Gene Expression Analysis in Rats Treated with Experimental Acetyl-Coenzyme A Carboxylase Inhibitors Suggests Interactions with the Peroxisome Proliferator-Activated Receptor Pathway

Acetyl CoA carboxylase (ACC) 2, which catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, has been identified as a potential target for type 2 diabetes and obesity. Small-molecule inhibitors of ACC2 would be expected to reduce de novo lipid synthesis and increase lipid oxidation. Treatment of ob/ob mice with compound A-908292 (S) ({(S)-3-[2-(4-isopropoxy-phenoxy)-thiazol-5-yl]-1-methylprop-2-ynyl}-carbamic acid methyl ester), a small-molecule inhibitor with an IC50 of 23 nM against ACC2, resulted in a reduction of serum glucose and triglyceride levels. However, compound A-875400 (R) ({(R)-3-[2-(4-isopropoxy-phenoxy)thiazol-5-yl]-1-methyl-prop-2-ynyl}-carbamic acid methyl ester), an inactive enantiomer of A-908292 (S) with approximately 50-fold less activity against ACC2, also caused a similar reduction in glucose and triglycerides, suggesting that the glucose-lowering effects in ob/ob mice may be mediated by other metabolic pathways independent of ACC2 inhibition. To characterize the pharmacological activity of these experimental compounds at a transcriptional level, rats were orally dosed for 3 days with either A-908292 (S) or A-875400 (R), and gene expression analysis was performed. Gene expression analysis of livers showed that treatment with A-908292 (S) or A-875400 (R) resulted in gene expression profiles highly similar to known peroxisome proliferator-activated receptor (PPAR)activators. The results suggest that, in vivo, both A-908292 (S) and A-875400 (R) stimulated the PPAR-dependent signaling pathway. These results were further supported by both an in vitro genomic evaluation using rat hepatocytes and immunohistochemical evaluation using 70-kDa peroxisomal membrane protein. Overall, the gene expression analysis suggests a plausible mechanism for the similar pharmacological findings with active and inactive enantiomers of an ACC2 inhibitor. The incidence of type 2 diabetes has dramatically increased over the past decade. This epidemic is largely attributed to the proliferation of key risk factors, which include a sedentary lifestyle, a high-fat diet, obesity, and the demographic shift to a more aged population. There is ample evidence that abdominal obesity and physical inactivity contribute significantly to the development of type 2 diabetes (Turkoglu et al., 2003; Steyn et al., 2004). At the cellular level, an increase in ectopic fat storage in nonadipose tissues such as skeletal muscle, liver, and pancreas is a strong predictor of the development of insulin resistance and type 2 diabetes (Sinha et al., 2002; Hulver et al., 2003). In addition, deregulation of fatty acid metabolism also contributes to the overall development of metabolic syndrome. Thus, targeting key enzymes that are critically involved in lipid synthesis, lipid oxidation, and/or triglyceride synthesis pathways with J.F.W. and Y.Y. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.126938. ABBREVIATIONS: ACC, acetyl CoA carboxylase; A-908292, {(S)-3-[2-(4-isopropoxy-phenoxy)-thiazol-5-yl]-1-methyl-prop-2-ynyl}-carbamic acid methyl ester; A-875400, {(R)-3-[2-(4-isopropoxy-phenoxy)-thiazol-5-yl]-1-methyl-prop-2-ynyl}-carbamic acid methyl ester; PPAR, peroxisome proliferator-activated receptor; LC, liquid chromatography; MS, mass spectrometry; ANOVA, analysis of variance; IVT, In Vitro Technologies; HMGCS2, 3-hydroxy-3-methylglutaryl-CoA synthase; PMP70, 70-kDa peroxisomal membrane protein; A-859610, {3-[2-(4-isopropoxy-phenoxy)thiazol-5-yl]-1-methyl-prop-2-ynyl}-carbamic acid methyl ester; GW7647, 2-(4-(2-(1-cyclohexanebutyl)-3-cyclohexylureido)ethyl)phenylthio)-2methylpropionic acid. 0022-3565/08/3242-507–516$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 324, No. 2 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 126938/3300851 JPET 324:507–516, 2008 Printed in U.S.A. 507 at A PE T Jornals on Sptem er 4, 2017 jpet.asjournals.org D ow nladed from small-molecule inhibitors would probably cause a reduction of fat accumulation in nonadipose tissues, notably in liver and skeletal muscles, and would therefore improve insulin sensitivity and overall lipid profiles. Acetyl CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA. Malonyl-CoA, in turn, can be used by fatty acid synthase for de novo lipogenesis and also acts as a potent allosteric inhibitor of carnitine palmitoyltransferase 1, a mitochondrial membrane protein that shuttles long-chain fatty acyl CoAs into the mitochondria where they are oxidized (Ruderman and Prentki, 2004). In rodents and in humans, there are two isoforms of ACC encoded by distinct genes, but sharing overall 70% amino acid identity. ACC1, a 265-kDa cytosolic protein, is highly expressed in lipogenic tissues, such as liver and adipose tissue, whereas the 280-kDa ACC2 protein is preferentially expressed in oxidative tissues like skeletal muscle, heart, and, to some extent, liver (Abu-Elheiga et al., 1997; Mao et al., 2003). ACC2 has a unique N terminus domain comprised of 114 amino acids that represent a putative transmembrane domain thought to be responsible for mitochondrial targeting (Abu-Elheiga et al., 2000). It has been shown that whole-body knockout of ACC2 in mice shows a favorable metabolic profile; the mice are resistant to high-fat diet-induced obesity and show decreased lipid content in both liver and adipose (Oh et al., 2005). The liver lipid content in the ACC2 / mouse was described in Abu-Elheiga et al. (2001). Small-molecule inhibitors of ACC2 have been synthesized at Abbott Laboratories (Abbott Park, IL). These compounds have been shown to lower malonyl-CoA in skeletal muscle and to a lesser extent in liver in Sprague-Dawley rats (Gu et al., 2006; Xu et al., 2007). A-859610 is a racemic mixture of A-908292, the active (S) enantiomer (IC50 0.023 M for human ACC2 and 30 M for human ACC1) and A-875400, the less active (R) enantiomer (IC50 1.082 M for human ACC2 and 30 M for human ACC1). In a previous study, in ob/ob mice, the racemate, A-859610, dosed at 30 mg/kg/day (b.i.d., p.o.) for 2 weeks was shown to lower serum glucose by approximately 30% (data not shown). We were surprised to find that in a 2-week repeat-dose study, both the active and inactive enantiomers of A-859610 showed a reduction in glucose and triglycerides to a similar extent, indicating a potential off-target effect. To better characterize the active and inactive enantiomers of A-859610, microarray analysis was performed on rats treated with either A-908292 (S) or A-875400 (R). The results suggest that both compounds behave as activators of the peroxisome proliferator-activated receptor (PPAR)pathway in vivo, which is most probably the underlying mechanistic basis for glucose and triglyceride lowering in ob/ob mice. These results are an example of how gene expression profiling can aid in determining the mechanistic action of certain pharmacological agents. Materials and Methods Animals and Treatment. ob/ob mice (6–7 weeks of age; The Jackson Laboratory, Bar Harbor, ME) were acclimated to the animal research facilities for 5 days. The ob/ob mice were obtained and treated as in previous publications (Zinker et al., 2002; Waring et al., 2003). Mice were fed a standard diet of Lab Diet Rodent Laboratory Diet 5105. The ob/ob mice were dosed with vehicle (n 10), A-875400 (R) (n 9), or A-908292 (S) (n 9) at 30 mg/kg/day (b.i.d., p.o.) for 2 weeks. For the malonyl-CoA rat study, normal SpragueDawley rats (n 6) were fasted for 4 h and then orally dosed with 10 and 50 mg/kg of either A-908292 (S) or A-875400 (R), followed by oral gavage of high-carbohydrate-enriched meals. For the gene expression analysis study, Sprague-Dawley rats [Crl: CD(SD)IGS BR] weighing between 201 and 221 g were obtained from Charles River Laboratories, Inc. (Portage, MI). Rats were fed a standard diet of Lab Diet Rodent Laboratory Diet 5001 pellets (PMI Nutrition International, Inc., St. Louis, MO). Rats were dosed with vehicle (sterile H2O containing 1% Tween) (n 3), A-875400 (R) (n 3) or A-908292 (S) (n 3) at 30 or 100 mg/kg/day b.i.d. for a period of 3 days. The dose volume for all treatment groups was 4 ml/kg/dose. All rats were fasted overnight after their last treatment and euthanized under CO2 anesthesia. In a separate study, Sprague-Dawley rats [Crl:CD(SD)IGS BR] weighing between 201–221 g were obtained from Charles River Laboratories, Inc. Rats were fed a standard diet of Lab Diet Rodent Laboratory Diet 5001 pellets (PMI Nutrition International, Inc.). Rats were dosed with vehicle (sterile H2O containing 1% Tween) (n 3) or bezafibrate at 200 mg/kg/day b.i.d. for a period of 3 days. The dose volume for all treatment groups was 4 ml/kg/dose. All rats were fasted overnight after their last treatment and euthanized under CO2 anesthesia. All tissues collected for gene expression analysis were immediately flash frozen in liquid nitrogen and subsequently stored at 70°C. For histopathology evaluation, livers from rats were fixed in 10% neutral buffered formalin. Blood samples drawn from the animals at necropsy were used to measure serum concentrations or activities of blood urea nitrogen, creatinine, alanine amino transferase, aspartate amino transferase, gamma glutamyltransferase, alkaline phosphatase, cholesterol, triglycerides, bilirubin, glucose, total protein, albumin, and globulins using an Abbott Aeroset clinical