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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1756
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 10 5559-5565
Copyright © 2005 by The Endocrine Society

The Role of Intramyocellular Lipids during Hypoglycemia in Patients with Intensively Treated Type 1 Diabetes

Elisabeth Bernroider, Attila Brehm, Martin Krssak, Christian Anderwald, Zlatko Trajanoski, Gary Cline, Gerald I. Shulman and Michael Roden

Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University of Vienna (E.B., A.B., M.K., C.A., M.R.), A-1090 Vienna, Austria; Institute of Biomedical Engineering, University of Technology (Z.T.), A-8020 Graz, Austria; Howard Hughes Medical Institute, Department of Internal Medicine, Yale University School of Medicine (G.C., G.I.S.), New Haven, Connecticut 06536-8012; and First Medical Department, Hanusch Hospital (M.R.), A-1140 Vienna, Austria

Address all correspondence and requests for reprints to: Dr. Michael Roden, First Medical Department, Hanusch Hospital, Heinrich Collin Strasse 30, A-1140 Vienna, Austria. E-mail: michael.roden{at}meduniwien.ac.at.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Endocrine defensive mechanisms provide for energy supply during hypoglycemia. Intramyocellular lipids (IMCL) were recently shown to contribute to energy supply during exercise.

Objective: The objective of this study was to assess the contribution of IMCL compared with lipolysis and endogenous glucose production (EGP) to insulin-mediated hypoglycemia counterregulation in patients with type 1 diabetes mellitus (T1DM).

Design and Setting: This was a prospective explorative study preformed in a university research facility.

Participants: Six well-controlled T1DM (age, 29 ± 4 yr; body mass index, 23.4 ± 1.0 kg/m2; hemoglobin A1c, 6.3 ± 0.1%) and six nondiabetic humans (controls; age, 28 ± 2 yr; body mass index, 23.4 ± 1.0 kg/m2; hemoglobin A1c, 5.1 ± 0.1%) were studied.

Interventions: We performed 240-min hypoglycemic (~3 mM)-hyperinsulinemic (0.8 mU/kg·min) clamps on separate days to measure: 1) systemic lipolysis ([2H5]glycerol turnover), EGP ([6,6-2H2]glucose), and local lipolysis in abdominal sc adipose tissue and gastrocnemius muscle (microdialysis); and 2) IMCL (by 1H nuclear magnetic resonance spectroscopy) in soleus and tibialis anterior muscle.

Main Outcome Measures: The main outcome measures were changes in IMCL during prolonged hypoglycemia.

Results: At baseline, EGP, glycerol turnover, and IMCL were not different between the groups. During hypoglycemia, hormonal counterregulation was blunted in T1DM (peak: glucagon, 68 ± 4 vs. 170 ± 37 pg/ml; cortisol, 16 ± 2 vs. 24 ± 2 µg/dl; epinephrine, 274 ± 84 vs. 597 ± 212 pg/ml; all P < 0.05 vs. control). T1DM had approximately 50% lower EGP (4.6 ± 0.6 vs. 10.9 ± 0.5 µmol/kg·min; P < 0.005), but approximately 40% higher glycerol turnover (374 ± 21 vs. 272 ± 19 µmol/kg·min; P < 0.01). Glycerol concentrations in muscle (T1DM, 302 ± 22 control, 346 ± 17 µmol/liter) and adipose tissue (264 ± 25 vs. 318 ± 25 µmol/liter) did not differ between groups. IMCL in soleus and tibialis anterior muscle did not change from baseline during hypoglycemia.

Conclusions: In well-controlled T1DM, impaired hypoglycemia counterregulation is associated with decreased glucose production and augmented whole body lipolysis, which cannot be explained by either hydrolysis of muscle triglycerides or increased abdominal sc adipose tissue lipolysis.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
HYPOGLYCEMIA AND THE loss of counterregulatory hypoglycemic warning symptoms are well-known complications in intensively treated type 1 diabetes mellitus (T1DM) (1, 2). Analyses of the Diabetes and Complications Trial demonstrated that patients practicing intensive insulin therapy suffer from about two episodes of symptomatic hypoglycemia per week. Therefore, strategies reducing hypoglycemic events are required that reverse the syndrome of hypoglycemia unawareness despite optimal glycemic control (3). The majority of previous studies focused largely on changes in glucose fluxes and their relationship to hormonal responses, whereas little attention has been paid to changes in substrates, other than glucose.

In humans, the deposition of triglycerides (TG) as energy stores and the liberation of free fatty acids (FFA) and glycerol via hydrolysis of TG occur not only in adipose tissue (4, 5, 6, 7). High rates of im lipolysis have been demonstrated in vivo (7, 8), and recent studies demonstrate that its regulation appears to differ from that in adipose tissue (9, 10, 11). In adipocytes, catecholamines accelerate lipolysis through all three ß-adrenoreceptor subtypes (ß1, ß2, and ß3), whereas in skeletal muscle tissue, the ß2 receptor subtype is of importance (10). In addition, hyperinsulinemia per se is not sufficient alone to suppress skeletal muscle lipolysis, but only in combination with hyperglycemia (11). Of note, differences also exist for the regulation of lipolysis in adipose tissue across the body. Lipolysis is more easily stimulated and less readily inhibited in the visceral than in the sc region (8, 12).

Intramyocellular lipid (IMCL) content, which can be rapidly determined by noninvasive 1H nuclear magnetic resonance spectroscopy (1H-NMRS) (4, 13), is another source of energy within the muscle (14). Employing this technique, it has been demonstrated that there is a substantial utilization of IMCL during exercise (14). The importance of IMCL stores as an indicator of whole-body insulin sensitivity in nondiabetic, diabetic, and insulin-resistant humans was also demonstrated (13, 15, 16, 17, 18).

Interestingly, IMCL content is increased in T1DM (17, 18). Because skeletal muscle lipolysis is not easily inhibited by hyperinsulinemia (11), IMCL could play an important role as a source of lipolysis during hypoglycemia in T1DM. Thus, we hypothesized that increased IMCL content could be a protective adaptation to frequent hypoglycemia associated with intensive insulin therapy in T1DM.

Previous studies demonstrated that lipolysis plays a critical role in hypoglycemic metabolic counterregulation. Increased lipolysis during hypoglycemia provides plasma FFA and glycerol. Increased plasma concentrations of FFA limit skeletal muscle glucose uptake during hypoglycemia, and glycerol is an effective precursor for gluconeogenesis and endogenous glucose production (EGP) (19). During prolonged hypoglycemia, lipolysis-mediated effects of catecholamines were estimated to account for as much as approximately 50% of the increased EGP and for about 85% of the reduced whole body glucose disposal in nondiabetic humans (20).

Thus, lipolysis seems to play an important role in hypoglycemic counterregulation, but its contribution in patients with T1DM is as yet unclear. Decreased (6), unchanged (21), as well as increased (22) lipolysis after hypoglycemia have been reported in T1DM, and studies of the potential contribution of IMCL stores as a source of lipolysis during hypoglycemia are not available.

Thus, this study was performed to assess: 1) IMCL content in both tibialis anterior (IMCL-TA) and soleus muscle (IMCL-S) using 1H-NMRS; 2) systemic lipolysis using the isotope tracer method; and 3) local lipolysis in adipose and skeletal muscle tissue, performing the microdialysis technique in healthy and well-controlled T1DM during prolonged moderate hypoglycemia.


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

Six male T1DM [age, 29 ± 4 yr; body mass index, 23.3 ± 0.9 kg/m2; hemoglobin A1c (HbA1c), 6.3 ± 0.1%; diabetes duration, 13 ± 2 yr] and six nondiabetic humans without a family history of diabetes [controls (CON); age, 28 ± 2 yr; body mass index, 23.4 ± 1.2 kg/m2; HbA1c, 5.1 ± 0.1%] were matched for sex, age, and body mass index. All T1DM were under excellent long-term glucometabolic control by applying multiple insulin injections employing the basal-bolus principle and algorithms derived from physiological knowledge (23) after 1 wk of intensive training. They were recruited from our diabetes out-patient service and had been seen by a trained physician at least four times per year since the onset of diabetes. The diabetic patients had no history of hypoglycemic episodes for at least 2 wk before the study and did not present with diabetes-related late complications, such as neuropathy, nephropathy, and proliferative retinopathy. Informed consent to the protocols, which were approved by the local ethics board, was obtained from all participants.

Study protocol

All participants were studied on two occasions at 4-wk intervals. They were instructed to ingest a carbohydrate-rich, weight-maintaining diet and to refrain from strenuous physical exercise for at least 3 d before the studies. The patients were admitted to the Clinical Research Center the night before the studies, and they had their last injection of long-lasting insulin at least 24 h before the study began. An antecubital vein was cannulated (Vasofix, Braun, Melsungen, Germany) for continuous infusion of regular insulin (Actrapid, Novo-Nordisk, Bagsvaerd, Denmark) to maintain euglycemia.

At 0700 h (–135 min), two microdialysis probes were inserted. At –120 min, a primed [5 min; 5 mg/kg fasting glucose (mg/dl) /90 (mg/d)] continuous (355 min; 0.05 mg/kg·min) infusion of D-[6,6-2H2]glucose (99% enriched; Cambridge Isotopes Laboratories, Andover, MA), a primed (1 min; 0.20 mg/kg) continuous (359 min; 0.02 mg/kg·min) infusion of D-[1,1,2,3,3-2H5]glycerol (99% enriched; Cambridge Isotopes Laboratories), and an infusion of mannitol were started. The D-[6,6-2H2]glucose and D-[1,1,2,3,3-2H5]glycerol infusions were used to assess glucose and glycerol turnover before (–5 min) and during the clamp tests (0–240 min), respectively. Mannitol (10%) was used as an extracellular marker to calculate the recovery of interstitial fluid. At 0 min (0835 h), a primed (0–8 min; 1.65 mU/kg·min) continuous (8–240 min; 0.8 mU/kg·min) infusion of regular insulin (Actrapid, Novo-Nordisk) was started. Plasma glucose concentrations were monitored at 5-min intervals and were clamped to 3 mmol/liter by a variable glucose infusion (20%, wt/vol) for the next 240 min. To reduce changes in plasma enrichment of D-[6,6-2H2]glucose during the clamp tests, the variable glucose infusion was enriched to about 2.0 ± 0.0% with D-[6,6–2H2]glucose (24, 25).

Adipose and skeletal muscle tissue microdialysis

The microdialysis probe (CMA60, CMA/Microdialysis, Solna, Sweden) and details of the microdialysis experiments were described in detail previously (26). Briefly, the active part of the microdialysis catheter consists of a chamber constructed of a semipermeable membrane with an inlet and an outlet. The chamber is connected to a high precision perfusion pump (CMA60, CMA/Microdialysis). The perfusion fluid leaves the chamber through the outlet and is collected in time fractions (dialysate) in closed microvials. One microdialysis catheter was percutaneously inserted into the sc tissue of the abdomen, 3–6 cm lateral of the umbilicus. The second microdialysis catheter was inserted in the medial part of the gastrocnemius muscle. The penetration of the muscular fascia was in most cases recognized, and the im location was confirmed by the development of muscular twitches during insertion. The catheters were continuously perfused with artificial extracellular fluid (135 mmol/liter NaCl, 3.0 mmol/liter KCl, 1.0 mmol/liter MgSO4, 1.2 mmol/liter CaCl2, 300 µmol ascorbate, and 1.0 mmol/liter Na3PO4 buffer, adjusted to pH 7.4) at a rate of 0.3 µl/min and collected every 60 min during baseline and throughout the study for the determination of glycerol. Glycerol is not reused to any great extent in adipose tissue; thus, changes in extracellular glycerol concentrations reflect changes in lipolysis, as discussed previously in detail (27).

1H-NMRS of IMCL

The participants were studied in the supine position with a 3.0-T/80-cm NMR spectrometer (15, 24) (Medspec, Bruker, Ettlingen, Germany) equipped with a whole-body gradient coil (BG-A55; 40 mT/m; <250 µsec). First, the calf muscle of the right leg was positioned in a standard 28-cm quadrature bird cage 1H volume coil for IMCL-S and IMCL-TA measurements. The STEAM sequence parameters (echo time, 20 msec; mixing time, 30 msec; repetition time, 6 sec; number of scans, 8 and 32) (28) were complemented by CHESS water suppression and applied on the volume of interest placed in soleus and tibialis anterior muscles (1.2 cm)3. IMCL-S and -TA were quantified from line-broadened and line-fitted processed spectra as the percent relation of the intensity of the -(CH2)n- group resonance (1.25 parts/million) to the intensity of the water resonance from nonwater-suppressed spectra of the same volume of interest.

Analyses

Plasma glucose concentrations were measured using glucose oxidase (glucose analyzer II, Beckman Coulter, Inc., Fullerton, CA). Plasma concentrations of FFA [intra- and interassay coefficients of variance (CVs), 4.3% and 5.7%] were determined by enzymatic methods (Wako Chemicals, Neuss, Germany). Plasma insulin (CV, <8%), C peptide (CV, <9%), glucagon (CV, <8%), and GH (CV, <7%) were measured by RIA (insulin: Pharmacia-Upjohn, Uppsala, Sweden; C peptide: CIS, Gif-Sur-Yvette, France; glucagon: Linco Research, Inc., St. Charles, MO). Plasma cortisol (CV, <6%) was determined after extraction and charcoal-dextran separation by RIA. Plasma catecholamines were analyzed by reverse phase HPLC using plasma catecholamine extraction tubes (ESA, Chelmsford, MA) for the isolation procedure. Inter- and intraassay CVs were less than 5%, for both compounds, respectively (29, 30). Gas chromatography-mass spectrometry analysis of enrichments of [6,6-2H]glucose and [1,1,2,3,3-2H]glycerol in plasma and infusates was performed using the pentaacetate derivative of glucose (24) (31) and the triacetate derivate of glycerol (31), respectively. Gas chromatography-mass spectrometry analysis was performed on a Hewlett-Packard 5890 gas chromatograph (Palo Alto, CA) equipped with a CP-Sil5 (25 m x 0.25 mm x 0.12 µm) (24).

Calculations

Glucose turnover. Basal rates of EGP were calculated by the following equation (32): basal EGP = IR ([enrichmentinf/enrichmentplasma] – 1), where IR is the basal [6,6-2H2]glucose infusion rate (milligrams per kilograms per minute), enrichmentinf is the percent enrichment of [6,6-2H2]glucose infusate, and enrichmentplasma is the percent basal plasma [6,6-2H2]glucose enrichment.

The rates of appearance and disappearance (Rd) of glucose during the clamp tests were calculated using Steele’s non-steady-state equations as modified for the use of stable isotopes (24, 33). EGP during the clamp test was calculated from the difference between the rate of appearance of glucose in plasma and the glucose infusion rates.

Glycerol turnover. Glycerol turnover (rate of appearance[glycerol]) at baseline and during the clamp was calculated using the following formula (31): IR x [(enrichmentinf/enrichmentplasma) – 1], where IR is the basal infusion rate, enrichmentinf is the glycerol infusate enrichment (percentage), and enrichmentplasma is the steady-state basal plasma glycerol enrichment (percentage).

Absolute glycerol concentrations in the dialysates. The absolute glycerol concentrations in adipose and skeletal muscle dialysates was calculated as glycerol (abs) = glycerol (dialysate)/recovery, with recovery calculated as the ratio of the mannitol concentration in plasma to the mannitol concentration in the sampled microdialysates.

Statistical analyses

All data are presented as the mean ± SEM. Data for T1DM and CON were compared using unpaired Student’s t test. One-way repeated measure ANOVA with post hoc testing by Newman-Keuls test was used for statistical comparisons of time courses within the different groups. Statistical significance was considered at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Plasma glucose, insulin, and glucose metabolism

The variable insulin infusion applied overnight in T1DM normalized plasma glucose concentration before the clamp test [T1DM, 5.4 ± 0.3 mmol/liter (98 ± 5 mg/dl); CON, 5.6 ± 0.2 mmol/liter (101 ± 3 mg/dl); not significant (NS)]. After the start of the clamp test, the plasma glucose concentration decreased to approximately 3 mmol/liter at about 45 min in both groups and was not different between groups throughout the duration of the clamp test (Fig. 1AGo).



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  		FIG. 1. Plasma concentrations of glucose (A) and insulin (B) in T1DM ({circ}) and CON ({blacksquare}) during the hyperinsulinemic hypoglycemic clamp test. To convert the glucose concentration from millimoles per liter to milligrams per deciliter, multiply by 0.05551. To convert the insulin concentration from microunits per liter to picomoles per liter, multiply by 6. #, P < 0.05, T1DM or CON vs. basal; *, P < 0.05, T1DM vs. CON.

 
Due to the overnight insulin infusion in T1DM, plasma concentrations of insulin were higher immediately before the start of the clamp test [T1DM, 10 ± 1 µU/ml (60 ± 9 pmol/liter); CON, 5 ± 1 µU/ml (28 ± 4 pmol/liter); P < 0.01; Fig. 1BGo]. Although an identical insulin infusion protocol was applied in both groups during the clamp test, plasma insulin concentrations were higher at 60 min in T1DM [T1DM, 60 ± 6 µU/ml (358 ± 36 pmol/liter); CON, 45 ± 3 µU/ml (269 ± 17 pmol/liter); P < 0.05], but did not differ between groups thereafter [mean, 120–240 min: T1DM, 51 ± 4 µU/ml (306 ± 27 pmol/liter); CON, 47 ± 4 µU/ml (280 ± 21 pmol/liter); P = 0.427; NS].

Glucose infusion rates required to maintain plasma glucose at approximately 3 mmol/liter during the clamp tests were about 2.1-fold higher in T1DM compared with CON (T1DM, 13.7 ± 2.1; CON, 6.4 ± 2.5 µmol/kg·min; P < 0.05; Fig. 2AGo). Before the clamp test, rates of EGP were not different between the groups (T1DM, 9.4 ± 1.3; CON, 10.7 ± 0.4 µmol/kg·min; NS; Fig. 2BGo). Throughout the period of hypoglycemia, rates of EGP were about 58% lower in T1DM compared with CON (mean, 60–240 min: T1DM, 4.6 ± 0.6; CON, 10.9 ± 0.5 µmol/kg·min; P < 0.005).



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  		FIG. 2. Glucose infusion rates (A) and rates of EGP (B) in T1DM ({circ}) and CON ({blacksquare}) during the hyperinsulinemic hypoglycemic clamp test. To convert rates from micromoles per kilogram per minute to milligrams per kilogram per minute, divide by 5.55. *, P < 0.05, T1DM vs. CON; §, P < 0.005, T1DM vs. CON.

 
Plasma FFA and whole-body glycerol turnover

Plasma FFA concentrations did not differ between the groups before the clamp test (T1DM, 293 ± 53; CON, 324 ± 29 µmol/liter; NS; Fig. 3AGo). After the start of the hypoglycemic clamp test, the initial decrease in the plasma FFA concentration was more pronounced in T1DM, leading to lower plasma FFA concentrations at 60 min in T1DM compared with CON (T1DM, 40 ± 8; CON, 193 ± 28 µmol/liter; P < 0.001). Thereafter plasma FFA concentrations similarly increased in both groups, reaching a level at the end of the clamp of 188 ± 69 µmol/liter in T1DM and 252 ± 55 µmol/liter in CON (T1DM vs. CON; NS; Fig. 3AGo).



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  		FIG. 3. Plasma concentrations of FFA (A; {circ}, T1DM; {blacksquare}, CON), rates of glycerol turnover (B; {circ}, T1DM; {blacksquare}, CON), and IMCL in soleus ({blacksquare}, CON; {circ}, T1DM) and tibialis anterior muscle ({blacktriangleup}, CON; {diamond}, T1DM) in T1DM and CON during the hyperinsulinemic hypoglycemic clamp test. #, P < 0.05, T1DM or CON vs. basal; *, P < 0.05, T1DM vs. CON; §, P < 0.001, T1DM vs. CON.

 
Before the clamp test, glycerol turnover was not different between both groups (T1DM, 251 ± 29; CON, 366 ± 70 µmol/min; NS). During hypoglycemia, glycerol turnover became approximately 40% higher in T1DM compared with CON (T1DM, 374 ± 21; CON, 272 ± 19 µmol/min; P < 0.01; Fig. 3BGo).

IMCL and regional lipolysis

At baseline, IMCL-TA (T1DM, 0.75 ± 0.10%; CON, 0.68 ± 0.14%; NS) and IMCL-S (1.83 ± 0.26 vs. 1.30 ± 0.08%; NS) were not different between T1DM and CON. In both muscles, IMCL remained unchanged during the hypoglycemic clamp test (Fig. 3CGo).

Before the start of the clamp test, the glycerol concentration in the dialysate of skeletal muscle (M) and sc adipose tissue (F) was not different (F-T1DM, 323 ± 7; F-CON, 308 ± 16 µmol/liter; M-T1DM, 296 ± 19; M-CON, 323 ± 17 µmol/liter; for both comparisons T1DM vs. CON, NS). During hypoglycemia, the glycerol concentrations in dialysate from skeletal muscle (T1DM, 302 ± 22; CON, 346 ± 17 µmol/liter; NS) and adipose tissue were unchanged compared with basal levels and were comparable in both groups (T1DM, 264 ± 25; CON, 318 ± 25 µmol/liter; NS; Table 1Go). At baseline, the absolute glycerol concentrations in the dialysates of skeletal muscle and sc adipose tissue were not different (F-T1DM, 1132 ± 557; CON, F-452 ± 28; M-T1DM, 577 ± 145; M-CON, 1999 ± 1481 µmol/liter; NS). Intramuscular glycerol remained unchanged during hypoglycemia (T1DM, 579 ± 89 vs. 706 ± 83 µmol/liter; NS), and the absolute glycerol concentration in the adipose tissue dialysates was similar in the two groups (T1DM, 1607 ± 560 vs. 1659 ± 1165 µmol/liter; NS; data not shown).


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  		TABLE 1. Dialysate glycerol concentrations from microdialysis performed in sc abdominal adipose tissue and gastrocenmius muscle in T1DM patients and in nondiabetic subjects (CON) before (0 min) and during a hyperinsulinemic-hypoglycemic clamp test

 
Counterregulatory hormone response

Fasting plasma glucagon concentrations were comparable in control subjects and T1DM patients. During hypoglycemia, hormonal counterregulation was blunted in T1DM compared with CON [T1DM vs. CON: peak values of glucagon, 68 ± 4 pg/ml (68 ± 4 ng/liter) vs. 170 ± 37 pg/ml (170 ± 37 ng/liter); Fig. 4AGo; peak values of cortisol: 16.2 ± 2.2 µg/dl (446 ± 60 nmol/liter) vs. 24.4 ± 1.7 µg/dl (674 ± 48 nmol/liter); Fig. 4CGo; all P < 0.05]. In both groups, basal plasma epinephrine concentrations were approximately 70 pg/ml (0.38 nmol/liter). In CON, a rapid and marked increase was seen early, reaching a peak level of 596.6 ± 211.5 pg/ml (3.26 ± 1.15 nmol/liter). The rise in epinephrine was delayed in the diabetic group, reaching a peak value of 274.3 ± 84.2 pg/ml (1.49 ± 0.46 nmol/liter) after 120 min (Fig. 4BGo). There was a similar increase in norepinephrine due to hypoglycemia in both groups [peak values of norepinephrine (T1DM vs. CON), 251 ± 35 pg/ml (1.48 ± 0.21 nmol/liter) vs. 244 ± 30 pg/ml (1.44 ± 0.18 nmol/liter; NS); data not shown].



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  		FIG. 4. Plasma concentrations of glucagon (A), epinephrine (B), and cortisol (C) in T1DM ({circ}) and CON ({blacksquare}) during the hyperinsulinemic hypoglycemic clamp test. To convert values in picograms per milliliter to nanograms per liter, multiply by 1.0. To convert values in picograms per milliliter to nanomoles per liter, multiply by 0.005911. To convert values in micrograms per deciliter to nanomoles per liter, multiply by 27.59. #, P < 0.05, T1DM or CON vs. basal; *, P < 0.05, T1DM vs. CON.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This study was undertaken to test the hypothesis that lipolysis could play a role in the defense against hypoglycemia during prolonged hypoglycemia in patients with T1DM treated intensively with insulin. We found impaired hormonal counterregulation and lower rates of EGP during hypoglycemia. IMCL did not differ between T1DM and CON before the clamp test. During the hypoglycemic clamp test, IMCL, abdominal sc adipose tissue, and skeletal muscle lipolysis did not change from preclamp values in T1DM and CON, but systemic glycerol turnover was increased in T1DM during the late phase of hypoglycemia.

Studies in humans with the insulin-clamp technique showed an inverse relationship between the rates of glucose and fat oxidation under euglycemic conditions. A similar relationship has been found during mild hypoglycemia in healthy subjects, where small increments in insulin produced marked changes in carbohydrate oxidation that were directly mediated by the availability of circulating FFA. This led to the postulation that the glucose-FFA cycle is a physiological mechanism that induces peripheral insulin resistance in the late phase of hypoglycemic counterregulation. Subsequently, studies in humans demonstrated that changes in lipolysis account for about 50% of the adrenergic contribution to increase EGP and approximately 85% of suppressed skeletal muscle glucose disposal during recovery of hypoglycemia (19, 20). Because indirect effects of catecholamines (stimulation of lipolysis) take place in the late period (after 180 min) of hypoglycemia (19, 20) we have chosen a clamp duration of 240 min, which is of clinical relevance especially because most T1DM suffer from nocturnal hypoglycemia. Consequently, the number of blood samples had to be reduced to allow for monitoring of the complete study period. Thus, the time resolution of changes in metabolites and hormones is limited.

Because 15–20% of systemic glycerol turnover during fasting is not from lipolysis of adipose tissue triglycerides (34) and the turnover rate in skeletal muscle is about 10 times more rapid than that in adipose tissue (8), we speculated that there could be significant lipolysis in skeletal muscle during hypoglycemia. Furthermore, it has been reported that im triglyceride content is increased in skeletal muscle of poor to moderately controlled T1DM (16, 18). We hypothesized that increased IMCL content could be a protective adaptation to frequent hypoglycemia associated with insulin therapy in T1DM. However, IMCL content was not increased in T1DM in the soleus or tibialis anterior muscle, and there was no detectable glycerol release from IMCL during hypoglycemia. Our NMR data were in accordance with the data from the microdialysis technique, where we also found no differences in glycerol release in skeletal muscle during hypoglycemia. The data for absolute glycerol concentrations resulted from substantial variability in the dialysate recovery of mannitol. It is of note that there are various limitations using the microdialysis technique. Several factors may have caused the variability in the recovery of mannitol in our study. We included lean subjects with minor sc adipose tissue, and the microdialysis catheter was perhaps not completely surrounded by fat tissue. Additionally, changes in local blood flow due to vasoconstriction through epinephrine, which was more pronounced in CON than in T1DM, could be a limiting factor. Lipolysis occurs not only in adipose tissue and skeletal muscle through activation of the hormone-sensitive lipase, but also from lipoprotein lipase, so glycerol turnover becomes a composite estimate of both processes. Furthermore, the sample sizes were small because of the long study duration. We cannot exclude that these limitations, including analytical variances, explain the differences in systemic lipolysis between CON and T1DM.

During the late phase of hypoglycemia, we found an increase in systemic glycerol turnover in T1DM, but not in CON, despite reduced counterregulatory hormone release. There was no increase in glycerol concentrations in skeletal muscle and adipose tissue dialysates during hypoglycemia. The fact that there was no difference in plasma FFA concentrations could be explained by increased FFA utilization (as energy supply to increase rates of gluconeogenesis) in T1DM during hypoglycemia. Similar to FFA concentrations, plasma glycerol concentrations were not different (T1DM, 278 ± 40; CON, 267 ± 34 µmol/liter at 240 min), perhaps due to an attempt to compensate the defects in hormonal hypoglycemia counterregulation. Higher rates of glycerol turnover in T1DM could be explained by resistance to the antilipolytic effect of insulin in poorly controlled type 1 diabetics (HbA1c, >9%), which show impaired stimulation of glucose and fat oxidation (35). To keep the antilipolytic effect of insulin low, we applied an insulin infusion, leading to plasma insulin concentrations in the lower physiological range (0.8 mU/kg·min). Using the microdialysis technique, Bolinder et al. (22) reported markedly enhanced lipolysis in the posthypoglycemic period in T1DM (HbA1c, 7.8%). In this study the plasma glucose concentration was not controlled, resulting in more severe hypoglycemia in the well-controlled patients. Moreover, the study did not last long enough to evaluate the lipolytic effect during prolonged moderate hypoglycemia, which is relevant to the clinical situation of hypoglycemia in diabetic patients. Despite a reduced catecholamine secretory response, the mechanism could be due to an increase in the number of high-affinity ß-adrenoceptors in fat or skeletal muscle cells (36). The increase in lipolysis could be due to insulin deficiency rather than catecholamine stimulation, because the glycerol level increased after withdrawal of exogenous insulin infusion (22). In contrast, a recent study has shown a defective stimulation of lipolysis in skeletal and adipose tissues in well-controlled T1DM (HbA1c, 6.9%) during hypoglycemia. Enoksson et al. (6) also performed the microdialysis technique to evaluate glycerol release in adipose and skeletal muscle tissues without using stable isotope infusion to determine systemic glycerol turnover. Because gender is a well-known factor influencing the hormonal response to hypoglycemia (37), differences in the lipolytic response between the present and the previous study could result from including only male participants in our study. In addition, the peak plasma epinephrine response during hypoglycemia was 1.7-fold higher in T1DM in the present study compared with that in the study by Enoksson et al. (6). In the present study the plasma epinephrine response of T1DM was delayed and less pronounced compared with that of CON, but was still present. This can possibly be attributed to the fact that none of the T1DM in the present study had suffered from symptomatic hypoglycemia at least 4 wk before the study.

In conclusion, in well-controlled T1DM, impaired hypoglycemia counterregulation is associated with decreased endogenous glucose production and augmented whole body lipolysis, which can not be explained by either hydrolysis of skeletal muscle triglycerides (IMCL) or increased abdominal sc adipose tissue lipolysis.


   Acknowledgments
 
We gratefully acknowledge the assistance of A. Hofer, H. Lentner, and the laboratory staff of the Division of Endocrinology and Metabolism.


   Footnotes
 
This work was supported by grants from the Austrian Science Foundation (Fonds zur Förderung der Wissenschaftlichen Forschung: P14298-GEN).

First Published Online July 5, 2005

Abbreviations: CON, Control; CV, coefficient of variance; EGP, endogenous glucose production; FFA, free fatty acid; HbA1c, hemoglobin A1c; 1H-NMRS, 1H nuclear magnetic resonance spectroscopy; IMCL, intramyocellular lipid; IMCL-S, IMCL content in soleus muscle; IMCL-TA, IMCL lipid content in tibialis anterior muscle; NS, not significant; T1DM, type 1 diabetes mellitus patient; TG, triglycerides.

Received September 2, 2004.

Accepted June 28, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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