pharmacia Biotech were used for binding. Immunoblotting was carried out
with rabbit-anti-PPAR
from Calbiochem at 1:1500.
20 °C.
Plasma leptin was assayed using the Linco leptin assay kit (Linco
Research, St. Charles, MO). Plasma glucose was measured by the glucose
oxidase method using the glucose analyzer II (Beckman, Brea, CA).
Plasma free fatty acids were determined using the Roche Molecular
Biochemicals kit. Plasma TG levels were measured by the glycerol
phosphate oxidase-Trinder triglyceride kit (Sigma).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Relationship of plasma leptin to total body
fat in Harlan Sprague-Dawley rats receiving diets with a fat content of
4 ( ) or 60% (
). A, plasma leptin levels
in the two groups. The inset provides a view of the daily
leptin profile for the first week of the study. B, total
body fat measured by MRS. Representative spectral tracings and
transrenal images are displayed. C, the relationship between
plasma leptin levels and body fat.
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Fig. 2.
A, comparison of the mean (± S.E.)
plasma leptin, TG, and free fatty-acid (FFA) levels in
normal Harlan Sprague-Dawley rats fed a diet containing either 4 (open bar) (n = 6) or 60% (shaded
bar) (n = 6) fat for 10 weeks. *,
p < 0.001. B, the mean (± S.E.) tissue TG
content of six normal Harlan Sprague-Dawley rats on a 60% fat diet at
4 weeks of age before starting the high fat diet (open bar)
and at 14 weeks of age after 10 weeks on 60% fat intake (shaded
bar). TG content in islets is expressed as
ng/islet. In liver, heart and skeletal muscle, it is
expressed as mg/g of wet weight of tissue. Body fat as
determined by MRS is expressed as g/animal (gram). *,
p < 0.001.
and its
target enzymes liver-CPT-1 and ACO might be expected (30). To determine
whether the in vivo protection against hepatic
overaccumulation of TG in normal rats on a high fat diet is mediated by
this mechanism, we semiquantified PPAR
protein and ACO and
liver-CPT-1 mRNAs in livers of normal rats receiving either a 60 or
a 4% fat intake. PPAR
protein and liver-CPT-1 mRNA were both
significantly greater in the former group, but ACO mRNA was not
different (Fig. 3, A and
B).
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Fig. 3.
Mechanism of antisteatotic protection of
liver during high fat feeding of normal rats. A,
PPAR protein measured by immunoprecipitation in the liver of four
rats that were fed a diet containing 4% fat and four rats that were
fed a 60% fat diet. B, mRNA of enzymes of fatty-acid
-oxidation, ACO and liver-CPT-1, quantified by northern
hybridization. *, p < 0.01
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Fig. 4.
Mechanism of antisteatotic protection of
islets during high fat feeding of normal rats. A,
comparison of rates of oxidation of [3H ]palmitate in
rats receiving a diet containing either 4 (open bar) or 60%
(shaded bar) fat. *, p < 0.01. B, comparison of the rates of incorporation of
[U-14C]glucose into lipids in normal Harlan
Sprague-Dawley (SD) rats on a 4 (open bar) or
60% (shaded bar) fat intake. These rates are significantly
less than in islets of hyperphagic fa/fa ZDF rats on a 6%
(striped bar) fat intake. *, p < 0.01; §,
p < 0.05. C, comparison of expression of
the lipogenic enzymes acetyl-CoA carboxylase (ACC),
fatty-acid synthase (FAS), and glycerol-PO4
acyltransferase (GPAT) in islets of normal Harlan
Sprague-Dawley rats that were fed either a 4 or 60% fat diet. As an
internal control for enzyme mRNA, TATA box-binding protein
(TBP) was employed. *, p < 0.005; ¶,
p < 0.01; §, p < 0.05.
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Fig. 5.
A, comparison of mean (± S.E.) plasma
leptin, TG, and free fatty acid (FFA) levels in rodents that
are either leptin-deficient (ob/ob mice) or unresponsive to
leptin because of loss-of-function mutation in its receptor gene
(db/db mice and fa/fa rats) (shaded
bar) and the corresponding wild-type (+/+) controls (open
bar). *, p < 0.01. B,
comparison of TG content in tissues of these rodents. All animals
received a diet containing 6% fat. *, p < 0.001.
-galactosidase was infused into age-matched ZDF
fa/fa rats as a control. The wild-type OB-Rb transgene introduced in vivo with an adenovirus vector was expressed
exclusively in the steatotic liver of the ZDF fa/fa rats
(Fig. 6A). None was detected
in any other tissues including the hypothalamus.
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Fig. 6.
A, effect of intravenous infusion of
obese ZDF fa/fa rats with AdCMV-OB-Rb or
AdCMV- -galactosidase on the expression of wild-type receptor in the
liver and hypothalamus. B, polymerase chain
reaction-restriction fragment length polymorphism (PCR-RFLP)
of OB-Rb in liver and hypothalamus of a lean wild-type (+/+) ZDF rat,
an untreated obese (fa/fa) ZDF rat, and an obese
fa/fa rat 4 weeks after treatment with either
AdCMV-
-galactosidase or AdCMV-leptin. Whereas in the untreated and
AdCMV-
-galactosidase-treated fa/fa rat only mutated OB-Rb
is present, in the AdCMV-leptin-treated fa/fa rat, the OB-Rb
matches that of the +/+ rat. C, plasma TG after AdCMV-OB-Rb
(
) or AdCMV-
-galactosidase (
). **, p < 0.01. D, triacylglycerol (TG) content of liver in obese
ZDF fa/fa rats after treatment with AdCMV-OB-Rb (black
bar) or AdCMV-
-galactosidase (open bar). *,
p < 0.05. E, heart TG and skeletal muscle
TG after AdCMV-OB-Rb (black bar) or AdCMV-
-galactosidase
(open bar).
-galactosidase controls and untreated controls (Fig.
5C), the result of a delay in the increase in liver TG
compared with the controls. TG contents of heart and skeletal muscle
were unaffected (Fig. 6B). Food intake was identical
in the two adenovirus-treated groups (29.8 ± 1.4 g/day
versus 29.8 ± 1.5 g/day). Because the liver was the
only site of expression of the normal OB-Rb in these ZDF
fa/fa rats and the only site of antisteatotic action, we
must assume that the elevated endogenous leptin levels, which averaged 24 ± 2 ng/ml in AdCMV-OB-Rb-treated rats and 28 ± 2 ng/ml
in controls, exerted a direct antisteatotic action on the liver. This
strongly implies that the function of hyperleptinemia of obesity is to prevent steatosis in tissues with functioning OB-Rb.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-oxidative metabolism of the surplus fatty acids (7). Whereas in the
liver there was an increase in PPAR
protein and CPT-1 mRNA, no
such changes could be detected in pancreatic islets despite a 30%
increase in the rate of [3H]palmitate oxidation. The
greater induction of FA
-oxidative enzymes in liver than in
extrahepatic tissues confirms a recent observation by Cook et
al. (31).
, acetyl CoA carboxylase, and fatty
acid synthase expression on a 6% fat intake (32). By contrast,
in normal rats receiving the 60% fat diet, these rates remained
in the low normal range and the lipogenic rate declined. When the
antilipogenic effect of leptin is lacking, lipogenesis is excessive and
is not reduced by lipid excess, as in normal islets (30,32).
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ACKNOWLEDGEMENTS |
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We thank Susan Kennedy for superb secretarial services. We also thank Drs. Cai Li and Daniel Foster for critical review of this manuscript. We thank Dr. Per Bo Jensen for expert help with multiplex reverse trnscriptase-PCR.
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FOOTNOTES |
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* This work was supported in part by the Department of Veterans Affairs Institutional Support (SMI 821-109), National Institutes of Health Grant DK02700-37, National Institutes of Health Juvenile Diabetes Foundation Diabetes Interdisciplinary Research Program, and Novo-Nordisc Corporation.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.
§ These authors contributed equally to this work.
** To whom correspondence should be addressed: Ctr. for Diabetes Research, University of Texas Southwestern Medical Ctr., 5323 Harry Hines Blvd., Dallas, TX 75390-8854. Tel.: 214-648-3488; Fax: 214-648-9191; E-mail: runger@mednet.swmed.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008553200
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ABBREVIATIONS |
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The abbreviations used are: TG, triacylglycerol; FA, fatty acid; ZDF, Zucker Diabetic Fatty; PCR, polymerase chain reaction; AdCMV, adenocytomegalovirus; ACO, acyl-CoA oxidase; liver-CPT-1, liver-carnitine palmitoyltransferase-1; PPAR, peroxisome proliferator-activated receptor; MRS, magnetic nuclear resonance spectroscopy.
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