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Daniel Beiroa,1,2 Monica Imbernon,1,2 Rosalía Gallego,3 Ana Senra,1 Daniel Herranz,4 Francesc Villaroya,2,5 Manuel Serrano,4 Johan Fernø,6 Javier Salvador,2,7 Javier Escalada,2,7 Carlos Dieguez,1,2 Miguel Lopez,1,2 Gema Frühbeck,2,7 and Ruben Nogueiras1,2
GLP-1 Agonism Stimulates Brown Adipose Tissue Thermogenesis and Browning Through Hypothalamic AMPK DOI: 10.2337/db14-0302
1Department of Physiology, CIMUS, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela, Spain 2CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn), Santiago de Compostela, Spain 3Department of Morphological Sciences, School of Medicine, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela, Spain 4Tumor Suppression Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain 5Department of Biochemistry and Molecular Biology and Institute of Biomedicine (IBUB), University of Barcelona, Barcelona, Spain 6Department of Clinical Science, K.G. Jebsen Center for Diabetes Research, University of Bergen, Bergen, Norway
hypothalamic sites and that these mechanisms might be clinically relevant.
GLP-1 is an incretin hormone released by L cells located in the ileum and colon into the bloodstream postprandially (1,2). Among its numerous physiological effects (3), GLP-1 increases insulin and decreases glucagon secretion in a glucose-dependent manner (4,5), slows gastric emptying (6), increases glucose disposal, and decreases appetite (7). Therefore, incretin hormone analogs acting on the GLP-1 receptor (GLP-1R) are considered the most promising new therapies for type 2 diabetes (T2D). Within the central nervous system (CNS), numerous neuronal populations express GLP-1R, including hypothalamic nuclei crucial for the regulation of energy balance (8). Furthermore, a large number of extrahypothalamic areas have GLP-1 binding sites (9). Indeed, it has been demonstrated that peripheral administration of GLP-1 leads to neuronal activation in various parts of the CNS (10), indicating the existence of brain sites accessible from the bloodstream. Finally, cells with GLP-1 mRNA are widely expressed in human brain areas (11).
7Department
of Endocrinology and Nutrition, Clínica Universidad de Navarra, Pamplona, Spain Corresponding author: Ruben Nogueiras, [email protected]. Received 21 February 2014 and accepted 30 April 2014. This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0302/-/DC1. © 2014 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 Publish Ahead of Print, published online June 10, 2014
OBESITY STUDIES
GLP-1 receptor (GLP-1R) is widely located throughout the brain, but the precise molecular mechanisms mediating the actions of GLP-1 and its long-acting analogs on adipose tissue as well as the brain areas responsible for these interactions remain largely unknown. We found that central injection of a clinically used GLP-1R agonist, liraglutide, in mice stimulates brown adipose tissue (BAT) thermogenesis and adipocyte browning independent of nutrient intake. The mechanism controlling these actions is located in the hypothalamic ventromedial nucleus (VMH), and the activation of AMPK in this area is sufficient to blunt both central liraglutide-induced thermogenesis and adipocyte browning. The decreased body weight caused by the central injection of liraglutide in other hypothalamic sites was sufficiently explained by the suppression of food intake. In a longitudinal study involving obese type 2 diabetic patients treated for 1 year with GLP-1R agonists, both exenatide and liraglutide increased energy expenditure. Although the results do not exclude the possibility that extrahypothalamic areas are also modulating the effects of GLP-1R agonists, the data indicate that long-acting GLP-1R agonists influence body weight by regulating either food intake or energy expenditure through various
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Liraglutide and Thermogenesis
In the hypothalamus, GLP-1 acts as a physiological satiety factor (7), and dose-dependent central administration of GLP-1 reduces feeding in rats (12). Central GLP-1 administration blunted fasting-induced neuropeptide Y and Agouti-related peptide levels and fasting-reduced proopiomelanocortin and cocaine- and amphetamineregulated transcript expression (13). Stimulation of the central GLP-1 system not only suppresses food intake but also regulates glucose homeostasis (14), behavioral responses to stress (15), and visceral illness (16). Moreover, brain GLP-1 modulates lipid metabolism in white adipose tissue (WAT) (17) and brown adipose tissue (BAT) thermogenesis (18) through the activation of the sympathetic nervous system. However, the molecular mechanisms mediating the actions of brain GLP-1 on adipose tissue as well as the brain areas responsible for these interactions are unknown. Furthermore, whether some of the beneficial effects exerted in the clinical setting by long-acting GLP-1 agonists could be mediated at the central level is yet unclear. One of these long-acting GLP-1 analogs is liraglutide, which injected once daily improves glycemic control in T2D with the additional benefits of weight loss and a low risk of hypoglycemia (19,20). As GLP-1R agonists start to be included in treatment guidelines, they are generally being recommended as second- or third-line therapies after the failure of one or more oral antidiabetic drugs (21). In the current study, we show that central stimulation of GLP-1R by the agonist liraglutide leads to body weight loss independent of reduction in food intake. Instead, this weight loss is caused by the activation of the thermogenic program in BAT. Specific injection of liraglutide in the hypothalamic ventromedial nucleus (VMH) is sufficient to cause food intake–independent weight loss and stimulation of BAT thermogenic activity. This regulatory mechanism depends on AMPK. RESEARCH DESIGN AND METHODS Animal Models
Swiss male mice (8–10 weeks old, 20–25 g) and SpragueDawley rats (8–10 weeks old, 200–250 g) were housed under conditions of controlled temperature (23°C) and illumination (12-h light/dark cycle). They were allowed ad libitum access to water and standard laboratory chow. Body composition was measured by a nuclear magnetic resonance imaging whole-body composition analyzer (EchoMRI, Houston, TX). Animals were killed by decapitation, and the tissues were removed rapidly, frozen immediately on dry ice, and kept at 280°C until analysis. All animal experiments and procedures were reviewed and approved by the Ethics Committee of the University of Santiago de Compostela in accordance with European Union norms for the use of experimental animals. Implantation of Intracerebroventricular Cannulae and Treatments
Rats and mice were anesthetized by an intraperitoneal injection of ketamine 80 and 8 mg/kg body weight,
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respectively, and xylazine 100 and 3 mg/kg body weight, respectively. Intracerebroventricular (ICV) cannulae were implanted stereotaxically in mice and rats, as described previously (22). Animals received vehicle, GLP-1 (1 mg/mouse), liraglutide (3 mg/mouse or 10 mg/rat), or AICAR (3 mg/mouse). Conditioned Taste Aversion
Conditioned taste aversion (CTA) was expressed as the percent saccharin preference ratio [(100 3 saccharin intake) / (saccharin intake + water intake)]. A saccharin preference ratio ,50% was considered a signal of CTA (23). Cold Exposure
Twenty-four hours after the liraglutide injection, animals were placed for 6 h in a room with a stable temperature of 4°C as previously described (24). Body temperature was recorded with a rectal probe connected to a digital thermometer, and the interscapular temperature was recorded with an infrared camera. Stereotaxic Microinjection
Sprague-Dawley rats were placed in a stereotaxic frame under ketamine-xylazine anesthetics. Liraglutide (10 mg/rat) was injected in the arcuate nucleus (ARC) (anterior to the bregma [AP], 22.85 mm; lateral to the sagittal suture [L], 60.3 mm; and ventral from the surface of the skull [V], 10.2 mm), in the lateral hypothalamus area (LHA) (AP, 22.85 mm; L, 62 mm; DV, 28.1 mm), in the VMH (AP, 22.85 mm; L, 60.6 mm; DV, 210mm), in the dorsomedial nucleus (DMH) (AP, 23.12 mm; L, 60.5 mm; DV, 28.6 mm), and in the paraventricular nucleus (PVH) (AP, 21.9 mm; L, 60.5 mm; DV, 28 mm) with a 25-gauge needle (Hamilton, Reno, NV) connected to a 1-mL syringe. Acetylsalicylic acid (Bayer, Leverkusen, Germany) 150 mg/kg was injected intraperitoneally after surgery as a painkiller. Liraglutide 5 mg and adenoviral vectors (green fluorescent protein [GFP] or AMPKa-CA [Viraquest, North Liberty, IA]) (25,26) were injected simultaneously into the VMH with a 25-gauge needle connected to a 2-mL syringe. Immunohistochemistry and Immunofluorescence
Rat brains were fixed, and diaminobenzidine immunohistochemistry and detection of GFP were performed as previously reported (22,25). RNA Isolation and Real-Time RT-PCR
Total RNA and real-time RT-PCR were performed as previously described (22). The primers and probes are described in Supplementary Table 1. Western Blot Analysis
Western blot was performed as previously described (22). Briefly, total protein lysates from epididymal WAT (30 mg) and BAT (15 mg) were subjected to SDS-PAGE, electrotransferred on a polyvinylidene difluoride membrane, and probed with the following antibodies: uncoupling protein (UCP) 1, UCP3, b-adrenoreceptor 1 (ADRb1), cell death–inducing DNA fragmentation factor a–like effector a (CIDEA), fibroblast growth factor 21 (FGF21), and PR
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domain containing 16 (PRDM16) (Abcam, Cambridge, U.K.); GAPDH (EMD Millipore, Billerica, MA); and a-tubulin (T-5168; Sigma-Aldrich, St. Louis, MO). Patient Selection
All clinical studies were approved from an ethical and scientific standpoint by the hospital’s ethics committee and were conducted in accordance with the principles of the Declaration of Helsinki, with patients giving their informed consent for participation. Twenty-five obese T2D patients matched for sex, age, and BMI were studied before (baseline) and after (12 6 3 months) antidiabetic treatment instauration with metformin in combination with either of the GLP-1 agonists exenatide or liraglutide. The initial dose of metformin was 500 mg b.i.d. orally, which was increased to 1,000 mg b.i.d. orally after 2 weeks. Exenatide treatment was initiated with 5 mg b.i.d. administered subcutaneously and increased to 10 mg b.i.d. after 2 weeks of good digestive tolerance. Liraglutide treatment was initiated with a single subcutaneous administration of 0.6 mg a day progressing to 1.2 mg a day after 2 weeks of adequate digestive tolerance. All patients were of Caucasian origin, attended the Endocrinology Department at the University Clinic of Navarra, and underwent a clinical assessment. Inclusion criteria were age between 20 and 80 years, hemoglobin A1c (HbA1c) levels $7.0%, and no previous treatment with insulin and/or a sulfonylurea. Exclusion criteria were lactating or pregnant women, uncontrolled treated or untreated hypertension, fasting C-peptide levels ,0.1 ng/mL, recurrent major hypoglycemia or hypoglycemic unawareness, known proliferative retinopathy or maculopathy requiring acute treatment, impaired renal function defined as a serum creatinine level $133 mmol/L for men and $124 mmol/L for women, history of chronic pancreatitis or idiopathic acute pancreatitis, known history of unstable angina, acute coronary event, heart failure, other significant cardiac event or stroke, thyroid disorders, malignant diseases, hematologic alterations, and concurrent medication likely to influence energy homeostasis. Indirect Calorimetry
Resting energy expenditure (REE) and respiratory quotient (RQ) were determined by indirect calorimetry after a 12-h overnight fast by using an open-air circuit–ventilated canopy measurement system (Vmax29; SensorMedics Corporation, Yorba Linda, CA) (27). After adjustment for body composition, the measured REE was compared with predicted REE according to age- and sex-specific equations (28). The physical activity level (PAL) was assessed by a questionnaire validated with doubly labeled water (29). On the basis of the REE determination and obtained PAL, the total energy expenditure for each individual was calculated.
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Statistical Analysis
For rodents, data are expressed as mean 6 SEM in relation (%) to the specific control (vehicle-treated rats). Statistical significance was determined by Student t test (for two groups) or ANOVA and post hoc two-tailed Bonferroni test (for more than two groups). P , 0.05 was considered significant. For human data, a mixed ANOVA with a repeated-measures design was performed to study between- and within-subject factors comparing temporal changes in values between the treatment groups. Moreover, a MANOVA was used to assess differences between groups during the overall study time course, using Pillai trace criterion as a test of significance. Comparisons between pre- and postdata within a same treatment group were further analyzed by two-tailed paired Student t tests. The calculations were performed by SPSS for Windows version 15.0 software (IBM Corporation, Chicago, IL). P , 0.05 was considered statistically significant. RESULTS Central GLP-1R Stimulation Suppresses Food Intake and Decreases Body Weight in Mice
A single ICV injection of liraglutide (0.3, 1, and 3 mg/ mouse) significantly decreased food intake and body weight after 24 h at doses of 1 and 3 mg/mouse, and these effects remitted at 48 h (Supplementary Fig. 1). We next compared the central effects of liraglutide versus GLP-1 at 1 mg/mouse, a dose adapted from previous studies (31,32). Whereas a single ICV injection of liraglutide (3 mg/mouse) suppressed feeding behavior after 24 h, GLP-1 failed to do so (Fig. 1A). ICV liraglutide-decreased food intake was not caused by aversive effects (Fig. 1B). ICV liraglutide also suppressed body weight independent of its food intake effect because vehicle pair-fed mice, which ate the same amount of food as liraglutide-treated mice, did not show a significant decrease in body weight relative to vehicle-treated mice (Fig. 1C). ICV liraglutide-treated mice showed increased energy expenditure when corrected for lean mass (Fig. 1D), without changes in locomotor activity (Fig. 1E) or RQ (Fig. 1F). Body composition of these mice was determined immediately after keeping them in the indirect calorimetric system. The reduction in body weight was accompanied by a decrease in circulating leptin levels but without any changes in free fatty acids, triglycerides, cholesterol, insulin, or glucose levels (Supplementary Table 1). To rule out the possibility that centrally infused liraglutide may leak out of the CNS into the circulation and elicit a response by directly acting at peripheral level, we administered liraglutide peripherally. At the same doses as those infused centrally, liraglutide did not change cumulative food intake (Fig. 1G) or body weight (Fig. 1H).
Blood Determinations
Central GLP-1R Stimulation Triggers BAT Thermogenic Activity in Mice
Blood determinations were performed as previously described (22,30).
Because it has been reported that central GLP-1 infusion increases BAT thermogenesis (18), we investigated the
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Figure 1—Effect of a 24-h ICV liraglutide (3 mg/mouse) or GLP-1 (1 mg/mouse) injection on cumulative food intake (A), CTA (B), body weight change (C), energy expenditure (D), locomotor activity (E), and RQ (F). Effect of a 24-h intraperitoneal liraglutide (3 mg/mouse) on cumulative food intake (G) and body weight change (H). Data are mean 6 SEM of 7–8 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle. #P < 0.05, ##P < 0.01. EE, energy expenditure; IP, intraperioteneal; LiCl, liraglutide; PF, pair-fed.
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effects of liraglutide on this issue. First, we found that core body temperature was not affected by ICV liraglutide at any of the studied time points when animals were housed at room temperature (Fig. 2A). However, BAT interscapular temperature increased significantly at 12 h after ICV liraglutide injection in the same animals housed at room temperature (Fig. 2B and C). Histomorphological analysis revealed smaller lipid droplets in BAT from mice 24 h after a single ICV injection of liraglutide (Fig. 2D). Accordingly, in BAT of centrally liraglutide-treated mice, we found higher gene expression levels of several thermogenic biomarkers, such as CIDEA, FGF21, bone morphogenic protein 7 (BMP7), PRDM16, and ADRb1 (Fig. 2E). Protein levels of UCP1, UCP3, ADRb1, FGF21, and PRDM16 were also significantly augmented after the ICV liraglutide injection (Fig. 2F). There were no differences in basal core body temperatures between vehicle and liraglutide-treated mice (Fig. 2G). However, liraglutide-treated mice showed increased core body temperature (Fig. 2G) compared with control mice when they were kept at 4°C. Central GLP-1R Stimulation Induces Browning of WAT in Mice
ICV liraglutide induced browning in WAT, evident 24 h after the liraglutide injection (Fig. 3A). UCP1 protein levels (Fig. 3B) and both UCP1 and PRDM16 mRNA expression (Fig. 3C) were significantly upregulated in WAT of mice treated with ICV liraglutide. GLP-1R Stimulation in the VMH Regulates Thermogenesis and Browning in Rats
We next aimed to investigate the hypothalamic area responsible for the actions of liraglutide on BAT and WAT. Thus, we specifically injected liraglutide in the ARC, LHA, PVH, DMH, or VMH of rats (Fig. 4A–E). C-FOS immunostaining was assessed to corroborate the efficiency of the injections in each area (Fig. 4A–E). We found that the specific activation of the GLP-1R in the ARC, LHA, and PVH decreased food intake and body weight of rats (Fig. 4A–C), whereas no effects were detected when liraglutide was injected in the DMH (Fig. 4D). At the molecular level, UCP1 was unaltered in BAT and WAT of rats injected with liraglutide in those nuclei (Fig. 4A– D). Of note, administration of liraglutide in the VMH led to a significant weight loss with no significant differences in food intake, and UCP1 levels were increased in BAT and WAT (Fig. 4E). AMPK Within the VMH Is Essential for the Central Actions of Liraglutide on BAT and WAT
Because previous reports have demonstrated a key role of AMPK within the VMH in the regulation of BAT thermogenesis (25,26), we first assessed hypothalamic pAMPK levels after ICV injection of liraglutide. We found that pAMPK and its downstream target pACC were significantly decreased in the whole hypothalamus of ICV liraglutidetreated mice compared with their controls (Fig. 5A). Therefore, to demonstrate the relevance of hypothalamic AMPK
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as a mediator of the actions of liraglutide, we used a pharmacological activator of AMPK, AICAR (25). At the dose used, ICV AICAR did not blunt the anorexigenic effect of ICV liraglutide (Fig. 5B) but prevented ICV liraglutideinduced weight loss (Fig. 5C). Second, AMPK activity was elevated by using an adenoviral vector encoding constitutively active (CA) AMPKa, with adenoviruses expressing GFP used as controls (25). The adenoviruses were injected stereotaxically into the VMH of rats together with liraglutide, and the specificity of these injections was corroborated by GFP immunostaining (Fig. 5D). Liraglutide injected specifically into the VMH decreased weight, but the liraglutide-induced weight loss was completely blunted when AMPKa-CA was overexpressed in the VMH (Fig. 5E). Identical to the effect on body weight, AMPK activation in the VMH also reduced the liraglutide-induced UCP1 expression in BAT of rats (Fig. 5F) and WAT (Fig. 5G). The SIRT1/p53 system interacts with AMPK at the central level, and both molecules are key mediators of the orexigenic action of ghrelin (33). Furthermore, GLP-1 inhibits ghrelin-stimulated neuronal activity in the hypothalamus as well as its effects on food intake (34). Therefore, we next examined whether SIRT1 or p53 might be relevant for the central actions of liraglutide. To this aim, we used mice with moderate overexpression of SIRT1 under the control of its natural promoter (35) and p53 null mice (33). The findings demonstrate that ICV injection of liraglutide decreased food intake and body weight similarly in both wild-type and SIRT1 transgenic mice (Fig. 5H and I) or p53 null mice (Fig. 5J and K). Long-Term GLP-1R Agonism Increases Energy Expenditure in Obese T2D Patients
In addition to the functional data on liraglutide from the mouse study, we analyzed data from a cohort of obese patients with T2D who had been treated for 1 year with the antidiabetic drugs metformin and metformin in combination with the GLP-1R agonists exenatide and liraglutide (Table 1). At baseline, all obese T2D groups exhibited an identical sex distribution with no significant differences in age, BMI, waist circumference, body composition, REE, and RQ (Table 1) as well as in biochemical and hormonal variables (Supplementary Table 1). After 1 year of antidiabetic treatment instauration, all study groups showed a decrease in fasting plasma glucose and insulin concentrations. No significant changes in total cholesterol, HDL cholesterol, and LDL cholesterol concentrations at the end of the 1-year study period were observed. After 12 months of antidiabetic treatment, the groups treated with metformin combined with exenatide or liraglutide showed a significant decrease in BMI and total body fat percentage and a significant increase in fatfree mass (FFM) (Table 1). None of the study groups significantly changed in RQ and PAL during the experimental period, which remained within the same sedentary range as before treatment started as well as between the diverse antidiabetic administration groups (Table 1). Although no
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Figure 2—Effect of ICV liraglutide (10 mg/rat) on core body temperature (A) and BAT interscapular temperature (B) in animals housed at room temperature after 6, 12, and 24 h of the injection. C: Representative pictures of BAT interscapular temperature in animals housed at room temperature after 12 h of the injection. D: Representative BAT histology pictures (hematoxylin-eosin) 24 h after liraglutide ICV injection (3 mg/mouse). Effect of a 24-h ICV liraglutide (3 mg/mouse) on mRNA expression of PGC1a, ADRb1, ADRb2, ADRb3, CIDEA, FGF21, BMP7, and PRDM16 (E) and protein levels of UCP1, UCP3, CIDEA, FGF21, and PRDM16 (F). Tubulin was used to normalize protein levels. G: Body core temperature measurements over a 6-h period at 4°C. Data are mean 6 SEM of 7–8 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle. PF, pair-fed.
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Figure 3—A: Representative WAT histology pictures (hematoxylin-eosin) 24 h after liraglutide ICV injection (3 mg/mouse). Effect of 24-h ICV liraglutide (3 mg/mouse) on protein levels of UCP1 (B) and mRNA expression of PGC1a, PPARg, PPARa, CEBPa, CEBPb, ADRb1, ADRb2, ADRb3, FGF21, UCP1, and PRDM16 (C). GAPDH was used to normalize protein levels. Data are mean 6 SEM of 7–8 animals per group. *P < 0.05, **P < 0.01 vs. vehicle. PF, pair-fed.
statistically significant differences between and within groups were observed for unadjusted REE, there was a clear tendency to be higher in patients treated with GLP-1 agonists. When the REE data were adjusted for FFM, we found a significant increase in REE in patients treated with metformin in combination with exenatide or liraglutide; however, patients treated only with metformin did not show changes in any of these parameters (Fig. 6 and Table 1).
DISCUSSION
We report that the CNS GLP-1 system directly activates BAT thermogenesis and browning of white adipocytes. The primary finding is that these actions are mediated by hypothalamic AMPK, specifically within the VMH. To our knowledge, these findings are the first to provide information about the brain site and mechanisms by which fat mass decreases in response to a stimulation of CNS
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Figure 4—Effect of the specific injection of liraglutide (10 mg/rat) in the ARC (A), LHA (B), PVH (C), DMH (D), and VMH (E) on C-FOS, 24-h food intake, 24-h body weight change, BAT UCP1 levels, and WAT UCP1 levels. Tubulin was used to normalize protein levels. Data are mean 6 SEM of 7–8 animals per group. *P < 0.05, **P < 0.01 vs. vehicle.
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Figure 5—A: Effect of 24-h ICV liraglutide (3 mg/mouse) on hypothalamic protein levels of pAMPK, pACC, and ACC. Food intake (B) and body weight change (C) in mice receiving an ICV injection of liraglutide (3 mg/mouse) after previous injection with the AMPK activator AICAR (5 mg/ mouse). Effect of the injection of adenoviral particles encoding for GFP or pAMPK-CA in the VMH of rats treated with liraglutide (10 mg/rat) (D) and on body weight change (E), BAT UCP1 protein levels (F), and WAT UCP1 protein levels (G). Effect of 24-h ICV liraglutide (3 mg/mouse) in wild type (WT) and SIRT1 transgenic (TgA) mice on food intake (H) and body weight change (I). Effect of 24-h ICV liraglutide (3 mg/mouse) in WT and p53 null (p53 KO) mice on food intake (J) and body weight change (K). GAPDH was used to normalize protein levels. Data are mean 6 SEM of 7–8 animals per group. *P < 0.05, **P < 0.01 vs. vehicle. KO, knockout; Lig, liraglutide; PF, pair-fed; Veh, vehicle.
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Table 1—Anthropometric characteristics and indirect calorimetry data of T2D patients before and after 1 year of antidiabetic treatment initiation Treatment variable Sex (male/female)
Metformin monotherapy Metformin in combination with GLP-1 agonists Statistical Premetformin Postmetformin Pre-exenatide Postexenatide Preliraglutide Postliraglutide significance 7/3
7/3
8/3
8/3
10/4
10/4
ns
65 6 11
—
67 6 14
—
66 6 15
—
ns
BMI (kg/m )
33.0 6 6.6
31.4 6 9.5
34.9 6 2.6
32.1 6 1.8*
35.3 6 2.2
32.0 6 2.1*
*
Waist (cm)
115 6 13
110 6 19
116 6 10
106 6 15
117 6 11
110 6 16
ns
39.0 6 16.9
37.9 6 9.4
39.5 6 4.3
35.8 6 3.7*
39.2 6 3.6
35.1 6 3.3*
*
63.3 6 17.0
67.1 6 9.3
60.7 6 4.2
64.3 6 3.4*
60.8 6 3.1
64.9 6 3.0*
Age (years) 2
Fat mass (%) FFM (%) REE (kJ/day)
7,670 6 1,030 7,469 6 1,230 7,595 6 832
8,240 6 901 7,623 6 1,014 8,326 6 1,107
* ns
74.9 6 10.0
74.1 6 11.7
75.7 6 8.4
79.1 6 8.7
76.0 6 10.3
80.9 6 11.2
REE (kJ/kg FFM/day) 121.0 6 16.7
114.3 6 18.8
120.6 6 5.1
135.7 6 6.0*
122.4 6 6.9
143.6 6 7.1*
*
RQ (vCO2/vO2)
0.85 6 0.02
0.84 6 0.03
0.85 6 0.04
0.85 6 0.05
0.83 6 0.06
0.84 6 0.04
ns
PAL
1.47 6 0.11
1.46 6 0.08
1.45 6 0.15
1.48 6 0.12
1.46 6 0.13
1.47 6 0.16
ns
REE (kJ/kg/day)
ns
Data are mean 6 SEM unless otherwise indicated. ns, nonstatistically significant differences by repeated-measures ANOVA, MANOVA, and two-tailed paired Student t test between pre- and postvalues within a same treatment group. *P , 0.05 by repeated-measures ANOVA and compared with baseline (pre) values within the same treatment group by two-tailed paired Student t test.
GLP-1 activity independently of anorexigenic actions. We also observed that obese T2D patients treated with metformin in combination with exenatide or liraglutide, which showed a reduction in BMI and fat mass, had an increased energy expenditure, suggesting that at the clinical level, the effects of GLP-1 agonists on body weight are at least partially mediated by increased energy expenditure. The mechanisms by which central GLP-1 stimulation reduces body weight seem to involve food intake (7), decreased lipid storage in WAT (17), and increased BAT thermogenesis (18). The diversity of the biological actions of CNS GLP-1 is likely caused by the wide expression of its receptor throughout the entire CNS (36). Therapies for T2D-based GLP-1R agonists are now commonly used in combination with other drugs and are under investigation for the treatment of human obesity (20). Liraglutide is a long-acting GLP-1 analog indicated for the treatment of T2D (37,38). In addition to its antidiabetic properties, the systemic administration of liraglutide induces weight loss in obese rats (39) and patients (20). Because previous studies found differences in the central actions of GLP-1 and long-acting GLP-1 analogs (31), we aimed to investigate the central effects of liraglutide on brown and white adipocyte metabolism. In agreement with previous findings (7,17,18), we found that the central stimulation of GLP-1R reduces food intake and body weight. Differently from previous studies that used a chronic activation of the brain GLP-1R (17,18), the present data show that a single ICV injection of liraglutide maintains its biological actions for 24 h. The decreased body weight in mice receiving a single ICV administration of liraglutide was independent of feeding behavior. Instead, the results show that increased energy expenditure, and more specifically BAT thermogenesis and WAT browning, can explain the liraglutide-induced
weight loss. Although previous reports indicated that brain GLP-1 induces BAT thermogenesis (18), the capacity of brain GLP-1 to enhance white-to-brown transdifferentiation was previously unknown. This effect is likely mediated by the increased activity of sympathetic fibers innervating WAT, as previously reported for BAT (18). Although GLP-1R is widely distributed throughout the rodent hypothalamus (36), the specific sites of action and the molecular pathways triggered by GLP-1 within the hypothalamus remain largely unknown. In the current study, we demonstrate that injection of liraglutide specifically into ARC, LHA, or PVH reduced both food intake and body weight. The reduced food intake observed after the administration of liraglutide in the ARC might seem controversial because a previous study showed that ARC GLP-1R regulates glucose homeostasis but not feeding (14). However, important methodological differences exist between these studies, such as the compounds used (GLP-1 vs. liraglutide) and the time at which food was weighed (2 vs. 24 h). Differently from the hypothalamic sites cited, the administration of liraglutide to the VMH significantly decreased body weight without concomitant reduction in food intake, suggesting that the stimulation of GLP-1R in this particular area reduces weight in a food intake–independent manner, which is corroborated by the significantly higher levels of UCP1 in the BAT and WAT of VMH in liraglutide-treated rats. Indeed, the VMH has been previously demonstrated to be an essential modulator of BAT metabolism, with the AMPK pathway as an important mediator controlling energy dissipation (25,26). Therefore, we hypothesized that the central effects of liraglutide on BAT and WAT could be mediated by AMPK in the VMH. The data show that ICV liraglutide administration decreased hypothalamic AMPK activity, measured as decreased levels of pAMPK. To investigate whether activation of AMPK could counteract the effect
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Figure 6—REE in obese T2D patients before and after the treatment for 1 year with metformin (A), metformin and exenatide (B), and metformin and liraglutide (C).
of liraglutide, we first pharmacologically activated central AMPK with the compound AICAR and then genetically activated AMPK specifically in the VMH by using adenoviralmediated targeting. In both cases, we found that the actions of liraglutide on thermogenesis and browning were abolished, indicating that VMH AMPK modulates the actions of brain GLP-1 on both BAT and WAT. Indeed, it is well established that the GLP-1R is also located in extrahypothalamic areas (8,9); therefore, the current results do not exclude the possibility that extrahypothalamic areas are also involved in the effects of GLP-1R agonists on BAT thermogenesis and energy expenditure. In particular, GLP-1 binding sites were found in the inferior olive and nucleus of the solitary tract (9); both areas have been described as involved in the control of thermogenesis (40,41). Although at the central level SIRT1/p53 and AMPK pathways are interacting to mediate the orexigenic actions of ghrelin (33), neither SIRT1 nor p53 modified liraglutide-induced hypophagia or weight loss, indicating that the brain SIRT1/p53 system does not interact with AMPK to mediate the actions of liraglutide on thermogenesis and browning. Finally, because it is known that BAT is active in humans (42–45), we investigated the clinical value of the current data and found that obese T2D patients treated with metformin in combination with the GLP-1R agonists exenatide or liraglutide showed an increase in energy expenditure. The HbA1c reductions observed were consistent with those from other studies of exenatide and liraglutide (20,46,47). In the patient study, both GLP-1 agonists were applied at doses known to have good digestive tolerance. Although previous reports showed that liraglutide failed to significantly affect energy expenditure in patients (46–49), in the current study, a significant increase in the adjusted REE and a reduction in BMI and the percentage of fat mass were evident.
The apparent discrepancies in the results can be explained by the length of the treatment period; in previous studies, patients were treated with GLP-1 agonists for 4 (48), 8 (46),
Figure 7—Schematic overview summarizing the physiological effects of the central injection of liraglutide on energy balance. Activation of GLP-1R in the VMH dephosphorylates AMPK and stimulates BAT thermogenesis and energy expenditure. Activation of GLP-1R in the LHA, PVH, and ARC decreases caloric intake. Combined, these parallel metabolic changes result in a decreased body weight.
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or 12 (47) weeks, whereas in the current study, patients were treated for a mean of 1 year. The 1-year clinical data demonstrate for first time in our knowledge that liraglutide, which is now under evaluation by the Food and Drug Administration as an antiobesity drug, increases energy expenditure. Although we cannot rule out the possibility that liraglutide-induced REE in humans is partially mediated by peripheral mechanisms, the present findings suggest that liraglutide uses a specific central pathway to increase energy expenditure and ultimately reduce body weight, and this pathway might be of clinical relevance. As summarized in Fig. 7, we have provided a combination of pharmacological and genetic evidence to demonstrate that the central stimulation of GLP-1R induces not only BAT thermogenesis, but also adipocyte browning in WAT. The molecular mechanism controlling these actions involves AMPK in the VMH. Funding. This work has been supported by grants from Ministerio de Economia y Competitividad (BFU2011-29102 to C.D. and RYC-2008-02219 and BFU2012-35255 to R.N.), Xunta de Galicia (10PXIB208164PR and 2012-CP070 to M.L. and EM 2012/039 and 2012-CP069 to R.N.), Fondo de Investigaciones Sanitarias (PI12/01814 to M.L. and FISPI12/00515 to G.F.), and CIBERobn. CIBERobn is an initiative of the Instituto de Salud Carlos III of Spain, which is supported by FEDER (European Fund for Regional Development) funds. This research has also received funding from the European Community’s Seventh Framework Programme under the following grants: to C.D., M.L., and R.N.: FP7/2007-2013: no. 245009: NeuroFAST, and R.N.: ERC StG-2011-OBESITY53-281408. Duality of Interest. No potential conflicts of interest relevant to this article were reported. Author Contributions. D.B., M.I., R.G., and A.S. contributed to the experiments and data analysis. D.H. contributed to the experiments. F.V., M.S., J.F., J.S., J.E., C.D., M.L., and G.F. contributed to the development of the analytical tools and discussion. R.N. contributed to the experimental design and writing of the manuscript. R.N. 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. Drucker DJ. The role of gut hormones in glucose homeostasis. J Clin Invest 2007;117:24–32 2. Göke R, Fehmann HC, Göke B. Glucagon-like peptide-1(7-36) amide is a new incretin/enterogastrone candidate. Eur J Clin Invest 1991;21:135–144 3. Campbell JE, Drucker DJ. Pharmacology, physiology, and mechanisms of incretin hormone action. Cell Metab 2013;17:819–837 4. Kreymann B, Williams G, Ghatei MA, Bloom SR. Glucagon-like peptide-1 7-36: a physiological incretin in man. Lancet 1987;2:1300–1304 5. Mojsov S, Weir GC, Habener JF. Insulinotropin: glucagon-like peptide I (7-37) co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas. J Clin Invest 1987;79:616–619 6. Willms B, Werner J, Holst JJ, Orskov C, Creutzfeldt W, Nauck MA. Gastric emptying, glucose responses, and insulin secretion after a liquid test meal: effects of exogenous glucagon-like peptide-1 (GLP-1)-(7-36) amide in type 2 (noninsulin-dependent) diabetic patients. J Clin Endocrinol Metab 1996;81:327– 332 7. Turton MD, O’Shea D, Gunn I, et al. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 1996;379:69–72 8. Shimizu I, Hirota M, Ohboshi C, Shima K. Identification and localization of glucagon-like peptide-1 and its receptor in rat brain. Endocrinology 1987;121: 1076–1082
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9. Göke R, Larsen PJ, Mikkelsen JD, Sheikh SP. Distribution of GLP-1 binding sites in the rat brain: evidence that exendin-4 is a ligand of brain GLP-1 binding sites. Eur J Neurosci 1995;7:2294–2300 10. Skibicka KP, Alhadeff AL, Grill HJ. Hindbrain cocaine- and amphetamineregulated transcript induces hypothermia mediated by GLP-1 receptors. J Neurosci 2009;29:6973–6981 11. Holst JJ. Incretin hormones and the satiation signal. Int J Obes (Lond) 2013; 37:1161–1168 12. Tang-Christensen M, Larsen PJ, Göke R, et al. Central administration of GLP-1-(7-36) amide inhibits food and water intake in rats. Am J Physiol 1996; 271:R848–R856 13. Seo S, Ju S, Chung H, Lee D, Park S. Acute effects of glucagon-like peptide-1 on hypothalamic neuropeptide and AMP activated kinase expression in fasted rats. Endocr J 2008;55:867–874 14. Sandoval DA, Bagnol D, Woods SC, D’Alessio DA, Seeley RJ. Arcuate glucagon-like peptide 1 receptors regulate glucose homeostasis but not food intake. Diabetes 2008;57:2046–2054 15. Kinzig KP, D’Alessio DA, Herman JP, et al. CNS glucagon-like peptide-1 receptors mediate endocrine and anxiety responses to interoceptive and psychogenic stressors [published correction appears in J Neurosci 2003;23:8158]. J Neurosci 2003;23:6163–6170 16. Kinzig KP, D’Alessio DA, Seeley RJ. The diverse roles of specific GLP-1 receptors in the control of food intake and the response to visceral illness. J Neurosci 2002;22:10470–10476 17. Nogueiras R, Pérez-Tilve D, Veyrat-Durebex C, et al. Direct control of peripheral lipid deposition by CNS GLP-1 receptor signaling is mediated by the sympathetic nervous system and blunted in diet-induced obesity. J Neurosci 2009;29:5916–5925 18. Lockie SH, Heppner KM, Chaudhary N, et al. Direct control of brown adipose tissue thermogenesis by central nervous system glucagon-like peptide-1 receptor signaling. Diabetes 2012;61:2753–2762 19. Marre M, Shaw J, Brändle M, et al.; LEAD-1 SU study group. Liraglutide, a once-daily human GLP-1 analogue, added to a sulphonylurea over 26 weeks produces greater improvements in glycaemic and weight control compared with adding rosiglitazone or placebo in subjects with type 2 diabetes (LEAD-1 SU). Diabet Med 2009;26:268–278 20. Astrup A, Rössner S, Van Gaal L, et al.; NN8022-1807 Study Group. Effects of liraglutide in the treatment of obesity: a randomised, double-blind, placebocontrolled study. Lancet 2009;374:1606–1616 21. Nathan DM, Buse JB, Davidson MB, et al.; American Diabetes Association; European Association for the Study of Diabetes. Medical management of hyperglycaemia in type 2 diabetes mellitus: a consensus algorithm for the initiation and adjustment of therapy: a consensus statement from the American Diabetes Association and the European Association for the Study of Diabetes. Diabetologia 2009;52:17–30 22. Imbernon M, Beiroa D, Vazquez MJ, et al. Central melanin-concentrating hormone influences liver and adipose metabolism via specific hypothalamic nuclei and efferent autonomic/JNK1 pathways. Gastroenterology 2013;144:636– 649 23. López M, Lelliott CJ, Tovar S, et al. Tamoxifen-induced anorexia is associated with fatty acid synthase inhibition in the ventromedial nucleus of the hypothalamus and accumulation of malonyl-CoA. Diabetes 2006;55:1327–1336 24. Czyzyk TA, Romero-Picó A, Pintar J, et al. Mice lacking d-opioid receptors resist the development of diet-induced obesity. FASEB J 2012;26:3483–3492 25. López M, Varela L, Vázquez MJ, et al. Hypothalamic AMPK and fatty acid metabolism mediate thyroid regulation of energy balance. Nat Med 2010;16: 1001–1008 26. Martínez de Morentin PB, Whittle AJ, Fernø J, et al. Nicotine induces negative energy balance through hypothalamic AMP-activated protein kinase. Diabetes 2012;61:807–817 27. Moreno-Navarrete JM, Catalán V, Ortega F, et al. Circulating omentin concentration increases after weight loss. Nutr Metab (Lond) 2010;7:27
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28. Frühbeck G, Alonso R, Marzo F, Santidrián S. A modified method for the indirect quantitative analysis of phytate in foodstuffs. Anal Biochem 1995;225: 206–212 29. Johansson G, Westerterp KR. Assessment of the physical activity level with two questions: validation with doubly labeled water. Int J Obes (Lond) 2008;32: 1031–1033 30. Catalán V, Gómez-Ambrosi J, Ramirez B, et al. Proinflammatory cytokines in obesity: impact of type 2 diabetes mellitus and gastric bypass. Obes Surg 2007; 17:1464–1474 31. Barrera JG, D’Alessio DA, Drucker DJ, Woods SC, Seeley RJ. Differences in the central anorectic effects of glucagon-like peptide-1 and exendin-4 in rats. Diabetes 2009;58:2820–2827 32. Hwa JJ, Ghibaudi L, Williams P, Witten MB, Tedesco R, Strader CD. Differential effects of intracerebroventricular glucagon-like peptide-1 on feeding and energy expenditure regulation. Peptides 1998;19:869–875 33. Velásquez DA, Martínez G, Romero A, et al. The central Sirtuin 1/p53 pathway is essential for the orexigenic action of ghrelin. Diabetes 2011;60:1177–1185 34. Riediger T, Eisele N, Scheel C, Lutz TA. Effects of glucagon-like peptide 1 and oxyntomodulin on neuronal activity of ghrelin-sensitive neurons in the hypothalamic arcuate nucleus. Am J Physiol Regul Integr Comp Physiol 2010;298: R1061–R1067 35. Pfluger PT, Herranz D, Velasco-Miguel S, Serrano M, Tschöp MH. Sirt1 protects against high-fat diet-induced metabolic damage. Proc Natl Acad Sci U S A 2008;105:9793–9798 36. Shughrue PJ, Lane MV, Merchenthaler I. Glucagon-like peptide-1 receptor (GLP1-R) mRNA in the rat hypothalamus. Endocrinology 1996;137:5159–5162 37. Buse JB, Rosenstock J, Sesti G, et al.; LEAD-6 Study Group. Liraglutide once a day versus exenatide twice a day for type 2 diabetes: a 26-week randomised, parallel-group, multinational, open-label trial (LEAD-6). Lancet 2009; 374:39–47 38. Garber A, Henry R, Ratner R, et al.; LEAD-3 (Mono) Study Group. Liraglutide versus glimepiride monotherapy for type 2 diabetes (LEAD-3 Mono): a randomised, 52-week, phase III, double-blind, parallel-treatment trial. Lancet 2009; 373:473–481
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39. Larsen PJ, Fledelius C, Knudsen LB, Tang-Christensen M. Systemic administration of the long-acting GLP-1 derivative NN2211 induces lasting and reversible weight loss in both normal and obese rats. Diabetes 2001;50:2530– 2539 40. Kotz CM, Grace MK, Briggs J, Levine AS, Billington CJ. Effects of opioid antagonists naloxone and naltrexone on neuropeptide Y-induced feeding and brown fat thermogenesis in the rat. Neural site of action. J Clin Invest 1995;96: 163–170 41. Uno T, Shibata M. Role of inferior olive and thoracic IML neurons in nonshivering thermogenesis in rats. Am J Physiol Regul Integr Comp Physiol 2001; 280:R536–R546 42. Wu J, Boström P, Sparks LM, et al. Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell 2012;150:366–376 43. Virtanen KA, Lidell ME, Orava J, et al. Functional brown adipose tissue in healthy adults. N Engl J Med 2009;360:1518–1525 44. Cypess AM, Lehman S, Williams G, et al. Identification and importance of brown adipose tissue in adult humans. N Engl J Med 2009;360:1509–1517 45. van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, et al. Coldactivated brown adipose tissue in healthy men. N Engl J Med 2009;360:1500– 1508 46. Harder H, Nielsen L, Tu DT, Astrup A. The effect of liraglutide, a long-acting glucagon-like peptide 1 derivative, on glycemic control, body composition, and 24-h energy expenditure in patients with type 2 diabetes. Diabetes Care 2004; 27:1915–1921 47. Bradley DP, Kulstad R, Racine N, Shenker Y, Meredith M, Schoeller DA. Alterations in energy balance following exenatide administration. Appl Physiol Nutr Metab 2012;37:893–899 48. Horowitz M, Flint A, Jones KL, et al. Effect of the once-daily human GLP-1 analogue liraglutide on appetite, energy intake, energy expenditure and gastric emptying in type 2 diabetes. Diabetes Res Clin Pract 2012;97:258–266 49. Tan TM, Field BC, McCullough KA, et al. Coadministration of glucagon-like peptide-1 during glucagon infusion in humans results in increased energy expenditure and amelioration of hyperglycemia. Diabetes 2013;62:1131–1138
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Daniel Beiroa,1,2 Monica Imbernon,1,2 Rosalía Gallego,3 Ana Senra,1 Daniel Herranz,4 Francesc Villaroya,2,5 Manuel Serrano,4 Johan Fernø,6 Javier Salvador,2,7 Javier Escalada,2,7 Carlos Dieguez,1,2 Miguel Lopez,1,2 Gema Frühbeck,2,7 and Ruben Nogueiras1,2
GLP-1 Agonism Stimulates Brown Adipose Tissue Thermogenesis and Browning Through Hypothalamic AMPK DOI: 10.2337/db14-0302
1Department of Physiology, CIMUS, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela, Spain 2CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn), Santiago de Compostela, Spain 3Department of Morphological Sciences, School of Medicine, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela, Spain 4Tumor Suppression Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain 5Department of Biochemistry and Molecular Biology and Institute of Biomedicine (IBUB), University of Barcelona, Barcelona, Spain 6Department of Clinical Science, K.G. Jebsen Center for Diabetes Research, University of Bergen, Bergen, Norway
hypothalamic sites and that these mechanisms might be clinically relevant.
GLP-1 is an incretin hormone released by L cells located in the ileum and colon into the bloodstream postprandially (1,2). Among its numerous physiological effects (3), GLP-1 increases insulin and decreases glucagon secretion in a glucose-dependent manner (4,5), slows gastric emptying (6), increases glucose disposal, and decreases appetite (7). Therefore, incretin hormone analogs acting on the GLP-1 receptor (GLP-1R) are considered the most promising new therapies for type 2 diabetes (T2D). Within the central nervous system (CNS), numerous neuronal populations express GLP-1R, including hypothalamic nuclei crucial for the regulation of energy balance (8). Furthermore, a large number of extrahypothalamic areas have GLP-1 binding sites (9). Indeed, it has been demonstrated that peripheral administration of GLP-1 leads to neuronal activation in various parts of the CNS (10), indicating the existence of brain sites accessible from the bloodstream. Finally, cells with GLP-1 mRNA are widely expressed in human brain areas (11).
7Department
of Endocrinology and Nutrition, Clínica Universidad de Navarra, Pamplona, Spain Corresponding author: Ruben Nogueiras, [email protected]. Received 21 February 2014 and accepted 30 April 2014. This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0302/-/DC1. © 2014 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 Publish Ahead of Print, published online June 10, 2014
OBESITY STUDIES
GLP-1 receptor (GLP-1R) is widely located throughout the brain, but the precise molecular mechanisms mediating the actions of GLP-1 and its long-acting analogs on adipose tissue as well as the brain areas responsible for these interactions remain largely unknown. We found that central injection of a clinically used GLP-1R agonist, liraglutide, in mice stimulates brown adipose tissue (BAT) thermogenesis and adipocyte browning independent of nutrient intake. The mechanism controlling these actions is located in the hypothalamic ventromedial nucleus (VMH), and the activation of AMPK in this area is sufficient to blunt both central liraglutide-induced thermogenesis and adipocyte browning. The decreased body weight caused by the central injection of liraglutide in other hypothalamic sites was sufficiently explained by the suppression of food intake. In a longitudinal study involving obese type 2 diabetic patients treated for 1 year with GLP-1R agonists, both exenatide and liraglutide increased energy expenditure. Although the results do not exclude the possibility that extrahypothalamic areas are also modulating the effects of GLP-1R agonists, the data indicate that long-acting GLP-1R agonists influence body weight by regulating either food intake or energy expenditure through various
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In the hypothalamus, GLP-1 acts as a physiological satiety factor (7), and dose-dependent central administration of GLP-1 reduces feeding in rats (12). Central GLP-1 administration blunted fasting-induced neuropeptide Y and Agouti-related peptide levels and fasting-reduced proopiomelanocortin and cocaine- and amphetamineregulated transcript expression (13). Stimulation of the central GLP-1 system not only suppresses food intake but also regulates glucose homeostasis (14), behavioral responses to stress (15), and visceral illness (16). Moreover, brain GLP-1 modulates lipid metabolism in white adipose tissue (WAT) (17) and brown adipose tissue (BAT) thermogenesis (18) through the activation of the sympathetic nervous system. However, the molecular mechanisms mediating the actions of brain GLP-1 on adipose tissue as well as the brain areas responsible for these interactions are unknown. Furthermore, whether some of the beneficial effects exerted in the clinical setting by long-acting GLP-1 agonists could be mediated at the central level is yet unclear. One of these long-acting GLP-1 analogs is liraglutide, which injected once daily improves glycemic control in T2D with the additional benefits of weight loss and a low risk of hypoglycemia (19,20). As GLP-1R agonists start to be included in treatment guidelines, they are generally being recommended as second- or third-line therapies after the failure of one or more oral antidiabetic drugs (21). In the current study, we show that central stimulation of GLP-1R by the agonist liraglutide leads to body weight loss independent of reduction in food intake. Instead, this weight loss is caused by the activation of the thermogenic program in BAT. Specific injection of liraglutide in the hypothalamic ventromedial nucleus (VMH) is sufficient to cause food intake–independent weight loss and stimulation of BAT thermogenic activity. This regulatory mechanism depends on AMPK. RESEARCH DESIGN AND METHODS Animal Models
Swiss male mice (8–10 weeks old, 20–25 g) and SpragueDawley rats (8–10 weeks old, 200–250 g) were housed under conditions of controlled temperature (23°C) and illumination (12-h light/dark cycle). They were allowed ad libitum access to water and standard laboratory chow. Body composition was measured by a nuclear magnetic resonance imaging whole-body composition analyzer (EchoMRI, Houston, TX). Animals were killed by decapitation, and the tissues were removed rapidly, frozen immediately on dry ice, and kept at 280°C until analysis. All animal experiments and procedures were reviewed and approved by the Ethics Committee of the University of Santiago de Compostela in accordance with European Union norms for the use of experimental animals. Implantation of Intracerebroventricular Cannulae and Treatments
Rats and mice were anesthetized by an intraperitoneal injection of ketamine 80 and 8 mg/kg body weight,
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respectively, and xylazine 100 and 3 mg/kg body weight, respectively. Intracerebroventricular (ICV) cannulae were implanted stereotaxically in mice and rats, as described previously (22). Animals received vehicle, GLP-1 (1 mg/mouse), liraglutide (3 mg/mouse or 10 mg/rat), or AICAR (3 mg/mouse). Conditioned Taste Aversion
Conditioned taste aversion (CTA) was expressed as the percent saccharin preference ratio [(100 3 saccharin intake) / (saccharin intake + water intake)]. A saccharin preference ratio ,50% was considered a signal of CTA (23). Cold Exposure
Twenty-four hours after the liraglutide injection, animals were placed for 6 h in a room with a stable temperature of 4°C as previously described (24). Body temperature was recorded with a rectal probe connected to a digital thermometer, and the interscapular temperature was recorded with an infrared camera. Stereotaxic Microinjection
Sprague-Dawley rats were placed in a stereotaxic frame under ketamine-xylazine anesthetics. Liraglutide (10 mg/rat) was injected in the arcuate nucleus (ARC) (anterior to the bregma [AP], 22.85 mm; lateral to the sagittal suture [L], 60.3 mm; and ventral from the surface of the skull [V], 10.2 mm), in the lateral hypothalamus area (LHA) (AP, 22.85 mm; L, 62 mm; DV, 28.1 mm), in the VMH (AP, 22.85 mm; L, 60.6 mm; DV, 210mm), in the dorsomedial nucleus (DMH) (AP, 23.12 mm; L, 60.5 mm; DV, 28.6 mm), and in the paraventricular nucleus (PVH) (AP, 21.9 mm; L, 60.5 mm; DV, 28 mm) with a 25-gauge needle (Hamilton, Reno, NV) connected to a 1-mL syringe. Acetylsalicylic acid (Bayer, Leverkusen, Germany) 150 mg/kg was injected intraperitoneally after surgery as a painkiller. Liraglutide 5 mg and adenoviral vectors (green fluorescent protein [GFP] or AMPKa-CA [Viraquest, North Liberty, IA]) (25,26) were injected simultaneously into the VMH with a 25-gauge needle connected to a 2-mL syringe. Immunohistochemistry and Immunofluorescence
Rat brains were fixed, and diaminobenzidine immunohistochemistry and detection of GFP were performed as previously reported (22,25). RNA Isolation and Real-Time RT-PCR
Total RNA and real-time RT-PCR were performed as previously described (22). The primers and probes are described in Supplementary Table 1. Western Blot Analysis
Western blot was performed as previously described (22). Briefly, total protein lysates from epididymal WAT (30 mg) and BAT (15 mg) were subjected to SDS-PAGE, electrotransferred on a polyvinylidene difluoride membrane, and probed with the following antibodies: uncoupling protein (UCP) 1, UCP3, b-adrenoreceptor 1 (ADRb1), cell death–inducing DNA fragmentation factor a–like effector a (CIDEA), fibroblast growth factor 21 (FGF21), and PR
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domain containing 16 (PRDM16) (Abcam, Cambridge, U.K.); GAPDH (EMD Millipore, Billerica, MA); and a-tubulin (T-5168; Sigma-Aldrich, St. Louis, MO). Patient Selection
All clinical studies were approved from an ethical and scientific standpoint by the hospital’s ethics committee and were conducted in accordance with the principles of the Declaration of Helsinki, with patients giving their informed consent for participation. Twenty-five obese T2D patients matched for sex, age, and BMI were studied before (baseline) and after (12 6 3 months) antidiabetic treatment instauration with metformin in combination with either of the GLP-1 agonists exenatide or liraglutide. The initial dose of metformin was 500 mg b.i.d. orally, which was increased to 1,000 mg b.i.d. orally after 2 weeks. Exenatide treatment was initiated with 5 mg b.i.d. administered subcutaneously and increased to 10 mg b.i.d. after 2 weeks of good digestive tolerance. Liraglutide treatment was initiated with a single subcutaneous administration of 0.6 mg a day progressing to 1.2 mg a day after 2 weeks of adequate digestive tolerance. All patients were of Caucasian origin, attended the Endocrinology Department at the University Clinic of Navarra, and underwent a clinical assessment. Inclusion criteria were age between 20 and 80 years, hemoglobin A1c (HbA1c) levels $7.0%, and no previous treatment with insulin and/or a sulfonylurea. Exclusion criteria were lactating or pregnant women, uncontrolled treated or untreated hypertension, fasting C-peptide levels ,0.1 ng/mL, recurrent major hypoglycemia or hypoglycemic unawareness, known proliferative retinopathy or maculopathy requiring acute treatment, impaired renal function defined as a serum creatinine level $133 mmol/L for men and $124 mmol/L for women, history of chronic pancreatitis or idiopathic acute pancreatitis, known history of unstable angina, acute coronary event, heart failure, other significant cardiac event or stroke, thyroid disorders, malignant diseases, hematologic alterations, and concurrent medication likely to influence energy homeostasis. Indirect Calorimetry
Resting energy expenditure (REE) and respiratory quotient (RQ) were determined by indirect calorimetry after a 12-h overnight fast by using an open-air circuit–ventilated canopy measurement system (Vmax29; SensorMedics Corporation, Yorba Linda, CA) (27). After adjustment for body composition, the measured REE was compared with predicted REE according to age- and sex-specific equations (28). The physical activity level (PAL) was assessed by a questionnaire validated with doubly labeled water (29). On the basis of the REE determination and obtained PAL, the total energy expenditure for each individual was calculated.
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Statistical Analysis
For rodents, data are expressed as mean 6 SEM in relation (%) to the specific control (vehicle-treated rats). Statistical significance was determined by Student t test (for two groups) or ANOVA and post hoc two-tailed Bonferroni test (for more than two groups). P , 0.05 was considered significant. For human data, a mixed ANOVA with a repeated-measures design was performed to study between- and within-subject factors comparing temporal changes in values between the treatment groups. Moreover, a MANOVA was used to assess differences between groups during the overall study time course, using Pillai trace criterion as a test of significance. Comparisons between pre- and postdata within a same treatment group were further analyzed by two-tailed paired Student t tests. The calculations were performed by SPSS for Windows version 15.0 software (IBM Corporation, Chicago, IL). P , 0.05 was considered statistically significant. RESULTS Central GLP-1R Stimulation Suppresses Food Intake and Decreases Body Weight in Mice
A single ICV injection of liraglutide (0.3, 1, and 3 mg/ mouse) significantly decreased food intake and body weight after 24 h at doses of 1 and 3 mg/mouse, and these effects remitted at 48 h (Supplementary Fig. 1). We next compared the central effects of liraglutide versus GLP-1 at 1 mg/mouse, a dose adapted from previous studies (31,32). Whereas a single ICV injection of liraglutide (3 mg/mouse) suppressed feeding behavior after 24 h, GLP-1 failed to do so (Fig. 1A). ICV liraglutide-decreased food intake was not caused by aversive effects (Fig. 1B). ICV liraglutide also suppressed body weight independent of its food intake effect because vehicle pair-fed mice, which ate the same amount of food as liraglutide-treated mice, did not show a significant decrease in body weight relative to vehicle-treated mice (Fig. 1C). ICV liraglutide-treated mice showed increased energy expenditure when corrected for lean mass (Fig. 1D), without changes in locomotor activity (Fig. 1E) or RQ (Fig. 1F). Body composition of these mice was determined immediately after keeping them in the indirect calorimetric system. The reduction in body weight was accompanied by a decrease in circulating leptin levels but without any changes in free fatty acids, triglycerides, cholesterol, insulin, or glucose levels (Supplementary Table 1). To rule out the possibility that centrally infused liraglutide may leak out of the CNS into the circulation and elicit a response by directly acting at peripheral level, we administered liraglutide peripherally. At the same doses as those infused centrally, liraglutide did not change cumulative food intake (Fig. 1G) or body weight (Fig. 1H).
Blood Determinations
Central GLP-1R Stimulation Triggers BAT Thermogenic Activity in Mice
Blood determinations were performed as previously described (22,30).
Because it has been reported that central GLP-1 infusion increases BAT thermogenesis (18), we investigated the
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Figure 1—Effect of a 24-h ICV liraglutide (3 mg/mouse) or GLP-1 (1 mg/mouse) injection on cumulative food intake (A), CTA (B), body weight change (C), energy expenditure (D), locomotor activity (E), and RQ (F). Effect of a 24-h intraperitoneal liraglutide (3 mg/mouse) on cumulative food intake (G) and body weight change (H). Data are mean 6 SEM of 7–8 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle. #P < 0.05, ##P < 0.01. EE, energy expenditure; IP, intraperioteneal; LiCl, liraglutide; PF, pair-fed.
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effects of liraglutide on this issue. First, we found that core body temperature was not affected by ICV liraglutide at any of the studied time points when animals were housed at room temperature (Fig. 2A). However, BAT interscapular temperature increased significantly at 12 h after ICV liraglutide injection in the same animals housed at room temperature (Fig. 2B and C). Histomorphological analysis revealed smaller lipid droplets in BAT from mice 24 h after a single ICV injection of liraglutide (Fig. 2D). Accordingly, in BAT of centrally liraglutide-treated mice, we found higher gene expression levels of several thermogenic biomarkers, such as CIDEA, FGF21, bone morphogenic protein 7 (BMP7), PRDM16, and ADRb1 (Fig. 2E). Protein levels of UCP1, UCP3, ADRb1, FGF21, and PRDM16 were also significantly augmented after the ICV liraglutide injection (Fig. 2F). There were no differences in basal core body temperatures between vehicle and liraglutide-treated mice (Fig. 2G). However, liraglutide-treated mice showed increased core body temperature (Fig. 2G) compared with control mice when they were kept at 4°C. Central GLP-1R Stimulation Induces Browning of WAT in Mice
ICV liraglutide induced browning in WAT, evident 24 h after the liraglutide injection (Fig. 3A). UCP1 protein levels (Fig. 3B) and both UCP1 and PRDM16 mRNA expression (Fig. 3C) were significantly upregulated in WAT of mice treated with ICV liraglutide. GLP-1R Stimulation in the VMH Regulates Thermogenesis and Browning in Rats
We next aimed to investigate the hypothalamic area responsible for the actions of liraglutide on BAT and WAT. Thus, we specifically injected liraglutide in the ARC, LHA, PVH, DMH, or VMH of rats (Fig. 4A–E). C-FOS immunostaining was assessed to corroborate the efficiency of the injections in each area (Fig. 4A–E). We found that the specific activation of the GLP-1R in the ARC, LHA, and PVH decreased food intake and body weight of rats (Fig. 4A–C), whereas no effects were detected when liraglutide was injected in the DMH (Fig. 4D). At the molecular level, UCP1 was unaltered in BAT and WAT of rats injected with liraglutide in those nuclei (Fig. 4A– D). Of note, administration of liraglutide in the VMH led to a significant weight loss with no significant differences in food intake, and UCP1 levels were increased in BAT and WAT (Fig. 4E). AMPK Within the VMH Is Essential for the Central Actions of Liraglutide on BAT and WAT
Because previous reports have demonstrated a key role of AMPK within the VMH in the regulation of BAT thermogenesis (25,26), we first assessed hypothalamic pAMPK levels after ICV injection of liraglutide. We found that pAMPK and its downstream target pACC were significantly decreased in the whole hypothalamus of ICV liraglutidetreated mice compared with their controls (Fig. 5A). Therefore, to demonstrate the relevance of hypothalamic AMPK
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as a mediator of the actions of liraglutide, we used a pharmacological activator of AMPK, AICAR (25). At the dose used, ICV AICAR did not blunt the anorexigenic effect of ICV liraglutide (Fig. 5B) but prevented ICV liraglutideinduced weight loss (Fig. 5C). Second, AMPK activity was elevated by using an adenoviral vector encoding constitutively active (CA) AMPKa, with adenoviruses expressing GFP used as controls (25). The adenoviruses were injected stereotaxically into the VMH of rats together with liraglutide, and the specificity of these injections was corroborated by GFP immunostaining (Fig. 5D). Liraglutide injected specifically into the VMH decreased weight, but the liraglutide-induced weight loss was completely blunted when AMPKa-CA was overexpressed in the VMH (Fig. 5E). Identical to the effect on body weight, AMPK activation in the VMH also reduced the liraglutide-induced UCP1 expression in BAT of rats (Fig. 5F) and WAT (Fig. 5G). The SIRT1/p53 system interacts with AMPK at the central level, and both molecules are key mediators of the orexigenic action of ghrelin (33). Furthermore, GLP-1 inhibits ghrelin-stimulated neuronal activity in the hypothalamus as well as its effects on food intake (34). Therefore, we next examined whether SIRT1 or p53 might be relevant for the central actions of liraglutide. To this aim, we used mice with moderate overexpression of SIRT1 under the control of its natural promoter (35) and p53 null mice (33). The findings demonstrate that ICV injection of liraglutide decreased food intake and body weight similarly in both wild-type and SIRT1 transgenic mice (Fig. 5H and I) or p53 null mice (Fig. 5J and K). Long-Term GLP-1R Agonism Increases Energy Expenditure in Obese T2D Patients
In addition to the functional data on liraglutide from the mouse study, we analyzed data from a cohort of obese patients with T2D who had been treated for 1 year with the antidiabetic drugs metformin and metformin in combination with the GLP-1R agonists exenatide and liraglutide (Table 1). At baseline, all obese T2D groups exhibited an identical sex distribution with no significant differences in age, BMI, waist circumference, body composition, REE, and RQ (Table 1) as well as in biochemical and hormonal variables (Supplementary Table 1). After 1 year of antidiabetic treatment instauration, all study groups showed a decrease in fasting plasma glucose and insulin concentrations. No significant changes in total cholesterol, HDL cholesterol, and LDL cholesterol concentrations at the end of the 1-year study period were observed. After 12 months of antidiabetic treatment, the groups treated with metformin combined with exenatide or liraglutide showed a significant decrease in BMI and total body fat percentage and a significant increase in fatfree mass (FFM) (Table 1). None of the study groups significantly changed in RQ and PAL during the experimental period, which remained within the same sedentary range as before treatment started as well as between the diverse antidiabetic administration groups (Table 1). Although no
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Figure 2—Effect of ICV liraglutide (10 mg/rat) on core body temperature (A) and BAT interscapular temperature (B) in animals housed at room temperature after 6, 12, and 24 h of the injection. C: Representative pictures of BAT interscapular temperature in animals housed at room temperature after 12 h of the injection. D: Representative BAT histology pictures (hematoxylin-eosin) 24 h after liraglutide ICV injection (3 mg/mouse). Effect of a 24-h ICV liraglutide (3 mg/mouse) on mRNA expression of PGC1a, ADRb1, ADRb2, ADRb3, CIDEA, FGF21, BMP7, and PRDM16 (E) and protein levels of UCP1, UCP3, CIDEA, FGF21, and PRDM16 (F). Tubulin was used to normalize protein levels. G: Body core temperature measurements over a 6-h period at 4°C. Data are mean 6 SEM of 7–8 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle. PF, pair-fed.
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Figure 3—A: Representative WAT histology pictures (hematoxylin-eosin) 24 h after liraglutide ICV injection (3 mg/mouse). Effect of 24-h ICV liraglutide (3 mg/mouse) on protein levels of UCP1 (B) and mRNA expression of PGC1a, PPARg, PPARa, CEBPa, CEBPb, ADRb1, ADRb2, ADRb3, FGF21, UCP1, and PRDM16 (C). GAPDH was used to normalize protein levels. Data are mean 6 SEM of 7–8 animals per group. *P < 0.05, **P < 0.01 vs. vehicle. PF, pair-fed.
statistically significant differences between and within groups were observed for unadjusted REE, there was a clear tendency to be higher in patients treated with GLP-1 agonists. When the REE data were adjusted for FFM, we found a significant increase in REE in patients treated with metformin in combination with exenatide or liraglutide; however, patients treated only with metformin did not show changes in any of these parameters (Fig. 6 and Table 1).
DISCUSSION
We report that the CNS GLP-1 system directly activates BAT thermogenesis and browning of white adipocytes. The primary finding is that these actions are mediated by hypothalamic AMPK, specifically within the VMH. To our knowledge, these findings are the first to provide information about the brain site and mechanisms by which fat mass decreases in response to a stimulation of CNS
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Figure 4—Effect of the specific injection of liraglutide (10 mg/rat) in the ARC (A), LHA (B), PVH (C), DMH (D), and VMH (E) on C-FOS, 24-h food intake, 24-h body weight change, BAT UCP1 levels, and WAT UCP1 levels. Tubulin was used to normalize protein levels. Data are mean 6 SEM of 7–8 animals per group. *P < 0.05, **P < 0.01 vs. vehicle.
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Figure 5—A: Effect of 24-h ICV liraglutide (3 mg/mouse) on hypothalamic protein levels of pAMPK, pACC, and ACC. Food intake (B) and body weight change (C) in mice receiving an ICV injection of liraglutide (3 mg/mouse) after previous injection with the AMPK activator AICAR (5 mg/ mouse). Effect of the injection of adenoviral particles encoding for GFP or pAMPK-CA in the VMH of rats treated with liraglutide (10 mg/rat) (D) and on body weight change (E), BAT UCP1 protein levels (F), and WAT UCP1 protein levels (G). Effect of 24-h ICV liraglutide (3 mg/mouse) in wild type (WT) and SIRT1 transgenic (TgA) mice on food intake (H) and body weight change (I). Effect of 24-h ICV liraglutide (3 mg/mouse) in WT and p53 null (p53 KO) mice on food intake (J) and body weight change (K). GAPDH was used to normalize protein levels. Data are mean 6 SEM of 7–8 animals per group. *P < 0.05, **P < 0.01 vs. vehicle. KO, knockout; Lig, liraglutide; PF, pair-fed; Veh, vehicle.
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Table 1—Anthropometric characteristics and indirect calorimetry data of T2D patients before and after 1 year of antidiabetic treatment initiation Treatment variable Sex (male/female)
Metformin monotherapy Metformin in combination with GLP-1 agonists Statistical Premetformin Postmetformin Pre-exenatide Postexenatide Preliraglutide Postliraglutide significance 7/3
7/3
8/3
8/3
10/4
10/4
ns
65 6 11
—
67 6 14
—
66 6 15
—
ns
BMI (kg/m )
33.0 6 6.6
31.4 6 9.5
34.9 6 2.6
32.1 6 1.8*
35.3 6 2.2
32.0 6 2.1*
*
Waist (cm)
115 6 13
110 6 19
116 6 10
106 6 15
117 6 11
110 6 16
ns
39.0 6 16.9
37.9 6 9.4
39.5 6 4.3
35.8 6 3.7*
39.2 6 3.6
35.1 6 3.3*
*
63.3 6 17.0
67.1 6 9.3
60.7 6 4.2
64.3 6 3.4*
60.8 6 3.1
64.9 6 3.0*
Age (years) 2
Fat mass (%) FFM (%) REE (kJ/day)
7,670 6 1,030 7,469 6 1,230 7,595 6 832
8,240 6 901 7,623 6 1,014 8,326 6 1,107
* ns
74.9 6 10.0
74.1 6 11.7
75.7 6 8.4
79.1 6 8.7
76.0 6 10.3
80.9 6 11.2
REE (kJ/kg FFM/day) 121.0 6 16.7
114.3 6 18.8
120.6 6 5.1
135.7 6 6.0*
122.4 6 6.9
143.6 6 7.1*
*
RQ (vCO2/vO2)
0.85 6 0.02
0.84 6 0.03
0.85 6 0.04
0.85 6 0.05
0.83 6 0.06
0.84 6 0.04
ns
PAL
1.47 6 0.11
1.46 6 0.08
1.45 6 0.15
1.48 6 0.12
1.46 6 0.13
1.47 6 0.16
ns
REE (kJ/kg/day)
ns
Data are mean 6 SEM unless otherwise indicated. ns, nonstatistically significant differences by repeated-measures ANOVA, MANOVA, and two-tailed paired Student t test between pre- and postvalues within a same treatment group. *P , 0.05 by repeated-measures ANOVA and compared with baseline (pre) values within the same treatment group by two-tailed paired Student t test.
GLP-1 activity independently of anorexigenic actions. We also observed that obese T2D patients treated with metformin in combination with exenatide or liraglutide, which showed a reduction in BMI and fat mass, had an increased energy expenditure, suggesting that at the clinical level, the effects of GLP-1 agonists on body weight are at least partially mediated by increased energy expenditure. The mechanisms by which central GLP-1 stimulation reduces body weight seem to involve food intake (7), decreased lipid storage in WAT (17), and increased BAT thermogenesis (18). The diversity of the biological actions of CNS GLP-1 is likely caused by the wide expression of its receptor throughout the entire CNS (36). Therapies for T2D-based GLP-1R agonists are now commonly used in combination with other drugs and are under investigation for the treatment of human obesity (20). Liraglutide is a long-acting GLP-1 analog indicated for the treatment of T2D (37,38). In addition to its antidiabetic properties, the systemic administration of liraglutide induces weight loss in obese rats (39) and patients (20). Because previous studies found differences in the central actions of GLP-1 and long-acting GLP-1 analogs (31), we aimed to investigate the central effects of liraglutide on brown and white adipocyte metabolism. In agreement with previous findings (7,17,18), we found that the central stimulation of GLP-1R reduces food intake and body weight. Differently from previous studies that used a chronic activation of the brain GLP-1R (17,18), the present data show that a single ICV injection of liraglutide maintains its biological actions for 24 h. The decreased body weight in mice receiving a single ICV administration of liraglutide was independent of feeding behavior. Instead, the results show that increased energy expenditure, and more specifically BAT thermogenesis and WAT browning, can explain the liraglutide-induced
weight loss. Although previous reports indicated that brain GLP-1 induces BAT thermogenesis (18), the capacity of brain GLP-1 to enhance white-to-brown transdifferentiation was previously unknown. This effect is likely mediated by the increased activity of sympathetic fibers innervating WAT, as previously reported for BAT (18). Although GLP-1R is widely distributed throughout the rodent hypothalamus (36), the specific sites of action and the molecular pathways triggered by GLP-1 within the hypothalamus remain largely unknown. In the current study, we demonstrate that injection of liraglutide specifically into ARC, LHA, or PVH reduced both food intake and body weight. The reduced food intake observed after the administration of liraglutide in the ARC might seem controversial because a previous study showed that ARC GLP-1R regulates glucose homeostasis but not feeding (14). However, important methodological differences exist between these studies, such as the compounds used (GLP-1 vs. liraglutide) and the time at which food was weighed (2 vs. 24 h). Differently from the hypothalamic sites cited, the administration of liraglutide to the VMH significantly decreased body weight without concomitant reduction in food intake, suggesting that the stimulation of GLP-1R in this particular area reduces weight in a food intake–independent manner, which is corroborated by the significantly higher levels of UCP1 in the BAT and WAT of VMH in liraglutide-treated rats. Indeed, the VMH has been previously demonstrated to be an essential modulator of BAT metabolism, with the AMPK pathway as an important mediator controlling energy dissipation (25,26). Therefore, we hypothesized that the central effects of liraglutide on BAT and WAT could be mediated by AMPK in the VMH. The data show that ICV liraglutide administration decreased hypothalamic AMPK activity, measured as decreased levels of pAMPK. To investigate whether activation of AMPK could counteract the effect
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Figure 6—REE in obese T2D patients before and after the treatment for 1 year with metformin (A), metformin and exenatide (B), and metformin and liraglutide (C).
of liraglutide, we first pharmacologically activated central AMPK with the compound AICAR and then genetically activated AMPK specifically in the VMH by using adenoviralmediated targeting. In both cases, we found that the actions of liraglutide on thermogenesis and browning were abolished, indicating that VMH AMPK modulates the actions of brain GLP-1 on both BAT and WAT. Indeed, it is well established that the GLP-1R is also located in extrahypothalamic areas (8,9); therefore, the current results do not exclude the possibility that extrahypothalamic areas are also involved in the effects of GLP-1R agonists on BAT thermogenesis and energy expenditure. In particular, GLP-1 binding sites were found in the inferior olive and nucleus of the solitary tract (9); both areas have been described as involved in the control of thermogenesis (40,41). Although at the central level SIRT1/p53 and AMPK pathways are interacting to mediate the orexigenic actions of ghrelin (33), neither SIRT1 nor p53 modified liraglutide-induced hypophagia or weight loss, indicating that the brain SIRT1/p53 system does not interact with AMPK to mediate the actions of liraglutide on thermogenesis and browning. Finally, because it is known that BAT is active in humans (42–45), we investigated the clinical value of the current data and found that obese T2D patients treated with metformin in combination with the GLP-1R agonists exenatide or liraglutide showed an increase in energy expenditure. The HbA1c reductions observed were consistent with those from other studies of exenatide and liraglutide (20,46,47). In the patient study, both GLP-1 agonists were applied at doses known to have good digestive tolerance. Although previous reports showed that liraglutide failed to significantly affect energy expenditure in patients (46–49), in the current study, a significant increase in the adjusted REE and a reduction in BMI and the percentage of fat mass were evident.
The apparent discrepancies in the results can be explained by the length of the treatment period; in previous studies, patients were treated with GLP-1 agonists for 4 (48), 8 (46),
Figure 7—Schematic overview summarizing the physiological effects of the central injection of liraglutide on energy balance. Activation of GLP-1R in the VMH dephosphorylates AMPK and stimulates BAT thermogenesis and energy expenditure. Activation of GLP-1R in the LHA, PVH, and ARC decreases caloric intake. Combined, these parallel metabolic changes result in a decreased body weight.
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or 12 (47) weeks, whereas in the current study, patients were treated for a mean of 1 year. The 1-year clinical data demonstrate for first time in our knowledge that liraglutide, which is now under evaluation by the Food and Drug Administration as an antiobesity drug, increases energy expenditure. Although we cannot rule out the possibility that liraglutide-induced REE in humans is partially mediated by peripheral mechanisms, the present findings suggest that liraglutide uses a specific central pathway to increase energy expenditure and ultimately reduce body weight, and this pathway might be of clinical relevance. As summarized in Fig. 7, we have provided a combination of pharmacological and genetic evidence to demonstrate that the central stimulation of GLP-1R induces not only BAT thermogenesis, but also adipocyte browning in WAT. The molecular mechanism controlling these actions involves AMPK in the VMH. Funding. This work has been supported by grants from Ministerio de Economia y Competitividad (BFU2011-29102 to C.D. and RYC-2008-02219 and BFU2012-35255 to R.N.), Xunta de Galicia (10PXIB208164PR and 2012-CP070 to M.L. and EM 2012/039 and 2012-CP069 to R.N.), Fondo de Investigaciones Sanitarias (PI12/01814 to M.L. and FISPI12/00515 to G.F.), and CIBERobn. CIBERobn is an initiative of the Instituto de Salud Carlos III of Spain, which is supported by FEDER (European Fund for Regional Development) funds. This research has also received funding from the European Community’s Seventh Framework Programme under the following grants: to C.D., M.L., and R.N.: FP7/2007-2013: no. 245009: NeuroFAST, and R.N.: ERC StG-2011-OBESITY53-281408. Duality of Interest. No potential conflicts of interest relevant to this article were reported. Author Contributions. D.B., M.I., R.G., and A.S. contributed to the experiments and data analysis. D.H. contributed to the experiments. F.V., M.S., J.F., J.S., J.E., C.D., M.L., and G.F. contributed to the development of the analytical tools and discussion. R.N. contributed to the experimental design and writing of the manuscript. R.N. 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. Drucker DJ. The role of gut hormones in glucose homeostasis. J Clin Invest 2007;117:24–32 2. Göke R, Fehmann HC, Göke B. Glucagon-like peptide-1(7-36) amide is a new incretin/enterogastrone candidate. Eur J Clin Invest 1991;21:135–144 3. Campbell JE, Drucker DJ. Pharmacology, physiology, and mechanisms of incretin hormone action. Cell Metab 2013;17:819–837 4. Kreymann B, Williams G, Ghatei MA, Bloom SR. Glucagon-like peptide-1 7-36: a physiological incretin in man. Lancet 1987;2:1300–1304 5. Mojsov S, Weir GC, Habener JF. Insulinotropin: glucagon-like peptide I (7-37) co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas. J Clin Invest 1987;79:616–619 6. Willms B, Werner J, Holst JJ, Orskov C, Creutzfeldt W, Nauck MA. Gastric emptying, glucose responses, and insulin secretion after a liquid test meal: effects of exogenous glucagon-like peptide-1 (GLP-1)-(7-36) amide in type 2 (noninsulin-dependent) diabetic patients. J Clin Endocrinol Metab 1996;81:327– 332 7. Turton MD, O’Shea D, Gunn I, et al. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 1996;379:69–72 8. Shimizu I, Hirota M, Ohboshi C, Shima K. Identification and localization of glucagon-like peptide-1 and its receptor in rat brain. Endocrinology 1987;121: 1076–1082
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