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Regulation of energy balance by the hypothalamic lipoprotein lipase 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: angiopoietin-like protein 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 proteins (Angptls) are a family of proteins that share two structural domains with the angiopoietins: the N-terminal coiled-coil domain (CCD) and the C-terminal fibrinogen-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