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Am J Physiol Endocrinol Metab 293: E1303-E1310, 2007. First published August 21, 2007; doi:10.1152/ajpendo.00325.2007
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Basal insulin, glucagon, and growth hormone replacement

Suzanne M. Breckenridge,1 Bharathi Raju,1 Ana Maria Arbelaez,1 Bruce W. Patterson,2 Benjamin A. Cooperberg,1 and Philip E. Cryer1

Divisions of 1Endocrinology, Metabolism, and Lipid Research and 2Geriatrics and Nutritional Science, Washington University School of Medicine, St. Louis, Missouri

Submitted 25 May 2007 ; accepted in final form 14 August 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Conclusions drawn from the pancreatic (or islet) clamp technique (suppression of endogenous insulin, glucagon, and growth hormone secretion with somatostatin and replacement of basal hormone levels by intravenous infusion) are critically dependent on the biological appropriateness of the selected doses of the replaced hormones. To assess the appropriateness of representative doses we infused saline alone, insulin (initially 0.20 mU·kg–1·min–1) alone, glucagon (1.0 ng·kg–1·min–1) alone, and growth hormone (3.0 ng·kg–1·min–1) alone intravenously for 4 h in 13 healthy individuals. That dose of insulin raised plasma insulin concentrations approximately threefold, suppressed glucose production, and drove plasma glucose concentrations down to subphysiological levels (65 ± 3 mg/dl, P < 0.0001 vs. saline), resulting in nearly complete suppression of insulin secretion (P < 0.0001) and stimulation of glucagon (P = 0.0059) and epinephrine (P = 0.0009) secretion. An insulin dose of 0.15 mU·kg–1·min–1 caused similar effects, but a dose of 0.10 mU·kg–1·min–1 did not. The glucagon and growth hormone infusions did not alter plasma glucose levels or those of glucoregulatory factors. Thus, insulin "replacement" doses of 0.20 and even 0.15 mU·kg–1·min–1 are excessive, and conclusions drawn from the pancreatic clamp technique using such doses may need to be reassessed.

octreotide; pancreatic clamp

THE PANCREATIC (OR ISLET) CLAMP TECHNIQUE involves the infusion of somatostatin (or the somatostatin analog octreotide) to suppress endogenous insulin, glucagon, and growth hormone secretion and replacement of basal levels of these hormones by intravenous infusion (1, 3, 12, 23, 24, 25, 27, 33). It has been used to assess the metabolic roles of these hormones and led, for example, to the conclusion that glucagon normally supports the postabsorptive plasma glucose concentration, since plasma glucose levels decreased when somatostatin and insulin were infused and glucagon was not replaced (27). Although there is a body of evidence that supports that view (26), much of that evidence is open to alternative interpretations. Clearly, such conclusions are critically dependent on the biological appropriateness of the selected doses of the replaced hormones.

The glycemic response to infusion of somatostatin is biphasic with an initial decrease in glucose production (19, 28, 31) and the plasma glucose concentration (20, 28, 31) followed by an increase in glucose production (19, 28, 31) and the plasma glucose concentration (20, 28, 31) in healthy humans. Like native somatostatin, infusion of the more potent somatostatin analog octreotide suppresses plasma insulin, glucagon, and growth hormone concentrations in humans (16).

Insulin has been infused peripherally in doses of 0.14 (25), 0.15 (19), 0.20 (1, 27), and 0.24 (24) mU·kg–1·min–1 in humans [and intraportally in a dose of 0.25 mU·kg–1·min–1 in dogs (12)] to replace basal insulin levels during the infusion of somatostatin. In the human studies peripheral insulin concentrations were intended to be raised approximately twofold on the basis of the rationale that, given ~50% extraction of insulin by the liver, it was necessary to double peripheral insulin concentrations to replace basal hepatic portal venous insulin levels, which were thought to be the sole determinant of endogenous glucose production and thus the postabsorptive plasma glucose concentration. However, that rationale could be questioned given more recent evidence that some of the effect of insulin to reduce endogenous glucose production is indirect, i.e., initially extrahepatic (and extrarenal), on adipose tissue to reduce nonesterified fatty acid levels (2), on muscle to reduce gluconeogenic precursor flux to the liver (and kidneys), on pancreatic {alpha}-cells to reduce glucagon secretion, and on the brain to increase parasympathetic firing to the liver (4) and thus the result of peripheral insulin actions. The relative contributions of the direct and indirect actions continue to be debated (6, 11). Clearly, it is important that insulin not be substantially overreplaced during pancreatic clamps. To our knowledge, the effects of putative basal insulin replacement doses alone (in the absence of somatostatin) in healthy humans has not been reported. Lewis et al. (18) found that an intravenous insulin dose of 0.7 units/h (0.17 mU·kg–1·min–1 if one assumes an average body weight of 70 kg) did not lower plasma glucose concentrations substantially in four patients with type 1 diabetes.

Glucagon has been infused peripherally in doses of 0.40 (23), 0.65 (1, 25), 0.75 (33), and 1.00 (3, 24) ng·kg–1·min–1 in humans [and intraportally in a dose of 0.50 ng·kg–1·min–1 in dogs (12)] to replace basal glucagon levels during the infusion of somatostatin. The use of the higher doses, infused peripherally, in the human studies was based on the assumption of ~25% extraction of glucagon by the liver and the premise that only portal levels are relevant to the actions of glucagon on glucose production. Clearly, it is important that glucagon not be substantially overreplaced during pancreatic clamps.

Growth hormone has been infused peripherally in doses of 2.0 (25), 3.0 (1), and 4.7 (24) ng·kg–1·min–1 in humans to replace basal growth hormone levels during the infusion of somatostatin. Growth hormone levels were held constant with the 3.0 ng·kg–1·min–1 dose (1); they appeared to be somewhat lower than baseline with the 2.0 ng·kg–1·min–1– dose (25). Since the plasma glucose-raising action of growth hormone is delayed for several hours (22), the need to replace growth hormone could be questioned. However, growth hormone has an initial insulin-like effect (22).

To assess the appropriateness of representative doses of insulin, glucagon, and growth hormone to produce putative replacement of basal hormone levels, we infused saline alone, insulin (initially 0.20 mU·kg–1·min–1) alone, glucagon (1.0 ng·kg–1·min–1) alone, or growth hormone (3.0 ng·kg–1·min–1) alone intravenously, in random sequence, for 4 h without somatostatin in healthy adults. We reasoned that if these were truly basal replacement doses they would have little effect on glucose kinetics and plasma glucose concentrations.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Subjects. Thirteen healthy individuals [7 women/6 men, mean (±SD) age 30 ± 8 yr, mean (±SD) body mass index 24 ± 3 kg/m2] gave their written, informed consent to participate in this study, which was approved by the Washington University Medical Center Human Research Protection Office and conducted in the outpatient facility of the Washington University General Clinical Research Center. Inclusion criteria included negative medical histories and normal physical examinations as well as normal fasting plasma glucose concentrations, hematocrits, and electrocardiograms.

Experimental design. All studies were performed in the morning after an overnight fast. Subjects were in the supine position throughout. To permit estimation of glucose kinetics, a primed (22.5 µmol/kg), constant (0.25 µmol·kg–1·min–1), intravenous infusion of [6,6-2H2]glucose was started at –180 min and continued through 300 min. Arterialized venous plasma samples, from a hand vein with that hand kept in a ~55°C plexiglas box, were drawn at 15-min intervals from –30 to 300 min for glucose concentration and isotope enrichment determinations and from –15 to 240 min and at 270 and 300 min for the other analytes detailed below. Heart rates and blood pressures were determined at the same time points, and the electrocardiogram was monitored throughout.

Intravenous infusions of saline, insulin (0.20 mU·kg–1·min–1), glucagon (1.0 ng·kg–1·min–1), or growth hormone (3.0 ng·kg–1·min–1) were administered from 0 to 240 min in random sequence. Subsequently, a subset (n = 10) of the subjects was restudied with infusion of insulin in a dose of 0.10 mU·kg–1·min–1 from 0 to 120 min and 0.15 mU·kg–1·min–1 from 120 to 240 min. Hormone infusates were prepared in saline containing 0.5 g/dl albumin.

Biochemical analytical methods. Plasma glucose concentrations were measured using a glucose oxidase method (Yellow Springs Analyzer 2; Yellow Springs Instruments, Yellow Springs, OH). Plasma insulin (17), C-peptide (17), glucagon (7), pancreatic polypeptide (10), growth hormone (29), and cortisol (8) were measured with radioimmunoassays. 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 (30). Serum nonesterified fatty acids (15) and blood lactate (21) were measured with enzymatic techniques.

Glucose tracer methodology. Plasma proteins were precipitated with ice-cold acetone and lipids extracted with hexane. The aqueous phase was dried by Speed-Vac centrifugation (Savant Instruments, Farmingdale, NY). Samples were derivatized with 10% heptafluorobutyric (HFB) anhydride in ethyl acetate (30 min at 70°C). The tracer-to-tracee ratio (TTR) of HFB-glucose was measured by gas chromatography-mass spectrometry using electron impact ionization (ions of mass/charge ratio 519 and 521 for natural and [6,6-2H2]glucose, respectively) on an Agilent 5973 system equipped with a 30 m x 0.32 mm HP-5MS column. Instrument response was calibrated using prepared glucose standards of known isotopic enrichment. Non-steady-state kinetic analysis to obtain the rates of appearance (Ra) and disappearance (Rd) of plasma glucose was performed (28,30) as follows:

Formula

Formula
where Ra(t) and Rd(t) are the rates of appearance and disappearance of unlabeled glucose, respectively, as functions of time (µmol·kg–1·min–1); F is the infusion rate of [6,6-2H2]glucose (µmol·kg–1·min–1); pV is the effective glucose volume of distribution (assumed to be 40 ml/kg); C is the concentration of unlabeled glucose (mmol/l); E is the isotopic enrichment (TTR); dE/dt is the rate of change of TTR; and dC/dt is the rate of change of unlabeled glucose concentration. Plasma glucose concentrations and TTR values were smoothed using a loess local polynomial smoothing function (Mathcad 11; Mathsoft Engineering & Education, Cambridge, MA) prior to calculations and to obtain the rates of change of TTR and concentration.

Statistical methods. Data are expressed as means ± SE, except where the standard deviation is specified. Condition and time-related data were analyzed by mixed-model repeated-measures analysis of variance. The Proc Mixed module of the SAS statistical package version 8.2 was used. P values <0.05 were considered to indicate significant differences.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Plasma concentrations of infused hormones. During saline infusion, plasma insulin concentrations tended to decline (Fig. 1), as did plasma glucose concentrations (Fig. 2) . Insulin infusion in a dose of 0.20 mU·kg–1·min–1 raised plasma insulin concentrations approximately threefold (P < 0.0001 vs. saline; Fig. 1) from 3.8 ± 0.7 µU/ml at 0 min to 13.8 ± 2.1 µU/ml at 30 min and 11.8 ± 2.2 µU/ml at 240 min. A dose of 0.10 mU·kg–1·min–1 raised plasma insulin concentrations approximately twofold (from 3.5 ± 0.5 µU/ml at 0 min to 7.5 ± 1.0 µU/ml at 120 min, P = 0.0005; Fig. 1). A dose of 0.15 mU·kg–1·min–1 raised plasma insulin concentrations further (to 10.2 ± 1.4 µU/ml at 240 min, P < 0.0001; Fig. 1). Glucagon infusion (1.0 ng·kg–1·min–1) appeared to raise plasma glucagon concentrations slightly, although not significantly (Fig. 1). The levels were 113 ± 5 pg/ml at 0 min and 131 ± 6 pg/ml at 240 min. Growth hormone infusion (3.0 ng·kg–1·min) did not raise plasma growth hormone concentrations (Fig. 1). The levels were 3.9 ± 3.0 ng/ml at 0 min and 3.2 ± 1.0 ng/ml at 240 min.


Figure 1
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  		Fig. 1. Mean (±SE) plasma insulin, glucagon, and growth hormone concentrations before, during (0–240 min), and after infusions of saline (shaded areas), insulin [0.20 ({circ}), 0.10 ({square}), and 0.15 mU·kg–1·min–1 ({blacksquare})], glucagon [1.0 ng·kg–1·min–1 ({triangleup})], and growth hormone [GH; 3.0 ng·kg–1·min–1 ({blacktriangleup})]. Compared with saline infusion, plasma insulin concentrations were raised during infusion of insulin in doses of 0.20 (P < 0.0001), 0.10 (P = 0.0005), and 0.15 (P < 0.0001) mU·kg–1·min–1.

 

Figure 2
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  		Fig. 2. Mean (±SE) plasma glucose concentrations before, during (0–240 min), and after infusions of saline (shaded area), insulin [0.20 ({circ}), 0.10 ({square}), and 0.15 mU·kg–1·min–1 ({blacksquare})], glucagon [1.0 ng·kg–1·min–1 ({triangleup})], and growth hormone [3.0 ng·kg–1·min–1 ({blacktriangleup})]. Compared with saline infusion, plasma glucose concentrations were reduced during infusion of insulin in doses of 0.20 (P < 0.0001), 0.10 (P = 0.0158), and 0.15 (P < 0.0001) mU·kg–1·min–1.

 
Plasma glucose concentrations and glucose kinetics. Insulin infusion in a dose of 0.20 mU·kg–1·min–1 lowered plasma glucose concentrations sharply (P < 0.0001 vs. saline; Fig. 2) from 88 ± 2 mg/dl at 0 min to 72 ± 4 mg/dl at 60 min, 71 ± 3 mg/dl at 120 min, and 65 ± 3 mg/dl at 240 min. Insulin infusion in a dose of 0.10 mU·kg–1·min–1 lowered plasma glucose concentrations (from 88 ± 2 to 76 ± 1 mg/dl at 120 min; P = 0.0158) and in a dose of 0.15 mU·kg–1·min–1 lowered plasma glucose concentrations further (to 67 ± 2 mg/dl at 240 min, P < 0.0001; Fig. 2).

Insulin infusion in a dose of 0.20 mU·kg–1·min–1 decreased the relative (to baseline) Ra initially (P = 0.0086 vs. saline; Table 1). Thus, glucose Ra was lower than glucose Rd, and the plasma glucose concentrations declined. Then, glucose Ra increased and matched plasma Rd. A similar, but smaller, glucose kinetic pattern appeared to develop initially during insulin infusions in lower doses (Table l), although that did not reach statistical significance (P = 0.0562). Glucose Ra and Rd drifted downward during infusion of saline (Table l). They were unaltered compared with saline during infusions of glucagon and of growth hormone in the doses studied (data not shown). Thus, glucose Ra and Rd time course profiles were superimposable during infusions of saline, glucagon, and growth hormone.


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  		Table 1. Mean relative Ra and Rd before, during (0 through 240 min), and after infusion of saline and infusions of insulin

 
Neuroendocrine and other metabolic responses. As plasma glucose concentrations decreased during infusion of insulin in a dose of 0.20 mU·kg–1·min–1, plasma C-peptide concentrations decreased sharply (P < 0.0001; Fig. 3) from 1.1 ± 0.1 ng/ml at 0 min to 0.7 ± 0.1 ng/ml at 60 min, 0.4 ± 0.1 ng/ml at 120 min, and 0.3 ± 0.0 ng/ml at 240 min. Indeed, as plasma glucose declined within the physiological range during infusion of insulin in a dose of 0.10 mU·kg–1·min–1, plasma C-peptide concentrations also declined (from 1.3 ± 0.1 ng/ml at 0 min to 0.7 ± 0.1 ng/ml at 120 min, P = 0.0038), and, as plasma glucose concentrations fell further during infusion of insulin in a dose of 0.15 mU·kg–1·min–1, plasma C-peptide concentrations decreased further (to 0.4 ± 0.0 ng/ml at 240 min, P < 0.0001; Fig. 3).


Figure 3
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  		Fig. 3. Mean (±SE) plasma C-peptide, glucagon, epinephrine, and pancreatic polypeptide concentrations before, during (0–240 min), and after infusions of saline (shaded areas) and insulin [0.20 ({circ}), 0.10 ({square}), and 0.15 mU·kg–1·min–1 ({blacksquare})]. Compared with saline infusion, plasma C-peptide concentrations decreased during infusion of insulin in doses of 0.20 (P < 0.0001), 0.10 (P = 0.0038), and 0.15 (P < 0.0001) mU·kg–1·min–1. Plasma glucagon concentrations increased during infusion of insulin in doses of 0.20 (P = 0.0059) and 0.15 (P = 0.0041) mU·kg–1·min–1. Plasma epinephrine concentrations increased during infusion of insulin in doses of 0.20 (P = 0.0009) and 0.15 (P = 0.0094) mU·kg–1·min–1. Plasma pancreatic polypeptide concentrations increased during infusion of insulin in a dose of 0.20 mU·kg–1·min–1 (P < 0.0001) and appeared to increase during infusion of insulin in a dose of 0.15 mU·kg–1·min–1.

 
Decrements in plasma glucose concentrations to subphysiological levels during infusion of insulin in a dose of 0.20 mU·kg–1·min–1 were associated with increments in the plasma concentrations of glucagon (P = 0.0059), epinephrine (P = 0.0009), and pancreatic polypeptide (P < 0.0001; Fig. 3). Plasma glucagon levels increased from 116 ± 6 pg/ml at 0 min to 144 ± 10 pg/ml at 240 min, epinephrine levels from 76 ± 9 to 292 ± 89 pg/ml, and pancreatic polypeptide levels from 73 ± 10 to 228 ± 41 pg/ml. Plasma norepinephrine concentrations (data not shown) increased (P < 0.0001) from 179 ± 7 to 226 ± 24 pg/ml. Plasma growth hormone (P = 0.0318) and cortisol (P = 0.0002) concentrations also increased (Table 2). Serum nonesterified fatty acid concentrations were suppressed (P = 0.0163) from 652 ± 54 µmol/l at 0 min to a nadir of 237 ± 59 µmol/l at 90 min and then rose to 532 ± 84 µmol/l at 240 min (Table 3). Blood lactate concentrations (443 ± 28 µmol/l at 0 min and 634 ± 112 µmol/l at 240 min) increased (P = 0.0117) (Table 3).


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  		Table 2. Mean plasma GH and cortisol concentrations before, during (0–240 min), and after infusions of saline and insulin

 

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  		Table 3. Mean serum NEFA and blood lactate concentrations before, during (0–240 min), and after infusions of saline and insulin

 
Decrements in plasma glucose concentrations within the physiological range during infusion of insulin in a dose of 0.10 mU·kg–1·min–1 were not associated with increments in the plasma concentrations of glucagon, epinephrine, or pancreatic polypeptide (Fig. 3), of growth hormone or cortisol (Table 2), or of norepinephrine (data not shown). Serum nonesterified fatty acid concentrations remained suppressed (P = 0.0463), but blood lactate concentrations tended to increase (Table 3). On the other hand, decrements in plasma glucose concentrations to subphysiological levels during infusion of insulin in a dose of 0.15 mU·kg–1·min–1 were associated with increments in the plasma concentrations of glucagon (P = 0.0041) and epinephrine (P = 0.0094); pancreatic polypeptide appeared to increase (P = 0.1185) (Fig. 3). Growth hormone increased (P = 0.0416), and cortisol tended to increase (P = 0.1436) (Table 2). Norepinephrine increased (P = 0.0009; data not shown).

Infusions of glucagon and of growth hormone were not associated with changes in the plasma concentrations of insulin or epinephrine (Table 4), the serum concentrations of nonesterified fatty acids, or the blood concentrations of lactate (Table 5) or any of the other measured neuroendocrine variables (data not shown).


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  		Table 4. Mean plasma insulin and epinephrine concentrations before, during (0–240 min), and after infusions of saline, glucagon, and GH

 

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  		Table 5. Mean serum NEFA and blood lactate concentrations before, during (0–240 min), and after infusions of saline, glucagon, and GH

 
Blood pressures and heart rates. None of the hormone infusions was associated with significant changes in systolic or diastolic blood pressures or in heart rates (data not shown) compared with infusion of saline aside from slightly higher heart rates (P = 0.0016) and slightly lower diastolic blood pressures (P = 0.0275) during infusion of insulin in a dose of 0.20 mU·kg–1·min–1.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
These data indicate that the intravenous insulin infusion doses ranging from 0.14 to 0.24 mU·kg–1·min–1 often used to attempt to produce "basal" insulin replacement during suppression of endogenous insulin (and glucagon and growth hormone) secretion in the pancreatic (or islet) clamp technique in humans (e.g., see Refs. 1, 19, 24, 25, and 27) are excessive in healthy young adults. Infusion of insulin alone in a dose of 0.20 mU·kg–1·min–1 raised plasma insulin concentrations approximately threefold and drove plasma glucose concentrations down to below the postabsorptive physiological range of ~72–108 mg/dl (5). That activated physiological defenses against hypoglycemia (5). As plasma glucose concentrations declined within the physiological range, insulin secretion as assessed by plasma C-peptide concentrations decreased sharply. As plasma glucose concentrations fell further to levels below the glycemic thresholds for activation of glucose counterregulatory systems, the plasma concentrations of glucagon and epinephrine, as well as those of growth hormone and cortisol, increased. Were it not for these glucose counterregulatory defenses, plasma glucose concentrations would undoubtedly have fallen to even lower levels. Furthermore, infusion of insulin in a lower dose of 0.15 mU·kg–1·min–1 also drove plasma glucose concentrations down to subphysiological levels and activated glucose counterregulatory systems. Clearly, conclusions drawn from use of the pancreatic clamp technique with such doses that might be critically dependent on an excessive insulin replacement dose, including our own (27), need to be reassessed in light of these findings.

The lowest insulin dose tested, 0.10 mU·kg–1·min–1, raised plasma insulin concentrations approximately twofold and lowered plasma glucose concentrations within the physiological range. As a result, endogenous insulin secretion, as assessed by plasma C-peptide concentrations, decreased. It also suppressed lipolysis as assessed by serum nonesterified fatty acid concentrations. Clearly, that too is a biologically active dose. However, over the time frame studied, unlike both of the higher doses, it did not cause subphysiological glucose levels and did not suppress insulin secretion completely. It would likely have had less of a plasma glucose-lowering effect in the absence of endogenous insulin secretion. Thus, a peripheral intravenous insulin dose of 0.10 mU·kg–1·min–1 would appear to be a more appropriate dose for portal venous insulin replacement with the pancreatic clamp technique in humans.

The glucagon and growth hormone doses tested did not alter plasma glucose concentrations or any of the measured glucoregulatory factors. Infusion of glucagon in a dose of 1.0 ng·kg–1·min–1 did not raise plasma glucagon concentrations significantly. Mean values at 240 min were only 16% higher than those prior to glucagon infusion. Growth hormone infusion in a dose of 3.0 ng·kg–1·min–1 did not alter plasma growth hormone concentrations measurably. However, we have no measure of any effects of these infusions on endogenous glucagon or growth hormone secretion. In any event, these doses are not excessive. Indeed, they are somewhat low.

Insulin lowered plasma glucose concentrations by suppressing glucose production rather than by stimulating glucose utilization. The glucose Ra decreased initially and was lower than the glucose Rd, and the plasma glucose concentrations, therefore, decreased. Then glucose Ra increased to match Rd, and the plasma glucose levels plateaued. The glucose kinetic method we used underestimates glucose Ra and Rd (9, 15) even during infusions of low doses of insulin (12). Therefore, our results are reported as those relative to baseline rather than as absolute rates. The observed decrement in glucose Ra was small compared with that during euglycemic clamps with similar doses of insulin (15), but the latter may well have also included a suppressive effect of glucose per se. Glucose turnover rates drifted downward during infusion of saline and were similar, with glucose Ra and Rd superimposable, during infusion of saline, of glucagon, and of growth hormone. Thus, in the doses tested, neither glucagon nor growth hormone raised plasma glucose concentrations.

These data confirm several principles of the physiology of glucose counterregulation, the mechanisms that normally prevent or rapidly correct clinical hypoglycemia, in humans (5, 13). First, the glycemic threshold for decrements in insulin secretion as plasma glucose concentrations decline lies within the postabsorptive plasma glucose concentration range. Second, the glycemic thresholds for increments in the secretion of the glucose counterregulatory hormones (glucagon, epinephrine, growth hormone, and cortisol) lie just below the physiological range. Third, in the setting of intact glucose counterregulatory systems, plasma glucose concentrations plateau at levels just below the physiological range and at levels higher than those required to produce symptoms of hypoglycemia despite ongoing hyperinsulinemia sufficient to decrease plasma glucose levels. In essence, a new steady state is established at plasma glucose concentrations low enough to maintain activation of glucose counterregulatory systems but higher than the glycemic threshold for symptoms of hypoglycemia.

In summary, these data indicate that putative basal intravenous insulin replacement doses of 0.20 mU·kg–1·min–1 and even of 0.15 mU·kg–1·min–1 are too high for use in the pancreatic (or islet) clamp technique in humans. A peripheral intravenous insulin dose of 0.10 mU·kg–1·min–1 would appear to be a more appropriate dose for portal venous insulin replacement. A glucagon replacement dose of 1.0 ng·kg–1·min–1 and a growth hormone replacement dose of 3.0 ng·kg–1·min–1 are not excessive; indeed, they are somewhat low. Thus, conclusions based on the use of this technique with excessive insulin replacement may need to be reassessed.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This work was supported, in part, by United States Public Health Service/National Institutes of Health Grants R37-DK-27085, MO1-RR-00036, P30-DK-56341, P60-DK-20579, and T32-DK-07120 and a fellowship award from the American Diabetes Association.


   DISCLOSURES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
P. E. Cryer has served on Advisory Boards for Novo Nordisk, Takeda Pharmaceuticals North America, MannKind, and Merck and and as a consultant for Amgen, TolerRx, and Marcadia Biotech in recent years. S. M. Breckenridge, B. Raju, A. M. Arbelaez, B. W. Patterson, and B. A. Cooperberg have nothing to disclose.


   ACKNOWLEDGMENTS
 
We acknowledge the assistance of the staff of the Washington University General Clinical Research Center in the performance of this study; the technical assistance of Krishan Jethi, Cornell Blake, Gene Wade Sherrow, Michael Morris, Zina Lubovich, Sharon Travis, Sharon O'Neill, Freida Custodio, Jennifer Shew, and Dr. Adewole Okunade; and the assistance of Janet Dedeke with the preparation of this manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. E. Cryer, Campus Box 8127, Washington University 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.


   REFERENCES
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