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Combined Hyperinsulinemia and Hyperglycemia, But
Not Hyperinsulinemia Alone, Suppress Human Skeletal
Muscle Lipolytic Activity in Vivo
VERONICA QVISTH, EVA HAGSTRO
¨M-TOFT, STAFFAN ENOKSSON, ROBERT S. SHERWIN,
STEFAN SJO
¨BERG, 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
Effects of circulating insulin and glucose concentrations on
skeletal muscle and adipose tissue lipolytic activity were in-
vestigated 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 hy-
poinsulinemia/moderate hyperglycemia (11 mmol/liter), hy-
perinsulinemia/ normoglycemia (5 mmol/liter), and hyperin-
sulinemia/moderate hyperglycemia, respectively. Local tissue
blood flow rates were measured with the
133
Xe clearance tech-
nique. In response to the change from hypo- to hyperinsulin-
emia, the fractional release of glycerol decreased from 159.6 ⴞ
17.8 to 85.1 ⴞ13.7
mol/liter (P<0.0001) in adipose tissue,
whereas it remained unchanged in skeletal muscle (44.6 ⴞ6.4
vs. 36.0 ⴞ7.4
mol/liter; not significant). When hyperinsulin-
emia was combined with hyperglycemia, fractional glycerol
release was further reduced in adipose tissue (64.5 ⴞ12.2
mol/liter; P<0.05), and in this situation it was also markedly
decreased in skeletal muscle (18.1 ⴞ4.8
mol/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 skel-
etal 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.
(J Clin Endocrinol Metab 89: 4693– 4700, 2004)
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 syn-
drome). 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 re-
ciprocal relationship between lipid and carbohydrate utili-
zation in muscle, and increased levels of FFA in the circu-
lation impair insulin-stimulated skeletal muscle glucose
disposal (1–7).
In humans, the source of FFA is generally assumed to be
hydrolysis (lipolysis) of triglycerides in adipose tissue de-
posits. However, FFA generated from hydrolysis of im trig-
lycerides may also be of importance, because several inves-
tigators have demonstrated a significant lipolytic activity in
human skeletal muscle (8–11). 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 sen-
sitivity has been observed in many studies, as recently re-
viewed (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 sug-
gested a similar antilipolytic effect of insulin in skeletal muscle
and adipose tissue (9, 10, 14). However, in more recent inves-
tigations 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 lipo-
lytic 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 se-
cretion. By infusing insulin and glucose, the patients were
clamped at standardized combinations of relative hypoinsu-
linemia/hyperglycemia, hyperinsulinemia/normoglycemia,
and hyperinsulinemia/hyperglycemia, respectively. Microdi-
alysis measurements of glycerol concentrations in situ and cal-
culation of the fractional release of glycerol (i.e. difference be-
tween interstitial and arterialized venous plasma glycerol) were
carried out simultaneously in the two tissues together with
determinations of local blood flow rates using the
133
Xe clear-
ance technique.
Abbreviations: FFA, Free fatty acid; I-A, interstitial and arterialized;
LPL, lipoprotein lipase.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the en-
docrine community.
0021-972X/04/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 89(9):4693– 4700
Printed in U.S.A. Copyright © 2004 by The Endocrine Society
doi: 10.1210/jc.2003-030656
4693
Subjects and Methods
Subjects
Ten men (age, 43 ⫾3 yr; body mass index, 23.8 ⫾0.4 kg/m
2
) with
insulin-dependent type 1 diabetes mellitus were investigated. All sub-
jects were plasma C peptide negative and were receiving continuous
insulin treatment (total insulin dose, 0.8 ⫾0.1 U/kg䡠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 A
1c
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 mi-
crodialysis catheter (CMA/60, CMA Microdialysis AB, Stockholm, Swe-
den) consists of a semipermeable polyamide membrane (30 ⫻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 contin-
uously 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% O
2
saturation). Retrograde catheters were
also placed in the cubital veins bilaterally for blood sampling and in-
fusion of insulin and glucose, respectively. After superficial skin anes-
thesia (EMLA, Astra, Sodertalje, Sweden), one microdialysis catheter
was inserted percutaneously into the medial portion of the gastrocne-
mius muscle in one leg. A second microdialysis catheter was inserted
into the periumbilical sc adipose tissue about 8 cm lateral to the um-
bilicus. 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 chlo-
ride at a flow rate of 0.3
l/min. Previous studies have shown that the
recovery of glycerol is almost complete (ⱖ95%) 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䡠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 through-
out 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 ex-
perimental protocol three separate 60-min steady state periods were
achieved (Fig. 1). 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 mod-
erate hyperglycemia.
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
133
Xe clearance technique (19) as previously de-
scribed in detail (15). In adipose tissue,
133
Xe (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 scintil-
lation 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,
133
Xe (0.3 MBq in 0.1
ml saline) was injected in the medial part of the contralateral gastroc-
nemius muscle after 25 min during each steady state period. After 5-min
FIG. 1. Study protocol.
4694 J Clin Endocrinol Metab, September 2004, 89(9):4693–4700 Qvisth et al. •Effect of Insulin and Glucose on Skeletal Muscle Lipolysis
equilibration, the residual activity was monitored for 10 min. In the
skeletal muscle, the
133
Xe decay curve gradually becomes multiexpo-
nential (20). Therefore, the
133
Xe 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
133
Xe washout
curve (20, 21). Adipose tissue and muscle blood flow (TBF) rates were
calculated according to the following formula: TBF ⫽k⫻
⫻100 ml/100
g䡠min, where kdenotes the rate constant of the decay of the residual
activity, and
is the tissue to blood partition coefficient. The values for
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-
2
H
5
]glycerol (0.02
mg/m
2
body surface area䡠min) was administered via an antecubital
vein. A retrograde cannula was inserted into a dorsal vein in the con-
tralateral 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 mus-
cle and into the periumbilical sc adipose tissue, respectively. The cath-
eters were continuously perfused (0.3
l/min) with an artificial extra-
cellular fluid, as described above. After a minimum of 120 min of
equilibration, dialysate samples were collected in 30-min fractions dur-
ing 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 en-
richment. Conversion of interstitial (I) to venous (V) glycerol concen-
trations was made according to the equation: [glycerol]
V
⫽([glyc-
erol)]
I
⫺[glycerol]
a
)⫻(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䡠min in both skeletal
muscle and adipose tissue, as previously suggested (22–24) and dis-
cussed 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 ⫽共关glycerol兴a⫻Ea兲
⫺共关glycerol兴V⫻Ed兲/共关glycerol兴a⫻Ea兲共%兲
Glycerol uptake ⫽共关glycerol兴a⫻Q兲
⫻关1⫺关glycerol兴V⫻Ed兲/共关glycerol兴a⫻Ea兲]] 共nmol/100 g䡠min兲
Glycerol release ⫽Q⫻([glycerol
V
⫺[glycerol]
a
)⫹glycerol uptake
(nmol/100 g䡠min), where E
a
and E
d
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 biolumi-
nescence (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-
2
H
5
]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 (Wil-
mington, 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 Scheffe´’s F test. Linear regression analysis was
performed using the method of least squares. P⬍0.05 was considered
statistically significant.
Results
Arterialized venous blood glucose and plasma free insulin
levels are shown in Fig. 2. Plasma free insulin was 87 ⫾12
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.
Qvisth et al. •Effect of Insulin and Glucose on Skeletal Muscle Lipolysis J Clin Endocrinol Metab, September 2004, 89(9):4693–4700 4695
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 sam-
pling period (B), and finally returned to 11.3 ⫾0.2 mmol/
liter during the last steady state sampling period (C).
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.
3A). Glycerol concentrations in plasma, adipose tissue, and
muscle were all significantly reduced during hyperinsulin-
emia and normoglycemia (P⬍0.0001, by ANOVA). In skel-
etal 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, hyperin-
sulinemic period, with no further reduction during the hy-
perglycemic, hyperinsulinemic period.
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. 3B. In skeletal muscle, the interstitial-plasma differ-
ence 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
FIG. 4. Relationship between the I-A glycerol difference in gastroc-
nemius muscle and the arterialized venous plasma glycerol concen-
tration during steady-state periods of hyperglycemia/hyperinsulin-
emia (A), normoglycemia/hyperinsulinemia (B), and hyperglycemia/
hyperinsulinemia (C), respectively. See Fig. 2 for additional details.
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 glyc-
erol level (I-A difference; B). Statistical differences between study
periods were calculated with ANOVA, repeated measurements, and
post hoc analysis by Scheffe´⬘s F test. *, P⬍0.05 between study periods
A and B; f,P⬍0.05 between study periods B and C. See Fig. 2 for
additional details.
4696 J Clin Endocrinol Metab, September 2004, 89(9):4693–4700 Qvisth et al. •Effect of Insulin and Glucose on Skeletal Muscle Lipolysis
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. 4).
For comparative purposes, the same type of data presen-
tation 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 postab-
sorptive state, no significant correlation was observed be-
tween the fractional glycerol release in the gastrocnemius
muscle and the arterialized venous plasma glycerol concen-
tration (Fig. 5A). During the triacylglycerol infusion, nega-
tive interstitial and arterialized (I-A) glycerol difference val-
ues were registered in five of the six subjects, indicating
definite uptake of glycerol in the muscle. In these five sub-
jects a strong negative relationship (r ⫽⫺0.84, ⬍0.05) was
evident between the I-A glycerol difference and the plasma
glycerol concentration (Fig. 5B).
Figure 6 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 re-
cordings. In adipose tissue, blood flow decreased by approx-
imately 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 hyperinsu-
linemic, normoglycemic and hyperinsulinemic, hyperglyce-
mic periods in the skeletal muscle.
The results of the methodological experiments, in which
regional glycerol kinetics were investigated in the basal state
in healthy subjects, are summarized in Table 1. After the
120-min equilibration period, the enrichment of glycerol in
arterialized venous plasma (8.0 ⫾1.2%) and in skeletal mus-
cle 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 ap-
preciable 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.
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䡠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.
FIG. 6. Effect of circulating insulin and glucose levels on blood flow
rates in adipose tissue and skeletal muscle. See Figs. 2 and 3 for
additional details.
TABLE 1. Glycerol kinetics in skeletal muscle and adipose tissue
Muscle Adipose tissue
Fractional extraction of
[
2
H
5
]glycerol (%)
32 ⫾9⫺2⫾41
Glycerol uptake (nmol/100 g䡠min) 33.8 ⫾10.9 ⫺10.4 ⫾67.1
Glycerol release (nmol/100 g䡠min) 80.7 ⫾15.3 255.5 ⫾34.7
Regional glycerol kinetics in skeletal muscle and adipose tissue
were investigated in four healthy, normal weight subjects in the basal,
postabsorptive state. [
2
H
5
]Glycerol was infused systemically, and
glycerol isotope enrichment was measured in arterialized venous
plasma and in skeletal muscle and adipose tissue microdialysates. For
further details, see Subjects and Methods. Values are the means ⫾
SEM.
Qvisth et al. •Effect of Insulin and Glucose on Skeletal Muscle Lipolysis J Clin Endocrinol Metab, September 2004, 89(9):4693–4700 4697
Discussion
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 endog-
enous insulin secretion, thereby enabling circulating insulin
and glucose levels to be experimentally controlled in a stan-
dardized 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 ef-
fective 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 de-
pends 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 concentra-
tions) should represent a more accurate measure of the local
lipolytic activity. Thus, as expected, we found that hyper-
insulinemia 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. Con-
sistent with recent observations (11, 15, 16), this finding def-
initely argues against an inhibitory effect of insulin on muscle
lipolysis in humans at physiological, normoglycemic condi-
tions. Instead, the decrease in skeletal muscle interstitial glyc-
erol 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 hy-
perglycemia, in contrast, a significant decrease in the frac-
tional 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 ex-
clude 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 ac-
tivity in adipose tissue as well as in skeletal muscle during
combined hyperinsulinemia and hyperglycemia. The molec-
ular 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 bind-
ing 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 phosphatidyl-
inositol 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 effective-
ness 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 unam-
biguous differentiation between in vivo glycerol release and
uptake in a single skeletal muscle in humans. Although var-
ious 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, be-
cause the enzyme responsible for glycerol utilization, glyc-
erokinase, is present in skeletal muscle, but not in adipose
tissue in humans (42). Moreover, in skeletal muscle, the cal-
culated uptake of glycerol was approximately 40% the cor-
responding 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
4698 J Clin Endocrinol Metab, September 2004, 89(9):4693–4700 Qvisth et al. •Effect of Insulin and Glucose on Skeletal Muscle Lipolysis
difference in glycerol observed in both skeletal muscle and
adipose tissue during combined hyperinsulinemia and hy-
perglycemia 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 any-
thing, the uptake of glycerol in muscle tissues is supposed to
be reduced in response to meal ingestion and hyperinsulin-
emia (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 be-
tween 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 intravas-
cular triglyceride hydrolysis in the two tissues may also be
of importance. It is not possible, using microdialysis, to dif-
ferentiate between intracellular (mediated by hormone-
sensitive lipase) and extracellular, LPL-induced lipolysis. In
response to insulin, however, the activity of LPL is stimu-
lated in adipose tissue, whereas the opposite occurs in skel-
etal muscle (43). Therefore, because the reduction in frac-
tional 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 in-
terstitial glycerol level (44), via delivery and removal of the
metabolite by the microcirculation. In this study, we used the
133
Xe washout technique for simultaneous determinations of
absolute blood flow rates in adipose tissue and skeletal mus-
cle. 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 repro-
ducibility 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 demon-
strated in various insulin-resistant conditions (45–47).
Hence, our present data may be explained by disturbed
vascular effects of insulin related to the diabetic state. Dif-
ferences 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䡠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. Never-
theless, the fact that muscle blood flow rates remained un-
changed 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 li-
polysis 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 glu-
cose stimulation. Moreover, from the data in this study we
cannot define with certainty the level of glycemia that po-
tentiates 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 in-
sulin on skeletal muscle lipolysis rates was registered in
either nonobese or obese subjects with normal glucose tol-
erance 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 to-
gether, 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 pa-
tients 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 ef-
fectiveness of the hormone. This mechanism may be impor-
tant for increased triglyceride deposition in skeletal muscle
(and adipose tissue), which is well documented in various
insulin-resistant conditions with subtle abnormalities in glu-
cose control.
Acknowledgments
We acknowledge the excellent technical assistance of B.-M. Leijon-
huvfud, K. Hertel, E. Sjo¨lin, and K. Wåhle´n. We also thank Gary W. Cline
and Frances Rife (Yale General Clinical Research Center) for assistance
with the stable isotope preparation and analyses.
Received April 15, 2003. Accepted June 4, 2004.
Address all correspondence and requests for reprints to: Dr. Jan
Bolinder, Department of Medicine, M 63, Karolinska University Hos-
pital-Huddinge, S-141 86 Stockholm, Sweden. E-mail: jan.bolinder@hs.se.
Qvisth et al. •Effect of Insulin and Glucose on Skeletal Muscle Lipolysis J Clin Endocrinol Metab, September 2004, 89(9):4693–4700 4699
This work was supported by grants from the Swedish Research Coun-
cil, the Swedish Diabetes Association, Karolinska Institute, NIH (RR-
00125), and the Yale Diabetes Endocrinology Research Center
(DK-45735).
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