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*Compound via MeSH
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*GLUCOSE
*LACTIC ACID
*TOLBUTAMIDE
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*Hormones
*Hypoglycemia
The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 3 1037-1046
Copyright © 1999 by The Endocrine Society

Original Studies

Counterregulatory Response to Hypoglycemia Differs According to the Insulin Delivery Route, But Does Not Affect Glucose Production in Normal Humans1

Gary F. Lewis2, André Carpentier3, Debra Bilinski, Adria Giacca and Mladen Vranic

Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada M5G 2C4

Address all correspondence and requests for reprints to: Dr. Gary Lewis, Toronto Hospital, General Division, 200 Elizabeth Street, Room EN 11–229, Toronto, Ontario, Canada M5G 2C4. E-mail: glewis{at}torhosp.toronto.on.ca


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The magnitude of the counterregulatory response to insulin-induced hypoglycemia is primarily determined by the degree of hypoglycemia. We examined whether the route of acute insulin delivery (portal or peripheral venous) is also important in determining the magnitude of the counterregulatory response to hypoglycemia in nine healthy nondiabetic men. Pancreatic insulin secretion, stimulated by an iv tolbutamide infusion (portal insulin study), was matched with an exogenous insulin infusion into the peripheral vein 4–6 weeks later (peripheral insulin study). Each study consisted of a 150-min baseline tracer equilibration period, a 180-min euglycemic hyperinsulinemic (portal or peripheral insulin delivery) period, a 60-min hypoglycemic period in which insulin secretion diminished during tolbutamide or was reduced during exogenous insulin, and a 30-min recovery period. Peripheral venous glucose concentrations were well matched in the portal and peripheral studies during euglycemia and hypoglycemia (glucose nadir, 2.9 ± 0.1 mmol/L in the portal and 2.7 ± 0.1 mmol/L in the peripheral; mean ± SEM; P = NS), and insulin concentrations were about 1.5-fold higher throughout the experiment in the peripheral vs. the portal insulin study due to the first pass extraction of insulin in the portal study. There was a much greater increment (P < 0.0001) in FFA in the portal vs. the peripheral study (area under the curve: portal, 19.5 ± 3.9 mmol/L·90 min; peripheral, 3.3 ± 1.1 mmol/L·90 min), whereas plasma glucagon and GH were higher in the peripheral study (P = 0.01 for glucagon; P = 0.015 for GH). There was no significant difference between studies in epinephrine and norepinephrine responses to hypoglycemia or stimulation of endogenous glucose production (area under the curve: portal, 636 ± 103 µmol/kg·90 min; peripheral, 705 ± 69 µmol/kg·90 min; P = NS). In summary, we have shown that the glucagon, GH, and FFA responses to hypoglycemia during insulin dissipation are affected by the route of insulin delivery and are not controlled exclusively by the nadir blood glucose level. The clinical importance of these observations in diabetic subjects as they relate to route of insulin delivery (portal or peripheral) during insulin dissipation remains to be determined.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THERE IS an increased incidence of hypoglycemia when attempts are made to institute tight glycemic control using currently available regimens of sc insulin administration in patients with type 1 diabetes (1). Symptomatic hypoglycemia occurs frequently in insulin-treated patients, and 36% of patients were found in one study to have experienced hypoglycemic coma in their lifetime (2). Up to 10% of patients practicing conventional insulin therapy and 25% of those practicing intensive therapy suffer at least one episode of severe, temporarily disabling hypoglycemia, often with seizure or coma, in a given year (1, 3, 4), and hypoglycemia causes recurrent and even persistent psychological morbidity in many patients with type 1 diabetes (1).

Currently, the majority of patients with insulin-treated diabetes are treated with insulin administered by sc injection or infusion, with absorption of the insulin into the systemic circulation. If adequate glycemic control is achieved, this nonphysiological route of insulin delivery is invariably associated with systemic hyperinsulinemia, because higher insulin concentrations are required to adequately insulinize the liver when insulin is not delivered by the portal route. It is well recognized that the glucose level is the primary determinant of the hormonal and metabolic counterregulatory responses to insulin-induced hypoglycemia, but there is emerging evidence that the peripheral venous insulin concentration may also play an important role in modifying the counterregulatory response to hypoglycemia (5). In addition, there is growing appreciation that glucose sensing by the portohepatic region plays an important role in modulating the response to hypoglycemia (6, 7, 8, 9). We were interested, therefore, in examining whether the counterregulatory responses to hypoglycemia induced by portal vs. peripheral venous insulin delivery differed.

To achieve the same rate of insulin delivery via the portal and the peripheral venous routes, we used our previously published method of noninvasively matching the rate of pancreatic insulin secretion with a peripheral venous insulin infusion in healthy nondiabetic humans (10, 11, 12, 13, 14). With this method the peripheral insulin concentrations are lower with portal vs. peripheral insulin delivery, due to an approximately 50% extraction of portally delivered insulin on first pass through the liver, with subsequent dilution in the greater systemic circulation. This is achieved with a programmed iv tolbutamide infusion, calculation of the insulin secretion rate from peripheral venous C peptide levels, followed by a euglycemic hyperinsulinemic clamp in the same individual 4–6 weeks later in which the exogenous insulin infusion rate is matched with the calculated rate from the earlier tolbutamide study. We have previously shown no independent in vivo effect of tolbutamide on glucose turnover, insulin sensitivity and clearance, intermediary metabolites, or glucagon when used acutely in this fashion (10, 12, 13). The present method allows us to compare the effects of matched hypoglycemia induced by an equal dose of insulin delivered via either the portal or peripheral venous route, resulting in vastly different peripheral venous insulin concentrations. As the portal insulin in this case results from stimulation of pancreatic insulin secretion, our method allowed us in addition to determine whether the intraislet paracrine effects of insulin on glucagon secretion or the suppressive effect of peripheral insulin (hormonal effect) on glucagon secretion are dominant.

Another unique feature of this model relates to the fact that we are using a glucose clamp during tolbutamide infusion to maintain euglycemia and then hypoglycemia. When the glucose infusion is reduced or discontinued, and glucose decreases into the hypoglycemic range, tolbutamide ceases to stimulate insulin secretion, and insulin levels decline rapidly. Thus, hypoglycemia is accompanied not by continuous hyperinsulinemia, but by rapidly decreasing amounts of insulin. This study thus allowed us to examine the effect not only of the counterregulatory response to hypoglycemia, but also of a simultaneous reduction of insulin concentrations during hypoglycemia. With the introduction of rapidly acting synthetic insulins such as Lys-Pro insulin, hypoglycemia is likely to occur more commonly in insulin-treated patients in the setting of rapidly dissipating plasma insulin concentrations.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Nine healthy nondiabetic men participated in the study. The mean age of the subjects was 21.1 ± 0.4 yr, and the body mass index was 24.7 ± 0.9 kg/m2. No subject had a history of systemic illness, and none was taking any medication at the time of the study. Informed written consent was obtained from all participants in accordance with the guidelines of the human subjects review committee of Toronto Hospital, University of Toronto (Toronto, Canada). Permission was granted by the Health Protection Branch, Health and Welfare Canada, for the use of iv tolbutamide (IND 034076, Dr. G. Lewis).

Experimental protocol

All subjects were studied on two occasions each, 6–8 weeks apart. In the first study hyperinsulinemia was induced by an iv tolbutamide infusion; this will be referred to as the portal (insulin) study. In the second study hyperinsulinemia was induced by an exogenous insulin infusion; this will be referred to as the peripheral (insulin) study. Each study consisted of a 150-min baseline tracer equilibration period (-150 to 0 min), a 180-min euglycemic hyperinsulinemic (portal or peripheral insulin delivery) period (0–180 min), a 1-h hypoglycemic period (180–240 min), and a 30-min recovery period (240–270 min).

Portal studies

Subjects were admitted to the Metabolic Test Center of Toronto Hospital after a 12-h overnight fast and did not eat until completion of the study that afternoon. At approximately 0800 h (-150 min) a primed (3.3 x 106 dpm) continuous infusion (0.33 x 106 dpm/min) of 3-[3H]glucose (New England Nuclear, Boston, MA) was started and maintained throughout the study. The tracer had been submitted to the high performance liquid chromatography purification procedure (15). After 110 min, five samples of arterialized venous blood were drawn every 10 min for basal determinations from an iv catheter placed in a dorsal hand vein of the opposite arm, which was maintained in a warming device. At 0 min, tolbutamide sodium, USP (Upjohn Company, Kalamazoo, MI; 3.5 g) in 250 mL normal saline was infused into a peripheral arm vein at a rate of 1 g/h for the first hour, 800 mg/h for the second hour, and 600 mg/h for the remainder of the study. This dose was empirically determined in earlier studies to produce sustained and steady rates of pancreatic insulin secretion during euglycemia in nondiabetic individuals (10). An aliquot of [3-3H]glucose was added to the 20% dextrose infusate (0.388 µCi/kg 3-[3H]glucose added to 500 mL dextrose solution) to minimize the decline in glucose specific activity during the clamp (hot Ginf method) (16, 17). Potassium chloride was infused at approximately 10 mEq/h in all subjects. Plasma glucose levels were measured every 5 min during the tolbutamide infusion. The values were used to adjust the rate of a 20% dextrose infusion to maintain constant euglycemia (glucose, 5.0–5.5 mmol/L) for the first 3 h of the hyperinsulinemic period (0–180 min). At 180 min the dextrose infusion rate was reduced initially by 50% and then by a further 50% every 5 min to lower the plasma glucose concentration in a controlled fashion to approximately 3.0 mmol/L over a 20- to 30-min period. An attempt was made to maintain this level of hypoglycemia until 240 min (it was not possible in most cases because of the hypoglycemia-induced inhibition of endogenous insulin secretion), in many cases by discontinuing the dextrose infusion during this time. At 240 min the dextrose infusion rate was increased rapidly to raise the plasma glucose concentration to euglycemia (5.0–5.5 mmol/L) for the final 30 min of the study (240–270 min). Blood samples were drawn for determinations of glucose, [3-3H]glucose specific activity, insulin, C peptide, free fatty acids (FFA), lactate, and counterregulatory hormones (glucagon, catecholamines, GH,and cortisol) at baseline and at frequent intervals throughout the study. Samples for measurement of FFA were drawn into chilled ethylenediamine tetraacetate tubes on ice containing 0.4 µm/mL blood of the lipase inhibitor m-aminophenylboronic acid (Sigma Chemical Co., St. Louis, MO).

Tolbutamide was discontinued after completion of the study (at 270 min), the subjects were permitted to eat, and the rate of the potassium chloride and 20% dextrose infusion was gradually reduced overnight, maintaining euglycemia at all times according to half-hourly blood glucose measurements drawn through a sampling iv catheter. In most cases the dextrose infusion was discontinued within 12 h of stopping the tolbutamide.

Peripheral insulin study

The study using an exogenous insulin infusion was performed 6–8 weeks later using crystalline human insulin (Novo Nordisk A/S Canada, Toronto, Canada) infused into a peripheral vein between 0–270 min. Plasma glucose levels were maintained in the euglycemic range for the first 3 h of the infusion (0–180 min) as described above.

For the first 3 h (0–180 min) the rate of infusion of exogenous insulin was matched in each individual to the calculated mean steady rate of pancreatic insulin secretion between 60–180 min of the earlier tolbutamide infusion. Over the first hour of the infusion (0–60 min) the insulin infusion rate was increased in increments of 25% of the calculated maximal rate every 15 min to mimic as closely as possible the gradual increase in insulin secretion seen in the earlier portal insulin study. Pancreatic insulin secretion had been calculated from peripheral plasma C peptide levels by deconvolution using a two-compartment mathematical model for C peptide distribution and metabolism as previously described (18) (the software program for calculation of insulin secretion was provided by Drs. K. Polonsky and J. Sturis, University of Chicago, Chicago, IL). The use of standard parameters for C peptide clearance and distribution has been shown to result in insulin secretion rates that differ in each subject by only 10–12% from those obtained with individual parameters, and there is no systematic over- or underestimation of insulin secretion (18). At 180 min the exogenous insulin infusion rate was decreased to match the mean endogenous insulin secretion rate calculated between 180–210 min and between 210–240 min in the earlier portal study. Between 240–270 min the exogenous insulin infusion was increased to match the mean calculated endogenous insulin secretion rate during the same time period in the earlier portal study.

Calculations

The specific activity of the infusate was calculated as previously described (12). Briefly, calculations were based on estimation of the parameters of the formula of Finegood et al. (16), modified to allow for incomplete suppression of glucose production (GP). GP was calculated as the endogenous rate of appearance measured with [3-3H]glucose; glucose utilization was the rate of disappearance measured with [3-3H]glucose (Rd). For glucose turnover calculations, a modified one-compartmental model (16) was used to account for the exogenously infused mixture of labeled and unlabeled glucose. Data were smoothed with the optimal segments routine (19), using the optimal error algorithm (20). With the hot Ginf method the monocompartmental assumption becomes minor because the nonsteady state part of the Steele’s equation is close to zero. At euglycemia, Rd corresponded to glucose utilization, and the plasma clearance rate of glucose (Rd/glycemia) corresponded to the glucose MCR.

Laboratory methods

Glucose was assayed enzymatically at the bedside using a Beckman Coulter Glucose Analyzer II (Beckman Coulter, Inc., Fullerton, CA). Insulin was measured by RIA using a double antibody separation method (kit supplied by Pharmacia Biotech, Uppsala, Sweden). C Peptide was measured by RIA using previously described techniques (21). Glucagon was measured by RIA with a double antibody procedure using a kit from Diagnostic Products Corp. (Los Angeles, CA). FFA levels were measured by a colorimetric method (kit supplied by Wako Industrials, Osaka, Japan). Triglycerides were measured as esterified glycerol using an enzymatic colorimetric kit (Boehringer Mannheim, Mannheim, Germany; catalogue no. 450032). Free glycerol was eliminated from the sample in a preliminary reaction followed by enzymatic hydrolysis of triglyceride with subsequent determination of the liberated glycerol by colorimetry. GH was measured by a two-site immunoradiometric assay (Pharmacia & Upjohn, Inc., Uppsala, Sweden), and cortisol was determined by a solid phase RIA (Diagnostic Products Corp.). Catecholamines (epinephrine and norepinephrine) were determined by a radioenzymatic assay (22). Lactate was measured by an enzymatic fluorometric method in deproteinized blood (23).

For the determination of [3-3H]glucose specific activity, plasma was deproteinized with Ba(OH)2 and ZnSO4. An aliquot of the supernatant was then evaporated to dryness to eliminate tritiated water. After the addition of water and liquid scintillation solution, the radioactivity from [3-3H]glucose was counted by liquid scintillation spectrometry. An external standard was used for quench corrections. Aliquots of the infused [3-3H]glucose and of the labeled glucose infusate were assayed together with the plasma samples.

Statistical methods

The data were expressed as the mean ± SEM. Two-way ANOVA for repeated measurements was performed for differences between experimental groups during the last 40 min of the basal period (-40 to 0 min) and the last 90 minutes (90–180 min, steady state period) of the portal or peripheral hyperinsulinemic period. To more accurately determine the response of each parameter to hypoglycemia, we eliminated prehypoglycemia differences in the parameters of interest by performing a two-way ANOVA of values after subtraction of the mean of the two values immediately preceding the hypoglycemic period (165 and 180 min). For the hypoglycemic/recovery period (180–270 min), the incremental or decremental areas under the concentration vs. time curves (i.e. areas above or below the mean of the two concentration measurements made immediately preceding the hypoglycemic period) were calculated; in some cases the peak or nadir value for each variable for the two experimental groups was calculated, and values were compared by paired analysis. Statistics from the latter analyses are reported when comparing responses in the portal and the peripheral insulin studies during the hypoglycemic phase. Calculations were performed with SAS software (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Mean values reported below for the baseline and the euglycemic hyperinsulinemic period are the means of -40 to 0 min and 90–180 min, respectively. The data for the hypoglycemic period (180–240 min) and recovery period (240–270 min) were pooled to analyze the counterregulatory response to hypoglycemia.

Insulin infusion/secretion rates (Fig. 1Go)

In the portal study the steady state insulin secretion rate in the last 90 min of the euglycemic hyperinsulinemic period (90–180 min) was 345 ± 11 pmol/min, and the exogenous insulin infusion rate in the peripheral insulin study was similar at 353 ± 7 pmol/min (P = NS). Hypoglycemia suppressed endogenous mean insulin secretion in the portal study to a nadir of 121 ± 23 pmol/min. By design, the exogenous insulin infusion rate was decreased similarly to a nadir of 156 ± 20 pmol/min (P = NS).



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  		Figure 1. Insulin infusion and secretion rates and plasma glucose, insulin, and C peptide concentrations. The calculated pancreatic insulin secretion rate in response to tolbutamide in the portal study (solid circles) and the exogenous insulin infusion rate in the peripheral study (open circles) are shown. Tolbutamide (in the portal study) or exogenous insulin (in the peripheral study) was infused from 0–270 min (the hyperinsulinemic period). By design, the peripheral insulin infusion rate was matched with the calculated insulin secretion rate in each individual. Euglycemia was maintained for the first 180 min of this period, followed by a 1-h hypoglycemic period (between 180–240 min) and a 30-min (240–270 min) euglycemic recovery period. Mean values and statistics for differences between portal and peripheral studies are presented in the text (see Results).

 
Peripheral venous glucose, insulin, and C peptide concentrations (Fig. 1Go)

Euglycemia (plasma glucose, 5.0–5.5 mmol/L) was maintained in both the portal and peripheral studies throughout the basal period and the initial 180 min of the hyperinsulinemic periods. Two methods were used to calculate the nadir blood glucose. Firstly, we considered the lowest blood glucose level for each subject regardless of the time at which the nadir blood glucose occurred and calculated the means for the portal and peripheral insulin studies, respectively. The mean value was 2.9 ± 0.1 mmol/L for the portal study and 2.7 ± 0.1 mmol/L for the peripheral insulin study. Secondly, the mean values depicted in Fig. 1Go were calculated as means by time for each study. The latter method resulted in a blood glucose nadir of 3.0 ± 0.3 mmol/L at 210 min for the portal study and 2.7 ± 0.1 mmol/L at 208 min for the peripheral study. With neither method was the difference between studies statistically significant. As shown in Fig. 1Go, it was not possible to maintain hypoglycemia at the target of approximately 3.0 mmol/L for the full 60-min period (180–240 min) as was originally intended, because the hypoglycemic state suppressed tolbutamide-stimulated endogenous insulin secretion. However, the dextrose infusion administered in the peripheral study was adjusted accordingly to match plasma glucose levels as closely as possible in the portal and peripheral studies. Consequently, there was no significant difference in plasma glucose between studies when we analyzed the glucose nadir, the glucose concentration vs. time curves, or the decremental area under the curves (AUCs).

The peripheral insulin levels rose from a basal value of 34 ± 3 to 207 ± 16 pmol/L between 90–180 min in the portal study, which was less than the rise from 35 ± 2 to 291 ± 12 pmol/L in the peripheral study (P < 0.0001). Peripheral insulin levels remained significantly higher in the peripheral vs. the portal study throughout the hypoglycemic and recovery periods (P < 0.0001), declining to a nadir of 58 ± 9 pmol/L in the portal study and 101 ± 17 pmol/L in the peripheral study.

C Peptide levels rose (P < 0.0001) from 0.42 ± 0.03 to 1.19 ± 0.02 nmol/L between 90–180 min in the portal study and decreased (P < 0.05) from 0.31 ± 0.02 to 0.22 ± 0.01 nmol/L in the peripheral insulin study. During hypoglycemia, C peptide declined from 1.18 ± 0.05 to a nadir of 0.70 ± 0.07 nmol/L at 232 ± 4 min in the portal study and was suppressed to 0.07 ± 0.01 nmol/L in the peripheral study (P < 0.0001 between studies).

[3-3H]Glucose specific activity, dextrose infusion rates (Ginf), endogenous GP (Ra), and glucose utilization (Rd; Fig. 2Go)

There was a greater increase in [3-3H]glucose SA in the portal (25 ± 2% increase in SA at 90–180 min; P < 0.0001) vs. the peripheral study (6 ± 1% increase; P < 0.0001 for difference between studies). The specific activity declined toward the basal level during hypoglycemia and in the recovery period in both studies, as illustrated in Fig. 2Go.



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  		Figure 2. [3-3H]Glucose specific activity (SA), dextrose infusion rate (Ginf), glucose utilization (Rd), and endogenous GP (Ra). Data from the portal studies are illustrated with solid circles, and the peripheral insulin studies are shown with open circles. Tolbutamide (in the portal study) or exogenous insulin (in the peripheral study) was infused from 0–270 min (the hyperinsulinemic period). Euglycemia was maintained for the first 180 min of this time period, followed by a 1-h hypoglycemic period (between 180–240 min) and a 30-min (240–270 min) euglycemic recovery period. Mean values and statistics for differences between portal and peripheral studies are presented in the text (see Results).

 
The Ginf continued to increase throughout the first 180 min of the hyperinsulinemic period in both studies. The rate necessary to maintain euglycemia was greater in the peripheral insulin study than in the portal study (P < 0.0001). By design, the Ginf was decreased in both studies to reduce plasma glucose to approximately 3.0 mmol/L for 1 h between 180–240 min. The Ginf remained greater in the peripheral than in the portal study throughout the hypoglycemic and recovery periods, reaching a nadir of 0.3 ± 1.0 µmol/kg·min in the portal study and 7.0 ± 1 µmol/kg·min in the peripheral study (P < 0.005).

The pattern of the glucose utilization rate (Rd) vs. time curves was similar to that of the Ginf curves. As expected, glucose utilization rose proportionally to the peripheral insulin levels and was greater (P < 0.0001) between 90–180 min in the peripheral study (42.5 ± 1.7 µmol/kg·min) than in the portal study (30.6 ± 1.7 µmol/kg·min). During the hypoglycemic and recovery periods Rd remained greater at all times in the peripheral vs. the portal studies (P < 0.01), reaching a nadir of 14.8 ± 1.3 µmol/kg·min in the portal study and 30.5 ± 2.5 µmol/kg·min in the peripheral study.

Endogenous GP (Ra) in the basal period was significantly lower in the portal (11.6 ± 0.3 µmol/kg·min) than in the peripheral study (12.5 ± 0.2 µmol/kg·min; P < 0.0001). The suppression of Ra between 90–180 min was similar in the peripheral (71.2 ± 0.04%; change in Ra, -8.9 ± 0.5 µmol/kg·min) and portal (69.0 ± 0.43%; change in Ra, -8.0 ± 0.5 µmol/kg·min, P = NS for portal vs. peripheral) studies. The early effect (initial 90 min of hyperinsulinemia) of insulin on Ra, however, tended to be greater in the portal study than in the peripheral insulin study, as illustrated in Fig. 2Go. After induction of hypoglycemia, there was a marked increase in endogenous GP in both the portal and peripheral insulin studies. The incremental area under the Ra vs. time curve was similar for the portal (636 ± 103 µmol/kg·90 min) and peripheral (705 ± 69 µmol/kg·90 min; P = NS) studies. Similarly, there was no difference in the peak Ra for the portal (16.6 ± 1.4 µmol/kg·min) and peripheral (15.7 ± 1.9 µmol/kg·min) studies.

Intermediary metabolites: FFA and lactate (Fig. 3Go)

FFA levels decreased significantly from baseline and were similar (P = NS) in the portal (baseline, 0.497 ± 0.044 mmol/L decreased to 0.087 ± 0.006 mmol/L; P < 0.0001) and peripheral (baseline, 0.442 ± 0.050 mmol/L decreased to 0.078 ± 0.014 mmol/L; P < 0.0001) studies. In response to hypoglycemia, FFAs increased to a far greater extent (P < 0.0001) in the portal (AUC, 19.5 ± 3.9 mmol/L·90 min) than in the peripheral (AUC, 3.3 ± 1.1 mmol/L·90 min) insulin study, and the peak FFA concentration was greater (P < 0.003) in the portal (0.575 ± 0.104 mmol/L) than in the peripheral (0.170 ± 0.028 mmol/L) study.



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  		Figure 3. Plasma FFA and lactate. Data from the portal studies are illustrated with solid circles, and the peripheral insulin studies are shown with open circles. Tolbutamide (in the portal study) or exogenous insulin (in the peripheral study) was infused from 0–270 min (the hyperinsulinemic period). Euglycemia was maintained for the first 180 min of this period, followed by a 1-h hypoglycemic period (between 180–240 min) and a 30-min (240–270 min) euglycemic recovery period. Mean values and statistics for differences between portal and peripheral studies are presented in the text (see Results).

 
Lactate levels increased to a greater extent (P < 0.0001) with hyperinsulinemia in the peripheral study (744 ± 80 µmol/L at baseline; increased to 950 ± 41 µmol/L between 90–180 min) than in the portal study (774 ± 50 µmol/L at baseline; and 719 ± 37 µmol/L between 90–180 min). In response to hypoglycemia, the peak lactate level was similar in the peripheral (1,301 ± 137 µmol/L) and portal (1,134 ± 147 µmol/L) studies, with a greater incremental AUC in the portal study (19,145 ± 6,851 µmol/L·90 min in the portal study vs. 12, 553 ± 4,098 µmol/L·90 min in the peripheral study; P = 0.03).

Counterregulatory hormone response to hypoglycemia (Fig. 4Go and Table 1Go)

Table 1Go lists the areas under the incremental concentration vs. time curves (AUC) between 180–270 min, peak plasma concentrations, and statistics for these variables determined by two-way ANOVA for glucagon, GH, cortisol, epinephrine, and norepinephrine. These parameters are graphically depicted in Fig. 4Go. In summary, there was a greater response of glucagon and GH in the peripheral study, but no difference in the cortisol, epinephrine, and norepinephrine responses to hypoglycemia.



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  		Figure 4. Plasma concentrations of counterregulatory hormone response (glucagon, GH, cortisol, epinephrine, and norepinephrine) to portal or peripheral insulin-induced hypoglycemia. Data from the portal studies are illustrated with solid circles, and those from the peripheral insulin studies are shown with open circles. Tolbutamide (in the portal study) or exogenous insulin (in the peripheral study) was infused from 0–270 min (the hyperinsulinemic period). Euglycemia was maintained for the first 180 min of this period, followed by a 1-h hypoglycemic period (between 180–240 min) and a 30-min (240–270 min) euglycemic recovery period. Mean values and statistics for differences between portal and peripheral studies are presented in Table 1Go.

 

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  		Table 1. Incremental AUC of counteregulatory hormones during the hypoglycemic period

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We have shown in this study that some hormonal and metabolic counterregulatory responses to insulin-induced hypoglycemia are dependent not only on the venous glucose concentration but also on the route of insulin delivery (portal or peripheral). Although our model directly tested the effects of portal vs. peripheral insulin delivery, the two routes of insulin delivery were associated with very different peripheral insulin concentrations. Whether it is the route of delivery per se or the associated difference in peripheral venous insulin concentrations that affects the counterregulatory response was not directly tested in the present study. We suggest the latter, based on the evidence discussed below indicating that plasma insulin concentrations may affect the counterregulatory response to hypoglycemia. The major differences in counterregulation between our portal and peripheral insulin studies were a greater increase in the FFA response in the portal study and a greater increase in glucagon and GH responses in the peripheral study. There was, however, no significant difference in the magnitude of stimulation of endogenous GP despite differences in the counterregulatory response, and this may be related to the fact that insulin levels dissipated to a relatively similar extent during hypoglycemia in portal and peripheral studies.

Previous studies that have investigated the role of peripheral insulin concentrations in modifying the counterregulatory response to hypoglycemia have not revealed consistent results. Some studies in nondiabetic (24, 25) and diabetic humans (26) have shown that antecedent hyperinsulinemia results in a diminished glucagon response to hypoglycemia, whereas others (27, 28) have shown no such effect. Mellman et al. (29) showed in normal subjects that brief antecedent exposure to hyperinsulinemia had no effect on the glucagon response to subsequent hypoglycemia, whereas more prolonged exposure resulted in selective blunting of the plasma glucagon response. Davis et al. (30) came to similar conclusions using a dog model, i.e. that the duration of prior nonhypoglycemic hyperinsulinemia and the level of insulin appear to play an important role in the counterregulatory response by suppressing glucagon release. Davis et al. showed that high supraphysiological insulin levels result in a significant increase in catecholamine and cortisol secretion, GP, and lipolysis despite equivalent hypoglycemia in dogs (30, 31), normal male humans (32), and female humans (33), but not in humans with established type 1 diabetes (34). They did not, however, compare the effect of insulin delivery route on the above parameters.

There are some important limitations in the design of this study that need to be considered when interpreting the results. In the portal study, as hypoglycemia is a potent inhibitor of endogenous insulin secretion, which overrides the acute stimulatory effect of tolbutamide, we were unable to lower blood glucose to a greater extent and were unable to maintain the hypoglycemic state for a full 30 min, as is usually done during hypoglycemic clamp studies. The full stimulation of counterregulatory hormone secretion, therefore, did not occur. Although we fully acknowledge this limitation of the experimental model used, we point out that this method is still the only noninvasive method currently available that allows investigators to acutely match portal and peripheral venous insulin delivery rates in humans to compare the effects of insulin delivered via the two routes on metabolic processes, such as the response to insulin-induced hypoglycemia.

We were able to determine in the present study whether glucagon secretion is inhibited by insulin predominantly by a paracrine (intraislet) or by an endocrine (arterial) mechanism. Histological studies of the microvasculature of the islets of Langerhans and islet perfusion studies have suggested that insulin released by ß-cells directly inhibits downstream glucagon-secreting {alpha}-cells (35, 36, 37, 38). Tolbutamide-induced pancreatic insulin would be expected to result in a paracrine effect of insulin on downstream {alpha}-cells. Insulin also acutely inhibits glucagon secretion in proportion to its peripheral plasma concentration, as demonstrated in studies previously performed in experimental animals (39) where there was a greater inhibition of glucagon by acute peripheral infusion vs. infusion of insulin directly into the portal vein. The paracrine effect was recently demonstrated in vivo in humans by demonstrating a lower glucagon response to hypoglycemia induced by tolbutamide vs. an exogenous insulin infusion (40) and in humans treated with insulin plus glibenclamide vs. insulin alone (41). The study by Peacey et al. (40) differed from ours in that they did not attempt to match either the peripheral or portal insulin levels or the insulin delivery rate, with glucagon levels being measured in response to matched hypoglycemic glucose concentrations. As the method used in the present study allowed us to precisely match the pancreatic insulin secretion rate with an exogenous insulin infusion, and the glucagon response to hypoglycemia was greater with peripheral insulin infusion despite the higher peripheral insulin concentrations, we can conclude that the paracrine effect of insulin in suppressing glucagon release during hypoglycemia is dominant in vivo. We do, however, acknowledge that small differences in the blood glucose level in a hypoglycemic clamp setting can result in significant differences in the stimulation of the counterregulatory hormone response (5), but the issue of whether a 0.2 mmol/L higher blood glucose nadir in the portal study could have been a significantly less potent stimulus for glucagon release is difficult to settle by referring to previously reported studies.

Our model relies on endogenous pancreatic insulin secretion to create portal hyperinsulinemia, whereas insulin infusion devices deliver insulin directly into the portal vein, distal to the pancreas. We cannot exclude the possibility that matched exogenous insulin infusion via the peripheral vs. the portal venous route may have a greater suppressive effect on the glucagon response to hypoglycemia, based on the higher peripheral insulin concentrations invariably associated with peripheral insulin delivery. Unfortunately, such a study cannot be performed noninvasively in humans at the present time. In accordance with the idea that chronic sc insulin administration may have such an effect, Saudek et al. (42) showed that ip insulin treatment reduced the incidence of hypoglycemic episodes compared to intensive sc insulin treatment, and Selam et al. (43) reported a reduction in the rates of significant hypoglycemia when ip insulin infusion replaced intensive control with sc insulin therapy. Shi’s group (44) recently showed that ip, but not sc, insulin treatment normalizes the responses of glucagon and GP to hypoglycemia in diabetic rats. Shi et al. (45) also recently showed that a severely impaired glucagon response to hypoglycemia in streptozotocin diabetic rats can be improved by 3–4 days of normalization of hyperglycemia with phlorizin (an agent that blocks renal tubular glucose reabsorption and corrects hyperglycemia independent of insulin), but not with sc insulin treatment.

In the present study there was a marked elevation of plasma FFAs during hypoglycemia induced by portal compared to peripheral insulin. This finding can best be explained by similar stimulation of peripheral tissue lipolysis with portal and peripheral insulin delivery, but greater reesterification of FFA in the presence of high insulin in the peripheral study. Davis et al. (34) observed a similar phenomenon in patients with type 1 diabetes mellitus, who had equivalent experimental hypoglycemia in the presence of differing peripheral insulin concentrations. FFAs have been shown to block GH release (46) either at the pituitary gland (47) or by stimulating hypothalamic somatostatin release (48), and the higher FFA levels in the portal study are one explanation for the lower GH response to hypoglycemia. This issue is somewhat complicated by the fact that GH also has a direct lipolytic effect on adipose tissue (49).

Blood lactate levels were significantly higher throughout the euglycemic and hypoglycemic hyperinsulinemic periods in the peripheral insulin study compared to those in the portal study, although the incremental AUC during hypoglycemia was greater with portal than peripheral insulin. The overall higher lactate levels with peripheral insulin are consistent with the greater stimulation of glycolysis secondary to the higher peripheral insulin levels in the peripheral study. Davis et al. also reported a significantly greater stimulation of lactate during hypoglycemia in the presence of higher peripheral insulin levels in patients with type 1 diabetes (34) and in normal controls (32).

Despite the differences in hormone and metabolite responses to hypoglycemia discussed above, there was no significant difference in glucose productin with portal vs. peripheral insulin delivery. A unique feature of our model is that insulin secretion is suppressed during tolbutamide infusion when plasma glucose levels decline into the hypoglycemic range, resulting in dissipation of plasma insulin concentrations. We carefully matched the exogenous insulin infusion rate in the peripheral insulin study, resulting in similar relative declines in plasma insulin concentrations in portal and peripheral studies. The counterregulatory response to hypoglycemia in the present study was less than that observed by others when a 0.1 U/kg iv bolus of insulin was administered to normal subjects (50) and was similar to that seen during prolonged hyperinsulinemia with a slow decline in plasma glucose concentrations (51). The fact that GP was stimulated to a similar extent in both portal and peripheral studies despite differences in counterregulatory hormone and intermediary metabolite responses and the fact that the actual magnitude of the increase in GP was marked despite a relatively small counterregulatory response suggest that the dissipation of insulin during hypoglycemia played an important role in stimulating GP.

Although not the major focus of the present study, the responses of insulin, glucose disappearance (Rd), GP, and glucose infusion (Ginf) rate during the euglycemic hyperinsulinemic period deserve comment. As expected, the peripheral insulin levels were about 1.5-fold higher throughout the experiment in the peripheral than in the portal study due to the first pass hepatic insulin extraction of portal insulin. Consequently, the Ginf required to maintain euglycemia and the Rd increased to a greater extent in the peripheral study and remained higher throughout the euglycemic and subsequent hypoglycemic periods. GP declined more rapidly in the portal vs. the peripheral study during the euglycemic hyperinsulinemic period, consistent with the idea that portal insulin inhibits GP directly at the liver, whereas peripheral insulin indirectly inhibits GP by a slower inhibition of peripheral lipolysis (12, 14).

In summary, we have shown that some of the counterregulatory hormone and intermediary metabolite responses to insulin-induced hypoglycemia are not controlled exclusively by the nadir blood glucose level. Other factors, such as the route of insulin delivery (portal or peripheral) as well as the peripheral insulin concentration, also play an important role in determining the magnitude of response. Dissipation of insulin during hypoglycemia plays an important role in stimulating GP. The tolbutamide infusion method used in the present study does not allow one to draw conclusions regarding potential differences in the counterregulatory response that may arise as a result of direct insulin delivery into the portal vein distal to the pancreas. If the availability of implantable devices with portal insulin delivery (i.e. ip pumps) becomes widely available in the future, the effect of the site of insulin delivery on the counterregulatory response to hypoglycemia will be important to assess.


   Acknowledgments
 
The expert technical assistance of Loretta Lam and Linda Szeto is acknowledged with appreciation.


   Footnotes
 
1 This work was supported by the Canadian Diabetes Association (to G.F.L., supported by a grant in memory of the late George and Vesta Davidge), the Juvenile Diabetes Foundation (Giacca 193135), and the Medical Research Council of Canada (Vranic and Giacca MT2917). Back

2 Recipient of a Career Investigator Award from the Heart and Stroke Foundation of Canada. Back

3 Supported by a Research Fellowship from the Juvenile Diabetes Foundation, New York. Back

Received September 30, 1998.

Revised November 23, 1998.

Accepted December 2, 1998.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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S. Banarer, V. P. McGregor, and P. E. Cryer
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