INHIBITION OF THE CENTRAL MELANOCORTIN SYSTEM DECREASES BROWN ADIPOSE TISSUE ACTIVITY

The melanocortin system is an important regulator of energy balance and MC4R deficiency is the most common monogenic cause of obesity. We investigated whether the relationship between melanocortin system activity and energy expenditure is mediated by brown adipose tissue (BAT) activity. Therefore, female APOE*3-Leiden. CETP transgenic mice were fed a Western-type diet for 4 weeks and infused intracerebroventricularly with the MC3/4R antagonist SHU9119 or vehicle for 2 weeks. SHU9119 increased food intake (+30%), body fat (+50%) and decreased energy expenditure by reduction in fat oxidation (-42%). In addition, SHU9119 impaired plasma clearance of VLDL-TG, which was solely explained by reduced TG uptake by BAT. In line with this, SHU9119 decreased uncoupling protein-1 protein levels in BAT (-60%) and induced large intracellular lipid droplets, indicative of severely disturbed BAT activity. Finally, SHU9119-treated mice pair-fed to the vehicle-treated group still exhibited these effects, indicating that MC4R inhibition impairs BAT activity independent of food intake. We conclude that inhibition of central MC3/4R signaling impairs BAT function, which is accompanied by reduced energy expenditure thereby promoting adiposity. We anticipate that activation of MC4R is a promising strategy to combat obesity by increasing BAT activity. effect BAT we inhibited the central melanocortin system using the MC3/4R synthetic antagonist SHU9119 in APOE*3-Leiden,CETP mice. We found that i.c.v. administration of SHU9119 decreased EE and BAT activity, concomitant with selectively impaired uptake of TG from plasma by BAT, independent of food intake.


INTRODUCTION
The hypothalamus is important in the regulation of energy balance. Activation of proopiomelanocortin neurons, e.g. by insulin or leptin, induces secretion of -melanocytestimulating hormone, which in turn stimulates melanocortin -3 and -4 receptors (MC3R/ MC4R) within the paraventricular nucleus to cause a negative energy balance (1). Accordingly, activation of central MC4R in rodent models results in anorexia and weight loss (2), whereas blockade or targeted gene disruption induces hyperphagia and obesity, even on regular chow diet (3,4). Loss-of-function mutations in MC4R are the most common monogenic form of obesity in humans and are associated with severe obesity in childhood (5). In addition, recent meta-analyses of genome-wide association studies identified common variants near MC4R to influence fat mass, obesity and obesity risk (6,7). These observations support an essential role for the melanocortin system in the regulation of energy homeostasis across mammalian species.
In addition to the effects of the melanocortin system on food intake, this system also affects energy balance via other pathways. This notion is supported by the observation that pharmacological inhibition of central MC4R by intracerebroventricular (i.c.v.) administration of the synthetic MC3/4R antagonist SHU9119 still increases body fat in pair-fed rats (8). Moreover, the peripheral effects of the central melanocortin system involves alterations in the activity of the sympathetic nervous system (SNS), as i.c.v. administration of the MC3/4R agonist MTII dose-dependently increases renal sympathetic activity in mice (9). Furthermore, ablation of neurons that produce agouti-related protein (AgRP), the endogenous antagonist for MC4R, in mice changes autonomic output into metabolic organs, accompanied by a changed respiratory exchange ratio (RER) indicating altered nutrient combustion (10). Additionally, chronic i.c.v. SHU9119 treatment in rats increases the RER (8), indicative of reduced lipid utilization. Interestingly, variants near and in the MC4R gene in humans are not only associated with an increased RER (8), but also with reduced total energy expenditure (EE) (11,12), underscoring the importance of the melanocortin system in the regulation of EE.
A recently discovered and highly important contributor to EE is brown adipose tissue (BAT). BAT contributes to EE by combusting high amounts of TG into heat, a process mediated by uncoupling protein-1 (UCP-1) (13). Interestingly, MC4R expressing neurons project onto BAT (14), indicating that BAT may mediate the association between MC4R signaling and EE. Therefore, the aim of this study was to evaluate the role of the melanocortin system in BAT activity. For this purpose, we inhibited melanocortin receptor signaling using the MC3/4R antagonist SHU9119 in APOE*3-Leiden.CETP transgenic mice.

Animals and diet
Female APOE*3-Leiden.CETP mice on a C57Bl/6J background (15) were bred at our institutional animal facility and housed under standard conditions with a 12-12 h light-dark cycle with ad libitum access to food and water unless stated otherwise. From 12-22 weeks after birth, they were fed a Western-type diet containing 15% (w/w) cacao butter and 0.1% cholesterol (AB Diets, Woerden, The Netherlands) for the duration of the study. After 4 weeks of run-in diet, mice were randomized into groups that received i.c.v. administration of artificial cerebrospinal fluid (vehicle) or SHU9119 (5 nmol/day; Bachem, Bubendorf, Germany) in vehicle during 14-17 days. Since SHU9119 induces hyperphagia (3), the effect of SHU9119 on BAT activity independent of food intake was also investigated by using an extra treated group that was pair-fed to the vehicle-treated group. All animal experiments were approved by the institutional ethics committee on animal care and experimentation at Leiden University Medical Center.

Surgical procedure
For continuous i.c.v. administration of SHU9119 vs. vehicle, mice were sedated using a mixture of dexmedetomidine (0.5 mg/kg), midazolam (5 mg/kg) and fentanyl (0.05 mg/kg), and cannulas (Brain Infusion Kit 3, ALZET Cupertino, CA, USA) were stereotactically placed in the left lateral ventricle of the brain (coordinates: -0.45 mm anteroposterior, -1.00 mm lateral and 2.50 mm dorsoventral from bregma). Osmotic mini-pumps (Model 1004, Alzet, CA) attached to the cannula via a catheter were implanted subcutaneously on the back slightly posterior to the scapulae. The skin was sutured and the sedation was antagonized with a mixture of antiparnezol (2.5 mg/kg), flumanezil (0.5 mg/kg) and naloxon (1.2 mg/kg). Buprenorphine (0.9 µg) was used as pain killer. After the surgery, mice were housed individually and food intake and body weight were monitored on a daily basis. By filling the catheters with vehicle, mice were allowed to recover for four days before actually receiving the assigned treatment for 17 days (collection of organs or VLDL production) or 14 days (indirect calorimetry and VLDL clearance).

Body composition
After 17 days of treatment, body composition (lean mass and fat mass) was determined in conscious mice using an EchoMRI-100 (EchoMRI, Houston, Texas).

Indirect calorimetry
During the first 5 days of treatment, oxygen uptake (V˙ O2), carbon dioxide production (V˙ CO2) and physical activity were measured in fully automatic metabolic cages (LabMaster System, TSE Systems, Bad Homburg, Germany). The average RER, EE, carbohydrate and fat oxidation rates were calculated as described previously (16).

Liver lipid staining and content
Liver samples were perfused with PBS, collected, snap frozen and stored at -80°C. Sections of 10 µm were cut, fixed in 4% paraformaldehyde and stained with Oil-red-O and Mayer's hematoxylin. Lipids were extracted according to a modified protocol from Bligh and Dyer (17). In short, small liver pieces were homogenized in ice-cold methanol (10 µL/mg tissue). 1.8 mL of CH3OH: CHCl3 (3:1, vol/vol) was added to 45 µL of homogenate. After vigorous mixing and centrifugation, the supernatant was dried and suspended in 2% Triton X-100. Concentrations of hepatic TG, total cholesterol (TC) and phospholipids (PL) were measured using commercially available enzymatic kits for TG (11488872, Roche Diagnostics, Germany), TC (11489232, Roche Diagnostics, Mannheim, Germany) and PL (3009, Instruchemie, Delfzijl, the Netherlands). Liver lipids were expressed per milligram of protein, which was determined using the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA).

VLDL production
After 17 days of treatment, after 4 h of fasting (from 8.00 h to 12.00 h), the VLDL production rate was assessed. Mice were sedated using a mixture of ventranquil (6.25 mg/kg), dormicum (6.25 mg/kg), and fentanyl (0.31 mg/kg). Subsequently, mice were injected intravenously (i.v.) with 100 µL PBS containing 150 µCi Tran 35 S label to measure de novo apoB synthesis and blood samples were taken via tail bleeding (t=0). 30 min after injection of the Tran 35 S label, the mice received an i.v. injection of 500 mg of tyloxapol (Triton WR-1339, Sigma Aldrich, Germany) per kg body weight as 10% (w/w) solution in PBS, to block VLDL-TG clearance by lipoprotein lipase (LPL)-mediated TG hydrolysis. Additional blood samples were taken at t=15, 30, 60 and 90 min after tyloxapol injection and used for determination of plasma TG concentration. After 120 min, the mice were exsanguinated via the retro-orbital plexus. VLDL was isolated from serum after density gradient ultracentrifugation and counted for incorporated 35 S-activity. VLDL particle size was determined using a Zetasizer (Malvern Instruments, Malvern, UK) and VLDL lipid composition was determined as described above.

BAT histology and UCP-1 analysis
After 17 days of SHU9119 treatment, a part of interscapular BAT was fixed in 4% paraformaldehyde in PBS (pH 7.4) for 24 h, dehydrated and embedded in paraffin. 10 µm sections were cut, rehydrated and stained with Mayer's hematoxylin and eosin. Another part of BAT was snap frozen and stored at -80°C. These BAT samples were homogenized in RIPA buffer, centrifuged and protein concentration was determined using the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Samples were diluted and denatured for 5 min at 95°C after adding Laemmli Sample Buffer (1:1, vol/vol; Serva, Heidelberg, Germany). Proteins within homogenates (1 µg protein) were separated on a 10% SDS-page gel and subsequently transferred onto blotting membrane. The blotting membranes were then washed with PBS+0.1% Tween (PBS+T), blocked with 5% milk powder in PBS+T and incubated O/N at 4°C with the first antibody (anti-UCP-1 rabbit polyclonal; 1:5,000; Ab U6382, Sigma Aldrich, Germany). After washing (PBS-T) the second antibody (anti-rabbit IgG HRP conjugate; 1:5,000; Promega, Madison, WI, USA) was added. After another wash with PBS-T and PBS, SuperSignal Western Blot Enhancer (Thermo Scientific, Rockford, IL, USA) was added to the blotting membranes after which they were analyzed with Bio-Rad Quantity One. C]CO) double-labeled VLDL-like emulsion particles (80 nm) were prepared as previously described (18). After 14 days of i.c.v. SHU9119 or vehicle treatment, mice were fasted for 4 h (from 8.00 h to 12.00 h) and injected i.v. with the radiolabeled emulsion particles (1.0 mg TG in 200 µL PBS) via the tail vein. At time points t=2, 5, 10 and 15 min after injection, blood was taken from the tail vein to determine the serum decay of both radiolabels. Immediately after the last blood withdrawal, mice were euthanized by cervical dislocation and perfused with ice-cold PBS for 5 min. Organs were harvested, weighed, and the uptake of 3 H and 14 C radioactivity was determined.

Statistical analysis
Differences between groups were determined using independent sample T-tests for normally distributed data and Mann-Whitney U tests for non-normal distributed data. Serum decay in the VLDL clearance experiment was analyzed using repeated measurements ANOVA with a Tukey's Post-Hoc test. Probability values less than 0.05 were considered statistically significant. Data are presented as means ± SEM.

SHU9119 reduces whole body fat oxidation independent of food intake
Since SHU9119 induced fat accumulation independent of food intake, we reasoned that SHU9119 likely affected EE. Therefore, we next assessed the effect of SHU9119 on energy metabolism. Fully automated metabolic cages were used during the first 5 days of treatment in order to prevent a potential confounding effect of differences in body weight. Indeed, in ad libitum fed mice, SHU9119 decreased EE (-10%; 23 These effects were not caused by an effect on carbohydrate oxidation (FIGURE 2C) but rather by a large reduction in fat oxidation (-43%; 5.1±1.0 vs. 8.9±0.3 cal/h/g FFM, p<0.001) (FIGURE 2D). SHU9119 also reduced activity of the animals (-46%; 67±6 vs. 123±5 A.U., p<0.05; not shown). Strikingly, the effects of SHU9119 in pair-fed mice, as compared to the control group, were essentially similar as in ad libitum fed mice with respect to EE (23.9±0.1 cal/h/g FFM; p<0.01), RER (0.91±0.01; p<0.01), fatty acid oxidation (5.3±0.5 cal/h/g FFM; p<0.001) and activity (75±3 A.U.; p<0.05). Apparently, SHU9119 reduced EE, mainly because of reduced fat oxidation and independent of food intake. FIGURE 1 -SHU9119 increases body weight and fat mass independent of food intake. APOE*3-Leiden.CETP mice were treated intracerebroventricularly with vehicle (n=21) or SHU9119 (5 nmol/day) while being fed ad libitum (n=21) or being pair-fed (pf) to the vehicle-treated group (n=22). Food intake A and body weight gain B were monitored on a daily basis. After 17 days of treatment, lean and fat mass were measured in a random selection of the mice (n=3-4 per group) using EchoMRI C . Part of the mice were used for the collection of organs, and weight of gonadal white adipose tissue was determined (n=10-11 per group) D .

SHU9119 induces hepatic steatosis, and this is due to increased food intake
Since the liver is an important player in TG storage and secretion, we evaluated the effect of SHU9119 on liver weight and TG content as well as on hepatic VLDL-TG secretion. SHU9119 induced hepatomegaly as evidenced by increased liver weight (+85%; 2.17±0.11 vs. 1.17±0.06 g, p<0.001) (FIGURE 3A) and aggravated hepatic steatosis, as shown by a selective increase in liver TG (+57%; 689±33 vs. 439±37 nmol/mg protein, p<0.001) (FIGURE 3B) and neutral lipid staining (FIGURE 3C). However, the effects of SHU9119 on the liver were fully attributed to the induction of hyperphagia, as hepatomegaly and hepatic steatosis were not induced under pair-fed conditions (FIGURES 3A-C). SHU9119 did not affect the VLDL-TG production rate in mice that were either fed ad libitum (3.39±0.14 mmol/L/h) or pair-fed (3.61±0.37 mmol/L/h) as compared to control mice (3.59±0.29 mmol/L/h) (FIGURES 3D, E). The VLDL-apoB production rate was slightly decreased in SHU9119-treated mice, but not in pair-fed SHU9119-treated CETP mice were treated intracerebroventricularly with vehicle (n=21) or SHU9119 (5 nmol/day) while being fed ad libitum (n=21) or being pair-fed (pf) to the vehicle-treated group (n=20). After 17 days of treatment, part of the mice were sacrificed (n=10-11 per group) to collect organs and determine liver weight A , and to determine liver content of triglycerides (TG), total cholesterol (TC) and phospholipids (PL) B . Frozen liver samples were sectioned and stained with a neutral lipid staining (Oil-red-O) and hematoxylin, and representative pictures are shown C . The remaining mice (n=8-10 per group) were 4 h fasted, consecutively injected with Trans 35 S label and tyloxapol, and blood samples were drawn up to 90 min after tyloxapol injection. Plasma TG concentration was determined and plotted as the increase in plasma TG relative to t=0 D . The rate of TG production was calculated from the slopes of the curves from the individual mice E . After 120 min, the total VLDL fraction was isolated by ultracentrifugation and the rate of newly synthesized VLDL-ApoB was determined F . The VLDL fractions were assayed for particle size G and lipid content H .
Values are means ± SEM. *p<0.05, ***p<0.001 compared to control.   In line with these observations, SHU9119 did not affect VLDL particle size (FIGURE 3G), VLDL composition (FIGURE 3H), or hepatic expression of the genes Apob, Mttp, Dgat2 involved in VLDL synthesis (not shown). Taken together, SHU9119 induced hepatic steatosis secondary to its induction of hyperphagia and without affecting VLDL-TG secretion.

SHU9119 induces brown adipose tissue dysfunction independent of food intake
Since BAT strongly contributes to fat oxidation and total energy expenditure, we subsequently determined the effect of SHU9119 treatment on BAT function. SHU9119 treatment increased BAT weight in ad libitum fed mice (+50%; 0.15±0.01 vs. 0.10±0.01 g, p<0.01) and tended to increase BAT weight in pair-fed animals (+24%; 0.13±0.01 g, p=0.06) (FIGURE 4A). Surprisingly, SHU9119 dramatically increased intracellular lipid droplet size in BAT in both ad libitum fed and pair-fed mice (FIGURE 4B), along with largely reduced UCP-1 protein levels in BAT (-61%; p<0.001 and -61%, p<0.001) (FIGURE 4C). These data imply that SHU9119 decreases BAT activity independent of food intake, which may result in a decreased capacity of BAT to take up VLDL-TG for subsequent combustion as well as decreased burning of intracellulary stored TG. Therefore, we next determined the effect of SHU9119 on the kinetics of i.v. injected  conditions (FIGURE 4E). At 15 min after injection, the distribution of radiolabels over the organs was assessed. In control mice, [ 3 H]TO uptake by BAT (31.6±8.0%/g) was much higher than the uptake by liver (~4-fold), muscle (~25-fold) and WAT (~25-fold). Interestingly, SHU9119 tended to selectively decrease the uptake of [ 3 H]TO by BAT in the ad libitum fed group, and significantly did so in mice pair-fed to the control group (-57%; 13.7±1.9% of injected dose/g; p<0.05) (FIGURE 4F). In the control group, as compared to the 3 H-label, the uptake of the 14 C-label was much lower in BAT (~10-fold), muscle (~4-fold), heart (~3-fold) and WAT (~3-fold), while the uptake of 14 C-label was higher in liver (~3-fold). This pattern is compatible with selective delipidation of the VLDL-like emulsion particles in plasma by the LPL-expressing tissues (i.e. uptake of 3 H activity), with subsequent uptake of the core remnant by the liver (i.e. uptake of 14 C activity). SHU9119 treatment tended to reduce the uptake of [ 14 C]CO in the liver of both ad libitum fed mice(-20%; 20.8±3.6 %/g; p=0.11) and pair-fed mice (-11%; 23.1±1.5 %/g; p=0.25) as compared to the control group (26.1±2.1 %/g), whereas it decreased the uptake of [ 14 C]CO by BAT (ad libitum fed: -39%; p=0.24 ; pair-fed: -57%; p<0.01) (FIGURE 4G).

DISCUSSION
The melanocortin system is an important regulator of energy balance and MC4R deficiency is the most common monogenic cause of obesity. BAT recently emerged as an important player in energy expenditure by combusting high amounts of TG towards heat. In addition, MC4R expressing neurons project onto BAT (14). Hence, the association between MC4R and energy expenditure may be mediated by BAT. In the current study we aimed to evaluate the direct effect of the melanocortin system on BAT activity. For this purpose, we inhibited the central melanocortin system using the MC3/4R synthetic antagonist SHU9119 in APOE*3-Leiden,CETP mice. We found that i.c.v. administration of SHU9119 decreased EE and BAT activity, concomitant with selectively impaired uptake of TG from plasma by BAT, independent of food intake.
Both in ad libitum as well in pair-fed conditions, SHU9119 treatment increased body weight and WAT mass. These data are in line with those of Nogueiras et al. (8), who attributed weight gain and adiposity upon SHU9119 treatment to an increase in both lipid uptake as well as TG synthesis for storage in WAT. We also showed that in ad libitum fed conditions, SHU9119 induced ectopic lipid deposition in the liver, manifested by hepatomegaly and hepatic steatosis. Hepatic steatosis did not develop in pair-fed mice, indicating that this effect is a direct consequence of SHU9119-induced hyperphagia. Similar effects on the liver are observed after 4 days of i.c.v. SHU9119 treatment in rats (19) and in MC4R deficient mice, which in addition develop steatohepatitis when fed a high-fat diet and have therefore been proposed as a novel mouse model for non-alcoholic steatohepatitis (NASH) (20). Although hepatic steatosis could promote the secretion of hepatic lipid as VLDL (21), SHU9119 did not increase the production, size or composition of newly synthesized VLDL. Our data corroborate those of Stafford et al. (22) who showed that a single i.c.v. injection of 15 µg SHU9119 does not affect VLDL-TG production in rats.
Because SHU9119 was able to increase body adiposity independent of a change in food intake, we reasoned that SHU9119 reduces EE. Indeed, studies with metabolic cages confirmed that inhibition of the central melanocortin system reduced EE. SHU9119 treatment attenuated locomotor activity to some extent, which may have contributed to this decreased EE. More specifically, SHU9119 selectively reduced fat oxidation, whereas carbohydrate oxidation remained unaffected. This reduction in fat oxidation and total EE occurred independently of food intake and before changes in body weight were observed, indicative of a causal relation between reduced energy expenditure and the induction of obesity. Likewise, a previous study has shown that 7 days of i.c.v. injections with SHU9119 in rats increased the RER and thereby decreased fat utilization independent of food intake (8). As locomotor activity was not affected in that study, reduced fat oxidation may be dominant over the effect of decreased locomotor activity in the decrease in EE. In addition, MC4Rdeficient humans also display an increase in RER (8). Taken together, we suggest that, in general, inhibition of the melanocortin system results in a shift towards decreased metabolic use of lipids leading to elevated fat deposition in WAT.
Since BAT is a highly active metabolic tissue involved in EE and regulation of weight gain, we next proposed that the reduction in fat oxidation could be largely attributed to decreased activity of BAT. Indeed, in both ad libitum and pair-fed conditions, analysis of BAT revealed that SHU9119 largely increased intracellular lipid stores and decreased the protein level of the UCP-1, both indicative of reduced BAT activity (23). These data corroborate previous findings showing that chronic i.c.v. treatment of ad libitum fed rats with SHU9119 lowered BAT temperature during the night (24). Moreover, 7 daily i.c.v. injections of AgRP, the endogenous antagonist for MC4R, decreased UCP-1 gene expression in pair-fed rats (25), while acute i.c.v. injections of GLP-1, which indirectly stimulates MC4R, increased BAT thermogenesis by increasing activity of the sympathetic fibers towards BAT (26). These data can explain previous observations that MC4R deficient mice are unable to increase UCP-1 levels in BAT upon a high fat diet or cold stimulus (14). Since the activity of BAT is dependent on output from the SNS from the hypothalamus (27,28), it is likely that inhibition of the melanocortin system reduces sympathetic output to BAT.
Recently, Bartelt et al. (13) identified BAT as a major organ involved in plasma VLDL-TG clearance, with 24 hours of cold induction resulting in normalisation of plasma TG levels in hypertriglyceridemic mice. In a first experiment, we did observe a large increase in plasma TG levels upon 17 days of SHU9119 treatment under ad libitum conditions (SUPPLEMENTAL FIGURE 1A), which would be consistent with reduced uptake of TG by BAT. However, in a subsequent study the SHU9119-induced increase in plasma TG only reached significance under pair-fed conditions (SUPPLEMENTAL FIGURE 1B). Likewise, i.c.v. infusion of the MC4R synthetic antagonist HS104 also failed to increase plasma TG levels in pair-fed rats (29). It should be noted that MC4R deficient mice have only modestly increased plasma TG levels compared to control mice (+30%) (30), implying that partial inhibition of MC4R by SHU9119 may be insufficient to significantly increase plasma TG levels. In heterozygous MC4R-deficient subjects, plasma TG levels are increased (1.7 vs. 1.3 mmol/L) (31), indicating that the melanocortin system does play a role in the regulation of plasma VLDL-TG levels in humans.
Recently, Perez-Tilve et al. (32) demonstrated that inhibition of the central melanocortin neurons by either ghrelin or SHU9119 in wild-type mice increased circulating cholesterol, related to a decreased hepatic expression of SR-BI involved in the selective hepatic uptake of HDL-cholesteryl esters. In our study in APOE*3-Leiden.CETP mice with a human-like lipoprotein metabolism, SHU9119 did not increase total cholesterol (SUPPLEMENTAL FIGURE 1) despite decreased hepatic SR-BI expression (SUPPLEMENTAL FIGURE 2). This is likely due to the expression of human CETP that provides an alternative route for the clearance of HDL-cholesterol, as CETP expression in SR-BI-deficient mice also precludes an increase in HDL-cholesterol (33). Likewise, humans with heterozygous MC4R deficiency also do not have increased HDLcholesterol levels (31), pointing to a species-dependent effect of MC4R function on HDLcholesterol levels.
In conclusion, inhibition of central MC3/4R signaling by SHU9119 reduces BAT activity thereby reducing the uptake and combustion of VLDL-TG by BAT. As a consequence, excess lipids are stored in WAT (FIGURE 5). We anticipate that MC4R agonists that are currently in develop ment to combat obesity, increase energy expenditure through activation of BAT.

SUPPLEMENTARY APPENDIX
SUPPLEMENTARY FIGURE 1 -Effect of SHU9119 on plasma lipid levels. A In a first experiment, APOE*3-Leiden. CETP mice were treated intracerebroventricularly with vehicle (n=10) or SHU9119 (5 nmol/day) (n=7). After 17 days of treatment, blood was drawn after a 4 h fast (from 8.00 h to 12.00 h) via tail vein bleeding in paraoxoncoated capillary tubes to prevent ex vivo lipolysis and assayed for triglycerides (TG), total cholesterol (TC) and phospholipids (PL) using commercially available enzymatic kits for TG (11488872, Roche Diagnostics, Germany), TC (11489232, Roche Diagnostics, Mannheim, Germany) and PL (3009, Instruchemie, Delfzijl, the Netherlands). B In a second experiment, mice were treated i.c.v. with vehicle (n=21) or SHU9119 (5 nmol/day) while being fed ad libitum (n=21) or being pair-fed (pf) to the vehicle-treated group (n=22). Plasma lipids were determined after 4 h of fasting.
Values are means ± SEM. *p<0.05, **p<0.01 compared to control. SUPPLEMENTARY FIGURE 2 -Effect of SHU9119 on hepatic SR-BI protein levels. APOE*3-Leiden.CETP mice were treated intracerebroventricularly with vehicle (n=21) or SHU9119 (5 nmol/day) while being fed ad libitum (n=21) or being pair-fed (pf) to the vehicle-treated group (n=20). After 17 days of treatment, part of the mouse groups were sacrificed (n=10-11 per group) to collect organs and determine hepatic SR-BI protein levels.