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Diabetes Volume 64, April 2015
Hyun-Kyong Kim,1 Mi-Seon Shin,2 Byung-Soo Youn,3 Gil Myoung Kang,1
So Young Gil,1 Chan Hee Lee,1 Jong Han Choi,1,2 Hyo Sun Lim,1 Hyun Ju Yoo,4 and
Min-Seon Kim1,2
Regulation of Energy Balance by the
Hypothalamic Lipoprotein Lipase
Regulator Angptl3
METABOLISM
Diabetes 2015;64:1142–1153 | DOI: 10.2337/db14-0647
Hypothalamic lipid sensing is important for the maintenance of energy balance. Angiopoietin-like protein 3
(Angptl3) critically regulates the clearance of circulating
lipids by inhibiting lipoprotein lipase (LPL). The current
study demonstrated that Angptl3 is highly expressed in
the neurons of the mediobasal hypothalamus, an important area in brain lipid sensing. Suppression of hypothalamic Angptl3 increased food intake but reduced
energy expenditure and fat oxidation, thereby promoting
weight gain. Consistently, intracerebroventricular (ICV)
administration of Angptl3 caused the opposite metabolic changes, supporting an important role for hypothalamic Angptl3 in the control of energy balance.
Notably, ICV Angptl3 significantly stimulated hypothalamic LPL activity. Moreover, coadministration of the
LPL inhibitor apolipoprotein C3 antagonized the effects
of Angptl3 on energy metabolism, indicating that LPL
activation is critical for the central metabolic actions of
Angptl3. Increased LPL activity is expected to promote
lipid uptake by hypothalamic neurons, leading to enhanced brain lipid sensing. Indeed, ICV injection of
Angptl3 increased long-chain fatty acid (LCFA) and
LCFA-CoA levels in the hypothalamus. Furthermore,
inhibitors of hypothalamic lipid-sensing pathways prevented Angptl3-induced anorexia and weight loss.
These findings identify Angptl3 as a novel regulator of
the hypothalamic lipid-sensing pathway.
Obesity is a growing global health concern that underlies
the development of type 2 diabetes, hypertension, and
1Appetite Regulation Laboratory, ASAN Institute for Life Sciences, Seoul, Republic
of Korea
2Division of Endocrinology and Metabolism, Department of Internal Medicine,
University of Ulsan College of Medicine, Seoul, Republic of Korea
3Department of Anatomy, Wonkwang University School of Medicine, Iksan, Republic
of Korea
4Biomedical Research Center, ASAN Institute for Life Sciences, Seoul, Republic of
Korea
Corresponding author: Min-Seon Kim, [email protected].
cardiovascular diseases. Although many factors contribute to the obesity epidemic, chronic positive imbalance
between energy intake and expenditure is thought to be
the major cause of common forms of human obesity. For
over a century, the hypothalamus has been considered to
be a controlling center of energy balance (1). Specialized
hypothalamic neurons sense the whole-body nutritional
state, which is crucial for the hypothalamic regulation of
energy balance (2).
Intracerebroventricular (ICV) administration of longchain fatty acid (LCFA) suppresses food intake and
glucose production (3), indicating that fatty acids can
signal nutrient availability to the central nervous system
(CNS) and thereby restrict the additional release of
nutrients into the circulation. Accumulating data suggest
that esterified LCFA (LCFA-CoA) is an important lipid
metabolite in hypothalamic lipid-sensing pathways (4).
Hypothalamic inhibition of carnitine palmitoyltransferase-1,
which increases neuronal LCFA-CoA levels by inhibiting
fatty acid oxidation, reduces food intake and glucose production (5). This effect is reversed by a blockade of LCFACoA formation, supporting a crucial role for LCFA-CoA in
hypothalamic lipid sensing (5). By contrast, hypothalamic
activation of AMPK, which decreases LCFA-CoA levels by
increasing fatty acid oxidation, stimulates food intake
(4,6).
Lipoprotein lipase (LPL) is a key enzyme in lipid
metabolism that hydrolyzes triglycerides (TGs) in circulating TG-rich lipoproteins and promotes the cellular uptake of chylomicron remnants, cholesterol-rich
Received 23 April 2014 and accepted 13 October 2014.
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0647/-/DC1.
H.-K.K. and M.-S.S. contributed equally to this study.
© 2015 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
diabetes.diabetesjournals.org
lipoproteins, and free fatty acids by adipose tissue and
skeletal muscle (7). LPL is highly expressed throughout
the brain, including the hypothalamus and hippocampus
(8). In the CNS, LPL modulates various aspects of neurobiology such as learning and memory by regulating neuronal lipid uptake (9). Notably, mice with a neuronspecific LPL deficiency develop obesity, indicating an important role of neuronal LPL in the control of energy
balance (10). In these mice, hypothalamic LCFA levels
are decreased, suggesting that LPL-dependent lipid uptake
in the hypothalamus is essential for the maintenance of
energy homeostasis.
The angiopoietin-like proteins (Angptls) are a family
of proteins that share two structural domains with the
angiopoietins: the N-terminal coiled-coil domain and the
COOH-terminal fibrinogen-like domain (11). Among
the eight Angptls, Angptls 3, 4, 6, and 8 are implicated
in glucose, lipid, and energy metabolism (11,12). Angptl8/
betatrophin controls TG trafficking from skeletal muscle
and heart to adipose tissues upon food intake (12).
Angptl6, also known as angiopoietin-related growth factor, controls peripheral fatty acid oxidation and energy
metabolism (13). Angptl4 regulates plasma TG levels by
directly inhibiting LPL activity and mediates the antiobesity effects of a germ-free condition (14,15). Moreover,
we previously showed that Angptl4 regulates feeding
behavior and energy metabolism via central mechanisms
(16), extending the biological roles for Angptls to the
CNS regulation of energy metabolism.
Angptl3 is known as an important regulator of circulating lipid metabolism (11,17). In the periphery,
Angptl3 mRNA is exclusively found in the liver, where
it is secreted into the circulation (18). Angptl3 potently
inhibits the activity of LPL and endothelial lipase (19).
Supporting these actions, Angptl3-deficient mice display
lower circulating TG levels (20,21), whereas Angptl3
administration results in increased plasma lipid levels
(17). In humans, loss-of-function mutations of Angptl3
are associated with familial combined hypolipoproteinemia
(22). Based on our preliminary data showing hypothalamic Angptl3 expression, we investigated a potential regulatory role for Angptl3 in the central regulation of energy
balance.
RESEARCH DESIGN AND METHODS
Peptides and Compounds
Full-length Angptl3 and Angptl4 were purchased from
AdipoGen (Incheon, Republic of Korea), and apolipoprotein
C3 (apoC3) was purchased from Sigma (St. Louis, MO).
Triacsin-C (Tri-C) and AICAR were purchased from Sigma
and Toronto Research Chemicals (Toronto, Ontario, Canada),
respectively. Heparin was obtained from Halim Pharmaceuticals (Seoul, Republic of Korea).
Kim and Associates
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Republic of Korea). Animals were allowed free access to
a standard chow diet (Cargill Agri Purina, Inc., Seoul, Republic of Korea) and water, unless otherwise indicated.
Animals were housed under conditions of controlled temperature (22 6 1°C) and a 12-h light-dark cycle, with the
light on from 0800 to 2000 h. All procedures were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals (National
Institutes of Health, Bethesda, MD) and were approved by
the Institutional Animal Care and Use Committee at the
ASAN Institute for Life Sciences (Seoul, Republic of Korea).
Agouti-related protein (AGRP) neuron–specific EGFPexpressing (AGRP-EGFP) mice were generated by crossing
AGRP-IRES-Cre mice (obtained from Dr. Joel. K. Elmquist,
University of Texas Southwestern Medical Center, Dallas,
TX) with EGFP-floxed mice (The Jackson Laboratory, Bar
Harbor, ME).
Cannulation and Injection
Stainless steel cannulae (26 gauge; Plastics One) were
implanted into the third cerebral ventricle of the mice and
rats, as described previously (23). After a 7-day recovery
period, correct positioning of each cannula was confirmed
by a positive dipsogenic response to angiotensin-2 (50 ng
per mouse and 150 ng per rat). All peptides and compounds were dissolved in 0.9% saline solution just before use, and were administered via the ICV-implanted
cannulae in a total volume of 2 mL. The majority of
feeding studies were performed just before the end of
the daily light cycle, unless otherwise indicated. Food intake and body weight were monitored for 24 h after the
injections.
Small Interfering RNA Study
Small interfering RNAs (siRNAs) specific for murine Angptl3
(catalog #E-065291-00-0020) and LPL (catalog #STOP130319-015) and a nontargeting scrambled siRNA (catalog
#SCRO-101125-010) were purchased from Dharmacon
(Chicago, IL) and injected into the bilateral mediobasal
hypothalamus (MBH) as previously described (23). Successful knockdown was confirmed by determining hypothalamic Angptl3 and LPL mRNA levels at the end of the
study. Gene knockdown was considered successful when
hypothalamic Angptl3 and LPL mRNA levels fell to ,30%
of the average levels of the control group. Animals showing
successful gene knockdown were included in the analysis.
Energy Expenditure
Energy expenditure (EE) and respiratory quotient (RQ) were
monitored using the Oxymax Comprehensive Laboratory
Animal Monitoring System (CLAMS; Columbus Instruments,
Columbus, OH) over 24–48 h after treatment. Animals were
acclimatized in metabolic cages for 3 days before treatment.
Animals
LPL Activity
Adult male C57BL/6 mice and Sprague-Dawley rats (8–10
weeks of age) were purchased from Orient Bio (Seoul,
Saline, Angptl3 (1 mg), Angptl4 (3.5 mg), apoC3 (0.1 mg),
and heparin (5–10 units) were injected via ICV-implanted
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Angptl3 and Hypothalamic Lipoprotein Lipase
cannulae just before the beginning of the dark period,
unless otherwise indicated. The MBH was collected at 2 h
postinjection. Immediately after collection, MBH tissue
was weighed and minced into small pieces. After rinsing
with cold PBS to remove red blood cells, the minced tissue
was homogenized in 60 mL of cold 20 mmol/L Tris buffer,
pH 7.5, containing 150 mmol/L NaCl. After centrifugation at 10,000g for 10 min at 4°C, the supernatant was
carefully collected and subjected to LPL activity assay following the manufacturer’s protocol (Cell Biolabs, San
Diego, CA). The cerebrospinal fluid (CSF) of rats was collected 2 h after the ICV injection of heparin (20 units), as
previously described (24). LPL activity was measured in
50-mL amounts of CSF.
AMPK Activity
The MBH was collected 2 h after ICV injection of saline,
Angptl3 (1 mg), or Angptl4 (3.5 mg) into overnight-fasted
mice during the early light phase. The tissue was then
flash frozen in liquid nitrogen. Total AMPK activity was
determined as previously described (25).
Measurement of LCFA, LCFA-CoA, and TGs
To measure LCFA, free fatty acids were extracted from 10–20
mg of hypothalamic tissue or from 80 mL of plasma using
400 mL methanol and isooctane. The extracted free fatty
acids were derivatized using BCl3-methanol (26). A standard
mixture of fatty acid methyl esters obtained from SigmaAldrich was used to generate calibration curves. Fatty acid
methyl ester was analyzed with a gas chromatography–
mass spectrometry system (7890A/5975C detectors; Agilent)
using a capillary column (HP-5MS; 30 m 3 0.25 mm 3
0.2 mm) and electron impact ionization. To measure
LCFA-CoA, samples were prepared from 10215 mg of
hypothalamic tissues, as previously described (27). The
dried samples were stored at 220°C and reconstituted
with 30 mL H2O/acetonitrile (50/50 volume for volume)
prior to analysis. Liquid chromatography–tandem mass
spectroscopy was performed using the 1290 Infinity
Binary HPLC system (Agilent), the Qtrap 5500 system
(ABSciex), and a reverse-phase column (2.1 3 150 mm;
ZORBAX 300Extend-C18; Agilent). Analytical conditions
were the same as those previously described (27). Hypothalamic TG contents were determined using a colorimetric assay (Abcam). MBH blocks were homogenized with
60 mL 5% NP-40. The homogenate was centrifuged, and
50 mL of the resulting supernatant was used in the assay.
Immunofluorescence Staining
Mice were perfused with 4% paraformaldehyde through
the heart under anesthesia for 15 min after a 5-h fast.
After postfixation and dehydration, coronal brain sections
(14 mm thick) were cut using a cryostat (Leica, Wetzlar,
Germany) and incubated with primary antibodies against
the Angptl3 N-terminal fibrinogen-like domain (1:200,
mouse; Abcam), microtubule-associated protein 2 (MAP2)
(1:800, chicken; Millipore), glial fibrillary acid protein
(GFAP) (1:300, rabbit; Abcam), apoC3 (1:100, rabbit; Santa
Cruz Biotechnology), b-endorphin (1:10,000, rabbit; Phoenix
Diabetes Volume 64, April 2015
Pharmaceuticals), c-fos (1:100, rabbit; Santa Cruz Biotechnology), LPL (1:100, rabbit; Santa Cruz Biotechnology), or
EGFP (1:1,000, rabbit; Invitrogen) at 4°C for 24 h. After
washing, slides were incubated with an Alexa Fluor 488–
conjugated goat anti-mouse antibody, an Alexa Fluor 546–
conjugated goat anti-rabbit antibody, or an Alexa Fluor
633–conjugated goat anti-chicken antibody (all from Invitrogen) at room temperature for 1 h. For nuclear staining, slides were treated with DAPI (1:10,000; Invitrogen)
for 10 min before mounting. Triple immunofluorescence
was examined by confocal microscopy (Leica).
mRNA Expression
Total RNA was extracted using TRIzol Reagent (Life Technologies). Hypothalamic mRNA expression levels of Angptls
and LPL were determined by real-time PCR or semiquantitative RT-PCR (the primer sequences are shown in Supplementary Table 1) and normalized to levels of GAPDH mRNA.
Immunoblotting
Tissue proteins were extracted from hypothalamic tissue
blocks, as previously described (16), and were subjected to
immunoblotting using primary antibodies against Angptl3
(1:2,000, mouse; Abcam), LPL (1:1,000, rabbit; Santa Cruz
Biotechnology), paired amino acid–converting enzyme
4 (PACE4) (1:1,000, rabbit; Santa Cruz Biotechnology),
proprotein convertase subtilisin/kexin type 5 (PCSK5)
(1:1,000, rabbit; Sigma), phosphorylated (Tyr705) and total signal-transduction-activated transcript-3 (Stat3), and
phosphorylated (Thr308) and total Akt (all from Cell Signaling Technology). Hypothalamic tissue band density
was measured with a densitometer (VersaDoc Multi Imaging Analyzer System; Bio-Rad, Hercules, CA).
Conditioned Taste Aversion Test
Conditioned taste aversion (CTA) tests were conducted
after sequential intraperitoneal injection of saline plus
ICV saline, intraperitoneal lithium chloride (127 mg/kg,
Sigma) plus ICV saline, and intraperitoneal saline plus ICV
Angptl3 (1 mg), using a previously described protocol (28).
Statistical Analysis
All data are expressed as the mean 6 SEM. Statistical
analysis was performed using SPSS version 17.0 software
(SPSS Inc., Chicago, IL). Comparisons between two groups
were analyzed using the Student t test. Comparisons
among three or more groups were conducted using repeated or one-way ANOVA followed by a post hoc least
significance difference test. A P value ,0.05 was considered statistically significant.
RESULTS
Angptl3 Is Abundantly Expressed in Hypothalamic
Neurons
Real-time PCR analysis was first used to demonstrate the
mRNA expression of Angptl3 and other Angptls in the
MBH of normal mice (Fig. 1A). Immunoblotting analysis
also revealed the presence of full-length Angptl3 (62 kDa)
in protein extracts of the hypothalamic arcuate nucleus
(ARC), the lateral hypothalamus (LH) area, and the
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Figure 1—Angptl3 is expressed in hypothalamic neurons. A: The mRNA expression of Angptls in the hypothalamus of C57BL/6 mice.
B: Angptl3 protein expression in the hypothalamic ARC, PVN, and LH in normal mice fasted for 5 h. C: MBH Angptl3 protein expression
according to feeding conditions. Mice were fasted for 24 h or refed for 1 and 2 h after a 24-h fasting before being killed (n = 324). D: Double
immunofluorescence staining for Angptl3 and the neuron marker MAP2 or the glial marker GFAP in the MBH, demonstrating an exclusive
neuronal expression of Angptl3. E: Dual staining for Angptl3 and AGRP-EGFP (AGRP) or POMC (b-endorphin) in the MBH, showing that
Angptl3 was expressed in POMC or AGRP neurons (indicated by white arrows).
paraventricular nucleus (PVN) collected from mice fasted
for 5 h using the punch biopsy technique (Fig. 1B). A higher
expression of Angptl3 was found in the ARC and LH compared with the PVN. To verify whether MBH Angptl3 protein expression is altered by nutrient availability (Fig. 1C),
mice were either kept under fasted conditions for 24 h before
being killed or allowed to resume food intake during the
beginning of the dark period after a 24-h fast. Hypothalamic
Angptl3 protein levels were higher in refed mice than in
fasting mice (Fig. 1C), indicating that hypothalamic Angptl3
expression increases during the postprandial period.
To determine whether Angptl3 is expressed in hypothalamic neurons or glial cells in the mouse MBH, double
immunofluorescence staining was performed using antibodies against Angptl3 and the neuronal marker MAP2 or
the glial marker GFAP. Angptl3 immunoreactivity almost
completely overlapped with MAP2 but not with GFAP
(Fig. 1D), suggesting that Angptl3 is mostly expressed in
hypothalamic neurons. High levels of Angptl3 expression
were observed in the soma of MBH neurons, while lower
expression was seen in neuronal processes (Fig. 1D).
The key appetite-regulating neuropeptides proopiomelanocortin (POMC) and AGRP are produced by a discrete
population of MBH neurons that play a primary role in
hypothalamic nutrient sensing (1,29). Double immunostaining for Angptl3 and the POMC product b-endorphin
revealed that .90% of POMC neurons expressed Angptl3.
Dual staining for Angptl3 and EGFP in AGRP-EGFP mice
revealed Angptl3 expression in ;40–50% of AGRP neurons
(Fig. 1E). Notably, a considerable proportion of Angptl3expressing neurons did not produce either POMC or AGRP.
Hypothalamic Angptl3 Is Involved in the Central
Regulation of Energy Metabolism
Given the abundant expression of Angptl3 in the MBH,
a brain area critical for the control of energy balance, we
hypothesized that Angptl3 may be involved in the hypothalamic regulation of energy balance. To test this hypothesis,
MBH Angptl3 expression was depleted by microinjecting
Angptl3 siRNA into the bilateral MBH. Successful Angptl3
knockdown in the MBH was confirmed by examining
hypothalamic Angptl3 mRNA and protein expression levels
at the end of the study (Fig. 2A and B). Compared with
controls injected with a nontargeting siRNA, mice with reduced hypothalamic Angptl3 expression consumed more
food and recovered more quickly from postsurgical weight
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Angptl3 and Hypothalamic Lipoprotein Lipase
Diabetes Volume 64, April 2015
Figure 2—Inhibition of hypothalamic Angptl3 alters energy metabolism. A: Microinjection of Angptl3 siRNA (siAngptl3) into the bilateral MBH
significantly reduced hypothalamic Angptl3 mRNA levels (n = 6). B: Angptl3 immunohistochemistry showing decreased Angptl3 expression in
the MBH of mice injected with Angptl3 siRNA. C: Reduced hypothalamic Angptl3 expression increased food intake and promoted recovery
from postsurgical weight loss (n = 6). D and E: Reduced EE and increased RQ in mice injected with Angptl3 siRNA compared with mice
injected with scrambled control siRNA (siControl) (n = 6). *P < 0.05 vs. control siRNA. Data are reported as the mean 6 SEM.
loss (Fig. 2C). Moreover, CLAMS revealed that these mice
displayed a decreased EE and an increased RQ during the
dark period when access to food was not restricted (Fig. 2D
and E). Considering that hypothalamic Angptl3 expression
increases upon food intake (Fig. 1C), hypothalamic Angptl3
may coordinate multiple metabolic responses to food intake, including satiety generation, stimulation of EE, and
transition of nutrient oxidation from fat to carbohydrate.
The metabolic effects of exogenous administration of
Angptl3 were then examined. Full-length mouse Angptl3
(0.3, 1, and 3 mg) was administered via an ICV-implanted
cannula in the beginning of the light phase in mice fasted
overnight. ICV injection of Angptl3 significantly suppressed
fasting-induced feeding in a dose-dependent manner
(Fig. 3A). The anorexigenic effect of a single ICV injection
of Angptl3 was sustained for at least 24 h. Moreover, ICV
injection of Angptl3 inhibited weight gain for 24 h postinjection (Fig. 3B). To test the possibility that ICV injection of Angptl3 may increase aversion to food, we
performed a CTA test after ICV injection of Angptl3. In
contrast to intraperitoneal injection of the established
CTA inducer lithium chloride, which decreased saccharine
consumption, ICV Angptl3 injection did not induce CTA
(Fig. 3C), suggesting that Angptl3-induced anorexia was
not due to a conditioned aversion to food.
To examine the effects of Angptl3 on whole-body
energy metabolism, Angptl3 (1 mg) was injected ICV
into freely fed mice just before the end of the light cycle,
and energy metabolism was monitored using the CLAMS.
Angptl3-treated mice displayed a higher EE and a lower
RQ in the dark period compared with saline-injected mice
(Fig. 3D), indicating that ICV Angptl3 stimulates EE and
promotes fat consumption in peripheral tissues. Taken together with data from loss-of-function and gain-of-function
studies, an increase in hypothalamic Angptl3 leads to a negative energy balance.
We further investigated whether hypothalamic neurons
are responsive to Angptl3 by using c-fos immunostaining
after ICV injection of Angptl3 (1 mg). The c-fos immunoreactive neurons were mostly found in the hypothalamic
ARC of the Angptl3-treated mice (Fig. 3E), suggesting that
the neurons primarily responsive to Angptl3 reside in this
area. Since the ARC neurons produce important metabolic
regulators such as POMC, AGRP, and neuropeptide Y (NPY),
we tested the possibility that Angptl3 may exert its actions
through regulation of these neuropeptides. Indeed, the
mRNA expression of NPY and AGRP was profoundly decreased by ICV injection of Angptl3 (1 mg), while POMC
mRNA expression was not altered (Fig. 3F). Thus, Angptl3
may regulate energy balance via the downregulation of hypothalamic NPY and AGRP.
Angptl3 Stimulates Hypothalamic LPL Activity
We further sought to identify the signaling pathways by
which Angptl3 regulates feeding behavior and body weight.
Hypothalamic AMPK activity was determined after ICV
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Figure 3—Central administration of Angptl3 causes a negative energy balance. A and B: The effects of ICV administration of Angptl3 in mice
fasted overnight on food intake and body weight (n = 6). ICV Angptl3 (1 and 3 mg) inhibited fasting-induced hyperphagia and weight gain. *P <
0.05 vs. saline. C: CTA tests after ICV injection of Angptl3 and intraperitoneal injection of lithium chloride (n = 5). *P < 0.05 vs. saline. NS, not
significant. D: The effects of ICV injection of Angptl3 on EE and RQ. Angptl3 (1 mg) was injected ICV just before the end of the daily light cycle
(n = 4). The arrow indicates the time of the ICV injection. E: C-fos immunohistochemistry in the mouse hypothalamus, which was analyzed 1 h
after an ICV injection of saline or Angptl3 (1 mg). F: Effects of Angptl3 on the mRNA expression levels of NPY, AGRP, and POMC. MBH was
collected 1 h after the ICV injection of saline or Angptl3 (1 mg) in overnight-fasted mice. *P < 0.01 vs. saline. Data are reported as the mean 6
SEM.
injection of Angptl3 (1 mg), as AMPK is an important
downstream mediator of Angptl4 in the hypothalamus
(16). Consistent with a previous report, ICV injection of
Angptl4 (3.5 mg) potently suppressed AMPK activity in
the MBH at 2 h postinjection. By contrast, ICV Angptl3
injection had no effect on hypothalamic AMPK activity
(Fig. 4A). The effects of ICV injection of Angptl3 on hypothalamic Stat3 and on phosphoinositide 3-kinase-Akt
signaling were also examined, as many appetite regulators
act through these signaling pathways (28,30). Hypothalamic Stat3 and Akt phosphorylation were not significantly
altered by ICV-injected Angptl3 (Supplementary Fig. 1),
indicating that these signaling pathways are not involved
in the Angptl3-mediated regulation of energy balance.
In the periphery, Angptl3 strongly inhibit LPL activity
(19). Hypothalamic LPL activity was therefore evaluated
2 h after ICV administration of Angptl3 in mice fasted for
5 h. Animals were kept under fasting condition until they
were killed to avoid any possible effects of food intake on
hypothalamic LPL activity. Contrary to its inhibitory effect on peripheral LPL, ICV injection of Angptl3 (1 mg)
increased LPL activity in the MBH by ;1.8-fold (Fig. 4B).
Consistently, hypothalamic LPL activity was found to be
lower in mice injected with Angptl3 siRNA than in mice
injected with nontargeting siRNA after a 5-h fast (Fig. 4C).
ICV injection of Angptl4 (3.5 mg) also caused a modest but
significant increase in hypothalamic LPL activity (Fig. 4B).
We further investigated the mechanisms of Angptl3mediated regulation of hypothalamic LPL activity. By
using immunoblotting and immunofluorescence staining,
we demonstrated that Angptl3 (1 mg) increased MBH LPL
protein levels when ICV injected in mice fasted for 5 h
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Figure 4—Angptl3 stimulates hypothalamic LPL activity. A: The effects of ICV injection of Angptl3 (1 mg) and Angptl4 (3.5 mg) on
hypothalamic AMPK activity (n = 5). **P < 0.005 vs. saline. B: The effects of ICV administration of Angptl3 (1mg) and Angptl4 (3.5 mg)
on hypothalamic LPL activity (n = 5). *P < 0.05, **P < 0.005 vs. saline. C: Reduced hypothalamic LPL activity in mice injected with Angptl3
siRNA in the bilateral MBH (n = 5). *P < 0.05 vs. control siRNA. siAngptl3, Angptl3 siRNA; siControl, control siRNA. D: LPL immunofluorescence staining in the hypothalamus 1 h after ICV injection of either Angptl3 or saline. E: Effects of ICV-injected Angptl3 (1 mg) on
hypothalamic LPL, PACE4, and PCSK5 protein expression (n = 3). F: Effects of ICV-injected Angptl3 (1 mg) or heparin (10 units) on
hypothalamic LPL, hepatic lipase (HPL), and endothelial lipase (EL) mRNA expression (n = 5). Data are reported as the mean 6 SEM.
just before lights went off, whereas it did not increase
hypothalamic LPL mRNA levels (Fig. 4D–F). In the periphery, Angptl3 enhances LPL cleavage induced by proprotein
convertases (furin, PACE4, and PCSK5) (31). Hypothalamic PACE4 and PCSK5 expression was unchanged after
ICV injection of Angptl3 (Fig. 4E). Therefore, Angptl3
upregulates and activates hypothalamic LPL via as yet
unidentified post-transcriptional regulation. On the other
hand, the LPL assay we used in this study can also measure other lipase activities. Hypothalamic expression of
hepatic lipase and endothelial lipase was not altered by
Angptl3 administration (Fig. 4F), suggesting that LPL is
a major hypothalamic lipase regulated by Angptl3.
To confirm the effects of hypothalamic LPL activation on
food intake and body weight, mice were treated with heparin,
which activates LPL by promoting LPL release from cells
(32). Indeed, MBH LPL activity increased after ICV injection
of heparin (10 units) (Fig. 5A). LPL activity in the CSF was
also increased after ICV injection of heparin (20 units) in
Sprague-Dawley rats (Fig. 5B). Similar to Angptl3, ICV injection of heparin suppressed nighttime food intake and
decreased body weight when injected before the beginning
of the dark period (Fig. 5C and D). These findings suggest
that hypothalamic LPL activation induced by Angptl3 and
heparin could lead to decreased food intake and body weight.
To determine whether hypothalamic LPL activity is
regulated by feeding conditions, hypothalamic LPL activity was measured under fasting and refeeding conditions.
Hypothalamic LPL activity increased after food intake
(Fig. 5E), consistent with the effects of Angptl3 on hypothalamic LPL activity.
Inhibition of Hypothalamic LPL Antagonizes the
Central Metabolic Effects of Angptl3
ApoC3, a known LPL inhibitor (33), was found to be
highly expressed in ependymal lining cells and MBH neurons (Fig. 6A). In addition, a certain population of hypothalamic neurons coexpressed Angptl3 and apoC3. Therefore,
apoC3 was next used to determine whether the inhibition of
hypothalamic LPL can block the effects of Angptl3 on energy
metabolism. In contrast to Angptl3 and heparin, ICV injection of apoC3 strongly suppressed hypothalamic LPL activity
(Fig. 6B). To investigate the effect of hypothalamic LPL inhibition on energy metabolism, apoC3 (30–1,000 ng) was
administered ICV to freely fed mice before the end of
the daily light cycle. ApoC3 treatment stimulated nighttime
food intake in a dose-dependent manner (Fig. 6C), although
these effects were not sustained for 24 h postinjection (data
not shown). The ability of apoC3 to block the effects of
exogenous Angptl3 on hypothalamic LPL and metabolic
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Figure 5—Hypothalamic LPL activation by heparin affects energy metabolism. A and B: ICV injection of heparin (5–10 units in mice or 20
units in rats) increases the hypothalamic LPL activity in mice and CSF LPL activity in rats. *P < 0.05 vs. saline. C and D: ICV administration
of the LPL activator heparin decreased nighttime food intake and body weight (n = 5). *P < 0.05 vs. saline. E: The MBH LPL activity in mice
fasted for 24 h or refed for 1 and 2 h before being killed (n = 5). *P < 0.05 vs. 24-h fast. Data are reported as the mean 6 SEM.
behavior was tested next. ApoC3 (0.1 mg) was administered to freely fed mice at the beginning of the dark cycle,
30 min prior to the injection of Angptl3 (1 mg). ApoC3
pretreatment markedly blunted ICV Angptl3–induced
changes to hypothalamic LPL activity, food intake, body
weight, EE, and RQ (Fig. 6D–F), implying that hypothalamic Angptl3 regulates energy metabolism by activating
hypothalamic LPL.
To further confirm a role for hypothalamic LPL as
a downstream mediator of Angptl3, hypothalamic LPL was
depleted by injecting an LPL-specific siRNA into the bilateral
MBH (Fig. 6G). Similar to mice with a neuron-specific depletion of LPL (10), mice with an siRNA-mediated reduction
in hypothalamic LPL had a higher body weight than mice
injected with a scrambled control siRNA (Fig. 6H). In these
mice, ICV Angptl3–induced anorexia and weight loss were
significantly blunted (Fig. 6H).
Angptl3 Regulates Feeding via Hypothalamic LipidSensing Pathway
Angptl3-induced hypothalamic LPL activation would be
expected to increase LCFA uptake by the hypothalamic
neurons (10). After uptake, LCFAs are esterified to LCFACoA by acyl CoA synthetase (ACS) and accumulate in hypothalamic neurons (4). Increased hypothalamic LCFA-CoA
signals to the hypothalamus that energy is in excess, leading
to reduced food intake, as depicted in Fig. 7A (4). Direct
measurement of LCFA and LCFA-CoA revealed that
ICV-injected Angptl3 significantly increased the ratios of
hypothalamic versus plasma levels of both saturated (C14:0
and C16:0) and unsaturated (C18:1n9, C20:4n6, and
C22:6n3) LCFAs (Fig. 7B). Furthermore, hypothalamic
stearoyl-CoA was significantly increased at 2 h after Angptl3
administration (Fig. 7C). Hypothalamic oleoyl-CoA levels
were also marginally elevated, while palmitoyl-CoA levels
were unaltered by Angptl3 treatment. The neuronal
malonyl-CoA levels have been suggested to be critical for
the hypothalamic regulation of energy homeostasis (34).
Notably, ICV-administered Angptl3 significantly increased
the hypothalamic malonyl-CoA levels but not the hypothalamic TG content (Fig. 7D and E). Thus, an increase in
hypothalamic malonyl-CoA and LCFA-CoA levels may contribute to the Angptl3-induced suppression of food intake.
In order to determine whether Angptl3 acts through
the hypothalamic lipid-sensing pathway, the ACS inhibitor Tri-C was administered prior to Angptl3 to inhibit the
formation of LCFA-CoA (Fig. 7A) (4,5). Consistent with
our hypothesis, the blockade of ACS by Tri-C significantly
attenuated the Angptl3-induced reduction in food intake
and body weight (Fig. 7F).
By contrast, AMPK activation inhibits the hypothalamic
lipid-sensing pathway by stimulating mitochondrial LCFACoA oxidation through sequential acetyl-CoA carboxylase
inhibition and carnitine palmitoyltransferase-1 activation (Fig. 7A) (4). To inhibit the hypothalamic lipid-sensing
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Angptl3 and Hypothalamic Lipoprotein Lipase
Diabetes Volume 64, April 2015
Figure 6—Inhibition of hypothalamic LPL blocks the central metabolic effects of Angptl3. A: Double immunofluorescence staining for
apoC3 and Angptl3 in the MBH of normal mice. B and C: ICV injection of apoC3 (100 ng) decreased hypothalamic LPL activity and
increased food intake (n = 5). *P < 0.05 vs. saline. D–F: Pretreatment with apoC3 blocked Angptl3-induced changes in LPL activity, food
intake, body weight, and energy metabolism (n = 526). *P < 0.05 vs. saline; †P < 0.05 vs. Angptl3 alone. G: Reduced hypothalamic LPL
mRNA levels in mice injected with LPL siRNA (siLPL) into the bilateral MBH (n = 425). *P < 0.01 vs. control siRNA (siControl). H: The effects
of ICV Angptl3 on food intake and body weight in mice injected with control siRNA (siCont) or siLPL (n = 526). *P < 0.05 vs. siControl plus
saline; †P < 0.05 vs. siControl plus Angptl3. Data are reported as the mean 6 SEM.
pathway, a low dose (0.2 nmol) of AICAR was centrally
administered prior to Angptl3 injection in mice that had
been fasted overnight. AICAR treatment alone did not further increase fasting-induced hyperphagia and weight gain,
as hypothalamic AMPK activity would already be elevated
in these fasted mice (Fig. 7G). Nevertheless, pretreatment
with AICAR negated the Angptl3-induced anorexia and
weight reduction (Fig. 7G). Consistent with these feeding
effects, prior administration of Tri-C and AICAR attenuated the Angptl3-induced increase in the hypothalamic
stearoyl-CoA content (Fig. 7H). These data highlight the
importance of hypothalamic lipid-sensing pathways in
the Angptl3-mediated regulation of energy balance.
DISCUSSION
Accumulating data suggest that hypothalamic lipid
sensing is important for the maintenance of energy
homeostasis. Neuronal LPL appears to modulate an
early and important step in hypothalamic lipid sensing
by controlling lipid uptake into the neurons. However, the
regulatory mechanisms of hypothalamic LPL activity are
not well understood.
In the current study, we demonstrated that Angptl3,
a well-known regulator of peripheral LPL, is highly
expressed in hypothalamic neurons including POMC and
AGRP neurons. Moreover, ICV administration of Angptl3
increased hypothalamic LPL activity, whereas the inhibition of hypothalamic Angptl3 suppressed it. This
finding was unexpected and contrasts with the inhibitory
effects of these Angptls on peripheral LPL (19). In the
periphery, LPL activity is modulated by cleavage of LPL
at the linker region between its N-terminal catalytic domain and COOH-terminal lipid-binding domain (31). LPL
cleavage promotes its dissociation from the cell surface,
thereby inactivating it. Angptl3 enhances LPL cleavage
induced by proprotein convertases such as furin, PACE4,
and PCSK5 (31). In our current study, Angptl3 administration significantly increased the hypothalamic LPL
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Kim and Associates
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Figure 7—Angptl3 acts through the hypothalamic lipid-sensing pathway. A: Diagram depicting the hypothalamic lipid-signaling pathways
through which Angptl3 regulates food intake. The ACS inhibitor Tri-C and the AMPK activator AICAR inhibit hypothalamic lipid sensing by
reducing intracellular accumulation of LCFA-CoA. B–E: The ratios of hypothalamic vs. plasma levels of LCFA and hypothalamic LCFA-CoA,
malonyl-CoA, and TG contents measured at 2 h after ICV injection of saline and Angptl3 in mice fasted for 5 h (n = 527). *P < 0.05 vs.
saline. FFA, free fatty acid. F–H: Pretreatment with Tri-C or AICAR blocked the ICV Angptl3-induced anorexia, weight loss, and elevation in
the stearoyl-CoA levels (n = 526). *P < 0.05 vs. saline; †P < 0.05 vs. Angptl3 alone. Data are reported as the mean 6 SEM.
protein expression, but had no effect on hypothalamic
LPL mRNA levels and PACE4 and PCSK5 protein levels.
Therefore, Angptl3 activates hypothalamic LPL through
as yet unidentified post-transcriptional regulation.
We have shown that altered hypothalamic Angptl3
expression affects multiple aspects of energy metabolism. The siRNA-mediated inhibition of hypothalamic
Angptl3 caused increased food intake, decreased EE, and
increased RQ, leading to a positive energy balance.
Conversely, ICV injection of Angptl3 induced anorexia
via a mechanism unrelated to taste aversion, and
stimulated EE and fat use during the dark period. ICV
injection of Angptl3 increased the c-fos expression level
in hypothalamic ARC, indicating that Angptl3 may act
primarily on ARC neurons. Furthermore, Angptl3 administration potently inhibited hypothalamic NPY and AGRP
expression, which may account for Angptl3-induced anorexia and weight loss.
Interestingly, the central metabolic effects of Angptl3
were similar to those of Angptl4 (16), indicating a functional similarity between these closely related Angptls (11).
Despite similarities in structure and function, Angptl3 and
Angptl4 act through different signaling pathways in the
hypothalamus. Indeed, Angptl4 potently suppressed hypothalamic AMPK activity, while Angptl3 did not.
Recently, the involvement of neuronal LPL in body weight
metabolism was demonstrated in mice with a neuron-specific
deficiency in LPL (10). These mice developed obesity,
which may be due to transient hyperphagia and reduced
EE. In line with these findings, inhibition of hypothalamic
LPL by ICV apoC3 and intra-MBH injection of LPL
siRNA increased food intake and attenuated Angptl3induced anorexia. By contrast, hypothalamic LPL activation induced by ICV administration of Angptl3 and
heparin caused anorexia and weight loss. These data
confirm a key role for hypothalamic LPL in controlling
energy balance. However, some discrepancy exists between our findings and the phenotypes in neuronal LPL
knockout mice (10). The metabolic effects of acute hypothalamic LPL inactivation may differ from those of
chronic neuronal LPL depletion. Otherwise, Angptl3
might act through mechanisms other than hypothalamic LPL.
Hypothalamic n-3 fatty acid levels were reduced in
mice lacking neuronal LPL (10). Consistent with these
levels, we found that hypothalamic LCFA and LCFA-CoA
levels increased after ICV injection of Angptl3. These findings support an important role for neuronal LPL in neuronal uptake of TG-rich lipoprotein-derived fatty acids.
Notably, LPL activity in the hypothalamus increased in
the postprandial state. Given that hypothalamic Angptl3
expression increased under the same condition, it can be
reasonably speculated that Angptl3 mediates postprandial
LPL activation in the hypothalamus.
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Angptl3 and Hypothalamic Lipoprotein Lipase
Interestingly, the LPL inhibitor apoC3 was expressed in
ependymal lining cells and MBH neurons. Hypothalamic
expression of these LPL regulators strongly implies the
presence of local mechanisms that regulate LPL activity
depending on nutrient availability. Along with increased
LCFA-CoA levels, Angptl3 treatment significantly elevated
the MBH content of malonyl-CoA, an important lipid
metabolite that affects systemic energy metabolism. Although the mechanism mediating this effect is unclear, the
anorexigenic effect of Angptl3 may be partly attributable to
increased hypothalamic malonyl-CoA levels.
Enhanced LPL activity may activate hypothalamic
lipid-sensing pathways by increasing neuronal uptake of
LCFA. Neuronal LCFA-CoA levels are critical for hypothalamic lipid sensing, and are affected by both LCFA esterification and mitochondrial oxidation of LCFA (Fig. 6A) (4). By
blocking LCFA esterification with Tri-C and by activating
LCFA oxidation with AICAR, we demonstrated that hypothalamic lipid-sensing pathways mediate the effects of
Angptl3 on food intake, body weight, and hypothalamic
LCFA-CoA levels. Since hypothalamic sensing of circulating
lipid levels also controls hepatic glucose production (4), it will
be interesting to investigate the effects of hypothalamic
Angptl3 on peripheral glucose metabolism.
In summary, this study identified hypothalamic
Angptl3 as a key regulator of energy balance, which acts
through hypothalamic LPL and lipid-sensing pathways.
Acknowledgments. The authors thank Julie Roda and Stephen Cooke of
Bioedit Corporation for their help in the preparation of the manuscript.
Funding. This study was supported by grants from the National Research
Foundation of Korea (NRF-2013R1A1A3010137 and 2013M3C7A1056024), the
ASAN Institute for Life Sciences (2013-326), and the Korean Diabetes Association
(to M.-S.S., 2012).
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
Author Contributions. H.-K.K. and M.-S.S. performed the experiments
and the data analysis and wrote the manuscript. B.-S.Y. and J.H.C. contributed to
the discussion. G.M.K., S.Y.G., C.H.L., and H.S.L. performed the experiments.
H.J.Y. performed the experiments and contributed to the discussion. M.-S.K.
wrote the manuscript and directed the study. M.-S.K. is the guarantor of this
work and, as such, had full access to all the data in the study and takes
responsibility for the integrity of the data and the accuracy of the data analysis.
References
1. Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Central
nervous system control of food intake and body weight. Nature 2006;443:
289–295
2. Jordan SD, Könner AC, Brüning JC. Sensing the fuels: glucose and lipid
signaling in the CNS controlling energy homeostasis. Cell Mol Life Sci 2010;67:
3255–3273
3. Obici S, Feng Z, Morgan K, Stein D, Karkanias G, Rossetti L. Central administration of oleic acid inhibits glucose production and food intake. Diabetes
2002;51:271–275
4. Yue JT, Lam TK. Lipid sensing and insulin resistance in the brain. Cell
Metab 2012;15:646–655
5. Obici S, Feng Z, Arduini A, Conti R, Rossetti L. Inhibition of hypothalamic
carnitine palmitoyltransferase-1 decreases food intake and glucose production.
Nat Med 2003;9:756–761
Diabetes Volume 64, April 2015
6. Namkoong C, Kim MS, Jang PG, et al. Enhanced hypothalamic AMPactivated protein kinase activity contributes to hyperphagia in diabetic rats. Diabetes 2005;54:63–68
7. Mead JR, Irvine SA, Ramji DP. Lipoprotein lipase: structure, function, regulation, and role in disease. J Mol Med (Berl) 2002;80:753–769
8. Eckel RH, Robbins RJ. Lipoprotein lipase is produced, regulated, and
functional in rat brain. Proc Natl Acad Sci U S A 1984;81:7604–7607
9. Wang H, Eckel RH. What are lipoproteins doing in the brain? Trends Endocrinol Metab 2014;25:8–14
10. Wang H, Astarita G, Taussig MD, et al. Deficiency of lipoprotein lipase in
neurons modifies the regulation of energy balance and leads to obesity. Cell
Metab 2011;13:105–113
11. Hato T, Tabata M, Oike Y. The role of angiopoietin-like proteins in angiogenesis and metabolism. Trends Cardiovasc Med 2008;18:6–14
12. Wang Y, Quagliarini F, Gusarova V, et al. Mice lacking ANGPTL8
(Betatrophin) manifest disrupted triglyceride metabolism without impaired glucose homeostasis. Proc Natl Acad Sci U S A 2013;110:16109–
16114
13. Oike Y, Akao M, Yasunaga K, et al. Angiopoietin-related growth
factor antagonizes obesity and insulin resistance. Nat Med 2005;11:400–
408
14. Sukonina V, Lookene A, Olivecrona T, Olivecrona G. Angiopoietin-like protein
4 converts lipoprotein lipase to inactive monomers and modulates lipase activity
in adipose tissue. Proc Natl Acad Sci U S A 2006;103:17450–17455
15. Bäckhed F, Manchester JK, Semenkovich CF, Gordon JI. Mechanisms underlying the resistance to diet-induced obesity in germ-free mice. Proc Natl Acad
Sci U S A 2007;104:979–984
16. Kim HK, Youn BS, Shin MS, et al. Hypothalamic Angptl4/Fiaf is a novel
regulator of food intake and body weight. Diabetes 2010;59:2772–2780
17. Koishi R, Ando Y, Ono M, et al. Angptl3 regulates lipid metabolism in mice.
Nat Genet 2002;30:151–157
18. Feng SQ, Chen XD, Xia T, et al. Cloning, chromosome mapping and expression characteristics of porcine ANGPTL3 and -4. Cytogenet Genome Res
2006;114:44–49
19. Shimizugawa T, Ono M, Shimamura M, et al. ANGPTL3 decreases very low
density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase. J Biol
Chem 2002;277:33742–33748
20. Fujimoto K, Koishi R, Shimizugawa T, Ando Y. Angptl3-null mice show low
plasma lipid concentrations by enhanced lipoprotein lipase activity. Exp Anim
2006;55:27–34
21. Köster A, Chao YB, Mosior M, et al. Transgenic angiopoietin-like (angptl)4
overexpression and targeted disruption of angptl4 and angptl3: regulation of
triglyceride metabolism. Endocrinology 2005;146:4943–4950
22. Minicocci I, Montali A, Robciuc MR, et al. Mutations in the ANGPTL3 gene
and familial combined hypolipidemia: a clinical and biochemical characterization.
J Clin Endocrinol Metab 2012;97:E1266–E1275
23. Kim MS, Pak YK, Jang PG, et al. Role of hypothalamic Foxo1 in the
regulation of food intake and energy homeostasis. Nat Neurosci 2006;9:
901–906
24. Nirogi R, Kandikere V, Mudigonda K, et al. A simple and rapid method to
collect the cerebrospinal fluid of rats and its application for the assessment of
drug penetration into the central nervous system. J Neurosci Methods 2009;178:
116–119
25. Kim HK, Shin MS, Youn BS, et al. Involvement of progranulin in hypothalamic glucose sensing and feeding regulation. Endocrinology 2011;152:4672–
4682
26. Dodds ED, McCoy MR, Rea LD, Kennish JM. Gas chromatographic quantification of fatty acid methyl esters: flame ionization detection vs. electron impact
mass spectrometry. Lipids 2005;40:419–428
27. Haynes CA, Allegood JC, Sims K, Wang EW, Sullards MC, Merrill AH Jr.
Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid
diabetes.diabetesjournals.org
chromatography-electrospray ionization tandem mass spectrometry. J Lipid
Res 2008;49:1113–1125
28. Gil SY, Youn BS, Byun K, et al. Clusterin and LRP2 are critical components
of the hypothalamic feeding regulatory pathway. Nat Commun 2013;4:1862
29. Parton LE, Ye CP, Coppari R, et al. Glucose sensing by POMC neurons
regulates glucose homeostasis and is impaired in obesity. Nature 2007;449:
228–232
30. Xu AW, Kaelin CB, Takeda K, Akira S, Schwartz MW, Barsh GS. PI3K integrates the action of insulin and leptin on hypothalamic neurons. J Clin Invest
2005;115:951–958
Kim and Associates
1153
31. Liu J, Afroza H, Rader DJ, Jin W. Angiopoietin-like protein 3 inhibits lipoprotein lipase activity through enhancing its cleavage by proprotein convertases.
J Biol Chem 2010;285:27561–27570
32. Braun JE, Severson DL. Release of lipoprotein lipase from cardiac myocytes
by low-molecular weight heparin. Lipids 1993;28:59–61
33. Jong MC, Hofker MH, Havekes LM. Role of ApoCs in lipoprotein metabolism:
functional differences between ApoC1, ApoC2, and ApoC3. Arterioscler Thromb
Vasc Biol 1999;19:472–484
34. Gao S, Moran TH, Lopaschuk GD, Butler AA. Hypothalamic malonyl-CoA and
the control of food intake. Physiol Behav 2013;122:17–24