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Glucagon receptor antagonism induces increased cholesterol absorption

Hong-Ping Guan1, Xiaodong Yang1, Ku Lu1, Sheng-Ping Wang1, Jose M. Castro-Perez1, Stephen Previs1,

Michael Wright2, Vinit Shah1, Kithsiri Herath1, Dan Xie1, Daphne Szeto3, Gail Forrest3, Jing Chen Xiao1,

Oksana Palyha1, Li-Ping Sun1, Paula J. Andryuk4, Samuel S. Engel4, Yusheng Xiong5, Songnian Lin5, David

E. Kelley1, Mark D. Erion1, Harry R. Davis†, Liangsu Wang1

Departments of 1Cardiometabolic Disease, 2Late Stage In Vitro Pharmacology, 3Late Stage In Vivo

Pharmacology, and 5Discovery Chemistry, Merck Research Laboratories, 2015 Galloping Hill Rd,

Kenilworth, NJ 07033

4Clinical Research Department, Merck Research Laboratories, 126 Lincoln Ave, Rahway, NJ 07065

†Current address: CVPath Institute, Inc. 19 Firstfield Road, Gaithersburg, MD 20878

Running title: The increase of plasma cholesterol induced by a novel anti-diabetic class of agents,

glucagon receptor antagonists, is primarily caused by elevated cholesterol absorption.

Correspondence: Hong-Ping Guan, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA. Tel: +1-908-

740-7401. Fax: +1-908-740-5034. E-mail: Hong-Ping_Guan@ merck.com

Abbreviations:

Gcgr, glucagon receptor; Insr, insulin receptor; G6pc, glucose-6-phosphatase catalytic subunit; Gck,

glucokinase; Pck1, phosphoenolpyruvate carboxykinase 1 (soluble); Hk1, hexokinase 1; Hk2, hexokinase

2; Hmgcr, HMG-CoA reductase; Hmgcs1, HMG-CoA synthase 1; Hmgcs2, HMG-CoA synthase 2;

Insig1, insulin induced gene 1; Insig2, insulin induced gene 2; Pcsk9, proprotein convertase

subtilisin/kexin type 9; Srebp-1c, sterol regulatory element-binding protein-1c; Srebp-2, sterol regulatory

element-binding protein-2; Ldlr, LDL receptor; Cyp27a1, Cytochrome P450 family 27 subfamily A

polypeptide 1; Cyp39a1, Cytochrome P450 family 39 subfamily A polypeptide 1; Cyp46a1, Cytochrome

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P450 family 46 subfamily A polypeptide 1; Cyp51, Cytochrome P450 family 51; Cyp7a1, Cytochrome

P450 family 7 subfamily A polypeptide 1; Cyp7b1, Cytochrome P450 family 7 subfamily B polypeptide

1; Cyp8b1, Cytochrome P450 family 8 subfamily B polypeptide 1.

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ABSTRACT

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes

(T2DM) plasma glucagon is characteristically elevated, contributing to increased

gluconeogenesis and hyperglycemia. Therefore, glucagon receptor antagonism has been

proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-

molecule glucagon receptor antagonist (GRA), MK-0893 for 12 weeks demonstrated in T2DM

dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg

dose. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-

cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report

findings that link MK-0893 with increased GLP-2 and cholesterol absorption. There was not

however, a GRA related modulation of cholesterol synthesis. These findings were replicated

using structurally diverse GRAs. To examine potential pharmacologic mitigation, co-

administration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice, abrogated the

GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results

provide a novel finding by which glucagon and hence glucagon receptor antagonism governs

cholesterol metabolism.

Supplementary keywords: Diabetes, Glucagon receptor antagonist, Cholesterol/Absorption,

Hypercholesterolemia, GLP-2, Bile acids

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INTRODUCTION

It is through mostly opposing actions that the pancreatic islets hormones insulin and

glucagon interact in the governance of hepatic glucose production and its uptake. In type 2

diabetes mellitus (T2DM), and as well in type 1 diabetes mellitus, fasting plasma glucagon is

generally elevated, inappropriate to prevailing hyperglycemia, and there is less suppression

during prandial metabolism (1). This imbalance in secretion of islet hormones is considered to

be a key aspect of the pathophysiology causing hyperglycemia (1, 2). Glucagon receptor

antagonism has accordingly drawn considerable interest as a novel pharmacological approach for

treating T2DM. Several glucagon receptor antagonists (GRAs) have advanced into human

clinical trials in patients with T2DM. MK-0893 (3), MK-3577 (4), LY2409021 (5, 6), and an

anti-sense oligo targeting the glucagon receptor (ISIS-GCGRrx) (7) have each demonstrated

efficacy in lowering fasting and postprandial hyperglycemia, leading to substantial reductions of

HbA1c thereby providing clinical proof of concept for efficacy of GRA.

In a 12-week, placebo-controlled, dose ranging clinical study in T2DM using the GRA

MK-0893, dose-response improvement of hyperglycemia was observed, with a reduction of

HbA1c of 1.5% at 80 mg per day, the top dose examined (Supplemental Fig. S1) (3). This

efficacy is substantial and arguably as or more effective than contemporary standard-of-care oral

agents for treatment of T2DM. Yet, in association with the dose-responsive improvements in

hyperglycemia, a dose-dependent increase in plasma LDL-cholesterol (LDL-c) was observed.

At the 80 mg dose, plasma LDL-c increased by 16.7% relative to baseline, significantly greater

than under placebo or metformin treatment arms (-3.1 and 2.2% changes respectively)

(Supplemental Fig. S1) (3). LDL-c and T2DM are recognized to adversely influence risk for

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cardiovascular disease, and increased LDL-c in the setting of T2DM is concerning as this could

potentiate risk for cardiovascular disease risk (8-10).

The current studies were undertaken using a preclinical rodent model, cholesterol isotopic

flux determinations, as well as further exploration of archived plasma samples from the clinical

trial with MK-0893 to elucidate the principal mechanism underlying increased plasma LDL-c. A

fundamental related question is whether the findings are unique to a specific GRA compound or

instead represent a mechanism-based response. To address this, several structurally distinct

GRAs were investigated for effects on cholesterol homeostasis. The findings yield novel

insights into glucagon physiology as well as GRA pharmacology and indicate a substantial effect

in the regulation of cholesterol absorption.

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METHODS AND MATERIALS

Animal studies. All mice used in the studies were purchased from Taconic (Germantown, New

York) at 10-12 weeks of age. Animals were maintained in a 12 h/12 h light-dark cycle with free

access to food and water in an environment with temperature maintained at 22°C. Four mice

were housed in a regular cage. Male humanized GCGR mice (hGCGR) were generated on

B6.129S6 background and had been back-crossed to C57BL/6 for more than 13 generations.

This strain of mouse showed no metabolic phenotypes in glucose and cholesterol (11). All tests

were performed in the same strain of mouse and comparisons were made between compounds

treatment and vehicle groups. Animals were maintained on regular rodent chow diet 7012 (5%

dietary fat; 3.75 kcal/g) (Teklad, Madison, WI) for 2 weeks before receiving compound

treatments. Compounds were dissolved in 0.5% methylcellulose and oral gavage (p.o.) dosing

volume was 10 ml/kg body weight.

Glucagon challenge assay was performed in mice under ad libitum as previously described (12).

Briefly, at 1 h post compound administration via p.o., glucagon dissolved in PBS was injected at

15 µg/kg i.p. followed by glucose measurements using glucometer (Life Scan) via tail bleeding

at 0, 12, 24, and 48 min post injection. For cholesterol absorption and synthesis studies, stable

isotope labelled cholesterol was prepared as described previously (13). Briefly, 2,2,3,4,4,6-D6-

cholesterol (Cambridge Isotope Laboratory, 92543-08-3) and 3,4-13C2-cholesterol (Sigma,

662291) were dissolved in USP ethanol at 20 mg/ml and filtered through a 0.2 µm solvent-

resistant filter (Sterlitech). The solutions were warmed to 37˚C for 5 min and added dropwise

over 1 min to two volumes of 20% Intralipid (Sigma I141) with gentle mixing. After incubation

at 37˚C for 5 min, the solutions were cooled down to room temperature for 15 min, and then

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passed through a 1.2 micron filter (Sigma). Solutions were stored at 4˚C for 1 day before

administration.

Clinical samples. Human serum samples used in this study were obtained from a randomized,

double-blind, placebo-controlled, crossover trial comprising a 1-wk screening period and 6-wk

washout of previous anti-hyperglycemic agents followed by a 2-wk, placebo run-in period and

then 12-wk treatment periods (clinical trial NCT00479466). Available serum samples for

placebo (n = 8) and monotherapy of MK-0893 60 mg (n = 46) and MK-0893 80 mg (n = 16)

were assayed for glucose, total cholesterol, LDL-c, campesterol, sitosterol, and bile acid

profiling. For GLP-1 measurement, 6 samples in MK-0893 60 mg group had insufficient serum

left, thus sample numbers were placebo (n = 8), MK-0893 60 mg (n = 40), and MK-0893 80 mg

(n = 16).

cAMP production assay. Cryopreserved human primary hepatocytes were purchased from

CellzDirect (presently Life Technologies, Hu8080). One vial of frozen primary hepatocyte

(approximately 5 million cells in total) was quickly thawed to 37˚C in a water bath and washed

in Cryopreserved Hepatocyte Recovery Medium (CHRM) (Life Technologies, CM7000) and re-

suspended in buffer containing HBSS (Life Technologies 14025), 0.1% BSA (Sigma A9205) and

1.2 mM IBMX (Sigma, I-5879). To assess antagonist activity, 4000 cells per well were pre-

incubated with compounds or 0.1% DMSO for 30 min and stimulated with glucagon (5 nM)

(Sigma, G2044) for an additional 30 min at room temperature. The assay was terminated with

the addition of Cisbio Dynamic 2 (62AM4PEC) detection reagents as per the manufacturer’s

instructions (Cisbio). cAMP was detected by a decrease in time-resolved fluorescence energy

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transfer (TR-FRET) using an EnVision plate reader (PerkinElmer). The IC50 values were

calculated using nonlinear regression curve fit analysis in Prism (GraphPad).

Measurement of plasma or serum GLP-1 and GLP-2. Whole blood of mice was collected in

EDTA-coated tubes and plasma was separated by centrifugation at 8,500 rpm at 4°C and stored

at –80°C until assayed. Human serum was collected following standard blood collection

procedure after overnight fasting. Plasma or serum levels of GLP-1 and GLP-2 were measured

using a total GLP-1 assay kit (Meso Scale Discovery) and mouse / human GLP-2 kit (Alpco).

Analysis of plasma lipid, apolipoprotein, PCSK9, and fecal cholesterol. A commercial

enzymatic colorimetric kit was used for the determination of plasma total cholesterol (Wako

cholesterol E kit) according to manufacturer's instructions (WakoUSA). Plasma level of PCSK9

was determined by PCSK9 dissociation-enhanced lanthanide fluorescence immunoassay

(DELFIA) as described elsewhere (14). Plasma or serum lipoprotein profile was assayed by

FPLC as described previously (15). Fecal cholesterol was measured by extracting lipids using

the Folch method (16), whereby fecal samples were homogenized with 5 ml of

chloroform:methanol (2:1, v:v). The homogenate was then filtered and washed with 2 ml of

0.9% saline, followed by centrifugation and drying of lower phase under nitrogen gas. The

extract was reconstituted with 10% Triton X in isopropanol and analyzed using a commercial

cholesterol kit (WakoUSA).

2H-Labeling of body water and Analysis of 2H-labeling of total plasma cholesterol and

apoprotein. The 2H-labeling of body water was determined using headspace analyses following

exchange with acetone as described by Shah et al (17). Briefly, 20 µL of sample (or standard)

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was reacted with 2 µL of 10 N NaOH and 4 µL of a 5% (v/v) solution of acetone in acetonitrile

for 4 h at room temperature. The instrument is programmed to inject 5 µL of headspace gas from

the GC vial in a splitless mode. Samples were analyzed using a 2.0 min isothermal run (Agilent

5973 MS coupled to a 6890 GC oven fitted with an Agilent DB-5MS column, 30m x 250µm x

0.15µm, the oven was set at 170 0C and helium carrier flow was set at 1.0 mL x min-1), acetone

elutes at ~ 1.4 min, the mass spectrometer was set to perform selected ion monitoring of m/z 58

and 59 (10 ms dwell time per ion) in the electron impact ionization mode.

The isotopic labeling of total cholesterol was determined using GCMS (18). Lipids were

saponified by heating plasma (50 µL) with 1N KOH in 80% methanol (200 µL) at 65 °C for 1

hour. Samples were acidified with 25 µL 6N HCl and then extracted in 125 µL chloroform

followed by vigorous vortexing for 20 sec. The samples were centrifuged at 3000 rpm for 5 min,

100 µL of chloroform (lower layer) was collected and evaporated to dryness under N2. Samples

were derivatized by reacting with 100 µL of pyridine:acetic anhydride (1:2, v:v) at 65 °C for 1

hour. Excess reagent was evaporated to dryness under N2, the acetylated derivative was

reconstituted in 50 µL ethyl acetate for analysis by GC-MS. All analyses were performed using

an Agilent 5973 MS coupled to a 6890 GC oven fitted with an Agilent DB-5MS column (30m x

250µm x 0.15µm). The instrument was programmed to inject 1 µL of sample using a 10:1 split

(helium carrier flow was set at 1.0 mL × min-1), the oven temperature was started at 150°C,

raised at 20°C × min-1 to 310°C and held for 6 min oven, cholesterol elutes at ~ 9 min, the mass

spectrometer was set to perform selected ion monitoring of m/z 368 and 369 (10 ms dwell time

per ion) in the electron impact ionization mode.

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Calculations and statistical analysis. To quantify the contribution of cholesterol synthesis one

fits the data using a precursor:product labeling ratio to the general equation (18):

Newly made cholesterol = [Product labeling / (Precursor labeling × n)] × Concentration

where n is the number of exchangeable hydrogens (assumed to equal 26 for cholesterol) (2).The

change in the ratio of m/z 369:368 (i.e. M+1/M0) was used to model the product labeling,

whereas the precursor labeling was assumed to equal plasma water, the concentration of total

circulating cholesterol was determined via enzymatic assay (19, 20). Plasma level of ApoB and

flux of ApoB were quantified by LC-MS/MS method as described previously (21).

Analysis of campesterol and sitosterol in plasma or serum samples of mice and humans.

Five µl of plasma or serum were mixed with 25 μl of internal standard mix (1 µg/ml of D6-

campesterol and D7-sitosterol prepared in ethanol) and 100 µl of 1 N KOH in glass inserts placed

on a deep 96-well polypropylene plate. The mixture was sealed and heated at 80˚C with shaking

@ 600 rpm for 1 hr on an R-shaker (Eppendorf). Samples were evaporated under nitrogen to

dryness. One hundred and fifty microliters of derivatization reagent (1000 mg of 2-methyl 6-

nitro benzoic anhydride, 300 mg of 4-dimethyl pyridine, and 800 mg of piconilic acid dissolved

in 2 ml of TEA and 12 ml of pyridine) was added to each tube and the plate was incubated at

80˚C for 1 hr. After incubation, 500 µl of Hexane was added to each tube, vortexed and

centrifuged @ 4000 rpm at room temperature for 10 min. Four hundred microliters of

supernatant was transferred to a new glass microtube, evaporated to dryness under a constant

flow of nitrogen at 45˚C and reconstituted in 80 µl of loading solution (80% acetonitrile, 20%

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water and 0.1% of formic acid). Samples were then loaded for LC/MS analysis. Contents of

campesterol and sitosterol were normalized to the internal controls in each sample. For each

assay, 5 pooled plasma samples from multiple subjects were used as quality controls (QC) and

each QC sample was injected in triplicates for LC/MS assay. QCs with variation of ± 15%

coefficient were deemed as acceptable.

Bile acid and intermediates analysis. One hundred and fifty µl of serum was transferred into a

2 ml 96-deep well plate followed by the adding 585 µl ice-cold acetonitrile containing 0.1 %

formic acid solution and 5 µl 60 ng/ml internal standard mixture made of d6-7α,12α dihydroxy-

4-cholesten-3-one and d7-7α-hydroxy-4-cholesten-3-one. The plate was sealed and vortexed for

1 minute followed by centrifuge at 4,000 rpm for 20 minutes at room temperature. After

centrifugation, 600 µl of supernatant was passed through under positive pressure through a

protein precipitation plate which retained phospholipids but eluted the bile acid intermediates

(Ostro plate, Waters Corp, Milford, MA). The eluent was collected and evaporated under a

constant flow of N2 at 45˚C. The samples were then reconstituted in 100 µl of 80% acetonitrile

+0.1 % formic acid/ 20% water. The resultant extract was injected (10 µl) onto an LC/MS-MS

system operated in positive ion mode electrospray (UPLC/ Waters TQS mass spectrometer,

Milford, MA). Isotopic dilution quantitation was conducted to obtain concentrations of 7α,12α

dihydroxy-4-cholesten -3-one and 7a-hydroxy-4-cholesten-3-one.

Western blotting. One piece of liver weighed at ~ 100 mg was homogenized in 500 µl of RIPA

buffer by using FastPrep™-24 (MP Biomedicals). After incubation on ice for 30 min,

homogenate was centrifuged at 14,000 rpm at 4˚C for 30 min. Protein concentration of

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supernatant was determined by BCA protein assay kit (Pierce) and the final concentration was

calibrated to 1 mg/ml with RIPA buffer. After mixing with 2 × loading buffer and heated at

70˚C for 5 min, samples were loaded at 20 µg per well to a 4-10% SDS-PAGE gel for

electrophoresis. After transferring the protein to PVDF membrane, LDLR was blotted by using a

rabbit monoclonal antibody (RbMAb) (Abcam ab52818). Loading control was blotted by using

β-actin polyclonal antibody (CellSignaling).

Real-time quantitative PCR analysis and gene profiling

Liver samples isolated from mice treated with vehicle and GRA compound(s) were kept in

RNAlater solution (Qiagen) until processing. Tissues were homogenized and total RNA was

isolated by using RNA Easy kit and QIACube instrument (Qiagen). Two micrograms total RNA

from each sample was reverse transcribed with a cDNA kit (Life Technologies), and mRNA

levels for the genes of interest were measured by RT-PCR with Taqman Universal Mastermix

reagents and Taqman primer/probe sets (Life Technologies), or SYBR Green Mastermix

reagents and custom-designed PCR array developed in collaboration with SABiosciences-Qiagen

(22). The relative amounts of specific target amplicons for each gene were estimated by a cycle

threshold (CT) value and were normalized to the copy number of housekeeping genes, with all

genes in vehicle group arbitrarily set at 1 (22). The P values were determined by two-tailed equal

variance Student's t-test, comparing the 2-ΔCT values of the vehicle and GRAs-treated groups.

Data analysis and statistics. All data are presented as mean ± SEM. For rodent results,

statistical analysis was performed by using 1-way ANOVA followed by unpaired 2-tailed

Student’s t-test to compare mean values between treatment groups and control group. For

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human results, due to uneven number of human samples in different groups, the %change of the

measurements (parameters at 12 weeks vs. day -1) was calculated for each individual and then

averaged. Statistical analysis was performed by using Mann-Whitney test.

Declaration. For rodent studies, all testing protocols were reviewed and approved by the Merck

Research Laboratories Institutional Animal Care and Use Committee in Rahway and Kenilworth,

New Jersey. The Guide for the Care and Use of Laboratory Animals was followed in the

conduct of the animal studies. Veterinary care was given to animals requiring medical attention.

Finally, the ARRIVE guidelines published by NC3Rs were followed for reporting the in vivo

experiments in animal research.

Clinical trial protocols of MK-0893 were reviewed and approved by an independent Institutional

Review Board (IRB) or Ethical Review Committee (ERC) before being initiated. For each site,

the IRB/ERC and Merck’s Consent Form Review department (U.S. studies) or local medical

director (non-U.S. studies) approved the patient informed consent form. Written informed

consent was received from participants prior to inclusion in the study. In all cases, Merck

clinical studies were consistent with standards established by the Declaration of Helsinki and in

compliance with all local and/or national regulations and directives.

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RESULTS

Selection and characterization of GRA test compounds. Three GRA compounds (MK-0893

(23), GRA1 (24), and GRA2 (25)) were demonstrated to potently block glucagon binding to the

glucagon receptor (GCGR) and were selected for use in the preclinical studies that comprise this

investigation. MK-0893 and GRA1 were developed by Merck and GRA2 is under development

by Eli Lilly. GRA2 possesses a biaryl benzyl ether core that is distinctly different from the tri-

substituted diazole core present in GRA1 and MK-0893 (Fig. 1). To assess relative potencies,

cryopreserved human hepatocytes were incubated with MK-0893, GRA1, or GRA2 and in the

presence of 5 nM glucagon followed by cAMP quantitation in the culture media. Glucagon

induced cAMP production dose-dependently, with an EC50 of 575 pM, which is comparable to

physiological levels of glucagon in blood (2). MK-0893, GRA1, and GRA2 alone had no effect

on cAMP production. In the presence of glucagon, each compound suppressed cAMP

production, with IC50s of 563 nM, 448 nM and 292 nM, respectively (Fig. 1). Comparing these

results with values earlier obtained from CHO cells stably overexpressing human GCGR

(hGCGR.CHO) (23, 24), IC50s of the compounds are right-shifted, likely reflecting over-

expression of GCGR in engineered cells versus human primary hepatocytes. Nonetheless, these

data indicate the three GRA compounds are comparably potent. To further characterize these

compounds, the compounds were compared for effects on in vitro binding of glucagon to GCGR,

β-arrestin recruitment, cellular level of Ca2+, and cAMP production in hGCGR.CHO cells

(Supplemental Table S1). No meaningful differences amongst the three selected GRAs were

observed across these parameters, indicating good similarity of pharmacology of antagonizing

GCGR-mediated signaling.

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Effect of GRA on cholesterol synthesis and clearance in hGCGR mice. The next prerequisite

was to identify a relevant preclinical model, and for this purpose, the hGCGR mouse model was

chosen (11). The selected GRAs have more potent and specific binding for human than mouse

GCGR (24). hGCGR mice are lean, healthy, and without abnormal plasma glucose and lipids

(11). To test for qualification of hGCGR mice for these investigations, the mice were maintained

on chow diet and treated with GRA1 at 30 mpk once daily (QD) for 5 days. Glucagon-induced

glucose excursion on day 1 after a single dose and ambient glucose on day 5 were measured as

indicators of acute and subchronic GCGR blockade. Glucose excursion induced by glucagon

after a single dose was suppressed (Fig. 2A), and ambient glucose decreased by 20% after 5 days

(Fig. 2B), demonstrating effective target engagement. On chow diet under GRA1 treatment,

plasma levels of cholesterol, LDL-c, and HDL-cholesterol (HDL-c) were increased by 19%,

33%, and 21%, respectively (compared to vehicle) (Fig. 2B, Fig. 2D, and Supplemental Fig.

S3), an increase at least comparable to the rise in cholesterol observed clinically in T2DM (3,

26). Plasma TG level was not affected by GRA1 treatment (Supplemental Fig. S3). Taken

together, it is concluded that hGCGR mice are a suitable preclinical model for investigating the

mechanism of action by which GRA induces a rise in plasma cholesterol, LDL-c, and HDL-c.

It had been reported that glucagon inhibits 3-hydroxy-3-methylglutaryl-CoA reductase

(HMGCR) and suppresses cholesterol synthesis (27). Therefore, we initially focused upon

testing the hypothesis that GRA increases cholesterol synthesis. Isotope tracer methodology was

used; hGCGR mice were administered D2O (20 ml/kg body weight i.p.) to label newly

synthesized cholesterol and determine the rate of its synthesis as well for palmitate and ApoB.

(28). As a positive pharmacological comparator for the assays of palmitate and cholesterol

synthesis, T0901317, an LXR α and β agonist, was used (29). T0901317 treatment induced

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increased plasma cholesterol, LDL-c, and HDL-c by 76%, 166%, and 21%, respectively and

markedly increased synthesis of cholesterol and palmitate (Fig. 2). GRA1 treatment, while

replicating the increase in plasma cholesterol earlier described, did not have a significant effect

on synthesis of cholesterol, palmitate, or ApoB (Fig. 2E). Liver cholesterol content was similar

between vehicle and GRA1 treatment (Supplemental Fig. S3). It was also examined in these

studies whether GRA would alter key components governing LDL-c clearance. GRA1 treatment

had no effect relative to vehicle treatment on plasma PCSK9 and plasma ApoB (Fig. 2B), and

did not change hepatic LDLR protein level (Fig. 2C). Consistent with unchanged LDLR protein

level, LDL-c clearance was not affected by treatments of MK-0893, GRA1, and GRA2

(Supplemental Fig. S5).

Effects of GRA on cholesterol absorption in hGCGR mice. The next set of studies were

undertaken to examine a potential effect of GRA1 on cholesterol absorption and its excretion.

An in vivo study performed in hGCGR mice on chow diet was adapted from a study design

previously published (28). Cholesterol absorption was measured using a stable isotope method

and cholesterol excretion was monitored by measuring fecal cholesterol. Stable isotope-labeled

cholesterol, 2,2,3,4,4,6-D6-cholesterol (15 mpk, p.o.) and 3,4-13C2-cholesterol (15 mpk i.v.),

were administered on day 4 followed by blood collection at 24, 48, and 72 hrs post injection.

Cholesterol absorption was calculated by the ratio of 2,2,3,4,4,6-D6-cholesterol to 3,4-13C2-

cholesterol in plasma at different time points. At 24-h prior to euthanasia, mice were dosed with

D2O (i.p., 20 ml/kg) for measurement of cholesterol and palmitate synthesis (Fig. 3A). After 9

days of treatment, GRA1 increased plasma level of total cholesterol by 18%; thus a highly

consistent response (Fig. 3B). Cholesterol absorption was found to be increased by 46% (Fig.

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3C). Similar increase of cholesterol absorption was observed for MK-0893 and GRA2

(Supplemental Fig. S5). It was again observed that there was not a significant change in rate of

cholesterol synthesis (Fig. 3D). Cholesterol excretion was not found to be significantly changed

(Fig. 3E).

GRA1 and GRA2 increase blood cholesterol and cholesterol absorption. A key aspect of

these studies was to address whether the finding of increased cholesterol absorption studies was

unique for a specific GRA compound or instead could be mechanism based. The isotope study

described above was repeated using the structurally distinct GRA2. A second aspect was to add

measurements of phytosterols, plant sterols, the plasma level of which are recognized as

biomarkers of intestinal cholesterol absorption (30). GRA1 and GRA2 were dosed (30 mpk each)

to achieve similar and robust target engagement, as assessed by blockade of a glucagon-induced

glucose excursion (Fig. 4A). After 5 days of treatment, GRA1 and GRA2 increased plasma total

cholesterol by 20% and 27.3% respectively (Fig. 4B). Both compounds increased cholesterol

absorption as indicated by treatment related increases in plasma phytosterols. Campesterol

increased 33.1% for GRA1 and 33.3% for GRA2, and plasma sitosterol 37.2% for GRA1 and

38.9% for GRA2 (Fig. 4B). Based on D2O labeling, no significant change in rate of cholesterol

synthesis was found for either GRA1 or GRA2. GRA2 slightly increased the plasma level of

lathosterol, however there was not a significant difference in the lathosterol-to-cholesterol ratio

on treatment with GRA1 or GRA2 (Fig. 4B), an indirect measurement of cholesterol synthesis

(31).

It was reported that plasma levels of GLP-1 and GLP-2 were dramatically increased in

GCGR-/- mice (32, 33). In hGCGR mice treated with GRA1 and GRA2, plasma levels of total

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GLP-1 were increased by 398% and 674% respectively, and GLP-2 were increased by 71% and

96% respectively (Fig. 4C); consistent with the pattern found in clinical samples from the MK-

0893 clinical trial (Fig. 5C). In a separate study in hGCGR mice, MK-0893 treatment increased

plasma level of total GLP-1 by 168% and of GLP-2 by 37% (Supplemental Fig. S7).

As a corollary experiment concerning whether the observed effects on cholesterol

homeostasis are compound-specific, we sought to exploit that MK-0893 achieves substantially

more robust binding to human than mouse GCGR by examining respective treatment effect upon

cholesterol in WT versus hGCGR mice. We posited that if the increase in cholesterol absorption

was an “off-target” effect, then resultant increases in plasma cholesterol would be similar in WT

and hGCGR mice. Doses of 3 and 10 mpk were used for MK-0893 in WT and hGCGR mice, but

robust target engagement (glucose response to glucagon challenge) was demonstrable at both

doses only in hGCGR mice. MK-0893 had no significant effect on plasma cholesterol or

phytosterols in WT mice, but demonstrated a significantly increased plasma level of campesterol

at 10 mpk and a trend for dose-dependent increase in plasma sitosterols in hGCGR mice

(Supplemental Fig. S2). Taken together, these studies bolster the concept that blockade of

GCGR induces an increase in cholesterol absorption and plasma cholesterol as a mechanism-

based effect.

MK-0893 increases phytosterols, GLP-1, GLP-2, and bile acids in T2DM. Having generated

a hypothesis based on pharmacologic interventions in hGCGR mice that GRA induces increased

cholesterol absorption, and importantly, established utility of using phytosterols as relevant

biomarkers of this process, we were then positioned for further examination of archived plasma

samples obtained during the MK-0893 clinical study (ClinicalTrials.gov Identifier:

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NCT00479466). In this trial, placebo, metformin, and MK-0893 at 20, 40, 60, and 80 mg were

chosen to treat T2DM (3). Archived samples from the placebo, MK-0893 60 mg 80 mg

treatment arms were assayed for glucose, total cholesterol, GLP-1, GLP-2, LDL-c, phytosterols,

bile acids, and bile acid metabolites (Fig. 5 and Supplemental Fig. S4). The 60 mg treatment

with MK-0893 decreased glucose by 31% (versus a 3.6% rise with placebo) and increased LDL-

c by 4.2% (versus a 4.8% decline with placebo), as shown in Fig. 5A. Serum campesterol and

sitosterol were significantly increased with MK-0893 treatment (Fig. 5D).

It is recognized that GRA or genetic knockout of GCGR evokes large compensatory

increases in plasma glucagon, together with increased expression for other peptides derived from

preproglucagon. It has been reported that GLP-2 enhances nutrient absorption and induces

epithelial cell proliferation and regeneration (34-37). We observed in the archived plasma

samples a treatment related increase of total GLP-1 (6-fold for 60 mg and 12-fold for 80 mg),

and a less pronounced increase, in plasma GLP-2 (1.4-fold for 60 mg and nearly 3-fold for 80

mg) (Fig. 5B-C). In the aqueous environment of the small intestine, bile acids have a critical

role in facilitating absorption of highly hydrophobic cholesterol. Though an effect of GRA on

bile acid metabolism has not previously been reported, the plasma samples were examined for

total bile acid concentration, which was found to be significantly increased by MK-0893 (Fig.

5E). In particular, there was an increase of cholic acid and in 7-α hydroxy-4-cholesten-3-one (7-

HCO), a biochemical intermediate in the rate-limiting reaction converting cholesterol to bile

acids. 7α,12α-dihydroxy-4-cholesten-3-one, an intermediate in cholic acid synthesis, showed a

trend for increase (Fig. 5E and Supplemental Fig. S4E-F). These results augment the finding

that GRA induces an increase in cholesterol absorption and suggest that GRA mediates an

increase of bile acid synthesis, increases GLP-2 and modulates bile acid composition, as factors

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that could potentially contribute to the mechanism for increasing intestinal absorption of

cholesterol.

Effect of GRA on hepatic gene expression in hGCGR mice. Based on these novel findings in

clinical samples from T2DM patients who were treated with MK-0893, we investigated further

effects of GRA on mRNA expression in hGCGR mice. As expected, GRA1 and GRA2

suppressed hepatic mRNA levels of glucose-6-phosphatase (G6pc), phosphoenolpyruvate

carboxykinase 1 (Pck1), and hexokinase 2 (Hk2) but increased glucokinase (Gck). However, we

observed that the effects of GRA1 and GRA2 on gene expression in the pathway of cholesterol

synthesis were marginal. Only 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) was found

to be slightly increased by both compounds (Fig. 4D). The lack of significant changes in mRNA

levels for Pcsk9, Srebp-1c, Srebp-2, and Ldlr is consistent with the limited impact of GRA1 and

GRA2 on cholesterol synthesis (as ascertained isotopically in earlier aspects of this study) and a

limited effect on components governing cholesterol clearance (Fig. 2E and Fig. 4B). On the

other hand, the gene encoding Cyp7a1, which catalyzes the rate-limiting step of bile acid

synthesis, was dramatically increased by GRA1 and GRA2 (Fig. 4D). Other genes controlling

bile acid synthesis, Cyp27a1 and Cyp46a1, were also induced by GRA1 and GRA2, though to a

lesser extent (Fig. 4D). In both acute and subchronic studies, GRAs had no effect on mRNA

levels of intestinal Npc1l1 and Abcg5/g8, neither did they affect mRNA levels of liver Abcg5/g8

(Fig. 4D and Supplemental Table S2).

GRAs-induced increase in plasma cholesterol is abolished by ezetimibe. Based on the

findings that GRA increases cholesterol absorption and that this is a mechanism-based effect, we

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next undertook a study of whether administration of ezetimibe, which acts to reduce cholesterol

absorption, would effectively mitigate GRA-induced increases in LDL-c. hGCGR mice on chow

diet were treated with vehicle and GRA1 in the absence or presence of ezetimibe for 9 days.

Ezetimibe did not alter the target engagement or glucose-lowering efficacy of GRA1 (Fig. 6A).

Ezetimibe alone had no effect on plasma cholesterol due to a large increase in rate of cholesterol

synthesis (Supplemental Fig. S6), a compensatory effect previously demonstrated (38), and this

was similar during co-administration with GRA. But its co-administration with GRA1 abrogated

the 14% increase of plasma cholesterol observed in the GRA1 treatment arm (Fig. 6B).

Ezetimibe, alone and combined with GRA1, strongly inhibited intestinal absorption of

cholesterol as measured using orally administered D6-cholesterol (Fig. 6C), thus eliminating the

increase of 29% that was observed under GRA1 treatment. In these studies, it was again

observed that despite a clear effect of GRA1 treatment to increase cholesterol absorption, little

net effect on fecal cholesterol excretion was detected, whereas ezetimibe, alone and in

combination with GRA1, caused a marked increase in fecal cholesterol excretion (Fig. 6D).

This pattern of findings observed using GRA1 in combination with ezetimibe was then

tested using both MK-0893 and GRA2 and highly similar results were obtained. MK-0893 and

GRA2 increased plasma total cholesterol by 17% and 14%, respectively, together with

significant increases in plasma campesterol and sitosterol. Ezetimibe alone and in combination

with MK-0893 and GRA2 significantly and similarly reduced plasma campesterol and sitosterol

and in the co-administration arms maintained plasma cholesterol at normal levels (Fig. 6E).

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DISCUSSION

The impetus for these investigations of the effect of GRA on cholesterol homeostasis in

hGCGR mice arose from clinical observations that dose-dependent increases in LDL-c occur in

T2DM patients treated with the GRA MK-0893. The 12-week clinical trial of MK-0893

achieved clear evidence that antagonizing endogenous glucagon can improve hyperglycemia in

T2DM, attaining impressive efficacy on HbA1c and with minimal risk of hypoglycemia. Yet, the

collateral finding of a dose-dependent, statistically significant increase in LDL-c raises a concern

of increased cardiovascular risk despite substantial improvement in hyperglycemia.

This was therefore a “bedside-to-bench” inspired translational study, undertaken to

determine whether the rise in plasma LDL-c was restricted to MK-0893 or instead is more likely

to be mechanism-based. Because our data support that the effect is mechanism-based, we sought

to determine amongst cholesterol synthesis, excretion, absorption, or plasma clearance of LDL-c,

which is the mechanism primarily responsible. The initial step was to identify an appropriate

preclinical rodent model, a necessity because the GRA compounds had been optimized for

potency against human GCGR and are much weaker against mouse GCGR, reflecting species-

differences in GCGR homology (11). The hGCGR mouse demonstrated elevation of plasma

cholesterol with three structurally distinct GRA compounds and to an extent similar to the rise

observed in clinical studies, establishing both that this was a suitable model and inferring that the

rise in plasma LDL-c is mechanism-based. On the basis chiefly of isotopic flux determinations

of endogenous cholesterol synthesis and separately, of intestinal cholesterol absorption, it was

determined that GRA raises LDL-c by increasing cholesterol absorption. Bolstering this

interpretation is the observation that GRA significantly increases plasma phytosterols,

recognized biomarkers of intestinal absorption of cholesterol, and comparable to the increase of

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cholesterol absorption detected by isotopic methodology. Furthermore, these findings on plasma

phytosterols in the hGCGR mouse enabled our studies to pivot from back to “bench-to-bedside”,

and using archived samples from the clinical study of MK-0893, significant increases of

phytosterols were observed during treatment with this GRA.

The concept that glucagon influences cholesterol metabolism has been previously

reported though with uncertainty as to the responsible mechanism. Reports have appeared in the

literature since the 1950’s, describing an association between glucagon action and cholesterol.

For example, destruction of α-cells increased plasma cholesterol in rabbits (39) and dogs (40)

while infusing glucagon prevented hypercholesterolemia in rats fed with a high cholesterol diet

(41). LDL-c and the rate of cholesterol synthesis in a patient with familial hypercholesterolemia

were reduced by portacaval shunt surgery, which was associated with marked increases of

plasma glucagon and bile acids (42). Furthermore, glucagon infusion into humans profoundly

reduced plasma levels of cholesterol (43). GCGR-/- mice were reported to have a significantly

higher plasma LDL-c than WT (32). Plasma cholesterol was reported to increase in diet-induced

obese mice treated with a monoclonal antibody inhibiting GCGR (44). In preclinical studies

with MK-0893, there was not a prominent signal of increased plasma cholesterol that was noted

(unpublished results), perhaps simply reflecting the lesser potency of this compound for rodent

compared to human GCGR. Regardless, with the hindsight afforded by the clinical trial data with

MK-0893, it is clear that the published literature does contain precedents for the observation that

GRA can influence cholesterol metabolism.

The mechanism underlying the prior findings was not rigorously addressed though the

findings were variously attributed, prominent amongst which is the hypothesis that antagonism

of glucagon can increase cholesterol synthesis. There is a rationale that blocking glucagon

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signaling and hence an increase in cAMP would release inactivation of HMGCR (27). In

addition, it has been reported that glucagon suppresses plasma PCSK9 (45) and increases hepatic

LDL receptor (LDLR) protein levels (46), presumably as a consequence of effects on the Srebp-

2 pathway (45). Knockdown of GCGR in db/db mice significantly increased LDL-c, which was

attributed to elevated hepatic lipogenesis and cholesterol synthesis (47). Therefore, in the

current studies it was initially evaluated whether GRA mediated an increase in cholesterol

synthesis or might decrease LDL-c clearance. Our findings do not support either hypothesis.

LDLR, PCSK9, and rates of cholesterol synthesis were not significantly changed during

treatment with multiple GRAs despite unequivocal induction of increased plasma cholesterol.

Instead, our studies reveal the novel finding that there is a strong effect of GRA to

increase absorption of cholesterol. It appears there are marked differences between GRAs

treatment and GCGR knockdown. While GRAs increased LDL-c and HDL-c, GCGR

knockdown only increased LDL-c without any effect on HDL-c. Mechanistically, GCGR

knockdown induced cholesterol synthesis (47) whereas GRAs induced cholesterol absorption

without any effect on cholesterol synthesis. Though our studies did obtain some insights as to

what may in turn contribute to increased absorption of cholesterol, it is acknowledged that there

are a number of aspects of this novel hypothesis that will require further investigation. One

factor might be the effect of GRA to raise secretion of the gut hormone GLP-2. It is known that

glucagon, GLP-1, and GLP-2 are dramatically increased in GCGR-/- mice (33, 48). Plasma

levels of total GLP-1 and GLP-2 were both significantly increased by subchronic treatment of

GRA1, GRA2, and MK-0893 in hGCGR mice. Dramatic inductions of plasma total GLP-1 and

GLP-2 were observed in T2DM patients treated with MK-0893 chronically. GLP-2 has been

reported to induce crypt cell proliferation and its effect on intestinal lipid absorption depend on

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nutritional status and presence of GLP-1 (34, 36, 37, 49). Although induction of GRAs on GLP-2

is predictable based on results from GCGR-/- mice, it appears that GRA does not mirror the effect

of glucagon on cholesterol metabolism. This, however, does not negate the published findings of

glucagon on Srebp-2 pathway including LDLR (46), PCSK9 (50), and cholesterol synthesis (29).

The induction of GRA on GLP-2 is so dramatic that it might have masked the activation on

Srebp-2 pathway caused by blockade of GCGR inasmuch as intestinal cholesterol absorption and

hepatic cholesterol synthesis are reversely affected by each other (51, 52).

Another set of observations obtained in the present studies of GRA treatment that we

posit to be a fruitful for further examinations concerns bile acids. Bile acids serve a critical role

in facilitating intestinal absorption of hydrophobic lipids, notably including cholesterol and as

well bile acids are derived from cholesterol and thus intimately associated with hepatic

cholesterol metabolism. The archived plasma samples from the clinical study with MK-0893

were assayed for bile acid concentration and it was found that these levels were increased,

including cholic acid and cholic acid precursors. Cholic acid supplementation enhances

cholesterol absorption in humans (50). In GRA-treated hGCGR mice, a consistent finding is

increased Cyp7a1 mRNA expression, the enzyme that is rate-limiting in bile acid synthesis.

Consistent with increased mRNA level of Cyp7a1 in hGCGR mice, its proximal product (7-

HCO) was increased in T2DM during GRA treatment. It might also be considered that bile acids

can stimulate secretion of GLP-1 and GLP-2 from L-cell via activating bile acid receptor, TGR5

(53). There is resurgent interest in the role of bile acids in metabolic signaling, particularly in the

context of diabetes mellitus (54), and the current findings indicate that glucagon agonism and

antagonism can be influential in governing bile acid homeostasis.

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Our studies indicate that the effect of MK-0893 to increase LDL-c derives mostly from a

net increase in absorption of cholesterol and that this is a mechanism-based rather than

compound specific effect. This conclusion is based upon consistent findings in the hGCGR mice

using three structurally diverse GRAs. Yet, while the data of our preclinical studies indicate a

likely mechanism based effect to increase plasma LDL-c via increased cholesterol absorption,

we do recognize that the reported clinical findings to date with effect of GRA on plasma LDL-c

are inconsistent. In the clinical trials of LY2409021 (5, 6) and with ISIS-GCGRrx (7), no

induction of LDL-c was reported. It remains uncertain why these findings may differ from what

was observed in the clinical trial with MK-0893. Perhaps it is noteworthy that clinical trials of

LY2409021 and ISIS-GCGRrx were not exclusively GRA monotherapy as was the situation with

the clinical dose-ranging study of MK-0893; instead many of the T2DM participants in those

studies were also receiving metformin treatment (5, 7). Metformin generally improves

hypercholesterolemia in humans, although the mechanism is still not well understood (55).

Clinical studies conducted using MK-0893 in combination with metformin treatment was found

to have increased efficacy in reducing hyperglycemia and substantially mitigated the increase in

LDL-c observed with GRA monotherapy in T2DM (26).

In keeping with a translational emphasis of the present studies, the effectiveness of

ezetimibe to abrogate a GRA-induced increase in cholesterol absorption and plasma LDL-c was

investigated. Ezetimibe, the molecular target for which is inhibition of NPC1L1, effectively

abolished the increase in cholesterol absorption caused by GRA and without interfering with

efficacy in reducing hyperglycemia. It is possible that GRA-induced hypercholesterolemia could

be mediated in part by an effect on Npc1l1, (56, 57), this remains to be more fully characterized

but regardless, we describe in a rodent model that blocking NPC1L1 with ezetimibe effectively

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suppresses the GRA-induced hypercholesterolemia. We postulate that co-administering GRAs

and ezetimibe will work equally or better in humans because unlike the exclusive expression of

Npc1l1 in intestine in mice, NPC1L1 is expressed abundantly in liver and intestine in humans

(58) and ezetimibe exerts its lipid lowering effect by blocking NPC1L1 in both liver and

intestine in humans (59).

In summary, we used various pharmacological tools to study the major cause of GRA-

induced hypercholesterolemia in mice and humans. Studies in mice demonstrated that increased

plasma cholesterol was mainly attributable to cholesterol absorption. Consistent with this

finding obtained in rodent, we report the novel finding that the increase of plasma LDL-c and

cholesterol was associated with increases in plasma phytosterols in humans. With respect to

potential mechanisms that mediate GRA-induced increase in cholesterol absorption, it was

observed that GRA-induced increases in GLP-2 and a trend of increase in bile acid

concentrations in patients with T2DM and as well in the rodent model, together with mRNA

expression of hepatic bile acid synthesis enzymes in mice. These are factors that can plausibly

contribute to increased cholesterol absorption. Finally, in keeping with the translational

emphasis of these studies, it was shown that ezetimibe effectively mitigated GRA-induced

cholesterolemia in mice. Thus combining GRA and ezetimibe may provide a feasible approach

to mitigating MK-0893-induced elevation of cholesterol in patients with T2DM. Collectively,

these results provide novel insights into the effect of glucagon and glucagon receptor antagonism

in governing cholesterol homeostasis.

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Acknowledgments / grant support

The authors thank Taro Akiyama and Peter Stein for critically reading the manuscript and

providing constructive suggestions. Doug Johns, Cai Li, Dan Kemp, Tom Roddy, Jing Li,

Kristian Jensen, Juliann Ehrhart, Heather Zhou, Seongah Han, Eric Muise, Brad Sherborne, and

Paul Carrington provided insightful suggestions to the study design and data interpretation.

This work was supported by Merck Research Laboratories, Merck & Co., Inc.

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Tilvis, and Y. A. Kesaniemi. 1989. Serum cholestanol and plant sterol levels in relation to cholesterol metabolism in middle-aged men. Metabolism 38: 136-140. 31. Lakoski, S. G., F. Xu, G. L. Vega, S. M. Grundy, M. Chandalia, C. Lam, R. S. Lowe, M. E. Stepanavage, T. A. Musliner, J. C. Cohen, and H. H. Hobbs. 2010. Indices of cholesterol metabolism and relative responsiveness to ezetimibe and simvastatin. The Journal of clinical endocrinology and metabolism 95: 800-809. 32. Gelling, R. W., X. Q. Du, D. S. Dichmann, J. Romer, H. Huang, L. Cui, S. Obici, B. Tang, J. J. Holst, C. Fledelius, P. B. Johansen, L. Rossetti, L. A. Jelicks, P. Serup, E. Nishimura, and M. J. Charron. 2003. Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice. Proceedings of the National Academy of Sciences of the United States of America 100: 1438-1443. 33. Ali, S., B. J. Lamont, M. J. Charron, and D. J. Drucker. 2011. 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FIGURE LEGENDS

Figure 1. GRAs suppress glucagon-induced cAMP production in human primary

hepatocytes. Human primary hepatocytes were thawed and washed with CHAMPS buffer

followed by cAMP production assay. The assay was done in triplicates. EC50 of glucagon on

cAMP production is 575 pM. IC50s of MK-0893, GRA1, and GRA2 are 563 nM, 448 nM and

292 nM, respectively. All compound treatments were performed in presence of 5 nM of

glucagon. Dose titration of MK-0893 alone is shown in the panel of MK-0893. All treatments

were done in duplicates. Dotted curve is a replication of glucagon dose response curve in the

panel of glucagon.

Figure 2. GRA1 increases plasma total cholesterol, LDL-c and HDL-c without effect on

cholesterol synthesis. Male hGCGR mice at 10-12 wks of age were treated with vehicle, GRA1

(30 mpk) and T0901317 (40 mpk) QD for 5 days (n = 8). (A) Glucagon-induced glucose

excursion after single dose on day 1 in mice under ad libitum. (B) Plasma levels of glucose,

cholesterol, PCSK9, and ApoB after 5 days of treatment in mice under ad libitum. (C) Liver

LDLR protein levels determined by western blotting. LDLR is shown as a single band at

molecular weight between 100 and 150 kDa of protein markers. (D) Lipoprotein profile

determined by FPLC on plasma samples obtained from hGCGR mice after 5 days of treatment.

(E) Synthesis of cholesterol, palmitate, and ApoB after 24-h incorporation of D2O prior to

euthanasia of mice followed by processing of plasma samples and measurement of stable

isotope-labeled cholesterol by LC/MS. All data are shown as the means ± SEM. Statistical

significance is calculated by unpaired two-tailed Student's t test. Asterisks denote statistical

significance of the compounds treatment groups compared to the vehicle group. *P ≤ 0.05, **P

≤ 0.01, ***P ≤ 0.001.

Figure 3. GRA1 increases plasma total cholesterol and cholesterol absorption in hGCGR

mice. (A) Experimental design for the effect of GRAs treatment on cholesterol excretion,

absorption, and synthesis: Male hGCGR mice at 10-12 wks of age were treated with vehicle,

GRA1 (30 mpk) QD for 9 days (n = 8). For cholesterol excretion measurement, feces were

collected for 1 day pre and for 1 day (day 3 to 4) following GRA1 treatment. Stable isotope-

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labeled cholesterol and water were administered as indicated as shown and blood samples were

collected in mice under ad libitum for plasma preparation and analysis. (B – E) Plasma total

cholesterol, cholesterol absorption calculated by the ratio of 2,2,3,4,4,6-D6-cholesterol to 3,4-

13C2-cholesterol, cholesterol synthesis from D2O (24-h incorporation), and cholesterol

excretion based on fecal total cholesterol measurement. All data are shown as the means ± SEM.

Statistical significance is calculated by unpaired two-tailed Student's t test. Asterisks denote

statistical significance of the GRA1 group compared to the vehicle group. *P ≤ 0.05, **P ≤

0.01, ***P ≤ 0.001.

Figure 4. Parallel comparisons of different GRA1 and GRA2 on cholesterol metabolism.

Male hGCGR mice at 10-12 wks of age were treated with vehicle, GRA1 (30 mpk) and GRA2

(30 mpk) QD for 5 days (n = 8). (A) Glucagon-induced glucose excursion after single dose on

day 1. (B) Plasma levels of cholesterol, campesterol, sitosterol, lathosterol, lathosterol :

cholesterol ratio (L:C), and cholesterol synthesis from D2O (µg/ml) (24-h incorporation) after 5

days of treatment. (C) Plasma levels of total GLP-1 and GLP-2 after 5 days of treatment. All

data are shown as the means ± SEM. Statistical significance is calculated by unpaired two-tailed

Student's t test. Asterisks denote statistical significance of the compounds treatment groups

compared to the vehicle group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (D) Percent changes of

hepatic genes related to metabolism of glucose, cholesterol, and bile acid in hGCGR mice treated

with GRA1 (30 mpk) and GRA2 (30 mpk) over vehicle (n = 8 per group, data are shown as

means ± SEM).

Figure 5. MK-0893 dose-dependently increases serum total cholesterol, LDL-c, GLP-1,

GLP-2, phytosterols, and bile acid pool in T2DM. Serum of T2DM patients on placebo or

MK-0893 (60 mg and 80 mg QD) for 12 wks were analyzed for serum levels of glucose, total

cholesterol, LDL-c, GLP-1, GLP-2, campesterol, sitosterol, bile acids, cholic acid, 7-HCO, and

7,12-diHCO at baseline (day -1) and week 12 post treatment. BA, bile acid; CA, cholic acid; 7-

HCO, 7 alpha-hydroxycholestenone; 7,12-diHCO, 7,12 alpha-dihydroxycholestenone. (A) The

frequency distributions of percent change in glucose and LDL-c for placebo (n = 39) and MK-

0893 60 mg (n = 46) after 12-wk treatment. (B) Plasma levels of GLP-1 and GLP-2 at baseline

(day -1) and week 12 in T2DM patients treated with placebo (n = 8), MK-0893 60 mg (n = 40 for

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GLP-1, n = 46 for GLP-2), and MK-0893 80 mg (n = 16). (C) Averaged percent changes of

plasma GLP-1 and GLP-2 on week 12 from baseline (day -1) in B. (D – E) Percent change of

plasma total cholesterol, LDL-c, campesterol, sitosterol, bile acids, cholic acid, 7-HCO, and

7,12-diHCO on week 12 from baseline (day -1) in T2DM patients treated with placebo (n = 8),

MK-0893 60 mg (n = 46), and MK-0893 80 mg (n = 16). The data in B - E are shown as the

means ± SEM. Statistical significance is calculated by nonparametric two-tailed Mann-Whitney

test. Asterisks denote statistical significance of the MK-0893 group compared to the placebo

group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 6. Induction of GRAs on cholesterol is abolished by combined treatment with

ezetimibe. Male hGCGR mice at 10-12 wks of age were treated with vehicle, GRA1 (30 mpk),

MK-0893 (30 mpk), GRA2 (30 mpk) with or without ezetimibe (10 mpk) for 9 days (n = 8). (A)

Glucagon-induced glucose excursion after single dose on day 1. (B) Plasma levels of total

cholesterol. (C) Cholesterol absorption calculated by the ratio of 2,2,3,4,4,6-D6-cholesterol to

3,4-13C2-cholesterol in plasma. N.D., non-detectable. (D) Cholesterol excretion determined by

the percent change in fecal total cholesterol on day 4 versus day 0. E: Plasma levels of glucose,

total cholesterol, campesterol, and sitosterol after 9-day treatment. All data are shown as the

means ± SEM. Statistical significance is calculated by unpaired two-tailed Student's t test.

Asterisks denote statistical significance of the compounds treatment groups compared to the

vehicle group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

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Log[Compound]M

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