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Regulation of energy balance by the hypothalamic regulator Angptl3

Hyun-Kyong Kim1*, Mi-Seon Shin2*, Byung-Soo Youn3, Gil Myoung Kang1, So Young Gil1,

Chan Hee Lee1, Jong Han Choi1,2, Hyo Sun Lim1, Hyun Ju Yoo4, Min-Seon Kim1,2

1Appetite Regulation Laboratory and 4Biomedical Research Center, Asan Institute for Life

Sciences, 2Division of Endocrinology and Metabolism, Department of Internal Medicine,

Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Korea,

3Department of Anatomy, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-

749, Korea

*Both authors contributed equally to this study.

Running title: Angptl3 and hypothalamic lipoprotein lipase

Correspondence and reprint requests:

Min-Seon Kim, M.D., Ph.D.

Division of Endocrinology and Metabolism

Asan Medical Center

University of Ulsan College of Medicine

88 Olympic-ro 43-gil, Songpa-ku, Seoul 138-736, Korea

Email: [email protected]

Tel: +82-2-3010-3245 ; Fax: +82-2-3010-6962

1

Diabetes Publish Ahead of Print, published online October 22, 2014 Diabetes Page 2 of 39

Abstracts

Hypothalamic lipid sensing is important for the maintenance of energy balance.

Angiopoietin-like peptide 3 (Angptl3) critically regulates the clearance of circulating lipids by inhibiting lipoprotein lipase (LPL). The present 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, co-administration 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.

Key words: -like 3, lipoprotein lipase, hypothalamus, lipid sensing, energy metabolism

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Introduction

Obesity is a growing global health concern that underlies the development of type 2

diabetes, hypertension and 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 as 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 long-chain fatty acid (LCFA) suppresses

food intake and glucose production (3), indicating that fatty acids can signal nutrient

availability to the CNS and thereby restrict the additional release of nutrients into the

circulation. Accumulating data suggest that esterified long-chain fatty acid (LCFA-CoA) is an

important lipid metabolite in hypothalamic lipid sensing pathways (4). Hypothalamic

inhibition of carnitine palmitoyltransferase-1 (CPT-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 LCFA-CoA formation, supporting a crucial role for LCFA-

CoA in hypothalamic lipid sensing (5). By contrast, hypothalamic activation of AMP-

activated protein kinase (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 (TG) in circulating TG-rich lipoproteins and promotes the cellular uptake of

chylomicron remnants, cholesterol-rich 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,

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mice with a neuron-specific 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 (Angptls) are a family of proteins that share two structural domains with the : the N-terminal coiled-coil domain (CCD) and the C-terminal -like domain (FLD) (11). Among the eight Angptls, Angptl 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 anti-obesity effects of 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 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.

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Research Design and Methods

Peptides and compounds

Full length Angptl3 and Angptl4 were purchased from Adipogen (Incheon, Korea), and

apolipoprotein C3 (ApoC3) was purchased from Sigma (St. Louis, MO). Triacsin-C (Tri-C)

and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) were purchased from Sigma

and from Toronto Research Chemicals (Toronto, Canada), respectively. Heparin was

obtained from Halim Pharmaceuticals (Seoul, Korea).

Animals

Adult male C57BL/6 mice and Sprague-Dawley rats (8−10 weeks-of-age) were purchased

from Orient Bio (Seoul, Korea). Animals were allowed free access to a standard chow diet

(Agripurina Inc, Seoul, Korea) and water unless otherwise indicated. Animals were housed

under conditions of controlled temperature (22 ± 1°C) and a 12 h light-dark cycle, with the

light on from 08:00 to 20:00 h. All procedures were conducted in accordance with the

Principles of Laboratory Animal Care (NIH, Bethesda, MD) and were approved by the

Institutional Animal Care and Use Committee at the Asan Institute for Life Sciences (Seoul,

Korea). AGRP (Agouti related protein)-neuron-specific eGFP-expressing (AGRP-eGFP) mice

were generated by crossing AGRP-IRES-Cre mice (obtained from Dr. Joel. K. Elmquist,

University of Texas Southwestern) with eGFP-floxed mice (Jackson Laboratory, Bar Harbor,

Maine)

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). Following a 7-day recovery

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period, correct positioning of each cannula was confirmed by a positive dipsogenic response to angiotensin-2 (50 ng per mouse). All peptides and compounds were dissolved in 0.9% saline just before use and administered via the-ICV-implanted cannulae in a total volume of 2

l. 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 following the injections.

siRNA study

Small inhibitory RNAs (siRNAs) specific for murine Angptl3 (E-065291-00-0020) and

LPL (STOP-130319-015) and a non-targeting scrambled siRNA (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. knockdown was considered successful when hypothalamic Angptl3 and LPL mRNA levels fell to less than 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

Lab Animal Monitoring System (CLAMS: Columbus Instruments, Columbus, OH) over

24−48 h following treatment. Animals were acclimatized in metabolic cages for 3 days before treatment.

LPL activity

Saline, Angptl3 (1 g), Angptl4 (3.5 g), ApoC3 (0.1 g) and heparin (5−10 units) were

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injected via ICV-implanted cannulae just before the beginning of the dark period, unless

otherwise indicated. The MBH was collected at 2 h post-injections. 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 l of cold 20 mM

Tris buffer (pH 7.5) containing 150 mM NaCl. Following centrifugation at 10000 g 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 U) as previously

described (24). LPL activity was measured in 50 l amounts of CSF.

AMPK activity

The MBH was collected 2 h after ICV injection of saline, Angptl3 (1 g), or Angptl4 (3.5

g) into overnight-fasted mice during the early light phase. The tissue was then flash frozen

in liquid nitorogen. Total AMPK activity was determined as previously described (25).

Measurement of LCFA, LCFA-CoA and triglycerides

To measure LCFA, free fatty acids were extracted from 10−20 mg of hypothalamic tissue

or from 80 l of plasma using 400 l methanol and isooctane. The extracted free fatty acids

were derivatized using BCl3-methanol (26). A standard mixture of fatty acid methyl esters

(FAME) obtained from Sigma-Aldrich was used to generate calibration curves. FAME was

analyzed with a gas chromatography-mass spectrometry (GC-MS) system (Agilent

7890A/5975C) using a capillary column (HP-5MS, 30 m × 0.25 mm × 0.2 µm) and electron

impact ionization. To measure LCFA-CoA, samples were prepared from 10−15 mg of

hypothalamic tissues as previously described (27). The dried samples were stored at -20°C

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and reconstituted with 30 µl of H2O/acetonitrile (50/50 vol/vol) prior to analysis. Liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was performed using the 1290

Infinity Binary HPLC system (Agilent), the Qtrap 5500 (ABSciex), and a reverse phase column (Zorbax 300 Extend-C18, 2.1 × 150 mm). Analytical conditions were the same as those previously described (27). Hypothalamic triglyceride contents were determined using a colorimetric assay (Abcam). MBH blocks were homogenized with 60 l of 5% NP-40. The homogenate was centrifuged and 50 l 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 following a 5 h fast. Following post-fixation and dehydration, coronal brain sections (14

m thick) were cut using a cryostat (Leica, Wetzlar, Germany) and incubated with primary antibodies against the Angptl3 N-terminal FLD (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), β-endorphin (1:10000, rabbit;

Phoenix Pharmaceuticals), c-fos (1:100, rabbit; Santa Cruz), LPL (1:100, rabbit; Santa Cruz), or eGFP (1:1000, 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 (1:10000, 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

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mRNA expression levels of Angptls and LPL were determined by real-time PCR or semi-

quantitative RT-PCR (the primer sequences shown in Supplemental Table 1) and normalized

to levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA.

Immunoblotting

Tissue protein were extracted from hypothalamic tissue blocks as previously described

(16) and subjected to immunobloting using primary antibodies against Angptl3 (1:2000,

mouse; Abcam), LPL (1:1000, rabbit; Santa Cruz), PACE4 (1:1000, rabbit; Santa Cruz),

PCSK5 (1:1000, 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 (CTA) test

CTA tests were conducted following sequential injection of intraperitoneal (IP) saline plus

ICV saline, IP lithium chloride (127 mg/kg, Sigma) plus ICV saline, and IP saline plus ICV

Angptl3 (1 g), using a previously described protocol (28).

Statistical analysis

All data are expressed as the means ± SEM. Statistical analysis was performed using

SPSS 17.0 software (SPSS Inc., Chicago, IL). Comparisons between two groups were

analyzed using the Student’s t-test. Comparisons between 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.

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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 (Figure 1A). Immunoblotting analysis also revealed the presence of full-length Angptl3 (62 kD) in protein extracts of the hypothalamic arcuate nucleus (ARC), the lateral hypothalamic area (LH) and the paraventricular nucleus

(PVN) collected from 5 h-fasted mice using the punch biopsy technique (Figure 1B). A higher expression of Angptl3 was found in the MBH and LH compared with the PVN. To verify whether MBH Angptl3 protein expression is altered by nutrient availability (Figure

1C), mice were either kept under fasted conditions for 24 h before sacrifice or allowed to resume food intake during the beginning of the dark period following a 24 h fast.

Hypothalamic Angptl3 protein levels were higher in re-fed mice than in fasting mice (Figure

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 (Figure 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 (Figure 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 β-

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endorphin revealed that more than 90% of POMC neurons expressed Angptl3. Dual staining

for Angptl3 and eGFP in AGRP-eGFP mice revealed Angptl3 expression in approximately

40-50% of AGRP neurons (Figure 1E). Notably, a considerable proportion of Angptl3-

expressing 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 (Figure 2 A and B). Compared with controls injected with a non-

targeting siRNA, mice with reduced hypothalamic Angptl3 expression consumed more food

and recovered more quickly from post-surgical weight loss (Figure 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 (Figure 2D, E). Considering that hypothalamic

Angptl3 expression increases upon food intake (Figure 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 g) was administered via 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 (Figure 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 post-injection (Figure 3B).

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To test the possibility that ICV Angptl3 may increase aversion to food, we performed a CTA test following ICV injection of Angptl3. In contrast to IP injection of the established CTA inducer lithium chloride, which decreased saccharine consumption, ICV Angptl3 injection did not induce CTA (Figure 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 g) was injected via 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 (Figure 3D), indicating that ICV Angptl3 stimulates energy expenditure 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 if hypothalamic neurons are responsive to Angptl3 by employing c-fos immunostaining following ICV injection of Angptl3 (1 g). The c-fos immunoreactive neurons were mostly found in the hypothalamic ARC of the Angptl3-treated mice (Figure

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 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 g) whilst POMC mRNA expression was not altered (Figure

3F). Thus, Angplt3 may regulate energy balance via the down-regulation of hypothalamic

NPY and AGRP.

Angptl3 stimulates hypothalamic LPL activity

We further sought to identify the signaling pathways via which Angptl3 regulates feeding

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behavior and body weight. Hypothalamic AMPK activity was determined following ICV

injection of Angptl3 (1 g), as AMPK is an important downstream mediator of Angptl4 in

the hypothalamus (16). Consistent with a previous report, ICV injection of Angptl4 (3.5 g)

potently suppressed AMPK activity in the MBH at 2 h post-injection. By contrast, ICV

Angptl3 had no effect on hypothalamic AMPK activity (Figure 4A). The effects of ICV-

injected Agnptl3 on hypothalamic Stat3 and on PI3K-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 Figure 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 sacrifice 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 g) increased LPL activity in the MBH by approximately 1.8-fold

(Figure 4B). Consistently, hypothalamic LPL activity was found to be lower in mice injected

with Angptl3 siRNA than in mice injected with non-targeting siRNA following a 5 h fast

(Figure 4C). ICV Angptl4 (3.5 g) also caused a modest but significant increase in

hypothalamic LPL activity (Figure 4B).

We further investigated the mechanisms of Angptl3-mediated regulation of

hypothalamic LPL activity. By using immunoblotting and immunofluorescence staining, we

demonstrated that Angptl3 (1 g) increased MBH LPL protein levels when ICV-injected in 5

h-fasted mice just before light-off while it did not increase hypothalamic LPL mRNA levels

(Figure 4D−F). In the periphery, Angptl3 enhances LPL cleavage induced by proprotein

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convertases [furin, paired amino acid converting enzyme-4 (PACE4), and proprotein convertase subtilisin/kexin type 5 (PCSK5)] (31). Hypothalamic PCAE4 and PCSK5 expression was unchanged following the ICV injection of Angptl3 (Figure 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 (Figure 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 following ICV injection of heparin (10 units)

(Figure 5A). LPL activity in the cerebrospinal fluid was also increased following ICV injection of heparin (20 U) in Sprague-Dawley rats (Figure 5B). Similar to Angptl3, ICV injection of heparin suppressed night time food intake and decreased body weight when injected before the beginning of the dark period (Figure 5C, 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 re-feeding conditions.

Hypothalamic LPL activity increased after food intake (Figure 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 (Figure 6A). In addition, a certain population of hypothalamic

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neurons coexpressed Angptl3 and ApoC3. Therefore, ApoC3 was next use to determine

whether 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 (Figure 6B). To investigate the effect of hypothalamic LPL

inhibition on energy metabolism, ApoC3 (30−1000 ng) was administered ICV to freely fed

mice before the end of the daily light cycle. ApoC3 treatment stimulated night time food

intake in a dose-dependent manner (Figure 6C) although these effects were not sustained for

24 h post-injection (data not shown). The ability of ApoC3 to block the effects of exogenous

Angptl3 on hypothalamic LPL and metabolic behavior was tested next. ApoC3 (0.1 g) was

administered to freely fed mice at the beginning of the dark cycle, 30 min prior to injection of

Angptl3 (1 g). ApoC3 pretreatment markedly blunted ICV Angptl3-induced changes to

hypothalamic LPL activity, food intake, body weight, EE, and RQ (Figure 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

(Figure 6G). Similar to mice with a neuron-specific depletion of LPL (10), mice with a

siRNA-mediated reduction in hypothalamic LPL had a higher body weight than mice injected

with a scrambled control siRNA (Figure 6H). In these mice, ICV Angptl3-induced anorexia

and weight loss were significantly blunted (Figure 6H).

Angptl3 regulates feeding via hypothalamic lipid sensing pathway

Angptl3-induced hypothalamic LPL activation would be expected to increase LCFA

uptake by the hypothalamic neurons (10). Following uptake, LCFAs are esterified to LCFA-

CoA by acyl CoA synthetase (ACS) and accumulates in hypothalamic neurons (4). Increased

hypothalamic LCFA-CoA signals to the hypothalamus that energy is in excess, leading to

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reduced food intake as depicted in Figure 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 (Figure 7B). Furthermore, hypothalamic stearoyl-CoA was significantly increased at 2 h after Angptl3 administration (Figure 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 triglyceride content (Figure 7D, 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 acetyl-CoA synthase (ACS) inhibitor Tri-C was administered prior to Angptl3 to inhibit the formation of LCFA-CoA (Figure 7A) (4; 5). Consistent with our hypothesis, blockade of ACS by Tri-C significantly attenuated the Angptl3-induecd reduction in food intake and body weight (Figure 7F).

By contrast, AMPK activation inhibits the hypothalamic lipid sensing pathway by stimulating mitochondrial LCFA-CoA oxidation through sequential acetyl-CoA carboxylase

(ACC) inhibition and CPT-1 activation (Figure 7A) (4). To inhibit the hypothalamic lipid sensing 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 be already elevated in these fasted mice (Figure 7G). Nevertheless, pretreatment with AICAR negated the Angptl3-induced anorexia and weight reduction

(Figure 7G). Consistent with these feeding effects, prior administration of Tri-C and AICAR

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attenuated the Angptl3-induced increase in the hypothalamic stearoyl-CoA content (Figure

7H). These data highlight the importance of hypothalamic lipid sensing pathways in the

Angptl3-mediated regulation of energy balance.

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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 present 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 C-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 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 utilization during the dark period. ICV injection of

Angptl3 increased the c-fos expression level in hypothalamic ARC, indicating that Angptl3 may primarily act on ARC neurons. Furthermore, Angptl3 administration potently inhibited

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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 similarly 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 energy expenditure. In line

with these findings, inhibition of hypothalamic LPL by ICV ApoC3 and intra-MBH injection

of LPL siRNA increased food intake and attenuated Angptl3-induced 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

activation may differ from those of chronic neuronal LPL depletion. Otherwise, Angptl3

might act through the mechanisms other than hypothalamic LPL.

Hypothalamic n3-fatty acid levels were reduced in mice lacking neuronal LPL (10).

Consistent with these, we found that hypothalamic LCFA and LCFA-CoA increased

following 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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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.

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

(Figure 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.

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Acknowledgement

This study was supported by grants from the National Research Foundation of Korea (NRF-

2013R1A1A3010137, and NRF-2013056024), the Asan Institute for Life Science (2013-326),

and the Korean Diabetes Association (to M.S.S., 2012). We thank Julie Roda and Stephen

Cooke of Bioedit Corporation for their help in the preparation of this manuscript. Dr. Min-

Seon Kim 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.

Author Contributions

H.-K.K., M.-S.S., G.M.K., S.Y.G., C.H.L., H.S.L., H.J.Y. performed experiments; H.-K.K., M.-

S.S. data analysis and wrote manuscript; B.-S.Y., J.H.C., H.J.Y. contributed to discussion; M.-

S.K. wrote the manuscript and directed to this work.

Conflicts of Interest: Nothing to declare.

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Figure Legends

Figure 1. Angptl3 is expressed in hypothalamic neurons

(A) The mRNA expression of Angptls in hypothalamus of C57BL/6 mice. (B) Angptl3 protein expression in the hypothalamic arcuate nucleus (ARC), paraventricular nucleus

(PVN), and lateral hypothalamus (LH) in normal mice fasted for 5 h. (C) MBH Angptl3 protein expression according to feeding conditions. Mice were fasted for 24 h or re-fed for 1 h and 2 h after a 24 h fasting before sacrifice (n = 3−4). (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 (eGFP) or POMC (β-endorphin) in the MBH, showing that Angptl3 was expressed in POMC or AGRP neurons (indicated by white arrows).

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 a recovery from post-surgical weight loss (n = 6). (D, E) Reduced energy expenditure and increased RQ in mice injected with Angptl3 siRNA than in mice injected with scrambled control siRNA

(siControl) (n = 6). *P < 0.05 vs. control siRNA. Data are the means ± SEM.

Figure 3. Central administration of Angptl3 causes a negative energy balance

(A, 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 g) inhibited fasting-induced hyperphagia

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and weight gain. *P < 0.05 vs. saline. (C) CTA tests after ICV injection of Angptl3 and IP

injection of lithium chloride (n = 5). *P < 0.05 vs. saline, NS; not significant. (D) The effects

of ICV injection of Angptl3 on energy expenditure and RQ. Angptl3 (1 g) was injected ICV

just before the end of the daily light cycle (n = 4). *P < 0.05 vs. saline. (E) c-fos

immunohistochemistry in the mouse hypothalamus, which was analyzed 1 h after an ICV-

injection of saline or Angptl3 (1 g). (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 g) in overnight-fasted mice. *P < 0.01 vs. saline. Data are the means ± SEM.

Figure 4. Angptl3 stimulates hypothalamic LPL activity

(A) The effects of ICV injection of Angptl3 (1 g) and Angptl4 (3.5 g) on hypothalamic

AMPK activity (n = 5). **P < 0.005 vs. saline. (B) The effects of ICV administration of

Angptl3 (1g) and Angptl4 (3.5 g), 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. (D) LPL

immunofluorescence staining in the hypothalamus 1 h after ICV injection of either Angptl3

or saline. (E) Effects of ICV-injected Angptl3 (1 g) on hypothalamic LPL, PACE4, and

PCSK5 protein expression (n = 2). (F) Effects of ICV-injected Angptl3 (1 g) or heparin on

hypothalamic LPL, hepatic lipase (HPL) and endothelial lipase (EL) mRNA expression (n =

5). Data are the means ± SEM.

Figure 5. Hypothalamic LPL activation by heparin affects energy metabolism

(A, B) ICV injection of heparin (5-10 U in mice or 20 U in rats) increases the hypothalamic

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LPL activity in mice and CSF LPL activity in rats. *P < 0.05 vs. saline. (C, D) ICV administration of the LPL activator heparin decreased night time food intake and body weight

(n = 5). *P < 0.05 vs. saline. (E) The MBH LPL activity in mice fasted for 24 h or re-fed for

1 h and 2 h before sacrifice (n = 5). *P < 0.05 vs. 24 h fast. Data are the means ± SEM.

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, 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

= 5−6). *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 = 4−5). *P

< 0.01 vs. control siRNA (siControl). (H) The effects of ICV Angpl3 on food intake and body weight in mice injected with control siRNA (siCont) or LPL siRNA (siLPL) (n = 5−6). *P <

0.05 vs. siControl + saline; †P < 0.05 vs. siControl + Angptl3. Data are the means ± SEM.

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 versus plasma levels of LCFA and hypothalamic LCFA-CoA, malonyl-CoA, and triglyceride contents measured at 2 h after ICV injection of saline and

Angptl3 in mice fasted for 5 h (n = 5−7). *P < 0.05 vs. saline. (F-H) Pretreatment with Tri-C

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or AICAR blocked the ICV Angptl3-induced anorexia, weight loss and elevation in the

stearoyl-CoA levels (n = 5−6). *P < 0.05 vs. saline; †P < 0.05 vs. Angptl3-alone. Data are the

means ± SEM.

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Supplementary Figure Legends

Supplementary Figure 1. The effects of ICV injection of Angptl3 (1 g) on the hypothalamic Akt and STAT3 signaling. Angptl3 (1 g) or saline was injected ICV in mice fasted for 5 h (n = 3). The MBH was collected 1 h post-injection and subjected to immunoblot assay for phosphorylated and total Akt or STAT3. NS: not significant. Data are the means ± SEM.

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Supplementary Table 1. Primers used for real time PCR assay

Transcript Forward primer Reverse primer

Angptl1 GCT GGT GGT AGA TGT GGA CG ACG TCT CTG GGG TAA CCT GG

Angptl2 CAA GTT CCA GCA CCT GGC TA GAT GTT CCC AAA CCC TTG CT

Angptl3 AAG ATG ACC TTC CTG CCG AC GCC CAA CCA AAA TTC TCC AT

Angptl4 GAC AAG ACT TCG AGG GGA AA CAA GTA GAG AAG GGC AGG GA

Angptl6 ACT GTC ATC CAG AGA CGG CA GCT CCG GTA ATG ACC TCC AT

Angptl7 AGC AAG CAC ATG GAG TCT CG GGC GGT AGC TGT TCA GTT CA

LPL CAA GCT GGT GGG AAA TGA TG AGG TGG ACG TTG TCT AGG GG

Hepatic lipase GCA GAC CAT CAT GCT ATG GG AGG TCA TCC AGA TTT TCG GG Endothelial GCC CAT TCC TGG TAC AAC CT AAA ATG TCA CTT TGC GCT GG lipase POMC ATG CCG AGA TTC TGC TAC AG TCC AGC GAG AGG TCG AGT CTA

NPY GGA CTG ACC CTC GCT CTA TCG CAG AGC GGA GTA GTA

AGRP ACA ACT GCA GAC CGA GCA GAC GCG GAG AAC GAG ACT

1