Glucagon receptor antagonism induces increased cholesterol absorption
2015; Elsevier BV; Volume: 56; Issue: 11 Linguagem: Inglês
10.1194/jlr.m060897
ISSN1539-7262
AutoresHong-Ping Guan, Xiaodong Yang, Ku Lu, Shengping Wang, José Castro‐Perez, Stephen F. Previs, Michael Wright, Vinit Shah, Kithsiri Herath, Dan Xie, Daphne Szeto, Gail Forrest, Jing Chen Xiao, Oksana Palyha, Liping Sun, Paula J. Andryuk, Samuel S. Engel, Yusheng Xiong, Songnian Lin, David E. Kelley, Mark D. Erion, Harry R. Davis, Liangsu Wang,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoGlucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. 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 glucagon-like peptide 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, coadministration 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, GCGR antagonism govern cholesterol metabolism. Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. 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 glucagon-like peptide 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, coadministration 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, GCGR antagonism govern cholesterol metabolism. It is through mostly opposing actions that the pancreatic islet hormones, insulin and glucagon, interact in the governance of hepatic glucose production and its uptake. In type 2 diabetes mellitus (T2DM), as well as in type 1 diabetes mellitus, fasting plasma glucagon is generally elevated, inappropriate to prevailing hyperglycemia, and there is less suppression during prandial metabolism (1Müller W.A. Faloona G.R. Aguilar-Parada E. Unger R.H. Abnormal alpha-cell function in diabetes. Response to carbohydrate and protein ingestion.N. Engl. J. Med. 1970; 283: 109-115Crossref PubMed Scopus (476) Google Scholar). This imbalance in secretion of islet hormones is considered to be a key aspect of the pathophysiology causing hyperglycemia (1Müller W.A. Faloona G.R. Aguilar-Parada E. Unger R.H. Abnormal alpha-cell function in diabetes. Response to carbohydrate and protein ingestion.N. Engl. J. Med. 1970; 283: 109-115Crossref PubMed Scopus (476) Google Scholar, 2Unger R.H. Cherrington A.D. Glucagonocentric restructuring of diabetes: a pathophysiologic and therapeutic makeover.J. Clin. Invest. 2012; 122: 4-12Crossref PubMed Scopus (489) Google Scholar). Glucagon receptor (GCGR) antagonism has accordingly drawn considerable interest as a novel pharmacological approach for treating T2DM. Several GCGR antagonists (GRAs) have advanced into human clinical trials in patients with T2DM. MK-0893 (3Engel S.S. Xu L. Andryuk P.J. Davies M.J. Amatruda J. Kaufman K. Goldstein B.J. Efficacy and tolerability of MK-0893, a glucagon receptor antagonist (GRA), in patients with type 2 diabetes (T2DM).Diabetes. 2011; 60: A85Google Scholar), MK-3577 (4Engel S.S. Reitman M. Xu L. Andryuk P.J. Davies M.J. Kaufman K. Goldstein B.J. Glycemic and lipid effects of the short-acting glucagon receptor antagonist MK-3577 in patients with type 2 diabetes.Diabetes. 2012; 61: A266Google Scholar), LY2409021 (5Kazda C.M. Headlee S.A. Ding Y. Kelly R.P. Garhyan P. Hardy T.A. Lewin A.J. The glucagon receptor antagonist LY2409021 significantly lowers HbA1c and is well tolerated in patients with type 2 diabetes mellitus: a 24-week phase 2 study.Diabetologia. 2013; 56: S391PubMed Google Scholar, 6Kelly R.P. Garhyan P. Raddad E. Fu H. Lim C.N. Prince M.J. Pinaire J.A. Loh M.T. Deeg M.A. Short-term administration of the glucagon receptor antagonist LY2409021 lowers blood glucose in healthy subjects and patients with type 2 diabetes.Diabetes Obes. Metab. 2015; 17: 414-422Crossref PubMed Scopus (83) Google Scholar), and an anti-sense oligo targeting the GCGR (ISIS-GCGRrx) (7Morgan E. Smith A. Watts L. Xia S. Cheng W. Geary R. Bhanot S. ISIS-GCGRRX, an antisense glucagon receptor antagonist, caused rapid, robust, and sustained improvements in glycemic control without changes in BW, BP, lipids, or hypoglycemia in T2DM patients on stable metformin therapy.Diabetes. 2014; 63: LB28Google Scholar) have each demonstrated efficacy in lowering fasting and postprandial hyperglycemia, leading to substantial reductions of HbA1c, thereby providing clinical proof of concept for the efficacy of GRAs. 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 (supplementary Fig. 1) (3Engel S.S. Xu L. Andryuk P.J. Davies M.J. Amatruda J. Kaufman K. Goldstein B.J. Efficacy and tolerability of MK-0893, a glucagon receptor antagonist (GRA), in patients with type 2 diabetes (T2DM).Diabetes. 2011; 60: A85Google Scholar). 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) (supplementary Fig. 1) (3Engel S.S. Xu L. Andryuk P.J. Davies M.J. Amatruda J. Kaufman K. Goldstein B.J. Efficacy and tolerability of MK-0893, a glucagon receptor antagonist (GRA), in patients with type 2 diabetes (T2DM).Diabetes. 2011; 60: A85Google Scholar). LDL-c and T2DM are recognized to adversely influence risk for cardiovascular disease, and increased LDL-c in the setting of T2DM is a cause for concern, as this could potentiate the risk for cardiovascular disease (8Colhoun H.M. Betteridge D.J. Durrington P.N. Hitman G.A. Neil H.A. Livingstone S.J. Thomason M.J. Mackness M.I. Charlton-Menys V. Fuller J.H. Primary prevention of cardiovascular disease with atorvastatin in type 2 diabetes in the Collaborative Atorvastatin Diabetes Study (CARDS): multicentre randomised placebo-controlled trial.Lancet. 2004; 364: 685-696Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar, 9Betteridge J. Benefits of lipid-lowering therapy in patients with type 2 diabetes mellitus.Am. J. Med. 2005; 118: 10-15Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 10Howard B.V. Robbins D.C. Sievers M.L. Lee E.T. Rhoades D. Devereux R.B. Cowan L.D. Gray R.S. Welty T.K. Go O.T. et al.LDL cholesterol as a strong predictor of coronary heart disease in diabetic individuals with insulin resistance and low LDL: The Strong Heart Study.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 830-835Crossref PubMed Scopus (240) Google Scholar). The current studies were undertaken using a preclinical rodent model and 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. All mice used in the studies were purchased from Taconic (Germantown, NY) 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 (hGCGR) mice were generated on a B6.129S6 background and had been backcrossed to C57BL/6 for more than 13 generations. This strain of mice showed no metabolic phenotypes in glucose and cholesterol (11Shiao L.L. Cascieri M.A. Trumbauer M. Chen H. Sullivan K.A. Generation of mice expressing the human glucagon receptor with a direct replacement vector.Transgenic Res. 1999; 8: 295-302Crossref PubMed Scopus (28) Google Scholar). All tests were performed in the same strain of mice and comparisons were made between the compound treatments 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 (po) dosing volume was 10 ml/kg body weight. A glucagon challenge assay was performed in mice under ad libitum feeding, as previously described (12Dallas-Yang Q. Shen X. Strowski M. Brady E. Saperstein R. Gibson R.E. Szalkowski D. Qureshi S.A. Candelore M.R. Fenyk-Melody J.E. et al.Hepatic glucagon receptor binding and glucose-lowering in vivo by peptidyl and non-peptidyl glucagon receptor antagonists.Eur. J. Pharmacol. 2004; 501: 225-234Crossref PubMed Scopus (37) Google Scholar). Briefly, at 1 h post compound administration via po, glucagon dissolved in PBS was injected at 15 μg/kg ip followed by glucose measurements using a glucometer (Life Scan) via tail bleeding at 0, 12, 24, and 48 min post injection. For cholesterol absorption and synthesis studies, stable isotope-labeled cholesterol was prepared as described previously (13Bosner M.S. Lange L.G. Stenson W.F. Ostlund Jr, R.E. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry.J. Lipid Res. 1999; 40: 302-308Abstract Full Text Full Text PDF PubMed Google Scholar). 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 US Pharmacopoeia grade 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 2 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 passed through a 1.2 micron filter (Sigma). Solutions were stored at 4°C for 1 day before administration. Human serum samples used in this study were obtained from a randomized double-blind placebo-controlled crossover trial comprising a 1 week screening period and a 6 week washout of previous anti-hyperglycemic agents followed by a 2 week placebo run-in period and then 12 week 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 glucagon-like peptide (GLP)-1 measurement, six samples in the 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). Cryopreserved human primary hepatocytes were purchased from CellzDirect (presently Life Technologies, Hu8080). One vial of frozen primary hepatocytes (approximately five million cells in total) was quickly thawed to 37°C in a water bath and washed in cryopreserved hepatocyte recovery medium (Life Technologies, CM7000) and resuspended in buffer containing HBSS (Life Technologies, 14025), 0.1% BSA (Sigma, A9205), and 1.2 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma, I-5879). To assess antagonist activity, 4,000 cells per well were preincubated 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 transfer using an EnVision plate reader (PerkinElmer). The IC50 values were calculated using nonlinear regression curve fit analysis in Prism (GraphPad). 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 a 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). A commercial enzymatic colorimetric kit was used for the determination of plasma total cholesterol (Wako cholesterol E kit) according to manufacturer's instructions (WakoUSA). The plasma level of proprotein convertase subtilisin/kexin type 9 (PCSK9) was determined by PCSK9 dissociation-enhanced lanthanide fluorescence immunoassay, as described elsewhere (14Ni Y.G. Di Marco S. Condra J.H. Peterson L.B. Wang W. Wang F. Pandit S. Hammond H.A. Rosa R. Cummings R.T. et al.A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo.J. Lipid Res. 2011; 52: 78-86Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The plasma or serum lipoprotein profile was assayed by fast-protein LC, as described previously (15Castro-Perez J. Briand F. Gagen K. Wang S.P. Chen Y. McLaren D.G. Shah V. Vreeken R.J. Hankemeier T. Sulpice T. et al.Anacetrapib promotes reverse cholesterol transport and bulk cholesterol excretion in Syrian golden hamsters.J. Lipid Res. 2011; 52: 1965-1973Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Fecal cholesterol was measured by extracting lipids using the Folch method (16Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), 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 the lower phase under nitrogen gas. The extract was reconstituted with 10% Triton X-100 in isopropanol and analyzed using a commercial cholesterol kit (WakoUSA). The 2H-labeling of body water was determined using headspace analyses following exchange with acetone, as described by Shah et al. (17Shah V. Herath K. Previs S.F. Hubbard B.K. Roddy T.P. Headspace analyses of acetone: a rapid method for measuring the 2H-labeling of water.Anal. Biochem. 2010; 404: 235-237Crossref PubMed Scopus (49) Google Scholar). Briefly, 20 μl of sample (or standard) 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 was 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 mass spectrometer coupled to a 6890 GC oven fitted with an Agilent DB-5MS column (30 m × 250 μm × 0.15 μm); the oven was set at 170°C and helium carrier flow was set at 1.0 ml/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 GC-MS (18Previs S.F. Mahsut A. Kulick A. Dunn K. Andrews-Kelly G. Johnson C. Bhat G. Herath K. Miller P.L. Wang S.P. et al.Quantifying cholesterol synthesis in vivo using (2)H(2)O: enabling back-to-back studies in the same subject.J. Lipid Res. 2011; 52: 1420-1428Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Lipids were saponified by heating plasma (50 μl) with 1 N KOH in 80% methanol (200 μl) at 65°C for 1 h. Samples were acidified with 25 μl 6 N HCl and then extracted in 125 μl chloroform followed by vigorous vortexing for 20 s. The samples were centrifuged at 3,000 rpm for 5 min and 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 h. Excess reagent was evaporated to dryness under N2 and the acetylated derivative was reconstituted in 50 μl ethyl acetate for analysis by GC-MS. All analyses were performed using an Agilent 5973 mass spectrometer coupled to a 6890 GC oven fitted with an Agilent DB-5MS column (30 m × 250 μm × 0.15 μm). 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; 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. To quantify the contribution of cholesterol synthesis to blood cholesterol level, the data was fit (using a precursor:product labeling ratio) to the general equation (18Previs S.F. Mahsut A. Kulick A. Dunn K. Andrews-Kelly G. Johnson C. Bhat G. Herath K. Miller P.L. Wang S.P. et al.Quantifying cholesterol synthesis in vivo using (2)H(2)O: enabling back-to-back studies in the same subject.J. Lipid Res. 2011; 52: 1420-1428Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar): newly made cholesterol = [product labeling/(precursor labeling × n)] × concentration, where n is the number of exchangeable hydrogens (assumed to equal 26 for cholesterol) (2Unger R.H. Cherrington A.D. Glucagonocentric restructuring of diabetes: a pathophysiologic and therapeutic makeover.J. Clin. Invest. 2012; 122: 4-12Crossref PubMed Scopus (489) Google Scholar).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 (19Turley S.D. Herndon M.W. Dietschy J.M. Reevaluation and application of the dual-isotope plasma ratio method for the measurement of intestinal cholesterol absorption in the hamster.J. Lipid Res. 1994; 35: 328-339Abstract Full Text PDF PubMed Google Scholar, 20Zilversmit D.B. Hughes L.B. Validation of a dual-isotope plasma ratio method for measurement of cholesterol absorption in rats.J. Lipid Res. 1974; 15: 465-473Abstract Full Text PDF PubMed Google Scholar). The plasma level and flux of ApoB were quantified by the LC-MS/MS method, as described previously (21Zhou H. Li W. Wang S.P. Mendoza V. Rosa R. Hubert J. Herath K. McLaughlin T. Rohm R.J. Lassman M.E. et al.Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water.J. Lipid Res. 2012; 53: 1223-1231Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Five microliters 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 at 600 rpm for 1 h on an R-shaker (Eppendorf). Samples were evaporated to dryness under nitrogen. Derivatization reagent [150 μl (1,000 mg of 2-methyl 6-nitro benzoic anhydride, 300 mg of 4-dimethyl pyridine, and 800 mg of picolinic acid dissolved in 2 ml of triethylamine and 12 ml of pyridine] was added to each tube and the plate was incubated at 80°C for 1 h. After incubation, 500 μl of hexane was added to each tube, vortexed, and centrifuged at 4,000 rpm at room temperature for 10 min. Supernatant (400 μl) 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% water, and 0.1% 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, five pooled plasma samples from multiple subjects were used as quality controls and each quality control sample was injected in triplicate for LC-MS assay. Quality controls with a variation of ±15% coefficient were deemed as acceptable. Serum (150 μl) was transferred into a deep 2 ml 96-well plate followed by the addition of 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 min followed by centrifugation at 4,000 rpm for 20 min at room temperature. After centrifugation, 600 μl of supernatant was passed (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 and 0.1% formic acid/20% water. The resultant extract (10 μl) was injected onto an LC-MS/MS system operated in positive ion mode electrospray (UPLC/TQS mass spectrometer; Waters Corp.). Isotopic dilution quantitation was conducted to obtain concentrations of 7α,12α dihydroxy-4-cholesten-3-one and 7α-hydroxy-4-cholesten-3-one. One piece of liver (∼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. The protein concentration of the 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 heating 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 polyvinylidene difluoride membrane, LDL receptor (LDLR) was blotted by using a rabbit monoclonal antibody (Abcam, ab52818). Loading control was blotted by using β-actin polyclonal antibody (Cell Signaling Technology). 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 an RNA Easy kit and QIACube instrument (Qiagen). Total RNA (2 μg) 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 Master Mix reagents and TaqMan primer/probe sets (Life Technologies) or SYBR Green Master Mix reagents and a custom-designed PCR array developed in collaboration with SABiosciences-Qiagen (22Jensen K.K. Previs S.F. Zhu L. Herath K. Wang S.P. Bhat G. Hu G. Miller P.L. McLaren D.G. Shin M.K. et al.Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification.Am. J. Physiol. Endocrinol. Metab. 2012; 302: E209-E217Crossref PubMed Scopus (16) Google Scholar). 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 a vehicle group arbitrarily set at one (22Jensen K.K. Previs S.F. Zhu L. Herath K. Wang S.P. Bhat G. Hu G. Miller P.L. McLaren D.G. Shin M.K. et al.Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification.Am. J. Physiol. Endocrinol. Metab. 2012; 302: E209-E217Crossref PubMed Scopus (16) Google Scholar). The P values were determined by two-tailed equal variance Student's t-test, comparing the 2−ΔCT values of the vehicle and GRA-treated groups. All data are presented as mean ± SEM. For rodent results, statistical analysis was performed by using one-way ANOVA followed by an unpaired two-tailed Student's t-test to compare mean values between treatment groups and the control group. For human results, due to an uneven number of human samples in different groups, the percent 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 the Mann-Whitney test. For rodent studies, all testing protocols were reviewed and approved by the Merck Research Laboratories Institutional Animal Care and Use Committees in Rahway and Kenilworth, NJ. 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 (https://www.nc3rs.org.uk/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 or ethical review committee before being initiated. For each site, the institutional review board/ethical review committee and Merck's consent form review department (US studies) or local medical director (non-US 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. Three GRA compounds [MK-0893 (23Xiong Y. Guo J. Candelore M.R. Liang R. Miller C. Dallas-Yang Q. Jiang G. McCann P.E. Qureshi S.A. Tong X. et al.Discovery of a novel glucagon receptor antagonist N-[(4-{(1S)-1-[3-(3, 5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbo nyl]-β-alanine (MK-0893) for the treatment of type II diabetes.J. Med. Chem. 2012; 55: 6137-6148Crossref PubMed Scopus (71) Google Scholar), GRA1 (24Mu J. Qureshi S.A. Brady E.J. Muise E.S. Candelore M.R. Jiang G. Li Z. Wu M.S. Yang X. Dallas-Yang Q. et al.Anti-diabetic efficacy and impact on amino acid metabolism of GRA1, a novel small-molecule glucagon receptor antagonist.PLoS One. 2012; 7: e49572Crossref PubMed Scopus (44) Google Scholar), and GRA2 (25Conner, S. E., Zhu, G., inventors; Eli Lilly and Company, assignee. Glucagon receptor antagonists, preparation and therapeutic uses. United States patent US7816557. 2010 Oct 19.Google Scholar)] were demonstrated to potently block glucagon binding to the 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 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 (2Unger R.H. Cherrington A.D. Glucagonocentric restructuring of diabetes: a pathophysiologic and therapeutic makeover.J. Clin. Invest. 2012; 122: 4-12Crossref PubMed Scopus (489) Google Scholar). MK-0893, GRA1, or GRA2 alone had no effect on cAMP production. In the presence of glucagon, each compound suppressed cAMP production, with IC50s of 563, 448, and 292 nM, respectively (Fig. 1). Comparing these results with values earlier obtained from CHO cells stably overexpressing human GCGR (hGCGR.CHO) (23Xiong Y. Guo J. Candelore M.R. Liang R. Miller C. Dallas-Yang Q. Jiang G. McCann P.E. Qureshi S.A. Tong X. et al.Discovery of a novel glucagon receptor antagonist N-[(4-{(1S)-1-[3-(3, 5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbo nyl]-β-alanine (MK-0893) for the treatment of type II diabetes.J. Med. Chem. 2012; 55: 6137-6148Crossref PubMed Scopus (71) Google Scholar, 24Mu J. Qureshi S.A. Brady E.J. Muise E.S. Candelore M.R. Jiang G. Li Z. Wu M.S. Yang X. Dallas-Yang Q. et al.Anti-diabetic efficacy and impact on amino acid metabolism of GRA1, a novel small-molecule glucagon receptor antagonist.PLoS One. 2012; 7: e49572Crossref PubMed Scopus (44) Google Scholar), IC50s of the compounds are right-shifted, likely reflecting overexpression 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
Referência(s)