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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 9 4693-4700
Copyright © 2004 by The Endocrine Society

Combined Hyperinsulinemia and Hyperglycemia, But Not Hyperinsulinemia Alone, Suppress Human Skeletal Muscle Lipolytic Activity in Vivo

Veronica Qvisth, Eva Hagström-Toft, Staffan Enoksson, Robert S. Sherwin, Stefan Sjöberg and Jan Bolinder

Departments of Medicine (V.Q., E.H.-T., S.S., J.B.) and Vascular Surgery (S.E.), Karolinska University Hospital-Huddinge, Karolinska Institute, S-141 86 Stockholm, Sweden; and Department of Internal Medicine (R.S.S.), Section of Endocrinology, Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Dr. Jan Bolinder, Department of Medicine, M 63, Karolinska University Hospital-Huddinge, S-141 86 Stockholm, Sweden. E-mail: jan.bolinder{at}hs.se.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Effects of circulating insulin and glucose concentrations on skeletal muscle and adipose tissue lipolytic activity were investigated in 10 type 1 diabetes patients with no endogenous insulin secretion. Microdialysis measurements of interstitial glycerol and determination of fractional glycerol release were carried out during standardized combinations of relative hypoinsulinemia/moderate hyperglycemia (11 mmol/liter), hyperinsulinemia/ normoglycemia (5 mmol/liter), and hyperinsulinemia/moderate hyperglycemia, respectively. Local tissue blood flow rates were measured with the 133Xe clearance technique. In response to the change from hypo- to hyperinsulinemia, the fractional release of glycerol decreased from 159.6 ± 17.8 to 85.1 ± 13.7 µmol/liter (P < 0.0001) in adipose tissue, whereas it remained unchanged in skeletal muscle (44.6 ± 6.4 vs. 36.0 ± 7.4 µmol/liter; not significant). When hyperinsulinemia was combined with hyperglycemia, fractional glycerol release was further reduced in adipose tissue (64.5 ± 12.2 µmol/liter; P < 0.05), and in this situation it was also markedly decreased in skeletal muscle (18.1 ± 4.8 µmol/liter; P < 0.0001). Skeletal muscle blood flow was unaltered over the respective study periods. Adipose tissue blood flow decreased by 50% in response to hyperinsulinemia (P < 0.0005), but no further change was seen when hyperinsulinemia was combined with hyperglycemia. It is concluded that in patients with type 1 diabetes, insulin does not exert an antilipolytic effect in skeletal muscle during normoglycemia. However, in response to combined hyperinsulinemia and hyperglycemia, the lipolytic activity in skeletal muscle is restrained in a similar way as in adipose tissue. This may be explained by a glucose-mediated potentiation of the antilipolytic effectiveness of insulin.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
SKELETAL MUSCLE INSULIN resistance, as reflected by attenuated insulin-mediated glucose utilization, is a characteristic feature of type 2 diabetes mellitus, obesity, dyslipidemia, and hypertension (i.e. the metabolic syndrome). Of several possible pathogenetic mechanisms, free fatty acids (FFA) are considered to play a significant role in the development of insulin resistance. Thus, there is a reciprocal relationship between lipid and carbohydrate utilization in muscle, and increased levels of FFA in the circulation impair insulin-stimulated skeletal muscle glucose disposal (1, 2, 3, 4, 5, 6, 7).

In humans, the source of FFA is generally assumed to be hydrolysis (lipolysis) of triglycerides in adipose tissue deposits. However, FFA generated from hydrolysis of im triglycerides may also be of importance, because several investigators have demonstrated a significant lipolytic activity in human skeletal muscle (8, 9, 10, 11). Recent data also suggest that skeletal muscle lipolysis rates differ between muscle groups with varying fiber composition (12). Moreover, an inverse relationship between im triglyceride content and insulin sensitivity has been observed in many studies, as recently reviewed (13). This might imply a link between dysregulation of the local lipolytic activity and development of insulin resistance in skeletal muscle.

The hormonal regulation of human skeletal muscle lipolysis is still not completely elucidated. For instance, findings in initial studies that measured interstitial glycerol levels in vivo suggested a similar antilipolytic effect of insulin in skeletal muscle and adipose tissue (9, 10, 14). However, in more recent investigations in which the fractional release of glycerol was assessed in situ, we (15, 16) and others (11) have not been able to confirm that insulin exerts an inhibitory effect on skeletal muscle lipolytic activity. To further evaluate this seemingly controversial issue, we evaluated the effect of insulin as well as the possible influence of the prevailing glucose level on skeletal muscle and adipose tissue lipolysis, bearing in mind that circulating glucose concentrations may affect overall lipolytic activity in vivo (17). For this purpose, we investigated 10 male patients with insulin-dependent (type 1) diabetes without endogenous insulin secretion. By infusing insulin and glucose, the patients were clamped at standardized combinations of relative hypoinsulinemia/hyperglycemia, hyperinsulinemia/normoglycemia, and hyperinsulinemia/hyperglycemia, respectively. Microdialysis measurements of glycerol concentrations in situ and calculation of the fractional release of glycerol (i.e. difference between interstitial and arterialized venous plasma glycerol) were carried out simultaneously in the two tissues together with determinations of local blood flow rates using the 133Xe clearance technique.


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

Ten men (age, 43 ± 3 yr; body mass index, 23.8 ± 0.4 kg/m2) with insulin-dependent type 1 diabetes mellitus were investigated. All subjects were plasma C peptide negative and were receiving continuous insulin treatment (total insulin dose, 0.8 ± 0.1 U/kg·24 h). Apart from the insulin therapy, they took no other medication. The duration of diabetes was 19 ± 2 yr, and the metabolic control was fairly good, as evidenced by their hemoglobin A1c levels (6.9 ± 0.4%; normal range, 3.4–5.0%). Eight of the patients had background diabetic retinopathy; otherwise, they had no other signs of diabetic micro- or macrovascular complications.

The ethics committee of the Karolinska Institute approved the study. The subjects were given a detailed description of the study before they gave their informed consent.

Microdialysis device

The principles of the microdialysis technique and the microdialysis device have been described in detail previously (18). Briefly, the microdialysis catheter (CMA/60, CMA Microdialysis AB, Stockholm, Sweden) consists of a semipermeable polyamide membrane (30 x 0.62 mm; molecular cut-off, 20,000 Da), which is glued to the end of double-lumen polyurethane tubing. The probe is connected to a microinfusion pump (CMA/100 microinjection pump, CMA Microdialyis AB) and is continuously perfused with a sterile perfusion fluid. The perfusate solution enters the catheter through the outer lumen, an exchange of metabolites takes place over the microdialysis membrane, and the solution leaves through the inner lumen from which it is collected. The composition of the sampled dialysate then mirrors the interstitial fluid.

Study protocol

The subjects were admitted to the hospital on the evening before the investigation. They were fasting from midnight and received a variable iv insulin infusion to maintain the blood glucose level at approximately 11 mmol/liter during the night. Venous blood glucose was monitored at the bedside every hour (HemoCue, Angelholm, Sweden). The subjects remained in a supine position throughout the study period.

The experiment started at 0730 h. A retrograde catheter was inserted in a dorsal vein in the hand that was placed in a heated box (63 C) for sampling of arterialized venous plasma. Arterialization was confirmed by blood gas analysis (>95% O2 saturation). Retrograde catheters were also placed in the cubital veins bilaterally for blood sampling and infusion of insulin and glucose, respectively. After superficial skin anesthesia (EMLA, Astra, Sodertalje, Sweden), one microdialysis catheter was inserted percutaneously into the medial portion of the gastrocnemius muscle in one leg. A second microdialysis catheter was inserted into the periumbilical sc adipose tissue about 8 cm lateral to the umbilicus. The catheters were continuously perfused with Ringer’s solution (Apoteksbolaget, Umea, Sweden) containing 147 mmol/liter sodium, 4 mmol/liter potassium, 2.3 mmol/liter calcium, and 156 mmol/liter chloride at a flow rate of 0.3 µl/min. Previous studies have shown that the recovery of glycerol is almost complete (≥95%) in skeletal muscle as well as in adipose tissue at this flow rate (9).

After a 120-min equilibration period, a 60-min basal sampling period was started during which the variable insulin infusion was continued to keep the arterialized venous blood glucose at about 11 mmol/liter. Thereafter, the insulin infusion was increased to 0.075 U/kg·h for 90 min to make blood glucose fall in a linear fashion from 11 to 5.5 mmol/liter. This rate of iv insulin administration was thereafter maintained throughout the rest of the experimental period. A variable iv glucose infusion was then added for the next 60 min to maintain the blood glucose at 5.5 mmol/liter. Over the following 90 min, the glucose infusion rate was increased to raise the blood glucose level gradually to 11 mmol/liter. In some patients, the insulin infusion rate had to be slightly decreased to raise the blood glucose concentration to 11 mmol/liter within the 90-min period. The last 60 min of the experiment, blood glucose was maintained at 11 mmol/liter with a variable glucose infusion. Thus, with this experimental protocol three separate 60-min steady state periods were achieved (Fig. 1Go). The first period (A) reflected a state of relative insulin deficiency and moderate hyperglycemia. The second period (B) reflected a combination of hyperinsulinemia and normoglycemia, whereas the last period (C) showed sustained hyperinsulinemia coupled with moderate hyperglycemia.



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  		FIG. 1. Study protocol.

 
Throughout the experiment, dialysate samples were collected in 10-min fractions for analysis of glycerol. Arterialized venous plasma and whole blood were drawn every 10 min for analysis of glycerol and glucose during the three steady state periods. Plasma free insulin was analyzed every 20 min during the three steady state periods. Blood glucose was monitored bedside (HemoCue) for adjustment of glucose and/or insulin infusion rates every 10 min throughout the investigation.

Adipose tissue blood flow and skeletal muscle blood flow rates were determined with the 133Xe clearance technique (19) as previously described in detail (15). In adipose tissue, 133Xe (1 MBq in 0.1 ml saline; Mallinckrodt, Petten, The Netherlands) was injected percutaneously into the sc tissue in the contralateral abdominal side of the umbilicus 90 min before the first sampling period. After 30 min of equilibration, the residual activity was continuously monitored externally with a scintillation detector (Mediscint, Oakfield Instruments Ltd., Oxford, UK). The fractional decay per minute was assessed for consecutive 30-min periods throughout the study period. In skeletal muscle, 133Xe (0.3 MBq in 0.1 ml saline) was injected in the medial part of the contralateral gastrocnemius muscle after 25 min during each steady state period. After 5-min equilibration, the residual activity was monitored for 10 min. In the skeletal muscle, the 133Xe decay curve gradually becomes multiexponential (20). Therefore, the 133Xe clearance technique cannot be used for estimating muscle blood flow continuously over extended time periods, but it can be correctly assessed from the initial part of the 133Xe washout curve (20, 21). Adipose tissue and muscle blood flow (TBF) rates were calculated according to the following formula: TBF = k x {lambda} x 100 ml/100 g·min, where k denotes the rate constant of the decay of the residual activity, and {lambda} is the tissue to blood partition coefficient. The values for {lambda} were set at 10 ml/g for adipose tissue and 0.7 ml/g for muscle (19, 21).

Glycerol kinetics

In methodological experiments, skeletal muscle and adipose tissue glycerol uptake and release were simultaneously investigated in four normal weight, healthy subjects (three women and one man; age, 25–28 yr). The investigations were carried out at the General Clinical Research Center, Yale University School of Medicine, and were approved by the human investigations committee at Yale. At 0800 h, after an overnight fast, a 6-h primed continuous infusion of [1,1,2,3,3-2H5]glycerol (0.02 mg/m2 body surface area·min) was administered via an antecubital vein. A retrograde cannula was inserted into a dorsal vein in the contralateral hand that was warmed for sampling of arterialized venous blood. A small volume of 0.9% NaCl was allowed to flow through the sampling cannula to maintain potency. Microdialysis catheters were inserted percutaneously into the medial head of the gastrocnemius muscle and into the periumbilical sc adipose tissue, respectively. The catheters were continuously perfused (0.3 µl/min) with an artificial extracellular fluid, as described above. After a minimum of 120 min of equilibration, dialysate samples were collected in 30-min fractions during the 4-h study period for analysis of glycerol and glycerol isotope enrichment. Arterialized venous blood samples were obtained at 15-min intervals for measurement of plasma glycerol and glycerol isotope enrichment. Conversion of interstitial (I) to venous (V) glycerol concentrations was made according to the equation: [glycerol]V = ([glycerol)]I – [glycerol]a) x (1 – e–PS/Q) + [glycerol]a where [glycerol]a is the glycerol concentration in arterialized venous plasma, Q represents the plasma flow rate, and PS denotes the permeability surface product area. The latter value was approximated to 5 ml/100 g·min in both skeletal muscle and adipose tissue, as previously suggested (22, 23, 24) and discussed in detail (16). The fractional extraction of the glycerol isotope, and rates of glycerol uptake and release were calculated according to the formula (25, 26): Fractional extraction = ([glycerol]a x Ea) – ([glycerol]V x Ed)/([glycerol]a x Ea) (%) Glycerol uptake=([glycerol]axQ)x[1–[glycerol]VxEd)/([glycerol]axEa)]] (nmol/100 g·min)

Glycerol release = Q x ([glycerolV – [glycerol]a) + glycerol uptake (nmol/100 g·min), where Ea and Ed represent the enrichment of glycerol in arterialized venous plasma and tissue dialysate, respectively.

Analyses

Dialysate glycerol was measured with an enzymatic fluorometric method using an automatic tissue dialysate sample analyzer (CMA/600, CMA Microdialysis). Plasma glycerol was determined with bioluminescence (27). Free insulin in serum was measured with a commercial RIA kit (Pharmacia Biotech, Uppsala, Sweden). The hospital’s routine clinical chemistry department determined plasma glucose and hematocrit.

Gas chromatography-mass spectrometry analysis of enrichments of [1,1,2,3,3-2H5]glycerol in plasma, infusate, and dialysates was performed using the triacetate derivative of glycerol (28), as described in detail previously (29). Gas chromatography-mass spectrometry analysis was performed with a Hewlett-Packard 5971A Mass Selective Detector (Wilmington, DE), operating in the electron ionization mode. Ions with m/z 145, 146, and 148 were monitored for molar percent enrichment in glycerol.

Statistics

Data are presented as the mean ± SEM. Comparisons of plasma, muscle, and adipose tissue data were performed by factorial ANOVA. Variations over time in the same individuals were calculated by one-factor ANOVA, corrected for repeated measurements. Post hoc analyses were performed using Scheffé’s F test. Linear regression analysis was performed using the method of least squares. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Arterialized venous blood glucose and plasma free insulin levels are shown in Fig. 2Go. Plasma free insulin was 87 ± 12 pmol/liter during the first sampling period (A). During the following two periods (B and C), insulin levels were 515 ± 25 and 428 ± 18 pmol/liter, respectively (P < 0.05). The blood glucose concentration was initially 11.3 ± 0.2 mmol/liter (A), decreased to 5.9 ± 0.1 mmol/liter during the second sampling period (B), and finally returned to 11.3 ± 0.2 mmol/liter during the last steady state sampling period (C).



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  		FIG. 2. Arterialized venous plasma concentrations of glucose and insulin during standardized iv administration of glucose and/or insulin in 10 patients with type 1 diabetes without endogenous insulin secretion. Values are the mean ± SEM.

 
In the fasting hypoinsulinemic state, glycerol levels were about 3.5 times higher in adipose tissue and 1.6 times higher in muscle compared with the arterialized venous plasma glycerol concentrations (P < 0.05, by factorial ANOVA; Fig. 3AGo). Glycerol concentrations in plasma, adipose tissue, and muscle were all significantly reduced during hyperinsulinemia and normoglycemia (P < 0.0001, by ANOVA). In skeletal muscle, glycerol concentrations continued to decrease when circulating glucose was raised from normoglycemic to hyperglycemic levels (P < 0.01, by ANOVA). Conversely, in adipose tissue and plasma, glycerol concentrations were maximally decreased during the normoglycemic, hyperinsulinemic period, with no further reduction during the hyperglycemic, hyperinsulinemic period.



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  		FIG. 3. Effect of circulating insulin and glucose levels on glycerol concentrations in arterialized venous plasma, adipose tissue, and skeletal muscle (A) and on the difference between the interstitial tissue glycerol concentration and the arterialized venous plasma glycerol level (I-A difference; B). Statistical differences between study periods were calculated with ANOVA, repeated measurements, and post hoc analysis by Scheffé's F test. *, P < 0.05 between study periods A and B; {blacksquare}, P < 0.05 between study periods B and C. See Fig. 2Go for additional details.

 
The fractional release of glycerol, the difference between the interstitial glycerol concentration in adipose and muscle tissue and that in the arterialized venous plasma, is shown in Fig. 3BGo. In skeletal muscle, the interstitial-plasma difference in glycerol remained unchanged between steady state periods A and B. It was then significantly reduced by 53% during the last steady state period when hyperinsulinemia was combined with hyperglycemia (P < 0.0001). In adipose tissue, the decrease in the interstitial-plasma difference in glycerol values was most apparent in the hyperinsulinemic, normoglycemic state (47%; P < 0.0001, by ANOVA), but a further reduction (13%; P < 0.05, by ANOVA) was also registered in the hyperinsulinemic, hyperglycemic state.

Using individual data, no significant relations were found between the fractional release of glycerol in skeletal muscle and the arterialized venous plasma glycerol concentration in steady state periods A–C (Fig. 4Go).



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  		FIG. 4. Relationship between the I-A glycerol difference in gastrocnemius muscle and the arterialized venous plasma glycerol concentration during steady-state periods of hyperglycemia/hyperinsulinemia (A), normoglycemia/hyperinsulinemia (B), and hyperglycemia/hyperinsulinemia (C), respectively. See Fig. 2Go for additional details.

 
For comparative purposes, the same type of data presentation was also calculated from a methodological study that was recently presented by our group (12). Briefly, in the latter study circulating plasma glycerol was raised about 9-fold above basal levels in six healthy subjects by a continuous 3-h infusion of a triacylglycerol emulsion. In the basal postabsorptive state, no significant correlation was observed between the fractional glycerol release in the gastrocnemius muscle and the arterialized venous plasma glycerol concentration (Fig. 5AGo). During the triacylglycerol infusion, negative interstitial and arterialized (I-A) glycerol difference values were registered in five of the six subjects, indicating definite uptake of glycerol in the muscle. In these five subjects a strong negative relationship (r = –0.84, <0.05) was evident between the I-A glycerol difference and the plasma glycerol concentration (Fig. 5BGo).



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  		FIG. 5. Relationship between the I-A glycerol difference in gastrocnemius muscle and the arterialized venous plasma glycerol concentration in six healthy subjects in the fasting state (A) and in response to triacylglycerol infusion (B). Muscle dialysate glycerol was measured in 15-min samples over 60 min in the fasting state and during a 3-h infusion of triacylglycerol emulsion (200 mg/ml) given at a rate of 1.85 ml/kg·h. Arterialized venous plasma glycerol was determined every 15 min. Data in B relate to the last 60 min of the triacylglycerol infusion when steady-state conditions were achieved. Individual data from one subject were excluded from the statistical calculation in B. In this subject, a positive I-A glycerol difference value was registered, showing net glycerol release (and not uptake) in response to triacylglycerol infusion.

 
Figure 6Go depicts adipose tissue and muscle blood flow rates. The blood flow rate was 4.4 times higher in adipose tissue compared with muscle tissue during the baseline recordings. In adipose tissue, blood flow decreased by approximately 30% during hyperinsulinemia and normoglycemia (P = 0.0005, by ANOVA). No further change was seen when hyperinsulinemia was coupled with hyperglycemia. The blood flow rate remained unchanged during the hyperinsulinemic, normoglycemic and hyperinsulinemic, hyperglycemic periods in the skeletal muscle.



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  		FIG. 6. Effect of circulating insulin and glucose levels on blood flow rates in adipose tissue and skeletal muscle. See Figs. 2Go and 3Go for additional details.

 
The results of the methodological experiments, in which regional glycerol kinetics were investigated in the basal state in healthy subjects, are summarized in Table 1Go. After the 120-min equilibration period, the enrichment of glycerol in arterialized venous plasma (8.0 ± 1.2%) and in skeletal muscle and adipose tissue dialysates (3.4 ± 0.2% and 2.8 ± 0.4%, respectively) remained stable during the 4-h sampling period (data not shown). In skeletal muscle, there was significant fractional extraction of the glycerol tracer (~30%), whereas in adipose tissue, the corresponding value did not differ significantly from zero. In skeletal muscle, there was an appreciable calculated uptake of glycerol, which amounted to about 40% of the calculated release of glycerol. In adipose tissue, the release of glycerol was approximately 3 times higher than that in skeletal muscle, whereas no uptake of glycerol was registered.


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  		TABLE 1. Glycerol kinetics in skeletal muscle and adipose tissue

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In this study we also investigated whether insulin possess an antilipolytic effect in human skeletal muscle by focusing on a possible role of the level of glycemia. This was carried out in patients with type 1 diabetes with no residual endogenous insulin secretion, thereby enabling circulating insulin and glucose levels to be experimentally controlled in a standardized way by iv administration of insulin and glucose. In the first part of the experiment, the patients were clamped at moderate hyperglycemia (11 mmol/liter) by a variable, low rate infusion of insulin to simulate a state of relative insulin deficiency. Thereafter and for the remaining period of the experiment, circulating insulin was increased well above the concentration of the hormone that possess a maximally effective antilipolytic effect (30, 31). Plasma glucose was set at normoglycemia (5 mmol/liter) and moderate hyperglycemia (11 mmol/liter), respectively.

In response to the change from hypo- to hyperinsulinemia, the interstitial glycerol concentration decreased significantly in both adipose tissue and skeletal muscle. Similar findings have previously been reported and were interpreted as being indicative of an antilipolytic effect of insulin in muscle (9, 10, 14). However, the interstitial concentration of glycerol depends not only on lipolytic activity in the tissue, but also on the delivery of glycerol from other sources via the arterial circulation. Consequently, the fractional release of glycerol (i.e. the I-A difference between plasma glycerol concentrations) should represent a more accurate measure of the local lipolytic activity. Thus, as expected, we found that hyperinsulinemia mediated a marked reduction (~50%) in the I-A glycerol difference in adipose tissue. In skeletal muscle, in contrast, it remained unchanged in response to insulin. Consistent with recent observations (11, 15, 16), this finding definitely argues against an inhibitory effect of insulin on muscle lipolysis in humans at physiological, normoglycemic conditions. Instead, the decrease in skeletal muscle interstitial glycerol after insulin stimulation is best explained by reduced arterial inflow of glycerol to the tissue bed as a result of insulin-induced antilipolysis in adipose tissue.

When hyperinsulinemia was combined with moderate hyperglycemia, in contrast, a significant decrease in the fractional release of glycerol was registered in both adipose tissue and skeletal muscle compared with the corresponding data obtained during hyperinsulinemia/normoglycemia. For practical reasons it was not possible to infuse insulin and glucose in such a way that study periods B and C could be evaluated in a randomized way. Therefore, we cannot exclude the possibility that the observed decrease in fractional glycerol release in the two tissue compartments between the respective study periods was due to the extended time of hyperinsulinemia, rather than being the result of the change from normo- to hyperglycemia. This appears less likely, however, because the glycerol concentrations in arterialized venous plasma, adipose tissue, and skeletal muscle remained stable during each of the experimental periods. Hence, our findings may indicate a different regulation of lipolytic activity in adipose tissue as well as in skeletal muscle during combined hyperinsulinemia and hyperglycemia. The molecular mechanism underlying this phenomenon is not known. However, many years ago we observed that the antilipolytic effect of insulin in isolated adipocytes was potentiated by glucose regardless of whether the cells had been exposed to high insulin and glucose concentrations in vitro (32) or in vivo (33); this was associated with increased insulin receptor binding affinity as well as stimulation of postbinding events. It has also been shown that the effectiveness of the inhibitory action of insulin on adipose tissue lipolysis is dependent on the prevailing lipolytic activity, so the antilipolytic effect of the hormone is more pronounced when the rate of lipolysis is augmented (34, 35), probably due to increased insulin receptor autophosphorylation and signal transduction through an insulin receptor substrate-1- and phosphatidylinositol 3-kinase-dependent pathway (35). Furthermore, at least in adipocytes, glucose enhances the lipolysis rate (36, 37), which may be attributed to increased expression of the rate-limiting enzyme for lipolysis, hormone-sensitive lipase (38, 39). This enzyme is also present in skeletal muscle (40). Thus, it is possible that our observed decrease in lipolytic activity in adipose tissue as well as in skeletal muscle in response to combined hyperinsulinemia and hyperglycemia was the result of a more pronounced antilipolytic effectiveness of insulin because of an augmented, glucose-mediated, elevation of lipolytic activity in both tissues. In theory, the decrease in fractional glycerol release in the two tissues could also have been due to increased local glycerol reutilization during combined insulin and glucose stimulation. To date there has been no method available that has allowed unambiguous differentiation between in vivo glycerol release and uptake in a single skeletal muscle in humans. Although various tracer techniques have been used for this purpose, these methods, even when combined with elaborate catherization techniques, can only determine the net substrate release or uptake in the various muscles (and other tissues) drained by the venous effluent. Bearing in mind that lipolytic activity may vary even between different skeletal muscle groups in humans (12), no definite conclusions about glycerol kinetics in a separate muscle tissue compartment can be made. In the present study, however, we introduced a novel approach to simultaneously investigate local tissue glycerol uptake and release using a combination of systemic administration of a stable glycerol tracer and measurement of enrichment of glycerol in tissue interstitial fluid sampled by microdialysis. By this method we registered significant fractional extraction of the glycerol tracer and definite calculated glycerol uptake in skeletal muscle. Hence, these findings are the first to show with certainty that uptake of glycerol takes place in a defined skeletal muscle group in humans, as previously shown in rodents (41). In adipose tissue, in contrast, we found no appreciable uptake of glycerol. This may be expected, because the enzyme responsible for glycerol utilization, glycerokinase, is present in skeletal muscle, but not in adipose tissue in humans (42). Moreover, in skeletal muscle, the calculated uptake of glycerol was approximately 40% the corresponding calculated release of glycerol. This is similar to that reported by Jensen (26), who estimated regional glycerol uptake and release across the thigh. Although caution should be exercised in extrapolating data, our findings relating to the calculated regional rates of glycerol uptake and release argue against the idea that the decrease in the interstitial-plasma difference in glycerol observed in both skeletal muscle and adipose tissue during combined hyperinsulinemia and hyperglycemia was due to an increase in glycerol uptake rather than suppression of the lipolytic activity. Hence, assuming a comparable relation between skeletal muscle glycerol uptake and release in the patients with type 1 diabetes and healthy subjects, the uptake of glycerol should have been increased at least 2- to 3-fold to account for the more than 50% decrease in fractional glycerol release found in skeletal muscle in the former study group. This appears unlikely because, if anything, the uptake of glycerol in muscle tissues is supposed to be reduced in response to meal ingestion and hyperinsulinemia (26). Furthermore, as mentioned above, no uptake of glycerol takes place in adipose tissue. Moreover, using data from a previously published methodological investigation (12) in which circulating plasma glycerol levels were elevated well above the basal interstitial glycerol concentration in skeletal muscle by a continuous triacylglycerol infusion, we found a strong negative correlation between the I-A glycerol difference in the gastrocnemius muscle and the arterialized venous plasma glycerol concentration (i.e. when uptake of glycerol constituted the main direction of glycerol movement in muscle tissue). By contrast, no apparent relationship between these variables was observed during any of the three experimental study periods in the present investigation. Thus, taken together, our findings are best explained by a suppression of lipolytic activity in both skeletal muscle and adipose tissue in response to combined hyperinsulinemia and hyperglycemia.

Differences in lipoprotein lipase (LPL)-mediated intravascular triglyceride hydrolysis in the two tissues may also be of importance. It is not possible, using microdialysis, to differentiate between intracellular (mediated by hormone-sensitive lipase) and extracellular, LPL-induced lipolysis. In response to insulin, however, the activity of LPL is stimulated in adipose tissue, whereas the opposite occurs in skeletal muscle (43). Therefore, because the reduction in fractional release of glycerol was evident in both tissues during combined hyperinsulinemia and hyperglycemia, it seems unlikely that differences in insulin-mediated LPL activity influenced our findings in a major way.

Local tissue blood flow is another determinant of the interstitial glycerol level (44), via delivery and removal of the metabolite by the microcirculation. In this study, we used the 133Xe washout technique for simultaneous determinations of absolute blood flow rates in adipose tissue and skeletal muscle. This method has been widely applied for recordings of nutritive blood flow in both tissues (19, 21). Accordingly, in a previous methodological study (16), we found the reproducibility and precision of this technique to be satisfactory. In the present study, no effect of insulin on the blood flow rate was registered in skeletal muscle, whereas in adipose tissue it decreased significantly, by about 30%, in response to insulin. These findings were unexpected, because previous data in healthy, nonobese subjects have shown an increase in muscle blood flow, but no change in adipose tissue blood flow after insulin stimulation (16, 45). However, defective insulin-mediated blood flow regulation has been demonstrated in various insulin-resistant conditions (45, 46, 47). Hence, our present data may be explained by disturbed vascular effects of insulin related to the diabetic state. Differences in study design are probably also of importance. For example, during the basal part of the present experiment (hyperglycemia/relative hypoinsulinemia), the recorded blood flow rate in adipose tissue (~6 ml/100 g·min) was approximately 3 times higher than that previously observed by our group in the postabsorptive state in healthy subjects (15, 16). Therefore, comparison of present and previous blood flow data should be performed with caution. Nevertheless, the fact that muscle blood flow rates remained unchanged in response to insulin imply that the observed changes in the fractional release of glycerol in skeletal muscle should not have been influenced by microcirculatory events.

It should be noted that the lipolytic activity might vary between different muscle groups, probably depending on the fiber composition (12). In the present study we preferred to examine gastrocnemius muscle, which possesses a high lipolysis rate (12). In consequence, we cannot exclude the possibility that the lipolytic activity in other muscle groups may be regulated differently in response to insulin and glucose stimulation. Moreover, from the data in this study we cannot define with certainty the level of glycemia that potentiates the antilipolytic effect of insulin in skeletal muscle or whether this phenomenon is confined to diabetic patients. However, in a previous study, no suppressive effect of insulin on skeletal muscle lipolysis rates was registered in either nonobese or obese subjects with normal glucose tolerance in response to elevated circulating levels of insulin and glucose (maximum arterialized venous plasma glucose concentration, <10 mmol/liter) after an oral glucose load (15). Thus, considering previous and present findings together, it would appear that elevation of circulating glucose levels at least above the normal postprandial range is needed to influence the antilipolytic effect of insulin in skeletal muscle.

In summary, the results of this experimental study in patients with type 1 diabetes mellitus add further evidence for the supposition that insulin does not exert an antilipolytic effect in skeletal muscle in vivo in humans during normal glycemic conditions. This is in contrast to the insulin effect on the regulation of adipose tissue lipolysis. In response to combined hyperinsulinemia and moderate hyperglycemia, in contrast, the lipolytic activity in skeletal muscle is reduced in a similar way as in adipose tissue, which may be explained by a glucose-mediated potentiation of the antilipolytic effectiveness of the hormone. This mechanism may be important for increased triglyceride deposition in skeletal muscle (and adipose tissue), which is well documented in various insulin-resistant conditions with subtle abnormalities in glucose control.


   Acknowledgments
 
We acknowledge the excellent technical assistance of B.-M. Leijonhuvfud, K. Hertel, E. Sjölin, and K. Wåhlén. We also thank Gary W. Cline and Frances Rife (Yale General Clinical Research Center) for assistance with the stable isotope preparation and analyses.


   Footnotes
 
This work was supported by grants from the Swedish Research Council, the Swedish Diabetes Association, Karolinska Institute, NIH (RR-00125), and the Yale Diabetes Endocrinology Research Center (DK-45735).

Abbreviations: FFA, Free fatty acid; I-A, interstitial and arterialized; LPL, lipoprotein lipase.

Received April 15, 2003.

Accepted June 4, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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