Mechanism, temporal patterns, and magnitudes of the metabolic responses to the KATP channel agonist diazoxide

Bharathi Raju and Philip E. Cryer

Division of Endocrinology, Metabolism, and Lipid Research, The General Clinical Research Center and the Diabetes Research and Training Center, Washington University School of Medicine, St. Louis, Missouri

Submitted 30 April 2004 ; accepted in final form 30 August 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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To assess the mechanism, temporal patterns, and magnitudes of the metabolic responses to the ATP-dependent potassium channel agonist diazoxide, neuroendocrine and metabolic responses to intravenous diazoxide (saline, 1.0 and 2.0 mg/kg) and oral diazoxide (placebo, 4.0 and 6.0 mg/kg) were assessed in healthy young adults. Intravenous diazoxide produced rapid, but transient, decrements (P = 0.0023) in plasma insulin (e.g., nadirs of 2.8 ± 0.5 and 1.8 ± 0.3 µU/ml compared with 7.0 ± 1.0 µU/ml after saline at 4.0–7.5 min) and C-peptide (P = 0.0228) associated with dose-related increments in plasma glucose (P = 0.0044) and serum nonesterified fatty acids (P < 0.0001). After oral diazoxide, plasma insulin appeared to decline, as did C-peptide, again associated with dose-related increments in plasma glucose (P < 0.0001) and serum nonesterified fatty acids (P = 0.0141). Plasma glucagon, as well as cortisol and growth hormone, was not altered. Plasma epinephrine increased (P = 0.0215) slightly only after intravenous diazoxide. There were dose-related increments in plasma norepinephrine (P = 0.0038 and P = 0.0005, respectively), undoubtedly reflecting a compensatory sympathetic neural response to vasodilation produced by diazoxide, but these would not raise plasma glucose or serum nonesterified fatty acid levels. Thus selective suppression of insulin secretion, without stimulation of glucagon secretion, raised plasma glucose and serum nonesterified fatty acid concentrations. These findings define the temporal patterns and magnitudes of the metabolic responses to diazoxide and underscore the primacy of regulated insulin secretion in the physiological regulation of postabsorptive carbohydrate and lipid metabolism.

glucose; insulin; glucagon; epinephrine; norepinephrine; adenosine 5'-triphosphate-dependent potassium channel


DIAZOXIDE IS AN ATP-dependent potassium (KATP) channel agonist that is used clinically to treat hyperinsulinemic hypoglycemia (2, 8, 10, 11, 13, 21, 25, 34). It opens potassium channels and hyperpolarizes plasma membranes, including those of pancreatic {beta}-cells and thus decreases insulin secretion (1, 2, 6, 7, 8, 10, 11, 13, 21, 23, 25, 34). However, diazoxide is a nonselective KATP channel agonist (6, 23), KATP channels are expressed widely (1, 6, 7), and alternative mechanisms of its plasma glucose-raising action have been proposed (10, 11). One example of the widespread actions of diazoxide is its vasodilating effect (3, 22). Indeed, the drug has been used clinically for the urgent treatment of hypertension (3, 22). After rapid intravenous injection, the onset of the effect to reduce vascular resistance is rapid (minutes) but, because the drug is >90% protein bound, prolonged (hours). Despite the initial studies in humans decades ago (8, 10, 11, 21) and the use of the drug clinically (1, 2, 3, 8, 10, 11, 13, 21, 25, 34), the precise mechanism, temporal patterns, and magnitudes of the metabolic responses to diazoxide have not been defined systematically. To do so, we measured neuroendocrine and metabolic responses to intravenous and oral diazoxide in healthy young adults and thus tested the hypothesis that suppression of insulin secretion, without stimulation of glucagon secretion, raises the postabsorptive plasma glucose concentration.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. Twelve individuals (6 women and 6 men; mean ± SD: age = 23 ± 3 yr and mean body mass index = 25 ± 3 kg/m2) gave their written consent to participate in the study of intravenous diazoxide. Twelve individuals (6 women and 6 men; mean ± SD: age = 25 ± 5 yr and mean body mass index = 27 ± 7 kg/m2) gave their written consent to participate in the study of oral diazoxide. The studies were approved by the Washington University Medical Center Human Studies Committee (Institutional Review Board) and conducted in the outpatient facilities of the Washington University General Clinical Research Center. Participants were judged to be healthy on the basis of a medical history and physical examination. None had a personal or family history of diabetes mellitus. All had normal fasting plasma glucose concentrations, serum creatinine concentrations, and hematocrits.

Experimental design. Studies were performed in the morning after an overnight fast. Aside from measurements of blood pressures and heart rates with the subjects supine and at 2 and 5 min of standing before and after each study, subjects were in the supine position throughout. Intravenous lines were inserted in a hand vein (with that hand kept in an ~55°C Plexiglas box for arterialized venous sampling) and an antecubital vein (for drug or saline injection and saline infusion). In the intravenous diazoxide study, arterialized venous samples were drawn, and blood pressures and heart rates were recorded (Propaq Encore; Protocol Systems, Beverton, OR) at –30, –15, 0, 2, 4, 7.5, 10, 12, 15, 22.5, 30, 45, and 60 min. Saline, 1.0 mg/kg diazoxide (Hyperstat; Schering-Plough, Kenilworth, NJ), or 2.0 mg/kg diazoxide were injected intravenously after the 0-min sample. Saline or 1.0 mg/kg diazoxide was given in random sequence in six subjects to establish the safety (with respect to blood pressure lowering) of that diazoxide dose. Next, saline, 1.0 mg/kg diazoxide, or 2.0 mg/kg diazoxide were given in random sequence in 10 additional subjects, and 2.0 mg/kg diazoxide were also given in two of the original subjects. In the oral diazoxide study, arterialized venous samples were drawn, and blood pressures and heart rates were recorded at –30, –15, 0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 210, and 240 min. Arginine hydrochloride, 5.0 g intravenously, was then administered, and samples were obtained 3, 5, and 7 min thereafter. Placebo, 4.0 mg/kg diazoxide (Proglycem; Schering-Plough), or 6.0 mg/kg diazoxide were administered orally after the 0-min sample. Placebo or 4.0 mg/kg diazoxide were given in random sequence in all 12 subjects to establish the safety (with respect to blood pressure lowering) of that diazoxide dose. Thereafter, 6.0 mg/kg diazoxide were given to six of the subjects. The electrocardiogram was monitored throughout all studies.

Analytic methods. Plasma glucose concentrations were measured with a glucose oxidase method (Yellow Springs Analyzer 2; Yellow Springs Instruments, Yellow Springs, OH). Plasma insulin (16), C-peptide (16), glucagon (9), pancreatic polypeptide (14), growth hormone (30), and cortisol (12) were measured with RIAs. The insulin, C-peptide, glucagon, and pancreatic polypeptide assays were performed with materials purchased from Linco Research (St. Louis, MO) and the cortisol assay with materials purchased from Diasorin (Stillwater, MN). An antibody provided by the National Institutes of Health was used for the growth hormone assay. Plasma epinephrine and norepinephrine were measured with a single isotope derivative (radioenzymatic) method (31). Serum nonesterified fatty acids (15) and blood lactate (19) and {beta}-hydroxybutyrate (26) were measured with enzymatic techniques.

Statistical methods. Data are expressed as means ± SE, except where the SD is specified. Baseline adjusted data were analyzed by mixed-model repeated-measures ANOVA. P values <0.0500 were considered to indicate significant differences. Condition or condition x time interaction P values are reported to indicate statistically significant differences among the three curves for those variables. Post hoc statistical testing was not performed.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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Intravenous diazoxide. Dose-related decrements in plasma insulin (P = 0.0023; Fig. 1) and increments in plasma glucose (P = 0.0044) and serum nonesterified fatty acids (P < 0.0001; Fig. 2) followed intravenous diazoxide administration. Plasma C-peptide also decreased (P = 0.0228; Table 1). Plasma glucagon (Fig. 3) was not altered. Plasma epinephrine (Fig. 4) increased (P = 0.0215) slightly, but only to a peak of 29 ± 5 pg/ml. There were dose-related increments (P = 0.0038) in plasma norepinephrine (Fig. 4). Plasma pancreatic polypeptide (Table 1) and plasma cortisol and growth hormone (data not shown) were not altered nor were blood lactate and {beta}-hydroxybutyrate (data not shown). Heart rate (Table 2) and systolic blood pressure (Table 3) were not altered significantly, but diastolic blood pressure (Table 3) decreased (P = 0.0072).



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Fig. 1. Mean ± SE plasma insulin before and after intravenous saline (shaded area), 1.0 mg/kg diazoxide ({circ}), or 2.0 mg/kg diazoxide ({bullet}). ANOVA, P = 0.0023.

 


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Fig. 2. Mean ± SE plasma glucose (ANOVA, P = 0.0044) and serum nonesterified fatty acids (NEFA; ANOVA, P = 0.0001) before and after intravenous saline (shaded areas), 1.0 mg/kg diazoxide ({circ}), or 2.0 mg/kg diazoxide ({bullet}).

 

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Table 1. Plasma C-peptide and pancreatic polypeptide before and after intravenous saline, 1.0 mg/kg diazoxide, or 2.0 mg/kg diazoxide

 


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Fig. 3. Mean ± SE plasma glucagon before and after saline (shaded area), 1.0 mg/kg diazoxide ({circ}), or 2.0 mg/kg diazoxide ({bullet}).

 


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Fig. 4. Mean ± SE plasma norepinephrine (ANOVA, P = 0.0008) and epinephrine (ANOVA, P = 0.0215) before and after intravenous saline (shaded areas), 1.0 mg/kg diazoxide ({circ}), or 2.0 mg/kg diazoxide ({bullet}).

 

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Table 2. Heart rate before and after intravenous saline, 1.0 mg/kg diazoxide, or 2.0 mg/kg diazoxide

 

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Table 3. Systolic and diastolic BP before and after intravenous saline, 1.0 mg/kg diazoxide, or 2.0 mg/kg diazoxide

 
Oral diazoxide. Apparent, but statistically nonsignificant, decrements in plasma insulin (Fig. 5) and C-peptide (Table 4) and significant dose-related increments in plasma glucose (P < 0.0001) and serum nonesterified fatty acids (P = 0.0141; Fig. 6) followed oral diazoxide administration. Plasma glucagon (Fig. 7) and epinephrine (Fig. 8) were not altered. There were dose-related increments (P = 0.0005) in plasma norepinephrine (Fig. 8). Plasma pancreatic polypeptide (Table 4) and plasma cortisol and growth hormone (data not shown) were not altered nor were blood lactate and {beta}-hydroxybutyrate (data not shown). Heart rate (Table 5) increased slightly (P = 0.0031); systolic and diastolic blood pressures (Table 6) were not altered significantly, although diastolic blood pressure tended to decrease. Diazoxide administration did not reduce the plasma glucagon (Fig. 7) or insulin (Fig. 5) responses to arginine.



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Fig. 5. Mean ± SE plasma insulin before and after oral placebo (shaded area), 4.0 mg/kg diazoxide ({circ}), or 6.0 mg/kg diazoxide ({bullet}). Arg, arginine (5.0 g iv).

 

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Table 4. Plasma C-peptide and pancreatic polypeptide before and after oral placebo, 4.0 mg/kg diazoxide, or 6.0 mg/kg diazoxide

 


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Fig. 6. Mean ± SE plasma glucose (ANOVA, P < 0.0001) and serum NEFA (ANOVA, P = 0.0141) before and after oral placebo (shaded areas), 4.0 mg/kg diazoxide ({circ}), or 6.0 mg/kg diazoxide ({bullet}). Arg, arginine (5.0 g iv).

 


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Fig. 7. Mean ± SE plasma glucagon before and after oral placebo (shaded area), 4.0 mg/kg diazoxide ({circ}), or 6.0 mg/kg diazoxide ({bullet}). Arg, arginine (5.0 g iv).

 


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Fig. 8. Mean ± SE plasma norepinephrine (ANOVA, P = 0.0005) and epinephrine before and after oral placebo (shaded areas), 4.0 mg/kg diazoxide ({circ}), or 6.0 mg/kg diazoxide ({bullet}). Arg, arginine (5.0 g iv).

 

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Table 5. Heart rate before and after oral placebo, 4.0 mg/kg diazoxide, or 6.0 mg/kg diazoxide

 

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Table 6. Systolic and diastolic BP before and after oral placebo, 4.0 mg/kg diazoxide, or 6.0 mg/kg diazoxide

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
These data document that both intravenous and oral diazoxide, compared with intravenous saline and oral placebo, respectively, cause dose-related increments in plasma glucose and serum nonesterified fatty acid concentrations in healthy individuals. Plasma insulin and C-peptide concentrations decreased sharply, but transiently, after intravenous diazoxide administration and appeared to decline, despite increments in circulating glucose and nonesterified fatty acid levels, after oral diazoxide administration. The data provide no plausible explanation for the diazoxide-induced glucose and nonesterified fatty acid elevations other than selective suppression of insulin secretion.

The plasma concentrations of glucagon, which can increase plasma glucose (but not serum nonesterified fatty acid) concentrations rapidly (4), did not increase after diazoxide administration. Glucagon is secreted in the hepatic portal circulation, exerts its glucose-raising action on the liver, and is partially cleared by the liver (4). Thus it is at least conceivable that there were diazoxide-induced increments in portal glucagon that were not detected in the peripheral circulation that we sampled. However, diazoxide does not stimulate glucagon release from the perfused rat pancreas (23). In contrast to glucagon, epinephrine is secreted in the peripheral circulation that we sampled. The plasma concentrations of epinephrine, which can increase plasma glucose and serum nonesterified concentrations rapidly (5), were unaltered after oral diazoxide administration and increased only slightly after intravenous diazoxide. The latter epinephrine levels were clearly not high enough to raise plasma glucose or serum nonesterified fatty acid levels (5). The plasma concentrations of growth hormone and cortisol, hormones that can increase plasma glucose and nonesterified fatty acid levels, albeit over several hours (20, 27), also did not increase after diazoxide administration.

Plasma norepinephrine concentrations (an index of sympathetic neural activation under these conditions in which there were essentially no increments in plasma epinephrine concentrations, a specific measure of adrenomedullary activation) increased in a dose-related fashion after both intravenous and oral diazoxide administration. Sympathetic neural activation was undoubtedly a central nervous system-mediated compensatory response to the vasodilatory effect of diazoxide that prevented the development of hypotension. However, plasma norepinephrine elevations to levels much higher than those that occurred after diazoxide administration are required to raise plasma glucose and serum nonesterified fatty acid concentrations (32), and neither pharmacological nor hemodynamic sympathetic neural activation raises postabsorptive plasma glucose or serum nonesterified fatty acid concentrations (17, 18, 24). Indeed, Navegantes et al. (24) not only found no increase in circulating glucose but also no increase in interstitial muscle or adipose tissue glucose concentrations during sympathetic neural activation induced by lower body negative pressure. They also found no increase in circulating glycerol despite an ~25–30% increase in the interstitial adipose tissue glycerol concentration; nonetheless, given the latter findings, it is conceivable that sympathetic neural activation contributed to the increase in circulating nonesterified fatty acid levels that we observed.

Because there were increments, rather than decrements, in plasma glucose concentrations, glucose autoregulation (glucose production as an inverse function of ambient glucose levels independent of hormonal and neural signals; see Ref. 29) cannot be invoked to explain the observed increments in plasma glucose concentrations that followed diazoxide administration. Increased nonesterified fatty acid levels per se do not raise plasma glucose concentrations in the time frame observed in the present study (28, 33), nor do increased plasma glucose levels raise serum nonesterified fatty acid concentrations.

These data also document the safety, with respect to blood pressure lowering, of the intravenous and oral doses of diazoxide administered. As noted earlier, diazoxide undoubtedly caused vasodilation, but hypotension did not occur because of a compensatory sympathetic neural response.

On the basis of these data, we conclude that the plasma glucose-raising (and the serum nonesterified fatty acid-raising) action of diazoxide is the result of selective suppression of insulin secretion. These data define the temporal patterns and magnitudes of the metabolic responses to diazoxide and underscore the primacy of regulated insulin secretion in the physiological regulation of postabsorptive carbohydrate and lipid metabolism. They document that suppression of insulin secretion, without stimulation of glucagon secretion, raises the postabsorptive plasma glucose concentration.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported, in part, by National Institutes of Health Grants R37 DK-27085, MO1 RR-00036, and P60 DK-20579 and a fellowship award from the American Diabetes Association.


    ACKNOWLEDGMENTS
 
We acknowledge the assistance of the nursing staff of the Washington University General Clinical Research Center in the performance of this study, the assistance of Krishan Jethi, Cornell Blake, Joy Brothers, Zina Lubovich, and Michael Morris in the analytic work, and the assistance of Janet Dedeke in the preparation of this manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. E. Cryer, Campus Box 8127, Washington Univ. School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110 (E-mail: pcryer{at}wustl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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