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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 2 521-528
Copyright © 2001 by The Endocrine Society

From the Clinical Research Centers

Troglitazone Amplifies Counterregulatory Responses to Hypoglycemia in Nondiabetic Subjects1

Ilan Gabriely, Robert Wozniak, Meredith Hawkins and Harry Shamoon

Department of Medicine (Division of Endocrinology and Metabolism), Diabetes Research Center, and General Clinical Research Center, Albert Einstein College of Medicine, Bronx, New York 10461

Address all correspondence and requests for reprints to: Ilan Gabriely, M.D., Diabetes Research Center, Albert Einstein College of Medicine, Belfer Building #701, 1300 Morris Park Avenue, Bronx, New York 10461. E-mail: gabriely{at}aecom.yu.edu


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
As insulin sensitizers, thiazolidinediones could affect the hormonal counterregulatory response to hypoglycemia via the modulatory effect of insulin on counterregulation. In addition, recent studies suggest that thiazolidinediones may influence key steps in glucose sensing and glucoregulatory hormone secretion. We therefore evaluated the effects of a short course of troglitazone on counterregulatory hormones in response to mild hypoglycemia in eight lean nondiabetic subjects. Subjects received either troglitazone (400 mg/day) or placebo for 7 days before stepped hypoglycemia clamp studies (5.0, 4.4, 3.9, and 3.3 mmol/L target plasma glucose steps, 50 min each). The glycemic thresholds for secretion of epinephrine (3.77 ± 0.05 mmol/L) and glucagon (3.83 ± 0.11 mmol/L) were reset to a higher plasma glucose concentration after troglitazone [4.05 ± 0.05 mmol/L (P = 0.003) and 4.10 ± 0.05 mmol/L (P = 0.03), respectively]. In addition, the magnitude of the rise in epinephrine and glucagon concentrations was higher with troglitazone (28% and 11%, respectively; P < 0.05 for both), whereas plasma norepinephrine, GH, and cortisol were comparable in both sets of studies. Endogenous glucose production, measured with [3-3H]glucose, rose by 33% (P < 0.05) in the troglitazone studies compared with 17% (P = NS) after placebo. We conclude that thiazolidinediones may induce an amplification of the counterregulatory response to hypoglycemia characterized by a shift in the glycemic threshold for and an increase in the magnitude of glucagon and epinephrine secretion, and subsequent activation of glucose production.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
COUNTERREGULATION of hypoglycemia involves a hierarchical hormonal response that parallels the depth and duration of hypoglycemia (1). The initiation of secretion of each counterregulatory hormone occurs at a specific plasma glucose level, or glycemic threshold (1, 2, 3). Glucagon and epinephrine are the major counterregulatory hormones that contribute to glucose counterregulation during moderate hypoglycemia; both activate endogenous glucose production (EGP) to overcome the insulin-induced suppression of EGP and ultimately restore plasma glucose concentrations to baseline (3, 4). Patients with type 1 diabetes suffer from an impaired counterregulatory hormonal response to hypoglycemia, characterized by severe blunting or absence of the glucagon response, and both a delayed threshold for and a reduced magnitude of epinephrine secretion (3, 5, 6). As a consequence, these patients have an increased risk for developing severe hypoglycemia (7, 8, 9, 10, 11).

Among the factors shown to modulate the counterregulatory response to hypoglycemia is the prevailing plasma insulin concentration (12, 13, 14, 15, 16, 17). Davis et al. demonstrated that in the dog, hypoglycemia induced at a higher insulin concentration resulted in a greater counterregulatory response compared with equivalent hypoglycemia induced by a lower insulin dose (12). The same researchers confirmed this observation in nondiabetic humans (13, 14). In parallel, Davis et al. (15, 16) and Lingenfelter et al. (17) demonstrated that plasma insulin positively modulates the counterregulatory response in patients with type 1 diabetes. Taken together, these studies suggest that insulin per se may modulate the counterregulatory hormonal response, although the precise mechanism of this modulatory effect is not well defined. We hypothesized that by using an insulin-sensitizing agent (i.e. a thiazolidinedione) we could mimic an enhanced insulin signal as an alternative experimental model to hyperinsulinemia and thus influence the counterregulatory hormonal response to hypoglycemia. In vitro (18) and in vivo (19, 20) studies have shown that thiazolidinediones augment insulin action on glucose metabolism; however, it is not known whether they amplify the insulin signal vis à vis the hypoglycemia counterregulatory response. Additionally, it has been recently demonstrated that thiazolidinediones may play an important role in glucose sensing (21, 22, 23, 24, 25), particularly by inhibiting KATP channel activity peripherally (21) and in the central nervous system (22, 23). Thus, by affecting key steps in the glucose-sensing apparatus, thiazolidinediones may also influence the effector arm of the hypoglycemia response and consequently modify the counterregulatory response.

The present study was therefore designed to evaluate the possible effects of troglitazone (a member of the thiazolidinedione class) on the counterregulatory response to stepped hypoglycemia in nondiabetic subjects.


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

We studied eight lean nondiabetic volunteers (six men and two women; age, 32 ± 4 yr; body mass index, 23.8 ± 1.11 kg/m2). All participants were in good health, were taking no medications, and had no family history of diabetes. Each subject participated in two stepped hypoglycemic clamp studies separated by an interval of 6 weeks. Each hypoglycemic clamp was preceded by 7 days of oral troglitazone (400 mg/day) or a placebo tablet given in a double masked manner in random order. Two subjects participated in a third (placebo) study at a reduced insulin infusion rate, as described below. Clamp studies were performed after an overnight fast, and the final oral troglitazone or placebo tablet was taken at 0700 h on the day of the study.

Informed written consent was obtained in accordance with policy of the committee on clinical investigations of the Albert Einstein College of Medicine.

Procedures

Subjects were admitted to the General Clinical Research Center for each experiment. At 0700 h on the day of study, two indwelling cannulas were inserted, one in an antecubital vein for infusions and the second in a retrograde fashion in a distal hand vein of the contralateral forearm for blood sampling. To obtain arterialized venous blood samples, this hand was maintained at 55 C in a thermoregulated Plexiglas box. At -120 min, a primed continuous infusion of high pressure liquid chromatography-purified [3-3H]glucose was initiated with a bolus of 21.6 µCi followed by continuous infusion of 0.15 µCi/min for the entire period of study.

The specific activity of infused dextrose was kept equivalent to plasma glucose specific activity by the addition of [3-3H]glucose to the infusate using the method of Finegood et al. to prevent negative Ra artifacts (26). At 0 min, a primed continuous infusion of insulin (Humulin Regular, Eli Lilly & Co., Indianapolis, IN) at a rate of 2.4 pmol/kg·min was initiated, and a variable infusion of 20% dextrose was begun to maintain the plasma glucose concentration at 5.0 mmol/L for 50 min (step 1 of the clamp). At 50 min and every 50 min thereafter, the plasma glucose concentration was decreased by 0.55 mmol/L decrements for 50 min each by reducing the dextrose infusion rate accordingly. Plasma glucose was clamped at the desired range by varying the dextrose infusion according to plasma glucose measured at 5-min intervals with targets of 4.4, 3.9, and 3.3 mmol/L. In two subjects a third study (placebo) was performed using a lower insulin infusion rate of 1.5 pmol/kg·min and the identical protocol. At the end of the clamp, all infusions were discontinued, and the subject was given a meal and discharged from the General Clinical Research Center.

Weekly safety evaluation of subjects included determinations of liver function, hematological indexes, and urinalysis. Fasting levels for serum fructosamine and hemoglobin A1c (HbA1c) were determined on the morning of each clamp study. During the clamps, blood samples were obtained for the determinations of plasma insulin, C peptide, glucagon, epinephrine, norepinephrine, cortisol, GH, free fatty acid, lactate, and glycerol, as well as for glucose kinetics.

Analytical methods

Plasma glucose was measured with a glucose analyzer (Beckman Coulter, Inc., Fullerton, CA) using the glucose oxidase method. Plasma [3-3H]glucose radioactivity was measured in duplicate on the supernatants of barium hydroxide-zinc sulfate precipitates of plasma samples after evaporation to dryness to eliminate tritiated water. Steele’s equation was used for calculation of glucose turnover as described previously (27). Values for EGP and glucose uptake, obtained at 10-min intervals, were averaged over the final 30 min of each glucose step for each subject.

The methods for measuring plasma insulin, C peptide, glucagon, epinephrine, norepinephrine, cortisol, and GH and determining their intra- and interassay variations have been previously reported (28). An additional commercial insulin-specific assay (29) was performed for all samples. Plasma free fatty acids, lactate, and glycerol were measured using a spectrophotometric or colorimetric technique (30, 31, 32).

The data in the text, figures, and tables are presented as the mean ± SEM. The glycemic thresholds for activation of counterregulatory hormone secretion were calculated as described previously (33). The area under the curve was calculated using the trapezoidal method. Insulin clearance rates were estimated from the prevailing plasma insulin concentrations and insulin infusion rates (34). Statistical analyses were performed using paired Student’s t tests. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
HbA1c, serum fructosamine, plasma glucose, insulin, C peptide, and glucose infusion rate (Figs. 1Go and 2Go)

HbA1c concentrations were in the normal range and identical after troglitazone or placebo treatment (5.9 ± 0.3% vs. 5.8 ± 0.3%, respectively; P = NS). Fasting serum fructosamine levels were also similar after troglitazone or placebo treatment and were equal to 316 ± 11 µmol/L after 7 days of troglitazone treatment and 314 ± 8 µmol/L (P = NS) after placebo administration. Body weight was unchanged during the course of the studies.



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  		Figure 1. Study protocol and plasma glucose concentrations at each glucose step in the troglitazone ({blacksquare}) and placebo ({circ}) studies.

 


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  		Figure 2. Plasma insulin (A) and C peptide (B) concentrations after troglitazone ({blacksquare}) and placebo ({circ}) treatments.

 
Plasma glucose concentrations during both clamps are shown in Fig. 1Go. Plasma glucose concentrations at 0 min were 4.88 ± 0.11 mmol/L in the troglitazone studies and 4.94 ± 0.11 mmol/L in the placebo studies (P = NS). Plasma glucose concentrations averaged for the final 30 min of each glucose step were 4.94 ± 0.02 mmol/L in the troglitazone studies and 4.94 ± 0.01 mmol/L in the placebo studies during the first clamp step (5.0 mmol/L target plasma glucose), 4.38 ± 0.01 and 4.49 ± 0.02 mmol/L, respectively, during the second clamp step (4.4 mmol/L target plasma glucose), 3.88 ± 0.01 and 3.88 ± 0.02 mmol/L, respectively, during the third step (3.9 mmol/L target plasma glucose), and 3.38 ± 0.01 and 3.38 ± 0.02 mmol/L, respectively, during the final clamp step (3.3 mmol/L target plasma glucose). In all studies there were no significant differences in plasma glucose between troglitazone and placebo studies.

Basal plasma insulin concentrations were nearly identical in both studies (Fig. 2AGo), averaging 28.2 ± 4.2 pmol/L in the troglitazone and 31.2 ± 5.4 pmol/L in the placebo studies (P = NS). However, despite an identical insulin infusion in both sets of studies (2.4 pmol/kg·min), plasma insulin concentrations after troglitazone were lower at each clamp step (Fig. 2AGo), averaging 168 ± 3.6 pmol/L in the troglitazone and 204 ± 5.4 pmol/L in the placebo studies (P < 0.05). Plasma insulin concentrations measured in an insulin-specific assay corresponding to these values were 162 ± 4.8 and 204 ± 1.8 pmol/L, respectively, virtually identical to the values measured in the polyclonal insulin assay. Plasma C peptide concentrations were comparable in both sets of studies at baseline (0.33 ± 0.03 nmol/L in the troglitazone studies and 0.36 ± 0.03 nmol/L in the placebo studies; P = NS) and remained very similar during the clamp, decreasing in both studies to 0.03 ± 0.01 nmol/L at the hypoglycemic nadir at 200 min (Fig. 2BGo).

As plasma insulin levels during the clamps were lower after troglitazone, we tested the possibility of an increase in the metabolic clearance of insulin (MCRi) in the troglitazone studies. Indeed, the calculated MCRi (based on the plasma insulin levels measured in the standard assay) were 11.2 ± 0.4 mL/kg·min after troglitazone and 8.8 ± 0.6 mL/kg·min after placebo (22% higher in the troglitazone studies) during the 5 and 4.4 mmol/L glucose steps, and were 13.5 ± 0.5 mL/kg·min after troglitazone and 10.6 ± 0.6 mL/kg·min after placebo (21% higher in the troglitazone studies) during the 3.9 and 3.3 mmol/L glucose steps (both P < 0.05).

Counterregulatory hormones (Figs. 3Go and 4Go and Table 1Go)

During the 5.0 and 4.4 mmol/L glucose steps, plasma epinephrine concentrations remained at or near basal values and were similar in both troglitazone and placebo studies (208.4 ± 34.3 pmol/L after troglitazone and 219.4 ± 36.5 pmol/L after placebo; P = NS). However, a further reduction in plasma glucose to 3.9 mmol/L was associated with an increment in plasma epinephrine in both sets of studies, although the threshold for epinephrine release occurred at a higher plasma glucose concentration in the troglitazone compared with the placebo studies (4.05 ± 0.05 and 3.77 ± 0.05 mmol/L, respectively; P = 0.003; Fig. 3AGo). Moreover, the average maximal raise in plasma epinephrine was significantly higher during the 3.9 and 3.3 mmol/L glucose steps in the troglitazone compared with the placebo studies, equaling 1124 ± 109 and 628 ± 104 pmol/L, respectively, during the 3.9 mmol/L glucose step, and 3247 ± 147 and 2543 ± 251 pmol/L respectively, during the 3.3 mmol/L glucose step (both P < 0.05; Fig. 3BGo). The area under the epinephrine curve during the entire duration of the clamps was also significantly higher in the troglitazone compared with the placebo studies (3176 ± 228 vs. 2139 ± 165 pmol/mL·h; P < 0.05).



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  		Figure 3. Plasma epinephrine concentrations over time (A) and averaged for the final 30 min of each glucose step (B). {blacksquare}, Studies after troglitazone; {circ} and {square}, studies after placebo. *, P < 0.05 vs. placebo.

 


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  		Figure 4. Plasma glucagon concentrations over time (A) and averaged for the final 30 min of each glucose step (B). {blacksquare}, Studies after troglitazone; {circ} and {square}, studies after placebo. *, P < 0.05 vs. placebo.

 

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  		Table 1. Plasma norepinephrine, GH, and cortisol at baseline and averaged for the final 30 min of each glucose step

 
Plasma glucagon concentrations were equivalent during the 5.0 and 3.9 mmol/L glucose steps in both studies (Fig. 4Go). However, the threshold for glucagon release occurred at a significantly higher plasma glucose concentration in the troglitazone studies compared with that in the placebo study (4.10 ± 0.11 and 3.83 ± 0.011 mmol/L, respectively; P = 0.03). Similarly, the magnitude of glucagon release during the 3.9 and 3.3 mmol/L glucose steps was significantly higher in the troglitazone compared with the placebo studies (74 ± 3 and 103 ± 2 ng/L in the troglitazone studies, respectively, and 63 ± 2 ng/L and 91 ± 5 ng/L in the placebo studies, respectively; P < 0.05 for both). The area under the glucagon response curve was higher in the troglitazone compared with the placebo studies (224 ± 21 vs. 184 ± 17 ng/mL·h; P < 0.05). Plasma norepinephrine, GH, and cortisol levels (Table 1Go) were comparable in both sets of studies.

To assess the potential impact of the difference in plasma insulin concentration in the two studies, we repeated an identical study with oral placebo preceding a stepped hypoglycemic clamp, but with 1.5 pmol/kg·min insulin infusion in two subjects. This insulin infusion was intended to result in the levels of plasma observed during the troglitazone studies. The mean plasma insulin concentration in these studies was 157.5 ± 6.1 and 164.5 ± 4.7 pmol/L (almost identical to those during the troglitazone studies); however, the threshold and magnitude of epinephrine and glucagon release were identical to those in the placebo studies in the same subjects (data not shown).

Plasma substrate concentrations (Table 2Go)

Plasma FFA levels were slightly lower in the troglitazone compared with placebo studies in the fasting state (362 ± 86 and 531 ± 115 µmol/L, respectively; P = NS). With the induction of hypoglycemia, plasma FFA fell gradually and comparably in the two sets of studies, and similarly, the small increases in plasma FFA concentrations at 3.9 and 3.3 mmol/L glucose steps were comparable. Plasma lactate and glycerol levels were also similar in both sets of studies.


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  		Table 2. Plasma lactate, free fatty acids (FFA), and glycerol at baseline and averaged for the final 30 min of each glucose step

 
Glucose kinetics (Fig. 5Go and Table 3Go)

Glucose infusion rates in the experimental protocol are depicted in Fig. 5AGo. During the first and second glucose steps, average glucose infusion rates were comparable in both studies (10.8 ± 0.5 µmol/kg·min in the troglitazone and 10.3 ± 0.5 µmol/kg·min in the placebo studies (P = NS). However, during the 3.9 mmol/L glucose step, the mean rate of glucose infusion was lower in the troglitazone studies (8.1 ± 0.5 vs. 9.4 ± 0.5 µmol/kg·min; P = NS), and during the 3.3 mmol/L glucose step, the average glucose infusion rate was 50% lower in the troglitazone compared with the placebo studies (2.3 ± 0.5 vs. 4.4 ± 0.5 µmol/kg·min; P < 0.05).



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  		Figure 5. Glucose infusion rates averaged for the final 30 min of each glucose step (A) and increments in EGP from the first glucose step (during which maximal suppression was achieved) averaged for the final 30 min of each glucose step (B). {circ}, Studies after troglitazone; {square}, studies after placebo. *, P < 0.05 vs. placebo.

 

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  		Table 3. Endogenous glucose production (EGP), glucose uptake, and plasma [3-3H]glucose specific activity at baseline and averaged for the final 30 min of each glucose step

 
Mean fasting EGP values were similar in the two studies (9.4 ± 0.5 µmol/kg·min with troglitazone and 9.9 ± 0.5 µmol/kg·min with placebo; P = NS). The average EGP calculated for the final 30 min of each clamp step is depicted in Table 3Go. During the 5.0 and 4.4 mmol/L glucose steps (after the initiation of insulin infusion), EGP was equally suppressed by approximately 35% in both studies. During the next two clamp steps (3.9 and 3.3 mmol/L), EGP rose to 76% and 94%, respectively, of basal in the troglitazone studies and to 67% and 83%, respectively, in the placebo studies. To evaluate the relative increase in EGP (eliminating the possible different suppressive effect of insulin), the recovery of EGP was calculated as the increase in EGP from the 5.0 mmol/L clamp step. The transition to activation of EGP from insulin-suppressed levels at euglycemia began earlier with troglitazone, at the 3.9 mmol/L step (0.49 ± 0.05 µmol/kg·min in the troglitazone and -0.61 ± 0.05 µmol/kg·min in the placebo studies), and EGP became significantly greater in the troglitazone studies at the 3.9 mmol/L step (1.94 ± 0.05 µmol/kg·min in the troglitazone and 0.66 ± 0.05 µmol/kg·min in the placebo studies; P < 0.05; Fig. 5BGo), coinciding with the earlier and more robust epinephrine and glucagon responses.

Glucose disposal rates were similar before insulin infusion in all studies. With the initiation of the clamp glucose uptake (Rd) was slightly and proportionally lower in the troglitazone studies during all glucose steps, although the difference was not significant. As expected, the MCR of glucose paralleled the Rd in all studies (data not shown).

[3-3H]Glucose specific activity was effectively maintained in both sets of studies during the clamps and did not vary by more than 20% (Table 3Go).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Three novel observations emerge from these studies. Our data demonstrate that a relatively short exposure to troglitazone amplified the counterregulatory hormonal response to hypoglycemia in nondiabetic individuals, both by shifting the glycemic thresholds for glucagon and epinephrine secretion (i.e. hormone release occurred at a higher plasma glucose concentration) and by increasing the magnitude of these counterregulatory hormones. In concert with these findings, we observed a more robust response of the measured EGP, suggesting that glucose counterregulation might be amplified by this mechanism. Troglitazone treatment was also associated with an enhancement of the clearance rate of exogenously infused insulin.

Prior studies have shown that hyperinsulinemia can magnify the hormonal response to hypoglycemia in dogs and humans (both nondiabetic and in type 1 diabetes subjects) (12, 13, 14, 15, 16, 17). Davis et al. (12) demonstrated that in conscious dogs, a higher dose of insulin during hypoglycemia resulted in amplification of the hormonal counterregulatory response (epinephrine, norepinephrine, cortisol, and pancreatic polypeptide) compared with equivalent hypoglycemia induced by a lower insulin dose. The same researchers demonstrated qualitatively similar results in humans (13, 14); in the latter studies, insulin was infused to steady state levels of either 486 ± 30 or 3054 ± 234 pmol/L, whereas plasma glucose was lowered to the same hypoglycemic plateau. The higher plasma insulin concentration was associated with a significantly greater increase in the magnitude of catecholamines and cortisol secretion; however, in all of these experiments, glucagon secretion did not increase with higher insulin levels (14). Similar results were observed in patients with type 1 diabetes (15, 16, 17). Conversely, other studies in humans reported a decrease in certain counterregulatory responses (e.g. glucagon) with higher insulin concentrations, suggesting that insulin may cause a decrease in the counterregulatory response to hypoglycemia (35, 36). Although all of these experiments suggest that insulin per se may modulate hypoglycemia counterregulation, the degree of hyperinsulinemia varied widely between studies, and different experimental designs preclude a coherent conclusion.

Thiazolidinediones act by binding to and activating the peroxisome proliferator-activated receptor-{gamma} (37). In skeletal muscle and adipocytes, thiazolidinediones increased basal and insulin-stimulated 2-deoxyglucose or glucose uptake (19, 38, 39), insulin binding to the plasma membrane, and the expression of glucose transporters GLUT1 and GLUT4 messenger ribonucleic acid and protein (40, 41, 42). Additionally, troglitazone prevented and reversed the insulin receptor kinase inhibition induced by high glucose levels in fibroblasts (43). Although the effects of troglitazone on insulin sensitivity in type 2 diabetes (44, 45, 46) and in patients with impaired glucose tolerance (47, 48) were induced after a few weeks of treatment, it has been clearly demonstrated that troglitazone can acutely increase in vitro insulin action (49). In addition, Olefsky et al. have shown in vivo that troglitazone (administered iv) induces an acute and significant increase in insulin-mediated glucose disposal and a significant decrease in hepatic glucose production, again emphasizing the acute effects of troglitazone (19).

We therefore used troglitazone in our studies to examine the possible role that enhancement of insulin signaling might play in hypoglycemia counterregulation. We hypothesized that an augmented insulin signal induced by troglitazone during hypoglycemia could enhance the counterregulatory hormonal response. Indeed, our experiments demonstrate that short exposure to troglitazone can amplify the hormonal response to mild hypoglycemia and possibly induce an earlier and greater increment in EGP. Thus, we speculate that troglitazone induced a resetting of the threshold for the counterregulatory hormones toward a higher plasma glucose level. This shift in glycemic thresholds occurred in the absence of evidence for any meaningful impact of troglitazone on indexes of chronic glycemia. The finding that troglitazone amplified the counterregulatory hormonal response, including both a significant shift in the thresholds and a greater magnitude for glucagon and epinephrine release, suggests that troglitazone may have a direct effect on hypoglycemia sensing.

Defective counterregulation in diabetes appears to be due in part to altered glycemic thresholds for activation of hormones and symptoms, which, in turn, may result from alteration in blood-brain barrier (BB) glucose transport (50, 51). Under conditions of hypoglycemia glucose transport becomes rate limiting for brain glucose metabolism (52). The major BBB glucose transport molecule is GLUT1, which has been shown to be up-regulated (messenger ribonucleic acid levels, protein, and activity) in certain experimental hypoglycemic conditions (51). In parallel, other studies have demonstrated that troglitazone enhances GLUT1 activity in vitro (53). Although there are no studies that examine BBB glucose transport activity in the presence of thiazolidinediones, GLUT1 is the major BBB and glial glucose transporter. It is conceivable that troglitazone may alter hypoglycemia counterregulation by enhancing BBB glucose transporter activity. Such a putative increase in BBB glucose transport could reduce the response to hypoglycemia (which is not consistent with our observation) or, vice versa, chronic brain hyperglycemia could modify the set-point for glucose sensing toward a higher value, inducing an increase in the counterregulatory response to hypoglycemia.

Another possible mechanism that can explain the effects of troglitazone on hypoglycemia counterregulation is the recent finding that thiazolidinediones may play an important role in the regulation of ionic channel activity (21, 22, 23, 24, 25). Using the patch-clamp technique Lee et al. (21, 23) demonstrated that troglitazone induces inhibition (closure) of type 2 K-ATP channel peripherally and in the tolbutamide-sensitive neurons in the rat ventromedial hypothalamus (the area in the brain considered to be the main site for central glucose sensing) (54). Additional studies (25) have shown that another thiazolidinedione (ciglitazone) stimulates the activity of large conductance K+ channels expressed in pituitary GH3 cells (which are involved in neural excitability and hormonal secretion). It is thus conceivable that by directly affecting key steps in the glucose-sensing system and/or in neurohormonal signaling, troglitazone may have influenced the hormonal counterregulatory response.

In concert with the significantly greater increase in the hormonal response during hypoglycemia after troglitazone treatment, the rate of glucose infusion was significantly lower, and EGP responded accordingly. As plasma insulin levels were lower after troglitazone (see discussion below) and may have affected EGP in these studies, we calculated the relative increments in EGP from the 5.0 mmol/L glucose step, the time point at which EGP was already maximally suppressed. Although EGP was suppressed by insulin (~35% in both studies), the recovery of EGP occurred earlier in the troglitazone studies and was significantly higher than that in placebo studies during the 3.3 mmol/L glucose step.

Another observation in our study was a lower plasma insulin concentration after troglitazone treatment despite the same insulin infusion rate in the placebo studies. The possibility of a suppressive effect of troglitazone on endogenous insulin secretion is not likely, as plasma C peptide levels were identical and equally suppressed in both studies. Additionally, C peptide clearance rates are slower than that those of insulin (55); thus, a difference in the insulin secretion rate would have been more evident in the measurement of plasma C peptide concentrations. Another explanation that might have accounted for the difference in plasma insulin levels could have been differential insulin processing reflected in our standard (polyclonal) insulin assay. However, insulin assayed using an insulin-specific monoclonal antibody method resulted in identical values. Finally, there was no significant difference in body weight with troglitazone, which could presumably influence plasma insulin concentrations. Taken together, these findings suggest that the insulin clearance rate is augmented by troglitazone treatment, consistent with our calculated MCRi. However, as the insulin clearance rate was not measured during the basal state we cannot confidently explain the difference in plasma insulin concentrations during the infusions of insulin by this mechanism.

Importantly, HbA1c and serum fructosamine concentrations were in the normal range and identical after troglitazone or placebo treatment, suggesting that there was no change in glucose homeostasis before the clamp studies. Although the observation that insulin clearance may be increased in nondiabetic subjects during troglitazone treatment may be of interest, it is unlikely to have played a role in the hormonal response to hypoglycemia. Indeed, as noted above, the literature suggests that a reduced counterregulatory hormonal response is associated with lower plasma insulin concentrations. However, to eliminate this possibility, we repeated placebo studies in two of the subjects using a lower insulin infusion to achieve the same plasma insulin concentrations as in the troglitazone studies. The counterregulatory hormonal response was identical to that in the other placebo studies, suggesting that the slightly reduced circulating insulin concentration during the troglitazone studies did not account for the difference we observed. Finally, it is possible that the lower plasma insulin concentrations during the troglitazone studies played a role in augmenting the EGP response to hypoglycemia. Although these differences in plasma insulin were small, a possible contribution of lower insulin levels to the enhanced EGP response cannot be entirely excluded.

In conclusion, this study describes a novel finding that the counterregulatory hormonal response to hypoglycemia can be enhanced or reset pharmacologically in nondiabetic subjects with troglitazone. Augmentation of the counterregulatory response was characterized by a shift in the glycemic threshold for epinephrine and glucagon secretion (which occurred at a higher plasma glucose level) and an increased magnitude of these hormones. These experimental results open a new avenue of research, suggesting that protective counterregulatory hormonal and hepatic responses to hypoglycemia can be modified pharmacologically. These data have implications for the treatment of both type 1 and type 2 diabetes and the prevention of hypoglycemia in both.


   Acknowledgments
 
We acknowledge the nursing staff of Albert Einstein General Clinical Research Center for their superb care of the subjects.


   Footnotes
 
1 This work was supported by a research grant from Parke-Davis and Grant RR-12248 (to Albert Einstein General Clinical Research Center). The results of this study were reported in abstract form at the Annual Scientific Sessions of the American Diabetes Association, San Antonio, TX, June 13, 2000. Back

Received August 15, 2000.

Revised October 23, 2000.

Accepted October 25, 2000.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Mitrakou A, Ryan C, Veneman T, Evron W, Cryer P, Gerich J. 1991 Hierarchy of glycemic thresholds for counterregulatory hormone secretion, symptoms, and cerebral dysfunction in normal humans. Am J Physiol Endocrinol Metab. 260:E64–E67.
  2. Schwartz NS, Clutter WE, Shah SD, Cryer P. 1987 The glycemic thresholds for activation of glucose counterregulatory systems are higher than the thresholds for symptoms. J Clin Invest. 79:777–781.
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