Pharmacological inhibition of diacylglycerol acyltransferase 1 reduces body weight and modulates gut peptide release-Potential insight into mechanism of action

Pharmacological Inhibition ofDiacylglycerol Acyltransferase 1 ReducesBody Weight and Modulates Gut PeptideRelease—Potential Insight intoMechanism of ActionJinqi Liu1, Judith N. Gorski1, Stephen J. Gold2, Dunlu Chen1, Shirley Chen1, Gail Forrest1, Yoshiki Itoh3,Donald J. Marsh4, David G. McLaren1, Zhu Shen1, Lisa Sonatore1, Ester Carballo-Jane1, Stephanie Craw1,Xiaoming Guan5, Bindhu Karanam1, Junichi Sakaki6, Daphne Szeto1, Xinchun Tong1, Jianying Xiao1,Ryo Yoshimoto7, Hong Yu1, Thomas P. Roddy1, James Balkovec1 and Shirly Pinto1

Objective: Investigation was conducted to understand the mechanism of action of diacylglycerol

acyltransferase 1 (DGAT1) using small molecules DGAT1 inhibitors, compounds K and L.

Design and Methods: Biochemical and stable-label tracer approaches were applied to interrogate the

functional activities of compounds K and L on TG synthesis and changes of carbon flow. Energy

homeostasis and gut peptide release upon DGAT1 inhibition was conducted in mouse and dog models.

Results: Compounds K and L, dose-dependently inhibits post-prandial TG excursion in mouse and dog

models. Weight loss studies in WT and Dgat1-/- mice, confirmed that the effects of compound K on body

weight loss is mechanism-based. Compounds K and L altered incretin peptide release following oral fat

challenge. Immunohistochemical studies with intestinal tissues demonstrate lack of detectable DGAT1

immunoreactivity in enteroendocrine cells. Furthermore, 13C-fatty acid tracing studies indicate that

compound K inhibition of DGAT1 increased the production of phosphatidyl choline (PC).

Conclusion: Treatment with DGAT1 inhibitors improves lipid metabolism and body weight. DGAT1

inhibition leads to enhanced PC production via alternative carbon channeling. Immunohistological studies

suggest that DGAT1 inhibitor’s effects on plasma gut peptide levels are likely via an indirect mechanism.

Overall these data indicate a translational potential towards the clinic.

Obesity (2013) 21, 1406-1415. doi:10.1002/oby.20193

IntroductionObesity is one of the primary risk factors in diabetes and cardiovas-

cular diseases. Of the several metabolic pathways identified in body

weight (BW) homeostasis (1-3), the triacylglycerol synthesis ma-

chinery has emerged as a potential pharmacological target for modu-

lating BW. Excess accumulation of triglyceride (TG) in blood and

tissues is a critical mediator in metabolic diseases. Abnormal levels

of TG in non-adipose tissues such as liver, skeletal muscle, and

myocardium present significantly higher risk associated with insulin

resistance and cardiovascular diseases (4). The two major pathways

that mediate TG biosynthesis are the glycerol phosphate pathway (5)

and the monoacylglycerol pathway (6). DGAT, which catalyzes the

joining of diacylglycerol (DG) with a fatty acyl CoA, mediates the

final step of TG synthesis in both pathways. Two Dgat genes have

been identified, Dgat1 and Dgat2. The expression patterns of both

genes are conserved across humans and mice (7,8). DGAT1, while

broadly expressed, is markedly enriched in intestine, where the

monoacylglycerol pathway is believed to be the major route for TG

synthesis.

Initial evidence demonstrating that DGAT1 inhibition could have a

beneficial metabolic effect came from studies of a mouse model

with genetic deletion of Dgat1. Dgat1 knock out (Dgat1�/�) mice

are resistant to diet-induced obesity (DIO) and have improved insu-

lin sensitivity (9,10). Dgat1�/� mice also showed markedly reduced

1 Merck Research Laboratories, Rahway, New Jersey, USA. Correspondence: Shirly Pinto ([email protected]) 2 Merck Research Laboratories,Kenilworth, New Jersey, USA 3 Sato Pharmaceuticals Co., Ltd., Japan 4 Merck Research Laboratories, West Point, Pennsylvania, USA 5 Department ofNeurodegeneration, GlaxoSmithKline Research and Development Center, Shanghai 201203, China 6 Mochida Pharmaceuticals Co., Ltd, Japan 7 MedicinalResearch Laboratories, Shionogi Co., Ltd, Japan

Disclosure: The authors declared no conflict of interest.

Additional Supporting Information may be found in the online version of this article.

Received: 22 October 2012 Accepted: 11 November 2012 Published online 30 November 2012. doi:10.1002/oby.20193

1406 Obesity | VOLUME 21 | NUMBER 7 | JULY 2013 www.obesityjournal.org

Original ArticleOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

Obesity

levels of intestinal TG synthesis and postabsorptive chylomicrone-

mia following an oral lipid challenge, indicating that Dgat1 defi-

ciency has a significant impact on intestinal TG metabolism in the

presence of a high dietary fat load (11). Interestingly, intestine-spe-

cific expression of Dgat1 in Dgat1�/� mice rescues their sensitivity

to DIO, indicating the key role of intestinal DGAT1 activity in con-

trolling BW homeostasis (12). Additionally, these results point to

the importance of the postprandial triglyceridemic response in deter-

mining susceptibility to high fat diet (HFD)-induced obesity. More

recently it has been demonstrated, from Dgat1�/� phenotyping stud-

ies, that DGAT1 mediates the prolongation of dietary fat-induced

increase in plasma gut peptide levels, and deficiency of Dgat1resulted in a delay of gastric emptying (GE) (13).

The positive metabolic phenotype of Dgat1�/� mice has led to sig-

nificant efforts among the pharmaceutical industry toward the devel-

opment of small molecule DGAT1 inhibitors for the treatment of

metabolic diseases. Preclinical efficacy of small molecule DGAT1

inhibitors was demonstrated in rodent proof of concept studies for

diminished postprandial hyperlipidemia and resistance to both DIO

and liver steatosis (14-20). More advanced DGAT1 inhibitors have

progressed into clinical development including, Novartis (LCQ-908),

Pfizer (PF 04620110), and AstraZeneca (AZ-7687) (15).

Despite relative progress in the field, additional structurally diverse

DGAT1 small molecule inhibitors are required to better understand

the mechanism whereby DGAT1 inhibition positively impacts me-

tabolism. In this report, we utilized novel, selective DGAT1 inhibi-

tors, compound K and L, to investigate the role of DGAT1 in induc-

ing weight loss and compared it to a marketed weight reducing

agent, orlistat. Furthermore, using an immunohistochemical based

approach, we aimed to understand the mechanisms by which attenu-

ation of postprandial TG synthesis, mediated by blockade of

DGAT1 activity, alters gut peptide release. Finally, we examined

the fate of DGAT1 substrates upon DGAT1 inhibition with tracer-

based approaches in cellular and in vivo systems.

Methods and ProceduresMaterialsImmunoassay kits from Meso Scale Discovery (MSD, MD); total

TG colorimetric assay (Thermo Scientific, NJ); anti-mouse

DGAT1 (Santa Cruz, CA); Cy3-conjugated secondary antibody

(Jackson ImmunoResearch, PA); anti-GLP-1 (Bachem, CA); Cy5-

conjugated secondary antibody (Jackson ImmunoResearch, PA);

40,6-diamidino-2-phenylindole (DAPI), diolein and oleoyl CoA

(Sigma-Aldrich, MO); 7-diethylamino-3-(40-maleimidylphenyl)-4-

methylcoumarin (CPM) (Invitrogen, CA).

MiceExperiments were performed in male lean and DIO C57BL/6 mice.

All WT mice were obtained from Taconic Farms (Germantown,

NY) and maintained on either standard rat diet or HFD in a 12-h

light/12-h dark cycle. Animal protocols used in these studies were

approved by the Merck Research Laboratories Institutional Animal

Care and Use Committee (Rahway, NJ). Dgat1�/� mice were

received from Tsukuba Research Institute, Japan (13). Six month

old mice were placed on HFD for 10 weeks prior to study.

CellsHT-29 and C2C12 cells were obtained from American Type Culture

Collection (ATCC, VA).

DGAT1 enzyme assayThe activity of DGAT1 from different species were measured using

DG/oleoyl CoA as substrates at Km concentrations in the presence

of CPM, which is weakly fluorescent until reacted with free thiols

of CoA released from oleoyl CoA after it is incorporated into DG to

form TG. IC50s were calculated using either Assay Data Analyzer

or GraphPad Prism4 software.

Cell-based functional assaysHT-29 and C2C12 cells were seeded in the presence or absence of

DGAT1 inhibitors followed by two hour incubation with 13C-labeled

oleic acid. The lipids were extracted with isopropanol containing

internal lipid standard followed by measurement using liquid chro-

matography-mass spectrometry (LC/MS).

Chronic dosing of compound K in miceMice were acclimated to nonspecific stress for 7 days before the

onset of the chronic dosing studies. DIO mice were dosed orally

with 0.1, 0.3, 1, 3, and 10 mg/kg of compound K for 14 or 28 days,

with BW and food intake (FI) monitored daily. Orlistat (Roche) was

dosed orally at 50 mg/kg.

Analysis of plasma samplesConcentrations of compounds in plasma were determined by LC-

MS/MS. Acetonitrile solution containing internal standard was added

for protein precipitation followed by centrifugation and separation of

the supernatants.

Body compositionWhole body composition analysis of conscious live mice was con-

ducted using quantitative magnetic resonance method (Echo Medical

Systems, TX).

Mouse post prandial TG excursion testLean C57BL/6 mice (10 week old, n ¼ 8/group) fasted overnight,

received 10 ml/kg corn oil via oral administration (PO) 60 min post

compound administration. Plasma was collected at various time

points. For gut peptide measurements, plasma was collected at 120

min post lipid challenge.

Dog post prandial TG excursion testLean beagle dogs (2-6 years old, n ¼ 6-8/group Marshall BioRe-

sources, NY) were fasted overnight and received 4 ml/kg heavy

cream (PO) 1 h post compound administration. Blood sampling was

taken at �1, 0, 1, 2, 4, 6 h relative to the lipid challenge.

Plasma gut peptide measurementsTotal and active GLP-1 levels were analyzed using immunoassay

kits from MSD. Analysis of PYY and gastric inhibitory polypeptide

(GIP) were conducted using milliplex mouse metabolic magnetic

Original Article ObesityOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

www.obesityjournal.org Obesity | VOLUME 21 | NUMBER 7 | JULY 2013 1407

bead panel (Millipore, MA). The data were analyzed using Bio-Plex

Manager 6.0 software.

Plasma triglyceride measurementsTG was measured using colorimetric assay from Thermo Scientific.

Fecal fat analysisFeces were collected the last two days of chronic treatment of com-

pound K in DIO mice. The fecal free fatty acids (FFAs) were

extracted with ethanol:distilled water:potassium hydroxide at ratio of

4:1:2. Following incubation at 70�C for 2 h, 6N HCl and petroleum

ether were added at ratio of 1:2. The upper phase after spinning was

dried and dissolved in isopropanol followed by measurement using

FFA kit (Wako Pure Chemicals, Japan).

ImmunohistochemistryParaffin-embedded mouse intestine sections were incubated over-

night with DGAT1 antibody (Ab) followed by incubation with Cy3-

conjugated secondary Ab. The tissue sections were then incubated

with GLP-1 or GIP Ab followed by incubation with Cy5-conjugated

secondary Ab. The nuclei were stained with DAPI.

Tracer-based plasma lipid profiling studyDIO mice (24-26 weeks old, n ¼ 7-8 per group) were chronically

dosed in feed (HFD) for 3 weeks with compound K. Following an

overnight fast, mice received an intravenous bolus of 13C-oleic acid

as a tracer. Plasma samples were collected at various time points.

Lipids were extracted and measured by LC/MS as described (21).

Statistical analysisStatistical analysis of data was conducted by Student’s t-test, one

way ANOVA using GraphPad Prism4 (Graph-Pad Software, CA).

Data are presented as means 6 standard errors of the mean (SEM)

and P values <0.05 were considered significant. GraphPad software

was also used to determine areas under curve (AUC).

ResultsIdentification of novel DGAT1 small moleculeinhibitorAn enzymatic-based assay, using human DGAT1 enriched membranes

from DGAT1 overexpressing Picchia, was utilized for screening

Merck’s small molecule libraries. Subsequent lead optimization effort

led to the identification of compound K as a potent inhibitor of

DGAT1 activity with a 50% inhibition (IC50) at 60 nM (Figure 1A)

and a related analog compound L with IC50 value of 24 nM (Figure

1B). Counter screens against closely related acyl transferases indicated

that compounds K and L are highly specific DGAT1 inhibitors. To

further understand compounds K and L activity across species, similar

enzymatic based-assays were performed using DGAT1 from different

species and demonstrated that compounds K and L were potently

active on DGAT1 from these preclinical species (Figure 1A and B).

To determine the functional activity of compound K inhibition in

cellular systems, two physiologically relevant cells, human intestinal

epithelial cells (HT-29) and mouse skeletal muscle cells (C2C12)

were evaluated. 13C-labeled oleic acid was used as the substrate at

Km concentration (100 lM). The incorporation of 13C-labeled oleic

acid into TG was measured through LC/MS analysis. Under these

conditions, compound K inhibited TG synthesis in a concentration-

dependent manner with IC50s at 150 nM and 35 nM for HT-29 and

C2C12 cells, respectively (Figure 1C and D).

In vivo effects of compound K demonstratepharmacological proof of conceptPharmacokinetic (PK) study of compound K was conducted to guide

in vivo efficacy studies. Compound K showed 16 ml/min/kg clear-

ance, 2 h half-life and 54% oral bioavailability in mice, which are

suitable for evaluation of in vivo efficacy. Compound K’s pharma-

cokinetic properties precipitated a series of pharmacological studies

to assess the effects of DGAT1 inhibition by compound K on

several metabolic parameters.

To determine the degree of compound K inhibition of DGAT1

required for the efficacy of BW loss, post prandial TG (PPTG)

excursion was used as a pharmacodynamic measure of gut DGAT1

inhibition. PPTG levels were measured in lean mice at 1, 2, and 3 h

after an oral corn oil bolus challenge, given 1 h after compound dos-

ing (Cmax). Under these conditions, a significant TG excursion was

observed in the plasma of vehicle-treated animals. Compound K

treatment led to dose-dependent attenuation in the PPTG relative to

vehicle group, where 1 mg/kg was determined to be the minimal

efficacious dose (MED) for maximal efficacy (Figure 2A).

FIGURE 1 Chemical structure and activity profile of compound K (A) and L (B).Compound concentration-dependent inhibition of triglyceride (TG) synthesis inhuman intestinal epithelial HT-29 (C) and mouse myoblastic C2C12 cells (D). Cellswere preincubated with various concentrations of compound K for 30 min followedby [13C-18] oleic acid incubation. A representative labeled TG species,TG13C18:1-13C18:1-13C18:1, was used to represent TG levels in the presence ofdifferent concentrations of compound K. Results are expressed as mean 6 SEM.IC50: 150 6 26 nM for HT-29 and 35 6 2 nM for C2C12. Compounds K and Lwere evaluated against other acyltransferases. IC50s: >7,5500 nM (humanDGAT2); >100,000 nM (human MGAT2, MGAT3, GPAT1); 1,933 nM (humanACAT1). The above study was repeated three or more times. DGAT2: diacylglyc-erol-O-acyl transferase 2; MGAT2: monoacylglycerol-acyl transferase 2; MGAT3:monoacylglycerol-acyl transferase 3; GPAT1: glycerol-3-phosphate acyltransferase1; ACAT1: acetyl-CoA-acyltransferase 1.

Obesity DGAT1 as a Key Regulator of Metabolic Pathways Liu et al.


We next investigated compound K’s effects on BW and FI in a DIO

mouse model. DIO mice were treated daily for 28 days with 0.1,

0.3, 1, or 3 mg/kg of compound K. Compound K treatment at 1 mg/

kg and 3 mg/kg led to 3.5% and 5% BW loss, respectively, relative

to vehicle group (Figure 2B). The BW change was predominantly

due to fat mass reduction (Figure 2C) with minimal effect on lean

mass (data not shown). Furthermore, compound K treatment yielded

modest but significant reductions in FI in the initial part of the study

(Figure 2D), suggesting that compound K reduces BW partially by

attenuating food consumption. Compound K-treated mice appeared

normal with no overt behavioral changes or alteration in locomotor

activities during the dosing period. PK analysis conducted at the

conclusion of the study demonstrated that plasma concentration of

compound K was 41 lM h as determined by the AUC at 3 mg/kg

dose. Trough total plasma level of compound K was 1.2 lM. Since

compound K is a highly-protein bound compound, the plasma-free

fraction of the compound at trough is below Ki. However, full target

engagement is observed at this drug dose (Figure 2A). Furthermore,

10 mg/kg dose (Figure 4) or 30 mg/kg (data not shown) did not

lead to greater weight loss, suggesting that maximum DGAT1

engagement is achieved at 3 mg/kg dose. These data implicate that

intestinal compound exposure levels may be a more accurate

predictive of efficacy over plasma.

To examine the contribution of DGAT1 pharmacological inhibition

to energy expenditure, the effect of acute DGAT1 inhibition was

evaluated. No significant effects were observed on oxygen consump-

tion (VO2) or respiratory quotient (RQ) between vehicle and

compound L treated groups over a period of 24 h (Supporting

Information Figure S1).

To confirm that the effect of compound K on BW is directly medi-

ated by inhibition of DGAT1 activity, WT and Dgat1�/� mice were

placed on HFD and were monitored for 7 weeks. As previously

reported, Dgat1�/� mice were shown to be resistant to diet-induced

obesity (Supporting Information Figure S2) (9). At the 10th week on

HFD, Dgat1�/� and WT mice were treated with daily dosing of 3

mg/kg compound K or vehicle for 14 days. In WT mice, the 3 mg/

kg dose of compound K led to approximately 3% reduction of BW

gain relative to vehicle-treated controls. However, weight gain under

FIGURE 2 Effects of compound K on postprandial TG excursion, BW loss, fat mass, and FI. Compound K reduced post-prandial TG excursion ina dose-dependent manner after acute dosing of compound K in lean mice (A). Change in percentage BW (B). Changes of fat mass after 28 daycompound K treatment (C) and daily FI (D) in DIO mice treated with daily PO dosing of either compound K at 0.1, 0.3, 1, and 3 mg/kg or vehiclefor 28 days. Results are expressed as mean 6 SEM (n ¼ 8 per group). *P < 0.05. The average BW is 46 g at the beginning of the study acrossall groups. The average BW is 50 g in vehicle-control group and 47.5 g in 3 mg/kg compound K treated group at the end of the study. The statis-tical analysis was performed using Student t test for TG excursion and one way ANOVA for BW and FI.



vehicle or compound K treated Dgat1�/� mice was virtually indis-

tinguishable (Figure 3A). Consistent with BW results, compound K

led to a significant 5% reduction in daily average FI in WT mice

with minimal effect observed in compound K treated Dgat1�/�

mice (Figure 3B). BW loss in the compound-treated WT mice was

at least partially due to the significant decrease in FI observed in

the first two days of treatment (Figure 3C). To rule out potential

confounding differences in compound K drug metabolism between

WT and Dgat1�/� mice, plasma compound levels were determined

on day 14 of treatment. Cmax and trough levels of compound K

were comparable between WT and Dgat1�/� groups (data not

shown). Taken together, these data support DGAT1 as the specific

biological target of compound K-mediated pharmacological effects

on weight loss.

Comparison of in vivo efficacy of compound Kwith orlistatTo understand the relative efficacy and tolerability of compound K

to a marketed drug targeting a related mechanism, a chronic weight

loss study was conducted comparing compound K to the pancreatic

lipase inhibitor, orlistat. Pancreatic lipase is the key enzyme media-

ting breakdown of dietary TG in intestine, an essential step for intes-

tinal fat absorption. By inhibiting intestinal TG breakdown, orlistat

prevents absorption of dietary fat thereby decreasing absorption of

dietary calories (22). DIO mice were treated with either compound

K or orlistat (50 mg/kg) for 14 days. Mice were monitored daily for

BW and FI. Compound K at 3 or 10 mg/kg led to approximately

4% BW loss, similar to that observed in our earlier studies, whereas

orlistat treated group lost roughly 6% compared to vehicle group

FIGURE 4 Comparison of compound K with orlistat suggests greater therapeutic margin. Changes of BW (A) and fecalFFA level (B) after 14-day chronic treatment with compound K (0.3, 1, 3, 10 mg/kg), orlistat (50 mg/kg) or vehicle in DIOmice. Fecal fatty acid contents trended higher with increasing doses of compound K, but significantly lower comparedto the orlistat-treated group. Results are expressed as fold changes versus vehicle (mean 6 SEM). ***P < 0.0001. Thestatistical analysis was performed using Student t-test for fecal fat output and one way ANOVA for BW measurements.

FIGURE 3 Dgat1�/� mice demonstrate mechanism-based BW loss. Changes of BW (A), accumulative FI after daily PO dosing with compound K (3 mg/kg) inWT and Dgat1�/� mice over the 14-day compound K treatment period (B), daily FI normalized as kcal/g/day (C). Compound K reduced FI and BW in WT, butnot in Dgat1�/� mice. Results are expressed as mean 6 SEM (n ¼ 8/group). *P<0.05. Both WT and Dgat1�/� mice were on HFD for 10 weeks prior to andduring 14 days of the study. The average starting BW at the time of the study for WT and Dgat1�/� mice was 45 g and 40 g, respectively. The statistical analy-sis was performed using Student t test for accumulative FI and one way ANOVA for BW and daily FI.



(Figure 4A). In contrast to compound K, orlistat resulted in a mod-

est but significant increase in cumulative FI (data no shown).

To determine the effects of orlistat or compound K on intestinal fat

absorption, fecal FFA was analyzed at the end of the study. Com-

pound K treatment led to a small, but dose-dependent increase in

fecal FFA (1.4- to 3-folds versus vehicle). In contrast, a profound

increase (11-fold versus vehicle) in fecal FFA was demonstrated

upon orlistat treatment (Figure 4B). These results indicate that

weight loss in the orlistat-treated group is likely due to fat

FIGURE 5 Postprandial gut peptide secretion and GE after compound L or K treatment in mouse and dog models. Two hour-postprandialmouse plasma levels of TG (A), active GLP-1 (B), PYY (C), and GIP (D). Increased in stomach weight 2 h after lipid load (E), suggesting adelay in GE after compound treatment. Results are expressed as mean 6 SEM (n ¼ 8/group). Time course of postprandial dog plasmalevels of TG (F), active GLP-1 (G), PYY (H), and GIP (I). Results are expressed as mean 6 SEM (n ¼ 6/group). *P < 0.05, **P < 0.005,***P < 0.0001. The statistical analysis was performed using Student t-test.



malabsorption predominantly, which was compensated by the

increase of energy intake. On the other hand, DGAT1 inhibition led

to decreased FI with only modest increase in fecal fat, suggesting

that weight loss is primarily due to a mechanism independent of fat

absorption.

Prolongation of lipid-induced gut hormonerelease in rodent and canine modelsIt has been previously demonstrated that Dgat1 inactivation in

mouse, rat, and canine models lead to modulation of gut peptide

secretion and delay in GE post-prandially (13,19,20). To further

investigate these observations, after pharmacological inhibition of

DGAT1 activity. PPTG and gut hormone secretion profile were

interrogated in rodent and canine models. In the rodent study, C57/

BL6 mice were treated with compound K or L, which has similar

potency on inhibiting DGAT1 activity and slightly weaker PK pro-

file compared to compound K (Figure 1B and data not shown). The

dosed mice were then challenged 1 h later with corn oil. Plasma TG

and the levels of gut hormones were monitored 2 h post challenge.

Compound K or L treatment completely suppressed plasma TG ele-

vation compared to vehicle-treated group (Figure 5A, data not

shown). However, compound K or L treatment led to increased lev-

els of plasma active and total GLP-1, as well as PYY (Figure 5B,

C, Supporting Information Figure S5 and data not shown). Interest-

ingly, GIP level was reduced upon compound L treatment (Figure

5D). In addition to modulation of gut peptide release, stomach

weight was significantly elevated in the compound-treated mice

(Figure 5E), indicating a delay in GE. Overall, these data suggest

that pharmacological inhibition of DGAT1 leads to similar effects

observed in the Dgat1�/� mice (13).

To determine whether the effects of compound K in rodents were

translatable to higher species, we investigated beagle dog model to

determine PPTG and gut hormone secretion. Compound K (1 mg/

kg) was administered orally to lean beagle dogs 1 h prior to lipid

challenge. Plasma TG and gut hormone levels were measured at �1,

0, 1, 2, 4, and 6 h relative to the time of lipid challenge. Consistent

with the results observed in mice, compound K treatment com-

pletely suppressed the elevation of plasma TG at all time points

tested (Figure 5F). On the other hand, plasma GLP-1 (total and

active forms) and PYY levels were markedly elevated and these ele-

vations were prolonged for up to 6 h after treatment (Figure 5G and

H and data not shown). In contrast to the elevation of GLP-1 and

PYY, GIP levels were initially attenuated, however, showed a signif-

icant increase relative to vehicle treated group at 6 h time point

(Figure 5I). These results demonstrate a tight homology in response

to DGAT1 inhibition between rodent and canine models, implying

that the mechanism of modulating gut hormone release via DGAT1

inhibition could be conserved across species.

Immunohistochemistry studies to interrogateDGAT1 expression in enteroendocrine cellsTo further understand the mechanisms underlying modulations of

gut peptide release mediated by DGAT1 inhibition, we conducted

studies to assess whether DGAT1 inhibition directly affects the

secretion of GLP-1 and PYY by enteroendocrine L cells and GIP

release from enteroendocrine K cells. An alternative mechanism for

DGAT1 effects on gut hormone release could be mediated by an

indirect signaling between the enterocytes and enteroendocrine cells.

An immunohistochemistry-based approach was utilized to address

this question. Dual-labeling immunohistochemistry studies were con-

ducted using antibodies against DGAT1 and GLP-1 or GIP. This

strategy permitted an assessment of the degree of DGAT1 immuno-

reactivity in enteroendocrine cells. We first tested the specificity of

antibodies against DGAT1 by comparing staining of the duodenum,

jejunum, and ileum of WT and Dgat1�/� mouse small intestine.

Expression of DGAT1 protein was detected throughout small intes-

tine. The highest DGAT1 expression was detected in the duodenum

relative to the proximal portion of the intestine, which is consistent

with mRNA levels (data not shown). More importantly, expression

of DGAT1 was only detected in the WT intestine and was entirely

absent along the intestine of Dgat1�/� mice (Figure 6A and B),

indicating that the antibody and the conditions used were specific

for the detection of DGAT1 expression along the gut.

To interrogate whether DGAT1 expression is detectable in the enter-

oendocrine cells, GLP-1 or GIP was utilized as L or K cell marker,

respectively. Antibodies against DGAT1 and GLP-1 or GIP were

used for the immunohistochemical staining to evaluate the colocali-

zation of DGAT1 with GLP-1 or GIP in target cells. It has been pre-

viously reported that GLP-1 positive cells are enriched at the distal

portion of intestine and GIP expressing cells are predominantly

located in the mid portion of small intestine (23). Similar observa-

tions were made in this study. However, survey of all sections

across entire intestine reveals lack of colocalization of DGAT1 with

GLP-1 (Figure 6C and D; Supporting Information Figure S3) or

with GIP (Supporting Information Figure S4). Taken together, these

data support the hypothesis of an indirect mechanism of communica-

tion between the enterocytes and enteroendocrine cells likely driving

the gut peptide release mediated by DGAT1 inhibition.

FIGURE 6 DGAT1 expression in enterocyte with non-detectable level in enteroendo-crine cells. Representative photomicrographs of DGAT1 immunoreactivity in duode-num sections from WT mice (A) and Dgat1�/� mice (B). DGAT1 expression wasonly observed in duodenum from WT mice. Dual-labeling analysis of DGAT1 andGLP-1 expression using jejunum sections from Dgat1�/� and WT mice at low (C)and high magnification (D). Multichannel image of the intestine demonstrates lackof colocalization of DGAT1 (red) and GLP-1 (green) in WT intestinal sections. Multi-ple sections were analyzed in these studies, and the representative sections areshown in this figure.



Alternative carbon channeling due to DGAT1inhibitionTo determine the carbon flow upon inactivation of DGAT1, we

interrogated the fate of DGAT1 substrates in response to DGAT1 in-

hibition. In contradiction to the expectation that the DGAT1 sub-

strate, DG, would accumulate following DGAT1 inhibition, levels of

DG in tissues of Dgat1�/� mice remain unchanged (24,25). Simi-

larly, acute treatment of pharmacological inhibitor of DGAT1

yielded unchanged levels of DG in the heart (24). This observation

led to the hypothesis that carbon flow is diverted to a different path-

way following DGAT1 inhibition. To address this hypothesis, HT29

and C2C12 cells were treated with various concentrations of com-

pound K followed by pulsing with 13C-labeled oleic acid. Newly

synthesized 13C labeled lipid species were measured at 2 h after

FIGURE 7 Alternative carbon channeling using a stable isotope as a tracer. HT-29 (A) and C2C12 cells (B) were pre-incubated with various concentrations of compound K for 30 min followed by the loading of [13C18]-oleic acid.Changes of levels of DG in the cells after compound K treatment were measured. One of labeled DG species,DG13C18:1-13C18:1, was shown to represent DG levels. A significantly different profile of DG was observed in HT-29compared with C2C12 cells. One of representative labeled PC species, PC13C18:1-13C18:1, was shown after com-pound K treatment for HT-29 (C) and C2C12 cells (D). PC levels were enhanced with increasing concentrations ofcompound K. Changes of other PC species including PC16:0-13C18:1 and PC13C18:1-C18:1 exhibited the similartrend (data not shown). Results are expressed as mean 6 SEM. IC50 for DG in C2C12 cells: 15 6 6 nM. EC 50 forDG in HT-29 cells: >5,000 nM. EC50 for PC in C2C12 cells: 14 6 2 nM. EC50 for PC in HT-29 cells: 130 6 7 nM.The above study was repeated three times. Diet-induced obese C57/BL6 mice were chronically dosed with vehicleor compound K in feed for three weeks. Mice were then fasted for 18 h followed by an intravenous bolus of intralipidcontaining 50 mg/kg [13C18]-oleic acid via tail vein injection. Plasma PC levels at 0, 15, 30, 60 min in vehicle andcompound K treated groups (E). Plasma PC levels area under curve (F) (1–60 min). PC levels were significantlyhigher in compound treated group compared with vehicle group. n ¼ 10 mice/group, **P < 0.005. The statisticalanalysis was performed using Student t-test.



tracer loading. Compound K treatment led to minimal accumulation

of DG in HT-29 cells at all concentrations (Figure 7A). Surprisingly,

modest but significant reduction in DG levels was observed in

C2C12 cells after compound K treatment (Figure 7B). While addi-

tion of compound K resulted in small to modest changes in DG con-

centration in both cells types, the levels of phosphatidylcholine (PC)

were elevated in these cells in a concentration-dependent manner

(Figure 7C and D), suggesting a divergent route for substrate carbon

flow during DGAT1 inhibition.

To determine whether the carbon re-channeling observed in cellular

system also occurs in vivo, 13C-labeled oleic acid was administrated

via intravenous bolus to mice treated with compound K (3 mg/kg)

for 21 days. Labeled lipid species from the plasma were analyzed

via LC/MS analysis. Consistent with the observations in the cellular

system, plasma DG levels were unaltered (data not shown), whereas

plasma PC levels were significantly increased to over 50% in com-

pound K treated group compared with vehicle control (Figure 7E

and F). Thus, both in vivo and in vitro data sets confirmed the

observations that DG is being shunted toward PC or potentially

other lipid synthetic pathways upon inhibition of DGAT1.

DiscussionIn this report, we describe potent selective DGAT1 inhibitors, com-

pound K and L, structurally diverse from previously reported com-

pounds. Compound K-treated mice exhibited mechanism based

weight loss and reduction of FI. PPTG was employed as a surrogate

target engagement marker at the end of the study, at which com-

pound K had reached trough levels in plasma. Interestingly, at doses

where statistically significant weight reduction occurred, TG excur-

sion was nearly completely inhibited. At the lower dose (0.3 mg/

kg), the level of TG excursion was decreased to approximately 50%,

whereas weight reduction was not observed at this dose. These data

suggest that nearly complete inhibition of DGAT1 is required to

achieve the beneficial effects on BW. Modest reduction of FI was

observed with compound K treatment, suggesting that FI, at least in

part, contributes to the weight loss. Although increased energy ex-

penditure was reported in Dgat1�/� mice (9,26), in our study, acute

treatment of compound L led to no significant effect on energy ex-

penditure. It remains possible that chronic DGAT1 inhibition could

lead to increased energy expenditure. However it does imply that

the pharmacological inhibition of DGAT1 may differentiate from

germline deletion of Dgat1. Further investigations are needed to elu-

cidate this possibility.

Although the mechanism by which DGAT1 inhibition leads to

weight loss is not fully understood, there have been several reports

interrogating the site of action of DGAT1 inhibition as it relates to

BW reduction. Lee et al (12) described a transgenic mouse model

with intestine-specific Dgat1 expression in a Dgat1 null background.

These mice were reported to attenuate the resistance of DIO com-

pared to Dgat1 null mice, suggesting that inhibition of DGAT1 in

the gut may be the primary driver for BW regulation. However, other

studies suggest that adipose could be a key site of action, as demon-

strated by the transplantation of Dgat1-deficient white adipose tissue

into mice prone to obesity and insulin resistance. The Dgat1�/� adi-

pose recipient WT mice acquired DIO resistance and increased insu-

lin sensitivity (27). Additionally, the reported prolonged release of

satiety hormones including PYY and GLP-1 as well as delayed GE

(13) could also be the factors contributing to the metabolically bene-

ficial effects of Dgat1 deficiency. Our studies demonstrated that

administration of compound K led to delay of GE, greater and pro-

longed release of GLP-1 and PYY as well as attenuation of GIP

release in rodent and canine models. The mechanism by which

DGAT1 inhibition leads to delay in GE remains elusive. It is possible

that GE delay is mediated directly by GLP-1 action, or perhaps

stimulated by higher levels of FFAs in the lumen. The delay in GE

and/or the prolongation of GLP-1 and PYY release could potentially

explain DGAT1 blockade-induced suppression of FI and loss of BW.

Further studies in mice with a deletion of GLP-1 receptor are under-

way to establish the correlation of magnitude and the duration of

delay in GE, elevation of GLP-1 with the efficacy of BW reduction

occurred following the treatment with DGAT1 inhibitor. It is encour-

aging to see a similar profile of gut peptide release and GE delay in

both mouse and canine models, indicating that the observation could

potentially be translatable to the clinic.

The exact mechanism of GLP-1/PYY elevation and GIP attenuation

through DGAT1 inhibition remains to be investigated. Immunohisto-

chemistry evaluation indicates that DGAT1 does not co-express with

enteroendocrine markers GLP-1 or GIP, suggesting that the pro-

longed secretion of GLP-1 and PYY or inhibition of GIP release is

likely not mediated by a direct inhibition of DGAT1 in the enteroen-

docrine cells. Rather, we propose that inhibition of TG synthesis via

DGAT1 blockade alters luminal distribution of lipids leading to

modulation of gut peptide secretion by an indirect mechanism. We

have observed delay of fat absorption with compound K treatment,

which could lead to higher levels of FFAs in the L cell enriched,

distal portion of the intestine. These FFAs could potentially serve as

agonistic factors to interact with G-protein coupled receptors with

GLP-1 and/or PYY secretagogue functions, including GPR119 and

GPR120, on L cells (28,29). These interactions may potentiate the

release of incretin peptides. On the other hand, attenuated levels of

FFAs in the proximal portion of intestine could contribute to the

reduced level of GIP. Additional studies are required to link the con-

nection between the inhibition of TG synthesis by DGAT1 inhibitor

and the alteration of incretin release.

The primary function of orlistat, marketed as a weight reducing

agent, is to prevent fat absorption which is also the cause of an

array of reported gastro-intestinal adverse effects (22). To compare

the effect of DGAT1 inhibitor to orlistat on lipid excretion, we con-

ducted a study for the direct comparison of compound K and orli-

stat’s effects on BW and lipid levels in feces. DIO mice were

administered chronically with either compound K at doses that led

to maximal weight loss efficacy or with orlistat at comparable clini-

cally efficacious dose. In comparison to compound K treated mice,

orlistat treated mice demonstrated slight increase in FI, which is

consistent with accelerated GE and enhanced energy intake observed

in previous human studies (22,30). Despite these observations, orli-

stat treatment led to significant weight loss that was comparable to

that obtained with compound K. As observed in the clinic, a pro-

found increase in fecal fat excretion was demonstrated in orlistat

treated group. In contrast, DGAT1 inhibition by compound K

yielded minor increases in fecal fat and this was observed only at

higher doses. With only modest weight loss effect, orlistat exhibits

minimal margin regarding risk versus benefit ratio clinically. Impor-

tantly, compound K’s markedly smaller effect on fecal lipids rela-

tive to orlistat occurred at doses with comparable BW loss. Our

studies suggest that a DGAT1 inhibitor’s effect on BW would be



commensurate with a clinically relevant dose of orlistat but with a

superior tolerability profile.

In contrast to the expectation that inhibiting DGAT1 will result in

accumulation of DG levels, we and others have demonstrated that

DG levels remain unaltered upon DGAT1 inhibition. The fate of the

carbons upstream of DGAT1 remains a critical question to address.

The utilization of a stable labeled tracer approach allowed us to in-

terrogate the outcome of carbon flow upon DGAT1 inhibition in

vitro and in vivo. HT-29 and C2C12 cells were selected to represent

gut epithelial cells from human origin and skeletal muscle cells

from mouse, respectively. We demonstrated that carbon flow is

diverted toward phospholipid upon DGAT1 inhibition. While DG

levels remain largely unchanged, different trend was observed in

these cells, implicating a possible differential capacity of metaboliz-

ing DG in intestinal and muscle derived cells. Consistently, the

EC50 of PC production is approximately tenfold more potent in

C2C12 cells than in HT-29 cells. In addition, mouse primary entero-

cytes were investigated in our studies and the trend was similar to

HT-29 cells (data not shown).

Blockade of DGAT1 activity promoted cellular PC synthesis in a

compound concentration-dependent manner. Consistent with these

observations, elevated PC levels in plasma was also demonstrated in

vivo after chronic treatment of compound K. These findings may

have significant physiological implications. Since increased DG

levels in skeletal muscle was shown to exacerbate muscle insulin

resistance (31), reduction of DG level due to alternative carbon

channeling, through DGAT1 inhibition, could lead to improved insu-

lin sensitivity. Moreover, PC is a crucial component of high density

lipoprotein (HDL), thus implying a potential additional benefit in

cardiovascular health via DGAT1 inhibition. The mechanism leading

to altered DG levels, increased production of PC, and potentially

other lipid species remains to be further investigated. It is likely that

the kinetics of DG production and/or consumption varies in different

cell types. Since DG has been shown to be a signaling molecule in

a variety of signaling pathways (32), it is possible that DG differen-

tially mediates cellular functions in these cell types.

Much of the interest in DGAT1 as a target for development of small

molecule inhibitors resulted from reports describing the metabolic

benefit of Dgat1�/� mice. The identification of selective and potent

small molecule inhibitors allows interrogation of the mechanism of

inhibition of DGAT1 and the effects on its upstream and down-

stream pathways. More importantly, the development of these and

other small molecule DGAT1 inhibitors are key steps toward greater

understanding the underlying mechanism of DGAT1 inhibition in

conjunction with potential translation to clinical efficacy.O

AcknowledgmentsAll work described here was performed by authors while employed

by Merck & Co., Inc. The authors would like to thank Timothy He

for his contribution to the studies related to this manuscript.

VC 2012 The Obesity Society

References1. Friedman JM. Obesity of the new millennium. Nature 2000;404:632-634.

2. Spiegelman BM, Flier JS. Adipogenesis and obesity: rounding out the big picture.Cell 1996;87:377-389.

3. Flier JS. Obesity wars: molecular progress confronts an expanding epidemic. Cell2004;116:337-350.

4. Capeau J. Insulin resistance and steatosis in humans. Diabetes Metab 2008;34:649-657.

5. Kennedy EP. Metabolism of lipids. Annu Rev Biochem 1957;26:119-148.

6. Kayden HJ, Senior JR, Mattson FH. The monoglyceride pathway of fat absorptionin man. J Clin Invest 1967;46:1695-1703.

7. Cases S, Smith SJ, Zheng YW, et al. Identification of a gene encoding an acylCoA: diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. ProcNatl Acad Sci USA 1998;95:13018-13023.

8. Cases S, Stone SJ, Zhou P, et al. Cloning of DGAT2, a second mammalian diacyl-glycerol acyltransferase, and related family members. J Biol Chem 2001;276:38870-38876.

9. Smith SJ, Cases S, Jensen DR, et al. Obesity resistance and multiple mechanisms oftriglyceride synthesis in mice lacking Dgat1. Nat Genet 2000;25:87-90.

10. Chen HC, Farese RV Jr. Inhibition of triglyceride synthesis as a treatment strategyfor obesity: lessons from DGAT-1 deficient mice. Arterioscler Thromb Vasc Biol2005;25:482-486.

11. Buhman KK, Smith SJ, Stone SJ, et al. DGAT1 is not essential for intestinal triac-ylglycerol absorption or chylomicron synthesis. J Biol Chem 2002;277:25474-25479.

12. Lee B, Fast AM, Zhu J, Cheng J-X, Buhman KK. Intestine-specific expression ofacyl CoA: diacylglycerol acyltransferase 1 reverses resistance to diet-induced he-patic steatosis and obesity in DGAT1�/� mice. J Lipid Res 2010;51:1770-1780.

13. Okawa M, Fujii K, Ohbuchi K, et al. Role of MGAT2 and DGAT1 in the releaseof gut peptides after triglyceride ingestion. Biochem Biophys Res Commun 2009;390:377-381.

14. Birch AM, Birtles S, Buckett LK, et al. Discovery of a potent, selective, and orallyefficacious pyrimidinooxazinyl bicyclooctaneacetic acid diacylglycerol acyltransfer-ase-1 inhibitors. J Med Chem 2009;52:1558-1568.

15. Birch AM, Buckett LK, Turnbull AV. DGAT1 inhibitors as anti-obesity and anti-diabetic agents. Curr Opin Drug Discov Dev 2010;13:489-496.

16. Fox BM, Iio K, Li K, et al. Discovery of pyrrolopyridazines as novel DGAT1inhibitors. Bioorg Med Chem Lett 2010;20:6030-6033.

17. King AJ, Segreti JA, Larson KJ, et al. In vivo efficacy of acyl CoA: Diacylglycerolacyltransferase (DGAT) 1 inhibition in rodent models of postprandial hyperlipid-emia. Eur J Pharmacol 2010;637:155-161.

18. Yamamoto T, Yamaguchi H, Miki H, et al. Coenzyme A: diacylglycerol acyltrans-ferase 1 inhibitor ameliorates obesity, liver steatosis, and lipid metabolism abnor-mality in KKAy mice fed high-fat or high-carbohydrate diets. Eur J Pharmacol2010;640:243-249.

19. King AJ, Judd AS, Souers AJ. Inhibitors of diacylglycerol transferase: a review of2008 patents. Expert Opin Ther Pat 2010;20:19-29.

20. Schober G, Arnold M, Birtles S, Buckett L, Turnbull AV, Langhans W. DGAT-1 in-hibition increases plasma glucagon-like peptide-1 (GLP-1) and peptide tyrosine-tyro-sine (PYY) levels in response to a high fat (HF) meal in rats. Appetite 2011;57:S39.

21. McLaren DG, He T, Wang S-P, et al. The use of stable-isotopically labeled oleicacid to interrogate lipid assembly in vivo: assessing pharmacological effects in pre-clinical species. J Lipid Res 2011;52:1150-1161.

22. O’Donovan D, Feinle-Bisset C, Wishart J, Horowitz M. Lipase inhibition attenuatesthe acute inhibitory effects of oral fat on food intake in healthy subjects. Br J Nutr2003;90:849-852.

23. Mortensen K, Christensen LL, Holst JJ, Orskof C. GLP-1 and GIP are colocalizedin a subset of endocrine cells in the small intestine. Regul Pept 2003;114:189-196.

24. Liu L, Yu S, Khan RS, et al. DGAT1 deficiency decreases PPAR expression and doesnot lead to lipotoxicity in cardiac and skeletal muscle. J Lipid Res 2011;52:732-744.

26. Streeper RS, Koliwad SK, Villanueva CJ, Farese RV. Effects of DGAT1 deficiencyon energy and glucose metabolism are independent of adiponectin. Am J PhysiolEndocrinol Metab 2006;291:E388-E394.

25. Chen HC, Smith SJ, Ladha Z, et al. Increased insulin and leptin sensitivity in micelacking acyl CoA: diacylglycerol acyltransferase. J. Clin Invest 2002;109:1049-1055.

27. Chen HC, Jensen DR, Myers HM, Eckel RH, Farese RV Jr. Obesity resistance andenhanced glucose metabolism in mice transplantation with white adipose tissuelacking acyl CoA: diacylglycerol acyltransferase 1. J Clin Invest 2003;111:1715-1722.

28. Oh DY, Talukdar S, Bae EJ, et al. GPR 120 is an omega-3 fatty acid receptor medi-ating potent anti-inflammatory and insulin-sensitizing effects. Cell 2010;142:687-698.

29. Cox HM, Tough IR, Woolston A-M, Zhang L, Nguyen AD, Sainsbury A. PeptideYY is critical for acyl ethanolamine receptor Gpr-119-induced activation of gastro-intestinal mucosal responses. Cell Metab 2010;11:532-542.

30. Ellrichmann M, Kapelle M, Ritter PR, et al. Orlistat inhibition of intestinal lipaseacutely increase appetite and attenuates postprandial glucagon-like peptide-1-(7-36)-amide-1, cholecystokinin and peptide YY concentrations. J Clin Endocrinol Metab2008;93:3995-3998.

31. Erion DM, Shulman GI. Diacylglycerol-mediated insulin resistance. Nat Med 2010;16:400-402.

32. Carraso S, Merido I. Diacylglycerol, when simplicity becomes complex. TrendsBiochem Ser 2007;32:27-36.



Documents

Pharmacological inhibition of diacylglycerol acyltransferase 1 reduces body weight and modulates gut peptide release-Potential insight into mechanism of action