Adrenergic mechanisms in recovery from hypoglycemia in man: adrenergic blockade WILLIAM L. CLARKE, JULIO V. SANTIAGO, EHUD BEN-GALIM, MOREY W. HAYMOND,
Metabolism Divisions, Departments of Pediatrics School of Medicine, St. Louis, Missouri 63110 CLARKE, WILLIAM L., JULIO V. SANTIAGO, LORRAINE THOMAS, EHUD BEN-GALIM, MOREY W. HAYMOND, AND Pnnrp E. CRYER. Adrenergic mechanisms in recovery from hypoglycemia in man: adrenergic blockade. Am. J. Physiol.
236(2):E147-E152,1979or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 5(2): El47-E152, 1979.- Simultaneous glucoseand alanine turnover rates, along with static measurements of counterregulatory hormonesand selectedmetabolic intermediates, were determined during insulin-induced hypoglycemia in normal subjectsinfused with saline, phentolamine (an alpha-adrenergic blocking agent), and propranolol (a betaadrenergic blocking agent) in separate experiments. In the control studiesplasmaglucosefell from 87 t 4 to 35 t 4 mg/dl 30 min after intravenous crystalline insulin (0.05 U/kg). Glucoseutilization doubled and then returned to baseline by 40min, whereasglucoseproduction fell initially, began to rise by 30 min, and reached maximal rates twice baseline by 40 min after insulin. Hypoglycemia was associatedwith increments in plasma epinephrine, norepinephrine, glucagon, growth hormone, and cortisol; blood lactate rose transiently after insulin injection, whereas blood beta-hydroxybutyrate was suppressedinitially but rose during glucose recovery. Despite hemodynamic and metabolic evidence of alpha- and beta-adrenergic blockade during the infusion of phentolamine and of propranolol, respectively, base-line and postinsulin plasmaglucoseconcentrations, glucoseutilization and production rates, and rates of glucoseformation from alanine did not differ from those of the control study. Thus, adrenergic mechanismsdo not appear to play a primary role in recovery from insulin-induced hypoglycemia in man. Rather, on the basisof other evidence, adrenergic mechanisms appear to play a secondary role in that they becomecritical to recovery from hypoglycemia only in the absenceof other counterregulatory mechanisms. catecholamines;epinephrine; norepinephrine; glucose kinetics; alanine kinetics; glucagon; cortisol; growth hormone
fromhypoglycemiaorof prevention of hypoglycemia have not been clearly defined either in animals or human subjects. Glucose recovery from insulin-induced hypoglycemia, the most widely studied model, cannot be attributed solely to the disappearance of insulin because in a previous study in our laboratory (8) glucose kinetic studies documented that glucose counterregulation was well under way at a time when mean plasma insulin concentrations were still tenfold over basal levels. Five hyperglycemic fac-
THE MECHANISMSOFRECOVERY
0363-6100/79/0000-0000$01.25
LORRAINE THOMAS, AND PHILIP E. CRYER
and Medicine,
Washington
University
tors (glucagon, epinephrine, norepinephrine, growth hormone, and cortisol) are released during hypoglycemia in man (8), whereas epinephrine, norepinephrine, and growth hormone have been shown to be released during relatively rapid physiological decrements (95-60 mg/dl) in the plasma glucose concentration in normal and diabetic subjects (3). Thus, these five hyperglycemic factors are potentially important glucose counterregulatory factors. Available evidence is consistent with an important role for the catecholamines in recovery from hypoglycemia. Not only is there major release of epinephrine and norepinephrine during hypoglycemia, but only the release of these catecholamines precedes or coincides with the onset of kinetically determined glucose counterregulation, whereas demonstrable release of glucagon, cortisol, and growth hormone follows the onset of ,glucase counterregulation (8). Although the hyperglycemic effect of the catecholamines is well known, it remains to be determined whether this effect is mediated through alpha- or beta-adrenergic mechanisms. Studies with isolated rat hepatocytes (11, 14) and perfused rat livers (6, 7, 12) suggest that catecholamine-stimulated glucose production is mediated through alpha-adrenergic mechanisms, whereas studies in other species (24) suggest a more important role for beta-adrenergic mechanisms. In human subjects, beta-adrenergic blockade has been reported to impair recovery from hypoglycemia (1) or to hav e no effect (25) on recovery from hypoglycemia as studied with static plasma glucose concentrations. Furthermore, studies in patients with spinal cord transections and resultant deficits in sympathetic neural outflow have yielded apparently conflicting results. Brodows and co-workers (2) observed a subnormal-to-absent rise in the plasma glucose concentration after administration of 2-deoxyglucose to such patients, whereas Palmer and co-workers (19) found normal glucose recovery from insulin-induced hypoglycemia in cord-sectioned patients. Thus, the relative role of adrenergic mechanisms among other potentially important glucose counterregulatory factors remains to be determined. To clarify the role of adrenergic mechanisms in recovery from hypoglycemia, glucose, and alanine (a major gluconeogenic precursor) turnover studies - along with static measurements of plasma glucagon, epinephrine,
Copyright 0 1979 the American Physiological Society
El47
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
El48
norepinephrine, cortisol, and growth hormone; and blood lactate and beta-hydroxybutyrate -were performed during saline infusion, alpha-adrenergic blockade with phentolamine, and beta-adrenergic-blockade with propranolol before and during insulin-induced hypoglycemia in normal human subjects. METHODS
Subjects. Four healthy adult male volunteers (ages 27-35) were studied on the Washington University Clinical Research Center on 3 separate days - while at complete bed rest. Informed consent was obtained in each case. Each was within 10% of ideal body weight (Metropolitan Life Insurance Co. tables) and was fasted overnight prior to study. Study protocol. After the insertion of intravenous needles, initial blood samples were obtained. A priming dose of [3-3H]glucose (0.15 &i/kg) and [UJ4C]alanine (0.05 &i/kg) (Amersham/Searle, Burlington Heights, IL) was infused followed by a continuous 5-h infusion of both isotopes (glucose 0.075 &i kg+ h-l, alanine 0.1 &i kg-l h-l). The specific activity of the [3-3H]glucose was 10 Ci/mmol and that of the [UJ4C]alanine was 164 mCi/mmol. Infusion rates were calibrated after each study and samples of the infusate were saved for counting. Venous samples were obtained at - 180, -90, -75, and -60 min, at which time a randomly selected continuous intravenous infusion of either saline, phentolamine (5 mg by rapid injection followed by infusion at 0.5 mg/min), or propranolol (1 mg by rapid injection followed by infusion at 0.1 mg/min> was begun through an independent intravenous site and continued for 3 h. Samples were obtained at -50, -40, -30, -15, and 0 min, at which time 0.05 U/kg of crystalline insulin was given intravenously. Venous samples were collected subsequently at 10-min i.ntervals over the next hour and every 15 min during the subsequent 5th h of the study. Blood pressure and pulse rate were recorded prior to obtaining each sample. Analytical methods. Each sample was divided into four separate chilled tubes. Samples for determination of metabolic intermediates and specific activities were mixed immediately with equal volumes of 3 M perchloric acid. Samples for catecholamine determination were placed into tubes containing a final concentration of of 5 n&I reduced glutathione, thoSe for determination glucagon and insulin into tubes containing 1,000 U Trasylol (FBA Pharmaceuticals, New York), and those for determination of plasma glucose, alanine, growth hormone, and cortisol into heparinized tubes. All samples were placed on ice, centrifuged at 4”C, and separated, and supernatants were stored at -2OOC (perchloric acid extracts were stored at 1800C). Plasma glucose was measured with a Beckman glucose analyzer. Plasma alanine (:13), blood lactate, and (17) were determined microfluobeta-hydroxybutyrate rometrically. Plasma catecholamines were measured utilizing a single isotope derivative method (5). Neither phentolamine nor propranolol cross-react in this assay (4). Insulin (9), growth hormone (21), and glucagon (16)
CLARKE
ET
AL.
were measured utilizing double-antibody radioimmunoassay techniques. Plasma cortisol was determined by a competitive protein binding technique (18). Glucose and alanine were recovered from the perchlorate samples for determination of specific ,activity utilizing serial ion exchange chromatography with Dowex 5OW-18 and AGI-X8 (Bid Rad Laboratories, Richmond, Ca) resins (15). Alanine was further isolated by ion exchange chromatography utilizing PA28 resin (Beckman, Palo Alto, Ca). Glucose recovery averaged 96%, whereas alanine recovery averaged 82%. Glucose and alanine fractions were diluted with 3 ml scintillation cocktail (Radiation Products, Inc., Elk Grove Village, IL) and counted for 100 min. All counts were corrected for 14C spillover. Glucose kinetics were calculated using a modification of Steele’s equations (23) for nonsteady-state conditions. Alanine conversion to glucose was determined using modifications of the glucose-lactate equations of Kreisberg (15). Statistical evaluation of the data was performed utilizing the paired t test. RESULTS
Prior to the initiation of adrenergic blockade, plasma glucose concentrations, glucose kinetics, glucose formation from alanine, plasma alanine concentrations, alanine kinetics, and plasma norepinephrine and epinephrine concentrations for the saline control did not differ significantly (Figs. l-3). Similarly, basal plasma cortisol, growth hormone, and glucagon levels (Fig. 5) and blood lactate and P-hydroxybutyrate concentrations were comparable for all three studies. Effects of alpha- and beta-adrenergic blockade on basal data. The administration of phentolamine resulted in a 16% increase in pulse rate and a 19% fall in
diastolic blood pressure, whereas propranolol produced a 13% decrease in pulse rate without altering supine blood pressure (Table 1). Plasma norepinephrine concentrations rose significantly from base-line values of 189 t 36 to 380 t 54 pg/ ml at -30 min (P c 0.02) during phentolamine administration and to 478 t 69 pg/ml at 0 min, whereas glucose and alanine concentrations and kinetics, as well as circulating concentrations of epinephrine, glucagon, cortisol, growth hormone, lactate, and beta-hydroxybutyrate were unaffected by the infusion of either phentolamine or propranolol (Figs. 3-5). Effects of insulin-induced hypoglycemia d luring alpha- and beta-adrenergic blockade. A&r the adminis-
tration of insulin, plasma glucose concentrations decreased over the first 30 min to nadirs of 35 t 4, 39 t 5, and 40 t 4 mg/dl during the saline, phentolamine, and propranolol studies, respectively. Plasma glucose concentrations subsequently increased to base-line levels by 90 min. During the adrenergic blockade studies, plasma glucose concentrations were similar to those obtained during saline infusion (Fig. 1). After insulin administration, no significant differences were noted in the rates of plasma insulin clearance between saline and adrenergic blockade studies. Mean plasma insulin concentrations were 171 t 12, 158 t 22, and 163 t 6
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
ADRENERGIC
MECHANISMS
IOOr
IN HYPOGLYCEMIA
El49
IN MAN
saline infusion. Glucose production from alanine decreased 20 min after insulin, but then rose significantly (P c 0.05) over th e f o11owing 10 min in all three studies. By 40 min glucose production from alanine was 268, 226, and 160% (saline, phentolamine, and propranolol,
I
400
W
40 I
40 I
PRODUCTION
I
II
c -I
I_
.
UTILIZATION
W zz
a1 a -60
0
60
I
o+’
I
I
I
I
I
11
1
I
J
;
a -I a w
I I
!
I
a
Z
aJ 0 E a
II
r
120
0 E 1
20ti O
-60
0
UTILIZATION
I I
60
120
FIG. 2. Mean (&SE) plasma alanine concentrations and alanine kinetics before and during (-60 through 120, denoted by dashed vertical lines) infusion of saline (solid lines), phentolamine (dashed lines), or propranolol (dotted lines). Insulin was injected at 0 min (arrows).
FIG. 1. Mean (*SE) plasma glucose concentrations, glucose kinetics, and glucose production from alanine in normal subjects before and during (-60 through 120 min, dashed vertical lines), infusion of saline (solid lines), phentolamine (dashed lines), or propranolol (dotted lines). Insulin was injected at 0 min (arrows).
pU/ml at 10 min and fell with half-times of disappearance of 6,8, and 7 min to levels below 20 pU/ml after 50 min in the saline, phentolamine, and propranolol studies, respectively. During all three study periods glucose utilization increased 98-166% above baseline by 10 min (P < 0.05), returned to basal base-line rates by 40 min, and remained constant throughout the rest of the study (Fig. 1). Glucose production was decreased by 20 min, increased at 30 min, and reached maximal production rates (219 t 29,216 t 64, and 184 t 42% above baseline for the saline control, phentolamine, and propranolol, respectively) at 40 min. Glucose production remained significantly elevated (P < 0.05) for the remainder of the study with values 53 t 12, 93 t 30, and 86 t 41% (saline, phentolamine, and propranolol) above base-line production rates 120 min after insulin (Fig. 1). Glucose production and utilization rates were similar in all three studies except that glucose utilization was slightly diminished during propranolol infusion at 20 min (P c 0.02) when compared with the same time point during
jr 0
0
2
L
a0 3I a
-60
0
60
120
FIG. 3. Mean (*SE) plasma cortisol, growth hormone, and glucagon concentrations before and during (-60 through 120, denoted by interrupted vertical lines) infusion of saline (solid lines), phentolamine (dashed lines), or propranolol (dotted lines). Insulin was injected at 0 min (arrows).
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
El50
CLARKE
TABLE
1. Hemodynamic
during
adrenergic
effects observed blockade pulse
Control study Baseline Saline Saline hypoglycemia
R.& per min
58 + 3 59 2 4 85 f 3
‘ygd mmHg
’
112 + 3 114 + 2 117 +, 11
Diastolic Blood Pressure, dg
74 2 2 72 + 3 1 f 1
ET AL.
to rise with recovery from hypoglycemia (P < 0.05 compared to saline control). Blood lactate concentrations rose significantly after insulin administration during all three studies (P < 0.05). The change in lactate concentrations during propranolol infusion was less than that observed during the saline control study (P < 0.05). Lactate concentrations had returned to baseline levels by 120 min in each study (Fig. 5). DISCUSSION
Alpha-adrenergic Baseline Phentolamine Phentolamine
blockade study hypoglycemia
Beta-adrenergic blockade study Baseline Propranolol Propranolol h-ypwlycemia a
_-
64 +, 4 74 2 10 108 f 10
106 f 4 116 + 3 111 + 21
69 + 1 53 2 7 42 + 4
64 + 1 54 It 2 55 2 4
110 + 2 107 & 13 114 + 7
70 A 3 70 + 3 76 f: 6
Elevated plasma catecholamine concentrations during the combined infusion of phentolamine and propran0101 (4) and during phentolamine alone (10) have been previously reported. In the present study, marked elevations in plasma norepinephrine but not epinephrine were observed during phentolamine infusion, whereas
c
--
Values are means + SE.
respectively) a.bove base-line conversion rates (P < 0.05) and remained elev pated for the duration of each study (Fig. 1). Alanine kinetics remained constant throughout this period and were not influenced by insulin, phentolamine, or propranolol (Fig. 2). Plasma cortisol concentrations rose 30 min after insulin and were significantly elevated above base-line concentrations by 50 min Cp < 0.05), but were unaffected by adrenergic blockade (Fig. 3). During the saline study, plasma growth hormone concentrations rose 30 min (P c 0.02) after insulin administration and reached maximal concentrations at 60 min (Fig. 3). In contrast, phentolamine administration blunted the plasma growth hormone response to hypoglycemia (P < 0.05 from 4090 min). Growth hormone concentrations during propranolol infusion were not statistically different from those of the saline control study. During the saline study, plasma glucagon concentrations rose to concentrations 41% above baseline at 40 min. Similar increments occurred during the phentolamine and proprano101studies (Fig. 3). Plasma epinephrine concentrations rose from 13 t 6 to 896 t 55 pg/ml 30 min after insulin (P c 0.02) and were not significantly different from saline control values during either the phentolamine or propranolol infusion (Fig. 4). Plasma norepinephrine responses, however, were strikingly dissimilar during phentolamine infusion. Norepinephrine concentrations rose identically in response to hypoglycemia during the saline control and propranolol infusions to levels 70% above baseline at 30 min (P < 0.02). The phentolamine infusion produced elevated base-line norepinephrine levels and rise (200% above time 0 values) in circulating norepinephrine after insulin administration, with significant elevations as early as 20 min. No diminution in this response was seen after the return of plasma glucose concentrations to preinsulin values. Blood beta-hydroxybutyrate concentrations decreased after insulin administration in all subjects (Fig. 5) and then rose to concentrations above base-line values during the saline control (P c 0.05) and phentolamine infusion (P < 0.01) studies. However, during proprano101infusion. beta-hvdroxvbutvrate concentrations failed
1200
I
w z
800
ho CL z4 00 Q, W
un
-60
0
60
120
4. Mean (*SE) plasma norepinephrine and epinephrine concentrations before and during (-60 through 120, denoted by dashed vertical lines) infusion of saline (solid lines), phentolamine (dashed lines) or propranolol (dotted lines). Insulin was injected at 0 min (arrows). FIG.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
ADRENERGIC
0 Ed
200= a
W
s a B
Sal9
s a Is
L
z
o--v -
200-
Q + 5f= oE Q
El51
IN MAN
BLOCKADE HYPOGLYCEMIA RECOVERY
-03
200
5- a E e x
IN HYPOGLYCEMIA
BASELINE
E
u a c2
MECHANISMS
sa13
s
2 0
1Q B
s
a
2> E
L
/3
s
a
P
FIG. 5. Mean (*SE) base-line blood beta-hydroxybutyrate and lactate concentrations and change ( *SE) from baseline during infusion of saline (s), phentolamine ((Y), or propranolol (p) before insulin (blockade alone), during hypoglycemia and during recovery from hypoglycemia. *, denotes significant differences (P < 0.05) from changes observed during saline control study.
0
neither catecholamine changed significantly during propranolol administration. Elevated norepinephrine concentrations during phentolamine infusion could be due to 1) a direct effect of alpha-adrenergic blockade on the central nervous system resulting in increased sympathetic neural activity, 2) reflex activation of sympathetic activity secondary to hemodynamic changes caused by postsynaptic alpha-adrenergic blockade and mediated through the central nervous system, or 3) a direct effect of alpha-adrenergic blockade of presynaptic alpha receptors on axon terminals of sympathetic postganglionic neurons (22) resulting in accelerated norepinephrine release. Because mechanisms involving central stimulation of the sympathetic nervous system would be expected to accelerate release of both adrenomedullary epinephrine and sympathetic neural norepinephrine, the finding of selective norepinephrine release during alpha-adrenergic blockade favors the third possibility. Previous studies (1,25> of the effect of beta-adrenergic blockade on glucose recovery from hypoglycemia have employed only static measurements of plasma glucose concentrations and have yielded conflicting results. We have examined the effect of both alpha- and beta-adrenergic blockade on glucose recovery from insulin-induced hypoglycemia in normal human subjects by employing sensitive measurements of glucose and alanine kinetics as well as frequent static determinations of the plasma concentrations of the currently recognized, potentially important glucose counterregulatory factors (epinephrine, norepinephrine, glucagon, cortisol, and growth hormone), in order to clarify the relative role of adrenergic mechanisms in recovery from hypoglycemia. Neither alpha-adrenergic blockade with phentolamine nor beta-adrenergic blockade with propranolol altered basal glucose or alanine kinetics or the kinetic responses to insulin-induced hypoglycemia in this study. Because only partial adrenergic blockade can be produced in human subjects, it is conceivable that insufficient blockade was produced to impact on glucose counterregulation. However, the appropriate hemodynamic changes during the infusion of phentolamine and
of propranolol, the blunted growth hormone response during recovery from hypoglycemia during phentolamine infusion, and the inhibition of a beta-hydroxybutyrate response to hypoglycemia during propranolol infusion attest to the production of adrenergic blockade. Thus, particularly in view of the sensitive methods employed, some impact of adrenergic blockade should have been discernible if adrenergic mechanisms play a primary role in the counterregulatory response to hypoglycemia. The lack of evidence supporting a primary role of adrenergic mechanisms in recovery from hypoglycemia in this study is consistent with recent observations in normal and-adrenalectomized subjects infused with somatostatin during insulin-induced hypoglycemia (un-- -published observations). In these studies, recovery Corn hypoglycemia in normal subjects was impaired during somatostatin infusion, an effect reversed by glucagon but not growth hormone replacement. That major glucose recovery, although impaired, did occur was attributed to augmented catecholamine release. However, as demonstrated by the failure of glucose recovery from hypoglycemia in somatostatin-infused adrenalectomized subjects, only in the absence of glucagon did an adrenal factor, probably epinephrine, become critical to recovery from hypoglycemia. Thus, adrenergic mechanisms appear to play a secondary role in recovery from insulin-induced hypoglycemia only i.n the absence of other counterregulatory mechanisms, notabl .y glucagon secretion. If so, one would expect adrenergic blockade to impair recovery from hypoglycemia only in the absence of glucagon release. Preliminary evidence (20) supports this hypothesis. The authors acknowledge the technical assistance of Suresh Shah, Rivka Levine, Joy Brothers, and Dan Dallas. This investigation was supported in part by National Institutes of Health Research Service Award lF32AM05544-01 and Grants AM 20579, HDAM06355 and RR00036 and by grants from the Diabetic Children’s Welfare Association and American Diabetes Association, Greater St. Louis Affiliate. Address requests for reprints to: J. V. Santiago: 500 South Kingshighway, Box 14871, St. Louis, MO 63178. Received 1 May 1978; accepted in final form 15 September 1978
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
El52
CLARKE
ET AL.
REFERENCES E . , AND R. ARKY. Role of beta-adrenergic receptors in counter-regulation to insulin-induced hypoglycemia. Diabetes
1. ABRAMSON,
17: 141-146,1968. 2. BRODOWS, R.
G., F. X. PI-SUNYER, AND R. G. CAMPBELL. control of counterregulatory events during glucopenia
J. Clin. Invest. 3. CLARKE, W.,
52: 1841-1844,
Neural in man.
1973.
J. SANTIAGO, AND P. CRYER. Catecholamine and growth hormone release with physiologic decrements in the plasma glucose concentration in normal and diabetic man (Abstract). CZin. Res. 25: 561A, 1977. 4. CRYER, P. E., M. W. HAYMOND, J. V. SANTIAGO, AND S. D. SHAH. Norepinephrine and epinephrine release and adrenergic mediation of smoking associated hemodynamic and metabolic events.
N. Engl. 5. CRYER,
J. Med.
295: 573-577,
1976.
P., A. SILVERBERG, J. SANTIAGO, AND S. SHAH. Plasma catecholamines in diabetes: the syndromes of hypoadrenergic and hyperadrenergic postural hypotension. Am. J. Med. 64: 407-
416, 1978. 6. EXT~N, J., AND S. HARPER.
catecholamines
on hepatic
Role of cyclic AMP in the actions of carbohydrate metabolism. Adv.
Cyclic Nucleotide Res. 5: 519-532, 1975. 7. EXTON, J,, G. ROBISON, E. SUTHERLAND,
AND C. R. PARK. Studies on the role of adenosine 3’,5’-monophosphate in the hepatic actions of glucagon and catecholamines. J. BioZ. Chem. 246:
6166-6177, 1971. 8. GARBER, A., P. CRYER, J. SANTIAGO, AND D. KIPNIS. The role of adrenergic
responses to insulin-induced
M. HAYMOND, A. PAGLIARA, mechanisms and hormonal hypoglycemia in man. J. CZin.
Invest. 58: 7-14, 1976. 9. HALES, C . , AND P. RANDLE . Immunoassay of insulin with lin-antibody precipitate. Biochem. J. 88: 137-146, 1963. 10. HALTER, J. B., AND D. PORTE. Plasma catecholamines
insu-
in the evaluation of sympathetic nervous system function in man (Abstract). Clin. Res. 25: 295A, 1977. 11. HUTSON, N., F. BRUMLEY, F. ASSIMACOPOULOS, S. HARPER, AND J. EXT~N. Studies on the a-adrenergic activation of hepatic glucose output. I. Studies on the ar-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthetase in isolated rat liver parenchymal cells. J. BioZ. Chem. 251: 5200-5208,1976. 12. JAKOB, A., AND S. DIEM.
Metabolic responses of perfused rat livers to alpha- and beta-adrenergic agonists, glucagon and
cyclic AMP. Biochim. Biophys. Acta 404: 57-66, 1975. I., A. PAGLIARA, AND D. KIPNIS. A microfluorometric enzymatic assay for the determination of alanine and pyruvate in plasma and tissues. J. Lab. CZin. Med. 80: 434-441, 1972. 14. KNEER, N., A. BOSCH, M. CLARK, AND H. LARDY. Glucose inhibition of epinephrine stimulation of hepatic gluconeogenesis by blockade of the a-receptor function. Proc. NatZ. Acad. Sci. 71:
13. KARL,
4523-4527, 15. KREISBERG,
1974.
R., A. SIEGAL, AND W. OWEN. Glucose-lactate interrelationships: effect of ethanol. J. CZin. Invest. 50: 175-185,197l. 16. LEICHTER, S., A. PAGLIARA, M. GREIDER, S. POHL, J. ROSAI, AND D . KIPNIS. Uncontrolled diabetes mellitus and hyperglucagonemia associated with an islet cell carcinoma. Am. J. Med. 58:
285-293,1975. 17. LOWRY, O., AND J. PASSONNEAU. A FZexibZe Systellt AnaZysis. New York: Academic, 1972. 18. MURPHY, B. Some studies of the protein-binding
of Enzymatic
of steroids and their application to routine micro- and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J. CZin. EndocrinoZ. Metab. 27: 973-990, 1967. 19. PALMER, J. P., D. P. HENRY, J. W. BENSON, D. G. JOHNSON, AND J. W. ENSINCK. Glucagon response to hypoglycemia in sympathectomized man. J. CZin. Invest. 57: 522-525, 1976. 20. RIZZA, R., P. CRYER, AND J. GERICH. Hormonal contributions to acute glucose counter-regulation in man (Abstract). CZin. Res. 26: 426A, 21. SCHALCH,
1978.
D., AND M. PARKER. A sensitive double antibody radioimmunoassay for human growth hormone in plasma. Na-
ture 203: 1141-1142, 1964. 22. STARKE, K., H. TAUBE, AND
systems in catecholaminergic
E. BOROWSKI. Presynaptic receptor transmission. B&hem. Pharma-
coZ. 26: 259-268, 1977. 23. STEELE, R., H . ROSTAMI
calculator
, AND N. ALTSZULER. A two-compartment for the dog glucose pool in the nonsteady state.
Federation Proc. 33: 1869-1976,1974. 24. SUTHERLAND, E., AND T. RALL.
The relation of adenosine-3’,5’phosphate and phosphorylase to the actions of catecholamines and other hormones. Pharmacol. Rev. 12: 265-300, 1960. 25. WALTER, R., R. DUDL, J. PALMER, AND J. ENSINCK. The effect of adrenergic blockade on the glucagon responses to starvation and hypoglycemia in man. J. CZin. Invest. 54: 1214-1220, 1974.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
Metabolism Divisions, Departments of Pediatrics School of Medicine, St. Louis, Missouri 63110 CLARKE, WILLIAM L., JULIO V. SANTIAGO, LORRAINE THOMAS, EHUD BEN-GALIM, MOREY W. HAYMOND, AND Pnnrp E. CRYER. Adrenergic mechanisms in recovery from hypoglycemia in man: adrenergic blockade. Am. J. Physiol.
236(2):E147-E152,1979or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 5(2): El47-E152, 1979.- Simultaneous glucoseand alanine turnover rates, along with static measurements of counterregulatory hormonesand selectedmetabolic intermediates, were determined during insulin-induced hypoglycemia in normal subjectsinfused with saline, phentolamine (an alpha-adrenergic blocking agent), and propranolol (a betaadrenergic blocking agent) in separate experiments. In the control studiesplasmaglucosefell from 87 t 4 to 35 t 4 mg/dl 30 min after intravenous crystalline insulin (0.05 U/kg). Glucoseutilization doubled and then returned to baseline by 40min, whereasglucoseproduction fell initially, began to rise by 30 min, and reached maximal rates twice baseline by 40 min after insulin. Hypoglycemia was associatedwith increments in plasma epinephrine, norepinephrine, glucagon, growth hormone, and cortisol; blood lactate rose transiently after insulin injection, whereas blood beta-hydroxybutyrate was suppressedinitially but rose during glucose recovery. Despite hemodynamic and metabolic evidence of alpha- and beta-adrenergic blockade during the infusion of phentolamine and of propranolol, respectively, base-line and postinsulin plasmaglucoseconcentrations, glucoseutilization and production rates, and rates of glucoseformation from alanine did not differ from those of the control study. Thus, adrenergic mechanismsdo not appear to play a primary role in recovery from insulin-induced hypoglycemia in man. Rather, on the basisof other evidence, adrenergic mechanisms appear to play a secondary role in that they becomecritical to recovery from hypoglycemia only in the absenceof other counterregulatory mechanisms. catecholamines;epinephrine; norepinephrine; glucose kinetics; alanine kinetics; glucagon; cortisol; growth hormone
fromhypoglycemiaorof prevention of hypoglycemia have not been clearly defined either in animals or human subjects. Glucose recovery from insulin-induced hypoglycemia, the most widely studied model, cannot be attributed solely to the disappearance of insulin because in a previous study in our laboratory (8) glucose kinetic studies documented that glucose counterregulation was well under way at a time when mean plasma insulin concentrations were still tenfold over basal levels. Five hyperglycemic fac-
THE MECHANISMSOFRECOVERY
0363-6100/79/0000-0000$01.25
LORRAINE THOMAS, AND PHILIP E. CRYER
and Medicine,
Washington
University
tors (glucagon, epinephrine, norepinephrine, growth hormone, and cortisol) are released during hypoglycemia in man (8), whereas epinephrine, norepinephrine, and growth hormone have been shown to be released during relatively rapid physiological decrements (95-60 mg/dl) in the plasma glucose concentration in normal and diabetic subjects (3). Thus, these five hyperglycemic factors are potentially important glucose counterregulatory factors. Available evidence is consistent with an important role for the catecholamines in recovery from hypoglycemia. Not only is there major release of epinephrine and norepinephrine during hypoglycemia, but only the release of these catecholamines precedes or coincides with the onset of kinetically determined glucose counterregulation, whereas demonstrable release of glucagon, cortisol, and growth hormone follows the onset of ,glucase counterregulation (8). Although the hyperglycemic effect of the catecholamines is well known, it remains to be determined whether this effect is mediated through alpha- or beta-adrenergic mechanisms. Studies with isolated rat hepatocytes (11, 14) and perfused rat livers (6, 7, 12) suggest that catecholamine-stimulated glucose production is mediated through alpha-adrenergic mechanisms, whereas studies in other species (24) suggest a more important role for beta-adrenergic mechanisms. In human subjects, beta-adrenergic blockade has been reported to impair recovery from hypoglycemia (1) or to hav e no effect (25) on recovery from hypoglycemia as studied with static plasma glucose concentrations. Furthermore, studies in patients with spinal cord transections and resultant deficits in sympathetic neural outflow have yielded apparently conflicting results. Brodows and co-workers (2) observed a subnormal-to-absent rise in the plasma glucose concentration after administration of 2-deoxyglucose to such patients, whereas Palmer and co-workers (19) found normal glucose recovery from insulin-induced hypoglycemia in cord-sectioned patients. Thus, the relative role of adrenergic mechanisms among other potentially important glucose counterregulatory factors remains to be determined. To clarify the role of adrenergic mechanisms in recovery from hypoglycemia, glucose, and alanine (a major gluconeogenic precursor) turnover studies - along with static measurements of plasma glucagon, epinephrine,
Copyright 0 1979 the American Physiological Society
El47
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
El48
norepinephrine, cortisol, and growth hormone; and blood lactate and beta-hydroxybutyrate -were performed during saline infusion, alpha-adrenergic blockade with phentolamine, and beta-adrenergic-blockade with propranolol before and during insulin-induced hypoglycemia in normal human subjects. METHODS
Subjects. Four healthy adult male volunteers (ages 27-35) were studied on the Washington University Clinical Research Center on 3 separate days - while at complete bed rest. Informed consent was obtained in each case. Each was within 10% of ideal body weight (Metropolitan Life Insurance Co. tables) and was fasted overnight prior to study. Study protocol. After the insertion of intravenous needles, initial blood samples were obtained. A priming dose of [3-3H]glucose (0.15 &i/kg) and [UJ4C]alanine (0.05 &i/kg) (Amersham/Searle, Burlington Heights, IL) was infused followed by a continuous 5-h infusion of both isotopes (glucose 0.075 &i kg+ h-l, alanine 0.1 &i kg-l h-l). The specific activity of the [3-3H]glucose was 10 Ci/mmol and that of the [UJ4C]alanine was 164 mCi/mmol. Infusion rates were calibrated after each study and samples of the infusate were saved for counting. Venous samples were obtained at - 180, -90, -75, and -60 min, at which time a randomly selected continuous intravenous infusion of either saline, phentolamine (5 mg by rapid injection followed by infusion at 0.5 mg/min), or propranolol (1 mg by rapid injection followed by infusion at 0.1 mg/min> was begun through an independent intravenous site and continued for 3 h. Samples were obtained at -50, -40, -30, -15, and 0 min, at which time 0.05 U/kg of crystalline insulin was given intravenously. Venous samples were collected subsequently at 10-min i.ntervals over the next hour and every 15 min during the subsequent 5th h of the study. Blood pressure and pulse rate were recorded prior to obtaining each sample. Analytical methods. Each sample was divided into four separate chilled tubes. Samples for determination of metabolic intermediates and specific activities were mixed immediately with equal volumes of 3 M perchloric acid. Samples for catecholamine determination were placed into tubes containing a final concentration of of 5 n&I reduced glutathione, thoSe for determination glucagon and insulin into tubes containing 1,000 U Trasylol (FBA Pharmaceuticals, New York), and those for determination of plasma glucose, alanine, growth hormone, and cortisol into heparinized tubes. All samples were placed on ice, centrifuged at 4”C, and separated, and supernatants were stored at -2OOC (perchloric acid extracts were stored at 1800C). Plasma glucose was measured with a Beckman glucose analyzer. Plasma alanine (:13), blood lactate, and (17) were determined microfluobeta-hydroxybutyrate rometrically. Plasma catecholamines were measured utilizing a single isotope derivative method (5). Neither phentolamine nor propranolol cross-react in this assay (4). Insulin (9), growth hormone (21), and glucagon (16)
CLARKE
ET
AL.
were measured utilizing double-antibody radioimmunoassay techniques. Plasma cortisol was determined by a competitive protein binding technique (18). Glucose and alanine were recovered from the perchlorate samples for determination of specific ,activity utilizing serial ion exchange chromatography with Dowex 5OW-18 and AGI-X8 (Bid Rad Laboratories, Richmond, Ca) resins (15). Alanine was further isolated by ion exchange chromatography utilizing PA28 resin (Beckman, Palo Alto, Ca). Glucose recovery averaged 96%, whereas alanine recovery averaged 82%. Glucose and alanine fractions were diluted with 3 ml scintillation cocktail (Radiation Products, Inc., Elk Grove Village, IL) and counted for 100 min. All counts were corrected for 14C spillover. Glucose kinetics were calculated using a modification of Steele’s equations (23) for nonsteady-state conditions. Alanine conversion to glucose was determined using modifications of the glucose-lactate equations of Kreisberg (15). Statistical evaluation of the data was performed utilizing the paired t test. RESULTS
Prior to the initiation of adrenergic blockade, plasma glucose concentrations, glucose kinetics, glucose formation from alanine, plasma alanine concentrations, alanine kinetics, and plasma norepinephrine and epinephrine concentrations for the saline control did not differ significantly (Figs. l-3). Similarly, basal plasma cortisol, growth hormone, and glucagon levels (Fig. 5) and blood lactate and P-hydroxybutyrate concentrations were comparable for all three studies. Effects of alpha- and beta-adrenergic blockade on basal data. The administration of phentolamine resulted in a 16% increase in pulse rate and a 19% fall in
diastolic blood pressure, whereas propranolol produced a 13% decrease in pulse rate without altering supine blood pressure (Table 1). Plasma norepinephrine concentrations rose significantly from base-line values of 189 t 36 to 380 t 54 pg/ ml at -30 min (P c 0.02) during phentolamine administration and to 478 t 69 pg/ml at 0 min, whereas glucose and alanine concentrations and kinetics, as well as circulating concentrations of epinephrine, glucagon, cortisol, growth hormone, lactate, and beta-hydroxybutyrate were unaffected by the infusion of either phentolamine or propranolol (Figs. 3-5). Effects of insulin-induced hypoglycemia d luring alpha- and beta-adrenergic blockade. A&r the adminis-
tration of insulin, plasma glucose concentrations decreased over the first 30 min to nadirs of 35 t 4, 39 t 5, and 40 t 4 mg/dl during the saline, phentolamine, and propranolol studies, respectively. Plasma glucose concentrations subsequently increased to base-line levels by 90 min. During the adrenergic blockade studies, plasma glucose concentrations were similar to those obtained during saline infusion (Fig. 1). After insulin administration, no significant differences were noted in the rates of plasma insulin clearance between saline and adrenergic blockade studies. Mean plasma insulin concentrations were 171 t 12, 158 t 22, and 163 t 6
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
ADRENERGIC
MECHANISMS
IOOr
IN HYPOGLYCEMIA
El49
IN MAN
saline infusion. Glucose production from alanine decreased 20 min after insulin, but then rose significantly (P c 0.05) over th e f o11owing 10 min in all three studies. By 40 min glucose production from alanine was 268, 226, and 160% (saline, phentolamine, and propranolol,
I
400
W
40 I
40 I
PRODUCTION
I
II
c -I
I_
.
UTILIZATION
W zz
a1 a -60
0
60
I
o+’
I
I
I
I
I
11
1
I
J
;
a -I a w
I I
!
I
a
Z
aJ 0 E a
II
r
120
0 E 1
20ti O
-60
0
UTILIZATION
I I
60
120
FIG. 2. Mean (&SE) plasma alanine concentrations and alanine kinetics before and during (-60 through 120, denoted by dashed vertical lines) infusion of saline (solid lines), phentolamine (dashed lines), or propranolol (dotted lines). Insulin was injected at 0 min (arrows).
FIG. 1. Mean (*SE) plasma glucose concentrations, glucose kinetics, and glucose production from alanine in normal subjects before and during (-60 through 120 min, dashed vertical lines), infusion of saline (solid lines), phentolamine (dashed lines), or propranolol (dotted lines). Insulin was injected at 0 min (arrows).
pU/ml at 10 min and fell with half-times of disappearance of 6,8, and 7 min to levels below 20 pU/ml after 50 min in the saline, phentolamine, and propranolol studies, respectively. During all three study periods glucose utilization increased 98-166% above baseline by 10 min (P < 0.05), returned to basal base-line rates by 40 min, and remained constant throughout the rest of the study (Fig. 1). Glucose production was decreased by 20 min, increased at 30 min, and reached maximal production rates (219 t 29,216 t 64, and 184 t 42% above baseline for the saline control, phentolamine, and propranolol, respectively) at 40 min. Glucose production remained significantly elevated (P < 0.05) for the remainder of the study with values 53 t 12, 93 t 30, and 86 t 41% (saline, phentolamine, and propranolol) above base-line production rates 120 min after insulin (Fig. 1). Glucose production and utilization rates were similar in all three studies except that glucose utilization was slightly diminished during propranolol infusion at 20 min (P c 0.02) when compared with the same time point during
jr 0
0
2
L
a0 3I a
-60
0
60
120
FIG. 3. Mean (*SE) plasma cortisol, growth hormone, and glucagon concentrations before and during (-60 through 120, denoted by interrupted vertical lines) infusion of saline (solid lines), phentolamine (dashed lines), or propranolol (dotted lines). Insulin was injected at 0 min (arrows).
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
El50
CLARKE
TABLE
1. Hemodynamic
during
adrenergic
effects observed blockade pulse
Control study Baseline Saline Saline hypoglycemia
R.& per min
58 + 3 59 2 4 85 f 3
‘ygd mmHg
’
112 + 3 114 + 2 117 +, 11
Diastolic Blood Pressure, dg
74 2 2 72 + 3 1 f 1
ET AL.
to rise with recovery from hypoglycemia (P < 0.05 compared to saline control). Blood lactate concentrations rose significantly after insulin administration during all three studies (P < 0.05). The change in lactate concentrations during propranolol infusion was less than that observed during the saline control study (P < 0.05). Lactate concentrations had returned to baseline levels by 120 min in each study (Fig. 5). DISCUSSION
Alpha-adrenergic Baseline Phentolamine Phentolamine
blockade study hypoglycemia
Beta-adrenergic blockade study Baseline Propranolol Propranolol h-ypwlycemia a
_-
64 +, 4 74 2 10 108 f 10
106 f 4 116 + 3 111 + 21
69 + 1 53 2 7 42 + 4
64 + 1 54 It 2 55 2 4
110 + 2 107 & 13 114 + 7
70 A 3 70 + 3 76 f: 6
Elevated plasma catecholamine concentrations during the combined infusion of phentolamine and propran0101 (4) and during phentolamine alone (10) have been previously reported. In the present study, marked elevations in plasma norepinephrine but not epinephrine were observed during phentolamine infusion, whereas
c
--
Values are means + SE.
respectively) a.bove base-line conversion rates (P < 0.05) and remained elev pated for the duration of each study (Fig. 1). Alanine kinetics remained constant throughout this period and were not influenced by insulin, phentolamine, or propranolol (Fig. 2). Plasma cortisol concentrations rose 30 min after insulin and were significantly elevated above base-line concentrations by 50 min Cp < 0.05), but were unaffected by adrenergic blockade (Fig. 3). During the saline study, plasma growth hormone concentrations rose 30 min (P c 0.02) after insulin administration and reached maximal concentrations at 60 min (Fig. 3). In contrast, phentolamine administration blunted the plasma growth hormone response to hypoglycemia (P < 0.05 from 4090 min). Growth hormone concentrations during propranolol infusion were not statistically different from those of the saline control study. During the saline study, plasma glucagon concentrations rose to concentrations 41% above baseline at 40 min. Similar increments occurred during the phentolamine and proprano101studies (Fig. 3). Plasma epinephrine concentrations rose from 13 t 6 to 896 t 55 pg/ml 30 min after insulin (P c 0.02) and were not significantly different from saline control values during either the phentolamine or propranolol infusion (Fig. 4). Plasma norepinephrine responses, however, were strikingly dissimilar during phentolamine infusion. Norepinephrine concentrations rose identically in response to hypoglycemia during the saline control and propranolol infusions to levels 70% above baseline at 30 min (P < 0.02). The phentolamine infusion produced elevated base-line norepinephrine levels and rise (200% above time 0 values) in circulating norepinephrine after insulin administration, with significant elevations as early as 20 min. No diminution in this response was seen after the return of plasma glucose concentrations to preinsulin values. Blood beta-hydroxybutyrate concentrations decreased after insulin administration in all subjects (Fig. 5) and then rose to concentrations above base-line values during the saline control (P c 0.05) and phentolamine infusion (P < 0.01) studies. However, during proprano101infusion. beta-hvdroxvbutvrate concentrations failed
1200
I
w z
800
ho CL z4 00 Q, W
un
-60
0
60
120
4. Mean (*SE) plasma norepinephrine and epinephrine concentrations before and during (-60 through 120, denoted by dashed vertical lines) infusion of saline (solid lines), phentolamine (dashed lines) or propranolol (dotted lines). Insulin was injected at 0 min (arrows). FIG.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
ADRENERGIC
0 Ed
200= a
W
s a B
Sal9
s a Is
L
z
o--v -
200-
Q + 5f= oE Q
El51
IN MAN
BLOCKADE HYPOGLYCEMIA RECOVERY
-03
200
5- a E e x
IN HYPOGLYCEMIA
BASELINE
E
u a c2
MECHANISMS
sa13
s
2 0
1Q B
s
a
2> E
L
/3
s
a
P
FIG. 5. Mean (*SE) base-line blood beta-hydroxybutyrate and lactate concentrations and change ( *SE) from baseline during infusion of saline (s), phentolamine ((Y), or propranolol (p) before insulin (blockade alone), during hypoglycemia and during recovery from hypoglycemia. *, denotes significant differences (P < 0.05) from changes observed during saline control study.
0
neither catecholamine changed significantly during propranolol administration. Elevated norepinephrine concentrations during phentolamine infusion could be due to 1) a direct effect of alpha-adrenergic blockade on the central nervous system resulting in increased sympathetic neural activity, 2) reflex activation of sympathetic activity secondary to hemodynamic changes caused by postsynaptic alpha-adrenergic blockade and mediated through the central nervous system, or 3) a direct effect of alpha-adrenergic blockade of presynaptic alpha receptors on axon terminals of sympathetic postganglionic neurons (22) resulting in accelerated norepinephrine release. Because mechanisms involving central stimulation of the sympathetic nervous system would be expected to accelerate release of both adrenomedullary epinephrine and sympathetic neural norepinephrine, the finding of selective norepinephrine release during alpha-adrenergic blockade favors the third possibility. Previous studies (1,25> of the effect of beta-adrenergic blockade on glucose recovery from hypoglycemia have employed only static measurements of plasma glucose concentrations and have yielded conflicting results. We have examined the effect of both alpha- and beta-adrenergic blockade on glucose recovery from insulin-induced hypoglycemia in normal human subjects by employing sensitive measurements of glucose and alanine kinetics as well as frequent static determinations of the plasma concentrations of the currently recognized, potentially important glucose counterregulatory factors (epinephrine, norepinephrine, glucagon, cortisol, and growth hormone), in order to clarify the relative role of adrenergic mechanisms in recovery from hypoglycemia. Neither alpha-adrenergic blockade with phentolamine nor beta-adrenergic blockade with propranolol altered basal glucose or alanine kinetics or the kinetic responses to insulin-induced hypoglycemia in this study. Because only partial adrenergic blockade can be produced in human subjects, it is conceivable that insufficient blockade was produced to impact on glucose counterregulation. However, the appropriate hemodynamic changes during the infusion of phentolamine and
of propranolol, the blunted growth hormone response during recovery from hypoglycemia during phentolamine infusion, and the inhibition of a beta-hydroxybutyrate response to hypoglycemia during propranolol infusion attest to the production of adrenergic blockade. Thus, particularly in view of the sensitive methods employed, some impact of adrenergic blockade should have been discernible if adrenergic mechanisms play a primary role in the counterregulatory response to hypoglycemia. The lack of evidence supporting a primary role of adrenergic mechanisms in recovery from hypoglycemia in this study is consistent with recent observations in normal and-adrenalectomized subjects infused with somatostatin during insulin-induced hypoglycemia (un-- -published observations). In these studies, recovery Corn hypoglycemia in normal subjects was impaired during somatostatin infusion, an effect reversed by glucagon but not growth hormone replacement. That major glucose recovery, although impaired, did occur was attributed to augmented catecholamine release. However, as demonstrated by the failure of glucose recovery from hypoglycemia in somatostatin-infused adrenalectomized subjects, only in the absence of glucagon did an adrenal factor, probably epinephrine, become critical to recovery from hypoglycemia. Thus, adrenergic mechanisms appear to play a secondary role in recovery from insulin-induced hypoglycemia only i.n the absence of other counterregulatory mechanisms, notabl .y glucagon secretion. If so, one would expect adrenergic blockade to impair recovery from hypoglycemia only in the absence of glucagon release. Preliminary evidence (20) supports this hypothesis. The authors acknowledge the technical assistance of Suresh Shah, Rivka Levine, Joy Brothers, and Dan Dallas. This investigation was supported in part by National Institutes of Health Research Service Award lF32AM05544-01 and Grants AM 20579, HDAM06355 and RR00036 and by grants from the Diabetic Children’s Welfare Association and American Diabetes Association, Greater St. Louis Affiliate. Address requests for reprints to: J. V. Santiago: 500 South Kingshighway, Box 14871, St. Louis, MO 63178. Received 1 May 1978; accepted in final form 15 September 1978
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
El52
CLARKE
ET AL.
REFERENCES E . , AND R. ARKY. Role of beta-adrenergic receptors in counter-regulation to insulin-induced hypoglycemia. Diabetes
1. ABRAMSON,
17: 141-146,1968. 2. BRODOWS, R.
G., F. X. PI-SUNYER, AND R. G. CAMPBELL. control of counterregulatory events during glucopenia
J. Clin. Invest. 3. CLARKE, W.,
52: 1841-1844,
Neural in man.
1973.
J. SANTIAGO, AND P. CRYER. Catecholamine and growth hormone release with physiologic decrements in the plasma glucose concentration in normal and diabetic man (Abstract). CZin. Res. 25: 561A, 1977. 4. CRYER, P. E., M. W. HAYMOND, J. V. SANTIAGO, AND S. D. SHAH. Norepinephrine and epinephrine release and adrenergic mediation of smoking associated hemodynamic and metabolic events.
N. Engl. 5. CRYER,
J. Med.
295: 573-577,
1976.
P., A. SILVERBERG, J. SANTIAGO, AND S. SHAH. Plasma catecholamines in diabetes: the syndromes of hypoadrenergic and hyperadrenergic postural hypotension. Am. J. Med. 64: 407-
416, 1978. 6. EXT~N, J., AND S. HARPER.
catecholamines
on hepatic
Role of cyclic AMP in the actions of carbohydrate metabolism. Adv.
Cyclic Nucleotide Res. 5: 519-532, 1975. 7. EXTON, J,, G. ROBISON, E. SUTHERLAND,
AND C. R. PARK. Studies on the role of adenosine 3’,5’-monophosphate in the hepatic actions of glucagon and catecholamines. J. BioZ. Chem. 246:
6166-6177, 1971. 8. GARBER, A., P. CRYER, J. SANTIAGO, AND D. KIPNIS. The role of adrenergic
responses to insulin-induced
M. HAYMOND, A. PAGLIARA, mechanisms and hormonal hypoglycemia in man. J. CZin.
Invest. 58: 7-14, 1976. 9. HALES, C . , AND P. RANDLE . Immunoassay of insulin with lin-antibody precipitate. Biochem. J. 88: 137-146, 1963. 10. HALTER, J. B., AND D. PORTE. Plasma catecholamines
insu-
in the evaluation of sympathetic nervous system function in man (Abstract). Clin. Res. 25: 295A, 1977. 11. HUTSON, N., F. BRUMLEY, F. ASSIMACOPOULOS, S. HARPER, AND J. EXT~N. Studies on the a-adrenergic activation of hepatic glucose output. I. Studies on the ar-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthetase in isolated rat liver parenchymal cells. J. BioZ. Chem. 251: 5200-5208,1976. 12. JAKOB, A., AND S. DIEM.
Metabolic responses of perfused rat livers to alpha- and beta-adrenergic agonists, glucagon and
cyclic AMP. Biochim. Biophys. Acta 404: 57-66, 1975. I., A. PAGLIARA, AND D. KIPNIS. A microfluorometric enzymatic assay for the determination of alanine and pyruvate in plasma and tissues. J. Lab. CZin. Med. 80: 434-441, 1972. 14. KNEER, N., A. BOSCH, M. CLARK, AND H. LARDY. Glucose inhibition of epinephrine stimulation of hepatic gluconeogenesis by blockade of the a-receptor function. Proc. NatZ. Acad. Sci. 71:
13. KARL,
4523-4527, 15. KREISBERG,
1974.
R., A. SIEGAL, AND W. OWEN. Glucose-lactate interrelationships: effect of ethanol. J. CZin. Invest. 50: 175-185,197l. 16. LEICHTER, S., A. PAGLIARA, M. GREIDER, S. POHL, J. ROSAI, AND D . KIPNIS. Uncontrolled diabetes mellitus and hyperglucagonemia associated with an islet cell carcinoma. Am. J. Med. 58:
285-293,1975. 17. LOWRY, O., AND J. PASSONNEAU. A FZexibZe Systellt AnaZysis. New York: Academic, 1972. 18. MURPHY, B. Some studies of the protein-binding
of Enzymatic
of steroids and their application to routine micro- and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J. CZin. EndocrinoZ. Metab. 27: 973-990, 1967. 19. PALMER, J. P., D. P. HENRY, J. W. BENSON, D. G. JOHNSON, AND J. W. ENSINCK. Glucagon response to hypoglycemia in sympathectomized man. J. CZin. Invest. 57: 522-525, 1976. 20. RIZZA, R., P. CRYER, AND J. GERICH. Hormonal contributions to acute glucose counter-regulation in man (Abstract). CZin. Res. 26: 426A, 21. SCHALCH,
1978.
D., AND M. PARKER. A sensitive double antibody radioimmunoassay for human growth hormone in plasma. Na-
ture 203: 1141-1142, 1964. 22. STARKE, K., H. TAUBE, AND
systems in catecholaminergic
E. BOROWSKI. Presynaptic receptor transmission. B&hem. Pharma-
coZ. 26: 259-268, 1977. 23. STEELE, R., H . ROSTAMI
calculator
, AND N. ALTSZULER. A two-compartment for the dog glucose pool in the nonsteady state.
Federation Proc. 33: 1869-1976,1974. 24. SUTHERLAND, E., AND T. RALL.
The relation of adenosine-3’,5’phosphate and phosphorylase to the actions of catecholamines and other hormones. Pharmacol. Rev. 12: 265-300, 1960. 25. WALTER, R., R. DUDL, J. PALMER, AND J. ENSINCK. The effect of adrenergic blockade on the glucagon responses to starvation and hypoglycemia in man. J. CZin. Invest. 54: 1214-1220, 1974.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.056.064.029) on December 16, 2018.
