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*Diabetes Type 1
*Hypoglycemia
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*EPINEPHRINE
*GLUCOSE
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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 4 1503-1511
Copyright © 2003 by The Endocrine Society

Defective Activation of Skeletal Muscle and Adipose Tissue Lipolysis in Type 1 Diabetes Mellitus during Hypoglycemia

Staffan Enoksson, Sonia K. Caprio, Frances Rife, Gerald I. Shulman, William V. Tamborlane and Robert S. Sherwin

Department of Medicine, Section of Endocrinology (S.E., R.S.S.), Pediatric Endocrinology (S.K.C., W.V.T.), the General Clinical Research Centers (F.R.), and Howard Hughes Medical Institute (G.I.S.), Yale University School of Medicine, New Haven, Connecticut 06510

Address all correspondence and requests for reprints to: Staffan Enoksson, M.D., Ph.D., Department of Vascular Surgery, C1:88, Huddinge University Hospital at the Karolinska Institute, 14186 Huddinge, Sweden. E-mail: staffan.enoksson{at}karo.ki.se.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The increased risk of hypoglycemia during intensified treatment of type 1 diabetes mellitus (T1DM) patients, who have a deficient glucagon secretory response, is largely attributed to the development of suppressed adrenomedullary responses. A consequence of this impairment of catecholamine secretion might be reduced lipolysis in major target tissues (muscle and adipose) and, in turn, increased glucose metabolism. To test this hypothesis, we used microdialysis to monitor glycerol (index of lipolysis) in the extracellular fluid of skeletal muscle and adipose tissue and assessed whole-body glucose use by measuring [6,6-2H2]glucose enrichment in plasma in seven intensively treated T1DM patients and eight nondiabetic subjects who received a 3-h insulin infusion (0.8 mU/kg·min) on two occasions: during mild-moderate hypoglycemia or euglycemia. In the hypoglycemic study, the rise in plasma epinephrine was approximately 50% less in the T1DM patients despite a greater fall in plasma glucose (to 3.0 vs. 3.5 mM in controls; P < 0.05). Moreover, the rate of glucose flux and the plasma-extracellular fluid glucose gradient in muscle was increased during hypoglycemia in T1DM subjects compared with controls. Glycerol levels in muscle, adipose, and plasma fell similarly in both groups in the first hour. Thereafter, tissue glycerol remained suppressed in the T1DM patients but rebounded significantly (P < 0.01) in the control subjects. The glycerol response in muscle and adipose tissue was significantly correlated with plasma epinephrine concentration (r = 0.73, P = 0.002; and r = 0.52, P = 0.04, respectively), and inversely correlated with whole-body glucose disposal (r = -0.51, P = 0.05; and r = -0.50, P = 0.05). To determine whether the absence of the lipolytic response is limited to deficient catecholamine release, we perfused muscle and adipose tissue in situ with the selective ß2-agonist terbutaline during hyperinsulinemic euglycemia. Local addition of agonist increased glycerol and blood flow in both muscle and adipose (P < 0.01 and P < 0.05, respectively) similarly in T1DM and control subjects. We conclude that deficient release of (rather than impaired responsiveness to) catecholamines in T1DM prevents the local fat breakdown within muscle and adipose tissue that normally occurs during mild-moderate hypoglycemia. This defect within peripheral tissues may lead to a delayed increase in glucose disposal that could contribute to the severity of hypoglycemia when it is prolonged.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PATIENTS WITH TYPE I diabetes mellitus (T1DM) lack the ability to secrete glucagon during hypoglycemia (1) and commonly develop impaired adrenomedullary responses, particularly during intensive insulin therapy (2). Diminished adrenergic responses to decrements in plasma glucose greatly increase the risk of severe hypoglycemia, which has emerged as the main obstacle to the benefits of intensive insulin therapy (3). Although it is now appreciated that antecedent iatrogenic hypoglycemia plays a critical role in the appearance of defective adrenergic responses in many patients receiving intensive insulin therapy (4), it is less certain how these treatment-induced changes impact at the tissue level, particularly responses within peripheral tissues. Inasmuch as catecholamines exert some of their insulin antagonistic effects in the periphery (2, 5, 6) and conventionally treated T1DM patients appear to counteract insulin hypoglycemia to a large extent by decreasing peripheral glucose uptake, it is surprising that little attention has been paid to the effects of more intensive treatment of diabetes on counterregulatory responses in muscle and adipose tissue (7, 8). By suppressing adrenergic responses, such therapy might promote hypoglycemia by accelerating peripheral glucose metabolism, thereby exaggerating the hypoglycemic effects of the glucagon-mediated defect in hepatic glucose production.

The insulin antagonist effects of catecholamines result from their capacity to mobilize substrates and fuel for gluconeogenesis from peripheral tissues as well as to inhibit glucose metabolism in muscle and adipose tissue (5, 9). An important mediator of these effects is ß-adrenergic stimulation of lipolysis. Mobilized nonesterified fatty acids (NEFA) and glycerol promote gluconeogenesis and suppress glucose oxidation and use in peripheral tissues (10). Indeed, it has been suggested that the critical role of catecholamines in T1DM during hypoglycemia is in large part due to its potent lipolytic effect (11). According to this scenario, insulin therapy might contribute to the increased risk of hypoglycemia by inhibiting catecholamine-induced lipolysis, which in turn increases glucose use in peripheral tissues, specifically muscle.

Although it is well known that adipose tissue is the principle source of lipid-derived fuel (NEFA and glycerol), recent studies have demonstrated that there is significant intramuscular lipolysis as well (12, 13, 14), and that its regulation appears to differ from that in adipose tissue (15, 16, 17). Moreover, increased muscle lipid content has been reported in T1DM and insulin-resistant patients (18, 19). It is not established how diabetes and its treatment affect the local changes in glucose and lipid metabolism in muscle during hypoglycemia. Moreover, it is uncertain whether the response to ß-adrenergic stimulation is altered by intensified insulin therapy in T1DM; decreased (20, 21), unchanged (22, 23), and increased responsiveness has been reported (24, 25).

The current study was undertaken to assess the consequences of the impaired sympathoadrenal responses on skeletal muscle and adipose tissue lipolysis and its effect on glucose counterregulation in T1DM. For this purpose, we used microdialysis of the extracellular water space in skeletal muscle and adipose tissue to monitor the local lipolytic response to insulin-induced mild-moderate hypoglycemia and local administration of selective ß2-agonist in healthy subjects and intensively treated T1DM patients showing impaired adrenergic responses to hypoglycemia. Whole-body glucose use was assessed by measuring [6,6-2H2]glucose enrichment in plasma, and the result was evaluated in relation to increments in plasma epinephrine, as well as to the glycerol response in plasma and peripheral tissues.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
The Human Investigation Committee of the Yale University School of Medicine approved the protocol for this study.

Subjects

Eight nondiabetic subjects (5 men and 3 women; age, 21 ± 3 yr; body mass index, 23 ± 0.8 kg/m2) and seven subjects with intensively treated T1DM (3 men and 4 women; age, 25 ± 2 yr; body mass index, 25 ± 2.1 kg/m2) were recruited for participation in the study. The duration of disease in the diabetic subjects was 14 ± 2 yr, and they were all receiving either continuous sc insulin infusion (6 subjects) or multiple injection therapy (1 subject), hemoglobin A1C 6.9 ± 0.3 vs. control 5.2 ± 0.1% (upper limit of normal, 6.4%). No patient had clinical evidence of autonomic or peripheral neuropathy. None was taking medication other than insulin. All experimental procedures were performed in the General Clinical Research Center of the Yale-New Haven Hospital. Written informed consent was obtained from each subject before the study.

Insulin-induced hypoglycemia

In the diabetic group, only short-acting insulin was administered sc for 48 h until the night before the study when the patients were admitted to receive a continuous iv insulin infusion to achieve euglycemia overnight. Studies were performed after a 10-h overnight fast. An infusion catheter was inserted into an antecubital vein and kept patent with 0.9% saline. A second catheter was placed in a retrograde fashion into a dorsal hand vein for blood sampling. The hand was placed in a heated box (~60 C) to arterialize venous blood. Basal samples were drawn for the determination of plasma glucose, lactate, glycerol, insulin, NEFA, catecholamine, glucagon, cortisol, and GH concentrations. Subsequently, substrates and hormones were collected hourly throughout the study. A primed continuous infusion of [6,6-2H]glucose (Cambridge Isotopes Laboratories, Andover, MA) was started. The tracer was allowed to equilibrate for 3 h. Blood samples were drawn at 10-min intervals during the final 40 min of the equilibration period (baseline) and during the final hour of the insulin infusion for the determination of enrichment of plasma [6,6-2H]glucose. During the equilibration period, normoglycemia was maintained in the diabetic subjects by means of a variable low-dose iv insulin infusion. At time zero, maintenance infusion of insulin was discontinued, and regular insulin was then infused in all subjects at a rate of 0.8 mU/kg·min for 180 min. Plasma glucose levels were measured every 5 min. Plasma glucose was allowed to fall to 3 mmol/liter (54 mg/dl). In subjects in whom the glucose concentration fell below the desired level (all diabetic subjects), 20% dextrose was infused to maintain plasma glucose above 2.8 mmol/liter.

Microdialysis

The principle of microdialysis has been described in detail previously (26). In brief, the active part of the microdialysis catheter consists of a chamber constructed of a semipermeable membrane with an inlet and an outlet. The dialysis chamber is connected to a high-precision perfusion pump via the inlet. Molecules in the perfusion fluid and the extracellular water equilibrate over the membrane; the perfusion fluid leaves the chamber through the outlet and is collected in timed fractions (dialysate) in closed microvials. In each subject, microdialysis catheters (CMA/60 CMA Microdialysis, Acton, MA) were inserted percutaneously into the periumbilical sc adipose tissue and the medial head of the gastrocnemius muscle. Before catheter insertion, the skin was superficially anesthetized with lidocaine (EMLA, Astra USA, Inc., Zeneca, Sweden). Subsequently, the catheters were continuously perfused with an artificial extracellular fluid (ECF; NaCl 135 mmol/liter, KCl 3 mmol/liter, MgCl2 1 mmol/liter, CaCl2 1.2 mmol/liter, ascorbate 200 mmol/liter, and NaPO2 buffer 2 mmol/liter, adjusted to pH 7.4) at a constant rate of 0.3 µl/min. After a minimum of 2 h of equilibration, the sample fluid, i.e. the dialysate, was collected every 30 min during baseline and throughout the study for the determination of glycerol, glucose, and lactate. Under the stated conditions, the recovery of glycerol, glucose, and lactate is reported to be approximately 90% in adipose and skeletal muscle tissue (13).

Insulin infusion with euglycemia and local perfusion with ß2-agonist

In a subsequent euglycemic clamp study, skeletal muscle and adipose tissue responsiveness to insulin and ß2-adrenergic stimulation was evaluated. Six of the subjects with intensively treated T1DM and six of the control subjects who participated in the former hypoglycemic study were able to take part in a second study. Two male control subjects, age 19 and 16 yr, together with one male diabetic patient, age 30, refrained from participation a second time for reasons unrelated to the study. The two studies were performed 6 wk apart in each subject. The glycemic control in the diabetic patients was not significantly changed between the two occasions (hemoglobin A1C, 6.9 ± 0.3 vs. 7.0 ± 0.2%). All diabetic subjects were admitted to the research facility in the evening before the study. The prestudy protocol was performed, as described above, for the hypoglycemic study. Two microdialysis catheters were inserted in each tissue in the morning immediately before the insulin infusion study, as described earlier. The catheters were perfused with artificial ECF at 2.0 µl/min supplemented with 50 mmol/liter of ethanol to investigate the blood flow locally in the tissue surrounding the microdialysis catheter during insulin infusion and ß2-adrenergic stimulation. To ensure rapid detection of changes in lipolysis and blood flow during ß2-adrenergic stimulation, samples were collected every 15 min for measurement of ethanol concentration in the perfusate and dialysate. Changes in the ratio of ethanol concentration, i.e. the outgoing dialysate vs. the ingoing perfusate, have been shown to reflect changes in local blood flow (27, 28, 29). After an equilibration period of more than 2 h, insulin was infused iv at a rate of 0.8 mU/kg·min for 150 min. In this study, mean plasma glucose concentration was maintained at approximately 5.0 mmol/liter (90 mg/dl) throughout the study by a variable-rate glucose infusion. The selective ß2-agonist terbutaline was added to the perfusion fluid at a concentration of 10-5 mol/liter in all catheters during the final hour of insulin infusion. We chose to use a selective ß2-adrenergic agonist because the ß2-adrenoceptor is reported to be the dominant adrenoceptor subtype mediating catecholamine-induced lipolysis and vasodilation in both adipose and skeletal muscle tissue (16, 30).

Analyses

Plasma glucose was measured by the glucose oxidase method (Glucose analyzer II, Beckman Instruments, Inc., Fullerton, CA). Free insulin was determined by a double-antibody RIA after precipitating plasma samples at the bedside with polyethylene glycol (31). Plasma glucagon, cortisol, and GH concentrations were measured using RIA kits (glucagon, Linco Research, Inc., St. Charles, MO; cortisol, Diagnostic Products Corp., Los Angeles, CA; GH, Sanofi Pharmaceuticals, Inc., Pasteur, MN). Plasma NEFA concentrations were determined using a microfluorometric method (32). Catecholamines were collected in iced tubes containing glutathione and assayed by HPLC with electrochemical detection. Plasma and dialysate glycerol and lactate concentrations, as well as dialysate glucose concentrations were determined with enzymatic fluorometric methods using a tissue sample analyzer allowing extremely small sample volumes (CMA/600, CMA Microdialysis, Acton, MA). Dialysate and perfusate ethanol was determined with an enzymatic method (YSI 2700 Select Analyzer, YSI, Inc., Yellow Springs, OH).

For determining glucose disposal, plasma glucose collected at 10-min intervals during baseline and in the last hour of [6,6-2H2]glucose infusion was derivatized as the pentaacetate, following Ba(OH)2/ZnSO4 deproteinization and semipurification by anioncation exchange chromatography (AG1-8X, AG50W-8X; Bio-Rad Laboratories, Inc., Richmond, CA), as previously described (33). Gas chromatography-mass spectrometry analysis was performed with a Hewlett-Packard 5890 gas chromatograph (HP-1 capillary column, 12 mm x 0.2 mm x 0.33 µm film thickness; Hewlett-Packard Co., Palo Alto, CA) interfaced to a Hewlett-Packard 5971 mass selective detector operating in the electron impact ionization mode. Selected ion monitoring was used to determine enrichment in various molecular ion fragments. [6,6-2H2]Glucose m + 2 enrichment was determined from the ratio of m/z 202 to 200 of fragment ion consisting of C2 to C6 (34).

Calculations

Glucose disposal rate was calculated as follows: glucose disposal rate = (f/bw) x [(enrichmentinf/enrichmentplasma) - 1], where f = [6,6-2H]glucose infusion rate (milligrams/minute), bw = body weight (kilograms), enrichmentinf = [6,6-2H]glucose infusate enrichment (%), and enrichmentplasma = steady-state plasma [6,6-2H]glucose enrichment (%). The term enrichment refers to the fraction of isotope of glucose to naturally occurring glucose, expressed as a percentage.

Statistics

Data are expressed as means ± SE of the mean (SEM). A two-way ANOVA with repeated measurements was used to evaluate differences between the groups over time. When the group-time interaction was significant, the differences between the groups were then assessed by the post hoc Student’s t test to localize differences after intervention.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Insulin-induced hypoglycemia

Plasma-free insulin and glucose. After an overnight fast (with basal insulin replacement in the diabetic patients), the baseline free insulin concentration in arterialized venous plasma was not significantly different between the nondiabetic controls (9 ± 1 µU/ml) and the diabetic patients (12 ± 1 µU/ml). During the subsequent insulin infusion, plasma insulin rose to the same extent in both groups, about 4-fold, and stabilized (Fig. 1AGo).



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  		Figure 1. Plasma levels of free insulin (A), glucose (B), and epinephrine (C), in eight healthy control subjects (open circles) and seven T1DM patients (filled circles) at baseline and during a 3-h insulin infusion (0.8 mU/kg·min). Values are means ± SEM. Plasma glucose was allowed to fall to 3 mmol/liter (54 mg/dl). In subjects in whom the glucose concentration fell below the desired level (all diabetic subjects), 20% dextrose was infused to maintain plasma glucose at 3 mmol/liter. Despite glucose supplementation, plasma glucose fell to a greater extent in the T1DM group (P = 0.001). The epinephrine response (area under the curve) in the diabetic patients was less than 50% of that in the control subjects (P = 0.02).

 
Baseline plasma glucose levels were likewise similar in the control subjects and the diabetic patients, 5.2 ± 0.11 and 5.5 ± 0.26 mmol/liter, respectively (P = 0.16; Fig. 1BGo). During insulin infusion, the plasma glucose concentration fell in the control subjects to 3.5 ± 0.06 mmol/liter and stabilized without infusion of exogenous glucose. In contrast, plasma glucose decreased to a greater extent (P = 0.001) in the diabetic group, i.e. 3.0 ± 0.03 mmol/liter, and exogenous glucose (2.6 ± 0.41 mg/kg·min) had to be infused in all seven diabetic subjects to prevent the development of more severe hypoglycemia.

Counterregulatory hormone responses. In both groups, basal plasma epinephrine concentration was approximately 30 pg/ml (Fig. 1CGo). In the control subjects, a rapid and marked increase was seen early, reaching peak level 386 ± 79 pg/ml at 180 min. The rise in epinephrine was considerably delayed in the diabetic patients. Furthermore, despite a greater fall in glucose in the diabetic patients, the epinephrine response was 42% of that in the control subjects (160 ± 45 pg/ml; P = 0.02 vs. controls). As shown in Table 1Go, fasting glucagon, norepinephrine, cortisol, and GH concentrations were comparable in the control subjects and the diabetic patients. During hypoglycemia, plasma glucagon was unchanged in the diabetic patients, whereas it increased 2-fold in the control subjects (Table 1Go). The maximum increase in norepinephrine level was similar in the two groups (Table 1Go). Stimulated levels of plasma GH and cortisol were greater in the control subjects than in the diabetic patients, however, only the increase in cortisol was statistically different between groups (Table 1Go).


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  		Table 1. Counterregulatory hormone responses in plasma during insulin infusion and hypoglycemia

 
Plasma glycerol and serum NEFA. Changes in plasma glycerol and serum NEFA levels during insulin-induced hypoglycemia are shown in Fig. 2Go, A and B. During basal conditions, plasma glycerol and NEFA levels were not significantly different between the study groups. During the first hour of insulin infusion, the plasma glycerol concentration decreased to the same extent in both the control subjects and the diabetic patients, 52% and 54%, respectively (Fig. 2AGo). Thereafter, as hypoglycemia was established after the second hour of insulin infusion, the plasma glycerol level rose toward baseline in the control group, whereas there was a delayed and impaired rebound in the diabetic group (P = 0.05 vs. control). The NEFA response followed the same trend (Fig. 2BGo). First, a parallel reduction in plasma NEFA concentration by approximately 70% in both groups, subsequently during hypoglycemia, a partial restoration of basal levels occurred in both groups, but to a much lesser extent in the diabetic patients (P = 0.05 vs. control).



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  		Figure 2. Plasma levels of glycerol (A) and NEFA (B) in eight healthy control subjects (open circles) and seven T1DM patients (filled circles) at baseline and during mild-moderate hypoglycemia. Basal glycerol and NEFA levels were comparable in the two groups. During hypoglycemia, T1DM patients showed a delayed and incomplete recovery in both glycerol and NEFA levels in plasma (P < 0.05 vs. control). For further details, see legend to Fig. 1Go.

 
Adipose and skeletal muscle tissue concentrations of glycerol, glucose, and lactate. There was no significant difference between control subjects and diabetic patients in baseline glycerol levels, or the immediate antilipolytic effect of insulin in either skeletal muscle or adipose tissue (Fig. 3Go, A and B). The extracellular concentrations of glycerol decreased in parallel in both groups in response to insulin, by 25% or more in skeletal muscle (P < 0.01) and by 50% in adipose tissue (P < 0.001). During hypoglycemia, there was a significant rebound in muscle and adipose tissue glycerol in the control subjects (P < 0.01). In striking contrast, ECF glycerol remained suppressed in both tissues in the diabetic patients in the face of hypoglycemia. As a result, the response of ECF glycerol over time was significantly reduced in the diabetic subjects, both in muscle (P < 0.001) and adipose tissue (P < 0.02). Simple linear regression analysis of the plasma epinephrine concentration during the final hour of insulin infusion against the glycerol response in skeletal muscle of all subjects from nadir to 180 min showed a significant correlation (r = 0.73; P = 0.002; Fig. 4AGo). The glycerol response in adipose tissue was also significantly correlated with plasma epinephrine (r = 0.52; P = 0.04). The correlation between changes in the plasma NEFA concentration vs. the plasma epinephrine level during the hypoglycemic period did not reach statistical significance.



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  		Figure 3. Effect of insulin infusion and hypoglycemia on the skeletal muscle concentration of glycerol, glucose, and lactate (A, C, and E, respectively) and the adipose tissue concentration of glycerol, glucose, and lactate (B, D, and F, respectively). Microdialysis of the gastrocnemius muscle and sc abdominal adipose tissue of eight healthy control subjects (open circles) and seven T1DM patients (filled circles) were performed at baseline and during 3 h of insulin infusion. The dialysate was collected in 30-min fractions, and the last collection in every hour is presented. Values are means ± SEM. The decrement in ECF glucose was especially marked and protracted in skeletal muscle of the diabetic patients (P = 0.046 vs. control). There was a significant rebound in muscle (P = 0.005) and adipose (P = 0.007) tissue glycerol in the control subjects, whereas it remained suppressed in both tissues in the diabetic patients.

 


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  		Figure 4. Relation between the plasma epinephrine concentration during the final hour of insulin infusion and the glycerol response in skeletal muscle of all subjects (n = 15) from nadir to 180 min (r = 0.73; P = 0.002; A), and between the glycerol response in skeletal muscle and plasma glucose clearance during the final hour of insulin infusion (r = -0.51; P = 0.05; B). The linear regression line is solid, and lines representing the 95% confidence interval are dashed.

 
Changes in extracellular levels of glucose are depicted in Fig. 3Go, C and D. Tissue concentrations of glucose decreased in parallel in both groups during insulin infusion, reaching lower levels than those in plasma. Notably, the drop in ECF glucose was more pronounced in the diabetic patients than in the control subjects, the difference being especially marked in skeletal muscle (P < 0.05). In the diabetic group, the change in ECF glucose level decreased over time in muscle to a greater extent than was seen in the controls (P < 0.01). A similar trend was seen in adipose tissue, however, the difference did not reach statistical significance between the groups. As a result, the plasma-ECF glucose gradient in skeletal muscle was greatly increased in the T1DM patients (from 0.7 ± 0.1 to 1.9 ± 0.2 mmol; P < 0.05), whereas it changed minimally (P = not significant) in the nondiabetic controls during hypoglycemia.

ECF lactate levels were also different in the two groups (Fig. 3Go, E and F). During the first 2 h of insulin infusion in the control subjects, ECF lactate increased over time in skeletal muscle (P = 0.05) and adipose tissue (P = 0.05) and tapered off slightly during the final hour. In contrast, in the diabetic patients, lactate levels remained unchanged during hypoglycemia in skeletal muscle, as well as in adipose tissue. The differences in absolute levels between the groups over time did not, however, reach statistical significance.

Glucose turnover. To permit an estimate of whole-body glucose turnover, dideuterated glucose was infused throughout the study. The coefficient of variation in plasma isotope enrichments during the final hour of the study was approximately 4% in both groups, indicating relative steady state conditions. Rates of glucose disappearance (Rd) and glucose clearance (i.e. Rd divided by the ambient glucose concentration) are given in Table 2Go. During hypoglycemia, both Rd and glucose clearance were significantly higher in the diabetic subjects than in the control group (P = 0.033 and P = 0.02, respectively). Interestingly, a significant inverse correlation was found between the glycerol response in skeletal muscle and plasma glucose clearance (r = -0.51; P = 0.05), i.e. decreased clearance with increasing glycerol release (Fig. 4BGo). A significant inverse correlation between the glycerol response and plasma glucose clearance was also found in adipose tissue (r = -0.50; P = 0.05). The relationship between whole-body glucose uptake and NEFA levels during the final hour was not significant when all subjects were studied together. However, when the diabetic patients were investigated separately, a correlation was apparent; but owing to the limited number of subjects, the correlation was only of borderline statistical significance (r = -0.72; P = 0.06; data not depicted).


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  		Table 2. Rate of glucose disappearance (Rd) and glucose clearance during hypoglycemia

 
Insulin infusion with euglycemia and local perfusion with ß2-agonist

Substrate and hormone concentrations. Fasting plasma glucose concentration in the diabetic subjects was slightly higher than in the control subjects, but there was no significant change during insulin infusion (Table 3Go). The baseline plasma-ECF glucose gradient (skeletal muscle) was identical in both groups (1.0 ± 0.1) and was not significantly altered during euglycemic hyperinsulinemia.


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  		Table 3. Substrate and hormone concentrations in plasma during insulin infusion and euglycemia

 
Basal plasma glycerol, NEFA, free insulin, epinephrine, and norepinephrine levels were comparable in the control subjects and the diabetic patients (Table 3Go). Free insulin levels rose similarly in both groups during the euglycemic clamp study and reached the same hyperinsulinemic plateau as in the hypoglycemic study. Plasma catecholamines did not significantly change during euglycemia. A similar decrease in plasma NEFA and glycerol amounting to approximately 90% and 50%, respectively, was seen in control subjects and diabetic patients in response to the unopposed antilipolytic effect of insulin. Likewise, the dialysate glycerol levels (which are lower due to the higher perfusion rates of artificial ECF in these studies) in peripheral tissues during euglycemic hyperinsulinemia was similar in the control subjects and the diabetic patients. The glycerol concentration in muscle and adipose tissue was suppressed by about 40% in both groups (Fig. 5Go). Subsequently, when the tissues were perfused with the selective ß2-agonist terbutaline, there was a significant rebound in glycerol levels in muscle and adipose tissue in all subjects. The rise in ECF glycerol produced by the ß2-agonist was, however, substantially greater in adipose compared with muscle tissue. Although the ß2-agonist-induced lipolysis in adipose tissue tended to be higher in the diabetic patients than in the control subjects, the difference between the groups did not reach statistical significance.



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  		Figure 5. Glycerol response in skeletal muscle and adipose tissue of six healthy control subjects (hatched bars) and six T1DM patients (shaded bars) during three phases of euglycemia. After 2 h of equilibration, a 2.5-h insulin infusion (0.8 mU/kg·min) was started. A variable infusion of 20% dextrose was administered to maintain euglycemia. Microdialysis of the gastrocnemius muscle and sc abdominal adipose was performed at baseline, during hyperinsulinemia, and during hyperinsulinemia plus local perfusion with ß2-agonist, which was added to the perfusion fluid during the final hour of insulin infusion. The dialysate was collected in 15-min fractions. The mean of two collections during each of the three phases of the study is presented. Values are means ± SEM.

 
Local blood flow (Table 4Go). Effects of insulin and ß2-agonist on the local blood flow in adipose and skeletal muscle tissue were investigated in the euglycemic study using the ethanol technique (35). The loss of ethanol from the perfusion medium to the extracellular tissue during microdialysis was measured. In the basal state, the ratio of outgoing and ingoing ethanol concentration was similar in the two groups. In adipose tissue, a significant decrease in ethanol ratio (i.e. increased blood flow) was induced by insulin, as well as by ß2-agonist in both control subjects and diabetic patients. In muscle, there was no detectable insulin-induced change in ethanol ratio in either group, whereas the addition of ß2-agonist to the perfusion medium caused a significant decrease in ethanol ratio in both control subjects and diabetic patients. A significant difference between the groups regarding the stimulatory effect on blood flow of insulin (adipose) or ß2-agonist (adipose and muscle) was not distinguishable (P = not significant).


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  		Table 4. Ethanol ratios in adipose and muscle tissue during three phases of the euglycemic study

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This human in vivo study was undertaken to investigate whether diminished lipolysis within muscle and adipose tissue was associated with the impaired defense against hypoglycemia seen in many T1DM patients treated intensively with insulin. Our patients, much like others with near-normal glycosylated hemoglobin levels treated intensively with insulin, showed defective glucose counterregulation, failed to release glucagon in response to hypoglycemia, and had impaired catecholamine responses to hypoglycemia (36, 37, 38). Their release of cortisol (and to a lesser extent GH) during hypoglycemia was reduced as well, in keeping with previous observations (38, 39). Not unexpectedly, these changes in counterregulatory hormone responses markedly reduced the rebound rise in circulating levels of glycerol and NEFA that act to promote glucose production via gluconeogenesis (40).

Our data demonstrate that the rebound release of glycerol (a marker of lipolysis) in both muscle and adipose tissue to baseline values that normally occurs during hypoglycemia was absent in our T1DM patients. This defect in lipolysis within muscle and fat during hypoglycemia might contribute to impaired hypoglycemia defense mechanisms in T1DM patients who lack glucagon responses and are therefore dependent on catecholamines that act predominantly on peripheral tissues (4, 5). The failure of NEFA to show a delayed rebound increase in T1DM patients demonstrating reduced adrenergic responses could act to facilitate glucose use and oxidation in peripheral tissues (e.g. muscle) during more prolonged periods of hypoglycemia (11, 40, 41, 42). In keeping with this scenario, our data show a strikingly lower ECF-glucose concentration in skeletal muscle in the T1DM subjects after a 2-h period of hypoglycemia (Fig. 3CGo), suggesting a substantially increased plasma-ECF gradient of glucose during hypoglycemia and increased glucose extraction. This is further supported by the finding of a greater increase in the Rd after 2 h of hypoglycemia in the diabetic patients, but not in the nondiabetic controls (Table 2Go). Moreover, a significant inverse correlation between glycerol release and glucose clearance was shown (Fig. 4BGo). These observations are consistent with earlier studies by Fanelli et al. (11), who reported in healthy subjects that the indirect effects of catecholamines on lipolysis become important contributors to hypoglycemic counterregulation, specifically in the late phase of mild-moderate hypoglycemia. In those studies, blockade of catecholamine-induced lipolysis increased glucose use during low-dose insulin infusion, and this effect on glucose metabolism was reversed by recreating the delayed rebound rise in plasma NEFA seen during hypoglycemia. Presumably, the replacement of NEFA in those experiments interfered with the gradual increase in insulin-stimulated glucose uptake that is normally seen for several hours when euglycemia is maintained by infusion of exogenous glucose (44). Increased free fatty acid (FFA) availability to muscle tissue has been clearly found to alter insulin signaling, thereby leading to insulin resistance (41, 42, 43). However, it is noteworthy that these effects have been found to occur at very high FFA levels (41, 42, 43, 44). Thus, one might question how the rebound elevation in FFA levels (~400 µm) reported here could lead to a reduction in peripheral glucose uptake. It is possible that although euglycemic hyperinsulinemia requires very high FFA levels to alter peripheral glucose metabolism, in the setting of more modest hyperinsulinemia and hypoglycemic counterregulation, small changes in FFA levels, possibly together with other substrates and hormones (i.e. GH and cortisol), are sufficient to impair glucose uptake. Thus, it is conceivable that the impaired lipolytic response may have contributed to the more sustained increase in peripheral glucose uptake and greater glucose decrement seen after several hours in our T1DM patients. On the other hand, it is very unlikely that lipolysis plays an important role in the critical immediate counterregulatory response in intensively treated T1DM patients as well as in healthy subjects; plasma glycerol and NEFA levels (Fig. 2Go) and muscle and adipose tissue glycerol levels (Fig. 3Go) were suppressed comparably at 1 h.

To investigate whether the observed defect in lipolytic response in muscle and adipose tissue during insulin-induced hypoglycemia could be explained in part by altered adrenergic sensitivity, we perfused muscle and adipose tissue with a selective ß2-agonist during euglycemic hyperinsulinemia. We found no significant difference in glycerol release between healthy subjects and the diabetic patients. Indeed, the lipolytic response in adipose tissue of the diabetic patients tended to be accentuated (Fig. 5BGo). Although there have been no previous studies looking at muscle, our data concerning the lipolytic response to adrenergic stimulation in adipose tissue may seem, at first glance, at odds with some previous reports (23, 24, 45). For example, Bolinder et al. (25), using a similar microdialysis technique to study the adrenergic regulation of lipolysis in T1DM, reported enhanced lipolysis in adipose tissue after hypoglycemia, despite a reduced epinephrine response, suggesting a compensatory increase in ß-adrenoceptor action. They used a rapid infusion of insulin, which was interrupted when blood glucose reached 2.8 mmol/liter. Thus, the increase in glycerol release in their study may be attributed to the development of relative insulin deficiency. It is noteworthy that other studies using different experimental approaches, such as the isoproterenol test, report a decreased, unchanged, or increased adrenergic response in T1DM (20, 21, 22, 23, 24, 25), perhaps in part depending on how successfully they had been able to avoid antecedent episodes of hypoglycemia (46, 47, 48). It is noteworthy in this regard that none of our tightly controlled T1DM subjects reported any autonomic symptoms during the hypoglycemic study, suggesting the presence of hypoglycemia unawareness. Other factors, such as dose and mode of insulin infusion (nonclamped conditions), as well as the endpoints evaluated, may also have contributed to the discrepancies in lipolytic outcome.

Changes in the local blood flow may also be involved in lipolysis, and hence, influence counterregulation, because vascular tone is regulated by both insulin and catecholamines (49, 50, 51). We did not find any difference between normal and diabetic subjects in sensitivity to the stimulatory effect of insulin or to the ß2- agonist terbutaline on local blood flow in either skeletal muscle or adipose tissue. This finding is in agreement with the reports from Bolinder et al. (25) and others (52, 53). Thus, differences in lipolysis between the nondiabetic and diabetic subjects appear not to be explained by microcirculatory variability.

In previous studies in normal subjects, we demonstrated a significant increase in alanine and lactate (as well as catecholamine) concentrations in skeletal muscle and adipose tissue ECF during hypoglycemia, suggesting that local generation of gluconeogenic substrates from peripheral tissues contribute to the counterregulatory response (54). In the present hypoglycemic study, alanine was not measured, but lactate increased in both tissues in the control subjects (Fig. 3Go). In the diabetic patients, however, lactate remained unchanged throughout the study in both muscle and fat. This finding implies that the production of gluconeogenic substrates in skeletal muscle and adipose tissue is partially dependent on an adequate adrenergic response, presumably by virtue of its effect on glycogen breakdown and glucose oxidation (42, 43).

In conclusion, in T1DM patients receiving intensive insulin therapy and glycosylated hemoglobin levels close to the normal range, deficient release of, rather than impaired responsiveness to, catecholamines abolishes the normal lipolytic response that occurs in peripheral tissues during mild-moderate hypoglycemia. Defective stimulation of lipolysis in skeletal muscle and adipose tissue was associated with an inappropriate increase in skeletal muscle glucose disposal when the period of hypoglycemia was prolonged. These pathophysiological changes may be important contributors to the development of hypoglycemia during intensive insulin treatment.


   Acknowledgments
 
We are grateful for the skilled assistance of the staff of the Yale General Clinical Research Center (GCRC) and the technical support from Ralph Jacob, Andrea Belous, and Aida Groszman of the GCRC Core Laboratory. We also thank Tony Ma for his help in the statistical analysis of the data.


   Footnotes
 
This work was supported by grants from the National Institutes of Health (DK20495, DK45735, and RR00125) as well as the Juvenile Diabetes Research Foundation Center for the Study of Hypoglycemia. S.E. was supported by a mentor-based fellowship from the American Diabetes Association, the Swedish Institute, and the foundation of Johan Throne Holst.

Abbreviations: ECF, Extracellular fluid; FFA, free fatty acid; NEFA, nonesterified fatty acids; Rd, rate of glucose disappearance; T1DM, type 1 diabetes mellitus.

Received June 28, 2002.

Accepted December 23, 2002.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Gerich JE, Langlois M, Noacco C, Karam JH, Forsham PH 1973 Lack of glucagon response to hypoglycemia in diabetes: evidence for an intrinsic pancreatic {alpha} cell defect. Science 182:171–173[Abstract/Free Full Text]
  2. Cryer PE, Gerich JE 1985 Glucose counterregulation, hypoglycemia, and intensive insulin therapy in diabetes mellitus. N Engl J Med 313:232–241[Abstract]
  3. The Diabetes Control and Complications Trial Research Group 1993 The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 329:977–986[Abstract/Free Full Text]
  4. Cryer PE 1994 Banting Lecture. Hypoglycemia: the limiting factor in the management of IDDM. Diabetes 43:1378–1389[Abstract]
  5. Lager I 1991 The insulin-antagonistic effect of the counterregulatory hormones. J Intern Med Suppl 735:41–47[Medline]
  6. Sacca L, Vigorito C, Cicala M, Corso G, Sherwin RS 1983 Role of gluconeogenesis in epinephrine-stimulated hepatic glucose production in humans. Am J Physiol 245:E294–E302
  7. Sacca L, Sherwin R, Hendler R, Felig P 1979 Influence of continuous physiologic hyperinsulinemia on glucose kinetics and counterregulatory hormones in normal and diabetic humans. J Clin Invest 63:849–857
  8. De Feo P, Perriello G, De Cosmo S, Ventura MM, Campbell PJ, Brunetti P, Gerich JE, Bolli GB 1986 Comparison of glucose counterregulation during short-term and prolonged hypoglycemia in normal humans. Diabetes 35:563–569[Abstract]
  9. Sherwin RS, Shamoon H, Hendler R, Sacca L, Eigler N, Walesky M 1980 Epinephrine and the regulation of glucose metabolism: effect of diabetes and hormonal interactions. Metabolism 29(11 Suppl 1):1146–1154
  10. Fanelli C, Calderone S, Epifano L, De Vincenzo A, Modarelli F, Pampanelli S, Perriello G, De Feo P, Brunetti P, Gerich JE, Bolli GB 1993 Demonstration of a critical role for free fatty acids in mediating counterregulatory stimulation of gluconeogenesis and suppression of glucose utilization in humans. J Clin Invest 92:1617–1622
  11. Fanelli CG, De Feo P, Porcellati F, Perriello G, Torlone E, Santeusanio F, Brunetti P, Bolli GB 1992 Adrenergic mechanisms contribute to the late phase of hypoglycemic glucose counterregulation in humans by stimulating lipolysis. J Clin Invest 89:2005–2013
  12. Maggs DG, Jacob R, Rife F, Lange R, Leone P, During MJ, Tamborlane WV, Sherwin RS 1995 Interstitial fluid concentrations of glycerol, glucose, and amino acids in human quadriceps muscle and adipose tissue. Evidence for significant lipolysis in skeletal muscle. J Clin Invest 96:370–377
  13. Hagstrom-Toft E, Enoksson S, Moberg E, Bolinder J, Arner P 1997 Absolute concentrations of glycerol and lactate in human skeletal muscle, adipose tissue, and blood. Am J Physiol 273(3 Pt 1):E584–E592
  14. Itani SI, Ruderman NB, Schomieder F, Boden G 2000 Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and Ik-B-2. Diabetes 51:2005–2011
  15. Bolinder J, Kerckhoffs DA, Moberg E, Hagstrom-Toft E, Arner P 2000 Rates of skeletal muscle and adipose tissue glycerol release in non-obese and obese subjects. Diabetes 49:797–802[Abstract]
  16. Hagstrom-Toft E, Enoksson S, Moberg E, Bolinder J, Arner P 1998 ß-adrenergic regulation of lipolysis and blood flow in human skeletal muscle in vivo. Am J Physiol 275(6 Pt 1):E909–E916
  17. Enoksson S, Degerman E, Hagstrom-Toft E, Large V, Arner P 1998 Various phosphodiesterase subtypes mediate the in vivo antilipolytic effect of insulin on adipose tissue and skeletal muscle in man. Diabetologia 41:560–568[CrossRef][Medline]
  18. Perseghin G, Scifo P, De Cobelli F, Pagliato E, Battezzati A, Arcelloni C, Vanzulli A, Testolin G, Pozza G, Del Maschio A, Luzi L 1999 Intramyocellular triglyceride content is a determinant of in vivo insulin resistance in humans: a 1H–13C nuclear magnetic resonance spectroscopy assessment in offspring of type 2 diabetic parents. Diabetes 48:1600–1606[Abstract]
  19. Ebeling P, Essen-Gustavsson B, Tuominen JA, Koivisto VA 1998 Intramuscular triglyceride content is increased in IDDM. Diabetologia 41:111–115[CrossRef][Medline]
  20. Berlin I, Grimaldi A, Landault C, Zoghbi F, Thervet F, Puech AJ, Legrand JC 1988 Lack of hypoglycemic symptoms and decreased ß-adrenergic sensitivity in insulin-dependent diabetic patients. J Clin Endocrinol Metab 66:273–278[Abstract]
  21. Berlin I, Grimaldi A, Bosquet F, Puech AJ 1986 Decreased ß-adrenergic sensitivity in insulin-dependent diabetic subjects. J Clin Endocrinol Metab 63:262–265[Abstract]
  22. Divertie GD, Jensen MD, Cryer PE, Miles JM 1997 Lipolytic responsiveness to epinephrine in non-diabetic and diabetic humans. Am J Physiol 272(6 Pt 1):E1130–E1135
  23. Fritsche A, Sturmvoll M, Haring HU, Gerich JE 2000 Reversal of hypoglycemia unawareness in a long-term type 1 diabetic patient by improvement of ß-adrenergic sensitivity after prevention of hypoglycemia. J Clin Endocrinol Metab 85:523–525[Abstract/Free Full Text]
  24. Hilsted J, Richter E, Madsbad S, Tronier B, Christensen NJ, Hildebrandt P, Damkjaer M, Galbo H 1987 Metabolic and cardiovascular responses to epinephrine in diabetic autonomic neuropathy. N Engl J Med 317:421–426[Abstract]
  25. Bolinder J, Sjoberg S, Arner P 1996 Stimulation of adipose tissue lipolysis following insulin-induced hypoglycaemia: evidence of increased ß-adrenoceptor-mediated lipolytic response in IDDM. Diabetologia 39:845–853[CrossRef][Medline]
  26. Tossman U, Ungerstedt U 1986 Microdialysis in the study of extracellular levels of amino acids in the rat brain. Acta Physiol Scand 128:9–14[Medline]
  27. Fellander G, Linde B, Bolinder J 1996 Evaluation of the microdialysis ethanol technique for monitoring of subcutaneous adipose tissue blood flow in humans. Int J Obes Relat Metab Disord 20:220–226[Medline]
  28. Hickner RC, Ekelund U, Mellander S, Ungerstedt U, Henriksson J 1995 Muscle blood flow in cats: comparison of microdialysis ethanol technique with direct measurement. J Appl Physiol 79:638–647[Abstract/Free Full Text]
  29. Hickner RC, Bone D, Ungerstedt U, Jorfeldt L, Henriksson J 1994 Muscle blood flow during intermittent exercise: comparison of the microdialysis ethanol technique and 133Xe clearance. Clin Sci (Colch) 86:15–25[Medline]
  30. Enoksson S, Shimizu M, Lonnqvist F, Nordenstrom J, Arner P 1995 Demonstration of an in vivo functional ß3-adrenoceptor in man. J Clin Invest 95:2239–2245
  31. Nakagawa S, Nakayama H, Sasaki T, Yoshino K, Yu YY 1973 A simple method for the determination of serum free insulin levels in insulin-treated patients. Diabetes 22:590–600[Medline]
  32. Miles J, Glasscock R, Aikens J, Gerich J, Haymond M 1983 A microfluorometric method for the determination of free fatty acids in plasma. J Lipid Res 24:96–99[Abstract]
  33. Shulman GI, Cline G, Schumann WC, Chandramouli V, Kumaran K, Landau BR 1990 Quantitative comparison of pathways of hepatic glycogen repletion in fed and fasted humans. Am J Physiol 259(3 Pt 1):E335–E341
  34. Caprioli RM, Seifert Jr WE 1973 Direct analysis of 18 O in glucose by mass spectrometry. Biochem Biophys Acta 297:213–219[Medline]
  35. Hickner RC, Rosdahl H, Borg I, Ungerstedt U, Jorfeldt L, Henriksson J 1991 Ethanol may be used with the microdialysis technique to monitor blood flow changes in skeletal muscle: dialysate glucose concentration is blood-flow-dependent. Acta Physiol Scand 143:355–356[Medline]
  36. Bolli G, de Feo P, Compagnucci P, Cartechini MG, Angeletti G, Santeusanio F, Brunetti P, Gerich JE 1983 Abnormal glucose counterregulation in insulin-dependent diabetes mellitus. Interaction of anti-insulin antibodies and impaired glucagon and epinephrine secretion. Diabetes 32:134–141[Abstract]
  37. De Feo P, Bolli G, Perriello G, De Cosmo S, Compagnucci P, Angeletti G, Santeusanio F, Gerich JE, Motolese M, Brunetti P 1983 The adrenergic contribution to glucose counterregulation in type I diabetes mellitus. Dependency on A-cell function and mediation through ß2-adrenergic receptors. Diabetes 32:887–893[Abstract]
  38. Simonson DC, Tamborlane WV, DeFronzo RA, Sherwin RS 1985 Intensive insulin therapy reduces counterregulatory hormone responses to hypoglycemia in patients with type I diabetes. Ann Intern Med 103:184–190
  39. Polonsky K, Bergenstal R, Pons G, Schneider M, Jaspan J, Rubenstein A 1982 Relation of counterregulatory responses to hypoglycemia in type I diabetics. N Engl J Med 307:1106–1112[Abstract]
  40. Caprio S, Amiel S, Tamborlane WV, Gelfand RA, Sherwin RS 1990 Defective free-fatty acid and oxidative glucose metabolism in IDDM during hypoglycemia: influence of glycemic control. Diabetes 39:134–141[Abstract]
  41. Caprio S, Gelfand RA, Tamborlane WV, Sherwin RS 1989 Oxidative fuel metabolism during mild hypoglycemia: critical role of free fatty acids. Am J Phys 256:E413–419
  42. Dresner A, Laurent D, Marcucci M, Griffin ME, Dufour S, Cline GW, Slezak LA, Andersen DK, Hundal RS, Rothman DL, Petersen KF, Shulman GI 1999 Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidylinositol 3-kinase activity. J Clin Invest 103:253–259[Medline]
  43. Griffin ME, Marcucci MJ, Cline GW, Bell K, Barucci N, Lee D, Goodyear LJ, Kraegen EW, White MF, Shulman GI 1999 Free fatty acid-induced insulin resistance is associated with activation of protein kinase C {tau} and alterations in the insulin signaling cascade. Diabetes 48:1270–1274[Abstract]
  44. Sherwin RS, Kramer KJ, Tobin JD, Insel PA, Liljenquist JE, Berman M, Andres R 1974 A model of the kinetics of insulin in man. J Clin Invest 53:1481–1492
  45. Wahrenberg H, Lonnqvist F, Engfeldt P, Arner P 1989 Abnormal action of catecholamines on lipolysis in adipocytes of type I diabetic patients treated with insulin. Diabetes 38:524–533[Abstract]
  46. Cranston I, Lomas J, Maran A, Macdonald I, Amiel SA 1994 Restoration of hypoglycaemia awareness in patients with long-duration insulin-dependent diabetes. Lancet 344:283–287[CrossRef][Medline]
  47. Korytkowski MT, Mokan M, Veneman TF, Mitrakou A, Cryer PE, Gerich JE 1998 Reduced ß-adrenergic sensitivity in patients with type-1 diabetes and hypoglycemia unawareness. Diabetes Care 21:1939–1943[Abstract]
  48. Trovik TS, Jaeger R, Jorde R, Sager G 1994 Reduced sensitivity to ß-adrenoceptor stimulation and blockade in insulin dependent diabetic patients with hypoglycaemia unawareness. Br J Clin Pharmacol 38:427–432[Medline]
  49. Creager MA, Liang CS, Coffman JD 1985 ß-Adrenergic-mediated vasodilator response to insulin in the human forearm. J Pharmacol Exp Ther 235:709–714[Abstract/Free Full Text]
  50. Baron AD, Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G 1995 Insulin-mediated skeletal muscle vasodilation contributes to both insulin sensitivity and responsiveness in lean humans. J Clin Invest 96:786–792
  51. Laakso M, Edelman SV, Brechtel G, Baron AD 1990 Decreased effect of insulin to stimulate skeletal muscle blood flow in obese man. A novel mechanism for insulin resistance. J Clin Invest 85:1844–1852
  52. Fernqvist-Forbes E, Linde B, Gunnarsson R 1988 Insulin absorption and subcutaneous blood flow in normal subjects during insulin-induced hypoglycemia. J Clin Endocrinol Metab 67:619–623[Abstract]
  53. Fernqvist-Forbes E, Gunnarsson R, Linde B 1989 Insulin-induced hypoglycaemia and absorption of injected insulin in diabetic patients. Diabetes Med 6:621–626[Medline]
  54. Maggs DG, Jacob R, Rife F, Caprio S, Tamborlane WV, Sherwin RS 1997 Counterregulation in peripheral tissues: effect of systemic hypoglycemia on levels of substrates and catecholamines in human skeletal muscle and adipose tissue. Diabetes 46:70–76[Abstract]



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