Inhibition of acetyl-CoA carboxylase (ACC), with its resultant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation, has the potential to favorably affect the multitude of cardiovascular risk factors associated with the metabolic syndrome. To achieve maximal effectiveness, an ACC inhibitor should inhibit both the lipogenic tissue isozyme (ACC1) and the oxidative tissue isozyme (ACC2). Herein, we describe the biochemical and acute physiological properties of CP-610431, an isozyme-nonselective ACC inhibitor identified through high throughput inhibition screening, and CP-640186, an analog with improved metabolic stability. CP-610431 inhibited ACC1 and ACC2 with IC 50 s of ϳ50 nM. Inhibition was reversible, uncompetitive with respect to ATP, and non-competitive with respect to bicarbonate, acetyl-CoA, and citrate, indicating interaction with the enzymatic carboxyl transfer reaction. CP-610431 also inhibited fatty acid synthesis, triglyceride (TG) synthesis, TG secretion, and apolipoprotein B secretion in HepG2 cells (ACC1) with EC 50 s of 1.6, 1.8, 3.0, and 5.7 M, without affecting either cholesterol synthesis or apolipoprotein CIII secretion. CP-640186, also inhibited both isozymes with IC 50 s of ϳ55 nM but was 2-3 times more potent than CP-610431 in inhibiting HepG2 cell fatty acid and TG synthesis. CP-640186 also stimulated fatty acid oxidation in C2C12 cells (ACC2) and in rat epitrochlearis muscle strips with EC 50 s of 57 nM and 1.3 M. In rats, CP-640186 lowered hepatic, soleus muscle, quadriceps muscle, and cardiac muscle malonyl-CoA with ED 50 s of 55, 6, 15, and 8 mg/kg. Consequently, CP-640186 inhibited fatty acid synthesis in rats, CD1 mice, and ob/ob mice with ED 50 s of 13, 11, and 4 mg/kg, and stimulated rat whole body fatty acid oxidation with an ED 50 of ϳ30 mg/kg. Taken together, These observations indicate that isozyme-nonselective ACC inhibition has the potential to favorably affect risk factors associated with the metabolic syndrome.

Administration of high-dose ethinylestradiol to rats decreases bile flow, Na,K-ATPase specific activity, and liver plasma membrane fluidity. By use of highly purified sinusoidal and bile canalicular membrane fractions, the effect of ethinylestradiol administration on the protein and lipid composition and fluidity of plasma membrane fractions was examined. In sinusoidal fractions, ethinylestradiol (EE) administration decreased Na,K-ATPase activity (32%) and increased activities of alkaline phosphatase (254%), Mg2+-ATPase (155%), and a 160-kDa polypeptide (10-fold). Steady-state and dynamic fluorescence polarization was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH) was significantly higher in canalicular compared to sinusoidal membrane fractions. Ethinylestradiol (5 mg/kg per day for 5 days) selectively increased sinusoidal polarization values. Similar changes were demonstrated with the probes 2- and 12-anthroyloxystearate. Time-resolved fluorescence polarization measurements indicated that EE administration for 5 days did not change DPH lifetime but increased the order component (r infinity) and decreased the rotation rate (R). However, 1 and 3 days after EE administration and with low doses (10-100 micrograms/kg per day for 5 days) the Na,K-ATPase, bile flow, and order component were altered, but the rotation rate was unchanged. Vesicles prepared from total sinusoidal membrane lipids of EE-treated rats, as well as phospholipid vesicles, demonstrated increased DPH polarization, as did intact plasma membrane fractions. Liver plasma membrane fractions showed no change in free cholesterol or cholesterol/phospholipid molar ratio, while esterified cholesterol content was increased with high-dose but not low-dose ethinylestradiol.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane proteins of transporting epithelia are often distributed between apical and basolateral surfaces to produce a functionally polarized cell. The distribution of Na',K+-ATPase [ATP phosphohydrolase (Na+/K+-transporting), EC 3.6.1.37] between apical and basolateral membranes of hepatocytes has been controversial. Because Na',K+-ATPase activity is fluidity dependent and the physiochemical properties ofthe apical membrane reduces its fluidity, we investigated whether altering membrane fluidity might uncover cryptic Na',K+-ATPase in bile canalicular (apical) surface fractions free of detectable Na',K+-ATPase and glucagon-stimulated adenylate cyclase activities. Apical fractions exhibited higher diphenylhexatriene-fluorescence polarization values when compared with sinusoidal (basolateral) membrane fractions. When 2-(2-methoxyethoxy)ethyl 8-(cis-2-noctylcyclopropyl)octanoate (A2C) was added to each fraction, Na',K+-ATPase, but not glucagon-stimulated adenylate cyclase activity, was activated in the apical fraction. In contrast, further activation of both enzymes was not seen in sinusoidal fractions. The A2C-induced increase in apical Na',K'-ATPase approached 75% of the sinusoidal level. Parallel increases in apical Na',K+-ATPase were produced by benzyl alcohol and Triton WR-1339. All three fluidizing agents decreased the order component of membrane fluidity. Na',K+-ATPase activity in each subfraction was identically inhibited by the monoclonal antibody 9-A5, a specific inhibitor of this enzyme.These findings suggest that hepatic Na',K+-ATPase is distributed in both surface membranes but functions more efficiently and, perhaps, specifically in the sinusoidal membranes because of their higher bulk lipid fluidity.Membrane proteins that polarize function across cell surfaces of transporting epithelia are generally distributed asymmetrically at the cellular apical or basolateral pole (1-3). For example, leucine aminopeptidase is present on the apical membranes of enterocytes, proximal renal tubules, and hepatocytes (3-8), whereas IgA receptor (secretory component) is found in basolateral surfaces of most epithelial cells (9). On the other hand, Na',K+-transporting ATPase [ATP phosphohydrolase (Na+/K+-transporting); EC 3.6.1.37] is generally believed to be localized to the basolateral surface of renal (10), intestinal (11), and cultured MDCK (kidney) cells (12, 13) but on the apical surface of the choroid plexus (14) and possibly on both poles of the exorbital and parotid gland cells (15-17).The localization of Na',K+-ATPase in hepatocytes has been controversial (18). With histochemical, biochemical, and cell-fractionation techniques, Na',K+-ATPase activity has been seen in the basolateral membrane (19-22), whereas immunocytochemistry using a Na',K+-ATPase-specific antibody has localized Na+/K+-pump sites to the bile canalicular (or apical) surface, as well as the sinusoidal (or basolateral) surface (23, 24). Contradictory results also have been reported with these techniques (25)(26)(27).Activities of many i...

Bile acids are efficiently recovered from the intestinal lumen by a Na(+)-dependent transport process that is localized in the ileal enterocyte brush-border membrane. To establish a cell culture model for this process, we examined the Na+ dependence of cholyltaurine (C-tau; taurocholate) transport across monolayers of differentiated Caco-2 cells grown on permeable filter inserts. Transport of [3H]C-tau was Na+ dependent (> 20-fold stimulation), saturable, and time linear for at least 60 min. The apparent Michaelis constant of [3H]C-tau transport was approximately 65 microM, and the maximal transport rate was approximately 800 pmol.min-1.mg protein-1. Transport of [3H]C-tau in the apical-to-basolateral direction was 17-fold greater than transport in the reverse direction. Lowered incubation temperature, various metabolic inhibitors, and various unlabeled bile acids inhibited [3H]C-tau transport. Caco-2 cells thus transport bile acids in a manner similar to that described for ileal brush-border membrane vesicles and isolated ileal enterocytes and are therefore an appropriate model for studying the molecular basis of ileal bile acid transport.

We have explored the use of steroidal glycosides as cholesterol absorption inhibitors which act through an unknown mechanism. The lead for this program was tigogenin cellobioside (1, tiqueside) which is a weak inhibitor (ED50 = 60 mg/kg) as measured in an acute hamster cholesterol absorption assay. Modification of the steroid portion of the molecule led to the discovery of 11-ketotigogenin cellobioside (5, pamaqueside) which has an ED50 of 2 mg/kg. Replacement of the cellobiose with other sugars failed to provide more potent analogs. However, large improvements in potency were realized through modification of the hydroxyl groups on the cellobiose. This strategy ultimately led to the 4", 6"-bis[(2-fluorophenyl)carbamoyl]-beta-D-cellobiosyl derivative of 11-ketotigogenin (51) with an ED50 of 0.025 mg/kg in the hamster assay, as well as the corresponding hecogenin analog 64 (ED50 = 0.07 mg/kg).