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
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ABSTRACT |
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It has been suggested that insulin-induced suppression of
endogenous glucose production (EGP) may be counteracted independently of increased epinephrine (Epi) or glucagon during moderate
hypoglycemia. We examined EGP in nondiabetic (n = 12)
and type 1 diabetic (DM1, n = 8) subjects while lowering plasma
glucose (PG) from clamped euglycemia (5.6 mmol/l) to values just above
the threshold for Epi and glucagon secretion (3.9 mmol/l).
Individualized doses of insulin were infused to maintain euglycemia
during pancreatic clamps by use of somatostatin (250 µg/h), glucagon
(1.0 ng · kg1 · min
1),
and growth hormone (GH) (3.0 ng · kg
1 · min
1)
infusions without need for exogenons glucose. Then, to achieve physiological hyperinsulinemia (HIns), insulin infusions were fixed at
20% above the rate previously determined for each subject. In
nondiabetic subjects, PG was reduced from 5.4 ± 0.1 mmol/l to 3.9 ± 0.1 mmol/l in the experimental protocol, whereas it was held constant
(5.3 ± 0.2 mmol/l and 5.5 mmol/l) in control studies. In the latter,
EGP (estimated by [3-3H]glucose) fell to values
40% of basal (P < 0.01). In contrast, in the experimental
protocol, at comparable HIns but with PG at 3.9 ± 0.1 mmol/l, EGP was
activated to values about twofold higher than in the euglycemic control
(P < 0.01). In DM1 subjects, EGP failed to increase in the
face of HIns and PG = 3.9 ± 0.1 mmol/l. The decrease from basal EGP
in DM1 subjects (4.4 ± 1.0 µmol · kg
1 · min
1)
was nearly twofold that in nondiabetics (2.5 ± 0.8 µmol · kg
1 · min
1,
P < 0.02). When PG was lowered further to frank
hypoglycemia (~3.1 mmol/l), the failure of EGP activation in DM1
subjects was even more profound but associated with a 50% lower plasma
Epi response (P < 0.02) compared with nondiabetics. We
conclude that glucagon- or epinephrine-independent activation of EGP
may accompany other counterregulatory mechanisms during mild
hypoglycemia in humans and is impaired or absent in DM1.
counterregulation; glucose turnover; endogenous glucose production
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INTRODUCTION |
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COUNTERREGULATION OF HYPOGLYCEMIA in nondiabetic subjects involves several sequential response components; the depth of hypoglycemia activates each compo-nent at a differing glycemic threshold as well as modulating its magnitude (35, 43). Among the important components of the counterregulatory cascade during moderate hypoglycemia are the secretion of glucagon and epinephrine, both of which activate endogenous glucose production (EGP) (9, 15, 16). A large body of evidence indicates that the defective secretion of these two hormones is critical to the persistent suppression of EGP and consequently the development of more severe hypoglycemia (8). Defective glucose counterregulation in type 1 diabetes is due, in large part, to the inability of EGP to overcome insulin-induced suppression and is associated with impaired secretion of glucagon and epinephrine (9, 28). The defect in epinephrine is manifested as both delay (with respect to the hypoglycemic threshold level for) and reduction (with respect to the magnitude) of hormone secretion. Recent studies from this and other laboratories suggest that extrahepatic tissues (e.g., skeletal muscle) may partially compensate for defective hepatic counterregulation (7, 9), although the mechanism underlying this effect also appears to be largely epinephrine dependent (22).
Because hormone-dependent hypoglycemia counterregulation is clearly impaired in type 1 diabetes, we hypothesized that hormone-independent activation of EGP during impending hypoglycemia might likewise be defective. The studies supporting the existence of alternative, nonhormonal mechanisms by which EGP might be activated [e.g., by direct neural innervation (17, 38) or by so-called "autoregulation" by glucose per se] are inconclusive (8, 23, 42). Saccà et al. (42) suggested that a nonhormonal signal stimulated EGP during a fall in plasma glucose between ~3.9 and ~3.1 mmol/l in nondiabetic human subjects. In addition, they concluded that either direct neural innervation or glucose per se might be operative (42). In type 1 diabetics, however, they found impairment of EGP but only under conditions associated with defective glucagon secretion (19, 42). Thus the role of defects in hormone-independent activation of EGP in the pathogenesis of hypoglycemia in type 1 diabetes has not been specifically examined.
In nondiabetic human subjects, Bolli et. al. (4), using steady-state
pancreatic/pituitary clamps, suggested that glucose per se did not
activate EGP except during severe hypoglycemia (~1.9 mmol/l).
However, since combined - and
-adrenergic blockade was used in
their experimental model, a role for an epinephrine-independent mechanism was not elucidated. Hansen et al. (23) also concluded that a
low glucose per se did not activate EGP during mild hypoglycemia (~3.6 mmol/l), although somatostatin without glucagon replacement was
used in those experiments, resulting in a glucagon-deficient metabolic
milieu that might have altered glucose fluxes. Moreover, all these
previous reports used isotopic techniques to evaluate glucose turnover
but were not designed to maintain constant glucose-specific activity
and did not use purified tracers. These methodological considerations
are germane, given that nonhormonal counterregulatory factors
contribute only a small portion (~20-30%) of the EGP response during hypoglycemia, and a small effect may go undetected unless experimental methods are optimized (14).
The present study was therefore designed to compare the possible role of nonhormonal activation of EGP in nondiabetic and type 1 diabetic subjects with the following experimental goals in mind: 1) to exclude the effect of hormonal counterregulation by controlling changes in glucagon and epinephrine secretion; 2) to replace insulin by use of exogenous insulin infusions to match each subject's basal requirement, and 3) to optimize tracer methodology by use of current standards such as purified tracer and stable isotope specific activity.
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MATERIALS AND METHODS |
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Subjects. We studied 12 healthy, nondiabetic subjects (seven men, five women) aged 28 ± 1 (SE) yr and eight C-peptide-deficient type 1 diabetic subjects (33 ± 3 yr), also equally gender distributed. The diabetic subjects had a mean duration of diabetes of 12 ± 2 yr and an Hb A1c of 8.4 ± 0.4% (range 6.8-10.2%). Body mass index in the nondiabetic subjects averaged 24.7 ± 0.9 kg/m2, and in diabetic subjects 26.5 ± 1.4 kg/m2. The diabetic subjects did not have proliferative retinopathy, autonomic neuropathy measured with cardiovascular function tests, urinary protein excretion >2,650 µmol/ 24 h, or a serum creatinine >124 µmol/l. Two subjects had microalbuminuria, and three had treated hypothyroidism and were euthyroid. None was taking any chronic medications other than insulin with the exception of two subjects with microalbuminuria who took an angiotensin-converting enzyme inhibitor that was discontinued 3 days before the studies. None of the subjects had had an episode of severe hypoglycemia (9) in the previous year or frequent mild-to-moderate (9) hypoglycemic episodes in the month before the study. Informed written consent was obtained in accordance with the guidelines of the Committee on Clinical Investigations of the Albert Einstein College of Medicine.
Study design. The study was designed to examine counterregulatory mechanisms regulating EGP while controlling changes in glucagon secretion, and this assessment was made at the 3.9 mmol/l plasma glucose step. This was achieved by clamping plasma glucagon concentrations during somatostatin infusion and by clamping plasma glucose just above the threshold for activation of epinephrine release (5, 35, 43). A deeper level of hypoglycemia (3.1 mmol/l) was then induced and represented a model of glucagon-independent but epinephrine-dependent counterregulation.
All studies were performed in the postabsorptive state after an overnight fast. Nondiabetic subjects were studied on two separate occasionsMethods. Plasma glucose was measured with a Beckman glucose analyzer (Fullerton, CA) by use of the glucose oxidase method. Plasma [3-3H]glucose radioactivity was measured in duplicate on the supernatants of barium hydroxide-zinc sulfate precipitates (Somogyi procedure) of plasma samples, after evaporation to dryness to eliminate tritiated water. Plasma tritiated water-specific activity was determined by liquid scintillation counting of the protein-free supernatant (Somogyi filtrate) before and after evaporation to dryness. Plasma insulin, C-peptide, glucagon, growth hormone, and cortisol were determined by RIA (44). Plasma epinephrine and norepinephrine were measured by means of an isotope derivative assay (44, 47). Plasma FFA and glycerol were measured by means of calorimetric enzymatic methods (39, 40), and plasma lactate was measured by an enzymatic assay (18). For indirect calorimetry, airflow and O2 and CO2 concentrations in the expired and inspired air were measured by a computerized open-circuit system (Deltatrac, Sensormedics, Yorba Linda, CA). Urinary nitrogen was measured by the Kjeldahl procedure (24). Hb A1c was measured by means of ion-exchange chromatography with an upper normal limit of 6.1%.
Analyses.
Rates of glucose appearance (Ra) and disappearance
(Rd) were calculated by use of Steele's steady-state
equation (48). Endogenous glucose production (EGP) was determined by
subtracting the rates of glucose infusion from the tracer-determined
Ra. Rates of glycolysis from plasma glucose were estimated
from the increment in tritiated water per unit time
(dpm · ml1 · min
1)
multiplied by body water mass (ml) per
[3-3H]glucose specific activity (dpm/mg), as
previously validated (41). Glycogen synthetic rates were estimated as
the difference between glucose disposal rate (Rd) and
glycolysis from plasma glucose (41). Carbohydrate oxidation (CHO) was
calculated from O2 consumption and CO2
production (corrected for protein oxidation) using the Lusk equation
(31). Data for glucose turnover, carbohydrate and lipid oxidation,
plasma hormones, and substrate concentrations represent the mean values
during the final 60 min of the baseline euglycemic period and the final
60 min of each of the three hyperinsulinemic periods. Statistical
analysis of the data over time was performed using PROC MIXED in SAS
System Version 6.12 (SAS Institute, Cary, NC) (29). The random effect
considered in this mixed model is the error measurement of individual
subjects, and the within-individual fixed effect is the difference
between groups. For averaged data, the two-sample Student's
t-test and the Wilcoxon ranked-sum tests were employed (49).
All data are presented as means ± SE.
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RESULTS |
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Plasma glucose and insulin, and glucose infusion rate.
Plasma glucose and insulin during the Experimental protocol in both
groups are shown in Fig. 1. Plasma glucose
concentrations during the basal euglycemic period were 5.7 ± 0.2 mmol/l in nondiabetics and 6.0 ± 0.4 mmol/l in type 1 diabetics
[P = not significant (NS)]. During the final 60 min of each
Experimental protocol step, plasma glucose averaged 5.4 ± 0.1, 3.9 ± 0.1, and 3.2 ± 0.1 mmol/l in nondiabetic subjects and 5.6 ± 0.1, 3.9 ± 0.1, and 3.1 ± 0.1 mmol/l in type 1 diabetics (all
P = NS between groups at each step). Plasma glucose
was maintained at desired targets with coefficients of variation (CVs)
for the 5.6, 3.9, and 3.1 mmol/l nominal targets averaging 2.8, 3.4, and 8.7%, respectively, in nondiabetic subjects, and 4.3, 3.7, and
2.2%, respectively, in type 1 diabetic subjects. During Control
studies (5.6 mmol/l nominal targets throughout), the CVs were 2.8, 3.0, and 2.2%, respectively, in nondiabetics. Plasma glucose levels during
the Control studies averaged 5.4 ± 0.1 mmol/l in the basal period,
5.3 ± 0.2 mmol/l in the first hyperinsulinemic period (corresponding
to 5.6 mmol/l in the Experimental protocol), and 5.5 ± 0.2 mmol/l in the second period (corresponding to 3.9 mmol/l in the
Experimental protocol).
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Plasma hormone and substrate concentrations.
Plasma glucagon (Fig. 2) and GH (Table
2) were clamped at basal concentrations
using somatotropin release-inhibiting hormone (SRIH) and replacement
infusions throughout all three study periods. Moreover, these levels
were not significantly different in the two study groups. Plasma
cortisol (Table 2), and epinephrine and norepinephrine (Fig. 2), on the
other hand, were free to change. At the 3.9 mmol/l glucose step all
three hormones remained at or near basal concentrations and were
similar in both diabetic and nondiabetic groups. In particular, plasma
epinephrine averaged 453 ± 98 pmol/l in nondiabetics and 267 ± 44 pmol/l in diabetics (P = NS), in neither group exceeding
thresholds previously reported to activate EGP (5). However, the
reduction of plasma glucose to 3.9 mmol/l from 5.6 mmol/l was
associated with an increment in plasma epinephrine in both groups (from
153 ± 33 pmol/l to 267 ± 44 pmol/l in subjects with
diabetes, and 142 ± 38 pmol/l to 453 ± 98 pmol/l in nondiabetics,
both P < 0.05).
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Glucose turnover.
Glucose uptake at baseline (not shown) was 13.8 ± 1.0 µmol · kg1 · min
1
in nondiabetic subjects and 12.5 ± 1.3 µmol · kg
1 · min
1
in type 1 diabetics (P = NS). The slightly greater baseline
values in the nondiabetics persisted at the hyperinsulinemic 5.6 mmol/l step (Fig. 3), but the difference was more
marked with nondiabetics having ~40% higher rates (P
< 0.01). When plasma glucose was lowered to 3.9 mmol/l,
glucose uptake fell in nondiabetics but was unchanged in diabetics and
still remained lower (P < 0.01). Finally, when the 3.1 mmol/l
step was achieved, glucose uptake rates in the two groups were nearly
identical. Glycolysis from plasma glucose, however, was similar in the
two groups at all plasma glucose levels; thus glycogen synthesis
accounted for most of the changes observed in glucose uptake (Fig. 3),
with rates in diabetics ~30% those of nondiabetics at the 5.6 mmol/l
and 3.9 mmol/l steps.
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Indirect calorimetry.
During the Experimental study in nondiabetic subjects, CHO was higher
than in diabetic subjects at the 5.6 mmol/l step and remained unchanged
at 3.9 mmol/l. However, at 3.1 mmol/l, there was a shift in substrate
utilization such that CHO fell by ~65% and lipid oxidation doubled
(Table 4). In diabetic subjects who started
out with a significantly higher rate of lipid oxidation at the 5.6 mmol/l step, CHO and lipid oxidation rates were relatively unchanged,
consistent with the stable plasma concentrations of FFA.
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DISCUSSION |
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We have examined the response of EGP to mild hypoglycemia under conditions of hyperinsulinemia together with basal glucagon. The data suggest that the insulin-induced suppression of EGP can be reversed in nondiabetic subjects with unchanged glucagon and minimal changes in epinephrine. Under conditions of euglycemia plus the same degree of hyperinsulinemia in nondiabetics, EGP was ~50% more suppressed than when the stimulus of 3.9 mmol/l glucose was present. This early defense against further hypoglycemia may fail in subjects with type 1 diabetes, since EGP at the same mild hypoglycemia was nearly twofold more suppressed than in nondiabetics. Thus we present evidence that glucagon- or epinephrine-independent activation of EGP may be an early step in glucose counterregulation, and this activation is impaired or absent in type 1 diabetes. The present study also confirms the importance of defective counterregulation in type 1 diabetes when plasma glucose levels fall below the normal threshold for epinephrine release (i.e., 3.1 mmol/l). Finally, the data suggest that the effect of modest hyperinsulinemia to suppress EGP cannot be overcome by intact epinephrine secretion alone, even in nondiabetic subjects at 3.1 mmol/l, and underscore the necessity of intact glucagon responses for the preservation of normal glucose counterregulation.
The mechanism of activation of EGP before and/or independently of the stimulation of glucagon and epinephrine release remains uncertain. The role of glucose per se on hepatic glucose metabolism has been studied primarily under conditions of suppression of EGP by hyperglycemia (2) and is most likely mediated through the modulation of the fluxes through hepatic glucokinase and glucose-6-phosphatase (33). Soskin et al. (46) first proposed that glucose per se regulated glucose output by the liver, although subsequent in vitro studies yielded conflicting results (21, 32, 45). Although not directly pertinent to hypoglycemia, in vitro studies strongly suggest that glucose per se can regulate hepatic glycogen synthesis and breakdown (26).
With reference to hypoglycemia, studies by Saccà et al. (42)
indicated that plasma glucagon and epinephrine did not increase significantly during hypoglycemia and low-dose (0.9 pmol · kg1 · min
1)
insulin infusion, suggesting the existence of nonhormonal activation of
EGP during moderate hypoglycemia in nondiabetic subjects. The interpretation of these data, however, is clouded by 1) lack of any increase in plasma epinephrine or glucagon during frank
hypoglycemia that is not consistent with the majority of data from a
number of laboratories (9, 35, 43); 2) glucose turnover
estimations that used nonpurified [3-3H]glucose
without maintenance of stable specific activity; 3) infusion of
insulin that was not matched to individual insulin requirements; and
4) glucagon secretion that was not controlled by SRIH.
Bolli et al. (4) addressed some of these deficiencies by using the
"islet clamp" technique. However, by use of SRIH to block changes
in islet hormones, metyrapone to prevent cortisol secretion, and -
and
-adrenergic blockade to paralyze the sympathetic activation of
EGP, they were unable to demonstrate that moderate hypoglycemia
contributed any stimulus to overcome insulin-induced suppression of
EGP. However, when severe hypoglycemia (plasma glucose ~1.7 mmol/l)
was produced, EGP appeared to be activated by hormone-independent
mechanism(s). Hansen et al. (23) also suggested that a small (<30%)
component of EGP activation could be attributable to the effects of
hypoglycemia per se. These conclusions are consistent with our
findings, though clearly the study by Bolli et al. was not designed to
evaluate the potential role of hormone-independent activation of the
liver at mild hypoglycemia. First, the plasma insulin concentrations in
their study were 30% higher than ours and their glucagon replacement
rates were 35% lower, both of which may have rendered the liver more
refractory to stimulation. Second, their studies had other
methodological differences (use of nonpurified tracer, no evidence of
maintenance of glucose-specific activity, and use of a model of
non-steady-state hypoglycemia). Finally, SRIH plus
- and
-adrenergic blockade would, of course, have inhibited both hormonal-
and neural-sympathetic activation of EGP. Finally, neither Bolli et al.
nor Hansen et al. studied this question in patients with diabetes.
In other nonhuman experimental models, the stimulatory effect of a low glucose concentration per se on hepatic glucose output could be distinguished from other potential inputs (e.g., neural). For example, in the perfused rat liver model, sympathetic nerve stimulation enhanced glucose output by activating glycogen phosphorylase activity by a calcium-dependent signaling mechanism (1). This effect may be most directly related to the effect of hypoglycemia because glycogenolysis is probably the first step in counteracting the effects of insulin on the liver (15, 30). Finally, recent studies indicate that the liver may indeed possess an intrinsic ability to sense hypoglycemia via a putative portal vein glucose sensor (25). Neural afferents that are sensitive to changes in glucose have been identified in the portal region in nonprimate animal models and have impulse discharges that increase under low glucose conditions (37). The mechanism of the inability of EGP to be activated by mild hypoglycemia in type 1 diabetes is obscure. Given the potential role of hepatic innervation on EGP noted above, one might be tempted to consider this to reflect a form of diabetic autonomic neuropathy. However, our subjects did not exhibit other findings of autonomic neuropathy when crude tests of autonomic function were used. Furthermore, studies in animals with sympathetic denervation mimicking autonomic dysfunction suggest that there is a compensatory hypersensitivity to glucagon and norepinephrine (27). Whether more subtle degrees of autonomic neuropathy in diabetes are responsible for this defect remains a possibility.
Several caveats regarding interpretation of our results are in order. First, the magnitude of the nonhormonal effect on EGP was small and may not be detectable under nonexperimental conditions. Also, although Control (euglycemia) studies in normal subjects were performed to address the question of nonhormonal activation of EGP, we did not perform such a study in DM1 subjects. However, in previous studies using the same pancreatic clamp technique and matching individual basal insulin requirements by exogenous infusion in nondiabetic and type 2 diabetic subjects (33), no time-dependent changes in EGP were noted. We also have been careful to compare nondiabetic with DM1 subjects for the equivalent experimental periods. Second, there is evidence that the central nervous system may directly influence other compensatory counteregulatory mechanisms (e.g., plasma FFA) (34). Plasma FFA concentrations in our studies were greater in subjects with type 1 diabetes and might have explained the difference in glucose uptake, particularly that seen during hypoglycemia (7). Third, there may be other compensating physiological mechanisms for activation of EGP in type 1 diabetes. For example, the liver may be more sensitive to secreted epinephrine in type 1 diabetes (6) independent of the role of hypoinsulinemia (3). In addition, we cannot entirely exclude the possibility that the difference in plasma epinephrine between diabetic and nondiabetic subjects could explain the difference in EGP, although a small increment in plasma epinephrine was seen in both groups. In a previous report (5), levels of plasma epinephrine that were required to significantly increase EGP averaged two- to threefold the levels in our study. Also, these studies do not permit conclusions regarding the potential role of other counterregulatory hormones on early activation of EGP (12, 13), although cortisol and growth hormone are generally thought to play a minor role in the early EGP response (20). Finally, it is possible that the differences in portal insulin concentrations accounted for differential suppression of EGP. In fact, plasma insulin concentrations were higher in nondiabetics compared with type 1 diabetic subjects, and the EGP response in the former may have been underestimated.
These studies in humans are consistent with the body of literature confirming the presence of nonhormonal factors that control EGP in hypoglycemia (36). They lead to several clinical implications of the data. First, a redundant, albeit subtle, mechanism for the early activation of EGP emphasizes the physiological relevance of overlapping counterregulatory mechanisms in protecting the organism from hypoglycemia (20). Second, the lack of an adequate response to a plasma glucose reduction down to 3.9 mmol/l in type 1 diabetes suggests that, in addition to the demonstrated risks of severe hypoglycemia conferred by decreased secretion of glucagon and epinephrine, one must add another component of defective counterregulation that could protect the EGP response.
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ACKNOWLEDGEMENTS |
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We are indebted to Anne Thomashunis of the GCRC for care of the subjects and to Robin Sgueglia for laboratory determinations.
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FOOTNOTES |
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The Albert Einstein Diabetes Research and Training Center (DK-20541), Core Radioimmunassay Laboratory, and the General Clinical Research Center (RR-12248) provided invaluable assistance.
This work was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases Grants: DK-20541 (L. Rosetti, H. Shamoon), DK-45024 (L. Rosetti) and DK-48321 (L. Rosetti).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Shamoon, Diabetes Research Center, Albert Einstein College of Medicine, Belfer Bldg. 701D, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: shamoon{at}aecom.yu.edu).
Received 14 May 1999; accepted in final form 25 October 1999.
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