QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.050468v1 306/2/646    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Velliquette, R. A. Articles by Ernsberger, P. Search for Related Content PubMed PubMed Citation Articles by Velliquette, R. A. Articles by Ernsberger, P.

ENDOCRINE AND REPRODUCTIVE

The Role of I₁-Imidazoline and ₂-Adrenergic Receptors in the Modulation of Glucose Metabolism in the Spontaneously Hypertensive Obese Rat Model of Metabolic Syndrome X

Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio

Received February 13, 2003; accepted May 13, 2003.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
We examined glucose metabolism after I₁-imidazoline (I₁R) and ₂-adrenergic receptor (₂AR) activation in an animal model of metabolic syndrome X. Fasted spontaneously hypertensive obese rats (SHROB) were given the I₁R/₂AR agonists moxonidine and rilmenidine or the ₂AR agonist guanabenz. Because of the dual specificity of moxonidine, its actions were split into adrenergic and nonadrenergic components by using selective antagonists: rauwolscine (₂AR) efaroxan (I₁R/₂AR), or 2-endo-amino-3-exo-isopropylbicyclo[2.2.1.]heptane (AGN 192403) (I₁R). Hyperglycemia induced by moxonidine, rilmenidine, and guanabenz resulted from inhibition of insulin secretion. Similar responses were observed after oral dosing and in lean littermates. Glucagon was reduced by the I₁R agonists (moxonidine, 32 ± 5%; rilmenidine, 24 ± 7%) but elevated by guanabenz (71 ± 32%). The hyperglycemic and hypoinsulinemic responses to moxonidine were blocked by rauwolscine. In contrast, rauwolscine potentiated the reduction in glucagon (39 ± 6%). AGN 193402 blocked the glucagon response without affecting hyperglycemia and hypoinsulinemia. Efaroxan blocked all responses to moxonidine. When SHROB rats were treated with moxonidine 15 min before an oral glucose tolerance test, the glucose area under the curve (AUC) was increased. Antagonizing the ₂AR component of moxonidine's action with rauwolscine improved glucose AUC 3-fold and facilitated the insulin secretory response and reduced glucagon secretion. Testing fasting glucose and insulin during 3 weeks of oral moxonidine revealed early hyperglycemia that later faded, and a progressive drop in fasting insulin. The acute hyperglycemia and hypoinsulinemia elicited by moxonidine and rilmenidine was mediated by ₂AR, whereas I₁R may reduce glucagon and increase insulin, particularly after a glucose load.

Chronic administration of I₁R agonists can reduce insulin resistance and improve glucose disposal rates in humans (Reid, 2001) and animals (Rosen et al., 1997; Henriksen et al., 1997; Ernsberger et al., 1999a). However, because all clinically used I₁R agonists activate both I₁R and ₂AR, it is not known which receptor is responsible for these beneficial therapeutic effects.

In contrast to these chronic effects, acute administration of imidazoline agents induces hyperglycemia and impairs glucose tolerance in humans (Metz et al., 1978; Barbieri et al., 1980) and in rats (Bock and Van Zwieten, 1971; Ditullio et al., 1984; Rosen et al., 1997) as a result of decreased plasma insulin levels. The fall in insulin secretion is believed to be due to the acute activation of -cell ₂ARs leading to decreases in cytosolic cAMP levels and inhibition of insulin secretion. However, a possible contribution of imidazoline receptors to the inhibition of insulin secretion by imidazolines has never been directly tested.

In vitro studies with imidazoline agonists have indicated a potential insulin secreting effect. This was first described for the imidazoline phentolamine, which was found to increase insulin secretion independently of ₂AR blockade (Schulz and Hasselblatt, 1988, 1989c). Furthermore, the imidazoline clonidine inhibited insulin release when given alone, as a result of its ₂AR agonist action, but elicited insulin release through a nonadrenergic mechanism after ₂AR blockade. More recent studies have identified selective imidazoline agents that stimulate insulin release from the pancreas while lacking adrenergic actions (Efanova et al., 1998; Efanov et al., 2001). The mixed agent moxonidine facilitated glucose induced insulin secretion in isolated rat -cells in a dose-dependent manner (Rosen et al., 1997). In HIT-T15 cells, clonidine stimulated glucose-induced insulin secretion only when cells were pretreated with pertussis toxin to block ₂AR-mediated responses (Hirose et al., 1997). In contrast, others have reported that moxonidine had no effect on insulin secretion in isolated rat -cells (Morgan et al., 1995).

Chronic imidazoline treatment improves glucose metabolism, whereas acute treatment impairs glucose homeostasis in both humans and rodents. Thus, chronic and acute effects of imidazolines seem to be opposite. Inhibition of insulin secretion induced by ₂AR activation has been documented for 30 years (Bock and Van Zwieten, 1971). Less information regarding the effects of ₂AR activation on plasma glucagon levels is available. However, the general consensus is that both acute in vitro and in vivo ₂AR activation seems to stimulate glucagon secretion (Gotoh et al., 1988; Hirose et al., 1992; Saito et al., 1992). The influence of acute I₁R activation by imidazoline agents on glucagon levels in vivo has not been reported.

Metabolic syndrome X is a cluster of metabolic diseases, including insulin resistance, glucose intolerance, obesity, hypertension, and hyperlipidemia (Reaven, 1993). Metabolic syndrome X frequently precedes type II diabetes and is a strong predictor of cardiovascular disease, yet specific pharmacotherapy has not been established. The obese spontaneously hypertensive rat (SHROB; Koletsky rat) is a unique animal model with genetic obesity superimposed on a background of genetic hypertension. The obesity mutation is a recessive trait, designated fa^k, which is a nonsense mutation of leptin receptor gene resulting in a premature stop codon in the leptin receptor extracellular domain. The SHROB rat lacks expression of any of the isoforms of the leptin receptor. SHROB shows marked elevations in fasting insulin, glucagon, triglycerides, and total cholesterol together with obesity, glucose intolerance, and hypertension, while maintaining normoglycemia. Thus, the SHROB is a leading animal model of metabolic syndrome X (Ernsberger et al., 1999b; Velliquette et al., 2002).

The aim of this study was to examine the role of I₁R and ₂AR in the control of glucose metabolism in a rodent model of metabolic syndrome X. In addition, we wanted to examine the effectiveness of I₁R agonists monotherapy in the treatment of metabolic syndrome X. We hypothesized that the in vivo hyperglycemia response of I₁R agonists was from the activation of pancreatic ₂AR, whereas pancreatic I₁R activation inhibits glucagon secretion and stimulates insulin secretion, leading to improved glucose homeostasis.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Animals. Adult male and female SHROB and lean SHR littermates aged between 16 and 24 weeks were housed individually and were provided food (Teklad 8664; Tekland, Madison, WI) and water ad libitum. SHROB (fa^k/fa^k) were bred in a closed colony from brother-sister mating of heterozygous carriers (Fa^k/fa^k), because obese animals or either sex cannot reproduce (Ernsberger et al., 1999b). Animals were on a 12-h light/dark cycle (lights on from 7:00 AM to 7:00 PM) 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.

Acute Drug Treatment. Moxonidine was provided by Solvay Pharmaceuticals (Hannover, Germany). Rilmenidine, guanabenz, efaroxan, rauwolscine, and AGN 192403 were obtained from Sigma-Aldrich (St. Louis, MO). All drugs were dissolved in 20% dimethyl sulfoxide and then diluted with 0.9% saline (final dimethyl sulfoxide concentration <5%). 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 in SHR as determined by telemetry (Ernsberger and Rao, 2000). In a few experiments, drugs were given orally by gavage to both SHROB and SHR (moxonidine, 2 and 8 mg/kg; clonidine, 0.8 mg/kg). Dose-response studies were examined in SHROB 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). All drugs were administered after an 18-h overnight fast. Tail blood was taken at baseline and 1, 2, and 4 h after i.p. injection, and at 2, 4, 8, and 12 h after oral gavage. In blocking experiments, antagonists were mixed with agonists and administered as one bolus.

To test whether acute I₁R activation modulated glucose tolerance, we injected SHROB with moxonidine (0.5 mg/kg), moxonidine + rauwolscine (0.5 + 7.5 mg/kg), or saline followed by a glucose challenge. Animals were fasted for 18 h and then administered drugs i.p. An oral glucose tolerance test (OGTT) was performed 15 min postinjection.

Chronic Drug Treatment. To test progressive changes in glucose and insulin during the course of chronic treatment, eight SHROB and eight SHR were given moxonidine orally by adding it to powdered rat chow before pelleting (0.2 mg/kg chow for SHROB and 0.13 mg/kg chow for SHR). Moxonidine free base was dissolved in 0.1% citric acid at 1 mg/ml to ensure even mixing. At day 20 of drug treatment, SHROB consumed 18.9 ± 1.4 g of chow and had a body weight of 492 ± 15 g. Thus, the ingested dose of moxonidine was 8.0 ± 0.4 mg/kg/day. SHR consumed 16.2 ± 1.1 g of chow and had a body weight of 223 ± 3 g. Thus, the ingested dose of moxonidine was 9.4 ± 0.6 mg/kg/day. Food was removed each day at 12:00 PM and a single tail blood sample (0.2 ml) was taken at 4:00 PM before reintroduction of food. Blood glucose and plasma insulin were determined as described below.

OGTT. As described previously (Ernsberger et al., 1999a), rats were given by oral gavage a 50% glucose solution at a dose of 6g/kg body weight. Blood (0.2 ml) was obtained from the tail vein of conscious animals at baseline and 30, 60, 120, 240, and 360 min after the glucose load. Plasma glucose, insulin, C-peptide, and glucagon were determined at each time point.

Biochemical Measurements. Glucose in whole blood or plasma was determined by colorimetric glucose oxidase assay (whole blood: One-Touch; Lifescan, Milpitas, CA; plasma: enzymatic glucose kit; Sigma-Aldrich). The remaining sample was chilled on ice, centrifuged for 20 min at 5000g at 4°C, and the plasma frozen at –70°C until assayed for glucose, insulin, C-peptide, and glucagon. Insulin, C-peptide, and glucagon radioimmunoassay kits were used with rat insulin, C-peptide, and glucagon standards and antibodies directed against rat insulin, C-peptide, and glucagon, respectively (Linco, St. Charles, MO). Assays were conducted in duplicate. Intra-assay coefficient of variation was 0.8% for glucose, 1.1% for glucagon, 2.9% for C-peptide, and 3.7% for insulin. The interassay variation was 2.3% for glucose, 7.2% for glucagon, and 6.2% for both C-peptide and insulin.

Statistical Methods. Results are presented as means ± standard error of the mean. Comparisons between groups were made using one- or two-way analysis of variance (ANOVA) or analysis of variance with repeated measures (REMANOVA) using Prism (GraphPad Software Inc., San Diego, CA) with post hoc analyses by Newman-Keuls test.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Acute Metabolic Response to ₂AR and I₁R Agonists in SHROB. The selective I₁R agonists moxonidine (0.5 mg/kg) and rilmenidine (2.5 mg/kg) both induced hyperglycemia after acute i.p. administration (Fig. 1A). Hyperglycemia was mirrored by a significant reduction in plasma insulin (Fig. 1B). Plasma C-peptide levels, an index of insulin secretion, also fell (Fig. 1C). These data imply that moxonidine and rilmenidine induced hyperglycemia primarily by reducing insulin secretion. In addition, plasma glucagon concentrations were significantly reduced after acute i.p. moxonidine and rilmenidine (Fig. 1D), an effect that would tend to blunt the hyperglycemic response. The effects of moxonidine were more short-lived than those of rilmenidine, consistent with the 1-h half-life of moxonidine in the rat (He et al., 2000).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Effects of acute i.p. moxonidine and rilmenidine on blood glucose (A), plasma insulin (B), C-peptide (C), and glucagon (D) in SHROB. After an 18-h fast, six to eight rats were injected i.p. with either 0.5 mg/kg moxonidine or 2.5 mg/kg rilmenidine. Tail blood (0.2 ml) was collected at baseline and 1, 2, and 4 h after injection. Both moxonidine and rilmenidine induced hyperglycemia while reducing plasma insulin and C-peptide. Plasma glucagon was also decreased.

Increasing doses of moxonidine over an 8-fold range elicited similar hyperglycemic effects, with the main effect of increasing doses to flatten the time course of the response (Fig. 2A; dose x time interaction, p < 0.0001). A dose-dependent reduction in plasma insulin concentration was observed (Fig. 2B). A significant dose-time interaction was observed in plasma insulin concentration (p < 0.0001), implying greater duration of action at higher doses. Plasma insulin concentration significantly overshot the baseline at 4 h for the two lower doses of moxonidine, which was not achieved at the two higher doses. A biphasic dose-response relationship for plasma glucagon concentrations was observed (Fig. 2C), wherein the lowest dose of moxonidine (0.5 mg/kg) reduced plasma glucagon concentration, whereas higher doses had the opposite effect. This pattern of responses raised the possibility that the I₁R may be responsible for the reduction of plasma glucagon at low doses, response, whereas ₂AR mediate the increases at higher doses, because I₁R effects are likely to dominate at low doses of moxonidine (I₁R K_i = 2 nM), whereas ₂AR effects may become overwhelming at higher doses (₂AR K_i = 75 nM) (Ernsberger et al., 1993).

View larger version (23K): [in this window] [in a new window]   Fig. 2. Dose-response relationship for the effects of i.p. moxonidine on blood glucose (A), plasma insulin (B), and glucagon (C) in SHROB (n = 6–8/dose). Procedure was the same as for Fig. 1. Moxonidine induced dose- and time-dependent hyperglycemia and fall in plasma insulin that was significant at all doses tested. Moxonidine had a biphasic effect on plasma glucagon. The low dose of moxonidine reduced plasma glucagon, whereas higher doses elevated plasma glucagon concentrations (significant effects of dose and time and their interaction; p < 0.0005; two-way ANOVA).

We next compared the effects of moxonidine with those of guanabenz, a potent ₂AR agonist (K_i = 7 nM) with low affinity for I₁R (K_i > 1000 nM) (Ernsberger et al., 1993). Guanabenz elicited a dose-dependent increase in blood glucose concentration (Fig. 3A). The hyperglycemia induced by guanabenz was mirrored by a dose-dependent reduction in plasma insulin levels (Fig. 3B). The reduction in plasma insulin concentration resulted from an inhibition of insulin secretion, because plasma C-peptide concentrations were decreased in parallel (data not shown). In contrast to moxonidine, an overshoot above baseline at the later time points was not detected. A significant dose-dependent increase in plasma glucagon concentrations was observed after acute i.p. guanabenz (Fig. 3C). These results with a relatively specific ₂AR agonist suggest that hyperglycemia, reductions in insulin secretion, and increases in glucagon secretion are mediated through the ₂AR.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Dose-response relationship for the effects of i.p. guanabenz on blood glucose (A), plasma insulin (B), and glucagon (C) in SHROB (n = 6–8/dose). Procedure was the same as for Figs. 1 and 2. Guanabenz induced hyperglycemia and a fall in plasma insulin in a dose- and time-dependent manner. Guanabenz significantly elevated plasma glucagon concentrations.

To determine whether these responses to imidazoline and nonimidazoline ₂AR agonists were specific for the i.p. route of administration, moxonidine and the nonselective I₁R and ₂AR agonist clonidine were given orally by gavage. Because the oral bioavailability of moxonidine in the rat is only 5% (He et al., 2000), 2- and 8-mg/kg doses were administered. Clonidine was given at 0.8 mg/kg because previous studies have shown that moxonidine has approximately 1/10 the antihypertensive potency of clonidine when given to SHR (Armah et al., 1988). Oral moxonidine markedly increased plasma glucose and decreased insulin, with a prolonged time course extending up to 12 h (Fig. 4). Clonidine elicited nearly identical effects at 1/10 the moxonidine dose. Thus, oral dosing with ₂AR agonists is capable of inducing significant hyperglycemia.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Effect in SHROB of oral moxonidine or clonidine on blood glucose (A) and plasma insulin (B). After an 18-h fast, eight SHROB were given by oral gavage either 2 or 8 mg/kg moxonidine or 0.8 mg/kg clonidine. Tail blood (0.2 ml) was collected at baseline and at each time point. Both moxonidine and clonidine induced hyperglycemia while reducing plasma insulin). The effect persisted throughout the 12-h observation period. Administration of vehicle (0.1% citric acid) had no effect on glucose or insulin (latter not shown).

Acute Metabolic Response to ₂AR and I₁R Agonists in SHR. To determine whether these responses to imidazoline and nonimidazoline ₂AR agonists were specific for the insulin-resistant SHROB animal, the experiments with orally administered agents were repeated in lean SHR littermates. As shown in Fig. 5, the rise in blood glucose showed a reduced overall amplitude, but was highly significant. Plasma insulin is by necessity plotted on a different scale for SHR than for SHROB. Insulin fell to nearly undetectable levels after either moxonidine or clonidine. The time course of the glucose and insulin responses was similar in SHR and SHROB, suggesting comparable pharmacokinetics. In both genotypes, not all groups had returned to baseline by 12 h postinjection. Because responses to oral and i.p. routes were similar, and because larger responses were observed in SHROB rats, further investigations were carried out with i.p. administration to SHROB.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Effect in SHR of oral moxonidine or clonidine on blood glucose (A) and plasma insulin (B). After an 18-h fast, eight SHR were given agents by oral gavage as described for Fig. 4. Both moxonidine and clonidine induced hyperglycemia while reducing plasma insulin. Administration of vehicle (0.1% citric acid) had no effect on glucose or insulin (latter not shown).

Effects of Selective Receptor Blockade. Selective antagonists were used to further test the receptor mechanisms responsible for the acute effects of i.p. moxonidine in SHROB. The selective ₂AR antagonist rauwolscine (7.5 mg/kg), the mixed I₁R and ₂AR antagonist efaroxan (0.6 mg/kg), or the selective I₁R antagonist AGN 192403 (10 mg/kg) (Munk et al., 1996; Schafer et al., 2002) was given with moxonidine (0.5 mg/kg). Coadministration of moxonidine with either of the antagonists active at ₂AR completely abolished the hyperglycemic effect of moxonidine alone (Fig. 6A; p < 0.0001 versus moxonidine only and p > 0.10 versus saline). Reductions in plasma insulin were also blocked by both rauwolscine and efaroxan (Fig. 6B). In fact, the addition of rauwolscine seemed to unmask an insulin secretory response induced by moxonidine, which is suggested by a significant increase in plasma insulin and C-peptide without a change in glycemia. In contrast, the selective I₁R antagonist AGN 192403 had no effect on glucose or insulin levels at 1 or 2 h relative to rats treated with moxonidine alone, but did increase glucose and decrease insulin at 4 h. In other words, AGN 192403 selectively inhibited the overshoot in plasma insulin levels observed at 4 h for moxonidine alone or moxonidine with selective ₂AR blockade, resulting in higher 4-h glucose levels. These results imply that the ₂AR is responsible for moxonidine's acute hyperglycemic response by suppressing insulin secretion.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Effect of acute i.p. moxonidine alone, or with the addition of either rauwolscine or efaroxan on blood glucose (A), plasma insulin (B), and glucagon (C) in SHROB (n = 6–8/dose). Moxonidine (0.5 mg/kg) alone or with antagonists rauwolscine (7.5 mg/kg), efaroxan (0.6 mg/kg), or AGN 192403 (10 mg/kg) was given together as a mixture. The addition of either rauwolscine or efaroxan blocked the hyperglycemic response to moxonidine (effect of antagonist, p < 0.0001; two-way ANOVA). Similarly, either rauwolscine or efaroxan, but not AGN 192403, blocked the reduction in plasma insulin concentration induced by moxonidine. In fact, rauwolscine seemed to unmask an insulin secretory response at later time points (p < 0.05, Newman-Keuls test). Efaroxan and AGN 192403 blocked the reduction in plasma glucagon induced by moxonidine, whereas in contrast rauwolscine actually potentiated the reduction in plasma glucagon (both p < 0.01; two-way ANOVA).

In contrast to the results for insulin, only the mixed I₁R and ₂AR antagonist efaroxan blocked the reduction in plasma glucagon concentration induced by acute i.p. moxonidine treatment (Fig. 6C). Selective blockade of ₂AR with rauwolscine actually potentiated moxonidine's effect on plasma glucagon. This further rules out a role for ₂AR in the inhibition of glucagon secretion and implicates I₁R. Furthermore, selective blockade of I₁R with AGN 192403 uncovered a stimulatory effect of moxonidine on glucagon levels, presumably mediated by unopposed activation of ₂AR.

Some studies, but not others, have reported that ₂AR antagonists can increase insulin secretion (Struthers et al., 1985; Hsu et al., 1987; Schulz and Hasselblatt, 1988). Efaroxan is an insulin secretagogue in vitro, allegedly via an agonist action at putative I₃-imidazoline receptors (Chan et al., 2001), but very high concentrations (10–100 µM) are required to elicit insulin secretion. Therefore, we tested the effects of rauwolscine and efaroxan when administered alone. As shown in Fig. 7, neither agent had any effect on glucose, insulin, or glucagon concentrations. Given that the doses used are sufficient to induce complete blockade of ₂AR, these results imply that ₂AR do not tonically participate in the regulation of glucose homeostasis in the basal state in the SHROB model.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Effects of 2AR antagonists alone on blood glucose (A), plasma insulin (B), and glucagon (C) in SHROB (n = 6). Rauwolscine (7.5 mg/kg) and efaroxan (0.6 mg/kg) had no effects when given alone.

Effects on Glucose Tolerance. To examine the role of I₁R and ₂AR in nutrient-induced changes in insulin secretion and glucose homeostasis in vivo, we preformed an OGTT after acute i.p. administration of selective I₁R and ₂AR agents. Acute i.p. moxonidine (0.5 mg/kg) significantly worsened the glucose response during an OGTT compared with saline-treated controls (Fig. 8). Plasma glucose AUC was significantly elevated after acute i.p. moxonidine compared with saline (Fig. 8B). Similar effects on plasma glucose responses were observed after 0.1 mg/kg guanabenz (data not shown). In contrast, antagonizing the ₂AR component of moxonidine with rauwolscine (7.5 mg/kg) significantly improved the glucose response during the OGTT (Fig. 8). This resulted in a significantly reduced plasma glucose AUC for the group treated with moxonidine plus rauwolscine versus saline-treated controls. This improved glucose response was entirely due to a lower glucose AUC beyond 60 min after the glucose load, because 0- to 60-min plasma glucose AUC was identical for moxonidine plus rauwolscine and saline (5.6 ± 0.7 and 5.4 ± 0.8 g/ml · min, respectively). In contrast, guanabenz plus rauwolscine was not different from saline (data not shown). These results suggest that the ₂AR component worsens and the I₁R component of moxonidine improves the glucose response during an OGTT in an animal model of impaired glucose tolerance.

View larger version (14K): [in this window] [in a new window]   Fig. 8. Effect of acute moxonidine pretreatment with and without rauwolscine on oral glucose tolerance. OGTT was conducted on 18-h fasted rats, 15 min after the acute i.p. administration of 0.5 mg/kg moxonidine. Rats were administered by oral gavage a 50% glucose solution at a dose of 6g/kg body weight. Blood (0.2 ml) was obtained from the tail vein of unrestrained, conscious animals at baseline and 30, 60, 120, 240, and 360 min after the glucose load. Acute i.p. moxonidine (0.5 mg/kg) significantly worsened the glucose response during an OGTT compared with saline-treated. In contrast, antagonizing the 2AR component of moxonidine's action with rauwolscine (7.5 mg/kg) significantly improved the glucose response during the OGTT relative to saline-treated controls (all comparisons p < 0.0001; two-way ANOVA.

The impairment in glucose tolerance observed after acute i.p. moxonidine was due to near complete inhibition of the insulin response during the first 60 min postglucose load (Fig. 9), which resulted in a negative plasma insulin AUC (–228 ± 81 ng/ml · min). The improved glucose response after acute moxonidine plus rauwolscine was mirrored by an increased plasma insulin response (Fig. 9). The enhanced plasma insulin response was reflected in an increased plasma insulin AUC compared with saline-treated (Fig. 9B). Similar to the effect on the plasma glucose response, the enhancement of insulin secretion was only observed 60 min postglucose load and beyond (7300 ± 1200 versus 2300 ± 1400 ng/ml · min; p < 0.01).

View larger version (15K): [in this window] [in a new window]   Fig. 9. Effect of acute moxonidine pretreatment with and without rauwolscine on the plasma insulin response to an oral glucose load. OGTT was conducted as described for Fig. 5. Acute i.p. moxonidine (0.5 mg/kg) significantly inhibited the insulin response to a glucose load compared with saline-treated controls. In contrast, antagonizing the 2AR component of moxonidine with rauwolscine (7.5 mg/kg) reversed the inhibition and significantly enhanced the insulin response relative to saline-treated controls (all comparisons p < 0.0001; two-way ANOVA).

The changes in the plasma insulin response during the OGTT were generally mirrored by changes in plasma C-peptide concentrations (Fig. 10), suggesting that the changes in plasma insulin concentrations were due to secretion. Total plasma C-peptide AUC was not significantly different between moxonidine and saline. Antagonizing the ₂AR component of moxonidine with rauwolscine significantly improved total plasma C-peptide AUC relative to both moxonidine alone and saline (Fig. 10B). Similar to the plasma insulin AUC, moxonidine's 0- to 60-min plasma C-peptide response was markedly different from the 60- to 360-min response (–45 ± 16 and 583 ± 88 nM · min, respectively). Antagonizing the ₂AR component of moxonidine with rauwolscine completely reversed the early inhibition of plasma C-peptide induced by moxonidine (AUC, –45 ± 16 versus 73 ± 5.3 nM · min; p < 0.001). In addition, the early insulin secretion response (0–60 min) was increased by moxonidine when the ₂AR component was blocked with rauwolscine (plasma C-peptide AUC, 73 ± 5.3 versus 28 ± 15 nM · min for the saline-treated group; p < 0.05). Moreover, the plasma C-peptide AUC with moxonidine plus rauwolscine was elevated during the late phase (60–360 min) of the OGTT compared with saline-treated controls (897 ± 123 versus 563 ± 28 nM · min; p < 0.05). These results suggest that moxonidine's ₂AR component inhibits and the I₁R component stimulates insulin secretory activity during an OGTT.

View larger version (15K): [in this window] [in a new window]   Fig. 10. Effect of acute moxonidine pretreatment with and without rauwolscine on the plasma C-peptide response to an oral glucose load. OGTT was conducted as described for Fig. 5. The effects of moxonidine with and without rauwolscine on C-peptide levels paralleled the effects on insulin shown in Fig. 6, implying that the changes in insulin level closely reflect changes in secretion rather than turnover or clearance.

In agreement with previous results (Velliquette et al., 2002), plasma glucagon showed a paradoxical rise in response to a glucose load in the SHROB model. As shown in Fig. 11, the rise in plasma glucagon immunoreactivity during an OGTT was attenuated after acute moxonidine treatment relative to animals receiving only saline before the glucose load. When the ₂AR component of moxonidine's action was blocked with rauwolscine, plasma glucagon AUC was significantly reduced compared with both moxonidine alone and saline (Fig. 11B). These results suggest that moxonidine's action to reduce plasma glucagon through an I₁R-mediated action can be observed during a glucose load as well as under fasting conditions.

View larger version (15K): [in this window] [in a new window]   Fig. 11. Effect of acute moxonidine pretreatment with and without rauwolscine on the plasma insulin response to an oral glucose load. OGTT was conducted as described for Fig. 5. Glucagon showed a paradoxical rise in response to a glucose load in SHROB-treated with moxonidine (0.5 mg/kg) or with saline. In contrast, antagonizing the 2AR component of moxonidine's action with rauwolscine (7.5 mg/kg) significantly attenuated the glucagon response during the OGTT.

Time Course of Hyperglycemic and Hypoinsulinemic Effects during Chronic Treatment. Because the acute and chronic effects of moxonidine on plasma glucose are in opposite directions, we characterized the time course of glucose and insulin concentrations during chronic oral treatment of SHROB and SHR with 8 mg/kg/day moxonidine (Fig. 12). Relative to baseline (day 0), the 1st day of oral moxonidine treatment increased blood glucose and decreased plasma insulin. This effect was observed even though the rats had no food, and thus no drug, for 4 h before sampling. This finding is consistent with the prolonged hyperglycemic and insulinopenic response to oral moxonidine (Figs. 4 and 5). After reaching a peak on day 3 in SHROB and day 5 in SHR, fasting blood glucose fell progressively until by day 12 it was no longer differed from baseline in either SHROB or SHR (p > 0.05, Newman-Keuls test and REMANOVA). In contrast, fasting insulin levels fell to nearly one-third of starting levels and remained reduced through the 20 days of treatment in SHROB, but showed little change in SHR. Thus, in SHROB by the 3rd week of treatment fasting normoglycemia was maintained with one-third the amount of circulating insulin, implying increased insulin sensitivity. In both SHROB and SHR, the hypoglycemic and hypoinsulinemic effects of moxonidine seem to show complete tachyphylaxis with chronic treatment.

View larger version (27K): [in this window] [in a new window]   Fig. 12. Progressive effect of chronic oral moxonidine on fasting glucose and insulin in SHROB (A) and SHR (B). Animals were treated with moxonidine at a dose of 8 mg/kg/day added to their food. Food was withdrawn at 12:00 PM, and a tail blood sample was taken at 4:00 PM before reintroduction of food. Blood glucose was increased during the 1st week (p < 0.05 Newman-Keuls after REMANOVA) but not by the 3rd week. In SHROB, plasma insulin was consistently reduced throughout the study period (p < 0.05 Newman-Keuls after REMANOVA).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we examined the impact of agents active on I₁R and ₂AR on glucose homeostasis in an animal model of metabolic syndrome X. Currently available imidazoline agents activate both I₁R and ₂AR, thus it is important to separate responses mediated by these receptors. Acute administration of the selective I₁R agonists moxonidine and rilmenidine induced hyperglycemia by reducing insulin secretion, as indicated by falls in both plasma insulin and C-peptide. The reduced insulin secretion was mediated by ₂AR activation and not the I₁R, as indicated by similar responses to the specific ₂AR agonist guanabenz and by the ability of the nonimidazoline ₂AR antagonist rauwolscine to block the effects. The beneficial reduction in plasma glucagon was mediated by the I₁R because the responses were neither reproduced by guanabenz nor blocked by the addition of rauwolscine, but were blocked by efaroxan, a mixed I₁R and ₂AR antagonist, or by the selective I₁R antagonist AGN 192403.

Rilmenidine, the first selective I₁R agonist to be used clinically, improves glucose tolerance and insulin resistance in patients with metabolic syndrome X (Reid, 2001). A double-blind comparison trial of rilmenidine against a calcium channel blocker showed that only rilmenidine improved glucose tolerance (De Luca et al., 2000). Furthermore, as reviewed in the Introduction, recent studies in humans and animals have shown that moxonidine and rilmenidine have therapeutic potential in insulin resistance and diabetes as well as in hypertension. Some previous studies have used a 90-day treatment period (Ernsberger et al., 1992a, 1996), whereas another group showed that 21 days of treatment was sufficient to improve insulin resistance in the Zucker rat model (Henriksen et al., 1997). The present study confirms that this treatment period is sufficient to reduce fasting insulin levels required to maintain normoglycemia.

We hypothesized that the apparent paradox of acute hyperglycemia verses chronic hypoglycemia could be explained by the dual activity of imidazoline agents. Acutely, peripheral ₂AR activation would predominate, leading to inhibition of insulin secretion and stimulation of glucagon secretion. Chronically, ₂AR may be partially desensitized and I₁R activation becomes manifest, leading to improved glucose homeostasis. The transition from early hyperglycemic response to later improvements in glucose homeostasis is illustrated in Fig. 12 when moxonidine was administered orally to SHROB and SHR. In SHROB, elevated blood glucose and reduced plasma insulin concentrations were observed for the first 12 days, followed by normalization of blood glucose with maintenance of reduced plasma insulin concentrations. The hyperglycemic response faded over time in SHR as well.

Acute I₁R and ₂AR activation interact with nutrient regulated insulin and glucagon secretion in vitro and in vivo. Pancreatic ₂AR activation is known to inhibit insulin secretion in the fasted and fed condition in vivo and in isolated pancreatic islets in vitro (Ditullio et al., 1984). In the present study, ₂AR activation induced profound hyperglycemia, far in excess of that elicited by a large glucose load. This was observed not only the hyperinsulinemic SHROB but also in lean SHR littermates. Both i.p. and oral routes of administration elicited hyperglycemia. These data suggest possible adverse effects of ₂AR agonists in type diabetics and others with compromised insulin function.

In contrast to the inhibitory effects of ₂AR activation, activation of an I₁R (or a related imidazoline site; Morgan et al., 1995) may facilitate insulin release. Clonidine (an I₁R and ₂AR agonist) in the presence of the nonimidazoline irreversible ₂AR antagonist benextramine increased insulin secretion in mice pancreatic islets compared with control (Schulz and Hasselblatt, 1989b). Similar effects on insulin secretion have been reported for phentolamine (an I₁R agonist and ₂AR antagonist) and antazoline (an I₁R and a weak ₂AR agonist) in vitro (Schulz and Hasselblatt, 1989a). In agreement with these in vitro findings, we observed that acute ₂AR activation impairs glucose-induced insulin secretion, yet acute I₁R activation potentiates glucose-induced insulin secretion in vivo.

The potential use of ₂AR antagonists to enhance insulin release has been debated (Hsu et al., 1987; Schulz and Hasselblatt, 1989c). In agreement with the lack of effect of either rauwolscine or efaroxan in the present study, most investigators have found little effect of ₂AR antagonists on insulin levels in vivo in humans and animals. Efaroxan has reported to increase insulin secretion from isolated pancreatic islets, although relatively high concentrations of 10 to 100 µM are required for this effect (Morgan et al., 1995). This action is independent of ₂AR and is thought to be mediated through an agonist effect at an imidazoline receptor subtype. Because efaroxan is an antagonist at I₁R, it has been proposed that efaroxan acts via a novel I₃R. However, it is not clear whether this proposed I₃R mechanism applies in vivo except at high doses (Mourtada et al., 1997a; Mayer and Taberner, 2002) In the present study, a dose of 0.6 mg/kg was sufficient to block both ₂AR and I₁R, yet had no effect on glucose, insulin, C-peptide, or glucagon when given alone. These data imply that ₂AR of the pancreatic islets are not tonically active in restraining insulin secretion under resting conditions.

Glucagon is the primary counter-regulatory hormone to insulin, and its levels rise during fasting and fall during the postprandial period. In metabolic diseases such as diabetes and metabolic syndrome X, fasting and postprandial plasma glucagon concentrations are elevated and a paradoxical postprandial rise in glucagon may be observed (Shah et al., 2000; Velliquette et al., 2002). The impact of ₂AR activation on glucagon secretion is controversial, with some studies reporting increases, others decreases, and yet others no response. However, previous in vivo studies have not tested specific ₂AR agonists but rather tested imidazolines or catecholamines, which activate multiple receptor types (Gotoh et al., 1988; Saito et al., 1992). Saito et al. (1992) reported that the increase in glucagon levels elicited by clonidine was mediated by ₂AR, because it was completely blocked by yohimbine. We have shown that the relatively specific ₂AR agonist guanabenz elevated plasma glucagon and that this effect of guanabenz and other ₂AR agonists can be blocked with rauwolscine. Thus, ₂AR agonists given in relatively low doses in vivo have significant effects on circulating levels of glucagon. The increased glucagon in combination with decreased insulin can account for the observed hyperglycemic response.

The effects of imidazoline agents on glucagon levels in vivo has not been studied previously. One in vitro study showed that the I₁R agonists phentolamine and antazoline both reduced arginine-induced glucagon secretion (Mourtada et al., 1997b). We found that low doses of moxonidine reduced and higher doses elevated plasma glucagon. This biphasic response is consistent with the selectivity of moxonidine for the I₁R (K_i = 2 nM) relative to ₂AR (K_i = 75 nM), which suggests that inhibitory effects of low doses may be mediated by I₁R and the stimulatory effect of high doses may be mediated by ₂AR. In support of this hypothesis, we observed that blockade of ₂AR potentiated the inhibitory action of moxonidine on glucagon levels, whereas the I₁R blocker AGN 192403 inverted the inhibitory response into a stimulatory one. Moreover, glucagon secretion evoked by a glucose load was inhibited by moxonidine and this effect was also potentiated by ₂AR blockade.

Previous studies of functional responses mediated by I₁R have focused on falls in blood pressure and ocular pressure mediated by sympathoinhibition (Szabo, 2002). A major limitation of these studies is that ₂ARs mediate identical sympathoinhibitory responses, thereby making separation of I₁R- and ₂AR-mediated actions difficult. In the present study, we found opposite effects of these two receptors on spontaneous and glucose-evoked glucagon secretion. Furthermore, ₂AR mediated a profound inhibition of insulin secretion, whereas I₁R mediated a small increase in insulin secretion after complete blockade of ₂AR. The stimulatory action of I₁R activation was most clearly noted after glucose load (Fig. 9). Thus, in contrast to their similar actions on blood and ocular pressure, I₁R and ₂AR mediate distinct and even opposing actions on glucose homeostasis. This is similar to the opposing actions of these two receptors on carotid body electrophysiological responses to hypoxia (Ernsberger et al., 1998).

There are several limitations to this study. Because testing was done in vivo, multiple target organs might be involved in the observed effects. For example, we cannot rule out a contribution from circulatory effects, including direct vasoconstriction mediated by ₂AR and sympathoinhibition mediated by both ₂AR and I₁R. Where possible, doses of each agent were matched for relative antihypertensive actions based on prior studies (Ernsberger and Rao, 2000). A further limitation is the lack of entirely specific agonists or antagonists at I₁R that do not also affect ₂AR. This was addressed by the use of both agonists and antagonists that act on ₂AR but not I₁R. The use of an animal model with a genetic deletion of the I₁R might be a preferred approach, given the recent cloning this signaling protein (Piletz et al., 2000). However, the I₁R protein is an intracellular signaling protein with multiple functions in cell migration and forms a signaling complex with insulin receptor substrate 4 (Sano et al., 2002). Genetic deletion of I₁R might produce multiple effects unrelated to the response to imidazoline drugs.

This study suggests that I₁R agonists, in addition to their role in central control of blood pressure, can improve glucose homeostasis by reducing glucagon levels and attenuating insulin resistance. The effectiveness of these agents would be improved by the coadministration of ₂AR antagonists or by the development of a new generation of I₁R agonists lacking ₂AR activity. Such agents might be particularly useful to improve glucose tolerance complicated by hypertension, as in metabolic syndrome X.

	Acknowledgements

 
We thank David Bedol and Suma Rao for dedicated technical assistance.

	Footnotes

 
This study was supported by Grant HL44514 from the National Institutes of Health and by a grant from Solvay Pharmaceuticals (Hannover, Germany).

DOI: 10.1124/jpet.103.050468.

ABBREVIATIONS: ₂AR, ₂-adrenergic receptor; I₁R, I₁-imidazoline receptor; SHROB, spontaneously hypertensive obese rat; SHR, spontaneously hypertensive rat; OGTT, oral glucose tolerance test; ANOVA, analysis of variance; REMANOVA, analysis of variance with repeated measures; AUC, area under the curve; AGN 192403, 2-endo-amino-3-exo-isopropylbicyclo[2.2.1.]heptane.

Address correspondence to: Dr. Paul Ernsberger, Department of Nutrition, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4906. E-mail: pre{at}po.cwru.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Armah BI, Hofferber E, and Stenzel W (1988) General pharmacology of the novel centrally acting antihypertensive agent moxonidine. Arzneim-Forsch 38: 1426–1434.[Medline]

Barbieri C, Ferrari C, Caldara R, Testori G, Dal Bo GA, and Bertazzoni A (1980) Clonidine-induced hyperglycemia: evidence against a growth hormone-mediated effect. J Pharmacol Exp Ther 214: 433–436.[Abstract/Free Full Text]

Bock JU and Van Zwieten PA (1971) The central hyperglycemic effect of clonidine. Eur J Pharmacol 16: 303–310.[Medline]

Buccafusco JJ, Lapp CA, Westbrooks KL, and Ernsberger P (1995) Role of medullary I₁-imidazoline and ₂-adrenergic receptors in the antihypertensive responses evoked by central administration of clonidine analogs in conscious spontaneously hypertensive rats. J Pharmacol Exp Ther 273: 1162–1171.[Abstract/Free Full Text]

Chan SL, Mourtada M, and Morgan NG (2001) Characterization of a KATP channel-independent pathway involved in potentiation of insulin secretion by efaroxan. Diabetes 50: 340–347.[Abstract/Free Full Text]

De Luca N, Izzo R, Fontana D, Iovino G, Argenziano L, Vecchione C, and 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.[CrossRef][Medline]

Ditullio NW, Cieslinski L, Matthews WD, and Storer B (1984) Mechanisms involved in the hyperglycemic response induced by clonidine and other ₂-adrenoceptor agonists. J Pharmacol Exp Ther 228: 168–173.[Abstract/Free Full Text]

Efanov AM, Zaitsev SV, Berggren PO, Mest HJ, and Efendic S (2001) Imidazoline RX871024 raises diacylglycerol levels in rat pancreatic islets. Biochem Biophys Res Commun 281: 1070–1073.[Medline]

Efanova IB, Zaitsev SV, Brown G, Berggren PO, and Efendic S (1998) RX871024 induces Ca²⁺ mobilization from thapsigargin-sensitive stores in mouse pancreatic beta-cells. Diabetes 47: 211–218.[Abstract]

Ernsberger P, Damon TH, Graff LM, Christen MO, and Schäfer SG (1993) Moxonidine, a centrally-acting antihypertensive agent, is a selective ligand for I₁-imidazoline sites. J Pharmacol Exp Ther 264: 172–182.[Abstract/Free Full Text]

Ernsberger P, Giuliano R, Willette RN, and Reis DJ (1990) Role of imidazole receptors in the vasodepressor response to clonidine analogs in the rostral ventrolateral medulla. J Pharmacol Exp Ther 253: 408–418.[Abstract/Free Full Text]

Ernsberger P, Ishizuka T, Liu S, Farrell CJ, Bedol D, Koletsky RJ, and Friedman JE (1999a) Mechanisms of antihyperglycemic effects of moxonidine in the obese spontaneously hypertensive Koletsky rat (SHROB). J Pharmacol Exp Ther 288: 139–147.[Abstract/Free Full Text]

Ernsberger P, Koletsky RJ, Collins LA, and 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, and Friedman JE (1999b) Molecular pathology of the obese spontaneous hypertensive Koletsky rat: a model of syndrome X. Ann NY Acad Sci 892: 272–288.[Abstract/Free Full Text]

Ernsberger P, Kou YR, and Prabhakar NR (1998) Carotid body I₁-imidazoline receptors: binding, visualization and modulatory function. Respir Physiol 112: 239–251.[CrossRef][Medline]

Ernsberger P and Rao S (2000) Moxonidine's antihypertensive actions do not involve ₂-adrenergic receptors in radiotelemetered SHR. J Hypertens Suppl 18: S234.

Gotoh M, Iguchi A, and Sakamoto N (1988) Central versus peripheral effect of clonidine on hepatic venous plasma glucose concentrations in fasted rats. Diabetes 37: 44–49.[Abstract]

He MM, Abraham TL, Lindsay TJ, Chay SH, Czeskis BA, and Shipley LA (2000) Metabolism and disposition of moxonidine in Fischer 344 rats. Drug Metab Dispos 28: 446–459.[Abstract/Free Full Text]

Henriksen EJ, Jacob S, Fogt DL, Youngblood EB, and Gödicke J (1997) Antihypertensive agent moxonidine enhances muscle glucose transport in insulin-resistant rats. Hypertension 30: 1560–1565.[Abstract/Free Full Text]

Hirose H, Maruyama H, Itoh K, Koyama K, Kido K, and Saruta T (1992) Alpha-2 adrenergic agonism stimulates islet glucagon release from perfused rat pancreas: possible involvement of alpha-2A adrenergic receptor subtype. Acta Endocrinol 127: 279–283.

Hirose H, Seto Y, Maruyama H, Dan K, Nakamura K, and Saruta T (1997) Effects of ₂-adrenergic agonism, imidazolines and G-protein on insulin secretion in cells. Metabolism 46: 1146–1149.[CrossRef][Medline]

Hsu WH, Schaffer DD, and Pineda MH (1987) Yohimbine increases plasma insulin concentrations of dogs. Proc Soc Exp Biol Med 184: 345–349.[Abstract]

Mayer G and Taberner PV (2002) Effects of the imidazoline ligands efaroxan and KU14R on blood glucose homeostasis in the mouse. Eur J Pharmacol 454: 95–102.[Medline]

Metz SA, Halter JB, and Robertson RP (1978) Induction of defective insulin secretion and impaired glucose tolerance by clonidine: selective stimulation of metabolic -adrenergic pathways. Diabetes 27: 554–562.[Medline]

Morgan NG, Chan SL, Brown CA, and Tsoli E (1995) Characterization of the imidazoline binding site involved in regulation of insulin secretion. Ann NY Acad Sci 763: 361–373.[Medline]

Mourtada M, Brown CA, Smith SA, Piercy V, Chan SL, and Morgan NG (1997a) Interactions between imidazoline compounds and sulphonylureas in the regulation of insulin secretion. Br J Pharmacol 121: 799–805.[CrossRef][Medline]

Mourtada M, Smith SA, and Morgan NG (1997b) Insulin secretagogues with an imidazoline structure inhibit arginine-induced secretion from isolated glucagon secretion from isolated rat islets of Langerhans. Biochem Biophys Res Commun 236: 162–166.[CrossRef][Medline]

Munk SA, Lai RK, Burke JE, Arasasingham PN, Kharlamb AB, Manlapaz CA, Padillo EU, Wijono MK, Hasson DW, Wheeler LA, et al. (1996) Synthesis and pharmacologic evaluation of 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane: a potent imidazoline1 receptor specific agent. J Med Chem 39: 1193–1195.[CrossRef][Medline]

Piletz JE, Ivanov TR, Sharp JD, Ernsberger P, Chang CH, Pickard RT, Gold G, Roth B, Zhu H, Jones JC, et al. (2000) Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization. DNA Cell Biol 19: 319–329.[CrossRef][Medline]

Reaven GM (1993) Role of insulin resistance in human disease (syndrome X): an expanded definition. Annu Rev Med 44: 121–131.[CrossRef][Medline]

Reid JL (2001) Update on rilmenidine: clinical benefits. Am J Hypertens 14: 322S–324S.[Medline]

Rosen P, Ohly P, and 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.

Saito M, Saitoh T, and Inoue S (1992) Alpha 2-adrenergic modulation of pancreatic glucagon secretion in rats. Physiol Behav 51: 1165–1171.[Medline]

Sano H, Liu SC, Lane WS, Piletz JE, and Lienhard GE (2002) Insulin receptor substrate 4 associates with the protein IRAS. J Biol Chem 277: 19439–19447.[Abstract/Free Full Text]

Schafer U, Burgdorf C, Engelhardt A, Kurz T, and Richardt G (2002) Presynaptic effects of moxonidine in isolated buffer perfused rat hearts: role of imidazoline-1 receptors and 2-adrenoceptors. J Pharmacol Exp Ther 303: 1163–1170.[Abstract/Free Full Text]

Schulz A and Hasselblatt A (1988) Phentolamine, a deceptive tool to investigate sympathetic nervous control of insulin release. Naunyn-Schmiedeberg's Arch Pharmacol 337: 637–643.[Medline]

Schulz A and Hasselblatt A (1989a) An insulin-releasing property of imidazoline derivatives is not limited to compounds that block alpha-adrenoceptors. Naunyn-Schmiedeberg's Arch Pharmacol 340: 321–327.[Medline]

Schulz A and Hasselblatt A (1989b) An insulin-releasing property of imidazoline derivatives is not limited to compounds that block ₂-adrenoceptors. Naunyn-Schmiedeberg's Arch Pharmacol 340: 321–327.

Schulz A and Hasselblatt A (1989c) An insulin-releasing property of imidazoline derivatives is not limited to compounds that block -adrenoceptors. Naunyn-Schmiedeberg's Arch Pharmacol 340: 321–327.

Shah P, Vella A, Basu A, Basu R, Schwenk WF, and Rizza RA (2000) Lack of suppression of glucagon contributes to postprandial hyperglycemia in subjects with type 2 diabetes mellitus. J Clin Endocrinol Metab 85: 4053–4059.[Abstract/Free Full Text]

Struthers AD, Brown DC, Brown MJ, and Schumer B (1985) Selective ₂-receptor blockade facilitates the insulin response to adrenaline but not to glucose in man. Clin Endocrinol 23: 539–546.[Medline]

Szabo B (2002) Imidazoline antihypertensive drugs: a critical review on their mechanism of action. Pharmacol Ther 93: 1–35.[CrossRef][Medline]

Tolentino-Silva FP, Haxhiu MA, Waldbaum S, Dreshaj IA, and Ernsberger P (2000) _2A-Adrenergic receptors are not required for central anti-hypertensive action of moxonidine in mice. Brain Res 862: 26–35.[CrossRef][Medline]

Velliquette RA, Koletsky RJ, and Ernsberger P (2002) Plasma glucagon and free fatty acid responses to a glucose load in the obese spontaneous hypertensive rat (SHROB) model of metabolic syndrome X. Exp Biol Med 227: 164–170.[Abstract/Free Full Text]

This article has been cited by other articles:

				Z. Sun and P. Ernsberger Marked Insulin Resistance in Obese Spontaneously Hypertensive Rat Adipocytes Is Ameliorated by in Vivo but Not in Vitro Treatment with Moxonidine J. Pharmacol. Exp. Ther., February 1, 2007; 320(2): 845 - 852. [Abstract] [Full Text] [PDF]

				J. K. Saha, J. Xia, J. M. Grondin, S. K. Engle, and J. A. Jakubowski Acute Hyperglycemia Induced by Ketamine/Xylazine Anesthesia in Rats: Mechanisms and Implications for Preclinical Models Experimental Biology and Medicine, November 1, 2005; 230(10): 777 - 784. [Abstract] [Full Text] [PDF]

				R. A. Velliquette, J. E. Friedman, J. Shao, B. B. Zhang, and P. Ernsberger Therapeutic Actions of an Insulin Receptor Activator and a Novel Peroxisome Proliferator-Activated Receptor {gamma} Agonist in the Spontaneously Hypertensive Obese Rat Model of Metabolic Syndrome X J. Pharmacol. Exp. Ther., July 1, 2005; 314(1): 422 - 430. [Abstract] [Full Text] [PDF]