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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 2 645-648
Copyright © 2001 by The Endocrine Society

Original Studies

Oral Glucose Augments the Counterregulatory Hormone Response during Insulin-Induced Hypoglycemia in Humans1

Rubina A. Heptulla, William V. Tamborlane, Tony Y.-Z. Ma, Fran Rife and Robert S. Sherwin.

Department of Pediatrics, Internal Medicine and the General Clinical Research Center, Yale University School of Medicine, New Haven, Connecticut 06520; and Department of Pediatrics Baystate Medical Center, Springfield, Massachusetts 01199

Address all correspondence and requests for reprints to: Rubina A. Heptulla, M.D., Department of Pediatrics, Division of Endocrinology, Baystate Medical Center, 3300 Main Street, Springfield, Massachusetts 01199. E-mail: rheptulla{at}yahoo.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been suggested that the counterregulatory hormone (CRH) response to acute hypoglycemia is triggered via glucose sensors situated in either the hypothalamus or the portohepatic area. If the latter were critical during hypoglycemia, one would anticipate that ingestion of glucose, by raising glucose levels in the portal circulation, should attenuate CRH responses previously described in animal studies. To evaluate the effect of raising portal, but not peripheral, glucose levels during insulin-induced hypoglycemia, we performed hypoglycemic clamp studies in five healthy adult males on two occasions. On one occasion, subjects received oral glucose (OG) (25 g) during hypoglycemia; and on one occasion, noncarbohydrate-containing drink of equal volume, while maintaining plasma glucose at 55 ± 2 mg/dL (3.08 mmol/L).

As a result, there were no significant differences in systemic plasma glucose levels between the two hypoglycemic clamp studies, and basal CRH concentrations were also similar. As expected, there was a brisk rise in all CRH during the control (hypoglycemia+noncarbohydrate drink) study. In the experimental study, administration of OG (hypoglycemia+OG), to raise intraportal glucose levels during systemic hypoglycemia, did not attenuate CRH responses. Indeed, OG enhanced the rise in epinephrine, glucagon, and GH.

Increases in cortisol and norepinephrine did not differ between the two studies.

Therefore, our data suggest that increasing the level of glucose in the portal vein above that in the systemic circulation, during hypoglycemia, enhances (rather than suppresses) CRH responses. Thus, ingestion of glucose may reverse hypoglycemia directly by provision of substrate, as well as indirectly by stimulating counteregulatory mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-INDUCED HYPOGLYCEMIA RESULTS in a prompt and coordinated counterregulatory hormone (CRH) response resulting from hormonal and neural activation (1). Although CRH responses to hypoglycemia have been extensively studied (2), the putative mechanisms of hypoglycemia detection have not been fully elucidated. Indeed, studies in animal models have provided conflicting results as to whether the preeminent locus of hypoglycemia detection resides in the brain (3, 4, 5) or in the portohepatic area (6, 7, 8).

A variety of experiments in rats suggest that the ventromedial hypothalamus (VMH) plays the central role in stimulating catecholamine and glucagon responses to hypoglycemia (9, 10, 11, 12). On the other hand, epinephrine and norepinephrine responses were reduced in studies in dogs in which systemic hypoglycemia was induced by insulin while euglycemia was maintained within the portal vein by direct intraportal infusion of exogenous glucose (6). The suppression of antiinsulin hormones by entry of glucose into the portal system might facilitate the switch from the fasted to fed state and serve to limit postprandial hyperglycemia (13). However, during hypoglycemia, it might have the adverse effect of reducing the capacity of oral glucose (OG) feeding to promote glucose recovery.

In comparison with animal models, there is paucity of data available regarding the locus of hypoglycemia detection in human subjects. In an experiment in nature, it has been recently shown that the counterregulatory response to hypoglycemia was markedly attenuated in a patient with hypothalamic sarcoidosis (14). Experiments examining the influence of portal vein glucose sensors in human subjects have been impeded by practical difficulties in trying to prevent reductions in glucose in the portal system while lowering plasma glucose systemically. In the current study, we have attempted to overcome this obstacle by introducing glucose into the portal vein via the oral route, while a stable and reproducible hypoglycemic stimulus was induced systemically using the hypoglycemic clamp technique (15). Surprisingly, CRH to hypoglycemia tended to be increased rather than diminished by OG administration, in comparison with control hypoglycemia studies without OG.


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

We studied five healthy, nondiabetic, lean (body mass index, 24 + 1 kg/m2) adult males (age, 26 + 1 yr) on two occasions. All subjects were seen in the outpatient department of Yale General Clinical Research Center, in the morning, after a 10- to 12-h overnight fast. A detailed medical history and physical examination were obtained for each subject. All subjects were in good health and taking no medications. The nature and purpose of the study was explained to them, and written voluntary consent to participate in the study was obtained . The Human Investigation Committee of Yale University School of Medicine approved the study protocols.

Procedures

To produce a standardized hypoglycemic stimulus, we performed a one-step hypoglycemic clamp procedure, as described by Simonson et al. (16), in all the subjects. Two cannulas were inserted, one in an antecubital vein for infusion of insulin and glucose and the other in the dorsal hand vein for blood sampling. The hand was placed in a heated (65 degree C) box to arterialize the venous blood sampling (17). The subjects were given continuous iv infusions of insulin (120 mU per square meter of body-surface area per minute), and target plasma glucose values were achieved by varying the rate of infusion of 20% glucose in water. Plasma glucose was measured, at the bedside, at 5-min intervals (Beckman Coulter, Inc. glucose analyzer, Beckman Coulter, Inc., Fullerton, CA). In all the subjects, plasma glucose concentrations were stabilized between 90 and 108 mg/dL (5.0 and 6.0 mmol/L) before the induction of hypoglycemia. The length of the euglycemic phase was 60 min in all subjects, the plasma glucose concentration was reduced to approximately 50–55 mg/dL (2.8–3.1 mmol/L), over a period of approximately 10 min, by reducing the rate of glucose infusion. Plasma glucose concentrations were then maintained at that level for a further 100 min.

During one hypoglycemic clamp study, a load of 25 g glucose was given orally as a cola-flavored drink at 80 min, the time when plasma glucose concentration had reached approximately 50 mg/dL (2.8 mmol/L). During the other clamp, patients were given a caffeine-free diet cola equal in volume and color to the OG drink at 80 min of the study (0–60 min euglycemia, 60–180 min hypoglycemia, 80 min administration of the oral drink)

Measurements

Blood samples were taken at 10-to 20-min intervals for measurements of plasma insulin, epinephrine, norepinephrine, cortisol, glucagon, and GH.

Plasma catecholamines were measured by high-performance liquid chromatography using an electrochemical detector; and plasma insulin, GH, cortisol, and glucagon were measured by double-antibody RIAs. All the samples from each subject were analyzed in a single assay, and all tests were run in duplicate. The intraassay variations for the GH were 4.2% (at 11.1 ng/mL); glucagon, 8.3%; and cortisol, 8.6%. For epinephrine and norepinephrine, the interassay variability levels were 17% and 16%, respectively. The intraassay variability values were 6% for epinephrine and 4% for norepinephrine.

Statistical analysis

The plasma glucose concentrations and hormone responses in the two studies were compared by ANOVA with repeated-measures design. When there was a statistically significant group-time effect, two-tailed paired t tests were used to localize the effects. Differences were regarded as statistically significant if P values were less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma insulin concentrations in the basal state [8 ± 1 vs. 7 ± 1 µU/mL (57 ± 7 vs. 50 ± 7 pmol/L)] and during the insulin infusion [178 ± 8 vs. 190 ± 10 µU/mL (1277 ± 57 vs. 1363 ± 70 pmol/L)] did not differ between the studies, regardless of whether or not OG was given during hypoglycemia. As shown in Fig. 1Go, there were also no significant differences (P < 0.8) between the two studies, with respect to basal glucose concentrations or in the fall in plasma glucose levels during both hypoglycemic clamps. Thus, virtually an identical hypoglycemic stimulus was achieved, whether or not the subject ingested OG.



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  		Figure 1. Data are expressed as means ± SE for plasma glucose concentrations. Shaded circles represent the hypoglycemia study along with noncarbohydrate drink, and open circles represent data of the hypoglycemia and OG study where patients received 25 g OG at 80 min. To convert plasma glucose values to millimoles per liter, multiply by 0.056.

 
As shown in Fig. 2Go, basal concentrations and the early (60–80 min) rise in plasma epinephrine, GH, and glucagon levels were similar in both studies. However, during the last 60 min of the hypoglycemic clamp with OG, the plasma concentrations of epinephrine [1212 ± 145 pg/mL (6605 ± 790 pmol/L)], GH (23 ± 3 µg/L), and glucagon (134 ± 23 ng/L) rose to values that were significantly higher than plasma epinephrine [824 ± 181 pg/mL (4490 ± 986 pmol/L), P < 0.015], GH (14 ± 3 µg/L, P < 0.015), and glucagon (86 ± 20 pg/mL, P < 0.05) levels observed during the hypoglycemic clamp without OG (Table 1Go). In contrast, as shown in Fig. 3Go, administration of OG did not significantly affect plasma cortisol (P < 0.08) or plasma norepinephrine (P < 0.16) responses to hypoglycemia.



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  		Figure 2. Data are expressed as means ± SE for plasma epinephrine, GH, and glucagon concentrations. Shaded circles represent the hypoglycemia study along with noncarbohydrate drink, and open circles represent data of the hypoglycemia and OG study where patients received 25 g OG at 80 min. To convert plasma epinephrine values to picomoles per liter, multiply by 5.458.

 

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  		Table 1. Counterregulatory hormone response to hypoglycemia with and without oral glucose

 


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  		Figure 3. Data are expressed as means ± SE for plasma cortisol and norepinephrine concentrations. Shaded circles represent the hypoglycemia study along with noncarbohydrate drink, and open circles represent data of the hypoglycemia and OG study where patients received 25 g OG at 80 min. To convert plasma norepinephrine values to picomoles per liter, multiply by 5.458.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present investigation was undertaken to examine the role of glucose sensors within the portal venous system in regulating CRH responses to hypoglycemia in human subjects. Animal experiments from our own laboratory have suggested the VMH is responsible for detection of hypoglycemia (9, 10, 11, 12); whereas other studies have shown that maintenance of euglycemia in the portal vein suppresses the CRH response to systemic hypoglycemia (6). In this study, we attempted to produce similar experimental conditions in human subjects by using the glucose clamp technique to cause an identical reduction in systemic plasma glucose concentrations during two studies: one with and one without ingestion of OG. Relatively large iv doses of insulin were used in both studies so that precise control of systemic glucose concentrations could be achieved in the face of ingestion of OG in amounts (25 g) within the range that might be used clinically to treat hypoglycemia.

Oral glucose was ingested during the experimental study after mild hypoglycemia was induced and CRH responses were initiated. Practical considerations precluded measurements of glucose concentrations in the portal vein in these subjects. However, in animal studies, ingestion of 40g OG leads to a rapid rise in glucose concentration and appearance of approximately 27 g glucose intraportally within 15–30 min of glucose ingestion (18, 19, 20). Under these conditions, one might estimate that we would have observed a detectable reduction in plasma epinephrine concentration vs. control experiments if a portal glucose sensor was operative in humans. However, ingestion of OG enhanced, rather than reduced, the adrenomedullary responses to hypoglycemia. Moreover, the ingestion of OG also increased GH and glucagon responses to hypoglycemia.

This study was not designed to investigate the mechanisms underlying alterations in CRH responses induced by OG. However, with respect to GH, it is noteworthy that ghrelin (a GH-releasing acetylated peptide from the stomach) has been shown to stimulate GH release independent of regulation by hypothalamic GHRH (21). Previous studies by Donovan et al. (7, 8) did not examine changes in GH levels during their hypoglycemia and portal euglycemia experiments. The increased glucagon responses with OG could be mediated by neural connections linking the portal venous system with the VMH (22). There may be other mediators of glucagon secretion (like endothelin-1) that may account for the increased glucagon response (23). In addition, the study design with hyperinsulinemic euglycemia before hypoglycemia induction may have also contributed to the reduction in glucagon response (24).

Regardless of the mechanisms involved, the data support the contention that the increased CRH responses were the result of absorption of glucose into the portal system. We controlled for nonmetabolic effects of volume and taste by having the subjects, during the control study, ingest a decaffeinated drink of the same taste and volume as they ingested during the experimental study. Moreover, the pattern of a 40-min delay in response for all three hormones is more consistent with the time required to absorb ingested glucose into the portal circulation than what might be expected from a more immediate neural response. It is also noteworthy that these responses were very consistent and selective, so that statistical significance was readily observed with a relatively small number of subjects. Ingestion of OG did not seem to alter plasma cortisol and norepinephrine responses to hypoglycemia, but further studies in larger numbers of subjects are needed to evaluate these responses more definitively.

In conclusion, although the results of the present study were surprising, the implications regarding clinical management of hypoglycemia are potentially important. Further studies are needed to determine whether similar effects are observed in patients with type 1 diabetes with or without autonomic neuropathy. If so, provision of OG to patients with diabetes during hypoglycemic events may have benefits that go beyond the simple provision of substrate.


   Footnotes
 
1 Supported by NIH Grants DK-20495, HD-30671, RR-06022, and RR-125. Back

Received July 20, 2000.

Revised October 16, 2000.

Accepted October 25, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gerich JE, Campbell PJ. 1988 Overview of counterregulation and its abnormalities in diabetes mellitus and other conditions. Diabetes Metab Rev. 4:93–111.[Medline]
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  3. Benzo CA. 1983 Minireview. The hypothalamus and blood glucose regulation. Life Sci. 32:2509–2515.[CrossRef][Medline]
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  8. Donovan CM, Hamilton-Wessler M, Halter JB, Bergman RN. 1994 Primacy of liver glucosensors in the sympathetic response to progressive hypoglycemia. Proc Natl Acad Sci. 91:2863–2867.[Abstract/Free Full Text]
  9. Borg MA, Borg WP, Tamborlane WV, Brines ML, Shulman GI, Sherwin RS. 1999 Chronic hypoglycemia and diabetes impair counterregulation induced by localized 2-deoxy-glucose perfusion of the ventromedial hypothalamus in rats. Diabetes. 48:584–587.[Abstract]
  10. Borg MA, Sherwin RS, Borg WP, Tamborlane WV, Shulman GI. 1997 Local ventromedial hypothalamus glucose perfusion blocks counterregulation during systemic hypoglycemia in awake rats. J Clin Invest. 99:361–365.[Medline]
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  13. Schmitt M. 1973 Influence of hepatic portal receptors on hypothalamic feeding and satiety centers. Am J Physiol. 225:1089–1095.[Free Full Text]
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  17. McGuire E, Helderman J, Tobin J, Andres R, Bergman R. 1976 Effects of arterial versus venous sampling on glucose kinetics in man. J Appl Physiol. 41:65–73.
  18. Abumrad NN, Cherrington AD, Williams PE, Lacy WW, Rabin D. 1982 Absorption and disposition of a glucose load in the conscious dog. Am J Physiol. 246:E398–406.
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