Naunyn-Schmiedeberg’s Arch Pharmacol (2006) 372: 300–312 DOI 10.1007/s00210-005-0024-3 ORIGINA L ARTI CLE Rodney A. Velliquette . Rachel Kossover . Stephen F. Previs . Paul Ernsberger Lipid-lowering actions of imidazoline antihypertensive agents in metabolic syndrome X Received: 19 September 2005 / Accepted: 21 November 2005 / Published online: 17 January 2006 # Springer-Verlag 2006 Abstract Agonists active at I1-imidazoline receptors (I1R) not only lower blood pressure but also ameliorate glucose intolerance, insulin resistance, and hyperlipidemia with long-term treatment. We sought to determine the possible mechanism for the lipid-lowering actions of imidazolines in a model of metabolic Syndrome X, the spontaneouslyhypertensive obese (SHROB) rat. The acute actions of moxonidine and rilmenidine, selective I1R agonists, were compared to a specific α2-adrenergic receptor agonist, guanabenz, with and without selective receptor blockers. Moxonidine and rilmenidine rapidly reduced plasma triglyceride (20±4% and 21±5%, respectively) and cholesterol (29±9% and 27±9%). In contrast, the specific α2adrenergic receptor agonist guanabenz failed to reduce plasma lipids. Blocking experiments showed that moxonidine’s actions were mediated by I1R and not α2adrenergic receptors. To evaluate a hepatic site of action, radioligand binding studies with liver plasma membranes confirmed the presence of I1R. Intraportal moxonidine reduced plasma triglycerides by 23±3% within 10 min. Moxonidine inhibited hepatic triglyceride secretion by 75% compared to vehicle treatment. Tracer studies with 2 H2O suggested that moxonidine inhibits de novo fatty acid synthesis. Thus, activation of I1R lowers plasma lipids, with the main site of action probably within the liver to reduce synthesis and secretion of triglycerides. More selective I1R agonists might provide monotherapy for hyperlipidemic hypertension. R. A. Velliquette . R. Kossover . S. F. Previs . P. Ernsberger (*) Department of Nutrition, Case Western Reserve University School of Medicine, 10900 Euclid Ave, Cleveland, OH 44106-4906, USA e-mail: pre@po.cwru.edu Tel.: +1-216-3684738 Fax: +1-216-3684752 Keywords Plasma triglycerides . Imidazoline receptors . Alpha2-adrenoceptors . Moxonidine . Rilmenidine . Guanabenz . Efaroxan . Rauwolscine Insulin resistance in humans commonly occurs along with hypertension and hyperlipidemia, a cluster of conditions known as metabolic syndrome X (Reaven 1988). Antihypertensive agents differ in their impact on insulin resistance and circulating lipids. We recently reported that certain centrally-acting antihypertensives active at the I1-imidazoline receptor (I1R) reduce insulin resistance, improve glucose tolerance, facilitate insulin secretion, reduce glucagon secretion and potentiate insulin signaling (Ernsberger et al. 1996, 1997; Friedman et al. 1998; Ernsberger et al. 1999a; Velliquette and Ernsberger 2003a,b). We carried these studies out in a unique animal model of metabolic Syndrome X, the obese spontaneously-hypertensive rat (SHROB; Koletsky rat), which expresses genetic obesity superimposed on a background of genetic hypertension resulting in extreme hyperlipidemia, hyperinsulinemia, and glucose intolerance (Ernsberger et al. 1999b). Although these agents also activate α2-adrenergic receptors (α2AR) in addition to I1R, stimulation of α2AR actually impairs glucose tolerance and insulin secretion (Velliquette and Ernsberger 2003b). Related imidazoline compounds have also been reported to promote glucose-stimulated insulin secretion (Zaitsev et al. 1996; Chan et al. 2001; Efendic et al. 2002). Long-term treatment with agents active on I1R affects another major component of metabolic Syndrome X, hyperlipidemia. Thus, moxonidine at 8 mg/kg/d for 90 d reduced triglycerides and cholesterol in hyperlipidemic SHROB, but had no effect in lean SHR littermates (Ernsberger et al. 1996). Moxonidine normalized elevated plasma triglycerides induced by fructose feeding (Rosen et al. 1997) and similar results have been obtained with another I1R agonist, rilmenidine (Penicaud et al. 1998). Lipid-lowering efficacy was equivalent with a lower dose (4 mg/kg/d) and a shorter treatment period (15d) (Velliquette and Ernsberger 2003a). Free fatty acids were 301 reduced by chronic moxonidine in SHROB and in SHR (Ernsberger et al. 1999a; Velliquette and Ernsberger, 2003a) as well as in Zucker fatty rats (Yakubu-Madus et al. 1999). The selective I1R agonists rilmenidine and moxonidine have been in clinical use for hypertension in Europe since 1989 and 1992 respectively (Ernsberger et al. 1997). Clinical trial data indicate that long-term treatment with I1R agonists reduces circulating triglycerides or cholesterol in hyperlipidemic humans (Yakubu-Madus et al. 1999; Haenni and Lithell 1999; De Luca et al. 2000; Jacob et al. 2004; Lumb et al. 2004). Rilmenidine has recently been reported to raise HDL cholesterol (Anichkov et al. 2005). Thus far, the mechanism for this apparent lipid-lowering action has not been characterized in the laboratory, nor have the cellular receptor or target organs been identified. All currently-available I1R agonists also activate α2AR. Several groups have claimed that all of the actions of moxonidine and rilmenidine can be entirely accounted for by their activity at α2AR (Zhu et al. 1999; Szabo 2002; Tan et al. 2002). Both I1R and α2AR mediate falls in blood pressure through inhibition of the sympathetic nervous system, making separation of their actions difficult. In mice with inactivated α2AAR, moxonidine’s antihypertensive action following intravenous injection was reduced (Zhu et al. 1999), whereas when moxonidine was directly administered into the brainstem in these same mice the antihypertensive action was completely preserved (Tolentino-Silva et al. 2000). Interpretation of the results is complicated by the fact that α2AR in peripheral blood vessels mediate a rise in blood pressure, while central nervous system α2AR mediate the opposite effect (Sawyer et al. 1985). In the present study, we compared changes in lipid metabolism following activation by I1R and α2AR. As we have recently shown for glucose metabolism, I1R and α2AR can mediate opposing effects, allowing clear separation of the contribution of the two receptors (Velliquette and Ernsberger 2003b). Materials and Methods Materials Rilmenidine, guanabenz, efaroxan, rauwolscine, oxymetazoline, epinephrine, naphazoline, oxymetazoline, phentolamine and tyloxapol were obtained from Sigma-Aldrich (St. Louis, MO, USA). Moxonidine free base was provided by Solvay Pharmaceutical (Hannover, Germany). [125I]pIodoclonidine was obtained from Perkin-Elmer (Boston, MA, USA). 2H2O (99.9 atom% excess) was purchased from Isotec (Miamisburg, OH, USA). Gas chromatography–mass spectrometry supplies were purchased from Agilent Technologies (Wilmington, DE, USA). Animals Adult male and female obese spontaneously-hypertensive rats (SHROB) were obtained from a closed colony that has been continuously inbred since 1973 (Ernsberger et al. 1999b). Because both male and female SHROB are sterile, the strain is propagated by mating lean heterozygous carriers of the mutant fak allele. No sex differences were noted in any experimental parameter, consistent with previous results (Ernsberger et al. 1999b). Animals were not used in any other experiments, were housed individually and were provided food (Teklad formula 8664; Teklad, Madison WI) and water ad libitum. Animals were on a 12:12-h light–dark cycle (lights on from 7:00 to 19:00) and were maintained at a constant temperature of 21°C. These procedures were carried out with the approval of the Case Western Reserve University Animal Care and Use Committee. Hepatic plasma membrane isolation Preliminary data showed a large amount of nonspecific binding of [125I]p-iodoclonidine in crude total particulate fractions from rat liver (not shown). Therefore, a plasma membrane fraction was isolated for radioligand binding assays, by using a modification of previous methods (Separovic et al. 1996). All steps were carried out on ice or at 4°C. Rat liver (2 g wet weight) was homogenized by using a polytron (Ultra-Turrax T25, IKA, Wilmington, NC) at setting 3 in 8 ml of 0.32 M sucrose containing a cocktail of protease inhibitors (Boehringer Mannheim), and buffered to pH 7.4 with 25 mM HEPES and Tris base. The homogenate was layered on a sucrose density gradient consisting of 7 ml 0.85 M sucrose and 7 ml 1.2 M sucrose. The gradient was centrifuged at 100,000× g for 2 h, and the interface fraction between 0.85 M and 1.2 M sucrose was collected and diluted to 20 ml in 50 mM Tris-HCl buffer, pH 7.7 containing 5.0 mM EDTA. The diluted plasma membrane fraction was centrifuged at 100,000× g for 1 h, and the pellet was flash frozen for later use in radioligand binding assays. [125I]p-Iodoclonidine binding assays Radioligand binding assays with [125I]p-iodoclonidine were performed in 96-well plates (Beckman Macrowell) as previously described (Ernsberger et al. 1995) using either liver plasma membranes as described above, or rat PC12 pheochromocytoma cell membranes prepared as previously described (Separovic et al. 1996). Membranes were slowly thawed and resuspended in Tris-HEPES buffer (5.0 mM; pH 7.7 at 25°C, containing 0.5 mM EDTA, 0.5 mM EGTA, and 0.5 mM MgCl2) at a concentration of about 0.1 mg protein/ml. Incubations were initiated by the addition of membrane to the well and were carried out for 30 min at 22°C. Nonspecific binding was defined in the presence of 10 μM oxymetazoline. Incubations were stopped by vacuum filtration using a cell harvester (Brandel M18) connected to a vacuum pump rated at 120 l/min (Edwards EM8). Samples were filtered over sheets of glass-fiber filter paper (Schleicher & Schuell #34), which were preincubated 302 for 4 h at 4°C in 0.03% polylethyleneimine to reduce nonspecific binding. Each sample well was washed four times with 4 ml ice-cold assay buffer, an operation completed in less than 12 s. Individual filters were placed in glass 12×75 mm tubes, and counted at 80% efficiency (Beckman). Acute drug treatment All drugs that were tested in vivo were dissolved in 20% DMSO and then diluted with 0.9% saline to the needed concentration. The final concentration of DMSO was <2%. Control animals received vehicle of identical composition. All drugs were administered ip after an 18-h overnight fast. In blocking experiments, antagonists were mixed together with agonists and administered as one bolus. Initial doses for moxonidine, rilmenidine, guanabenz, efaroxan and rauwolscine were 0.5, 2.5, 0.3, 0.6 and 7.5 mg/kg respectively. Initial doses for moxonidine, rilmenidine and guanabenz were set based on equal hypotensive effects (Ernsberger et al. 1997). The dose of rauwolscine was that sufficient to block the fall in insulin elicited by α2AR agonists, and the dose of efaroxan was that sufficient to block the fall in plasma glucagon levels elicited by moxonidine (Velliquette and Ernsberger 2003b). Dose response studies were examined for moxonidine (0.5, 1.0, 2.0 and 4.0 mg/kg) and guanabenz (0.03, 0.1, 0.3 and 1.0 mg/kg). Tail blood (200 μL) was taken at baseline and at 1, 2 and 4 h after ip injection. In a separate group of rats matched for age and sex, blood pressure and heart rate responses to agonists and antagonists were tested using a tail-cuff method as previous described (Ernsberger et al. 1997). After three sessions of habituation to the test procedure on separate days, blood pressures were measured after an 18-h overnight fast, then drug treatments were administered ip as above. Blood pressure and heart rate were measured again at 1 h and 2 h post-injection. In the case of guanabenz, blood pressure responses only appeared after 4 h, so this is the timepoint that was used. The delayed response to guanabenz presumably reflects slower penetration into the brain (Meacham et al. 1980). To examine whether imidazolines had direct effects on the liver, SHROB were anesthetized with ketamine (100 mg/kg) and acepromazine (5 mg/kg) and the liver was exposed by a midline incision. Injections of moxonidine (0.5 mg/kg) and guanabenz (0.3 mg/kg) were made with a 30-gauge needle directly into the portal vein. Tail blood samples (0.2 mL) were taken at baseline, and 10 and 60 min after injection. Plasma triglyceride and total cholesterol were measured at each time point. Triglyceride secretion study Tyloxapol (Triton WR 1339) is a detergent that inhibits the activity of lipoprotein lipase (Swift et al. 2001). Tyloxapol thus blocks the catabolism of circulating triglyceride-rich lipoproteins and promotes their accumulation in the plasma over time, which permits estimation of the rate of triglyceride secretion. Tyloxapol was dissolved at a concentration of 150 mg/ml in reagent grade water warmed to 37°C and injected into the tail vein at a dose of 500 mg/kg 20 min prior to ip injection of vehicle or moxonidine. Tail blood (100 μL) was obtained at baseline and at 30, 60, 120 and 180 min after the ip injection. Plasma triglyceride concentrations were determined at each time point, and the slope of the rise in plasma triglycerides for each animal was used to determine its triglyceride secretion rate. Animals were sacrificed after collection of the last blood sample and not used for any subsequent studies. De novo lipogenesis study In order to determine whether lipogenesis was affected by moxonidine treatment, we used 2H2O as a probe to follow newly-synthesized lipids. 2H2O was administered ip at 25 ml/kg body weight to 18 h-fasted SHROB rats. The 2H label rapidly equilibrates with total body water. Body water is then incorporated into newly-synthesized fatty acids, which are included within newly-synthesized triglycerides and secreted into the plasma. Since the labeling of body water with 2H2O is constant, the rate of the accumulation of 2 H label in triglyceride-bound palmitate quantitatively reflects the contribution of de novo lipogenesis to triglyceride synthesis. Moxonidine (1 mg/kg) or vehicle was given ip 90 min after 2H2O administration, and tail blood (200μL) was sampled at baseline and at 40, 80 and 120 min after drug injection. Plasma samples were analyzed for the incorporation of 2H into plasma water and triglyceride palmitate. The 2H-labeling of body water was determined by exchange with acetone as described (Brunengraber et al. 2003). The 2H-labeling of acetone was determined using an Agilent 5,973N-MSD equipped with an Agilent 6,890 GC system. A DB17-MS capillary column (30 m×0.25 mm× 0.25 μm) was used in all analyses. The temperature program was: 60°C initial, increase by 20°C/min to 100°C, increase by 50°C/min to 220°C and hold for 1 min. The split ratio was 40:1 with a helium flow of 1 ml/min. Acetone elutes at ∼1.5 min. The mass spectrometer was operated in the electron impact mode. Selective ion monitoring of m/z 58 to 60 was performed using a dwell time of 10 ms per ion. To assay 2H-labeling of triglyceride-bound palmitate, a known quantity of plasma was hydrolyzed and extracted. Fatty acids were derivatized using diazomethane. Briefly, diazomethane was prepared and used as previously described (Brunengraber et al. 2002). Fatty-acid methyl esters were formed by dissolving the extracted fatty acids in 50 μl of methanol and adding ∼300 μl of etherdiazomethane. The sample was allowed to react at room temperature for 45 min. Excess solvent was removed by evaporation to dryness. The fatty-acid methyl esters were then dissolved in 100 μl of chloroform and analyzed by gas chromatography with electron impact ionization mass 303 spectrometry. The 2H-enrichment of palmitate was determined by monitoring m/z 270 and 271. Biochemical measurements Blood samples were immediately placed on ice, centrifuged for 20 min at 5,000 g at 4°C and plasma frozen at -20°C until assayed for plasma triglyceride, total cholesterol and free fatty acids. Plasma triglycerides and total cholesterol were determined by colorimetric, enzymatic assay (Sigma-Aldrich, St. Louis, MO, USA). Plasma free fatty acids were determined by an enzymatic kit (Wako Chemicals, Richmond, VA, USA). All assays were conducted in duplicate. Intra-assay coefficient of variation was 2.5% for total cholesterol, 2.4% for triglyceride and 4.8% for free fatty acids. The interassay variation was 5% for total cholesterol, 3.7% for triglyceride and 7.2% for free fatty acids. Statistical methods Results are presented as means ± standard error of the mean. Comparisons between groups were made using oneor two-way analysis of variance (ANOVA) by time and dose, or analysis of variance with repeated measures (REMANOVA) using Prism (Graph Pad Software, San Fig. 1 Effects of acute ip moxonidine and rilmenidine on plasma lipid levels in 18 h-fasted SHROB. Data represent the mean and standard error of the net change from baseline for six animals per condition. a Plasma triglyceride levels. b Plasma total cholesterol levels. c Plasma free fatty acids concentration. Moxonidine was given at 0.5 mg/kg and rilmenidine at 2.5 mg/kg. Moxonidine and rilmenidine both significantly reduced plasma triglycerides and total cholesterol (P<0.001). For plasma free fatty acids, rats treated with Diego, CA, USA) with post-hoc analyses by NeumanKeuls test. A significant effect of drug dosage was defined as a significant contribution in the analysis of variance (P< 0.05 by F-test). Unpaired t-test with Welch-correction was used to compare two means. Results Agonist effects on plasma lipids Both moxonidine and rilmenidine significantly decreased plasma triglycerides following acute ip administration to 18 h-fasted SHROB rats (Fig. 1a). A rapid reduction in plasma total cholesterol concentration was also observed (Fig. 1b). In contrast, neither moxonidine nor rilmenidine had an effect on plasma free fatty acids relative to vehicletreated (Fig. 1c). All three groups showed a tendency toward gradual elevation of free fatty acid levels with continued fasting. Moxonidine was administered ip to lean normolipidemic SHR at low (0.5 mg/kg) and high (2.0 mg/kg) doses. Neither dose had any significant effects on plasma triglycerides, total cholesterol, or free fatty acids. As a representative example, triglycerides were completely unchanged 4 h after the high dose (84.6±9.3 versus 82.1± 9.8 mg/dL, N=6, P>0.50). Thus, the lipid-lowering action moxonidine or rilmenidine did not differ from vehicle-treated controls, which tended to increase over time with continued fasting. Baseline values were (a) 476±71, 475±54 and 520±38 mg/dL; (b) 225±38, 195±37, 195±58 mg/dL; and (c) 1.9±0.1, 1.7±0.1 and 1.8± 0.1 mM for vehicle, moxonidine, and rilmenidine groups respectively. Baseline values did not differ between groups. *P<0.05; **P<0.01 304 of moxonidine is only observed in the hyperlipidemic SHROB rats and not in the normolipidemic SHR. A significant dose-dependent reduction in plasma triglyceride concentrations was observed after acute ip moxonidine treatment (Fig. 2a). A significant dose–time interaction was observed in that the duration of the response increased with dose (2-way ANOVA, P<0.0001). The time course of drug action is consistent with the 2 h plasma half-life of moxonidine in rats (He et al. 2000). For plasma total cholesterol, a biphasic dose response relationship was observed, with low doses decreasing and high doses increasing cholesterol levels (Fig. 2b). This result Fig. 2 Dose response relationship for the effects of acute ip moxonidine on (a) plasma triglyceride levels and (b) plasma total cholesterol in 18 h-fasted SHROB, and effects of selective antagonists on the response to 0.5 mg/kg moxonidine (c,d). Data represent the mean and standard error of the net change from baseline for six animals per dose. Moxonidine reduced plasma triglyceride levels in a doseand time-dependent manner (P<0.001). Moxonidine’s effects on cholesterol levels were biphasic depending on dose. The addition of rauwolscine (7.5 mg/ kg) potentiated the peak response of plasma triglycerides to moxonidine, whilst efaroxan (0.6 mg/kg) blocked the response. The addition of efaroxan but not rauwolscine blocked the reduction in total cholesterol levels. Baseline values were (a) 475±54, 349±33, 440±62 and 481±68 mg/dL for 0.5, 1, 2, and 4 mg/kg groups respectively; (b) 195±37, 224±62, 314±68 and 294±70 mg/dL for 0.5, 1, 2, and 4 mg/kg groups respectively; (c) 475±54, 579± 71 and 605±75 mg/dL for the moxonidine, moxonidine plus rauwolscine and moxonidine plus efaroxan groups respectively; and (d) 195±37, 112±40 and 117±18 mg/dL for the moxonidine, moxonidine plus rauwolscine and moxonidine plus efaroxan groups respectively. Baseline values did not differ between groups. *P<0.05; **P<0.01, ***P<0.001 implies two opposing processes, one predominating at low doses and another that is increasingly apparent at higher doses. Conceivably, these opposing processes might be mediated by different receptors. To further evaluate the role of sympathoinhibition and of α2AR in plasma lipid responses, we tested guanabenz, a selective α2AR agonist structurally similar to moxonidine but lacking an imidazoline ring. Guanabenz increased plasma triglyceride concentrations at all doses tested at 4 h (Fig. 3a; P<0.01). No significant dose response relationship was observed for plasma total cholesterol after acute ip guanabenz (Fig. 3b). However, cholesterol did rise over time 305 Fig. 3 Dose response relationship for the effects of acute ip guanabenz (GBZ) on (a) plasma triglyceride levels and (b) plasma total cholesterol in 18 h-fasted SHROB; and (c) blockade of the action of guanabenz by rauwolscine (7.5 mg/kg). Data represent the mean and standard error for six animals per dose. Numbers in parentheses in inset are number of separate trials. Guanabenz significantly elevated plasma triglycerides at all doses tested at 4 h. The overall effect on cholesterol was also a significant increase. Dose-dependence could be demonstrated for the triglyceride changes, but not for cholesterol. Baseline values were: (a) 581± 61, 406±53, 455±29 and 495±60 mg/dL for 0.03, 0.1, 0.3, and 1.0 mg/kg groups respectively; and (b) 177±16, 135±14, 134±15 and 254±53 mg/dL for 0.03, 0.1, 0.3, and 1.0 mg/kg groups respectively. Baseline values did not differ between groups. *P<0.05; **P<0.01, ***P<0.001 in the guanabenz-treated groups (P=0.006, two-way ANOVA). Thus, guanabenz did not share the lipid-lowering actions of moxonidine and rilmenidine, even though all three agents are potent sympatholytic agents and α2AR agonists. Effects of selective antagonists To further establish the receptor mechanisms responsible for the effects of moxonidine, we tested the effects of selective antagonists. The selective α2AR antagonist Fig. 4 Effect of selective antagonists administered alone on triglycerides (a), cholesterol (b), and free fatty acids (c). Baseline values did not differ between groups. No significant effects of any treatment were detected (P<0.05), but free fatty acids increased over time. *P<0.05 306 rauwolscine (7.5 mg/kg) or the dual I1R and α2AR antagonist efaroxan (0.6 mg/kg) were given with moxonidine (0.5 mg/kg). These doses were selected on the basis of previous studies showing rauwolscine blockade of α2ARmediated hyperglycemic responses and efaroxan blockade of I1R-mediated decrease in plasma glucagon (Velliquette and Ernsberger 2003b). The moxonidine-induced reduction in plasma triglyceride concentrations was blocked by efaroxan (P<0.001), whilst the addition of rauwolscine potentiated the effect of moxonidine on plasma triglyceride concentrations (Fig. 2c; p<0.001). The moxonidine-induced reduction in plasma total cholesterol concentrations was also blocked by the addition of efaroxan (P<0.001) but not by rauwolscine (Fig. 2d). These results implicate I1R in the lipid-lowering actions of acute ip moxonidine in the SHROB model. The apparent action of 0.1 mg/kg guanabenz to raise triglycerides at 4 h after injection could not be blocked by rauwolscine (vehicle: 455±29 mg/dl (N=24); 0.1 mg/kg guanabenz: 606±58 (N=14); and 0.1 mg/kg guanabenz plus 7.5 mg/kg rauwolscine: 594±49 (N=22)). Thus, any possible effect of guanabenz on triglycerides does not appear to be mediated by α2AR and may be nonspecific. The ability of 0.1 mg/kg guanabenz to raise cholesterol, in contrast, was antagonized by rauwolscine in the same group of rats, as shown in Fig. 3, implying that guanabenz may have a modest acute cholesterol-raising action mediated by α2AR. Figure 4 shows the effects of the antagonists given alone on plasma lipids. There were no significant effects on any parameter. Thus, the effects observed represent true antagonism and not counteraction. Free fatty acids increased over time in all groups, including vehicle controls, presumably as a result of continued fasting. Blood pressure responses In order to test the hypothesis that changes in circulating lipids were mediated by hemodynamic changes, we measured blood pressure and heart rate by tail cuff in a separate group of SHROB trained to the injection and blood pressure measuring procedure, and treated with agonist and antagonist agents in a manner identical to the previous group. Baseline systolic blood pressures were 196.0±0.84 mmHg (N=108 trials) and did not differ between treatment groups (P>0.50). As shown in Fig. 5, moxonidine (0.5 mg/kg) and rilmenidine (2.5 mg/kg) elicited the expected antihypertensive action at 1 h and 2 h after injection. Co-administration of the dual I1R and α2AR antagonist efaroxan (0.6 mg/kg) not only completely blocked the action of moxonidine but inverted the response into a pressor action. The selective α2AR antagonist rauwolscine (7.5 mg/kg) did not alter the response to 3 Fig. 5 Effect of agonists and antagonists on systolic blood pressure. Separate groups of SHROB not subjected to blood sampling were administered drugs at the identical doses described in Figs. 1, 2, 3, 4. Change in blood pressure is shown at 1 h (a), 2 h (b), and 4 h (c). Guanabenz had no significant effect at 1 h or 2 h, so only 4 h data are shown. Columns which do not share a letter label are significantly different (P< 0.05, 2-way ANOVA by repeated measures and Newman-Keuls test) 307 %Total Specific Binding 100 Rilmenidine 50 Efaroxan Oxymetazoline Naphazoline Epinephrine 0 0 -9 -8 -7 -6 -5 log[Drug] Fig. 6 Competition binding with [125I]p-iodoclonidine in rat liver plasma membranes. Each data point represents the mean and standard error of four to six experiments conducted in triplicate or quadruplicate. Competition curves represent the best fit to a singlecomponent logistic equation. Two-site models did not yield a better fit. Liver plasma membranes isolated by sucrose density gradient were incubated with 0.5 nM [125I]p-iodoclonidine and increasing concentrations of competing drug. Nonspecific binding was defined in the presence of 10 μM oxymetazoline moxonidine, confirming previous findings that α2AR do not contribute to the action of moxonidine (Ernsberger et al. 1997; Tolentino-Silva et al. 2000). Efaroxan given by itself had no effect on blood pressure, but rauwolscine Fig. 7 The effect of portal vein administration of moxonidine (0.5 mg/kg) or guanabenz (0.3 mg/kg) on plasma triglyceride levels (a) and net change in plasma triglyceride levels after acute iv tyloxapol and ip moxonidine or vehicle in 18 h fasted SHROB (b,c). Data represent the mean and standard error for 6 animals per group. a Moxonidine but not guanabenz reduced plasma triglycerides, an action which was fully apparent 10 min post injection. This rapid effect, reproducing the effect of higher ip doses, suggests a direct hepatic action. b Moxonidine significantly reduced the tyloxapol- elicited a large depressor response, probably reflecting blockade of vascular α2AR (Sawyer et al. 1985). Guanabenz is shown separately as it had no significant effect on blood pressure at 1 h or 2 h (data not shown), as expected on the basis of slower brain penetration of this drug. At 4 h, a large fall in pressure was seen (Fig. 5c). Both α2AR antagonists, efaroxan and rauwolscine, antagonized the antihypertensive effect. Baseline heart rates in the conscious restrained SHROB were 400.2±2.9 bpm (N=108 trials) and did not differ between treatment groups (P>0.50). No significant change in heart rate was induced by vehicle, moxonidine, guanabenz or efaroxan (data not shown). Rilmenidine decreased heart rate (fall of 48±11 bpm at 1 h; fall of 42±7 bpm at 2 h), while rauwolscine increased it (rise of 33±5 bpm at 1 h; rise of 41±6 bpm at 2 h). The effect on heart rate of moxonidine and rauwolscine in combination did not differ from that of rauwolscine alone (rise of 36±6 bpm at 1 h; rise of 37±5 bpm at 2 h). Characterization of I1-imidazoline binding sites in liver plasma membrane Figure 6 shows the results of competition binding assays carried out to characterize I1-imidazoline binding in liver plasma membrane. Each of the compounds tested showed induced triglyceride accumulation, implying that moxonidine inhibits triglyceride secretion. c Triglyceride secretion rate after ip moxonidine or vehicle treatment in 18 h-fasted SHROB. Moxonidine inhibited hepatic triglyceride secretion by 75%. Baseline values were: (a) 379±82 and 378±38 mg/dL for the moxonidine and guanabenz groups, respectively, and 389±66 and 284±12 mg/dL for the moxonidine and vehicle and groups, respectively. Baseline values did not differ between groups. *** P<0.001 308 Since binding studies indicated that I1R are present in rat liver plasma membrane, we hypothesized that the lipidlowering actions of I1R agonists may be reproduced by direct administration into the liver. Intraportal injection of moxonidine significantly reduced plasma triglyceride conTable 1 Rank-ordered affinities of various compounds at rat liver plasma membrane I1R binding sites Compound Rilmenidine Efaroxan Phentolamine Oxymetazoline Guanabenz Naphazoline Epinephrine Rat Liver Plasma Membranes Ki in nM ± SE 14.3±1.7 20.4±2.7 82±12 154±18 260±42 610±48 14,400±1,300 Rat PC12 Cells Ki in nM ± SE 19.1±2.5 20.8±1.6 64±8 26.6±3.2 140±68 523±85 >100,000 Values are in nM ± the standard error of the estimate derived from analysis of the averaged results of four to six experiments, each carried out in triplicate or in quadruplicate. Competition curves were analyzed by using nonlinear curve fitting to a logistic equation (Prism, GraphPAD Software) Action of moxonidine on hepatic triglyceride secretion To test whether hepatic triglyceride secretion or peripheral degradation were affected by acute ip moxonidine treatment, lipoprotein lipase was inhibited with intravenous tyloxapol prior to acute ip vehicle or moxonidine treatment. Results showed the expected progressive rise in plasma triglyceride concentration over time in SHROB given vehicle after tyloxapol (Fig. 7b). The increase in plasma triglyceride concentrations induced by tyloxapol was significantly attenuated by moxonidine treatment (P<0.001). The secretion rate of plasma triglyceride was estimated using plasma volumes measured by plasma 2H2O dilution. Plasma triglyceride secretion rate after moxonidine treatment was reduced by 75% (0.22±0.03 versus 0.88± 0.15 mg/min in vehicle-treated control) (Fig. 7c, inset). 125 H-Palmitate in Plasma Lipid, %Change Administration of agonist into the hepatic portal circulation centrations within 10 min post-injection, and this effect was maintained at 60 min (Fig. 7a). This response was not reproduced by intraportal injection of the selective α2AR agonist guanabenz. Surprisingly, total cholesterol concentration was not affected by intraportal moxonidine injection (data not shown), implying that the action of moxonidine on plasma cholesterol levels may occur at a site outside the liver. Vehicle (slope = 0.81±0.11) 100 Moxonidine (slope = -0.48±0.10) 75 50 * 25 *** *** 0 -25 2 dose-dependent inhibition of specific [125I]p-iodoclonidine binding that was best fit by a single-site competitive binding model. Rilmenidine was the most potent, followed closely by efaroxan. Epinephrine produced very little inhibition even at very high concentrations, implying a lack of α2AR binding sites. Affinity values are given in rank order in Table 1. For comparison, competition studies with these agents were also carried in membrane fractions from a common cellular model system rat pheochromocytoma cells (PC12 cells). Affinity values did not differ between PC12 cell and rat liver plasma membrane, with the exception of oxymetazoline, which was about five-fold less potent in liver than in PC12 cells. Not shown are the results of competition experiments with moxonidine, which gave inconsistent and partial inhibition of specific binding. We hypothesize that the inconsistent activity of moxonidine in these preparations is due to the rapid metabolism of moxonidine by rat liver (He et al. 2000), presumably by microsomal enzymes expected to be present in plasma membrane fractions. (Interestingly, moxonidine is not metabolized in the human liver, but is primarily excreted unchanged in the urine (Weimann and Rudolph, 1992)). Rilmenidine is not metabolized by rat liver, which may explain its high relative potency in this preparation. The affinity of guanabenz is higher than that previously reported in bovine brainstem, but is closer to the value obtained in human platelet plasma membranes (Piletz et al. 1996). Thus, the hepatic [125I]p-iodoclonidine binding site closely resembles the I1R characterized in other target tissues. -50 -75 0 30 60 90 Time, min 120 Fig. 8 Illustrates the percent change in labeling of triglyceride palmitate after vehicle or moxonidine (1 mg/kg) treatment in 18 hfasted SHROB (N=4 rats per group). Moxonidine significantly reduced hepatic de novo synthesis of palmitate incorporated into plasma triglycerides, *P<0.05; ***P<0.001. Baseline values were 2.09±0.65 and 2.05±0.46% deuterium enrichment for the moxonidine and vehicle groups respectively. Baseline values did not differ between groups 309 Effect of moxonidine on de novo synthesis of plasma triglyceride fatty acids Since the studies with tyloxapol indicated reduced secretion of plasma triglycerides from the liver, we investigated the role of de novo lipid synthesis in the mechanism of action of moxonidine. Fig. 8 illustrates the percent change in basal lipogenesis as measured by using 2H2O incorporation into plasma triglyceride palmitate. Acute ip moxonidine significantly reduced basal lipogenesis in fasted SHROB compared to vehicle treated controls (P< 0.001). Discussion Acute administration of sympatholytic agents active at I1R and α2AR reduced plasma triglycerides and cholesterol in a hyperlipidemic animal model. These actions were mediated by the I1R, since they were not mimicked by guanabenz, a sympatholytic agent selective for α2AR, nor were they blocked by the selective α2AR antagonist rauwolscine. Furthermore, the lipid-lowering actions of moxonidine were not blocked but slightly potentiated by rauwolscine. In contrast, the dual I1R and α2AR antagonist efaroxan completely blocked these effects. Overactivity of the sympathetic nervous system may contribute to hyperlipidemia in hypertension, and thus sympatholytic agents should reduce circulating lipids (Ernsberger et al. 1998). Unexpectedly, in the present study we found that guanabenz, a centrally-acting sympatholytic agent, failed to reduce plasma lipids. In fact, guanabenz tended to raise plasma cholesterol and triglycerides. However, the lipid-raising effects were modest and were dose-dependent only for triglycerides, and were reversible by an α2AR antagonist only in the case of cholesterol. Moreover, moxonidine had no lipid-lowering effect in normolipidemic SHR. Thus, the ability of agents to lower plasma triglycerides does not correlate with their ability to inhibit sympathetic activity. Whilst chronic treatment with moxonidine lowered triglycerides, cholesterol and FFA, the non-imidazoline sympatholytic agent, α-methyldopa, was inactive despite equivalent bloodpressure lowering (Velliquette and Ernsberger 2003a). Thus sympathoinhibition alone cannot account for the actions of this class of drugs on lipid metabolism. It might be argued that the lipid-lowering effect produced by moxonidine was the result of a reduction in blood pressure. Many antihypertensives, such as angiotensin-converting enzyme inhibitors, have a slight lipidlowering effect (Brook 2000; Lindholm et al. 2003). However, other antihypertensives such as β-AR antagonists and thiazide diuretics may actually raise plasma lipid levels. Moreover, in the present study the antihypertensive agent guanabenz failed to lower triglyceride and cholesterol levels. Indeed, in considering the effects of agonists and antagonists alone and in combination on blood pressure and on plasma lipids, there exists no clear correlation between hemodynamic actions and metabolic responses. Having identified the I1R as a potential target for the lipid-lowering action of imidazolines, the question then arose as to the site of action. The antihypertensive actions of these agents are mediated within the brainstem (Ernsberger et al. 1997). However, both I1R and α2AR mediate similar reductions in sympathetic outflow via a brainstem action, whereas these receptors elicit opposing actions on plasma lipids. Given the central role of the liver in controlling levels of plasma lipids, and the recent discovery within this organ of mRNA for a gene candidate for the I1R (Piletz et al. 1999), we investigated a hepatic site of action. Radioligand binding studies confirmed the presence of high-affinity I1R binding sites in plasma membrane fractions from rat liver. Moreover, binding affinities closely agreed with those obtained in PC12 cells, an established cell model for I1R. The affinity values for rat liver plasma membrane are also in good overall agreement with past studies of I1R binding affinity in bovine brainstem and canine prostate, among others. As expected on the basis of prior literature showing that liver α2AR are confined to Kupfer cells (Zonnenchein et al. 1990; Zhou et al. 2001; Cussac et al. 2001), α2AR could not be detected in plasma membrane fractions. To further evaluate a hepatic site of action, moxonidine was directly injected into the portal vein. A maximal triglyceride response was elicited within 10 min and sustained for at least 60 min. Guanabenz, despite its potency at α2AR and at I2-imidazoline binding sites, had no effect. These results are consistent with a hepatic site of action for moxonidine’s triglyceride-lowering effect. In the fasted state, the liver controls plasma triglycerides primarily by regulating their secretion into the circulation. Thus, the rate of triglyceride secretion was estimated by blocking triglyceride breakdown with tyloxapol, an inhibitor of lipoprotein lipase. Following tyloxapol administration, the rate of triglyceride accumulation in the plasma was profoundly inhibited by moxonidine treatment. These data suggest that the primary action of moxonidine is not on triglyceride breakdown by lipoprotein lipase, since its effects persisted following inhibition of this enzyme. Instead, moxonidine appears to inhibit the production and secretion of triglycerides by the liver. One source of fatty acids for plasma triglycerides is newlysynthesized fatty acids created by lipogenesis locally within the liver, or in adipocytes followed by transport to the liver. Our studies were done in fasted animals with low rates of lipogenesis. Nonetheless, a detectable fraction of the palmitate present in plasma triglycerides was derived from de novo synthesis, as shown by the incorporation of label from body water spiked with deuterium. In control animals, labeled palmitate increased in abundance linearly with time. In contrast, the labeled fraction of palmitate in plasma triglyceride actually fell in moxonidine-treated animals. One explanation for this phenomenon might be an increased supply of unlabeled palmitate liberated from adipocytes. However, moxonidine treatment had no acute effect on plasma free fatty acids (see Fig. 1c). Thus, moxonidine may be a potent inhibitor of hepatic lipogenesis. 310 The acute actions of I1R agonists reported here are mainly consistent with chronic effects reported previously. Thus, treatment with moxonidine and rilmenidine for a period of weeks has been reported to reduce plasma triglyceride and cholesterol in hyperlipidemic animals (Ernsberger et al. 1996, 1997; Henriksen et al. 1997; Penicaud et al. 1998; Ernsberger et al. 1999a). Chronic treatment with either rilmenidine or moxonidine reduced plasma triglyceride concentrations in the high-fructose-fed animal model of hyperlipidemia (Henriksen et al. 1997; Penicaud et al. 1998). The primary mechanism of hyperlipidemia in the fructose-fed animal is increased lipogenesis, and thus the lipid-lowering effect of I1R agonists in this model is consistent with the present data showing that moxonidine is an inhibitor of de novo lipogenesis. However, not all of the chronic effects are reproduced acutely. Notably, long-term treatment reduces plasma free fatty acids, whereas acute treatment does not. Thus, effects on free fatty acids may develop gradually, similar to the actions of imidazolines on insulin resistance which manifest over 2 weeks (Velliquette and Ernsberger 2003a). Do these findings apply to hyperlipidemic humans? Moxonidine accelerated cardiovascular events and mortality in congestive heart failure (Cohn et al. 2003), but the excess deaths occurred within the first few months and were probably independent of atherosclerotic progression. Instead, the high dose of moxonidine, ten times that used for control of hypertension, may have profoundly inhibited sympathetic activity, leading to a lethal drop in cardiac output. Few clinical trials have been carried out with imidazolines specifically on hyperlipidemic subjects. Our data indicate hypertensive animals with normal triglyceride and cholesterol levels show no lipid-lowering response to imidazolines, consistent with a lack of a similar effect in human essential hypertension. However, most trials show nonsignificant trends toward a decrease (Prichard and Graham 2000), and moxonidine in three studies (Haenni and Lithell, 1999; Anichkov and Shostak 2004; Lumb et al. 2004) and rilmenidine in several studies significantly reduced triglycerides and either total or LDL cholesterol, or reduced the overall atherogenic profile (U.K.Working Party on Rilmenidine, 1990). Guanabenz may slightly decrease plasma cholesterol in hypertensive humans (Ames 1986). These chronic effects were not reproduced acutely in the present study, suggesting that the lipid-lowering actions of this agent may develop over time. Remarkably, guanabenz has been shown to decrease the synthesis of both triglycerides and cholesterol in isolated rat liver cells (Capuzzi and Cevallos 1984). However, very high concentrations of guanabenz were required, and these effects may be nonspecific. Similarly, an I1R agonist was shown to reduce cholesterol synthesis in hepatocytes, but the IC50 of 8,000 nM was over 1,000-fold higher than the Ki at I1R (Venteclef et al. 2005). In the present study, doses of guanabenz sufficient to cause α2AR stimulation (Velliquette and Ernsberger 2003b) and to lower blood pressure have no cholesterollowering action, and even produce a significant increase which could be blocked by an α2AR antagonist. The liver has been studied as a model system for the I2imidazoline binding site (Zonnenchein et al. 1990; Remaury et al. 1999). These binding sites are expressed in mitochondria and are mainly present on the enzyme monoamine oxidase (Remaury et al. 1999). The density of hepatic I2-sites is about 100 times greater than that of I1R sites. In the present study, we isolated plasma membrane fractions to minimize contamination from mitochondria. This allowed the identification of I1R binding sites. Any contribution from I2-sites to the binding results can be excluded, because efaroxan lacks affinity for I2-sites yet showed high affinity for [125I]piodoclonidine binding sites. Furthermore, a functional contribution from I2-sites can be ruled out by the lack of effect of guanabenz, which has low nanomolar affinity for hepatic I2sites (Zonnenchein et al. 1990), and by the potent blockade of all responses by efaroxan. Although our data implicate the liver as a site of action for the lipid-lowering action of I1R agonists, we cannot rule out actions on other tissues, such as adipocytes. No effect of moxonidine or rilmenidine on lipolysis was detected in vivo, as free fatty acid levels did not change. The effects of imidazolines on lipolysis in isolated rat and human adipocytes has been studied extensively, and all the actions can be accounted for by activation of α2AR (Carpene et al. 1990; Carpene et al. 1995). We have recently confirmed— using SHROB adipocytes—the lack of any effect of I1R agonists on lipolysis following blockade of α2AR (data not shown). Nonetheless, it is conceivable that imidazolines may have insulin-sensitizing effects in adipocytes. Most of the established lipid-lowering agents have some degree of hepatic toxicity. Toxicity was not directly evaluated in this study, but liver enzymes have been studied extensively in humans treated with moxonidine and do not change (Schachter et al. 1998; Elliott 1998; Schachter 1999). Moreover, hepatotoxic effects have not been reported for any imidazoline at concentrations in the therapeutic range. Indeed, of the centrally-acting antihypertensive agents only alpha-methyldopa has been linked to occasional increases in liver enzymes, and this compound is an amino acid structurally unrelated to the imidazolines (Webster and Koch 1996). In fact, clonidine and moxonidine have been suggested for the treatment of portal hypertension in cirrhotic patients (Esler and Kaye 1998). At high doses, moxonidine behaves as a typical α2AR agonist with regard to sedative actions (Ernsberger et al. 1997), central respiratory depression (Haxhiu et al. 1995), stimulation of glucagon secretion (Velliquette and Ernsberger, 2003b), and inhibition of insulin secretion (Velliquette and Ernsberger 2003b). In the present study, high doses of moxonidine increased plasma cholesterol similar to the effect of the selective α2AR agonist guanabenz. Thus, characterization of responses mediated by I1R requires careful titration of the dose of imidazoline agents to avoid the masking of I1R effects by opposing responses mediated by α2AR. Adrenergic-mediated control of lipid metabolism has been characterized for α1 and β-AR, whilst little acute in vivo information is available for α2AR-mediated lipid responses (Brook 2000). The elevation of plasma triglyce- 311 ride and cholesterol in response to α2AR agonists observed in the present study has not been previously reported. This may be because α2AR agonists were not tested in hyperlipidemic models. Hyperlipidemia and hypertension commonly coexist, and contribute synergistically to cardiovascular risk. Tandem reductions in these risk factors could significantly impact overall risk. Future development of specific I1R agents devoid of α2AR activity may have applications in metabolic disorders associated with hypertension. Acknowledgements We thank Anthony DiVito and Janean Johnson, B.S. for their technical assistance. Supported by HL44514 from the NIH and the Mount Sinai Health Care Foundation of Cleveland. Preliminary studies were partly supported by a grant from Solvay Pharmaceuticals, Hannover, Germany. Submitted in partial fulfillment of the requirements for a doctorate in Nutrition from Case Western Reserve University School of Medicine to R.A.V. References Ames RP (1986) The effects of antihypertensive drugs on serum lipids and lipoproteins. II. Non-diuretic drugs. Drugs 32:335– 357 Anichkov DA, Shostak NA (2004) Effect of moxonidine on parameters of lipid metabolism in patients with metabolic syndrome. Kardiologiia 44:13–15 Anichkov DA, Shostak NA, Schastnaya OV (2005) Comparison of rilmenidine and lisinopril on ambulatory blood pressure and plasma lipid and glucose levels in hypertensive women with metabolic syndrome. Curr Med Res Opin 21:113–119 Brook RD (2000) Mechanism of differential effects of antihypertensive agents on serum lipids. Curr Hypertens Rep 2:370–377 Brunengraber DZ, McCabe BJ, Kasumov T, Alexander JC, Chandramouli V, Previs SF (2003) Influence of diet on the modeling of adipose tissue triglycerides during growth. Am J Physiol Endocrinol Metab 285:E917–E925 Brunengraber DZ, McCabe BJ, Katanik J, Previs SF (2002) Gas chromatography-mass spectrometry assay of the (18)O enrichment of water as trimethyl phosphate. Anal Biochem 306:278– 282 Capuzzi DM, Cevallos WH (1984) Inhibition of hepatic cholesterol and triglyceride synthesis by guanabenz acetate. J Cardiovasc Pharmacol 6(Suppl 5):S847–S852 Carpene C, Galitzky J, Larrouy D, Langin D, Lafontan M (1990) Non-adrenergic sites for imidazolines are not directly involved in the alpha 2-adrenergic antilipolytic effect of UK 14304 in rat adipocytes. Biochem Pharmacol 40:437–445 Carpene C, Marti L, Hudson A, Lafontan M (1995) Nonadrenergic imidazoline binding sites and amine oxidase activities in fat cells. Ann N Y Acad Sci 763:380–397 Chan SL, Mourtada M, Morgan NG (2001) Characterization of a KATP channel-independent pathway involved in potentiation of insulin secretion by efaroxan. Diabetes 50:340–347 Cohn JN, Pfeffer MA, Rouleau J, Sharpe N, Swedberg K, Straub M, Wiltse C, Wright TJ (2003) Adverse mortality effect of central sympathetic inhibition with sustained-release moxonidine in patients with heart failure (MOXCON). Eur J Heart Fail 5:659– 667 Cussac D, Schaak S, Denis C, Flordellis C, Calise D, Paris H (2001) High level of alpha2-adrenoceptor in rat foetal liver and placenta is due to alpha2B-subtype expression in haematopoietic cells of the erythrocyte lineage. Br J Pharmacol 133:1387– 1395 De Luca N, Izzo R, Fontana D, Iovino G, Argenziano L, Vecchione C, Trimarco B (2000) Haemodynamic and metabolic effects of rilmenidine in hypertensive patients with metabolic syndrome X. A double-blind parallel study versus amlodipine. J Hypertens 18:1515–1522 Efendic S, Efanov AM, Berggren PO, Zaitsev SV (2002) Two generations of insulinotropic imidazoline compounds. Diabetes 51(Suppl 3):S448–S454 Elliott HL (1998) Moxonidine: pharmacology, clinical pharmacology and clinical profile. Blood Press Suppl 3:23–27 Ernsberger P, Friedman JE, Koletsky RJ (1997) The I(1)-imidazoline receptor: From binding site to therapeutic target in cardiovascular disease. J Hypertens 15(Suppl 1):S9–S23 Ernsberger P, Ishizuka T, Liu S, Farrell CJ, Bedol D, Koletsky RJ, Friedman JE (1999a) Mechanisms of antihyperglycemic effects of moxonidine in the obese spontaneously hypertensive Koletsky rat (SHROB). J Pharmacol Exp Ther 288:139–147 Ernsberger P, Koletsky RJ, Friedman JE (1999b) Molecular pathology in the obese spontaneous hypertensive Koletsky rat: a model of syndrome X. Ann N Y Acad Sci 892:272–288 Ernsberger P, Koletsky RJ, Collins LA, Bedol DL (1996) Sympathetic nervous system in salt-sensitive and obese hypertension: Amelioration of multiple abnormalities by a central symaptholytic agent. Cardiovasc Drugs Ther 10:275–282 Ernsberger P, Koletsky RJ, Friedman JE (1998) Contribution of sympathetic nervous system overactivity to cardiovascular and metabolic disease. Rev Contemp Pharmacother 9:411–428 Ernsberger P, Piletz JE, Graff LM, Graves ME (1995) Optimization of radioligand binding assays for I1-imidazoline sites. Ann N Y Acad Sci 763:163–168 Esler M, Kaye D (1998) Increased sympathetic nervous system activity and its therapeutic reduction in arterial hypertension, portal hypertension and heart failure. J Auton Nerv Syst 72:210–219 Friedman JE, Ishizuka T, Liu S, Farrell CJ, Koletsky RJ, Bedol D, Ernsberger P (1998) Anti-hyperglycemic activity of moxonidine: metabolic and molecular effects in obese spontaneously hypertensive rats. Blood Press Suppl 3:32–39 Haenni A, Lithell H (1999) Moxonidine improves insulin sensitivity in insulin-resistant hypertensives. J Hypertens 17(Suppl 3): S29–S35 Haxhiu MA, Dreshaj IA, Erokwu B, Collins LA, Ernsberger P (1995) Effect of I1-imidazoline receptor activation on responses of hypoglossal and phrenic nerve to chemical stimulation. Ann N Y Acad Sci 763:445–462 He MM, Abraham TL, Lindsay TJ, Chay SH, Czeskis BA, Shipley LA (2000) Metabolism and disposition of moxonidine in Fischer 344 rats. Drug Metab Dispos 28:446–459 Henriksen EJ, Jacob S, Fogt DL, Youngblood EB, Gödicke J (1997) Antihypertensive agent moxonidine enhances muscle glucose transport in insulin-resistant rats. Hypertension 30:1560–1565 Jacob S, Klimm HJ, Rett K, Helsberg K, Haring HU, Godicke J (2004) Effects of moxonidine vs. metoprolol on blood pressure and metabolic control in hypertensive subjects with type 2 diabetes. Exp Clin Endocrinol Diabetes 112:315–322 Lindholm LH, Persson M, Alaupovic P, Carlberg B, Svensson A, Samuelsson O (2003) Metabolic outcome during 1 year in newly detected hypertensives: results of the Antihypertensive Treatment and Lipid Profile in a North of Sweden Efficacy Evaluation (ALPINE study). J Hypertens 21:1563–1574 Lumb PJ, McMahon Z, Chik G, Wierzbicki AS (2004) Effect of moxonidine on lipid subfractions in patients with hypertension. Int J Clin Pract 58:465–468 Meacham RH, Ruelius HW, Kick CJ, Peters JR, Kocmund SM, Sisenwine SF, Wendt RL (1980) Relationship of guanabenz concentrations in brain and plasma to antihypertensive effect in the spontaneously hypertensive rat. J Pharmacol Exp Ther 214:594–598 312 Penicaud L, Berthault MF, Morin J, Dubar M, Ktorza A, Ferre P (1998) Rilmenidine normalizes fructose-induced insulin resistance and hypertension in rats. J Hypertens Suppl 16:S45–S49 Piletz JE, Jones JC, Zhu H, Bishara O, Ernsberger P (1999) Imidazoline receptor antisera-selected cDNA clone and mRNA distribution. Ann N Y Acad Sci 881:1–7 Piletz JE, Zhu H, Chikkala DN (1996) Comparison of ligand binding affinities at human I1- imidazoline binding sites and the high affinity state of alpha-2 adrenoceptor subtypes. J Pharmacol Exp Ther 279:694–702 Prichard BN, Graham BR (2000) I1 imidazoline agonists. General clinical pharmacology of imidazoline receptors: implications for the treatment of the elderly. Drugs Aging 17:133–159 Reaven GM (1988) Banting lecture 1988. Role of insulin resistance in human disease. Diabetes 37:1595–1607 Remaury A, Ordener C, Shih J, Parini A (1999) Relationship between I2 imidazoline binding sites and monoamine oxidase B in liver. Ann N Y Acad Sci 881:32–34 Rosen P, Ohly P, Gleichmann H (1997) Experimental benefit of moxonidine on glucose metabolism and insulin secretion in the fructose-fed rat. J Hypertens 15(Suppl 1):S31–S38 Sawyer R, Warnock P, Docherty JR (1985) Role of vascular alpha 2adrenoceptors as targets for circulating catecholamines in the maintenance of blood pressure in anaesthetised spontaneously hypertensive rats. J Cardiovasc Pharmacol 7:809–812 Schachter M (1999) Moxonidine: a review of safety and tolerability after seven years of clinical experience. J Hypertens 17(Suppl 3):S37–S39 Schachter M, Luszick J, Jager B, Verboom C, Sohlke E (1998) Safety and tolerability of moxonidine in the treatment of hypertension. Drug Saf 19:191–203 Separovic D, Kester M, Ernsberger P (1996) Coupling of I1imidazoline receptors to diacylglyceride accumulation in PC12 rat pheochromocytoma cells. Mol Pharmacol 49:668–675 Swift LL, Valyi-Nagy K, Rowland C, Harris C (2001) Assembly of very low density lipoproteins in mouse liver: evidence of heterogeneity of particle density in the Golgi apparatus. J Lipid Res 42:218–224 Szabo B (2002) Imidazoline antihypertensive drugs: a critical review on their mechanism of action. Pharmacol Ther 93:1–35 Tan CM, Wilson MH, MacMillan LB, Kobilka BK, Limbird LE (2002) Heterozygous alpha 2A-adrenergic receptor mice unveil unique therapeutic benefits of partial agonists. Proc Natl Acad Sci U S A 99:12471–12476 Tolentino-Silva FP, Haxhiu MA, Waldbaum S, Dreshaj IA, Ernsberger P (2000) α2A-adrenergic receptors are not required for central anti- hypertensive action of moxonidine in mice. Brain Res 862:26–35 U.K.Working Party on Rilmenidine (1990) Rilmenidine in mild to moderate essential hypertension: A double-blind, randomized, parallel-group, multicenter comparison with methyldopa in 157 patients. Curr Ther Res 47:194–210 Velliquette RA, Ernsberger P (2003a) Contrasting metabolic effects of antihypertensive agents. J Pharmacol Exp Ther 307:1104– 1111 Velliquette RA, Ernsberger P (2003b) The role of I(1)-imidazoline and alpha(2)-adrenergic receptors in the modulation of glucose metabolism in the spontaneously hypertensive obese rat model of metabolic syndrome X. J Pharmacol Exp Ther 306:646–657 Venteclef N, Guillard R, Issandou M (2005) The imidazoline-like drug S23515 affects lipid metabolism in hepatocyte by inhibiting the oxidosqualene:lanosterol cyclase activity. Biochem Pharmacol 69:1041–1048 Webster J, Koch HF (1996) Aspects of tolerability of centrally acting antihypertensive drugs. J Cardiovasc Pharmacol 27 (Suppl 3):S49–S54 Weimann H-J, Rudolph M (1992) Clinical pharmacokinetics of moxonidine. J Cardiovasc Pharmacol 20(Suppl 4):S37–S41 Yakubu-Madus FE, Johnson WT, Zimmerman KM, Dananberg J, Steinberg MI (1999) Metabolic and hemodynamic effects of moxonidine in the Zucker diabetic fatty rat model of type 2 diabetes. Diabetes 48:1093–1100 Zaitsev SV, Efanov AM, Efanova IB, Larsson O, Östenson CG, Gold G, Berggren PO, Efendic S (1996) Imidazoline compounds stimulate insulin release by inhibition of KATP channels and interaction with the exocytotic machinery. Diabetes 45:1610–1618 Zhou M, Yang S, Koo DJ, Ornan DA, Chaudry IH, Wang P (2001) The role of Kupffer cell alpha(2)-adrenoceptors in norepinephrine-induced TNF-alpha production. Biochim Biophys Acta 1537:49–57 Zhu QM, Lesnick JD, Jasper JR, MacLennan SJ, Dillon MP, Eglen RM, Blue DR, Jr. (1999) Cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild-type and D79N alpha2A-adrenoceptor transgenic mice. Br J Pharmacol 126: 1522–1530 Zonnenchein R, Diamant S, Atlas D (1990) Imidazoline receptors in rat liver cells: a novel receptor or a subtype of alpha 2adrenoceptors? Eur J Pharmacol 190:203–215