A Role for Hepatic Leptin Signaling in Lipid
Metabolism via Altered Very Low Density Lipoprotein
Composition and Liver Lipase Activity in Mice
Frank K. Huynh,1 Ursula H. Neumann,1 Ying Wang,2 Brian Rodrigues,2 Timothy J. Kieffer,1,3
and Scott D. Covey4
Obesity is highly associated with dyslipidemia and cardiovascular disease. However, the
mechanism behind this association is not completely understood. The hormone leptin
may be a molecular link between obesity and dysregulation of lipid metabolism. Leptin
can affect lipid metabolism independent of its well-known effects on food intake and
energy expenditure, but exactly how this occurs is ill-defined. We hypothesized that since
leptin receptors are found on the liver and the liver plays an integral role in regulating
lipid metabolism, leptin may affect lipid metabolism by acting directly on the liver. To test
this hypothesis, we generated mice with a hepatocyte-specific loss of leptin signaling. We
previously showed that these mice have increased insulin sensitivity and elevated levels of
liver triglycerides compared with controls. Here, we show that mice lacking hepatic leptin
signaling have decreased levels of plasma apolipoprotein B yet increased levels of very low
density lipoprotein (VLDL) triglycerides, suggesting alterations in triglyceride incorporation into VLDL or abnormal lipoprotein remodeling in the plasma. Indeed, lipoprotein
profiles revealed larger apolipoprotein B-containing lipoprotein particles in mice with
ablated liver leptin signaling. Loss of leptin signaling in the liver was also associated with a
substantial increase in lipoprotein lipase activity in the liver, which may have contributed
to increased lipid droplets in the liver. Conclusion: Lack of hepatic leptin signaling results
in increased lipid accumulation in the liver and larger, more triglyceride-rich VLDL particles. Collectively, these data reveal an interesting role for hepatic leptin signaling in modulating triglyceride metabolism. (HEPATOLOGY 2013;57:543-554)
D
espite the well-accepted link between obesity,
diabetes, and dyslipidemia, the molecular
mechanisms that drive this association are not
understood. The hormone leptin is a potential link
between obesity and abnormal lipid metabolism. Leptin is secreted from adipose tissue and acts on the
hypothalamus to reduce food intake and increase
energy expenditure.1,2 Thus, leptin-deficient ob/ob
mice and leptin receptor-deficient db/db mice are
hyperphagic and obese. However, these mice also display hypertriglyceridemia,3 hypercholesterolemia,3 hepatic steatosis,4 and impaired lipid tolerance.5 Several
studies suggest that these effects on lipid metabolism
are independent of leptin’s effects on food intake and
obesity. For example, restricting food intake in ob/ob
mice cannot improve lipid metabolism as effectively as
leptin treatment.6,7 In addition, lipodystrophic mice
and humans, which have little to no adipose tissue and
are hypoleptinemic, also display hyperlipidemia and
hepatic steatosis, and these symptoms are ameliorated
by leptin.8,9 Clearly, leptin has effects on lipid metabolism independent of its effects on body weight.
The manner by which leptin directly affects lipid
metabolism is not well understood. We hypothesized
Abbreviations: Ad-b-gal, adenovirus expressing b-galactosidase; Ad-Lepr-b, adenovirus expressing isoform b of the leptin receptor; apoB, apolipoprotein B; HL,
hepatic lipase; LPL, lipoprotein lipase; mRNA, messenger RNA; VLDL, very low density lipoprotein.
From the 1Departments of Cellular and Physiological Sciences and 4Biochemistry and Molecular Biology, Life Sciences Institute, the 2Molecular and Cellular
Pharmacology Group, Faculty of Pharmaceutical Sciences, and the 3Department of Surgery, University of British Columbia, Vancouver, British Columbia, Canada.
Received March 27, 2012; accepted August 13, 2012.
Supported by a grant from the Canadian Institutes of Health Research. T. J. K. received a Senior Scholarship from the Michael Smith Foundation for Health
Research. F. K. H. received scholarships from the Natural Sciences and Engineering Research Council of Canada and the Canadian Diabetes Association. U. H. N.
received the University of British Columbia Summer Student Research Program award sponsored by the Kinsley Brotherton McLeod Endowment and the Florence
& George Heighway Endowment Fund.
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that because the liver plays a role in lipid metabolism,
leptin acts directly on the liver to exert some of its metabolic effects. Indeed, leptin receptors are found in the
liver,10,11 and leptin administration to ob/ob mice elicits
many changes in the expression of genes involved with
lipid metabolism in the liver.6,8,12 Furthermore, leptin
treatment in ob/ob mice can reverse hepatic steatosis,7
potentially due to direct effects of leptin on the liver.13,14
To address the direct effects of leptin on the liver,
Cohen et al.15 knocked out leptin receptors specifically in
hepatocytes. Surprisingly, they found no accumulation of
hepatic lipids, but other aspects of lipid metabolism were
not explored. We also generated mice with a loss of hepatic leptin signaling wherein the leptin signaling domain
is removed specifically from hepatocytes.13 These mice
were protected from age- and diet-related glucose intolerance and had increased hepatic insulin sensitivity.13 Further, these mice had elevated liver triglyceride and cholesterol levels,13 indicating an alteration in hepatic lipid
metabolism. We have now discovered that mice lacking
hepatic leptin signaling have larger apolipoprotein B
(apoB)-containing lipoproteins and elevated triglyceride
levels in very low density lipoprotein (VLDL) particles.
This is accompanied by decreased plasma apoB, higher
lipoprotein lipase (LPL) activity in the liver, and lower
non-LPL activity compared with controls. Taken together, these data reveal a novel role for hepatic leptin signaling in regulating triglyceride metabolism.
Materials and Methods
Animals. Leprflox/flox AlbCre and Leprflox/flox AlbCre
ob/ob mice were generated as described.13,16 Leprflox/flox
AlbCre ob/ob mice were treated with 0.6 lg/day mouse
recombinant leptin (National Hormone and Peptide
Program, Torrance, CA) via mini-osmotic pumps
(Alzet, Palo Alto, CA). Db/db mice were treated intravenously with 1 109 pfu of an adenovirus expressing
either the long signaling isoform of the mouse leptin
receptor (Ad-Lepr-b) or b-galactosidase (Ad-b-gal) as a
control. Ob/ob mice were treated with 1.5 lg/g leptin
via intraperitoneal injections or 0.6 lg/day leptin via
miniosmotic pumps. Procedures were performed in
accordance with the University of British Columbia
Animal Care Committee guidelines.
HEPATOLOGY, February 2013
Triglyceride Secretion. Four-hour fasted mice were
injected intraperitoneally with 1 g/kg of poloxamer-407
(Sigma-Aldrich, Oakville, Ontario, Canada) followed by
an intraperitoneal injection of 0.6 U/kg or 0.725 U/kg
insulin (Novolin; Novo Nordisk, Mississauga, Ontario,
Canada). Plasma samples were taken throughout the
experiment for triglyceride measurements.
Plasma Analytes. Cholesterol levels were measured
using a Cholesterol E kit (Wako Chemicals USA, Richmond, VA), free fatty acids using a HR Series NEFAHR(2) kit (Wako Chemicals USA), triglycerides using a
Serum Triglyceride Determination kit (Sigma-Aldrich),
and insulin using a Ultrasensitive Mouse Insulin ELISA
(ALPCO Diagnostics, Salem, NH).
Lipoprotein Profiles. Equal volumes of plasma
from mice of the same genotype were pooled and 200
lL of the pooled plasma was applied to a Superose 6L
HR 10/30 column (GE Healthcare, Baie d’Urfe, Quebec, Canada) with 154 mM NaCl, 1 mM ethylene
diamine tetraacetic acid (pH 8). Fractions were assayed
using modified protocols of the Cholesterol E kit and
Serum Triglyceride Determination kit.
Western Blotting. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed on 1 lL
of plasma or 15 lL of fast protein liquid chromatography eluate using a 4%-15% gradient gel. Polyvinylidene fluoride membranes were probed with an apoB
antibody that detects both apoB48 and apoB100
(K23300R; Meridian Life Science, Saco, ME).
Quantitative Polymerase Chain Reaction. Quantitative polymerase chain reaction for apoB, hepatic
lipase, and LPL is described in detail in the Supporting Information.
Lipase Activity. Liver lysates were prepared and
assessed for LPL and non-LPL activity as described in
the Supporting Information.
Microscopy. Livers were fixed in 4% paraformaldehyde overnight and stored in 70% ethanol. Sections
(5 lm) were stained with hematoxylin and eosin and
visualized under oil immersion.
Oral Lipid Tolerance. Four-hour fasted mice
were given 5 lL/g olive oil via oral gavage. Plasma
samples were taken over 5 hours and assayed for
triglycerides.
Address reprint requests to: Timothy J. Kieffer, Department of Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall,
Vancouver, British Columbia, Canada V6T 1Z3, E-mail: [email protected]; fax: 604-822-2316; or Scott D. Covey, Department of Biochemistry and Molecular
Biology, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3. E-mail: scott.covey@
ubc.ca; fax: 604-822-5227.
C 2012 by the American Association for the Study of Liver Diseases.
Copyright V
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hep.26043
Potential conflict of interest: Nothing to report.
Additional Supporting Information may be found in the online version of this article.
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Fig. 1. Mice lacking hepatic leptin signaling have increased hepatic triglyceride secretion after an insulin bolus. Mice were fasted for 4 hours,
injected with poloxamer-407 at t ¼ 0, and then were either (A) not injected (n ¼ 5 for Leprflox/flox AlbCre, n ¼ 4 for Leprflox/flox AlbCreþ), (B)
injected with 0.6 U/kg insulin (n ¼ 6 for Leprflox/flox AlbCre, n ¼ 8 for Leprflox/flox AlbCreþ), or (C) injected with 0.725 U/kg insulin (n ¼ 5
for Leprflox/flox AlbCre, n ¼ 9 for Leprflox/flox AlbCreþ) at t ¼ 2 hours. Plasma triglyceride levels were monitored throughout the experiment.
Data are expressed as the mean 6 SEM. P values were determined via two-way analysis of variance with a Holm-Sidak post hoc test.
Results
We first determined whether triglyceride output
from the liver was altered in the fasting state. To evaluate VLDL triglyceride secretion from the liver, we
injected fasted mice with poloxamer-407. Poloxamer407 was a potent inhibitor of triglyceride uptake (Fig.
1A), but the accumulation in plasma triglycerides
occurred at similar rates in Leprflox/flox AlbCreþ mice
and their Leprflox/flox AlbCre littermate controls.
Because insulin suppresses VLDL triglyceride secretion17 and the livers of Leprflox/flox AlbCreþ mice are
more sensitive to the effects of insulin,13 we examined
whether a bolus of insulin could differentially affect
VLDL triglyceride secretion in these mice. In response
to insulin, there was a decreased rate of plasma triglyceride accumulation in both Leprflox/flox AlbCreþ mice
and littermate controls (Figs. 1B,C). Surprisingly, in
Leprflox/flox AlbCreþ mice, there were elevated levels of
plasma triglycerides after insulin injection compared
with controls (Figs. 1B,C), suggesting that insulin
mediated suppression of triglyceride secretion is muted
in mice lacking hepatic leptin signaling.
We next investigated the effects of hepatic leptin signaling on fasting plasma triglycerides under more
strenuous metabolic conditions. Leprflox/flox AlbCre mice
were crossed onto an obese, hyperinsulinemic ob/ob
background to generate ob/ob mice lacking functional
hepatic leptin receptors (Leprflox/flox AlbCre ob/ob mice).
Unlike Leprflox/flox AlbCreþ mice, which could have developmental differences compared with their littermate
controls due to a life-long loss of hepatic leptin signaling, Leprflox/flox AlbCreþ ob/ob mice are equivalent to
their littermate controls until treated with exogenous
leptin. At 7 weeks of age, Leprflox/flox AlbCre ob/ob mice
were treated with low dose leptin so as to maintain
obesity and hyperinsulinemia (Supporting Fig. 1). This
dose of leptin lowered plasma cholesterol and triglycerides in ob/ob mice with and without hepatic leptin signaling (Fig. 2A-C). However, plasma triglyceride levels
in Leprflox/flox AlbCreþ ob/ob mice did not decrease as
much as in their Leprflox/flox AlbCre- ob/ob controls
(Fig. 2C). By the last day of leptin treatment, the
Leprflox/flox AlbCreþ ob/ob mice had 36% higher plasma
triglycerides than their littermate controls (Fig. 2C).
The effects of leptin treatment persisted even after leptin therapy ceased, with plasma triglyceride levels in
both groups only returning to near pre-leptin levels 50
days after the leptin pump was removed (Fig. 2C),
indicating leptin treatment in ob/ob mice has longterm effects on lipid metabolism.
Because the effect on plasma triglycerides in Leprflox/flox
AlbCre ob/ob mice was subtle, we sought to reproduce
these results in a complementary mouse model. We
treated leptin receptor-deficient db/db mice with AdLepr-b, which confers liver-selective expression18 and
restores phospho-STAT3 signaling in the liver.16 Upon
treatment with Ad-Lepr-b, the db/db mice remained
obese and hyperinsulinemic (Supporting Fig. 2). Also,
db/db mice treated with Ad-Lepr-b and control db/db
mice treated with Ad-b-gal both had a response to the
virus itself independent of the Lepr-b or b-gal constructs.
We attribute this to an acute phase immune response
to the virus, which has been shown to have effects on
lipid metabolism.19 Nonetheless, we observed no differences in plasma cholesterol and free fatty acids between
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Fig. 2. Hepatic leptin signaling has a subtle but consistent effect on plasma triglycerides in obese, hyperinsulinemic mice. (A-C) Seven-weekold male Leprflox/flox AlbCre ob/ob mice were treated with 0.6 lg/day mouse recombinant leptin for 28 days via mini-osmotic pump starting on
day 1. Mice were tracked for 4-hour-fasted plasma cholesterol (A), free fatty acids (B), and triglycerides (C) (n 5 for both groups). (D-F) Male
db/db mice were treated with Ad-b-gal or Ad-Lepr-b at 12 weeks of age and tracked for 4-hour-fasted plasma cholesterol (D), free fatty acids
(E), and triglycerides (F) (n 6 for both groups). Data are shown as the mean 6 SEM. P values were determined via two-way analysis of variance with a Holm-Sidak post hoc test.
Ad-Lepr-b– and Ad-b-gal–treated db/db mice (Fig. 2DE). Although both virus-treated groups had an increase
in plasma triglycerides, db/db mice treated with
Ad-Lepr-b had lower fasting plasma triglycerides than the
Ad-b-gal–treated controls between 1 and 3 weeks postinfection, with Ad-Lepr-b treated mice reaching 31% lower
plasma triglycerides 12 days postinfection (Fig. 2F).
These data are similar to those of Lee et al.,14 who
treated fa/fa rats with an adenovirus expressing b-gal or
Lepr-b and also saw a marked increase in plasma triglycerides in the b-gal–treated animals compared with the
Lepr-b–treated animals. Collectively, the data show that
under obese, hyperinsulinemic conditions, hepatic leptin
signaling is required for maintaining normal plasma
triglyceride levels.
Because leptin has been implicated in regulating the
amount of triglyceride incorporation into VLDL,17 we
evaluated lipoprotein profiles in Leprflox/flox AlbCreþ
mice and their littermate controls. Mice lacking he-
patic leptin signaling had no alterations in the distribution or amount of cholesterol (Fig. 3A). Interestingly, Leprflox/flox AlbCreþ mice had elevated
triglycerides in fractions consistent in size with VLDL
particles (Fig. 3B). We next performed western blots
for apoB, since each VLDL particle is associated with
one apoB molecule.20 The western blots showed that
particles containing apoB in the plasma of Leprflox/flox
AlbCreþ mice eluted earlier than apoB-containing particles in Leprflox/flox AlbCre mice, suggesting larger
apoB-containing particles (Fig. 3C-F). Therefore, mice
lacking hepatic leptin signaling have more triglyceriderich VLDL particles and larger apoB-containing lipoprotein particles.
Because there appeared to be a slight decrease in
total apoB levels in mice lacking hepatic leptin signaling (Fig. 3F), we measured total apoB levels in whole
plasma from individual mice. Indeed, plasma apoB100
levels were 18% lower in Leprflox/flox AlbCreþ mice
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Fig. 3. Attenuation of hepatic leptin signaling results in increased VLDL triglycerides and larger apoB-containing lipoprotein particles. Pooled
4-hour-fasted plasma samples from 17- to 30-week-old male Leprflox/flox AlbCreþ mice and littermate controls (n 3 for each trial) were subjected to fast protein liquid chromatography, and the fractions were assayed for cholesterol (A) and triglycerides (B). Data represent the average
of two trials in (A) with plasma from a total of eight Leprflox/flox AlbCre mice and 11 Leprflox/flox AlbCreþ mice. In (B), the average of three trials
is shown, representing plasma from a total of 14 Leprflox/flox AlbCre mice and 14 Leprflox/flox AlbCreþ mice. (C) Four-hour-fasted plasma samples (n 3) were pooled from 18- to 27-week-old Leprflox/flox AlbCreþ mice and their littermate controls. The pooled samples were fractionated
and then western blots for apoB were performed on fractions 6-37. Mean pixel intensity of the apoB100 band (D), apoB48 band (E), and combined apoB100 and apoB48 bands (F) are shown for each fraction. Data are expressed as the mean 6 SEM. *P 0.05 (Student t test).
compared with controls (Figs. 4A,B), with a similar
but nonsignificant trend for plasma apoB48 levels
(Figs. 4A,C). Because apoB can come from the small
intestine as well as the liver, we measured hepatic
apoB transcript levels to see whether changes in the
liver could account for the decreased plasma apoB levels. Hepatic apoB messenger RNA (mRNA) levels
were 24% lower in Leprflox/flox AlbCreþ mice, suggesting that decreased plasma apoB can be accounted for
by decreased hepatic expression of apoB (Fig. 4D).
Accordingly, hepatic apoB mRNA levels in db/db mice
were 26% lower than C57BL/6 controls, and upon reexpression of functional leptin receptors in the liver,
hepatic apoB transcript levels returned to wild-type
levels (Fig. 4E). Thus, functional hepatic leptin signaling is positively correlated with plasma apoB levels.
Our data indicate that mice specifically lacking hepatic leptin signaling have less total plasma apoB,
larger apoB-containing lipoprotein particles, and
increased amounts of triglycerides in VLDL-sized particles. It is possible that a reduction in lipase activity
could explain some of these observations, since
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HEPATOLOGY, February 2013
given that LPL is not normally expressed in adult
mouse liver.22,23 To determine whether a loss of hepatic leptin signaling induces the liver to produce
LPL, we measured hepatic LPL mRNA levels and
found no difference in transcript levels between
Leprflox/flox AlbCreþ mice and their littermate controls
(Fig. 5B). The contribution of hepatic LPL to total triglyceride lipase activity in the liver increased from
17% in control mice to 57% in mice lacking hepatic
leptin signaling (Fig. 6C). Overall, these alterations to
LPL activity resulted in increased total triglyceride
lipase activity in the livers of Leprflox/flox AlbCreþ mice
(Fig. 6C). Because overexpression of LPL in different
tissues can cause lipid accumulation,22 we performed a
histological examination of livers from Leprflox/flox AlbCre mice and observed that loss of hepatic leptin signaling led to enlarged lipid droplets (Fig. 7). These
Fig. 4. Attenuation of hepatic leptin signaling decreases plasma
and hepatic apoB levels. (A) Western blots for apoB were performed
on 4-hour-fasted plasma samples from 25- to 30-week-old Leprflox/flox
AlbCre mice (n 6). Quantification of apoB100 and apoB48 for all
samples by densitometry is shown in (B) and (C) (n 6). (D) ApoB
transcript levels were measured in the livers of 22-week-old male
Leprflox/flox AlbCre mice following a 4-hour fast (n ¼ 9). (E) Hepatic
apoB transcript levels were measured in C57BL/6 controls and db/db
mice 4 weeks postinfection with either Ad-b-gal or Ad-Lepr-b (n 5).
Data are expressed as the mean 6 SEM. P values were determined
via Student t test.
patients with hepatic lipase (HL) deficiency display
abnormally large lipoprotein particles.21 Indeed, mice
lacking leptin signaling in the liver had 23% lower HL
mRNA (Fig. 5A) and a trend toward lower non-LPL
activity levels in the liver (Fig. 6A) compared with
controls. However, there was a substantial 4.5-fold
increase in LPL activity in the liver of mice lacking
liver leptin signaling (Fig. 6B). This was surprising
Fig. 5. Hepatic leptin signaling can regulate hepatic lipase and
lipoprotein lipase mRNA levels in the liver. (A, B) Hepatic lipase and
lipoprotein lipase transcript levels were measured in the livers of
22-week-old male Leprflox/flox AlbCre mice following a 4-hour fast (n 8). (C, D) Hepatic lipase and lipoprotein lipase transcript levels were
measured in the livers of 16-week-old C57BL/6 controls (n ¼ 5) and
db/db males 4 weeks postinfection with Ad-b-gal or Ad-Lepr-b (n 7).
Data are expressed as the mean 6 SEM. P values were determined via
Student t test.
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Fig. 6. Hepatic leptin signaling regulates
lipase activity levels in the liver. (A, B) NonLPL (A) and LPL (B) activity levels were
assessed in liver lysates from 22-week-old
male Leprflox/flox AlbCre mice (n ¼ 6 per
group). (C) Total triglyceride lipase activity in
the liver. (D-F) Non-LPL (D), LPL (E), and
total lipase activity (F) levels were measured
in liver lysates from C57BL/6 controls (n ¼
5) and db/db mice 4 weeks postinfection
with either Ad-b-gal or Ad-Lepr-b (n ¼ 7).
Data are expressed as the mean 6 SEM. P
values were obtained by Student’s t-test.
data clearly reveal a role for hepatic leptin signaling in
regulating lipase activity in the liver.
Similar to mice that have a liver-specific loss of leptin
signaling, Ad-b-gal-treated db/db mice also had a 30%
decrease in non-LPL activity in the liver compared with
C57BL/6 controls (Fig. 6D), and this correlated with a
decrease in hepatic HL mRNA (Fig. 5C). When functional leptin receptors were overexpressed in the livers of
db/db mice, non-LPL activity increased even beyond levels seen in wild-type mice (Fig. 6D). Furthermore, control db/db mice had a two-fold increase in LPL activity
levels, and when db/db mice were treated with Ad-Leprb, LPL activity returned to wild-type levels (Fig. 6E).
We also observed that in the total lack of leptin signaling, hepatic LPL activity contributed to 60% of total
triglyceride lipase activity in the liver, and when leptin
signaling was selectively restored to the liver, hepatic
LPL activity contributed only 20% to total triglyceride
lipase activity, which is similar to wild-type levels (Fig.
6F). These data from two complementary models reveal
a novel role for hepatic leptin signaling in modulating
lipase activity in the liver. However, the manner (transcriptional versus posttranscriptional) by which lipase activity in the liver is regulated in mice with a life-long
loss of hepatic leptin signaling and mice with an
induced gain of hepatic leptin signaling is different
(Figs. 5 and 6). Nonetheless, the functional end result is
that with a loss of hepatic leptin signaling, non-LPL
lipase activity is decreased and LPL activity is increased.
To determine whether these effects of leptin on
apoB transcription and lipase activity in the liver are
due to direct or indirect actions of leptin, we treated
ob/ob mice with acute leptin injections as well as
chronic leptin infusions, which restored leptin signaling to all tissues. Acute leptin injections increased
apoB mRNA in the liver by nearly 60%, but chronic
low-dose leptin treatment had no effect (Supporting
Fig. 3A). Further, while liver-selective restoration of
leptin signaling in db/db mice decreased hepatic LPL
expression back toward wild-type levels (Fig. 5D),
acute leptin injections into ob/ob mice increased hepatic LPL mRNA (Supporting Fig. 3C). Therefore,
the increase in hepatic LPL mRNA in ob/ob mice after
acute leptin treatment is likely a result of leptin action
outside of the liver. Interestingly, we previously
observed that a whole body loss of leptin signaling has
distinct effects, in fact opposite, from a liver specific
loss of leptin signaling with respect to glucose homeostasis.13 Notably, chronic low-dose leptin did not
change hepatic LPL mRNA expression in ob/ob mice
(Supporting Fig. 3C), further highlighting the differences between acute versus chronic actions of leptin that
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Fig. 7. Loss of hepatic leptin signaling results in hepatic lipid accumulation. (A-C) Representative images from three male Leprflox/flox AlbCre
littermate controls. (D-F) Representative images from three male Leprflox/flox AlbCreþ mice. (Original magnification 100; hematoxylin and eosin
stain.)
have also been observed by others.24 Collectively, these
results suggest that the direct and indirect effects as
well as acute versus chronic effects of leptin on liver
lipid metabolism are distinct.
Similar to Ad-b-gal–treated db/db mice, which
showed decreased hepatic HL mRNA levels compared
with wild-type controls (Fig. 5C), ob/ob mice also had
decreased hepatic HL transcript levels (Supporting Fig.
3B). Liver HL mRNA levels were restored almost to
wild-type levels by acute leptin injections as well as
chronic low-dose leptin to ob/ob mice (Supporting
Fig. 3B). However, these effects of leptin on hepatic
HL transcript levels appear to be independent of direct
hepatic leptin signaling, because restoration of functional leptin signaling selectively in the livers of db/db
mice did not restore wild-type hepatic HL mRNA levels (Fig. 5C).
Interestingly, these changes in hepatic LPL and HL
mRNA levels in leptin-treated ob/ob mice did not translate into corresponding changes in hepatic LPL or nonLPL activity levels (Supporting Fig. 4). Ob/ob mice had
decreased LPL activity in the liver despite elevated LPL
mRNA. Furthermore, wild-type LPL activity levels
were unable to be restored by leptin in ob/ob mice despite a marked increase in LPL mRNA expression after
acute leptin injections (Supporting Fig. 4B). Similarly,
despite changes in HL mRNA levels, non-LPL activity
levels in the liver were largely unchanged by loss of leptin signaling in the ob/ob mice (Supporting Fig. 4A).
Thus, the regulation of lipase activity in the liver by
leptin seems to involve both transcriptional and posttranscriptional mechanisms.
Because altered lipase activity can affect triglyceride
clearance and leptin may act on the liver to promote
postprandial triglyceride clearance,25 we performed an
oral lipid tolerance test on mice with a loss of leptin
signaling in the liver. These mice had no alterations in
lipid tolerance compared with controls (Supporting
Fig. 5). However, when we treated obese, hyperinsulinemic Leprflox/flox AlbCreþ ob/ob mice with leptin, lipid
tolerance was not improved to the same extent as in
their littermate controls (Fig. 8A). Interestingly, the
effects of leptin on lipid tolerance seemed to persist
even after leptin therapy was ceased, indicating again
that leptin treatment in ob/ob mice has long-term
effects on lipid metabolism (Fig. 8B). We also assessed
lipid tolerance in db/db mice treated with Ad-Lepr-b
or Ad-b-gal. Lipid tolerance in the mice that received
Ad-Lepr-b was improved compared with control mice
that received Ad-b-gal (Figs. 8D,E). These data further
suggest that lipid metabolism is differentially affected
by a loss of hepatic leptin signaling in lean mice compared with hepatic leptin signaling in obese, hyperinsulinemic mice.
Discussion
It is well-established that leptin affects lipid metabolism, but whether these effects are a result of direct
leptin action on the liver has not been fully addressed.
We investigated lipid metabolism in four complementary mouse models and found a previously unreported
role for hepatic leptin action in modulating lipase activity in the liver. Although it has been reported that
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Fig. 8. Hepatic leptin signaling acutely
improves lipid tolerance in ob/ob and db/db
mice. (A-C) Seven-week-old male Leprflox/flox
AlbCre ob/ob mice were treated with 0.6 lg/
day mouse recombinant leptin for 28 days
via mini-osmotic pump starting on day 1.
Oral lipid tolerance tests were performed by
fasting the mice for 4 hours and then administering an oral gavage of 5 lL/g olive oil.
Mice were subjected to an oral lipid tolerance
test after 7 days of leptin treatment (A), 9
days after pump removal (B), and 75 days
after pump removal (C) (n 5). (D, E) Male
db/db mice were subjected to an oral lipid
tolerance test (D) 9 days before and (E) 7
days after treatment with either Ad-b-gal or
Ad-Lepr-b (n 6). Data are expressed as
the mean 6 SEM. P values were determined
via two-way analysis of variance with a HolmSidak post hoc test.
HL mRNA levels in the liver are decreased in ob/ob
mice and restored with whole body leptin treatment,12
we now report that this is not associated with
increased non-LPL lipase activity in the liver. However,
in mice with a liver-specific loss or gain of leptin signaling, our data do support a role for leptin signaling
specifically in the liver to positively regulate non-LPL
activity. Further, we also report a novel finding that
leptin resistance specifically in the liver leads to a
marked increase in hepatic LPL activity. Because an
overexpression of LPL in tissues can cause increased
lipid uptake and lipid accumulation,22 we speculate
that the elevation of LPL activity in Leprflox/flox
AlbCreþ mice contributes to their elevated hepatic
triglycerides.13
LPL activity has a complex mechanism of regulation, including transcriptional, posttranscriptional,
translational, and/or posttranslational mechanisms
depending on nutrient status and tissue.26 Adding to
this complexity, our data show that in db/db mice, the
loss of leptin signaling caused an elevation of hepatic
LPL activity through transcriptional changes, but in
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HUYNH ET AL.
Leprflox/flox AlbCreþ mice, the increased LPL activity
was mediated through posttranscriptional mechanisms.
Furthermore, because insulin can regulate LPL activity
in adipose and muscle,26 leptin regulation of hepatic
LPL activity may be indirect through the effects of leptin on hepatic insulin signaling. Additionally, because
leptin treatment in ob/ob mice was unable to fully normalize lipase activity in the liver, secondary extrahepatic effects of leptin signaling also appear to contribute to the regulation of lipase activity in the liver.
Although it is clear that loss of hepatic leptin signaling
can increase hepatic LPL mRNA, the exact mechanism
by which leptin regulates lipase activity in the liver
remains to be determined.
Leprflox/flox AlbCreþ mice have increased hepatic insulin sensitivity,13 and insulin is an important regulator
of lipid metabolism in the liver as evidenced by its
role in decreasing plasma apoB levels.17 Consistent
with this, our data show that in mice lacking hepatic
leptin signaling, increased hepatic insulin sensitivity is
associated with decreased plasma apoB levels even in
the fasting state. Although it is possible that this effect
on apoB is mediated directly by leptin signaling independent of insulin, we speculate that it is actually the
effect of leptin on insulin signaling that mediates
changes in apoB, since leptin itself does not affect
plasma apoB levels.17,27 Interestingly, in ob/ob mice,
leptin was able to increase hepatic apoB mRNA only
when leptin was administered at a dose and route of
administration that has been shown to lower plasma
insulin levels12,28 and not at a dose that did not affect
plasma insulin levels (Supporting Fig. 1D). In association with decreased plasma apoB levels, Leprflox/flox
AlbCreþ mice had increased triglycerides in VLDL
particles, suggesting that these mice may have fewer
VLDL particles in total but more triglycerides per
VLDL particle. We hypothesize that the increased
incorporation of triglycerides in these mice is due in
part to elevated liver triglycerides, leading to increased
substrate availability. This can result in more triglyceride incorporation into each VLDL particle,17,29
leading to enlarged, more triglyceride-rich VLDL
particles.29 Intriguingly, overexpression of HL in a
rat liver cell line resulted in secretion of triglyceridepoor VLDL,30 and patients with HL deficiency
have been shown to have triglyceride-rich lipoproteins
that are also larger in size.21 Therefore, although we
cannot rule out the involvement of other non-LPL lipases in the liver, decreased HL activity in Leprflox/flox
AlbCreþ mice likely contributes to altered lipid loading, leading to enlarged, triglyceride-rich VLDL
particles.
HEPATOLOGY, February 2013
Our observations are compatible with the theory
that insulin is responsible for modulating the number
of VLDL particles, while hepatic leptin action can
modulate the amount of triglycerides available for
incorporation into each VLDL particle through the
effects of leptin on increasing fatty acid oxidation.17 In
our model of increased hepatic insulin sensitivity with
extreme hepatic leptin resistance, there is less plasma
apoB, indicating fewer VLDL particles, and increased
hepatic triglycerides, which may lead to larger, more
triglyceride-rich VLDL particles. According to this
model, one might expect that hepatic triglyceride
secretion would be suppressed more in Leprflox/flox
AlbCreþ mice because they are more insulin-sensitive
than controls.13 Surprisingly, while we did observe insulin-mediated suppression of hepatic triglyceride
secretion in both groups of mice, mice lacking hepatic
leptin signaling had higher plasma triglycerides than
controls after insulin (Fig. 1). This may be due to
enhanced lipogenic effects of insulin in the Leprflox/flox
AlbCreþ mice, which together with the lack of leptin
signaling can result in even more substrate availability
during hyperinsulinemic conditions, allowing for more
triglycerides per VLDL particle. Interestingly, liver insulin receptor knockout mice have increased plasma
apoB yet decreased plasma triglycerides and no alterations in liver triglycerides.31 Thus, insulin signaling in
the liver may also have effects on triglyceride loading
onto VLDL independent of its effects on substrate
availability and our data suggest that leptin signaling
in the liver may serve to counter this effect.
Despite evidence of major changes in hepatic lipid
metabolism genes upon leptin treatment in models of
leptin deficiency,6,8,12 our data suggest that indirect
effects are involved. Consistent with the literature,14,32
our data do support the fact that direct leptin action
on the liver plays an antisteatotic role, but the level of
hepatic steatosis in Leprflox/flox AlbCreþ mice was not as
severe as in livers of db/db mice. Furthermore, unlike
db/db mice with a global loss of leptin signaling, lean
mice with a liver-specific loss of leptin signaling have
normal total fasting plasma triglycerides and cholesterol levels.13 It appears that although mice with a hepatocyte-specific loss of leptin signaling have increased
incorporation of triglycerides into VLDL particles,
they do not develop hypertriglyceridemia due to their
concurrent reduction in hepatic apoB production.
However, in more metabolically stressed obese, hyperinsulinemic mice, we did observe a more pronounced
perturbation in fasting plasma triglycerides and lipid
tolerance. Interestingly, patients with metabolic syndrome have a higher proportion of large VLDL than
HEPATOLOGY, Vol. 57, No. 2, 2013
healthy patients, even in patients with normal plasma
triglyceride levels.33 Therefore, subtle effects of hepatic
leptin resistance on lipid metabolism could have a
major impact on health.
Our model of hepatic leptin resistance shows that
loss of leptin signaling in the liver can contribute to
the development of hepatic steatosis and large, triglyceride-rich lipoproteins. Given that obese humans are
leptin-resistant, our data suggest that defects in lipid
metabolism seen in obesity may stem in part from resistance to leptin action in the liver. Although the
effects of liver leptin signaling on lipid metabolism
appear subtle, our data show that these effects are
more pronounced in obese and hyperinsulinemic
states. Intriguingly, polymorphisms in the LEPR,34
HL,35 and LPL36 genes have been linked with familial
combined hyperlipidemia, the most common genetically linked hyperlipidemia in humans. Thus, alterations to HL and LPL activity in the liver due to hepatic leptin resistance may result in increased risk of
dyslipidemia and perhaps contribute to the development of metabolic syndrome.
Acknowledgments: We thank Streamson C. Chua
(Albert Einstein College of Medicine) for his generous
contribution of the Leprflox/flox mice and A. F. Parlow
(National Hormone and Peptide Program) for providing mouse recombinant leptin. We also thank Martin
G. Myers (University of Michigan) and Christopher J.
Rhodes (University of Chicago) for providing the AdLepr-b virus.
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