Differing physiological effects of epinephrine in type 1 diabetes and nondiabetic humans

Deanna Aftab Guy,1 Darleen Sandoval,2 M. A. Richardson,2 Donna Tate,2 Paul J. Flakoll,4 and Stephen N. Davis2,3

2Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, and 1Division of Pediatric Endocrinology, Department of Pediatrics, Vanderbilt University School of Medicine, and 3Nashville Veteran Affairs Medical Center, Nashville, Tennessee; and 4Center for Food Science and Human Nutrition, Iowa State University, Iowa City, Iowa

Submitted 14 July 2004 ; accepted in final form 2 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Acute increases of the key counterregulatory hormone epinephrine can be modified by a number of physiological and pathological conditions in type 1 diabetic patients (T1DM). However, it is undecided whether the physiological effects of epinephrine are also reduced in T1DM. Therefore, the aim of this study was to determine whether target organ (liver, muscle, adipose tissue, pancreas, cardiovascular) responses to epinephrine differ between healthy subjects and T1DM patients. Thirty-four age- and weight-matched T1DM (n = 17) and healthy subjects (n = 17) underwent two randomized, single-blind, 2-h hyperinsulinemic euglycemic clamp studies with (Epi) and without epinephrine infusion. Muscle biopsy was performed at the end of each study. Epinephrine levels during Epi were similar in all groups (4,039 ± 384 pmol/l). Glucose (5.3 ± 0.06 mmol/l) and insulin levels (462 ± 18 pmol/l) were also similar in all groups during the glucose clamps. Glucagon responses to Epi were absent in T1DM and significantly reduced compared with healthy subjects. Endogenous glucose production during the final 30 min was significantly greater during Epi in healthy subjects compared with T1DM (8.4 ± 1.3 vs. 4.4 ± 0.6 µmol·kg–1·min–1, P = 0.041). Glucose uptake showed almost a twofold greater decrease with Epi in healthy subjects vs. T1DM ({Delta}31 ± 2 vs. {Delta}17 ± 2 nmol·kg–1·min–1, respectively, P = 0.026). Glycerol, {beta}-hydroxybutyrate, and nonesterified fatty acid (NEFA) all increased significantly more in T1DM compared with healthy subjects. Increases in systolic blood pressure were greater in healthy subjects, but reductions of diastolic blood pressure were greater in T1DM patients with Epi. Reduction of glycogen synthase was significantly greater during epinephrine infusion in T1DM vs. healthy subjects. In summary, despite equivalent epinephrine, insulin, and glucose levels, changes in glucose flux, glucagon, and cardiovascular responses were greater in healthy subjects compared with T1DM. However, T1DM patients had greater lipolytic responses (glycerol and NEFA) during Epi. Thus we conclude that there is a spectrum of significant in vivo physiological differences of epinephrine action at the liver, muscle, adipose tissue, pancreas, and cardiovascular system between T1DM and healthy subjects.

epinephrine; catecholamines; metabolic target organ; glucose clamp technique


THE LOSS OF THE GLUCAGON RESPONSE to hypoglycemia with increasing duration of disease in patients with type 1 diabetes (T1DM) results in epinephrine assuming the role of the principal counterregulatory hormone against this stress. Unfortunately, epinephrine responses during hypoglycemia are reduced in intensively treated T1DM patients (3). However, it is undecided whether the physiological effects of the hormone are also lowered in T1DM. This has clinical relevance because strategies aimed at increasing epinephrine levels during episodes of hypoglycemia may be only partially successful if the hormone has reduced physiological effects in T1DM patients.

Previous studies investigating responses to epinephrine in healthy humans have resulted in conflicting results. Although some studies have found that epinephrine stimulates glucagon release (39), others have shown decreased (38) or minimal effects (35, 41). Similarly, epinephrine has been reported to result in either increases (17) or no change of endogenous glucose production (EGP; see Ref. 35) under hyperinsulinemic euglycemic conditions. However, there is no debate regarding epinephrine’s ability to decrease glucose uptake (Rd) and muscle glycogen synthase and increase muscle glycogen phosphorylase, plasma lactate, glycerol, and free fatty acids in healthy humans (13, 35, 37, 39).

Epinephrine infusion studies in T1DM subjects have also provided somewhat conflicting results. Shamoon et al. (42) found no effect of epinephrine on glucagon secretion in either T1DM subjects or healthy subjects, whereas Gerich et al. (22) and Berk et al. (6) found a significantly greater increase in plasma glucagon levels in T1DM during epinephrine infusion compared with healthy subjects. Limited information exists concerning the effects of epinephrine in the presence of hyperinsulinemic euglycemia in T1DM (13, 17, 35, 37). Only one study has utilized the hyperinsulinemic euglycemic clamp to compare responses to epinephrine infusion in healthy subjects and T1DM (13). Cohen et al. (13) found a greater increase in EGP and lipolysis in T1DM compared with healthy subjects but no change in glucagon values in either group. No data are available comparing the effects of epinephrine infusion during hypoglycemia in humans. However, data from Amiel et al. (3) reveal that, during equivalent mild hypoglycemia, epinephrine levels were significantly increased in a group of T1DM with average glucose control compared with a group of healthy subjects. Thus, in a scenario where other neuroendocrine hormone responses were either limited and/or similar, the above data imply a relatively reduced effect of epinephrine in T1DM.

Taken together, the above studies provide rather inconsistent information regarding 1) the in vivo metabolic effects of epinephrine in T1DM and 2) whether the physiological effects of the hormone differ in T1DM patients and healthy subjects. The experimental designs used in previous studies have been varied and have used differing levels of epinephrine, insulin, and glucose. These three important variables will interact and potentially significantly influence results. In the present study, a dose of epinephrine that would simulate levels found during moderate hypoglycemia in healthy nondiabetic humans and T1DM patients with average glycemic control was used (15). This level of epinephrine produces easily measurable metabolic responses (carbohydrate and lipid substrate flux), even in the presence of moderate physiological hyperinsulinemia. Hyperglycemia, which will oppose and therefore limit epinephrine’s metabolic and neuroendocrine effects, can be prevented by using a hyperinsulinemic euglycemic clamp design. The confounding variable of hyperinsulinemia may be controlled for by performing a series of hyperinsulinemic euglycemic clamps that will establish the effects of insulin per se and provide a baseline for epinephrine’s physiological effects. Therefore, to test the hypothesis that epinephrine has differing biological effects in T1DM and nondiabetic humans, we have used a combined integrative approach of glucose clamping, indirect calorimetry, muscle biopsy, and isotopic turnover techniques to measure diverse target tissue responses to the hormone.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects

We studied 17 T1DM patients (9 males/8 females) age 33 ± 2 yr with a body mass index (BMI) 24.3 ± 1 kg/m2 and Hb A1C of 8.2 ± 0.1% and 17 healthy subjects (8 males/9 females) age 31 ± 2 yr with BMI 22.7 ± 1 kg/m2 and Hb A1C of 5.0 ± 0.5%. None of the patients gave a history of hypoglycemic unawareness and all had similar duration of disease. No patients had any clinical evidence of autonomic neuropathy (as evidenced by history and bedside autonomic testing) or any other tissue-specific complication of diabetes. All patients and healthy subjects had normal blood count, plasma electrolytes, and liver and renal function. All gave written informed consent. Studies were approved by the Vanderbilt University Human Subjects Institutional Review Board. All subjects were asked to avoid exercise and to consume their usual weight-maintaining diet for 3 days before the study. We instructed each patient to be particularly careful to avoid any hypoglycemia in the period before a study. All patients performed intensive self-monitoring of blood glucose (SMBG) before each meal, at bedtime, and on two occasions at 3:00 AM for 2 wk before a study. Patients called in their SMBG results so that appropriate changes in insulin and dietary intake could be implemented. An experiment was not conducted unless all readings were 4.5 mmol/l or more. On the day preceding an experiment, intermediate-acting or long-acting insulin was discontinued and replaced by injections of regular insulin before breakfast and lunch. T1DM patients and healthy subjects were admitted to the Vanderbilt Clinical Research Center at 5:00 PM on the evening before each experiment. At this time, the T1DM patients received their usual evening meal, and a continuous low-dose intravenous infusion of insulin was started to normalize plasma glucose. This infusion was adjusted so that plasma glucose levels remained between 5.0 and 6.7 mmol/l overnight. All patients and healthy subjects were studied after a 10-h overnight fast. Subjects were blinded to the order of the experiments and randomly assigned to either protocol 1 (No Epi) or protocol 2 (Epi; Fig. 1). Each protocol was separated by a minimum of 2 mo.



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Fig. 1. Schematic of hyperinsulinemic euglycemic clamp protocol in overnight-fasted, euglycemic, type 1 diabetes mellitus (T1DM) subjects and healthy subjects.

 
Experimental Design

Euglycemic clamp procedure. Upon admission, all subjects had two intravenous cannulas inserted under 1% lidocaine local anesthesia. One cannula was placed in a retrograde fashion in a vein in the back of the hand. The hand was placed in a heated box (50–60°C) so that arterialized blood could be obtained (1) The other cannula was placed in the contralateral arm so that 20% glucose and potassium chloride (20 mmol/l) in normal saline could be infused via a variable-rate volumetric infusion pump (Imed, San Diego, CA). Purified tritiated glucose, deuterated glycerol and insulin, and epinephrine (Epi) were infused via precalibrated infusion pumps on the day of the study (Harvard Apparatus, South Natick, MA). Each subject remained awake in the supine position throughout each of the clamp procedures.

Protocol 1 (control study: No Epi). On the morning of No Epi, after the insertion of the venous cannulas, a primed (18 µCi) constant infusion (0.18 µCi/min) of [3-3H]glucose was started to measure glucose kinetics. A period of 90 min was allowed to elapse followed by a 30-min basal period and a 120-min hyperinsulinemic-euglycemic experimental period. In addition, a simultaneous infusion (0.1 µmol·kg–1·min–1) of [2H5]glycerol was infused and continued throughout the 240-min experiment to measure glycerol flux. A low-dose intravenous insulin infusion was continued in the T1DM subjects to maintain euglycemia until time 120 (min). At that time, a primed continuous infusion of insulin was administered at a rate of 9 pmol·kg–1·min–1 from time 120 until time 240 in all subjects (44). Plasma glucose was measured every 5 min, and a variable infusion of 20% dextrose was adjusted so that plasma glucose levels were held constant at ~5 mmol/l for the duration of the study (16). At time 240, a percutaneous muscle biopsy was performed under euglycemic conditions (Fig. 1).

Protocol 2 (Epi). Protocol 2, or Epi, was identical to protocol 1 (No Epi) except for the addition of a continuous infusion of epinephrine at a rate of 0.06 µg·kg–1·min–1 from time 120 to 240.

Muscle biopsy. Percutaneous muscle biopsy was performed according to the Bergstrom procedure and is described elsewhere (5). The skin and fascia 12–16 cm above the patella on the lateral aspect of the thigh were anesthetized using a eutopic mixture of topical anesthetic cream (lidocaine) applied 1 h before the procedure followed by a local infusion of subcutaneous 1% xylocaine without epinephrine and suprafascial infusion of 2% xylocaine without epinephrine. A small incision was made using a 10-blade scalpel through the skin and underlying fascia. A 5-mm-diameter side-cutting Bergstrom percutaneous muscle biopsy needle was inserted 2–4 cm beyond the fascia in the muscle to obtain a sample of the vastus lateralis muscle. Approximately 200 mg of muscle were obtained during each protocol.

Indirect calorimetry. Whole body carbohydrate metabolism and fat flux was assessed using indirect calorimetry. Air flow and O2 and CO2 concentrations in expired and inspired air were measured by a computerized open-circuit system (DeltaTrak; Sensormedics, Yorba Linda, CA). Urea nitrogen was measured by the Kjeldahl procedure (3). Rates of carbohydrate and fat oxidation were calculated from O2 consumption and CO2 production (corrected for protein oxidation) with the equations described by Frayn (20). Nonoxidative glucose disposal was calculated by subtracting the difference between the rates of total glucose disposal (Rd) and oxidative glucose disposal.

Analyses

The collection and processing of blood samples have been described elsewhere (11). Blood samples for glucose flux were taken every 10 min throughout the control period and every 15 min during the experimental period. Blood for hormones and intermediary metabolites was drawn two times during the control period and every 15 min during the experimental period.

Plasma glucose concentrations were measured in triplicate using the glucose oxidase method with a glucose analyzer (Beckman, Fullerton, CA). Glucagon was measured according to the method of Aguilar-Parada et al. (2) with an interassay coefficient of variation (CV) of 15%. Insulin and free insulin measurements (in T1DM) were determined as previously described with an interassay CV of 11% (47). Catecholamines were determined by high-pressure liquid chromatography (HPLC) with an interassay CV of 12% for epinephrine and for norepinephrine (9). We made the following two modifications to the procedure for catecholamine determination: we used a five-point rather than a one-point standard calibration curve, and we spiked the initial and final samples of plasma with known amounts of epinephrine and norepinephrine so that accurate identification of the relevant respective catecholamine peaks could be made. Cortisol was assayed by using the Clinical Assays Gamma Coat RIA kit with an interassay CV of 8%. RIA with a CV of 8% determined growth hormone (30). Pancreatic polypeptide was measured by RIA using the method of Hagopian et al. (28) with an interassay CV of 8%. Lactate, glycerol, alanine, and 3-hydroxybutyrate were measured on deproteinized whole blood using the method of Lloyd et al. (36). Nonesterified fatty acids (NEFAs) were measured using the WAKO kit adopted for use on a centrifugal analyzer (29).

Ra, EGP, and Rd were calculated according to the methods of Wall et al. (46). EGP was calculated by determining the total Ra (which comprises both EGP and exogenous glucose infused to maintain the desired euglycemia) and subtracting from it the amount of glucose infused. It is now recognized that this approach is not fully quantitative because underestimates of total Ra and Rd can be obtained. The use of highly purified tracer and the taking of measurements under steady-state conditions (i.e., constant specific activity) in the presence of a low glucose flux minimizes the major problems. To maintain constant glucose specific activity, the rate of exogenous glucose tracer was increased proportionally to increases in glucose flux.

Determination of [2H5]glycerol enrichment and turnover was performed as described previously (19). Briefly, plasma was deproteinized with 4% perchloric acid, and the supernatant was passed over cation and anion exchange resins. The eluate was dried overnight at 50°C, the glycerol fraction was derivatized with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide containing 1% tert-butyldimethylchlorosilane (Regis Chemical, Morton Grove, IL), and samples were analyzed by GC-MS for determination of plasma glycerol enrichment. The major fragments monitored for the tert-butyldimethylsilyl derivatives of glycerol and [2H5]glycerol were the [M-57] ion fragments, mass-to-charge ratios 377 and 382, respectively. Plasma rates of glycerol appearance and disappearance were calculated using the Steele equations. The steady-state equation (i.e., isotope infusion divided by isotopic enrichment) was used during the baseline period when substrate concentrations and enrichment were stable.

Cardiovascular parameters (pulse and systolic, diastolic, and mean arterial pressure) were measured noninvasively using a Dinamap (Critikon, Tampa, Fl) every 10 min throughout each study. Autonomic and neuroglycopenic symptoms characteristic of hypoglycemia were quantified using a previously validated semiquantitative questionnaire (14). Each patient and healthy subject was asked to quantify on a scale of 1 to 10 (1 being no symptoms and 10 being the most) his or her experience of the symptoms once during the control period and every 15 min during the experimental period. Symptoms measured included tiredness, confusion, hunger, dizziness, difficulty thinking, blurred vision, sweating, tremors, agitation, sensation of heat/thirst, and palpitations. The ratings of the first six symptoms were summed to get the neuroglycopenic score, whereas the ratings from the last five symptoms provided an autonomic symptom score.

Materials

HPLC-purified [3-3H]glucose (New England Nuclear, Boston, MA) was used as the glucose tracer (11.5 mCi·mmol–1·l–1). Human regular insulin was purchased from Eli Lilly (Indianapolis, IN). The insulin infusion solution was prepared with normal saline and contained 3% (vol/vol) of the subjects’ own plasma.

Epinephrine-injectable solution (1:1,000, 1 mg/ml, 1 ml ampule) was prepared with normal saline for continuous infusion. [2H5]glycerol (250 mg/vial) was purchased from Isotech (Sigma Adlrich).

Muscle Enzyme Analysis

Glycogen synthase activity was measured using the technique described by Guinovart et al. (25). Glycogen phosphorylase activity was measured using the technique described by Golden et al. (24). Glycogen content of muscle was determined by enzymatic microdetermination, as described by Bruss and Black.(8).

Statistical Analysis

Data are expressed as means ± SE, unless otherwise stated. Statistical comparisons between groups were performed by use of standard parametric two-way ANOVA for repeated measurements when appropriate. This was coupled with Duncan’s post hoc test to delineate the time when statistical significance was reached. A P value <0.05 indicated significant difference.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insulin and Glucose

Insulin and glucose levels were similar in both groups during the hyperinsulinemic-euglycemic clamps. Steady-state insulin levels were 519 ± 48, 511 ± 40, 514 ± 32, and 519 ± 21 pmol/l in the T1DM patients during No Epi and Epi and healthy subjects during No Epi and Epi, respectively. Plasma glucose levels were maintained at 5.3 ± 0.1, 5.3 ± 0.1, 5.2 ± 0.1, and 5.2 ± 0.1 mmol/l during the clamp procedure in the T1DM patients during No Epi and Epi and healthy subjects during No Epi and Epi, respectively (Fig. 2).



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Fig. 2. Glucose and insulin levels during hyperinsulinemic (9 pmol·kg–1·min–1) euglycemia with (Epi) and without (No Epi) epinephrine infusion in overnight-fasted, euglycemic, T1DM subjects and healthy subjects. Levels were similar during No Epi and Epi studies between all 3 groups. Values are means ± SE.

 
Epinephrine and Norepinephrine

Steady-state epinephrine levels during Epi were similar in both groups during the 2-h infusion (4,139 ± 351 pmol/l in T1DM and 4,020 ± 304 pmol/l in the healthy subjects). These levels differed significantly (P < 0.001) from No Epi in T1DM (161 ± 27 pmol/l) and in healthy subjects (172 ± 23 pmol/l). There were similar significant increases of norepinephrine during No Epi ({Delta}0.28 ± 0.1 nmol/l) and Epi ({Delta}0.28 ± 0.1 nmol/l) in healthy subjects. However, the norepinephrine response to Epi was significantly less in T1DM compared with healthy subjects ({Delta}0.04 ± 0.1 vs. 0.28 ± 0.1 nmol/l, P = 0.011; Fig. 3).



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Fig. 3. Plasma epinephrine and norepinephrine responses to hyperinsulinemic (9 pmol·kg–1·min–1) euglycemia in overnight-fasted, euglycemic, T1DM subjects and healthy subjects with and without epinephrine infusion. *Epinephrine levels were significantly greater in Epi vs. No Epi in both groups (P < 0.05). {dagger}Norepinephrine response ({Delta}) to epinephrine was significantly greater in healthy subjects vs. T1DM during EPI. Values are means ± SE.

 
Glucagon, Cortisol, Growth Hormone, and Pancreatic Polypeptide

The relative incremental glucagon responses to Epi vs. No Epi during the final 30 min were reduced in T1DM compared with healthy subjects (–4 ± 2 vs. ± 16 ± 1 ng/l, P = 0.033; Table 1).


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Table 1. Effect of epinephrine infusion on neuroendocrine responses during hyperinsulinemic euglycemia in individuals with T1DM and healthy subjects

 
Cortisol responses increased similarly in both groups during Epi. Growth hormone responses were minimally affected regardless of whether epinephrine was present or not. Pancreatic polypeptide increased similarly and minimally in both groups during the final 30 min of each protocol.

Glucose Kinetics

Glucose specific activity (dpm/mmol) was in a steady state (CV ≤3%) during the basal period and final 30 min of each study (Table 2). EGP during No Epi was similar in T1DM and healthy subjects. EGP during (Fig. 4) Epi was significantly reduced in T1DM compared with healthy subjects (4.4 ± 0.6 vs. 8.4 ± 1.3 µmol·kg–1·min–1; P = 0.041). Glucose uptake during the final 30 min was significantly reduced in T1DM compared with healthy subjects during No Epi (33 ± 3 vs. 51 ± 3 µmol·kg–1·min–1, respectively; P = 0.007.) There was also an approximately twofold decrease in Rd in response to epinephrine (compared with No Epi) in the healthy subjects vs. T1DM ({Delta} –33 ± 3 vs. –17 ± 2 µmol·kg–1·min–1, respectively; P = 0.026).


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Table 2. Effect of epinephrine infusion on glucose specific activity during hyperinsulinemic euglycemia in individuals with T1DM and healthy subjects

 


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Fig. 4. Effect of epinephrine infusion on glucose kinetics during hyperinsulinemic (9 pmol·kg–1·min–1) euglycemia in overnight-fasted, euglycemic T1DM subjects and healthy subjects. Top: endogenous glucose production (EGP) during the final 30 min of clamp study with and without epinephrine infusion. Bottom: glucose utilization (Rd) during the final 30 min of clamp study with and without epinephrine infusion. *EGP and glucose utilization responses to epinephrine were significantly lower in T1DM subjects vs. healthy subjects (P < 0.05). Values are means ± SE.

 
Indirect Calorimetry and Glucose Disposal

There was less of a reduction in total glucose disposal with epinephrine in T1DM patients compared with healthy subjects (P = 0.026). Nonoxidative glucose metabolism was reduced (16 ± 1 µmol·kg–1·min–1) in T1DM during Epi (34 ± 3 µmol·kg–1·min–1) in healthy subjects (P = 0.034). Oxidative glucose metabolism was significantly lower during both No Epi and Epi in T1DM compared with healthy subjects (13 ± 1 µmol·kg–1·min–1 during No Epi in T1DM vs. 17.1 ± 2 µmol·kg–1·min–1 in healthy subjects; P = 0.029 and 11 ± 2 µmol·kg–1·min–1 during Epi in T1DM vs. 19.3 ± 2 µmol·kg–1·min–1 in healthy subjects; P = 0.034; Fig. 5).



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Fig. 5. Effect of epinephrine infusion on glucose disposal during hyperinsulinemic (9 pmol·kg–1·min–1) euglycemia in overnight-fasted, euglycemic T1DM subjects and healthy subjects. *Nonoxidative glucose metabolism was significantly greater with epinephrine infusion in T1DM subjects vs. healthy subjects (P < 0.05). {dagger}Oxidative glucose metabolism was significantly greater during both Epi and No Epi in healthy subjects vs. T1DM subjects (P < 0.05).

 
Whole body lipid oxidation during Epi, however, was greater (P = 0.025) in T1DM (0.6 ± 0.1 mg·kg–1·min–1) compared with healthy subjects (0.2 ± 0.1 mg·kg–1·min–1)

Cardiovascular Parameters

Heart rate, diastolic blood pressure, and systolic blood pressure changes in response to epinephrine are summarized in Table 3. There was a significantly greater increase (P = 0.006) in systolic blood pressure in response to epinephrine in the healthy subjects compared with T1DM. Diastolic blood pressure responses to epinephrine were greater in T1DM, with a threefold decrease compared with healthy subjects. Heart rate increased similarly in both groups, but mean arterial blood pressure increased in healthy subjects but decreased in T1DM (P < 0.001; Table 3).


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Table 3. Effects of epinephrine infusion on cardiovascular parameters during hyperinsulinemic euglycemia in individuals with T1DM and healthy subjects

 
Intermediary Metabolism

Blood lactate increased similarly during Epi in both groups. Blood alanine remained relatively unchanged in both groups with epinephrine infusion. There was a significantly greater increase (P = 0.045) of glycerol in response to Epi in T1DM (37 ± 9 µmol/l) compared with healthy subjects (17 ± 8 µmol/l). The relative responses of glycerol turnover, NEFA, and blood {beta}-hydroxybutyrate were also significantly greater in T1DM during Epi vs. No Epi (P = 0.045, 0.005, and 0.008, respectively) compared with healthy subjects (Table 4).


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Table 4. Effect of epinephrine infusion on intermediary metabolites during hyperinsulinemic euglycemia in individuals with T1DM vs. healthy subjects

 
Muscle Biopsy Assays

Skeletal muscle glycogen synthase activity during No Epi was 22 ± 6% in T1DM and 10 ± 2% in healthy subjects. Synthase activity during Epi was 6 ± 1% in T1DM and 7 ± 1% in healthy subjects. There was thus a greater reduction in synthase activity during Epi in T1DM compared with healthy subjects (P = 0.026).

Glycogen phosphorylase activity was similar in both groups during No Epi (18 ± 7% in T1DM and 16 ± 4% in healthy subjects) and Epi (17 ± 4% in T1DM and 14 ± 6% in healthy subjects), with little or no response to Epi in either group. Total muscle glycogen content (expressed as µmol glucose/g muscle tissue) also did not differ significantly between protocols or between groups (14.9 ± 2 in T1DM and 16.7 ± 2 in healthy subjects during No Epi and 15.1 ± 4 in T1DM and 14.8 ± 3 in healthy subjects and during Epi).

Autonomic Symptoms

Total symptom score, consisting of the sum of autonomic and neuroglycopenic symptoms, did not differ during No Epi in either group. During Epi, total symptom score increased significantly (P = 0.045) more in healthy subjects (16 ± 1 to 22 ± 1) compared with T1DM (15 ± 1 to 18 ± 2).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The integrated in vivo physiological effects of epinephrine on neuroendocrine, metabolic, and cardiovascular responses in T1DM and healthy humans have not been fully defined. This study examined these effects independently of the confounding variable of hyperglycemia by using a combined approach incorporating isotopic tracers, fuel flux, muscle biopsy, and the hyperinsulinemic euglycemic clamp technique. Our data demonstrate that patients with T1DM have decreased neuroendocrine (glucagon and norepinephrine), metabolic (EGP, Rd, and glucose oxidation), cardiovascular (mean arterial pressure), and symptomatic responses but increased lipolytic responses to moderately elevated physiological levels of epinephrine compared with healthy subjects. These data reveal that T1DM have a wide spectrum of altered physiological responses to epinephrine compared with healthy subjects.

Each of the two participating groups (T1DM and healthy subjects) underwent two separate hyperinsulinemic euglycemic clamps with and without epinephrine. This experimental design controlled for the three important variables of epinephrine, insulin, and glycemia. Additionally, the physiological insulin levels used in the euglycemic clamp resulted in a complete suppression of EGP, a fivefold increase in glucose disposal, and a large reduction in lipolysis. These metabolic scenarios provided an optimal baseline from which the magnitude of the various metabolic effects of epinephrine could be easily determined (e.g., the ability of epinephrine to increase EGP could be easily determined because the rate of EGP in the control experiment was effectively 0).

Furthermore, an additional goal of this study was to determine the metabolic effects of epinephrine in the presence of physiological hyperinsulinemia. This has clinical applications, since daily insulin levels in T1DM often peak into the mid-physiological range. Therefore, our present approach differs fundamentally from earlier studies (6) that studied epinephrine action under low or euinsulinemic conditions and in the presence of hyperglycemia.

Increased duration of T1DM is associated with absent glucagon responses to hypoglycemia (23). Hence, epinephrine assumes the critical role as a glucose counterregulatory hormone in T1DM. Epinephrine exerts its metabolic effects via direct actions on target tissues and indirectly via actions on other neuroendocrine hormones. An important indirect neuroendocrine response of epinephrine involves a stimulatory effect upon glucagon secretion. In the present study, epinephrine’s ability to stimulate glucagon was absent in T1DM. Previous studies have reported either exaggerated (22), moderate (6), or even absent glucagon responses to epinephrine infusion in T1DM (13). Glucagon levels fell significantly during hyperinsulinemic euglycemia during No Epi in both groups. However, in the healthy subjects, epinephrine could overcome the blunting effects of insulin on the {alpha}-cell and maintain glucagon at baseline levels during the glucose clamps. This ability of epinephrine to overcome insulin action at the {alpha}-cell was lost in T1DM. The relative difference in glucagon values between groups was 20 ng/l. This difference in systemic glucagon values would have been amplified even further (2- to 3-fold) in the portal vein because of hepatic extraction of the hormone. Thus hepatic sinusoidal levels (and thus effects on hepatic glucose production) would have been significantly reduced in the T1DM group during epinephrine infusion. Epinephrine increases glucagon secretion via stimulation of {beta}2-adrenoreceptors on the {alpha}-cells of the pancreas (31). Therefore, the blunted glucagon response to epinephrine in T1DM is likely because of decreased {beta}-receptor sensitivity. Furthermore, although unknown, it is intriguing to speculate that part of the mechanism responsible for the lack of glucagon response seen during hypoglycemia in T1DM may be due to defective {beta}2-adrenoreceptor signaling in pancreatic {alpha}-cells.

Epinephrine also had significantly reduced effects on glucose kinetics in T1DM compared with healthy subjects. Epinephrine’s ability to increase EGP and inhibit insulin-mediated glucose uptake (Rd) were both reduced in T1DM. Epinephrine infusion has been demonstrated to increase both hepatic and renal glucose release in healthy subjects and T1DM (10, 45). However, we are unable to partition the relative effects of epinephrine on hepatic and renal glucose production in T1DM and healthy subjects in this study. Previous animal work has demonstrated that epinephrine increases hepatic glucose production directly by acting upon both {beta}2- and {alpha}1-adrenergic receptors (18, 43). Studies performed in healthy humans during hyperinsulinemic euglycemia demonstrated that {beta}-blockade completely prevented epinephrine’s stimulatory effect upon glucose production (17). Therefore, {beta}-adrenergic receptors appear to play the major role in regulating EGP responses to epinephrine in humans. In addition, the blunted response of glucagon during epinephrine infusion in T1DM would also have contributed significantly to the reduced EGP observed in these individuals compared with the healthy subjects.

Glucose production in response to epinephrine is derived from varying degrees of both glycogenolysis and gluconeogenesis. It has been demonstrated in the dog that epinephrine and glucagon have additive effects on both glycogenolytic and gluconeogenic flux (26). The gluconeogenic flux resulting from both glucagon and epinephrine appears to be dependent on the presence of gluconeogenic precursors (27). Chu et al. (12) have demonstrated that the majority of the increased EGP occurring during epinephrine infusion is the result of hepatic gluconeogenesis and that elevated NEFA levels inhibits epinephrine’s glycogenolytic action at the liver. Interestingly, despite similar increases in lactate and alanine and even greater increases in glycerol and NEFA responses (potent substrates and fuel for gluconeogenesis), the effect of epinephrine on EGP was decreased in T1DM compared with healthy subjects. This suggests a reduced ability of epinephrine to stimulate gluconeogenesis in our T1DM patients.

T1DM patients also had a diminished ability to restrict glucose uptake during epinephrine infusion compared with healthy subjects. This is relevant, since limitation of glucose disposal is the major mechanism by which epinephrine defends blood glucose during prolonged hypoglycemia. Mechanisms responsible for this altered response in T1DM patients are controversial but may be the result of a reduction in transport and/or phosphorylation of glucose (32, 33). Parenthetically, it is relevant to note that glucose uptake was significantly lower in T1DM compared with healthy subjects, even during the hyperinsulinemic euglycemic clamp without epinephrine infusion. T1DM also had significantly less glucose oxidation during both control (No Epi) and Epi studies. This indicates that our T1DM patients were insulin resistant, no doubt because of "glucose toxicity" induced by moderate glycemic control. In this present study, we have determined the relative reduction of Rd occurring between the No Epi (control experiments) and Epi in healthy subjects and T1DM. We believe this approach is more clinically relevant, since it is the epinephrine-driven reduction in glucose Rd that is critical in the defense against insulin-induced hypoglycemia in T1DM. We further examined epinephrine’s effect on glucose disposal by measuring skeletal muscle glycogen metabolism. Our present results demonstrate that there was a greater reduction of glycogen synthase in the T1DM patients compared with the healthy subjects (although absolute levels of the enzyme were similar in both groups). There was no indication that glycogen phosphorylase (and thus glycogen breakdown) was differentially affected by epinephrine in T1DM or healthy subjects. Our present results therefore support the study of Laurent et al. (35) by demonstrating that epinephrine limits insulin stimulated glucose utilization via a reduction in the rate of glycogen synthesis, which is resultant on inhibition of glucose transport and/or phosphorylation.

Whole body lipid oxidation, glycerol turnover, lipolysis, and ketogenesis were increased by epinephrine to a greater extent in T1DM compared with healthy subjects. Epinephrine’s mechanism of action at adipose tissue receptors is dose dependent (34). At low levels, epinephrine is thought to exert its effect on the higher-density {alpha}2-receptors, inhibiting lipolysis. At higher levels (as used) in this study, epinephrine exerts its effect on {beta}-adrenergic receptors, stimulating lipolysis. Microdialysis studies have revealed a significantly greater lipolytic response in T1DM during hypoglycemia compared with healthy subjects and confirmed the increase is mediated via {beta}-adrenergic receptors (7, 21). Increased free fatty acids are an important mechanism responsible for limiting glucose uptake and stimulating EGP. Both of these important physiological effects were reduced during Epi infusion in the T1DM patients. It is therefore worth noting that the magnitude in the deficits of glucose flux apparent during EPI infusion in T1DM could have been much greater if it were not for the increased adipose tissue response to the hormone. Therefore, it appears likely that an upregulation of epinephrine’s effect on adipose tissue may help offset the reduced action of the catecholamine on glucose kinetics.

A spectrum of cardiovascular responses, including systolic, diastolic, and mean arterial blood pressure, also differed between the two groups. Epinephrine’s cardiovascular physiological effects are to increase systolic and decrease diastolic blood pressure. Despite similar increases in heart rate during Epi in both groups, there was a greater systolic blood pressure response in the healthy subjects and a significantly greater decrease in diastolic pressure in T1DM. These changes resulted in an overall increase in mean arterial pressure in the healthy subjects but a decrease in mean arterial pressure in T1DM. The reason for this difference is unclear. Although our patients had no history or evidence of autonomic neuropathy, they exhibited a significantly blunted increase in norepinephrine in response to epinephrine. This suggests that reduced autonomic function may exist that cannot be detected by usual bedside tests of autonomic function. In addition, our results indicate that some aspects of autonomic function cannot be normalized despite moderately elevated physiological levels of epinephrine. Thus blunted neurocardiac sympathetic activation may have contributed to the attenuated response in systolic blood pressure in T1DM during epinephrine infusion. Insulin exerts positive effects on cardiac output (4). Previous studies have demonstrated that, under euglycemic conditions, insulin can increase cardiac output in T1DM (40). Our present results are consistent with those findings, since there were no differences in cardiovascular parameters during the No Epi hyperinsulinemic-euglycemic clamps between the healthy subjects and T1DM groups. The similar cardiovascular responses during the control studies in T1DM and nondiabetic groups illustrate the reduced effects of epinephrine per se rather than insulin on the heart and/or peripheral vasculature in the T1DM patients. Adrenergic symptoms were also significantly reduced in T1DM compared with healthy subjects. This demonstrates that the brain is another organ resistant to epinephrine action. Total symptom scores in our T1DM patients were much lower than previously observed during hypoglycemia, despite similar epinephrine levels (15). This indicates that 1) the genesis of hypoglycemic symptoms is multifactorial, with epinephrine only contributing ~20% to the total increase in symptom scores during hypoglycemia, and 2) patients with T1DM are at increased risk of severe hypoglycemia not only because of an inability to mount a full metabolic response to epinephrine but also because physiological warning symptoms are also reduced.

In summary, this study has demonstrated a broad spectrum of differential physiological effects of epinephrine in T1DM. Responses at the liver, muscle, pancreas, adipose tissue, autonomic nervous system, and cardiovascular system were altered in T1DM. Patients with T1DM had reduced EGP, Rd, glucagon, and symptom and systolic blood pressure and increased lipolytic responses compared with healthy subjects. These findings may have implications in the treatment and/or prevalence of severe hypoglycemia in T1DM. Efforts to improve glucose counterregulation should consider strategies aimed at improving epinephrine’s actions at target organs as well as increasing the plasma level of the hormone during hypoglycemia.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institutes of Health Grants PPG 2PO1-HL-56693, Vanderbilt GCRC 5M01-RR-00095, and DRTC 5P60-DK-020593.


    ACKNOWLEDGMENTS
 
We acknowledge Wanda Snead and Eric Allen for contribution in measurement of neuroendocrine hormones and Masakazu Shiota for assistance in muscle enzyme assays. We also recognize the expert care of the Vanderbilt General Clinical Research Center (GCRC) nurses.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. N. Davis, 715 PRB, Division of Diabetes, Endocrinology & Metabolism, Vanderbilt Univ. School of Medicine, Nashville, TN 37232-6303

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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