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Triglycerides Induce Leptin Resistance at the
Blood-Brain Barrier
William A. Banks,
1
Alan B. Coon,
1
Sandra M. Robinson,
1
Asif Moinuddin,
1
Jessica M. Shultz,
1
Ryota Nakaoke,
1,2
and John E. Morley
1
Obesity is associated with leptin resistance as evi-
denced by hyperleptinemia. Resistance arises from im-
paired leptin transport across the blood-brain barrier
(BBB), defects in leptin receptor signaling, and block-
ades in downstream neuronal circuitries. The mediator
of this resistance is unknown. Here, we show that milk,
for which fats are 98% triglycerides, immediately inhib-
ited leptin transport as assessed with in vivo, in vitro,
and in situ models of the BBB. Fat-free milk and in-
tralipid, a source of vegetable triglycerides, were with-
out effect. Both starvation and diet-induced obesity
elevated triglycerides and decreased the transport of
leptin across the BBB, whereas short-term fasting de-
creased triglycerides and increased transport. Three of
four triglycerides tested intravenously inhibited trans-
port of leptin across the BBB, but their free fatty acid
constituents were without effect. Treatment with gem-
fibrozil, a drug that specifically reduces triglyceride
levels, reversed both hypertriglyceridemia and impaired
leptin transport. We conclude that triglycerides are an
important cause of leptin resistance as mediated by
impaired transport across the BBB and suggest that
triglyceride-mediated leptin resistance may have
evolved as an anti-anorectic mechanism during starva-
tion. Decreasing triglycerides may potentiate the ano-
rectic effect of leptin by enhancing leptin transport
across the BBB. Diabetes 53:1253–1260, 2004
Leptin is a 16-kDa protein secreted by fat cells (1)
that regulates feeding and energy expenditures
by acting at sites primarily within the central
nervous system (2– 4). Obesity in humans and
rodents is almost always associated with a resistance to,
rather than a deficiency of, leptin (5–7). Resistance is
associated with impaired transporter, receptor, postrecep-
tor, and downstream neuronal circuitry functions in ani-
mal models of obesity (9 –13). Leptin is transported across
the blood-brain barrier (BBB) by a saturable transporter
(8), and impaired transport can be acquired, may precede
receptor/postreceptor defects, worsens with increasing
obesity, and is to some extent reversible (14 –16). The
relation between cerebrospinal fluid and serum levels of
leptin in obese humans (17,18) suggests that defective
BBB transport accounts for more of the overall resistance
to leptin than the receptor/postreceptor defects (19).
The obesity-related defect in leptin BBB transport has
two aspects (10). First, circulating substances cause an
immediate impairment. Leptin itself, which is elevated in
obesity, is likely one of these circulating substances.
Second, an unidentified mechanism impairs transport in
obese mice even when BBB transport is assessed by brain
perfusion, a method that removes the immediate effects of
blood-borne substances. Fasting or leptin administration
can partially reverse these defects in leptin transport (16).
Starvation, like obesity, is accompanied by a decreased
BBB transport rate of exogenous leptin (20). Whereas it is
difficult to explain the evolutionary advantage of de-
creased leptin transport in obesity, an advantage is obvi-
ous in starvation. Decreasing the amount of the anorectic
protein reaching the central nervous system should en-
hance the drive for seeking food. The mechanism of the
starvation-induced impairment in transport is unknown
but cannot be caused by leptin itself because its levels
decrease with fasting (21).
Here, we postulate that triglycerides may underlie the
impairment in BBB transport in both obesity and starva-
tion. Triglycerides are decreased with fasting but are
elevated with starvation and tend to be elevated with
obesity. Supporting this hypothesis is the observation that
mice with impaired triglyceride synthesis are protected
against development of both diet-induced obesity and
obesity-induced leptin resistance (22). Thus, hypertriglyc-
eridemia could explain impaired transport of leptin across
the BBB in both starvation and obesity.
RESEARCH DESIGN AND METHODS
Radioactive labeling of leptin. Mouse recombinant leptin (a gift from
Amgen, Thousand Oaks, CA) was radioactively labeled with
131
I (Amersham
Pharmacia, Piscataway, NJ) by the lactoperoxidase method, and the I-Lep was
purified on a column of G-10 Sephadex. Specific activity was ⬃100 –125 Ci/g.
Measurement of leptin transport across the BBB in mice. All studies
were approved by the local animal care and use committee, were performed
in an Association for Assessment and Accreditation of Laboratory Animal
Care–approved facility, and used adult male CD-1 mice from our colony. Mice
were anesthetized with urethane (4.0 g/kg i.p.), and the left jugular vein and
right carotid artery were exposed. A total of 0.2 ml of Ringer’s lactated
solution (LR) with 1% BSA containing 10
6
cpm of I-Lep was injected into the
jugular vein. Blood was collected from the carotid artery, and the whole brain
was removed 10 min after the jugular injection, a time when the radioactivity
represents intact I-Lep (8). Blood was centrifuged at 5,000gfor 10 min at 4°C,
and the serum was collected. The whole brain was cleaned of large vessels
From the
1
Department of Internal Medicine, Division of Geriatrics, Geriatric
Research, Education, and Clinical Center, Veterans Affairs Medical Center, St.
Louis University School of Medicine, St. Louis, Missouri; and the
2
Department
of Pharmacology, Nagasaki University School of Medicine, Nagasaki, Japan.
Address correspondence and reprint requests to William A. Banks, 915 N.
Grand Blvd., St. Louis, MO 63106. E-mail: bankswa@slu.edu.
Received for publication 30 June 2003 and accepted in revised form 3
February 2004.
BBB, blood-brain barrier; DMOG, 1,2-dimyristoyl-3-oleoyl-rac-glycerol;
DPOG, 1,2-dipalmitoyl-3-oleoyl-rac-glycerol; DSOG, 1,2-distearoyl-3-oleoyl-
rac-glycerol; FFA, free fatty acid; LR, Ringer’s lactated solution; MBEC, mouse
brain endothelial cell.
© 2004 by the American Diabetes Association.
DIABETES, VOL. 53, MAY 2004 1253
and weighed after discarding the pituitary and pineal gland. Levels of
radioactivity in brain and serum were measured in a ␥-counter, and brain/
serum ratios (l/g) were calculated.
Mouse brain perfusion studies. Mice were anesthetized with urethane (4.0
g/kg i.p.). The thorax was opened, the heart was exposed, both jugulars were
severed, and the descending thoracic aorta was clamped. A 26-gauge butterfly
needle was inserted into the left ventricle of the heart, and the buffer of
Zlokovic et al. (23) containing I-Lep [2(10)
6
cpm/ml] was infused at a rate of
2 ml/min for 5 min (24). The exact counts per minute infused was determined
on a 10-l aliquot of perfusion fluid. After perfusion, the vascular space of the
brain was washed out by injecting 20 ml LR in ⬍1 min through the left
ventricle of the heart. The brain was removed as above, and brain/perfusion
ratios were calculated. In other mice, 10% whole milk or 10% intralipid was
included in the perfusate, and brains were collected after 1–5 min of perfusion
without vascular washout. The unidirectional influx rate (K
i
) was calculated
by regressing the brain/perfusion ratio against perfusion time.
Mouse brain endothelial cell monolayers. The protocol for isolating
mouse brain endothelial cells (MBECs) was modified from that for rat brain
endothelial cells (25–27). Brains from anesthetized CD-1 mice were cleaned of
meninges and homogenized with a handheld scalpel. The homogenate was
digested in a collagenase solution (1 mg/ml collagenase type 2 in 288 units/ml
of DNase I; Sigma, St. Louis, MO) at 37°C for 1 h. Neurons, astrocytes, and
Schwann cells were removed by centrifuging in Dulbecco’s modified Eagle’s
medium solution (Sigma) containing 20% BSA. The partially purified mixture
was digested again (1 mg/ml collagenase/dispase with 288 units/ml DNase I at
37°C for 30 min). Finally, the endothelial cells were purified on a 33% Percoll
gradient (Amersham Biosciences) centrifuged at 1,000gfor 10 min.
The MBECs were placed in culture dishes (Falcon) coated with 0.1 mg/ml
collagen type 1 (Sigma) and 0.1 mg/ml fibronectin (Sigma) and incubated at
37°C with 5% CO
2
(27,28) in endothelial cell culture medium (20% plasma-
derived serum [Quad Five, Ryegate, MT] containing 1 ng/ml basic fibroblast
growth factor [Sigma] and Dulbecco’s modified Eagle’s medium) (25,29). Cell
culture medium was changed every 2–3 days. MBECs were typically 70 –80%
confluent by day 7.
MBECs (4.0 ⫻10
4
cells/insert) cultured to 70 –80% confluence were added
to Transwell culture inserts (Coster, 24-well format, 3470) and cultured for 3
more days. Transwells had a culture plate (abluminal side) volume of 0.6 ml,
an insert volume of 0.1 ml, and polyester membrane pores of 0.4 m.
Transendothelial electrical resistance was used to confirm confluence of
monolayers on the day of study. I-Lep was added to the luminal chamber with
or without 10% milk. The abluminal chamber was sampled 1, 2.5, and 5 min
after adding the I-Lep. The percentage of material transported per minute
(PMT/min) was calculated with the following (30):
PMT/min ⫽100共cpm in luminal sample兲/关共cpm in abluminal chamber兲/t]
where tis time in minutes. The “cpm in abluminal chamber”was corrected by
subtracting the cpm that had entered the luminal chamber and adding the cpm
present in the MBEC inserts at the end of the experiment.
Measurement of serum leptin levels. The murine leptin radioimmunoassay
used (Linco, St. Charles, MO) has a 50% cross-reactivity with rat leptin and no
cross-reactivity with human or bovine leptin.
Administration of milk and intralipid. A total of 2.5 ml LR, whole milk, or
intralipid (Pharmacia & UpJohn, Peapack, NJ) was injected intraperitoneally
into CD-1 mice. I-Lep (10
6
cpm in 0.2 ml LR) was injected 0.5–24 h later into
the right jugular vein. Brain/serum ratios were calculated 10 min after
intravenous injection of I-Lep as described above. In other mice, the uptake by
brain of intravenous I-Lep was measured 4 h after the intraperitoneal injection
of fat-free milk, whole milk, intralipid, or LR. In other mice, 0.2 ml fat-free
milk, whole milk, intralipid, or LR containing 10
6
cpm of I-Lep was given
intravenously 10 min before harvesting brain and blood.
Preparation of triglyceride and free fatty acid emulsions. Triglycerides
or free fatty acid (FFA) and L-␣-phosphatidylcholine (all from Sigma) were
each dissolved in chloroform, mixed, and dried under a stream of nitrogen gas.
Zlokovic’s buffer was added, and the material was vigorously mixed, homog-
enized, and alternatively frozen in liquid nitrogen and thawed in a warm water
bath for 12 cycles. Material was either immediately diluted to the desired
concentration with LR containing I-Lep and injected intravenously or stored at
⫺20°C for use within 48 h. Oleate was purchased in liquid form, dried, and
used immediately after dissolving in chloroform. On the day of study,
anesthetized mice received intravenous injections of 0.2 ml LR containing 1%
BSA and 10
6
cpm I-Lep with or without an FFA or triglyceride emulsion.
Carotid artery blood and brains were obtained 10 min later, and results were
expressed as brain/serum ratios.
Administration of gemfibrozil. Mice were weighed and fed 1 ml/kg vegeta-
ble oil with or without gemfibrozil (1 g/kg) twice per day for 5 days. On the
morning after the day of the last dose, mice were anesthetized and given
intravenous I-Lep, and brain and arterial blood was collected 10 min later as
described above. Triglyceride levels were measured on arterial serum with a
kit (Sigma).
Diet-induced obesity. Mice were weighed and kept on regular food (4.5% fat,
5001 Rodent Diet; PMI Nutrition International, Brentwood, MO) or switched to
breeder food (10% fat, Teklad Mouse Breeder Diet; Harlan Teklad, Madison,
WI) for an average of 17 weeks. Brain and blood samples were collected 10
min after intravenous I-Lep as described above. Triglyceride levels were
measured on serum. This experiment was repeated, except that each dietary
group was randomized into 16-h fasted and nonfasted groups.
Statistical analysis. Means are reported with their standard errors and n.
Two groups were compared by a ttest. More than two groups were compared
by ANOVA followed by a Newman-Keuls post hoc test. Regression lines were
computed by the least-squares method, and their slopes were compared with
the software package in Prism 4.0 (GraphPad, San Diego, CA).
RESULTS
Starvation for 48 h impaired transport of intravenously
administered I-Lep, decreasing brain/serum ratios from
19.1 ⫾1.28 l/g (n⫽10) to 15.1 ⫾1.01 l/g (n⫽8) (Fig.
1A;P⬍0.05). About 10 l/g of the brain/serum ratio
represents vascular space, so the decrease in I-Lep uptake
was from ⬃9.1 to 5.1, or ⬃44%. In contrast to intravenous
injection results, brain perfusion found no difference in
leptin transport between fed and 48-h starved mice (Fig.
1B). Starvation for 48 h increased serum triglyceride levels
(165 ⫾9 mg/dl, n⫽5) in comparison to nonfasted (135 ⫾
10, n⫽6, P⬍0.05) or 16-h fasted (96 ⫾7, n⫽7, P⬍
0.001) mice (Fig. 1C).
The effect of triglycerides on I-Lep transport was tested
by injecting bovine whole milk (98% of fat content being
triglycerides) or intralipid, a source of plant triglycerides
and FFA, into the peritoneal cavity. Brain/serum ratios are
expressed relative to the time-matched mice injected with
LR (Fig. 2A,n⫽5–6 mice/point). Milk produced an
immediate long-lasting impairment in leptin transport,
whereas intralipid had no statistically significant effect.
Two-way ANOVA showed an effect for treatment
[F(2,69) ⫽30.5, P⬍0.001], time [F(6,69) ⫽5.62, P⬍
0.001], and interaction [F(12,69) ⫽2.86, P⬍0.005].
Newman-Keuls post hoc test showed that the 30-min, 2-h,
and 4-h values for milk differed from the time-matched
controls (all at P⬍0.01). Two-way ANOVA showed that
serum triglyceride levels (Fig. 2B) were elevated after
intraperitoneal injection of milk with significant effects for
treatment [F(1,55) ⫽21.4, P⬍0.0001] and time [F(1,55) ⫽
6.26, P⬍0.0005] but not interaction. For serum leptin
levels, two-way ANOVA showed a significant effect for
treatment [F(1,56) ⫽10.1, P⬍0.005], a trend for time
[F(1,56) ⫽2.25, P⫽0.07], and no effect for interaction
(Fig. 2C). Newman-Keuls post hoc test found no time-
matched differences between mice treated with milk ver-
sus LR for either triglycerides or leptin.
Brain perfusion found that whole milk, but not in-
tralipid, inhibited the transport of I-Lep across the BBB
(Fig. 3). Transport rates for I-Lep perfused in buffer (K
i
⫽
2.74 ⫾0.79 l䡠g
⫺1
䡠min
⫺1
,n⫽18, r⫽0.655, P⬍0.005)
or intralipid (K
i
⫽2.29 ⫾0.85 l䡠g
⫺1
䡠min
⫺1
,n⫽13, r⫽
0.631, P⬍0.05) were not different. K
i
for I-Lep in milk was
not measurable (n⫽18, r⫽0.217, P⬎0.4) and differed
from buffer [F(1,31) ⫽6.64, P⬍0.05] or intralipid
[F(1,26) ⫽4.51, P⬍0.05] perfusions.
Whole milk inhibited leptin transport across MBEC
monolayers (Fig. 4), decreasing the percentage of material
TRIGLYCERIDES, LEPTIN RESISTANCE, AND THE BBB
1254 DIABETES, VOL. 53, MAY 2004
transported per minute from (3.42 ⫾0.085)10
⫺4
to (3.05 ⫾
0.06)10
⫺4
(n⫽24/group, P⬍0.001).
Nonfat milk was compared with whole milk and in-
tralipid by injecting animals intraperitoneally 4 h before
the intravenous injection of I-Lep or by coinjecting them
with the intravenous I-Lep. Only whole milk had an effect
on I-Lep uptake (Fig. 5).
Triglycerides (7.2 mg/mouse, an amount equal to the
total triglyceride content in 0.2 ml milk) were included in
the intravenous injection of I-Lep given 10 min before
collection of brain and blood. Three triglycerides (triolein,
1,2-dipalmitoyl-3-oleoyl-rac-glycerol [DPOG], and 1,2-dis-
tearoyl-3-oleoyl-rac-glycerol [DSOG]) each inhibited I-Lep
transport (all P⬍0.001), but 1,2-dimyristoyl-3-oleoyl-rac-
glycerol (DMOG) had no effect (Fig. 6A). An inverse linear
relation existed between the log dose of intravenous
triolein and brain uptake of I-Lep [Fig. 6B,n⫽7–8
mice/dose, Y⫽72.3 ⫺12.0X,r⫽(⫺0.827), n⫽7, P⬍
0.05].
The FFA derivable form triolein, DPOG, and DSOG were
tested for their ability to inhibit I-Lep transport (Fig. 6A).
Doses tested of palmitate (0.4 mg/mouse), stearate (0.4
mg/mouse), and oleate (0.72 mg/mouse) were those esti-
mated to produce a level in blood 10 –20 times higher than
the level seen for FFAs in starvation. FFAs were injected
intravenously with I-Lep, and brain and serum samples
were collected 10 min later. These FFAs had no effect on
leptin transport. Oleate tested at the dose of 7.2 mg/mouse,
the dose at which triolein was effective, was without
effect.
Obesity was induced by placing male CD-1 mice on
breeder food (10% fat, Teklad Mouse Breeder Diet; Harlan
FIG. 1. Starvation-induced inhibition of leptin transport across the
BBB is caused by a circulating factor. A: Replication of classic work
(20) showing that the leptin transporter is inhibited with starvation. B:
With brain perfusion, a method that negates the effect of circulating
substances, the transporter returns to normal. C: Triglyceride levels in
serum in control mice, mice fasted for 16 h, and mice fasted for 48 h. IV,
intravenous. *P<0.05; **P<0.01.
FIG. 2. Leptin resistance to BBB transport is induced by milk fats. A:
Intraperitoneal injection of whole milk, but not intralipid, induced an
impairment in leptin transport across the BBB. Intraperitoneal injec-
tion of milk increased serum triglycerides (B) and serum leptin levels
(C)by⬃40%. **P<0.01.
W.A. BANKS AND ASSOCIATES
DIABETES, VOL. 53, MAY 2004 1255
Teklad) for 17 weeks and comparing them to littermates
left on a regular food (4.5% fat, 5001 Rodent Diet; PMI
Nutrition International). Mice fed breeder food weighed
⬃44% more than mice fed regular food. In the initial study
(experiment 1), 16 mice were used per group. This study
was repeated (experiment 2) with half the mice fasted for
16h(n⫽8/group). With the high-fat diet, serum triglyc-
erides increased and leptin transport decreased. Fasting
decreased serum triglycerides and increased leptin trans-
port in both mice fed breeder food and mice fed regular
food (Fig. 7). Triglyceride levels and leptin transport were
inversely related among these groups: Y⫽22.6 ⫺0.044X,
r⫽⫺0.860, P⬍0.05).
Short-term administration of gemfibrozil reduced tri-
glyceride levels to ⬍100 mg/dl in four of six mice fed
regular food (Fig. 8A,n⫽7 control, n⫽6 vehicle P⬍
0.05). These four mice had a statistically significant in-
crease in leptin transport in comparison to mice fed
vehicle only (Fig. 8B,P⬍0.05). A statistically significant
correlation existed between the means for serum triglyc-
erides and brain/serum ratios for leptin (Fig. 8C;r⫽1.0,
n⫽3, P⬍0.01). A decrease in body weight seen in these
four mice was not statistically significant (Fig. 8D).
DISCUSSION
Obesity is associated with leptin resistance caused by
impaired leptin transport across the BBB, defects in
leptin-receptor signaling, and blockades in downstream
neuronal circuitries. The inability of obese mice to re-
spond to peripherally administered leptin while respond-
ing to centrally administered leptin is likely caused by a
defect in leptin transport across the BBB. It is unclear
what causes defective transport of leptin in either obesity
or starvation. Because serum triglycerides are elevated in
both starvation and obesity, we postulated that triglycer-
ides inhibit leptin transport across the BBB. Here, we
showed that starvation-induced inhibition of leptin trans-
port was caused by a circulating factor; that the fat
component of milk (which is 98% triglycerides) as well as
specific triglycerides could induce inhibition of leptin
transport across the BBB in vivo, in situ, and in vitro; that
the FFAs comprising those triglycerides were ineffectual;
that manipulation of triglyceride levels with diet or fasting
in normal or obese mice had an inverse effect on leptin
transport; and that reduction of triglycerides by pharma-
cological intervention reversed the impairment in leptin
transport. Taken together, these findings show that trig-
lycerides directly inhibit the transport of leptin across the
BBB and so could be a major cause of leptin resistance at
the BBB.
We fasted mice for 48 h to determine whether starvation
impairs the transport of intravenously administered I-Lep.
We confirmed that short-term fasting decreased serum
triglyceride levels, whereas 48 h of fasting (starvation)
increased them. The level of reduction in the brain uptake
of intravenous administered I-Lep was almost identical to
the results found by others (20). That group further
showed that longer fasts progressively impaired leptin
transport across the BBB to the point of total inhibition
after 5 days of starvation. This inhibition of leptin trans-
port and the accompanying decrease in levels of leptin in
the blood (21) are likely adaptive because they would
reduce the anorectic signal in starvation.
In contrast to intravenous injection, brain perfusion
FIG. 3. Brain perfusion with intralipid and milk. The slope of the
relation between brain/perfusion ratios and time measures K
i
for
I-Lep. Intralipid had no effect on BBB transport, but whole milk
prevented any measurable transport.
FIG. 4. In vitro assessment of milk in a monolayer model of the BBB
using cultured mouse brain endothelial cells. Addition of milk to the
buffer inhibited transport of leptin. PMT, percentage of material
transported. **P<0.001.
FIG. 5. Intraperitoneal (IP) and intravenous (IV) milk fat inhibits
leptin transport. With IP injection, fat-free milk and intralipids were
ineffective, whereas whole milk inhibited leptin transport. Milk given
intravenously (0.2 ml milk administered intravenously with the I-Lep,
with brain and serum samples collected 10 min later) was immediately
effective in reducing I-Lep transport, whereas fat-free milk and in-
tralipid were not. *P<0.05; **P<0.01.
TRIGLYCERIDES, LEPTIN RESISTANCE, AND THE BBB
1256 DIABETES, VOL. 53, MAY 2004
found no difference in leptin uptake between starved and
fed mice. Because the intravenous and brain perfusion
methods usually give similar results except when a circu-
lating factor has an acutely reversible effect on transport
(31–33), the results show that starvation inhibits leptin
transport by releasing a blood-borne factor.
We more directly tested triglycerides by injecting bovine
whole milk or intralipid into the peritoneal cavity. The fat
in milk is 98% triglycerides (34), whereas the fat in
intralipid is a soybean oil–based source of triglycerides
containing the essential FFAs linolenic and linoleic acid,
purified egg phospholipids, and glycerol. Milk increased
serum triglyceride and leptin levels by ⬃40% and produced
an immediate long-lasting impairment in leptin transport
across the BBB. Serum triglycerides showed a time-depen-
dent decline during the course of the study in both milk-
and vehicle-injected animals, probably related to diurnal
rhythm. However, at those times when leptin transport
was inhibited, serum triglycerides in milk-injected animals
were higher than the vehicle-injected animals’highest
value (baseline). The increase in serum leptin levels was
likely produced by the mouse because pasteurization
significantly reduces leptin levels in milk (35), and our
immunoassay was species specific. The increase in serum
leptin from ⬃4.5 to 6.5 ng/ml during the period of leptin
inhibition is likely too low to explain the inhibition in
leptin transport. Previous work shows that this would
result in only about a 10% decrease in the leptin transport
rate (33). Additionally, leptin levels were highest at 6 h, a
time when transport was no longer significantly inhibited.
In fact, the 6-h serum leptin level for vehicle-treated mice
was a little higher than the serum leptin level in milk-
treated mice at 2 h, the time of greatest inhibition in leptin
transport. Milk also inhibited leptin transport in the in situ
brain perfusion model and in the in vitro brain monolayer
model of the BBB, conditions where leptin secretion could
not occur.
In comparison to milk, intralipid was without effect,
suggesting that plant triglycerides and essential FFAs do
not inhibit leptin transport. Milk given intravenously was
immediately effective at less than one-tenth the intraperi-
toneal dose. The immediacy of effect after intravenous
injection suggests that triglycerides rather than a degrada-
tion product (e.g., FFAs) affected transport. Nonfat milk,
which contains the same concentration of proteins and
phospholipids as whole milk and has only the triglycerides
removed (34), was without effect. These results show that
inhibition was not caused by leptin remaining in pasteur-
ized milk (35,36). They also show that animal-derived
triglycerides impair leptin transport across the BBB, but
not essential FFAs, plant-derived triglycerides, or milk
proteins.
We directly tested the ability of triglycerides to inhibit
leptin transport across the BBB. Three of four commer-
cially available triglycerides (triolein, DPOG, and DSOG)
inhibited uptake of I-Lep when injected intravenously at a
dose that equaled the total triglyceride content of milk
FIG. 6. Triglycerides and leptin transport. A: Triglycerides but not
FFAs induce resistance to leptin transport across the BBB. Triglycer-
ides or FFAs were given intravenously with the I-Lep and brain and
blood samples collected 10 min later. Three triglycerides (triolein,
DPOG, and DSOG) inhibited leptin transport across the BBB. DMOG
was ineffective in mice. Triglycerides were given at a dose of 7.2
mg/mouse to replicate the dose of total triglycerides in a volume of 0.2
ml whole milk. Palmitate (0.4 mg/mouse), stearate (0.4 mg/mouse), and
oleate (0.72 mg/mouse) were ineffective at doses calculated to produce
blood levels exceeding those seen in starvation. Oleate was also
ineffective at the doses at which triolein was effective (7.2 mg/mouse).
**P<0.01. B: Triolein has a dose-dependent effect on I-Lep transport.
Triolein and I-Lep were injected together, and brain and blood samples
were collected 10 min later.
FIG. 7. Effect of high-fat (breeder food) diet and 16 h of fasting on
triglyceride levels and leptin transport. I-Lep was injected intrave-
nously, and brain and blood samples were collected 10 min later.
Triglyceride levels were measured in serum. The relation between
triglyceride levels and brain/serum ratios was statistically significant
(rⴝⴚ0.860, nⴝ6, P<0.05). The regular and breeder groups were
tested twice: first as the initial experiment (experiment 1) and again as
controls to the fasting groups (experiment 2).
W.A. BANKS AND ASSOCIATES
DIABETES, VOL. 53, MAY 2004 1257
(Fig. 6A). A dose-response curve suggests that, at least in
the case of triolein, lower doses are also effective. DMOG,
the triglyceride that did not inhibit leptin transport, illus-
trates that the sn-1 position is important for the inhibitory
effect. Myristate, as a medium-chain FFA, is only produced
in by mammary alveolar cells; therefore, triglycerides
containing it may not reflect diet or obesity (37). Addition-
ally, it would not be expected to circulate in significant
amounts in blood. These results suggest that leptin trans-
port will be inhibited by triglycerides endogenous to
blood.
We ruled out the possibility that FFAs hydrolyzed from
the triglycerides were inhibiting leptin transport. The
FFAs (palmitate, stearate, and oleate) that could be hydro-
lyzed from the triglycerides were without effect at doses
that would have produced blood levels higher than those
seen in starvation. We also tested oleate at the same dose
as triolein, but found it was without effect. Because the
molecular weight of triolein is only ⬃75% fatty acid with
the remainder comprised of the glycerol backbone, we
tested oleate at a molarity at least 30% higher than could
be achieved with total hydrolyzation of triolein. This
shows that the triglycerides themselves and not the FFAs
derived from them are responsible for inhibiting leptin
transport.
We tested the pathophysiological relevance of hypertri-
glyceridemia by studying the effects of dietary-induced
obesity on the relation between triglycerides and I-Lep
uptake. We also tested the ability of a 16-h fast to affect the
relation between triglycerides and I-Lep uptake in these
groups of mice. As Fig. 7 illustrates, diet-induced obesity
increased triglycerides and reduced I-Lep uptake by the
brain. In both lean and diet-induced obese mice, fasting
reduced triglycerides and increased I-Lep uptake by the
brain.
Gemfibrozil is selective for reduction of serum triglyc-
eride levels and is used clinically for the treatment of
hypertriglyceridemia. Short-term administration of gemfi-
brozil reduced triglyceride levels to ⬍100 mg/dl in four of
six mice (Fig. 8). These responders had a statistically
significant increase in leptin transport in comparison to
mice fed vehicle (P⬍0.05). This showed that reduction of
triglyceride levels by pharmacological treatment could
enhance leptin transport across the BBB.
These results show that serum triglycerides have a rapid
and immediate effect on the transport of leptin. As such,
they explain the inhibition in leptin transport seen with
starvation. They also likely contribute to the inhibition
seen with obesity. Triglycerides could produce their effect
on leptin transport by binding leptin in the circulation or
by acting directly on the leptin transporter. Other BBB
transporters are known to be regulated by uncompetitive
and noncompetitive mechanisms (38,39), and leptin trans-
port is altered by ␣
1
-adrenergic agonists, glucose, and
insulin (40,41). It may be that the leptin transporter
possesses a regulatory site controlled by triglycerides.
The ability of triglycerides to inhibit leptin transport into
the brain completes a negative feedback loop between
leptin action and triglycerides. Leptin promotes triglycer-
ide hydrolysis and FFA oxidation and inhibits FFA synthe-
sis (42,43,44), therefore decreasing triglyceride levels.
The importance of leptin in reducing triglyceride levels
FIG. 8. Effect of gemfibrozil on serum triglyceride levels and leptin transport. I-Lep was injected intravenously, brain and blood samples were
collected 10 min later, and triglyceride levels were measured in serum. A: In comparison to mice that received the vegetable oil vehicle only,
gemfibrozil responders (four of six treated mice) had lower triglyceride levels (**P <0.01). B: Gemfibrozil responders had higher leptin
transport rates than vehicle (*P <0.05) or control (*P <0.05) mice. C: A significant correlation (P<0.01) existed between brain/serum ratios
for leptin and serum triglyceride levels for the three groups. D: The weight loss for the gemfibrozil group was not statistically significant.
TRIGLYCERIDES, LEPTIN RESISTANCE, AND THE BBB
1258 DIABETES, VOL. 53, MAY 2004
is dramatically illustrated in patients with lipodystrophy
and lipoatrophy. These patients have little or no fat mass
and, as a result, have little or no leptin. They also have very
severe hypertriglyceridemia that is reversed by treatment
with leptin (45). The ability of triglycerides to induce
leptin resistance would counter the leptin-induced shift
toward use of triglycerides as an energy source and so
help to conserve fat stores. This would make evolutionary
sense because hypertriglyceridemia has probably more
often represented starvation than obesity. Healthy ba-
boons living in the wild have fat stores and serum leptin
levels that are a fraction of those seen in Western humans
and laboratory animals (46), but when supplied with
abundant calories, develop a condition resembling the
metabolic syndrome X (47), including the development of
hyperlipidemia (48). These studies in wild baboons are
consistent with the hypothesis that ancestral levels of
leptin were much lower than those seen in Western
civilization and that starvation was a more probable threat
than obesity.
The usefulness of leptin resistance in obesity is less
clear than its obvious utility in starvation. Starvation-
induced hypertriglyceridemia may have been so dominant
an evolutionary pressure that leptin resistance induced by
obesity-related hypertriglyceridemia was never selected
against. Alternatively, it may be that the anorectic effect of
leptin must be overridden to maintain an adequate intake
of water-soluble vitamins, minerals, electrolytes, and other
substances less efficiently stored than fat.
Our results also provide a mechanism to explain previ-
ous findings of why mice unable to synthesize triglycerides
are more sensitive to leptin (22). Mice lacking acyl coen-
zyme A:diacylglycerol acyltransferase 1, a critical enzyme
needed to synthesize triglycerides, are more sensitive to
infusions of leptin. Without this enzyme, obesity did not
develop in a strain of mice normally resistant to leptin but
did in a strain that is leptin deficient. This shows that leptin
is critical to the mechanism by which lack of triglycerides
protects from diet-induced obesity. The results presented
here provide one mechanism by which lowering triglycer-
ides can increase leptin sensitivity.
In conclusion, these studies show that serum triglycer-
ides impair the ability of the BBB to transport leptin.
Triglycerides are likely a major cause of the leptin resis-
tance seen in both starvation and obesity (9,10,16,20).
Lowering triglycerides may be therapeutically useful in
enhancing the effects of leptin on weight loss.
ACKNOWLEDGMENTS
This study was supported by VA Merit Review R01
N541863 and RO1 AA12743.
The authors thank Dr. Harold M. Farrell, Jr., Agricultural
Research Service, U.S. Department of Agriculture, for help
in determining the fat content of milk.
REFERENCES
1. Zhang Y, Proenca R, Maffel M, Barone M, Leopold L, Friedman JM:
Positional cloning of the mouse obese gene and its human homologue.
Nature 372:425–432, 1994
2. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D,
Lallone RL, Burley SK, Friedman JM: Weight-reducing effects of the plasma
protein encoded by the obese gene. Science 269:543–546, 1995
3. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P: Recombinant mouse
OB protein: evidence for a peripheral signal linking adiposity and central
neural networks. Science 269:546 –549, 1995
4. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T,
Collins F: Effects of the obese gene product on body weight regulation in
ob/ob mice. Science 269:540 –543, 1995
5. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce
MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL, Caro JF: Serum
immunoreactive-leptin concentrations in normal-weight and obese hu-
mans. N Engl J Med 334:292–295, 1996
6. Pelleymounter MA, Cullen MJ, Healy D, Hecht R, Winters D, McCaleb M:
Efficacy of exogenous recombinant murine leptin in lean and obese 10- to
12-mo-old female CD-1 mice. Am J Physiol 275:R950 –R959, 1998
7. Heymsfield SB, Greenberg AS, Fujioka K, Dixon RM, Kushner R, Hunt T,
Lubina JA, Patane J, Self B, Hunt P, McCamish M: Recombinant leptin for
weight loss in obese and lean adults: a randomized, controlled, dose-
escalation trial. JAMA 282:1568 –1575, 1999
8. Banks WA, Kastin AJ, Huang W, Jaspan JB, Maness LM: Leptin enters the
brain by a saturable system independent of insulin. Peptides 17:305–311,
1996
9. Hileman SM, Pierroz DD, Masuzaki H, Bjorbaek C, El Haschimi K, Banks
WA, Flier JS: Characterization of short isoforms of the leptin receptor in
rat cerebral microvessels and of brain uptake of leptin in mouse models of
obesity. Endocrinology 143:775–783, 2002
10. Banks WA, DiPalma CR, Farrell CL: Impaired transport of leptin across the
blood-brain barrier in obesity. Peptides 20:1341–1345, 1999
11. El Haschimi K, Pierroz DD, Hileman SM, Bjorbaek C, Flier JS: Two defects
contribute to hypothalamic leptin resistance in mice with diet-induced
obestiy. J Clin Invest 105:1827–1832, 2000
12. Yamashita T, Murakami T, Iida M, Kuwajima M, Shima K: Leptin receptor
of Zucker fatty rat performs reduced signal transduction. Diabetes 46:
1077–1080, 1997
13. Wilsey J, Zolotukhin S, Prima V, Scarpace PJ: Central leptin gene therapy
fails to overcome leptin resistance associated with diet-induced obesity.
Am J Physiol 285:R1011–R1020, 2003
14. van Heek M, Compton DS, France CF, Tedesco RP, Fawzi AB, Graziano
MP, Sybertz EJ, Strader CD, Davis J: Diet-induced obese mice develop
peripheral, but not central, resistance to leptin. J Clin Invest 99:385–390,
1997
15. Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM:
Physiological response to long-term peripheral and central leptin infusion
in lean and obese mice. Proc Natl Acad SciUSA94:8878 –8883, 1997
16. Banks WA, Farrell CL: Impaired transport of leptin across the blood-brain
barrier in obesity is acquired and reversible. Am J Physiology 285:E10 –
E15, 2003
17. Caro JF, Kolaczynski JW, Nyce MR, Ohannesian JP, Opentanova I,
Goldman WH, Lynn RB, Zhang PL, Sinha MD, Considine RV: Decreased
cerebrospinal-fluid/serum leptin ratio in obesity: a possible mechanism for
leptin resistance. Lancet 348:159 –161, 1996
18. Schwartz MW, Peskind E, Raskind M, Boyko EJ, Porte D Jr: Cerebrospinal
fluid leptin levels: relationship to plasma levels and adiposity in humans.
Nat Med 2:589 –593, 1996
19. Banks WA: Is obesity a disease of the blood-brain barrier? Physiological,
pathological, and evolutionary considerations. Current Pharmaceutical
Design 9:801–809, 2003
20. Kastin AJ, Akerstrom V: Fasting, but not adrenalectomy, reduces transport
of leptin into the brain. Peptides 21:679 –682, 2000
21. Kolaczynski JW, Considine RV, Ohannesian J, Marco C, Opentanova I,
Nyce MR, Myint M, Caro JF: Responses of leptin to short-term fasting and
refeeding in humans: a link with ketogenesis but not ketones themselves.
Diabetes 45:1511–1515, 1996
22. Chen HC, Smith SJ, Ladha Z, Jensen DR, Ferreira LD, Pulawa LK, McGuire
JG, Pitas RE, Eckel RH, Farese RV Jr: Increased insulin and leptin
sensitivity in mice lacking acyl CoA:diacylglycerol acyltransferase 1. J Clin
Invest 109:1049 –1055, 2002
23. Zlokovic BV, Lipovac MN, Begley DJ, Davson H, Rakic L: Slow penetration
of thyrotropin-releasing hormone across the blood-brain barrier of an in
situ perfused guinea pig brain. J Neurochem 51:252–257, 1988
24. Shayo M, McLay RN, Kastin AJ, Banks WA: The putative blood-brain
barrier transporter for the -amyloid binding protein apolipoprotein J is
saturated at physiological concentrations. Life Sci 60:L115–L118, 1996
25. Abbott NJ, Hughes CCW, Revest PA, Greenwood J: Development and
characterization of a rat brain capillary endothelial culture: towards an in
vitro blood-brain barrier. J Cell Sci 103:23–37, 1992
26. Deli MA, Joo F: Cultured vascular endothelial cells of the brain. Keio J Med
45:183–198, 1996
27. Kis B, Kaiya H, Nishi R, Deli MA, Abraham CS, Yanagita T, Isse T, Gotoh
W.A. BANKS AND ASSOCIATES
DIABETES, VOL. 53, MAY 2004 1259
S, Kobayashi H, Wada A, Niwa M, Kangawa K, Greenwood J, Yamashita H,
Ueta Y: Cerebral endothelial cells are a major source of adrenomedullin.
J Neuroendocrinology 14:283–293, 2002
28. Domotor E, Sipos I, Kittel A, Abbott NJ, Adam-Vizi V: Improved growth of
cultured brain microvascular endothelial cells on glass coated with a
biological matrix. Neurochem Int 33:473–478, 1998
29. Demeuse P, Kerkhofs A, Struys-Ponsar C, Knoops B, Remacle C, van den
Bosch de Aguilar P: Compartmentalized coculture of rat brain endothelial
cells and astrocytes: a syngenic model to study the blood-brain barrier.
J Neurosci Methods 121:21–31, 2002
30. Maresh GA, Maness LM, Zadina JE, Kastin AJ: In vitro demonstration of a
saturable transport system for leptin across the blood-brain barrier. Life
Sci 69:67–73, 2001
31. Smith QR, Momma S, Aoyagi M, Rapoport SI: Kinetics of neutral amino
acid transport across the blood-brain barrier. J Neurochem 49:1651–1658,
1987
32. Zlokovic BV, Begley DJ, Djuricic BM, Mitrovic DM: Measurement of solute
transport across the blood-brain barrier in the perfused guinea pig brain:
method and application to N-methyl-␣-aminoisobutyric acid. J Neurochem
46:1444 –1451, 1986
33. Banks WA, Clever CM, Farrell CL: Partial saturation and regional variation
in the blood to brain transport of leptin in normal weight mice. Am J
Physiol 278:E1158 –E1165, 2000
34. Christie WW: Composition and structure of milk lipids. In Advanced Dairy
Chemistry. Volume. 2: Lipids. Fox PF, Ed. Chapman and Hall, 1994, p.
1–36
35. Resto M, O’Connor D, Leef K, Funanage V, Spear M, Locke R: Leptin levels
in preterm human breast milk and infant formula. Pediatrics 108:E15, 2001
36. Smith-Kirwin SM, O’Connor DM, De Johnston J, Lancey ED, Hassink SG,
Funanage VL: Leptin expression in human mammary epithelial cells and
breast milk. J Clin Endocrinol Metab 83:1810 –1813, 1998
37. Neville MC, Picciano MF: Regulation of milk lipid secretion and composi-
tion. Ann Rev Nutr 17:159 –184, 1997
38. Banks WA, Kastin AJ: Modulation of the carrier-mediated transport of the
Tyr-MIF-1 across the blood-brain barrier by essential amino acids. J Phar-
macol Exp Ther 239:668 –672, 1986
39. Banks WA, Kastin AJ, Fasold MB: Differential effect of aluminum on the
blood-brain barrier transport of peptides, technetium and albumin. J Phar-
macol Exp Ther 244:579 –585, 1988
40. Banks WA: Enhanced leptin transport across the blood-brain barrier by
␣1-adrenergic agents. Brain Res 899:209 –217, 2001
41. Kastin AJ, Akerstrom V: Glucose and insulin increase the transport of
leptin through the blood-brain barrier in normal mice but not in strepto-
zotocin-diabetic mice. Neuroendocrinology 73:237–242, 2001
42. Steinberg GR, Bonen A, Dyck DJ: Fatty acid oxidation and triacylglycerol
hydrolysis are enhanced after chronic leptin treatment in rats. Am J
Physiol 282:E593–E600, 2002
43. Reidy SP, Weber JM: Accelerated substrate cycling: a new energy-wasting
role for leptin in vivo. Am J Physiol 282:E312–E317, 2002
44. Matsuoka N, Ogawa Y, Masuzaki H, Ebihara K, Aizawa-Abe M, Satoh N,
Ishikawa E, Fujisawa Y, Kosaki A, Yamada K, Kuzuya H, Nakao K:
Decreased triglyceride-rich lipoproteins in transgenic skinny mice overex-
pressing leptin. Am J Physiol Endocrinol Metab 280:E334 –E339, 2001
45. Oral EA, Simha V, Ruiz E, Andewelt A, Premkumar A, Snell P, Wagner AJ,
DePaoli AM, Reitman ML, Taylor SI, Gorden P, Garg A: Leptin-replacement
therapy for lipodystrophy. N Engl J Med 346:570 –578, 2002
46. Banks WA, Phillips-Conroy JE, Jolly CJ, Morley JE: Serum leptin levels in
wild and captive populations of baboons (Papio): implications for the
ancestral role of leptin. J Clin Endocrinol Metab 86:4315–4320, 2001
47. Hansen BC, Saye J, Wennogle LP: The Metabolic Syndrome X: Conver-
gence of Insulin Resistance, Glucose Intolerance, Hypertension, Obesity,
and Dyslipidemia: Searching for the Underlying Defects. New York, New
York Academy of Sciences, 1999, p. 1–336
48. Banks WA, Altmann J, Sapolsky RM, Phillips-Conroy JE, Morley JE: Serum
leptin levels as a marker for a syndrome X-like condition in wild baboons.
J Clin Endocrinol Metab 88:1234 –1240, 2003
TRIGLYCERIDES, LEPTIN RESISTANCE, AND THE BBB
1260 DIABETES, VOL. 53, MAY 2004
