Transgenic overexpression of neuromedin U promotes leanness and hypophagia in mice.

Recent work has shown that neuromedin U (NmU), a peptide initially identified as a smooth muscle contractor, may play a role in regulating food intake and energy homeostasis. To further evaluate this putative function, we measured food intake, body weight, energy expenditure and glucose homeostasis in transgenic mice that ubiquitously overexpress murine proNmU. NmU transgenic mice were lighter and had less somatic and liver fat, were hypophagic, and had improved insulin sensitivity as judged by an intraperitoneal insulin tolerance test. Transgenic mice had higher levels of hypothalamic NPY, POMC and MCH mRNA. There was no difference in O2 consumption between genotypes; however, NmU transgenic mice displayed a modest increase in respiratory quotient during food deprivation and refeeding. There were no behavioral disturbances in the NmU transgenic mice that could account for the results (e.g. changes in locomotor activity). When placed on a high-fat diet, transgenic mice remained lighter than wild-type mice and ate less, but gained weight at a rate similar to wild-type mice. Despite the increased weight gain with high-fat feeding, glucose tolerance was significantly improved in the transgenic mice. These findings support the hypothesized role of NmU as an endogenous anorexigenic peptide.

Ezetimibe, a potent cholesterol absorption inhibitor, inhibits the development of atherosclerosis in ApoE knockout mice.

Ezetimibe (SCH58235) is a potent, selective, cholesterol absorption inhibitor. The objective of this study was to determine whether ezetimibe reduces plasma cholesterol and inhibits atherogenesis in apolipoprotein E knockout (apoE-/-) mice. Cholesterol absorption was inhibited by >90% at doses of ezetimibe >3 mg/kg in apoE-/- mice. Atherosclerosis and lipoprotein changes were determined in apoE-/- mice fed a high-fat (0.15% cholesterol) "western" diet, a low-fat (0.15% cholesterol) diet, or a semisynthetic cholesterol-free diet with or without ezetimibe (5 mg/kg per day) for 6 months. Ezetimibe reduced plasma cholesterol levels from 964 to 374 mg/dL, from 726 to 231 mg/dL, and from 516 to 178 mg/dL in the western, low-fat, and cholesterol-free diet groups, respectively. The reductions occurred in the very low density and low density lipoprotein fractions, whereas high density lipoprotein cholesterol levels were increased by ezetimibe treatment. Ezetimibe reduced aortic atherosclerotic lesion surface area from 20.2% to 4.1% in the western diet group and from 24.1% to 7.0% in the low-fat cholesterol diet group. Ezetimibe reduced carotid artery atherosclerotic lesion cross-sectional area by 97% in the western and low-fat cholesterol groups and by 91% in the cholesterol-free group. Ezetimibe inhibits cholesterol absorption, reduces plasma cholesterol, increases high density lipoprotein levels, and inhibits the progression of atherosclerosis under western, low-fat, and cholesterol-free dietary conditions in apoE-/- mice. Although apoE-/- mice are more hypercholesterolemic than are humans and low density lipoprotein reductions with ezetimibe are not as pronounced clinically, ezetimibe may inhibit atherogenesis in individuals consuming restricted-fat or western diets.

Ezetimibe selectively inhibits intestinal cholesterol absorption in rodents in the presence and absence of exocrine pancreatic function.

1. Ezetimibe potently inhibits the transport of cholesterol across the intestinal wall, thereby reducing plasma cholesterol in preclinical animal models of hypercholesterolemia. The effect of ezetimibe on known absorptive processes was determined in the present studies. 2. Experiments were conducted in the hamster and/or rat to determine whether ezetimibe would affect the absorption of molecules other than free cholesterol, namely cholesteryl ester, triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid. In addition, to determine whether exocrine pancreatic function is involved in the mechanism of action of ezetimibe, a biliary anastomosis model, which eliminates exocrine pancreatic function from the intestine while maintaining bile flow, was established in the rat. 3. Ezetimibe reduced plasma cholesterol and hepatic cholesterol accumulation in cholesterol-fed hamsters with an ED(50) of 0.04 mg kg(-1). Utilizing cholesteryl esters labelled on either the cholesterol or the fatty acid moiety, we demonstrated that ezetimibe did not affect cholesteryl ester hydrolysis and the absorption of fatty acid thus generated in both hamsters and rats. The free cholesterol from this hydrolysis, however, was not absorbed (92 - 96% inhibition) in the presence of ezetimibe. Eliminating pancreatic function in rats abolished hydrolysis of cholesteryl esters, but did not affect the ability of ezetimibe to block absorption of free cholesterol (-94%). Ezetimibe did not affect the absorption of triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid in rats. 4. Ezetimibe is a potent inhibitor of intestinal free cholesterol absorption that does not require exocrine pancreatic function for activity. Ezetimibe does not affect the absorption of triglyceride as a pancreatic lipase inhibitor (Orlistat) would, nor does it affect the absorption of vitamin A, D or taurocholate, as a bile acid sequestrant (cholestyramine) would.

Comparison of the activity and disposition of the novel cholesterol absorption inhibitor, SCH58235, and its glucuronide, SCH60663.

Previous studies described the metabolism-based discovery of a potent, selective inhibitor of intestinal absorption of cholesterol, SCH58235 (Ezetimibe). Here we demonstrate that the phenolic glucuronide (SCH60663) of SCH58235, was more potent at inhibiting cholesterol absorption in rats than SCH58235, when administered by the intraduodenal route. To understand the increased potency of the glucuronide, the metabolism and distribution of SCH58235 and SCH60663 were studied in bile duct-cannulated rats. One minute after intraduodenal delivery of SCH58235, significant levels of compound were detected in portal plasma; >95% was glucuronidated, indicating that the intestine was metabolizing SCH58235 to its glucuronide. When intraduodenally delivered as SCH58235, the compound was glucuronidated, moved through the intestinal wall, into portal plasma, through the liver, and into bile. However, when delivered as SCH60663, >95% of the compound remained in the intestinal lumen and wall, which may explain its increased potency. Significant inhibition of cholesterol absorption and glucuronidation of SCH58235 occurred when SCH58235 was intravenously injected into bile duct-cannulated rats. Autoradiographic analysis demonstrated that drug related material was located throughout the intestinal villi, but concentrated in the villus tip. These data indicate that (a) SCH58235 is rapidly metabolized in the intestine to its glucuronide; (b) once glucuronidated, the dose is excreted in the bile, thereby delivering drug related material back to the site of action and (c) the glucuronide is more potent than the parent possibly because it localizes to the intestine. Taken together, these data may explain the potency of SCH58235 in the rat (ID(50) = 0.0015 mg kg(-1)) and rhesus monkey (ID(50) = 0.0005 mg kg(-1)).

Effect of chronic central administration of glucagon-like peptide-1 (7-36) amide on food consumption and body weight in normal and obese rats.

Glucagon-like peptide (7-36) amide (GLP-1) acutely inhibits food and water consumption in rats after intracerebroventricular (icv) administration. To assess the potential for desensitization of these effects, we investigated the effects of chronic icv administration of GLP-1 on food consumption and body weight in Sprague-Dawley (SD) rats and Zucker (fa/fa) obese rats. In vitro functional densensitization of the GLP-1 receptor was not observed after overnight exposure of Rin m5F insulinoma cells to GLP-1 at concentrations up to 10 nM. Administration of GLP-1 to SD rats (30 microg icv twice a day for 6 days) resulted in significant reductions in 24-hour food consumption each day (25 +/- 1%). Continuous icv infusion of GLP-1 for 7 and 14 days significantly inhibited cumulative food consumption and reduced body weight in SD rats. In the genetically obese Zucker rat, chronic dosing with GLP-1 (30 microg icv) once a day for 6 days caused significant reductions in food consumption each day and a reduction in body weight. These results indicate that the GLP-1 pathways in the central nervous system controlling food consumption do not desensitize after chronic exposure to GLP-1 and suggest that agonists of the central GLP-1 receptor may be effective agents for the treatment of obesity.

The relationship of tissue localization, distribution and turnover to feeding after intraperitoneal 125I-leptin administration to ob/ob and db/db mice.

Brain and whole body localization and distribution of 125I-leptin was determined after intraperitoneal administration to ob/ob and db/db mice, and was compared to inhibition of food intake. Food intake was not significantly inhibited at3 hours post-injection, but was decreased significantly at 6 h (p < 0.0007) and 24 h (p < 0.02) in ob/ob mice, times at which > 97 % of the radioactive dose was found in the urine. The highest concentrations of 125I-leptin at all time-points were found in the serum, liver and kidneys. These findings were verified by whole body autoradiography. Virtually no 125I-leptin was found in the CNS at later timepoints in either ob/ob or db/db mice. Coronal sectioning of entire brains from ob/ob and db/db mice revealed 125I radioactivity localized to the choroid plexus and in the ventricular space, but not in other CNS regions. No differences in localization, accumulation, or clearance of 125I-leptin in ob/ob vs. db/db mice were found in any of the tissues studied. The present studies demonstrate that the inhibitory effect of leptin on food intake in the ob/ob mouse persists for up to 24 hours after a single dose, despite the complete degradation and elimination of the labeled leptin during the first several hours after injection.

Hypocholesterolemic activity of a novel inhibitor of cholesterol absorption, SCH 48461.

The amount of cholesterol that circulates in the plasma as lipoproteins can be affected by the balance of cholesterol metabolism within and between the intestines and liver. In the present report, we describe a novel hypocholesterolemic agent and document its pharmacological effects in animal models of hypercholesterolemia. The oral administration of (3R,4S)-1,4-bis-(4-methoxyphenyl)-3-(3-phenylpropyl)-2-azetidinone (SCH 48461) reduced plasma cholesterol concentrations in cholesterol-fed hamsters, rats and rhesus monkeys with ED50s of 1, 2 and 0.2 mg/kg per day, respectively, SCH 48461 was also highly effective in reducing hepatic cholesteryl ester accumulation in cholesterol-fed hamsters and rats after 7 days of treatment. In one 3 week study, rhesus monkeys were fed a 0.25% cholesterol/22% saturated fat diet with or without SCH 48461. At the end of the 3 week period the control group's VLDL + LDL-cholesterol increased to 180 Mg/dl from a baseline of approximately 65 mg/dl while plasma apolipoprotein B levels had doubled. Animals treated daily with 1 mg/kg SCH 48461 maintained their baseline levels of VLDL + LDL-cholesterol, HDL-cholesterol, and plasma apolipoproteins B and A-I. After 3 weeks the diets of the two groups were switched. Within 1 week SCH 48461 (1 mg/kg per day) rapidly reversed the elevated VLDL + LDL-cholesterol levels of the previous control group to near baseline values. SCH 48461 exerted its hypocholesterolemic effect through the inhibition of cholesterol absorption. A dose of 10 mg/kg per day inhibited cholesterol absorption in cholesterol-fed hamsters by 68% while a similar reduction was achieved in chow-fed monkeys with 3 mg/kg per day. This latter dose inhibited cholesterol absorption in cholesterol-fed monkeys by 95%. Treatment of cholesterol-fed monkeys with 10 mg/kg per day SCH 48461 significantly increased fecal neutral sterol excretion (52 vs. 32 mg/kg) but had no effect on acidic sterol excretion. Using a 2-h absorption model in cholesterol-fed hamsters, SCH 48461 caused a 46% inhibition of unesterified [14C]cholesterol accumulation in the intestinal wall and a 90% inhibition of cholesteryl ester formation at a dose of 10 mg/kg. Similar data were observed when the plasma radioactivity was assessed, indicating inhibition of both free (61%) and esterified (85%) cholesterol appearance. In contrast, CI-976, a potent acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, did not affect the uptake of free cholesterol into the intestines while inhibiting cholesterol esterification (98% inhibition).(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of acyl CoA: cholesterol acyltransferase inhibition on the uptake, esterification and secretion of cholesterol by the hamster small intestine.

Acyl CoA: cholesterol acyltransferase (ACAT) inhibitors are known to inhibit cholesterol absorption and are under investigation to reduce hypercholesterolemia. These studies examine the effect of an ACAT inhibitor 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)-dodecanamide (PD128042) on the uptake, metabolism and secretion of cholesterol by the hamster intestinal wall in a short-term model. Preliminary studies in this model indicated that the uptake of 14C-cholesterol and its subsequent esterification 2 hr postoral dosing occurs primarily in the duodenal and jejunal segments of the small intestine and most of the radiolabeled cholesterol and cholesteryl ester in the plasma was associated with chylomicrons. In both single- and multiple-dose studies, PD128042 (50 mg kg-1 day-1) did not inhibit intestinal uptake of [14C]-cholesterol but [14C]-cholesteryl ester formation was inhibited. The free [14C]-cholesterol appearing in plasma was not affected despite a large reduction in [14C]-cholesteryl ester. In contrast, cholestyramine (1 g kg-1 day-1) inhibited the uptake of the radiolabeled free cholesterol and the appearance of cholesteryl ester in the intestine and plasma. The effects of PD128042 on cholesterol and cholesteryl ester mass associated with scraped intestinal mucosa were consistent with the effects observed with the use of the radiolabeled cholesterol. In addition, PD128042 did not affect the uptake of appearance of radiolabeled triglyceride in the intestinal wall after oral gavage of 3H-trioleoylglycerol. Taken together, the data suggest that ACAT inhibition reduces cholesterol absorption by limiting cholesteryl ester incorporation into chylomicrons and has no effect on the intestinal processing of free cholesterol to be secreted into plasma.

Demonstration of a direct effect on hepatic acyl CoA: cholesterol acyl transferase (ACAT) activity by an orally administered enzyme inhibitor in the hamster.

Orally active inhibitors of acyl CoA:cholesterol acyl transferase (ACAT), such as Lederle CL277082 (LE), are known to reduce plasma and hepatic cholesteryl ester levels, although the mechanisms are not well understood. Several groups have reported the inhibition of cholesterol absorption upon oral ACAT inhibitor administration. In this study, we used 7-day dietary and drug treatments of hamsters to examine the possible effects of LE on hepatic ACAT. ACAT assays were performed using liver homogenates in the absence and presence of a saturating level of exogenously added cholesterol. LE (100 mg/kg/day) treatment of chow or 0.5% cholesterol-fed animals caused reductions in ACAT activity without additional cholesterol as compared with non-treated animals. When a saturating level of cholesterol was added to the assays, reductions in ACAT activity upon LE treatment of chow- or cholesterol-fed animals were also observed. Treatment of cholesterol-fed animals with cholestyramine in the diet reduced ACAT activity in the absence of added cholesterol. However, ACAT activities similar to those of non-treated animals were observed at a saturating level of cholesterol. This latter effect demonstrates that inhibition of cholesterol absorption reduces cholesterol delivery to the liver but does not reduce cholesterol esterifying capacity since cholestyramine is not absorbed and has no direct effect on the liver. The decreased ACAT activity in homogenates from LE-treated animals could also be mimicked in a dose-dependent manner by the addition of exogenous LE to liver homogenates from non-treated animals. These results indicate that hepatic ACAT activity is regulated by the availability of free cholesterol, and that orally administered LE has a direct effect on hepatic ACAT activity in the liver. In addition, the data are consistent with LE activity in the liver as being responsible, in part, for the reduced hepatic and plasma cholesteryl esters in treated animals.

NG-nitro-L-arginine methyl ester does not affect balloon catheter-induced intimal hyperplasia in rats.

The L-arginine derived NO-cGMP pathway's role in the response of the arterial wall to balloon catheter injury was examined. Rats were given the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (10 mg/kg po twice daily) or vehicle for 6 days before and 2 weeks after balloon catheter injury. NG-nitro-L-arginine methyl ester treatment increased blood pressure and inhibited acetylcholine responses in aortic rings but did not alter the lesions produced by balloon injury. Our results suggest that the L-arginine derived NO-cGMP pathway does not play a significant role in the response of the artery wall to balloon injury in the rat.